Volume 2, Issue 2

i
Aims and Scope

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Volume 2, Issue 2 April - June, 2014
ii

Editorial Board
Senior Editor-in-Chief
Prof. Nazeer Khan
Executive Editors
Syed Salman Ahmed,
Sajid Ali
Editor-in-Chief
Asfandyar Sheikh
Managing Editor
Syed Arsalan Ali
Assistant Editor-in-Chief
Haris Sheikh

Assistant Managing Editor
Shanawer Khan

Prof. Asaad Javaid,
Dr. Ye Yang,
Dr. Abdul Hafeez Baloch,
Dr. Mansoor Husain,
Dr. Muzaffar H Qazilbash,
Dr. Tasneem Z Naqvi,
Dr. Asim A Shah,
Dr. Samina Abidi,
Senior Editors
Dr. Rashid Mazhar,
Dr. Mosaddiq Iqbal,
Prof. Javed Akram,
Prof. Abdul Bari Khan,
Prof. Ashraf Ganatra,
Dr. Raza Ur Rehman,
Dr. Waris Qidwai,
Dr. Muhammad Ishaq Ghori,
Dr. Akber Agha,
Dr. Adnan Mustafa Zubairi,
Dr. Saqib Ansari,
Dr. Mohsina Ibrahim,
Dr. Qamaruddin Nizami,
Dr. Samra Bashir,
Dr. Nabeel Manzar,
Muhammad Ashar Malik
Section Editors
Ali Sajjad,
Hafiz Muhammad Aslam,
Syed Askari Hasan,
Muhammad Uzair Rauf,
Syed Mumtaz Ali Naqvi,

Statistics Editors
Mehwish Hussain,
Syed Ali Adnan
Editors
Dr. Hussain Muhammad
Abdullah,
Asfandyar Khan Niazi,
Muhammad Danish Saleem,
Iqra Ansari

Production Editors
Muhammad Hamid
Chaudhary,
Adnan Salim,
Bushra Mufti,
Parisa Aijaz,
Mariam Farooqi
Assistant Editors
Gulrayz Ahmed,
Raza Mahmood Hussain,
Uzair Ahmed Siddiqui,
Maheen Anwer,
Anum Saleem,
Hira Hussain Khan,
Imran Jawaid,
Hina Azhar Usmani,
Hira Burhan,
Quratulain Ghori,
Bushra Iqbal,
Maria Rahim
Layout Editor
Shahzad Anwar

v

Table of Contents
FrontPage i
Editorial Board ii
Call for Papers iii
Health Poster iv
Table of Contents v

Editorial

Novel anticancer agents in clinical and preclinical trials
Adnan Salim
38

Original Articles

Adverse neonatal and maternal outcomes in Pakistani tertiary care
hospitals: A prospective, observational study
Sarah Saleem, Elizabeth M McClure, Janet Moore, Samina Iqbal, Syed Hasan Ala, Fariha Khawaja,
Omrana Pasha, Robert L Goldenberg
40

Incidental gallbladder carcinoma in laparoscopic cholecystectomy: Five
years local experience
Wagih Mommtaz Ghnnam, Turki Maed Al Salem Elbeshry, Jaweed Rafiq Malek, Emad Shebl
Emarra, Mohammed Eid Alzahrany, Ahmad Ali Alqarni, Ammar Ahmad Khattab
47

Impact of SCARB2 on pro-inflammatory cytokines in tissues of EV71-
infected mice
Jian Li, Zhenliang Han, Qingxin Geng, Peipei Liu, Tiegang Lv, Dandan Xin, Yuanyuan Wang, Fei Lei,
Long Song, Zongbo Chen
52

Comparative effect of antiplatelet drugs in streptozotocin-induced diabetic
nephropathy in experimental rats
Taruna Katyal, Jitender Negi, Monika Sachdeva, R D Budhiraja
59

Dorsal hippocampus histaminergic and septum GABAergic neurons work in
anxiety related behavior: Comparison between GABAA and GABAB
receptors
Leila Chodari, Shahrbanoo Oryan, Ramesh Ahmadi, Ghorbangol Ashabi
64

Examination of bone marrow mesenchymal stem cells seeded onto poly(3-
hydroxybutyrate-co-3-hydroxybutyrate) biological materials for myocardial
patch
Junsheng Mu, Hongxing Niu, Fan Zhou, Jianqun Zhang, Ping Hu, Ping Bo, Yan Wang
70

vi

Comparison of linear, logarithmic and mel-frequency filter-bank energy
cepstra in automatic seizure detection using radial basis function neural
network
Chandrakar Kamath
82

Evaluation of foramen magnum in sex determination from human crania by
using discriminant function analysis
Deepali Jain, O P Jasuja, Surinder Nath
89

Mineral content analysis and investigation of antimicrobial activities of
Evolvulus alsinoides (L.) L. against clinical pathogens
Duraisamy Gomathi, Manokaran Kalaiselvi, Ganesan Ravikumar, Kanakasabapathi Devaki,
Chandrasekar Uma
93

Flexural and tensile strengths of three restorative materials used in
pediatric dentistry
Marcia Pereira Alves Dos Santos, Lucianne Cople Maia
97

Comparison of squash smears and frozen sections versus paraffin sections
in the intra-operative diagnosis of central nervous system lesions
Hephzibah Rani, Padmaja Kulkarni, Udupi Shastry Dinesh, Ravikala Vittal Rao, Sateesh Melkundi
101

Research knowledge and behavior of health workers at Federal Medical
Centre, Bida: A task before learned mentors
Ibrahim Taiwo Adeleke, Adedeji Olugbenga Adekanye, Abdullahi Daniyan Jibril, Fausat Fadeke
Danmallam, Henry Eromosele Inyinbor, Sunday Akingbola Omokanye
105

Medical students' perception about teaching-learning and academic
performance at Nobel Medical College, Biratnagar, Nepal
Mukhtar Ansari, Attique ur Rahman Mufti, Salman Khan
110

Short Reports

Microalbuminuria: An early marker of diabetic kidney disease
Zahra Ali, Muhammad Adnan, Saira Bashir, Iffat Shabbir, Tayyaba Rahat
114

A dermatological approach to the feet of soccer players
Asli Feride Kaptanolu, Hasan Ula Yavuz, Kaya Ser
117

Pattern of deliveries during three calendar years in rural India
Rajiv Mahendru, Sunita Siwach, Richa Kansal, Vijayata Sangwan, Ritika Kaur, Pooja Gupta
120

vii

Magnetic resonance imaging cholangiographic evaluation of normal
common bile duct size
Mustafa Fatih Erko, Sevil Alkan, Sinan Soylu, Aylin Okur
122

Review

Do socioeconomic inequalities lead to deceptive measurement of obstetric
morbidity in India?
Kshipra Jain, Mayank Prakash
124

Case Reports

Bilateral mucoepidermoid carcinoma of parotid
Mayank Baid, Vikram Chaturvedi, Jayesh Jha
131

Bilateral granulomatous mastitis after local nandrolone injection
Sadaf Alipour, Akram Seifollahi
134

Unusual presentation of a case of fallopian tube carcinoma
Latika Sahu, Gouri Gandhi, Krishna Agarwal, Sunita Dubey, Preeti Yadav, Richa Gupta
137

Ileoileal intussusception in a young adult secondary to a mucinous
adenocarcinoma
Archana Shetty, Mudasser Rehan, Chowdappa Vijaya
141

Phocomelia A case study
Soniya B Parchake, Nilesh Keshav Tumram, A P Kasote, M M Meshram, Pradeep G Dixit
144

Rectopopliteal fecal fistula developed through an intra-abdominal adhesion
Mustafa Emiroglu, Abdullah Inal, Ismail Sert, Cem Karaali, Cengiz Aydn
146

Atypical mesothelial hyperplasia mimicking mesothelioma in patient with
metastatic papillary carcinoma of thyroid
Mutahir A Tunio, Mushabbab AlAsiri, Syed Azfer Husain, Nagoud Mohamed Omar Ali, Shomaila S
Akbar
148

Primary eosinophilic granuloma presenting as bilateral otitis media and
mastoiditis
Purnima Aggarwal, Uma Debi, Geetanjli Jindal
151

Peripheral ossifying fibroma
S V S G Nirmala, Ramasub Bareddy, Sivakumar Nuvvula, Swetha Alahari, Sandeep Chilamakuri
153

Lipoma of retromandibular space
Anand Gupta, Varun Chopra, Gurvanit Lehl, Shivani Jindal
156
viii

Opinions and Debates

Antidepressants in the management of bipolar depression - An appraisal
Siddharth Sarkar
159

Pre-treatment evaluation: Setting a foundation in the management of drug-
resistant tuberculosis
Saurabh RamBihariLal Shrivastava, Prateek Saurabh Shrivastava, Jegadeesh Ramasamy
164

Essays

Stem cells from gynecological tissue: Trash to treasure
S Indumathi, B Padmanav, D Sudarsanam, B Ramesh, M Dhanasekaran
166

Evolutionary context of hypertensive disorders in human pregnancy
Abhay Kumar Pandey, Anjali Rani, Shripad B Deshpande, B L Pandey
168

Therapeutic spectrum of diuretics in different diseases
Muhammad Majid Aziz, Muhammad Ikram Ur Rehman, Muhammad Wajid, Muhammad Ali Raza,
Javed Ahmed
170

Letters to Editor

Anesthetic considerations and implications for non-cardiac surgery in a
patient presenting with aorto-occlusive disease
Teena Bansal, Manish Bansal, Sarla Hooda
173

Extra-abdominal breast fibromatosis: A rare breast pathology in medical
practice
Mehmet Yildirim, Nkhet Eliyatkn, Hakan Postaci, Nazif Erkan
176

Sarcomatoid lung cancer: a rare, aggressive form of non-small cell lung
cancer with an initially indolent presentation in one of the youngest
documented patients
Arpan Patel, Joshi Sumendra, Dinesh Ananthan, Hayas Haseer Koya
178

A new comprehensive method for treatment of severe intra-uterine
adhesions
Sefa Kelekci, Serpil Aydogmus, Emine Demirel, Mustafa Sengul
180

Organophosphate poisoning presenting as bradycardia
Hari Krishan Aggarwal, Deepak Jain, Shivraj Goyal, Shaveta Dahiya, Ashima Mittal
182

ix

Appendices

Instructions to Authors ix
Sponsorship Information xiii

38 Novel anticancer agents in clinical and preclinical trials

Vol 2, No 2
Open Access Editorial
Novel anticancer agents in clinical and preclinical trials
Adnan Salim
1
Editorial
Billions of people worldwide are affected with various forms of can-
cer of virtually any part of the human body. Despite vast amounts of
funds being poured into cancer research, the production of a single
drug, or a group of drugs, which may cure cancer remains an elusive
dream. The future is not so bleak, though. Many drugs have been
approved recently which combat the cancerous growth and alleviate
quality of life of the patient. Countless others are under trials. What
follows is an attempt to summarize a few.
Akt, or protein kinase B (PKB), is a serine/threonine protein kinase
which acts as a mediator in many cellular processes. Three members
in the Akt family have been identified until now, of which Akt1 is the
molecule playing a key role in cell survival and metabolism. It acts
mainly via the activation of receptor tyrosine kinases (RTK), and pro-
duces such effects as inhibition of apoptosis, promotion of cell cycle
progression and stimulation of angiogenesis. Miltefosine is the only
Akt inhibitor which has been approved (that too for leishmaniasis),
while several others show promise in their pre-clinical trials. These
have been divided into different classes according to their mode of
actions, and some, like Perifosine have failed too. [1].
Poly(ADP-ribose) Polymerases (PARPs) are a group of 17 proteins
which play a role in apoptosis, genetic maintenance, inflammatory
responses and regulation of gene transcription. PARP inhibitors were
developed as agents that seem to target cancer cells when they are
undergoing DNA repair [2]. Olaparib (AZD2281) showed anti-tumor
effects in patients with BRCA1/2 mutated cancers. Patients showed
40% response rate in platinum sensitive ovarian cancer with
germline BRCA1/2 mutations [3]. Rucaparib, another PARP inhibitor
showed promising results with chemopotention when used with te-
mozolomide for metastatic melanoma [4].
c-Met is a proto-oncogene that encodes hepatocyte growth factor
receptor (HGFR) [5]. It plays an important role in embryonic devel-
opment, organ morphogenesis and healing reactions [6]. Met is a
membrane receptor stimulating cell motility, invasion, protection
from apoptosis and angiogenesis. Dysregulated activity of c-Met can
cause a wide variety of cancers, including colorectal, gastric carci-
noma, liver, thyroid, breast, pancreas, renal cell, ovary, prostate and
melanoma [7]. c-Met inhibitors are quite recent drugs. Foretinib
XL880 completed phase 2 clinical trial with indications for head and
neck, gastric and renal cell carcinoma and is still experimental [8].
Cabozantinib (XL184) was approved by the U.S. Food and Drug Au-
thority in November 2012 for the treatment of medullary thyroid
cancer. There are several drugs of this category undergoing trials and
there is promise shown that these used in conjunction with other

1
Dow Medical College, Dow University Of Health Sciences, Baba-e-Urdu Road,
Karachi, Pakistan
Correspondence: Adnan Salim
Email: adnan.salim@mednifico.com
chemotherapeutic agents will significantly alter the course of the dis-
ease [7].
Imatinib, a tyrosine Kinase Inhibitor, is being used widely for the
treatment of chronic myeloid leukemia (CML) [9]. Nilotinib, Da-
satinib, Bosutinib and Ponatinib are newer drugs of this class ap-
proved for the treatment of imatinib resistant or intolerant CML [10,
11].
Histone de-acetylase inhibitors (HDIs) are yet another class of futur-
istic anti-cancer drugs bring used. [12]. Vorinostat (SAHA) and ro-
midepsin (ISTODAX) are FDA approved for the treatment of cutane-
ous T cell lymphoma. Use of HDIs as other types of cancer shows
moderate effects [13, 14].
Vismodegib, a hedgehog pathway inhibitor has been recently ap-
proved for treatment of advanced basal cell carcinoma [15]. Cyclo-
pamine is the prototype inhibitor of the Sonic Hedgehog (Shh) path-
way and is currently undergoing preclinical and clinical studies as an
agent in treatment of basal cell carcinoma, medulloblastoma and
rhabdomyosarcoma [16, 17]. Saridegib, a synthetic analog of cyclo-
pamine, has shown encouraging results in phase I trial of advanced
solid tumors [18, 19].
Heat Shock Protein (HSP) inhibitors, drugs which inhibit molecular
chaperones, though still in phase II clinical trials, show promise in the
treatment of a variety of malignancies [20].
Many rounds of preclinical and clinical trials are still needed to de-
termine accurately the potential of anticancer medicines. While
many may show promise, there is still the question of their therapeu-
tic indices and toxicity profiles. Some of these agents may stop or
revert the growth of a tumor but may adversely affect the patients
health otherwise. Chemotherapy is an exciting and ever-growing
field of research and intense work is being done which promises
hope for health professionals and for the affected.
Competing interests: The authors declare that no competing interests exist.
Received: 28 March 2014 Accepted: 30 March 2014
Published Online: 30 March 2014
References
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8(1):49.
2. Carey LA, Sharpless NE: PARP and cancer--if it's broke, don't fix it. The New
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3. Kummar S, Chen A, Parchment RE, Kinders RJ, Ji J, Tomaszewski JE, Doroshow
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4. Usmani H, Hussain S, Sheikh A: PARP inhibitors: current status and implications
for anticancer therapeutics. Infectious Agents and Cancer 2013, 8(1):46.
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Beachy PA: Effects of oncogenic mutations in Smoothened and Patched can
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40 Adverse neonatal and maternal outcomes in Pakistani hospitals

Vol 2, No 2
Open Access Original Article
Adverse neonatal and maternal outcomes in Pakistani tertiary care hospitals: A prospective,
observational study
Sarah Saleem
1
, Elizabeth M McClure
2
, Janet Moore
2
, Samina Iqbal
3
, Syed Hasan Ala
4
, Fariha Khawaja
1
, Omrana Pasha
1
, Robert L Goldenberg
5

Introduction
Approximately 2.9 million neonatal deaths and 280,000 maternal
deaths occur globally every year [1]. The majority of these deaths
occur in low and middle-income countries (LMIC). Infections, as-
phyxia, and the consequences of prematurity are leading causes of
neonatal mortality [1-3]. Hemorrhage, infection and preeclamp-
sia/eclampsia are leading causes of maternal mortality. Both neona-
tal and maternal mortality are largely preventable with appropriate
care.
In LMIC with high neonatal mortality rates, approximately one-half
of the neonatal deaths are due to infections acquired at home or in
the hospital [2, 4-6]. While the majority of births occur at home, high
risk of infection-related mortality has also been associated with in-
adequate care among facility births. Relatively simple, inexpensive
interventions such as clean delivery and cord care, exclusive breast-
feeding, and hand-washing with clean water and soap should pre-
vent most infection-related neonatal deaths [3, 7-9]. However, inad-
equate antibiotic use also contributes to neonatal sepsis risk and to
higher mortality rates [10].
Medical complications such as preeclampsia and placental abruption
during pregnancy and the intrapartum and postpartum periods in-
crease the likelihood of adverse outcomes for mothers and new-
borns [11-14]. Infants born to mothers with intrapartum hemor-
rhage, fever, prolonged labor and convulsions have higher risks of
mortality compared to those born to women without complications
[15, 16]. Clinical expertise and advanced technologies have signifi-
cantly reduced neonatal and maternal mortality in higher income
countries (HIC). In LMIC, even when simple, inexpensive interven-
tions are available, low quality services, lack of training and health
provider absenteeism are factors contributing to poor services and
high maternal and neonatal mortality at health facilities [17-24]. On

1
Department of Community Health Sciences, Aga Khan University, Karachi Pakistan
2
Department of Statistics and Epidemiology, Research Triangle Institute, Durham,
NC, US
3
Department of Obstetrics, Sobhraj Maternity Hospital, Karachi, Pakistan
4
Department of Obstetrics, Qatar Tertiary Care Hospital, Karachi Pakistan
the other hand, over medicalization of maternal and newborn care
are also becoming problematic in LMIC.
In addition to gaps in health care, socio-demographic and other
household factors clearly contribute to newborn mortality and mor-
bidity. Newborn care practices, such as applying dung to the umbil-
ical cord, have been associated with increased risk for morbidity and
mortality in many LMIC [16-18]. Clean birth care, umbilical cord care
and appropriate hand-washing, in particular, have often been cited
as relatively simple practices that may significantly reduce newborn
mortality [22, 25]. Additionally, the mothers ability to make timely
and appropriate health-seeking choices directly or indirectly affects
her survival and that of her neonate [16-18].
Given the facility and home practices associated with mortality, our
goal was to define the extent of maternal and neonatal mortality and
morbidity occurring in hospital births in urban Pakistan and to de-
termine some of the risk factors related to these outcomes. To do
this, we conducted secondary analyses of data collected as part of a
randomized trial of chlorhexidine vaginal wipes during labor and in-
fant wipes immediately after delivery to prevent perinatal morbidity
and mortality [26].
Methods
From 2005 to 2008, three large tertiary level hospitals serving the
poor, urban population of Karachi, Pakistan participated in a ran-
domized controlled trial of chlorhexidine vaginal and infant wipes
[26]. Each woman in the treatment group received a vaginal wipe
with 0.6% chlorhexidine in labor and the newborn was wiped with
chlorhexidine after birth. Exclusions were known contraindication to
cervical examination, active genital herpes, vulvo-vaginal ulceration,
face presentation, fetal death or planned cesarean delivery. Since
5
Department of Obstetrics and Gynecology, Columbia University, New York, NY, US
Correspondence: Sarah Saleem
Email: sarah.saleem@aku.edu
Abstract
Background: Neonatal and maternal death rates remain high in low-income countries, with little improvement with increasing facility births.
We sought to examine risks for neonatal mortality/morbidity among low-risk hospital deliveries in a low-resource setting.
Methods: Deliveries at tertiary hospitals in Karachi, Pakistan from 2005-2008 were prospectively enrolled with follow-up to 42 days
postpartum.
Results: Of 5,008 women enrolled, 98% were followed 28 days post-delivery. 4.3% of infants had severe illness or neonatal death. 28-day
neonatal mortality rate was 19.1/1,000 births. Newborn death/severe illness was associated with being unbooked, low birth weight, newborn
intensive care, and lack of cord/neonatal skin cleansing and hand-washing before handling the baby.
Conclusion: Although most were low-risk and all were hospital deliveries, maternal and perinatal morbidity and mortality rates were high.
Lack of cord care and maternal hand washing was significantly associated with adverse outcomes. Results suggest that in addition to
improving hospital care, simple interventions such as hand-washing may reduce maternal and newborn morbidities. (El Med J 2:2; 2014)
Keywords: Neonatal Mortality, Neonatal Morbidity, Maternal Mortality, Low-resource Countries, Maternal Risk Factors
Saleem S, McClure EM, Moore J et al 41
there were no significant differences in neonatal infection-related se-
vere morbidity or mortality between the treatment groups, the en-
tire population was combined for this secondary analysis. To reduce
potential confounding from twins, only data from the first born was
considered in this analysis.
The primary outcome for this secondary analysis was severe neonatal
morbidity and neonatal death through 28 days after birth. To deter-
mine the outcome, newborns were followed in-hospital until dis-
charge or death, while an independent field team examined the
mothers and the newborns in home visits on days 7 and 28. The In-
tegrated Management of Childhood Illness (IMCI) screening tool was
used to identify severe illness in neonates [27]. Mothers were inter-
viewed regarding fever, lower abdominal pain, and vaginal discharge
and were examined by trained study doctors. A supplementary ques-
tionnaire on household assets, maternal hygienic practices, and new-
born care practices was completed during the 7 or 28 day home visit
on the final 4,132 study subjects enrolled. This questionnaire ad-
dressed infant cord and skin cleansing, and maternal hand-washing.
Because of the small number of positive responses for several ques-
tions and the overlap in responses, for the final regression analysis,
a single variable was created consisting of appropriate responses to
maternal cord and skin cleansing, and hand-washing prior to han-
dling the baby.
Data was analyzed using SAS version 9.3. For descriptive analyses,
means and standard deviations were calculated for continuous vari-
ables and proportions and percentages were calculated for categor-
ical variables. To determine predictors for neonatal severe illness and
death, logistic regression was used. Crude odds ratios (95% confi-
dence intervals) were generated for each of the risk factors including
maternal and demographic characteristics, intrapartum events, cord
care and hand-washing practices. Risk factors with significant unad-
justed odds ratios were included in a stepwise multivariate logistic
regression analysis to select a final model best describing the effect
of various factors on the risk of neonatal severe illness and mortality.
The institutional review boards at Drexel University (Philadelphia, PA,
USA), RTI International (Durham, North Carolina, USA), and at Aga
Khan University (Karachi, Pakistan) approved the study as did each
of the three study hospitals. The trial was monitored by an independ-
ent data and safety committee established for the Global Network
by the NICHD. The women provided written informed consent prior
to study enrollment.
Results
A total of 5,008 women/infants were enrolled in the study and 5,004
had delivery information available. The 28-day follow-up was ob-
tained for 4,895 infants (97.8%). Ninety three (1.9%) infants were lost
to follow-up (figure 1).
The mean maternal age at enrollment was 25.4 years (SD 4.6) while
the mean and standard deviation (SD) for gravidity was 2.8 (SD 2.0).
Data from the supplemental socio-demographic interview which col-
lected information from the study population on education, employ-
ment, income, and household characteristics are summarized in ta-
ble 1.

Figure 1: Enrolment summary of the research participants
Table 1: Maternal characteristics (n=5,008)
Variable Results
Maternal age, mean (SD)
25.4 (4.6)
Gravidity, mean (SD)
2.8 (2.0)
Number of living children, mean (SD)
1.5 (1.8)
Number of miscarriages, mean (SD)
0.2 (0.6)
Mother received formal schooling*, n (%)
2760 (66.8)
Mother unemployed*, n (%)
4003 (96.9)
Husband unemployed*, n (%)
90 (2.2)
Monthly income*, Rupees, median (range)
5000 (1000-60000)
Rooms in household*, mean (SD)
2.6 (1.5)
Residents of household*, mean (SD)
9.1 (5.0)
*n=4,132
Table 2 presents data related to the delivery hospitalization. 10% of
the study subjects were unregistered to deliver in the study hospitals
and 3.8% were referred from non-tertiary facilities. Upon admission,
7% of women had a known co-morbidity, including hypertension,
hepatitis B and C, tuberculosis, asthma, and diabetes. Of the vaginal
deliveries, 33.7% received an episiotomy and nearly 7% were by for-
ceps or vacuum extractor. 8.3% of the deliveries were cesarean.
Nearly 51% of all women had artificial rupture of membranes
(AROM) to augment labor. More than 11% and 0.4 % of women had
meconium-stained and foul-smelling amniotic fluid, respectively.
While in the hospital, most (94.1%) women received antibiotics.
Table 3 presents the maternal, fetal and neonatal outcomes of 5,004
mothers with delivery data, all singleton infants, and the first born of
61 twins at delivery and at the 7 and 28 day visits. At delivery, 16
infants were stillborn (0.3%). The remaining 4,988 infants (99.7%)
were live births. Approximately 9% of these infants weighed less
than 2500 grams. More than 9% (n=467) of the newborns were re-
ferred to a Newborn Intensive Care Unit (NICU) or another hospital
for an Apgar score <8 at 5 minutes (4.0%) or for other signs of illness.

Vol 2, No 2
On day 7 of follow-up, a total of 4,943 mothers/infants (99%) re-
ceived a home visit. Thirty four infants had died in the hospital and
an additional 45 infants had died between discharge and the day 7
visit for a total of 79. Two mothers were reported dead by day 7, one
in the hospital, and one after discharge. A total of 4,895 mothers/in-
fants (98%) were seen at day 28. Between day 7 and day 28, 16 ad-
ditional infants died, for a total of 95 neonatal deaths (a neonatal
mortality rate of 19.1 per 1,000 live births). By day 28, 212 neonates
had died or were diagnosed with or remained hospitalized for severe
illness. One additional woman died between day 7 and day 28, for a
total of 3 maternal deaths by day 28. Fifty three women (1.1%) were
noted to have a wound or episiotomy infection or dehiscence prior
to 28 days post-delivery.
Table 2: Maternal hospital admission and delivery (n=5,004)
Variable n (%)
Reason for
hospital
admission
Registered case for delivery 4,313 (86.2)
Not registered for delivery 500 (10.0)
Referred from other hospitals 188 (3.8)
Known maternal morbidity at admission 348 (7.0)
Type of
delivery
Vaginal delivery without episiotomy 2,556 (51.2)
Vaginal delivery with episiotomy 1,684 (33.7)
Vaginal delivery with forceps 86 (1.7)
Vacuum assisted delivery 239 (4.8)
Cesarean section 415 (8.3)
Assisted breech delivery 12 (0.2)
Rupture of
membranes
Artificial 2,542 (50.9)
Spontaneous 2,456 (49.1)
Meconium stained liquor 565 (11.4)
Foul smelling vaginal discharge 18 (0.4)
Received antibiotics 4,708 (94.1)

Table 3: Maternal and newborn outcomes (n=5,004)
Outcomes n (%)
Birth
outcomes
Live birth 4,988 (99.7)
Stillbirth 16 (0.3)
Male infant 2,652 (53.2)
Birth weight < 2500 g 460 (9.2)
Apgar score < 8 at 5 minutes 200 (4.0)
Newborn referred to NICU
or other hospital
467 (9.4)
Day 0-7
outcomes
Neonatal death 79 (1.6)
Maternal death 2 (0.04)
Day 7-28
outcomes
Cumulative
28 day
outcomes
Primary neonatal outcome:
death or severe illness
212 (4.3)
Neonatal severe illness 117 (2.4)
Maternal wound or episiotomy
dehiscence/infection
53 (1.1)

Table 4 shows the frequency of various neonatal cord and skin care
practices and maternal hand-washing in the last 4,085 study enrol-
lees. Most mothers reported washing hands before eating or using
the toilet, but nearly 23% of the women reported not usually wash-
ing their hands before handling the baby. 92% of the mothers
cleaned their newborns umbilical cord stump: most often with oil
(78%), water (57%), or antibiotic powder or antiseptic lotions (23%)
(data not shown). Approximately 96% of the mothers reported using
one or more substances to clean or massage the skin. Water (95%)
was most commonly used followed by oil (90%). Other substances
used less frequently were talcum powder, indigenous medicines,
topical corticosteroids and baby lotion.
Table 4: Cord care and hand-washing (n=4,085)
Care n (%)
Usually washes hands before
eating
Yes 4,036 (98.8)
No 49 (1.2)
Usually washes hands after
using toilet
Yes 4,064 (99.5)
No 21 (0.5)
Usually washes hands before
handling baby
Yes 3,159 (77.3)
No 926 (22.7)
Cleansing of cord (water, oil or
other)
Yes 3,725 (92.0)
No 322 (8.0)
Cleansing of skin (water, oil or
other)
Yes 3,882 (95.9)
No 165 (4.1)

Table 5 provides the unadjusted odds ratios (OR) for potential risk
factors for neonatal severe illness and mortality. Neonates of primi-
gravida women (unadjusted OR 1.6, 1.1-2.2) were at increased risk of
severe illness and death as compared to neonates of multiparous
women (p=0.01). Known co-morbidities, extent of maternal school-
ing, and employment status were not associated with severe illness
and neonatal mortality. Neonates whose births were assisted by in-
struments (unadjusted OR 2.8, 95 % CI, 1.9-4.2), or through cesarean
section (unadjusted OR 2.5, 95 % CI 1.7-3.7) were at increased risk
compared to normal vaginal deliveries. Neonates of mothers who
were referred from another hospital (unadjusted OR 3.3, 95 % CI 2.0-
5.3) or whose mothers were not registered (unadjusted OR 1.7, 1.1-
2.5) were at greater risk of developing severe illness or death com-
pared to neonates whose mothers were registered to deliver in the
study hospitals. Those who were born with meconium-stained liquor
(unadjusted OR 3.0, 95% CI 2.2-4.1), had birth weight < 2500 g (un-
adjusted OR 3.4, 95 % CI 2.4-4.7), males (unadjusted OR 1.4, 95 % CI
1.1-1.9), those with low Apgar scores at 5 minutes (unadjusted OR
26.2, 95% CI 18.8-36.6), and those who were referred to the NICU
(unadjusted OR 23.0, 95 % CI 17.0-31.1) were at increased risk of se-
vere illness or death compared to their counterparts.
Neonates of mothers who did not wash hands before eating (unad-
justed OR 4.2, 95 % CI 1.9-9.6), did not wash their hands after using
the toilet (unadjusted OR 4.2, 95 % CI 1.2-14.4), or before handling
the baby (unadjusted OR 2.8, 95 % CI 2.0-3.8) were at increased risk
of developing severe illness and mortality as compared to neonates
whose mothers usually washed hands at those times. Infants who
did not receive cord cleansing were at increased risk of the primary
outcome (unadjusted OR 6.4, 95% CI 4.4-9.4) as were infants with
lack of skin cleansing (unadjusted OR 16.2, 95% CI 10.9, 24.2).
Vol 2, No 2
Table 5: Odds ratios (unadjusted) for demographic and maternal characteristic and severe newborn illness or death by day 28
Variables Severe illness or death by day 28 Unadjusted
OR (95% CI)
P-value
No
+
[N (%)] Yes
@
[N (%)]
Maternal medical/socio-demographic
Known co-morbidities Yes 319 (6.8) 20 (9.4) 1.42 (0.88, 2.28) 0.1483
No 4,345 (93.2) 192 (90.6) Reference
Gravidity One 1,660 (35.5) 97 (45.8) 1.56 (1.11, 2.20) 0.0099
Two to three 1,630 (34.8) 63 (29.7) 1.03 (0.71, 1.50)
More than three 1,390 (29.7) 52 (24.5) Reference
Mother received
formal schooling
Yes 2,618 (66.9) 116 (67.1) Reference 0.9643
No 1,296 (33.1) 57 (32.9) 0.99 (0.72, 1.37)
Mother's employment
status
Employed 118 (3.0) 7 (4.0) Reference 0.4415
Not employed 3,795 (97.0) 166 (96.0) 0.74 (0.34, 1.60)
Husband's employ-
ment status
Employed 3,827 (98.0) 165 (95.4) Reference 0.0206
Not employed 77 (2.0) 8 (4.6) 2.41 (1.14, 5.07)
Type of delivery Normal vaginal delivery 4,000 (85.6) 146 (69.2) Reference <0.0001
Assisted delivery 302 (6.5) 31 (14.7) 2.81 (1.88, 4.22)
Cesarean section 370 (7.9) 34 (16.1) 2.52 (1.71, 3.71)
Select health care factors
Reason for admission Registered case for delivery 4,063 (86.9) 160 (75.8) Reference <0.0001
Not registered for delivery 451 (9.6) 30 (14.2) 1.69 (1.13, 2.52)
Referred from other hospital 163 (3.5) 21 (10.0) 3.27 (2.02, 5.29)
Rupture of membranes Artificial 2,392 (51.1) 98 (46.4) 0.91 (0.66, 1.24) 0.1562
Spontaneous rupture at hospital 1,573 (33.6) 71 (33.6) Reference
Spontaneous rupture at home 712 (15.2) 42 (19.9) 1.31 (0.88, 1.93)
Meconium stained
liquor
Yes 499 (10.8) 55 (26.6) 2.99 (2.16, 4.12) <0.0001
No 4,120 (89.2) 152 (73.4) Reference
Birth weight* < 2500 g 402 (8.6) 51 (24.2) 3.39 (2.43, 4.72) <0.0001
2500 g 4,274 (91.4) 160 (75.8) Reference
Newborn gender Male 2,475 (52.9) 130 (61.6) 1.43 (1.08, 1.90) 0.0133
Female 2,206 (47.1) 81 (38.4) Reference
Apgar score at 5
minutes
< 8 115 (2.5) 84 (39.8) 26.22 (18.81, 36.55) <0.0001
8 4,559 (97.5) 127 (60.2) Reference
Baby referred to NICU Yes 322 (6.9) 133 (63.0) 23.04 (17.04, 31.15) <0.0001
No 4,351 (93.1) 78 (37.0) Reference
Maternal hand washing
Usually washes hands
before eating
Yes 3,838 (99.0) 159 (95.8) Reference 0.0006
No 40 (1.0) 7 (4.2) 4.22 (1.86, 9.58)
after using toilet
Yes 3,861 (99.6) 163 (98.2) Reference 0.0235
No 17 (0.4) 3 (1.8) 4.18 (1.21, 14.41)
before handling baby
Yes 3,039 (78.4) 94 (56.6) Reference <0.0001
No 839 (21.6) 72 (43.4) 2.78 (2.02, 3.81)
Cleansing of cord
(water, oil or other)
Yes 3,601 (92.9) 92 (67.2) Reference <0.0001
No 274 (7.1) 45 (32.8) 6.43 (4.41, 9.37)
Cleansing of skin
(water, oil or other)
Yes 3,758 (97.0) 91 (66.4) Reference <0.0001
No 117 (3.0) 46 (33.6) 16.24 (10.89, 24.21)
*28 missing birth weight;
+
N=4,683;
@
N=212
Table 6a presents the multivariate analysis of risk factors for the in-
fant dying, or having severe illness by day 28 on all subjects in the
study. Referrals for delivery from other hospitals (adjusted OR 2.4
95% CI 1.2-4.5), mothers who were unregistered (unadjusted OR 1.9
95% CI 1.2-3.0), birth weight < 2500 g (adjusted OR 1.9, 95% CI 1.2-
2.8), had low Apgar scores at 5 minutes (adjusted OR 7.3, 95% CI 4.9-
10.9) and referral to an NICU or other hospital (adjusted OR 11.7, 95
% CI 8.2-16.5) remained independent risk factors for neonatal severe
illness and mortality after adjustment for confounding factors. Table
6b presents the multivariate analysis of risk factors for the infant dy-
ing, or having severe illness by day 28 on all 4,032 subjects whose
mothers answered the questionnaire, including data on hand-wash-
ing, cord and skin care. A single variable was created that included
responses to the questions on cord and skin cleansing and maternal
Vol 2, No 2
Table 6(a): Adjusted odds ratios for factors associated with neonatal severe illness or death before 28 days (total population).
Variables Severe illness or death by day 28 Adjusted*
OR (95% CI)
P-value
No
+
[N (%)] Yes
@
[N (%)]
Reason for admission Registered case for delivery 4,056 (87.0) 160 (76.2) Reference 0.0015
Birth weight < 2500 g 400 (8.6) 51 (24.3) 1.86 (1.24, 2.79) 0.0026
2500 g 4,264 (91.4) 159 (75.7) Reference
Apgar score at 5
minutes
< 8 114 (2.4) 83 (39.5) 7.29 (4.88, 10.88) <0.0001
8 4,550 (97.6) 127 (60.5) Reference
No 4,342 (93.1) 78 (37.1) Reference
*Adjusted for listed factor;
+
N=4,664;
@
N=210
Table 6(b): Adjusted odds ratios for factors associated with neonatal severe illness or death before 28 days (population with
questionnaire data only).
Variables Severe illness or death by day 28 Adjusted*
OR (95% CI)
P-value
No
+
[N (%)] Yes
@
[N (%)]
Reason for admission Registered case for delivery 3,460 (89.5) 131 (79.4) Reference 0.0210
Birth weight < 2500 g 335 (8.7) 43 (26.1) 1.88 (1.19, 2.99) 0.0074
2500 g 3,532 (91.3) 122 (73.9) Reference
Apgar score at 5
minutes
< 8 93 (2.4) 65 (39.4) 7.39 (4.64, 11.78) <0.0001
8 3,774 (97.6) 100 (60.6) Reference
No 3,606 (93.3) 59 (35.8) Reference
Cleansing of cord,
cleansing of skin and
usually washes hands
before handling baby
Yes 2,813 (72.7) 62 (37.6) Reference <0.0001
No 1,054 (27.3) 103 (62.4) 3.33 (2.30, 4.83)
*Adjusted for listed factor;
+
N=3,867;
@
N=165
hand-washing before handling the baby. In this model, referrals for
delivery from other hospitals (adjusted OR 2.6 95% CI 1.0-6.4), moth-
ers who were unregistered (unadjusted OR 1.8 95% CI 1.0-3.2), had
a low birth weight (adjusted OR 1.9, 1.2, 3.0), had low Apgar scores
at 5 minutes (adjusted OR 7.4, 95% CI 4.6-11.8) and referral to an
NICU or other hospital (adjusted OR 11.9, 95 % CI 8.0-17.7) remained
independent risk factors for neonatal severe illness and mortality, af-
ter adjustment for confounding factors. Failure to provide cord and
skin cleansing, and hand-washing prior to handling the baby com-
pared to those mothers who usually performed each of these tasks
was associated with an adjusted OR for the primary outcome of
death or severe illness of 3.33 (2.30, 4.83).
Discussion
In this observational study of more than 5,000 relatively low risk
women presenting for delivery in three urban Pakistani hospitals, the
maternal and perinatal outcomes were substantially worse than
those seen in HIC. Although these women presented to the hospitals
with a live fetus and did not appear to require emergency measures,
16 of their fetuses were stillborn, 95 were neonatal deaths and an-
other 117 had severe illness. Additionally, three women lost their
lives. The maternal and neonatal mortality rates were 5-fold or
greater than those seen in HIC.
Experts propose that effective care at the time of birth is a litmus test
of health system performance [22]. It seems likely that better prena-
tal and labor and delivery care for the mother and improved neona-
tal care for the newborn in Pakistani tertiary level hospitals could
improve these outcomes, thereby reducing the high burden of ma-
ternal and neonatal mortality and morbidity. This should include the
use of appropriate technology, presence of skilled staff and imple-
mentation of standard evidence based treatment protocols. Linking
different levels of the health system with communities has been pro-
posed to avoid delays in care [22]. In our study, women who were
not registered to deliver in a hospital or were referred from another
facility were at higher risk of having adverse outcomes for them-
selves and for their newborns.
The over medicalization of maternal care, which has been a concern
among HIC for decades, is becoming a concern for LMIC as well. Prac-
tices fitting this description include routine episiotomy incisions, in-
judicious use of oxytocics, overuse of cesarean sections and artificial
rupture of membranes (AROM) [29]. Unnecessary use of episiotomy
incisions and cesarean sections and their associated risks to mothers
and babies have frequently been noted in the literature [30, 31]. In
our study, one in every three women had an episiotomy incision,
which included women with previous births. Approximately 94% of
the women received prophylactic antibiotics either in the intrapar-
tum or in the post natal period.
More than half the women had AROM. AROM is generally recom-
mended when there is fetal distress or some maternal indication so
that the delivery process can be expedited [30, 32]. We did not col-
lect information on the indications for AROM for this study, but we
believe that the high rate of this practice suggests unnecessary in-
tervention. In a recently published study of 26 hospitals from eight
developing countries, records were reviewed retrospectively to iden-
tify adverse events associated with healthcare management rather
than the underlying disease process [30]. Reported adverse events
ranged from 2.5% to 18.4% per country with nearly 30% resulting in
death of the patient. About 34% of the adverse events followed ther-
apeutic errors in relatively non-complex clinical situations. Inade-
quate training and supervision of clinical staff or the failure to follow
policies or protocols contributed to most adverse events.
In our study, nearly one percent of women remained hospitalized for
more than a week, usually due to complications resulting from sur-
gical and instrumental intervention. In addition, a number of other
infections and dehiscence of episiotomy and cesarean section
wounds occurred after returning to their homes. Therefore, there is
a need to ensure that evidence-based practices are used and used
correctly in hospitals in LMIC.
Some elements of home-based maternal/neonatal care practices
were also explored. Care practices involving the umbilical stump and
skin were generally appropriate, but those infants that did not re-
ceive cord care or cleansing had worse outcomes. Recent papers
suggest that 4% chlorhexidine cord treatment may play an im-
portant role in reducing omphalitis and subsequent systemic infec-
tions [26, 33-35]. Most women reported washing their hands prior to
eating or after using the toilet, but those that did not had worse
outcomes. Nearly a fourth of women did not routinely wash their
hands prior to handling the baby. Failure to do so was also associ-
ated with poor newborn health outcomes.
Potential limitations of the study include that these data were de-
rived from a randomized trial of chlorhexidine targeted at predomi-
nantly low risk mothers and their newborns. The mortality and mor-
bidity rates, while high, would likely have been higher if the popula-
tion of all births was studied. In addition, although the percentage
of mothers and infants lost to follow-up was small, it may be that
some of those lost had died. The actual mortality rates might there-
fore be higher. It would have been interesting to know the cause of
death for mothers, fetuses and newborns, but actual or verbal au-
topsies were not feasible in this setting. We also did not seek infor-
mation on maternal wound care practices for episiotomy and cesar-
ean section incisions. Nevertheless, we believe the data presented
here illustrates some of the practices related to the high maternal
and perinatal mortality rates in LMIC hospitals and emphasizes that
simply moving women from their homes into hospitals for delivery
will not automatically reduce those mortality rates.
In summary, women at apparently low-risk for obstetric complica-
tions who delivered in tertiary level hospitals, all too often faced mor-
bidity, mortality and loss of their newborns. Women mostly faced
morbidities as a sequel of treatment they received while at the hos-
pital. Factors associated with neonatal severe illness and death were
maternal transfers, unregistered status of mother, depressed babies,
and mothers who did not wash hands before handling their babies.
Conclusion
Multiple approaches are required to reduce neonatal and maternal
mortality in facility based care. Improving the quality of care at de-
livery and in the NICU, high quality training of practitioners, appro-
priate use of technology and use of evidence based practices are
needed. At the household level, efforts to make women aware of the
need for clean care practices and especially hand-washing are re-
quired. More research is needed in urban areas to determine the
household factors associated with severe illness and mortality in ne-
onates.
Authors Contribution: SS led the study design and provided overall study
oversight, wrote the initial draft of the manuscript; EMM and RLG participated in
the study design, study oversight and monitoring, and helped write the manuscript;
JM provide statistical monitoring of the study and statistical analyses for the
manuscript; SI, HA and FK provided study design input and oversaw study
implementation; OP and LLW assisted in protocol development, study monitoring
and editing the manuscript. All authors read and approved the final manuscript.
Acknowledgement: This study was funded by grants from the Eunice Kennedy
Shriver National Institute of Child Health and Human Development (NICHD) (grants
U01 HD040607, U01 HD040636), the Bill and Melinda Gates Foundation and Aga
Khan University. CLINICALTRIALREGISTRATION: clinicaltrials.gov, NCT00121394.
Received: 20 January 2014 Accepted: 27 February 2014
Published Online: 27 February 2014
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Ghnnam WM, Elbeshry TMAS, Malek JR et al 47

Incidental gallbladder carcinoma in laparoscopic cholecystectomy: Five years local experience
Wagih Mommtaz Ghnnam
1
, Turki Maed Al Salem Elbeshry
2
, Jaweed Rafiq Malek
2
, Emad Shebl Emarra
2
, Mohammed Eid Alzahrany
2
,
Ahmad Ali Alqarni
2
, Ammar Ahmad Khattab
2

Introduction
Carcinoma of the gallbladder (GBC) is the fifth most common cancer
of digestive tract and the most common malignancy of the biliary
tract. It is an aggressive disease because symptoms and signs usually
appear late and most patients are seen at an advanced stage (which
is due to both the anatomic position of the gallbladder, and the
vagueness and non-specificity of symptoms) with a poor prognosis
[1, 2]. More than 80% of gallbladder cancers are adenocarcinomas
[3]. The clinical manifestations of GBC are generally indistinguishable
from those associated with cholecystitis or cholelithiasis. Around
90% of GBC have accompanying cholelithiasis [4]. Stones and
chronic inflammation are the risk factors for GBC. However, only 0.5
3% of patients with cholelithiasis will actually develop GBC [5].
Cholecystectomy is the most common major abdominal procedure
performed worldwide. This procedure is being performed at an in-
creasing rate according to hospital records [6]. Reports from many
regions of Saudi Arabia show a striking increase in occurrence of gall-
stones during the last two decades [7]. In the era of laparoscopic
cholecystectomy (LC), despite advancements in various diagnostic
procedures, preoperative diagnosis of GBC is an exception rather
than the rule, occurring in fewer than 20% of patients [8]. The inci-
dence of incidental GBC is 0.3-5% of all cholecystectomies. Incidental
GBC has dramatically increased and is now the major way patients
present with GBC. GBC not only presents a diagnostic dilemma but
also poses a difficult treatment option in the era of laparoscopic chol-
ecystectomy [9, 10].
In this single-center study, we report our experience with gallbladder
cancer incidentally diagnosed during or after laparoscopic cholecys-
tectomy performed for gallstone disease.
Patients and Methods
We evaluated the medical records of patients with gallstone disease
who underwent LC in the Department of General Surgery of our Gen-

1
Mansoura University, Egypt
2
Khamis Mushayt General Hospital, Saudi Arabia
Correspondence: Wagih Mommtaz Ghnnam
Email: wghnnam@gmail.com
eral Hospital over the past five years. Routine preoperative assess-
ment was performed in all patients, including liver function tests and
abdominal ultrasonography. Exclusion criterion was existence of
gallbladder polyps detected during preoperative ultrasonography.
All operations were carried out by the authors using the standard
four-port, two-hand technique [11}. Post-operatively all gall bladder
specimens were sent for histopathological examination. Tumor stag-
ing was based on the 7
th
edition of the American Joint Committee
on Cancer (AJCC) manual (Table 1) [12].
Recorded data included patients demographics, details of operative
procedures, perioperative outcomes, tumor histopathology, follow-
up, and long-term survival. All the cases were further consulted with
the oncologists for further adjunctive therapy and postoperative fol-
low-up was done. Follow-up data were obtained for all patients
through them. Statistical analysis was done using SPSS 19 software.
The study was ethically approved by the ethical committee of our
hospital.
Results
During the period from December 2007 to December 2012, we per-
formed 1982 LC procedures. Nearly all patients had cholecystitis or
cholelithiasis. Out of those cases, 10 cases were found to have GBC.
Among them, eight were females and two were males, giving a male
to female ratio of 1:4. These patients were between the ages of 56
and 91 years old. The mean age was 73.6 years (Table 2). Nausea,
vomiting and pain in the right upper quadrant (RUQ) of the abdo-
men was the common clinical presentation in all the ten cases. Two
patients presented with the features of acute cholecystitis, with a
positive Murphys sign. In the remaining patients, the symptoms
were more chronic in nature and of a longer duration, which ranged
from 6 months to 5 years. None of the ten cases were clinically sus-
pected to have GBC preoperatively although all had preoperative
routine ultrasound imaging (Figure 1).

Abstract
Background: Carcinoma of the gallbladder is the most common malignancy of the biliary tract. Most of the cases are diagnosed as an
incidental case among patients undergoing cholecystectomy. The objectives of this study were to report the rate of incidental carcinoma of
gallbladder in patients undergoing cholecystectomy and to study the demographic profile and prognosis of these patients in our locality.
Methods: A retrospective study was carried out in our general hospital during 2007-2012. The hospital records and histopathology reports
of 1982 patients who had undergone elective laparoscopic cholecystectomy were studied.
Results: Out of 1982 cases of cholecystectomy, gallbladder cancer was detected in 10 (0.5%) cases and was more common in females (M:F
ratio = 2:8) .The mean age of occurrence was 73.6 years. Most of the cases diagnosed, were at their early stages and only two of them were
in pT3 pathological stage. Five of those patients survived to date with a mean follow up duration of 26 months.
Conclusion: The rate of incidental carcinoma of gallbladder is 0.5% in our locality and nearly half of patients were early stage with acceptable
five year survival rates. Routine postoperative histopathology of gall bladder is mandatory. (El Med J 2:2; 2014)
Keywords: Gallbladder Carcinoma, Laparoscopic Cholecystectomy, Incidental
48 Incidental gallbladder carcinoma in laparoscopic cholecystectomy
Vol 2, No 2
Table 1: TNM staging of gallbladder cancer [12]
Primary
Tumor (T)
TX Primary tumor cannot be assessed
T0 No evidence of primary tumor
Tis Carcinoma in-situ
T1 Tumor invades lamina propria or mus-
cle layer
T1a Tumor invades lamina propria
T1b Tumor invades the muscle layer
T2 Tumor invades the perimuscular con-
nective tissue; no extension beyond the
serosa or into the liver
T3 Tumor perforates the serosa (visceral
peritoneum) and/or directly invades
the liver and/or one other adjacent or-
gan or structure, such as the stomach,
duodenum, colon, or pancreas, omen-
tum or extra hepatic bile ducts
T4 Tumor invades main portal vein or he-
patic artery or invades multiple extra
hepatic organs or structures
Regional
Lymph
Nodes (N)
NX Regional lymph nodes cannot be as-
sessed
N0 No regional lymph node metastasis
N1 Regional lymph node metastasis
Distance
Metastasis
MX Distant metastasis cannot be assessed
M0 No distant metastasis
M1 Distant metastasis

All cases had gallbladder stones. None of those patients were diag-
nosed for GBC preoperatively. Three cases needed conversion to
open cholecystectomy and two of them were suspected during con-
version, so extended cholecystectomy was done for both of them
with excision of a wedge shaped area of the liver from the gallblad-
der bed (cleared later by histopathology [Figure 2]) with resection of
visible and palpable lymph nodes.
Out of the 10 cases of incidental GBC, two patients died during hos-
pitalization due to unrelated causes (acute myocardial infarction and
massive cerebral hemorrhage). Two more cases died after 8 and 13
months of diagnosis due to causes unrelated to GBC. One patient
died due to metastatic disease 3 months after surgery. Five patients
(50%) have survived to date with a mean follow up duration of 26
months. The details of the 10 cases of incidental GBC has been de-
scribed in (Table 2).

Since, our hospital is a secondary care center, the cases were referred
to an oncology center after histological diagnosis of malignancies for
further treatment but we did establish contact with patients and rel-
atives for estimation of the survival and mortality. The majority of
the patients were in early pathological stages (pT1-3) and none was
in pT4 stage (Figures 3-6).

Figure 1: Pre-operative ultrasonographic picture of one case of GBC with thick
walled gallbladder and multiple gall stones.
Figure 2: Attached hepato-parenchymal tissue. There is no tumor invasion.
Figure 3: Intact mucosa is seen on the right. Abrupt area of tumor in which
neoplastic structures invade the adventitia/serosa layer through the fragmented
muscular layer.

Figure 4: Mucinous adenocarcinoma with mucin secretion (mucin pools).
Figure 5: Gross picture of one case of GBC with diffuse wall thickening more in
the neck and multiple gallstones.
Table 2: TNM staging of gallbladder cancer* [12]
Case Age Sex Diagnosis
(duration in years)
Ultrasound
Findings
Operative
Findings
Operation
Type
Histological
Diagnosis
Tumor
Stage
Survival
1 91 F CCC(4) CCC,TW,GBD LC No susp SLC WDA pT2 Died
2 90 F CCC(3) CCC,TW,GBD LC No susp SLC MDA pT3 Died
3 74 F CCC(1) CCC,TW,GBD OC Susp C+LR+LN PDA pT2 Died
4 56 F CCC(0.5) CCC,TW,GBD LC No susp SLC WDA pT1a Alive
5 62 F CCC(4) CCC,TW,GBD LC No susp SLC WDA pT1a Alive
6 69 M CCC(3) CCC,TW,GBD LC No susp SLC WDA pT1b Alive
7 77 M A&CCC(5) CCC,TW,GBD,PC OC Susp C+LR+LN MDA pT3 Died
9 64 F CCC(2) CCC,TW,GBD,PC OC No susp SLC PDA pT1b Died
*A&CCC=Acute on top of chronic calcular cholecystitis; CCC=Chronic calcular cholecystitis; GBD=Gallbladder distended; Susp=Suspicious; TW=Thick wall gallbladder; PCF=Pericholecystic fluid;
LC=Laparoscopic cholecystectomy; OC=Open cholecystectomy; SLC=Simple laparoscopic cholecystectomy; C+LR+LN=Cholecystectomy+localized liver resection+lymph nodes excision;
WDA=Well differentiated adenocarcinoma; MDA=Moderately differentiated adenocarcinoma; PDA=Poorly differentiated adenocarcinoma

Discussion
Carcinoma of the gallbladder is a rare gastrointestinal malignancy.
However, it is reported to be the most frequent carcinoma of the
extra-hepatic biliary tract. Symptoms are non-specific and the diag-
nosis is often made at an advanced stage at operation for routine
cholecystectomy. Thus, the outcome for GBC remains largely dismal
[13].
We reported 7 cases of GBC out of 47 cases reported in Saudi Arabia
during the period from 2007 to 2011 [14]. Our results agree with
literatures in that GBC is two to six times more common in women
than men. Incidence increases with age and more than 75% of pa-
tients with this malignancy are older than 65 years [15, 16]. Literature
review revealed that 0.3% to 4.6% of the patients who underwent
cholecystectomy for presumed benign disease were found to have
GBC with more common incidence in eastern than western coun-
tries. The proportion of GBC detected in patients undergoing chole-
cystectomy for cholelithiasis in our study was 0.5% and is quite com-
parable with other literature (Table 3) [17-20]. In the present retro-
spective study, reviewing 10 patients with GBC surgical treatment
remains the only treatment modality associated with a benefit in
terms of survival. Simple cholecystectomy may be sufficient for pa-
tients with pT1a tumors by the TNM classification with cure rates
ranging from 73 to 100%, even those diagnosed following LC [2].
Eight patients in our study had cholecystectomy alone as a definitive
treatment for GBC, the lesion was (pT1a stage) in 4 patients, pT1b
stage in two patients and pT2 in two more patients. For pT1b tumors
(tumor invading the muscular layer) the benefit of radical resection
is controversial. We performed simple cholecystectomy in those pa-
tients and later they were referred to oncologist for further treat-
ment. No further surgery was required and no recurrence was noted
during the follow up period. Some authors advocate radical resec-
tion on the basis of evidence of lymph node metastases in these pa-
tients as well as disease in gallbladder fossa. Thus, performing a lym-
phadenectomy with excision of at least a wedge of liver tissue from
segments IV b and V may be indicated in pT1b Tumors [22].
The benefit of radical resection may be particularly pronounced
among patients with muscle layer invasion as they have higher inci-
dence of lymph node metastases. Excision of at least 2cm of liver
tissue from the gallbladder fossa with lymphadenectomy were per-
formed in two patients with tumor invading the muscle layer (pT3)
proved by histologic examination of the cholecystectomy specimen.
During recent years, extended operations combining a resection of
the liver with wide lymph node dissection have improved the long-
term survival [23, 24]. However, there are still many controversies
regarding the type of surgical treatment for each stage of the disease
[25].
The widespread use of laparoscopic techniques has led to an in-
crease in referrals for cholecystectomy. As a consequence, the inci-
dental finding of GBC at an earlier stage has altered the management
and the outcome of the disease. However, GBC remains a lethal dis-
ease associated with a dismal prognosis. Controversies exist on the
optimal treatment of this unexpected finding during routine laparo-
scopic cholecystectomy. The management is difficult because no
guidelines have been established and some authors have reported
worse overall prognosis when patients have not been adequately
treated during the first operation. Simple cholecystectomy is gener-
ally considered as an adequate treatment for pT1b patients, but this
is still a matter under discussion. Shirai has reported a favorable out-
come regardless of the type of operation done (cholecystectomy
alone or extended cholecystectomy) in early stages [13]. Advanced

Figure 6: Perineural invasion (blue stars indicate nerve).
50 Incidental gallbladder carcinoma in laparoscopic cholecystectomy
Vol 2, No 2
Table 3: Comparison between other studies and our study
Author Year N Cancer (%) F/M ratio Mean Age pTis, pT1 pT2, pT3, pT4 Median
Survival
(months)
Sarli et al [33] 2000 2300 9 (0.39) 6/3 62.3 4 5 12
Daphna et al [17] 2002 1697 6 (0.35) 5/1 70 1 5 8
Antonakis et al [34] 2003 5539 11 (0.2) 8/3 57 0 11 8.1
Yamamoto et al [35] 2005 1663 9 (0.54) 4/5 73 4 5 19
Shimizu et al [36] 2006 1195 10 (0.84) 7/3 61.4 4 6 62.5
Amanullah et al [18] 2007 428 8 (1.9) 7/1 47 5 3 12
Yokomuro et al [20] 2007 84 4 (4.7) 2/2 75 2 2 NA
Kwon et al [38] 2008 1793 38 (2.12) 21/17 66 20 18 68
Tantia et al [16] 2009 3205 19 (0.59) 14/5 56 16 3 18.4
Choi et al [38] 2009 3145 33 (1.05) 24/9 63 12 21 46.3
Zhang et al [39] 2009 10466 20 (0.19) 16/4 65.7 8 12 43
Morera et al [19] 2009 372 6 (1.1) 2/4 63.8 2 4 23
Ghimire et al [40] 2011 783 10 (1.28) 7/3 63.8 8 2 8
Cavallaro et al [41] 2012 1490 19 (1.2) 11/8 68 7 12 38
Ioannidis et al [42] 2013 1536 14 (0.9) 11/3 69.4 5 9 NA
Our study 2013 1982 10 (0.5) 8/2 73.6 6 4 26
stage of the disease, because of delayed diagnosis, leads to its poor
prognosis [26-30].
Gallbladder carcinomas are epithelial in origin and they account for
98% of all the gallbladder malignancies. Among these, adenocarci-
nomas account for 90% of all the carcinomas of the gallbladder [31].
In our series, all were adenocarcinoma and one had mucinous areas
(Figure 4). The sub mucosal spread of the infiltrating carcinomas ap-
pears grossly as focal or diffuse areas of wall thickening, nodularity
or induration in the gallbladder wall. Similar gross features were seen
in all the 10 cases of incidental GBC in this series, and none of them
showed any intraluminal growth or mass lesion (Figure 5).
In as many as 50% of cases, GBC are discovered at pathologic analysis
after simple cholecystectomy for presumed gallstone disease. Five-
year survival is 50% for patients with pT1 tumors. Patients with pT2
tumors have a 5-year survival rate of 29%, which appears to improve
with more radical resection. Patients with lymph node metastases
(pN1) or locally advanced tumors (PT4) rarely experience long-term
survival. The site-specific prognostic factors include histologic type,
histologic grade, and vascular invasion. Papillary carcinomas have
the most favorable prognosis. Unfavorable histologic types include
small cell carcinomas and undifferentiated carcinomas. Lymphatic
and/or blood vessel invasion indicate a less favorable outcome. His-
tologic grade also correlates with outcome [12].
The importance of a histological examination of the post-cholecys-
tectomy specimens cannot be over-emphasized. The non-specific
clinical features and the sonographic findings of early GBC make the
pre-operative diagnosis difficult and an incidental GBC has been rec-
orded in every reported series of laparoscopic cholecystectomy
cases. Thus we agree with other surgeons who recommend surgery
even for the patient with asymptomatic gallbladder stones because
of its likelihood to harbor occult malignancy [32].
Conclusion
Although GBC incidence in our locality is low, yet prognosis is dismal
and survival rate does not exceed 50% in early-stage treated cases.
Thus, routine histopathological examinations of all cholecystectomy
patients is recommended. Simple or extended cholecystectomy in
our experience is the treatment of choice for early GBC discovered
accidently in laparoscopic cholecystectomy and further surgery may
be needed according to multifactorial issues particularly histopatho-
logical stage and grade. The study cases are few, yet they are the
available. However, further studies are needed to fully establish
guidelines for incidentally discovered GBC during laparoscopic chol-
ecystectomy.
Received: 12 December 2013 Accepted: 10 January 2014
Published Online: 10 January 2014
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52 Scavenger receptor B2 in enterovirus 71-infected mice

Vol 2, No 2
Impact of SCARB2 on pro-inflammatory cytokines in tissues of EV71-infected mice
Jian Li
1
, Zhenliang Han
1
, Qingxin Geng
2
, Peipei Liu
1
, Tiegang Lv
1
, Dandan Xin
1
, Yuanyuan Wang
1
, Fei Li
1
, Long Song
1
, Zongbo Chen
1

Introduction
Enterovirus 71 (EV71), a neurotropic virus with undefined pathogen-
esis, has caused significant morbidity and mortality throughout the
world, especially in the Asia-Pacific region since it was first detected
in 1969 in the United State, including Singapore, South Korea, Ma-
laysia, Japan, Vietnam, Taiwan, and China mainland [1-10]. EV71, to-
gether with coxsackievirus A16 (CVA16) infections are generally as-
sociated with hand, foot and mouth disease (HFMD), but EV71 infec-
tion occasionally progress to severe neurological disease, including
aseptic meningitis, poliomyelitis-like paralysis, and possibly fatal en-
cephalitis in neonates, especially brain stem encephalitis associated
with pulmonary edema and cardiac insufficiency which are the pri-
mary manifestations in patients with neurologic involvement [11,
12]. Numerous animal models have been developed to study the
pathogenesis of EV71 infection using the mouse-adapted strain of
EV71 in innate immunodeficient mice [13-15]. The EV71 BrCr strain
was demonstrated to induce neurological manifestation of tremor,
ataxia, and brain edema in cynomolgus monkeys [16]. Moreover,
EV71 BrCr infected mice also developed limbs paralysis and enceph-
alitis [17].
SCARB2 (also known as Lysosomal Integral Membrane Protein II,
LIMP II, LGP85 or CD36b like-2) is composed of 478 amino acids and
belongs to the CD36 family, which includes CD36 and scavenger re-
ceptor B, member 1 (SR-B I and its splicing variant SR-B II) [18, 19].
SCARB2 is one of the most abundant proteins in the lysosomal mem-
brane and participates in membrane transportation and the reorgan-
ization of the endosomal/lysosomal compartment [19-21]. SCARB2
shuttles between these compartments and the plasma membrane
[19]. SCARB2 is a type III double-transmembrane protein with a large
extracellular domain (when it is present at the cell surface) and short
cytoplasmic domains at the amino- and carboxy-terminus [18].
SCARB2 is expressed in a variety of tissues, including neurons in the

1
Pediatric Department of the Affiliated Hospital of Qingdao University Medical
School, China
2
Department of Cardiology, Jinan Central Hospital, Shandong University, China
CNS. SCARB2 deficiency in mice causes ureteric pelvic junction ob-
struction, deafness, and peripheral neuropathy, and SCARB2 defi-
ciency in humans causes action myoclonus-renal failure syndrome
(AMRF) [22, 23]. The role of SCARB2 appears to be connected to the
TNF--dependent and early activation of Listeria macrophages
through internal signals linking the regulation of late trafficking
events with the onset of the innate Listeria immune response [24].
Animal models have been developed to detail the pathogenesis of
EV71 infection. However, the majority of the research has been de-
voted to understanding the neurotropism and neuropathogenesis of
EV71, whereas the immunopathogenesis aspect of the viral infection
has remained largely unknown. It was proposed that overwhelming
virus replication combined with the induction of massive pro-inflam-
matory cytokines is responsible for the pathogenicity of EV71 [25-
27]. Indeed, high levels of interleukin-1 (IL-1), IL-6, IL-10, IL-13,
gamma interferon (IFN-), and tumor necrosis factor alpha (TNF-)
in the serum and cerebral spinal fluid (CSF) from EV71-infected pa-
tients have been consistently reported [25, 27, 28]. In particular, CSF
levels of IL-1, IL-6, and TNF- were found significantly elevated in
patients with pulmonary edema (PE) and encephalitis, demonstrat-
ing a strong correlation between pro-inflammatory cytokine produc-
tion and clinical severity in EV71 infections [26, 29]. EV71-infected
neonate mouse model also sustained high levels of IL-6 [30].
SCARB2-deficient mice display a macrophage-related defect in Lis-
teria innate immunity. They produce less acute phase pro-inflamma-
tory cytokines/chemokines, MCP-1, TNF-, and IL-6, but normal lev-
els of IL-12, IL-10, and IFN- and 25-fold increase in susceptibility to
Listeria infection [24].
In this study, we assessed the expression of SCARB2 and the produc-
tion of pro-inflammatory cytokine during EV71 infection in the neo-
natal mouse. Our results indicate that EV71 infection leads to the
Correspondence: Zongbo Chen
Email: chenzbdr001@163.com

Abstract
Background: Scavenger receptor class B, member 2 (SCARB2) participates in early innate immune responses to infection. Our aim in this
study was to explore the expression and role of mouse SCARB2 (mSCARB2) in different tissues in EV71-infected mice.
Methods: ICR mice were inoculated intraperitoneally (i.p.) with EV71 0.1 ml 10
7.5
TCID50 /ml. The control mice were injected i.p. with the
same volume RD cell lysate. Mice were sacrificed by aether anesthesia at day 4, 8 and 12 post infection (p.i.). Their brain, brainstem, spinal
cord, cerebellum, lung and heart were dissected out for determining the number of copies of viral RNA by quantitative real-time PCR (qRT-
PCR). Detection of expression of mSCARB2 was performed by immunohistochemistry and qRT-PCR. Cytokines quantification was done by
ELISA.
Results: The viral loads in central nervous system (CNS) were higher than in lung or/and heart. The expression of mSCARB2 increased in
tissues of EV71-infected mice. However, the levels of mSCARB2 increased in CNS were higher than in lung or/and heart within a certain
period of time, particularly in brain stem and brain. In addition, local TNF-, IL-6 and IL-1 levels of production were consistent with
mSCARB2 levels of expression in tissues of EV71-infected mice. However, it presented a positive correlation between relative mSCARB2
mRNA level and TNF-, IL-6 and IL-1 levels in local tissues at day 4 and 8 p.i.
Conclusion: Our data revealed that the elevated local mSCARB2 may modulate pro-inflammatory cytokines induction in local tissues,
particularly, in CNS of EV71-infected mice. (El Med J 2:2; 2014)
Keywords: Enterovirus 71, Mice, mSCARB2, Pro-inflammatory Cytokines
Li J, Han Z, Geng Q et al 53
expression of SCARB2 increased in different tissues, which correlated
with the local elevated levels of pro-inflammatory cytokine induc-
tion, especially in CNS.
Materials and Methods
Cells and viruses
Human rhabdomyosarcoma (RD) cells (purchased from the Chinese
Academy of Sciences Cell Bank, Shanghai, China.) were maintained
in Dulbecco's Modified Eagle's Medium (DMEM, Gibco) containing
10-15% fetal bovine serum (FBS, Gibco), 2 mM L-glutamine, 100 IU
of penicillin, and 100 g of streptomycin/ml at 37
o
C, 5% CO2. Non-
mouse-adapted EV71 strain BrCr (a kind gift from Institute of Medical
Biology, Chinese Academy of Medical Sciences & Peking Union Med-
ical College, Kunming, China) was propagated in RD cells. Once the
cells displayed cytopathic effect (CPE), they were harvested, and cel-
lular debris was removed by centrifugation at 10,000g for 30 min.
To prepare virus stocks, virus were propagated for one more passage
in RD cells. The virus was purified by Amicon Ultra 100 K device
(Millipore) at 4,000g for 40 min. The 50% tissue culture infective
dose (TCID50) was determined in RD cells using the Reed and Muench
formula, and working virus stocks at 10
7.5
TCID50 per ml [31].
Animals and treatments
ICR mice were purchased from Laboratory Animal & Animal Experi-
ment Center, Qingdao, China. They were housed under specific path-
ogen-free conditions. All institutional guidelines for animal care and
use were strictly followed throughout the experiments. One-day-old
ICR mice were inoculated intraperitoneally (i.p.) with EV71 0.1 ml
10
7.5
TCID50 /ml. The control mice were injected i.p. with the same
volume RD cell lysate and kept in separate cages. Their weight gain
or loss and clinical signs, including ruffled fur, hunchback, wasting,
limb weakness, limb paralysis, twitch, moribund and death were
monitored daily up to 14 days after inoculation. The clinical score
was graded as follows: 0, healthy; 1, weakness in hind limbs; 2, pa-
ralysis in a single limb; 3, paralysis in more than two limbs; 4, death
[32]. In addition, mice per group were sacrificed by aether anesthesia
at day 4, 8, and 12 post infection, respectively. After perfusion with
PBS containing EDTA, their brain, cerebellum, brainstem, spinal cord,
heart and lung were immediately dissected out for the extraction of
RNA, for the extraction of protein or for immunohistochemical exam-
inations, respectively. The experimental protocol was approved by
the Animal Care and Use Committee of the Institute of Laboratory
Animal Science of Chinese Academy of Medical Sciences.
Virus detection in mice
For this study, quantitative real-time PCR (qRT-PCR) was used to de-
termine the number of copies of viral RNA present in detected tis-
sues. Total RNA was extracted from individual brain, cerebellum,
brainstem, spinal cord, heart and lung using an RNAiso Plus Kit
(Takara, Dalian, China) according to manufacturers' instruction. Next,
total RNA was reverse-transcribed with random hexamers using a
Reverse Transcription kit (Thermo Scientific). The cDNA was sub-
jected to quantitative PCR in a 50-l reaction mixture (Thermo Sci-
entific DyNAmo SYBR Green qRT-PCR Kit) with primers of EV71-S (5'-
GCAGCCCAAAAGAACTTCAC-3') and EV71-A (5'-ATTTCAGCAGCTT-
GGAGTGC-3') for EV71/BrCr of nucleotides 2372-2598, and the con-
ditions consisted of a denaturation step at 95
o
C for 15 min and 40
cycles of thermal cycling of 95
o
C for 10 s and 60
o
C for 60 s [14, 33].
The EV71 virus fragment of nucleotides 2372-2598 was used as real-
time PCR standard by adjusting to a concentration gradient of 110
7

copies/l, 110
6
copies/l, 110
5
/l, and 110
4
copies/l, and the
DNA fragment with known copies was used as standard to calculate
the copy number of virus RNA in the infected tissues. Quantitative
real-time RT-PCR was performed using the Mxpro-Mx3000P system.
Immunohistochemical staining
The tissues from sacrificed mice were rinsed in 10% buffered forma-
lin and then embedded in paraffin. Four micrometer sections were
slided (Leica RM 2235) and placed on poly-L-lysine-coated glass
slides before fixing with 3.7% paraformaldehyde. The sections were
blocked by endogenous peroxidase for 10 min, nonspecific protein
binding sites were also blocked for 10 min. The sections were incu-
bated with mSCARB2 antibody (Abcam discover more) 1:100 for 1
h, and then were incubated with secondary antibody IgG-Biotin and
Streptavidin-HRP (Streptavidin-HRP Kit, CWbio.Co.Ltd, Beijing, China)
for 10 min at room temperature, respectively. A red to brown perox-
idase stain was developed using the DAB Chromogenic Reagent kit
(CWbio.Co.Ltd, Beijing, China), and the sections were examined with
a light microscope after counterstaining with hematoxylin.
Detection of mSCARB2 gene expression
To examine mSCARB2 expression, total RNA from different tissues of
EV71-infected mice and controls using an RNAiso Plus Kit (Takara,
Dalian, China) following the manufacturer instructions were isolated.
Total RNA was converted into cDNA by the reaction of reverse tran-
scription (RT) using a Reverse Transcription kit (Thermo Scientific).
The cDNA was subjected to quantitative PCR (Thermo Scientific Dy-
NAmo SYBR Green qRT-PCR Kit) with a Rotor-Gene RG-3000 System.
The primers were mSCARB2-L1 (5'-TCTGCTGTCACCAATAAGGC-3')
and mSCARB2-R1 (5'-CCAGATCCACGACAGTCAAC-3'). The conditions
consisted of a denaturation step at 95
o
C for 15 min and 40 cycles of
thermal cycling of 95
o
C for 10 s and 60
o
C for 60 s. The GAPDH was
used as an internal control. The relative gene expression was calcu-
lated using the 2
-Ct
as described previously [34]. Each sample was
run in triplicate.
Cytokine quantification
Various tissues were harvested from sacrificed animals at indicated
time point, weighed, and homogenized in 500 l of 1phosphate-
buffered saline (PBS) immediately. The homogenates were centri-
fuged at 13,000g for 10 min at 4
o
C, and the supernatant was col-
lected and stored frozen at -80
o
C until further analysis. The levels of
cytokines were measured using a Solid Phase Sandwich ELISA kits
(Mouse TNF-, IL-6 and IL-1 Quantikine, R&D Systems), and follow-
ing the manufacturer's instructions. Sensitivities of the TNF-, IL-6
and IL-1 assays according to manufacturer protocol were 7.21
pg/ml, 1.8 pg/ml and 4.8 pg/ml, respectively. Intraassay and interas-
say coefficients of variation were: TNF-: 3.9% and 6.2%; IL-6: 3.9%
and 8.9%; IL-1: 4.6% and 6.6%.
Statistical analysis
All statistical analyses were done with GraphPad Prism, version 5.0
(GraphPad 4 Software, San Diego, CA), for Mac. Kaplan-Meier survival
curves were analyzed by a log rank test. Clinical score curves were
analyzed by the Kruskal Wallis test. Other experiments were analyzed
Vol 2, No 2
by Student's t-test or by one-way analysis of variance (ANOVA) fol-
lowed by Tukey's multiple comparison tests. Pearson's correlation
was used to analyze the relation between pro-inflammatory cyto-
kines and mSCARB2. A P-value of <0.05 was considered as statisti-
cally significant.
Results
EV71 infection in mice
Survival rate and disease score curves were used to display the num-
ber of mice that died or became sick. The mice infected with virus
were monitored daily for 14 days after inoculation with virus. In this
study, infected mice developed severe symptoms. Fatigue in the
hind limbs occurred at day 1-2 p.i., followed by paralysis in a single
limb or/and paralysis in more than two limbs at days 3-7 p.i., or
showed other signs of encephalitis such as hunched posture, leth-
argy, or ataxia, and death occurred at 2-7 p.i.. The healthy mice in
the cell lysate control group did not have a single mouse dead.
Among the observed three groups (A, B, C), the survival curves were
not significantly different (Figure 1A). But 7-8 days later, the survi-
vors' symptoms gradually restored. In the three groups (A, B, C), the
clinical scores were not significantly different (Figure 1B). However,
their body weights appeared to grow slowly.
EV71 strain BrCr displays neurotropism in ICR mice
The viral loads in spinal cord, brain stem, cerebellum, brain, heart
and lung from animals i.p. infected with EV71 strain BrCr were mon-
itored by qRT-PCR. At day 1 p.i., viral RNA were only detected in spi-
nal cord, but were not detected in brain stem, cerebellum, brain,
heart and lung. The number of copies of EV71 RNA detected at day
4 p.i. were in lung (3.99 0.13 log10 copies/mg tissue), heart (3.11
0.12 log10 copies/mg tissue), brain (5.31 0.30 log10 copies/mg tis-
sue), brain stem (6.17 0.18 log10 copies/mg tissue), spinal cord (5.59
0.12 log10 copies/mg tissue), and cerebellum (4.51 0.26 log10 cop-
ies/mg tissue). However, the virus was gradually eliminated (Figure
2 A, B, C, D, E, F). A histopathological examination of the infected
mice in different time was carried out. Marked lesions and/or obvious
signs of inflammation were observed for the brain, brain stem, spinal
cord, cerebellum, but heart and lung showed less lesions and/or
signs of inflammation (data not shown).

Different tissues of EV71-infected mice express mSCARB2
We conducted immunohistochemical studies to determine the ex-
pression of mSCARB2 in different tissues at different time point. Ex-
pectedly, mSCARB2 immuno-reactivity was not only observed in
lung, heart, brain, brain stem, spinal cord and cerebellum cells, but
also the obvious immuno-reactivity was observed at day 4 p.i. com-
pared to controls, and gradually decreased in later days (Figure 3).
These results suggested that expression of mSCARB2 increased in
these tissues after mice with EV71 infection.
To quantitatively measure mSCARB2 mRNA in different tissues and
to determine whether mSCARB2 expression localized to specific tis-
sues in EV71-infected mice, we tested the mSCARB2 gene expression
in different tissues at different time point by qRT-PCR, and found that
the mSCARB2 mRNA levels were elevated in all selected tissues at
day 4 p.i., but the mSCARB2 mRNA levels were higher in brain stem
(P<0.001), brain (P<0.01), spinal cord (P<0.01) and cerebellum
(P<0.05) than in lung or/and heart. However, at day 8 p.i., the
mSCARB2 mRNA levels obviously decreased; still in brain stem
(P<0.001), brain (P<0.05) were higher compared to lung or/and
heart. At day 12 p.i., only the mSCARB2 mRNA level in brain stem

Figure 1: One-day-old ICR mice were inoculated i.p. with EV71. The survival
rates (A) and clinical scores (B) of the infected mice (A group, n=6) were
monitored over a 14-day period. Results are representative of 3 independent
experiments.

A B
C D
E F
Figure 2: Viral loads in tissues from ICR mice infected with EV71 via The i.p.
route. One-day-old mice were inoculated i.p. with EV71. At day 2, 4, 6, 8 and 12
p.i., animals (n=6) were euthanized, and virus titers in the lung (A), heart (B),
brain (C), brain stem (D), spinal cord (E), and cerebellum (F) were determined
by qRT-PCR. Results are expressed as log viral RNA copies per milligram of
tissue and values are means SEM of triplicate experiments.
were observed higher than in lung or/and heart (P<0.05) (Figure 4
A). Figure 4 A also showed that the mSCARB2 mRNA levels in brain
stem and brain were higher than in spinal cord and cerebellum.
These results suggest that the expression of mSCARB2 increased in
CNS of EV71-infected mice, especially, in brain stem and brain (Fig-
ure 4A).

Lung
C D4
D8 D12
Heart
C D4
D8 D12
Brain
C D4
D8 D12
Brainstem
C D4
D8 D12
Spinal Cord
C D4
D8 D12
Cerebellum
C D4
D8 D12
Figure 3: Detection of mSCARB2 in different tissues by immunohistochemistry.
One-day-old ICR mice were inoculated i.p. with EV71. The animals (n=3) were
sacrificed at day 4, 8 and 12 p.i., and paraffin sections of the lung, heart, brain,
brain stem, spinal cord and cerebellum were stained with monoclonal
antibody against mSCARB2. Observation were made at a magnification of
1020. Scale bars, 50m.
C = Control; D4 = Day 4 p.i.; D8 = Day 8 p.i.; D12 = Day 12 p.i.
Vol 2, No 2
The expression of mSCARB2 protein was detected in selected tissues
using western blot, and the results showed moderate signals
(~70~85 KDa band) at day 4 p.i. in brain stem, brain, spinal cord,
cerebellum, lung and heart, weaken band at day 8 and 12 p.i., and
controls. These results further confirmed the mSCARB2 gene expres-
sion tested by qRT-PCR. The protein expression of mSCARB2 had a
similar trend with gene expression (Figure 4 B, C, D, E).

Local levels of pro-inflammatory cytokines elevated in EV71-
infected mice
Enhanced cytokine production has been proposed to contribute to
EV71 pathogenesis in both humans and mice [26, 28, 30]. Local TNF-
, IL-6 and IL-1 levels were significantly higher in the various tis-
sues homogenates prepared from EV71-infected animals at day 4 p.i.
than in those from age-matched non-infected controls. Meanwhile,
TNF-, IL-6 and IL-1 levels were significantly higher in CNS (brain,
brain stem, spinal cord and cerebellum) than in lung or/and heart
(Figure 5A, B, C). At day 8 p.i., these pro-inflammatory cytokines lev-
els decreased in all tested tissues, but still higher in CNS than in lung
or/and heart (Figure 5A, B, C), and at day 12 p.i., these pro-inflamma-
tory cytokines further declined, however, IL-6 and IL-1 levels in
brain stem and brain presented higher compared to in lung or/and
heart (Figure 5A, B, C).
Carrasco-Marn E et al have reported that LIMP-2 (SCARB2)-deficient
mice infected with listeria monocytogenes (LM) had a 10-fold re-
duced concentration of acute phase pro-inflammatory cytokines,
TNF-, MCP-1, and IL-6 compared with the levels seen in WT mice
[24]. In this study, we found that local mSCARB2 expression were
consistent with TNF-, IL-6 and IL-1 production in the brain, brain
stem, spinal cord, cerebellum, lung and heart from EV71-infected
mice. Surprisingly, it presented a positive correlation between rela-
tive mSCARB2 mRNA level and TNF-, IL-6 and IL-1 levels in local
tissues at day 4 p.i. and at day 8 p.i., till at day 12 p.i., it showed no
correlation (Table 1). These results suggested that the elevated pro-
inflammatory cytokines in a certain range in local tissues induced
higher expression of mSCARB2.

Discussion
In this study, one-day-old ICR mice were infected by the EV71 BrCr
strain in vivo, and we used these models to assess the expression of
mSCARB2 in CNS, lung and heart. The survival rates and clinical
scores of infected mice were used to measure clinical symptoms or
activities. After infection with EV71, virus was detected within various
tissues by qRT-PCR. Our results indicate that one-day-old ICR mice
are susceptible to EV71 infection which leads to CNS infection, as
observed for humans. Upon infection via the peritoneal route, ICR
mice consistently displayed hunchback, limb weakness, and limb pa-
ralysis prior to death. Similar to human manifestations of EV71 en-
cephalomyelitis, the virus exhibited a strong tropism for the CNS of
ICR mice, with the numbers of viral RNA copies in CNS (brain stem,
brain, spinal cord and cerebellum) being higher than in lung or/and
heart, especially in brainstem. The copies in brain coincided with the
severity or even death of the animals [11]. In addition, all sick mice
exhibited massive neuronal damage, increased levels of cytokines, as
reported previously for severe cases of human EV71 disease [35].
Expression of mSCARB2 moderately increased in CNS, lung and heart
in EV71 (BrCr)-infected mice, and the expression of mSCARB2 was
higher in CNS than in lung or/and heart at day 4 p.i., especially, in
brain stem and brain, and at day 8 and 12, the expression of
mSCARB2 decreased. The TNF-, IL-6 and IL-1 production signifi-
cantly increased in the CNS of EV71 infected mice in comparison with
lung and heart at day 4 p.i. At day 8 and 12 p.i., the levels of TNF-,
IL-6 and IL-1 production decreased. Interestingly, the expression of
mSCARB2 in various tissues of EV71-infected mice has a similar trend
to the production of TNF-, IL-6 and IL-1. Surprisingly, our data
revealed a positive correlation between relative mSCARB2 mRNA
A
Figure 4: Detection of mSCARB2 in different tissues by qRT-PCR. One-day-old
ICR mice were inoculated i.p. with EV71. (A) mSCARB2 gene expression, the
animals (n=6, at each time points) were sacrificed at day 4, 8 and 12 p.i..
Values are mean SEM of triplicate experiments. The results were normalized
to GAPDH. *** p<0.001, ** p<0.01, * p<0.05 vs. lung or/and heart.

A B
C
Figure 5: TNF-, IL-6 and IL-1 levels in different tissues from EV71-infected
mouse. One-day-old ICR mice were inoculated i.p. with EV71. At day 4, 8, and
12 p.i., six mice per time point were sacrificed, and various tissues were
harvested and homogenized. The levels of TNF- (A), IL-6 (B) and IL-1 (C) in
different tissues homogenates from EV71-infected animals and controls were
quantified individually by ELISA. The results are expressed as the mean SEM.
*** p<0.001, ** p<0.01, * p<0.05 vs. lung or/and heart. Data are representative
of three independent experiments.
Table 1: Correlations of local levels of pro-inflammatory cytokines and expression of mSCARB2 in EV71-infected mice
Time Cytokines /
Tissues
mSCARB2 Pearson correlation/Significance (two-tailed)
Brainstem Brain Spinal Cord Cerebellum Heart Lung
Day 4 p.i.
TNF- (pg/mL) 0.933 0.829 0.899 0.825 0.989 0.913
0.007 0.041 0.015 0.043 <0.01
a
0.011
IL-1 (pg/mL) 0.958 0.693 0.861 0.903 0.989 0.920
0.003 0.127 0.028 0.014 <0.01
a
0.009
IL-6 (pg/mL) 0.955 0.830 0.918 0.910 0.959 0.920
0.003 0.041 0.010 0.012 0.003 0.009
Day 8 p.i.
TNF- (pg/mL) 0.983 0.839 0.915 0.938 0.973 0.962
<0.01
a
0.037 0.011 0.006 0.001 0.002
IL-1 (pg/mL) 0.968 0.820 0.981 0.960 0.982 0.922
0.002 0.046 0.001 0.002 <0.01
a
0.009
IL-6 (pg/mL) 0.978 0.828 0.980 0.959 0.992 0.935
0.001 0.042 0.001 0.002 <0.01
a
0.006
Day 12 p.i.
TNF- (pg/mL) -0.518 0.396 0.665 -0.154 0.586 -0.501
0.293 0.473 0.149 0.771 0.222 0.311
IL-1 (pg/mL) -0.784 -0625 -0.623 -0.702 -0.583 -0.459
0.065 0.184 0.187 0.120 0.224 0.359
IL-6 (pg/mL) -0.580 -0.726 -0.493 -0.684 -0.423 -0.641
0.227 0.102 0.323 0.134 0.403 0.170
a
Correlation is significant at the 0.01 level (2-tailed)
level and TNF-, IL-6 and IL-1 levels in local tissues at day 4 p.i. and
at day 8 p.i., but at day 12 p.i., it showed no correlation.
Carrasco-Marn et al presented evidence for the specific role of LIMP-
2/SCARB2 in the innate immune response to listeria monocytogenes
and in phagocytosis. LIMP-2 tightly controls the number of cytosolic
LM and the induction of acute phase pro-inflammatory cytokines
such as MCP-1, TNF-, and IL-6. However, the production of late pro-
inflammatory cytokines, such as INF- and IL10, was not regulated
by LIMP-2/SCARB2 [24]. In infection, two cytokines involved in mac-
rophages (M) activation: TNF- and INF-. TNF- acts as an early
signal in innate immunity, INF- is a late signal. It has been claimed
that exogenous action of TNF- promotes an early activating state
in Ms that triggers the cytosolic microbicidal mechanisms [36-38].
In EV71 infection, SCARB2 may also participates in exogenous M
activation, the early signals modulated by TNF-.
Conclusion
Taken together, we assume that in EV71 infected mice, the elevated
local mSCARB2 may regulate the early innate immune response to
EV71, or even modulate pro-inflammatory cytokines induction;
mSCARB2 may also act as the invasive receptor for the enterovirus
71 although no experimental evidence has ever been provided to
support this hypotheses, because human SCARB2 (hSCARB2) have
been identified as cellular receptors for EV71, and mSCARB2 exhib-
ited 85.8% amino acid identity and 99.9% similarity to hSCARB2 [39,
40]. The elevated expression of mSCARB2 in EV71-infected mice may
play other roles, which are not clear now.
Funding: This study was financially supported by grants from the Natural Science
of China (no. 31171212).
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Katyal T, Negi J, Sachdeva M et al 59

Comparative effect of antiplatelet drugs in streptozotocin-induced diabetic nephropathy in
experimental rats
Taruna Katyal
1
, Jitender Negi
1
, Monika Sachdeva
1
, R D Budhiraja
1

Introduction
Diabetes mellitus is a complex metabolic disease characterized by
insulin deficiency or development of insulin resistance that leads to
hyperglycemia and altered glucose, fat and protein metabolism [1].
Diabetic nephropathy is a multistage clinical syndrome characterized
by thickening of the glomerular basement membrane and mesangial
expansion with progression into glomerulosclerosis, tubular necrosis
and interstitial fibrosis, which ultimately leads to renal failure [2].
Moreover, diabetic nephropathy is also associated with the hyperac-
tivity of the sorbitol-aldose reductase pathway, hyperactivity of hex-
osamine biosynthetic pathway, activation of protein kinase C and
mitogen activated protein kinases (MAPK) and overexpression of
growth factors and cytokines i.e. transforming growth factor-, vas-
cular endothelial growth factor, platelet-derived growth factor and
insulin-like growth factor which further lead to the progression of
the disease [2].
Platelets have been reported to play a role in the progression and
development of diabetic nephropathy. Platelets levels has been
found to be increased in diabetic patients. Further increased glucose
level is responsible for abnormalities in endothelial and platelet func-
tion, which further contribute to the cellular events that increase the
risk of diabetic complications. Hyperglycemia causes non-enzymatic
glycation of platelet glycoproteins, causing changes in their structure
and conformation, as well as alteration of membrane lipid dynamics
[3]. In diabetes, oxidative stress is increased that favors atherothrom-
botic processes, thrombogenesis, and endothelial dysfunction [4].
Chronic hyperglycemia results in reactive oxygen species (ROS) pro-
duction directly via glucose metabolism and auto-oxidation, or indi-
rectly by formation of advanced glycation end products. ROS acti-
vate signaling molecules within endothelial cells, including PKC and

1
Indo Soviet Friendship College of Pharmacy, India.
Correspondence: Taruna Katyal
Email: tarunakatyal@gmail.com
nuclear factor-kB (NF-kB), resulting in progression of disease [1]. Tak-
ing into account above mentioned various downstream signaling
pathways, these collectively play an important role in the multifari-
ous pathogenesis of diabetic nephropathy. However, further studies
are needed to investigate the ameliorative role of platelets in dia-
betic nephropathy. As such, we designed this study to investigate
the role of platelets in diabetic nephropathy.
Antiplatelet drugs are used for a number of pathological conditions.
These are particularly recommended to be regularly taken by dia-
betic patients, because these patients are sensitive to hyperglycemia
and likely to suffer stroke, heart attack and various other diabetic
related complications. Salicylates are the first choice of antiplatelet
drugs. However, salicylates are reported to cause nephrotoxicity in
clinical doses. Therefore, the present study has been undertaken to
study the comparative effect of salicylates and other antiplatelet
drugs in streptozotocin (STZ) induced nephropathy in experimental
rats.
The Institutional Animal Ethical Committee approved the experi-
mental protocol used in the present study. Age matched young
wistar rats weighing about 200240 g were employed in the present
study. Rats were fed on standard chow diet and water, ad libitum.
They were acclimatized in institutional animal house and were ex-
posed to normal cycles of day and night. STZ was obtained from
Sigma-Aldrich Ltd., St. Louis, USA. Marketed formulations of aspirin,
cilostazol and dipyridamole were used. All other chemicals used in
the present study were of analytical grade.

Abstract
Background: Platelets have been reported to play a role in the progression and development of diabetic nephropathy (DN). The present
study has been designed to investigate the comparative effect of some antiplatelet drugs in streptozotocin (STZ) induced nephropathy in
rats.
Methods: Diabetic nephropathy was induced by administering single dose of streptozotocin in wistar rats. DN was clinically assessed by
estimation of various biochemical parameters and histopathological studies of renal tissue. DN was assessed by measuring serum creatinine,
blood urea nitrogen, proteinuria, renal cortical collagen content, lipid profile, serum nitrite/nitrate ratio, renal TBARS, reduced glutathione
levels.
Results: Administration of these antiplatelet drugs (aspirin, dipyridamole, cilostazol) did not treat the diabetic induced nephropathy, but
did retard the progression of the disease to a great extent. Treatment with high dose of cilostazol (25mg/kg, i.p.) shows significant
improvement in (BUN, serum creatinine, total urine protein, ratio of kidney wt/body wt, TBARS, reduced glutathione) renal parameters
studied for DN.
Conclusion: It may be therefore concluded that renoprotective effect of cilostazol is due to its antiplatelet property and through the
inhibition of various intracellular pathways. (El Med J 2:2; 2014)
Keywords: Diabetic Nephropathy, Antiplatelet Drugs
60 Streptozotocin-induced diabetic nephropathy
Vol 2, No 2
Assessment of diabetes
Experimental diabetes mellitus was induced in rats by single injec-
tion of STZ (50 mg/kg IP) dissolved in freshly prepared ice cold citrate
buffer of pH 4.5. The blood sugar level was monitored once daily for
first week after administration of STZ. Then, at the end of the exper-
imental protocol (6 weeks after administration of STZ), the blood
samples were collected and serum was separated. The serum glu-
cose concentration was estimated by glucose oxidase peroxidase
(GOD-POD) method using the commercially available kit (Crest Bios
stems, Goa, India) [5].
Assessment of diabetic nephropathy
Diabetes mellitus induced nephropathy was assessed biochemically
by estimating serum creatinine, blood urea nitrogen and proteinuria
by using the commercially available kit (Crest Bios stems, Goa, India)
[6-8].
Estimation of renal oxidative stress
The development of oxidative stress in the kidney was assessed by
estimating renal thiobarbituric acid reactive substances (TBARS) and
reduced form glutathione (GSH).
Preparation of renal homogenate
The kidney was dissected and washed with ice cold isotonic saline
and weighed. The kidney was then minced, and a homogenate (10%
w/v) was prepared in chilled 1.15% KCl. The homogenate was used
for estimating TBARS, GSH and total protein.
Estimation of TBARS
The renal TBARS, an index of lipid peroxidation, were estimated ac-
cording to the method described earlier [9]. The reaction mixture
was prepared by mixing 0.2 ml of tissue homogenate, 0.2 ml of 8.1%
sodium dodecyl sulphate, 1.5 ml of 20% acetic acid solution adjusted
to pH 3.5 with NaOH, and 1.5 ml of 0.8% aqueous solution of thio-
barbituric acid (TBA). The reaction mixture was made up to 4.0 ml
with distilled water, and then heated in water bath at 95C for 60
min. After cooling in tap water, 1.0 ml of distilled water and 5.0 ml
of the mixture of n-butanol and pyridine (15:1 v/v) were added to
reaction mixture and shaken vigorously. After centrifugation at 4000
rpm for 10 min, the organic layer was taken and its absorbance at
532 nm was measured. The standard curve using 1,1,3,3-tertrameth-
oxyopropane was plotted to calculate the concentration of TBARS
and the results were expressed as nmol/mg of protein.
Estimation of reduced GSH
The GSH level in the kidney was estimated using the method previ-
ously described by Ellman [1, 10].
Estimation of renal hypertrophy and fibrosis
The renal hypotrophy and fibrosis was accessed by measuring the
kidney weight and body weight ratio.
Estimation of bleeding time (BT)
BT was measured by cutting the tail-tip as described by Chan in 1993
[11]. The tail of each anesthetized rat was cut 2 mm from the end
using a sharp pair of surgical scissors and then immersed immedi-
ately into the cylinder with 100 ml isotonic saline at 37C. Bleeding
time was measured from the moment the tail was surgically cut until
bleeding completely stopped.
Experimental protocol
Eight groups were employed in the present study and each group
comprised 6 rats. Group I (normal control) rats were maintained on
standard food and water and no treatment was given. Group II (dia-
betic control) rats were administered STZ (50 mg/kg, IP, once) dis-
solved in citrate buffer (pH 4.5). Group III (aspirin low dose) diabetic
rats after 4 weeks of STZ were administered low dose of aspirin (50
mg/kg, PO) for 2 weeks. Group IV (cilostazol low dose) diabetic rats
after 4 weeks of STZ were administered low dose of cilostazol (12.5
mg/kg, PO) for 2 weeks. Group V (dipyridamole low dose) diabetic
rats after 4 weeks of STZ were administered low dose of dipyr-
idamole (50 mg/kg, PO) for 2 weeks. Group VI (aspirin high dose)
diabetic rats after four weeks of STZ were administered high dose of
aspirin (100 mg/kg, PO) for 2 weeks. Group VII (cilostazol high dose)
diabetic rats after 4 weeks of STZ were administered high dose of
cilostazol (25 mg/kg, PO) for 2 weeks. Group VIII (dipyridamole high
dose) diabetic rats after 4 weeks of STZ were administered high dose
of dipyridamole (100 mg/kg, PO) for 2 weeks.
All values were expressed as mean standard deviation. The data
obtained from various groups were statistically analyzed using one
way ANOVA, followed by Tukey's multiple comparison tests. The P
value of less than 0.05 was considered to be statistically significant
and the P values were two tailed.
Results
Administration of STZ (50 mg/kg, IP, once) produced hyperglycemia
after 72 h (serum glucose 180 mg/dl). After 7 days of STZ administra-
tion, the rats showed blood glucose level of greater than 260 mg/dl
were selected and were named as diabetic rats. Aspirin (50 mg/kg,
PO and 100 mg/kg, PO), cilostazol (12.5 mg/ kg, PO and 25 mg/kg,
PO) and dipyridamole (50 mg/kg, PO and 100 mg/kg, PO) were ad-
ministered to diabetic rats after 4 weeks of single injection of STZ
and their treatments were continued for 2 weeks. All the parameters
were assessed at the end of 6 weeks in normal and diabetic rats with
or without drug treatments.
Effect of pharmacological interventions on serum glucose
A marked increased in serum concentration of glucose was noted in
diabetic rats when compared with age matched normal rats. Treat-
ment with aspirin (50 mg/kg, PO, 2 weeks) did not alter the serum
glucose concentration in diabetic rats. However, treatment with as-
pirin (100 mg/kg, PO, 2 weeks) slightly reduced the glucose level.
Treatment with cilostazol (12.5 mg/kg, PO, 2 weeks) showed no ef-
fect on the serum glucose level similar to the aspirin but cilostazol
(25 mg/kg, PO, 2weeks) showed a significant lowering in the blood
glucose level compared to the diabetic rats. Furthermore, treatment
with dipyridamole (50 mg/kg, PO, 2 weeks) showed significant low-
ering in the glucose level which further continued with the dose of
(100 mg/kg, PO, 2 weeks). All the results are given in figure 1.
Effect of various Pharmacological interventions on BUN
The concentration of BUN was noted to be significantly increased
(p<0.05) in diabetic rats when compared with age matched normal
rats. Treatment with aspirin (50 mg/kg, PO, 2 weeks) and aspirin (100
mg/kg, PO, 2 weeks) did not alter the BUN concentration in diabetic
rats. Furthermore, on treating the diabetic rats with the cilostazol
(12.5 mg/kg, PO, 2 weeks) and cilostazol (25 mg/kg, PO, 2 weeks), a
significant lowering in the level of the BUN was seen and this lower-
ing was dose dependent. Moreover, on treating the diabetic rats with
the dipyridamole (50 mg/kg, PO, 2 weeks) and aspirin (100 mg/kg,
PO, 2 weeks) also lowered the BUN level significantly. On comparing
the effect of all the three drugs on the BUN, dipyridamole lowered
the BUN to the most significant level and the maximum lowering in
the BUN level was seen at the dose of dipyridamole (100 mg/kg, PO,
2 weeks) (table 1).
1 0
100
200
300
400
500
Normal control
Diabetic control
Asprin treated(50mg/kg)
N
o
r
m
a
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a
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e
d (
5
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Cilostazol(12.5mg/kg)
C
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o
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a
z
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1
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b
Dipyridamole ( 50mg/kg)
D
i
p
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r
i
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a
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(
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g
/
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Aspirin (100mg/kg)
A
s
p
i
r
i
n
T
r
e
a
t
e
d
(
1
0
0
m
g
/
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)
Cilostazol (25mg/kg)
C
i
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o
s
t
a
z
o
l
(
2
5
m
g
/
k
g
)
Dipyridamole(100mg/kg)
D
i
p
y
r
i
d
a
m
o
l
e
(
1
0
0
m
g
/
k
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)
b,c,d,e
b,c,d,e
b,c,d,e
a
SERUM GLUCOSE
S
e
r
u
m

g
l
u
c
o
s
e

(
m
g
/
d
l
)
Figure 1: Effect of antiplatelet drugs on serum glucose
Effect of various pharmacological interventions on serum
creatinine
The concentration of serum creatinine was noted to be significantly
increased (p<0.05) in diabetic rats when compared with age-
matched normal rats. Treatment with aspirin (50 mg/kg and 100 mg/
kg, PO, 2 weeks) and cilostazol (12.5 mg/kg and 25 mg/kg, PO, 2
weeks) showed a significant lowering in the level of the serum cre-
atinine and this lowering increased with the increase in the drug.
Dipyridamole (100 mg/kg, PO, 2 weeks) had a significant lowering in
the serum creatinine, but at the dose of (50 mg/kg, PO, 2 weeks) the
effect on the serum creatinine was not significant (table 1).
Effect of various pharmacological interventions on total proteins
A significant increase (p<0.05) in the proteins excreted in the urine
was noted in diabetic rats when compared with age matched normal
control rats. Treatment with aspirin (50 mg/kg, PO, 2 weeks) had no
effect on the level of the protein excreted in the urine, but with the
increase in the dose i.e. at aspirin (100 mg/kg, PO, 2 weeks), showed
significantly reduced total proteins in diabetic rats. In the similar
manner, cilostazol (12.5 mg/kg and 25 mg/kg, PO, 2 weeks) and di-
pyridamole (50 mg/kg, PO and 100 mg/kg, PO, 2 weeks) showed sig-
nificant lowering in the levels of the protein excreted in urine. Ci-
lostazol (25 mg/kg, PO, 2 weeks) was found to be statistically signif-
icant (table 1).
Effect of various pharmacological interventions on renal TBARS
Marked increase in renal tissue TBARS concentration was noted in
kidney of STZ administered diabetic rats as compared to normal rats.
Treatment with aspirin (50 mg/kg, PO, 2 weeks) significantly lower-
ing in the levels of the TBARS which further continued to decrease
with the increase of the dose i.e. aspirin (100 mg/kg, PO, 2 weeks).
Moreover, with the treatments with cilostazol (12.5 mg/kg and 25
mg/kg, PO, 2 weeks) and dipyridamole (50 mg/kg, PO and 100
mg/kg, PO, 2 weeks), there was also a significant decrease in the
TBARS levels when compared with normal rats. Furthermore, the
treatment with cilostazol (25 mg/kg, PO, 2 weeks) was found to be
of maximal statistical significance (figure 2).
0.0
0.5
1.0
1.5
2.0
Normal control
Diabetic control
Aspirin treated(50mg/kg)
N
o
r
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a
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cilostazol(12.5mg/kg)
C
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.5
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a
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k
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)
Aspirin (100mg/kg)
A
s
p
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1
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k
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)
C
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)
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)
b
b,c
b,e
b,c
b,c
a
b,d,e,f,g
TBARS
T
B
A
R
S

Figure 2: Effect of antiplatelet drugs on TBARS
Effect of various pharmacological interventions on reduced GSH
Marked decrease in the level of the reduced form of GSH was noted
in kidney of STZ-administered diabetic rats as compared to diabetic
rats. The level of the reduced glutathione was significantly increased
by all the three drugs. The maximum increase in the level of the re-
duced glutathione was noted in cilostazol (25 mg/kg, PO, 2 weeks)
in comparison to the other drugs (figure 3).
0
10
20
30
Normal Control
Diabetic Control
N
o
r
m
a
l
C
o
n
t
r
o
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i
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t
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t
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(
5
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)
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1
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.5
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r
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a
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(
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)
Aspirin (100mg/kg)
A
s
p
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(
1
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)
C
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a b,c,d
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c
REDUCED GLUTATHION
R
e
d
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e
d

g
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u
t
a
t
h
i
o
n
e
(
n
m
o
l
/
m
g
)

Figure 3: Effect of antiplatelet drugs on reduced GSH
Effect of various pharmacological interventions on kidney
weight / body weight (%)
A significant increase in kidney weight / body weight (%) was noted
in diabetic rats when compared with normal rats. Treatment with all
the three drugs lowered the kidney weight / body weight (%). Treat-
ment with cilostazol (25 mg/kg, PO, 2 weeks) significantly decreased
the level of the kidney weight / body weight (%) in diabetic rats as
compared to other drugs (figure 4).
Effect of various pharmacological interventions on bleeding
time
There was a significant decrease in the bleeding of the diabetic rats
in comparison to the normal rats. Treatment with all the drugs in-
creased the bleeding time because of its antiplatelet action. Aspirin
at a dose of 100 mg/kg showed the most significant increase in the
bleeding time in comparison to the other drugs (table 1).
62 Streptozotocin-induced diabetic nephropathy
Vol 2, No 2
Table 1: Effect of antiplatelet drugs on various pharmacological interventions
Bleeding Time (seconds) Blood Urea Nitrogen Serum Creatinine Total Proteins in Urine
Normal Controls 119.00 8.50 20.30 3 0.496 0.044 5.30 0.78
Diabetic Controls 61.16 6.01 99.57 17 1.226 0.220 104.26 7.50
Aspirin (50 mg/kg) 91.50 6.80 92.83 7 0.850 0.050 96.61 8.87
Cilostazol (12.5 mg/kg) 79.66 4.13 75.82 7 0.701 0.084 79.68 6.75
Dipyridamole (50 mg/kg) 75.00 3.79 72.00 6 1.131 0.160 86.91 5.14
Aspirin (100 mg/kg) 105.83 8.90 89.88 11 0.660 0.044 81.20 6.36
Cilostazol (25 mg/kg) 99.66 8.10 65.78 4 0.651 0.061 62.37 5.25
Dipyridamole (100 mg/kg) 94.16 9.19 58.69 6 0.980 0.091 73.85 6.97
Figure 4: Effect of antiplatelet drugs on kidney weight / body
weight
Discussion
The present study investigated the possible involvement of some
anti-platelet drugs in diabetic nephropathy. Estimation of blood glu-
cose has been used as a marker of hyperglycemia. Hyperglycemia
induces oxidative stress and decreases antioxidant defense system,
which has been assessed in various studies to estimate the degree
of oxidative stress [12]. Increase in serum creatinine, BUN, and pro-
teinuria have been documented to be an index of nephropathy. In
the present study, the serum creatinine, BUN, and proteinuria were
noted to be significantly increased in STZ-induced diabetic rats com-
pared to normal rats. Kidney weight / body weight (%) and collagen
deposition is reported to be a marker of renal hypertrophy and fibro-
sis in diabetic nephropathy [13, 14]. In the present study, kidney
weight / body weight (%) and total collagen deposition was found
to be increased in renal cortex, in the diabetic rat.
The renal structural and functional abnormalities in diabetic rats ob-
served in the present study indicate the development of nephropa-
thy. Treatment with the anti-platelet drugs i.e. aspirin, dipyridamole
and cilostazol, partially prevented the development of nephropathy
in diabetic rats by inhibiting the platelet aggregation and decreasing
inflammatory mediators and thrombus formation in the rat renal
cells. It can be concluded from previous studies that hyper-reactive
platelets are implicated in the pathogenesis and progress of vascular
complications in diabetes. Platelet abnormalities in diabetes can be
explained by both increased intrinsic platelet activation and de-
creased influences of endogenous inhibitors of platelet function.
These abnormalities, in close interaction with inflammation, endo-
thelial dysfunction and coagulation, seem to contribute to the en-
hanced thrombotic potential in diabetes. The present study supports
the idea that hyperglycemia contributes to the platelet hyper-reac-
tivity in diabetes.
The abnormal metabolic state in diabetes is responsible for abnor-
malities in endothelial and platelet function, which may contribute
to the cellular events that cause atherosclerosis and subsequently
increase the risk of the diabetic nephropathy [15]. Hyperglycemia-
induces oxidative stress, which is responsible for enhanced peroxi-
dation of arachidonic acid to form biologically active F2-isopros-
tanes. The activated platelets further activate many secondary fac-
tors like (platelet-derived growth factor (PDGF), transforming growth
factor- (TGF-), vascular endothelial growth factor (VEGF), basic fi-
broblast growth factor (bFGF), platelet derived epidermal growth
factor (PDEGF) and IGF-1 (insulin-like growth factor-1). -granules
contain platelet factor- 4 (PF-4), plasminogen activator inhibitor-1
(PAI-1), -thromboglobulin, fibrinogen, fibronectin, thrombospon-
din and von Willebrand factor (vWF) [16]. These factors together lead
to the progression of diabetic nephropathy.
Hyperglycemia causes activation of nuclear factor kappa B (NFB)
(p65 levels), a transcription factor, which further induces the synthe-
sis of other inflammatory cytokines e.g. tumor necrosis factor-
(TNF-) in the kidney. Aspirin exerts its effects through inhibition of
cyclooxygenase enzymes and through certain cyclooxygenase-inde-
pendent actions as well such as inhibition of degradation of inhibitor
of kappa B (IB) [17]. Aspirin exerts its anti-inflammatory effect by
lowering the levels of NFB (p65 levels) in the kidney. There are re-
ports which show that long term and high dose treatment of aspirin
reduce type 1 diabetes [18]. Aspirin is also expected to reduce the
levels of TGF- in the kidney, and as a result is helpful in the diabetes
and diabetic-related complications.
Cilostazol has a beneficial effect on lipids. It has been reported to
decrease the triglyceride levels and increase the levels of HDL cho-
lesterol in blood [19, 20]. Recent studies have suggested that cAMP
and cGMP regulate several signaling pathways involved in the devel-
opment and progression of renal disease, including mitogenesis, in-
flammation, and extracellular matrix synthesis [21, 22]. It has been
reported that it mainly acts by preventing the platelet aggregation
and dilation of blood vessels via increases in tissue cAMP levels [23].
It also inhibits the ROS generation induced by PKC activation by in-
hibiting the PI3 kinase dependent pathway. Our results further con-
firm that cilostazol improves oxidative stress and ameliorates the
early stage of diabetic nephropathy. Dipyridamole has been re-
ported to reduce the amount of proteinuria and is believed to have
0.0
0.2
0.4
0.6
0.8
Normal control
Diabeti c Control
N
o
r
m
a
l
C
o
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t
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D
i
a
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r
o
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A
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p
i
r
i
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a
t
e
d
(
5
0
m
g
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k
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C
i
l
o
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t
a
z
o
l
(
1
2
.5
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)
D
i
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a
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(
5
0
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g
/k
g
)
Aspirin (100mg/kg)
A
s
p
i
r
i
n
(
1
0
0
m
g
/
k
g
)
C
i
l
o
s
t
a
z
o
l
(
2
5
m
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k
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D
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i
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(
1
0
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b
b,e
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Kidney Wt./Body Wt.
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W
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/

B
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W
t
a renoprotective effect by inhibition of intraglomerular coagulation
and suppression of platelet derived growth factors and cytokines [24,
25]. Dipyridamole has also been considered an inhibitor of cGMP
phosphodiesterase (PDE), and it enhances the action of nitric oxide
[26-28]. Thus, dipyridamole maintains renal microcirculation and
may also show a renoprotective effect via NO action. Our study re-
sults indicated a significant decrease in the levels of the TBARS in the
dipyridamole treated group in comparison to the diabetic control.
Our study results also showed a significant increase in the level of
reduced glutathione in the dipyridamole treated group compared to
the diabetic rats. These findings support the previous studies that
showed that dipyridamole reduces the oxidative stress in the rat kid-
ney and decreases the generation of the free radicals.
Conclusion
On the basis of the above discussion and our study results, it can be
safely concluded that diabetes-induced nephropathy is associated
with the increase of the activated platelets, which leads to the acti-
vation of various pathways which in turn results in diabetic compli-
cations including nephropathy. Treatment with anti-platelets may
not completely treat the diabetic nephropathy, but our findings and
the literature reveals that it retards to a great extent the progression
of the disease. In the present study we have used three drugs at dif-
ferent dose levels. Our results show that all the drugs are effective in
the treatment of nephropathy, but cilostazol is the most effective in
comparison to the others. Thus, although aspirin is very commonly
used antiplatelet drug, other drugs can be used in the setting of al-
lergy or resistance to aspirin or other contraindications. However,
further laboratory and clinical studies are needed to completely elu-
cidate this effect.
Acknowledgement: We express our gratitude to Shri. Parveen Garg, Honorable
Chairman, ISF College of Pharmacy, Moga, Punjab, India for his inspiration and
constant support for this study.
Received: 13 December 2014 Accepted: 1 February 2014
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64 GABAergic neurons work in anxiety related behavior

Vol 2, No 2
Dorsal hippocampus histaminergic and septum GABAergic neurons work in anxiety related behavior:
Comparison between GABAA and GABAB receptors
Leila Chodari
1
, Shahrbanoo Oryan
2
, Ramesh Ahmadi
3
, Ghorbangol Ashabi
4

Introduction
Anxiety disorder is the most common psychiatric disorder in the
world [1]. Hence, scientists today focus more on improving anxiety
disorders in clinical and/or laboratory settings. It should be noted
that many regions of brain regulate anxiety related behaviors which
include limbic system, prefrontal cortex and amygdala. The im-
portant role of septum and hippocampus in controlling anxiety has
been widely reported in different studies [2, 3]. Anatomic and func-
tional connections between hippocampus and septum have also
been investigated by researchers [4, 5]. The septum and hippocam-
pus act in concert to control anxiety and memory related behaviors.
Glutamatergic, GABAergic, cholinergic and opioidergic neurons have
been found between septum and hippocampus [6, 7]. These neurons
and their activations interfere with anxiety behaviors.
Ablation or pharmacological inhibition of septum area reduces rats
anxiety-related behaviors in the elevated plus-maze test, suggesting
that the septum normally plays an excitatory role in the control of
anxiety [8, 9]. However, far less is known about the effects of inhib-
iting hippocampal activity on rats anxiety reactions in these tests
and the integrated role of the hippocampus and septum in regulat-
ing the expression of anxiety. One possibility is that the hippocam-
pus makes a direct contribution to the excitatory role of the septum
in regulating anxiety. The major hippocampal sub-regions, CA1, CA3
and the dentate gyrus have different anatomical relationships with
the medial septum (MS). While each of these sub-regions receives
ipsilateral projections from MS, only CA1 and CA3 project to the cho-
linergic and non-cholinergic neurons into MS [10-13]. Reciprocal
connections with MS suggest that CA1 and CA3 might be important
components of the medial septum-hippocampal system.

1
Tabriz University of Medical Sciences, Iran
2
Kharazmi University, Iran
3
Islamic Azad University, Iran
4
Ahvaz Jundishapur University of Medical Sciences, Iran
Histaminergic neurons in the nucleus tubermammillaris of the pos-
terior hypothalamus innervate wide parts of the brain and spinal
cord [14, 15]. Anxiety related stress may cause histamine release [16].
Histamine has strong effects on excitability in the hippocampus by
acting on histamine H2 receptors and also affects the hippocampus
indirectly through its projections on the MS which provide the cho-
linergic and non-cholinergic input to the hippocampus [17-19]. Our
previous studies indicated that GABAergic system blockade in the
MS attenuates anxiety-like behavior [6]. Herein, we aimed to investi-
gate the histaminergic and GABAergic associations between dorsal
hippocampus and MS, respectively. Moreover, the present study
compares the power of two kinds GABA receptors, GABAA and GABAB
receptors, in MS in anxiety related behavior of male rats.
Animals
Male Wistar rats (Pasteur Institute, Tehran, Iran) weighing 220 20g
at the time of surgery, were examined. The animals were housed four
per cage, in a room with a 12:12 light/dark cycle (lights on 07:00 h)
and controlled temperature (23 1
C). Animals had free access to

food and water and they were allowed to adapt to the laboratory
conditions for at least 1 week prior to surgery. Seven animals were
used in each group of experiments. The study was approved by Eth-
ics committee of the Kharazmi University which corresponds to the
national guidelines for animal care and use.
Stereotaxic surgery and microinjections
Rats were anesthetized intra-peritoneally with ketamine hydrochlo-
ride (50 mg/kg) and xylazine (4mg/kg) and placed in a Stoelting ste-
Correspondence: Ghorbangol Ashabi
Email: as_habi@yahoo.com
Abstract
Background: Integration of septum and hippocampus has an important role in anxiety modulation and inhibition. Our previous studies
indicated that GABAergic system blockade in the medial septum (MS) attenuates anxiety-like behavior. Herein, we aimed to investigate
the histaminergic and GABAergic associations between dorsal hippocampus and MS, respectively. Moreover, the present study compares
the power of two kinds GABA receptors, GABAA and GABAB receptors, in MS in anxiety related behavior of male rats.
Methods: In the present study we investigated the intrahippocampal CA1 (intra-CA1) microinjection of histamine and intra-septal
GABAergic agents on anxiety-related behavior in rats, using elevated plus-maze test of anxiety.
Results: Intra-CA1 administration of histamine increased open arm time (%OAT) and open arm entry (%OAE). Intra-septal administration
of the GABAA agonist muscimol (10 ng/rat) increased %OAE and %OAT but not locomotor activity. Administration of muscimol (2.5 and 5
ng/rat) had no effect on anxiety, while co-administration of sub-effective dose of histamine (1 g/rat) into CA1 and sub-effective dose of
muscimol (2.5ng/rat) into medial septum area increased %OAE and %OAT. Administration of GABAA receptor antagonist confirmed GABAA
function in the medial septum. In contrast, injection of GABAB receptor agonist decreased OAT and OAE percentage. Microinjection of
GABAB receptor antagonist revealed that this GABA receptor increased the anxiety in the rats by a different mechanism.
Conclusion: The effect of histamine on anxiety in CA1 may be modulating through septal GABAergic system and GABAA receptor is
involved in the process. (El Med J 2:2; 2014)
Keywords: Histamine, Muscimol, Bicuculline, Anxiety, Baclofen, Septo-hippocampus, Rat
Chodari L, Oryan S, Ahmadi R et al 65
reotaxic instrument (Stoelting Co, Illinois, and USA). The stainless
steel guide cannula (22 gauge) was implanted in the dorsal hippo-
campus and medial septum. Stereotaxic coordinate for cannula im-
plantation in the dorsal hippocampus was: anterocaudal: -3 to -3.5
mm (depending on body weight) anterior to bregma, 1.8 to 2mm
lateral to the midline, and -2.8 to -3mm ventral of the dorsal surface
of the skull, according to the atlas of Paxinos and Watson [20]. The
guide cannulas were anchored to the skull with two jewelers and
acrylic dental cement. After surgery, rats were allowed seven days to
recover from the operation.
The drug solutions were injected over a 1 minute period by means
of internal cannula (27 gauge) connected by polyethylene tubing 25
ml Hamilton syringe and the cannula was left in place for an addi-
tional 1 min before being slowly withdrawn. The medial septum was
injected with 1 l solution over a 1 minute period.
Elevated plus-maze
The method is basically the same as described by Pellow et al, 1985
[21]. The elevated plus-maze is a wooden, cross-shaped maze, con-
sisting of four arms arranged in the shape of a plus sign. Two of the
arms have no side or end walls (open arms, 5010 cm). The other
two arms have side walls and end walls, but are open on the top
(closed arms, 501040 cm). Where the four arms intersect, there is
a square platform of 1010 cm. The maze was elevated to a height
of 50 cm. In order to elevate total arm entries on the maze, rats were
placed in a wooden test arena (505035 cm) for 5 min prior to
maze testing. Seven days after implantation, the effects of intra-CA1
injection of drugs were tested in the elevated plus-maze.
Animals were randomly allocated to treatment conditions and were
tested in counterbalanced order. The rats were individually placed in
the center of the maze facing a closed arm and allowed 5 min of free
exploration. The number of entries into open arms, the number of
entries into closed arms and the total time spent in the open arms
and total time spent in the closed arms were measured. Entry was
defined as all four paws in the arms and measured by hand counter.
The percentage of open arms entries and open arm times as the
standard anxiety indices were calculated as follow: (a) %OAT (the
ratio of times spent in the open arm to total times spent in any arms
100); (b) %OAE (the ratio entries into open arms into total entries
100); (c) Total closed arm entries were measured as a relative pure
index of locomotor activity [22].
Drugs
The drugs used in the present study were histamine dihydrochloride,
baclofen and CGP34358, muscimol (Sigma, USA) and bicuculline
(Ciba-Geiry, Switerland). All drugs were dissolved in sterile 0.9% sa-
line, just before the experiment. Histamine was injected in a volume
of 0.5 l in each side of CA1 (1 l/rat) although muscimol and bicu-
culline were unilaterally injected in a volume of 1 l into medial sep-
tum. Total doses of the drugs were expressed as g/rat. Control ani-
mals received 0.9% saline.
Drug treatment
Experiment 1: effects of histamine in the CA1 on anxiety-like behavior
In this experiment, four groups of rats received saline (0.5 l/side, 1
l) or 3 different doses of histamine (1, 5 and 10 g/rat, 0.5 l/side).
The test session was performed 5 min after intera-CA1 injections.
%OAT, %OAE and locomotor activity were measured as described in
the materials and method section.
Experiment 2: effects of histamine in the CA1 with muscimol in the MS on
anxiety-like behavior
In this experiment, four groups of rats received saline (1 l/rat) or 3
different doses of muscimol (2.5, 5 and 10 ng/rat). After that, four
groups of rats received saline-saline (0.5 l/side, 1 l/rat intra hippo-
campal and 1 l/rat intra septal) or subeffective doses of histamine
(1 g/rat, 0.5 l/side) and muscimol (2.5 ng/rat) in the CA1 and MS,
respectively. The test session was performed 5 min after intera-CA1
and intra-MS injections. %OAT, %OAE and locomotor activity were
measured.
Experiment 3: effects of histamine in the CA1 with bicuculline in the MS on
In this experiment four groups of rats received saline (0.5 l/side , 1
l/rat) or different doses of bicuculline (10, 20 and 30 ng/rat , intra
medial septal). Four other groups of animals received histamine (10
mg/rat, intra-hippocampal) or different dose of bicuculline (10, 20
and 30 ng/rat , intra-medial septal) in the same time. The test session
was performed 5 min after intra-hippocampal and intra septal injec-
tions.
Experiment 4: effects of histamine in the CA1 with baclofen in the MS on
In this experiment four groups of rats received saline (0.5 l/side, 1
l/rat) or different doses of baclofen (0.1, 0.5 and 1 ng/rat, intra-me-
dial septal). The test session was performed 5 min after intra-hippp-
campal and intra-septal injections.
Experiment 5: effects of histamine in the CA1 with CGP35348 in the MS on
In this experiment four groups of rats received saline (0.5 l/side, 1
l/rat) or different doses of baclofen (0.1, 0.5 and 1 ng/rat, intra-me-
dial septal). Four other groups of animals received histamine (10
mg/rat, intra-hippocampal) or different dose of CGP35348 (5, 10 and
15 ng/rat, intra-medial septal) in the same time. The test session was
performed 5 min after intra-hippocampal and intra-septal injections.
One-way ANOVA was used for comparison between the effects of
different doses of histamine, muscimol and baclofen with its vehicle.
Two-way ANOVA was used for evaluation of interactions between
drugs. Following a significant F-value, post-hoc analysis (Tukey test)
was performed for assessing specific group comparisons. Differences
with P<0.05 between experimental groups at each point were con-
sidered statistically significant.
Results
Histology
Figure 1 illustrates the approximate point of the drugs injections in
the CA1 and MS respectively. The histological results were plotted
on representative sections taken from the rat brain atlas of Paxinos
and Watson. Data from the animals with the injection sites located
outside the CA1 and MS were not used in the analysis.
Vol 2, No 2

Effects of histamine injection into CA1 on anxiety-like behavior
Supplementary figure 1 shows the effects of intraCA1 injection of
histamine (1, 5 and 10 g/rat) on anxiety-related parameters in the
elevated plus-maze. A one-way ANOVA revealed that histamine at
dose of 10 g/rat (0.5 l/side) increased %OAT [F(3,24)=8.7, P<0.001]
and %OAE [F(3,24)=5.3, P<0.01] indicating an anxiolytic response by
histamine. No significant change in the locomotor activity was ob-
served following administration of histamine [F(3,24)=0.8, P>0.05] .
This data indicates that histamine administration into CA1 induces
an anxiolytic effect.

The effects of histamine injection into CA1 and muscimol
injection into MS on anxiety-like behavior
Figure 2 shows the effects of MS injections of muscimol (2.5, 5 and
10 ng/rat) on anxiety-related parameters in the elevated plus-maze
test. A one-way ANOVA revealed that muscimol at dose of 10 ng/rat
(1l/rat) also increased %OAT [F(3,24)=16.3, P<0.001] and %OET
[F(3,24)=9.60, P<0.001] indicating anxiolytic response by muscimol.
No significant change in the locomotor activity was observed follow-
ing administration of muscimol [F(3,24)=3.52,P>0.05]. Effects of sim-
ultaneous injection of histamine and muscimol in the CA1 and MS
respectively on anxiety-like behavior are shown in figure 3. A one-
way ANOVA revealed that combined infusions of histamine 1 g/rat
(0.5 l/side) and muscimol 2.5 ng/rat (1 l/rat) into CA1 and MS
respectively increased %OAT [F(3,24)=85.51,P<0.001] and %OAE
[F(3,24)=26.6<0.001], but not locomotor activity [F(3,24)=1.07, P>
0.05]. The data indicates that histamine and muscimol have a syner-
gistic anxiolytic effect.

Effects of histamine injection into CA1 and bicuculline injection
into MS on anxiety-like behavior
Effects of histamine and bicuculline injection into CA1 and MS re-
spectively on anxiety-like behavior are shown in figure 4. A two-way
ANOVA indicated a difference between the responses induced by
bicuculline in the absence or presence of histamine. The data
showed that intra-septal infusions of bicuculline selectively antago-
nizes the anxiolytic effects of intra-hippocampal histamine in the el-
evated plus-maze. No significant change in the locomotor activity
was observed following administration of histamine alone or hista-
mine plus bicuculline.

Figure 1: The approximate placement of injection cannulae within the CA1 are
indicated by circles. Representative sections of the CA1 and medial septum
were taken from the rat brain atlas of Paxinos and Watson (1986).

Supplementary figure 1: The effects of intra-CA1 injection of histamine on
anxiety. Rats were injected with saline (1 l/rat; 0.5 l/ bilateral) or histamine
(1, 5 and 10 g/rat; 0.5 l bilateral). The test was performed 5 min after intra-
CA1 injections. Each bar is mean S.E.M. %OAT (A), %OAE (B) or locomotor
activity (C). N=7. **P<0.01, ***P<0.001, when compared to the saline treated
rats.

Figure 2: The effects of intramedial septum injection of muscimol on anxiety.
Rats were injected with saline (1 l/rat) or muscimol (2.5, 5 and 10 ng/rat).The
test was performed 5 min after intra-medial septum injections. Each bar is
mean SEM. %OAT (A), % OAE (B) or locomotor activity (C). N=7. ***P< 0.001,
when compared to the saline treated rats.


Effects of baclofen injection into MS on anxiety-like behavior
Figure 5 shows the effects of MS injections of baclofen (0.1, 0.5 and
1 ng/rat) on anxiety-related parameters in the elevated plus-maze. A
one-way ANOVA revealed that baclofen at the dose of 1 ng/rat (1
l/rat) could attenuate %OAT [F(3,48)=10.3, P<0.001] and %OAE
[F(3,48)=96.55, P<0.001] indicating anxiolytic response by baclofen.
No significant change in the locomotor activity was observed follow-
ing administration of baclofen [F(3,48)=1.01,P>0.05].

Effects of histamine injection into CA1 and CGP35348 (GABAB
antagonist) injection into MS on anxiety-like behavior
Herein, we investigated the effect of GABAB antagonist on anxiety
like behavior to identify the exact role of GABAB on the anxiety (fig-
ure 6). We administrated 3 different doses of CGP35348 (5, 10 and
15 ng/rat, intra-medial septum). Administration 15 ng/rat could im-
prove %OAT and %OAE ([F(3,48)=33.3, P<0.001], [F(3,48)=21.48,
P<0.001], respectively) in the rats. Simultaneous injection of
CGP35348 (15 ng/rat in MS) and histamine (1g /rat into CA1) could
increase the anxiolytic behavior synergically.
Discussion
The present results indicated that elevation of histamine level in the
hippocampus and/or stimulating GABAergic receptors in the medial
septum decreases anxiety as measured in the elevated plus maze
test. Furthermore, the data suggests that concomitant stimulating
hippocampal histaminergic and septal GABAergic receptors attenu-
ate anxiety related behavior. Herein, we compared the anxiolytic po-
tential of two GABA receptors, GABAA and GABAB receptors. The
study has confirmed that stimulating GABAA agonist receptor using

Figure 3: The effects of intra-CA1 injection of histamine and intra-septum
injection of muscimol on anxiety. Rats were injected with saline-saline(1 l/rat)
or saline(1l/rat)- histamine(1g/rat) or saline(1l/rat)-muscimol(2.5 ng/rat) or
histamine(1g/rat)-muscimol(2.5ng/rat).The test was performed 5 min after
intra-CA1 injections and intra-septum injections. Each bar is mean S.E.M.
%OAT (A) , %OAE (B) or locomotor activity (C).N=7. ***P<0.001, when
compared to the saline treated rats.

Figure 4: The effects of intra CA1 injection of histamine (10g/rat) and intra
MS injection of bicuculline (10, 20 and 30 ng/rat) on anxiety. Rats were
injected with saline (1l/rat) or histamine (1, 5 and 10 g/rat , 0.5 bilateral) in
CA1 and with bicuculline in MS. The test was performed 5 min after intra-CA1
and intra-MS injections. Each bar is mean S.E.M. %OAT (A), % OAE (B) or
locomotor activity (C). N=7. ***P< 0.001, when compared to the saline treated
rats. +++P< 0.001, when compared to the saline/histamine treated rats.

Figure 5: The effects of intra-MS of baclofen (0.1, 0.5 and 1 ng/rat) on anxiety.
Rats were injected with saline (1l/rat) or baclofen (0.1, 0.5 and 1 ng/rat) in
MS. The test was performed 5 min after intra-MS injections. Each bar is mean
S.E.M. %OAT (A), % OAE (B) or locomotor activity (C). N=7. ***P< 0.001, when
compared to the saline treated rats.
Vol 2, No 2
muscimol in the medial septum in connection with hippocampal his-
taminergic system has synergic effect on the anxiolytic related be-
havior. In contrast, GABAB receptor agonist, baclofen, did not have
any significant changes alone and/or with simultaneous injection of
histamine in the hippocampus. Administration of GABAA and GABAB
receptors antagonists, bicuculine and CGP34358 respectively, estab-
lished the anxiety regulative effects of these drugs.

The anxiolytic effects induced by stimulating the hippocampal hista-
minergic system are consistent with previous data indicating that in-
tra-hippocampal infusions of histaminergic antagonists could in-
crease anxiety [23]. Our results are also consistent with these data
indicating histaminergic agents induce anxiolytic effects. The finding
indicates that probably histamine in hippocampus through activa-
tion of GABAergic pathways toward septum and inhibition leads to
decrease of anxiety. With respect to the septum, we have repeatedly
found that ablation or pharmacological inhibition of this area re-
duces rats anxiety-related behaviors in the elevated plus-maze and
the shock-probe burying tests, suggesting that the septum normally
plays an excitatory role in the control of anxiety [3, 9]. It has been
reported that the GABA receptor mechanism can influence anxiety
behavior in rats [24, 25]. In addition, the study confirmed that stim-
ulating septal GABAergic receptors with a directly acting GABAA ag-
onist, muscimol, can increase %OAT and %OAE but not locomotor
activity. Thus, our study provides clear evidence that higher dose of
muscimol in the MS decrease anxiety. The previous study also indi-
cates that administration of muscimol into lateral and basal amyg-
dale nuclei prior to anxiety conditioning or testing also reduces
freezing in rats [26]. These findings indicate that probably muscimol
through inhibition septum reduces anxiety. Our finding is also con-
sistent with data indicating that muscimol injection into MS de-
creases anxiety [27].
Overall, these results suggest that increasing histamine activity in the
dorsal hippocampus reduces anxiety. The similarity of the anxiolytic
effects of muscimol infused into the lateral and medial septum is
consistent with lesion studies [28]. In addition, the present study
shows that simultaneous stimulation of both hippocampal hista-
minergic and septal GABAergic receptors with sub-effective doses of
above drugs results in a larger anxiolytic effect compared to the in-
dependent stimulation of each of these receptor systems.
Our central finding indicates that intra-septal infusions of bicuculline,
a GABAA antagonist, selectively antagonizes the anxiolytic effect of
intra-hippocampal histamine in the elevated plus-maze. It must be
mentioned that if intra-septal bicuculline was anxiogenic, then this
could mask any anxiolytic actions of intra-hippocampal histaminer-
gic agent, regardless of whether these latter effects were ultimately
due to reduced neurotransmission in a hippocampal projection tar-
get such as the septal nucleus or some other site. The results from
our experiments strongly suggest that the hippocampus and the MS
regulate open-arm exploration.
To investigate whether GABAB receptors are involved in anxiety re-
lated behavior, we injected baclofen (GABAB agonist) to the dorsal
hippocampus. Baclofen decreased %OAT and %OAE in the elevated
plus maze. Moreover, injection of CGP35348 (GABAB antagonist) re-
versed these changes in the rats. Concomitant injection of histamine
and CGP35348 can induce anxiolytic effect more than a solitary in-
jection of CGP35348. This different role of GABAB receptors might
depend on their receptor site in the neuronal synapses [29].
In summary, anxiolytic effect of GABAA receptor system in the MS
region in the elevated plus-maze has been investigated. Histaminer-
gic and GABAA systems act synergically for controlling anxiety. On
the other hand, GABAB has anxiogenic effects in the medial septum.
Taken together, it seems that anxiolytic effect of septohippocampus
system may be modulated through GABAA receptors.
Conclusion
In summary, this report indicates that GABAA receptor has more ef-
fective potency in the septohippocampus system in the anxiety re-
lated behavior. In contrast, it has been showed that GABAB receptor
has an anxiolytic effect in the medial septum. Hence, it can be con-
cluded that hippocampal histaminergic systems act together via sep-
tum GABAergic system for regulating anxiety in male rats.
Funding: This work was supported financially by Kharazmi University of Tehran.

Figure 6: The effects of intra-CA1 injection of histamine (10 g/rat) and intra-
MS injection of CGP35348 (5, 10 and 15 ng/rat) on anxiety. Rats were injected
with saline (1 l/rat) or histamine (1, 5 and 10 g/rat, 0.5 bilateral) in CA1 and
with CGP35348 in MS. The test was performed 5 min after intra-CA1 and intra-
MS injections. Each bar is mean S.E.M. %OAT (A), %OAE (B) or locomotor
activity (C). N=7. **P< 0.01, when compared to the saline treated rats. +++P<
0.001, when compared to the saline/CGP35348 treated rats.
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70 BMSCs seeded onto biological materials

Vol 2, No 2
Examination of bone marrow mesenchymal stem cells seeded onto poly(3-hydroxybutyrate-co-3-
hydroxybutyrate) biological materials for myocardial patch
Junsheng Mu
1
, Hongxing Niu
1
, Fan Zhou
2
, Jianqun Zhang
1
, Ping Hu
3
, Ping Bo
1
, Yan Wang
3

Introduction
Cell therapy is believed to be a promising candidate treatment for
severe heart failure [1]. Various types of cell sources, including skel-
etal myoblasts, bone marrow mononuclear cells and mesenchymal
stem cells, have been examined in both basic and clinical studies [2].
Although there have been reports showing that transplanted cells
improve the function of the ischemic heart, the effects of cell therapy
are variable in clinical trials and beneficial effects and type of cells
suitable for myocardial repair are still unknown [3, 4]. The efficiency
of cell transplantation is hindered by a low rate of engraftment of
transplanted cells, caused by leakage during injections and massive
cell death within the host [5, 6]. Biological scaffolds are expected to
circumvent the loss of grafted cells as they confer a three-dimen-
sional (3D) microenvironment for the cells that supports their sur-
vival, proliferation and function [7]. Many types of scaffolds have
been designed to date; however, there are many limitations that re-
main to be addressed in terms of their correct matching with en-
grafted cells, compatibility with host tissue, and clinical safety.
Self-assembling nanopeptides consist of alternating hydrophilic and
hydrophobic amino acid residues that can adopt -sheet structures
and form a stable three-dimensional hydrogel, consisting of > 99.5%
water depending upon pH, salt, and time [8]. Hydrogels have been
shown to promote cell survival, proliferation and differentiation of
many different cell types in vitro, including neural stem cells, osteo-
cytes, and endothelial cells [9-11]. When self-assembling peptides
are injected into tissue, they form a nanofiber network, which creates
three-dimensional microenvironments for endogenous cells, leading
to angiogenesis and axonal regeneration [12, 13].
In this manuscript, we examined 3D patch material capable of imi-

1
Beijing Institute of Heart, Lung and Blood Vessel Diseases, China
2
General Hospital of the Chinese Peoples Armed Police Forces, China
3
Tsinghua University, China
tating aspects of the extracellular stroma, and providing a suitable
environment for the survival and proliferation of seed cells. The op-
timal myocardial patch material should provide good biocompatibil-
ity, biodegradability and safety, reduce cell loss following transplan-
tation and promote cell growth for tissue repair [14, 15]. Current
studies are developing poly(3-hydroxybutyrate-co-3-hydroxybutyr-
ate) [P(3HB-co-3HB)] biomaterials for medical applications. However,
little research has been performed into the combination of P(3HB-
co-3HB) biomaterials with BMSCs for their application as a patch ma-
terial for myocardial regeneration. This study aimed to isolate and
expand BMSCs in vitro, which will be subsequently seeded onto three
patch biomaterials to observe the influence of these biomaterials on
cell growth and proliferation. From the results of this study we hope
that a biological material with good compatibility and physical prop-
erties suitable for BMSC growth and proliferation will be identified
for in vivo applications [16].
Materials
The experiment was conducted from July 2011 to April 2012 in the
Cardiovascular Disease Research Institute of Beijing and in the key
laboratory for cardiovascular remodeling. BSL-C57 mice were bought
from Wei Tong Lihua Experimental Animal Laboratory, Beijing. Fetal
calf serum and low glucose Dulbeccos modified Eagles medium
(LG-DMEM) were purchased from Gibco Life Technologies. Antibod-
ies were purchased from BD Biosciences and DAPI reagents were
purchased from Sigma. Biological materials composed of a thin film
of PU, P(3HB-co-3HB) and PPC were provided and detected by the
High Polymer Institute Laboratory of the Chemical Department in
Qinghua University. The porosity was analyzed using a micrometrics
Correspondence: Junsheng Mu
Email: wesleymu@hotmail.com
Abstract
Background: The implantation of bone marrow mesenchymal stem cells (BMSCs) into the heart has been reported to be effective for
myocardial infarction. However, it is unknown as to what methods are most suitable for supporting stem cell growth in a myocardial patch.
We used a new polymer material composed of poly(3-hydroxybutyrate-co-3-hydroxybutyrate) [P(3HB-co-3HB)] co-cultured with BMSCs to
create a myocardial patch.
Methods: BMSCs were obtained from healthy male BSL-C57 mice. The cells were treated with 5-azacytidine to investigate their
differentiation into cardiomyocytes. The cells were seeded for 24-hours onto P(3HB-co-3HB) biological material films (n=8). Cell-biomaterial
constructs were fixed and analyzed using different methods. BMSCs were CD34
-
, CD45
-
, CD90
+
(low) and CD73
+
. The cells were stained
with anti-cardiac troponin T (cTnT) and anti-connexin 43 (CX43) antibodies after 5-azacytidine treatment.
Results: Scanning electron microscopy showed that BMSC morphology was normal and cell numbers were more abundant on the P(3HB-
co-3HB) material surfaces. The growth curve of BMSCs on the biomaterial patches showed the P(3HB-co-3HB) material permitted good
stem cell growth.
Conclusion: Owing to its excellent biocompatibility and biodegradability properties, in particular its porosity, P(3HB-co-3HB) is highlighted
as an optimal material to support myocardial cell growth and myocardial patch formation in patients with myocardial infarction. (El Med
J 2:2; 2014)
Keywords: Myocardial Regeneration, Bone Marrow Mesenchymal Stem Cells, Myocardial Infarction, Poly(3-hydroxybutyrate-co-3-
hydroxybutyrate)
Mu J, Niu H, Zhou F et al 71
ASAP 2020 instrument and the BET method was used to determine
the surface area (31.2899 m
2
/g).
Methods
Cell Isolation and cultivation
Four-week-old C57 mice were euthanized by cervical dislocation.
Both tibial and femoral bones were excised and separated. Muscle
tissues were removed and the bone marrow cavity was exposed un-
der aseptic conditions. The protocol was approved by Capital Medi-
cal University Institutional Animal Care and Use Committee. All ani-
mal welfare measures were taken to ameliorate suffering. Sterile
DMEM was used to repeatedly flush cells from the bone marrow cav-
ity, prior to filtering through a 200 mesh nylon net to remove large
tissue fragments. The cell suspension was collected in liquid and cen-
trifuged at 1000 rpm for 5 min. Cells were re-suspended and cultured
in LG-DMEM containing 10% FBS. The culture medium was changed
after 24 hours and every other day thereafter. Cells were incubated
in 5% CO2 at 37C, with a volume fraction of 0.05. Original generation
cells (P0) were harvested on reaching 90% confluence by digestion
with 0.25% EDTA-pancreatic enzymes and passaged at a ratio of 1:2
for subsequent cultures (P1-P5). Culture medium was changed every
other day and cell growth was observed using an inverted micro-
scope.
Flow cytometry
BMSCs (P5) were harvested using 0.25% EDTA-Pentazyme and ad-
justed to a cell concentration of 110
7
/ml with phosphate-buffered
saline (PBS). Cells were added to five flow tubes containing 100l of
cell-suspension. PE-anti-CD90, PE-anti-CD34, FITC-anti-CD45 and
FITC-anti-CD73 were each added to one tube of cells and the remain-
ing tube represented a blank control [17-20]. After stirring, cells were
incubated at 4C for 30 min, washed twice with PBS and analyzed
using flow cytometry.
Differentiation in vitro and immunohistochemistry
BMSCs of all five passages were re-suspended after trypsin treatment
and washed three times with PBS. The cells were re-suspended in
complete medium and seeded into a 6-well dish at a density of 5
10
5
cells/dish. 24 hours after seeding, the medium was changed to
complete medium containing 5-azacytidine (10 mol/l). After incu-
bating for another 24 hours, the medium was changed to complete
medium without 5-azacytidine. The medium was changed twice a
week thereafter until the experiment was terminated two weeks af-
ter drug treatment. After completing the protocol, the cells were
mounted for microscopic examination, and the cells were prepared
for immunohistochemical analysis using fluorescence microscopy.
Induced and uninduced cells grown on glass coverslips in a 24-well
plate, were fixed in 4% formaldehyde for 10 min. washed three times
with PBS for 3 min each at room temperature. Non-specific binding
was prevented by several washes with PBS. Then, cells were perme-
abilized with 0.2% Triton X-100 for 10 min, and washed three times
with PBS for 10 min at room temperature. After blocking with 5%
BSA in PBS for 0.5 h at room temperature, the cells were incubated
with primary antibodies directed against anti-cTnT or anti-CX43 over-
night at 4C. After three washes in PBS for 5 min each, cells were
incubated with goat anti-rabbit IgG/RBITC or goat anti-rabbit
IgG/FITC as the secondary antibody for 45 min at room temperature.
Finally, after washing three times with PBS, they were incubated with
0.5g/mL DAPI for 10 min, washed with PBS three times, and
mounted for microscopic examination under fluorescence micros-
copy. Cells with green or red antibody staining in the cytoplasm were
considered positive for the protein in question. Cells with blue stain-
ing in the nucleus were considered healthy.
Preparation and identification of a myocardial patch
Myocardial patch biomaterials and puncher were sterilized with
epoxyethane. Circular discs (0.5 cm diameter) were punched out and
soaked in culture medium. Patches were rinsed three times in PBS
before placing them flattened on a 96-hole board for use. BMSCs
(P5) were seeded onto the patches at a density of 1 10
5
cells per
construct.
A) Preparation for scanning electronic microscopy (SEM): Cell-constructs
were cleaned three times with deionized water and fixed with 500L
0.25% glutaraldehyde at 4C. With the cell patches totally sub-
merged, samples were analyzed under the microscope.
B) Preparation for fluorescent staining and cell counting: BMSCs were re-
suspended at 1 10
5
cells/mL and transferred in triplicate into the
24-hole board (500L/hole). Cells were cultured on the board for 24
h in the incubator at 5% CO2 and 37C. Medium was removed after
cells had adhered to the surface. Cells were fixed with paraformalde-
hyde for 10 min, washed three times with PBS and placed on a glass
slide. DAPI was dropped onto the patch to immerse the cell-con-
struct, after which it was covered with a glass coverslip. Slides were
mounted reversely under a fluorescent microscope for observation
and photography. Average cell counts were obtained from 10 fields
of vision for each material.
Data processing
Cell counts were analyzed with SPSS 12.0 statistics software and var-
iance analysis was determined for inter-group comparison. A value
of P < 0.05 was found to be statistically significant. Flow cytometry
results were analyzed using EXPOTM 32 ADC software.
Results
BMSC Morphology
Original generation BMSCs were observed in various shapes includ-
ing circular, elliptical, rod and shuttle-like morphologies. The cells
grew in colonies after 4-6 days, extending outwards in a radial or
vortex arrangement. By 2-3 weeks, approximately 80-90% conflu-
ence was achieved. Non-adherent cells were removed with media
changes and the cells were distributed more evenly on the surface,
with spindle-like morphology and rapid growth (Figure 1).
Flow cytometry
BMSCs (P3) were analyzed using flow cytometry to characterize their
cell surface marker profile. As show in Figure 2, BMSCs were CD34
-

and CD45
-
, CD90
+
(low) and CD73
+
(Figure 2).
Differentiation into cardiomyocytes
To determine the morphological changes in BMSCs induced by 5-
azacytidine treatment, phase-contrast microscopy and / or im-
Vol 2, No 2
munostaining with anti-CX43 or anti-cTnT antibodies were per-
formed. The morphological differentiation from MSCs to myogenic-
like cells evolved gradually. During exposure to 5-azacytidine, some
adherent cells died, while the surviving cells began to proliferate and
differentiate. One week later, approximately 30% of all of the remain-
ing adherent cells had enlarged and assumed ball-like or stick-like
morphologies. Within 2 weeks, cells had connected with adjoining
cells and formed myotube-like structures. The cells looked like long
spindle-shaped fibroblastic cells when observed under a microscope.

Figures 3 and 4 shows fluorescence microscopy images of BMSCs
immunostained with antiCX43 or anti-cTnT antibodies at two weeks
after 5-azacytidine treatment. CX43-positive or cTnT-positive cells
gradually connected with neighboring CX43-positive or cTnT-posi-
tive cells. The uninduced cells were negative for both of these cardi-
omyocyte markers.
SEM analysis
Using SEM, the patch was observed to have a normal surface, com-
prising high numbers of granule-like cell aggregates with typical cell
morphology. P(3HB-co-3HB) is a 3D network-structure with a fiber
intertexture and even pore size, into which the BMSCs grew well with
normal morphology and an absence of cell lysate or debris (Figure
5).
Fluorescence microscopy and cell counting
Cell-seeded patches, stained with DAPI (Figure 6), were mounted
and examined under a microscope to obtain average cell counts and
growth curves on the biomaterial patches (Figure 7).

Figure 1: Inverted microcopy image of BMSCs (P5) at: 100 and 200.
A
B
C
D
Figure 2: Flow cytometry for surface characteristics of BMSCs. BMSCs were CD34
-

and CD45
-
negative, CD90
+
(low) and CD73
+
.

Discussion
BMSCs are relatively primitive, with a strong potential for multi-line-
age cell differentiation. BMSCs have been shown to differentiate into
cardiac cells, through transverse differentiation and cell integration,
and into endothelial and smooth muscle cells, directly participating
in the formation of blood vessels [21, 22]. The paracrine action of
BMSCs activates an endogenous rehabilitation mechanism to repair
the damaged heart and this function has drawn scientific attention
towards their clinical application in tissue engineering and gene
therapy [23]. Due to advantages in repairing cardiac muscle and as-
sociated vessels, MSCs have been the preferable cell source for car-
diac tissue engineering applications. The formation of cellularized
cardiac muscle is the primary goal for tissue engineering treatments
of myocardial infarct. However, reduced blood flow and heart me-
chanical activity may cause loss of transplanted cells [24]. It is there-
fore necessary to identify materials with excellent physical character-
istics, biological compatibility and biodegradability properties to
provide a good environment for transplanted cells, for cell survival
and growth in the infarct area, ultimately improving post-treatment
conditions.

We have established BMSCs that can be induced to differentiate into
cardiomyocytes in vitro by 5-azacytidine induction. CX43 and cTnT
are known early markers of myogenic differentiation and are also in-
volved in muscle cell contraction. Other markers include alpha-car-
diac actin and beta-MHC. In this study, CX43 and cTnT were found in
the cytoplasm of the cells induced with 5-azacytidine (Figures 3 and
4). In the ventricular muscle of small mammals, there is a develop-
mental switch from expression of CX43 and cTnT, which is the pre-
dominant fetal form [25, 26]. Differentiated BMSCs mainly expressed
CX43 and cTnT, indicating activation of the transcriptional cascade
regulating cardiomyocyte differentiation.
Many 3D materials have been investigated for myocardial scaffolds,
including fibrin glue, poly(lactic-coglycolic) acid (PLGA), gelatin hy-
drogels, and hyaluronic acid (HA) hydrogels [7]. Once the cells adapt
to the 3D milieus, the cells migrate, proliferate and function accord-
ing to the multi-directional molecular and mechanical signals similar
to organized tissue. However, there are substantial challenges for ex-
isting materials, including stimulation of the host inflammatory re-
sponse, toxic degradation, and excessively large pore sizes and fiber
diameters [27]. Self-assembling nanopeptides, such as P(3HB-co-
3HB), are an artificial material containing no animal-derived proteins.
Hydrogels formed by self-assembling peptides have very small pore
sizes that promote endothelial adhesion and capillary formation,
while still allowing the rapid migration of cells [28]. This study also
determined the porosity and surface area using the BET method of
P(3HB-co-3HB). When implanted into the myocardium, nanofibers
rapidly polymerize at physiological pH and osmolality, resulting in
entrapment of the transplanted cells and prevention of leakage. In
addition, the rigidity of the hydrogel can be tuned by the concentra-
tion of the nanopeptide solution, enabling us to deliver BMSCs easily.
These properties of nanopeptides, especially its porosity, may en-
hance the engraftment of a larger number of cells.
Self-assembling nanopeptides can be modified in a variety of ways
A B
Figure 3(A): Induced cells: Under fluorescence microscopy, cells with the CX43
green antibody binding in the cytoplasm, cells with blue reaction product in the
nuclear region.
Figure 3(B): Uninduced cells: Under fluorescence microscopy, dew cells with the
CX43 green antibody binding in the cytoplasm, cells with blue reaction product
in the nuclear region (x400).
A B
Figure 4(A): Induced cells: Under fluorescence microscopy, cells with cTnT red
antibody binding in the cytoplasm. Cells with blue reaction product in the
nuclear region.
Figure 4(B): Uninduced cell: Under fluorescence microscopy, few cells with cTnT
red antibody binding in the cytoplasm. Cells with blue reaction product in the
nuclear region (x400).

Figure 5: SEM images of P(3HB-co-3HB) polymer biomaterial patches.
(A) x100; (B) x300; (C) x1000.

Figure 6: Fluorescent microscopy, DAPI staining of BMSCs on P(3HB-co-3HB)
patches. (A) 48h 400; (B) 72h 400; (C) 96h 400.

Figure 7: Growth curve on P(3HB-co-3HB) biomaterial patches.
Vol 2, No 2
that allow for cell-specific signals to be delivered. For example, nan-
ofiber scaffolds containing RGD-binding sequence have been shown
to significantly promote the proliferation of mouse pre-osteoblasts
[29]. Moreover, alkaline phosphatase (ALP) activity and osteocalcin
secretion, which are early and late markers for osteoblastic differen-
tiation, were also significantly increased [29]. Silva et al. synthesized
self-assembling peptides containing an IKVAV laminin motif, in
which neuronal precursors differentiated into neurons with exten-
sive processes, while very few cells differentiated into astrocytes [30].
These tailor-made peptides have the ability to modulate bidirec-
tional signals between nanopeptides and transplanted cells, ena-
bling us to create a 3D cardiac graft, which substitutes the scar tissue
after myocardial infarction, leading to prevention of cardiac remod-
eling and improvement of cardiac function.
We have shown that P(3HB-co-3HB) can be a useful biomaterial,
which assures the safe and effective delivery of cells into the myo-
cardium. Therefore, P(3HB-co-3HB) may be suitable for supporting
long-term proliferation, engraftment and differentiation of trans-
planted cells in infarcted heart tissue. P(3HB-co-3HB) is a new third
generation polyhydroxyalkanoates (PHA) material that has received
wide attention due to its good physical properties, in particular its
porosity. P(3HB-co-3HB) is a novel material synthesized by the Poly-
mer Research Institute in the Department of Chemical Engineering,
Tsinghua University, which has been shown to have better biological
compatibility and biodegradability properties than polyhydroxy-
butyrate (PHB).
Following initial culturing, BMSCs are heterogeneous in shape, with
circular, elliptical, rod and shuttle-like morphologies. During culture,
non-adherent cells were removed and remaining adherent cells
grew into characteristic MSCs, with spindle-shaped morphology.
BMSCs (P3) were CD34
-
and CD45
-
, CD90
+
(low) and CD73
+
. These
cell surface marker characteristics are similar to those reported for
MSCs. Twenty-four hours after seeding BMSCs onto the patch bio-
materials, cell growth and morphology on the three biomaterials
were observed using SEM. Results indicated that there were higher
cell numbers, fewer cell debris and good cell morphology on the
P(3HB-co-3HB) scaffold compared with the other materials, indicat-
ing that it was most effective at supporting growth and morphology
of BMSCs. P(3HB-co-3HB) is highly porous material that is effective
for good cell attachment, tissue in-growth and cell maintenance.
These findings were supported by cell counting using DAPI staining
and by fluorescent microscopy. P(3HB-co-3HB) has excellent tissue
compatibility and biodegradability properties and can be well ab-
sorbed in the body.
However, there were some limitations of this study. Although cells
can survive in vitro on the P(3HB-co-3HB) myocardial patches, the
influence of degradation and absorption of the patch materials
within the body and the influence of the transplanted patch on the
immune system is yet to be determined. Further studies are required
to assess the feasibility of using P(3HB-co-3HB) as a cardiac patch in
the clinic as well as its biotoxicity and in vivo tissue repair function.
Conclusion
In summary, we have investigated a number of potential biomateri-
als for application as a myocardial patch. In this study, BMSCs were
readily transplanted onto P(3HB-CO-3HB) patch biomaterials. How-
ever, due to the non-absorbability property of the former, it has lim-
ited in use in the fields of medical and biological materials. P(3HB-
CO-3HB) is a novel material with good biological tissue compatibility
and biodegradability that has good potential for application as a my-
ocardial patch. Further studies are required to determine the feasi-
bility of using P(3HB-co-3HB) as a myocardial patch in vivo, as well
as determining its biotoxicity and tissue repair function.
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76 Myeloperoxidase in neonatal sepsis

Vol 2, No 2
Plasma myeloperoxidase enzyme assay in cases of neonatal sepsis
Eman Abdel Ghany
1
, Hannah Abul Ghar
1
, Eslam Madbooly
1
, Rania Mohamed Samy
2

Introduction
Sepsis neonatorum is the term used to describe any systemic bacte-
rial infection documented by a positive blood culture in the first
month of life. Bacterial sepsis in the neonate is a clinical syndrome
characterized by systemic signs of infection accompanied by bacte-
remia [1]. Approximately 99% of the 4 million annual neonatal
deaths occur in low and middle income countries, and 36% in others
are attributed to serious infections; in high mortality settings this
proportion may approach 50% [2].
Neonatal sepsis can be classified into two subtypes depending upon
whether the onset of symptoms is before 72 hours of life: early onset
neonatal sepsis (EONS) or late onset sepsis (LONS). These definitions
have contributed greatly to diagnosis and treatment by identifying
which microorganisms are likely to be responsible for sepsis during
these periods and the expected outcomes of infection [3].
At the time of term birth, the immune system has not fully matured.
The inexperienced adaptive immune system must still develop spec-
ificity and memory, which is completed only in the early childhood
years. As such, normal term neonates rely heavily on their innate im-
mune response but this too is immature. Immaturity of the immune
system is more pronounced in infants born preterm [4].
Polymorphonuclear leukocytes (PMNL) are the first cell type in hu-
man beings activated in host immune defense against infection.
These cells driven by chemotactic gradients migrate to inflammatory
loci, where they recognize and phagocytose bacteria and other ex-
trinsic microorganisms by release of hydrolytic enzymes and bacte-
ricidal proteins pre-stored in granules as well as newly generated re-
active oxygen species (ROS) [5]. PMNLs isolated from premature hu-
man neonates have impaired phagocytosis, decreased capacity to

1
Pediatrics Department, Faculty of Medicine, Cairo University, Egypt
2
Clinical Pathology Department, Faculty of Medicine, Cairo University, Egypt
Correspondence: Eman Abdel Ghany
Email: dreman_75@yahoo.com
generate oxygen radicals, and deficient intracellular bacterial killing
[6].
Vascular leakage and recruitment of circulating PMNLs to the site of
injury represent the early phase of the host defense mechanism and
response to tissue injury or sepsis. Clinically, the increased number
of PMNLs in blood is generally used to determine the development
of inflammation/sepsis [7]. A more feasible and quantitative ap-
proach is the use of the biochemical assay of PMNLs associated MPO
enzyme activity. This enzyme is highly enriched in the azurophilic
granules of PMNLs recruited to injured tissue to mediate the acute
phase of the inflammatory response [8]. Circulating lipopolysaccha-
rides released from bacteria may activate both neutrophils and mon-
ocytes. The activated neutrophils release MPO a major granule en-
zyme in neutrophils which accounts for 5% of the total neutrophil
protein [5]. MPO enzyme promotes oxidative stress in numerous in-
flammatory pathologies. It uses hydrogen peroxide to catalyze the
production of strong oxidants including reactive oxygen species and
free radicals that have bactericidal action [9].
The isolation of microorganisms from blood and/or CSF still remains
the gold standard for definitive diagnosis. For years, investigators
have sought a test or panel of tests able to identify septic neonates
accurately and rapidly while awaiting culture results, in order to ob-
tain an early diagnosis and develop a specific effective treatment for
a successful outcome. This study aimed: 1) to determine the diag-
nostic utilities [sensitivity, specificity, positive predictive value (PPV),
and negative predictive value (NPV)] of myeloperoxidase (MPO) en-
zyme for detection of neonatal sepsis; 2) to define the optimal cutoff
value for MPO level using the receiver operating characteristics
(ROC) curve so that it may be used as a reference with which future
studies can be compared.

Abstract
Background: Myeloperoxidase (MPO) is a heme enzyme of azurophilic granules in neutrophils that has a strong oxidative activity. MPO
has been demonstrated to be a major pathway for O2-dependent microbicidal activity.
Methods: This study was conducted on 84 neonates. 41 with culture proven sepsis and 43 healthy neonates who served as control.
Complete blood count, C-reactive protein quantitative assay and blood gases were done in cases group, MPO enzyme level measured by
ELISA was done and compared in both groups.
Results: MPO enzyme level was statistically higher in case group compared to control group (P-value < 0.001). There was no statistically
significant difference in whether the infected were pre-term or full term. MPO enzyme levels were statistically higher in cases with sepsis
without septic shock compared to cases with septic shock (P-value < 0.001). MPO level showed positive correlations with total leukocyte
count and absolute neutrophil count. MPO assay (using the cutoff value of 146.5 ng/ml) had a sensitivity of 80% and specificity of 83.9%
with a positive predictive value of 92.9%, negative predictive value of 61.5% and accuracy of 82.9%.
Conclusion: MPO enzyme level increases significantly in neonates with sepsis. MPO assay is not affected by gestational age but can be
affected by deterioration to septic shock. Early diagnosis of sepsis cannot rely on a single laboratory test and clinical decision remains to
have the upper hand in diagnosis. (El Med J 2:2; 2014)
Keywords: Myeloperoxidase, Neonatal Sepsis, Septic Shock, Systemic Inflammatory Response Syndrome
Ghany EA, Ghar HA, Madbooly E et al 77
Methods
This cross-sectional study was carried out in neonatal intensive care
units (NICU) of Cairo University Hospital (Kasr El Aini) and New Chil-
dren's Teaching Hospitals (El-Monira) between January and August
2013, with the approval of the Ethical Committee of NICU and par-
ents of the neonates. Data was confidentially preserved according to
the revised Helsinki Declaration of Bioethics [10].
Patients
The study was carried on forty one newborns with confirmed neo-
natal infection. It included preterm (PT) and full term (FT) neonates
of both gender with clinical symptoms and signs of sepsis within 1
st

month of life and positive blood culture. Exclusion criteria for enrol-
ment into this study were congenital anomalies, chromosomal ab-
normalities, inborn errors of metabolism and neonates with surgical
emergencies.
Forty three healthy stable newborns were enrolled in this study as a
control group. They were born to healthy mothers with negative
medical and obstetric history. All were free on clinical examination,
their blood samples were taken within the first 28 days of life for
MPO measurement. The collection of the blood specimens coincided
with other routine blood sampling procedures, such as hematocrit,
electrolytes, or glucose measurement.
Methods
All septic newborns were subjected to the following:
Comprehensive history taking including:
1. Antenatal history: maternal diseases (diabetes and hyperten-
sion), maternal infections (TORCH infections) and maternal med-
ications during present pregnancy.
2. Obstetric history: includes mode of delivery and premature rup-
ture of membrane (PROM) > 24 hours.
3. Natal history: gestational age, neonatal sex and birth weight.
4. Postnatal history: resuscitation data and Apgar score at 1 and 5
minutes, respiratory distress and cyanosis, onset of sepsis, severe
sepsis (sepsis complicated by organ dysfunction), occurrence of
septic shock (tachycardia with signs of decreased perfusion), du-
ration of hospital stay and outcome (survival or mortality).
Clinical examination for neonates:
1. Assessment of gestational age (GA) through analysis of maternal
dates and Ballard scores [11].
2. Assessment of general condition and reflexes (Moro / suckling).
3. Assessment of vital signs (respiratory rate, heart rate, blood pres-
sure, temperature, capillary refilling time) at time of sampling.
4. Complete examination including cardiac, chest, abdominal, and
neurological laying stress on tolerance to oral feeding, ab-
dominal distension, residual gastric aspirate, oliguria, jaundice,
cyanosis, convulsions, bleeding tendency and signs of septic
shock (tachycardia, decreased peripheral pulses, altered alert-
ness, cool extremities, reduced urinary output and lately hypo-
tension).
Laboratory investigations:
1. Complete blood count with differential leukocytic count (Abott
Cell - Dyn3700 - Abbott diagnostics - USA).
2. Analysis of plasma myeloperoxidase enzyme level by enzyme-
linked immunosorbent assay (ELISA) (MPO ELIZA kit - Immundi-
agnostik AG - Germany): 1ml peripheral blood sample on EDTA
vacutainer tube was collected. The sample was taken at the time
of sepsis confirmation. Freshly collected EDTA blood was centri-
fuged to collect plasma. The assay utilizes the two-site sand-
wich technique with two selected polyclonal antibodies that
bind to human MPO.
3. C-reactive protein (CRP) quantitative assay (NEPHSTAR CRP kit-
Goldsite Diagnostics - China): normal range of CRP concentra-
tion of healthy infant is < 5 mg/L.
4. Measurement of venous blood gases: Metabolic acidosis is de-
fined as an arterial blood pH < 7.35 (if venous sample pH < 7.32)
with plasma bicarbonate < 22 mEq/L (if venous sample < 19
mEq/L).
5. Blood culture for aerobic and anaerobic organism and antibiotic
sensitivity testing: 1 to 5 mL of blood was drawn from venipunc-
ture using sterile needle and then blood was injected in the
blood culture bottle for Bactec microbial detection system (Bac-
tec 9050, Becton - Dickinson).
Statistical methods
Data analysis was performed using Statistical Package for Social Sci-
ences (SPSS) version 17. Numerical data was summarized using me-
dian and ranges. Categorical data was summarized as percentages.
Comparisons between two groups with respect to normally distrib-
uted numeric variables were done using the t-tests. Non-normally
distributed variables were compared by Mann-Whitney test. Com-
parisons between septic shock cases, septic cases without shock and
controls were performed by KruskalWallis test followed by the post
hoc Bonferroni test. To measure the strength of the association be-
tween MPO and other factors, Spearmans correlation coefficients
were used. The receiver operator characteristic (ROC) curve was used
to display the relationship between sensitivity and specificity [12].
All p-values were two-sided. P-values < 0.05 were considered signif-
icant.
Results
Among the total of 41 neonates in the case group, there were 23
(56.1%) males and 18 (43.9%) females. 14 (34.1%) neonates were
delivered normally (NVD) and 27 (65.9%) neonates by cesarean sec-
tion (CS). 8 (19.5%) neonates were delivered after history of PROM
and 33 (80.5%) neonates with no history of PROM. Among the total
of 43 neonates in the control group, there were 30 (69.8%) males
and 13 (30.2%) females that represent. 20 (46.5%) neonates were
delivered by NVD and 23 (53.5%) neonates by CS. 3 (7%) neonates
were delivered after history of PROM and 40 (93%) neonates with no
history of PROM that represent. Table 1 shows comparison between
the case and control groups regarding GA, postnatal age, weight and
Apgar score at 1 and 5 minutes and revealed that there was a signif-
icant difference between the two groups regarding these variables,
p-value < 0.001. The following data were found in study group of
neonates with sepsis: 1) PT neonates (61%) were more than FT ones
(39%); 2) LOS is of higher prevalence (78%) than EOS (22%); 3) Re-
garding outcome of neonates; 73.2% were discharged while 26.8%
died. Table 2 shows the laboratory data (CBC with differential count,
blood gases and CRP) of the case group.
Vol 2, No 2
Table 1: Demographic data of the case and control groups
Item Cases (n=41) Controls (n=43) P-value
Mean SD* Minimum Maximum Mean SD* Minimum Maximum
GA
@
(weeks) 34.1 4.1 28.0 41.0 38.0 2.1 30.0 40.0 <0.001
Postnatal age (days) 15.0 7.7 3.0 28.0 9.1 4.3 3.0 21.0 <0.001
Weight (Kg) 2.1 1.0 0.9 5.0 3.2 0.6 1.3 4.2 <0.001
Apgar 1 min 3.7 1.9 1.0 7.0 6.4 1.4 3.0 8.0 <0.001
Apgar 5 min 6.8 1.8 2.0 9.0 8.8 0.5 7.0 9.0 <0.001
*SD=Standard Deviation;
@
GA=Gestational Age

Table 2: Laboratory data of the case group
Variable Mean Standard Deviation Median Minimum Maximum
Blood Count Platelets (1000/ml) 132.6 91.4 - 17.0 413.0
Total leukocyte count (1000/ml) 16.0 11.5 - 3.4 44.0
Absolute neutrophil count (/mm
3
) - - 6.12 1.51 31.68
Absolute band cells count (1000/mm
3
) - - 1.08 0.1 14.08
Band (%) 13.0 8.4 - 1.0 34.0
Absolute segmented cells count (1000/mm
3
) - - 4.3 0.93 26.84
Segmented cells (%) 44.5 18.1 - 16.0 82.0
Immature to total neutrophil ratio (I/T ratio) 0.2 0.1 - 0.0 0.6
Blood Gases pH 7.3 0.1 - 7.1 7.6
pCO2 46.4 12.1 - 16.0 80.0
pO2 45.7 23.1 - 20.0 124.0
HCO3 24.1 6.7 - 9.2 35.0
Base excess - - -0.6 -16.0 12.7
C-reactive Protein - - 24 6 192
Table 3 shows thrombocytopenia in 61% of cases, leukocytosis in
26.8% of cases, leukopenia in 7.3% of cases, shift to left in 63.4% of
cases and metabolic acidosis in 29.3% of cases.
Table 3: Thrombocytopenia, total leukocyte count, shift to
left and metabolic acidosis frequency in the case group
Variable N %
Thrombocytopenia
(<150,000/l)
No 16 39.0%
Yes 25 61.0%
Total leukocyte count
Normal 27 65.9%
Leukopenia* 3 7.3%
Leukocytosis
@
11 26.8%
Shift to the left
(I/T ratio 0.2)
Yes 26 63.4%
No 15 36.6%
Metabolic acidosis Yes 12 29.3%
No 29 70.7%
*Leukopenia <5000/mm
3
;
@
Leukocytosis >20,000/mm
3

Table 4 demonstrates the frequencies of organisms isolated from
blood culture of neonates with sepsis. The highest frequency was for
Klebsiella that was isolated from 17 neonates (41%).
Figure 1 shows a comparison between cases and control groups re-
garding MPO level. MPO level differs significantly in both groups. In
the case group; the median was 238 ng/ml while in the control
group; it was 67 ng/ml with p-value < 0.001.

Table 4: Blood culture organism frequency
Frequency Percentage
Klebsiella 17 41.5%
Pseudomonas 10 24.4%
Acenitobactear 3 7.3%
CoNS 3 7.3%
Candida 3 7.3%
Klebsiella and Pseudomonas 2 4.9%
MRSA* 1 2.4%
E.coli 1 2.4%
Klebsiella & MRSA 1 2.4%
Total 41 100%
*Methicillin-resistant Staphylococcus aureus

Figure 1: Comparison of MPO level in the case and control
groups
A comparison regarding MPO level between PT neonates (median =
238 ng/ml) and FT neonates (median = 232 ng/ml) among case
group was done and it was found that MPO level was not signifi-
cantly different (p-value=0.979).
Figure (2) revealed that MPO level was lower in neonates with septic
shock (median=100 ng/ml) than other cases in group of sepsis (me-
dian = 276 ng/ml) with statistically significant difference (p-value <
0.001).

Figure 2: Comparison of MPO level in neonates with septic shock
and other cases with sepsis
Figure 3 revealed no statistically significant difference in MPO level
between neonates with septic shock (median = 100 ng/ml) and con-
trol group (median = 67 ng/ml) while both groups when compared
to the group of other cases of sepsis (not in septic shock) (median =
276 ng/ml) show statistically significant difference (p-value < 0.001).

Figure 3: Comparison of MPO level in neonates with septic
shock, other cases and control group
NB.: Samples (numbered according to their number in master sheet) whose results
were plotted outside the ranges of septic shock group and control group in figure
3 is considered to be extreme values.
Table 5 revealed no statistically significant difference of MPO level
between neonates with severe sepsis (median = 187, minimum = 32,
maximum = 635) compared to other cases (median = 262, minimum
= 50, maximum = 588) as p-value was not significant (0.629). Figure
4 revealed moderate positive correlation between MPO level and
TLC with r-value (0.503) and p-value (0.001). A moderate positive
correlation was found between MPO level and absolute neutrophil
count with r-value (0.536) and p-value (<0.001) (figure 5). Table 6
and figure 6 revealed the diagnostic utilities of MPO assay (using the
cutoff value of 146.5 ng/ml); it had a sensitivity of 80%, specificity of
83.9% with a positive predictive value of 92.9%, negative predictive
value of 61.5% and accuracy of 82.9% as per the ROC curve.

Figure 4: Correlation of MPO level with total leukocyte count

Figure 5: Correlation of MPO level with absolute neutrophil
count

Figure 6: ROC curve for MPO as a predictor for diagnosis of sepsis
Table 5: Demographic data of the case and control groups
Item Neonates in case group (n=41) P-value
Severe sepsis (n=22) Other cases (n=19)
Median Minimum Maximum Median Minimum Maximum
MPO (ng/ml) 187 32 635 262 50 588 0.629

Table 6: Diagnostic utilities of MPO assay
Area Standard Error P-value Asymptotic 95% Confidence Interval
Lower Bound Upper Bound
0.865 0.066 0.001 0.735 0.994
Vol 2, No 2
Discussion
The present cross-sectional study was done on neonates in whom
infection was confirmed by positive blood culture and were then re-
cruited into the study. CBC, CRP, venous blood gases and plasma
MPO enzyme level were measured.
Regarding maternal risk factors of neonatal sepsis in our study;
PROM accounted for 19.5% in cases diagnosed with sepsis. In the
study by Leal et al, PROM was reported for 32.7% [13]. These differ-
ences may be attributed to large sample size (11,790 neonates) com-
pared to our sample size. In agreement with the study of Fahmey,
the highest frequency pathogen among culture-proven cases was
for Klebsiella that was isolated from 41.5% neonates in our study and
42.8% in the comparative study [14].
Currently, we need a predictive marker in diagnosis of sepsis that
follows two main strategies: 1) starting an antibacterial therapy as
early as possible in a case of suspected sepsis; and 2) initiating ther-
apy only in infected patients. Thus, a high sensitivity and negative
predictive value of approximate 100%, and a good specificity and
positive predictive value in excess of 85% are recommended. In ad-
dition, standardized cut-off values are crucial, making results compa-
rable between laboratories [15].
Total leukocyte count (>20000 or <5000), differential leukocyte
count and morphology, total neutrophil count, total non-segmented
neutrophil count, neutrophil ratios and platelet count are the indices
most commonly used. These hematological counts and ratios
showed a limited accuracy with wide range of sensitivity (1790%)
and specificity (31100%), due to the relatively long period neces-
sary to become positive and the significant influence of non-specific
factors. However, I/T ratio of >0.2 may reach a sensitivity of 90% and
negative predictive value of 98% [16].
Our study revealed that MPO enzyme level was higher in cases of
sepsis compared to controls. This comes in agreement with Kothari
and his colleagues [5]. MPO enzyme activity was higher in cases of
sepsis (mean, 2.4 1.8) than control group (mean 0.32 0.11) and
the difference was statistically significant (p-value <0.01), while in
SIRS group (mean 1.86 1.2 in) the level of MPO was slightly lower
than that of sepsis with no significant difference despite of statisti-
cally significant (p-value <0.01) when compared to control group,
noting that MPO specific activity was expressed as nanomoles of
H2O2 degraded per milligram protein per 10 minutes.
In accordance of Mhl et al study, MPO enzyme activity was higher
in cases of sepsis than control group (cases of SIRS due to acute burn
injury patients not infected) with the difference being significant
[17]. In their study, daily samples were withdrawn from cases and
control groups. Comparison between both groups revealed that the
p-value < 0.05 on first and second days of intensive care unit admis-
sion and this value changed on third day to be < 0.01.
In the study by Yunanto et al, MPO enzyme activity was assessed not
only in the blood but also in the saliva and was found to be higher
in neonates with sepsis than healthy ones with statistically significant
difference in both conditions (p-value<0.001) [18].
In agreement with Kothari et al, MPO enzyme level was expected to
increase with the progression of sepsis and development of septic
shock, but it was constantly observed to be on the lower side in pa-
tients with advanced stages of septic shock [5]. As MPO enzyme level
reflects the neutrophil function, neutrophil count was found to be
low in patients with septic shock that may be explained by pancyto-
penia in these patients due to bone marrow suppression in advanced
stages of septic shock.
Spearmans correlation coefficients between MPO level and labora-
tory data showed moderate positive correlation of MPO level with
TLC and absolute neutrophil count. These correlations are explained
by the fact that MPO is a major granule enzyme in neutrophils, ac-
counting for 5% of the total neutrophil protein as mentioned by Ko-
thari and his colleagues [5].
In our study, MPO assay (using the cutoff value of 146.5 ng/ml) had
a sensitivity of 80% and specificity of 83.9% with a PPV of 92.9%, NPV
of 61.5% and accuracy of 82.9%. Compared to other markers of neo-
natal sepsis, Adib and his colleagues reported that CRP (using the
cutoff value of 12 mg/l) had a sensitivity of 45% and specificity of
95% with a PPV of 30%, NPV of 30% while procalcitonin (PCT) (using
the cutoff value of 1.1 ng/ml)was concluded to be a better marker
than CRP in the diagnosis of neonatal sepsis due to 70% sensitivity,
80% specificity, 80% PPV and 75% NPV [19]. The increase in the se-
rum concentration of CRP is rather slow during the first 14-48 h of
infection and this may negatively affect the sensitivity of the test. In
addition, increase in CRP concentration in non-infected clinical con-
ditions such as meconium aspiration, prolong rupture of membranes
are thought to affect the specificity of the test while PCT is detecta-
ble in the plasma as early as 2 h after the exposure to the bacterial
products.
Advantages of using MPO as a diagnostic marker are: 1) The meas-
urement can be quantitative and thus enables comparison of re-
sults among different centers; 2) The MPO level is not affected by
gestational age; 3) It can be done in saliva rather than blood sample
rendering it noninvasive utility in the diagnosis of neonatal sepsis
[18].
As with any other study, there are certain points of weakness in our
study: 1) It would have been better to do assay on serum samples to
avoid factors that may cause MPO concentration shift in plasma sam-
ples such as the time between serum collection and analysis as well
as repeated freeze-thaw cycles; 2) We didn't perform the diagnostic
utilities of the hematologic parameters (CBC and CRP) in comparison
with that of MPO for more confirmative data in control group; 3) Our
study had a relatively small number of cases compared to others.
Conclusion
MPO can be used as a marker of neonatal sepsis with considerable
diagnostic utility. MPO assay in addition to other markers of neona-
tal sepsis such as CRP may be of better value in early diagnosis of
neonatal sepsis.Further studies on larger scale should be done to
clarify the importance of MPO and assess diagnostic value of combi-
nation of MPO and other sepsis markers in achieving higher sensitiv-
ity, specificity, PPV, and NPV.
Authors Contributions: Hanna Abul Ghar: conception and design of study, critical
revision of the article and final approval of the version to be published. Eman Abdel
Ghany: study design, data collection and interpretation of data, drafting of the
article and final approval for publishing. Eslam Madbooly: data acquisition and an
analysis and critical revision for important intellectual content. Rania Samy:
laboratory work of the study, analysis and interpretation of data and final approval
of the article.
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82 Filter-bank energy cepstra in seizure detection

Comparison of linear, logarithmic and mel-frequency filter-bank energy cepstra in automatic seizure
detection using radial basis function neural network
Chandrakar Kamath
1

Background
Epilepsy is the most prevalent chronic neurological disorder, which
afflicts about 1-3% of the world population [1]. Epileptic patients suf-
fer from recurrent unprovoked epileptic seizures, which are episodic
and rapidly evolving temporary events. Epilepsy can be controlled,
but not cured with anti-epileptic medication. Until now, not much is
understood about the occurrence and mechanism underlying the
epileptic seizure. Long term inpatient/ambulatory electroencephalo-
gram (EEG), lasting from a few hours to several days which contain
interictal and ictal hallmarks of epilepsy, is required clinically to di-
agnose, monitor and localize the epileptogenic zone [2-3]. The tradi-
tional methods rely on well-trained clinical neurophysiologists who
visually inspect the entire lengthy EEG signals. However, this is a
costly, tedious and a time-consuming process. Therefore, many au-
tomated epileptic detection systems have been proposed in the re-
cent years [4]. Such automated systems reduce the time taken to
review off-line the long-term EEG recordings considerably and facili-
tate the neurologist to diagnose and treat more patients in a given
time.
The entire process of epileptic seizure detection can be generally
subdivided into two main stages: (1) feature extraction and (2) clas-
sification. Selecting an optimal set of significant features plays an im-
portant role in developing a good classification system. Different
methods have been used to extract diverse features, including those
which capture frequency, energy, and structural content of the sig-
nal, for the task of epileptic seizure detection [5-8]. In a recent study,
we had found that the overall performance of both the composite
vectors of the traditional cepstrum (CEP) deteriorated compared to

1
Manipal Institute of Technology, India
Correspondence: Chandrakar Kamath
Email: kamath. chandrakar@gmail.com
that of the baseline vector in the seizure detection and classification
of EEG segments [9]. However, there are not many studies that have
explored to a sufficient depth other features used in different do-
mains of signal processing, for example, features such as filter-bank
based cepstrum (FBE-CEP), being tried for seizure detection. FBE-
CEPs have been used for speech recognition and analysis [10-15].
However, to the best of our knowledge, this is the first study where,
frequency scaled FBE-CEPs are applied on the EEG database by An-
drzejak and investigated for unbalanced EEG data classification in
eight classification problems, discussed below [16]. We also compare
the performance of our approach with those of other researchers
who have used the same database.
There are two variants in the approach adopted in automated detec-
tion of seizures. The first is based on a set of heuristic rules and
thresholds. The second is based on classifier which employs pattern
recognition techniques. In the former approach, the results depend
upon a single operating point and hence, there is not much control
over the accuracy. On the other hand, the latter permits the classifier
to adapt to the desired performance and meet the requirements.
Hence, we go in for the latter approach. The rationale behind choos-
ing radial basis function neural network (RBFNN) is that: (1) the ear-
lier literature shows that RBFNN is a more suitable classifier in medi-
cal applications because of its simplicity and faster learning abilities
due to locally tuned neurons [17]; (2) RBFNN is also suitable from
the point of view of its high speed, high accuracy, strong tolerance
to input noise, and real-time property in updating network structure
[17].

Abstract
Background: Epilepsy is a neurological disorder affecting a very large number of people worldwide. A large fraction of the epilepsy
patients have poorly controlled epilepsy. The conventional method relies on experienced neurophysiologists, who visually examine the
continuous long-term inpatient/ambulatory electroencephalogram (EEG) signal. This is a tedious and time-consuming, and not a cost
effective procedure. Several automated epileptic detection and classification systems have been proposed and such systems facilitate the
neurologist to diagnose and treat more patients in a given time. There are not many studies that have explored to an adequate depth the
features used in other areas of signal processing, for example, the seldom used feature such as filter-bank energy cepstrum (FBE-CEP),
being tried for seizure detection.
Methods: Epileptic seizures are abnormal transient recurrent discharges in the brain with signatures manifesting in the EEG recordings
by frequency changes and increased amplitudes. We employed static and dynamic features derived from FBE-CEP to capture these
changes in amplitude and frequency. We compared the diagnostic performance of the linear, logarithmic, and mel-frequency, baseline
FBE-CEP and its two composite vectors in epileptic seizure detection on a standard publicly available EEG database. The comparison was
tried on eight different classification problems in the medical field related to epilepsy, using radial basis function neural network.
Results: All the three FBE-CEP methods, irrespective of frequency scaling, showed excellent overall performance.
Conclusion: The static and dynamic features derived from FBE-CEPs outperform those derived from CEP, suggesting their suitability in
epilepsy seizure detection. (El Med J 2:2; 2014)
Keywords: Electroencephalogram, Epilepsy, Filter-bank Energy Cepstrum, Radial Basis Function Neural Network, Seizure Detection
Kamath C 83
Methods
EEG Records
The EEG data used for this work is from publicly available Bonn Uni-
versity EEG database [16]. The choice of this database is based on
the rationale that many seizure detection methods have employed
this database and it becomes easy to compare the end results. The
database consists of five sets (designated Z, O, N, F and S), each con-
taining 100 single channel EEG segments of 23.6 second duration.
These segments have been picked from continuous multi-channel
EEG recordings after removal of any artifacts like muscle activity or
eye movements, making sure that they fulfilled stationarity require-
ments. Sets Z and O contain segments taken from surface EEG re-
cordings acquired from five healthy volunteers using a standard 10-
20 electrode placement scheme. The subjects were awake and re-
laxed with their eyes open for set Z and eyes closed for set O, respec-
tively. The segments for sets N, F, and S were acquired from five ep-
ileptic patients undergoing pre-surgical diagnosis. The type of epi-
lepsy identified was temporal lobe epilepsy with the epileptogenic
focus as the hippocampal formation. These recordings were taken
from intracranial electrodes as they offer the most precise access to
the emergence of seizures. Sets N and F contained only activity
measured during seizure free intervals (interictal epileptiform activ-
ity), with segments in set N recorded from hippocampal formation
of the opposite hemisphere of the brain and those in set F recorded
within epileptogenic zone. On the other hand, set S contained only
seizure activity (ictal intervals), with all segments recorded from sites
exhibiting ictal activity. The patients had attained complete seizure
control after resection of one of the hippocampal formations which
was confirmed to be the epileptogenic zone. All the EEG signals were
recorded using the same 128-channel amplifier system using an av-
erage common reference. The data were digitized at 173.61 samples
per sec with 12 bit resolution. The bandpass filter setting was at 0.53-
40 Hz (12 dB/octave). Each single channel EEG segment has 4096
samples.
In this work, we cover the six classification problems (CPs) proposed
by Guo et al and Tzallas et al [18-20]. To encompass other discrimi-
nations in the medical field related to epilepsy, we have included
two more CPs.
1. In the first CP, two classes, normal (includes only set Z) and sei-
zure (includes set S) are examined. In this CP, 200 EEG segments
are used.
2. In the second classification, two classes, namely, non-seizure (Z,
N, and F) and seizure (S) are examined. In this CP, the dataset
includes 400 EEG segments.
3. In the third problem, again, two classes, non-seizure (Z, O, N, and
F) and seizure (S) are examined. In this CP, 500 EEG segments
are used.
4. In the fourth CP, three classes are examined, normal (Z), non-
seizure (F), and seizure (S). In this case, 300 segments are used.
5. The fifth CP takes care of five datasets comprising 500 EEG seg-
ments into three classes, normal (Z and O), non-seizure (N and
F), and seizure (S).
6. The sixth CP handles five datasets comprising 500 EEG segments
into five individual classes, eyes-open (Z), eyes-closed (O), non-
seizure interictal (N), non-seizure interictal (F), and seizure (S).
7. In the seventh CP, three datasets comprising 300 EEG segments
into two classes, non-seizure (N and F) and seizure (S) are exam-
ined.
8. Finally, in the eighth classification problem, three classes com-
prising 300 EEG segments, normal (Z), non-seizure (N), and sei-
zure (S) are examined.
The first three CPs were proposed by Guo et al, the next three CPs
were proposed by Tzallas et al, while the seventh and eighth are
proposed by us [18-20]. These CPs have been chosen such that they
are close to clinical applications.
Cepstrum derived from log magnitude spectrum
Cepstrum (CEP) analysis is a nonlinear signal processing technique
with a variety of applications in areas such as speech and image pro-
cessing. It is possible to compare two relatively long time series with
only a few cepstral coefficients. This implies that if two cepstral series
are close then the corresponding signals have a similar evolution in
time.
The real CEP is defined as the inverse Fourier transform of the log
magnitude spectrum as given by:
Cr[k] = IDFT {log |DFT {x[n]}|} (1)
where Cr[k] represents k
th
order real cepstral coefficient, x[n] is the
discrete time signal whose cepstrum is to be computed. If the inverse
Fourier transform is replaced by discrete cosine transform (DCT), the
resulting equation becomes:
C[k] = DCT {log |DFT {x[n]}|} (2)
where C[k] represents k
th
order pseudo cepstral coefficient.
The advantages are that: (1) DCT has better energy compaction
properties than the DFT and hence decreases memory requirements;
(2) it reduces the computational complexity drastically without de-
grading the information content in the CEP and hence, decreases
execution time; and (3) DCT produces highly uncorrelated features.
The resulting sequence of coefficients C[k], called pseudo CEP, is an
approximation to the CEP, and in reality simply represents an orthog-
onal and compact representation of the log magnitude spectrum.
The difference between cepstral coefficients of different time series
can serve as a similarity measure among these time series. The
cepstral coefficients decay rapidly to zero and hence, only the first
few coefficients are needed to capture most of the dynamic infor-
mation in the time series. This property of cepstral coefficients helps
in reducing the dimensionality. Also, the number of coefficients to
be retained does not depend upon the length of the time series.
Moreover, the higher order coefficients represent the excitation pro-
cess which is less useful. The coefficient C[0] is similar to log energy
(or DC component) of the signal and represents the segment energy.
It is, usually, not treated as a cepstral coefficient and is dropped.
Filter-bank energy Cepstrum (FBE-CEP)
In filter-bank based systems, the signal magnitude spectrum is di-
vided into a few subbands using a multirate filter-bank. The filter-
bank uses triangular filters spread over the whole frequency range
from zero up to Nyquist frequency. The center frequencies and the
Vol 2, No 2
bandwidths are determined by the frequency scaling of the filter-
bank. In filter-bank based automatic speech recognition systems, fil-
ter-banks with linear, logarithmic, and Mel scale have been used [10-
15]. The cepstra derived from these filter-bank energies are respec-
tively designated as linear-frequency FBE-CEP, logarithmic frequency
FBE-CEP and Mel-frequency FBE-CEP. The magnitude spectrum of
the signal is computed and warped to the frequency scale of the
corresponding filter-bank followed by the usual log and DCT com-
putation using eqn. (2) to obtain the FBE-CEPs of the EEG segment
under consideration. We designate the resulting cepstrum by CFB[k].
The coefficient CFB[0] is similar to log energy (or DC component) of
the signal. Like CEP, in this study, we do not account for CFB[0]. There
are two major differences between traditional CEP and FBE-CEP: (1)
the CEP coefficients are derived from the entire full-band signal spec-
trum while the FBE-CEP coefficients are derived from the spectrum
of the filter-bank energy, (2) the frequency scale for CEP computa-
tion is always linear, while that for FBE-CEP can be linear, logarithmic
or Mel-scale.
Radial basis function neural network (RBFNN)
In this work, we employ radial basis function neural network (RBFNN)
for the classification of normal, non-seizure and seizure segments
through FBE-CEPs derived from EEG signals. RBFNN has advantages
of easy design, good generalization, strong tolerance to input noise,
and online learning ability. The properties of RBF networks make it
very suitable to design flexible control systems [17].
RBFNNs are nonlinear hybrid networks, which usually contain a sin-
gle layer of hidden neurons. The general architecture of a typical
RBFNN is shown in figure 1. There are three layers: an input layer, a
hidden layer, and an output layer. Each input neuron corresponds to
an element from the input vector and is connected to the k hidden
layer neurons. Each hidden neuron is connected to the output neu-
rons. The number of neurons in the output layer is equal to the num-
ber of possible classes n in the CP. The input layer broadcasts the
coordinates of the input vector to each of the nodes in the hidden
layer. Each node in the hidden layer then produces an activation
based on the associated radial basis function. Finally, each node in
the output layer computes a linear combination of the activations
from the hidden nodes. The output nodes from a RBFNN can be de-
scribed as:
Cj(x) = i wji ||x i || i 1 i k and 1 j n (5)
where Cj(x) represents the function corresponding to the j
th
output
unit or class-j and is a linear combination of k radial basis functions
with center i and bandwidth i. wj is the weight vector of class-j
and wji is the weight of j
th
class and i
th
center. The commonly used
basis function in the RBFNN to solve pattern recognition problems is
a Gaussian function and with this the eqn. (5) becomes:
Cj(x) = i wji exp (||x i ||
2
/ (2 i
2
)) 1 i k and 1 j n (6)
From eqn. (6) it can be observed that the output of RBFNN depends
upon total number of neurons k, the weights between the output
and the hidden layer wji, the centers of neurons i and the band-
widths of the neurons i. This implies that the performance of RBFNN
is determined by the selection of the right parameters. RBFNN can
be trained in different ways. In one of the conventional methods, the
training begins with a predetermined network structure. Then the
centers and the bandwidths are trained. Again, several methods are
proposed to find the centers of which clustering based methods are
popular.

Figure 1: A typical structure of RBFNN
In this work, we use MATLAB toolbox which greatly simplifies the
implementation of the required RBFNN. The RBFNN uses a radial ba-
sis layer which requires a parameter, spread constant, to be fixed. It
is important that the spread constant be large enough that the radial
basis layer neurons respond to overlapping regions of the input
space, but not so large that all the neurons respond in essentially the
same manner. From the MATLAB manual it is found that for the case
of RBFNN the default value of spread constant, s = 1.
Results
Now, we compare the diagnostic capability of the three FBE-CEPs:
(1) linear scale FBE-CEP, (2) logarithmic scale FBE-CEP, and (3) Mel
scale FBE-CEP and their composite vectors in the above eight CPs
using RBFNN. Empirically, we had found that repeating the same pro-
cedure as in [9], an analysis window length, W 868 samples (5.0
seconds), a spread constant, s 5 for RBFNN and a number of
cepstral coefficients, N 9 leads to optimum results in all the three
cases. In this work, a 1000-sample sliding window with 50% overlap
between consecutive windows, N = 9, and s = 1, is used in the com-
putation of FBE-CEPs. Distance-based classifiers demand normaliza-
tion of the data and hence, feature vectors are normalized before
they are applied to RBFNN. We adopted leave-one-record-out cross-
validation method. In specific, we ran 10 runs of a 10-fold cross-vali-
dation (with 10 runs for each fold split), thus having a total of 100
RBFNN runs to average to produce the final result. With each new
fold split, the EEG data segments are randomized.
First, we compare the results of the performance of the three FBE-
CEP baseline vectors in the general EEG seizure detection. The com-
parison is tried on each of the abovementioned eight different CPs
which have been widely used in the literature related to epilepsy.
Typical EEG segments, one from each dataset (in the order Z, O, N, F
and S), are shown in figure 2. Figures 3, 4, and 5 show the first 9
coefficients corresponding to the baseline vector and the two com-
posite vectors (from top to bottom) of the three FBE-CEPs (linear,
log, and Mel scale, respectively), for the same EEG segments shown
in figure 2, in the same order. Though there appears to be some sim-
ilarity in the shape of the corresponding plots, the feature values are
Kamath C 85
different. The similarity is an outcome of the lower and narrow fre-
quency range (0.5 to 40 Hz) of the EEG signal, compared to that of
speech signal where the frequency range is higher and broad (300
to 3000 Hz). Descriptive results of RBFNN analysis using FBE-CEP
baseline vectors for discriminating different CPs are depicted in Ta-
ble 1. It is found that the linear FBE-CEP baseline feature vector
shows the good performance in CPs 3, 4, and 8, log FBE-CEP baseline
vector shows the good performance in CP 1 only, while Mel FBE-CEP
baseline feature vector shows the good performance in CPs 2, 5, 6,
and 7.

Figure 2: Typical EEG segments from each of the five sets (Z, O,
N, F, and S), from top to bottom

Figure 3: The first 9 baseline and composite vector coefficients
for linear scale FBE-CEP method for the same EEG segments
shown in figure 2 in the order Z, O, N, F, and S

for log scale FBE-CEP method for the same EEG segments shown
in figure 2 in the order Z, O, N, F, and S

Table 1: Percentage average accuracy of RBFNN analysis
using linear, logarithmic, and Mel scale FBE-CEP methods
(W=1000, N=9 and s=1) for baseline vectors in
discriminating eight classification problems (CPs)
CP
Linear scale
FBE-CEP
Logarithmic
scale FBE-CEP
Mel scale
FBE-CEP
1 90.34 90.35 90.13
2 93.03 86.93 93.18
3 93.68 87.91 82.90
4 89.50 79.81 87.02
5 83.25 73.91 93.98
6 72.00 63.86 72.32
7 93.64 84.27 93.81
8 81.37 76.25 81.12

0 100 200 300 400 500
-200
0
200
0 100 200 300 400 500
-200
0
200
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Vol 2, No 2

for Mel scale FBE-CEP method for the same EEG segments shown
in figure 2 in the order Z, O, N, F, and S
The results of RBFNN analysis using composite cepstral vectors for
discriminating different CPs in the three FBE-CEP methods are shown
in Tables 2 and 3. The first composite vector includes velocity vector
together with the static cepstral vector. The second composite vec-
tor includes velocity and acceleration vectors together with the static
cepstral vector. It is found that the first composite feature vector of
linear FBE-CEP shows the best performance in all the CPs, that of log
FBE-CEP shows the best performance in CPs 1, 2, 4, 7, and 8, while
that of Mel FBE-CEP shows the best performance in all the CPs. The
second composite cepstral vectors in all the three FBE-CEP methods
show the best performance. This is in agreement with applications
in other domains of signal processing where the composite vectors,
in general, enhance the performance. For example, the composite
cepstral vectors of MFCC and LFCC add to improved performance in
speech processing [10-14]. In a recent study, on the other hand, we
had found that the overall performance of both the composite vec-
tors of the traditional CEP deteriorated compared to that of the base-
line vector in the seizure detection and classification of EEG seg-
ments [9]. It was interesting to note that the baseline CEP vector
alone showed the best performance. The composite CEP vectors, in-
stead of at least maintaining best performance, showed a degraded
performance. This implied that the velocity and acceleration CEP fea-
tures were hurting the performance, probably because of the non-
linearities introduced in the EEG significantly affected the computa-
tion of derivatives. However, in contrast to this behavior, the baseline
and composite vectors in all the three FBE-CEP methods, irrespective
of the frequency scale (linear, log, or Mel), maintain 100% overall
accuracy in all the eight CPs close to clinical applications.
(W=1000, N=9 and s=1) for composite vector-1 in
CP
Linear scale
FBE-CEP
Logarithmic
scale FBE-CEP
Mel scale
FBE-CEP
1 100.00 100.00 100.00
2 100.00 100.00 100.00
3 99.98 99.60 99.98
4 100.00 100.00 100.00
5 100.00 99.57 100.00
6 99.98 99.81 99.98
7 100.00 100.00 100.00
8 100.00 100.00 100.00

(W=1000, N=9 and s=1) for composite vector-2 in
CP
Linear scale
FBE-CEP
Logarithmic
scale FBE-CEP
Mel scale
FBE-CEP
1 100.00 100.00 100.00
2 100.00 100.00 99.97
3 99.98 100.00 99.98
4 100.00 100.00 100.00
5 100.00 100.00 100.00
6 99.98 99.98 100.00
7 100.00 100.00 100.00
8 100.00 100.00 100.00

Discussion
Table 4 provides a comparison between our method and other
methods (classification accuracy) proposed in the literature for epi-
leptic seizure detection. Only those methods that have used the
same database by Andrzejak et al have been compared [16]. In the
first CP (serial numbers: 1-8), the results obtained by Tzallas, Subasi,
Wang, Iscan, Orhan and our method are the best (100%). In the sec-
ond problem (serial numbers: 9-11), our method shows the best re-
sults (100.0%). For the third CP (serial numbers: 12-15), the result
found by Orhan and us are the best (100%). For the fourth, fifth, and
eighth CPs (serial numbers: 16-18, serial numbers: 19-21 and serial
numbers: 23-25), only our method showed an average accuracy of
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Kamath C 87
Table 4: A comparison of classification accuracy achieved by our method and best performed others method for eight
classification problems
Serial
No.
Number
of classes
Researcher
(year)
Method Dataset CA* (%)
1 2 Tzallas et al. (2007) [19, 20] Time-frequency analysis and ANN Z,S 100.0
2 2 Subasi and Gursoy (2010)
[21]
Principal component analysis, Independent
component analysis, Linear discriminant analysis
and support vector machines
Z,S 100.0
3 2 Guo et al. (2010) [18] Discrete wavelet transform, line length feature and
MLPNN
Z,S 99.6
4 2 Guo et al. (2011) [22] Genetic programming based feature extraction and
k-nearest neighbors classifier
Z,S 99.0
5 2 Wang et al. (2011) [23] Wavelet transform and Shannon entropy Z,S 100.0
6 2 Iscan et al. (2011) [24] Cross-correlation, power spectral density, support
vector machines, Linear discriminant analysis and k-
nearest neighbors classifier
Z,S 100.0
7 2 Orhan et al. (2011) [25] Wavelet transform, k-nearest neighbors classifier
and ANN
Z,S 100.0
8 2 This work (2013) Log FBE-CEP feature vectors and RBFNN Z, S 100.0
9 2 Ocak (2009) [26] Discrete wavelet transform and approximate
entropy
ZNF, S 96.65
MLPNN
ZNF, S 97.75
11 2 This work (2013) Log FBE-CEP feature vectors and RBFNN ZNF, S 100.0
12 2 Tzallas et al. (2007) [19, 20] Time-frequency analysis and ANN ZONF, S 97.73
MLPNN
ZONF, S 97.77
and ANN
ZONF, S 100.0
15 2 This work (2013) Log FBE-CEP feature vectors and RBFNN ZONF, S 100.0
16 3 Guler et al. (2005) [27] Lyapunov exponents, recurrent neural
network (RNN)
Z, F,S 96.79
17 3 Tzallas et al. (2007) [19, 20] Time-frequency analysis and ANN Z, F,S 99.28
18 3 This work (2013) Log FBE-CEP feature vectors and RBFNN Z, F,S 100.0
19 3 Guo et al. (2010) [18] Wavelet transform, line length, and ANN ZO, NF, S 97.77
and ANN
ZO, NF, S 95.60
21 3 This work (2013) Log FBE-CEP feature vectors and RBFNN ZO, NF, S 100.0
22 2 This work (2013) Log FBE-CEP feature vectors and RBFNN NF, S 100.0
23 5 Guler et al. (2007) [27 Wavelet transform, Lyapunov exponents-Support
vector machine
Z, O, N, F, S 99.28
24 5 Ubeyli (2010) [29] Lyapunov exponents and PNN Z, O, N, F, S 98.05
25 5 This work (2013) Log FBE-CEP feature vectors and RBFNN Z, O, N, F, S 99.8
*CA = Classification accuracy
Vol 2, No 2
100.0%. In the seventh CP (serial number: 22), the new CP appended
by us in this paper, the results are excellent (100%). All these results
collectively show a tremendous improvement in our approach over
the other epilepsy detection methods. The above comparison also
implies that an automated system developed based on this ap-
proach should provide feedback to the experts for quick and accu-
rate EEG classification.
A limitation of our method (like many who have used the same da-
tabase) is that the database used has already been preprocessed by
the removal of artifacts by visual inspection. Nevertheless, the results
of this study provide sufficient evidence of the application of this
approach to capture diagnostically significant information. Hence,
the method is well suited for implementation not only in epilepsy
detection system, but also in applications, such as seizure warning
systems, closed loop seizure control systems, or delivering abortive
responses/monitoring patients using implantable therapeutic de-
vices [30].
Conclusions
A comparison of the EEG epileptic seizure detection based on fre-
quency scaled FBE-CEP methods is presented. In the literature it is
found that in the applications such as speech analysis and recogni-
tion, the velocity and acceleration features do enhance the perfor-
mance. However, our previous study showed that in the case of EEG
discrimination using CEP method, the velocity and acceleration fea-
tures were hurting the performance. The chief finding of this study
is that unlike CEP method, in the FBE-CEP methods, the composite
vectors do perform on par with the baseline vector in the discrimi-
nation of EEG segments in a variety of CPs close to clinical applica-
tions. An automated system developed based on FBE-CEP method
should provide feedback to the clinical neurophysiologists for quick
and accurate EEG discrimination.
Competing interests: The author declares that no competing interests exist.
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Jain D, Jasuja OP, Nath S 89

Evaluation of foramen magnum in sex determination from human crania by using discriminant function
analysis
Deepali Jain
1
, O P Jasuja
1
, Surinder Nath
2

Introduction
In forensic and anthropological sciences, cranial analyses whether
morphognostic or morphometric, have played an important role in
sex determination. In most of the forensic studies, the skeleton will
be incomplete and makes gender identification difficult. The fora-
men magnum is one of the primary centers of ossification on the
cranial base during growth and development, and is located inferior
to the sagittal suture on the cranial base. Characteristics of foramen
magnum have identifying features for sexing [1]. The foramen mag-
num is a three dimensional aperture within the basal central region
of the occipital bone. The anterior border of the foramen magnum
is formed by the basilar process of the occipital bone, the lateral bor-
der by the left and right ex-occipitalis and posterior border is formed
by the supra-occipital part of the occipital bone [2].
In humans, the foramen magnum is farther underneath the head
than in great apes. Thus in humans, the neck muscles do not need
to be as robust in order to hold the head upright. Comparisons of
the position of the foramen magnum in early hominid species are
useful to determine how comfortable particular species was when
walking on two limbs (bipedality) rather than four. The location of
the foramen magnum plays a crucial role in our understanding of
human evolution. Usually, the location of the foramen magnum is
linked to bipedal behavior or the lack thereof. Due to the thickness
of the cranial base and its relatively protected anatomical position,
this area of skull tends to withstand both physical insults and inhu-
mation somewhat more successfully than many other areas of the
cranium.
The skull and particularly the skull base, has been analyzed with var-
ying results and levels of success [3]. Many studies have been con-
ducted on different populations on sexual dimorphism with respect
to the cranial base, including structures such as occipital condyle,
mastoid process and foramen magnum [4-17]. A number of studies
have investigated the utility of this anatomical region of cranium for
sex assessment employing morphometric traits using discriminant
function analysis.
Makaju, for example, worked on 300 samples of CT scan image of
head [18]. He studied the antero-posterior diameter, transverse di-
ameter, area, shape of foramen magnum and also the presence of
the accessory hypoglossal canal in the posterior margin of foramen
magnum based on the ethnicity of Nepal. It was concluded that CT
scan image of head can provide valuable measurements of the fora-
men magnum and could be used for sexual dimorphism and neuro-
surgery when other methods are inconclusive. Singh et al also ana-
lyzed the foramen magnum of human skulls [19]. Fifty adult skulls of
known sex were included in the study. Six standard parameters were
measured and analyzed by discriminant function analysis. The accu-
racy of sex prediction based on discriminant function analysis ranged
from 66% to 70% and was found a useful parameter for sex determi-
nation.
Uthman et al studied 88 samples for his study [20]. Foramen mag-
num sagittal diameter, transverse diameter, area and circumference
were measured. Foramen magnum circumference and area were the
best discriminant parameters with an overall accuracy of 67% and
69.3%, respectively. Raghavendra babu et al studied sexual dimor-
phism of the antero-posterior diameter, transverse diameter and
area of foramen magnum in a population of coastal Karnataka region
using statistical considerations [21]. The predictability of foramen
magnum measurements in sexing of crania was 65.4% for the antero-
posterior diameter. For the area of foramen magnum that was calcu-
lated using the formula derived by Routal and Teixeria, the predicted
probabilities were observed to be 81.6% and 82.2%, respectively [22,
23].

1
Punjabi University, India
2
Delhi University, India
Correspondence: O P Jasuja
Email: opjasuja@gmail.com

Abstract
Background: Many studies have been conducted on different populations on sexual dimorphism with respect to the cranial base. The
present research was undertaken to study the accuracy and reliability of the foramen magnum in sex determination by using discriminant
function analysis.
Methods: 140 adult skulls (70 of either sex) were included in the study. Length, breadth index and area of foramen magnum were subjected
to discriminant function analysis.
Results: The accuracy of sex classification was up to 75.7% using a single variable. The least value was obtained from the index of the
foramen magnum i.e. 57.9%. Three discriminant function equations were calculated based on these measurements. In stepwise analysis,
breadth of foramen magnum was found to be more discriminating variable providing an accuracy of 75.7%.
Conclusion: It can be concluded from the results that the measurements of foramen magnum of cranium provides valuable results and
suitable for sex determination when other methods are inconclusive. (El Med J 2:2; 2014)
Keywords: Cranium, Sex Determination, Sexual Dimorphism, Morphometric Analysis, Foramen Magnum, Discriminant Function Analysis,
Stepwise Analysis, Direct Analysis
90 Foramen magnum in sex determination
Vol 2, No 2
Galdames analyzed 211 human skulls using antero-posterior and
transverse diameters of foramen magnum [10]. All the dimensions
were found to be higher and accurately classified only in 66.5%
skulls. Gapert also evaluated morphometric variables of the region
of the foramen magnum [5]. The results demonstrated that signifi-
cant sexual dimorphism exists within these parameters. The correctly
classified crania with in this population ranged from 65.8% for uni-
variate functions to 70.3% for multivariate functions within the cra-
nial sample.
Therefore, the present study has been conducted to augment data
in this direction and to assess the level of sexual dimorphism present
in foramen magnum region of human skulls and to find out the ac-
curacy level of differentiating between males and females.
The study sample comprised of 140 crania (70 males and 70 females).
Only the skulls with no apparent deformity were included in the
study. Juvenile skulls were also excluded from the study. Initial ex-
amination of all the crania was done following the non-metric obser-
vations to categorize them into male and female category [24]. Data
was collected from Department of Anthropology, Delhi University,
University College of Medical Sciences, Delhi, Lady Harding Medical
College, Delhi, Holy Family Hospital, Delhi and Department of Foren-
sic Medicine and Anatomy, All India Institute of Medical Sciences.
Two direct and three indirect measurements pertaining to foramen
magnum were taken on each crania in accordance with the standard
measurement techniques recommended by Martin and Singh [25-
26].
Operational Definitions, Formulae and Procedures
Landmarks
Basion (ba): It is the point where the anterior margin of the foramen
magnum is cut by the mid-sagittal plane. This point lies exactly op-
posite the opisthion (o).
Opisthion (o): It is the point where the posterior margin of the fora-
men magnum cuts the mid-sagittal plane.
Length or antero-posterior diameter of foramen magnum (LFM)
It is measured as the straight distance between basion (ba) and opis-
thion (o).
Breadth or transverse diameter
of foramen magnum (BFM)
It is measured as the straight
distance between two points
of the foramen magnum on
most laterally placed mar-
gins.
Foramen magnum index
Breadth of foramen magnum
/ length of foramen magnum
x 100.
Area of foramen magnum
Area of foramen magnum is
calculated from length and
breadth of foramen magnum
utilizing different formulae
given by Routal and Tiexeria
[22, 23].
Formula given by Tiexeria: Area = ([LFM + BFM] / 4)
2
.
Formula given by Routal: Area = LFM * BFM * / 4.
Statistical Analysis
The data was analyzed using the SPSS 16.0 program. Descriptive sta-
tistics, including means, standard deviation, standard error and test
of significance were performed for each of the measurements. To
assess the level of significance in the mean values between the sexes,
t-test was applied. Stepwise, univariate and multivariate direct discri-
minant function analyses were performed to calculate specific discri-
minant function formulae for all parameters, which can also be used
on fragmentary remains. A leave one out classification procedure
was applied to demonstrate the accuracy rate of the original sample
and the one created by cross-validation.
Results
Table 1 presents the mean values, standard error of mean and stand-
ard deviation for all the five measurements (direct and indirect) of
male and female crania. It was observed that all the dimensions were
higher in males than females. On subjecting the data to test of
Table 1: Mean, standard error of mean, standard deviation, sex differences and original average accuracy percentages of the
cranial measurements
Measurements Males Females t-values OAA* (%)
Mean S.E.* S.D.* Mean S.E.* S.D.*
Length of foramen magnum 3.62 0.03 0.30 3.40 0.03 0.27 7.09** 62.90%
Breadth of foramen magnum 3.13 0.02 0.24 2.83 0.02 0.20 10.71** 75.70%
Foramen magnum index 86.69 0.79 6.66 83.45 0.73 6.11 3.02** 57.90%
Area of foramen magnum (Teixeria) 9.09 0.15 1.29 7.75 0.12 1.07 7.08** 70.00%
Area of foramen magnum (Routal) 8.95 0.15 1.26 7.59 0.12 1.02 7.19** 68.60%
*S.E. = Standard Error of Mean; S.D. = Standard Deviation; OAA = Original Average Accuracy
**Significant at 0.01% level

Figure 2: Basal view of the cranium
depicting the foramen magnum as defined
by the landmarks basion (ba) and opisthion
(o) and showing the measurements (1 and
2) used for the study.
Jain D, Jasuja OP, Nath S 91
significance (t-test), it was noted that the apparent variations ob-
served in the mean values of all the parameters revealed significant
sex differences.
Direct analysis was also employed to obtain the correct prediction
accuracies for all the single variables. On the basis of single variables,
the accuracy of sex classification reached from 57.9% to 75.7%. The
highest accuracy was obtained with the breadth of foramen mag-
num, i.e. 75.7% and the least was obtained with the index of the
foramen magnum i.e. 57.9%. Table 2 shows the result of stepwise
analysis. In the process variable breadth of foramen magnum was
selected and yielded an overall accuracy of 75.7%.
Table 2: Stepwise discriminant analysis
Variable Wilks
Lambda
Equivalent
f-ratio
Degrees of
Freedom
Breadth of
foramen magnum
0.687 62.943 1,138
Table 3 represents all the three discriminant functions which were
calculated to find out the accuracy level of sex determination. The
raw (unstandardized) coefficient was used to calculate discriminant
scores for all functions. The discriminant score was obtained by mul-
tiplying each measurement by its raw coefficient, summing them
and then adding the constant. For example, from function 1, the dis-
criminant score is calculated as:
D = (LFM * 2.819) + (BFM * 2.609) + (Index * 0.081) + [Area (t) *
-0.684] + [(Area (r) * 0.437] + (-22.486)
Sectioning point was the mean of male and female centroids. The
value of sectioning point obtained for all the discriminant function
equations was zero. If the discriminant function score was smaller
than the sectioning point or when the value was in negative, then it
was considered to be of female, while a value greater than the sec-
tioning point or the value being positive indicated a male. Three
combinations were tried using direct approach and provided the ac-
curacy up to 76.4%.
Sectioning point = 1 / 2 * male centroid + female centroid
A cross-validation using leave-one-out method was employed to
check how well the subjects were allocated to the groups. Objective
of discriminant analysis was to rank the variables according to their
contribution of two groups. Percentage of accuracy of sex determi-
nation was almost same for all the three functions. Original average
accuracy percentage was higher in function 1, i.e. 76.4%. Function 2
and function 3 had same original accuracy percentage i.e. 75.7%.
Discussion
The analysis of the foramen magnum characteristics is important in
the determination of sex for forensic purposes. Several authors have
emphasized on the value of foramen magnum and worked on vari-
ous parameters for the purpose of sex identification, using human
cranial remains. It is evident from the results that males display larger
mean values than females for all the variables. Significant sexual di-
morphism is found for all the parameters. This finding is similar to,
and yet different from, various studies conducted in the past.
Macaluso et al studied sex differences in linear and area dimensions
of foramen ovale and external carotid canal in documented French
sample [17]. The results, however, did not demonstrate very high
level of sexual dimorphism in the cranial base foramania of this sam-
ple. The cross-validated sex classification accuracy rates obtained for
univariate and multivariate discriminant functions ranged from only
Table 3: Unstandardized coefficients, centroids, sectioning points and average accuracies of all the discriminant function
equations
Functions and
Variables
Raw
Coefficients
Centroids Sectioning
Points
Average Accuracies (%)
O C
F1

LFM 2.819
M = 0.675
F = -0.675

* [0.675 + (-0.675)] = 0

76.5% 74.3%
BFM 2.609
Index 0.081
Area (t) -0.684
Area (r) 0.437
(constant) -22.486
F2
LFM 0.138
M = 0.671
F = -0.671
* [0.671 + (-0.671)] = 0 75.7% 75.0% BFM 4.350
(constant) -13.464
F3
Index 0.074
M = 0.699
F = -0.699
* [0.699 + (-0.699)] = 0 75.7% 75.7% Area (r) 0.808
(constant) -12.980
Stepwise
BFM 4.449 M = 0.671
F = -0.671
* [0.671 + (-0.671)] = 0 75.7% 75.7%
(constant) -13.274
*O = original group correctly identified; C = cross-validated group cases correctly identified; LFM = length of foramen magnum; BFM = Breadth of foramen magnum; M = male; F = female
92 Foramen magnum in sex determination
Vol 2, No 2
54.7% to 72%. Gapert et al also worked on foramen magnum param-
eters on skull base of 135 adult cranial bases from St. Brides docu-
mented skeletal collection in London [5]. Pooled age discriminant
function permitted 71.9% correct prediction. The statistical analysis
also showed no significant age effect on any of the variables, sug-
gesting that a separation by age is not necessary for the develop-
ment of sex determination methods. The results of all these studies
are almost similar to the results of the present study.
In the study by Uthman et al, foramen magnum circumference and
area were the best discriminant parameters that could be used to
study sexual dimorphism with an overall accuracy of 67% and 69.3%,
respectively. By using multivariate analysis, 90.7% of foramen mag-
num dimensions of males and 73.3% of foramen magnum dimen-
sions of females were sexed correctly. Uysal et al investigated the
value and accuracy of the measurements of the foramen magnum
by using 3D computed tomography using Fishers linear discriminant
functions test [27]. The length and width of foramen magnum diam-
eters were found to be statistically different in each sex with 81%
accuracy. The accuracy percentages of these studies are higher than
the accuracy percentage of the present study. In contrast, in the
study by Galdames et al all the dimensions were found to be higher,
and in mens skull, the foramen magnum size had low discriminating
power and was accurately classified only in 66.5% [10]. This percent-
age is lower than the results of the present study.
Results of our study indicated that the foramen magnum of cranium
is suitable for sex determination. The variations in the results ob-
tained from different populations can be a consequence of differ-
ences in robusticity of the skeleton due to genetic, environmental
and nutritional differences [28].
Conclusion
The present study successfully identified the sex via foramen mag-
num in 75.7% cases. Breadth of foramen magnum was selected in
stepwise analysis, providing an average accuracy of 75.7%. On the
basis of single measurements, up to 75.7% accuracy was attained i.e.
by breadth of foramen magnum which was followed by the area of
the foramen magnum i.e. 68.6%. Index of the foramen magnum was
not proven as a very good variable for sex determination. Three dis-
criminant function equations were calculated for sex differentiation.
The highest original average accuracy percentage was obtained for
function 1 i.e. 76.4%. Function 2 and function 3 showed same origi-
nal average accuracy values.
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Analysis of Foramen Magnum for Sex Determination in South Indian
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2. Scheuer L and Black S: The Juvenile Skeleton. The Elsevier; Londan. 2004.
3. Ukoha U, Egwu OA, Okafor IJ, Anyabolu AE, Ndukwe GU and Okpala I: Sexual
Dimorphism in the Foramen Magnum of Nigerian Adult. Int J Biol Med Res.
2011, 2(4): 878-881.
4. Wescott DJ and Moore-Jansen PH: Metric variation in the human occipital
bone: forensic anthropological applications. J Forensic Sci. 2001, 46(5): 1159-
1163.
5. Gapert R, Black S, Last J: Sex determination from the occipital condyle:
Discriminant function analysis in an eighteenth and nineteenth century British
sample. Am J Phys Anthropol. 2008, 138(4): 384-394.
6. Paiva LAS and Segre M: Sexing the human skull through the mastoid process.
Rev Hosp Clin. 2003, 58(1): 15-20.
7. Kemkes A and Gobel T: Metric assessment of the Mastoid Triangle for sex
determination: A Validation Study. J Forensic Sci. 2006, 51(5): 985-989.
8. Singh RP, Verma SK and Tyagi AK: Determination of sex by measurement of
area of Mastoid triangle in human skull. Ind Int J Forensic Med Toxicol. 2008,
6(2):29-43.
9. Nagaoka T, Shizushima A, Sawada J , Tomo S, Hoshino K, Sato H and Hirata
K: Sex determination using mastoid process measurements: standards for
Japanese human skeletons of the medieval and early modern periods.
Anthropol sci. 2008, 116(2):105-113.
10. Galdames ICS, Russo PP, Matamala DAZ and Smith RL: Sexual dimorphismin
the foramen magnum dimensions. Int J Morphol. 2009, 27(1): 21-23.
11. Saini V, Srivastava R, Raj Rk, Shamal SN, Singh TB and Tripathi SK: Sex
estimation from the mastoid process among North Indians. J Forensic sci. 2011,
57(2):434-439.
12. Gupta AD, Banerjee A, Kumar A, Rao SR and Jose J: Discriminant function
analysis of mastoid measurements in sex determination. J Life sci. 2012, 4(1):
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20(4): 255-259.
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process measurements in Brazilian skulls. Int j Morphol. 2008, 26(4): 941-944.
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Human Foramen Magnum: A study of Computerized Tomographic in Nepalese
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51.

Gomathi D, Kalaiselvi M, Ravikumar G et al 93

Mineral content analysis and investigation of antimicrobial activities of Evolvulus alsinoides (L.) L.
against clinical pathogens
Duraisamy Gomathi
1
, Manokaran Kalaiselvi
2
, Ganesan Ravikumar
1
, Kanakasabapathi Devaki
1
, Chandrasekar Uma
3

Introduction
Plants have been an important source of medicine for thousands of
years. Even today, the World Health Organization estimates that up
to 80 percent of people still rely mainly on traditional remedies such
as herbs for their medicines. This notion caries special significance
for India, as its civilization is very ancient and the country as a whole
has long been known for its rich resources of medical plants [1]. The
use of traditional medicines holds great promise as an easily availa-
ble source as effective medicinal agents to cure a wide range of ail-
ments among the people, particularly in tropical developing coun-
tries like India. This can be elucidated by the fact that people in these
countries consume several plants or plant derived formulations to
cure helminthic infections and for treatment of wounds [2, 3].
Evolvulus alsinoides (L.) L. (belonging to Convolvulaceae family) is an
important medicinal plant employed for different ailments in India
traditionally. It grows in open and grassy places almost throughout
the India and other subtropical countries of the world. The oldest
reports found of use of Evolvulus alsinoides are from India and sur-
rounding regions. The herb has been used to treat dysentery. Mo-
hammedan physicians used the plant as a general tonic to
strengthen the brain and memory and to treat fever. It was used to
treat bowel problems and to promote conception. The entire plant
was considered an astringent and found useful for treating hemor-
rhages. There are a variety of other medical applications, including
its use as an adaptogenic, antiphlogistic, antipyretic, antiseptic, aph-
rodisiac, febrifuge, stomachic, tonic, vermifuge, against asthma,
bronchitis, scrofula, syphilis, or in controlling night emissions and
to promote wound healing [4]. However, it has some phytochemicals
that are effective against the maladies for which people use them.

1
Karpagam University, India
2
Kongunadu Arts and Science College, India
3
Hawasaa University, Ethiopia
The isolation of evolvin, kaempferol-3-O--D glucopyranoside, cou-
marin etc. from E. alsinoides have been previously reported [5, 6].
Trace elements to be pharmacologically effective or essential, may
need to be combined/chelated with some ligand, in order to be
physiologically absorbed to prevent or cure impairment caused by
deficiency of the element. Active constituents of medicinal plants are
metabolic products of plant cells and as such, a number of trace el-
ements play an important role in the metabolism [7]. In this regard,
screening of elemental composition of the widely used medicinal
plants is highly essential. Therefore, the present study aimed to in-
vestigate the trace element composition and antimicrobial activity
of whole plant of Evolvulus alsinoides in order to assess its medicinal
value.
Collection of plant material
The whole plant of Evolvulus alsinoides (L.) L. used for the investiga-
tion was obtained from Coimbatore District, Tamilnadu, India. The
plant was authenticated by Dr. P. Satyanarayana, Botanical Survey of
India, TNAU Campus, Coimbatore, India. The voucher number is
BSI/SRC/5/23/2011-12/Tech.-514. Fresh plant material was washed
under running tap water, air dried and powdered.
Preparation of plant extract
100 g of dried plant powder was extracted in 500 ml of ethanol in
an orbital shaker for 72 hrs. Repeated extraction was done with the
same solvent till clear colorless solvent was obtained. Obtained ex-
tract was evaporated and stored at 0-4
o
C in an air tight container.
Correspondence: Chandrasekar Uma
Email: umachandrasekaran29@gmail.com
Abstract
Background: Medicinal plants are the richest bio-resource of drugs of traditional systems of medicine, modern medicines, nutraceuticals,
food supplements, folk medicines, pharmaceutical intermediates and chemical entities for synthetic drugs. Use of herbal products as
antimicrobial agents may provide the best alternative to the wide and injudicious use of synthetic antibiotics. Trace elements play a vital
role in the medical value of plants as curative and preventive agents in combating disease, nutritive and catalytic disorders. The aim of the
study was to investigate the trace element analysis by energy-dispersive x-ray spectrometer (EDX) and antimicrobial activity of ethanolic
extract of Evolvulus alsinoides.
Methods: Elemental analysis of Evolvulus alsinoides whole plant sample was done by using scanning electron microscope (SEM) with an
EDX and for the antimicrobial activity was done by using standard procedures.
Results: Our results showed the presence of carbon, oxygen, chloride, potassium and calcium in whole plant material of Evolvulus
alsinoides. The ethanolic extract of plant material showed significant antibacterial activity against Bacillus, Pseudomonas, Proteus, Klebsiella,
Streptococcus, Staphylococcus and Escherichia coli and also showed the good antifungal activity against all the fungi (Aspergillus flavus,
Candida albicans, Candida tropicans, Rhizopus and Fuserium) tested.
Conclusion: The data obtained in elemental analysis and antimicrobial activity of this plant indicates the promising potential in the
treatment of various diseases as a medicine. (El Med J 2:2; 2014)
Keywords: Evolvulus alsinoides, Antibacterial, Antifungal, EDX
94 Antimicrobial activities of Evolvulus alsinoides
Vol 2, No 2
Elemental analysis
The ethanolic extract derived from the whole plant material of
Evolvulus alsinoides were subjected to elemental analysis using scan-
ning electron microscope (SEM) with an energy dispersive x-ray
spectrometer (EDX).
Disc preparation
6 mm (diameter) discs were prepared from Whatmann No. 1 filter
paper. The discs were sterilized by autoclave at 12C. After the steri-
lization, the moisture discs were dried on hot air oven at 50C. The
ethanolic extract was dissolved in dimethyl sulfoxide to get a con-
centration of 5, 10 and 15 mg/ml and the discs were flooded with
20l of sample solution in each concentration. The plain discs are
used as a control and novobiocin was used as a standard.
Antibacterial and antifungal activity of Evolvulus alsinoides
The antibacterial and antifungal activity studies were carried out by
disc diffusion technique [8]. Sterile nutrient agar plates and potato
dextrose agar plates were prepared. Bacterial test organisms like Ba-
cillus, Pseudomonas, Proteus, Klebsiella, Streptococcus, Staphylococcus
and Escherichia coli species were spread over the nutrient agar plates
by using separate sterile cotton buds. Then, fungal test organism like
Aspergillus flavus, Candida albicans, Candida tropicans, Rhizopus and
Fuserium were spread over the potato dextrose agar plates. After the
microbial lawn preparation, the discs (preparation is mentioned in
disc preparation in this section) were placed on the organism inocu-
lated plates with equal distance. Then, the plates were incubated at
room temperature for 72 hrs. The diameter of the minimum zone of
inhibition was measured in cm.
Results
The mineral content of Evolvulus alsinoides is shown in table 1, which
demonstrates the presence of various elements like carbon, oxygen,
chloride, potassium and calcium in our plant extracts. The concen-
trations of carbon and oxygen are high when compared with other
elements (figure 1).
The results of the antimicrobial assay of the ethanolic extract of
Evolvulus alsinoides indicated that the plant exhibited antimicrobial
activity against the tested microorganisms at three different concen-
trations of 5, 10 and 15 mg/ml. The antibacterial activity of plant ma-
terial was investigated against various bacterial species like Bacillus,
Pseudomonas, Proteus, Klebsiella, Streptococcus, Staphylococcus and
Escherichia coli and Novobiocin was used as a standard which is
shown in figure 2.

Figure 2: Antibacterial activity of Evolvulus alsinoides (L.) L.
The standard antibiotic shows very good antibacterial activity
against all species except Proteus and Klebsiella. Our plant material
exhibited the inhibitory activity against Bacillus, Pseudomonas and
Klebsiella species at 5mg/ml, Staphylococcus and E. coli at 10 mg/ml
and Streptococcus, Proteus species at 15 mg/ml. Figure 3 shows the
antifungal activity of ethanolic extract of plant material. The poten-
tial sensitivity of the extract was obtained against all the fungi like
Aspergillus flavus, Candida albicans, Candida tropicans, Rhizopus,
Figure 1: EDX Spectra of Evolvulus alsinoides
Gomathi D, Kalaiselvi M, Ravikumar G et al 95
Table 1: Elemental detection of whole plant of Evolvulus alsinoides
Element App. conc. Intensity conc. Weight % Weight % Sigma Atomic %
Carbon 141.98 1.1967 52.49 0.88 59.89
Oxygen 56.28 0.5374 46.34 0.87 39.69
Chloride 0.53 0.8258 0.28 0.05 0.11
Potassium 1.61 1.0434 0.68 0.07 0.24
Calcium 0.45 0.9738 0.21 0.06 0.07

Fuserium and the zone of inhibition presented below. Based on our
results the tested plant material has antibacterial activity and also
good antifungal activity when compared with standard antibiotic.

Figure 3: Antifungal activity of Evolvulus alsinoides (L.) L.
Discussion
Medicinal plants are the richest bio-resource of drugs of traditional
systems of medicine, modern medicines, nutraceuticals, food supple-
ments, folk medicines, pharmaceutical intermediates and chemical
entities for synthetic drugs [9]. Thus, to provide a scientific justifica-
tion for these traditional remedies, the present study was planned to
assess their trace elements and antimicrobial potential using etha-
nolic extract against some clinically important microorganisms.
Medicinal plants play an important role in traditional medicine and
are widely consumed as home remedies. The past decade has seen
a significant increase in the use of herbal medicine due to their min-
imal side effects, easy availability and acceptability to the majority of
the population of developing countries. Since times immemorial,
plant based drugs have been in use in the amelioration of various
ailments ranging from common cold to cancer.
Relatively high levels of essential elements, such as iron, manganese,
zinc, and calcium have been demonstrated to influence the retention
of toxic elements in animals and human beings. Only scanty reports
are available on the role of micronutrients which play an important
role in the formation of active constituents responsible for their cu-
rative properties [10]. In recognition of the important role that major
and trace elements play in health and disease of human body, in the
building up and restoration phenomenon, it was observed that dur-
ing the last few years remarkable progress has occurred in this area
of health sciences. Elements research has definitely been part of this
explosion of scientific knowledge. Direct correlation between ele-
mental content of medicinal plants and their curative ability is not
yet understood in terms of modern pharmacological concepts. So,
the quantitative estimation of various trace element concentrations
is important for determining the effectiveness of the medicinal
plants in treating various diseases and also to understand their phar-
macological action [11].
Potassium is accumulated within human cells by the action of the
Na
+
, K
+
ATPase pump and it is an activator of some enzymes, in par-
ticular co-enzyme for normal growth and muscle function. It helps in
protein and carbohydrate metabolism. It is the principle cation of the
intracellular fluid, but it is also a very important constituent of the
extracellular fluid because it influences muscle activity particularly
that of the cardiac muscle. Potassium deficiency causes nervous dis-
order, diabetes and poor muscular control resulting in paralysis. Cal-
cium is essential for healthy bones, teeth and blood. The health of
the muscles and nerves depends on calcium. It is required for the
absorption of dietary vitamin B, for the synthesis of the neurotrans-
mitter acetylcholine, for the activation of enzymes such as the pan-
creatic lipase. It helps to regulate the activity of skeletal muscle, heart
and many other tissues. Deficiency of calcium causes rickets, osteo-
malacia and scurvy [12].
In recent years, antimicrobial activity of many substances has been
screened because of its great medicinal relevance. Infections have
increased to a great extent and resistant against antibiotics, becomes
an ever increasing therapeutic problem [13]. The presence of anti-
fungal and antimicrobial substances in the higher plants is a good
inspiration for novel drug compounds as plants derived medicines
have made significant contribution towards human health. The po-
tential for developing antimicrobials from higher plants appears re-
warding as it may lead to the development of phytomedicine against
microbes [14]. Phytomedicine has been used for the treatment of
diseases as in done in cases of Unani and Ayervedic system of medi-
cines, a natural blueprint for the development of new drugs. As such,
much of the exploration and utilization of natural products as anti-
microbial arise from microbial sources [15]. The present study con-
firms the presence good antifungal and antibacterial activity of
Evolvulus alsinoides, which has been supported by Saraswathy and
Dhanalekshmi [16].
The disc diffusion assay revealed that the ethanolic extract of Evolvu-
lus alsinoides showed broad spectrum of antimicrobial activity. Gram
negative organisms were more susceptible to the extracts of Evolvu-
lus alsinoides. The extract showed excellent antimicrobial activity
against Salmonella species and Staphylococcus aureus were found to
be more resistant to the extract when compared with other organ-
isms like E. coli, Klebsiella spp., Pseudomonas aeruginosa and Vibrio
cholerae. Evolvulus alsinoides extract also has got significant antifun-
gal activity for Candida species, Aspergillus flavus, Aspergillus niger
and Trichophyton mentagrophyte in agar dilution method was re-
ported by Dhanalekshmi et al [5]. The ethanol extract of Evolvulus
96 Antimicrobial activities of Evolvulus alsinoides
Vol 2, No 2
alsinoides has in vitro broad spectrum antimicrobial activity and thus,
extracts from this plant can be used to control infections caused by
Salmonella typhi, Escherichia coli, Klebsiella pneumoniae and Staphy-
lococcus aureus. Opportunistic infections such as bronchopneumo-
nia, bacterial endocarditis and meningitis caused by Micrococcus spp.
and Pseudomonas aeruginosa may also find treatment with the ex-
tracts of this medicinal plant [17].
Ogueke et al reported that the plant is used in the treatment of sores,
boils, wounds and control of dysentery and diarrhea among the Igbo
ethnic group in Nigeria [18]. In the current investigations the aque-
ous as well as the methanolic extracts of this plant exhibited antimi-
crobial properties, thus confirming previous records that the plant
has antibacterial properties on certain bacterial species [19].
Conclusion
The elemental results show that Evolvulus alsinoides contains essen-
tial elements of vital importance in human metabolism. The data ob-
tained from this study will be helpful in the formulation of new drugs
with different plants combinations, which can be used in curing
many diseases. This study also reveals that the plant extract has wide
antibacterial as well as antifungal activity. Therefore, the plant mate-
rial may be used in the treatment of infectious diseases like enteric
fever, cholera and fungal skin diseases.
Acknowledgement: We, the authors are thankful to our Chancellor, Advisor, Vice
Chancellor and Registrar of Karpagam University for providing facilities and
encouragement..
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pp: 350.

Dos Santos MPAD, Maia LC 97

Flexural and tensile strengths of three restorative materials used in pediatric dentistry
Marcia Pereira Alves Dos Santos
1
, Lucianne Cople Maia
1

Introduction
In order to improve their physical and mechanical properties, dental
composites have been technologically evolving since the 1970s,
which made them a restorative material indicated for restoring pos-
terior teeth [1, 2]. This technological advancement has not only al-
lowed resin composites to improve, but also contributed to the de-
velopment of other adhesive restorative materials such as resin-
modified glass-ionomer cements and polyacid-modified resin com-
posites [3, 4]. All these materials allow minimal dental cavity prepa-
rations because of their adhesive properties. In addition, some of
them are able to promote fluoride ion changes as well [5]. These
composites also have colors similar to those of natural teeth, thus
making them alternative restorative materials [6].
However, all materials are submitted to a series of laboratory tests in
order to assure their clinical applicability prior to commercialization
[7]. All these tests follow well-defined and standardized norms, such
as ISO 4049 regarding adhesive restorative materials [8, 9]. For me-
chanical evaluations, certain tests like three-point flexural strength
and tensile strength are largely used [10-13]. Their results are extrap-
olated to the clinical practice and consequently, they have been as-
sociated to the clinical performance of restorations.
In spite of several studies about mechanical properties of the adhe-
sive restorative materials in the dental literature consulted, there is
no previous report comparing Freedom, Vitremer and TPH Spec-
trum at the same time. Therefore, the objective of this study was to
compare these three adhesive restorative materials commonly indi-
cated for restoring primary molars in pediatric dentistry by using
flexural and tensile strength tests according to ISO 4049 norms.
Five samples of each restorative material (Table 1), namely, Freedom
(Group I), Vitremer (Group II), and TPH Spectrum (Group III), were

1
Faculdade de Odontologia, Universidade Federal do Rio de Janeiro, Brazil
Correspondence: Lucianne Cople Maia
Email: rorefa@terra.com.br
made by using appropriate black Teflon matrices according to ISO
4049 for three-point flexural and uniaxial tensile strength tests, total-
ling ten specimens of each restorative material. The samples were
handled according to the manufacturers instructions and room tem-
perature. For Groups I and II, the material insertion was performed
by using a syringe provided by the respective manufacturers,
whereas Group III had their materials inserted with metal spatula.
The matrix was filled with two increments, with the first layer being
deeper (approximately 1.0 mm) and the second one filling the matrix
completely. Photopolymerization was incrementally performed ac-
cording to the 7.0 mm diameter of the light curing tip so that 1.0-
mm increment in thickness could be obtained for a whole matrix
measuring 26.0 mm in length. The incremental area was divided into
four sections which were lightcured during 40s at light intensity of
500mW/cm
2
. A radiometer was used for gauging such a light inten-
sity. Following the last increment, all the materials were covered with
polyester strip and then lightcured again as described above.
Table 1: Main composition of the restorative materials studied
and their respective manufacturers
Product
(n=10)
Manuf* Main composition FPMS
+

(m)
Freedom SDI Strontium-silicate filler
particles and non-Bis-GMA
matrix
1.0
Vitremer 3M Espe Fluoroaluminosilicate filler parti-
cles, polycarboxylic acid, water,
HEMA and photoactivators
20-50
TPH
Spectrum
Dentsply Barium-silicate filler particles,
pyrogenic silica and urethane-
modified Bis-GMA matrix
0.8
*Manuf=Manufacturer
+
FPMS=Filler particle mean size

Abstract
Background: This in vitro study aimed to compare the flexural strengths (FS) by using the three-point method as well as the uniaxial
tensile strengths (TS) of three restorative materials commonly used in pediatric dentistry: Freedom/SDI (Group I), Vitremer/3M (Group II),
and TPH Spectrum/Dentsply (Group III).
Methods: For FS and TS, five samples of each material were specifically made upon black Teflon matrices according to ISO 4049. The
materials were incrementally inserted, lightcured (40s 500mW/cm
2
), and stored in moist and dark environment at 37
o
C for 12 hours. A
universal testing machine was used at crosshead speed of 0.5 mm/min and 50 kgf load. All results were analyzed with ANOVA and Tukey
tests at 5% significance. For FS, the mean values ( SD) (in MPa) were: GI = 111.00 43.49, GII = 114.80 42.60 and GIII = 344.90 68.90.
Results: For TS, the mean values obtained from GI, GII, and GIII were, respectively, 12.51 4.59, 9.93 3.98, and 33.09 10.69. The
performance of GIII was statistically better regarding both FS and TS than GI and GII (P<0.05). No statistical difference was found between
GI and GII regarding both FS and TS (p>0.05). Descriptive analysis showed that GI and GII were brittle compared to the GIII, which had
suffered plastic deformation before fracture.
Conclusion: The TPH material was more resistant than the Freedom and Vitremer ones, whose flexural and tensile strengths were found
to be similar. (El Med J 2:2; 2014)
Keywords: Adhesive Restorative Materials, Flexural Strength, Tensile Strength, ISO 4049, SEM
98 Three restorative materials used in pediatric dentistry
Vol 2, No 2
The samples were removed from the matrices, kept under moist and
dark environment at 37
o
C for 12 hours, and then posteriorly pre-
pared by means of abrasive sandpapers so that the required dimen-
sions for strength tests could be obtained (ISO 4049). A universal
testing machine (EMIC DL 10000) was used for performing the flex-
ural and tensile strength tests at crosshead speed of 0.5 mm/min and
50kgf load. Room temperature and relative humidity during the test
procedures were, respectively, 23.2
o
C and 73%. After the samples
were disrupted, the strength results regarding Groups I, II, and III
were statistically evaluated through non-parametric tests at 5% sig-
nificance.
With respect to the type of fracture resulting from the flexural
strength test, the inner section corresponding to the fractured area
was descriptively analyzed by using a scanning electronic micro-
scope (secondary electron) at 50x and 2Kx magnifications.
Results
Assuming a normal distribution, the flexural strength of Groups I, II,
and III had mean values (in MPa) and standard deviations of 111.00
43.49, 114.80 42.60, and 344.90 68.90, respectively. For tensile
strength, the mean values obtained from GI, GII, and GIII were, re-
spectively, 12.51 4.59, 9.93 3.98, and 33.09 10.69. GIII was sta-
tistically significant different from GI and GII in both tests (p<0.05)
(Table 2).
Descriptively, GI and GII showed similar surface aspects characterized
by V-shaped longitudinal areas (Figures 1a and 2a) throughout their
surfaces. At 2Kx magnification (Figures 1b and 2b), however, it was
observed that the Freedom restorative material presented fractures
between filler particles and matrix (Figure 1b), whereas the Vitremer
ones had fractures involving both matrix and filler particle (Figure
2b). The TPH Spectrum restorative material showed U-shaped frac-
tures (Figure 3a) not clearly distinguishable (Figure 3b), which might
characterize greater plastic deformation before the fracture.
Discussion
The importance of comparing these three types of restorative mate-
rials is based on the supposition that it is not clearly defined which
of them should be chosen for restoring posterior deciduous teeth.
Although clinical studies are mostly indicated to explain such a ques-
tion, methods for testing mechanical resistance have been employed
and also associated with the clinical success of these restorative ma-
terials regarding their durability [14, 15].
The present study followed the ISO 4049 standard in order to allow
replication of the experiments, since the results can be compared to
patterns established by the American Dental Association (ADA).
However, Yap and Teoh have suggested that flexural strength tests
using the three-point method should be carried out with samples
measuring 12mm x 2mm x 2mm in length [16]. According to the
authors, such dimensions are more suitable for clinical situations in
addition to reducing the costs and time as well as to allowing more
uniform photopolymerization.

Table 2: Results for Freedom (GI), Vitremer (GII), and THP Spectrum (GIII) restorative materials by using ANOVA and Tukeys test.
Flexural Strength Tensile Strength
GI GII GIII GI GII GIII
GI - p=0.993 p=0.008* - p=0.836 p=0.002*
GII p=0.993 - p=0.002* p=0.836 - p=0.001*
GIII p=0.000* p=0.000* - p=0.002* p=0.001* -
*P value significant at <0.05
Figure 1(a): Fractograph (50x) of inner area following disruption of the sample
(Freedom) submitted to flexural strength test. Note longitudinal crack
throughout the sample
Figure 1(b): Fractograph (2Kv) of the crack area. Note deep V-shaped fissure and
presence of filler particles of several sizes. The shift of a large filler particle in
the resin matrix can be observed

GI 1A GI 1B
(Vitremer) submitted to flexural strength test
Figure 2(b): Fractograph (2Kv) of the crack area. Note deep fissure throughout
the whole material and void spaces within the matrix, which indicates
incorporation of air bubbles resulting from material manipulation

GII 2A GII 2B
(TPH Spectrum) submitted to flexural strength test. Note slight depression in the
surface, a characteristic indicating greater plastic deformation before the
fracture (50x)
Figure 3(b): Fractograph (2Kv). This fracture pattern indicates that TPH spectrum
restorative materials have higher resistance

GIII 3A GIII 3B
Dos Santos MPAD, Maia LC 99
Tensile strength tests can be either diametrically or uniaxially per-
formed as occurred in the present study [17]. According to Anusav-
ice, the uniaxial tensile test is rather difficult to perform because a
perfect alignment of the samples is required during their placement
[13]. Such a fact was not observed in the present study as the ma-
jority of the samples had fractures in the middle of the material-ma-
trix interface, thereby validating the tests [13]. In addition, the results
obtained in the present work are corroborated by previous studies
[18-20].
The composite may have presented higher performance due to its
better chemical interaction involving matrix, silane, and load, besides
the increased load of inorganic particles, which confers greater flex-
ural and tensile strength upon this material [2, 22]. This finding was
confirmed by descriptive analysis as the materials resistance is found
to be inversely proportional to the square of the depth of the surface
failure [13]. As can be seen in the present study, the composite pre-
sented low-depth surface failure (Figure 3a and 3b).
According to El Kalla and Garcia-Godoy, compomers have character-
istics also seen in a composite, thereby being more resistant than
resin-modified glass-ionomer cements [23]. Nevertheless, such a
finding was not observed in the present study since both materials
showed similar resistance. Concerning the compomer, this may be
explained by the possible failure at the interface involving matrix,
silane and glass particles (Figure 1b). Polyacids were incorporated
into the compomer in order to promote acid reaction a character-
istic feature of resin-modified glass-ionomer cements 24. Conse-
quently, it is thought that such a chemical bond is not strong
enough, which might have decreased the cements resistance.
Despite the improved mechanical properties of the resin-modified
glass-ionomer cements compared to the conventional ionomer ones
25, the former possess large irregular particles that (Figure 2b) can
decrease their load capacity. As a result, cracks, fissures, and fracture
rapidly occur (Figure 2A) [10]. It should be observed, however, that
the resin-modified glass-ionomer cement evaluated in the present
study was manually manipulated, a fact which might have contrib-
uted to their inferior performance as air bubbles could be incorpo-
rated into the material, thereby decreasing their resistance (Figure
2b).
Even the human dental enamel has tensile strength value of 10MP,
that is, similar to that of compomers and resin-modified glass-iono-
mer cements. According to Anusavice, the human enamel does not
suffer fracture because it is supported by the dentin, which ends up
absorbing the load [13]. Therefore, based on the results found in the
present study, the Freedom and Vitremer materials can be consid-
ered less resistant than the TPH Spectrum one, for they are more
likely to suffer fractures when loads are applied on them. On the
other hand, clinical studies have shown that such restorative materi-
als have been successfully used for repairing deciduous molar teeth
[25]. Hence, the clinical durability of these dental restorations should
be evaluated based on the physical characteristics of each material.
Conclusion
Considering the methodology used in the present study, one can
conclude that both strength tests showed that TPH Spectrum restor-
ative material was superior to the Freedom and Vitremer ones, alt-
hough both latter materials had similar behavior.
Acknowledgement: We would like to thank Professor Hlio Sampaio and Thais
Canavarro Abdala.
Financial Support: We also would like to thank the Jos Bonifcio Foundation for
financial support (grant number 11615-7).
Received: 19 January 2014 Accepted: 29 March 2014
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Rani H, Kulkarni P, Dinesh US et al 101

Comparison of squash smears and frozen sections versus paraffin sections in the intra-operative
diagnosis of central nervous system lesions
Hephzibah Rani
1
, Padmaja Kulkarni
1
, Udupi Shastry Dinesh
1
, Ravikala Vittal Rao
1
, Sateesh Melkundi
2

Introduction
Brain tissue by virtue of its high water content is like a sea of water
wrapped in tubes of fat with a bit of protein thrown in to hold it all
together [1]. This inherently soft nature of brain tissue and high wa-
ter content results in poor quality frozen sections due to ice-crystal
artefacts. At the same time, this soft texture facilitates smear prepa-
ration which reveals the cytomorphology in detail. For intra-opera-
tive diagnosis of brain lesions, the neuropathologists use squash
smear, imprints and frozen section techniques. Implementing cytol-
ogy smears for intra-operative diagnosis of brain tumors is now well
established in many neurosurgical centers [1-3]
The aims of the present study were to evaluate the diagnostic accu-
racy and limitations of squash smears and frozen sections in the di-
agnosis of intracranial lesions, in comparison with paraffin sections
and also to study the discordant cases.
A total of two hundred and twenty one neurosurgical specimens in-
cluding brain and spinal lesions were received in the Department of
Pathology from January 2010 to December 2012, of which the neu-
rosurgeon sent 110 cases for intra-operative consultation. These
cases were studied retrospectively. Both frozen and squash prepara-
tion were performed on all cases and stained with hematoxylin &
eosin (H&E). Squash smears were prepared by crushing (squashing)
a small bit of tissue, about 1 cubic mm size, in between two micro-
scopic glass slides and pulling it apart with gentle pressure to make

1
Department of Pathology, SDM College of Medical Sciences and Hospital, India
2
Department of Neurosurgery, SDM College of Medical Sciences & Hospital, India
Correspondence: Hephzibah Rani
Email: dr.hephzibah@gmail.com
it a thinner smear. Smears were immediately fixed in 95% alcohol
and stained with rapid hematoxylin and eosin stain. Smears were
stained in hematoxylin stain for 1 min followed by differentiation in
1% alcohol, and dipped in eosin for 1-2 dips, washed in water, and
then mounted. In some cases smears were even stained with Pap
and May-Grnwald (MGG) stains, wherever tissue was adequate
enough. Frozen sections of 6 to 10 micrometer thickness were taken
in Leica CE/CN cryostat. Sections were stained with H&E stain. Sec-
tions were stained with hematoxylin for 1 min, differentiated in 1%
alcohol, dipped in eosin (1-2 dips), washed in water and mounted.
The clinical (age, location, chief complaints) and radiological data
was retrieved along with squash smear, frozen section and paraffin
section slides. An intra-operative diagnosis was made after examina-
tion of the squash smears and frozen section slides. The diagnosis
given on squash smears and frozen section was then compared with
final diagnosis given on paraffin-embedded sections. Immunohisto-
chemistry was performed in a few diagnostically challenging cases.
Accuracy of squash versus paraffin section diagnosis and combined
squash + frozen section diagnosis was determined.
Results
Of the 110 cases of open biopsies, 68 were males and 42 were fe-
males. The ages ranged from a minimum of 7 months to a maximum
of 72 years. Majority of the cases were astrocytomas (32.7%) fol-
lowed by meningiomas (21.8%) and schwannomas (10.9%). The di-
agnoses on squash smears correlated with final diagnoses (paraffin
sections) in 103 cases (93.6%). However, concordance between intra-

Abstract
Background: Utility of squash cytology is well established in the intra-operative diagnosis of brain tumors, thereby guiding the
neurosurgeon in the surgical management. A comparative study between squash smears, frozen sections and paraffin sections was done
with an aim to study the diagnostic accuracy as well as limitations of these techniques. The present study was also undertaken to evaluate
the discordant cases in intraoperative consultation of central nervous system (CNS) lesions.
Methods: A total of 221 neurosurgical specimens were received in the Department of Pathology over three years duration, of which
intra-operative consultation was requested in 110 cases. Both frozen and squash preparation was performed on all cases and stained with
hematoxylin and eosin. An intra-operative diagnosis was made after examination of the squash smears and frozen section slides. The
diagnosis given on squash smears and frozen section were then compared with final diagnosis given on paraffin-embedded sections.
Immunohistochemistry was performed in a few diagnostically challenging cases.
Results: This study compares the diagnostic accuracy of squash smears and frozen sections from 110 surgical specimens. The 110 cases
included thirty six astrocytomas (32.7%), twenty four meningiomas (21.8%), twelve schwannomas (10.9%), two hematolymphoid tumors
(1.8%), eight medulloblastomas (7.3%), three oligodendrogliomas (2.7%), four pituitary adenomas (3.6%), four ependymomas (3.6%), four
metastatic tumors (3.6%), one paraganglioma (0.9%), two pineal parenchymal tumors (1.8%) and ten infections (9.1%). The accuracy of
squash smears was 93.6%, and when combined with frozen sections the accuracy increased to 97.3%.
Conclusion: Squash smears are simple, rapid, economical and very effective tool in the diagnosis of brain tumors. However, in few matrix
rich/fibrous tumors like meningiomas and schwannomas, frozen sections are more useful. Hence, overall the combined use of squash
smear and frozen section study increases the intra-operative diagnostic accuracy of CNS lesions. (El Med J 2:2; 2014)
Keywords: CNS Tumors, Frozen Section, Intra-operative Consultation, Squash Smear
102 Comparison of squash smears and frozen sections
Vol 2, No 2
Table 1: Comparison of the accuracy of squash alone and squash + frozen section
Serial
number
Paraffin section
diagnosis
Total number
of cases
Squash smear
diagnosis
Squash smear
accuracy
Squash + frozen
diagnosis
Squash + frozen
accuracy
1 Astrocytoma 36 36 100 36 100
2 Meningioma 24 21 87.5 23 95.83
3 Schwannoma 12 12 100 12 100
4 Oligodendroglioma 3 1 33.33 1 33.33
5 Ependymoma 4 4 100 4 100
6 Medulloblastoma 8 8 100 8 100
7 Lymphoma 1 1 100 1 100
8 Plasmacytoma 1 1 100 1 100
9 Pituitary adenoma 4 4 100 4 100
10 Metastatic 4 4 100 4 100
11 Paraganglioma 1 0 0 0 0
12 Pineal tumours 2 2 100 2 100
13 Infections 10 9 90 10 100
Total 110 103 93.63 106 96.36
operative (squash + frozen) and paraffin section diagnosis was seen
in 106/110 cases giving an accuracy of 96.4% (table 1).
Diagnostic discordance was seen in 4/110 (3.63%) cases (table 2).
These cases were studied in detail and causes for discordance were
analyzed.
Table 2: Discordant cases in this study
Intra-operative diagnosis Final diagnosis Cases
Low grade astrocytoma Oligodendroglioma 2
Schwannoma Fibrous Meningioma 1
Myxopapillary ependymoma Paraganglioma 1
Total 4
Discussion
The information that a surgeon needs during intra-operative consul-
tation of brain lesions include adequacy and representativeness of
the tissue, whether it is neoplastic or infectious and if neoplastic,
whether glial or non-glial tumor, and finally the grade of the tumor.
In general, any kind of histopathological information that influences
a surgeons decision to extend or alter the surgery is a valid indica-
tion for intra-operative consultation in CNS lesions [3-5]. Freezing the
brain tissue results in ice crystal artefacts, which makes intra-opera-
tive diagnosis on frozen sections even more challenging to the
pathologists. With the advent of stereotactic biopsies, the amount of
tissue available is very tiny and the pathologists need a technique
that is quick, reliable and which reveals good cellular morphology [2,
4, 5].
To overcome the problems associated with traditional frozen section
technique, there is a need for a better technique. Enter, squash
smears. The inherently soft nature of brain tissue facilitates easy
smear preparation and reveals detailed cytological features thereby
aiding in intra-operative diagnosis [3, 4].
Glial tumors were easily identified by the presence of characteristic
fibrillary background [1, 2, 4]. In our study, squash smears showed
100% accuracy rates for astrocytomas. Frozen sections were not of
additional help in the diagnosis of glial tumors, since the fine fibril-
lary glial tissue is not appreciated well due to freezing artefacts.
Lymphomas are characterized by completely discohesive smears.
Large, monomorphic lymphoid tumor cells were seen without any
fibrillar background. The cells have large nuclei with prominent nu-
cleoli and scant to moderate cytoplasm. Tingible body macrophages
and lymphoglandular bodies were seen which were more obvious in
MGG stain. Immunocytochemistry was positive for CD45 in intact tu-
mor cells. Immunohistochemistry on paraffin sections showed posi-
tivity for CD45, CD20 and negativity for CD3. The frozen sections
showed tumor cells in sheets, but cytologic details were not clear [5-
7]. Hence, even in this case frozen section was not very advanta-
geous.
Squash smears, apart from removing the freezing artefacts, have sev-
eral other advantages. No special equipment is needed unlike the
frozen section which requires cryostat. A very tiny bit of tissue is suf-
ficient to prepare the smears as a result of which sufficient tissue
remains for paraffin section and other tests like immunohistochem-
istry, genetic studies or electron microscopy wherever required. It
also provides biophysical information as to whether the tissue is
smooth or rubbery in consistency, which aids in the diagnosis. For
example, schwannomas and meningiomas are firm to rubbery in
consistency and are resistant to smearing in contrast to glial tumors
and lymphomas, which smear smoothly and evenly without any re-
sistance. Above all, squash smears reveal good nuclear details which
is very essential in rendering proper intra-operative diagnosis [4-6].
As with any other technique, squash smears have a few limitations.
They are not very useful in firm tissues producing cohesive lesions
like schwannomas, meningiomas and metastatic lesions; in such
Rani H, Kulkarni P, Dinesh US et al 103
cases, frozen sections are advantageous and reveal better tissue ar-
chitecture. Smears are also not useful for evaluating the margin sta-
tus [1, 3, 5, 6]. Frozen sections need expensive equipment (cryostat),
technical expertise, electricity, high cost of maintenance, excessive
tissue sampling and are relatively time consuming. Apart from a few
tumors like schwannomas, meningiomas and metastatic lesions, fro-
zen sections do not have any additional advantage over squash in
the diagnosis [1, 5, 8].
In our study, the concordance rate between intra-operative diagno-
sis and final diagnosis given on paraffin sections was 96.36% com-
paring favorably with several other similar studies as shown in table
3. The clinical and radiological correlation was available in full detail
in all our cases, which is partly contributory for such high concord-
ance rate.
Table 3: Comparison of present study with similar studies
Authors Cases CR* (%) DR* (%)
Savargaonkar P et al [6] 103 94.0 6.0
Asha T et al [8] 178 87.0 13.0
Rao S et al [3] 120 96.0 4.0
Kini JR et al [10] 100 86.0 14.0
Sundaram S et al [11] 3057 89.0 11.0
Bleggi Torres LF et al [12] 650 97.3 2.7
Present study 110 96.36 3.63
*CR = Concordance rate; DR = Discordance rate
Two cases of oligodendrogliomas were misdiagnosed as fibrillary as-
trocytoma on both squash and frozen section (figure 1). MRI showed
an ill-defined space occupying lesion in the left frontal cortex, which
was non-enhancing on contrast. Smears showed cells with mild nu-
clear atypia against a felt background, thin walled blood vessels and
no fibrillary background. The presence of calcifications would prob-
ably have given some clue to the diagnosis which was absent in both
these cases. In permanent sections, the nuclei were seen floating in
a clear bubble, and lying retracted away from their cytoplasmic
membrane, giving the characteristic fried egg appearance. A study
conducted by Roessler et al has reported similar discrepancies due
to the presence of variable cytoplasmic processes and lack of mo-
notonous appearing cells [9]. Anaplastic oligodendroglioma was
mistaken for anaplastic astrocytoma in a similar study conducted by
Kini et al [10].
One case of fibroblastic variant of meningioma was misdiagnosed as
schwannoma. Both meningiomas and schwannomas pose technical
difficulty. They are tough and are resistant to smearing. Meningio-
mas invariably shed a few cells in a smear, but the cells are usually
tightly bound to a clump in case of schwannomas, giving rise to
twisted rope pattern (figures 2 and 3). In the absence of character-
istic whorls and psammoma bodies and presence of only spindle
shaped cells in fascicles, frozen sections becomes necessary to dif-
ferentiate them from schwannoma [3, 8]. In a similar study by Rao et
al, fibrous meningioma were misdiagnosed as schwannoma due to
thick squash and freezing artefacts [3]. Diagnostic difficulty in spin-
dle cell lesions has also been described by Plesec et al [13].
One case of paraganglioma of cauda equina was misdiagnosed as
myxopapillary ependymoma. Smears were cellular and showed cells
with eccentrically situated bland nuclear chromatin and thin walled
blood vessels against a myxoid like background. Based on the loca-
tion, benign cells and the myxoid like background, the intra-opera-
tive diagnosis rendered was myxopapillary ependymoma. Frozen
section showed severe freezing artefacts. Paraffin sections revealed
the characteristic Zellballen pattern. Immunohistochemical study
was strongly positive for synaptophysin and neuron-specific enolase
(NSE). Cauda equina is one of the rare sites for paraganglioma and
only few case reports have been reported in literature [14]. Although
ependymomas are a differential diagnosis at this site, the most im-
portant cytologically distinguishing features are largely discohesive
smears, monotonous cells with salt and pepper chromatin, and ab-
sence of fine glial processes in the background in case of paragan-
glioma.
One case of tuberculoma was missed on squash smear because of
sampling error since the smear showed only necrosis. However, fro-
zen section showed epithelioid granulomas and caseation necrosis
as well. A case of reactive gliosis secondary to tuberculoma was mis-
taken for low grade astrocytoma on smear cytology, partly due to
sampling error in a similar study conducted by Kini et al [10].

Figure 1(a): Smears from a case of lymphoma showing
discohesive cells. Monomorphic lymphoid tumor cells with
large nuclei, prominent nucleoli and scant cytoplasm seen.
Background shows lymphoid globules and absence of
fibrillary pattern. (H&E x40)

Figure 1(b): Immunocytochemistry on squash smears
showing positivity for CD45 in intact tumor cells
104 Comparison of squash smears and frozen sections
Vol 2, No 2

Conclusion
Squash smears are simple, rapid, economical, do not require costly
equipment or technical expertise and are a very effective tool in the
diagnosis of majority of brain tumors. However, in few matrix rich
tumors like meningiomas and schwannomas, frozen sections are
more useful. Hence, the authors conclude that the combined use of
squash smear and frozen section study increases the intra-operative
diagnostic accuracy of CNS lesions.

References
1. Joseph JT: Diagnostic Neuropathology Smears. Ist edition. Chapter 1.
Philadelphia: Lippincott Williams and Wilkins; 2007.
2. Lopes MBS, Vandenberg SR: Tumours of the Central Nervous System. Ed.
Fletcher CDM. In: Diagnostic histopathology of tumors. 3rd ed, Philadelphia:
Churchill Livingstone; 2007.
3. Rao S, Rajkumar A, Ehtesham MD, Duvuru P: Challenges in neurosurgical
intraoperative consultation. Neurol India, 57:2009:464-468.
4. Prayson RA, Goldblum JR: Neuropathology (A volume in the foundations in
diagnostic pathology series), Philadelphia: Churchill Livingstone; 2005.
5. Mills SE: Sternberg's diagnostic surgical pathology. 4 th ed. Philadelphia:
Lippincott Williams and Wilkins; 2004.
6. Savargaonkar P, Farmer PM: Utility of intra-operative consultations for the
diagnosis of central nervous system lesions. Ann Clin Lab Sci 2001, 31:133-9.
7. Herrlinger U, Schabet M, Bitzer M, Petersen D, Krauseneck P: Primary central
nervous system lymphoma: From clinical presentation to diagnosis. Journal of
Neuro-Oncology 1999, 43:219226.
8. Asha T, Shankar SK, Rao TV, Das S: Role of Squash- Smear Technique for Rapid
Diagnosis of Neurosurgical Biopsies A Cytomorphological Evaluation. Indian
J Pathol Microbiol 1989, 32:152-60.
9. Roessler K, Dietrich W, Kitz K: High Diagnostic Accuracy of Cytologic Smears of
Central Nervous System tumours. A 15- Year experience based on 4172
patients. Acta Cytol 2002, 46:667-4.
10. Kini JR, Jeyraj V, Jayaprakash CS, Indira S, Naik CNR: Intraoperative
consultation and smear cytology in the diagnosis of brain tumours.
Kathmandu University Medical Journal 2008, 6(4):453-457.
11. Goel D, Sundaram C, Paul TR, Uppin SG, Prayaga AK, Panigrahi MK, et al:
Intraoperative cytology (squash smear) in neurosurgical practice-pitfalls in
diagnosis experience based on 3057 samples from a single institution.
Cytopathology 2007, 18:300-8.
12. Bleggi-Torres LF, de Noronha L, Gugelmin ES , Sebastio APM , Werner B,
Maggio EM, et al: Accuracy of the smear technique in the cytological diagnosis
of 650 lesions of the central nervous system. Diagnostic Cytopathol 2001,
24(4):293-5.
13. Plesec TP, Prayson RA. Frozen section discrepancy in the evaluation of central
nervous system tumors. Arch Pathol Lab Med 2007, 131:1532-40.
14. Gelabert Gonzalez M: Paragangliomas of the lumbar region. Report of two
cases and review of the literature. J.Neurosurg.Spine 2005, 2:354-365.

Figure 2(a): Squash smears from oligodendroglioma showing
rounder and monotonous cells in a felt like background with thin
walled blood vessels than the elongated nuclear membranes of
astrocytomas. (H&E x10)

Figure 2(b): Section showing the characteristic fried egg
appearance in oligodendroglioma (H&E x40)
Figure 3: Squash smear from meningioma showing the
characteristic whorled pattern of meningothelial cells. (H&E x40)

Figure 4: Smears from schwannoma displaying cohesive cells with
frayed rope / snapped rope appearance at its end. (Pap x10)
Adeleke IT, Adekanye AO, Jibril AD et al 105

Research knowledge and behavior of health workers at Federal Medical Centre, Bida: A task before
learned mentors
Ibrahim Taiwo Adeleke
1
, Adedeji Olugbenga Adekanye
2
, Abdullahi Daniyan Jibril
3
, Fausat Fadeke Danmallam
4
, Henry Eromosele Inyinbor
5
,
Sunday Akingbola Omokanye
1

Background
Humans are born with the inherent need to explore their environ-
ment and to determine the reasons and concepts behind existing
phenomena. Research is the systematic and rigorous process of in-
quiry which aims to describe phenomena and to develop and test
explanatory concepts and theories. It is generally understood in
terms of the positivist tradition, a tradition that in many ways uncon-
cealed. Ultimately, it aims to contribute to a scientific body of
knowledge. In the healthcare context, research aims at improving
health, health outcomes and health services [1].
Interestingly, literature has revealed that research, especially clinical
research has been acknowledged by stakeholders as having positive
impact on institutions [2]. These include prestige and publication-
related effects on health facilities, enhanced patient referrals to hos-
pitals and health care delivery systems. Others consist of facilitated
faculty recruitment and subsequent retention, improved intellectual
quality of life and increased research revenues [2]. The quality of
healthcare relates to its effectiveness at improving patients health
status and how well it meets predefined and agreed standards about
how the care should be provided. Health services research aims to
produce reliable and valid research data on which to base appropri-
ate, effective, cost-effective, efficient and acceptable health services
in the broadest sense [1].
It is an undisputable fact that medical research requires human par-
ticipation. However, recruiting participants for such researches is
quite challenging. This has been of serious concern and has domi-
nated discussions among medical researchers. Essentially, research-
ers have an ethical obligation to publish their findings, and health
researchers and managers share the common goals of improving the
effectiveness and efficiency of health service delivery, with limited

1
Department of Health Information, Federal Medical Centre, Bida, Nigeria
2
Department of Surgery, Federal Medical Centre, Bida, Nigeria
3
Department of Obstetrics and Gynecology, Federal Medical Centre, Bida, Nigeria
4
Department of Administration, Federal Medical Centre, Bida, Nigeria
resources to improve health outcomes [3]. Ethical requirements
mandate researchers to believe that they owe an obligation to their
participants in presenting their results in a manner that will not del-
eteriously affect communities and/or individuals [4].
Although healthcare industry in Nigeria is characterized by paper-
based practice and inadequately IT-skilled professionals, it is worth-
while to note that in this millennium, researches including healthcare
research cannot thrive without passionate romance with information
and communication technology [5]. It is generally recognized that
the key to growth, development and survival, which research aims
to promote, depends on exploitation of knowledge resources which
include information technology and human knowledge and skills [6].
Of particular importance in this paper is the presumed attitude of
health workers at Federal Medical Centre, Bida (FMCB) most of
whom were considered as having indifferent dispositions towards
research. This was the assumption made by a group of researchers
in this hospital under the aegis of the Centre for Health and Allied
Researches having been practically involved in research works for
some years. They made a critical assessment of the willingness of
staff to respond to some research questions especially during data
collection for a study on confidentiality and discovered that most of
them were not willing [7]. This might be due to poor research orien-
tation or due to a perception that the hospital is primarily a service
providing institution which does not require much of research.
The hospital is a tertiary institution with the mandate to provide
qualitative services as well as training through the conduct of perti-
nent researches. In furtherance of their efforts to ascertain these as-
sumptions, efforts to access abstract or titles of published articles by
selected individual employee of the hospital revealed very limited
5
Department of Medical Microbiology, Federal Medical Centre, Bida, Nigeria
Correspondence: Ibrahim Taiwo Adeleke
Email: ibratadeleke_aliseyin@yahoo.com
Abstract
Background: Clinical research has been acknowledged to have positive impacts on healthcare systems. However, health workers at Federal
Medical Centre, Bida (FMCB) were presumably adjudged as indisposed to research. Thus, a scientific study became imminent. The present
study sought to determine research knowledge and behavior of health workers at FMCB.
Methods: A 25-point questionnaire was administered to 247 stratified randomly selected employees of the hospital.
Results: Most participants (99.4%) believed that research is relevant to healthcare services. A good number (73.1%) of them selected
abstract as the first section of journal articles, but not many (32.5%) selected research idea as the first step in research methodology. In
terms of knowledge about tools for health related research, most participants (83.8%) said they could operate computer system, but only
few (25.6%) could recognize SPSS as software for data analysis.
Conclusion: Health workers at FMCB acknowledged the impacts of health services research. However, not many possessed adequate
knowledge of research methodology and the right computing skills. Evolving a better research culture would require the few researchers
in the hospital to play continuous mentorship role along with the need for the hospital management to provide an enabling environment
promoting a positive research culture. (El Med J 2:2; 2014)
Keywords: Abstracts, Health Services Research, Health Information Technology, Research Design, Research Ethics
106 Research knowledge and behavior of health workers
Vol 2, No 2
number of published articles credited only to a few. These search
efforts deployed some relevant search engines like Pubmed, Google
Scholar and AJOL. In an attempt to correct the observed research
despondency, the researchers proposed a research sensitization
workshop to the hospital management. It was believed that the ef-
fort would enlighten the majority who seem to be ignorant of re-
search and reawaken the few researchers who tend to decline scien-
tifically due to the environment they find themselves. Hence, the
present study was conducted based on the assumptions from FMCB
in order to determine the level of research knowledge and attitude
of health workers in the hospital.
Methods
Background to the study area
The study was carried out at FMCB between December 2009 and
March 2010. The hospital, which is a tertiary health institution with
200 beds, became a tertiary health hospital in 1997 after 70 years of
existence in two capacities as a colonial hospital first, and a general
hospital later [8].
Study design
This was a cross-sectional study on research behavior of health work-
ers at FMCB.
Study population
At the time of the study, FMCB had a total complement of 700 staff.
These include 310 nurses and hospital attendants, 125 administrative
staff, 83 doctors, 57 health information management professionals
and aides, 43 medical laboratory scientists, technicians and assis-
tants, 41 accountants / auditors and executive officers (accounts), 16
pharmacists and technicians, 8 dental technologists / therapists /
nurses, 8 radiographers / darkroom attendants, 7 medical social
workers and 2 physiotherapists.
Data collection tools
A pretested 25-point semi-structured self-administered question-
naire was deployed. The questionnaire elicited data on basic demog-
raphy of participants, opinion on relevance of research to healthcare
services, previous research experience and future research inten-
tions, journal reading patterns, IT skills and utilization, research re-
lated sites and their knowledge of research environment.
Sampling technique
The study deployed stratified random sampling method. As such,
each group sample size was computed as a ratio of the total popu-
lation.
Sample size
Included in the selection were 110 nurses, 44 administrators / exec-
utive officers, 30 doctors, 20 health records officers, 15 medical la-
boratory scientists, 15 accountants / executive officers, 6 pharma-
cists, 3 dental technologists / therapists / nurses, 3 radiographers and
1 physiotherapist. Online sample size calculation software (www.sur-
veysystem.com/sscalc.htm) was used to compute the sample size,
keeping a 95% confidence level and a confidence interval of 5%. Ta-
ble 1 depicts the population of each category of staff, sample size
chosen at random and the participation rates.
Table 1: Total, sample size and rate of participation
Staff Categories /
Department Total Sample
Actual
Participants %
Nursing 310 110 59 53.6
Administration 125 44 20 45.5
Doctors 83 30 30 100
HIM* 57 20 12 60
Medical Laboratory 43 15 14 93.3
Finance and Audit 41 15 15 100
Pharmacy 16 6 6 100
Dental 8 3 3 100
Radiology 8 3 1 33.3
Physiotherapy 2 1 0 0
*HIM=Health Information Management

Inclusion and exclusion criteria
Those hospital employees who hold educational qualification below
higher national diploma or first degree as at the time of the study
were excluded from the study. Exception to this were nurses who
were assumed to have undergone one research project in spite of
the weight of their qualifications.
Data analysis and management
The statistical software SPSS v16.0 (2007) was used to analyze the
data. Discrete data was expressed as proportions and percentages,
while continuous variables were expressed as mean standard de-
viation. Chi square (
2
) was used to express association between cat-
egorical variables and test of statistical significance (P-value) was set
at P=0.05.
Ethics consideration
The ethics approval for this study was granted by the Research Ethics
and Review Committee of FMCB. Informed consent was obtained
from every participant before questionnaire was administered.
Results
Response rate
A total of 160 questionnaires were returned out of 247 distributed,
giving an overall response rate of 64.8%.
Socio-demographic characteristics
Table 2 indicates that there were female participants almost as much
as their male counterparts in this study and they had spent an aver-
age of 5 years in their practice. By profession, 59.4% represent clinical
support professionals, 21.9% represent non-clinical departments and
18.8% of the participants were core clinical (medical doctors). Major-
ity (142, 88.8%) possess first degree or lower certificates.
Participants research knowledge, skills and attitude towards
research tools
From the findings, the vast majority of the participants (159, 99.4 %)
believed that research is relevant to healthcare services. Table 3 pre-
sents participants knowledge-base in research and cross tabulation
to compare this according to the different professions (Table 4).

Table 2: Participants' socio-demographic characteristics
Indices N %
Sex Male 81 50.6
Female 79 49.4
Profession Nursing 59 36.7
Administration 20 12.5
Medicine & Surgery 30 18.8
Health Information Management 12 7.5
Finance & Audit 15 9.4
Medical Laboratory Science 14 8.8
Pharmacy 6 3.8
Dental Technology/Therapy 3 1.9
Radiography 1 0.6
Education
level
Professional Diploma 43 26.9
HND/1
st
Degree 99 61.9
Master 11 6.9
PhD/Fellow 6 3.8
No response 1 0.6
Years in
practice
< 3 years 64 40.0
3- <6 years 44 27.5
6- <9 years 14 8.8
9- <12 years 16 10.0
> 12 years 22 13.8
Majority of the participants (150, 93.8 percent) were positively dis-
posed to the relevance and very important roles of mentors in the
conduct of research. A fair knowledge of the arrangement of journal
articles was displayed by participants. Over 73 percent of the partic-
ipants selected abstracts as the first section of research article with
highest score of 100 percent by HIM professionals and dental staff,
while the lowest (0 percent) was by radiographers (Table 4). As
would be seen in Figure 1, most of the participants (134, 85.9 per-
cent) affirmed that they could operate computer system. However,
very poor knowledge of relevant research sites and software was
demonstrated by the participants (Table 3).
Only a few (10.0 percent) and just one quarter (41, 25.6 percent) of
the participants could identify with AJOL (African Journal Online) as
the platform for mainly African researches and SPSS (Statistical Pack-
age for Social Sciences; now: Statistical Product and Service Solu-
tions) as the software for data analysis respectively. Further analysis
(Table 4) shows that HIM professionals who were most correct in the
selection of AJOL and SPSS scored as low as 41.7 percent and 58.3
percent respectively. Other poor areas of research knowledge
among the participants were in research methodology where only
32.5 percent agreed that research idea precedes other research pro-
cesses and secondly, only 36.3 per cent of the participants accepted
that research ethics committee is not a bottleneck to research.

Table 3: Participants knowledgebase of research
Statement N % who
selected
correctly
% wrong
selection /
no knowledge
%
Undecided
The first section in journal articles (abstract) 160 73.1 26.3 0.6
The first step in the chain of research process (research idea) 160 32.5 66.3 1.3
The only software suitable for research data analysis in the list (SPSS) 160 25.6 72.5 1.9
The website that indexes mainly African related researches in the list (AJOL) 160 10.0 86.2 3.8
Research ethics committees or advocates are bottlenecks to research (False) 160 36.3 59.9 3.8
Research mentors are relevant to research (Very relevant/relevant) 160 93.3 3.1 3.1

Table 4: Participants' knowledge base of research by department (correct responses in percent)
Statement N* A* Do* H* F* L* P* De* R*
The first section in journal articles' publication (abstract) 59.3 75.0 83.3 100 73.3 92.9 50.0 100 0
The first step in the chain of research process (research idea) 25.4 40.0 33.3 25.0 20.0 50.0 50.0 100 0
The only software suitable for research data analysis in the list
(SPSS)
10.2 20.0 50.0 58.3 0 50.0 33.3 0 0
The website that indexes mainly African related researches in the
list (AJOL)
3.4 5.0 16.7 41.7 0 7.1 33.3 0 0
Research ethics committee or advocates are bottlenecks to
research (False)
27.1 35.0 56.7 33.3 20.0 50.0 50.0 33.3 0
Research mentors are relevant to research (Very relevant/
relevant)
98.3 95.0 86.7 91.7 86.7 92.9 100 100 100
*N = Nursing; A = Administration; Do = Medicine & Surgery; H = Health Information Management; F = Finance & Audit; L = Medical Laboratory Science; P = Pharmacy;
D = Dental Technology/Therapy; R = Radiography
108 Research knowledge and behavior of health workers
Vol 2, No 2

Figure 1: Participants skills and attitude towards research tools
Gender distribution on research knowledge and attitude
Although, the divide is not too prominent except in knowledge of
SPSS, Figure 2 depicts a clear indication that male participants have
better research knowledge and commendable research attitude than
their female counterparts according to the selected variables. It is
worthwhile to mention that these variables were selected at random
before being tested.

Figure 2: Gender distribution on research knowledge & attitude
Factors associated with research knowledge and behavior of
participants
Table 5 presents some significant factors affecting behaviors of the
participants towards research. Previous research experience had sta-
tistical significance on participants previous publication, their wishes
to be future investigators and their journal reading patterns
(P=0.000; P=0.012; P=0.039). Likewise, education level had associa-
tion with participants wishes to be future investigators and their
journal reading sequence (P=0.000; P=0.050). In the same vein, there
is association between profession and participants frequency of
journal reading. However, there is no association between years of
experience and any of the research behaviors.
Discussion
Health service research aims to improve health, health outcomes and
health services and there must be continued production of useful
clinical research that can be rapidly applied for the benefit of pa-
tients and taxpayers who fund the research in the first place [1, 9].
Our study comes in agreement with these assertions as a substantial
majority (99.4 percent) of the participants believed that research is
very relevant to healthcare services. It is noteworthy here to mention
the work of Khalil et al conducted few years ago, which is in tandem
with our findings that people recognized the value of medical re-
search and have great deal of trust with regard to it and their partic-
ipation in research [4].
The gender divide among participants was not too pronounced in
our study. However, more men possessed research knowledge and
good research behavior than their female counterparts. Although, it
is beyond the scope of this study to establish what could be respon-
sible, Bakken et al reported that the family-professional role conflict
that many women face combined with paucity of women role mod-
els in research contribute to lower self-efficacy for women in demon-
strating research skills [10].
The study attempted to specifically determine factors that influence
health workers research behavior and found out that previous re-
search experience most times, level of education at times, and pro-
fession occasionally, all do have their influences. Marsh and Brown
Table 5: Factors associated with participants attitude towards research
Variable % Correct
Response
Mean Standard
Deviation
Pearson 2 P-value
Experience Had published or presented research outcome before 29.4 1.76 0.43 2.366 0.669
Wished to be a future investigator 93.1 1.16 0.46 8.524 0.384
Read professional journals 46.8 2.83 1.54 16.96 0.656
Whether research wastes precious time 68.8 1.79 0.66 5.154 0.953
Level of
Education
Had published or presented research outcome before 29.4 1.76 0.43 1.676 0.795
Profession Had published or presented research outcome before 29.4 1.76 0.43 1.58 0.991
Previous
Research
Experience
Had published or presented research outcome before 29.4 1.76 0.43 23.001 0.000
134
132
73
22
25
83
0
50
100
150
Can operate
computer
Have access to
the Internet
Read professional
journals regularly
Yes No
0
10
20
30
40
50
60
70
Abstract as first
section of a journal
article
Research idea as
the first step in
research
SPSS as a suitable
software for data
analysis
63
29
31
54
23
10
Male Female
have over the years reported that significant differences in attitude
towards research depend on educational background [11]. They said
that the higher the educational degree, the more positive the mean
attitude to research. Similarly, the findings from Marsh & Brown,
Bjorkstrom and recently, that of Sabzwari et al revealed that partici-
pants who had previous research experience displayed more positive
attitudes towards research than those with no research experience
[11-13]. Our study shows that almost all health workers who had pre-
vious research experience (96.0 percent) wished to conduct investi-
gation in future and a majority of them selected abstract as the first
section of journal articles. Whereas, majority (88.1 percent) of those
without experience wished to be future investigators and a little
above half (53.7 percent) of them could select abstract correctly.
This study reveals that participants lacked adequate IT skills and they
seldom access internet services despite their earlier answer to have
had computer knowledge and internet access. Very poor knowledge
of SPSS as a software for research data analysis can testify to poor
computer usage as a researcher. Worst still, inability to identify AJOL
as the site mainly for African related studies shows poor usage of the
internet services for research. This finding is in tandem with Olalude
where utilization of the internet services for research was low among
researchers in Sub-Saharan Africa [14].
Studies by Khalil et al and that of Sabzwari et al discovered that the
morale of many participants was poor and must be improved and
that many of them demonstrated confusion with certain research
methodologies [4, 13]. This study discovers that most health workers
(67.5 per cent) did not know the steps in research methodology.
Conclusion
The study reveals that health workers at Federal Medical Centre, Bida
placed a high value on health services research and have good atti-
tude towards it. However, they possessed inadequate knowledge of
research methodology. Male health workers have better research
knowledge and attitudes than their female counterparts. Most of
them are capable of using computer systems and do have access to
the internet, but lacked the right computing skills and seldom made
use of the internet services especially for research. In view of the
above, the few researchers in the hospital would have to play con-
tinuous mentorship role for knowledge expansion and the hospital
management should encourage re-orientation of staff on research
through sensitization programs and enabling environment.
Recommendations
Based on our findings, we make the following recommendations:
1. The management of Federal Medical Centre Bida (and similar in-
stitutions) should create an enabling environment for the devel-
opment of health services research among their staff. Relevant
measures include:
a. Sensitization on the importance of research to healthcare
services through periodical workshops and advocacy.
b. Funding and adequate support for identified researchers
and research protocols.
c. Promotion of research through implementation of pertinent
findings especially those with peculiar relevance.
d. Subscription to relevant local and international journals and
making such available to staff in order to enhance reading
habit among healthcare providers.
2. Mentorship roles should be played by the available researchers
in the hospital. This will further strengthen health services re-
search and improve healthcare services to the Nigerian teeming
patients.

Authors Contribution: AIT conceived of the study, initiated its design, participated
in data analysis and coordination, interpreted the data and drafted the manuscript.
AAO participated in its design, coordination, data analysis and revised the
manuscript. JAD participated in its design, recruitment of participants, data analysis
and revised the manuscript. DFF participated in its design, coordination, data
analysis and revised the manuscript. IHE participated in its design, coordination,
data analysis and revised the manuscript. OSA participated in its design,
recruitment of participants, data analysis and revised the manuscript. All authors
have read and approved the final manuscript.
Acknowledgement: The insightful comment of Prof MA Tiamiyu of African
Regional Centre for Information Science, University of Ibadan is gratefully
acknowledged. We specifically thank Messrs Adebisi AA, Anamah TC, Adeleye JA,
Zakari IB, Hassan MW, Abodunrin OA and Mrs Ajuwon BE of the Department of
Health Information Federal Medical Centre, Bida for their assistance during data
collection. The authors wish to thank all staff of Federal Medical Centre, Bida who
completed the survey questionnaire.
Received: 13 December 2013 Accepted: 30 March 2014
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110 Medical students' perception about teaching-learning

Vol 2, No 2
Medical students' perception about teaching-learning and academic performance at Nobel Medical
College, Biratnagar, Nepal
Mukhtar Ansari
1
, Attique Ur Rahman Mufti
2
, Salman Khan
3

Introduction
Teaching-learning is a crucial component of any academic activity. It
is vital to have an effective teaching-learning for better performance
of students. There may be various factors contributing to the quality
of teaching-learning such as teaching-learning methods and envi-
ronment, class size and duration of teaching. More supportive the
teaching-learning environment and satisfaction of the teachers and
students, the better the outcome [1]. Class size and duration of
teaching also affect the performance of students. Smaller the class
size and shorter the duration of teaching, the better the quality and
performance of the students [2, 3].
Apart from these, students centered factors such as difficulty in un-
derstanding, the medium of instruction, psychological pressure,
background of the student and self-assessed depression also affect
their performance [4]. Modern methods involve a participatory ap-
proach of teaching-learning such as Problem Based Learning (PBL)
and small group discussions, which are more suitable than lecture
based teaching [5, 6]. Moreover, a lecture combined with discussion
is more relevant than conventional lectures. Lectures mainly facili-
tate to recall facts, whereas discussions not only produce higher level
comprehension but also encourage students active participation
and self-confidence [7, 8].
In Nepal, medical education is a highly demanded and respected
field of education. Students in Nepalese medical schools are enrolled
through three different mechanisms such as through passing the en-
trance examinations conducted either by the Ministry of Education,
Nepal or the respective universities, or through direct enrollment of
foreign students without entrance examinations. Hence, students

1
National Medical College Teaching Hospital and Research Centre, Nepal
2
University Kebangsaan Malaysia, Malaysia
3
Nepalgunj Medical College, Nepal
schooling preceding medical school matters for their medical school-
ing performance [9]. In general, the quality of medical education in
Nepal is somewhat dubious. There may be several factors but stu-
dents premedical schooling background, quality of teaching-learn-
ing and examination system are utmost. Quality of education during
students life reflects the quality of care/treatment in their profes-
sional life. More effectively the students learn and grasp the things
today, the better they can perform tomorrow in their professional
life [10].
The objective of this study was to gather students opinion about
teaching-learning activities at Nobel Medical College, Biratnagar, Ne-
pal and to suggest improvements in the quality of teaching-learning
activities.
Design
The study was a cross-sectional and descriptive in nature.
Study duration and location
The study was carried out during February and March 2012 at Nobel
Medical College, Biratnagar, Nepal. Nobel Medical College is a private
medical college in the eastern region of Nepal and is affiliated to
Kathmandu University (KU), Nepal.
Study population
The participants of the study were medical (MBBS) students of sec-
ond, fourth, sixth, seventh and ninth semesters. At the time of study,
first, third, fifth and eighth semesters did not exist. Therefore, stu-
dents of second, fourth, sixth, seventh and ninth semesters were only
Correspondence: Mukhtar Ansari
Email: mukhtaransari@hotmail.com
Abstract
Background: Effective teaching-learning is an important prerequisite for academic excellence. The study was aimed at determining
students perception about the effectiveness of teaching-learning methods and improving the academic performance.
Methods: This cross-sectional study was carried out among 385 medical students of Nobel Medical College, Biratnagar, Nepal during
February and March, 2012. The instrument used was a self-administered pretested questionnaire containing both closed and open ended
questions. The questionnaire was distributed among the students and their responses were collected and entered in SPSS 11.5 for windows
(Chicago Inc) for analyses.
Results: Nearly two third (64%) of the students were male and majority (61%) of the students were from three zones such as Bagmati,
Koshi and Janakpur of Nepal. Small group interactive discussions (64%), Problem Based Learning classes (65%) and use of multimedia
were found the most effective approaches for improving the academic performance. Burden of multi-subjects (53.5%), lack of seriousness
(28%), irregular study habit (17.4%) and fears of examinations (85%) were found the major causes of students poor performance in
examinations. There were statistically significant association between gender and fortnightly class test as a factor for improving academic
performance (p<0.001), use of transparency projector and power point projector as important means of teaching methods (p=0.010,
0.009) and studying many subjects as a reason for poor performance (p=0.030).
Conclusion: Multimedia combined with Problem Based Learning and small group interactive discussions were found the most efficient
approaches for better academic performance. (El Med J 2:2; 2014)
Keywords: Academic Performance, Medical Colleges, Medical Students, Nepal, Teaching-learning
Ansari M, Mufti AUR, Khan S 111
included in this study. The total duration of MBBS course is of four
and half years i.e. nine semesters and each semester comprises of six
months. Basic medical science subjects such as pharmacology, phys-
iology, pathology, biochemistry, microbiology, anatomy and com-
munity medicine are taught during the first two years of the course.
The latter two and half years are allocated for teaching clinical sub-
jects such as medicine, surgery, pediatrics, obstetrics and gynecol-
ogy, orthopedics, ENT, ophthalmology and dermatology.
Sample size and sampling procedure
A sample size of 385 subjects was recruited in the study. Nobel Med-
ical College has a history of about five years. Only 60 seats were per-
mitted by the Nepal Medical Council for admission in the first batch
and the numbers of seats allocated were gradually increased to 100
and 150 for second batch and third batch, and onwards. Therefore,
there were variable numbers of students in various semesters. Cen-
sus method was adopted for sampling the subjects but responses
were received only from 385 subjects.
Instrument
The instrument was a pretested self-administered questionnaire con-
taining both closed as well as open ended questions concerned with
the objectives of the study.
Procedure
The instrument was pretested among 10 randomly selected subjects
and necessary amendments such addition of options and modifica-
tion of questions were made. The modified version of the question-
naire was distributed among the students and their responses were
gathered. Before collecting the data, students consents were taken
and they were informed to express their responses independently as
they perceive or think. The study received ethical clearance from the
Research and Ethics Committee of Nobel Medical College, Biratnagar,
Nepal.
Data analyses
The data for closed ended responses were pre-coded in the ques-
tionnaire, whereas data for open ended responses were coded latter.
The data were entered in SPSS 11.5 for windows (Chicago Inc) and
analyzed for descriptive and inferential statistics.
Results
Nearly two third (64%) of the students were male and majority (48%)
of the students belonged to the age group of 20-21 years. Location
wise, about 61% of the students were from three zones such as Bag-
mati, Koshi and Janakpur of Nepal. Looking over the parents occu-
pation, most (42%) of them were involved in business and official
works. About two thirds of students were from 2
nd
and 4
th
semesters
and the remaining students were from 6
th
, 7
th
and 9
th
semesters. De-
tails of demographic characteristics have been shown in Table 1.
Table 2 illustrates students emphasis mainly on two components
such as regular interactive discussions and Problem Based Learning
(PBL) classes to have better impact on improving academic perfor-
mance of the students. However, majority of the students did not
stand in favor of any type of examinations such as surprise test, fort-
nightly test or monthly test.
Table 1: Demographic characteristics of medical students of
Nobel Medical College, Biratnagar, Nepal (n=385)
Characteristics N (%)
Gender Male 247 (64.2%)
Female 138 (35.8%)
Age 18-19 years 71 (18.4%)
20-21 years 184 (47.8%)
22-23 years 92 (23.9%)
24-25 years 38 (9.9%)
Origin of
Students
Bagmati 94 (24.4%)
Koshi 82 (21.3%)
Janakpur 58 (15.1%)
Sagarmatha 40 (10.4%)
India 25 (6.5%)
Others 86 (22.3%)
Parents
Occupation
Business 93 (24.2%)
Official Works 68 (17.7%)
Farming 49 (12.7%)
Teaching 44 (11.4%)
Government Job 41 (10.6%)
Doctor and Other
Health Professionals
25 (6.5%)
Others 65 (16.9%)

Table 2: Medical students perception about the techniques to
improve their academic performance (n=385)
Particulars Yes (%) No (%)
Surprise class test 59 (15.3%) 326 (84.7%)
Regular interactive discussions 246 (63.9%) 139 (36.1%)
Fortnightly class test 143 (37.1%) 242 (62.9%)
Monthly class test 57 (14.8%) 328 (85.2%)
Regular practical viva-voce 199 (51.7%) 186 (48.3%)
Regular PBL classes 249 (64.7%) 136 (35.3%)

Table 3 explains the students views toward teaching-learning meth-
ods. Among the five teaching-learning approaches investigated, dic-
tating lecture notes was found not effective or the least effective. On
the other hand, use of multimedia (power point projector) was
opined as the most effective method of teaching-learning approach.
Table 3: Medical students opinion about different teaching-learning methods (n=385)
Teaching medium/methods Not Effective (%) Just OK (%) Effective (%) Most Effective (%)
Use of black board & chalk 66 (17.1%) 156 (40.6%) 102 (26.5%) 61 (15.8%)
Use of white board & marker 44 (11.4%) 96 (24.9%) 154 (40.0%) 91 (23.6%)
Use of transparency projector 56 (14.5%) 147 (38.2%) 124 (32.2%) 58 (15.1%)
Use of PowerPoint projector 27 (7.0%) 72 (18.7%) 153 (39.7%) 133 (34.5%)
Dictating lecture notes 158 (41.0%) 98 (25.5%) 91 (23.6%) 38 (9.9%)
112 Medical students' perception about teaching-learning
Vol 2, No 2
Table 4 illustrates students perception about teaching 150 students
in a class. Although students had mixed opinion about teaching a
large number of students in a class, larger fraction of students artic-
ulated that handling such a large class is difficult and students mainly
of back rows have to suffer. Therefore, the class should be broken
into two sections.
Table 4: Medical students opinion about difficulties in
teaching large class size (n=385)
Particulars Yes (%) No (%)
Handling of class is difficult 232 (60.3%) 153 (39.7%)
Class becomes non interactive 197 (51.2%) 188 (48.8%)
Students of back rows have to
suffer
229 (59.5%) 156 (40.5%)
Class should be broken into
two sections
230 (59.7%) 155 (40.3%)
Students perception about conducting fortnightly written test
and seminar
About 60% of the students were in favor of conducting both fort-
nightly written tests as well as seminars as they help in self-assess-
ment of the students and increase their confidence level. On the con-
trary, about 40% of the students opined to have either fortnightly
written tests or fortnightly seminars but not the both.
Students perception about poor performance in fortnightly
written test
Although an array of views emerged out about the poor perfor-
mance of the students in fortnightly written tests, the crucial factors
were dealing with many subjects at a time (53.5%), lack of serious-
ness in study (28.1%), lack of regular study habit (17.4%), ineffective
teaching (7.0%) and vast syllabus (4.2%).
The association of gender, origin of the students and their parents
occupation with students perception about improving academic
performance, teaching-learning methods, teaching large class size
and fortnightly tests and seminars respectively were calculated.
However, statistically significant associations were found only in the
cases depicted in Table 5.
Discussion
The objective of this study was to explore medical students percep-
tion about teaching-learning activities and to suggest improvements
in academic performance at medical schools. In Nepal, medical edu-
cation is a highly attractive profession and the quality of teaching-
learning activities is one of the most important factors for quality
medical education [11]. Nearly two third (64%) of the students were
male which indicates either males are more directed or parents pro-
mote their sons more compared to daughters towards medical edu-
cation.
At the time of enrolment in first year of MBBS, the minimum age of
the students in general was 18 years or more and about three fourths
of students were from first two years of the course. This may be the
reason for large fraction of the students representing the age group
20-21 years. Although students studying at Nobel Medical College
were from various part of the nation including the neighboring
country India, about 61% of the students were from three zones of
Nepal such as Bagmati, Koshi and Janakpur. Koshi is the zone in
which Nobel Medical College is situated; Janakpur is a nearby histor-
ical zone; Bagmati zone represents the capital city.
In terms of improving the academic performance, students prefer-
ences were towards experimental methods of learning rather than
theoretical. This may be due to the reason that interactive discus-
sions encourage greater participation, improve the grasping ability
and self-confidence [8, 11, 12]. On the contrary, the trivial curiosity
of the students towards any type of tests or examinations such as
surprise test (15.3%), fortnightly test (37.1%) or monthly test (14.8%)
indicates that students harbored a fear of examinations, in general.
Examinations are considered as one of the important stress factors
among the students [13]. However, a study conducted by Larsen et
al found that tests or examinations promote better retention of in-
formation among the students [14].
While inquiring the students opinion about the effectiveness of five
teaching-learning methods such as PowerPoint projectors, black-
board-chalk, whiteboard-marker, overhead (transparency) projectors
and dictation, PowerPoint projectors (multimedia projectors) were
found the most effective. This may be due to the reason that multi-
media incorporate elements such as text, images and even audio-
video which makes it easier for the teacher to represent the things
as well as it is more receptive to the students [15]. On the other hand,
dictating lecture notes mainly in large class size was the least effec-
tive method of teaching-learning as lecturing lacks two way commu-
nication and may be considered boring leading to less students par-
ticipation. But, lecturing (the most common form of teaching) can
be made effective if the teacher or presenter is able enough to pre-
sent the information in the form of text and diagrams or photos in
addition to capturing the attention of students and improving their
participation [16].
*Association (2-tailed) was significant at level of 0.05; P-value was calculated by Pearsons chi-square test
Table 5: Association of gender, origin and parents occupation with students perception about improving academic performance
and teaching-learning activities (n=385)
Characteristics Students Perception
2
df P value
Gender Improving academic performance Fortnightly class tests 13.562 1 <0.001*
Teaching methods Use of Transparency projector 11.240 3 0.010*
Use of PowerPoint projector 11.641 3 0.009*
Poor performance Many subjects at a time 4.688 1 0.030*
Students Origin Conducting fortnightly tests Both fortnightly class tests and seminars 33.226 16 0.007*
Fortnightly written tests 34.820 16 0.004*
Parents Occupation Poor Performance Lack of a serious attitude 18.726 8 0.016*
Ansari M, Mufti AUR, Khan S 113
In general, medical schools of Nepal have a large number of students
per class, which is negatively linked with students performance. Stu-
dents were mostly of the opinion that class size should be reduced,
as larger classes are difficult to handle, non-interactive and poorly
communicative. This reflects students temptations for more partici-
patory or problem-solving classes which are more effective than tra-
ditional, didactic lectures [17]. Reducing class size can be another
suitable option but if class size is reduced, financial and technical
burdens on management authority may hike up and may not be
practically feasible.
There can be various approaches to assess the students and their
academic performance. Although the practice of fortnightly tests
(FNT) is one of the popular concepts in developed nations for as-
sessing the students, Nobel Medical College has recently introduced
this concept in the form of fortnightly written test and seminar.
When students were asked about the relevance of fortnightly tests,
there were mixed opinions about conducting fortnightly written
tests and seminar. A major fraction (60%) of the students was in favor
of both with the justification that fortnightly written tests and semi-
nars help in self-assessment of the students and increase the confi-
dence level. Similarly, about 40% of the students expressed positive
views about either fortnightly written tests or fortnightly seminars,
but not the both. Although students participation and performance
was good in fortnightly seminars, their performance in fortnightly
written tests was poor.
Although the findings of our study were interesting, there were cer-
tain limitations. The study was conducted in a single medical college
of Nepal and thus the findings cannot be generalized. Therefore, it is
recommended to conduct such studies at different medical schools
of Nepal and other countries and make comparisons. It is further rec-
ommended that the medical colleges of Nepal and the concerned
authorities should comply with and adopt the study findings in their
institutions for better quality medical education.
Conclusion
The students preferences for better retention of material and aca-
demic performance were towards modern method of teaching-
learning such as PowerPoint slides, combined with Problem Based
Learning and discussions mainly in small groups.
Authors Contribution: Mukhtar Ansari: designed, carried out and edited the
study. Attique ur Rahman Mufti and Salman Khan: designed and edited the study.
Acknowledgements: The authors would like to thank Prof. Dr. J.N. Bhagawati,
Principal of Nobel Medical College, Biratnagar, Nepal for facilitating this study. We
are also grateful to the students for their active participation. We also would like to
express our gratitude to Dr. S. P. Patel, biostatistician, National Medical College,
Birgunj, Nepal for his valuable suggestions and cooperation in statistical analysis of
the data.
Received: 29 November 2013 Accepted: 10 January 2014
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114 Microalbuminuria: An early marker of diabetic kidney disease

Vol 2, No 2
Open Access Short Report
Microalbuminuria: An early marker of diabetic kidney disease
Zahra Ali
1
, Muhammad Adnan
1
, Saira Bashir
1
, Iffat Shabbir
1
, Tayyaba Rahat
1

Introduction
With a worldwide increase in the diabetes epidemic, diabetic
nephropathy is a common consequence of long standing poor gly-
cemic control [1]. Diabetic nephropathy is the leading cause of end-
stage renal disease (ESRD) globally and contributes to the total dis-
ease burden [2]. The earliest clinically detectable stage of diabetic
kidney disease is microalbuminuria (MA). It is characterized by in-
creasing rates of urinary albumin excretion, starting from normoal-
buminuria, which progresses to microalbuminuria, macroalbuminu-
ria and eventually to ESRD [3].
Excretion of small amount of albumin in the urine has been docu-
mented to predict renal failure and cardiovascular morbidity as well
as mortality among diabetes, hypertensive and general population
as well [4-6]. Without specific interventions, 2040% of type 2 dia-
betic patients with microalbuminuria progress to overt nephropathy
[3]. As microalbuminuria can be reversed and the future develop-
ment of overt diabetic nephropathy significantly reduced, screening
for microalbuminuria and timely therapeutic intervention has be-
come standard of care worldwide [7].
According to recommendations by American Diabetes Association
(2009) for nephropathy screening and treatment for urine albumin
excretion should be tested annually in all type 2 diabetic patients,
starting at diagnosis, although implementation of routine screening
for renal disease is still far below recommended goals [8]. Therefore,
the present study was carried out to determine the frequency of mi-
croalbuminuria among type 2 diabetic subjects and to study the risk
factors associated with microalbuminuria.

1
Pakistan Medical Research Council Research Center, Fatima Jinnah Medical
College, Lahore, Pakistan
Correspondence: Zahra Ali
Email: alixahra@yahoo.com
Findings
Methodology
One hundred and thirty subjects with type 2 diabetes were screened
for microalbuminuria on their scheduled visits to the diabetic clinic
of Pakistan Medical Research Council (PMRC) Research Center, La-
hore between January to June 2013. Subjects presenting with
macroalbuminuria, hematuria, pyuria and urinary tract infections
were excluded from the study. Type II diabetics of both genders with
minimum one year history of disease were selected.
Demographic data of each subject such as age, gender, duration of
diabetes, weight, blood pressure, smoking habit, family history of di-
abetes, renal disease and hypertension was recorded on a specified
proforma designed for this study. Urine specimen was collected for
testing microalbumin and was determined by using semi quantita-
tive dry immunochemical screening strips. (Micral II test strips)
(Roche diagnostic GmbH Mannheim Germany). Microalbuminuria
was defined as a urinary albumin >20 mg/l. Biochemical indicator
included blood sugar random. Blood urea and creatinine were esti-
mated as per standard procedures using enzymatic kits.
Results
A total of 130 type 2 diabetic patients of both genders attending the
diabetic clinic of PMRC Research Center were prospectively recruited.
Of them 91 patients were selected for the study as 39 were excluded
because they had frank proteinuria. Out of remaining ninety one
subjects 33% were male and 67% were female. The mean age of the
subjects was 50.41 9.79 years. History of co-morbidity showed that
80.2% of the subjects were hypertensive. The overall prevalence of

Abstract
Background: Diabetic nephropathy is the leading cause of end-stage renal disease globally and contributes to the total disease burden.
The study was carried to estimate the occurrence of microalbuminuria in type II diabetics and to find its association with blood pressure,
duration of disease and anthropometric measurements.
Findings: The cross-sectional study was conducted for six months in Pakistan Medical Research Council Research Center, Fatima Jinnah
Medical College, Lahore. A total of 91 subjects who met the inclusion criteria were selected. A comprehensive questionnaire including
age, education, socio-economic status, medical history, blood pressure and anthropometric measurements was used as a study tool.
Random blood sample was used for the estimation of plasma glucose, blood urea and serum creatinine, while first morning urine sample
was collected for the qualitative screening of microalbuminuria. Data analysis was done using Statistical Package for Social Sciences
version 15 (SPSS-15).
The mean age of 91 selected subjects was 50 9 years and male to female ratio was 2:1. 80.2% subjects were diabetic hypertensive and
remaining 19.8% had only diabetes. The overall prevalence of microalbuminuria was 58.2%. Prevalence of microalbuminuria among males
was 30.2% and among females 69.8%. Correlation analysis showed that plasma glucose level, duration of diabetes and systolic blood
pressure correlated with microalbuminuria at P<0.05. While age, BMI, waist to hip ratio were not statistically correlated with microalbumin.
Among the 53 patients having microalbuminuria, serum urea and creatinine was found to be raised in 13.2%.
Conclusion: Microalbumin has associations with systolic blood pressure, elevated plasma glucose level and duration of diabetes. Hence,
diabetic patients should be subjected to routine screening for microalbuminuria at least once in a year. (El Med J 2:2; 2014)
Keywords: Microalbumin, Diabetic Nephropathy, Glycemic Control
Ali Z, Adnan M, Bashir S et al 115
microalbuminuria was 58.2%. Prevalence of microalbuminuria
among males was 30.2% and among females 69.8%.
Table 1 shows distribution of anthropometric measurements and
blood pressure. According to BMI criteria of overweight and obesity,
females were significantly more overweight than males. Systolic
blood pressure in females was significantly higher than in males with
p-value=0.005. Waist circumference values were found to be higher
than the normal cut offs (90 cm for males and 80 cm for females)
in more females than males indicating the presence of abdominal
obesity.
Table 1: Distribution of anthropometric measurements and
blood pressure
Parameter Male Female P-value
Height (cm) 167.087.49 153.985.61 <0.001*
Weight (kg) 70.2515.91 65.8411.57 0.167
BMI (kg/m
2
) 24.994.64 27.764.69 0.009*
Waist (cm) 95.4211.55 105.699.18 <0.001*
Hip (cm) 95.339.55 103.909.07 <0.001*
Systolic (mmHg) 129.5015.78 139.7516.08 0.005*
Diastolic(mmHg) 85.678.88 85.3610.72 0.891
*P value significant at P<0.05
Correlation analysis showed, plasma glucose level, duration of dia-
betes and systolic blood pressure correlated with microalbuminuria
at p<0.05. (Table 2). Among the 53 patients having microalbuminuria
serum urea and creatinine was found to be raised in 13.2%.
Table 2: Correlation analysis of microalbuminuria and
different variables in diabetic patients
r P-value
Age (years) 0.067 0.527
Duration (years) 0.225 0.032*
BSR (mg/dl) 0.219 0.037*
BMI (kg/m
2
) 0.092 0.386
Systolic (mmHg) 0.230 0.029*
Diastolic (mmHg) 0.043 0.683
Waist Hip ratio 0.099 0.348
*P value significant at P<0.05
Discussion
Diabetes mellitus is a major health problem in Pakistan with its prev-
alence ranging from 7.6% to 11.0% [9]. Microalbuminuria is the first
clinical detectable sign of involvement of the kidney. It affects be-
tween 20-40% of subjects 10-15 years after the onset of diabetes.
Once it is present, it progresses to proteinuria over 5-10 years in 20-
50% subjects [10].
This cross-sectional study presents data on prevalence and associa-
tions of microalbuminuria with various parameters in type-2 diabetes
mellitus. In the present study the overall prevalence of MA was 58.2%
which is much higher when compared to the study by Ghai et al,
where prevalence was reported at 25% and another studies from Pa-
kistan reported 34% and 28% [11, 12]. Various epidemiological and
cross-sectional studies have reported marked variation in the preva-
lence of MA. The prevalence rate of MA was considerably high (61%)
among diabetic patients in the UAE [9].
No correlation was found between age and microalbuminuria in the
present study which was similar to findings reported by Lutale et al
and Afkhami et al [13, 14]. However, some studies in contrast to our
results have shown correlation between age and microalbuminuria
[15]. No correlation was found between microalbuminuria and BMI
in our study, similar to a finding of the study by Erasmus [18]. How-
ever S Ghosh and Ruilope et al have reported a correlation of micro-
albuminuria with BMI [16, 17]. The microalbuminuria positive group
had a higher systolic blood pressure compared to the microalbumi-
nuria negative group (p<0.001) which has been observed by others
studies [7].
In the present study, statistically significant correlation was found
between the prevalence of microalbuminuria and the duration of di-
abetes that is consistent with findings of past studies (p value=0.032
as shown in Table 2). The frequency of microalbuminuria increased
with the increase in duration of diabetes [19]. Huraib et al, Varghese
et al, and Mather et al also reported a significant correlation between
microalbuminuria and the duration of diabetes [20-22]. Cross tabu-
lation of albumin excretion with glycemic status and hypertension
showed that plasma blood glucose (p=0.020) seemed to be influenc-
ing the levels of albumin in urine. Meigs has also established the as-
sociation of glycemic control with microalbuminuria [23].
Conclusion
In conclusion, the prevalence of microalbuminuria in type 2 diabetes
mellitus in this cross-sectional study across Lahore is 58.2%. The risk
factors are similar to those reported from other studies. Because of
the adverse impact of proteinuria on survival in subjects with type 2
diabetes, screening and intervention programs should be imple-
mented early at the stage of microalbuminuria and risk factors
should be evaluated and treated.
Received: 29 December 2014 Accepted: 29 March 2014
References
1. Venugopal S, Iyer UM: Risk Factor Analysis and Prevalence of Microalbuminuria
among Type 2 Diabetes Mellitus Subjects: The Need for Screening and
Monitoring Microalbumin. Asian J Exp Biol Sci 2010, 1(3): 652-659
2. Naqvi SAJ: Nephrology services in Pakistan, Nephrol Dial Transplant 2000, 15:
769-71.
3. American Diabetes Association: Nephropathy in diabetes (Position Statement).
Diabetes Care 2004, 27(1): 79-83.
4. Naidoo DO: The link between microalbuminuria, endothelial dysfunction and
cardiovascular disease in diabetes. Cardiovasc J South Africa 2002, 13(4): 194-
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5. Romundstad S, Holman J, Hallman H, Kvenild K, Ellekjaer H: Microalbuminuria
and all cause mortality in treated hypertensive individuals: Does sex matter?
The Nord-trondelag health study (HUNT), Norway. Circulation. 2003, 108(22):
2783-9.
6. Bello AK, Peters J, Wight J, de Zeeuw D, El Nahas M: A Population-Based
Screening for Microalbuminuria among Relatives of CKD Patients: The Kidney
Evaluation and Awareness Program in Sheffield (KEAPS).. Am J Kidney Dis.
2008, 52(3): 434-43.
116 Microalbuminuria: An early marker of diabetic kidney disease
Vol 2, No 2
7. Ahmedani MY, Hydrie MZI, Iqbal A, Gul A, Mirza WB, Basit A: Prevalence of
Microalbuminuria in Type 2 Diabetic Patients in Karachi: Pakistan A Multi-
center Study. JPMA 2005, 55(9): 382-386.
8. American Diabetes Association (2009): Standards of Medical Care in Diabetes-
2009 (Position Statement). Diabetes Care, 32(Suppl. 1):S13-S61.
9. International Diabetes Federation: Prevalence estimates of diabetes mellitus
(DM), 2010 - MENA. IDF Diabetes Atlas . 2010. International Diabetes
Federation. 3-9-2010.
10. Ritz E, Orth SR: Nephropathy is patients with type 2 diabetes mellitus N Engl.
J Med 2000, 341: 1127-33
11. Lutale J, Hrafnkell T, Zulfiqarali A, vetvik K: Microalbuminuria among Type 1
and Type 2 diabetic patients of African origin in Dar es Salaam, Tanzania. BMC
Nephrology 2007, 8: 210.
12. Afkhami-ardehani M, Modarresi M, Amirchaghmaghi E: Prevalance of
microalbuminuria and its risk factors in type 2 diabetic patients. Indian J
Nephrol 2008, 18(3): 112-7.
13. TanzaniaS Ghosh, I Lyaruu, K Yeates: Prevalence and factors associated with
microalbuminuria in type 2 diabetic patients at a diabetes clinic in northern.
African Journal of Diabetes Medicine 2012, 20(2): 43-46.
14. Ruilope LM, Segura J: Predictors of the evolution of microalbuminuria.
Hypertension 2006, 48: 8323.
15. Meigs J.B, Agostino RB, Nathan DM, Rafai N, Wilson P: Longitudinal association
of glycaemia and microalbuminuria. Diabetes Care 2002, 25(6): 977-83.
16. Mokdad AH, Ford ES, Bowman BA, Dietz WH, Vinicor F, Bales VS, Marks JS:
Prevalence of obesity, diabetes, and obesity related health risk factors. JAMA
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17. Body mass index calculator. Available at:
http://www.kcnet.com/~marc/bmi.html. [Accessed Oct 10, 2008]
18. Erasmus RT, Okesina B: Microalbuminuria in a clinic population of type
2Melanesian diabetics: relationship to glycaemic control, blood pressure and
duration of diabetes. Cent Afr J Med. 1999, 45(11): 291-4.
19. Naz S, Sadaruddin A, Khannum A, Osmani R: Frequency of microalbuminuria
in diabetic patients of Islamabad and Rawalpindi. Pak J Med Res 2007, 46(3):
704
20. Huraib S, Abu-Aisha H, Sulimani RA, Famuyiwa FO, Al-Wakeel J, Askar A,
Sulimani F: The pattern of diabetic nephropathy among Saudi patients with
NIDDM. Ann Saudi Med 1995, 15(2): 120-4
21. Varghese A, Deepa R, Rema M, Mohan V: Prevalence of microalbuminuria in
type 2 diabetes mellitus at a diabetes center in southern India. Postgrad Med
J 2001, 77: 399402.
22. Mather HM, Chaturvedi N, Kehely AM: Comparison of prevalence and risk
factors for microalbuminuria in south Asians and European with type 2
diabetes mellitus. Diabetes Med 1998, 15: 6727.
23. Meigs, JB, Agostino RB, Nathan DM, Rafai N, Wilson P: Longitudional
association of glycaemia and microalbuminuria. Diabetes Care 2002, 25: 977-
83.

Kaptanolu AF, Yavuz HU, Ser K 117

A dermatological approach to the feet of soccer players
Asli Feride Kaptanolu
1
, Hasan Ula Yavuz
1
, Kaya Ser
1

Introduction
Dermatological diseases are common in sportsmen. Therefore, phy-
sicians practicing sports medicine must be aware of the risks of pos-
sible skin disorders associated with different types of sports. Various
studies exist that discuss the relation of skin lesions with type of
sports such as athletics, swimming, wrestling and also soccer players.
Soccer is a sport in which the foot is susceptible to acute and chronic
mechanical injuries, as well as increased sweating and occlusion dur-
ing trainings [1]. Also many factors such as training environment, cli-
mate and education levels may contribute the occurrence of these
lesions. Present literature has only limited data on the evaluation of
possible dermatological conditions in soccer players' feet and asso-
ciated factors. Most of the studies on soccer players involve skin
health as a whole, and discusses aspects such as traumatic lesions,
infections, contact dermatitis, environmental encounters and exac-
erbation of pre-existing dermatoses [2, 3]. Therefore, the aim of this
study was to evaluate possible dermatological disorders and associ-
ated factors in soccer players.
Findings
This cross sectional study was conducted in North Cyprus. Forty
young Turkish Cypriot soccer players were included in the study with
their consent, as well as the consent of their families and team. De-
mographic features such as age, education level were recorded,
along with foot care features (such as wearing of socks, usage of dif-
ferent products, washing and drying attitudes and factor related to
shoes). Feet of all players were examined by a dermatologist and a
sports physician. All the lesions were recorded, along with the site of
occurrence, symmetry and diagnosis. Further mycological, bacterio-
logical and other screening tests were done to clarify the diagnosis.
Diagnoses were classified into 4 groups: bacterial infection, fungal
infections, dermatoses and traumatic lesions. Microbiological results
were also classified as: (a) No colonization in culture medium; (b)
Bacterial (normal flora); (c) Pathogenic bacteria; and (d) Mycoses.
Statistical analysis were done by using SPSS software program.

1
Near East University Hospital, North Cyprus
Correspondence: Kaya Ser
Email: kayasuer@mynet.com
We questioned the foot care habits and determined the frequency
of skin and nail lesions in young soccer players. 40 players joined the
study. The mean age, height, and weight means of the subjects were
15.2 2.3 years, 166.8 11.5 cm and 56.1 12.9 kg, respectively. The
average years in sports were 5.9 3.0 years. 26 players. Twenty six
players (65.0%) had type 2 skin characteristics, while 13 players
(32.5%) and 1 player (2.5%) had type 3 and type 4, respectively. The
foot care habits of the players have been summarized in table 1,
whereas table 2 gives the results of the physical examination which
was performed to determine possible skin and nail lesions.
Table 1: Foot care habits of the players
Foot care habits Yes No
Using the same spikes in
every training and match
21 (52.5%) 19 (47.5%)
Washing the feet everyday 33 (82.5%) 7 (17.5%)
Drying the feet after washing 26 (65%) 14 (35%)
Changing the socks everyday 29 (72.5%) 11 (27.5%)
Using foot care products 2 (5%) 38 (95%)

There was no relation between the types of skin or nail lesions (trau-
matic, viral, fungal or bacterial) and foot care habits of the players.
The only exception was that increased number of traumatic nail
changes were seen in the subjects who did not dry their feet after
washing (p=0.028) (table 3). Nail changes were present in 60% of the
subjects, with a dominance of traumatic lesions (55%). These trau-
matic nail changes were ingrown nail (17.5%), splinter hemorrhages
(12.5%), discoloration (15%) and hyperkeratinization (10%).
Skin and nail samples were taken to search for the reproduction of
bacterias and fungi. Fungal pathogen reproduction was seen in 13
(32.5%) players while bacterial pathogen reproduction was seen in 3
(7.5%) players (table 4). The fungal microorganisms were: Epidermo-
phyton floccosum (n: 9), Alternia (n: 3), Trichophyton rubrum (n: 1),

Abstract
Background: Soccer is a sport in which the feet are very susceptible to injuries and dermatological diseases. Present literature does not
have much data about the possible dermatologic conditions occurring in soccer players and the associated factors. Therefore, the aim of
this study was to evaluate the dermatologic foot problems and associated factors in soccer players.
Findings: We evaluated forty young soccer players and recorded dermatologic conditions localized to their feet. The skin and nail lesions
were recorded by the site of occurrence, symmetry and diagnosis. Further mycological, bacteriological or other screening tests were done
to clarify the diagnosis. Diagnoses were classified into 4 groups: bacterial infections, fungal infections, dermatoses and traumatic lesions.
Associated factors were also evaluated in detail. The results of our study revealed that there were at least two dermatological problem in
the feet of soccer players and the commonest problems were foot odor, fungal infections and traumatic lesions, respectively.
Conclusion: As dermatological foot lesions are common in soccer players, regular evaluation of soccer players feet should be essential so
as to identify skin problem, to treat them early and effectively in order to avoid deficits in performance. (El Med J 2:2; 2014)
Keywords: Feet, Soccer, Skin, Nail, Trauma, Infections
118 A dermatological approach to the feet of soccer players
Vol 2, No 2
Aspergillus fumigatus (n: 4), respectively. The bacteria found in soccer
players feet were as follows: Corynebacterium species, coagulase-
negative Staphylococci, Staphylococcus aureus and Clostridium spe-
cies. Table 5 provides a summary of the results from this study.
Table 2: Distribution of skin and nail lesions seen during
physical examination
Physical examination Positive Negative
Bacterial
lesions
Skin
6 (15%) 34 (85%)
Nail
0 (0%) 40 (100%)
Viral
lesions
Skin
1 (2.5%) 39 (97.5%)
Nail
0 (0%) 40(100%)
Fungal
lesions
Skin
7 (17.5%) 33(82.5%)
Nail
2 (5%) 38 (95%)
Traumatic
lesions
Skin
26 (65%) 14 (35%)
Nail
22 (55%) 18 (45%)

Table 3: Traumatic nail changes and drying of feet after
washing

Drying feet after washing
Total
Yes No
Nail
changes
+ve 15 (37.5%) 3 (7.5%) 18 (45.0%)
-ve 11 (27.5%) 11 (27.5%) 22 (55.0%)
Total 26 (65.0%) 14 (35.0%) 40 (100.0%)

Table 4: Fungal and bacterial reproduction
Reproduction
in Laboratory
Pathogen Non-pathogen Negative
reproduction
Bacterial 3 (7.5%) 23 (57.5%) 14 (35.0%)
Fungal 13 (32.5%) 4 (10.0%) 23 (57.5%)

Table 5: Foot problems
Complaint Frequency Percentage
Foot odor 19 47.5
Itching 7 17.5
Blisters 8 20.0
Callosities 13 32.5
Nail problems 8 20.0

Discussion
Foot health is a very important issue for soccer players. Skin of the
foot has as much importance as the other tissues of foot, partly due
to the barrier function to environmental threats. Dermatological
problems localized to the feet and nails may decrease the perfor-
mance of player posing problems such as itching, pain or secondary
infections.
Our results showed that there were at least two dermatological prob-
lems in the feet of a soccer player. Foot odor was the most common
problem in soccer players. Foot odor can be related with the bacte-
rial microflora of the foot. It is a well-known fact that staphylococci
and aerobic coryneform bacteria are associated with foot odor [4, 5].
In our group, most of the players had pitted keratolysis as a reason
of odor, and isolated microorganisms were a mixed sera of bacteria.
Corynebacteria and Staphylococcus spp. were suspected as patho-
genic and compatible with clinical findings, whereas Aspergillus was
accepted as a contamination. Pitted keratolysis is a bacterial infec-
tion of soles with typical rotten odor, plantar pits on the hyperkera-
totic areas and occasional maceration [5, 6]. Bacterial infections in
feet not only create lesions on the colonization site, but may also
cause other infections such as cellulitis, erysipelas or even toxic shock
syndrome [7]. In this study, we did not find any severe infections;
however, it should be kept in mind that development of frictional
blisters in addition to prolonged occlusion of feet may create an en-
tering site for other bacteria as well.
Fungal infections of both skin and nail were found in 22.5% clinically
and 32.5% after laboratory examination. Our results are similar to the
report of Purim et al, who report the rate of feet dermatophytosis as
21.74% in soccer players [3]. However, Ergn et al reported 12.4%
fungal infections in the feet of soccer players, with a 9.5% predomi-
nance of nail localization [8]. In our study, fungal infections of the
skin were more common than those of the nails. This might be re-
lated with the hot and humid climate of our island, where excess
sweating in daily life is inevitable. Also, we found Epidermopyhton
floccosum as the most common fungi in nail lesions even though
Trichophyton rubrum is generally accepted as the most common der-
matophyte involved in onychomycosis [9]. However, in the study of
Ergun et al, Candida species was reported as most common causa-
tive agent of the onychomycosis in soccer players [8]. Viral infections,
especially human papilloma virus (HPV) may also be seen in the
sportsmen. For soccer players, plantar verrucas may cause pain in the
feet. Also, they can be misdiagnosed or underestimated by creating
a clinical image of corn or callus [8]. In our study, we observed plan-
tar verruca in only 2.5% of the study population.
Traumatic blisters and callosities were also common. Blisters are
caused by constant friction between the skin and the shoes or socks.
Several factors can influence the production of these blisters. Moist
epidermis is more vulnerable to form blisters after it is exposed to
shearing forces. Very dry skin or very wet skin decreases the frictional
forces. Increased temperatures also are related to increasing proba-
bility of blister production [2, 10]. Hence, the high frequency of blis-
ters in our group might be due to the hot and humid climate of the
island, in addition to frictional forces acting on the feet.
Other dermatoses can be also seen in soccer players. Irritant contact
dermatitis, allergic contact dermatitis especially due to shoe insoles
or exacerbation of a skin disease with friction (e.g. Koebner phenom-
enon in psoriasis or vitiligo) are possible [2, 10, 11]. In our study,
however, we did not observe any kind of dermatitis or rash related
with a dermatological disease.
Kaptanolu AF, Yavuz HU, Ser K 119
Sports have been reported to have a high correlation of nail pathol-
ogies [12]. Moreover, nail changes in soccer players are often re-
ported to be common [2]. In our study, nail changes were observed
in 60% of the subjects, with a dominance of traumatic lesions. Our
results are not comparable with other studies as we were unable to
find significant epidemiologic data about the nail pathologies of
football players in the recent literature. Ergn et al reported the types
of onychomycosis in soccer players as subungual hyperkeratosis
(14.6%), yellow discoloration (10.2%), onycholyis (8%) and black dis-
coloration (3.6%), respectively [8]. In our study, the occurrence of
other nail lesions due to trauma was found to be as high as 55%.
Acute blunt forces resulting from kicking the ball or hitting the
ground, may also effect the nail. Subungual hematomas which lead
to black discoloration of the nail, should be followed more carefully
as the differential diagnosis includes malignant melanoma [2, 12].
Occasionally, strong forces may remove the nail entirely. Traumas of
nails also predispose to fungal infections and secondary bacterial in-
fections resulting with paronychia [8]. In our study, the only excep-
tional finding was a significant relation in the increased number of
traumatic nail changes in the subjects who did not dry their feet after
washing. This finding may suggest that moistened nails are more
vulnerable to trauma. These lesions in feet of a soccer player are im-
portant as they can cause down-time, performance deficits and may
cause pain.
Conclusion
In conclusion, regular dermatologic evaluation and recording the
soccer players' feet heath is essential. Coach and team physicians
must be aware of foot lesions, easily identify the problem, treat the
affected feet early and take the necessary precautions in order to
avoid decreases in performance. Also, educative activities emphasiz-
ing foot healthcare should be organized by the team coaches.
References
1. Purim KS, de Freitas CF, Leite N: Feet dermatophytosis in soccer players. An
Bras Dermatol. 2009. 284(5):550-2.
2. Adams BB: Dermatologic disorders of the athlete. Sports Med 2002,
232(5):309-21.
3. Derya A, Llgen E, Metin E: Characteristics of sports-related dermatoses for
different types of sports: a cross-sectional study. J Dermatol 2005, 32(8):620-
5.
4. Marshall J. Holland KT, Gribbon EM: A comparative study of the cutaneous
microflora of normal feet with low and high levels of odour. J Appl
Bacteriol.1988, 65:61-8.
5. Millet CR, Halpern AV, Reboli AC, Heymann WR: Bacterial Diseases. In:
Dermatology. Editors: Bolognia JL, Jorizzo JL,Schaffer JW. 3rd edition. 2012.
USA. Elsevier Saunders. 1201-3.
6. Kaptanogu AF, Yuksel O, Ozyurt S: Plantar pitted keratolysis: a study from non-
risk groups. Dermatology Reports 2012, 4(1):e4.
7. Taylor CM, Riordan FAI, Graham C: New football boots and toxic shock
syndrome. BMJ. 2006, 332(7554): 13761378.
8. Ergun M, Ertam I: Futbolcularda yzeyel mantar infeksiyonu sklnn
aratrlmas. TURKDERM. 2001, 35 (4): 312-314.
9. Elewski BE, Hughey LC, Sobea JO, Hay R. Fungal diseases. In: Dermatology.
Editors: Bolognia JL, Jorizzo JL, Schaffer JW. 3rd edition. 2012. USA. Elsevier
Saunders. 1251-1284.
10. Metelitsa A, Barankin B, Lin AN: Diagnosis of sports-related dermatoses. Int J
Dermatol 2004, 43:113-119.
11. Weiss G, Shemer A, Trau H: The Koebner phenomenon: review of the literature.
J Eur Acad Dermatol Venereol 2002, 16(3):241-8.
12. Rzonca EC, Lupo PJ: Pedal nail pathology: biomechanical implications. Clin
Podiatr Med Surg 1989, 6(2):327-37.
13. Zara I, Trojjet S, Mokni M, El Euch D, Laabidi H, Mezlini S, Ben Osman Dhahri
AB: Dermatologic disorders of the athlete: a report of 30 cases. Tunis Med.
2008, 86(10):865-8.

120 Pattern of deliveries in rural India

Vol 2, No 2
Pattern of deliveries during three calendar years in rural India
Rajiv Mahendru
1
, Sunita Siwach
1
, Richa Kansal
1
, Vijayata Sangwan
1
, Ritika Kaur
2
, Pooja Gupta
2

Introduction
It is a well-known fact that ovulation in humans occurs in a cyclical
fashion and a fertile woman has the potential of conception follow-
ing unprotected coitus at the time of ovulation. Humans have the
potential of year round reproduction in a cyclical pattern with stud-
ies reporting seasonal effect, though with conflicting views, on con-
ception even on the rate of ectopic pregnancy or spontaneous abor-
tion [1-4]. Seasonal variation of various obstetric events has been
reported by certain studies, as also the incidence of pre-eclampsia
and eclampsia, although the seasonal variation noticed in direct ob-
stetric mortality has not been found to be statistically significant [1,
5-7, 10, 11].
However, many of these studies had no reference to seasonal varia-
tion in the delivery rate, as a possible variable that may have resulted
in an apparent seasonal difference in the incidence of these condi-
tions. Considering the dearth of information on the seasonal distri-
bution of deliveries, it becomes necessary at this point in time, to
document such a variation, if any, and thus provide some evidence
on its existence or otherwise.
Findings
The delivery data included in the study for the past three calendar
years, 2009-2011, was collected from birth registration center in one
of the rural zones of Ambala District of Haryana Province (India). This
included both vaginal (spontaneous as well as instrumental) and
caesareans deliveries occurring after viable period of 28 weeks ges-
tation conducted in (i) a teaching Institute; (ii) government setup
hospital; and (iii) domiciliary environment of that particular area.
Conceptions resulting in spontaneous abortions, ectopic gestations,
and gestational trophoblastic diseases were excluded. This review
was approved by the departmental ethical committee.
A three-year record of deliveries at these centers was collected. The
extracted data was then fed into a computer and an analysis was

1
BPS Government Medical College, India
2
Maharishi Markandeshwar Institute of Medical Sciences and Research, India
Correspondence: Rajiv Mahendru
Email: dr.rmahendru@gmail.com
performed, based on the grouping of the delivery rates per month.
The monthly delivery rates were standardized to a uniform 30-day
month, to eliminate the disparity that may result from the differences
in the number of days in the various months of the year, and the
analysis was repeated. The SPSS version 16 statistical software was
used to analyze the results.
There were 43,191 total deliveries during the period of study. The
monthly distribution of total deliveries showed a slightly sinusoidal
pattern, the peak spanning during the months of August, September
and October in all of the three calendar years (figure 1). On the con-
trary, nadir was noticed twice: during the months of January, and
thereafter, in April and May of those years.

Figure 1: Month-wise delivery distribution
The percentage distribution of monthly deliveries is shown in (Table
1). It shows that more than 30% of the annual deliveries occur in the
months of August to October (monthly average = 10%) as against
16.80% during the months of January and April-May (monthly aver-
age = 5.60%). In addition, it shows the significance level of the

Abstract
Background: Seasonal variation of various obstetric events has been reported by certain studies. The objective of this study was to
understand the delivery pattern during the calendar months in rural India.
Findings: The study was carried in a retrospective manner by collecting month-wise delivery data for three calendar years from registration
center in a rural area of Ambala District of Haryana Province (India). This included deliveries occurring after viable period of 28 weeks
gestation conducted in (i) a teaching Institute; (ii) government set-up hospital; and (iii) domiciliary environment of that particular area.
During the study period, 43,191 deliveries were recorded with an average monthly rate of 1,199 births. Sinusoidal pattern was observed in
the monthly distribution of deliveries peaking during August-October and decline in January and April-May. There was a statistically
significant difference between the highest and lowest rates.
Conclusion: Results of this study may be beneficial in health system planning and in the interpretations of seasonal variations in other
reproductive parameters. (El Med J 2:2; 2014)
Keywords: Birth Data, Seasonal Variation, Rural Births, India
Mahendru R, Siwach S, Kansal R et al 121
monthly average deliveries compared to the computed overall aver-
age monthly delivery rate of 8.85 percent. The months of August,
September and October are shown to have significantly higher
monthly delivery rates and the months of January and April-May
have significantly lower average monthly delivery rates compared to
the computed monthly average at the 99% confidence interval.
Table 1: Month-wise distribution of deliveries in percentage
Months 2009 2010 2011 Significance*
January 5.58 5.68 5.84 Significant
February 8.61 8.83 8.74 N.S.
March 8.99 8.77 8.85 N.S.
April 5.09 5.65 5.37 Significant
May 6.05 5.52 5.56 Significant
June 8.94 8.99 9.08 N.S.
July 9.10 8.85 8.99 N.S.
August 9.86 9.95 10.04 Significant
September 10.23 10.42 9.88 Significant
October 10.07 9.82 9.82 Significant
November 8.67 9.01 8.91 N.S.
December 8.81 8.51 8.92 N.S.
*Statistical Significance: N.S. = Not Significant
Discussion
These findings relate favorably with other reports not only from India
but also from different parts of the world, which have reported a
significant variation in the monthly delivery rates. The findings of the
present study is consistent with the reports by Yadava et al, who
stated the maximum indices of deliveries in the months of August to
October and lowest from April to June in India. They extrapolated
this to indicate the maximum conception rates in the winter season
[12]. Studies from Norway and Australia have reported seasonal var-
iations in birth rates [5, 6]. The recent National vital statistics report
of the United States, reports that births peak historically in August
and decline in February [13]. Another study has reported a signifi-
cant bimodal seasonal trend in the estimated monthly number of
conceptions [4].
Different reasons have been adduced for the seasonal variations ob-
served in these studies. Seasonal variation in coital activity among
couples is the possible factor influencing the seasonal variation in
conception and delivery. Increase in cohabitation is expected in the
cold seasons as in the months of December, January. Seasonal vari-
ations in sperm quality and also in ovarian activity have been pro-
posed [14].
The effect of photoperiod, melatonin, and temperature are other
mechanisms that have also been proposed [15]. Despite these, dif-
ferent geographical locations will experience different months of
peak and nadir in the number of deliveries, as a result of differences
in altitudes and climatic conditions, as seen in the current study com-
pared with others from different climatic regions [2, 5, 6, 13, 16]. Sea-
sonal variation in the pattern of deliveries as observed in the study
under consideration is also corroborated by the latest research in Ni-
geria [17].
Conclusion
The knowledge gained of the seasonal variation in conception and
delivery will be important in various aspects of health system plan-
ning, especially those concerning reproductive health issues. In
batching for assisted reproduction, the findings could assist in pre-
dicting periods of improved successful outcome. It will also assist in
the better understanding of these reproductive issues and the fac-
tors that affect their occurrence. Knowledge of seasonal variation in
the delivery rates may also be a necessary denominator in validating
the apparent seasonal variations in various reproductive health sta-
tistics and when planning for a more efficient service delivery.
References
1. Phillips JK, Bernstein IM, Mongeon JA, Badger GJ: Seasonal variation in
preeclamsia based on timing of conception. Obstet Gynecol 2004, 104:1015-
20.
2. Eskandar M, Archibong E, Sadek A, Sobande A: Ectopic pregnancy and
seasonal variation: A retrospective study from the south western region of
Saudi Arabia. Bahrain Med Bull 2002, 24:63-5.
3. Goldenberg M, Bider D, Seidman DS, Lipitz S, Mashiach S, Oelsner G: Seasonal
pattern in tubal pregnancy. Gynecol Obstet Invest 1993, 35:149-51.
4. Warren CW, Gld J, Tyler CW, Smith JC, Allen L: Seasonal variation in
spontaneous abortions. Am J Public Health 1980, 70:1297-9.
5. Odegard W: Season of birth in the population of Norway, with particular
reference to the September birth maximum. Br J Psychiatry 1977, 131:339-44.
6. Mathers CD, Harris RS: Seasonal distribution of births in Australia. Int J
Epidemiol 1983, 12:326-31.
7. Subramaniam V: Seasonal variation in the incidence of preeclampsia and
eclampsia in tropical climatic conditions. BMC Womens Health 2007, 7:18
8. Makhseed M, Musini MV, Ahmed MA, Monem RA: Influence of seasonal
variation on pregnancy-induced hypertension and/or preeclampsia. Aust N Z
J Obstet Gynaecol 1999, 39:196.
9. Tam WH, Sahota DS, Lau TK, Li CY, Fung TY: Seasonal variation in pre-eclamptic
rate and its association with the ambient temperature and humidity in early
pregnancy. Gynecol Obstet Invest 2008, 66:22-6.
10. Okafor UV, Efetie RE, Ekumankama O: Eclampsia and seasonal variation in the
tropics-a study in Nigeria. Pan Afr Med J 2009, 2:7.
11. Etard JF, Kodio B, Ronsmans C: Seasonal variation in direct obstetric mortality
in rural Senegal: Role of malaria? Am J Trop Med Hyg 2003, 68:503-4.
12. Yadava KN, Dube D, Marwah SM: A study of seasonal trends in delivery and
medical termination of pregnancy. J Obst Gynaecol India 1979, 29:256-7.
13. Martin JA, Hamilton BE, Ventura SJ, Menacker F, Park MM. Births: Final data for
2000. National vital statistics report. Hyattsville, Maryland: National center for
health statistics 2002, 50:8,45.
14. Levine RJ, Bordson BL, Matthew RM, Brown MH, Stanley JM, Star TB:
Deterioration of semen quality during summer in New Orlaens. Fertil Steril
1988, 49:900-7.
15. Cagnacci A, Volpe A: Inluence of melatonin and photoperiod on animal and
human reproduction. J Endocrinol Invest 1996, 19:382-411.
16. Paonnenberg T, Aschoff J: Annual rhythms of human reproduction and
environmental correlations. J Biol Rhythms 1990, 5:217-39.
17. Enabudoso EJ, Okpighe AC, Gharoro EP, Okpere EE: Delivery rate in Benin City,
Nigeria: Are there seasonal variations?. Niger J Clin Pract 2011, 14:129-31.

122 MRI evaluation of normal common bile duct size

Vol 2, No 2
Magnetic resonance imaging cholangiographic evaluation of normal common bile duct size
Mustafa Fatih Erko
1
, Sevil Alkan
2
, Sinan Soylu
3
, Aylin Okur
4

Introduction
Magnetic resonance imaging (MRI) cholangiography has an im-
portant role in the diagnosis of biliary disorders with new develop-
ments in MRI technique. Most biliary diseases presents as common
bile duct (CBD) dilatation. After the progress in CBD dilatation, as a
result intrahepatic and extrahepatic biliary dilatation occurs and
these dilatations can be assessed radiologically. On the other hand,
in cholecystectomized patients these dilatations can occur without
any pathology physiologically. At this point, the most important
question is what the limit of a normal CBD size and does it really
differ after cholecystectomy? If it dilates after cholecystectomy, what
is the upper limit of CBD pointing to any pathology? The size of nor-
mal CBD has been reported in many studies by different techniques
previously but to our knowledge the size of the common bile duct
has not been measured by MRI cholangiography.
Many studies have been conducted in the past to ascertain the di-
ameters of CBD. In an autopsy study, the diameters of the duct
ranged from 4 to 12 mm, with an average of 7.39 1.64 mm [1]. The
upper limit of normal for the CBD has been reported as 410 mm on
ultrasound [2-6]. On the other hand, post-cholecystectomy dilatation
of the CBD has been a disagreement in previous literature. In some
studies there was no significant change in CBD after cholecystec-
tomy, but some investigators reported significant dilatation [5-9].
The aim of this study was, therefore, to determinate the normal di-
ameter of common bile duct and to compare with post-cholecystec-
tomy patients by MRI cholangiography.
Findings
The mean diameter of the normal CBD was measured by MRI chol-
angiography in 73 patients without cholecystectomy and in 31 pa-
tients with cholecystectomy. The patients with any pancreatic and

1
Numune Hastanesi Radyoloji Blm 58040 Sivas, Turkey
2
Numune Hastanesi Enfeksiyon Hastalklar Blm 58040 Sivas, Turkey
3
Numune Hastanesi Genel Cerrrahi Blm 58040 Sivas, Turkey
4
Bozok niversitesi Tp Fakltesi Radyoloji Anabilimdal 66200 Yozgat, Turkey
biliary disorders were excluded. All measurements were performed
on MRI slices including the CBD, ampullary and periampullary area
as shown Figure 1. All MRI examinations were performed on a whole-
body 1.5 T unit (Magnetom Symphony, Siemens, Germany, 2007) by
using a 4-channel sensitivity-encoding (SENSE) abdominal coil. The
imaging parameters for T2W-TSE slices were 3810 ms/114 ms/1 (rep-
etition time [TR] / echo time [TE] / number of signal average [NSA]),
150 flip angle, 259 x 118 matrix, 800-cm field of view (FOV), 130 kHz
bandwidth, and 13 echo train length, slice thickness 5 mm, with an
acquisition time of 4 minutes 26 seconds. SPSS 15.0 software was
used to analyze the data.

Correspondence: Mustafa Fatih ERKO
Email: drerkoc@gmail.com
Abstract
Background: Regardless of the etiology of biliary diseases, intrahepatic or extrahepatic biliary dilatation is usually observed as a result. To
investigate the possible reasons, primarily, we have to determine the dilatation. At this point, the most important question is what is the
limit of a normal common bile duct (CBD) size and does it really differ after cholecystectomy? The size of normal CBD has been measured
and reported in previous studies by various imaging methods but to our knowledge, it has not been measured by MRI cholangiography
before. The aim of this study was, therefore, to determinate the normal diameter of CBD and to compare with post-cholecystectomy patients
by MRI cholangiography.
Findings: MRI data was acquired for a six month period retrospectively. A total of 104 patients who had undergone MRI cholangiography
due to any reason were divided into two groups: with cholecystectomy (31 patients) and without cholecystectomy (73 patients). The mean
diameter of normal CBD in those without and with cholecystectomy patients was 4.28 mm and 7.92 mm respectively. Mean of CBD
diameters of post-cholecystectomy patients was significantly greater than the control group (p<0.001).
Conclusion: In conclusion, the mean diameter of normal CBD shows a significant increase in cholecystectomized patients. (El Med J 2:2;
2014)
Keywords: MRI Cholangiography, Common Bile Duct
A
B
Figure 1: The locations of measurements on MRI slices:
(a) Patients without cholecystectomy; (b) Post-cholecystectomy patients
Erko MF, Alkan S, Soylu S et al 123
Results
The mean diameter of normal CBD in without and with cholecystec-
tomy patients was 4.28 1.62 mm and 7.922.45 mm, respectively.
Mean of CBD diameters of post-cholecystectomy patients was signif-
icantly greater than the control group (p<0.001).
Discussion
The size of normal CBD has been reported in many studies by differ-
ent techniques previously. Some authors reported normal CBD size
as 4-10 mm on ultrasound [2-6]. In an ultrasonographic study, Pa-
rulekar et al reported the mean diameter of CBD 4.1 mm [3]. In our
study we found the mean diameter of normal CBD 4.28 mm which
is similar to the results of Parulekar. There is a mismatch on asset of
dilatation of CBD in post-cholecystectomy patients in previous stud-
ies. Hunt et al. found a negligible increase in CBD in post-cholecys-
tectomy patients which was not statistically significant [10[. On the
other hand, Freng B. et al and other investigators found a significant
increase inversely [9-13]. Some investigators have found no signifi-
cant increase in prospective studies [5, 8, 14].
Kaim et al found a significant increase in CBD after cholecystectomy
(mean 8.7 mm) [6]. Our findings of the mean CBD of 7.92 mm (range
4.3-13.8 mm) in cholecystectomized patients is similar to their find-
ings.
Conclusion
In this study, we found the mean diameter of normal CBD 4.28 mm
and 7.92 mm in cholecystectomized patients. It is clearly shown that
there is a significant increase in CBD size on cholecystectomy pa-
tients. Further studies including endoscopic retrograde colesistopan-
creotography (ERCP) correlations are necessary to explain the etiol-
ogy of increase in CBD size after cholecystectomy.
References
1. Mahour GH, Wakim KG, Ferris DO: The common bile duct in man: its diameter
and circumference. Ann Surg 1967, 165(3): 4159.2
2. Cooperberg PL: High-resolution real-time ultrasound in the evaluation of the
normal and obstructed biliary tract. Radiology 1978, 129(2): 47780.
3. Parulekar SG. Ultrasound evaluation of common bile duct size. Radiology 1979,
133(3 Pt 1): 7037.
4. Graham MF, Cooperberg PL, Cohen MM, Burhenne HJ: The size of the normal
common hepatic duct following cholecystectomy: an ultrasonographic study.
Radiology 1980, 135(1): 1379.
5. Mueller PR, Ferrucci JT Jr, Simeone JF, et al: Postcholecystectomy bile duct
dilatation: myth or reality? AJR Am J Roentgenol 1981, 136(2): 3558.
6. Kaim A, Steinke K, Frank M, et al: Diameter of the common bile duct in the
elderly patient: measurement by ultrasound. Eur Radiol 1998, 8(8): 14135.
7. Perret RS, Sloop GD, Borne JA: Common bile duct measurements in an elderly
population. J Ultrasound Med 2000, 19(11): 72730.
8. Puri SK, Gupta P, Panigrahi P, Kumar N, Gupta S, Chaudhary A:
Ultrasonographic evaluation of common duct diameter in pre and post
cholecystectomy patients. Trop Gastroenterol 2001, 22(1): 234.
9. Feng B, Song Q. Does the common bile duct dilate after cholecystectomy?
Sonographic evaluation in 234 patients. AJR Am J Roentgenol 1995, 165(4):
85961.
10. Hunt DR, Scott AJ: Changes in bile duct diameter after cholecystectomy: a 5-
year prospective study. Gastroenterology 1989, 97(6): 14858.
11. Kaude JV: The width of the common bile duct in relation to age and Stone
disease: an ultrasonographic study. Eur J Radiol 1983, 3(2): 1157.
12. Wu CC, Ho YH, Chen CY: Effect of aging on common bile duct diameter: a
realtime ultrasonographic study. J Clin Ultrasound 1984, 12(8): 4738.
13. Qvist CF: The influence of cholecystectomy on the normal common bile duct.
Acta Chir Scand 1957, 113(1): 304.
14. Skalicky M, Dajcman D, Hojs R: Effect of cholecystectomy for gallstones on the
surface of the papilla of Vater and the diameter of the common bile duct. Eur
J Gastroenterol Hepatol 2002, 14(4): 399404.

124 Measurement of obstetric morbidity in India

Vol 2, No 2
Open Access Review
Do socioeconomic inequalities lead to deceptive measurement of obstetric morbidity in India?
Kshipra Jain
1
, Mayank Prakash
1

Introduction
In recent years the issue of socioeconomic inequalities in the health
sector has gained considerable attention. Various researches have
reflected upon the mechanism through which health gets affected
by socioeconomic characteristics. Although there is no direct rela-
tionship between the two, but certainly the understanding of
healthy living conditions, various morbidities, treatment seeking be-
havior etc gets influenced by the intermediary factors which include
individuals level of education, wealth status, surrounding conditions
etc, and these intermediary factors are unequally distributed in the
society with the poor section having lesser access to resources mak-
ing them more vulnerable [1]. The condition of women in develop-
ing countries does not reflect an encouraging situation. A few of the
indicators at world level reflect the poor and miserable condition of
women. For instance, vast majority of poor people are women; two-
thirds of the worlds illiterates are female; out of millions of school
age children not in school, majority of them are girls; HIV/AIDS is
rapidly becoming a womans disease [2].
Maternal mortality is increasingly becoming a cause of concern. Its
importance was highlighted as early as in 1978 during the Alma Ata
Declaration, which was carried forward to International Conference
on Population & Development (1994) and most recently Millennium
Development Goals (2000). The fifth Millennium Development Goal
(MDG) initially articulated one target: to reduce maternal mortality
ratio (MMR) by three quarters by 2015 [3]. More recently, a second
target to achieve universal reproductive health was added to the

1
International Institute for Population Sciences, India
Correspondence: Mayank Prakash
Email: mayankprakash12@gmail.com
fifth MDG. Despite the fact that 189 countries have signed the Mil-
lennium Declaration, a United Nations progress report from 2008
stated: Maternal mortality has remained unacceptably high across
much of the developing world, constituting the area of least progress
among all MDGs [3]. At the time of inception of these goals, MMR
in India was around 301 per 100,000 live births which has declined
substantially to 212 in the year 2009 [4]. Given the current pace of
MMR decline India seems to be far away from achieving this goal
within the stipulated time. This reflects the extent and magnitude of
the problem in India.
Maternal mortality is the end result of lack of proper care, diet, health
behavior etc required during pregnancy, childbirth and the postpar-
tum period that often make women suffer from various complica-
tions and morbidities. Obstetric morbidity is one such morbidity. Nu-
merous studies have highlighted major obstetric morbidities among
women from the time of conception, in terms of antepartum and
postpartum hemorrhage, sepsis, eclampsia, toxemia, bleeding, con-
vulsion puerperium etc. [5-8]. Pregnancy-related complications are
found to be the leading causes of not only maternal deaths, but also
disability among women. At the same time, poor infrastructure and
ineffective public health services are also responsible for low and in-
adequate obstetric care [5].
Apart from the environmental factors, individual level factors are also
responsible for lack of proper treatment of obstetric morbidities. A
woman feels discomfort and shy because of physical symptoms and

Abstract
Background: The extent of maternal mortality is an indicator of disparity and inequity in access to appropriate health care and nutrition
services throughout a lifetime, and particularly during pregnancy and childbirth. According to WHO, 99% of maternal deaths occur in
developing countries. Three quarters of maternal mortalities result from the direct obstetric complications. However, the ambiguity and
variability in existing literature on the magnitude of socio-economic inequality in obstetric morbidity is leading to complexity for priority
setting in health policy. The present study aims at assessing how far self-reported obstetric morbidity misleads measurement of socio-
economic inequalities in India and to assess whether refined inequality measures can help to overcome this problem.
Methods: The study has used data from the most recent round of National Family Health Survey (NFHS-3). We have used concentration
indices to access the socio-economic inequalities in self-reported obstetric morbidity and later implied regression based decomposition
analysis to find out the contributions of socio-economic predictor.
Results: Findings reveal that self-reporting obstetric morbidity survey data is potentially misleading in measuring socio-economic
differentials with simple bivariate analysis as greater number of obstetric complications persists in socio-economically advantaged
groups which may be not true in reality. However, application of sophisticated regression based decomposition analysis demonstrates
that, to some extent, we can overcome the problem of measuring socio-economic inequality, as decomposition analysis shows incidence
of obstetric complication are greater among disadvantaged groups.
Conclusion: Primarily, the inequalities arise from poverty, education and residing in rural areas. However, the contribution of education
to the incidence of obstetric morbidity is negative. This could be because of differences in the educational level of women that potentially
affects their awareness and understanding about obstetric complications. The findings thus suggest that education is a key determinant
of bias in self-reporting obstetric morbidity. Finally, we conclude that logical explanations of the results are needed when we infer these
results for health policy interventions, as decomposition analysis has also failed to overcome the problem of self-reporting morbidities
among illiterate women. (El Med J 2:2; 2014)
Keywords: Obstetric Morbidity, Socio-economic Inequalities, India
Jain K, Prakash M 125
social isolation, which often leads to concealment of the morbidity.
In some cases, they are being abandoned or divorced by their hus-
bands compelling them to live away from their family [9]. The situa-
tion worsens as these morbidities and complications become the re-
sult of synergistic effects of malnutrition, poverty, illiteracy, unhy-
gienic living conditions, infections and unregulated fertility [10]. This
means that women belonging to deprived socio-economic groups
are most likely to suffer from such complications [11]. However, this
fact cannot be reflected in the averages which usually form the basis
for policy formulation [12-14]. Ensuring good maternal health is the
basic right of a woman but lack of medical care and ignorance to-
wards her health has made pregnancy a risky event in developing
countries, particularly in India. Drawing on this premise, the present
study aims to addresses whether socioeconomic inequalities mislead
the measurement of self-reported obstetric morbidity in India.
What is obstetric morbidity?
Obstetric morbidity is one of the major components in the larger
domain of reproductive morbidity. Reproductive morbidity refers to
the morbidity or dysfunction of the reproductive tract, or any mor-
bidity which is a consequence of reproductive behavior including
pregnancy, abortion, childbirth or sexual behavior. Obstetric morbid-
ity, being a sub domain of reproductive morbidity, is defined as
morbidity in a woman who has been pregnant (regardless of site or
duration of the pregnancy) resulting from any cause related to or
aggravated by the pregnancy or its management, but not from acci-
dental or incidental causes [15].
The lifetime risk of maternal death is commonly used to measure the
obstetric risk in women [16]. With regards to the biomedical causes
of maternal deaths, more than 70 percent of maternal deaths are
from direct obstetric complications making it as one of the major
causes of maternal death. Although from the footsteps of the Cairo
conference, there has been some concern on the reproductive mor-
bidity, but very little attention has been given to obstetric morbidity,
in spite of it being one of the important causes of maternal deaths.
Information on obstetric morbidity could provide the evidence nec-
essary for planning safe motherhood outreach activities so as to im-
prove the maternal health. However, there is little reliable infor-
mation available for the same. There exist socioeconomic differen-
tials in the incidence and prevalence of obstetric morbidity in India
which also affects the reporting of morbidity.
Socioeconomic differentials in reporting of obstetric morbidity
There have been some studies that have tried to understand the dif-
ferentials in reporting of the morbid condition. Jain and Parasuraman
(2002) in their study based on NFHS-2 data found that Madhya Pra-
desh and Bihar had the highest percentage of obstetric morbidity in
the country [17]. A large proportion of women experienced different
types of obstetric complications in these states. The extent of obstet-
ric complications increased with an increase in the womans age and
birth order and decreased with an increase in standard of living and
education. This implies that the better off section of the population
has less reporting of obstetric morbidity. However, contrary to it, a
study by Sontakke et al (2009) based on NFHS-3 data, pointed that
a significant proportion of women in Kerala (considered a demo-
graphically developed state) suffered from pregnancy related prob-
lems but these figures were lowest in Andhra Pradesh, which is con-
sidered demographically weaker state than Kerala [11]. This study
demonstrates that socio-economic and demographic factors have a
significant positive effect on reporting of obstetric morbidity.
Another study based on District Level Household and Facility Survey
(DLHS-2) data revealed that women in the younger age group, bet-
ter-educated, urban women and those with a higher standard of liv-
ing had more obstetric morbidity in Odisha, a direct contrast to the
study of Jain and Parasuraman [18]. This study reflects positive asso-
ciation between obstetric morbidity and socioeconomic status. How-
ever a study conducted among women in slums of Mumbai revealed
that neither the womens educational levels nor the sanitary condi-
tions in which they live are related to gynecological morbidity, as
women with a high income were more likely to report their problems
as compared to their counterparts [19]. Similarly, working women
were more likely to report their problems than non-working women
[19].
In an influential editorial, Sen (2002) also compared aggregated self-
reported morbidity rates and life expectancy between Kerala and Bi-
har, with Kerala reporting considerably higher rates of morbidity de-
spite experiencing the highest level of longevity, while Bihar with
low levels of longevity reported lower rates of morbidities [20]. The
argument was that although Bihars low life expectancy figures re-
flected its disease burden, the meager provision of health facilities in
the state coupled with its high percentage of illiterate population,
perhaps, accounted for its poor perception of illness. Conversely, Ker-
ala, with high levels of literacy and adequate health provision, is bet-
ter positioned to identify the perceived morbidities. Narayan (2000)
pointed out that womens perceptions regarding obstetric morbid-
ity, feeling of low self-esteem, embarrassment, and guilt were some
social barriers to reporting and utilization of services for obstetric
care [21]. There are thus no clear cut findings that can reflect
whether there is a positive or negative association of socioeconomic
factors with reporting of obstetric morbidities. The discussion re-
flects the ambiguity and variability in existing literature on the mag-
nitude of socio-economic inequality in obstetric morbidity. This
makes priority setting in health policy for obstetric morbidity diffi-
cult.
In recent times, there has been growing concern about the mislead-
ing measurement of socio-economic differentials in self-reporting
obstetric morbidity [14, 22]. Majority of the poor and illiterate
women do not report morbidities and do not go to health facilities
as they fail to realize that they have a morbid condition. In most of
the cases, true magnitude of the socioeconomic inequality of the
problem is not reflected [22]. In few literatures, the socioeconomic
characteristics are positively associated with reporting of obstetric
morbidity and in some it is inversely related. Therefore, systematic
investigation of self-reported obstetric morbidity is crucial for im-
proving maternal health in India. On the other hand, cautioning the
misleading measurements of socioeconomic inequality is important
for a true assessment of the problem.
To our knowledge there has been scarce or virtually no studies that
have attempted to understand socioeconomic differentials in report-
ing of obstetric morbidity and the relative contribution of a womans
Vol 2, No 2
socioeconomic status in the reporting of her morbid condition. This
study is a first attempt to decompose the existing inequalities at all
India level using sophisticated regression based decomposition anal-
ysis in order to understand the factors that are contributing to such
inequalities. Therefore, the aim of present study is to explore the
pathways leading to socioeconomic inequality in self-reported ob-
stetric morbidity in India by using improved health inequality
measures.
Methods and Materials
The present study has used data from third wave of National Family
Health Survey-3 (NFHS-3) conducted in 2005-06 [23]. NFHS is con-
sidered equivalent to the Worldwide Demographic Health Survey
(DHS), which is the standardized survey of over 80 countries with
over 240 surveys in the world. The survey is coordinated by Interna-
tional Institute for Population Sciences (IIPS) and Macro International
under the tutelage of Ministry of Health and Family Welfare, India. It
has received ethical approval from IIPS, Mumbai. The study has cov-
ered 124,385 women in the age group 15-49 years in India. The ur-
ban and rural samples within each state were drawn separately fol-
lowing a multi-stage sampling design. In each state the rural sample
was selected in two stages and urban sample in three stages.
The present study focuses on currently married women having at
least one birth during five years preceding the survey. The women
file of NFHS3, which has information on specific problems that the
women experienced during their pregnancies, is used in this study.
For the most recent birth in the five years preceding the survey,
women were asked whether, at any time during the pregnancy, they
had experienced any of the following problems:
Difficulty with vision during the day light
Night blindness, convulsions (not from fever)
Swelling of legs, body or face
Excessive fatigue
Vaginal bleeding
Every woman who had given birth in the five years preceding the
survey was asked if she had the following symptoms of possible
postpartum complications at any time during the two months after
the birth of her most recent child:
Excessive vaginal bleeding
Very high fever
The above-mentioned information is used to compute the following
dependent variables:
1. Pregnancy related problems, based on the problems faced dur-
ing pregnancy
2. Post-pregnancy problems, based on the problems faced after
childbirth
3. Any obstetric morbidity, based on pregnancy-related and post-
pregnancy problems
The analysis of the study was performed in three stages. In the first
stage, obstetric morbidity was examined using average group differ-
entials by socio-economic and demographic characteristics. In the
second stage, we estimated concentration indices as measures of in-
equality. This was a leading-edge inequality measure to assess dis-
proportionate and unequal distribution of health among population
sub-groups with different socioeconomic status. Finally, the concen-
tration index of obstetric morbidity was decomposed to find the per-
centage contribution of different socioeconomic predictors to total
inequality. All the statistical analyses in this paper were carried out
by using STATA 10.1 (STATA crop LP, College Station, Texas, USA)
and Microsoft Excel program. The following steps as proposed by
Wagstaff (2002b) and Hosseipoor et al (2006) explain the computa-
tion of concentration indices and their decomposition to the predic-
tors [24, 25]:
The obstetric morbidity variable is regressed against its predic-
tors through an appropriate model for finding the coefficients of
the explanatory variable (k).
Mean of the obstetric morbidity variable and each of its predic-
tor is calculated ( and Xk).
Concentration indices for obstetric morbidity variable and pre-
dictors (C and Ck) as well as the generalized CI of error term (GC)
where, yi and are the values of the predictors for the i
th
individ-
ual, and the determinant mean respectively is calculated.
The absolute contribution of each predictor is calculated by mul-
tiplying the obstetric morbidity variable elasticity with respect to
that predictor and its CI - (kXk/)Ck.
The percentage contribution of each determinant is calculated
by dividing its absolute contribution by CI of obstetric morbidity
variable (kXk/) Ck/C to quantify precise contribution of each
predictor included in the model to measure inequality in the ob-
stetric morbidity variable.
The mathematical equations for computation of concentration index
and its decomposition are given in the following section.
Methodology for computing concentration index
The concentration index is a ground-breaking inequality measure to
assess disproportionate and unequal distribution of health among
population sub-groups with different socioeconomic status. It is
computed using the following equation:
2
cov ( , ) w i i C y R

In the above equation yi and Ri are, respectively, the health status of
the i
th
woman and the fractional rank of the i
th
woman (for weighted
data) in terms of the index of household economic status; is the
(weighted) mean of the health of the sample and covw denotes the
weighted covariance. The value of the concentration index varies be-
tween 1 and +1. Its negative values imply that a variable is concen-
trated among disadvantaged section of the society, while the oppo-
site is true for its positive values. When there is no inequality, the
concentration index will be zero.
Decomposition of Concentration Index
The method proposed by Wagstaff (2002b) and Hosseipoor et al
(2006) was further used to decompose socio-economic inequalities
in obstetric morbidity into its determinants [24, 25]. The analysis
showed that for any linear regression model, the health variable of
interest, Y, is linked to a set of k health determinants, Xk where is
an error term. The relationship between Yi and Xki is given in the fol-
lowing equation:
= o +X
k
[
k
X
k
+e

Given the relationship between Yi and Xki the concentration index for
Y, C, can be written as:
C = _
[
k
X
k
p
_C
k
k
+
0C
z
p
= C
+
0C
z
p

Where, is the mean of Y, Xk is the mean of Xk, Ck is the concentration
index for Xk. In the last term (which can be computed as a residual),
GC is the generalized concentration index for .
Definition of variables considered for decomposition analysis
A long-standing issue in the literature on health inequality is whether
or not all inequalities should be measured or solely those which
show some systematic association with indicators of socioeconomic
standing should be measured [26-29]. Keeping this in mind, the pre-
dictor variables are specifically chosen that can systematically ex-
plain a major part of inequalities. The decomposition analysis is con-
fined to five critical socioeconomic predictors: place of residence,
household economic status, womens educational status, religion
and caste. All the socioeconomic covariates are dichotomized as in-
dicated in the Table 1 below coded 1 and 0; one was assigned to
disadvantaged group. Place of residence is coded as rural/non-rural,
economic status as poor/non-poor, education of women as illit-
erate/literate, religion as Muslim/non-Muslim and caste as Scheduled
Caste/Tribe (SC/ST) or non-SC/non-ST.
Table 1: Variables
Health variable
(Yes=1, Otherwise=0)
Predictive variables
(Yes=1, Otherwise=0)
Obstetric Morbidity Place of Residence: Rural
Household economic status: Poor
Womens educational status: Illiterate
Religion: Muslim
Caste: Scheduled Caste/Scheduled Tribe
In this study, poor includes poorest and poorer population and non-
poor includes middle, richer and richest population. Illiterate women
include all currently married women with no education and literate
includes women with primary, secondary or higher education. Non-
Muslim includes Hindu and other religious groups and non-SC/ST in-
cludes other backward classes (OBCs) and other forward castes. Such
categorization is done as non-Muslim and non-SC/ST, in India, are
assumed to be socially better off than their counter parts.
Results and Discussion
A woman suffers from various problems during and post-pregnancy.
For analytical purpose, the pregnancy problems are separately
clubbed as pregnancy-related problems and post-delivery problems
as mentioned above. However, in order to understand the severity
of the problem it is important to assess the percentage of women
who are suffering from these problems separately. Table 2 presents
the percentage of currently married women by types of pregnancy
related problems. The results reveal that approximately 50 percent
of women suffers from excessive fatigue and one fourth of women
suffers from leg, body or face swelling. Less than five percent of
women report vaginal bleeding. With regards to post-delivery prob-
lems, there is not much difference in the percent of women report-
ing massive vaginal bleeding and very high fever (approximately 13
percent). Overall the percent of women reporting any type of ob-
stetric complications ranges from less than five percent to 50 per-
cent. There are various factors responsible for such fluctuations. The
physiology and understanding of morbid condition is different for
every individual and this affects the reporting pattern of morbidity.
In order to understand such differentials, bivariate and regression
based decomposition analysis has been carried out.
Table 2: Percentage of currently married women by types of
problems reported, India, 2005-06*
Obstetric Problems India
Pregnancy-related
Problems
Difficulty with daylight vision 6.3
Night blindness 8.9
Convulsions not from fever 10.3
Leg, body or face swelling 25.1
Excessive fatigue 47.8
Vaginal bleeding 4.4
Post-delivery
Problems
Massive vaginal bleeding 12.4
Very high fever 13.5
Number of women (weighted) 39.677
*Note: Among women who had a live birth in the five years preceding the survey,
percentage of women who experienced specific health problems during pregnancy for
the most recent live birth.
Table 3 presents the percentage of currently married women who
reported any pregnancy related complications, post-pregnancy com-
plications or obstetric morbidity across socioeconomic standing.
Overall, there is higher reporting of pregnancy related complications
as compared to post-pregnancy complications. Obstetric morbidity
captures both pregnancy-related complications as well as post preg-
nancy complications. More than 50 percent of women report preg-
nancy related complications and any obstetric morbidity, whereas
less than one fourth of women report any post-pregnancy complica-
tions.
More or less, there is an inverse relationship in the reporting of mor-
bidity and age of the women, number of children ever born and
wealth status. There is more reporting among women in the younger
age group and it decreases with the increase in the age of women.
Similarly there is higher reporting of morbidity among women who
have given birth to first child. However, with regard to education of
women there is a positive relationship among the reporting of any
pre-pregnancy complication or obstetric morbidity. With the in-
crease in the education level of women there is an increase in the
reporting of morbidity. Among religion, Muslim women suffer maxi-
mum from the pregnancy-related complications.
Overall, the results show a movement in the reverse direction from
the conventional pattern with regard to some of the demographic

Vol 2, No 2

factors. This, perhaps, is because sometimes womans own under-
standing of her health may not be in accordance with the appraisal
of medical experts and thus she fails to realize that she is suffering
from morbidity and does not report it. An educated woman has a
better understanding of her health conditions in comparison to an
illiterate woman, and this could be one reason of higher reporting
with the increase in the educational level of woman. Similarly,
women of higher parity may not report because she might have
learnt from her previous experiences. However, bivariate analysis is
not able to give justifications for such unpredictable and unexpected
results. There is a need to employ some sophisticated statistical
methods to explore the grounds for such results.
Regression based decomposition analysis has been carried out
among currently married women who report obstetric morbidity to
explore the pathways and mechanisms that lead to such inequalities.
62% women report obstetric complications in India. Furthermore,
47% of women are illiterate and 38% belong to poor household eco-
nomic status. Majority of women belong to rural areas (69%).
The model explains a major part of the inequalities in reporting of
obstetric morbidity. It shows that the inequalities are more among
the poor and depressed groups of society. Poverty makes the largest
contribution, however illiteracy seems to be contributing in a direc-
tion against conventional belief that obstetric complications are
more among illiterate than literate women. It empirically proves that
an educated woman has a better understanding and is more vigilant
towards her health condition. Rural areas also contribute signifi-
cantly to the total inequalities. The Muslims are showing a very neg-
ligible impact on the inequalities. Caste is not showing any contribu-
tion to the inequalities. This means that belonging to SC/ST group is
not affecting the reporting pattern of a morbid condition. The de-
composition outcomes demonstrated that most of the inequalities
are explained by the selected socioeconomic predictors, that is, place
of residence, wealth status, education, religion and caste. A very
small value of the unexplained part (residual i.e. -0.003) proved that
the selection of predictors was correct and it has explained maxi-
mum part of inequalities in reporting of obstetric morbidity by
women. (Table 4)
To summarize most predictable socioeconomic inequalities seem to
arise from three socioeconomic predictors: poverty, illiteracy of
women and belonging to rural areas at national level. However it is
to be noted that illiteracy is contributing in the direction opposite to
what is being contributed by other two predictors.
Table 3: Percentage of currently married women aged 15-49 by various obstetric complications India, 2005-06*
Background Characteristics Pregnancy-related Problems Post-pregnancy Problems Obstetric Morbidity
Age 15-19 60.20 23.30 65.60
20-34 57.20 20.80 61.80
35-49 57.70 20.70 62.40
Children Ever Born 1 59.80 20.90 64.40
2-3 55.50 19.70 60.00
More than 3 58.50 23.20 63.40
Residence Urban 55.90 15.70 59.70
Rural 58.00 22.90 63.00
Education No education 56.70 23.00 61.70
Primary 59.50 22.60 64.40
Secondary 57.40 18.70 61.80
Higher 58.90 14.00 62.30
Religion Hindu 56.10 20.50 60.80
Muslim 63.30 23.40 68.10
Others 58.80 20.90 62.50
Caste Scheduled caste 56.00 21.30 60.50
Scheduled tribe 57.40 21.70 62.40
Others 58.30 19.20 62.60
Wealth Index Poorest 59.00 25.80 63.80
Poorer 58.40 22.70 64.20
Middle 55.50 20.60 60.30
Richer 56.30 18.20 60.50
Richest 57.60 15.30 60.90
Total 57.40 21.00 62.10
*Note: 1. Among women who had a live birth in the five years preceding the survey, percentage of women who experienced specific health problems during pregnancy for the most recent
live birth.
2. The association is tested significance with Pearson Chi-square at p<0.001 level significance for all the categories.
3. Index of household economic group are computed based on NFHS-3 wealth index (IIPS & Macro Internationals 2006), which is based on 33 assets and housing characteristics, each house-
hold asset is assigned a weight (factor score) generated through principle component analysis, and the resulting assets scores are standardized in relation to normal distribution with mean
of zero and standard deviation of one. Then the sample is divided into quintiles.

Conclusion
India accounts for 130,000 maternal death among estimated 536,000
maternal death worldwide annually [30]. It accounts for 22% of the
global burden of maternal death [31]. This reflects the disappointing
progress made in the last 30 years in reducing maternal mortality in
India. Obstetric morbidity does have an effect on health of a woman,
but unlike other morbidities, it has a negative psychological effect as
well. It is very crucial to estimate the correct magnitude of the preg-
nancy-related and post-pregnancy complications.
This study examines how far socioeconomic factors affect the self-
reporting of obstetric morbidity. Results reveal that self-reporting
obstetric morbidity survey data is potentially misleading in the meas-
urement of socioeconomic differentials with simple bivariate analy-
sis, as findings show a large number of obstetric complications per-
sist among younger, literate and other caste women. According to
Sen (2002), an individuals assessment of their health is directly con-
tingent on their social experience, and this leads to less reporting of
illness among socially disadvantaged individuals as they fail to per-
ceive the presence of illness or health deficits [20]. Majority of the
poor and illiterate women do not report morbidities and do not go
to health facilities as they fail to realize and accept that they have a
morbid condition. However, the application of regression based ine-
quality decomposition analysis helps in overcoming the self-report-
ing bias up to a certain extent. Primarily, the inequalities arise from
poverty, education and residing in rural areas. However, the contri-
bution of education to the incidence of obstetric morbidity is nega-
tive. This could be because of differences in the awareness levels of
literate and illiterate women which affect the reporting of the mor-
bidity and hence, the results.
The findings, thus, suggest that education is a key determinant of
bias in self-reporting obstetric morbidity. Improvement of education
is important for two reasons: first, it improves knowledge about the
problem of obstetric morbidity, which helps in seeking timely health
care. Second, education helps in reporting the problem accurately,
which helps in measurement of the magnitude of problem. However,
poor economic status and rural place of residence are also major
causes of obstetric morbidity. Therefore, results provide crucial in-
sights for specific health interventions in terms of obstetric morbid-
ity.
Finally, we conclude that logical explanations of the results are
needed when we infer these results for health policy interventions,
as decomposition analysis has also failed to overcome the problem
of self-reporting morbidities among illiterate women. India needs to
improve health knowledge of women on pregnancy-related prob-
lems for accurate measurement of the problem and improve treat-
ment seeking behavior, as the consciousness of ill health is certainly
very acute which leads to deceptive measurement.
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Table 4: Effects and contribution of predictor variables based on decomposition analysis for currently married women for
obstetric morbidity in India, 2005-06*
Predictors Mean Marginal Effect CI Contribution to C % Contribution
Rural 0.6927 0.022 -0.2675 -0.0066 70.55
Poor 0.3859 0.033 -0.7207 -0.0148 158.84
Illiterate 0.4720 -0.045 -0.3553 0.0122 -130.61
Muslim 0.1320 0.039 -0.0136 -0.0001 1.21
SC/ST 0.2765 0.000 -0.2141 0.0000 0.00
Obstetric Morbidity 0.6211 -0.0059 -0.0093 100.00
Residual 0.0034
*Note: 1. Among women who had a live birth in the five years preceding the survey, percentage of women who experienced specific health problems during pregnancy for the most recent
live birth.
2. Marginal effect is statistically significant at p<0.001.
Vol 2, No 2
(EAG) and South Indian states. Journal of biosocial science 2013, 45(2):167-
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Baid M, Chaturvedi V, Jha J 131

Open Access Case Report
Bilateral mucoepidermoid carcinoma of parotid
Mayank Baid
1
, Vikram Chaturvedi
1
, Jayesh Jha
1

Introduction
Mucoepidermoid carcinoma (MEC) is the most common salivary
gland malignancy [1]. It develops commonly in the major salivary
glands, most often in the parotid (45-70%) [1]. The second most
common site of occurrence is the palate (18%). Bilateral MEC is very
rare [2]. We report here a case of synchronous bilateral mucoepider-
moid carcinoma of parotid in a 65 years old female.
Case Presentation
A 65 years old female presented at the outpatient department of our
surgical oncology unit with bilateral swelling in the parotid region
for 1 year with sudden increase in the size of left sided swelling for
3 months. Patient had no symptoms except mild pain on the left
side. She had no history of chest pain, backache, headache or loss of
consciousness.
On examination, patient had mild pallor. Pulse rate was 76 per mi-
nute, blood pressure 130/90, respiratory rate 18 per minute, temper-
ature within normal limits and normal spine. On local examination a
large 8cm x 6cm hard swelling was found over left parotid region
with necrosis of the superficial skin and multiple palpable lymph
nodes on the left side of neck. On the right side a 2cm x 2cm hard
swelling was found in the parotid region, but the lymph nodes were
within normal limit.
Investigations revealed hemoglobin level of 9.9g/dl and total white
blood cell count of 9,500. Fine needle aspiration cytology (FNAC) was
performed from the swelling on both the sides which showed bilat-
eral mucoepidermoid carcinoma of the parotid. Contrast enhanced
CT scan of the paranasal sinus revealed a large variegated mass at
left parotid area with parapharyngeal involvement, with bilateral cer-
vical lymphadenopathy and bulky right parotid (figure 1). MR angio-
gram of the major neck artery revealed no obvious abnormality, in-
ternal carotid on both sides were normal. But branches of left exter-
nal carotid artery and left internal jugular vein were compressed and
encased by the mass.

1
Medical College, Kolkata, India
Correspondence: Mayank Baid
Email: drmayankbaid@gmail.com

The patient, at presentation, had no facial nerve deformity or altera-
tion in the voice (figure 2A). She underwent left sided radical pa-
rotidectomy with modified radical neck dissection (figure 2B) fol-
lowed by cervical flap coverage on left side (figure 2C). Left external
carotid artery was ligated at origin. Facial nerve, hypoglossal nerve
and spinal accessory nerve of the left side were excised en block with
the specimen. On the right side, facial nerve sparing superficial pa-
rotidectomy was done. Histopathological report revealed a case of

Abstract
Background: Mucoepidermoid carcinoma (MEC) is the most common malignant neoplasm of parotid. Bilateral mucoepidermoid carcinoma
is extremely rare. We report here a case of synchronous bilateral mucoepidermoid carcinoma of parotid.
Case Presentation: A 65 years old female presented at the outpatient department of our surgical oncology unit with bilateral swelling in
the parotid region for 1 year with sudden increase in the size of left sided swelling for 3 months. She underwent left sided radical
parotidectomy with excision of left facial, hypoglossal and spinal accessory nerve en block with the tumor and modified neck dissection
followed by cervical flap coverage. On the right side, facial nerve preserving superficial parotidectomy was done. Histopathology revealed
it to be high grade MEC and patient received adjuvant radiotherapy. At 1 year follow-up, she had no evidence of recurrence or metastasis.
Conclusion: Bilateral mucoepidermoid carcinoma is a rare tumor of the parotid for which, surgery forms the mainstay of therapy, although
postoperative radiation is thought to be effective. Radiation is reserved for primary treatment of malignant tumors in patient who are poor
surgical candidates, or who do not want to undergo surgery. (El Med J 2:2; 2014)
Keywords: Mucoepidermoid Carcinoma, Parotid

Figure 1: Contrast enhanced CT scan of the paranasal sinus showing a large
variegated mass at left parotid area with parapharyngeal involvement, with
bilateral cervical lymphadenopathy and bulky right parotid.
132 Bilateral mucoepidermoid carcinoma of parotid
Vol 2, No 2
high grade bilateral mucoepidermoid carcinoma (figure 2D). Patient
received 60Gy adjuvant radiotherapy. One year after surgery on fol-
low-up, patient had no evidence of metastasis or recurrence on ul-
trasonography.

Discussion
Mucoepidermoid carcinoma is the most common salivary gland ma-
lignancy [1]. It develops most commonly in the major salivary glands,
most often the parotid about (45-70%) [1]. This tumor displays a uni-
form age distribution between the ages of 20 and 70 years, with a
slight peak in occurrence in the 5
th
decade. There is a 3:2 female
predilection [1].
Mucoepidermoid carcinomas are usually classified as low-grade or
high-grade tumors. However, some authors also include an interme-
diate-grade as well. Low-grade tumors have a higher proportion of
mucous cells to epidermoid cells. These lesions behave more like be-
nign neoplasms, but are still nevertheless capable of local invasion
and metastasis. High-grade mucoepidermoid carcinomas have a
higher proportion of epidermoid cells, and it may be difficult to dif-
ferentiate this entity from squamous cell carcinoma. High-grade tu-
mors are aggressive neoplasms with a high propensity for metasta-
sis. Most cases present as painless mass. Facial paralysis, nodal me-
tastasis and local tissue invasion may be indicative of aggressive dis-
ease.
Fine Needle Aspiration (FNA) is helpful in diagnosis, but highly de-
pends on skill of the cytopathologist [3]. Incisional biopsy should be
avoided to prevent tumor violation, tumor spillage and facial nerve
injury. Contrast enhanced computed tomography (CECT) scan and
magnetic resonance imaging (MRI) scan are helpful in detailing the
extent of disease.
Surgery is the mainstay of therapy [4]. Clinical stage is an important
prognostic factor for MEC [5]. Besides tumor grade, the other im-
portant variable affecting survival is adequacy of surgical excision
margins. Close or positive margin show poor prognosis [5, 6]. Pa-
tients with positive margins are more likely to have locoregional re-
currence regardless of tumor grade than those with negative resec-
tion margin. Neck dissection is reserved for apparent neck disease.
Low grade mucoepidermoid carcinoma may be treated with surgery
alone.
Mucoepidermoid carcinoma has been considered a radioresistant tu-
mor, although postoperative radiation is thought to be effective.
Postoperative radiotherapy for MEC patients with positive surgical
margin has been reported to decrease local failure [7]. Radical sur-
gery followed by postoperative radiotherapy for salivary gland ma-
lignancies has improved local control, but it is difficult to control pa-
rotid gland cancer by radiotherapy alone [8].
Radiation is reserved for primary treatment of malignant tumors in
patient who are poor surgical candidates, or who do not want to
undergo surgery as well as post-operative treatment of high grade
or recurrent disease. Doses to the primary tumor bed are in range of
50 to 70Gy. Garden et al reported updates on their experience using
postoperative radiotherapy in parotid malignancies, highlighting lo-
cal and regional control [9]. They concluded that when radiotherapy
is used, there were 9% local recurrence and 90% control rates at 10
years.
Currently, there is no prognostically useful regimen of chemotherapy
[10]. However, histologic high-grade MEC needs chemotherapy as
adjunctive treatment to prevent local recurrence or distant metasta-
sis, so the possibility of a new regimen of chemotherapy containing
molecular target agents should be considered.
Guzzo et al and Clode et al reported a 5-year overall survival rate of
approximately 60% for mucoepidermoid carcinoma of parotid [5].
The high 5-year survival rate observed is probably due to aggressive
surgical treatment and postoperative radiotherapy in majority of pa-
tients.
Conclusion
In conclusion, this report presents a case of 65 years old female with
histology-proven high grade bilateral mucoepidermoid carcinoma of
parotid, who underwent left sided radical parotidectomy and modi-
fied neck dissection and facial nerve preserving superficial parotidec-
tomy on right side. Bilateral mucoepidermoid carcinoma is a rare tu-
mor of the parotid for which, surgery forms the mainstay of therapy,
although postoperative radiation is thought to be effective. Radia-
tion is reserved for primary treatment of malignant tumors in patient
who are poor surgical candidates, or who do not want to undergo
surgery.
Acknowledgement: The authors thank the patient for providing consent and for
her cooperation.

Figure 2(A): Clinical photograph of the patient with bilateral parotid swelling;
Figure 2(B): Intraoperative photograph of the left side of the patient after radical
parotidectomy and neck dissection;
Figure 2(C): Photograph of the patient at the end of surgery viewed from the left
side;
Figure 2(D): Histopathological slide of the tumor.
Baid M, Chaturvedi V, Jha J 133
References
1. Goode RK, El-Naggar AK. Mucoepidermoid Carcinoma. WHO Organization
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GA, Katsilieris I, Patsouris E: Mucoepidermoid carcinoma of the salivary glands.
Review of the literature and clinicopathological analysis of 18 patients. Oral
oncology 2007, 43(2):130-136.
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103(12):2544-2550.
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Postoperative radiotherapy for malignant tumors of the submandibular gland.
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958.
10. Laurie SA, Licitra L: Systemic therapy in the palliative management of
advanced salivary gland cancers. Journal of clinical oncology : official journal
of the American Society of Clinical Oncology 2006, 24(17):2673-2678.

134 Bilateral granulomatous mastitis after local nandrolone injection

Vol 2, No 2
Bilateral granulomatous mastitis after local nandrolone injection
Sadaf Alipour
1
, Akram Seifollahi
1

Introduction
Granulomatous mastitis (GM) is a rare inflammatory benign breast
disease which was first defined by Kessler and Wolloch in 1972 [1-3].
Etiology of the disease is still unrecognized, although its association
with various disorders or body milieu changes have been described
and discussed in the literature. Hormonal imbalance has been re-
garded as one of the probable causes, but generally includes female
sex hormones [2]. To our knowledge, development of GM in relation
to testosterone or its derivatives has not still been reported. We in-
troduce a case of bilateral GM following intramammary nandrolone
injection.
Case Presentation
A thirty-two years old woman came to our breast clinic with the com-
plaint of bilateral breast lumps which she had noticed one month
earlier. She had regular menstrual cycles, the history of two full-term
normal pregnancies with 4 years of breastfeeding as well as an abor-
tion which had occurred 5 months ago. Her first pregnancy was at
the age of 18 and she had never used oral contraceptives. She was a
passive smoker since her marriage because of the positive smoking
history of her husband. About two months prior to her attendance
and with the purpose of breast enlargement, she had received injec-
tions of three vials of 25 milligram nandrolone decanoate on each
breast in a one week period. The injection had been done directly
into the breast tissues on the anterior aspect. After one month, she
had detected a lump in the left breast first, and then after one week,
another in the right breast. The masses had progressively enlarged.
On clinical examination, there were about 25mm, firm masses at 12
oclock of the right and 9 oclock of the left breast. Both had nodular
surfaces and a lobulated, irregular contour, and both were mobile.
Ultrasonography showed 24 and 21 mm masses with irregular bor-
ders in the right and left side, respectively. No other pathology was
detected. Because of the young age of the patient, only mediolatero-
oblique view mammography was asked. These revealed only irregu-
lar asymmetric densities on both sides (figure 1).
Core needle biopsy was undertaken, and more than 6 cylindrical
specimens were caught from each mass. The histologic examination

1
Tehran University of Medical Sciences, Iran
Correspondence: Sadaf Alipour
Email: sadafalipour@yahoo.com
revealed granulomas composed of epithlioid histiocytes; Langhans
giant cells accompanied by lymphocytes and plasma cells mainly
centered in the lobules (figure 2). The final result was in favor of
granulomatous mastitis.

Abstract
Background: Granulomatous mastitis is a rare inflammatory disease of the breast. Several theories have been postulated for the etiology of
the disease.
Methods: In the present article, a case of bilateral granulomatous mastitis after intramammary injections of nandrolone decanoate in a 32
years woman is introduced. The clinical and radiologic pictures are presented. Pathogenesis, presentation, diagnosis, imaging, histopathology
and treatment of granulomatous mastitis as available in the present literature are briefly discussed.
Conclusion: The present case is in favor of a positive association between granulomatous mastitis and the anabolic steroid. This new topic
warrants further studies. (El Med J 2:2; 2014)
Keywords: Granulomatous Mastitis, Breast Mass, Nandrolone, Anabolic Steroids

Figure 1: Mammography: no specific abnormality,
only irregular asymmetries

Figure 2: Histologic slide: central granulomas filled with
epitheloid histiocytes and Langhans giant cells
Alipour S, Seifollahi A 135
Treatment with prednisolone at a dose of 10mg daily was started.
One month later, the masses persisted but were much softer and had
decreased in size on clinical exam. The treatment is continuing while
the patient is being followed.
Discussion
The prevalence of bilateral granulomatous mastitis has not been de-
fined in previous studies. A study of 1106 cases of benign conditions
of the breast revealed the figure of 1.8% for GM [1], while in a study
in Turkey, it constituted 6.8% of their surgical benign specimens [3].
The disease usually occurs in childbearing ages in the 3
rd
and 4
th
dec-
ades of life, but a range of 11 to 83 years has been reported in the
literature [1, 3, 4]. Almost all affected women reported in different
series have had a history of pregnancy and lactation, recent in many
cases. The etiology is unknown; association of the disease with preg-
nancy, breastfeeding and oral contraceptives (OCP) has been men-
tioned [1, 3-6]. Imoto et al focus on an association of the disease with
hormonal changes. In their review, they insist on the time period be-
tween a few months to 8 years from delivery to disease presentation,
with recent exogenous estrogen suppression of lactation, and the
history of OCP use in one third of cases as well [2].
The disease is seen more frequently in Hispanics and Asians, alt-
hough occurrence in all races has been seen and ethnic disposition
has not been established [1, 3]. Many other factors have been pos-
tulated as causative, but never proven: local irritants, various infec-
tious agents (viral, mycotic and parasitic), other hormonal disorders
(increased serum prolactin), diabetes, smoking, alpha-1 antitrypsin
deficiency [1, 3, 6]. An immune-related pathophysiology due to ooz-
ing of secretions from milk ducts into lobules during pregnancy and
lactation has been widely considered [1-3]. Reports of association of
the disease with autoimmune disorders such as erythema nodosum,
sarcoidosis, Wegeners granulomatosis, giant cell arteritis, or poly-
artheritis nodosa strengthens the theory [1, 3, 6]. The disease has
also been seen after local trauma and consumption of anti-depres-
sant drugs, perhaps due to hyperprolactinemia, as well as other gran-
ulomatous disorders as granulomatous thyroiditis, prostatitis and or-
chitis [2, 6].
GM is usually unilateral in any breast region but the subareolar [1, 2].
Bilateral disease has seldom been seen, although a 25% bilateral oc-
currence has been reported [1, 3, 4]. It usually presents as a progres-
sive, ill-defined firm lump which can be painful or tender, and may
invade the underlying chest wall mass with galactorrhea, skin ulcers
and lumpy indurations [1, 3-5]. It ultimately shows an infectious clin-
ical picture with hyperemia and inflammation in the skin, abscesses
and chronic fistulae [1, 4, 7]. Axillary lymphadenopathy is not fre-
quent, but nipple retraction and skin involvement or peau dorange
can occur with disease progression [1, 3]. This presentation fre-
quently mimics breast malignancies and even cases of inadvertent
mastectomy because of misdiagnosis have been reported [1-4]. Its
differential diagnosis with breast cancer is important because it may
simulate it even in imaging and cytology [2, 3].
GM is a diagnosis of exclusion and definitive diagnosis usually in-
volves histopathological diagnosis by wide tissue biopsy or excision
[3]. The microscopic picture usually shows discrete non-caseating
granulomas with infiltration of histiocytes, polymorphonuclear and
lymphocytic leukocytes, plasma cells and multinucleated giant cells
of the foreign body and Langerhans type in lobular units. Micro-
abscesses and necrosis are also usually seen [1-4, 8].
There is controversy upon the best treatment strategy, although
non-operative plans should be considered first [1-4]. The use of an-
tibiotics has not been confirmed in studies, but is nevertheless rec-
ommended in some situations, and these should be one of the first
administered treatments in case of abscesses [1-5]. Simple observa-
tion and follow-up is one of the approaches in non-complicated
cases. A spontaneous recovery rate of 50% has been reported in 2 to
24 months [1].
Earlier literature advocates for treatment of GM by surgical excision
followed by corticosteroid therapy [2]. If operation has to take place,
then wide excision with negative margins would result in less recur-
rence and better results than more limited surgery. The consequence
of any type of surgery is frequently a deformed breast with fistula
tracts and unpleasant scars [1]. Appropriate care of the wounds and
local infiltrations are useful. Occasionally, radical surgery such as
mastectomy is unavoidable [4].
Including anti-inflammatory drugs, colchicines or methotrexate in
the treatment plan has produced positive results. [4, 5] Corticoster-
oids are actually the most commonly prescribed and most effective
recognized treatment. These are administered with regards to the
course of the disease: in patients in need of surgery, they can down-
size the lesion pre-operatively, or they may be used post-operatively
for resistant and complicated cases [3]. Continuation of steroid ther-
apy till complete resolution has also been mentioned [3, 5]. High
steroid doses are generally used and the appropriate length of ther-
apy may be from a few weeks to several months [1]. Recurrence fre-
quently occurs after steroid tapering (up to 50%) [4, 5]. Methotrex-
ate and azathioprine have proved helpful in some of these cases [1,
4]. Steroids or methotrexate may eliminate the need for surgery [6].
A large review has demonstrated complete resolution of the lesions
in 6 to 12 months with one or several of the above treatment plans
[1].
The appearance of GM in radiologic images is nonspecific and may
imitate breast cancer closely [3, 9]. In a review of 14 cases where all
the patients had unilateral granulomatous mastitis, half mimicked
malignancy in imaging assessment [10]. Ultrasonography is probably
superior to mammography for differentiating GM from cancer [3];
the former usually reveals hypoechoic mass lesions, nodular struc-
tures, inhomogeneous hypoechogenity with internal hypoechoic
tubular structures, multiple abscesses, peripheral hyperechoic le-
sions, heterogeneous hypo- and hyperechoic areas, parenchymal
distortion, or an edematous pattern involving nearly the entire
breast, while focal asymmetric densities without clear borders, ill-de-
fined spiculated lumps, and bilateral multiple ill-defined nodules are
seen in the latter [1, 3, 9, 11]. As expected, there is a short supply of
literature about MRI findings of GM. This modality has the ability to
estimate the extent of the lesion, as well as to demonstrate aspects
of its morphology and it may be helpful in follow-up of the disease
in the long term [1, 3]. In the MRI study of 9 patients affected by the
disease, the most common detected change was focal or diffuse
asymmetrical signal intensity alterations, hypo- and hyperintense in
136 Bilateral granulomatous mastitis after local nandrolone injection
Vol 2, No 2
T1W and T2W images, respectively. There was no significant mass
effect, but nodular changes were observed in the images. These
same lesions showed vascular pathology in color Doppler imaging
and as mass-like, ring-like, and nodular enhancements in dynamic
contrast-enhanced mammography [11]. Actually, imaging cannot
make a definitive diagnosis of GM by any presently existing modality.
The patient presented in this article was in childbearing age and her
pregnancy history matched the general features seen in GM. She had
even a recent abortion which could be a triggering factor for her
disease. However, she had also self-administered nandrolone in her
breasts. Nandrolone is one of the synthetic derivatives of testos-
terone which, as an anabolic substance, is widely and illegally used
for bodybuilding [12-14]. This and similar androgen derivatives have
predominantly positive effects on skeletal muscle with regards to
size and strength. The National Household Survey on Drug Abuse in
1991 showed that more than 1000000 people in the United States of
America used these compounds, with a lifetime use of 0.9% and 0.1%
for men and women, respectively [14]. Most of the users are non-
athletes and recreational bodybuilders whose intention for andro-
gen use is cosmetic improvement of their physiques [13, 14]. The
drugs are supplied by healthcare systems in more than 40% of cases
[13]. Use of supraphysiologic doses of the anabolic steroids have sev-
eral sequelae. Some of the most commonly seen are acne, sleepless-
ness, mood and sexual disturbance as well as gynecomastia in men
[14].
Conclusion
The time relationship between injections and GM presentation in the
present case is in favor of a positive association between GM and the
drug. It appears that this new topic warrants further warning and
studies because of the presently frequent illicit and self-administered
use of steroid anabolics for cosmetic purposes.
Received: 29 November 2013 Accepted: 23 January 2014
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1. Patel RA, Strickland P, Sankara IR, Pinkston G, Many W Jr, Rodriguez M:
Idiopathic granulomatous mastitis: case reports and review of literature. J Gen
Intern Med 2010, 25:270-273.
2. Imoto S, Kitaya T, Kodama T, Hasebe T, Mukai K. Idiopathic granulomatous
mastitis: case report and review of the literature. Jpn J Clin Oncol 1997, 27:274-
277.
3. Ocal K, Dag A, Turkmenoglu O, Kara T, Seyit H, Konca K: Granulomatous
mastitis: clinical, pathological features, and management. Breast J 2010,
16:176-182.
4. Garcia-Rodiguez JA, Pattullo A: Idiopathic granulomatous mastitis: a
mimicking disease in a pregnant woman: a case report. BMC Res Notes 2013,
6:95.
5. Diesing D, Axt-Fliedner R, Hornung D, Weiss JM, Diedrich K, Friedrich M:
Granulomatous mastitis. Arch Gynecol Obstet 2004, 269:233-236.
6. Bellavia M, Damiano G, Palumbo VD, Spinelli G, Tomasello G, Marrazzo A et al:
Granulomatous Mastitis during Chronic Antidepressant Therapy: Is It Possible
a Conservative Therapeutic Approach? J Breast Cancer 2012, 15:371-372.
7. Ozel L, Unal A, Unal E, Kara M, Erdogdu E, Krand Oet al: Granulomatous
mastitis: is it an autoimmune disease? Diagnostic and therapeutic dilemmas.
Surg Today 2012, 42:729-733.
8. Tse GM, Poon CS, Ramachandram K, Ma TK, Pang LM, Law BK et al: Cheung.
Granulomatous mastitis: a clinicopathological review of 26 cases. Pathology
2004, 36:254-257.
9. Yilmaz E, Lebe B, Usal C, Balci P: Mammographic and sonographic findings in
the diagnosis of idiopathic granulomatous mastitis. Eur Radiol 2001, 11:2236-
2240.
10. Ozturk, E, Akin M, Can MF, Ozerhan I, Kurt B, Yagci G et al: Idiopathic
granulomatous mastitis. Saudi Med J 2009, 30:45-49.
11. Ozturk M, Mavili E, Kahriman G, Akcan AC, Ozturk F: Granulomatous mastitis:
radiological findings. Acta Radiol 2007, 48:150-155.
12. Basaria S: Androgen abuse in athletes: detection and consequences. Journal
of Clinical Endocrinology & Metabolism 2010, 95:1533-1543.
13. Melnik BC: Androgen abuse in the community. Curr Opin Endocrinol Diabetes
Obes 2009, 16:218-223.
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Med Sci Sports Exerc 2006, 38:644-651.
Sahu L, Gandhi G, Agarwal et al 137

Unusual presentation of a case of fallopian tube carcinoma
Latika Sahu
1
, Gouri Gandhi
1
, Krishna Agarwal
1
, Sunita Dubey
1
, Preeti Yadav
1
, Richa Gupta
1

Introduction
Primary fallopian tube carcinoma (PFTC) is the least common of all
gynecological cancers, accounting for approximately 0.14%1.8% of
female genital malignancies [1-3]. PFTC is extremely rare, with a re-
ported incidence of 0.41 per 100,000 women [4]. The etiology of this
cancer is unknown. Hormonal, reproductive, and possibly genetic
factors thought to increase epithelial ovarian cancer (EOC) risk might
also increase PFTC risk. High parity has been reported to be protec-
tive, and a history of pregnancy and the use of oral contraceptives
decreases the PFTC risk significantly [2, 5]. No statistically significant
correlation has been found between PFTC and age, race, weight, ed-
ucation level, pelvic inflammatory disease, infertility, previous hyster-
ectomy, endometriosis, lactose intolerance or smoking [5].
Here we report a case of primary fallopian tube carcinoma which was
diagnosed post-operatively and was treated as advanced ovarian
malignancy preoperatively with neoadjuvant chemotherapy, fol-
lowed by interval debulking surgery. The patient developed a
chronic non-healing ulcer at the fine needle aspiration cytology
(FNAC) site which is very unusual and has been rarely reported in
literature.
Case Presentation
A 45 year old perimenopausal woman was admitted to our gynecol-
ogy ward with a lower abdominal lump and generalized abdominal
pain since one and a half month. Patient had no menstrual com-
plaints. On physical examination, ascites was found. On vaginal ex-
amination, cervix was normal and uterus was anteverted, normal
sized and mobile. A left sided firm to hard irregular adnexal mass of
9cm x 8cm size and nodularity in the pouch of Douglas was found.
Clinical diagnosis of stage-III/IV ovarian malignancy was made.
Ultrasonography of pelvis revealed lobulated solid cystic mass pre-
sent in bilateral adnexal region with increased vascularity. Left ad-
nexa was 10cm x 8cm in size, right adnexa 4cm x 3cm and uterus

1
Maulana Azad Medical College, India
Correspondence: Latika Sahu
Email: latikasahu@gmail.com
was normal in size. Bilateral ovaries were not separately seen. CT ab-
domen revealed large predominantly solid lobulated mass, 9.5cm x
8cm x 6.5cm size in bilateral adnexal region covering abdomen and
pelvis with mesenteric and omental deposits with left lung and spi-
nal process metastasis. CT scan chest showed left upper lung nodule
of size 1.9cm x 1.7cm and left-sided gross pleural effusion. No pelvic
lymphadenopathy was seen. Free fluid was present in the abdomen.
Paracentesis fluid cytology was negative for malignancy. Ultrasound
guided FNAC from the mass revealed malignant epithelial neoplasia.
Tumor marker CA125 was raised (562 U/ml), while AFP, -hCG, CEA
were within normal limits. Her hemogram and other biochemical pa-
rameters were within normal limits. Provisional diagnosis of ad-
vanced malignant ovarian neoplasm was made.
FNAC revealed malignant epithelial neoplasia. Patient received 4 cy-
cles of neo-adjuvant chemotherapy (paclitaxel and carboplatin every
3 weeks). Few days following FNAC while on chemotherapy, she de-
veloped a small boil at the site of needle insertion on the left lower
abdomen, which developed into an ulcer and gradually increased to
a size of 3cm x 2cm and became a non-healing ulcer (figure 1). As
she was already started on chemotherapy, the ulcer edge biopsy
showed chronic inflammatory granulation tissue with no evidence of
malignancy. After chemotherapy, her hemogram was within normal
limit. LFT, RFT, chest x-ray and pap smear were also normal. CA125
was 78 U/ml (reference range <35U/L). Ultrasonography of the ab-
domen revealed free fluid. Liver, gallbladder, spleen, pancreas and
kidneys were bilaterally normal. No lymphadenopathy was seen.
Laparotomy and total abdominal hysterectomy with bilateral sal-
pingo oophorectomy, infracolic omentectomy, bilateral pelvic lym-
phadenectomy and para-aortic lymph node dissection was done and
wide excision of the ulcer with skin and underneath tissue was per-
formed at the same setting with primary closure. Her post-operative
period was uneventful. Histopathology revealed poorly differenti-
ated fallopian tube carcinoma, and skin margins that were negative

Abstract
Background: Primary fallopian tube carcinoma (PFTC) accounts only for <1% of all female genital cancers. The diagnosis of PFTC is rarely
considered preoperatively and is usually first appreciated at the time of operation or by a pathologist.
Case Presentation: Here we are presenting a case report of 45 years old lady with fallopian tube carcinoma, who presented to us with
features of advanced ovarian malignancy. FNAC from tumor showed epithelial malignancy and she received neo-adjuvant chemotherapy
followed by staging laparotomy and interval debulking surgery. She developed a non-healing ulcer at FNAC site which was excised during
laparotomy. On laparotomy, 200cc ascitic fluid was drained. Uterus, right ovary and tube appeared normal. Left side tube showed growth
of 3cm x 2cm. Total abdominal hysterectomy, bilateral salpingo-oophorectomy and infracolic omentectomy was done. Histopathological
examination revealed left side poorly differentiated primary fallopian tube carcinoma. Uterus and right tube with ovary were free of tumor.
She was advised to get 3 cycle of chemotherapy postoperatively.
Conclusion: In our patient, FNAC gave a positive diagnosis of malignancy and we were able to operate her malignancy properly following
chemotherapy. (El Med J 2:2; 2014)
Keywords: Fallopian Tube Carcinoma, Non-healing Ulcer, Chemotherapy
138 Unusual presentation of a case of fallopian tube carcinoma
Vol 2, No 2
for malignancy. Peritoneal washings showed no malignant cells.
Gross examination of specimen showed 3cm x 2cm solid growth
seen at fimbrial end of left fallopian tube. A normal looking left ovary
was seen separately. Uterus, right ovary and right fallopian tube were
normal looking (figure 2a) and resected skin ulcer has a 2cm margin
(figure 2b).

Cut section revealed areas of necrosis. Histological sections of the
solid area showed widespread areas of necrosis with dense chronic
inflammatory background and scattered foamy histiocytes (arrows)
with necrosis in background (figure 3). Occasional atypical cell left
after chemotherapy (figure 4) suggestive of fallopian tube carcinoma
were also seen. Omentum and lymph node sections showed no tu-
mor deposits. Patient received 2 cycles of adjuvant chemotherapy.
CA125 was 12 U/L after the chemotherapy. Skin ulcer site also healed
well. Ultrasound of abdomen and pelvis at 3/6/12 months was nor-
mal. Patient is doing fine at present without any recurrence.
Discussion
The true incidence of PFTC has been underestimated because PFTC
may have been mistakenly identified as ovarian tumors during initial
surgery and/or during microscopic examination by a pathologist, as
the histological appearance of these tumors are identical [6]. PFTC
most frequently occurs between the fourth and sixth decades of life,
with a median age of occurrence of 55 years (range: 1788 years)
[1].
Patients with PFTC appear to have a shorter history of symptoms
than those with epithelial ovarian cancers (EOC). These symptoms
are not specific. Latzkos triad of symptoms, consisting of intermit-
tent profuse serosanguinous vaginal discharge, colicky pain relieved
by discharge and abdominal or pelvic mass has been reported in
15% of cases [7]. Hydrops tubae profluens, a pathognomonic feature,
implies intermittent discharge of clear or blood-tinged fluid sponta-
neously or on pressure followed by shrinkage of an adnexal mass
and occurs in 5% of patients. PFTC is rarely asymptomatic, in contrast
to EOC [1]. However, a diagnosis of PFTC may be suspected in cases
of postmenopausal bleeding or spotting with negative diagnostic
curettage.

PFTC has been described in high-risk breastovarian cancer families
with germ-line BRCA-1 and BRCA-2 mutations [8, 9]. Some studies
suggest that the frequency and structure of the chromosomal
changes (BRCA-1 or BRCA-2 mutations) observed in PFTC have simi-
larities with those found in breast, serous ovarian, and uterine carci-
nomas, and consequently, a common molecular pathogenesis has
been claimed [10]. In patients with PFTC, a full history of malignancy
within the family should be obtained and genetic counseling of the
patient and relatives considered. Molecular biology studies have
shown that PFTC is characterized by an extremely unstable pheno-
type with highly scattered DNA ploidy patterns and frequent p53
gene alterations. Mutations of BRCA-1 or BRCA-2 genes and also al-
terations in p53 have also been shown a worse outcome in PFTC [11].
CA125 is a useful tumor marker for the diagnosis, assessment of re-
sponse to treatment, and detection of tumor recurrence during fol-
low-up. Although CA125 per se is not diagnostic for PFTC, >80% of

Figure 1: Non-healing ulcer on the left lower abdomen at FNAC site.

Figure 2(a): Gross specimen, posterior aspect showing 3cm x 2cm mass in left
fallopian tubes fimbrial end. Both ovaries normal.

Figure 2(b): Resected ulcer.
Figure 3: Scattered foamy histiocytes with necrosis in background (arrow).
(H&E X 400).
Figure 4: Occasional atypical cell left after chemotherapy (arrow). (H&E X 400)
Sahu L, Gandhi G, Agarwal et al 139
patients have elevated pre-treatment serum CA125 levels detected
more frequently in advanced or recurrent disease. It is an early and
sensitive marker for tumor progression during follow-up. It has been
reported that the lead time (elevated serum CA-25 levels prior to
clinical/radiological diagnosis of recurrence) is 3 months (range: 0.5-
7 months) [7].
Pap smear positivity occurs in 10%36% of cases. Pap smear shows
abnormal, suspicious, or poorly differentiated cells or glands alter-
nating with negative smear [7]. The discrepancy between an abnor-
mal Pap smear and negative findings on colposcopy, cervical biopsy,
and endometrial curettage should be considered suspicious for
PFTC. Studies have found a fivefold higher bilateral occurrence in
infertile patients than in fertile patients, and have reported a better
prognosis in nulliparous women [1].
Because it is difficult to differentiate PFTC from EOC, patients with at
least one of the following criteria should have the diagnosis of PFTC:
(a) The main tumor is in the tube and arises from the endosalpinx;
(b) Histologically, the pattern reproduces the epithelium of the mu-
cosa and often shows a papillary pattern; (c) If the wall is involved,
the transition between benign and malignant epithelium should be
demonstrable; and (d) The ovaries and endometrium are either nor-
mal or contain less tumor than the tube. In our case, the histopatho-
logical picture after chemotherapy showed occasional atypical cells
with histiocytes in the background of widespread necrosis.
Tubal carcinoma spreads in much the same manner as EOC, princi-
pally by the transcelomic exfoliation of cells that implant throughout
the peritoneal cavity (80% of patients with advanced disease). Tumor
spread can also occur by means of contiguous invasion, transluminal
migration, and hematogenous dissemination. Bilateral tubal involve-
ment has been reported in 10%27% of cases. Metastases to the
para-aortic lymph nodes have been documented in 33% of the pa-
tients with all stages of disease. On routine lymphadenectomy, 42%
59% of patients show lymph node metastases, with almost equal in-
volvement of the para-aortic and pelvic lymph nodes [7]. Compared
with EOC, nodal spread is more common in PFTC, and lymph node
sampling is a mandatory procedure of surgical staging. The Interna-
tional Federation of Gynecology and Obstetrics (FIGO) EOC staging
system has been adapted to apply to PFTC. In general 20%25% of
patients have stage I 20% have stage II, 45%50% have stage III, and
5%10% have stage IV disease [7].
Surgery is the treatment of choice for PFTC. Surgical principles are
the same as those used for ovarian cancer. Aggressive cytoreductive
surgery with removal of as much tumor as possible is warranted in
patients with advanced disease. If it is impossible to achieve optimal
debulking despite maximum effort, surgery should be attempted
again after a few courses of chemotherapy. Very aggressive forms of
surgery should only be considered in highly individualized patients.
Considering the strong tendency for lymphatic spread of the tumor,
a systematic pelvic and para-aortic lymphadenectomy is preferred to
lymph node sampling. In advanced disease, the bulk of extra-tubal
disease and post-operative residual disease >2 cm are adverse prog-
nostic factors. In young patients who want to retain fertility, limited
surgery can be considered for patients with an in situ carcinoma and
in those women with stage I and grade I carcinoma [7].
All patients beyond stage IA and IB are treated with platinum-based
combinations, identical to EOC patients. In addition, early-stage pa-
tients with tumors infiltrating the serosa or with pre- or intra-opera-
tively ruptured tumors should receive chemotherapy. Hormonal
agents like progestational agents increasingly have been used in
PFTC with no firm recommendations [1].
Most recurrences are extrapelvic, and mostly extraperitoneal, usually
in combination with intraperitoneal recurrence reported commonly
in the first 23 years but have also occurred many years later. Be-
cause there is no effective second-line or salvage chemotherapy, re-
current disease is associated with a very poor prognosis. Generally,
the reported 5-year survival rate is about 65%. Stage, patient age,
advanced disease, residual tumor after initial surgery are the most
important prognostic factors for survival. As in the case of EOC, sec-
ond-look laparotomy does not have a role in the management of
PFTC. In addition, there is no curative second-line therapy for those
patients with positive findings at second-look laparotomy. Radio-
therapy could possibly be considered either as adjuvant therapy for
early-stage patients, for stage III residual negative patients, or in the
relapse setting [1].
Image-guided (ultrasonography/CT/MRI) FNAC is being increasingly
used as a rapid, inexpensive, patient-friendly, and efficient method
with minimal morbidity for the pre-surgical diagnosis of ovarian
masses as well as for planning and evaluation of treatment for ad-
vanced inoperable malignant ovarian tumors, for evaluation of re-
current and metastatic tumors. FNAC appears to have a similar diag-
nostic accuracy and a safe diagnostic procedure in comparing be-
nign and malignant neoplasms as open biopsy [12]. Complications
of FNAC are rare, with the incidence of major complications generally
being 0.05%. The major drawback is that FNAC can lead to rupture
and spillage of tumor cells into the peritoneal cavity and can poten-
tially cause upstaging of a malignant tumor [13]. Another concern
relates to needle-tract seeding of malignant cells. This risk is ex-
tremely low, with reported incidence of 0.009% [14]. FNAC of a solid
ovarian mass is more useful for starting neo-adjuvant chemotherapy
to avoid suboptimal surgical cytoreduction in women with advanced
epithelial cancer [15]. Induction or neoadjuvant chemotherapy fol-
lowed by interval debulking surgery form an alternative to primary
debulking surgery, in the management of epithelial ovarian cancer,
in patients with advanced stage disease (stage III to IV). Neo-adju-
vant chemotherapy, however, requires a prior cytological diagnosis
with FNAC [16].
Conclusion
In our patient FNAC gave a positive diagnosis of malignancy and we
were able to operate her malignancy properly following chemother-
apy. She developed a non-healing ulcer at the site of FNAC which is
a very rare complication. But with wide margin excision of that skin
ulcer, the lesion did not did not have much effects on the patients
health.

140 Unusual presentation of a case of fallopian tube carcinoma
Vol 2, No 2
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surgery for advanced epithelial ovarian cancer. Cochrane Database Syst Rev
2009 Jan 21;(1):CD006014.

Shetty A, Rehan M, Vijaya C 141

Ileoileal intussusception in a young adult secondary to a mucinous adenocarcinoma
Archana Shetty
1
, Mudasser Rehan
1
, Chowdappa Vijaya
1

Introduction
Intestinal intussusceptions represent a rare cause of intestinal ob-
struction in adults (1%), with the most frequent localization being
the ileocecal region. The initial diagnosis is often missed or delayed
as the presentation is non-specific [1]. In 90% of the adult cases, an
organic cause can be identified, majority being benign tumors and
the malignancy rate being commoner in colonic intussusceptions We
report a case of mucinous adenocarcinoma of the ilium presenting
as ileoileal intussusceptions in a young adult female.
Case Presentation
A 23 year young female presented to our surgical outpatient depart-
ment with 5 days history of colicky pain in the right lower abdominal
quadrant, aggravated by food intake and associated with vomiting.
She also had history of constipation since two days. Physical exami-
nation showed pallor with tachycardia and normal blood pressure.
The abdomen was moderately distended with tenderness in right
iliac fossa. No palpable mass was identified, and auscultation re-
vealed sluggish bowel sounds.
Blood tests showed anemia (Hb = 7.3g/dl) and leukocytosis (WBC
count = 14,200/cmm). Erect X ray abdomen showed dilated loops of
small intestine. Ultrasound abdomen was inconclusive as the find-
ings were obscured by gas shadows. CT scan with oral contrast
showed a sausage shaped mass in long axis and a target sign mass
in the transverse axis in the right lower quadrant of the abdomen,
leading to the diagnosis of ilio ileal intussusception with features of
intestinal obstruction. Lead point was not identified in our CT scan
(Figure 1).
Emergency midline laparotomy was performed and it revealed ileoil-
eal intussusception for which segmental ileal resection was done
with end to end anastomosis. The apex was formed by what looked
like a polypoidal friable growth measuring 4cm x 3cm across, extend-
ing up to the serosa (figure 2). Histopathology of the mass showed
a moderately differentiated mucinous adenocarcinoma infiltrating
the muscularis mucosae without invasion of the serosa (figure 3).
Pools of mucin were seen.

1
Sapthagiri Institue of Medical Sciences and Research Center, India
Correspondence: Archana Shetty
Email: archanacshetty@yahoo.com

Lymph nodes, adjacent bowel and surgical margins were negative
for tumor deposits. Patient was discharged on the 10
th
post-opera-
tive day after an uneventful recovery. Patient was well at the 4 weeks
follow-up and was referred for chemotherapy.

Abstract
Background: Intestinal intussusceptions represent a rare cause of intestinal obstruction in adults, with the most frequent localization being
the ileocecal region. The initial diagnosis is often missed or delayed as the presentation is non-specific.
Case Presentation: A 23 year young female presented to our surgical OPD with 5 days history of colicky pain in the right lower abdominal
quadrant, aggravated by food intake and associated with vomiting. Emergency midline laparotomy was performed, and it revealed ileoileal
intussusception for which segmental ileal resection was done with end to end anastomosis.
Conclusion: In adults, majority of the intussusceptions are secondary to an underlying pathology, with approximately 65% due to malignant
or benign neoplasms. The incidence of malignancy is particularly high with colonic intussusceptions. (El Med J 2:2; 2014)
Keywords: Ileoileal Intussusception, Mucinous Adenocarcinoma

Figure 1: Abdominal computed tomography showing a target sign mass in
transverse axis (white arrow) in right lower quadrant of the abdomen,
characteristic of intussusceptions

Figure 2(a): Pulling the telescoped segment of intestine (black arrow) post-
surgery.
Figure 2(b): Cut section of the intestinal loop, showing the polypoidal mass
which was the lead point of the intussusception (white arrow)

2(a) 2(b)
142 Ileoileal intussusception in a young adult
Vol 2, No 2

Discussion
Intestinal intussusceptions were first described by Barbette in 1674.
John Hunter, in 1789, presented cases of this condition and defined
it intussusception. The first successful surgical treatment was pub-
lished in 1871 by Hutcinson [1].
Intussusception is commonly seen in children and has been reported
as the second most common abdominal emergency, trailing only ap-
pendicitis. Adult intussusceptions, however, account for only 5% of
all cases of intussusceptions and only 1% of all cases of bowel ob-
struction in adults, with the mean age of incidence being 56 years
[2]. This entity can be classified into 4 distinct categories: ileocecal
enteric, iliocolic, and colocolic [3]. Ileocolic is the commonest type.
The index case was a young adult female with an ileoileal type of
intussusception, both of the characteristics being rare.
Although laboratory findings are not diagnostic, the presence of leu-
kocytosis, which was seen in our case also, is important with regard
to strangulation [4]. Findings of conventional invagination triad in
children - pain, abdominal mass and intestinal bleeding - are rarely
observed in adults [4]. The clinical presentation of adult intussuscep-
tions is variable, with non-specific symptoms (acute, intermittent and
more often chronic), making the pre-operative diagnosis a challenge
[5]. The classic features on ultrasound include the target and dough-
nut sign on transverse view and the pseudokidney sign in longi-
tudinal view.
Although ultrasound has been used to evaluate intussusceptions, its
major disadvantage is masking by gas filled loops of bowel and op-
erator dependency [6]. The pre-operative diagnostic accuracy of ul-
trasonography is 78.5%. In cases of palpable abdominal mass, the
diagnostic accuracy of ultrasonography is even better [7]. Barium ex-
amination may be useful in colonic or ileocolic intussusceptions, in
which a cup shaped filling defect is characteristic. However, barium
studies are contraindicated if there is possibility of perforation or is-
chemia [6]. Abdominal CT has been reported to be the most useful
tool for diagnosis of intestinal intussusceptions. It is less invasive and
superior to other contrast studies, ultrasonography or colonoscopy.
The characteristic sign on CT is the target mass or sausage sign
with enveloped eccentrically located area of low density. The density
of the intussusceptum within the lumen gives this characteristic sign
[6, 8]. Also, CT is excellent in revealing the site, level and cause of
intestinal obstructions and in indicating possible bowel ischemia. In
can give additional information, such as metastasis or lymphade-
nopathy, which may indicate an underlying pathology [7].
In adults 80-90% of the intussusceptions are secondary to an under-
lying pathology, with approximately 65% due to malignant or be-
nign neoplasms. The incidence of malignancy is particularly high
with colonic intussusceptions. In a study of 47 cases of intussuscep-
tions over a period of 11 years by Cakir et al, benign causes included
ileum polyp (41%), idiopathic (13%), parasitic (2%), Meckels diver-
ticula (2%), ileal fibroma (2%) and jejunum polyp (2%). Malignant
causes were cecal adenocarcinoma (26%), sigmoid adenocarcinoma
(4%), ileal mucinous adenocarcinoma (2%), ileal adenocarcinoma
(2%) and rhabdomyosarcoma (2%). Our patient had ileal mucinous
adenocarcinoma, incidence of which is rare [4].
Treatment of adult intussusceptions is varied. Downsides of reduc-
tion prior to resection include the theoretical risk of intraluminal tu-
mor seeding, venous embolization of malignant cells spilling of suc-
cus through inadvertent perforation and anastomotic complication
in cases of an edematous and weakened bowel [8, 9]. Moreover, re-
duction should not be attempted if there are signs of inflammation
or ischemia of the bowel wall and over age of 60 years [7]. It is pos-
tulated that in ileoileal intussusceptions, vitality must be evaluated,
and resection done only in deteriorated vitality, unlike colonic invag-
inations, where resection must be done without the trial of reduction
due to high risk of malignancy [4].
Accordingly in adults, definitive surgical resection remains the rec-
ommended treatment in nearly all cases because of its non-specific
nature, varying duration of symptoms, the large proportion of struc-
tural anomalies and the relatively high risk of malignancy [10]. Even
in our case, it was only after resection that the malignant polypoidal
mass was identified which was not picked up even on CT imaging.
Conclusion
Adult intussusception, causing intestinal obstruction, is rare and is
most commonly due to organic pathology. This entity presents with
non-specific symptoms and is difficult to diagnose pre-operatively.
CT scanning proved to be the most useful diagnostic method. Alt-
hough the malignancy rate in colonic intussusceptions is higher,
even cases in small bowel can harbor adenocarcinomas, as in our
case. A high index of suspicion must be kept in mind while dealing
with cases of adult intussusceptions, and en block surgical resection
is recommended because of the frequency of neoplasms and bowel
ischemia.
References
1. Patrizi G, Rocco Di G, Giannotti D, Casella G, Mariolo CG, Bernieri MG, Redler
A: Double ileo-ceco-colic invagination due to right colon carcinoma: clinical
presentation and management. European Review for Medical and
Pharmacological Sciences 2013;17:2267-2269.

Figure 3: H&E stain microscopy of the tumor showing malignant glands
infiltrating the muscularis mucosae (short arrow), with luminal mucin (long
arrow).
Shetty A, Rehan M, Vijaya C 143
2. Shaheen K, Eisa N, Alraiyes AH, Alraies MC, Merugu S: Telescoping intestine in
an adult. Case Rep Med. 2013;2013:292961.
3. Alexander R, Traverso P, Bolorunduro OB, Ortega G, Chang D, Cornwell EE 3rd,
Fullum TM: Profiling adult intussusception patients: comparing colonic versus
enteric intussusception. Am J Surg. 2011 Oct;202(4):487-91.
4. Cakir M, Tekin A, Kucukkartallar T, Belviranli M, Gundes E, Paksoy Y:
Intussusception: as the cause of mechanical bowel obstruction in adults.
Korean J Gastroenterol. 2013:25;61(1):17-21.
5. Herculanoa R, Coutoa G, Monizb L, Santosa S, Matosa L: Ileocecal
intussusception secondary to cecal adenocarcinoma in the adult. GE J Port
Gastrenterol. 2013;20(2):91-92
6. Yakan S, Calskan C, Makay O, Denecl AG, Korkut MA: Intussusception in
adults: clinical characteristics, diagnosis and operative strategies. World
journal of gastroenterology: WJG 2009, 15(16):1985.
7. Singhal S, Singhal A, Arora PK, Tugnait R, Tiwari B, Malik P, Dhuria AS, Varghese
V, Bharali MD, Chandrakant S, Pushkar, Panwar V, Ballani A, Gupta N,Ramteke
VK: Adult ileo-ileo-caecal intussusception: case report and literature review.
Case Rep Surg. 2012;2012:789378.
8. Eisen LK, Cunningham JD, Aufses AH Jr: Intussusception in adults: institutional
review. J Am Coll Surg. 1999;188:390395.
9. Namikawa T, Okamoto K, Okabayashi T, Kumon M, Kobayashi M, Hanazaki K:
Adult intussusception with cecal adenocarcinoma: Successful treatment by
laparoscopy-assisted surgery following preoperative reduction. World J
Gastrointest Surg 2012;4(5):131-4.
10. Che JH, Wu JS: Single port laparoscopic right hemicolectomy for ileocolic
intussusception. World J Gastroenterol 2013, 7;19(9):1489-93.

144 Phocomelia A case study

Vol 2, No 2
Phocomelia A case study
Soniya B Parchake
1
, Nilesh Keshav Tumram
2
, A P Kasote
3
, M M Meshram
3
, Pradeep G Dixit
2

Introduction
Phocomelia is an extremely rare congenital disorder involving the
limbs (dysmelia). tienne Geoffroy Saint-Hilaire coined the term in
1836 [1]. Although various numbers of factors can cause phocomelia,
the prominent roots come from the use of the drug thalidomide and
from genetic inheritance. The occurrence of this malformation in an
individual results in various abnormalities to the face, limbs, ears,
nose, vessels and many other underdevelopments.
According to National Organization for Rare Disorders (NORD): when
phocomelia is transmitted [in its familial genetic form] it is seen as
an autosomal recessive trait and the mutation is linked to chromo-
some 8 [2]. If a person inherits one normal gene and one gene for
the disease, the individual will become a carrier for the disease; how-
ever, they normally do not show symptoms. The chance for two car-
rier parents to both supply the defective gene and produce a child
with symptoms is 25 percent with each pregnancy [2].
Case Presentation
History
A newly born female infant was brought to the ananthashram (or-
phanage) in our city. The parents were unknown and had aban-
doned the female infant on the stairs of the ananthashram.
Clinical Details
The female infant was about one day old and was brought to the
ananthashram in very weak condition. She was having rudimentary
upper limbs and lower limbs. She had pansystolic murmur in paras-
ternal region and was diagnosed with ventricular septal defect. His-
tory of phocomelia with acyanotic heart disease with small muscular
ventricular septal defect was noted. No past history of intake of any
drug or any congenital anomalies in parents of the deceased child

1
Department of Anatomy, Government Medical College, Solapur, Maharashtra,
India
2
Department of Forensic Medicine and Toxicology Government Medical College,
Nagpur, Maharashtra, India

was found as the child was deserted by their parents.
Investigations
Hematological: Peripheral smear - normocytic normochromic with
no parasite seen. Total leucocyte count 10000/cubic mm, poly-
morphs 68%, lymphocytes 26%, eosinophils 2%, monocytes 1%. RBC
4.14 x 106/l, Hb 13.0 g/dl, mean corpuscular volume 94.9 fl, mean
corpuscular hemoglobin 31.4 pg, mean corpuscular hemoglobin
concentration 33.0 g/dl, platelet count 144 x 103/l, MPV 7.4 fl, PDW
12.5 fl.
Infantogram: Both upper limbs showed single forearm with rudi-
mentary phalyngeal bones. Lower limbs showed rudimentary
phalyngeal bones with long femur, spine normal. 2D echo showed
small midmuscular ventricular septal defect of 2 mm size. PSG was
30 mm Hg with evidence of pulmonary hypertension. Ultrasonogra-
phy of abdomen did not show any congenital anomalies. NSG was
within normal limit.
Autopsy findings: Autopsy was done within 24 hours of the death of
the female infant. She was 9 days old female infant having rudimen-
tary upper limb and lower limb [Figures 1, 2 and 3]. Weight was 1.5
kg, head circumference was 33 cm, and chest circumference was 31
cm. During postmortem, external examination showed rudimentary
upper and lower limbs. No other significant abnormality was noted.
On internal examination no major organ defect were observed. Evi-
dence of bronchopneumonia was observed in both lungs. Cause of
death was attributed to bronchopneumonia.
Discussion
Typically the symptoms of phocomelia syndrome are undeveloped
limbs and absent pelvic bones; however, various abnormalities can
occur to the limbs and bones [3]. Usually the upper limbs are not
3
Department of Anatomy, Government Medical College, Nagpur, Maharashtra,
India
Correspondence: Nilesh Keshav Tumram
Email: ntumram@rediffmail.com
Abstract
Background: Phocomelia syndrome is a rare birth defect especially of the upper limbs. The bones of the upper arm and in some cases
other appendages may be extremely shortened and even absent. In rare cases, there is absence of the upper bones of both the arms and
legs so that the hands and feet appear attached directly to the body. Here we present a rare case of phocomelia.
Case Presentation: A newly born female infant was brought to the orphanage in our city in very weak condition. She had pansystolic
murmur in parasternal region and was diagnosed with ventricular septal defect. Both upper limbs showed single forearm with rudimentary
phalyngeal bones. Lower limbs shows rudimentary phalyngeal bones with long femur, spine normal. The child did not survive and died
shortly after presentation. Autopsy was done within 24 hours of the death of the female infant, and no significant abnormality other than
rudimentary upper and lower limbs was noted. Evidence of bronchopneumonia was observed in both lungs. Cause of death was attributed
to bronchopneumonia.
Conclusion: The present case had only deformity of the upper and lower limbs with acyanotic heart disease with small muscular ventricular
septal defect. The case highlights the morphological defect in such individuals and challenges that may exist due to the neglect of such a
child by their parents and the need for special care for such individuals having congenital defects if they survives. (El Med J 2:2; 2014)
Keywords: Phocomelia, Rudimentary Limbs, Developmental Disorder
Parchake SB, Tumram NK, Kasote AP et al 145
fully formed and sections of the hands and arms may be missing.
Short arm bones, fused fingers, and missing thumbs will often occur.
Legs and feet are also affected similar to the arms and hands. Indi-
viduals with phocomelia will often lack thigh bones, and the hands
or feet may be abnormally small or appear as stumps due to their
close attachment to the body [2].

According to NORD, individuals carrying phocomelia syndrome will
generally show symptoms of growth retardation previous to and af-
ter birth. The syndrome can also cause mental deficiencies in infants.
Infants born with phocomelia will normally have a petite head with
sparse hair that may appear silvery-blonde. Hemangiomas, the
abnormal buildup of blood vessels, will possibly develop around the
facial area at birth and the eyes may be set widely apart, a condition
known as orbital hypertelorism. The pigment of the eyes will be a
bluish white [2]. Phocomelia can also cause an undeveloped nose
with slender nostrils, disfigured ears, irregularly petite jaws (also
known as micrognathia), and a cleft lip with cleft palate [4].
According to NORD, severe symptoms of phocomelia include: a fis-
sure of the skull and a projecting brain known as (encephalocele), an
accumulation of spinal fluid under the skull also known as hydro-
cephalus causing vomiting and migraines, an abnormally shaped
uterus (bicornuate), Inability to clot blood efficiently due to a low
amount of platelets running through the blood, Malformations in the
kidney and heart, shortened neck and abnormalities in the urethra.
Conclusion
The present case had only deformity of the upper and lower limbs
with acyanotic heart disease with small muscular ventricular septal
defect. The case highlights the morphological defect in such individ-
uals, the challenges that may exist due to the neglect of such a child
by their parents and the need for special care for such individuals
having congenital defects if they survive.
References
1. Zimmer, Carl (15 March 2010). "Answers Begin to Emerge on How Thalidomide
Caused Defects". New York Times. Retrieved 12 November 2012. "The word
phocomelia means seal limb. It describes an extremely rare condition in
which babies are born with limbs that look like flippers."
2. "Phocomelia Syndrome". National Organization for Rare Disorders. 11 October
2007.
3. Olney RS, Joyme HE, Roche F, Ferguson K, Hintz S, Madan A: Limb/Pelvis
Hypoplasia. Aplasia With Skill Defect (Schinzel Phocomelia): Distinctive
Features And Prenatal Detection. American Journal of Medicine 103 (4): 205
301.
4. Hunt, Katherine Susan (2002). "Roberts SC phocomelia". Gale Encyclopedia of
Genetic Disorders, Part I. Detroit: The Gale Group Inc.

Figure 1: Female infant with rudimentary upper limbs and
lower limbs in phocomelia

Figure 2: Female infant with a rudimentary lower limb in
phocomelia

Figure 3: Female infant with a rudimentary upper limb in
phocomelia
146 Rectopopliteal Fecal Fistula

Vol 2, No 2
Rectopopliteal fecal fistula developed through an intra-abdominal adhesion
Mustafa Emiroglu
1
, Abdullah Inal
2
, Ismail Sert
3
, Cem Karaali
1
, Cengiz Aydn
1

Introduction
Enterocutaneous fistulas (ECF) create a tract through gastrointestinal
(GI) tract and skin. Theoretically, GI tract can drain from any point to
anywhere in human body from cervical area to popliteal area. Fistu-
las arising from retroperitoneal space develop in a long time interval.
Along with the well- known ECF etiologies (i.e. inflammatory bowel
diseases, malignancies, anastomotic dehiscence, diverticuli, ab-
dominal trauma, tuberculous peritonitis etc) adhesions between pa-
rietal peritoneum and GI tract may lead to a long and unexpected
fistula tract [1, 2]. Here, we present the properties of a fistula which
was developed by adhesion of upper rectum to femoral canal and
obturator foramen, lysis of bowel wall with subsequent perforation,
tracking down interfacial space between femoral muscles and even-
tually draining from popliteal fossa, in a patient who had undergone
uro-oncologic surgery two years earlier. We could not find a similar
case with enterocutaneous fistula secondary to intra-abdominal ad-
hesion on our literature search.
Case Presentation
A 58-year-old female patient underwent radical cystectomy with ileal
pouch reconstruction surgery two years earlier, for urinary bladder
carcinoma. Patient had adjuvant chemotherapy without radiother-
apy. Patient was admitted to urology clinic for fever reaching 39C,
pain and swelling on right leg, limitation of mobility and general clin-
ical deterioration. Patient had co-morbidities of diabetes mellitus
with fasting blood glucose 286 mg/dL and obesity with a body mass
index calculated as 36. On admission, blood pressure was 90/60
mmHg and blood test revealed leukocytosis in complete blood
count. Patient was managed with intravenous fluid replacement and
intravenous antibiotic treatment. On second day of admission, ery-
thema, swelling and emphysema became evident in popliteal fossa.
Patient was consulted to general surgery clinic for gas gangrene. X-
ray examination demonstrated air in subdermal space in popliteal
fossa. Popliteal fossa was laid open by skin incision, and debrided.
Wound care was carried out by hydrogen peroxide washouts twice
daily. Tissue sample cultures were positive for Str. fecalis and E. coli.

1
Izmir Tepecik Training and Research Hospital, Department of Surgery, Turkey
2
University of Erzincan, Mengucek Gazi Training and Research Hospital, Depart-
ment of Surgery, Turkey
3
Denizli Acpayam State Hospital, Department of Surgery, Turkey
Antibiotherapy was modified in accordance with results of antibio-
grams of cultures. On 5
th
day of admission, a fecal discharge was ob-
served on popliteal fossa. Patient was tolerating oral feeding well.
Abdomen was soft and non-tender at physical examination. Ab-
dominal ultrasonography could not show any pathological finding.
On 7
th
day of admission, fasting blood glucose was 160 mg/dL, blood
pressure was 115/75 mmHg, body temperature was 38C and white
blood cell count was within normal limits. Erythema and swelling in
the popliteal area was reduced but pain and swelling on right leg
persisted. Decision was made for surgical exploration on 7
th
day of
admission. No bowel preparation was done preoperatively.
Peritoneal space was clean at exploration. Moderate adhesions
among bowels were observed. Upper rectum adhered to right fem-
oral canal and obturator foramen, and this area was intensely in-
flamed. Recurrent malignancy was excluded by two separate frozen
section pathological examinations which revealed inflammation. Ad-
hesiolysis with sharp dissection demonstrated a closed perforation
in upper rectum. Perforated section was communicating with gluteal
space through obturator foramen and femoral canal. Saline solution
infused from right obturator foramen oozed from right popliteal
fossa. Fistula tract was washed out with copious povidoneiodine
solution and saline solution. Catheters were placed in order to con-
tinue washouts postoperatively. Adherent and perforated segments
of upper rectum were resected and a diverting colostomy was cre-
ated with an Hartmann closure (Figure 1). Fistula tract was washed
out twice daily for one hour each, postoperatively. Washouts were
continued for 10 days. On postoperative 5
th
day, inflammatory find-
ings on right leg began to regress and patient was mobilized. Patient
was discharged on postoperative 15
th
day with complete regression.
Discussion
Both, operation records in patients file and our interview with surgi-
cal staff confirmed that there was no colon or rectum injury in prior
uro-oncologic surgery. But there had been extensive dissection in
pelvic anterior and side walls. At exploratory surgery, there were
dense adhesions and granulation tissue on rectum. Inflammation
Correspondence: Mustafa Emiroglu
Email: musemiroglu@gmail.com
Abstract
Background: A number of etiological factors may result in an enterocutaneous fistula formation. Some fistulas may drain from long and
unexpected tracts.
Case Presentation: A 58-year-old female patient who had undergone radical cystectomy with ileal pouch reconstruction surgery two years
earlier for urinary bladder carcinoma was admitted for septic clinical condition and a rectopopliteal fecal fistula formation. Patient was
improved with a diversion surgery with Hartmann closure and proper wound care. This case is unique by being the sole enterocutaneous
fistula which is developed through an intra-abdominal adhesion. Rectopopliteal enteroatmospheric fistulas are rare clinical conditions.
Conclusion: Enterocutaneous fistulas may complicate any intra-abdominal condition, especially in comorbid patients who have had a major
abdominal surgery. A septic clinical presentation with erythema and subcutaneous emphysema is suggestive of enteric fistula formation. (El
Med J 2:2; 2014)
Keywords: Enterocutaneous Fistula, Intra-abdominal Adhesion, Rectopopliteal Fistula, Enteroatmospheric Fistula
Emiroglu M, Sert I, Inal A et al 147
and mesothelial ischemia is key component in peritoneal adhesion
formation. We assume that the ongoing inflammation in adhesion
tissue may lead to degeneration in rectal wall with a resulting perfo-
ration. Peristalsis and intra-colonic pressure drives the fistula tract
down to the popliteal fossa through intrafascial planes of femoral
muscles. This process generally takes one to five years to occur [1, 3,
5].

Our literature search introduced only three cases of enterocutaneous
popliteal fistula [3, 8, 9]. Our case is unique by being the sole enter-
ocutaneous popliteal fistula which occurred due to an intra-ab-
dominal adhesion (Table 1). Other similar fistulas result from anasto-
motic dehiscence, or diverticular disease [3, 8, 9]. There are also re-
ports of enterocutaneous fistulas in umbilical or sacral areas which
result from diverticular disease [4, 5, 6]. Our case has showed no ev-
idence of diverticular disease both in surgical exploration and post-
operative colonoscopy. A report of an enterocutaneous umbilical fis-
tula arising from tuberculosis peritonitis was also found [7]. Our case
had no evidence for tuberculosis.
Conclusion
Immunocompromised patients or patients with co-morbidities who
have undergone intra-abdominal surgery may develop a long fistula
tract which is due to adhesions to retroperitoneum and/or perfora-
tions. Subcutaneous emphysema in gluteal or femoral region is sug-
gestive of gastrointestinal communication.
Table 1: Review of literature for comparison of similar colocutaneous fistulas

Diagnosis Cause Tract Treatment
Dordea et al.(3) Spontaneous Anastomotic leak Recto-popliteal End colostomy
Pracyk et al.(4) Spontaneous Diverticula Sigmoid-umbilicus Sigmoidectomy
Hurlow et al.(6) Spontaneous Diverticula Sigmoid-sacral area Left hemicolectomy
Shscherba et al.(7) Spontaneous Tuberculosis Colon-umbilicus -
Drabble et al.(8) Drainage Diverticula Sigmoid-popliteal Hartmann closure
Benzione et al.(9) - Diverticula Sigmoid-popliteal Hartmann closure
Emiroglu et al. Drainage Adhesion Recto-popliteal Hartmann closure
References
1. Datta V, Engledow A, Chan S, Forbes A, Cohen CR, Windsor A: The
management of enterocutaneous fistula in a regional unit in the United
kingdom: a prospective study. Dis Colon Rectum 2010, 53(2):192199.
2. Berry SM, Fischer JE: Classification and pathophysiology of enterocutaneous
fistulas. Surg Clin North Am. 1996, 76(5):10091018.
3. Dordea M, Venkatsubramaniam AK, Green SE Varma JS: Delayed rectal
anastomotic dehiscence presenting as a colocutaneous fistula in the popliteal
fossa. Can J Surg. 2008, 51(3):E65-6.
4. Pracyk JB, Pollard SG, Calne RY: The development of spontaneous colo-
umbilical fistula. Postgrad Med J 1993, 69(815):750-1.
5. Orangio GR: Enterocutaneous fistula: medical and surgical management
including patients with Crohns disease. Clin Colon Rectal Surg. 2010,
23(3):169-175.
6. Hurlow RA, Bliss BP: Diverticular fistula between sigmoid colon and sacral cleft.
Proc R Soc Med. 1976, 69(3):226-8.
7. Shcherba BV, Rogozov LI: Umbilical fistula in a patient with asymptomatic
course of tuberculous peritonitis. Probl Tuberk 1988, (9):70.
8. Drabble EH, Greatorex RA: Colocutaneous fistula between the sigmoid colon
and popliteal fossa in diverticular disease. Br J Surg. 1994, 81(11):1659.
9. Benziane K, Boudier P, Kalfon M: Right sigmoid-popliteal fistula in diverticular
disease. Gastroenterol Clin Biol 1997, 21(11):895-7.

Figure 1: Postoperative view of enterocutaneous fistula after drainage and a
diverting colostomy
148 Atypical mesothelial hyperplasia mimicking mesothelioma

Vol 2, No 2
Atypical mesothelial hyperplasia mimicking mesothelioma in patient with metastatic papillary
carcinoma of thyroid
Mutahir A Tunio
1
, Mushabbab AlAsiri
1
, Syed Azfer Husain
2
, Nagoud Mohamed Omar Ali
3
, Shomaila S Akbar
1

Introduction
The pleural epithelial lining is composed of flat cells that are incon-
spicuous on conventional histopathological examination [1]. Con-
stant irritation of the pleural surfaces leads to mesothelial prolifera-
tion in form of simple mesothelial hyperplasia (SMH) or atypical mes-
othelial hyperplasia (AMH). Both types of hyperplasia have been
found in pleural effusion, infection, pulmonary infarcts, pneumotho-
rax, surgery and underlying lung carcinoma [2]. SMH is generally not
of diagnostic concern and there is no specific management required
[3]. AMH is a more aggressive mesothelial proliferation than simple
hyperplasia and is characterized by cellular atypia and hypercellular-
ity [4]. Contrary to SMH, AMH is often a diagnostic and therapeutic
concern as histopathological features resemble that of early non-in-
vasive malignant mesotheliomas or metastatic adenocarcinomas to
the pleura and the approach to these settings is entirely different [5].
Here-in we report on the clinico-pathological features of a hitherto
unrecognized AMH arising in the pleural cavity of an 80-year-old
Saudi woman with diagnosis of papillary carcinoma of thyroid with
pulmonary metastasis.
Case Presentation
An 80 years old Saudi woman presented in emergency room with
shortness of breath. She had this complaint for 2 days and it had
been progressively increasing over one day causing her difficulty in
breathing on sitting and lying postures. Her previous medical history
revealed that she had been treated for papillary carcinoma of thyroid
stage T3N1M1 (lungs) two years back with total thyroidectomy. His-
topathology revealed papillary thyroid carcinoma of left lobe with
positive margins and positive extra-thyroid extension and there were
three positive left level III lymph nodes out of eight retrieved lymph
nodes. Then she was given radioactive iodine (RAI) ablation 150 mCi
three months later. She remained fine when she developed bilateral
pulmonary metastasis one year after initial diagnosis and she was

1
Radiation Oncology, Comprehensive Cancer Center, King Fahad Medical City,
Riyadh-59046, Saudi Arabia
2
Thoracic Surgery, Comprehensive Cancer Center, King Fahad Medical City, Riyadh-
59046, Saudi Arabia
given another RAI ablation of 200 mCi. Since last 9 months, her dis-
ease was stable and she was on regular follow up with thyroid clinic.
She had no other medical illness or history of smoking.
On physical examination, she was found irritated, dyspneic without
any signs of anemia and oxygen saturation was 90% at room air.
There was decreased air entry on right chest with dull percussion;
however, there was no palpable lymphadenopathy and examination
of heart, nervous system and abdomen was unremarkable. Chest x-
ray showed right side pleural effusion extending into fissure with un-
derlying atelectasis. Her baseline serum creatinine was mildly high:
95 micromoles/liter (normal; 44-80), and serum lactate dehydrogen-
ase was also high 390 U/L (normal: 135-214). However other electro-
lytes, hematology and liver function tests were within normal limits.
On day 1, after performing diagnostic pleural tap, pigtail catheter
was inserted and 1400 ml pleural fluid was drained. Pleural fluid cul-
tures were negative for mycobacteria and cytology showed no met-
astatic papillary thyroid carcinoma cells, but numerous mesothelial
cells were observed, for which pleural biopsy was requested (Figure
1). On day 2, her dyspnea improved and computed tomography (CT)
of neck, chest and abdomen was done, which showed stable bilat-
eral pulmonary metastasis, pleural effusion of right middle and lower
lung lobes and new development of right pleural masses with largest
one of size 2.5 x 7.5 cm with interval increase in size of paratracheal
lymph nodes (Figure 2). Differential diagnosis for pleural mass was
malignant mesothelioma or metastatic pleural disease of thyroid pri-
mary carcinoma.
On Day 3, a pleural biopsy was performed, which showed florid mes-
othelial proliferation with cellular atypia, no necrosis and no stromal
invasion. Immunohistochemistry showed positivity for Calretinin and
negativity for desmin, p53, TTF-1, EMA, thyroglobulin and CK5/6 (Fig-
ure 3), which ruled out the metastatic papillary thyroid carcinoma or
3
Anatomic Pathology, Comprehensive Cancer Center, King Fahad Medical City,
Riyadh-59046, Saudi Arabia
Correspondence: Mutahir A Tunio
Email: mkhairuddin@kfmc.med.sa
Abstract
Background: Atypical mesothelial hyperplasia (AMH) is characterized by rapid proliferation of mesothelial cells with cellular atypia
secondary to pleural irritation. AMH is a diagnostic challenge and it is not always easy to differentiate from mesothelioma and metastatic
carcinoma to pleura. Only few related case reports have been published so far.
Case Presentation: Herein we present a case of an 80 year old Saudi woman, who was treated one year ago with total thyroidectomy and
radioactive iodine ablation twice for metastatic papillary carcinoma of thyroid, presented with right pleural effusion which was positive for
mesothelial cells and computed tomography of chest showed right pleural masses suspected for mesothelioma or metastatic papillary
carcinoma, which turned out to be AMH on immunohistochemistry.
Conclusion: AMH is rare benign manifestation which can be associated with underlying malignancy. Radioactive iodine therapy could be a
possible cause. The radiological features of AMH resemble that of mesothelioma or metastatic carcinoma to pleura and
immunohistochemistry is confirmatory. (El Med J 2:2; 2014)
Keywords: Papillary Thyroid Cancer, Atypical Mesothelial Hyperplasia, Rare
Tunio MA, AlAsiri M, Husain SA et al 149
mesothelioma and confirmed the diagnosis of atypical mesothelial
hyperplasia.

Patient was planned for pleurodesis but she had persistent pleural
fluid secretion of 500-600 ml every 24 hours, so she kept on sympto-
matic treatment. On Day 15, CT chest showed worsening of medias-
tinal lymphadenopathy coalescing to form a mass with right bron-
chus invasion with possibility of new primary of lung (Figure 4), but
patient was not fit for the biopsy. On Day 16, her general condition
deteriorated secondary to electrolytes imbalance and progressive
metastatic disease and subsequently she died on day 21 of initial
presentation of dyspnea.

Figure 1: Pleural fluid cytology showing numerous clusters of mesothelial cells
A
B
Figure 2: Computed tomography of chest showing:
(a) Right pleural masses with largest one of size 2.5 x 7.5 cm;
(b) Interval increased size of paratracheal lymph nodes
A
B
C
Figure 3: Pleural biopsy showing florid mesothelial proliferation with cellular
atypia, no necrosis and no stromal invasion. Immunohistochemistry (IHC) showed
positivity for Calretinin and negativity for desmin, p53, TTF-1, EMA, thyroglobulin
and CK5/6

Figure 4: Last computed tomography of chest showing coalescent mediastinal
lymphadenopathy or lung mass
150 Atypical mesothelial hyperplasia mimicking mesothelioma
Vol 2, No 2
Discussion
Atypical mesothelial hyperplasia (AMH) is a diagnostic challenge and
when confronted with AMH, pathologists and physicians need to
know as much clinical and radiologic information as is available be-
cause the differential diagnosis of AMH depends on whether one is
dealing with a mesothelioma or metastatic adenocarcinoma to
pleura [6]. Important histopathological key features which favor
AMH instead of a mesothelioma or metastasis are: (1) no stromal
invasion by mesothelial cells; (2) absence of necrosis; (3) increased
typical mitoses; and (4) absence of p53 positivity on IHC and focal
positivity of EMA [7]. However diagnostic dilemma is that in small
biopsy it is difficult to assess the stromal invasion and necrosis and
florid proliferation of mesothelial cells in AMH may result in bulky
nodules or masses of pleura which mimic a mesothelioma on imag-
ing. Therefore it is important to perform panel of IHC, as negativity
of cytokeratin 5/6, Calretinin, WT-1 and p53 rules out the diagnosis
of mesothelioma and negativity of EMA, CEA (colonic and ovarian
origin), CD15, TTF-1 (lung origin) and thyroglobulin (thyroid origin)
rules out metastatic adenocarcinoma, as seen in our patient [8].
Pathogenesis of AMH in our patient is not exactly known as AMH but
can be explained by first, new development of bronchogenic carci-
noma adjacent to AMH in our patient on her last CT chest imaging
which was considered as coalescent mediastinal lymphadenopathy
and biopsy was not taken (Figure 4). This hypothesis can be sup-
ported by case series of cases by Yokoi T and Mark EJ who reported
that in all eight cases of AMH there was an underlying bronchogenic
carcinoma in the lung subjacent to AMH [9]. Second reason could be
RAI ablation given twice in our patient, although no case has been
reported documenting AMH secondary to RAI ablation, but one case
of AMH was seen four weeks following radiation therapy for a Pan-
coast tumor [10].
The treatment of AMH is subjected to the underlying cause. However
our case addressed two main questions: (i) Is AMH an indicator of
underlying bronchogenic carcinoma? (ii) Can RAI ablation or radia-
tion therapy cause AMH? Currently there are no clear answers due
to rarity of AMH and lack of reporting cases or case series emphasiz-
ing the pathogenesis and diagnosis of AMH.
Received: 14 March 2014 Accepted: 28 March 2014
References
1. Cagle PT: Pleural histology. In: Light RW, Lee YCG, eds. (2003) Pleural Disease:
An International Textbook. London, England: Arnold Publishers, 249-55.
2. Churg A, Galateau-Salle F: The separation of benign and malignant mesothelial
proliferations. Arch Pathol Lab Med 2012, 136:1217-26.
3. Nicolas MM, Nazarullah A, Jagirdar JS: Nodular histiocytic and mesothelial
hyperplasia. Int J Surg Pathol 2011, 19:781-2.
4. Cagle PT, Churg A: Differential diagnosis of benign and malignant mesothelial
proliferations on pleural biopsies. Arch Pathol Lab Med 2005, 129:1421-7.
5. Krismann M, Thattamparambil P, Simon F, Johnen G: Differential diagnosis of
preneoplastic lesions of the pleura and of early mesothelioma:
immunohistochemical and morphological findings. Pathologe 2006, 27:99-
105.
6. Laga AC, Allen T, Bedrossian C, Laucirica R, Cagle PT. (2005) Reactive
mesothelial hyperplasia. In: Cagle PT, editor-in-chief. The Color Atlas and Text
of Pulmonary Pathology. New York, NY: Lippincott Williams & Wilkins,5,587-8.
7. Churg A, Roggli V, Colby TV, and the US Canadian Mesothelioma Panel: The
separation of benign and malignant mesothelioma proliferations. Am J Surg
Pathol 2000, 24:1183-1200.
8. Cury PM, Butcher DN, Fisher C, Corrin B, Nicholson AG: Value of the
mesothelium-associated antibodies thrombomodulin, cytokeratin 5/6,
calretinin, and CD44H in distinguishing epithelioid pleural mesothelioma from
adenocarcinoma metastatic to the pleura. Mod Pathol 2000, 13:107-12.
9. Yokoi T, Mark EJ: Atypical mesothelial hyperplasia associated with
bronchogenic carcinoma. Hum Pathol 1991, 22:695-9.
10. Jagirdar J, Frydman C, Sakurai H, Dumitrescu O: Mesothelial papillary
proliferation of the pleura associated with radiation therapy: does it have a
role in the pathogenesis of mesothelioma? Mt Sinai J Med 1989, 56:147-9.

Aggarwal P, Debi U, Jindal G 151

Primary eosinophilic granuloma presenting as bilateral otitis media and mastoiditis
Purnima Aggarwal
1
, Uma Debi
1
, Geetanjli Jindal
2

Introduction
Eosinophilic granuloma is an uncommon benign bone tumor which
has frequently been documented in the pediatric age group. Its inci-
dence is less than 1 per 200,000 population. Males are twice as com-
monly affected as females. It is usually found at flat and long bones.
The skull and vertebral spine is often affected [1]. Temporal bone
involvement is common whenever eosinophilic granuloma arises
from skull [2-12]. However, bilateral temporal bone involvement is
rare and only less than 20 cases are reported in the literature [2-7].
These patients usually present with aural discharge and conductive
hearing loss. Computed tomography scan can be diagnostic but re-
quire confirmation by histopathology and or immunohistochemistry
on tissue sample. Surgical excision, radiotherapy and chemotherapy,
either alone or in combination, are the main treatment options [8].
Case Presentation
A 3-years-old boy presented in otolaryngology clinic with a history
of bilateral aural discharge with gradually increasing swelling of peri-
auricular region bilaterally for 3 months duration. There was no his-
tory of any constitutional symptoms. On clinical examination there
were firm tender masses behind the ears on both sides. Floating
teeth were also found. A CT scan of skull revealed osteolytic lesions
in mastoid and squamous portions of both temporal bones with peri-
auricular soft tissue swelling (Figures 1 and 2). Osteolytic lesions also
involved the middle cranial fossa. Facial nerve was spared bilaterally.
A biopsy from the peri-auricular swelling revealed it to be a case of
eosinophilic granuloma. On subsequent radiological evaluation, re-
ticulonodular lesions were found in the chest. Patient was given
chemotherapy consisting of vinblastine, methotrexate and predniso-
lone. Patient responded well to treatment. Follow-up MR/CT imaging
obtained 3 months after chemotherapy showed no local recurrence.
Discussion
Eosinophilic granuloma is one of the three clinical variants of Lang-
erhans cell histiocytosis or Histiocytosis X and is characterized by id-
iopathic proliferation of histiocytes producing focal or systemic man-
ifestations. The localized form of langerhans cell histiocytosis, in

1
Department of Radiodiagnosis, Government Medical College, Chandigarh, India.
2
Department of Pediatrics, Government Medical College, Chandigarh, India.
Correspondence: Purnima Aggarwal
Email: sudhir_ortho@yahoo.com
which the disease is limited to bones, lymphatic nodes or the lung,
is commonly referred to as eosinophilic granuloma [8].

Abstract
Background: Simultaneous bilateral involvement of the temporal bones is a rare manifestation of eosinophilic granuloma, a disease
characterized by solitary or multiple foci of osteolytic damage.
Case Presentation: A young child initially presented with bilateral otitis media, with subsequent rapidly developing bilateral masses in the
mastoid region. Computed tomography demonstrated extensive bony destruction which, after a course of vinblastine, methotrexate and
prednisolone improved dramatically on follow-up scans.
Conclusion: We present an unusual case of bilateral temporal bone destruction due to eosinophilic granuloma in a young child along with
the literature review. (El Med J 2:2; 2014)
Keywords: Eosinophilic Granuloma, Bilateral, Temporal Bone, CT Scan

Figure 1: Axial post-contrast CT shows
inhomogeneous enhancement of destructive soft
tissue mass in bilateral mastoid bone

Figure 2: Axial CT with bone window irregular
geographic and punched out border with complete
loss of portions of the mastoid cortex
152 Primary eosinophilic granuloma
Vol 2, No 2
Eosinophilic granuloma is the most benign lytic lesion of bone usu-
ally with an excellent prognosis and other disorders include Hand-
Schller-Christian disease and Letterer-Siwe disease in order of in-
creasing severity. The differential diagnosis of the subgroups is made
according to the clinical manifestations such as visceral organ or
bone involvement. Childhood histiocytosis constitutes a diverse
group of disorders characterized by an intense proliferation of cells
of monocyte-macrophage system of bone marrow origin [13]. The
writing group of the Histiocyte Society divides histiocytosis syn-
dromes in children into three classes: class I is Langerhans' cell histi-
ocytosis; class II (non-LC histiocytosis) includes the familial and virus-
associated hemophagocytic syndromes, sinus histiocytosis with mas-
sive lymphadenopathy (Rosai-Dorfman), juvenile xanthogranuloma,
and reticulohistiocytoma; class III consists of the malignant histio-
cytic diseases [14].
More recently, a revised classification schema included division into
(1) dendritic cell disorders: Langerhans cell histiocytosis (LCH), sec-
ondary dendritic cell processes, juvenile xanthogranuloma, and soli-
tary histiocytoma with a dendritic phenotype; (2) macrophage-re-
lated disorders: primary and secondary hemophagocytic syndrome,
Rosai-Dorfman disease, and solitary histiocytoma with a macrophage
phenotype; and (3) malignant histiocytic disorders: monocyte re-
lated leukemia, extramedullary monocytic tumor, and dendritic cell
or macrophage-related histiocytic sarcoma [15]. Langerhans' cell his-
tiocytosis (LCH) occurs with an estimated incidence of about two to
five cases per million yearly [16].
The etiology of eosinophilic granuloma remains unclear to date but
uncontrolled proliferation of langerhans cells, previous inflamma-
tions or tumors and autoimmune disorders are suspected [1]. It is
currently believed that clonal accumulation and proliferation of
CD1a-positive langerhans cells are causative [9]. Eosinophilic granu-
loma is frequently confused with infectious diseases or neoplastic
conditions of the temporal bone. The temporal bone involvement
can be confused with more common disorders such as aural polyps
or chronic suppurative otitis media [10]. Although initially silent, the
disease if untreated, may destroy the bony labyrinth and spread to
the middle and posterior cranial fossae.
Only a surgically obtained biopsy leads to definitive diagnosis by his-
topathology including immunohistochemical detection of S-100 and
CD1a antigens in the tissue samples with or without the appearance
of intracytoplasmic organelles or Birbeck granules on electron mi-
croscopy [8, 11, 12]. Early detection is important to manage eosino-
philic granuloma properly and to minimize the complications or se-
quels of treatment. Surgical excision, radiotherapy and chemother-
apy, either alone or in combination, are the main treatment options
[11]. A systematic approach with regular CT and MRI follow-up is
suggested.
Conclusion
In conclusion, eosinophilic granuloma although a very rare disease,
it should be included in the otolaryngologist's differential diagnosis
of lytic lesions of the temporal bone.
References
1. Kitsoulis PV, Paraskevas G, Vrettakos A, Marini A: A case of eosinophilic
granuloma of the skull in an adult man: a case report. Cases J 2009, 2:9144.
2. Levy R, Sarfaty SM, Schindel J: Eosinophilic granuloma of the temporal bone.
Two cases with bilateral involvement. Arch Otolaryngol 1980, 106:167-71.
3. Yetiser S, Karahatay S, Deveci S: Eosinophilic granuloma of the bilateral
temporal bone. Int J Pediatr Otorhinolaryngol 2002, 62:169-73.
4. Ginsberg HN, Swayne LC, Peron DL, et al: Bilateral temporal bone involvement
with eosinophilic granuloma. Comp Med Imaging Graph 1998, 12:107-10.
5. Arcand P, Caouette H, Dufour JJ: Eosinophilic granuloma of the temporal
bone: Simultaneous bilateral involvement. J Otolaryngol 1985, 14:375-8.
6. McCaffrey TV, McDonald TJ: Histiocytosis X of the ear and temporal bone:
Review of 22 cases. Laryngoscope 1979, 89:1735-42.
7. Barton CP, Horlbeck D: Eosinophilic granuloma: Bilateral temporal bone
involvement. Ear Nose Throat J 2007, 86:342-3.
8. Hellmann M, Stein H, Ebmeyer J, Sudhoff H: Eosinophilic granuloma of the
temporal bone. Case report and literature review. Laryngorhinootologie 2003,
82:258-61.
9. Skoulakis CE, Drivas EI, Papadakis CE, Bizaki AJ, Stavroulaki P, Helidonis ES:
Langerhans cell histiocytosis presented as bilateral otitis media and
mastoiditis. Turk J Pediatr 2008, 50:70-3.
10. Appling D, Jenkins HA, Patton GA: Eosinophilic granuloma in the temporal
bone and skull. Otolaryngol Head Neck Surg 1983, 91:358-65.
11. Bayazit Y, Sirikci A, Bayaram M, Kanlikama M, Demir A, Bakir K: Eosinophilic
granuloma of the temporal bone. Auris Nasus Larynx 2001, 28:99-102.
12. Wanifuchi N, Ishizuka Y, Yabe T: A case of eosinophilic granuloma in the
temporal bone. Auris Nasus Larynx 1991, 18:17-26.
13. Singh H, Kaur S, Yuvarajan P, Jain N, Maini L: Unifocal Granuloma of Femur due
to Langerhans' Cell Histiocytosis: A Case Report and Review of the Literature.
Case reports in medicine 2010, 2010.
14. Chu T, DAngio GJ, Favara B: Histiocytosis syndromes in children. Lancet. 1987,
1(8526):208209.
15. Favara BE, Feller AC, Pauli M, et al. Contemporary classification of histiocytic
disorders. Medical and Pediatric Oncology. 1997, 29(3):157166.
16. Leonidas JC, Guelfguat M, Valderrama E: Langerhans cell histiocytosis. Lancet.
2003, 361(9365):12931295.

Nirmala SVSG, Bareddy R, Nuvvula S et al 153

http://www.mednifico.com/index.php/elmedj/article/view/113 Vol 2, No 2
Peripheral ossifying fibroma
S V S G Nirmala
1
, Ramasub Bareddy
1
, Sivakumar Nuvvula
1
, Swetha Alahari
1
, Sandeep Chilamakuri
1

Introduction
Solitary gingival enlargements in children are relatively common
findings and are usually the result of a reactive response to local irri-
tation [1]. Peripheral ossifying fibroma (POF) is a reactive lesion char-
acterized by the growth of non-neoplastic mass in the gingiva [2-6].
Its color may resemble that of a normal mucosa or may be slightly
reddish and its surface may be either intact or ulcerated [2]. Alt-
hough this lesion is thought to be relatively common, it accounts for
less than 1% of all oral biopsies performed. The lesion usually doesnt
exceed 2.0 cm and involves predominantly the anterior region of the
mandible affects more commonly females and it is more frequently
found during the second decade of life [2-8].
The etiology of the peripheral ossifying fibroma is unknown, alt-
hough some authors have suggested that the lesion is associated
with inflammatory hyperplasia of the periodontal ligament [4, 8].
Others speculate about a possible hormonal influence since prepu-
bertal patients are rarely affected and the disease incidence falls sig-
nificantly after the third decade of life [8]. Histologically, the lesion
consists of fibrous proliferation associated with formation of miner-
alized tissue, which can resemble both cement and dystrophic calci-
fication. When former is observed, the lesion is called peripheral ce-
mento ossifying fibroma [4, 5]. The objective of the present article is
to report a case of POF occurring in the maxillary anterior region of
a 13 year old girl.
Case Presentation
A healthy 13 year old girl reported to the department of Pedodontics
with a slow growing painless swelling behind her front teeth. Ac-
cording to the patient, the reddish purple lump had been present
for approximately 1 month and her father stated that it had just re-
cently become visible between the front teeth. As reported by the
patient, the growth was interfering with her bite and felt uncomfort-
able. Occasional bleeding was reported while brushing. During con-
sultation, it became apparent that the patients father was very con-
cerned about the pathogenesis of the lesion. According to the father,
their family physician had discussed the possibility of the lesion be-
ing cancer, which had raised the fathers anxiety level considerably.

1
Department of Pedodontics & Preventive Dentistry, Narayana Dental College and
Hospital, Nellore, Andhra Pradesh, India.
Correspondence: S V S G Nirmala
Email: nimskrishna2007@gmail.com
Family history, past health history and dental history were not rele-
vant. Clinical examination revealed a palatally visible exophytioc le-
sion, nodular with an irregular surface (Figure 1) pedunculated in
between 21 and 22 and it measured approximately 2 x 2 cm.

The lesion appeared reddish pink with areas of white. It was slightly
pedunculated with what appeared to be a broad based attachment.
The lesion was not fluctuant, nor did it blanch with the pressure, but
had a rubbery consistency. It was tender to firm pressure, but not to
light palpation. Bleeding on probing was observed. The tooth was
not tender on percussion and vitality test was positive. The differen-
tial diagnosis consisted of irritation fibroma, pyogenic granuloma
and peripheral giant cell granuloma (PGCG). The differential diagno-
sis was discussed with the patient and her father in an attempt to
alleviate fears of malignant lesion.
Complete hemogram was performed which showed all blood counts
to be within normal limits. Written consent was acquired for the pro-
cedure, the patient was scheduled for a thorough full mouth scaling.
Under local anesthesia, whole growth was excised completely using
both a scalpel and an electrocautery device. The tissue was submit-
ted to the oral pathology division for histopathological diagnosis.
Microscopic examination of the excised tissue revealed a gingival

Abstract
Background: Peripheral ossifying fibroma is a solitary growth on the gingiva which is thought to arise from the periodontal ligament. We
report a case of peripheral ossifying fibroma in the maxillary anterior region of a 13 year old girl.
Case Presentation: The patient presented with a gingival lesion in the maxillary left anterior region of the mouth since 2 weeks. Following
thorough full mouth scaling, an excisional biopsy was done and the specimen was sent for histopathological examination. Healing of the
surgical site was uneventful. Based on the clinical and histopathological findings a final diagnosis of peripheral ossifying fibroma was made.
Conclusion: The diagnosis warrants for frequent recall interval for monitoring recurrence. This report highlights the importance of definitive
diagnosis in order to provide appropriate treatment. (El Med J 2:2; 2014)
Keywords: Maxillary Gingiva, Peripheral Ossifying Fibroma, Pyogenic Granuloma
Figure 1: Intraoral picture showing the lesion
154 Peripheral ossifying fibroma
Vol 2, No 2
nodule that was partly ulcerated and partly lined with hyperkeratin-
ized stratified squamous epithelium with a normal maturation pat-
tern. Much of the nodule consisted of hypercellular well vascularized
fibrous connective tissue containing plump mesenchymal cells as
well as numerous multinucleated giant cells. The specimen also ex-
hibited a fairly large area of immature bone formation but no evi-
dence of malignancy (Figure 2). Based on histopathological and clin-
ical examination, the diagnosis stated was peripheral ossifying fi-
broma maintaining the nature and clinical appearance of the growth.

The patient presented for a follow up examination 20 days postop-
eratively. The surgical site appeared to be healing well. There was no
evidence of recurrence of the lesion and the child was asymptomatic
(Figure 3).

Discussion
Intraoral ossifying fibromas have been described in the literature
since the late 1940s. Many names have been given to similar lesions,
such as epulis, peripheral fibroma with calcification, peripheral ossi-
fying fibroma, calcifying fibroblastic granuloma, peripheral ce-
mentifying fibroma, peripheral fibroma with cementogenesis and
peripheral cemento-ossifying fibroma [2, 4, 9-11]. The sheer number
of names used for fibroblastic gingival lesions indicates that there is
much controversy surrounding the classification of these lesions [6,
12].
It has been suggested that POF represents a separate clinical entity
rather than a transitional form of pyogenic granuloma, PGCG or POF,
as well as similar clinical and histologic features. These lesions many
simply be varied histologic responses to irritation [4]. Gardner stated
that POF cellular connective tissue is so characteristic that a histo-
logic diagnosis can be made with confidence, regardless of the pres-
ence or absence of calcification [9]. Buchner and Hansen hypothe-
sized that early POF presents as ulcerated nodules with little calcifi-
cation, allowing easy misdiagnosis as a pyogenic granuloma [13].
Several publications address the issue of histologic differentiation in
depth, but this is beyond the scope of this article [3, 6, 9, 13].
When presented clinically with a gingival lesion, it is important to
establish a differential diagnosis. In this case, the clinical features led
to a differential diagnosis of irritation fibroma, pyogenic granuloma
or PGCG. Although it is also important to maintain a high index of
suspicion, discussion with family members should be tactful to pre-
vent undue distress during the waiting period between differential
diagnosis and definitive histopathological diagnosis.
Because the clinical appearance of these various lesions can be re-
markably similar, classification is based on their distinct histologic
differences. The POF must be differentiated from the peripheral
odontogenic fibroma (PODF) described by the World Health Organ-
ization [3, 13]. Histologically, the PODF has been defined as a fibro-
blastic neoplasm containing odontogenic epithelium [8]. Despite a
preponderance of literature supporting differentiation, some authors
continue to argue that the POF (or peripheral cement ossifying fi-
broma) is the peripheral counterpart of the central cemento-ossify-
ing fibroma [11].
POF, as discovered in this case, is a focal, reactive, non-neoplastic
tumor-like growth of soft tissue often arising from the interdental
papilla [2, 3, 9]. It is a fairly common lesion, comprising nearly 3% of
oral lesions biopsied in one study, approximately 1%2% in other
studies [8-11]. In 1993, Das and Das obtained similar results, with
1.6% POFs among 2,370 intraoral biopsies [12]. POF may present as
a pedunculated nodule, or it may have a broad attachment base [2,
5, 14]. These lesions can be red to pink with areas of ulceration, and
their surface may be smooth or irregular. Although they are generally
< 2 cm in diameter, size can vary [5, 13]. Reports range from 0.23.0
cm to 4 mm8 cm and some lesions may be as large as 9 cm in di-
ameter [1, 13-15]. Cases of tooth migration and bone destruction
have been reported, but these are not common. However in the pre-
sent case tooth migration was there.
The female to male ratio reported in the literature varies from 1.22:1
to 1.7:1 [1, 8, 13, 16]. By most reports, the majority of the lesions
occur in the second decade, with a declining incidence in later years
[2, 13, 16]. There are 2 reported cases of POF present at birth, pre-
senting clinically as congenital epuli [17, 18]. In a 2001 study, Cuisia
and Brannon reported that only 134 out of 657 diagnosed POFs
(20%) were in the pediatric population (019 years), with 8% in the
first decade [14]. In a retrospective study of 431 cases in the Chinese
population by Zhang and others, the mean age of incidence of POF

Figure 2: Histological picture

Figure 3: Intra oral picture showing healing of the lesion
Nirmala SVSG, Bareddy R, Nuvvula S et al 155
was found to be 44 years, which is contradictory to previously pub-
lished literature [19]. POF appears to be more common among white
people than black and slightly less common among those of His-
panic origin [14]. The case presented by us was female patient and
Asian origin.
The lesion may be present for a number of months to years before
excision, depending on the degree of ulceration, discomfort and in-
terference with function [1, 8]. Approximately 60% of POFs occur in
the maxilla, and they occur more often in the anterior than the pos-
terior area with 55%60% presenting in the incisor-cuspid region [2,
3, 8, 9, 13, 14, 16, 19]. The finding is in accordance with our finding.
POFs are believed to arise from gingival fibers of the periodontal lig-
ament as hyperplastic growth of tissue that is unique to the gingival
mucosa [2, 3, 20]. This hypothesis is based on the fact that POFs arise
exclusively on the gingiva, the subsequent proximity of the gingiva
to the periodontal ligament and the inverse correlation between age
distribution of patients presenting with POF and the number of miss-
ing teeth with associated periodontal ligament [8, 14, 19, 20]. In a
study of 134 pediatric patients with POF, in only two cases was POF
intimately associated with primary teeth, bringing into question the
reactivity of the lesion. The exfoliation of primary teeth and eruption
of their successors should result in an increased incidence of perio-
dontal ligament-associated reactive lesions [3, 6, 8, 9, 11]. In the pre-
sent case the lesion was associated with the permanent teeth.
Hormonal influences may play a role, given the higher incidence of
POF among females, increasing occurrence in the second decade
and declining incidence after the third decade [8]. In an isolated case
of multicentric POF, Kumar and others noted the presence of a lesion
at an edentulous site in a 49-year-old woman, which once again
raises questions regarding the pathogenesis of this type of lesion
[12]. In the present case it may be due to hormonal influence. Treat-
ment consists of conservative surgical excision and scaling of adja-
cent teeth [2, 8, 9]. The rate of recurrence has been reported at 8.9-
20% [2-4, 13, 14]. Therefore, regular follow-up is required.
Conclusion
In children, peripheral ossifying fibroma can exhibit an exuberant
growth rate and reach significant size in a relatively short period of
time. Early recognition and definitive surgical intervention result in-
less risk of tooth and bone loss.
References
1. Flaitz, CM: Peripheral giant cell granuloma: a potentially aggressive lesion in
children. Pediatr Dent 2000, 22(3):232-3.
2. Bhaskar SN, Jacoway JR: Peripheral fibroma and peripheral fibroma with
calcification: report of 376 cases. J Am Dent Assoc 1966, 73(6):131220..
3. Buchner A, Hansen LS: The histomorphologic spectrum of peripheral ossifying
fibroma. Oral Surg Oral Med Oral Pathol 1987, 63(4):45261.
4. Eversole LR, Rovin S: Reactive lesions of the gingiva. J Oral Pathol 1972,
1(1):308.
5. Neville BW, Damm DD, Allen CM, Bouquot JE: Oral and maxillofacial pathology.
2nd ed. Philadelphia: WB Saunders Co; 2002. p. 4512.
6. Shafer WG, Hine MK, Levy BM: A text book of oral pathology; 4 th ed.
Philadelphia. W.B Saunders 1987.
7. Zain RB, Fei YJ: Fibrous lesions of the gingiva: a histopathologic analysis of 204
cases. Oral Surg Oral Med Oral Pathol 1990, 70(4):46670.
8. Kenney JN, Kaugars GE, Abbey LM: Comparison between the peripheral
ossifying fibroma and peripheral odontogenic fibroma. J Oral Maxillofac Surg
1989, 47(4):37882.
9. Gardner DG: The peripheral odontogenic fibroma: an attempt at clarification.
Oral Surg Oral Med Oral Pathol 1982, 54(1):408.
10. Lee KW: The fibrous epulis and related lesions. Granuloma pyogenicum,
Pregnancy tumour, fibro-epithelial polyp and calcifying fibroblastic
granuloma. A clinico-pathological study. Periodontics 1968, 6(6):27792.
11. Feller L, Buskin A, Raubenheimer EJ: Cemento-ossifying fibroma: case report
and review of the literature. J Int Acad Periodontol 2004, 6(4):1315.
12. Kumar SK, Ram S, Jorgensen MG, Shuler CF, Sedghizadeh PP: Multicentric
peripheral ossifying fibroma. J Oral Sci 2006, 48(4):23943.
13. Buchner A, Hansen LS: The histomorphologic spectrum of peripheral ossifying
fibroma. Oral Surg Oral Med Oral Pathol 1987, 63(4):45261.
14. Cuisia ZE, Brannon RB: Peripheral ossifying fibroma a clinical evaluation of
134 pediatric cases. Pediatr Dent 2001, 23(3):2458.
15. Poon CK, Kwan PC, Chao SY: Giant peripheral ossifying fibroma of the maxilla:
report of a case. J Oral Maxillofac Surg 1995, 53(6):6958.
16. Skinner RL, Davenport WD Jr, Weir JC, Carr RF: A survey of biopsied oral lesions
in pediatric dental patients. Pediatr Dent 1986, 8(3):1637.
17. Yip WK, Yeow CS: A congenital peripheral ossifying fibroma. Oral Surg Oral
Med Oral Pathol 1973, 35(5):6616.
18. Kohli K, Christian A, Howell R: Peripheral ossifying fibroma associated with a
neonatal tooth: case report. Pediatr Dent 1998, 20(7):4289.
19. Zhang W, Chen Y, An Z, Geng N, Bao D: Reactive gingival lesions: a
retrospective study of 2,439 cases. Quintessence Int 2007, 38(2):10310.
20. Miller CS, Henry RG, Damm DD: Proliferative mass found in the gingiva. J Am
Dent Assoc 1990, 121(4):55960.

156 Lipoma of retromandibular space

Vol 2, No 2
Lipoma of retromandibular space
Anand Gupta
1
, Varun Chopra
2
, Gurvanit Lehl
2
, Shivani Jindal
2

Introduction
Lipomas are common benign tumors of mesenchymal origin that
may occur in any region of the body where fat is present. They are a
common tumor of skin, but less common in the head and neck ac-
counting for only 1-4.4% of all benign tumors [1]. Lipomas are slow
growing in nature and histopathologically they are nearly always be-
nign. They are usually seen in the posterior neck region and presen-
tation over anterior neck region is rare [2]. Clinically, lipomas are
non-tender, soft and mobile masses. On clinical examination, most
subcutaneous lipomas may be suspected with a high degree of ac-
curacy, while deep-seated or infiltrating lipomas require imaging for
further assessment [3].
This paper presents probably the first case of lipoma occurring at
retromandibular region which is an unusual site of its occurrence in
head and neck.
Case Presentation
A 35 year old male patient reported to the Department of Dentistry,
Government Medical College Hospital, Chandigarh, with a painless
swelling on left side of face for last two years. On examination, the
swelling was 6cm x 4cm in size extending from tragus of the ear up
to the upper border of thyroid cartilage and anteroposteriorly from
posterior border of ramus to anterior border of sternocleidomastoid
muscle (Figure 1). The swelling was soft, mobile and non-tender on
palpation. On intraoral examination no oral extension of the swelling
was seen.
Ultrasonography suggested lipomatous lesion. Magnetic resonance
imaging (MRI) revealed a well-defined, oval-shaped, hyperintense
signal in T1-and T2-weighted images involving the retromandibular
space, posterior to the ramus of mandible on left side (Figure 2a).

1
Department of Dentistry, Lady Hardinge Medical College, India
2
Department of Dentistry, Government Medical College and Hospital, Chandigarh,
India
Superiorly there was indentation on the superficial lobe of the pa-
rotid gland. No infiltration was seen into the parotid gland.

3
Department of Pathology, Government Medical College and Hospital, Chandigarh,
India
Correspondence: Anand Gupta
Email: dranandkgmc2@gmail.com
Abstract
Background: Lipomas are a common tumor of skin but less common in the head and neck accounting for only 1-4.4% of all benign tumors.
Of the variety of lipomatous benign tumors that occur, over 80 percent are ordinary lipomas and only about 13 percent of these occur in
the head and neck region, most commonly in the posterior neck. Rarely, lipomas can occur in the antrerior neck, infratemporal fossa, oral
cavity, pharynx, larynx and parotid gland. Lipomas involving submandibular and parapharayngeal spaces have been reported. To the best
of our knowledge after searching the English Literature on MEDLINE database, no case has been reported involving this region
(retromandibular space).
Case Presentation: We report a case of 40 year old male who presented with solitary swelling over left retromandibular region which
confirmed to be lipoma on histopathological examination after surgical excision. Postoperatively patient developed a sialocele after 2 weeks
which was managed successfully by aspiration and antisialagogues. Patient recovered completely after two weeks and no recurrence was
seen till follow up of two years.
Conclusion: Lipomas are common tumors of the head and neck region. They commonly occur at the site where fat is present and which
is not required for metabolic needs of the body. Untreated lipomas can result in aesthetic and functional disease. (El Med J 2:2; 2014)
Keywords: Lipoma, Face, Retromandibular Space, Sialocele

Figure 1: Preoperative profile photograph of the patient
showing the location of swelling over the left
retromandibular region

Figure 2(a): MRI T1w axial scan showing the
hyperechoic area posterior to the left
mandibular ramus region
Gupta A, Chopra V, Lehl G et al 157
The tumor was excised in toto, via extraoral submandibular approach
under general anesthesia (Figure 2b). Intraoperatively, it was found
to be abutting close to the superficial lobe of parotid gland as sug-
gested by MRI and the tumor was excised completely by dissecting
a plane between the gland and tumor.

Histopathological examination revealed lobules of mature fat cells
surrounded by thin fibrous connective tissue septa. Adipose cells
were uniform, round, with clear cytoplasm and eccentrically placed
nucleus mimicking signet ring appearance, confirming the diagnosis
of lipoma (Fig. 3a, 3b).

Postoperatively in the second week, patient developed a soft and
painless swelling near the operated site. The collection was aspirated
and the provisional diagnosis of sialocele was made which got con-
firmed after biochemical investigation of the salivary secretion. The
quantity decreased on second aspiration after 1 week and patient
was put on antisialagogue (Tab. Glycopyrrolate 1 mg 6 hourly) per
orally for 1 week. Patient recovered completely after two weeks and
had no further complication. No recurrence was seen till the last fol-
low up at 2 years.
Discussion
Lipomas are common tumors of the head and neck region. They
commonly occur at the site where fat is present and which is not
required for metabolic needs of the body. Untreated lipomas can re-
sult in aesthetic and functional disease [4]. Of the variety of lipoma-
tous benign tumors that occur, over 80 percent are ordinary lipomas
and only about 13 percent of these occur in the head and neck re-
gion, most commonly in the posterior neck [2,5]. Rarely, lipomas can
occur in the anterior neck, infratemporal fossa, oral cavity, pharynx,
larynx and parotid gland.[6] Lipomas involving submandibular and
parapharayngeal spaces have also been reported [7]. To the best of
our knowledge, after searching the Literature in MEDLINE database,
no case has been reported involving this region (retromandibular
space). Reporting the presence of lipoma at such an unusual location
becomes important to include it in the differential diagnosis of pain-
less swellings over the retromandibular region of the head and neck.
The diagnosis of lipoma is usually made on the basis of clinical ex-
amination, radiographic findings and the correlation with the histo-
logical features once biopsy is done. Ultrasonography offers a cost
effective alternative compared to CT and MRI which depicts lipoma
to be hyperechoeic relative to adjacent muscle and echogenic lines
running at right angles to ultrasound beam. MRI can also accurately
diagnose lipomas preoperatively, with typical signal intensity pat-
terns simulating that of subcutaneous fat (i.e. high signal intensity
on T1-weighted images and intermediate intensity on T2-weighted
images with a weak signal on fat suppressed images) [8]. Moreover,
the margin of a lipoma is clearly defined by MRI as a black rim, en-
abling one to distinguish lipomas from surrounding adipose tissue,
a distinction that cannot be made from CT images [9].
Furlong et al reported a large series of 125 lipomas of oral and max-
illofacial region. They classified them according to the specific ana-
tomic sites of oral and maxillofacial region like the parotid region (n
= 30), buccal mucosa (n = 29), lip (n = 21), submandibular region (n
= 17), tongue (n = 15), palate (n = 6), floor of mouth (n = 5) and
vestibule (n = 2). Histologically, classic lipomas comprised the ma-
jority, with the exception of the parotid region and the lip, where
spindle cell lipoma was the most common. Additional variants in-
cluded were fibrolipoma and chondroid lipoma [10].
Recurrence rate of lipoma is low and excision is treatment of choice.
Postoperative complications like sialocele may occur if the duct leaks
but no fistula forms. This may also result when the glandular sub-
stance of the parotid is disrupted but the parotid duct is intact. This
condition usually resolves by usage of conservative modality based
on regular aspiration of the content and compression dressing. Some

Figure 2(b): Photograph of the in-toto excised specimen of the tumor

Figure 3(a): Low power view shows an encapsulated tumor
composed of lobules of mature adipose tissue. These lobules are
separated by delicate fibrovascular septae. (10X, H&E)

Figure 3(b): High power view shows the lobules of adipose tissue
composed of mature adipocytes. These adipocytes have abundant
clear cytoplasm which is pushing the nucleus towards the periphery.
(20X, H&E)
158 Lipoma of retromandibular space
Vol 2, No 2
authors choose to employ anticholinergic agents to suppress glan-
dular function during healing or in an attempt to close a fistula or
resolve a sialocele spontaneously. Commonly used agents are pro-
pantheline bromide and glycopyrrolate, which inhibits the action of
acetylcholine at the postganglionic nerve endings of the parasym-
pathetic nervous system [4, 6, 7].
Received: 17 February 2014 Accepted: 28 March 2014
References
1. Fergnani ER, Pires FR, Falzoni R, Lopes M, Vorgas PA: Lipomas of the oral cavity
, Clinical findings, histological classification and proliferative activity of 46
cases. Int J Oral Maxillofac Surg 2003, 32:49-53.
2. Barnes L: Tumors and tumor like lesions of the head and neck. In: Barnes L,
editor. Surgical Pathology of the Head and Neck. 1st ed. New York: Dekker,
1985. pp 747-58.
3. El-Monem MH, Gaafar AH, Magdy EA: Lipomas of the head and neck:
Presentation variability and diagnostic work up. J Laryngol Otol 2006, 120:47-
55.
4. Kumarswamy SV, Nanjappa M, Keerthi R, Singh S: Lipomas of oral cavity case
reports with review of literature. J Maxillofac Oral Surg 2009, 8:394-397.
5. Enzinger FM, Weiss SW: Benign lipomatous tumours. In: Enzinger FM, Weiss
SW, eds. Soft tissue tumours. 3rd ed. St Louis: Mosby, 1995. pp 381-430.
6. Som PM, Scherl MP, Rao VM, Biller HF: Rare presentations of ordinary lipomas
of the head and neck: a review. AJNR Am J Neuroradiol. 1986, 7:657-64.
7. Rogers J, Patil Y, Strickland Marmol L, Pandhya T: Lipomatous tumours of
paraphrayngeal space: Case series and review of literature. Arch Otolarngol
Head Neck Surg 2010, 136:621-624.
8. Tien RD, Hesselink JR, Chu PK, Szumowski J: Improved detection and
delineation of head and neck lesions with fat suppression spin-echo MR
imaging. AJNR Am J Neuroradiol. 1991, 12:19-24.
9. Kimura Y, Ishikawa N, Goutsu K, Kitamura K, Kishimoto S: Lipoma in the deep
lobe of the parotid gland: a case report. Auris Nasus Larynx. 2002, 29:391-3.
10. Furlong MA, Fanburg-Smith JC, Childers ELB: Lipoma of the oral and
maxillofacial region: Site and subclassification of 125 cases. Oral Surgery, Oral
Medicine, Oral Pathology, Oral Radiology, and Endodontology 2004, 98:441-
450.

Sarkar S 159

http://www.mednifico.com/index.php/elmedj/article/view/140 Vol 2, No 2
Open Access Opinion and Debate
Antidepressants in the management of bipolar depression - An appraisal
Siddharth Sarkar
1

Introduction
The issue about whether and when to use antidepressants in bipolar
depression is a clinical question that has generated a lot of passion-
ate debate [1, 2]. There seems to be a discrepancy in the opinions of
experts across the Atlantic Ocean about the pros and cons of using
antidepressants for management of bipolar depression [35]. This is
amply reflected by the greater volume of discussion and opinion re-
lated scientific literature, rather than hardcore clinical data in the
form of randomized controlled trials. The debate as of present exists
with no final winner still as the use of antidepressants in bipolar de-
pression has both pros and cons. This essay attempts to offer evi-
dence to either of the viewpoints and offer some suggestions based
on literature.
The debate exists as there are benefits and potential harms of use of
antidepressants in bipolar depression. The pros of using antidepres-
sants in this disorder understandably include quick and effective re-
lief from symptoms of depression. This should translate into reduc-
ing the distress due to psychiatric problem, lowering the suicidal risk
and improving the quality of life. In fact, one of the major causes of
death in patients with bipolar disorder is suicide, and effectively
treating depression may mitigate the risk of suicide in these patients.
The cons that have been suggested about the use of antidepressants
include concerns of cycle acceleration and inducing switch. Manic
switch have been documented with almost all antidepressants and
is a potential cause of concern when being used in patients with
bipolar depression, especially when used without cover of mood sta-
bilizer.
But before exploring into the evidence, a few caveats pertinent to
literature on management of these patients need to be kept in mind.
The broad rubric of bipolar disorder encompasses a heterogeneous
patient population (as in Figure 1A to 1F) [6, 7]. Some patients may
have many manic and few depressive episodes while some primarily
depressive episodes with one hypomanic episode. Also, the fre-
quency and duration of episodes vary across the patients who are
overall labelled as having bipolar depression. Some patients may
have two episodes in ten years while others may have ten episodes
in two years. Additional disorders like substance use and medical ill-
nesses may complicate the picture and influence management deci-

1
Department of Psychiatry, JIPMER, Puducherry, India
Correspondence: Siddharth Sarkar
Email: sidsarkar22@gmail.com
sions as well as prognosis. The psycho-social and occupational dys-
function (and premorbid function) may vary across the patients.
Hence evaluation of literature would require some degree of lump-
ing of heterogeneous patient population with their own idiosyncra-
sies and circumstances, quite akin to considering oranges and apples
as same bipolar disorder fruits (Figure 1). Nonetheless, clinical deci-
sion making is facilitated and enhanced by the presence of scientific
data to endorse one course of action over another.

The pros
The major pro of use of antidepressant in bipolar depression pertains
to reduction in symptoms of depression. However, the evidence
needs to be looked at more closely. The first presumption is that an-
tidepressants are more efficacious than placebo for treatment of bi-
polar depression. There is evidence from randomized controlled tri-
als and meta-analyses to suggest that antidepressants do work bet-
ter than placebo in bipolar depression [810]. However, such evi-
dence is based upon older literature and involved tricyclics and mon-
omanine oxidase inhibitors (MAOIs). Recent studies have relied upon
active comparators or adjunctive medications in the form of antipsy-
chotics and mood stabilizers, to avoid denying the standard of care
to the patients [11, 12]. These studies also support the efficacy of

Abstract
The issue about whether and when to use antidepressants in bipolar depression is a clinical question that has generated a lot of passionate
debate. There seems to be a discrepancy in the opinions of experts across the Atlantic Ocean about the pros and cons of using antidepressants
for management of bipolar depression. This is amply reflected by the greater volume of discussion and opinion related scientific literature,
rather than hardcore clinical data in the form of randomized controlled trials. The debate as of present exists with no final winner still as the
use of antidepressants in bipolar depression has both pros and cons. This essay attempts to offer evidence to either of the viewpoints and
offer some suggestions based on literature. (El Med J 2:2; 2014)
Keywords: Bipolar Disorder, Depression, Management, Antidepressants, Antipsychotics, Controversy
Figure 1: Various facets of bipolar disorder
160 Antidepressants in the management of bipolar depression
Vol 2, No 2
antidepressants vis-a-vis placebo as an add-on treatment. Whether
antidepressants are related to lesser suicidal ideations and attempts
has not been assessed systematically in bipolar patients, though
studies of unipolar depression suggest that antidepressants are ef-
fective in reducing suicidality. By extension of unipolar depression, it
can be suggested that antidepressants are also effective in reducing
suicidality in bipolar depression. However, use of selective serotonin
reuptake inhibitors (SSRIs) has been associated with exacerbation of
suicidal ideations and attempts in the initial period, though this as-
sociation has also been inconclusive [13, 14]. Lithium on the other
hand has also been suggested to reduce suicidality in bipolar pa-
tients. Thus, whether antidepressants help with respect to suicidality
in patients with bipolar depression needs to be seen.
The second corollary is that antidepressants are more efficacious
than other treatment of bipolar disorder (including mood stabilizers
and antipsychotics). EMBOLDEN II was a double blind randomized
controlled study which provided evidence that quetiapine was supe-
rior to paroxetine and placebo in patients being treated for bipolar
depression [15]. This industry sponsored trial suggested SSRIs may
be less efficacious than antipsychotics in reducing the symptoms of
depression. In a previous study of lithium maintained patients, ami-
triptyline was found to be equivalent to sulpiride in relieving depres-
sion [16]. Thus, establishing superiority of antidepressants over other
pharmacological treatment of bipolar depression may be difficult.
A few studies have compared mood stabilizers as monotherapy with
antidepressants. A comparative study of fluoxetine and lithium in
patients of bipolar II disorder suggested that fluoxetine was more
effective than lithium and placebo in delaying a relapse [17]. Another
open label study in patients with bipolar II disorder suggested that
venlafaxine was better than lithium as a monotherapy in controlling
symptoms of depression [18]. But again, these trials were confined
to bipolar II disorder and may not be generalizable for the whole
gamut of bipolar disorders as in Figure 1.
Summarizing, antidepressants seem to work better than placebo in
bipolar depression, but evidence of superiority over other medica-
tions seem to be inconclusive.
The cons
The major drawback that has been suggested about the use of anti-
depressants in bipolar depression is the emergence of manic switch.
Though a manic switch is difficult to concretely operationalize, most
authorities consider switch as emergence of mania within 2 months
of initiation of an antidepressant or escalation of dose. Definitively
ascribing a causal relationship of antidepressant with mania or hy-
pomania may be cumbersome due to presence of confounders and
natural course of the illness. Nonetheless, switch has been suggested
to be present with almost all antidepressants, only the propensity
varies. Mania induced by an antidepressant may put forth an addi-
tional burden on the healthcare system as well as the family, and
may destabilize the patients condition. Hence whenever possible, it
would be prudent to avoid such a switch, but not at the cost of un-
der-treatment of the patient.
The rates of switch to mania and/or hypomania with the use of anti-
depressants seem to be higher in bipolar I disorder than bipolar II
[19]. Such rates of switch are greater than what is encountered in
patients of major depression. Thus, while switch rates of about 14%
can be expected in the acute phase of treatment for bipolar I depres-
sion, the rates drop to about 7% for bipolar II disorder. Also, it has
been found that rates of antidepressant induced switch in the
maintenance period are almost double of that in the acute phase of
treatment [19]. The switch evolves to exclusively hypomanic epi-
sodes in bipolar II, while it emerges with equal frequency to mania
and hypomania in bipolar I disorder. It must also be remarked that
the presence of substance use disorder like alcohol or cannabis de-
pendence may enhance the propensity of a manic switch [2]. Such a
scenario is far likely to be encountered in the clinical setting than the
research scenarios, and hence the rates of manic switch may be
higher in real-world setting.
The choice of the type of antidepressant may also determine the risk
of manic switch [8]. It has been seen that tricyclic antidepressants
are at a greater risk of inducing switch than SSRIs [8, 20, 21]. A com-
parative study suggested the risk of switch to be higher with ven-
lafaxine, compared to sertaline (an SSRI) and bupropion [22]. Certain
other risk factors have been identified as risk factors for manic switch
with antidepressants in patients with bipolar depression. These in-
clude among others lower age of onset of illness, hyperthymic tem-
perament, history of suicide attempts, greater number of past manic
episodes, past history of switch and lower rate of response to anti-
depressants [21, 23-25]. So a careful consideration of these factors
may be required before prescription of antidepressants in bipolar
depression.
Apart from switch, use of antidepressant has been suggested to
cause cycle acceleration and destabilization of the course of illness
especially for rapidly cycling bipolar disorder [2, 26]. However, such
a relationship has been questioned in a critical appraisal which sug-
gests that the association of use of antidepressants and increase in
cycle frequency may not be causal in relationship [27].
Antidepressant alone or in conjunction with mood
stabilizer?
Use of a combination of mood stabilizer along with an antidepres-
sant in the acute phase has been studied in many trials. Nemeroff et
al found that for low serum levels of lithium (but within the clinically
acceptable range), use of mood stabilizer along with imipramine or
paroxetine led to greater improvement when lithium was used with
a placebo [12]. But such an effect was not found for higher serum
levels of lithium. It has also been seen that addition of an antidepres-
sant to a mood stabilizer yields greater benefits in treating bipolar
depression than addition of another mood stabilizer [28].
If combination of mood stabilizers and antidepressants are used in
patients with bipolar depression, the next logical question would be
for how long. It has been suggested that antidepressants should be
used for inducing remission of depression and is not required for the
continuation phase. A trial of mood stabilizer with adjunctive antide-
pressant treatment did not find use of antidepressants to be associ-
ated with better outcome in attaining stable remission over placebo
[29]. Two other previous reports of add on imipramine to lithium did
not reveal superiority of the combination in preventing recurrence
of affective episode, suggesting lack of efficacy in maintenance
Sarkar S 161
phase [30, 31]. However, research suggests that in particular set of
subjects, the efficacy of antidepressants continue in the maintenance
phase also. One of the largest non-industry funded studies on bipo-
lar depression, the STEP BD suggests towards continuation of anti-
depressants in patients who are treated for bipolar depression with
combination of mood stabilizer with antidepressants [32]. The ques-
tion of duration of combination drug use has not been fully settled
and requires a further look at the merits of using each of medications
based on the characteristics of the case in hand
Alternatives to antidepressants
The question arises that when one decides not to use antidepres-
sants, then what choices are available without limitations similar to
that of antidepressants. Antipsychotics provide a viable alternative
for managing depression in patients having bipolar disorders. Quet-
iapine has been tested in large placebo controlled trials, and has
been shown to be efficacious than placebo in terms of outcomes of
depression. The BipOLar DEpRession [BOLDER] study which recruited
over 500 patients suggested that quetiapine at doses of 300mg and
600mg was better than placebo for inducing remission measured on
Montgomery-Asberg Depression Rating Scale [33]. Similarly, a study
from Spain in patients with rapid cycling bipolar disorder also found
quetiapine more efficacious than placebo in inducing remission [34].
Calabrese et al found quetiapine in doses of 300mg and 600mg to
be more efficacious in relieving depression than placebo [35]. The
advantage of using antipsychotics in patients with bipolar depres-
sion relates to negligible propensity to switch, and control of mixed
affective and manic symptoms as they emerge. However, using them
as pharmacoprophylaxis for long duration of time entails the unfa-
vorable side effect profile and lower efficacy than mood stabilizers
for preventing recurrence of episodes [36].
Another line of evidence points towards use of mood stabilizers only
for patients with bipolar depression. Lithium, valproate and lamotro-
gine have been effectively used in the treatment of bipolar depres-
sion [37, 38]. The advantages of using lithium and valproate lies in
their prophylactic efficacy against manic episodes also, and hence
can be used for a longer term. Other modalities of treatment like
rTMS and electroconvulsive therapy has also been tried in patients
with bipolar depression with fair success and are treatment alterna-
tives available in therapeutic armamentarium [39, 40].
The special characteristics patients
Though efficacy data exists for the treatment of bipolar depression
with antidepressants, the application to the clinical practice does re-
quire taking into account the characteristics of the patients. Also, ef-
ficacy data do not translate to the clinical practice as many patients
would not fulfil the stringent inclusion criteria that a number of the
clinical trials require for patient enrolment. For safety purposes, effi-
cacy trials often exclude patients with severe liver, cardiac and renal
disorders, as also patients with concomitant substance use disorders.
Pregnant women, elderly and children are also commonly excluded.
For example, if we consider example of an alcohol dependent male
with bipolar depression and chronic kidney disease, difficulties
would be encountered while using efficacy data directly. Lithium
would be contraindicated in such a case and valproate would require
extreme caution due to hepatic effects. Hence one may consider ti-
trating up doses of SSRIs in such a patient if the previous episodes
have been of primarily depressive polarity. Such clinical conundrums
may not be just one off rarity, but are often encountered in clinical
settings and necessitate trade-offs. The guidelines offer general sug-
gestions that are applicable to most patients, but need to be used
based upon unique characteristics of the patient.
Alluding to the different forms of bipolar disorder themselves, rele-
vant modifications in the treatment would be beneficial. Referring to
the Figure 1, the patient with life history tagged as Figure 1A would
benefit by avoiding antidepressants, while those in Figure 1B and 1C
would merit use of antidepressants, probably also for long duration
as prophylaxis along with a mood stabilizer. Patients referred to in
Figure 1D and 1F would benefit from short duration of antidepres-
sant, with Figure 1E benefitting from mood stabilizer in combination
with antipsychotic. So, one size fits all tenet cannot be promulgated
for patients with bipolar depression.
Clinical guidelines may offer suggestions for treatment in such situ-
ations also, but their premise too is often based upon evidence base.
When evidence is lacking, expert consensus supply the recommen-
dations for clinical practice. The recent practice guidelines formu-
lated by a conclave of experts in the field of bipolar disorder have
been developed using the Delphi method [41]. A deeper look into
the methodology reveals that out of the 25 initial statements, less
than half were finally endorsed by more than 80% of the experts.
This suggests that there seems to difference in opinion of the world-
wide experts too about how to treat bipolar depression, presumably
due to the lack of rigorous hardcore scientific evidence.
Conclusions
The authors take on the management of bipolar depression and the
role of antidepressants based on the available guidelines and scien-
tific literature is summarized in table 1. Primacy should be given to
the history of the patient and the salient features of the case.
Given the present scenario, it is appealing to speculate about what
should be done in the future. The present state of conflicting litera-
ture is unlikely to pass away soon, probably because of heterogene-
ity in the construct of bipolar disorder itself. Just routine recommen-
dation about bigger and fancier multi centric trials is unlikely to re-
solve issues and give clear-cut answers about whether and to what
extent antidepressants should be used in bipolar depression. More
compelling substantiation of evidence can accrue from using
measures of properties of bipolar depression into composite scores
and testing against response parameters. Contextualizing the facets
like duration of illness, number of episodes, polarity of the episodes,
severity of episodes (needing hospitalization/ECT), response to med-
ications etc into an index can help in drawing comparative conclu-
sions about treatment response (like that done for polarity index)
[42]. Presently evidence do note some data like number of episodes
but present it in a non-comparative manner. Reducing the features
of the bipolar illness into dimensions (like that done for schizophre-
nia) with temporal and treatment characteristics as a dimension can
help elucidate which antidepressant (with what other medication)
shows benefits.
Just a prescription of antidepressant to a patient of bipolar depres-
sion may not yield desired results. Ensuring adherence to the medi-
162 Antidepressants in the management of bipolar depression
Vol 2, No 2
cation along with psychotherapeutic intervention when deemed
necessary can improve the outcomes of the patient. Probably it
would be correct to conjecture that judicious use of antidepressants
by a skilful clinician and a compliant patient can improve the out-
come.
Table 1: Conclusions on use of antidepressants in bipolar dis-
order
Bipolar depression,
acute phase
Optimize mood stabilizer
Start antidepressant if:
Severe depression
Bipolar II disorder
Past history of multiple episodes of
depression
Past history of good response to an-
tidepressants
Consider SSRIs or bupropion
Avoid antidepressant if:
Past history of manic switch
Past history of multiple manic epi-
sodes
Concomitant substance use disorder
Consider antipsychotic like quetiapine
and olanzapine
Consider antidepressant monotherapy if:
Bipolar II disorder with infrequent
hypomanic episodes
Bipolar depression,
maintenance phase
Optimize mood stabilizer, consider dis-
continuation of antidepressant, except
when:
Past history of depressive relapse
when exclusively on mood stabilizer
Bipolar II disorder with frequent re-
lapses
Consider antidepressant monotherapy for
maintenance if:
Bipolar II disorder with infrequent
hypomanic episodes

Competing interests: The author declares that no competing interests exist.
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164 Pre-treatment evaluation

Vol 2, No 2
Open Access Opinion and Debate
Pre-treatment evaluation: Setting a foundation in the management of drug-resistant tuberculosis
Saurabh RamBihariLal Shrivastava
1
, Prateek Saurabh Shrivastava
1
, Jegadeesh Ramasamy
1

Opinion and Debate
The global emergence of resistance to the anti-tuberculosis (TB)
drugs has posed a serious challenge to the efforts of program man-
agers and the health workers across the world to reduce the magni-
tude of TB [1]. A confirmed multidrug-resistant (MDR) TB case should
suffice three criteria namely: (1) Sputum culture positive for Myco-
bacterium tuberculosis; (2) Resistant to isoniazid and rifampicin; and
(3) the drug sensitivity testing (DST) done in a Revised National TB
Control Program (RNTCP) certified culture and DST laboratory [1, 2].
Global TB control report, 2012 released by the World Health Organi-
zation has revealed that India is contributing the maximum number
of MDR-TB cases to the global burden [3]. However, the accurate
magnitude of drug resistant TB cases is difficult to estimate as a ma-
jor proportion of general population does not seek public health sec-
tor for their morbidities and thus a significant number of diagnosed
drug resistant TB cases remains un-notified [3, 4].
From a public health perspective, early diagnosis of drug-resistant
TB cases and initiation of the patient on second-line anti-TB drugs is
the key strategy as MDR-TB is associated with poor cure rate, poor
treatment success rate, high default rate, fatal and non-fatal compli-
cations, transmission to contacts and deaths [5, 6]. Treatment com-
pliance rate throughout the course of treatment (viz. 24-27 months)
has been identified as the key indicator to predict an outcome of a
MDR-TB case [1, 2].
The primary step to ensure a good outcome is to initiate the treat-
ment regimen based on the results of a pre-treatment evaluation
(PTE) performed at the drug resistant TB center (DR-TB center) by a
DR-TB center committee consisting of specialists from different fra-
ternities of medicine. The basic motive of performing a PTE is to rec-
ognize those patients who are more prone to experience harmful
drug reactions owing to consumption of potentially toxic second-
line MDR-TB drugs. PTE essentially consists of eliciting a comprehen-
sive and exhaustive history, meticulous and elaborative clinical as-
sessment, anthropometric measurements - height (for adjusting
therapeutic doses of renal-toxic drugs) and weight (for designating
a suitable weight band of treatment), complete blood profile, blood

1
Shri Sathya Sai Medical College and Research Institute, India
Correspondence: Saurabh RamBihariLal Shrivastava
Email: drshrishri2008@gmail.com
sugar, liver function tests, renal function tests - blood urea and serum
creatinine, thyroid stimulating hormone levels, urine routine, chest
x-ray and pregnancy test (for all women in the childbearing age-
group). PTE for extensively DR-TB cases in addition to above param-
eters, includes electrocardiogram, serum electrolyte levels, and sur-
gical appraisal. Further, all drug resistant TB cases are offered HIV
counseling and testing services and a psychiatric assessment is war-
ranted if patient is depressed or addicted to drugs/alcohol. However,
other than the clinical examination and a range of laboratory inves-
tigations, the place of counseling in PTE for the patient and their
family members is the most indispensable element which consists of
information about the nature and duration of therapy, adverse ef-
fects and consequences of irregular treatment. All the women in the
reproductive age-group are informed to adopt contraceptive
measures during the course of treatment as some of the second-line
TB drugs are teratogenic [2].
At the outset when treatment was initiated for drug-resistant TB
cases in the country, the only option for conducting PTE was to ad-
mit the patient at the DR-TB center for an initial period to gauge the
response to treatment and if tolerated, subsequently discharge them
for further treatment near to the patients residence. Although, this
strategy was put forth to adjust the therapeutic schedule based on
the observed adverse effects, if any, during the period of hospitali-
zation, but later on owing to the bed constraints at the DR-TB center
and the unwilling nature of patients to get admitted at the DR-TB
center, the program introduced the strategy of out-patient PTE. This
was done to keep up the Government commitment to not refuse
treatment for any reason. Hence, the local District TB Officer was
made accountable to ensure that all the preliminary tests that are
done as a part of PTE will be done in the nearby government hospital
and once reports are obtained, patient visits the DR-TB center for PTE
on an out-patient basis [2].
Altogether, PTE plays a critical role in ensuring a successful outcome
to the treatment of drug-resistant TB as a comprehensive PTE can

Abstract
The global emergence of resistance to the anti-tuberculosis (TB) drugs has posed a serious challenge to the efforts of program managers
and the health workers all across the world to reduce the magnitude of TB. From a public health perspective, early diagnosis of drug-resistant
TB cases and initiation of the patient on second-line anti-TB drugs is the key strategy as MDR-TB is associated with poor cure rate, poor
treatment success rate, high default rate, fatal and non-fatal complications, transmission to contacts and deaths. The primary step to ensure
a good outcome is to initiate the treatment regimen based on the results of a pre-treatment evaluation (PTE) performed at the drug resistant
TB center by an expert committee. Altogether, PTE plays a critical role in ensuring a successful outcome to the treatment of drug-resistant
TB as a comprehensive PTE can build a key foundation by averting the probable drug induced side effects right at the start of the treatment.
(El Med J 2:2; 2014)
Keywords: Tuberculosis, Multi-drug Resistant, Pre-treatment Evaluation, India
Shrivastava SR, Shrivastava PS, Ramasamy J 165
build a key foundation by averting the probable drug induced side
effects right at the start of the treatment.
References
1. Managing the RNTCP in your area - A training course (Modules 1-4).
[http://tbcindia.nic.in/documents.html].
2. Guidelines for PMDT in India [http://tbcindia.nic.in/documents.html]
3. World Health Organization. Global Tuberculosis Control Report 2012. Geneva:
WHO press; 2012.
4. National family health survey (NFHS-3), 2005-06
[http://www.measuredhs.com/pubs/pdf/SR128/SR128.pdf]
5. Garcia de la Osa Mde L, Garcia Silvera E, Solano Leal M, Milanes Virelles MT:
Response to therapy in multiple drug resistant tuberculosis patients. Rev
Cubana Med. Trop 2012; 64(2):153-62.
6. Unsal E, Guler M, Ofluoglu R, Capan N, Cimen F: Factors associated with
treatment outcome in 64 HIV negative patients with multidrug resistant
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166 Stem cells from gynecological tissue

Vol 2, No 2
Open Access Essay
Stem cells from gynecological tissue: Trash to treasure
S Indumathi
1
, B Padmanav
2
, D Sudarsanam
1
, B Ramesh
3
, M Dhanasekaran
4

Essay
Despite advances in biomedical research even in modern decades,
millions of people still suffer from devastating diseases. One of the
most surprising discoveries of recent decade that are believed to re-
solve these issues is the identification of stem cells in almost every
adult tissues/organs. One potential reason that this discovery has
gained importance is due to the fact that human development oc-
curs from stem cells. Stem cells in recent years have configured re-
generative medicine with their wide range of therapeutic potentials.
This revolutionary change has increased the demand of stem cells
for myriad of diseases. Their ability to treat so many diseases rests
on their unique properties of self-renewal and differentiation.
Stem cells from gynecological tissues are gaining importance, due to
their dynamic tissue regeneration capacity throughout the reproduc-
tive phase of women [1, 2]. Based on the dynamic tissue remodeling
in all compartments of the uterus, during the menstrual cycle and
pregnancy, it has been suggested that adult stem cells of the endo-
metrium plays a role in uterine tissue maintenance and function
marked by its high self-renewal and regenerative potential [3, 4].
Similar to the endometrial stem cell research, tubectomy and hyster-
ectomy surgical discards of fallopian tube also lag far behind the area
of stem cell research. The embryological origin of fallopian tube is
same as that of endometrium and has found to undergo cyclical en-
docrine-mediated changes such as cell growth and regeneration
necessary to maintain gamete viability, fertilization, embryologic de-
velopment and translocation to the uterus [5]. Recently, these po-
tential sources have been proved as an accessible, non-invasive ra-
ther potent source of stem cells for therapeutic interventions [6].
Menstrual blood has become the most convenient source in the
search for endometrial stem cells because collecting menstrual
blood is easy and non-invasive and endometrial stem/progenitor
cells are shed in menstrual blood [7-10]. This is due to the fact that
menstrual blood includes the apical portion of the endometrial
stroma. Each menstrual cycle is associated with vascular prolifera-
tion, glandular secretion and the endometrial growth. Absence of
progesterone, the demise of corpus luteum and the subsequent fall
in circulating progesterone lead to vasoconstriction, necrosis of the

1
Department of Zoology and Biotechnology, Loyola College, Chennai, India
2
Department of Stem cells, National Institute of Nutrition, Hyderabad, India
3
Manipal Institute of Regenerative Medicine, Bangalore, India
4
Ree Laboratories Pvt. Ltd, Mumbai, India
endometrium and menstruation. For these reasons, reliable studies
on menstrual blood derived stem cells are in process. They are re-
ported to provide great promise for use in tissue repair and treat-
ment of diseases, due to the plasticity and longevity of the cells. Alt-
hough menstrual blood has proven to be a unique and novel source
of stromal cells from the endometrial functionalis, putative adult
stem or progenitor cells that are responsible for the cyclical regener-
ation of the endometrium functionalis, every month reside in the ba-
salis region of the endometrium, as described earlier [6, 11].
Study of these stem cells from the basalis layer of the endometrial
tissue is still in its infancy. Based on the dynamic tissue remodelling
in all compartments of the uterus, during the menstrual cycle and
pregnancy, it has been suggested that adult stem cells from the en-
dometrial tissue play a vital role. Hence, a thorough characterization
of the uterine/endometrial stem cells derived from the endometrial
tissue biopsy of the inner lining of the uterus is of utmost im-
portance. Once a mechanical or functional characteristic platform
has been constructed, it then becomes easier to understand the
complex mechanisms underlying the morphogenesis and physiolog-
ical generation of the female reproductive tract, to improve the un-
derstanding of the pathophysiology of gynecologic diseases such as
endometrial cancer, fibroids, endometriosis and pregnancy loss as
well as determine the possible roles of endometrial stem/progenitor
cells of the female reproductive tract to these gynecologic diseases,
thereby considering them a possible therapeutic target for treat-
ment of wide horizon of diseases in regenerative medicine.
Similarly, fallopian tube has the capacity to undergo dynamic endo-
crine-induced changes during the menstrual cycle, including cell
growth and regeneration, in order to provide the unique environ-
ment required for the maintenance of these aforesaid functions.
With these attributes, human fallopian tubes, which are discarded
during surgical procedures of women submitted to sterilization or
hysterectomies, are considered to be a rich source of mesenchymal
stromal cells as specified above. The epithelial cells isolation from
lining of the inner surface of the Fallopian tube was first described
by Henriksen and co-workers to establish a method to culture these
Correspondence: S. Indumathi
Email: indu.stemcell@gmail.com
Abstract
Stem cell research has opened new avenues for developing therapeutic options for targeting disease as well to understand the underlying
principles of several pathological conditions when these stem cells go awry. This has increased the quest for identifying ideal stem cells and
their role in regeneration. The gynecological redundant sources such as endometrium and fallopian tube seem to have been neglected as a
source of stem cell for therapeutic application. The present essay unravels the potency of stem cells derived from these sources, thereby
bringing into light their therapeutic application from trash to a treasure. With their immense potency, these stem cells can also be banked for
off-the-shelf allogeneic stem cell therapy. (El Med J 2:2; 2014)
Keywords: Stem Cells, Endometrium, Menstrual Blood, Fallopian Tube, Stem Cell Therapy
Indumathi S, Padmanav B, Sudarsanam D et al 167
cells as a model for more specific studies of their properties [12].
However their stem cells status remains to be seen.
A great breakthrough has been achieved by the identification and
isolation of the stem cells from endometrium and fallopian tube. A
major advantage of being able to identify the cell surface markers of
epithelial and stromal population of the endometrium and fallopian
tube is that their features can be characterized in non-cultured cells,
and their utility in cell based therapies for regenerative medicine can
be evaluated in pre-clinical disease models. Recently, this was
achieved by our team and the detailed phenotypic characterization
of the endometrial and fallopian tube derived cells has been demon-
strated [13]. Besides, a proven record on its long term self-renewal
capacity and multi-differentiation ability of these sources have also
been demonstrated. These include its differentiation into adipocytes,
osteocytes, smooth muscle cells, endothelial cells, chondrocytes and
neuronal cells [2, 3, 9, 10, 14-16]. Thus, it is apparent that stem cells
from these trash sources could be of great therapeutic value.
We draw focus to the oft-ignored stem cell source and elucidate its
significance as a cutting edge source for therapeutics, due to its tre-
mendous regenerative capacity and remodeling throughout repro-
ductive life. However, further investigations are mandatory on its
therapeutic efficacy as well on its off-the-shelf banking capacity.
References
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13. Dhanasekaran M, Indumathi S, Lissa RP, Harikrishnan R, Rajkumar JS,
Sudarsanam D: A comprehensive study on optimization of proliferation and
differentiation potency of bone marrow derived mesenchymal stem cells
under prolonged culture condition. Cytotechnology 2013, 65(2):187-197.
14. Kato K, Yoshimoto M, Kato K et al: Characterization of side population cells in
human normal endometrium. Human Reproduction 2007, 22: 1214-1223.
15. Tsuji S, Yoshimoto M, Kato K et al: Side population cells contribute to the
genesis of human endometrium. Fertil Steril 2008, 90: 1528-1537.
16. Masuda H, Matsuzaki Y, Hiratsu E et al: Stem cell- Like properties of the
Endomertial side population: Implication in Endometrial regeneration. Plos
One 2010, 5(4).

168 Evolutionary context of hypertensive disorders in human pregnancy

Vol 2, No 2
Open Access Essay
Evolutionary context of hypertensive disorders in human pregnancy
Abhay Kumar Pandey
1
, Anjali Rani
2
, Shripad B Deshpande
1
, B L Pandey
3

Introduction
Physiology of mother has vital bearing on pregnancy, and its out-
come. The duration of pregnancy compels unparalleled adaptations
in lifetime, which are tuned to render healthy offspring. The process
exhibits evolution by acquiring and deleting factors contributing to
reproductive success and failure, respectively. Pregnancy-induced
hypertensive disorder is near exclusively human malady with pla-
centa the apparent pathologic locus. Research continues to unravel
mysteries of etiopathogenesis of eruption and ramifications of the
problems. Humans are indeed predisposed to ischemia/hypoxia dis-
turbance of uteroplacental circulation. Further, gender differences
are apparent in humans in autonomic nervous regulation of circula-
tion [1]. Determinants of risk impose burden on balancing physio-
logical mechanisms of uteroplacental circulation to a threshold when
essentially abnormal molecular mechanisms set in. Evolutionary
framework is briefly appraised including impact of high altitude hy-
poxic insult on uteroplacental circulation. Research findings may bet-
ter be understood for clinical relevance in context of the evolution-
ary structure and hemodynamics.
The Placenta
Placentae are defined anatomically on the basis of fetal villous struc-
ture, geometry of maternal fetal blood flow, shape or interhemal bar-
rier structure. Number of cell layers that separate maternal and fetal
blood is traditional basis for placenta classification [2]. The hemo-
chorial placenta in humans characterizes invasive trophoblast in in-
timate relation with maternal blood. Its formation begins with the
blastocyst breeching endometrium, invading inward and deeper in
endometrial tissue to anchor the conceptus. Fetal trophoblast cells
differentiate into villous or extra-villous cell types. These may fuse
forming multinucleated layer in contact with endometrium or fur-
ther migrate through the tissue to form extra-villous cytotropho-
blast. Some of the later cells serve paracrine signals while others ex-
press cell surface antigens enabling migration up the spiral blood
vessels. The process of endovascular migration involves angiopoietin
2 and its receptor Tie 2 [3]. Erosion of smooth muscle of maternal

1
Department of Physiology, Institute of Medical Sciences, Banaras Hindu
University, Varanasi-221005 (UP), India.
2
Department of Obstetrics and Gynecology, Institute of Medical Sciences, Banaras
Hindu University, Varanasi-221005 (UP), India.
spiral arteries causes fall in resistance facilitating high uteroplacental
blood flow when blood pressure remains unchanged. There is also
increased flow and sheer stress in upstream uterine artery, stimulat-
ing its growth and further increase in placental blood flow [4]. In the
humans, trophoblast cells are highly dispersed throughout the
uterus. Area of placenta occupies larger portion of uterus and larger
number of uterine arteries. This suggests functional significance. Im-
plantation in humans is particularly deep with complete embedding
of blastocyst in uterine stroma, and there is early timing of blastocyst
implantation than in other primates [5].
Pregnancy Hemodynamics
In pregnancy, after implantation there is annual 40% increase in
plasma volume and urine output and ventilation increases by about
25%. Uteroplacental blood flow supplying oxygen and nutrients for
developing fetus thus increase. The bipedalism narrows the dimen-
sions of true pelvis in humans, which homes uterus, bladder, internal
genital, and much of lower intestine. Expanding uterus and its con-
tents occupy much of the abdominal cavity. This associates compres-
sion of blood vessels especially inferior vena cava. This causes re-
duced cardiac output near term, especially in supine posture, with
tone of abdominal muscles adding to compression in bipedal sys-
tem. There is also chronic elevation in sympathetic tone to oppose
gravity for ensuring adequate cerebral perfusion. This is additional
cause of reduced venous return and decrease in cardiac output. Ina-
bility to overcome raised sympathetic tone and consequent reduc-
tion in plasma volume, form the link between latter and increased
pre-eclampsia risk [6]. Vasodilatation and structural remodelling of
uteroplacental vasculature is critically needed in humans therefore,
to ensure the exponential rise in blood flew near term. The charac-
teristics as early and deep invasiveness, greater placental area and
larger number of maternal arteries accessed by fetal cells thus ap-
pear as evolutionary adaptations for pregnancy in bipedal humans.
Bipedalism and risk in human pregnancy
3
Department of Pharmacology, Institute of Medical Sciences, Banaras Hindu Uni-
versity, Varanasi-221005 (UP), India.
Correspondence: Abhay Kumar Pandey
Email: abhay.physiology@gmail.com
Abstract
The erect posture and bipedalism in humans has evolved at cost of compromise of abilities in meeting some challenging demands of
physiology. Counter adaptive mechanisms have emerged and keen understanding of their pliability is crucial to health and management of
disease. Several features of human placenta are distinctive in providing advantage for uteroplacental blood supply. The depth and extent of
rooting of fetal trophoblastic cells in the maternal tissues and timing of implantation are particularly of scientific interest. Stressors upon
such system and their impact at molecular level constitute risk for unique diseases of human pregnancy, the hypertension and preeclampsia.
The study of such mechanisms is crucial to advance diagnostic and therapeutic strategies, and calls for very natural experimentation.
Reproduction at high altitude implies particular stress and strain on mechanisms toward ensuring high uteroplacental blood flow for feto-
maternal wellbeing. The context deserves intense examination toward medical advance in dreaded disease of pregnancy-induced
hypertension and preeclampsia. (El Med J 2:2; 2014)
Keywords: Pregnancy-induced Hypertension, Preeclampsia, Uteroplacental Circulation, Placenta, Human Female
Pandey AK, Rani A, Deshpande SB et al 169
Preeclampsia associates reduction in uteroplacental perfusion with
incomplete trophoblast invasion. Around tenth of primigravida preg-
nancies are afflicted by preeclampsia as unique human disease. In-
complete trophoblast invasion and tissue hypoxia trigger release of
reactive oxygen species or other toxic entities in placental circula-
tion, damaging endothelium and impairing normal heightened vas-
odilator response of pregnancy. The disorder appears to be based in
placenta. Although conflict between fetal and maternal genome is
one of the proposed base, the changes leading to maternal hyper-
tension following poor placental perfusion remain unique to hu-
mans. Preeclampsia is more likely to result in preterm delivery and
intrauterine growth restriction of fetus with reduced chances for sur-
vival. Biomechanical constrains imposed by bipedalism appear to
evoke compensatory early implantation and deep widespread inva-
sion of fetal trophoblast cells into maternal uterine vessels and pre-
dispose to pregnancy complication of preeclampsia.
Wisdom of studies at high altitude
High altitude associates lowered oxygen availability, interference
with implantation and compromise of development of fetal blood
supply. All these increase risk of preeclampsia and intrauterine
growth restriction [7, 8]. The consequent intrauterine growth re-
striction causes fetal programming increasing risk of cardiovascular
diseases in later life [9]. Individuals migrating from low to high alti-
tudes experience reproductive difficulty and high altitude increases
incidence of preeclampsia and intrauterine growth restriction of fe-
tus with increased risks to survival in uterus, in infancy and in adult
life. Hypoxic pathophysiology in newborns with pulmonary hyper-
tension and right heart failure is prominent killer [8]. Altitude in-
duced increase in preeclampsia incidence is known [7]. Magnitude
of altitude associated reduction in birth weight varies in relation to
the duration of residence at high altitude. Birth weight fell least in
longest resident groups and progressively more in shorter resident
populations. No protection is incurred by being born and raised at
high altitude [8]. Hill tribes, however, exhibit higher common iliac
flow near term and a greater increase in uterine artery diameter and
hence uteroplacental oxygen delivery as compared to new residents
of high altitude and suffer less risk of preeclampsia and intrauterine
growth restriction [8]. Identification of the mechanisms responsible
for evolutionary adaptation may facilitate search for solutions to se-
rious public health problem of preeclampsia and intrauterine growth
restriction.
References
1. Gandhi S, Singh J, Kiran: Gender and autonomic nervous system. Indian
Journal of Fundamental and Applied Life Sciences 2011, 1:172-179.
2. Mooris FH Jr, Boyd RD, Mahencran D: Placental transport. In KnobilE, Neil JD
editors, The Physiology of reproduction 2nd Ed. New York, Raven Press, 1994;
pp. 813-861.
3. Goldman-wohl DS, Ariel I, Greenfield C, Lavy Y, Yagel S: Tie-2 and angiopoietin-
2 expression at the fetal maternal interface: a receptor ligand model for
vascular remodelling. Mol Hum Reprod 2000, 6: 81-87.
4. Rockwell LC, Keyes LE, Moore LG: Chronic hypoxia diminishes pregnancy
associated DNA synthesis in guinea pig uteroplacental arteries. Placenta 2000,
21: 313-319.
5. Enders AC, Schifke S: Implantation in non-human primates and in the human.
In Dukolow WR, Rovin J editors Comparative Primate Biology, Vol 3,
Reproduction and Development, New York, Alan R Liss, 1986; pp.291-310.
6. Bernstein IM, Meyer MC, Oslo G, Ward K: Intolerance to volume expansion: a
theoretical mechanism for development of preeclampsia. Obstet Gynecol
1998, 92:306-308.
7. Palmer SK, Moore LG, Young DA, Creegor B, Berman JC, Zamudio S: Altered
blood pressure course during normal pregnancy and increased preeclampsia
at high altitude in colarado. Am J Obster Gynecol. 1999, 180: 1161-1168.
8. Moore LG, Young DY, Droma TS, Zhuang JG, Zamudio S: Tibetan protection
from intrauterine growth restriction and reproductive loss at high altitude. Am
J Hum Biol 2001, 13: 635-644.
9. Barker DJP, Bull AR, Osmoud C, Simmonds SJ: Fetal and placental size at risk
of hypertension in adult life. In Barker DJP, editor fetal and infant origins of
adult disease. BMJ, 1992, 175-186.

170 Therapeutic spectrum of diuretics in different diseases

Vol 2, No 2
Open Access Essay
Therapeutic spectrum of diuretics in different diseases
Muhammad Majid Aziz
1
, Muhammad Ikram Ur Rehman
1
, Muhammad Wajid
1
, Muhammad Ali Raza
1
, Javed Ahmed
1

Introduction
Diuretics have major role in numerous disorders such as congestive
heart failure, nephrosis, respiratory acidosis and in hypertension. Di-
uretics not only decrease the severity of the disease, but also cure
the disease [1].
Uses in Hypertension
Worldwide prevalence of hypertension varies in different geograph-
ical areas. It is reported that India has the minimum ratio and Poland
has the maximum ratio of hypertension in the world. In India, hyper-
tension rate in males is 3.4% and in females is 6.8%, while in Poland
this ratio is 68.9% in males and 72.5% in females. Only 46% of the
population has awareness of the disease. It is perhaps the biggest
health challenge both in under-developed and developed countries
because the treatment is commonly inadequate [2].
According to the new criteria of hypertension diagnosis (>140/90
mm Hg), prevalence of hypertension is 15-35% in urban area of Asia
in adult population. While in the villages the ratio of hypertension is
two to three times lesser than that in urban areas [3]. In the case of
Pakistan, the Baloch ethnicity has the maximum ratio, which is 25.3%
in men and 41.4% in women. Punjabis have the minimum ratio,
which is 17.3% in men and 16.4% in women. For Sindhis, it is 19% in
men and 9.9% in women, for Pashtuns, 23.7% in men and 28.4% in
women, for Muhajirs, 24.1% in men and 24.6% in women [4]. In low-
income settlements of Karachi, the prevalence of hypertension has
been observed to be 26%. The rate of occurrence of hypertension is
34% in males and 24% in females [5].
Diuretics have conventionally been used in the treatment of heart
failure with fluid accumulation. Diuretics are used with the usual
medications such as angiotensin-converting enzyme (ACE) inhibi-
ters. In hypertension, diuretics are suggested as first-line therapy. It
is found that low-doses of diuretics are the most effective first-line
treatment for prevention of mortality and morbidity due to cardio-
vascular disease [6]. Thiazide diuretics are better for the prevention
of one or more major forms of cardiovascular diseases. Thiazide diu-
retics are preferred for antihypertensive therapy because these are
economical [7].
Uses in Edema
In Australia, the self-reported prevalence of edema is 1.6% [8].
Edema is caused by an imbalance in the filtration system between

1
Department of Pharmacy, Faculty of Pharmacy and Alternative Medicine, The
Islamia University of Bhawalpur, Pakistan
Correspondence: Muhammad Majid Aziz
Email: pharmajid82@yahoo.com
the capillary and interstitial spaces. The kidneys have an important
character for the adjustment of extracellular fluid volume by elimi-
nating sodium and water. Major reasons of edema include venous
obstacles, augmented capillary permeability, and higher plasma vol-
ume. Secondary reasons include sodium and water accumulation.
Using diuretics, avoiding sodium content in food and appropriate
management of the disease are the most important treatment mo-
dalities. Loop diuretics are used as single or in combination therapy
[9]. Retention of 3 to 4 liters of water in the extracellular spaces
causes edema. Additional extracellular fluid volume can be reduced
by the use of diuretics [10]. Diuretics are also very useful in the treat-
ment of edema-forming states [11].
Uses in Mild Heart Failure
Statistical data of mild heart failure indicates that 4.9 million U.S cit-
izens have mild to moderate clinical heart failure [12]. Rate of self-
reported congestive heart failure is 1.1% in the adult population of
US and the prevalence is 2% when evaluated on clinical criteria. Pa-
tients of left heart ventricular failure will be treated with vasodilators
along with diuretics, bronchodilators and narcotics. In right heart fail-
ure, low doses of diuretics are used in the reduction of excessive fluid
[13].
Uses in Acute Pulmonary Edema
Acute pulmonary edema is the accumulation of extravascular fluid in
the lungs. It is an important reason of lungs stiffness. Alveoli of the
lungs filled with water and breathing becomes very difficult. Acute
pulmonary edema is one of the most common life-threatening
health emergencies. Prevalence of acute pulmonary edema in hos-
pitalized patients has been found to be 1.0% and it is estimated that
the contribution of acute pulmonary edema to death is 0.2% [14].
Pulmonary pressure is decreased by intravenous administration of
loop diuretics within 530 min as diuretics exert vasodilating effects
[15]. Pulmonary edema and peripheral congestion decreases by the
use of diuretics. Right and left ventricular filling pressure is reduced
by the reduction in plasma with the use of diuretics. Diuretics reduce
extracellular fluid volume, total body water and sodium [16].
Uses in Liver Cirrhosis
The end-stage of numerous special chronic liver ailments is liver cir-
rhosis. Liver cirrhosis affects major organs and systems such as gas-
trointestinal tract and nutrition, respiratory, urinary, cardiovascular
and skeletal system. During 1995, mortality rate due to liver cirrhosis

Abstract
Diuretics have major role in numerous disorders such as congestive heart failure, nephrosis, respiratory acidosis and in hypertension. Diuretics
not only decrease the severity of the disease, but also cure the disease. This essay highlights some of their uses. (El Med J 2:2; 2014)
Keywords: Diuretics
Aziz MM, Rehman MIU, Wajid M et al 171
in Sweden, Finland and Denmark was 6.7, 10.6, and 16.7 per 100.000
people, respectively. During 2001-2005, incidence of liver cirrhosis
was 26.5 in males and 11.8 in females per 100.000 individuals [17].
In the Western world, alcoholic liver diseases and hepatitis C are the
most common causes of liver cirrhosis. In many areas of Asia and
Africa, hepatitis B is common cause of liver cirrhosis. The prevalence
rate of liver cirrhosis is 0.15% in the USA [18]. The prevalence is three
times greater in men than women [19].
In Pakistan, HCV is the most common cause of liver cirrhosis, HBV
being the second. In Pakistan patients with dual infection of HBV and
HCV comprise 8%. Seroprevalence of HCV in general population of
Pakistan is 4.7%. The maximum rate of HCV is observed in Faisalabad
and Lahore i.e. 16%. The ratio of HCV prevalence in Karachi has been
found to be minimum i.e. 1.6%. In blood donors. Seroprevalence ra-
tio is 3.03% in Pakistan. Islamabad has the maximum ratio (12.5%)
and Multan minimum ratio (0.3%) [20].
Diuretic therapy and salt restriction is necessary for treatment of mild
to moderate ascites of liver cirrhosis. Mild to moderate ascites is
treated with spironolactone alone. Initial dose is 100-200 mg/dl. A
combination therapy is important with furosemide in those patients
who do not respond to spironolactone monotherapy. Loop diuretics
alone are less effective as compare to spironolactone and are not
suggested. Ascites is treated by albumin infusion and diuretics. Pa-
tients with sodium flow less than 80 mmol/24hr need diuretics. The
therapy should be withdrawn when serum sodium is less than 120-
125 mmol/L. Diuretic therapy and sodium restriction are essential for
the prevention of re-occurrence of ascites [21].
Uses in Nephrotic Syndrome
Nephrotic syndrome is characterized by small pores in the podocytes
of the glomerulus, resulting in glomerular inflammation which leads
to proteinuria and hematuria. It can occur at any part of life. In fe-
males, it is usually asymptomatic and has slight practical impairment.
Males usually develop signs and symptoms of renal obstruction. Glo-
merular diseases have varying spectrum in different areas of the
world. Genetics, ecological contact and chemical agents have a no-
ticeable effect on the prevalence of nephrotic syndrome. In industri-
alized nations, nephropathy is most common [22]. The use of diuret-
ics alone is safe and effective, in patients with nephrotic syndrome
[23].
Uses in Glaucoma
Glaucoma is the second most common cause of irreversible blind-
ness globally. In 2010, glaucoma affected 8.4 million people in the
world. It is estimated that in 2020, almost 80 million people will be
affected with glaucoma [24]. Acute angle closure glaucoma (AACG)
is a common ophthalmic emergency, which requires suitable man-
agement to minimize the visual loss. Reduction in intraocular pres-
sure is very important requirement which is attained by using medi-
cal therapy such as carbonic anhydrase inhibitors, beta-blockers and
osmotic diuretics [25].
Uses in Acute Mountain Sickness
Population living above 16,000 feet height mainly suffers from acute
mountain sickness (AMS) e.g. residents of Gilgit in Pakistan. Two to
three month stay at high altitude and smoking have a role in its de-
velopment. The occurrence of AMS is the highest in 1 to 20 years age
group. The severity of AMS decreases with growing age [26]. Aceta-
zolamide has been suggested for prevention and treatment of AMS.
However, other diuretics have also been proved effective in this con-
dition. Exact mechanism by which diuretics may affect in the AMS is
not defined still [27].
Uses in Recurrent Stone Formation
In the USA, the prevalence of kidney stones is 12% in male and 5%
in female. Major cause of the kidney stone is calcium oxalate, which
causes the stone formation in 80% of individuals. Calcium phosphate
causes stone formation in 10%, uric acid 9%, and other reasons com-
prising the remaining 1% include cystine formation, drug related
stones and ammonium acid urate [28]. Nutritional control of calcium
and oxalate, combined with thiazide and potassium citrate, ade-
quately control the hypercalciuria, and also reduces the urinary oxa-
late, decreases concentration of calcium oxalate in urine, practically
eradicating recurrent stone formation [29].
References
1. Tierney, (2003) Diuretics, Current Medical Diagnose and Treatment Edition No.
42 Lang Medical Books McGraw Hill: 383-384.
2. Kearney PM, Whelton M, Reynolds K, Whelton PK, He J: Worldwide prevalence
of hypertension: a systematic review. Journal of hypertension 2004, 22(1):11-
19.
3. Singh RB: Preventive cardiology in medical hospital and research centre. J Hum
Hypertens 2000, 14(10):749-763.
4. Jafar TH, Levey AS, Jafary FH, White F, Gul A, Rahbar MH, Khan AQ, Hattersley
A, Schmid CH, Chaturvedi N: Ethnic subgroup differences in hypertension in
Pakistan. Journal of hypertension 2003, 21(5):905-912.
5. Safdar S, Omair A, Faisal U, Hasan H: Prevalence of hypertension in a low
income settlement of Karachi, Pakistan. JPMA The Journal of the Pakistan
Medical Association 2004, 54(10):506-509.
6. Psaty BM, Lumley T, Furberg CD, Schellenbaum G, Pahor M, Alderman MH,
Weiss NS: Health outcomes associated with various antihypertensive therapies
used as first-line agents: a network meta-analysis. JAMA : the journal of the
American Medical Association 2003, 289(19):2534-2544.
7. Major outcomes in high-risk hypertensive patients randomized to angiotensin-
converting enzyme inhibitor or calcium channel blocker vs diuretic: The
Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial
(ALLHAT). JAMA : the journal of the American Medical Association 2002,
288(23):2981-2997.
8. National Health Survey: Summary of results of 2006 Australia. Australian
Bureau of Statistics 2006: 5-85.
9. O'Brien JG, Chennubhotla SA, Chennubhotla RV: Treatment of edema.
American family physician 2005, 71(11):2111-2117.
10. Andre G: The Ins & Outs of diuretic therapy. The Can J Cont Med Edu 2004,
14(10): 55-60.
11. Ellison DH: Diuretic drugs and the treatment of edema: from clinic to bench
and back again. American journal of kidney diseases : the official journal of the
National Kidney Foundation 1994, 23(5):623-643.
12. Adams KF, Jr.: New epidemiologic perspectives concerning mild-to-moderate
heart failure. The American journal of medicine 2001, 110 Suppl 7A:6s-13s.
13. Nieminen MS, Bohm M, Cowie MR, Drexler H, Filippatos GS, Jondeau G, Hasin
Y, Lopez-Sendon J, Mebazaa A, Metra M et al: Executive summary of the
guidelines on the diagnosis and treatment of acute heart failure: the Task
Force on Acute Heart Failure of the European Society of Cardiology. European
heart journal 2005, 26(4):384-416.
14. Stein PD, Henry JW: Prevalence of acute pulmonary embolism among patients
in a general hospital and at autopsy. Chest 1995, 108(4):978-981.
172 Therapeutic spectrum of diuretics in different diseases
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15. Wilson JR, Reichek N, Dunkman WB, Goldberg S: Effect of diuresis on the
performance of the failing left ventricle in man. The American journal of
medicine 1981, 70(2):234-239.
16. Follath F: Do diuretics differ in terms of clinical outcome in congestive heart
failure? European heart journal 1998, 19 Suppl P:P5-8.
17. Steingerour AG: Liver cirrhosis epidemiological and clinical aspects, section of
gastroenterology and hepatology, department of internal medicine
Sahlgrenska university hospital Goteborg University Goteborg, Sweden, 2010:
9-11.
18. Schuppan D, Afdhal NH: Liver cirrhosis. Lancet 2008, 371(9615):838-851.
19. Catherine MF: The Epidemiology of Cirrhosis and Abnormal Liver Function Cell
Lines, J Ethnopharmacol 2010, 104: 407409.
20. Umar M, Bushra HT, Ahmad M, Data A, Ahmad M, Khurram M, Usman S, Arif M,
Adam T, Minhas Z et al: Hepatitis C in Pakistan: a review of available data.
Hepatitis monthly 2010, 10(3):205-214.
21. Biecker E: Diagnosis and therapy of ascites in liver cirrhosis. World journal of
gastroenterology : WJG 2011, 17(10):1237-1248.
22. Richard AP: Developing diuretics, modern drug discovery. Am Chem Soc 2003:
19-20.
23. Kapur G, Valentini RP, Imam AA, Mattoo TK: Treatment of severe edema in
children with nephrotic syndrome with diuretics alone--a prospective study.
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of a protocol for acute treatment. Eye (London, England) 1999, 13 ( Pt 5):613-
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26. Luqman M, Alamgir W, Farooq M: Acute severe mountain sickness, Pakistan
Armed Forces Medical Journal 2006, 3: 251-254.
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prophylaxis and treatment of acute mountain sickness. Journal of applied
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28. Coe FL, Evan A, Worcester E: Kidney stone disease. The Journal of clinical
investigation 2005, 115(10):2598-2608.
29. Pak CY, Heller HJ, Pearle MS, Odvina CV, Poindexter JR, Peterson RD:
Prevention of stone formation and bone loss in absorptive hypercalciuria by
combined dietary and pharmacological interventions. The Journal of urology
2003, 169(2):465-469.

Bansal T, Bansal M, Hooda S 173

Open Access Letter to Editor
Anesthetic considerations and implications for non-cardiac surgery in a patient presenting with aorto-
occlusive disease
Teena Bansal
1
, Manish Bansal
1
, Sarla Hooda
1

Introduction
Patients with vascular diseases are at a high risk for perioperative
cardiovascular complications [1]. Their cardiovascular function is of-
ten compromised preoperatively and they are frequently subjected
to extensive and prolonged surgeries. Numerous investigators have
postulated that intensive cardiovascular monitoring should be of
benefit to these patients and some have even attributed improved
cardiovascular outcome to such monitoring [2]. Here we report a
patient posted for emergency laparotomy for intestinal obstruction
presenting with aorto-occlusive disease.
Case Report
A 50 year old male, weighing 60 kg, was scheduled for emergency
laparotomy for subacute intestinal obstruction. He had a history of
dyspnea grade III and loss of consciousness 2 days prior to admis-
sion. There was no history of drug allergy. On general physical exam-
ination his pulse was 110/min in right radial artery, no palpable pulse
in femoral area or distally and blood pressure was 160/90 mm Hg.
Systemic examination revealed normal heart sounds and chest was
clear bilaterally. He had adequate mouth opening, normal neck and
temporomandibular joint movements, Mallampati class I on oropha-
ryngeal examination. His hemoglobin (Hb) was 9.8 gm/dL and other
investigations like bleeding time (BT), clotting time (CT), urine exam-
ination, blood urea, blood sugar and serum electrolytes were within
normal limits. His electrocardiography (ECG) showed QS pattern with
T wave inversion in leads II, III, avF (inferior chest leads), indicative of
old inferior wall myocardial infarction. His CT scan of abdomen
showed atheromatous plaque in aorta below superior mesenteric ar-
tery and inferiorly aorta was reduced in caliber. Also the right kidney
was atrophic. He was not on any medication previously.
As it was not a dire emergency, cardiothoracic and vascular surgery
opinion was taken. Cardiac surgeon advised CT angiography. CT an-
giography revealed insignificant narrowing of abdominal aorta by an
atheromatous plaque starting from origin of superior mesenteric ar-
tery and below. Aortic plaque was blocking right renal artery with no
flow in right renal artery and because of this reason right kidney was
atrophic. Left kidney was normal. There was total obliteration of
aorta by thrombus/plaque below the origin of left renal artery up to
aortic bifurcation. Bilaterally, common and external iliac artery were

1
Department of Anesthesiology & Critical Care, Pt. B.D. Sharma University of Health
Sciences, Rohtak (Haryana), India
Correspondence: Teena Bansal
Email: aggarwalteenu@rediffmail.com
totally blocked. Collaterals were seen in pelvis. Echocardiography
was done which revealed ejection fraction 50% with hypokinesia of
inferior wall. Cardiologist opinion was taken and he opined that no
active cardiac intervention was required.
General anesthesia along with lumbar epidural analgesia was
planned for the procedure. Lumbar epidural analgesia was planned
as it gives good analgesia, reduces the requirement of opioids and
stress response attenuation and also produces intense postoperative
analgesia. The anesthetic procedure was explained to the patient
and high risk written informed consent was obtained. In the operat-
ing room, intravenous line was secured with 16 G cannula using
ringer lactate. Monitoring of ECG by five leads, pulse oximetry (SpO2)
and non-invasive blood pressure (NiBP) was instituted. Emergency
cardiac drugs and defibrillator were kept ready. Lumbar epidural
catheter was placed under aseptic conditions. Rapid sequence induc-
tion was planned.
After pre-oxygenation for 3 minutes, anesthesia was induced with
injection of fentanyl (2 g/kg) IV, Inj. thiopentone sodium (5 mg/kg)
IV and intubation of trachea was facilitated using succinylcholine 1.5
mg/kg IV with Sellicks maneouver. To suppress the stress response
to laryngoscopy and intubation injection xylocard 1 mg/kg was
given prior to thiopentone sodium. Anesthesia was maintained with
1% isoflurane and O2. Intermittent doses of vecuronium bromide as
and when required were administered. Blood pressure, heart rate,
ECG (lead II and chest lead), SpO2, EtCO2 (end tidal carbon dioxide),
temperature, urine output were monitored throughout the in-
traoperative period. Intraoperative analgesia was provided with 12
ml of 0.25% bupivacaine (plain). After relief of intestinal obstruction,
67% N2O in O2 was started. Monitoring of ischemia should be done
by transesophageal echocardiography but as it was not available in
our emergency O.T., so we could not monitor. At the end of surgery
residual neuromuscular blockade was reversed with neostigmine
0.05 mg/kg and glycopyrrolate 0.01 mg/kg intravenously. Surgery
lasted for 2 hours and was uneventful. Postoperatively, monitoring
of NiBP, pulse, SpO2, ECG and temperature was done for 72 hours
and there were no fresh changes in ECG. Postoperative analgesia was
given with 0.0625% bupivacaine (plain) through epidural catheter.

Abstract
Patients with peripheral vascular disease continue to challenge anesthesiologists as these patients often have associated coronary artery
disease. There is always a danger of myocardial ischemia and cardiac morbidity both intra-operatively and postoperatively in this group of
patients. Here we report a patient posted for emergency laparotomy for intestinal obstruction presenting with aorto-occlusive disease. (El Med
J 2:2; 2014)
Keywords: Aorto-occlusive Disease, Non-cardiac Surgery, Atherosclerosis
174 Considerations for non-cardiac surgery in aorto-occlusive disease
Vol 2, No 2
Discussion
Atherosclerosis is often a generalized disease, affecting not only the
coronary circulation, but other parts of the vascular system as well.
Vascular diseases most commonly encountered in patients with cor-
onary atherosclerosis are carotid disease, abdominal aortic aneurysm
and obliterative atherosclerosis in aorto-iliac segment [3]. Patients
with vascular disease have a high incidence of co-existence of coro-
nary artery disease [4]. The increased prevalence of underlying cor-
onary artery disease explains the increased risk of cardiac complica-
tions in patients with peripheral vascular disease which has been re-
ported to range from 3 to 50 percent [5]. Patients with peripheral
vascular disease have a 3 to 5 times overall greater risk of cardiovas-
cular ischemic events, such as myocardial infarction, ischemic stroke
and death than those without this disease. Critical limb ischemia is
associated with a very high intermediate term morbidity and mortal-
ity due mostly to cardiovascular events. Therefore, cardiac risk as-
sessment should be done by bedside echocardiography to deter-
mine EF and wall motion abnormality. When considering the cardiac
status of such a patient, left ventricular function is an important ele-
ment as decreased left ventricular function alters the long term prog-
nosis.
Risk factors associated with development of atherosclerosis are dia-
betes mellitus, hypertension, smoking, dyslipidemia, hyperhomocys-
teinemia and a family history of premature atherosclerosis [6]. Pa-
tients should be evaluated for risk factors and their health should be
optimized prior to surgery if not a dire emergency. Anesthetic man-
agement of such a patient with peripheral vascular disease is similar
to anesthetic management of non-cardiac surgery in a cardiac pa-
tient [7]. The anesthetic goals are: i) stabilize hemodynamics; ii) pre-
vent MI by optimizing myocardial oxygen supply and reducing oxy-
gen demand; iii) monitor for ischemia; iv) treat ischemia or infarction
if it develops; v) normothermia; and vi) avoidance of significant ane-
mia. The most frequently encountered intraoperative problems are
myocardial ischemia and acute arterial occlusion. A perioperative is-
chemic process may provoke an impairment of cardiac function with
possible heart failure and/or dysrhythmia. Ischemia is the result of an
imbalance between myocardial oxygen supply and demand [8].
Maintenance of hemodynamic stability throughout the perioperative
period decreases the incidence of myocardial ischemia and improves
the postoperative cardiac status of the patient with coronary artery
disease by avoiding the deleterious effects of ischemic episodes re-
lated to hemodynamic changes. Thus, the major recommendations
of Merins editorials remains valid: Keep the determinants of myo-
cardial oxygen consumption as close to the pre-anesthetized angina
free value as possible, while maintaining coronary perfusion pres-
sure [9].
Myocardial ischemia should be monitored by 12 lead ECG and TEE.
Characteristic ECG changes will often occur during myocardial ische-
mia and will be detected with careful ECG monitoring. In the anes-
thetized patient, the detection of Ischemia by ECG becomes even
more important because the hallmark symptom of angina i.e. pain is
not available. The presence of ST segment depression usually indi-
cates sub-endocardial ischemia while ST segment elevation suggests
transmural Ischemia [10]. If only one lead can be displayed, V5
should be used because lead V5 has the greatest sensitivity of 75%
of detecting ischemia intra-operatively. Combining leads II and V5
which can detect 96% of ischemic events are suggested optimal
leads for detecting intraoperative myocardial Ischemia. The sensitiv-
ity is further increased by combining leads II, V4 and V5.
RWMAs (regional wall motion abnormalities) detected with two di-
mensional echocardiography have been shown to be the earliest and
most sensitive sign of myocardial Ischemia. A 25% decrease in coro-
nary blood flow produces an RWMA without ECG changes and a 50%
decrease is required to cause ECG signs of ischemia. An 80 percent
reduction of coronary blood flow causes akinesis and a 95 percent
decrease causes dyskinesia [11].
Management of intraoperative ischemia [7]:
If patient is hemodynamically stable:
a) Beta blockers (IV metoprolol up to 15 mg)
b) IV nitroglycerin
c) Heparin after consultation with surgeon
If patient is hemodynamically unstable:
a) Support with inotropes
b) Use of intraoperative balloon pump may be necessary. This
was not possible in this case because the patient had pe-
ripheral vascular disease involving aorta.
c) Urgent consultation with cardiologist to plan for earliest
possible cardiac catheterization
Acute occlusion of a previously patent extremity artery is a dramatic
event characterized by pulselessness, pain, pallor, paresthesia and
paralysis (the five Ps). Absence of pulses and pallor are early man-
ifestations and can be monitored under general anesthesia. The sud-
den onset of pain is very common and it may be intense. Motor
weakness and paresthesia are usually late manifestations of severe
ischemia. Acute ischemia needs to be evaluated rapidly because ir-
reversible tissue injury can occur within 4 to 6 hours [12]. Therefore,
pulses should be checked hourly. Immediate surgical revasculariza-
tion is generally indicated in the profoundly ischemic extremity.
Myocardial ischemia and cardiac morbidity occur most frequently in
the postoperative period. Patients should be carefully monitored for
signs and symptoms of ischemia. The determinants of myocardial
oxygen supply and demand should be optimized to prevent ische-
mia before it develops. Hypothermia is associated with an increased
incidence of myocardial ischemia and cardiac morbidity. Therefore,
body temperature should be carefully monitored. It is important to
control the stress response in the postoperative period. This includes
preventing the potential triggers for myocardial ischemia (pain, ane-
mia, hypothermia, hemodynamic extremes and ventilatory insuffi-
ciency).
To conclude, patients with peripheral vascular disease continue to
challenge the anesthesiologist, given the significant physiologic
stress superimposed on a relatively elderly patient with a high inci-
dence of coexisting disease. Clinical studies provide insight into the
preoperative assessment and optimization of cardiac risk, diagnosis,
prevention and treatment of myocardial ischemia in these patients.
Bansal T, Bansal M, Hooda S 175
References
1. Mangano DT: Perioperatve cardiac morbidity. Anesthesiology 1990; 72:153-84.
2. Rao TK, Jacobs KH, El-Etr AA: Reinfarction following anesthesia in patients with
myocardial infarction. Anesthesiology 1983; 59:499-505.
3. Moharana M, Agarwal S,Pratap H, Singh A, Tamagond S, Satsangi D K: Efficacy
and safety of beating heart coronary revascularization coupled with ascending
aorto-bifemoral grafting: analysis of short term results. IJTCVS 2010; 26:11-4.
4. Sukhija R, Aronow WS, Yalamanchili K, Sinha N, Babu S: Prevalence of coronary
artery disease, lower extremity peripheral arterial disease and cerebrovascular
disease in 110 men with an abdominal aortic aneurysm. Am J Cardiol 2004;
94:1358-9.
5. Cooperman M, Pflug B, Martin EW Jr et al: Cardiovascular risk factors in patients
with peripheral vascular disease. Surgery 1978; 84:305.
6. Hines RL, Marschall KE. Vascular disease. Stoeltings Anesthesia and co-existing
disease. 5th ed. 2009; 145.
7. Kaul TK, Tayal G: Anaesthetic considerations in cardiac patients undergoing
non cardiac surgery. IJA 2007; 51(4):280-6.
8. Kloner RA, Braunwald E: Observations on experimental myocardial ischemia.
Cardiovas Res 1980; 14:371-95.
9. Merin RG. Is anaesthesia beneficial for the ischemic heart? I, II and III.
Anesthesiology 1980; 53:439-40.
10. Chaitman BR, Hanson JS: Comparative sensitivity and specificity of exercise
electrocardiographic lead systems. Am J Cardiol 1981; 47:1335-49.
11. Eisenberg MJ, London MJ, Leung JM et al: Monitoring for myocardial ischemia
during non-cardiac surgery: a technology assessment of transesophageal
echocardiography and 12 lead electrocardiography JAMA 1992; 268:210-6.
12. Stewart MT: Assessment of peripheral vascular disease. In Hurst JW, Schlant
RC, Rackley CE, et al. (eds): The Heart.NewYork, Mcgraw-Hill, 1990, p368.
176 Extra-abdominal breast fibromatosis

Vol 2, No 2
Extra-abdominal breast fibromatosis: A rare breast pathology in medical practice
Mehmet Yildirim
1
, Nkhet Eliyatkin
2
, Hakan Postaci
2
, Nazif Erkan
1

Introduction
Breast fibromatosis (desmoid tumor), also called extra-abdominal
desmoid tumor is rarely seen in surgical practice. Currently, the re-
ported incidence of breast fibromatosis is 0.2% [1]. They usually oc-
cur in the 3
th
or 4
th
decade of life. In the WHO system for classification
of breast tumors, fibromatosis (aggressive) is included in the list of
mesenchymal tumors. Breast fibromatosis may develop within the
breast parenchyma or from the aponeurosis of the pectoralis major
muscle. Preoperatively, it is difficult to distinguish from breast cancer
with clinical findings and imaging studies [2].
Case Presentation
A 41-year-old female presented with a palpable lump in her left
breast. She had a history of oral contraceptives use for 30 months.
Upon physical examination, we detected a non-tender solitary mass
of 5 5 cm in dimension in the upper external quadrant of the left
breast. Ultrasound examination revealed a solid hyperechoic mass
with internal homogenous echo pattern. Mammography imaging
was classified as BI-RADS-3, suspicious for complicated cystic lesion
of breast, and patient was referred for surgical evaluation (Figure 1).

Lumpectomy was performed. Macroscopically, the lesion appeared
firm, yellow-colored and had a thick capsule. Microscopically, the
main cellular component consisted with spindle cells without pleo-
morphic cells and cellular atypia. Fibrous tissue stained positive with
Mason tri-chrome. Histopathological examination of the specimen

1
Department of Surgery, Izmir Bozyaka Education and Research Hospital, Turkey
2
Department of Pathology, Izmir Bozyaka Education and Research Hospital, Turkey
Correspondence: Mehmet Yildirim
Email: mehmetyildi@gmail.com
established the diagnosis of a fibromatosis with immunohistochem-
ical expression vimentin, CD34, actin and CD3 being positive, and ER,
PR, CK7 and S100 being negative in the spindle cells (Figure 2). No
further surgery was performed and she has not had recurrence after
3 years of the surgery.

Discussion and Conclusion
The etiologic factors of breast fibromatosis are unknown. It appears
to arise in the breast either sporadically, after blunt breast trauma or
a previous breast surgery, such as silicone breast implants. In addi-
tion, Gardner syndrome, familial adenomatous syndrome and use of
contraceptives has been reported as predisposing conditions [2].
These tumors may manifest initially as a palpable unilateral mass as
large as 10 cm and may show a rapid increase in size [3]. It is often
difficult clinically to distinguish from breast cancer if they are present
with skin dimpling and retraction.
Sonographically, fibromatosis appears as a hypoechoic mass mim-
icking breast malignant lesion with spiculated margin. On mammo-
graphically, breast fibromatosis typically manifests as an ill-defined
mass with spiculated margins but unlike breast cancer, it doesnt
contain calcifications [4]. In our case, we defined a mass with a
smooth border indicating complicated breast cyst. Imaging features
overlap between benign and malignant lesions, but tumors with a
maximal diameter greater than 3 cm generally should arouse suspi-

Abstract
Fibromatosis is a rare entity of the breast. Ultrasound and mammography are not helpful for diagnosis. The ideal treatment is complete excision
of the mass with clear margins. (El Med J 2:2; 2014)
Keywords: Fibromatosis, Breast, Extra-abdominal

Figure 1: Mammography imaging was classified as BI-RADS-3, suspicious for
complicated cystic lesion of breast
Figure 2: Microscopically, the main cellular component consisted with spindle
cells without pleomorphic cells
Yildirim M, Eliyatkin N, Postaci H et al 177
cion about their possible malignancy. Typical histological appear-
ance includes main cellular component consisted of spindle cells.
Complete surgical resection is ideal treatment because of high lo-
cally recurrence even several years after incomplete excision. Hor-
monal drugs, radiotherapy and chemotherapy have been used for
treatment [5].
In conclusion, fibromatosis should be considered in the differential
diagnosis of breast masses. Complete excision must be done, but
recurrence risk should be kept in mind.
References
1. Schwarz GS, Drotman M, Rosenblatt R, Milner L, Shamonki J, Osborne MP:
Fibromatosis of the breast: case report and current concepts in the
management of an uncommon lesion. Breast J 2006, 12(1): 66-71.
2. Greenberg D, McIntyre H, Ramsaroop R, Arthur J, Harman J: Aggressive
fibromatosis of the breast:a case report and literature review. Breast J 2002,
8(1): 55-7.
3. Yamaguchi H, Sakakibara T, Hino M, Ryu M, Senuma K, Nakai K, et al: A case of
fibromatosis of the breast. Breast Cancer 2002, 9(2): 175-8.
4. Glazebrook KN, Reynolds CA: Mammary Fibromatosis. AJR 2009, 193: 856-60.
5. Privette A, Fenton SJ, Mone MC, Kennedy AM, Nelson EW: Desmoid tumor: a
case of mistaken identity. Breast J 2005, 11(1): 60-4.
178 Sarcomatoid lung cancer

Vol 2, No 2
Sarcomatoid lung cancer: a rare, aggressive form of non-small cell lung cancer with an initially indolent
presentation in one of the youngest documented patients
Arpan Patel
1
, Joshi Sumendra
1
, Dinesh Ananthan
1
, Hayas Haseer Koya
1

Introduction
Out of all histopathological types of lung cancer, sarcomatoid carci-
nomas (SCL) are one of the rarest, and are associated with a very
poor prognosis [1]. Sarcomatoid carcinomas account for less than
1% of non-small cell lung cancers [2]. There are five histological var-
iants: pleomorphic, spindle cell, giant cell, carcinosarcoma, and pul-
monary blastoma. SCL usually appears around 60 years of age, and
is six times more common in males [3]. Due to its rarity, there is no
specific treatment aimed for this cancer. [4]. The pleomorphic type,
a variant of SCL, has documented unusual early distant metastasis
and a median duration of survival of 3 months [5]. None of the pub-
lished literature has shown the severity and grim prognosis of sarco-
matoid lung cancer with a male as young as 31.
Case Presentation
Our patient was an incarcerated 31-year-old African American male
with past medical history of untreated HIV and chronic cough. He
was admitted due to altered mental status for five days and ques-
tionable masses in the lingula and lung. He had a positive smoking
history for about 15 pack years, and no family history for cancer.
First CT scan of the chest showed large parenchymal masses, first
one right next to the left ventricle that measured 6 cm by 3.4 cm,
and another mass just posterior to the wall of the left ventricle that
was measured to be 8.5 cm (Figure 1). IR guided biopsy of the lingu-
lar lesion was done at our facility, which showed initial signs of ma-
lignancy -sarcomatoid lung cancer, pleiotropic variant (Figure 2).
He was started on CT simulation for a course of palliative radiother-
apy. From this point on serial CTs showed significant progression of
the cancer. On August 18
th
, CT for staging showed lesions in the left
hepatic lobe that abut the anterior chest well (2.5 cm x 2.5 cm), right

1
Department of Medicine, SUNY Upstate Medical University, United States
Correspondence: Arpan Patel
Email: patelar@upstate.edu
lobe, and enlarged peri-arotic lymph nodes. On September 15
th
, CT
thorax showed enlarged lymph nodes not previously seen in the ax-
illary, prevascular, pretracheal, and precarinal regions. In the span of
under a month the left lingular mass was shown to be extending into
the left cardial space. The right lung was clear at this point. Four days
later CT of the abdomen showed extension of the masses through
the left diaphragm, as well as increase in size of previous enlarged
lymph nodes, with more clusters of enlarged lymph nodes. CT was
completed again 13 days later, which showed shift of the mediasti-
num due to extensive left pleural disease, new lymph nodes in the
subcarinal area, and new extension of a mass into the left hemidia-
phragm, new liver metastasis and new nodules in the right lung. Ten
days after the last imaging study, and failed attempts at radiotherapy
our patient eventually got discharged with hospice care.

Abstract
Background: Sarcomatoid lung cancer is a very infrequent form of aggressive non-small cell lung cancer accounting for less than 1 percent
of all lung cancers. Due to its rarity, no set standard of treatment has been devised. This cancer has a median age of diagnosis around 60.
Our patient is the youngest documented case of sarcomatoid carcinoma.
Case Presentation: A 31-year-old African American male with a history of HIV, CD4 of 25 came in for evaluation of anemia and chronic dry
cough. Upon initial workup he had a left lower lung mass on chest x-ray. Follow-up CT scan confirmed 6 x 3.4 cm mass next to the left
ventricle of the heart, another mass posterior to the LV (8.5 cm) and a third one in the lingula (5 cm). Brain MRI did now show any metastatic
lesions. The lingulal mass was biopsied and was shown to be pleomorphic type of sarcomatoid cancer. Reimaging two weeks later showed
an increase in size of all lesions and direct extension into the upper abdomen, diaphragm, pericardium and liver metastasis. The patient
was seen a month later for palliative radiation to the left lower lung.
Conclusion: This case serves illustration of the aggressive nature of sarcomatoid lung cancers and rapid progression. Due to rarity and
chemo-resistant nature of this histology of lung cancer, standard of care is not established. Patient in discussion underwent palliative
radiotherapy of his left lung, though due to significant decline in performance status and pulmonary decomposition he chose to undergo
comfort measures and subsequently died shortly after. (El Med J 2:2; 2014)
Keywords: Sarcomatoid Lung Cancer, Indolent, Young

Figure 2: CT scan showing pleomorphic sarcomatoid mass invading
the pericardium
Patel A, Sumendra J, Ananthan D et al 179

Discussion
A young 31-year-old male, with only a positive smoking history and
HIV was incidentally diagnosed with an unusual lung cancer. This
patients sarcomatoid lung cancer in the matter of three months
spread from initially his lingular and left lung mass to his liver, and
physically started invading his pericardium and hemidiaphragm.
With no standard of care, with no viable chemotherapy and failed
radiotherapy, our patient passed away in a mere 3 months after de-
tection of his cancer.
References
1. Yendamuri S, Caty L, Pine M, Adem S, Bogner P, Miller A, Demmy TL, Groman
A, Reid M: Outcomes of sarcomatoid carcinoma of the lung: a Surveillance,
Epidemiology, and End Results Database analysis. Surgery 2012, 152(3):397-
402.
2. Vieira T, Duruisseaux M, Ruppert AM, Cadranel J, Antoine M, Wislez M:
[Pulmonary sarcomatoid carcinoma]. Bulletin du cancer 2012, 99(10):995-
1001.
3. Jiang M, Cao D, Yang Y, Gou H: [Clinical analysis of sarcomatoid carcinoma
of the lung]. Zhongguo fei ai za zhi = Chinese journal of lung cancer 2006,
9(6):547-549.
4. Paleiron N, Tromeur C, Gut-Gobert C, Andre N, Quiot JJ, Quintin-Roue I,
Grassin F, Mondine P, Leroyer C: [Pulmonary sarcomatoid carcinoma: Clinical
and prognostic characteristics, a case report]. Revue de pneumologie
clinique 2012, 68(1):27-30.
5. Chang YL, Lee YC, Shih JY, Wu CT: Pulmonary pleomorphic (spindle) cell
carcinoma: peculiar clinicopathologic manifestations different from ordinary
non-small cell carcinoma. Lung cancer (Amsterdam, Netherlands) 2001,
34(1):91-97.

Figure 2: Histopathological slide showing biopsy of lung mass with typical
sarcomatoid features
180 Treatment of severe intra-uterine adhesions

Vol 2, No 2
A new comprehensive method for treatment of severe intra-uterine adhesions
Sefa Kelekci
1
, Serpil Aydogmus
1
, Emine Demirel
2
, Mustafa Sengul
2

Introduction
Asherman syndrome is an acquired condition characterized by the
formation of adhesions in the uterine cavity. Women with this dis-
ease often struggle with infertility, menstrual irregularities and recur-
rent pregnancy losses [1]. Most patients have adhesive bands that
bridge the anterior and posterior uterine walls. These adhesions are
usually avascular strands of fibrous tissue with varying amounts of
white blood cell infiltration [2]. Adhesions that begin as thin endo-
metrial strands progress to thicker, more fibrous bands quickly, sug-
gesting the frequency and severity of intra-uterine adhesions (IUA)
may be reduced by using the prophylactic measures discussed here
immediately after curettage [3].
These adhesions become denser as time passes, prompting some in-
vestigators to advise early intervention [4]. These types of adhesions
are denser and may be vascular. Muscular adhesions carry a poor
prognosis because some endometrial basalis is needed for the func-
tional layer to proliferate following therapy [3]. The system of the
American Society of Reproductive Medicine consists of three stages
of disease, based upon the extent of cavity involvement (<1/3, 1/3-
2/3, >2/3), the type of adhesion seen at the time of hysteroscopy
(filmy, filmy and dense, dense) and the patients menstrual pattern.
Women with the most severe Asherman syndrome have dense ad-
hesions affecting at least two-thirds of the uterine cavity [5]. Those
with extensive basal layer damage, called endometrial sclerosis, have
little or no functioning endometrium and thus a direr situation.
Our aim was to describe a special comprehensive approach to
women with severe IUA and oligo-amenorrhea, including preopera-
tive, operative and postoperative care.
Case Report
We performed a specific approach described below in a woman with
severe intrauterine adhesion. After a failure of previous hyster-
oscopic adhesiolysis, a 40 year old woman was admitted to outpa-
tient clinics with oligo-amenorrhea. Day 3 hormonal profile was
within normal limits. On 13
th
day of spotting transvaginal ultrasound

1
Department of Obstetrics and Gynecology, Izmir Katip Celebi University, Faculty
of Medicine, Izmir, Turkey
2
Department of Obstetrics and Gynecology, zmir Katip Celebi University, Atatrk
Education and Research Hospital, zmir, Turkey
revealed an endometrial thickness of 1 mm and lack of endometrial
line continuity and hyperechogenic dense adhesions in isthmic por-
tion of uterus (Figure 1a). After endogen estradiol (E2) level reached
200 pg/dl, we did transabdominal ultrasound directed hysteroscopic
sharp and blunt adhesiolysis. After creating an endometrial cavity,
we inserted triangle balloon stent (Balloon Uterine Stent, Cook
reland Ltd.) for a week. We treated patient with 2 x 2 mg estradiol
hemihidrate and azitromycin 1000 mg a day.

We removed the balloon stent and left it alone due to possibility of
reformation of new adhesions. After 1 week we performed office hys-
teroscopy to evaluate endometrial cavity and insertion of intra-uter-
ine device (IUD). New filmy adhesions were dissected by blunt and
aqua dissection and insertion of IUD (Figure 1b). Estradiol hemihid-
rate 4 mg/day for 22 days and medroxy-progesterone acetate 5
mg/day for last 5 days of estradiol course were prescribed for 1
month. After 3 months of operation, IUD was withdrawn. The patient
had been eumenorrheic for 12 months and she had a 7 week intra-
uterine pregnancy one year after operation (Figure 1c).
Discussion
n this study we didnt use prophylactic antibiotics. We did trans-
abdominal ultrasound directed hysteroscopic sharp and blunt adhe-
Correspondence: Emine Demirel
Email: er_em.dr@hotmail.com
Abstract
Background: The aim of this paper is to describe a special comprehensive approach to women with severe intra-uterine adhesions and
oligo-amenorrhea.
Case Presentation: A 40 year old woman was admitted with oligo-amenorrhea. After estradiol (E2) levels reached 200 pg/dl, we did
transabdominal ultrasound directed hysteroscopic adhesiolysis. We inserted a triangle balloon for a week. The Intra-uterine device was left
in place for 13 months, while the intrauterine catheters were removed after 12 weeks. We treated patient with 2 x 2 mg estradiol
hemihidrate and azitromycin daily.
Conclusion: The patent has been eumenorrheic and she had a 7 week intra-uterine pregnancy one year after the operation. (El Med J 2:2;
2014)
Keywords: Asherman Syndrome, Hysteroscopy, Intrauterine Catheter

Figure 1(a): Severe intra-uterine adhesions
Kelekci S, Aydogmus S, Demirel E et al 181
siolysis. A triangle balloon catheter was placed during surgery in-
stead of a round balloon, to minimize pressure points and tissue ne-
crosis that may occur with a Foley catheter balloon. After creating
endometrial cavity we inserted triangle balloon stent for a week. The
balloon catheter was removed within 1 week of surgery, and a cop-
per IUD immediately placed.

The use of estrogen before and after surgery provides several poten-
tial benefits [7]. Before surgery, estrogen promotes maximal endo-
metrial growth, and allows surgery to be performed in the prolifera-
tive phase [6, 8]. Furthermore, as the hysteroscopic repair is per-
formed under abdominal ultrasound guidance, prolonged estrogen
use will help achieve maximal endometrial thickness, and this may
help improve intraoperative visualization by abdominal ultrasound.
Because our patient had enough ovarian reserve (endogen E2:200
pg/dl), there was no need for preoperative treatment with estrogen
[9]. After surgery, continued proliferation is needed to stimulate the
endometrium to cover the denuded uterine cavity.
Conclusion
The main outcome measure of this study was to restore normal men-
ses in patient. The patient has been eumenorrheic for 12 months and
she had a 7 week intrauterine pregnancy one year after operation.
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6. Myers EM, Hurst BS: Comprehensive management of severe Asherman
syndrome and amenorrhea. Fertil Steril 2012, 97: 160-64.
7. American College of Obstetricians and Gynecologists Committee on Practice
Bulletins. Antibiotic prophylaxis for gynecologic procedures. Obstet Gynecol
2006, 108: 225234.
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adhesion (synechia uteri) under roentgenographic view. Fertil Steril 1981,
36(3): 333338.
9. Coccia ME, Becattini C, Bracco GL, Pampaloni F, Bargelli G, Scarselli G: Pressure
lavage under ultrasound guidance: a new approach for outpatient treatment
of intrauterine adhesions. Fertil Steril 2001, 75(3): 601606
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operative hysteroscopy in the prevention of intrauterine adhesions, a
prospective controlled study. Middle Eastern Fertil Soc J 2005, 10: 1359.
11. Vesce F, Jorizzo G, Bianciotto A, Gotti G: Use of the copper intrauterine device
in the management of secondary amenorrhea. Fertil Steril 2000, 73: 1625.
12. Orhue AA, Aziken ME, Igbefoh JO: A comparison of two adjunctive treatments
for intrauterine adhesions following lysis. Int J Gynaecol Obstet 2003, 82: 49
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15. Zikopoulos KA, Kolibianakis EM, Platteau P et al: Live delivery rates in subfertile
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16. McComb PF, Wagner BL: Simplified therapy for Ashermans syndrome. Fertil
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Reprod 1999, 14: 12301233.
19. Thomson AJM, Abbot JA, Kingston A, Lenart M, Vancaillie TG: Fluoroscopically
guided synechiolysis for patients with Ashermans syndrome: menstrual and
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20. Zikopoulos KA, Kolibianakis EM, Platteau P, de Munck L, Tournaye H, Devroey
P, et al: Live delivery rates in subfertile women with Ashermans syndrome
after hysteroscopic adhesion lysis using the resectoscope or the versapoint
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23. Charles M, March MD: Ashermans Syndrome. Semin Reprod Med 2011, 29: 83-
94.

Figure 1(b): Triangle balloon insertion

Figure 1(c): Intra-uterine pregnancy
182 Organophosphate poisoning presenting as bradycardia

Vol 2, No 2
Organophosphate poisoning presenting as bradycardia
Hari Krishan Aggarwal
1
, Deepak Jain
1
, Shivraj Goyal
1
, Shaveta Dahiya
1
, Ashima Mittal
1

Introduction
Indias is an agricultural economy which involves the use of different
insecticides. Amongst them, organophosphate (OP) compounds are
most frequently used due to their relative efficacy and cost. Due to
wide availability, these compounds are also notorious for their high
toxic potential. Their mode of administration can be inhalational or
by ingestion. These agents have varied systemic effects due to their
action on both muscarinic and nicotinic receptors. Cardiac complica-
tions that often accompany poisoning with these compounds may
be serious and often fatal. These complications are potentially pre-
ventable, if they are recognized early and treated adequately. Here
we present a case report of a patient presenting with bradycardia as
the sole manifestation of organophosphate poisoning.
Case Presentation
A 17 years old male student presented in the accident and emer-
gency department with history of dizziness and ingestion of dichlo-
rovos, an OP insecticide. There was no history of blurring of vision,
salivation, lacrimation, urination, diarrhea, vomiting or seizures. On
examination, patient was anxious, with a pulse rate of 38/min, blood
pressure of 120/70 mmHg, respiratory rate of 14/min. Chest auscul-
tation was normal. Cardiovascular, neurological and abdominal ex-
amination was normal with normal sized pupils and no fasciculation
or tremors. Biochemical investigations including complete hemo-
gram, renal and liver function tests, blood sugar and serum electro-
lytes were within reference range. His blood gas analysis was normal.
ECG showed bradycardia with normal intervals (figure 1).
On the basis of history of OP poisoning and bradycardia with dizzi-
ness, patient was started on injection atropine and pralidoxime infu-
sion along with continuous vital monitoring with chest auscultation
and pupillary size charting. Patients heart rate improved and his
lungs remained clear to auscultation. Patients heart rate oscillated
between normal sinus rhythm and bradycardia with no additional
signs of any OP toxicity. Subsequently in 3 days, atropine injection
was stopped and pralidoxime was subsequently tapered off and pa-
tient heart rate reverted to normal. Echocardiography was done
which revealed no abnormality.
Discussion
OP compounds exert their toxic effects by blockage of both nicotinic
and muscarinic receptors. Muscarinic effects include bradycardia, hy-
potension, rhinorrhea, bronchorrhea, bronchospasm, cough, severe

1
Pt BD Sharma University of Health Sciences, India
Correspondence: Deepak Jain
Email: jaindeepakdr@gmail.com
respiratory distress, hypersalivation, nausea and vomiting, ab-
dominal pain, diarrhea, fecal incontinence, genitourinary inconti-
nence, blurred vision, miosis, increased lacrimation, diaphoresis etc.
Nicotinic effects include muscle fasciculations, cramping, weakness,
and diaphragmatic failure. Autonomic nicotinic effects include hy-
pertension, tachycardia, mydriasis, and pallor. CNS effects include
anxiety, emotional lability, restlessness, confusion, ataxia, tremors,
seizures, and coma [1].
The mechanism of cardiac toxicity of OP is still not well elucidated.
Three phases of cardiac toxicity were described by Ludomirsky. The
stages are: phase I of briefly increased sympathetic activity; phase II
of prolonged increase in parasympathetic activity; and phase III in
which QT prolongation is followed by torsades de pointes ventricular
tachycardia and then ventricular fibrillation [2]. Other mechanisms
may be hypoxia, electrolyte derangements, and direct toxic effect on
myocardium [3].
Hypertension and sinus tachycardia, which may be seen in OP and
carbamate poisoning, are nicotinic effects, while hypotension and si-
nus bradycardia are cholinergic manifestations [1]. Sinus bradycardia
is considered to be an early manifestation of cholinergic poisoning
and the presence of hypertension and sinus tachycardia to be a man-
ifestation of severe poisoning [4]. ECG manifestations of OP cover a
wide spectrum and include prolonged QTC interval, sinus tachycar-
dia, sinus bradycardia, ventricular tachycardia, ventricular fibrillation
and nonspecific ST-T changes [5]. Other features are first degree AV
block and atrial fibrillation [6].
In conclusion, cardiac complications are common in organophos-
phate poisoning which are not fully appreciated by physicians. These
findings are due to different pathophysiological mechanisms of
which hypoxia, electrolyte derangements and acidosis are major fac-
tors. Once the condition is recognized promptly, the patient must be
transferred to an intensive care setting. Thus. intensive supportive
care, atropine and meticulous respiratory care are keys to manage-
ment of organophosphate poisoning. In our case, isolated manifes-
tation of bradycardia as a sole manifestation of OP poisoning is very
rare and is reported in few case reports, those too, associated with
other signs and symptoms. This case was discussed to bring into
light that isolated bradycardia can be a presenting manifestation of
OP poisoning.

Abstract
Organophosphate poisoning is very common in India. Its presentations are very vivid. Here we present a case of organophosphate poisoning
in a young male with only bradycardia as sole manifestation of poisoning. (El Med J 2:2; 2014)
Keywords: Organophosphate, Poisoning, Bradycardia
Aggarwal HK, Jain D, Goyal S et al 183

References
1. Organophosphate Toxicity Clinical Presentation
[http://emedicine.medscape.com/article/167726]
2. Ludomirsky A, Klein H, Sarelli P, Becker B, Hoffman S, Taitelman U, et al: Q-T
prolongation and polymorphous (torsade de pointes) ventricular arrhythmias
associated with organophosphorus insecticide poisoning. Am J Cardiol 1982;
49:1654-8.
3. Saadeh AM, Farsakh NA, al-Ali MK: Cardiac manifestations of acute carbamate
and organophosphate poisoning. Heart. 1997; 77(5): 461-4.
4. Namba T, Nolte CT, Jackrel J, Grob D: Poisoning due to organophosphate
insecticides. Acute and chronic manifestations. Am J Med 1971; 50:475-92.
5. Vijayakumar S, Fareedullah M, Ashok Kumar E, Mohan Rao K: A prospective
study on electrocardiographic findings of patients with organophosphorus
poisoning. Cardiovasc Toxicol. 2011; 11(2): 113-7.
6. Paul UK, Bhattacharyya AK: ECG manifestations in acute organophosphorus
poisoning. J Indian Med Assoc. 2012; 110(2): 98.
Figure 1: Electrocardiogram of the patient

ix

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C). Animals had free access to

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