Referensi

D ENGUE
BULLETIN
Volume 28 December 2004
DENGUE BULLETIN
Volume 28, 2004
WORLD HEALTH ORGANIZATION

South-East Asia Region Western Pacific Region
Dengue Bulletin
Volume 28 December 2004
World Health Organization

South-East Asia Region Western Pacific Region
ISBN 92 9022 256 5
World Health Organization 2004
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Acknowledgements
Editor, Dengue Bulletin, WHO/SEARO, gratefully thanks the following for peer reviewing
manuscripts submitted for publication.
In-house Review:
Nand L. Kalra: Reviewed the manuscripts in respect of format check, content, conclusions
drawn, including condensation of tabular and illustrative materials for clear, concise and
focused presentation and bibliographic references. He was also involved in the final stages
of printing of the Bulletin.
Peer Reviewers
1. Dana A. Focks
John W. Hock Company
P.O. Box 12852
Gainesville, FL 32604
USA
E-mail: DAFocks@JohnWHock.com
2. Duane J. Gubler
Director
Asia-Pacific Institute of Tropical Medicine and Infectious Diseases
Leahi Hospital
3675 Kilauea Ave
Honolulu, Hawaii 96816
USA
E-mail: dgubler@hawaii.edu
3. Oon Chong Teik

Tropical Medicine & Infectious Diseases
Mt Elizabeth Hospital
Singapore 228510
E-mail: oonchongteik@hotmail.com
Dengue Bulletin Vol 28, 2004 iii

4. Scott Halstead
Uniformed Services University of the Health Sciences
Bethesda, Maryland
USA
E-mail: halsteads@erols.com
5. Siripen Kalyanarooj
WHO Collaborating Centre for Case Management
of Dengue/DHF/DSS
Queen Sirikit National Institute of Child Health (Childrens Hospital)
Bangkok
Thailand
E-mail: sirip@health.moph.go.th
6. Suchitra Nimmannitya
WHO Collaborating Centre for Case Management
of Dengue/DHF/DSS
Queen Sirikit National Institute of Child Health (Childrens Hospital)
Bangkok
Thailand
E-mail: sujitran@health.moph.go.th
7. Kevin Palmer
Regional Office for the Western Pacific
P.O. Box No. 2932
12115 Manila
Philippines
E-mail: palmerk@wpro.who.int
8. Michael Nathan
Headquarters
20 Avenue Appia
1211 Geneva 27
Switzerland
E-mail: nathanm@who.int
The quality and scientific stature of the Dengue Bulletin is largely due to the conscientious
efforts of the experts and also due to the positive response of contributors to comments
and suggestions.
iv Dengue Bulletin Vol 28, 2004

From the Editors Desk
O ver the decades dengue/dengue haemorrhagic fever has emerged as a global public
health problem with countries in Asia and the Pacific sharing more than 70 % of the
disease burden. In some of these countries, DHF is gaining hyper-endemicity causing deaths
among children. During 2004, Indonesia reported a major dengue outbreak encompassing
Central Java, Sumatra and some outer islands. Till the end of July 2004, 69,017 cases of
DF/DHF and 770 deaths were registered by Indonesian health authorities. During this
epidemic DEN-3 was the predominant serotype.
Sri Lanka also reported a major outbreak with 12,400 cases and 71 deaths as of 23
August 2004. A majority of the cases were reported from five cities: Colombo, Kandy,
Gampaha, Kalutara and Kurunegala.
In the South-East Asia Region, Bhutan and Nepal continued to enjoy dengue-free status
till 2003 because of their sub-mountainous location. However, during August 2004, Bhutan
recorded the first-ever outbreak of DF/DHF in Phuntsholing (population 27,000), a border
town with India. During this outbreak a total of 2,544 DF/DHF cases with no deaths were
reported. More than 93 % of those affected were persons above 5 years of age. This sent a
strong signal to the adjoining DF-free north-eastern part of India and Nepal to take appropriate
preventive action.
DengueNet, the WHO Global Surveillance System for management of epidemiological

and virological surveillance data for early detection, planning and response, was launched in
the South-East Asia and Western Pacific countries during 2004. Each country identified
institutions which would participate in the programme.
The current Volume 28 (2004) of the Dengue Bulletin includes contributions from the
South-East Asia Region (13), the Western Pacific Region (7), the American Region (5) and the
European Region (4).
A supplement, featuring experiences from different countries in social mobilization and

communication for dengue prevention and control, is also being issued along with this volume.
We now invite contributions for Volume 29 (2005). The deadline for the receipt of
contributions is 30 June 2005. Contributors are requested to follow the instructions carefully
while preparing the manuscript. Contributions accompanied by computer diskettes using MS
Word for Windows should be sent to the Editor, Dengue Bulletin, WHO/SEARO, Mahatma
Gandhi Road, IP Estate, Ring Road, New Delhi-110 002, India, or by e-mail as a file
attachment to the Editor at dengue@whosea.org. Readers desirous of obtaining copies of the
Dengue Bulletin may contact the respective WHO Regional Offices in New Delhi or Manila or
the WHO Country Representative in their country of residence.
Dr Chusak Prasittisuk
Regional Adviser
Vector-borne Disease Control
Regional Office for South-East Asia
New Delhi, India
Contents
1. Annual Changes of Predominant Dengue Virus Serotypes in Six Regional 1

Hospitals in Thailand from 1999 to 2002
Surapee Anantapreecha, Sumalee Chanama, Atchareeya A-nuegoonpipat,
Sirirat Naemkhunthot, Areerat Sa-ngasang, Pathom Sawanpanyalert
and Ichiro Kurane
2. A Retrospective Study of the 1996 DEN-1 Epidemic in Trinidad: 7

Demographic and Clinical Features
T.U. Brown, K. Babb, M. Nimrod, C.V.F. Carrington, R.A. Salas
and M.A. Monteil
3. Ecological Study of Rio de Janeiro City DEN -3 Epidemic, 2001-2002 20

Maria Lucia F. Penna
4. Sero-epidemiological and Virological Investigation of Dengue Infection 28

in Oaxaca, Mexico, during 2000-2001
A. Cisneros-Solano, M.M.B. Moreno-Altamirano, U. Martnez-Soriano,
F. Jimenez-Rojas, A. Daz-Badillo and M.L. Muoz
5. Spatial and Temporal Dynamics of Dengue Haemorrhagic Fever 35

Epidemics, Nakhon Pathom Province, Thailand, 1997-2001
Wutjanun Muttitanon, Pongpan Kongthong, Chusak Kongkanon,
Sutee Yoksan, Narong Nitatpattana, Jean Paul Gonzalez and Philippe Barbazan
6. Sporadic Prevalence of DF/DHF in the Nilgiri and Cardamom Hills of 44

Western Ghats in South India: Is it a Seeding from Sylvatic Dengue Cycle
A Hypothesis
Nand Lal Kalra and Chusak Prasittisuk
7. Autoimmunity in Dengue Virus Infection 51

Chiou-Feng Lin, Huan-Yao Lei, Ching-Chuan Liu, Hsiao-Sheng Liu,
Trai-Ming Yeh, Shun-Hua Chen and Yee-Shin Lin
8. Inhibition of the NS2B-NS3 Protease Towards a Causative Therapy for 58

Dengue Virus Diseases
Gerd Katzenmeier
Dengue Bulletin Vol 28, 2004 v

Contents
9. Prognostic Factors of Clinical Outcome in Non-Paediatric Patients with 68

Dengue Haemorrhagic Fever/Dengue Shock Syndrome
Jaime R. Torres, Jos M. Torres-Viera, Hiplito Garca, Jos R. Silva,
Yasmn Baddour, Angel Bajares and Julio Castro M.
10. Dengue Haemorrhagic Fever with Encephalopathy/Fatality at Petchabun 77

Hospital: A three-year Prospective Study (1999-2002)
Prasonk Witayathawornwong
11. A New Tool for the Diagnosis and Molecular Surveillance of Dengue 87
Infections in Clinical Samples
C. Domingo, G. Palacios, M. Niedrig, M. Cabrerizo, O. Jabado,
N. Reyes, W.I. Lipkin and A. Tenorio
12. Clinical and Laboratory Observations Associated with the 2000 Dengue 96
Outbreak in Dhaka, Bangladesh
Monira Pervin, Shahina Tabassum, Md. Mobarak Ali, Kazi Zulfiquer Mamun
and Md. Nazrul Islam
13. Current Status of Dengue Diagnosis at the Center for Disease Control, 107
Taiwan
Pei-Yun Shu, Shu-Fen Chang, Yi-Yun Yueh, Ling Chow, Li-Jung Chien,
Yu-Chung Kuo, Chien-Lin Su, Tsai-Ling Liao, Ting-Hsiang Lin
and Jyh-Hsiung Huang
14. Detection of Dengue Virus Serotype-specific IgM by IgM Capture ELISA in 118
the Presence of Sodium thiocyanate (NaSCN)
Masaru Nawa, Tomohiko Takasaki, Mikako Ito, Ichiro Kurane
and Toshitaka Akatsuka
15. Genetic Influences on Dengue Virus Infections 126

J.F.P. Wagenaar, A.T.A. Mairuhu and E.C.M. van Gorp
16. Identification and Phylogenetic Analysis of DEN-1 Virus Isolated in 135

Guangzhou, China, in 2002
Jun-lei Zhang, Rui Jian, Ying-jie Wan, Tao Peng and Jing An
vi Dengue Bulletin Vol 28, 2004

Contents
17. Induction of Cytotoxic T Lymphocytes by Immunization with Dengue 145

Virus Derived, Modified Epitope Peptide, Using Dendritic Cells as a
Peptide Delivery System
Yoshiki Fujii, Hideyuki Masaki, Takanori Tomura, Kiyohiro Irimajiri
and Ichiro Kurane
18. Molecular Characterization of Brazilian Dengue Viruses 151

Marize Pereira Miagostovich, Flvia Barreto dos Santos
and Rita Maria Ribeiro Nogueira
19. Unusual Emergence of Guate98-like Molecular Subtype of DEN-3 161

during 2003 Dengue Outbreak in Delhi
Manoj Kumar, S.T. Pasha, Veena Mittal, D.S. Rawat, Subhash Chandra Arya,
Nirmala Agarwal, Depesh Bhattacharya, Shiv Lal and Arvind Rai
20. The Animal Models for Dengue Virus Infection 168

Tao Peng, Junlei Zhang and Jing An
21. Philippine Species of Mesocyclops (Crustacea: Copepoda) as a Biological 174

Control Agent of Aedes aegypti (Linnaeus)
Cecilia Mejica Panogadia-Reyes, Estrella Irlandez Cruz
and Soledad Lopez Bautista
22. Susceptibility of Aedes aegypti to Insecticides in Viet Nam 179

Vu Duc Huong, Nguyen Thi Bach Ngoc, Do Thi Hien
and Nguyen Thi Bich Lien
23. Ovipositioning Behaviour of Aedes aegypti in Different 184

Concentrations of Latex of Calotropis procera: Studies on
Refractory Behaviour and its Sustenance across Gonotrophic Cycles
Manju Singhi, Vinod Joshi, R.C. Sharma and Keerti Sharma
24. Community-based Assessment of Dengue-related Knowledge 189

among Caregivers
Khynn Than Win, Sian Za Nang and Aye Min
25. Students Perceptions about Mosquito Larval Control in a Dengue- 196

Endemic Philippine City
Jeffrey L. Lennon
Dengue Bulletin Vol 28, 2004 vii

Contents
Short Notes
1. Sero-surveillance in Delhi, India An Early Warning Signal for 207

Timely Detection of Dengue Outbreaks
D. Bhattacharya, Veena Mittal, M. Bhardwaj, Mala Chhabra, R.L. Ichhpujani
and Shiv Lal
2. Detection of Dengue Virus in Wild Caught Aedes albopictus (Skuse) 210

around Kozhikode Airport, Malappuram District, Kerala, India
B.P. Das, L. Kabilan, S.N. Sharma, S. Lal, K. Regu and V.K. Saxena
3. Entomological Investigations for DF/DHF in Alwar District, Rajasthan, India 213

Kalpana Baruah, Avdhesh Kumar and V.R. Meena
4. Essentiality of Source Reduction in both Key and Amplification Breeding 216

Containers of Aedes aegypti for Control of DF/DHF in Delhi, India
B.N. Nagpal, Aruna Srivastava, M.A. Ansari and A.P. Dash
5. Breeding of Dengue Vector Aedes aegypti (Linnaeus) in Rural Thar Desert, 220
North-western Rajasthan, India
B.K. Tyagi and J. Hiriyan
Book Reviews
1. A Review of Entomological Sampling Methods and Indicators for 223

Dengue Vectors
2. DengueNet in India 224
3. WHO/WPRO/SEARO Meeting on DengueNet Implementation in South- 226

East Asia and the Western Pacific, Kuala Lumpur, 11-13 December 2003
Instructions to Contributors 232
viii Dengue Bulletin Vol 28, 2004

Annual Changes of Predominant Dengue Virus
Serotypes in Six Regional Hospitals in Thailand
from 1999 to 2002
Surapee Anantapreecha* #, Sumalee Chanama*, Atchareeya A-nuegoonpipat*,
Sirirat Naemkhunthot*, Areerat Sa-ngasang*, Pathom Sawanpanyalert*
and Ichiro Kurane**
*National Institute of Health, Department of Medical Sciences, Ministry of Public Health,
88/7, Tivanond Road, Muang, Nonthaburi, 11000, Thailand
**Department of Virology I, National Institute of Infectious Diseases Toyama, Shinjuku-ku,
Tokyo 162-8640, Japan
Abstract
A virological study was conducted in six hospitals spread across Thailand from 1999 to 2002. All four
dengue serotypes were identified, of which DEN-1 was the most predominant. The predominant dengue
serotypes changed every 1-2 years in three of the six hospitals and the predominant serotypes were
different in different hospitals. DEN-1 was predominant in two hospitals during three years of the study
period (2000-2002). DEN-4 was not isolated, or accounted for only a small percentage of the total
isolates, except in one hospital in 2002.
Keywords: Dengue virus, dengue virus serotypes, Thailand.
Introduction occurred in a pattern of 2-year cycle, and

subsequently in irregular cycles as the
The dengue virus consists of four serotypes: disease spread throughout the country. The
DEN-1, DEN -2, DEN-3 and DEN-4. The largest outbreak was reported in 1987, with
clinical manifestations range from 174,284 reported cases, an incidence rate
undifferentiated febrile illness to classic of 325 cases per 100,000 population. It was
dengue fever (DF), dengue haemorrhagic followed by another outbreak in 2001,
fever (DHF) and to dengue shock syndrome which reported an incidence rate of 224
(DSS) [1-3] . Dengue virus infections are now cases per 100,000 population[2,4,5] . In 2002,
important public health problems in many 114,800 dengue cases were reported with
tropical and subtropical areas in the world. an incidence rate of 183 cases per 100,000
In Thailand, a dengue outbreak first population. It is important to determine
occurred in Bangkok in 1958. It then which of the four serotypes causes the
#
E-mail: surapee@dmsc.moph.go.th; Tel.: 66 29510000 Ext. 99219, 99220; Fax: 66 25915449
Dengue Bulletin Vol 28, 2004 1

Dengue Virus Serotypes in Thailand
dengue epidemic each year, and whether Figure 1. Location of six provinces where
the dominant serotype is the same or the sentinel sites were chosen
different in each region in the country. In
the present study, we analysed the
predominant dengue serotypes at the six
regional hospitals located in the four regions
of Thailand north, north-east, central and
south regions in each year from 1999 to
2002.
Materials and methods

Collection of blood specimens
A total of 5,160 and 3,619 blood specimens
were collected at acute and convalescent
stages, respectively, from suspected dengue
cases who visited Lampang Hospital in
Lampang (north), Maharat Nakhon
Ratchasima Hospital in Nakhon Ratchasima 1 = Lampang
2 = Nakhon Ratchasima
(north-east), Pathum Thani Hospital in
3 = Pathum Thani
Pathum Thani, Chareonkrung Pracharak 4 = Bangkok
Hospital in Bangkok, Ratchaburi Hospital in 5 = Ratchaburi and
Ratchaburi (central) and Hadyai Hospital in 6 = Songkhla
Songkhla (south) from 1999 to 2002 (Figure
1). Blood specimens were drawn into tubes
with ethylene diamine tetaacetate (EDTA)
anticoagulant and centrifuged. The number
of specimens ranged from 1 to 3 per patient.
The first samples were collected on the day Virus isolation and determination
of hospitalization and the second samples of dengue virus serotypes
were collected on the day of discharge.
Ten microlitres of acute phase-buffy coat
Third samples, if any, were collected samples were inoculated onto monolayer
between 10-14 days after the onset of of C6/36 cells in a 24-well plate with
symptoms. Plasma and buffy coat were rocking for 90 minutes at room
separated at each hospital, kept in liquid temperature [6] . The inocula were discarded
nitrogen and then transported to the and replaced by Leibovitz 15 medium (L-
Arbovirus Section of the National Institute of 15, Gibco BRL) containing 1% heat-
Health, Nonthaburi. Specimens were kept inactivated fetal bovine serum. After 7 days
at -70 C for virus isolation. of incubation at 28 C, the cultured media
were collected, the infected cells were
2 Dengue Bulletin Vol 28, 2004

stained by IFA, and dengue virus serotypes Results and discussion

were determined[7,8] . DEN -1 (16007 strain),
DEN -2 (16681 strain), DEN-3 (16562 The numbers of samples tested and found
strain) and DEN-4 (1032 strain) were used positive for virus isolation are shown in
as virus controls in IFA. Table 1.
Table 1. Number of samples used for the analysis
Convalescent Virus isolation

Year Hospital Acute sample
sample positive
1999 Lampang 128 108 74

Maharat Nakhon Ratchasima 26 18 6
Pathum Thani 0 0 0
Chareonkrug Pracharak 0 0 0
Ratchaburi 35 11 31
Hadyai 1 1 0
2000 Lampang 35 33 15
Pathum Thani 25 14 4
Ratchaburi 438 419 238
Hadyai 113 28 22
2001 Lampang 262 238 150

Hadyai 450 231 276
2002 Lampang 339 236 95

Chareonkrung Pracharak 135 128 65
Hadyai 326 103 77
All the four dengue serotypes were The predominant dengue virus
detected in the six hospitals during this serotypes in each hospital were analysed
study. When the total numbers of isolates when more than nine isolates were obtained
were analysed, DEN-1 was the predominant in a year. In the Lampang Hospital, the
serotype (48%), followed by DEN -2 (27%), predominant serotypes were DEN-2 (44%)
DEN-3 (20%) and DEN -4 (5%). in 1999, DEN-1 (86%) in 2000, DEN-1

(57%) in 2001 and DEN-2 (79%) in 2002. In predominant in four hospitals (Maharat
the Maharat Nakhon Ratchasima Hospital, Nakhon Ratchasima, Pathum Thani,
the predominant serotypes were DEN-1 Chareonkrung Pracharak and Hadyai) and
(47%) in 2001 and DEN-1 (57%) in 2002. In two hospitals (Lampang and Ratchaburi),
the Pathum Thani Hospital, the respectively. These results suggest that DEN -
predominant serotypes were DEN-1 and 1 was the most predominant serotype from
DEN-2 (41% each) in 2001 and DEN-1 1999 to 2002 in Thailand; however, the
(47%) in 2002. In the Chareonkrung predominant serotypes were different in
Pracharak Hospital, the predominant different hospitals.
serotypes were DEN-1 (50%) in 2000, DEN -
1 (61%) in 2001 and DEN -1 (65%) in 2002. The results of the present study are
In the Ratchaburi Hospital, the predominant generally consistent with those previously
serotypes were DEN-1 and DEN-2 (32% reported. The analysis of dengue virus
each) in 1999, DEN-3 (52%) in 2000, DEN- isolates at Bangkok Childrens Hospital from
1 (38%) and in 2001 and DEN -2 (74%) in 1973 to 1999 showed changes in dengue
2002. In the Hadyai Hospital, the virus serotypes from year to year[9] . Although
predominant serotype was DEN-1 (95%, our study leads to a similar conclusion, the
79% and 44%) in 2000, 2001 and 2002, study was conducted in six regional
respectively. hospitals throughout the country and not
only in central Bangkok.
The predominant serotypes were
compared among six hospitals in each of the Each dengue serotype may possess
study years (Table 2). In 1999 when data unique characteristics in a dengue epidemic
were analysed for two hospitals (Lampang and disease severity. There were
and Ratchaburi), DEN-2 was predominant associations between DEN-1, DEN-2 and
in one hospital (Lampang), and DEN-1 and DEN-3 and moderately severe dengue
DEN-2 were equally predominant in the epidemic years (1984-85, 1989-90, 1997),
other hospital (Ratchaburi). In 2000 when and between DEN-3 and severe dengue
data were analysed for four hospitals epidemic years (1987 and 1998) [9] . In that
(Lampang, Chareonkrung Pracharak, sense, it is important to collect more
Ratchaburi and Hadyai), DEN-1 and DEN-3 information on the predominant serotypes
were predominant in three hospitals and levels of epidemics. Although the
(Lampang, Chareonkrung Pracharak and present study generated information on
Hadyai) and one hospital (Ratchaburi), isolates only from the patients who visited
respectively. In 2001, DEN-1 was the six hospitals, they are located far away
predominant in five hospitals (Lampang, from each other in four regions of
Maharat Nakhon Ratchasima, Chareonkrung Thailand: north, north-east, central and
Pracharak, Ratchaburi and Hadyai) and south. It is thus assumed that the results of
DEN-1 and DEN-2 were equally this study demonstrate, to a certain extent,
predominant in one hospital (Pathum Thani). the general features of dengue epidemics
In 2002, DEN-1 and DEN -2 were in Thailand.

Table 2. Proportion of each of the four dengue serotypes determined by virus isolation
in the six hospitals
Year Hospital Total DEN-1 (%) DEN-2 (%) DEN-3 (%) DEN-4 (%)
1999 Lampang 74 31 (42) 33 (44) 8 (11) 2 (3)
Maharat Nakhon 6 4 (67) 2 (33) 0 (0) 0 (0)
Ratchasima
Pathum Thani 0 0 (0) 0 (0) 0 (0) 0 (0)
Chareonkrug 0 0 (0) 0 (0) 0 (0) 0 (0)
Pracharak
Ratchaburi 31 10 (32) 10 (32) 8 (26) 3 (10)
Hadyai 0 0 (0) 0 (0) 0 (0) 0 (0)
2000 Lampang 15 13 (86) 1 (7) 0 (0) 1 (7)
Maharat Nakhon 3 2 (67) 1 (33) 0 (0) 0 (0)
Ratchasima
Pathum Thani 4 1 (25) 2 (50) 1 (25) 0 (0)
Chareonkrug 10 5 (50) 4 (40) 1 (10) 0 (0)
Pracharak
Ratchaburi 238 99 (42) 9 (4) 125 (52) 5 (2)
Hadyai 22 21 (95) 0 (0) 1 (5) 0 (0)
2001 Lampang 150 85 (57) 35 (23) 26 (19) 1 (1)
Maharat Nakhon 176 83 (47) 68 (39) 16 (9) 9 (5)
Ratchasima
Pathum Thani 143 58 (41) 59 (41) 19 (13) 7 (5)
Chareonkrug 99 60 (61) 25 (25) 11 (11) 3 (3)
Pracharak
Ratchaburi 256 98 (38) 43 (17) 96 (37) 19 (7)
Hadyai 276 219 (79) 4 (2) 39 (14) 14 (5)
2002 Lampang 95 10 (11) 75 (79) 8 (8) 2 (2)
Maharat Nakhon 292 166 (57) 70 (24) 49 (17) 7 (2)
Ratchasima
Pathum Thani 32 15 (47) 8 (25) 7 (22) 2 (6)
Chareonkrug 65 42 (65) 16 (25) 3 (5) 4 (5)
Pracharak
Ratchaburi 170 21 (12) 126 (74) 12 (7) 11 (7)
Hadyai 77 34 (44) 13 (17) 13 (17) 17 (22)
Acknowledgements Dr Vitaya Jiwariyaves and Ms Vanna

Pengruangrojanachai of Ratchaburi Hospital,
We thank Dr Piyaporn Bowonkiratikachorn Dr Paiboon Vechpanich and Mr Prayuth
and Ms Souvapa Pongsathaporn of Kaewmalang of Maharat Nakhon Ratchasima
Charoenkrung Pracharak Hospital, Hospital, Dr Wilaiwan Gulgonkarn,
Dr Wandhana Sritubtim and Mr Suthichai Dr Aroonrat Suwanarat and Mr Somchai
Pongmonjit of Pathum Thani Hospital, Niyomthai of Lampang Hospital and

Dr Suda Chubuppakarn and Ms Raruay This work was partly supported by grants
Jitsakulchaidej of Hadyai Hospital, and other from the Department of Medical Sciences,
doctors, nurses and laboratory staff for Ministry of Public Health, Thailand, and the
assisting us with the collection of samples. Japan Health Science Foundation.
References
[1] George R and Lum LCS. Clinical spectrum of [7] Henchal EA, Gentry MK, McCrown JM and
dengue infection. In Gubler D and Kuno G Brandt WE. Dengue virus specific and
(Eds.) Dengue and dengue hemorrhagic flavivirus group determinants identified with
fever. Colorado: US Department of Health monoclonal antibodies by indirect
and Human Services, 1997: 89-113. immunofluorescence. Am J Trop Med Hyg,
[2] World Health Organization. Monograph on 1982, 31: 830-836.
dengue/dengue haemorrhagic fever. WHO, [8] Henchal EA, McCown JM, Seguin MC,
Regional Office for South-East Asia, New Gentry MK and Brandt WE. Rapid
Delhi 1993. identification of dengue virus isolates by
using monoclonal antibodies in an indirect
[3] World Health Organization. Dengue
immunofluorescence assay. Am J Trop Med
haemorrhagic fever. Diagnosis treatment,
prevention and control, 2nd edition. Hyg, 1983, 32: 164-169.
Geneva: WHO, 1997. [9] Nisalak A, Endy TP, Nimmannitya S,
Kalayanarooj S, Thisayakorn U, Scott RM,
[4] Office of the Permanent Secretary for Public
Health. Annual Epidemiological Surveillance Burke DS, Hoke CH, Innis BL and Vaughn
Report 1993. Ministry of Public Health, DW. Serotype-specific dengue virus
circulation and dengue disease in Bangkok,
Thailand.
Thailand from 1973 to 1999. Am J Trop
[5] Office of the Permanent Secretary for Public Med Hyg, 2003, 68(2): 191-202.
Health. Annual Epidemiological Surveillance
Report 2001. Ministry of Public Health,
Thailand.
[6] Igarashi A. Isolation of Singhs Aedes
albopictus cell clone sensitive to dengue and
chikungunya viruses. J Gen Virol, 1978, 40:
531-544.

A Retrospective Study of the 1996 DEN-1 Epidemic in
Trinidad: Demographic and Clinical Features
T.U. Brown*, K. Babb*, M. Nimrod*, C.V.F. Carrington*, R.A. Salas**
and M.A. Monteil*#
*Faculty of Medical Science, University of the West Indies, St. Augustine, Trinidad
**Caribbean Epidemiology Centre, Port-of-Spain, Trinidad
Abstract
A retrospective analysis of the 1996 DEN-1 epidemic in Trinidad was undertaken to better understand
the clinical and demographic expression of dengue infection in the island during one of the larger
epidemics in the past 10 years and following the reintroduction of DEN-1 into the island in 1991 after a
gap of 14 years. A total of 393 laboratory-confirmed cases were identified. Of these, notes for 157
patients were available for analysis. The epidemic was island-wide, though most cases occurred in the
most densely populated county of St. George. There was a slight predominance of females (51.6%)
among the cases, and while all age groups were affected, older children and adults comprised the
majority. South Asians among the population predominated. Overall, 27 clinical symptoms were
reported. The most common were: fever (98.7%), generalized pain (96.2%) and anorexia (63.1%). Rash,
arthralgia, retro-orbital pain and haemorrhage (all mentioned in the WHO clinical description for dengue
fever) were reported in <50% of cases. Gastrointestinal symptoms were also very common and occurred
in over two-thirds of cases at presentation. Bleeding manifestations were reported in 30% of patients and
commonly involved the gastrointestinal tract. Features of DHF were noted in only six (4%) patients and
there was one fatality. Deficiencies in documented clinical and laboratory monitoring of patients,
coupled with a lack of population-specific laboratory reference ranges, may contribute to under-
diagnosis of DHF in Trinidad.
Keywords: Dengue, DHF, demographic analysis, clinical analysis, Trinidad.
Introduction and potentially fatal form of dengue

infection known as dengue haemorrhagic
The dengue viruses (DEN), of the fever/dengue shock syndrome (DHF/DSS) is
Flaviviridae family, are mosquito-transmitted manifested, primarily through increased
viruses that can cause dengue fever (DF). DF vascular permeability and shock. Dengue
is an acute febrile illness characterized by exists as four distinct serotypes, DEN-1-4.
intense headaches, retro-orbital pain, Infection with one serotype gives life-long
myalgia, arthralgia, anorexia and rash. immunity for that virus but not to the others.
Additionally, in a minority of cases, a severe Data suggest that secondary infection with a
#
E-mail: mmonteil@tstt.net.tt

A Retrospective Study of the 1996 DEN-1 Epidemic in Trinidad: Demographic and Clinical Features
serotype different from the primary infection case of DHF/DSS[10] . Since then, increasingly
enhances the risk of developing larger outbreaks have been documented
DHF/DSS[1,2] . The pathological processes throughout the 1980s and the 1990s,
that lead to DHF/DSS remain poorly caused by DEN -2 and DEN-4. The first
elucidated, but epidemiological studies have cases of DHF/DSS were reported during the
highlighted viral and host factors that are 1993 DEN-1 epidemic[11] , and this was
associated with the development of followed in 1996 by yet another DEN-1
DHF/DSS[3] . outbreak which was characterized by a
larger number of reported and confirmed
An estimated 100 million people across
cases of DHF/DSS[12] .
the globe contract dengue annually with
about 250,000 persons developing We present here a retrospective analysis
DHF/DSS[4] . In the past 20 years, DF and of the 1996 DEN-1 epidemic in Trinidad.
DHF/DSS have become significant public This study was done, firstly, to observe the
health problems in the Caribbean and in clinical and demographic expression of the
Latin America. The discontinuation of the dengue infection in the Trinidad population
Pan American Health Organization (PAHO)- during one of the largest epidemics of the
initiated Aedes aegypti eradication past 10 years. In particular, we wished to
programmes in the 1970s caused a re- assess the ethnic distribution of the disease
introduction of the mosquito vector into since there had been previous anecdotal
virgin areas and a resurgence of dengue reports of a more severe disease in persons
outbreaks throughout the region. of South Asian ancestry. Secondly, the study
Furthermore, prior to the 1980s, dengue was done to gain an understanding of the
outbreaks in the Americas were caused by clinical and laboratory infrastructure for
single serotypes and were geographically dengue in Trinidad and Tobago as part of
restricted and largely self-limiting, with no the development of our ongoing dengue-
reports of DHF/DSS[5] . The first cases of related clinical research in the island.
DHF/DSS documented in this region were
identified during the Cuban DEN-2
epidemic in 1981[6] and the pattern of the Methods
disease has changed dramatically since A retrospective analysis of the clinical and
then[4,5] . Given the strong evidence linking demographic features of patients with
the disease severity with secondary laboratory-confirmed dengue from the 1996
heterologous infection[7-9] , increased DEN-1 epidemic in Trinidad, West Indies,
hyperendemicity is widely thought to be was conducted. Patients were identified
one of the most significant factors from the laboratory database of The
contributing to increases in the disease Caribbean Epidemiology Centre in Trinidad
severity[3] in the region where all the four (CAREC). The CAREC is a subsidiary of the
serotypes are now present. Pan American Health Organization (PAHO)
The first dengue outbreak in over 20 and offers infectious disease testing of sera
years in the island of Trinidad was reported from patients with suspected dengue
in 1977. That outbreak caused by DEN-1 infections. Since 1975, CAREC has
resulted in a few DF cases with no reported systematically conducted the isolation and

typing of dengue viruses in support of viral patients (40%). Information was incomplete
surveillance programmes[12] . in 60 (15%) of these cases. The following is
an analysis of the clinical and demographic
A total of 1,200 samples from
data available from the records of the 157
symptomatic suspected dengue cases in
dengue-confirmed cases.
Trinidad and Tobago were sent to CAREC in
1996 for laboratory confirmation, of which
393 were found positive. We identified Geographical and temporal
these confirmed cases of dengue infection distribution of dengue
and the medical institutions from which the virus infections
samples had originated. The demographic,
clinical and laboratory information related One hundred and fifty-four patients
to each sample was collected from the addresses were available. The estimated
respective medical records of each patient. county-specific incidence rates (per 10,000
population) based on these addresses
Data were entered into a computerized ranged from 0.60 to 1.87, with the extreme
Excel database (Microsoft Excel 2000) and values occurring in the rural counties of
analysed according to demographic Nariva/Mayaro and St. Andrews/St. David
groupings such as ethnicity, county of respectively (Figure 1). The highest
residence, age and gender and by proportion of these 154 confirmed cases
frequencies of clinical symptoms. The (46%) occurred in the most densely
incidence rates per 10,000 persons were populated county of St. George, in which
calculated using the 1990 census data[13] . the capital, Port-of-Spain, is situated. There
Proportional data were tested using the Chi was no significant difference in the rates of
square test and statistical significance was infection among all the counties (P=0.513;
established at P<0.05. All statistical analyses power of test with =0.05:0.0.301).
of the demographic and clinical data were
A review of the admission dates to
done using SigmaStat Statistical Software
health care facilities for 136 dengue-
for Windows Version 2.03 (Copyright
confirmed patients showed that there was a
1992-1997 SPSS Inc.). Ethical approval for
gradual increase in the number of cases
this project was obtained from the Ethics
from April to June, followed by a large rise
Committee of the Faculty of Medical
in July (29 cases) to a maximum in the
Sciences, University of the West Indies, St.
month of August (51 cases). In 1996, the
Augustine.
rainfall pattern (Figure 2) consisted of a large
increase in the rainfall (171.7 mm) between
Results April and May, followed by peak
precipitation in June (333.1 mm). A
The names of the 393 laboratory-confirmed comparison of dengue admissions with the
dengue patients were obtained from the monthly precipitation suggests a direct
CAREC database for 1996. Of these, 258 relationship between the rainfall and the
(66%) could be traced to the source number of cases reported, with a two-
institution or the treating doctor. Clinical month lag between the peak rainfall and the
records were obtained for a total of 157 peak number of admissions.

Figure 1. Map of Trinidad showing county-specific incidence rates (per 10,000 persons) for
dengue infections in 1996 (values in lower half of boxes) based on addresses from 154
confirmed cases. Values in the upper half of boxes show the proportion of the 157 cases
found in each county. County divisions are according to the Regional Health Authorities of
Trinidad & Tobago
Figure 2. Monthly admissions of 136 laboratory confirmed dengue cases and precipitation
levels for the year 1996. Rainfall data obtained from the records of the Trinidad & Tobago
Meteorological Office, Piarco, Trinidad
60 350
333.1
51 300
50
287.4
No. of confirmed cases
45 250
Rainfall 1996 (mm)
40
202.6 203.6
207.1 200
174.4
30
150
148.4
20 120.6
16
100
15
10 51.7 9
28.9 8 50
2 18.9 5 30.9
1 2 2
2
0 0
Sep
Dec
Apr
Jan
Oct
Nov
Jun
Feb
May
Aug
Jul
Mar
Dengue cases Rainfall

Incidence rates of dengue rate in children (15 yrs) was not

infections in relation to significantly different from that in adults
(>15 yrs). An analysis of the estimated
gender, age and ethnicity incidence rates by county showed that there
Gender and age data were available for all was variation in the age groups with the
157 cases. There was a slight female highest incidence rates for each county. In
predominance of 51.6%. three counties, the highest estimated
incidence rates occurred in persons 40 years
The highest proportion of the cases
and over; Caroni (50-59 yrs) (2.9%), Victoria
occurred in the 30-34-year age group
(40-49 yrs) (2.09%) and St. Andrews/St.
(15.9% of the cases), followed by 5-9 and
David (40-49 yrs) (3.65%). However,
10-14-year olds (14.0% and 14.6%
estimated highest incidence rates were
respectively) (Figure 3). The differences
observed in younger patients in the counties
among the groups were found to be highly
of St George (0-9 yrs) (1.89%) and St.
significant (P<0.001; power of test with
Patrick (10-19 yrs) (2.46%).
=0.05: 0.998). The estimated incidence
Figure 3. Age distribution of 157 confirmed dengue cases
18%
15.9%
14.6%
16%
14.0%
14%
% of confirmed cases
12%
8.9%
8.9%
10%
8.3%
7.0%
8%
5.7%
6%
4.5%
3.8%
3.8%
4%
2.5%
1.9%
2%
0%
60+
10-14
15-19
20-24
25-29
30-34
35-39
40-44
45-49
50-54
55-59
0-4
5-9
Age group

Self-reported ethnicity was available for counties. A statistically significant difference

136 patients. Of these, 50% were South was achieved in the county, St. Patrick,
Asians, 35% were Africans, 11% were mixed, where the number of South Asian patients
and 4% were Chinese and Europeans. As was higher than the African patients
seen in Figure 4, a comparison of the (P=0.005; power of test with =0.05:
incidence rates by ethnicity for the whole 0.807), and in the whole group where the
sample and by county showed that the incidence rate for South Asians was
incidence rates of dengue infection were significantly higher than the mixed
the greatest among South Asians for the population (P=0.016; power of test with
group as a whole and in three of the four =0.05: 0.70).
Figure 4. Incidence rates (per 10 000 persons) for 136 confirmed dengue infection patients
by ethnicity for whole population and individual counties.
County Victoria was excluded since ethnicity was reported in <50% of cases in that county
2.44
2.32
2.5
Incidence Rate
1.76
2
1.58
1.46
1.42
1.28
1.27
1.5
1.11
1.03
0.84
0.72
1
0.56
0.21
0.5
0
0
Whole Pop. St. George Caroni St. Patrick St. Andrew/ St.
David
County
South Asians Africans Mixed
Summary of clinical and gastrointestinal (GIT) complaints (nausea,

haemorrhagic manifestations vomiting, diarrhoea and anorexia) were
commonly reported (66.2%). Symptoms
Overall, 24 clinical symptoms were reported highlighted in black indicate the symptoms
in the medical records retrieved. The listed in the WHO clinical description of
frequency of these symptoms at dengue fever[14] . Four of these symptoms,
presentation is shown in Figure 5. Multiple arthralgia, retro-orbital (R/O) pain, rash and
symptoms at the time of admission were haemorrhage, were recorded in <50% of
generally reported. In this cohort, cases.

Figure 5. Frequency distribution of clinical symptoms at presentation in 157 dengue

confirmed patients. Symptoms highlighted in black are part of the WHO clinical
description of dengue fever
Fever, 156
Anorexia, 99 Pain, 151
Myalgia, 98
Headaches, 95
Vomiting, 90
Dehydration, 56
Upper resp., 52
Arthralgia, 50
R/O pain, 50
Chills, 45
Rash, 42
Diarrhoea, 38
Haemorrhage, 34
Adenopathy, 25
Conjunctivitis, 25
Dizziness, 20
Petechiae, 17
Malaise, 15
Rhinorrhoea, 14
Itching, 11
Hepatomegaly, 7
Short breath, 6
Photophobia, 4
0 10 20 30 40 50 60 70 80 90 100
Percentage of patients
In 34 patients (21.7%), haemorrhagic stool and rectal bleeding occurred in 50% of

manifestations (HM) other than petechiae the patients.
were reported. The reported haemorrhagic
Six cases (approximately 4% of the 157
symptoms and their frequency of
cases) could be defined as of DHF based on
occurrence are illustrated in Figure 6. More
the WHO criteria for the diagnosis of
than one form of bleeding was noted in
DHF/DSS[14] . Of these, five were female;
some patients. In keeping with the high
five were South Asians and one was mixed;
frequency of gastrointestinal complaints
four were children and one case (29-year
reported, GIT bleeding including bloody
old, South Asian female) died.

Figure 6. Type and frequency of haemorrhagic symptoms among 34 dengue confirmed

patients who presented with haemorrhagic manifestations. Symptom highlighted in
black is part of the WHO clinical description for DHF
Gastrointestinal
bleeding, 29.4%
Gingival bleeding,
29.4%
Bloody stool, 17.7%
Urinary tract
bleeding, 14.7%
Nasal bleeding,
11.8%
Not specified, 5.9%
Heavy menstrual
flow, 5.9%
Rectal bleeding, 2.9%
Bloodstained sputum,
2.9%
Discussion densely populated county of St. George.

There was a slight predominance of females
We reviewed the clinical and demographic and the infection occurred in all age groups.
features of 157 dengue-confirmed patients The estimated incidence rates were
from a DEN-1 epidemic in Trinidad. This generally higher in patients of South Asian
epidemic followed the reintroduction of this ethnicity. Gastrointestinal symptoms were
serotype into the island in 1991 after an the most common clinical manifestations at
absence of 14 years and the first presentation. Haemorrhagic manifestations,
documentation of DHF in the island in 1993. when present, also commonly involved GIT.
Approximately 1,200 samples from Six patients met the clinical and laboratory
suspected symptomatic patients were sent criteria for DHF and, of these, one died.
for laboratory confirmation and, of these,
393 (approx. 33%) proved to be positive. However, the 157 (40%) available
records closely reflected the distribution of
Clinical and demographic data reported
here are from 40% of the confirmed cases patients from public and private hospitals,
private doctors and health centres seen for
from that outbreak. The outbreak was
island-wide with the estimated incidence the 258 names that were traced to their
respective sample sources. In the sample of
rates being the highest in rural communities,
though most cases occurred in the most 258 patients, public hospitals accounted for

71%, private hospitals 16.7%, and health plastic containers in which water can collect.
centres and general practitioners 6.2% each. The main mosquito vector for dengue
In the sample of 157 patients for whom viruses in the Caribbean and Latin America
medical records were obtained, public is Aedes aegypti, which breeds primarily in
hospitals accounted for 67%, private relatively clean, stagnant domestic water
hospitals 17.2%, health centres 6.4% and containers. Local research has shown that
general practitioners 10%. Over 80% of the important breeding sites for Aedes in
patients were seen in hospitals. Trinidad and Tobago include outdoor drums,
water tanks, tyres and small discarded
The majority of cases (46%) in the
bottles and cans[16] , which get filled easily
sample lived in the most densely populated
with stagnant fresh water during the rainy
county in Trinidad, St. Georges where
season. The lag between the peak rainfall
36.5% of the total population of Trinidad
and the number of confirmed dengue cases
resides and where the Capital, Port-of-Spain
might reflect the period of the mosquito-
(POS), and its environs are located[13] .
vector population expansion.
Dengue, as a mosquito-borne infection,
would be expected to spread rapidly in All age groups were affected but over
populous areas, so the presence of a large 63% of them were adults (>15 years, the
proportion of patients in and around POS is cut off age for paediatric patients in
consistent with the experience elsewhere of Trinidad). Since DEN-1 had been
dengue infection as an urban reintroduced in Trinidad in 1991, it is
phenomenon[15] . Interestingly, our estimates expected that children aged 5 years or less
of county-specific dengue incidence (based would constitute a susceptible population.
on the sample of 157 patients) yielded rates Infection in this group would be primary in
that did not differ significantly between rural which the majority would be asymptomatic.
and urban counties in Trinidad. This This may account for children aged 0-4
suggests that despite the variation in years comprising only 8% of the sample.
population density, DEN-1 infection was Despite the 1992/93 DEN-1 outbreak in
fairly evenly spread across the island. The which over 3,060 cases were reported,
island of Trinidad is merely 4,828 km 2 and there were still many adults in 1996 who
the similarity of the incidence rates across were susceptible to DEN-1. In total, there
the island may reflect a combination of a were 3,588 reported cases that year.
DEN-1 nave population and easy However, based on the CAREC experience,
accessibility to all parts of the island. only 33% of the clinically suspected dengue
cases at that time were actually confirmed
The DEN-1 1996 epidemic occurred in
by laboratory tests, suggesting thereby that
the rainy season with most admissions taking
even during that outbreak, many cases with
place in July and August, 1-2 months after
clinical features consistent with dengue may
the peak rainfall for that year in June.
in fact not have been of dengue infection.
Rainfall is considered to be a risk factor for
Clinical symptoms of dengue are known to
the development of dengue outbreaks,
overlap with many other infections such as
especially where there is improper storage
measles, hepatitis A, rubella and
of water or the presence of receptacles in
leptospirosis [17-19] .
the domestic environment such as discarded

The high proportion of cases in the 30- increased incidence in South Asians is
34, 5-9 and 10-14-year age groups suggests interesting and merits further analysis with
the possibility of spread between children larger cohorts of dengue-confirmed cases.
and parents. In Trinidad and Tobago, 25%
The range of symptoms reported in the
of the heads of households are 30-39 years
157 Trinidadian dengue-confirmed patients
of age with an average of 1.99 children per
is similar to those described for patients with
household[13] . Trinidadian women often start
acute dengue in other parts of the world[18] .
their families in late teenage or early
Recent-onset fever and generalized body
twenties, so parents aged 30-34 years will
pains occurred in almost all patients.
have children in the age groups 5-9 or 10-
Headache, anorexia and myalgia, which
14 years.
often occur in the prodrome, were noted in
The racial composition of this sample 60% or more of the cases. Gastrointestinal
differs from that reported for the general and upper respiratory symptoms were also
population, which is composed of South common complaints at presentation.
Asians (40.3%), Africans (39.6%), mixed Gastrointestinal symptoms have been
(18.4%) and the rest (1.6%)[13] . Thus, there is described as predominant clinical
an overrepresentation of South Asians (50%) manifestations in epidemics in which there
in our sample with fewer persons of African is an adult-susceptible population[21] . The
origin (35%) and of mixed race (11%). Since 1996 epidemic was almost wholly due to
hospitalized patients account for 84% of the DEN-1 and the bulk of the study population
study population, it may reflect an increased comprised older children and adults.
number of South Asians being hospitalized
The symptoms of classic dengue as
for dengue infection as compared with
described by WHO occurred in fewer than
other racial groups. Teelucksingh[20] has
expected numbers of patients. For example,
previously reported a higher incidence of
retro-orbital pain and arthralgia were
more severe dengue infection and mortality
reported in 32% of patients and rash only
in South Asian people (colloquially referred
noted in 27%. While this may be due to a
to as East Indians) in Trinidad.
true low prevalence of these symptoms in
These data cannot exclude differences the study population, the cultural expression
in the environmental factors that might also of symptomatology may also contribute to a
increase the exposure of South Asian people falsely low detection of these symptoms. For
to more mosquito bites. South Asians have example, in Trinidad, all pains in the head
traditionally been the predominant racial region, including orbital pain, are commonly
group in agriculture and animal rearing in referred to as headache. Similarly, joint
Trinidad. In rural communities on the island pains may often be included in muscle
there is relatively poor piped water supply, pain or be part of a more generalized body
resulting in more water storage, thus pains. The rash, which is usually transient,
providing ideal domestic breeding sites for may be easily missed and even more so in
Aedes aegypti[16] . Therefore, farmers in rural dark-skin complexions.
areas of Trinidad may be at an increased
Haemorrhagic manifestations, including
risk of mosquito bites and consequent
petechiae, were noted in 51 (32.5%)
dengue infection. The tendency towards an
patients. This is consistent with other reports

of the prevalence of haemorrhage in dengue the hospitals that were visited. For example,
fever[18] . In keeping with the global chest X-rays were performed in fewer than
experience, the bleeding manifestations 10% of the patients admitted to private
occurred at many sites. Despite the bleeding hospitals but in over 60% of the patients in
manifestations in almost a third of the one public hospital. Serial blood tests were
patients, the WHO criteria for DHF were performed in approximately 20% of dengue
fulfilled only by 6 (4%) patients[14] . Of these, cases in private hospitals but in 40-80% of
five were female and one was male, five patients admitted to three public
were South Asian and one was of mixed institutions[22] .
ethnicity and four were children and two
Furthermore, there are no locally
were adults. One patient, a 29-year-old
developed reference ranges for haematocrit
female of South Asian ethnicity died. The
by age and sex in use in many laboratories
female gender and children were more
in Trinidad where the reference ranges
prone to the development of DHF/DSS in
developed in primarily the Caucasian
dengue-endemic areas [3] . While the 1996
populations have been adopted for use.
epidemic was predominantly DEN-1, DEN-
Moreover, haemoglobinopathies such as
2 was already endemic in the island[12] .
thalassaemia and sickle cell traits are not
Further, the predominance of persons of
uncommon among the Trinidadian
South Asian ancestry in this group was also
population and these persons tend to have
consistent with the observation that certain
normal haemtocrit levels below the
races were more susceptible to DHF/DSS
reference ranges in use. Consequently, such
than others.
patients presenting with 20% or more
The number of cases of DHF/DSS may increase in haemoconcentration may fall
have been underestimated. One of the within the adopted reference range, and if
essential WHO criteria for establishing the there is no repeat of haematocrit
diagnosis of DHF/DSS is the proof of concentration in the convalescent period,
increased vascular leakage such as an evidence of haemoconcentration would
increase of at least 20% in average have been completely missed and the
haematocrit for age and sex; a drop in patient wrongly diagnosed as DF instead of
haematocrit of 20% or more following a mild form of DHF[22] .
treatment, or clinical evidence of the same
In summary, the DEN-1 epidemic in
such as pleural effusion, ascites or
Trinidad in 1996 was characterized by a
hypoproteinaemia[14] . However, since the
large number of clinically suspected but
plasma leakage can be transient and only
unconfirmed cases of dengue infection.
evident by laboratory testing in milder forms
Laboratory analysis of a third of all reported
of DHF cases (e.g. Grade 1 and 2 DHF), it is
cases revealed that most of these cases were
possible to miss patients with these forms of
not of dengue. Furthermore, the variability
DHF if clinical monitoring of the
in the data available from clinical records,
haematocrit levels or tests such as lateral wherever these could be found, resulted in
decubitus chest X-rays to detect small less than optimal information retrieval for
pleural effusions are not performed. The further analysis. From the available data set,
frequency of chest X-rays and serial blood the Trinidadian dengue-infected patients
tests in this cohort of patients varied with were mainly older children and adults from

rural and urban communities. South Asians cases but features of DHF were noted in
were predominant. A wide range of clinical only 4%. Improvement in clinical diagnosis
manifestations was recorded, but the most and record-keeping is urgently required to
common were gastrointestinal. Bleeding underpin future clinical research in Trinidad.
manifestations occurred in over 30% of DF
References
[1] Halstead SB, Chow J and Marchette NJ. [9] Thein S, Aung MM, Shwe TN, Aye M, Zaw
Immunologic enhancement of dengue virus A, Aye K, Aye KM and Aaskov J. Risk factors
replication. Nature New Biology, 1973, 243: in dengue shock syndrome. American
24-26. Journal of Tropical Medicine and Hygiene,
[2] Morens DM, Halstead SB and Marchette NJ. 1997, 56(5): 566-572.
Profiles of antibody-dependent enhancement
[10] Le Maitre. Dengue fever epidemic in
of dengue virus type 2 infections. Microbial
Trinidad & Tobago: 1977-1978. Dissertation
Pathogenesis, 1987, October, 3(4): 231-237.
for the Diploma in Public Health, University
[3] Halstead SB. Epidemiology of dengue and of the West Indies, Mona, Jamaica, 1978;
dengue haemorrhagic fever. In: Gubler DJ, Chapter 2: 11-18.
Kuno G (Eds.), Dengue and dengue
haemorrhagic fever. New York: CAB [11] Teelucksingh S, Mangray AS, Barrow S,
International, 1997: 23-44. Jankey N, Prabhakar P and Lewis M.
Dengue haemorrhagic fever / dengue shock
[4] Gubler DJ. Dengue and dengue
haemorrhagic fever: Its history and syndrome: an unwelcome arrival in Trinidad.
resurgence as a global public health problem. West Indian Medical Journal, 1997, 46(2):
In: Gubler DJ, Kuno G (Ed.), Dengue and 38-42.
dengue haemorrhagic fever. New York: CAB [12] Campione-Piccardo J, Ruben M, Vaughan H
International, 1997: 1-23. and Morris-Glasgow V. Dengue viruses in
[5] Gubler DJ. Dengue and dengue the Caribbean. Twenty years of dengue virus
haemorrhagic fever. Clinical Microbiology isolates from the Caribbean Epidemiology
Review, 1998, 11(3): 480-496. Centre. West Indian Medical Journal, 2003,
[6] Pinheiro FP and Corber SJ. Global situation 52(3): 191-198.
of dengue and dengue haemorrhagic fever, [13] Central Statistical Office of the Government
and its emergence in the Americas. World of Trinidad & Tobago. http://www.cso.gov.tt.
Health Statistics Quarterly, 1997, 50(3-4):
161-169. [14] World Health Organization Regional Office
for South-East Asia. Prevention and control
[7] Halstead SB. Pathogenesis of dengue:
of dengue and dengue haemorrhagic fever:
challenges to molecular biology. Science,
1988, 239 (4839): 476-481. comprehensive guidelines. http://w3.whosea.org/
dengue/searono29/pdff.htm.
[8] Kliks SC, Nisalak A, Brandt WE, Wahl L and
Burke DS. Antibody-dependent [15] Kuno G. Factors influencing the transmission
enhancement of dengue virus growth in of dengue viruses. In: Gubler DJ, Kuno G
human monocytes as a risk factor for (Ed.), Dengue and dengue haemorrhagic
dengue haemorrhagic fever. American fever. New York: CAB International, 1997,
Journal of Tropical Medicine and Hygiene, 61-88.
1989, 40(4): 444-451.

[16] Focks DA and Chadee DD. Pupal survey: an [20] Teelucksingh S, Lutchman G, Udit A and
epidemiologically significant surveillance Pooransingh S Childhood dengue shock
method for Aedes aegypti: an example using syndrome in Trinidad. West Indian Medical
data from Trinidad. American Journal of Journal, 1999, 48(3): 115-117.
Tropical Medicine and Hygiene, 1997, [21] World Health Organization Regional Office
56(2): 159-167. for South-East Asia. Risk factors of
[17] Dietz VJ, Nieburg P, Gubler DJ and Gomez I. DEN/DHF epidemics in SEAR countries.
Diagnosis of measles by clinical case http://w3.whosea.org/Den1/risk.htm.
definition in dengue endemic areas: [22] Nimrod M, Brown TU, Babb K, Carrington
implications for measles surveillance and CVF, Salas RA and Monteil MA. An analysis
control. Bulletin World Health Organization, of laboratory parameters from confirmed
1992, 70(6): 745-750. cases of dengue haemorrhagic fever in the
[18] George R and Lum LCS. Clinical spectrum of 1996 Trinidad epidemic. West Indian
dengue infection. In: Gubler DJ, Kuno G Medical Journal, 2003, 52 (Suppl. 1): 17.
(Ed.), Dengue and dengue haemorrhagic
fever. New York: CAB International, 1997:1-
22.
[19] Levett PN, Branch SL and Edwards CN.
Detection of dengue infection in patients
investigated for leptospirosis in Barbados.
American Journal of Tropical Medicine and
Hygiene, 2000, 62(1): 112-114.

Ecological Study of Rio de Janeiro City
DEN-3 Epidemic, 2001-2002
Maria Lucia F. Penna#
Escola Nacional de Saude Publica, Fundao Oswaldo Cruz, Rua Leopoldo Bulhes 1480,
DENSP, 21041-210 Rio de Janeiro, RJ, Brazil
Abstract
Dengue virus serotype 3 was introduced in the Rio de Janeiro metropolitan area in January 2001 which
produced a large epidemic during 2001 and 2002. This study looks into the relationship between the
urban socioeconomic organization and the dengue attack rate during the epidemic in Rio. This study
uses secondary data published in the data website of the city administration, including variables related
to sanitation, use of the city area, tax collection, population density, education, income and life
expectancy. The model includes as predictors the proportion of households with a well, the proportion
of the available area in the city used for commerce and services in general, the proportion of city taxes
collected from industries, the mean per capita income and the residential area per inhabitant, all with a
statistical significant level of less than 0.05. The model explains 71% of the attack rate variance. The
variables included in the model indicated that dengue distribution in the city was related to peoples
socioeconomic status and the city organization. Variables that correlate with the movement of people
across towns have emerged as the most significant.
Keywords: Dengue virus, socioeconomic status, urban organization, Rio de Janeiro.
Introduction introduced in the country in 1990 and 2001,

respectively[2] . Once introduced in the Rio
After decades of freedom from dengue virus de Janeiro metropolitan area, DEN -3 caused
infection, Brazil experienced an outbreak of a large epidemic during 2001 and 2002. In
DEN-1 and DEN-4 in areas close to the 2003, DEN-1, DEN-2 and DEN-3 were in
borders with Venezuela in 1981-82. circulation in all except the two southern
Intensive vector control measures states of the country, with 341,092 cases
successfully controlled this outbreak and reported (Figure 1) [3] .
checked its spread to other areas in the
Despite the use of a variety of control
country[1] . In 1986, DEN-1 was introduced
in the Rio de Janeiro metropolitan area, strategies, dengue control is a major public
health challenge in the world. Demographic
which spread to other areas, causing a
massive epidemic that later covered the changes occurring in developing countries
due to widespread rural-urban migration
entire country. DEN -2 and DEN-3 were also
#
E-mail: mlpenna@ensp.fiocruz.br; Tel.: 55 21 38829212; Fax: 55 21 38829124

Ecological Study of Rio de Janeiro City DEN-3 Epidemic, 2001-2002
since the 1960s have resulted in of Aedes aegypti in the 70s is no longer
overcrowded cities with multiple deficiencies, applicable to the reality of the social,
particularly in housing and basic sanitation. demographic, economic and political
The strategy that resulted in the eradication situation in South American countries[4-6].
Figure. Circulation of dengue virus in the Brazilian states, 2003
None
DEN-1 and -3
DEN-1 and -2
DEN-1, -2 and -3
Predicting the risk of dengue correctly control measures to be more effective with
based on sociocultural factors has been the responsibilities divided between the
goal of many authors [7,8] , but the dynamics government and communities as per
of dengue transmission in urban settings are priorities established on the basis of reliable
still poorly understood[9] . The understanding data.
of the virus transmission dynamics requires a
The present study looked into the
theoretical framework that includes
relationship between the urban
individuals, households and behavioural risk
socioeconomic organization and the dengue
factors as well as the administrative aspects
attack rate during the DEN-3 epidemic in
of city management services, use of city
Rio de Janeiro city in 2001-2002. It is
space and movement of people across the
mainly an exploratory study, based on
metropolis. This understanding can help
available secondary data, that is likely to

throw some light on how urban settings residence (HOUSE), for industry
contribute to dengue transmission. (INDUSTRY), for commerce or service
(COMMERCE); the proportion of unused
areas (UNUSED); the proportion of the total
Methods area of parks and squares (PARKS); the
amount of city tax collected per inhabitant
Description of study area (TAX); the proportion of city tax of
Rio de Janeiro city is located at -2254'23" commercial origin (COMTAX), industrial
south latitude and -4310'21" west longitude, origin (INDTAX) and service origin
(SERVTAX); the mean per capita income
at the seashore, with an urban area of 1,255
(INCOME); the life expectancy (LE); the
km 2, including inland and continental
proportion of literacy among inhabitants
waters. The municipal area was divided into
(LITERACY); and the proportion of children
26 administrative regions (ARs) in 1999
aged 7 to 15 years attending school
which were used as spatial units for analysis.
The climate is tropical, hot and humid, with (SCHOOL) as independent variables. All
proportions were presented as percentages
local variations due to altitude differences,
and income and taxes as 1,000 reais
vegetation and proximity to the sea. The
(Brazilian currency). The independent
mean annual temperature is 22 C, with
variable was the attack rate for DEN-3
high daily means in summer (30 to 32 C).
epidemic, calculated by the number of
Rainfall is 1,200 to 1,800 mm per year,
concentrated in summer from December to reported dengue cases during 2001-2002
March. The city has the lowest rate of divided by the population estimate for
January 2001, per 100,000 inhabitants.
population growth among the Brazilian
capital cities, 6.9% between 1991 and 2000.
Statistical methods
Database A multiple regression model was adjusted to
the data using stepwise forward approach,
This study used secondary data published in
with F to enter = 1 and F to leave = 0.95,
the website of the city administration[10] ,
and the author interference to limit the
including the proportion of households
number of steps in order to prevent a
belonging to the sewage collection system
(SEWAGE), the proportion of households saturated model. The independent variable
belonging to the water supply system was given a log transformation. The software
used was Statistica, from Statsoft[11] .
(WATER), the proportion of households with
a well (WELL), the proportion of households
with waste collection by the city authority Results
(WASTE), the number of inhabitants per
household (INH/HOUSE), the area of Table 1 shows the correlation coefficients
residential properties (square metres) per between the independent variable and all
inhabitant (M2/INH), the proportion of those variables presented in the model,
households in slums (SLUMS), the along with their range. The model included
proportion of the total area built for as predictors the proportion of households

with a well, the proportion of the available partial correlation coefficients for the
area used for commerce and services, the variables included in the model, which is a
proportion of city taxes collected from measure of the association of each variable
industries, the mean per capita income and when the other variables are controlled for,
the built household area per inhabitant making it possible to rank their effect. The
(Table 2), all with a statistical significant level residues fitted well into a normal
less than 0.05. The model explains 71% of distribution and presented no correlation
the attack rate variance. Table 3 shows the with the predictors.
Table 1. Descriptive statistics and correlation coefficient with log (attack rate)
Variable Mean Minimum Maximum Correlation
WASTE 1 0.93 1 0.09

SWAGE 1 0.30 1 0.09
WATER 1 0.88 1 -0,30
WELL 0 0.00 0 0,39
M2/INH 24 5.56 56 0.20
SLAM 15 0.00 41 0.04
INH/HOUSE 3 2.36 4 -0.05
LE 72 65.99 78 -0.01
LITERACY 96 90.74 99 -0.38
SCHOOL 90 67.66 113 -0.36
INCOME 670 212.21 2229 -0.13
HOUSE 69 9.89 89 -0.26
COMMERCE 12 3.40 39 0.27
INDUSTRY 7 0.11 21 0.01
UNUSED 981 0.99 19729 0.02
PARKS 2 0.00 25 -0.02
COMTAX 0 0.00 3 -0.20
INDTAX 0 0.00 1 0.30
SERVTAX 99 94.30 100 0.03
TAX 396 2.78 7157 0.19

Table 2. Regression summary

(Standard regression coefficient, regression coefficient, t and P value)
Standard
Standard
Beta error of B t (20) P level
error of B
Beta
Intercept 6.360585 0.184526 34.46979 0.000000

WHELL 0.33653 0.145309 7.640343 3.299001 2.31596 0.031294
COMMERCE 0.34087 0.136198 0.024121 0.009638 2.50272 0.021111
INDTAX 0.46826 0.126483 2.084555 0.563072 3.70211 0.001410
INCOME -1.06086 0.273221 -0.001142 0.000294 -3.88279 0.000925
M /INH
2
1.12201 0.294558 0.048058 0.012616 3.80912 0.001099
R= 0.84282066; R2=0.71034667; F(5,20)=9.8096; P<0.00007; Standard error of estimate=0.35169
Table 3. Partial and semi-partial correlation
Beta in Partial correlation Semi -partial correlation
WELL 0.33653 0.459859 0.278711

COMMERCE 0.34087 0.488354 0.301187
INDTAX 0.46826 0.637673 0.445527
INCOME -1.06086 -0.655600 -0.467271
M /INH
2
1.12201 0.648419 0.458405
Discussion dealt with by the logarithmic transformation

of the attack rate[12] , as shown by the
The exploratory aspect of this study justifies residual analysis.
the presentation of a high number of urban
and socioeconomic variables in the model. The proportion of households with a
These variables present high covariance that well is really a proxy variable of the
induces the use of the forward stepwise discontinuity of water supply. The
proportion of households that have access
method. To avoid a saturated model, the
to the citys water supply network is 95.07%
author restrained the number of steps and
for the entire city, indicating a high coverage
assured that only significant variables were
kept in the model by using a relatively high of the citys water supply network. But the
value of F to leave. The rupture of the water supply is not equally effective in all
homocedasticy assumption due to the the administrative regions (ARs), with some
areas having chronic problems, mainly
nature of data (proportions) was successfully
discontinuous supply. The presence of a

well, although small, only in 1.12% of the affecting the presence of potential breeding
households (maximum of 11.85% in sites for the vector. This discussion
Paqueta AR and 11.06% in Guaratiba AR) is reinforces the relevance of multi-level
a proxy variable for the discontinuity analysis in the evaluation of dengue risk
problem, for it is a solution that involves factors.
financial expenses which is only justified in
The inclusion in the model of the
the face of an important and lasting problem
proportion of the area used for commerce
of supply. These results point to the fact that
and services and the proportion of city taxes
water supply is an important issue in dengue
paid by industry shows that urban
control, but only in the context of lack of or
organization plays an important role in the
irregular water supply where the population
distribution of dengue. These two variables
is forced to resort to storage, which creates
are proxy variables for the movement of
breeding sites for the vector and not in the
people across ARs. The presence of
usual context of adequate supply as
commerce and general services was
suggested by other authors [13] . .
represented by the proportion of the area
The model included mean per capita and the presence of industry as the
income as a protective factor meaning that proportion of city taxes, because those are
low socioeconomic status of residents of an the variables correlated to the intensity of
AR is a risk factor for dengue transmission. A the movement of people. The luxury
study in Brazil found no association commerce and services may collect more
between the socioeconomic status and taxes than the popular commerce and
dengue risk at the individual level[14] and services, but the areas of popular commerce
suggested that the previous finding of such and services have a much bigger flow of
an association at the aggregate level[15,16] was people. On the other hand, the relative size
a fallacy. However, it should be noted that of the industrial area is related to the type of
studies that focus on factors at individual industry, as for instance big industrial
level are insufficient to address the storage areas, and not to the production or
ecological links in the causal chain. the number of workers. Industries that
Ecological studies may be, as pointed out by collect more taxes have a larger production
Koopman and Languini[17] , the only way to and are more likely to have a higher number
study risk factors for infectious diseases. The of workers. Other authors [18] indicate that
risk of dengue virus infection is not the probability of being reached by a new
dependent on the physiological dengue virus is correlated to the intensity of
characteristics of individuals, but on the communication among people and the
environmental characteristics of the area density of traffic and the road network. It is
where the group of individuals live. This important to note that the diffusion of the
includes other individuals, the natural epidemic by proximity may be less
environment and the way it is transformed important than the diffusion caused by the
by humans. The mean per capita income circulation of people in central areas in big
has to be interpreted as an environmental cities, which is supported by the present
measure concerning the area and not as an finding. This area initially imports infected
aggregate measure of individuals, because it individuals from the initial foci of the virus
impacts the neighbouring households as where it is newly introduced, resulting in
well as the nearby public areas, thus higher local transmission, which, in turn,

results in exportation of infected individuals people across cities and its significance in
to other areas, reinforcing the epidemic all dengue epidemics.
over the city. This fact clearly indicates the
priority of mosquito control in areas with
high levels of population mobility, such as Conclusion
the Rio de Janeiro city central area. The current efforts to control dengue
The model also included the area of demand a more comprehensive approach,
residential property per inhabitant as a risk including health education, community
factor for dengue. This variable is closely participation, garbage disposal and proper
correlated with the socioeconomic status urban planning, besides chemical and
represented by the mean per capita income biological mosquito control measures. In
(R=0.864605) that is controlled in the order to improve the efficiency of control
model, meaning that, for the same efforts, these activities have to concentrate
socioeconomic status, a bigger area of on areas and populations at higher risk,
residential property per inhabitant results in which implies early identification of higher
bigger dengue risk. This is possibly due to incidence periods and areas and their
the existence of empty spaces in properties, characteristics[5] .
such as backyards, thus creating greater
In Rio de Janeiro, emphasis has been
opportunities for the existence of the vector placed on bringing about behavioural
breeding sites. This hypothesis has to be changes in the communities to make an
investigated for its importance in the impact on the determinants and risk factors
selection of priorities for vector control in of dengue through educational interventions.
private residences as well as in defining the Health authorities and the press attributed
contents of educational interventions.
the main responsibility of the problem to
The results of the present study allow public behaviour, not clearly distinguishing
the establishment of priorities in vector between the responsibilities of the
control and educational interventions, government and that of the private citizen[6] .
highlighting the importance of the flow of
References
[1] Osanai CH, Travassos-da-Rosa APA, Amaral [3] Secretaria de Vigilncia em Sade. Dengue
S, Passos ACD and Tauil PL. Surto de Boletim semana 16; 2004 online
dengue em Boa Vista, Roraima. Revista do http://dtr2001.saude.gov.br/svs/epi/dengue/
Instituto de Medicina Tropical de Sao Paulo, boletim/pdfs/Boletim%20semana%2016%20
1983, 1: 53-54. 2004.pdf.
[2] Nogueira RM, Miagostovich MP, Schatzmayr [4] Tauil PL. Urbanizao e ecologia do dengue.
HG, dos Santos FB, de Araujo ES, de Filippis Cad Sade Pblica, 2001, 17(1): 99-102.
AM, de Souza RV, Zagne SM, Nicolai C, [5] Pan American Health Organization. A
Baran M and Teixeira Filho G. Dengue in blueprint for action for the next generation:
the State of Rio de Janeiro, Brazil, 1986- dengue prevention and control. Washington:
1998. Mem Inst Oswaldo Cruz, 1999, 94(3): PAHO, 1999, 1-14.
297-304.

[6] Penna MLP. Dengue control: a challenge for [13] Forattini Oswaldo Paulo and e Brito
the public health system in Brazil. Cad Marylene de. Reservatrios domiciliares de
Sade Pblica, 2003, 19(1): 305-309. gua e controle do Aedes aegypti. Rev
Sade Pblica, 2003, 37: 676-677.
[7] Hayes JM, Garcia Rivera, Flores Reyna,
Suarez Rangel, Rodrguez Mata, Coto [14] Teixeira M, Barreto ML, Costa M, Ferreira
Portillo, Baltrons Orellana, Mendoza LD, Vasconcelos PF and Cairncross S.
Rodrguez, De Garay, Jubis Estrada, Dynamics of dengue virus circulation: a
Hernndez Argueta, Biggerstaff BJ and Rigau silent epidemic in a complex urban area.
Perez. Risk factors for infection during a Trop Med Int Health, 2002, 7: 757-762.
severe dengue outbreak in El Salvador in
[15] Vasconcelos PF, de Menezes, Melo LP,
2000. Am J Trop Med Hyg, 2003, 69: 629-
Pesso ET, Rodrguez SG, da Rosa, Timbo MJ,
633.
Coelho IC and Montenegro F. A large
[8] Ashford DA, Savage HM, Hajjeh RA, epidemic of dengue fever with dengue
McReady J, Bartholomew DM, Spiegel RA, hemorrhagic cases in Cear State, Brazil,
Vorndam V, Clark GG, Gubler DG. 1994. Rev Inst Med Trop Sao Paulo, 37:
Outbreak of dengue fever in Palau, Western 253-255.
Pacific: risk factors for infection. Am J Trop
[16] Figueiredo LT, Cavalcante SM and Simoes
Med Hyg, 2003, 69: 135-140.
MC. Dengue serologic survey of
[9] Cmara tecnica e cientifica de schoolchildren in Rio de Janeiro, Brazil, in
assessoramento ao controle do dengue. 1986 and 1987. Bull Pan Am Health Organ,
Recomendaes Secretaria de Estado de 1990, 24: 217-225.
Saude. Secretaria de Estado de Sade do Rio
[17] Longini IM Jr, Koopman JS, Haber M and
de Janeiro, Rio de Janeiro, RJ, 2002, 1-11.
Cotsonis GA. Statistical inference for
[10] Instituto Municipal de Urbanismo Pereira infectious diseases. Risk-specific household
Passos, Secretaria Municipal de Urbanismo, and community transmission parameters.
Cidade do Rio de Janeiro. Website: Am J Epidemiol, 1988, 128(4): 845-859.
http://www.armazemdedados.rio.rj.gov.br/,
[18] Muttitanon W, Kongthong P, Kongkanon
April 2004.
C, Yoksan S, Gonzalez JP and Barbazan P.
[11] StatSoft Inc. Electronic Statistics Textbook. Spatial and temporal dynamics of dengue
Tulsa, OK: StatSoft. http://www.statsoft.com/ haemorrhagic fever epidemics In:
textbook/stathome.html, 2004. Conference Proceedings of Map Asia
2002, GIS Development, 2002,
[12] Selvin S. Epidemiologic analysis: a case-
http://www.gisdevelopment.net/application/
oriented approach. Oxford University Press,
health/planning/healthp0010c.htm.
New York, 2001: 321.

Sero-epidemiological and Virological Investigation of
Dengue Infection in Oaxaca, Mexico, during 2000-2001
A. Cisneros-Solano*, M.M.B. Moreno-Altamirano**#, U. Martnez-Soriano***,
F. Jimenez-Rojas, A. Daz-Badillo and M.L. Muoz
*Escuela Nacional de Medicina y Homeopata
**Escuela Nacional de Ciencias Biolgicas-Instituto Poltcnico Nacional
***Universidad Autnoma Benito Jurez de Oaxaca

Lab. Est. Salud Pblica de Oaxaca

Centro de Investigacin y Estudios Avanzados-IPN
Abstract
A sero-epidemiological-cum-virological investigation was carried out in Oaxaca, Mexico, during 2000-
2001 to assess the incidence of dengue infection and the circulating viruses.
A total of 200 serum samples reportedly from dengue patients, based on clinical diagnosis, were
collected from Oaxacas Central Laboratory of Public Health (in the capital city of the state of
Oaxaca). The samples were initially collected from ten regional health centres located across Oaxaca.
The sample population for the study included both sexes and age groups with clinical signs compatible
with dengue infection. All samples were tested for the presence of dengue virus, mainly by MAC-
ELISA and RT-PCR.
Ninety-four out of 100 serum samples suspected of dengue were confirmed to be positive. Thirty-
two were found positive by MAC-ELISA and 58 were positive by RT-PCR. In addition, the RT-PCR
analysis showed that the prevalent serotype in the localities in the study area was DEN-2. However,
one isolate of DEN-1 and another of DEN-4 were also detected. The number of infected females was
higher than that of infected males and the most affected age group was of people aged under 35
years. The study also highlighted that the sensitivity and specificity of diagnostic tools were crucial for
epidemiological studies.
Keywords: Serodiagnosis, MAC- ELISA, RT-PCR, DEN- 2, Oaxaca, Mexico.
Introduction by Gubler[3] , factors such as demographic

and social changes are responsible for the
In recent years, dengue fever (DF) / dengue re emergence of dengue. Mexico is
haemorrhagic fever (DHF) has emerged as considered an endemic country for dengue
major health problem in Mexico. In 1960, and it is reported that major epidemics of
the Aedes aegypti mosquito was eradicated DEN-1 occurred on the eastern coast of
but it reappeared in 1965[1,2]. As pointed out Mexico during 1979-1980. In 1984-1985,
#
E-mail: mmoreno@encb.ipn.mx, morsan846698716@aol.com; Tel. (5255) 55729-6300 Ext. 62370,
Fax (5255) 55396-3503

Dengue Investigation in Oaxaca, Mexico
dengue was diagnosed in 25 of the 32 states Oaxaca is located in the subtropical

of Mexico. By then, DEN-1, DEN-2 and region of Mexico at about 1,600 metres
DEN-4 were present in the country, and in above sea level (Figure 1A). There is high
1995, DEN-3 was circulating as well. Several demographic pressure and migration to
cases of DHF were also confirmed[4] . In different urban zones is common, resulting
subsequent years dengue achieved in the establishment of scattered human
endemicity in the country. settlements with deficient public services. All
these factors contributed to the propagation
As per the records of the Mexican
of the Aedes aegypti mosquito, resulting in
Health Office[5] (Secretara de Salud, SS), a
dengue outbreaks every year in most of
higher number of dengue cases were
Oaxacas communities[6] .
recorded in the states of Nuevo Len,
Tamaulipas, Veracruz and Oaxaca during
1998-2001.
Figure 1. Oaxaca, Mexico

(A) United States of Mxico. Oaxaca is located in the west coast
(B) Oaxaca is divided by the health authorities in six jurisdictions (I-VI)

To assess the dengue situation, days of the onset of fever and were
epidemiological studies were undertaken to processed for anti-dengue IgM detection
make an estimate of the incidence of using IgM capture ELISA (MAC-ELISA) as
dengue virus infection and the circulating described by Vorndam et al.[8] Samples from
serotypes in some selected endemic areas of healthy donors were obtained at about the
Oaxaca during 2000-2001. same time.
As a routine practice and with the idea
Materials and methods of recording epidemic data, the suspected
dengue samples already clinically diagnosed
in community health centres were sent to
Population study
the Central Laboratories in the city of
The state of Oaxaca is located on the west Oaxaca (Laboratorio Estatal de Salud
coast of Mexico. The Mexican Health Office Pblica del estado de Oaxaca, Secretara de
has divided it into six jurisdictions: (I) Salud). In this laboratory, the presence of
Central Valleys, (II) Tehuantepec isthmus, dengue virus was confirmed by MAC-ELISA
(III) Tuxtepec, (IV) The Coast, (V) The and RT-PCR.
Mixteca, and (VI) The Sierra. The presence
of dengue virus has been registered in all six Dengue virus isolates
jurisdictions (Figure 1B). This study was
carried out in ten municipalities distributed Aedes albopictus C6/36 cells were grown in
in five jurisdictions in the state of Oaxaca. 48-well tissue culture plates as described by
The study population included both sexes Igarashi[9] . Briefly, 2X10 5 cells were plated in
and all age groups [7] . Two population groups 1 ml of minimum essential medium (Gibco-
were included in this study: one group BRL, Grand Island, N.Y.) supplemented with
consisting of 200 serum specimens from 7% fetal bovine serum (Sigma Chemical Co.,
patients manifesting signs and symptoms of St. Louis, Mo) and 1% glutamine, vitamins
dengue infection, and another group of 50 and nonessential amino acids. After 24
serum samples from healthy controls, all hours of culture, 100 l of every sera diluted
from the same jurisdictions. 1:10 was added to the corresponding well.
The mixture was then gently shaken and
incubated for 60 minutes at room
Sample collection and diagnosis
temperature. Cells were then washed with
of dengue serum-free medium and cultured at 28 C
Human sera were obtained from 200 with complete medium for at least 10 days.
patients presenting clinical manifestations of Cells were harvested for RT-PCR diagnosis.
dengue and tested for anti-dengue IgM
antibodies. Serum samples were collected RNA extraction
by venipuncture, using Vacutainer tubes
Total RNA was extracted either from 100 l
(Becton-Dickinson). The clinical samples
of serum or from cultured cells by using
corresponded with dengue cases reported
Trizol LS (GIBCO BRL, Gaithersburg, MD.)
during 2000-2001. Dengue-infected
according to the manufacturers
samples were obtained during the first five

recommendations. Ethanol-precipitated RNA reaction mixtures were electrophoresed and

was recovered by centrifugation and air-dried. visualised under UV light after ethidium
The RNA pellet was re -suspended in 50 l of bromide staining of the gels.
Diethyl-pyrocarbonate (Sigma)-treated water
(DEPC water) and used as a template for RT-
PCR. Results
Diagnosis of the samples by MAC-
RT-PCR
ELISA
Synthetic oligonucleotide primer pairs were
Two hundred serum samples initially
designed based on published sequence data
reported as suspected positive for dengue,
for each of the four serotypes of dengue [10,11] .
based on clinical reports from the hospital
Four fragments of an expected size of 482
where patients were hospitalised, were
bp (DEN -1), 392 bp (DEN -4), 290 pb (DEN -
submitted for diagnosis based on anti-
3) and 119 bp (DEN-2) were obtained by
dengue IgM antibodies detection by MAC-
using the SuperScripTM One Step RT-PCR kit
ELISA. From these, only 34 samples were
in conjunction with PlatinumR Taq
positive for IgM antibodies . As expected,
polymerase (Invitrogen, Life Technologies).
the 50 negative-control samples resulted
A mixture of 5 l of RNA, 25 M of sense
negative for anti-dengue IgM antibodies
and anti-sense PCR primers, and DEPC
(Table 1).
water to a total volume of 50 l was
incubated at 85 C for 5 minutes and then
chilled on ice. The tubes-reaction mixture
Diagnosis by RT-PCR
containing 2X PCR buffer containing 0.4 Once the serum samples were tested for
mM of each dNTP, 2.4 mM MgSO 4 and anti-dengue IgM antibodies, the results were
Super ScriptTM RT/platinumR Taq Mix, as confirmed by RT-PCR. In this case, only 25
recommended by the manufacturer (In samples from healthy donors were tested.
vitrogen TM Life Technologies), was added By this method 58 samples proved to be
to the RNA and primers -containing tube. positive for dengue, i.e. 24 more than by
The reverse transcription reaction was MAC-ELISA. Interestingly, all samples
performed at 50 C for 30 minutes. positive for MAC-ELISA were also positive
Thermocycling began with a hot start at by RT-PCR. Those samples showing
94 C for 2 minutes followed by 40 cycles positivity for DEN by RT-PCR were further
of annealing at 55 C for 30 seconds, and tested for the four serotypes (DEN-1, -2, -3
extension at 72 C for one minute and and -4). It was found that the main
denaturing at 94 C for 15 seconds. circulating serotype in Oaxaca during 2000-
The PCR conditions for serotype 2001 was DEN-2. Two other serotypes
assessment were as follows: 40 cycles of (DEN-1 and DEN -4) were also found (only
denaturing at 94 C for 30 seconds, one case each) (Table 1).
annealing at 55 C for 1 minute, and

extension at 72 C for 1 minute and, a final Out of 200 samples, originally sent, only 100 samples
were found in good condition for evaluation by MAC-
extension at 72 C for 7 minutes. The ELISA or RT-PCR. Other samples deteriorated under
transportation/storage conditions.

Table 1. Positivity for dengue by MAC-ELISA and RT-PCR

From one hundred samples tested, from patients with clinical diagnosis of dengue,
36% proved positive by MAC-ELISA and 61% by RT-PCR. DEN-2 was the prevailing
circulating serotype
Date of Jurisdiction Locality Number MAC- RT-PCR Serotype Serotype

collection of cases ELISA
Nov 2000 II Salina Cruz 18 13+/5- 9+/9- DEN-2 DEN-2
Nov 2000 III Tuxtepec 9 2+/7- 5+/4- DEN-2 DEN-2
Nov 2000 II Tehuantepec 10 4+/6- 5+/5- DEN-2 DEN-2
Jul 2000 III Temascal 8 3+/5- 5+/3- DEN-2 DEN-2
Nov 2000 II Juchitan 3 2+/1- 2+/1- DEN-2 DEN-2
Total cases 24+/24- 26+/22-
May-Jun IV Huatulco 21 6+/15- 12+/9- DEN-2 DEN-2
2001
Jun-Nov I Oaxaca 7 2+/5- 7+/0- DEN-2 DEN-2
2001
Apr 2001 II Juchitan 2 0+/2- 2+/0- DEN-2 DEN-2
May 2001 II Salina Cruz 1 0+/1- 1+/0- DEN-2 DEN-2
Feb 2001 III Tuxtepec 7 2+/5- 2+/5- DEN-2 DEN-2
Feb 2001 V Tonal 2 0+/2- 2+/0- DEN-2 DEN-2
Feb 2001 V Huajuapan 6 0+/6- 6+/0- DEN-2 DEN-2
Total cases 10+/36- 32+/14-
Table 2. Distribution of dengue cases Prevalence of infection by

by age and sex age and sex
Age An analysis by age and sex revealed a higher
Male Female
(in years) prevalence (61%) of infection in females
1 0 1 than in males (39%) and that the most
affected group of people was the under-35-
2-4 0 2
year-olds (Table 2).
5-9 7 10
10-14 5 6
Discussion
15-20 2 3
The Aedes aegypti mosquitos adaptability to
21-30 0 5 changing environmental conditions has
>30 6 6 contributed significantly to the increase in
dengue epidemics in the world. Mexico is
Total 20 (38%) 33 (62%)
considered an endemic country where the

four serotypes of the dengue virus are in and storage in order to send them to the
circulation. This study provides some insight reference laboratory for adequate diagnosis.
into the dengue epidemic situation in It is worth noting that in several Mexican
Oaxaca state, Mexico, in an attempt to states, health authorities are working on
contribute to the prevention and control of vector control as well as on facilities for
outbreaks of DF/DHF. sample collections to be sent to the
Instituto de Referencia Epidemiolgica
From the 200 serum samples collected
(InDRE) in Mexico City for a proper
from suspected dengue patients initially
diagnosis. It is still, however, a long way for
considered for the study, only 100 could be
good quality medical care to reach most
used. The remaining 100 samples were
Mexicans.
presumably subjected to non-appropriate
storage conditions. From the 100 suspected This report shows that by MAC-ELISA,
samples tested, nearly 100% proved to be 36% of the tested samples were found
positive for dengue (36% by MAC-ELISA positive for dengue, whereas by RT-PCR up
and 61% by RT-PCR). However, the data to 64% of the samples proved to be positive.
reported here could be an underestimation Although the sensitivity and specificity
considering the several factors that could reported for MAC-ELISA is reported to be
influence the laboratory determination good enough for a diagnosis system, it is
outcome, such as sample handling and the likely that as a result of inadequate handling
diagnosis systems performed at local and storage conditions, some samples
hospitals. In some localities of Oaxaca, the reported as negative could in fact be
diagnosis for dengue was being positive for dengue when tested by RT-PCR.
simultaneously carried out with the No false positive results were found. This
diagnosis for rubella and toxoplasma in a raises the question as to how many
monoclonal antibodies-based multiplex laboratory assays must be carried out on a
assay. In rural communities, however, only suspected dengue sample before reporting it
the presence of anti-dengue IgM antibodies as negative.
was tested.
The use of RT-PCR makes it possible to
In this regard, it is possible that some identify the dengue serotype involved; in
patients presenting an early secondary this regard this study shows that in Oaxaca,
infection in the absence of strong clinical Mexico, the prevalent serotype of dengue
manifestations had undetectable levels of virus was DEN -2, although isolated cases of
anti-dengue IgM antibodies, since IgG is the DEN-1 and DEN-4 infections were also
prevalent Ig isotype at this stage of the found. Some other local reports had also
infection. For these cases, it would be mentioned the presence of DEN-3 and
necessary to consider some other diagnosis several cases of DHF.
techniques such as virus isolation or RT-PCR.
Unfortunately, these are difficult to carry out
in rural hospitals due to high costs and lack Acknowledgements
of suitably trained personnel. We thank Dr F. Javier Snchez-Garca for
Additional effort is needed to ensure critically reviewing the manuscript. MMBMA
appropriate sample collection, handling is an EDI/IPN fellow.

References
[1] Novo S. Breve historia y antologa de la [7] Martnez-Soriano U. Tipificacin Molecular
fiebre amarilla. Salud Pblica de Mxico, del virus del dengue en el estado de Oaxaca.
1995, 37: S99-S102. B.Sc. Thesis, 2002, 19-58.
[2] Gmez H. Monografa sobre la [8] Vorndam V and Kuno G. Laboratory
epidemiologa del dengue en Mxico. diagnosis of dengue virus infections. In:
Secretara de Salud, 1992, Vol. 43. Gubler DJ and Kuno G (Eds.), Dengue and
dengue hemorrhagic fever. CAB
[3] Gubler JD. Epidemic dengue/dengue
International, New York, NY, 1997, 313-333.
haemorrhagic fever as a public health, social
and economic problem in the 21st century. [9] Igarashi A. Mosquito cell cultures and the
Trends in Microbiology, 2002, 10: 100-103. study of arthropod-borne togaviruses.
Advances in Virus Research, 1985, 30: 21-39.
[4] Briseo-Garca B, Gmez-Dantes H, Argott-
Ramrez E, Montesano R, Vzquez-Martnez [10] Lanciotti RS, Calisher CH, Gubler DJ, Chang
AL, Ibanez-Bernal S, Madrigal-Ayala G, GJ and Vorndam V. Rapid detection and
Ruiz-Matus C, Flisser A and Tapia-Conyer R. typing of dengue viruses from clinical
Potential risk for dengue haemorrhagic samples by using reverse transcriptase-
fever: the isolation of serotype dengue-3 in polymerase chain reaction. Journal of
Mexico. Emerging Infectious Disease, 1996, Clinical Microbiology, 1992, 30: 545-551.
2(2): 133-135.
[11] Harris E. A low cost approach to PCR. Ed.
[5] Anonymous. Gaceta informativa de la Oxford University Press, USA, 1998, 96-105.
Secretara de Salud, SSA-Oaxaca, 1999.
[6] Cisneros AS, Martnez US, Cruz GM, Tovar
RG, Moreno MG, Das-Badillo A and Muoz
ML. Dengue fever in the state of Oaxaca,
Mexico. The American Society of Tropical
Medicine and Hygiene, 50 th Meeting, 2001,
Atlanta, Georgia, USA.

Spatial and Temporal Dynamics of
Dengue Haemorrhagic Fever Epidemics,
Nakhon Pathom Province, Thailand, 1997-2001
Wutjanun Muttitanon*, Pongpan Kongthong**, Chusak Kongkanon**,
Sutee Yoksan***, Narong Nitatpattana***, Jean Paul Gonzalez
and Philippe Barbazan #
*Asian Journal of Geoinformatics; Space Technology Application and Research Program,

Asian Institute of Technology. P.O. Box 4, Klong Luang, Pathumthani, Thailand
**Department of Geography, Faculty of Education, Ramkhamhaeng University, Bangkok 10110, Thailand
***Center for Vaccine Development (CVD), Institute of Science and Technology for Research and
Development, Mahidol University, Nakhon Pathom 73170, Thailand

Research Center for Emerging Viral Diseases (RCEVD) IRD Center for Vaccine Development,
Institute of Science and Technology for Research and Development, Mahidol University,
Nakhon Pathom 73170, Thailand

Institut de Recherche pour le Dveloppement (IRD) Ur034, 213 rue La Fayette, 75480,
Paris cedex 10, France
Abstract
Several environmental factors modulate the distribution of dengue fever (DF), such as climate, density of
vector and human populations in urban areas and distribution of herd immunity. In order to identify
geographical variables involved in the spread of a DHF process, a Geographic Information System (GIS)
has been built to create links between geo-referenced data including medical records and
socioeconomic and environmental data. Applied to a retrospective analytical study of DHF epidemics in
Nakhon Pathom province 1 ( 997-2001), the GIS allowed a mapping of spatial variations of DHF
incidence, the recognition of different temporal incidence patterns and the quantification of the dispersal
of outbreaks among defined spatial units. The analysis showed that the diffusion process of these
epidemics was of a contagious type as the distance between epidemic areas (sub-districts) was
significantly lower than the average distance between every sub-district. This result indicates that these
epidemics were likely to be due to the spread of a new or rare virus serotype, from its emergence
location in the province to areas with a sufficient density of vectors and a similar limited immune
protection against this serotype.
Keywords: Dengue haemorrhagic fever, dengue virus, transmission, Geographic Information System, spatial analysis.
#
E-mail: fnpbb@diamond.mahidol.ac.th; Tel./Fax: (66) 2 441 01 89

Spatial and Temporal Dynamics of DHF Epidemics in Thailand
Introduction dispersion, largely a function of the stochastic

movement of incubating/infectious humans
Dengue fever (DF) is a viral disease with a and the transport via vehicles of virus-positive
worldwide distribution in all tropical areas. females[4].
It is caused by the dengue virus (genus
Flavivirus, family Flaviviridae) which presents The understanding of the mechanism of
the inter-community spread of DHF during
four antigenic forms or serotypes: DEN -1,
DEN-2, DEN -3 and DEN-4. In Thailand, epidemic periods is a primary factor likely to
lead to an evaluation of the risk of virus
Dengue haemorrhagic fever (DHF) a severe
form of dengue fever has been endemic transmission and disease dispersal[5] .
Moreover, it would provide some guidance
since 1958, with a cumulative total of
1,369,542 cases till date[1] . Epidemics occur on the distance from the spatial origin of an
epidemic at which preventive control
with a periodicity of between two and four
years; these epidemics are of significant measures should be applied.
concern for the public health authorities. In At a monthly time-scale, the main
most of the areas where serotype geographical factors involved in dengue
identifications were performed, two or three transmission (urbanization, demography,
serotypes were found to be co-circulating[2] . cultural and social characteristics) are
stable[6] . A change in the pattern of monthly
The dengue virus is an arbovirus
(arthropod-borne virus) transmitted by the DHF transmission, such as the emergence of
epidemics in an endemic area, should then
mosquito Aedes aegypti (L.). Control of the
spread of the disease focuses on vector rather be related to factors evolving with
time: climate, density of vectors, emergence
control strategies based mainly on the
elimination of potential breeding sites[3] . A of a new or rare virus serotype, each type of
factor inducing a specific pattern of diffusion
major attribute of the virus transmission is its
anthropophilic behaviour, as females mainly of the disease[7,8] . The emergence of a new
serotype in a given population is likely to
bite humans and lay eggs in man-made
containers near houses (for example, water exhibit particular spatial characteristics. The
outbreak would begin where the serotype
jars, cans, used tyres). The short flight range
of the vector, less than 1 km, contributes to first arrived and then move to places where
a low specific herd immunity (towards this
the limited spread of the disease by an
infected female. Most of the infections by serotype) and a sufficient density of
mosquito allow a high level of transmission.
dengue viruses are not severe and present
asymptomatically, allowing infected patients The spread of a new serotype is then likely
to follow the main model of contagious
to maintain normal activities.
diffusion described for the spread of other
Two types of viral spread can be types of moving phenomena[9] . Applied to
described: (i) the diffusion of human the diffusion of an infectious disease, it
infections as a function of the spatial means that the probability for an area to be
distribution of houses and the limited flight reached by a contagious disease will be
range of infectious or infected Aedes aegypti inversely correlated to the distance to the
females (intra-communal, contagious/ formerly contaminated areas, leading to
continuous); and (ii) inter-communal clusters of epidemic areas.

In order to test the validity of this model Department. DHF cases were defined
in the frame of dengue dispersal, a study according to WHO criteria[10] .
was conducted to describe the spread of
significantly higher levels of incidence rate Population and study area
(of epidemic significance) among sub-
districts. The study, done in a province of Nakhon Pathom province is a part of the
Thailand, covering the period 1997-2001, central plain region in Thailand
included two DHF epidemics. encompassing the latitude of 13 3845.6 N
to 14 1037.2 N and the longitude of
99 5110.8 E to 100 176 E. It covers
Materials and methods 2,164 sq km, has a population of 774,276
inhabitants and includes 7 districts and 106
Data collection sub-districts (Figure 1a). The population
density ranges from 153 to 623
Data on clinically diagnosed DHF cases inhabitants/sq km. The average surface area
were recorded at the Ministry of Public of sub-districts is 20.4 sq. km. The provincial
Health, the demographic data were health department reported 14,079 DHF
provided by the Administrative Department cases during 1983-2001; two DHF
of the Ministry of Interior, and the epidemics occurred in 1997-1998 and
geographical maps by the Royal Thai Survey 2000-2001 (Figure 2).
Figure 1. District scale approach: (i) Administrative limits of districts and sub-districts;
density of population; main roads; (ii) Average incidence observed before the epidemics
(cases/100,000 people), January 1992 June 1997; (iii) Ratio of the incidence during the
first three months of the DHF epidemic compared to the average incidence from
January 1992 June 1997
a) Density of population b) Average Incidence c) Ratio of Incidence

5 0 10 20 km
(inhabitants / ha) 01/1992 - 06/1997 07 09 1997 vs
(cases /100,000) aver. 01/1992 - 06/1997
Main roads
> 600 22 - 46 1.6 2.8
Districts 350 - 450 8 - 10 1.3 1.4
Sub-districts 150 - 350 3-7 0.3 0.7

Figure 2. Monthly DHF incidence in Nakhon Pathom province, Thailand, from January
1992 to August 2001
300
200
cases
100
0
1-92 7-92 1-93 7-93 1-94 7-94 1-95 7-95 1-96 7-96 1-97 7-97 1-98 7-98 1-99 7-99 1-00 7-00 1-01 7-01
month
Method of analysis In a contagious model for an infectious

disease, the spatial entities close to an
The study aimed to describe the spatial- infected one were assumed to be more at
temporal dynamics at a monthly time-scale risk to become infected than the distant
of a DHF epidemic among Nakhon ones. Applied to the diffusion of an
Pathoms 106 sub-districts considered as the epidemic phenomenon, it meant that the
spatial units. As a first step, epidemics were distance between the new epidemic sub-
defined at the province level as periods of districts and the former ones (observed
time (at least two consecutive months) when distance) should be shorter than the average
the incidence is higher than the average, (expected) distance between all the sub-
plus one standard deviation of the monthly districts. The distance between sub-districts
incidence of each month (i.e. January, was defined as the Euclidian distance
February, etc.). The average was calculated between their centroids. The expected
over the entire 1983-2001 period[11] . distance was the average distance between
each ESD and every other sub-district. The
During these epidemic months (EMs), observed distance was the average distance
epidemic sub-districts (ESDs) were those between each ESD and every other ESD,
where the monthly incidence was during the same month (cluster study), or
significantly higher than in other sub-districts. from one month to the next (spread study).
The threshold for a significantly higher
H0 (null hypothesis) = the average
incidence was leveled at the average
observed distance (between ESD) was not
monthly incidence (per 100,000
different from the average expected distances.
inhabitants) plus one standard deviation,
observed among every sub-district during H1 = average observed distance
that EM. <average expected distance.

The Z test was used to compare the drowned at 5 km; 10 km; 15 km; 20 km
average distances. and out of 20 km. This number is compared
to the number of sub-districts centroids
The method was applied to the study of
distributed in these surfaces to build a
two phenomena: (i) the occurrence of
relative risk index.
clusters of ESD during one month; and (ii)
the spread of the epidemic among sub- Relative risk index =
number of ESD in a circle
districts from one month to the next. A total number of ESD
number of sub - districts in a circle
cluster is defined here as an aggregation of
total number of sub - districts
ESD (during one EM) of sufficient size and
concentration to be unlikely to have
occurred by chance, i.e. if the average Results
distance (between these ESD) is shorter than
the average distance between all sub- At the district scale, the DHF incidence was
districts. The spread of the epidemic is higher in the central-west part of the
based on the comparison of observed and province. The epidemic broke out in the
expected distances during two consecutive northern district with a medium density of
EMs, i.e. the average distance between ESD population (Figures 1b and 1c). At the sub-
during one epidemic month (EMm) and ESD district scale, the maximum DHF incidence
during the next epidemic month (EMm+1), rate reached 540 cases per 100,000
versus the average distance between ESD inhabitants in July 1997.
during EMm and every sub-district during Nineteen EMs were identified in
EMm+1. Nakhon Pathom province from January
The (discrete) distance, at which an 1997 to August 2001 (Table); the number of
epidemic can spread in one month, was EMs in one sub-district ranged from 0
estimated by summing the number of ESD month (in 27 sub-districts) to a maximum of
centroid during EMm+1 observed inside 11 months.
circles centred on each ESD during EMm and
Table. Chronological distribution of epidemic months from January 1997 to August 2001 in
Nakhon Pathom province, Thailand
Year Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec
1997
1998
1999
2000
2001
= Epidemic month

A total of 49 ESDs were identified significantly lower (P<0.05) than the

during the first outbreak (1997-1998) and average expected distance, characterizing
61 during the second outbreak (2000- the occurrence of clusters of ESDs according
2001); 31 sub-districts were epidemic- to the H1 hypothesis.
affected during the two outbreaks. The
Spread study: The distance observed
probability of one sub-district being
between ESDs during EMm and ESDs during
epidemic-affected during the first outbreak
EMm+1, was significantly smaller than the
to be epidemic-affected during the second
expected distance (Figure 3), characterizing
outbreak as well is not significantly different
the contagious spread of DHF among ESDs
from a random distribution (P<0.05).
according to the H1 hypothesis.
Cluster study: During EM, 78.67% of
the average observed distances were
Figure 3. Study of the spread of DHF epidemic among sub-districts: observed distances are
calculated between epidemic-affected sub-districts during one month and the epidemic-
affected sub-districts during the next month; expected distances are calculated between all
the sub-districts (see text for details)
97-98 epidemic endemic 00-01 epidemic

25 20
15
nb of tambon
distance (km)
10
0 0
Aug Oct Dec Feb Apr Nov Jan Mar May
97 97 97 98 98 month 00 01 01 01
number of epidemic tambon observed distance expected distance
As a consequence, the distribution of Discussion

ESDs during EMm+1 in surfaces drowned
round ESD during EMm showed a significant The method used for the identification of an
(P<0.05) aggregation within the first two epidemic month in the province[11] allowed
circles (5 and 10 km). a precise framing of epidemics, defined as
periods during which the incidence was

significantly higher than the observed distribution of all sub-districts having been
average over the complete time series of epidemic-affected at least during one month
data (19 years). Meanwhile, this method (67% of the sub-districts) was not
could not be used directly at the sub-district significantly different from the spatial
scale because of the lack of long-time data distribution of all sub-districts (average
series on the incidence at this scale. distances not different, P=0.95). Meanwhile,
Moreover, the variance of the DHF the results implied a high degree of spatial
incidence in most of the sub-districts was auto-correlation, meaning that neighbouring
very high because of the low values of sub-districts shared similar characteristics,
incidence often recorded (during the study such as the level of immunity for the
period a null monthly incidence was different serotypes (due to a similar
reported in 66% of the 5,936 months X sub- epidemiological history) or the density of the
districts). Similarly, the village scale (from 3 vector.
to 24 villages per sub-district) could not be
As shown in Figure 3, the observed
used as the spatial unit as the addresses of
distances are smaller than the expected
patients were often consistent only at the
ones, but exhibit similar monthly variations.
sub-district scale and many students and
This was mainly because of a border effect,
pupils did not live in their village.
the propagation in sub-districts located in
We assumed in this study that sub- neighbouring provinces not being taken into
districts could be considered as account. During the months where ESDs
homogeneous small areas and that human were located on the periphery of the
displacements were sufficient to produce a province, the average distance to other sub-
homogenization of the population, allowing districts was larger than during the months
consideration of the sub-district as a unit where ESDs were located near the centre of
towards DHF transmission. An epidemic the province, as several neighbouring sub-
pattern can then be identified in any sub- districts located in other provinces
district, whatever its density of population: (epidemic or not) were missing in the
the distribution of ESD was not correlated to calculation. The absolute level of expected
the density of population (Pearsons and observed distances was then directly
correlation = -0.24, P = 0.71). Moreover, dependent on the location of the ESD in the
we used the incidence rate per 100,000 province.
inhabitants to reduce the bias related to the
The spread of the epidemic between
size of the population.
sub-districts followed Hagerstrands model
The geographical heterogeneity of the that has been used to describe many types
environment, e.g. the density of of phenomena, such as the spread of new
urbanization or the road network, could also ideas [9] or the waves of innovation which
be at the origin of clusters of ESDs. lose their energy when the distance from
Meanwhile, after the two epidemics, ESDs the source increases[12] . In public health
were found to be uniformly distributed over research it has been applied to infectious
the entire province, and the spatial influenza[13] . Applied to the DHF epidemic

in Nakhon Pathom, it means that during the DHF is endemic in Thailand and the
epidemic periods the ESDs were the origin different serotypes are largely distributed, as
of the emergence of epidemics in at least two or three serotypes are generally
neighbouring sub-districts during the next found at the same time in the same area[2,16] .
month. The probability of this emergence at Meanwhile, during epidemic periods the
m+1 significantly decreased with the relative prevalence of the serotypes varies,
distance from the former ESD. This model is as the 2000 epidemic in Bangkok that was
of a contagious type and may be opposed to due mainly to the serotypes DEN-1 and
a random or homogeneous model. In the DEN-2 (each reaching 42% of total
homogeneous models the occurrence of an isolations), whereas the 1994 epidemic was
epidemic could be due to a global due mainly to the rise in DEN -4 (36%). But
phenomenon, such as an increase in as serology and isolation of viruses are rarely
temperature, which should have been performed, the emergence of a DHF
observed in any sub-district, leading to a epidemic cannot be forecast by using these
random distribution of ESD [14] and an methods. Indirect methods, such as the
observed distance not different from the statistical identification of epidemic months,
expected distance. are then necessary to identify early the
emergence of DHF epidemics.
Inside human communities (villages) it
has been shown that the spread of DHF The epidemiology of DHF in Thailand is
viruses from one house to neighbouring changing[17] . This approach of the
houses due to the displacement of infected displacement of epidemics is likely to
vectors or hosts follows a pattern similar to contribute to the localization of the origins
what we have described between sub- of outbreaks and the delineation of areas at
districts[15] . Meanwhile, among communities risk during epidemics, as well as to help
separated by several kilometers, the spread public health authorities to focus vector
of viruses cannot be due to the active control activities on selected areas.
dispersal of mosquitoes or to their transport
by car, which is much more rare than the
displacement of infected hosts. More than Acknowledgements
80% of infections by dengue virus are The study and the preparation of this paper
unapparent or not severe, allowing healthy was supported by the Institut de Recherche
carriers to travel. The presence of sufficient pour le Dveloppement (IRD)-Ur034,
densities of vectors in destination France, by a fellowship to the Center for
communities is also necessary to allow the Vaccine Development, Institute of Science
transmission of the virus after it has been and Technology for Research and
imported. Development, Mahidol University, Thailand,
The contagious distribution and spread and by the Department for Technical and
of the two DHF epidemics among the sub- Economic Cooperation, Thailand. We thank
districts strongly suggests that they were due Professor Natth Bramapavarati for his
to the emergence of a new or rare serotype. constant support.

References
[1] Annual epidemiological surveillance report, [9] Hagerstrand T. The propagation of innovation
1983-2002. Bangkok: Ministry of Public waves. Lund studies in Geography, Series B,
Health, Department of Disease Control, 1952, 4.
Bureau of Epidemiology, 2002.
[2] Vaughn DW, Green S, Kalayanarooj S, Innis haemorrhagic fever: diagnosis, treatment
BL, Nimmannitya S, Suntayakorn S, and control, 2 nd edition. Geneva: 1997.
Rothman AL, Ennis FA and Nisalak A.
[11] Barbazan P, Yoksan S and Gonzalez JP.
Dengue in the early febrile phase: viremia
Dengue Hemorrhagic Fever epidemiology in
and antibody responses. Journal of
Thailand: Description and forecast of
Infectious Diseases, 1997, 176: 322-330.
epidemics. Microbes and Infection, 2002,
[3] World Health Organization. Dengue 1(4): 699-705.
prevention and control. Executive Board,
[12] Gould PR. Spatial diffusion. Washington, US,
109 th Session, 2001, EB109/16.
Association of American Geographers, 1969.
[4] Meade MS, Florin JW and Gesler WM.
Medical Geography. London, UK: The [13] Cliff AD, Haggett P and Ord JK. Spatial
aspects of influenza epidemic. London, Pion
Guilford Press, 1988.
Limited. 1986.
[5] Cuzick J and Elliott P. Small-area studies:
purpose and methods. Chapter 2 of [14] Jackson EK. Climate change and global
Geographical & Environmental Epidemiology infectious disease threats. The Medical
(Eds. P. Elliott, J. Cuzick, D. English and R. Journal of Australia, 1995, 163(4): 570-573.
Stern). 2nd ed. New York: Oxford University [15] Morrison AC, Getis A, Santiago M, Rigau-
Press, 1997. Perez JG and Reiter P. Exploratory space-
time analysis of reported dengue cases
[6] Kuno G. Review of the factor modulating
during an outbreak in Florida, Puerto Rico,
dengue transmission. Epidemiologic Reviews,
1991-1992. American Journal of Tropical
1995, 12(2): 321-335.
Medicine and Hygiene, 1998, 58(3): 287-298.
[7] Focks DA, Daniels E, Haile DG and Keesling
[16] Burke DS, Nisalak A, Johnson DE and Scott
JE. A simulation model of the epidemiology
of urban dengue fever: literature analysis, RM. A prospective study of dengue
model development, preliminary validation, infections in Thailand. American Journal of
and samples of simulation results. American Tropical Medicine and Hygiene, 1988,
Journal of Tropical Medicine and Hygiene, 38(1): 172-180.
1995, 53(5): 489-506. [17] Chareonsook O, Foy HM, Teeraratkul A and
[8] Mayer JD. The role of spatial analysis and Silarug N. Changing epidemiology of dengue
hemorrhagic fever in Thailand.
geographic data in the detection of disease
Epidemiology and Infection, 1999, 122:
causation. Social Science and Medicine,
161-166.
1983, 17(16): 1213-1221.

Sporadic Prevalence of DF/DHF in the Nilgiri and
Cardamom Hills of Western Ghats in South India: Is it a
Seeding from Sylvatic Dengue Cycle A Hypothesis
Nand Lal Kalra* and Chusak Prasittisuk**#
*A-38, Swasthaya Vihar, Vikas Marg, Delhi 110 092
**Regional Office for South-East Asia, World Health Organization, New Delhi, India
Abstract
The Western Ghats of south India, encompassing the Nilgiri and Cardamom hills, are the wettest region
of the country. Hills rising upto 3,000 metres receive over 200 cm of rainfall from both the south-west
monsoon (June to September) and north-eastern monsoon (October to January). The eastern slopes of
the Nilgiri Hills (200-500 metres) are bounded by Coimbatore and Erode districts of Tamil Nadu;
whereas the western slopes of the Nilgiri and Cardamom hills are in the state of Kerala. The countryside
has rich forests of teak and sandalwood, interspersed by groves of coconut, rubber, pepper, cardamom
and banana plantations. Apart from the rich flora, monkeys M ( acaca radiata) maintain a strong
association in orchards with humans competing for food.
The emergence of DF/DHF in this hilly region is a recent occurrence. An epidemiological team from
the National Institute of Communicable Diseases (NICD) investigated the first-ever reported outbreak in
Coimbatore in 1998. In all, 20 serological positive (IgM) cases were recorded by the city corporation.
Five cases came from urban towns and 15 cases from rural areas of the two districts of Coimbatore and
Erode. Rural cases were scattered in distantly located villages. Pyramid characterization and clustering of
cases was conspicuously absent. No attempt was made to link urban cases to central/peripheral wards,
nor the history of movement of patients two weeks prior to the onset of fever was investigated. No
increase in fever rate was observed. DF cases did not show any relationship with presence/absence of
Aedes breeding. Aedes aegypti detected in urban centres failed to amplify the infection.
Kerala state also reported 116 cases in 1997, from Kottayam district. Out of these, 14 cases were
confirmed serologically. After a lull of 4 years, 70 probable cases out of 877 were reported from the four
districts famous for rubber plantations. Entomological investigations recorded only Aedes albopictus in
these areas. Considering the high experimental susceptibility of both species of monkeys, viz. Macaca
mulatta and Macaca radiata to yellow fever virus, detection of dengue antigen in field collected Aedes
albopictus in Kozhikode (Kerala) and the evidence of transovarian transmission in Aedes albopictus
reared from soils of tree holes at Jodhpur Rajasthan (western India) lend support to the hypothesis that
DF in the Western Ghats of South India exists as enzootic monkey Aedes albopictus monkey cycle
and causes epizootics among rural human population either during periodic amplification of the enzootic
cycle or as occupational hazards to the people working in orchards.
Keywords: DF/DHF, sporadic cases, Macaca radiata, dengue antigen in Aedes albopictus, transovarial transmission,
sylvatic cycle, Nilgiri and Cardamom hills, south India.
#
E-mail: chusakp@whosea.org

A Suspected Sylvatic Cycle in the Nilgiri and Cardamom Hills in South India
Introduction Figure. Physical map of Kerala and

adjoining areas of Tamil Nadu
The Western Ghats of south India
encompass two southern states i.e. Tamil
Nadu and Kerala (Figure). The crests of the
Nilgiri and Cardamom Hills (rising upto
3,000 metres altitude) separate these two
states. The eastern slopes of the Nilgiri Hills
have a gentle slope and include two
important district towns of Tamil Nadu, viz.
Erode and Coimbatore, situated at an
altitude varying between 200 to 500 metres;
each with over one million population. This
part of the region receives rains from both
the south-west and north-east monsoon.
The south-west monsoons become weak,
being on the leeward side of the hills, but
maximum rains come from the north-east
monsoon, the total being <100 cm. The
forests are tropical monsoon, which are
famous for teak and sandalwood, with
patches of arecanut palms[1].
The western side of the Cardamon hills
encompasses the state of Kerala. Strong
winds of the south-west monsoon lead to
the formation of heavy sand dunes in
coastal areas and the rain water coming
from the steep hills results in formation of
shallow lagoons, all along the coast, at
places connected to the sea. These lagoons
are connected by canals. These backwaters
are the characteristics of Kerala state. The
banks of these backwaters and sand dunes
are dotted with coconut trees[1].
The Region receives heavy rains (>200 Dengue transmission cycles
cm) during the south-west monsoon. Hence, Transmission of dengue viruses occurs in two
the whole region is very wet and supports cycles, viz. enzootic and epidemic cycles.
luxuriant growth. Large tracts of forests have The enzootic cycle is a primitive sylvatic cycle
been cleared for raising cash crops, viz. maintained by lower primates (monkeys) and
arecanut palms, rubber, banana, pepper canopy dwelling Aedes mosquitoes, as
and cardamom plantations[1]. reported from South-Asia[2], Africa[3] and Sri

Lanka [4]. Current epidemiological evidence DF in hilly regions of Tamil Nadu

suggests that these viruses do not regularly
move out of the forests to urban centres but During 1998, a team from the National
at times are involved in an epidemic cycle in Institute of Communicable Diseases (NICD),
small rural villages or islands [3]. A number of Delhi, investigated the first-ever reported
Aedes species may act as reservoirs [2]. outbreak in Coimbatore [8], a town situated
at an altitude varying from 300 to 500
The epidemic cycle is confined to large
metres on the eastern slopes of the Nilgiri
urban centres. The viruses are maintained in
Hills. The epidemiological characteristics of
the Aedes aegypti human Aedes aegypti
the outbreak are summed up below:
cycles with periodic/cyclic epidemics.
Generally all serotypes circulate and give In all, 20 serologically positive and
rise to hyperendemicity. Virus is maintained compatible to DF/DHF cases were
either transovarially by the vectors or by reported. Eighty percent (16/20) were
continuous low-grade transmission in children below 10 years and two
susceptible hosts added to the population. patients aged 16 and 5 died of DHF.
DF/DHF in urban cycles is characterized by
iceberg or pyramid phenomenon. At the Fourteen cases were males.
base most of the cases are symptom-less, Seventeen cases came from Coimbatore
followed in increasing rarity, by district and three cases from rural areas
undifferentiated fever, DF, DHF or DSS[5].
of adjoining Erode district.
Occurrence of multiple cases in a single
household or clustering of cases in a locality Only five cases came from urban
is yet another characteristic of this disease[2]. Coimbatore town and the rest (12
cases) were from rural areas of
Coimbatore district.
DF/DHF in India
Rural cases were scattered in distantly
In recent years the first outbreak of DF/DHF located villages. Clustering of cases and
was reported from Kolkata (earlier known as pyramid phenomenon was conspicuously
Calcutta) in 1963[6]. Since then, more than absent. No attempt was made to link
60 outbreaks have been reported from all urban cases to central or peripheral
over the country[7]. Aedes aegypti, invariably wards/zones nor the movement history
has been found to be associated with these of patients two weeks prior to the onset
epidemics. All the four serotypes, DEN-1, 2, of fever was investigated.
3 and 4, are now circulating in the country.
No relationship could be established
between outbreak and increased fever
DF/DHF in Nilgiri/Cardamom rate.
hilly areas of south India Eighty-nine percent of blood samples
from healthy contact persons from
Since 1996-97, there have been reports of urban and rural areas showed dengue
sporadic occurrence of DF/DHF cases in
virus IgG antibodies.
Tamil Nadu and Kerala.

Entomological investigation recorded DF in Kerala

Aedes aegypti in all areas surveyed in
urban and rural areas, but failed to Epidemiological data
amplify the infection.
As per investigations undertaken by the
During the 2003 outbreak in Centre for Research in Medical Entomology
Coimbatore town 23 cases of DF were (CRME) [9], the state of Kerala started
recorded. Distribution once again reporting DF for the first time in 1997.
followed the same pattern, i.e. 5 cases Distribution of DF cases are included in
from urban towns and 18 cases from Table 1.
rural areas (Source: VBDC, New Delhi)
Table 1: Distribution of DF cases in Kerala state during 1997-2001
No. of suspected No. serologically

Year Deaths Districts
cases positive
1997 116 14 4 Kottayam
1998 67 0 13 Kottayam
1999 1 0 0 Kottayam
2000 0 0 0 Kottayam
2001 877 70 1 4 districts*
* Four districts included Kottayam, Idukki, Ernakulam and Thiruvananthapuram famous for rubber planatations
Table 2: Results of Aedes survey of 4 districts of Kerala state during 2001-2003
House index Container index

S. Name of Breteau
No. No. No. No. Remarks
No. district % % index
ex. +ve ex. +ve
Types of
1. Alappuzha 150 40 26.7 201 63 31.3 42.0
containers
and sites of
2. Ernakulam 24 10 41.7 54 41 75.9 170.8 detection of
Aedes
breeding, viz.
3. Kottayam 70 24 34.2 125 29 23.2 41.4
domestic/peri-
domestic;
4. Kozhikode 3,311 895 26.23 17,912 1,077 6.01 31.57 extradomestic
not mentioned
Pool rearing of larval breeding collected from all localities indicated Aedes albopictus as the major species.
Aedes aegypti was encountered in very few places and in scanty numbers (Source: NICD, Delhi, 2004)

Entomological data albopictus, when subjected to IFA test,

showed the presence of dengue antigen,
Entomological investigation initiated by a
thereby confirming the transovarial
CRME team in 2 localities of Kottayam
cycle of the virus [13].
district yielded only Aedes albopictus, and
Aedes aegypti was not detected. Sylvan
environment of rubber plantations was Hypothesis
detected as the unique habitat of the Aedes
albopictus[10,11]. Occurrence of DF cases in peripheral and
rural areas of Coimbatore and Erode districts
Results of yet another entomological in Tamil Nadu and non-amplification of
study carried out by the National Institute of infection by Aedes aegypti and sporadic
Communicable Diseases (NICD) field station occurrence of DF cases in Kerala in the
located at Kozhikode, in DF affected absence of Aedes aegypti points out to
districts during 2001-2003, are included in
either spillover of enzootic foci of dengue
Table 2.
during periodic amplification of the sylvatic
cycles or occupational hazards in the
Antigen detection of dengue virus presence of vertical transmission as
During May 2004, a pool of landing evidenced by the Kozhikode and Jodhpur
collection of Aedes albopicuts (dessicated), studies.
collected from the fringe of forested villages, Both the Nilgiri and Cardamom hills are
600 metres away from Kozhikode (earlier infested with Macaca radiata, the
known as Calicut) International Airport,
bonnet monkeys. Enzootic cycle of
yielded dengue antigen (processed at CRME,
simian malaria caused by Plasmodium
Madurai) [12].
cynomolgi and Plasmodium inui,
transmitted by Anopheles elegans
Vertical transmission by (Anopheles dirus group) [14,15], has been
Aedes albopictus detected in the Nilgiri hills. Whereas in
A recent study has been carried out at the Kerala, a similar simian foci has been
Desert Medicine Research Centre (DMRC), detected at Nilambur district of the
Jodhpur (Rajasthan), an institution under the western slope in Macaca radiata, while
Indian Council of Medical Research, on in Alappuzha (district in the central
possible existence of Aedes albopictus plains, earlier known as Alleppey)
monkey Aedes albopictus cycle. The monkeys were found negative for lack
highlights of the study included that: of Anopheles elegans population[16].
In a desert ecosystem, both Aedes Both the monkey species, viz. Macaca
aegypti and Aedes albopictus breed in mulatta and Macaca radiata have been
tree holes in zoo and monumental found to be highly susceptible to yellow
parks, harbouring monkeys, outside the fever virus (flaviviruses) under
city limits. experimental conditions[17]. This lends
support to the susceptibility of these
Viable eggs retrieved from the soil of
monkeys to dengue virus as well.
tree holes were reared to adults. Aedes

Lack of vectorial competence of Aedes Gubler[20], proposed that at some point

albopictus in the amplification of urban in the past, probably with the clearing
dengue epidemic has recently been of the forests and development of
demonstrated during the investigation human settlements, dengue viruses
of the first-ever DF outbreak at moved out of the jungles and into a
Phuentsholing, Bhutan in 2004[18]. rural environment where they were,
Entomological investigations revealed and still are transmitted to humans by
that Aedes aegypti occupied domestic peri-domestic mosquitoes such as Aedes
habitats breeding primarily in storage albopictus.
containers inside houses, while Aedes
In view of the aforesaid, the land use in
albopictus bred in tree holes, 55-gallon
the Nilgiri hills of Tamil Nadu and in the
drums and used tyres in the extra-
Cardamom hills in Kerala is under pressure
domestic habitats. The overlapping
of deforestation to be replaced with cash
zone was the peridomestic areas where
crops. This has brought monkey populations
both species shared breeding in flower
much closer to human settlements.
vases/trash. A large-scale source
Therefore, there is a need for indepth sero-
reduction/larvicidal campaign supported
epidemiological and entomological studies
by deltamethrin fogging in residential
with backup of virology support using
areas largely eliminated Aedes aegypti
molecular tools for genomic sequencing of
and the cases came down to single digits
viruses obtained from simian and human
within a month, while Aedes albopictus
sources. Validation of the hypothesis is of
still maintained high larval indices.
great epidemiological significance as it
Rudnik and Lim [19], while working in would require radical changes in developing
Malaysia, isolated DEN-1, 2 and 3 vector control strategies for Aedes
viruses from monkeys and also albopictus-transmitted DF.
proposed that the rural dengue vector
Aedes albopictus, may introduce sylvatic
virus into the human population. Acknowledgements
Studies in Sri Lanka proved that dengue The author gratefully acknowledges
virus causes epizootics among Dr Duane J. Gubler for critically reviewing
macaques, rather than being enzootic the manuscript.
as observed elsewhere [4].
References
[1] L Dudley Stamp. Asia a regional and [4] de Silva AM, Dittus WP, Amersinghe PH and
economic geography. Reprinted Indian Amersinghe FP. Serologic evidence for an
edition. New Delhi: B.I. Publications, 1991. epizootic dengue virus infecting toque
[2] Gubler DJ. Dengue and dengue macaques (Macaca sinica) at Polonnaruwa,
haemorrhagic fever, Clinical Microbiol Sri Lanka. Am J Trop Med Hyg, 1999, 60(2):
Review, 1998, 11(3): 480-496. 300-306.
[3] Ricco-Hesse R. Molecular evolution and [5] Guzman MG and Kouri G. Dengue: an
distribution of dengue viruses type 1 and 2 update. The Lancet Infectious Diseases,
2002, 2: 33-42.
in nature. Virology, 1990, 174: 479-493.

[6] Ramakrishnan SP, Gelfand HM, Bose PN, [13] Joshi V, Singhi M and Mourya DT. Studies
Sehgal PN, Mukherjee RN. The epidemic on determination of possible role of Aedes
of acute haemorrhagic fever, Calcutta, albopictus mosquitoes in maintenance of
1963 epidemiological inquiry. Ind Jour urban cycle of dengue. Desert Medicine
Med Res, 1964, 52: 633-650. Research Centre (Indian Council of
Medical Research), Jodhpur. Annual Report
[7] Yadava RL and Narasimham MVVL.
2002-2003: 58-65.
Dengue/dengue haemorrhagic fever and its
control in India. WHO/Dengue Newsletter, [14] Ramakrishnan SP and Mohan BN. An
1992, 17: 3-8. enzootic focus of malaria in Macaca radiata
radiata, Geoffroy, of Nilgiris Madras state,
[8] Singh J, Balakrishnan N, Bhardwaj P,
India. Ind J Malariology, 1962, 16: 87-94.
Muthadevi P, George EG, Subramani K,
Soundararajan K, Appavoo NC, Jain DC, [15] Pattanayak S. A note on natural simian
Ichhpujani RL, Bhatia R and Sokhey J. malaria infection in Kerala state, India. Ind
Silent spread of DF/DHF to Coimbatore J Malarilogy, 1963, 17: 293-294.
and Erode districts in Tamil Nadu, India
[16] Choudhury DS. Investigations on simian
1988. Need for effective surveillance to
malaria in India and its potential as a
monitor and control the disease. Epidemiol
source of zoonosis. Ind J Malariology, 1981,
Infect, 2000, 125: 195-200.
18: 28-34.
[9] Tyagi BK, Hiriyan J and Tewari SC.
[17] Kalra NL and Sharma VP. Yellow fever
Investigation on dengue fever in Kerala.
threat. Current Science, 1996, 71(12): 948.
Centre for Research in Medical Entomology
(Indian Council of Medical Research), [18] World Health Organization. WHO mission
Madurai Annual Report 2001-2002: 66-68. to Bhutan assistance to national
authorities for control of dengue outbreak
[10] Hiriyan J, Tewari SC and Tyagi BK. Aedes
at Phuentsholing, Bhutan (Draft report), 8-9
albopictus breeding in plastic cups and tea-
August 2004, WHO/SEARO, New Delhi.
vendor spots in Ernakulam city, Kerala state,
India. Dengue Bulletin, 2003, 27: 197-198. [19] Rudnik A and Lim TW (Eds.). Dengue fever
studies in Malaysia. Bulletin from the
[11] Sumodan PK. Potential of rubber
Institute for Medical Research, Malaysia,
plantations as breeding sources for Aedes
1986, 23: 1-241.
albopictus in Kerala, India. Dengue Bulletin,
2003, 27: 197-198. [20] Gubler DJ. Dengue and dengue
haemorrhagic fever: its history and
[12] Das BP, Kabilan L, Sharma SN, Lal S, Regu
resurgence as a global public health
K, and Saxena VK. Detection of dengue
problem. In: Gubler DJ and Kuno G (Eds.)
virus in wild caught Aedes albopictus
Dengue and dengue haemorrhagic fever.
(Skuse) around Kozhikode airport,
Wallingford: CAB International, 1997: 1-23.
Malappuram district, Kerala, India. Dengue
Bulletin, 2004, 28 (in press).

Autoimmunity in Dengue Virus Infection
Chiou-Feng Lin*, Huan-Yao Lei*, Ching-Chuan Liu**, Hsiao-Sheng Liu*,
Trai-Ming Yeh***, Shun-Hua Chen* and Yee-Shin Lin* #
*Department of Microbiology and Immunology, National Cheng Kung University Medical College,
Tainan, Taiwan
**Department of Paediatrics, National Cheng Kung University Medical College, Tainan, Taiwan
***Department of Medical Technology, National Cheng Kung University Medical College, Tainan, Taiwan
Abstract
Dengue haemorrhagic fever (DHF) is a complicated disease associated with viral and immune
pathogenesis. There is still no effective vaccine to prevent the progression of DHF because of its
undefined pathogenic mechanisms. The generation of autoimmunity in dengue virus (DEN) infection has
been implicated in dengue pathogenesis. Based on our previous studies showing antibodies (Abs) against
DEN nonstructural protein 1 (NS1) cross-reacted with human platelets and endothelial cells, a
mechanism of molecular mimicry may contribute to autoantibody (autoAb) production. Here, the
generation of autoAbs against human endothelial cells in patients infected with different DEN serotypes is
shown. The levels of autoAbs present in different disease stages of DHF and the induction of endothelial
cell apoptosis by patient sera were also determined. The results suggest that autoimmune responses are
implicated in dengue disease pathogenesis and cause concern in vaccine development.
Keywords: Dengue haemorrhagic fever, dengue virus serotype, autoimmunity, autoantibody, endothelial cells.
Introduction Presently, the severity of dengue disease is

primarily predicted according to the effect
Infection with dengue virus (DEN) causes of antibody-dependent enhancement (ADE)
dengue fever (DF) an important in different serotype cross-infections[2,5,6]. In
arthropod-borne viral disease in terms of order to effectively control the progression
morbidity and mortality[ 1,2] and may result in of the disease, development of an effective
severe dengue haemorrhagic fever and vaccine against DEN infection is needed.
dengue shock syndrome (DHF/DSS). There are several vaccine candidates
Globally, about 2.5 billion people are at risk undergoing clinical trials[7-10] . Nevertheless,
of the infection[3]. A recent dengue outbreak the role that antibodies (Abs) may play in
in Indonesia led to a 1.1% case-fatality rate increasing the severity of dengue
in 58,301 cases by April 2004[4] . All four infections [2,7,10]
remains a matter of concern
DEN serotypes were present in this outbreak. in vaccine development.
#
E-mail: yslin1@mail.ncku.edu.tw

In addition to the ADE of DEN infection, were 1:25 diluted and incubated with cells at
autoantibody (autoAb) production may also 4 C for 1 hour. After being washed three
be involved in dengue diseases[11-15] . We times with PBS, the cells were incubated with
demonstrated that the autoAbs generated in 20 l of fluorescein isothiocyanate (FITC)-
DEN infection induced endothelial cell conjugated anti-human IgG or IgM
damage[13] and inflammatory activation (in (PharMingen, San Diego, CA) at 4 C for 1
press). A mechanism of molecular mimicry in hour. The binding activity of Abs to cells was
which Abs directed against DEN analysed using flow cytometry (FACScan; BD
Biosciences, San Jose, CA) with excitation set
nonstructural protein 1 (NS1) is, at least in
at 488 nm.
part, responsible for the autoimmunity. The
relationships of the autoAb levels with
Cell death detection
dengue serotypes and disease severity are
examined in this study. For cell viability determination, cells were
stained with eosin Y and counted using light
microscopy. Apoptosis-induced DNA strand
Materials and methods breaks were analysed by terminal
deoxynucleotidyl transferase-mediated
Patient sera dUTP nick-end-labeling (TUNEL) reaction
DEN-2 and DEN-3 patient sera were using the ApoAlert DNA Fragmentation Assay
collected during the outbreaks in southern Kit (Clontech, Palo Alto, CA). After incubation
Taiwan from 1997 to January 1999[16] . DEN- with patient sera for 24 hours, endothelial
4 patient sera were obtained from the cells (1106) were fixed and stained
Department of Dengue Hemorrhagic Fever, according to the manufacturers instructions,
Childrens Hospital No. 1, Ho Chi Minh City, and then analysed using flow cytometry.
Viet Nam. The disease severity was based on
the WHO definition[3]. Normal control sera Statistical analysis
from five healthy individuals were used as The statistical difference was analysed using
background. unpaired Students t-tests in SigmaPlot
version 4.0 for Windows (Cytel Software
Cell cultures Corporation, Cambridge, MA).
Human umbilical cord vein endothelial cells
(HUVEC) were cultured in modified M-199
medium as described previously[13] . For Results
experiments, 1,000 U/ml trypsin and 0.5 Generation of autoAbs in dengue
mM ethylenediaminetetraacetic acid (EDTA)
were used to detach cells. patients infected with different
serotypes and at different disease
Binding activity detection stages
After detachment, cells were suspended at Our previous studies demonstrated the
5105 for flow cytometry. The cells were presence of anti-platelet and anti-
washed briefly with phosphate-buffered endothelial cell autoAbs in dengue patient
saline (PBS) and fixed with 1% formaldehyde sera[12,13] . The levels of these autoAbs were
in PBS at room temperature for 10 minutes,
then washed again with PBS. Patient sera
deoxyuridine triphosphate

higher in DHF/DSS than in DF patient sera. 4 (Table 1). There was no significant
The dysfunction of platelets and endothelial difference between different serotype
cells caused by the autoAbs was also shown. infections. The levels of autoAbs were
The cross-reactivity of patient sera with higher in DHF/DSS than in DF patient sera.
endothelial cells was the highest in the acute In addition, the levels of IgM isotype of
stage (3-7 days after fever onset) and autoAbs were higher than those of IgG. The
subsequently decreased in the convalescent DEN-1 serotype was not tested because we
(1-3 weeks after acute phase) and later (8-9 had no DEN-1-infected patient sera. We
months) stages. In our previous study, next investigated the endothelial cell cross-
patient sera were collected from an reactivity of DHF patient sera at different
outbreak of DEN-3 infection. In this study, disease grades. DHF patient sera collected
we further examined the autoAb levels from Grades I to IV with DEN -4 infection,
produced by patients infected with different according to the WHO definition, were
DEN serotypes and the relationship tested. There was no significant difference
between the autoAb levels and disease between the four grades of DHF in both
severity. The results showed that the levels anti-endothelial cell IgM and IgG (Table 1).
of anti-endothelial cell Abs, as determined Due to our limited sample sizes of patient
by both the percentages of endothelial cells sera, especially of Grades I and IV, we were
reactive with patient sera IgM or IgG and unable to determine whether there was any
the mean fluorescence intensity, were correlation of autoAbs with disease severity.
similar in patients infected with DEN -2, 3 or
Table 1. Anti-endothelial cell IgM/IgG levels in the sera of dengue patients infected with
different dengue serotypes and at different disease grades
% of endothelial cells reactive
Mean fluorescence intensity
with patient sera
IgM IgG IgM IgG
Mean (SD) Mean (SD) Mean (SD) Mean (S D)
Normal (n=5) 4.7 (0.5) 2.6 (0.6) 12.8 (1.5) 6.6 (0.8)
DEN-2 infection
DF (n=5) 38.6 (1.4)*** 14.8 (5.1)* 62.5 (4.1)*** 14.1 (2.1)*
DEN-3 infection
DF (n=6) 35.1 (3.7)*** 12.7 (2.1)* 65.7 (5.7)*** 15.7 (1.7)*
DHF/DSS (n=5) 54.6 (4.4)*** 23.7 (1.9)** 74.9 (4.6)*** 24.6 (3.6)**
DEN-4 infection
DF (n=5) 35.9 (1.6)*** 15.1 (3.5)* 66.6 (6.3)*** 10.0 (2.3)*
DHF (n=36) 50.5 (9.5)*** 24.9 (8.3)** 72.1 (13.9)*** 11.1 (4.4)*
Grade I (n=1) 66.9 17.8 83.4 8.8
Grade II (n=26) 51.7 (9.0)*** 25.5 (7.7)* 73.6 (10.4)*** 11.4 (4.3)*
Grade III (n=8) 45.4 (8.6)*** 24.7 (10.6)* 64.7 (7.0)*** 13.9 (6.4)*
Grade IV (n=1) 42.2 17.9 63.0 11.6
Students t-tests: *P<0.05 vs Normal; **P<0.01 vs Normal; ***P<0.001 vs Normal.

Induction of endothelial cell percentages of apoptotic cells from one set

of duplicate cultures are shown in the Figure
apoptosis by sera of dengue below. The results indicated that cell
patients infected with different apoptosis was induced by all patient sera
serotypes and the cells underwent a higher percentage
of apoptosis when induced by DHF patient
Anti-endothelial cell autoAbs caused cell
sera than by D F patient sera. Healthy-
damage which was characterized by
control sera showed only the background
apoptosis [13] . The ability of patient sera with
level. Cell viability detected using eosin Y
different DEN serotype infections to induce
staining showed an inverse relationship with
endothelial cell apoptosis was tested.
the percentages of apoptosis (Table 2).
HUVEC were treated with a 1:25 dilution of
There was no significant difference in
dengue patient or healthy-control sera for
endothelial cell apoptosis induced by
24 hours, and cell apoptosis was measured
patient sera with different serotype
using TUNEL reaction followed by flow
infections.
cytometric analysis. The histogram and the
Figure. Dengue patient sera induced endothelial cell apoptosis
Normal DEN-2 DF
DEN-3 DF DEN-3 DHF
DEN-4 DF DEN-4 DHF

Table 2. Endothelial cell apoptosis induced by sera of dengue patients infected with
different dengue serotypes
% of endothelial cell
% of cell viability
apoptosis
Normal (n=5) 94.1 (3.5) 6.9 (2.8)
DEN-2 infection
DF (n=5) 75.2 (5.4)** 21.9 (5.6)**
DEN-3 infection
DF (n=6) 81.7 (5.1)** 19.5 (7.1)**
DHF/DSS (n=5) 66.6 (10.8)*** 29.7 (9.3)***
DEN-4 infection
DF (n=5) 72.0 (9.5)** 22.0 (5.6)**
DHF (n=5) 63.3 (15.1)*** 37.2 (5.5)***
Students t-tests: **P<0.01 vs Normal; ***P<0.001 vs Normal.
Discussion serotypes. The percentages of endothelial

cells reactive with DHF/DSS patient sera
DHF is a life-threatening disease with poorly were higher than those with DF patient sera.
defined pathogenic mechanisms[1,2,5,10] . An However, there was no difference in anti-
ADE effect in different serotype cross- endothelial cell Ab levels at different DHF
infection is frequent[2,5,6]. Patients may disease grades. The sample size of patient
develop severe complications of progressive sera needs to be increased to gain an insight
DHF. Vascular leakage and haemorrhagic into the role of anti-endothelial cell Abs in
diathesis are the hallmarks in DHF patients. A DHF pathogenesis. These autoAbs exerted
number of studies have demonstrated similar effects in the induction of endothelial
abnormal immune responses caused by DEN cell apoptosis of patients infected with
infection, including cytokine and chemokine different DEN serotypes.
production, complement activation and
In dengue pathology, various cytokines
immune cell activation[10,11,17-20] . In addition,
autoimmune responses may be involved in and chemokines including TNF-, IL-6, IL-8,
DHF pathogenesis [12-15,21] . Dengue patients and RANTES have been detected in patient
produced Abs which cross-reacted with sera with DHF/DSS and in DEN-infected
human platelets and endothelial cells[12,13] . endothelial cell culture supernatants[17,18,20] .
Anti-NS1 produced after DEN infection may, Our recent studies also demonstrate that
at least in part, account for the cross- anti-NS1 Abs can stimulate cytokine and
reactivity of patient sera with endothelial cells. chemokine production (in press). Therefore,
In this study, we further showed that the
levels of anti-endothelial cell Abs were similar
regulated upon activation normal T cell expressed and
in patients infected with different DEN
secreted

both immune activation and apoptosis cross-reactivity of anti-NS1 to host antigens

occur in endothelial cells after stimulation and cells and a haemorrhage-like hallmark
by autoAbs. in mice. This, taken together with our
findings, suggests that a potential pathogenic
There are no dengue vaccines available.
effect of DEN NS1 vaccine should be taken
Yet, several potential vaccines, including
into consideration. The possible approaches
life-attenuated whole DEN and DNA
include gene modifications of DEN NS1 to
vaccines, are undergoing clinical trials[7-10] . It
truncate or mutate the epitopes that may
is hoped that a fusion or a chimera dengue
cause the pathogenic effects.
vaccine will be developed to provide
protection against all serotypes of DEN
infection. In addition, DEN NS1 protein Acknowledgement
used as a vaccine candidate in mice showed
resistance to fatal DEN encephalitis [22] . We thank Dr N.T. Hung from the
Passive administration of anti-NS1 Abs also Department of Dengue Haemorrhagic Fever,
conferred protection in mice when Childrens Hospital No. 1, Ho Chi Minh City,
challenged with lethal doses of DEN [23] . Viet Nam for providing the DEN-4 patient
However, these previous studies only sera. The authors acknowledge the editorial
monitored the survival rates of mice but did assistance of Bill Franke. This work was
not examine the potential histopathological supported by grant NSC92-3112-B006-003
effects. Studies by Falconar[21] showed the from the National Science Council, Taiwan.
References
[1] Gubler DJ. Dengue and dengue [7] Pang T. Vaccines for the prevention of
hemorrhagic fever. Clinical Microbiology neglected diseases dengue fever. Current
Reviews, 1998, 11: 480-496. Opinion in Biotechnology, 2003, 14: 332-
336.
[2] Halstead SB. Dengue. Current Opinion in
Infectious Diseases, 2002, 15: 471-476. [8] Pugachev KV, Guirakhoo F, Trent DW and
Monath TP. Traditional and novel
approaches to flavivirus vaccines.
haemorrhagic fever: diagnosis, treatment,
International Journal of Parasitology, 2003,
prevention and control. 2nd edition.
33: 567-582.
Geneva: WHO, 1997.
[9] Edelman R, Wasserman SS, Bodison SA,
[4] World Health Organization. Dengue fever in
Putnak RJ, Eckels KH, Tang D, Kanesa-
Indonesia-update 4. 11 May 2004.
Thasan N, Vaughn DW, Innis BL and Sun W.
[5] Halstead SB. Pathogenesis of dengue: Phase I trial of 16 formulations of a
challenges to molecular biology. Science, tetravalent live-attenuated dengue vaccine.
1988, 239: 476-481. American Journal of Tropical Medicine and
Hygiene, 2003, 69: 48-60.
[6] Halstead SB. Neutralization and antibody-
dependent enhancement of dengue viruses. [10] Rothman AL. Dengue: defining protective
Advance in Virus Research, 2003, 60: 421- versus pathologic immunity. Journal of
467. Clinical Investigation, 2004, 113: 946-951.

[11] Lei HY, Yeh TM, Liu HS, Lin YS, Chen SH [19] Lin YW, Wang KJ, Lei HY, Lin YS, Yeh TM,
and Liu CC. Immunopathogenesis of dengue Liu HS, Liu CC and Chen SH. Virus
virus infection. Journal of Biomedical replication and cytokine production in
Science, 2001, 8: 377-388. dengue virus-infected human B lymphocytes.
Journal of Virology, 2002, 76: 12242-12249.
[12] Lin CF, Lei HY, Liu CC, Liu HS, Yeh TM,
Wang ST, Yang TI, Sheu FC, Kuo CF and Lin [20] Hung NT, Lei HY, Lan NT, Lin YS, Huang KJ,
YS. Generation of IgM anti-platelet Lien LB, Lin CF, Yeh TM, Ha DQ, Huong
autoantibody in dengue patients. Journal of VTQ, Chen LC, Huang JH, My LT, Liu CC
Medical Virology, 2001, 63: 143-149. and Halstead SB. Dengue hemorrhagic fever
in infants: a study on clinical and cytokine
[13] Lin CF, Lei HY, Shiau AL, Liu CC, Liu HS,
profiles. Journal of Infectious Diseases, 2004,
Yeh TM, Chen SH and Lin YS. Antibodies
189: 221-232.
from dengue patient sera cross-react with
endothelial cells and induce damage. [21] Falconar AK. The dengue virus
Journal of Medical Virology, 2003, 69: 82- nonstructural-1 protein (NS1) generates
90. antibodies to common epitopes on human
blood clotting, integrin/adhesin proteins and
[14] Lin YS, Lin CF, Lei HY, Liu HS, Yeh TM, binds to human endothelial cells: potential
Chen SH and Liu CC. Antibody-mediated
implications in haemorrhagic fever
endothelial cell damage via nitric oxide.
pathogenesis. Archives of Virology, 1997,
Current Pharmaceutical Design, 2004, 10:
142: 897-916.
213-221.
[22] Zhang YM, Hayes EP, McCarty TC, Dubois
[15] Lin CF, Lei HY, Shiau AL, Liu HS, Yeh TM,
DR, Summers PL, Eckels KH, Chanock RM
Chen SH, Liu CC, Chiu SC and Lin YS.
and Lai CJ. Immunization of mice with
Endothelial cell apoptosis induced by dengue structural proteins and nonstructural
antibodies against dengue virus
protein NS1 expressed by baculovirus
nonstructural protein 1 via production of
recombinant induces resistance to dengue
nitric oxide. Journal of Immunology, 2002,
virus encephalitis. Journal of Virology, 1988,
169: 657-664.
62: 3027-3031.
[16] Lei HY, Huang JH, Huang KJ and Chang C.
[23] Henchal EA, Henchal LS and Schlesinger JJ.
Status of dengue control programme in
Synergistic interactions of anti-NS1
Taiwan - 2001. Dengue Bulletin, 2002, 26: monoclonal antibodies protect passively
14-23.
immunized mice from lethal challenge with
[17] Rothman AL and Ennis FA. Immuno- dengue 2 virus. Journal of General Virology,
pathogenesis of dengue hemorrhagic fever. 1988, 69: 2101-2107.
Virology, 1999, 257: 1 -6.
[18] Avirutnan P, Malasit P, Seliger B, Bhakdi S
and Husmann M. Dengue virus infection of
human endothelial cells leads to chemokine
production, complement activation, and
apoptosis. Journal of Immunology, 1998,
161: 6338-6346.

Inhibition of the NS2B-NS3 Protease Towards a
Causative Therapy for Dengue Virus Diseases
Gerd Katzenmeier#
Laboratory of Molecular Virology, Institute of Molecular Biology and Genetics, Mahidol University,
Salaya Campus, Phutthamonthon No. 4 Rd., Nakornpathom 73170, Thailand
Abstract
The high impact of diseases caused by dengue viruses on global health is now reflected in an increased
interest in the identification of drug targets and the rationale-based development of antiviral inhibitors
which are suitable for a causative treatment of severe forms of dengue virus infections dengue
haemorrhagic fever and dengue shock syndrome. A promising target for the design of specific inhibitors is
the dengue virus NS3 serine protease which in the complex with the small activator protein NS2B
catalyses processing of the viral polyprotein at a number of sites in the nonstructural region. The NS3
protease is an indispensable component of the viral replication machinery and inhibition of this protein
offers the prospect of eventually preventing dengue viruses from replication and maturation. After nearly
a decade of mainly genetic analysis of flaviviral replication, recent studies have contributed substantial
biochemical information on polyprotein processing including the 3-dimensional structure of the dengue
virus NS3 protease domain, the mechanism of co-factor-dependent activation and sensitive in vitro
assays which are needed for studies on substrate specificity and the development of high-throughput
assays for inhibitor screening. This review discusses recent biochemical findings which are relevant to the
design of potential inhibitors directed against the dengue virus NS3 protease.
Keywords: Dengue virus, NS2B/NS3, polyprotein, protease, inhibitor, treatment.
Viral polyprotein processing nucleotides and encodes a single

polyprotein precursor of 3,391 amino acid
Dengue viruses, members of the Flaviviridae residues[3] . Individual viral proteins are
family, possess single-stranded, positive arranged in the order C-prM-E-NS1-NS2A-
sense RNA genomes and generate mature NS2B-NS3-NS4A-NS4B-NS5. Proteolytic
viral proteins by co- and post-translational cleavages in the N-terminal region of the
proteolytic processing of a polyprotein viral polyprotein are mediated by a host
precursor catalysed by host cell and virus- signal peptidase and yields three structural
encoded proteases [for review see refs. 1, 2 proteins C, prM and E, which constitute the
and references herein]. The genomic RNA virion[4] . Before the virion exits the cell, prM
of dengue virus serotype 2 contains 10,723 is cleaved by a cellular furin-type
#
E-mail: frkgz@mahidol.ac.th; Tel.: (662)-800-3624-1237; Fax: (662)-441-9906

Inhibition of NS2B/NS3 Protease
prohormone convertase in the post-Golgi Cleavage sites recognized by the

acidic compartment to yield the M protein[5] . NS2B/NS3 protease consist of dibasic
Cleavages at the NS1/NS2A and residues Lys-Arg, Arg-Arg and Arg-Lys at the
NS4A/NS4B junctions are catalysed by a (nonprime) P1 and P2 positions of the
signalase bound to the membranes of the cleavage site sequence followed by short
ER [6,7] . Proteolytic cleavages in the chain residues such as Gly, Ala and Ser at
nonstructural region of the polyprotein are the (prime) P1 position. The non-
mediated by a heterodimeric complex of canonical NS2B/NS3 site contains a Gln
NS3 with the activator protein NS2B which residue at the P2 position (Figure 1).
catalyses in cis (intramolecular) cleavages at
the NS2A/NS2B and NS2B/NS3 sites and in The NS3 protease domain
trans (intermolecular) cleavages at the
NS3/NS4A and NS4B/NS5 polyprotein The existence of a trypsin-like serine
junctions[8-10] . Additional cleavages protease domain in the N-terminal region of
mediated by the NS3 protease within the C, the flaviviral NS3 proteins was originally
NS2A, NS4A and within a conserved C- predicted by sequence comparisons between
terminal portion of NS3 itself have been cellular and virus-encoded proteases[14]. The
described in the literature [11-13] . NS2B-NS3 endopeptidases of the Flavivirus
genus which at present comprises at least 68
known members, are now commonly
Figure 1. The 3-dimensional structure of designated as flavivirin (EC 3.4.21.91) [15,16] .
the dengue virus NS3 protease The dengue virus 69 kDa NS3 protein is a
(shown here is a superimposition of a multifunctional protein with a serine
ribbon-presentation of the C trace on a protease domain located within the N-
space-filling surface model. Residues of terminal 167 amino acid residues[17] and
the catalytic triad (His51, Asp75, Ser135) activities of a nucleoside triphosphatase
are shown as sticks. The figure was (NTPase) and RNA helicase in the C-
generated by Deepview Swiss-Pdb Viewer) terminal moiety[18] . A catalytic triad
consisting of residues His51, Asp75 and
Ser135 was identified by site-directed
mutagenesis experiments and replacement
of the catalytic serine by alanine resulted in
Ser135 an enzymatically inactive NS3 protein[19] .
His51 The NS3 protease is an essential component
for maturation of the virus and viable virus
was never recovered from infectious cDNA
Asp75
clones carrying mutations in the NS3
sequence which abolished protease
activity[20] . Interaction of the helicase
portion of NS3 with the viral RNA-
dependent RNA polymerase NS5 may
promote the association of the viral
replicase complex to the membranes of the
ER [21] .

Figure 2. Schematic of dengue virus polyprotein processing

(shown here are sites on the flavivirus polyprotein cleaved by host-encoded proteases and
the virus-encoded two-component protease NS2B-NS3)
C prM E NS1 NS2A NS4BNS5

NS2BNS3 NS4A
NS2B/NS3 protease
Signal peptidases
Furin protease
Unidentified protease
The 3-dimensional structure of the N- natural polyprotein substrates. Although the

terminal 185 residues of the dengue virus dengue virus NS3 protease exhibits NS2B-
NS3 protease domain (NS3pro) was independent activity with model substrates
resolved at a resolution of 2.1 [22] . The for serine proteases, enzymatic cleavage of
overall folding of the protein resembles the dibasic peptides is markedly enhanced with
6-stranded -barrel conformation typical for the NS2B-NS3 co-complex and the
chymotrypsin-like serine proteases. presence of the NS2B activation sequence is
Interestingly, the structure of the dengue indispensable for the cleavage of
virus NS3 protease is closer to that of the polyprotein substrates in vitro[24] . The initial
hepatitis C virus NS3/NS4A co-complex characterization of the co-factor
than to the unliganded HCV NS3 protease, requirement for the dengue virus NS3
an observation which is suggestive of major protease had revealed that the minimal
structural differences in the co-factor- region necessary for protease activation was
dependent activation mechanism of the two located in a 40-residue hydrophilic segment
proteases[23] . The substrate binding site of of NS2B[25] . The hydrophobic flanking
NS3pro is relatively shallow and contourless regions of the 14 kDa NS2B protein are
and specific enzyme-substrate interactions likely to be involved in targeting the
were not predicted to extend beyond the protease complex to the membranes of the
P2 and P2 positions of the substrate ER where genome replication occurs. Fusion
peptide in the absence of the NS2B co- of the NS2B core sequence to the NS3
factor[22] (Figure 2). protease domain yielded a catalytically
active NS2B(H)-NS3p protein, which, upon
expression in E. coli and subsequent
The NS2B co-factor refolding, displayed autoproteolytic
processing at the NS2B/NS3 site conducive
The presence of a small activating protein or
co-factor is a prerequisite for optimal activity to the formation of a non-covalent adduct[24] .
Incorporation of a flexible nonamer linker,
of the flaviviral NS3 proteases with their
Gly4 -Ser-Gly4, between the NS2B core

segment and the protease domain resulted reaction with the synthetic substrate peptide
in a cleavage-resistent protease with GRR-AMC.
optimized enzymatic activity against
For the HCV NS3-NS4A protease
hexapeptide substrates representing native
complex, large structural rearrangements
polyprotein cleavage junctions[26] . A
leading to a catalytic triad, which is
recombinant construct representing the full-
conformationally optimized for proton
length NS2B co-factor linked to the NS3
shuttle during catalysis, were observed as a
protease domain was enzymatically active
result of co-factor binding[30] . No 3-
with peptide substrates derived from the
dimensional structure is available for the
polyprotein; however, this protein was
dengue virus NS2B-NS3 co-complex and
completely resistant to proteolytic self-
the precise mechanism of co-factor-
cleavage[27,28] .
dependent activation is not fully elucidated
An essential requirement for the correct as yet. In particular, it is an open question as
association of the co-factor with the to whether binding of the substrate
protease is the presence of hydrophobic contributes to the formation of an induced
residues which act as anchor for the fit conformation as observed with the HCV
protease co-factor interaction. Recently, NS3 protease[31,32] .
based on mutagenesis experiments and
sequence comparisons of known flaviviral
co-factors, the x3 - motif was proposed Substrate specificity
as the common structural element involved So far, only very limited efforts have been
in co-factor binding to the protease[29] . The undertaken to analyse the precise substrate
x3 - motif is comprised of two bulky requirements and determinants of cleavage
hydrophobic residues separated by three efficiency for the dengue virus NS3 protease.
unspecified residues and it was This is surprising in the light of the fact that
hypothesized that additional residues development of inhibitors against serine
located at the N-terminus of the activation proteases usually starts with optimal peptide
sequence would contribute to the stringent substrates derived from the nonprime side
specificity of the protease for the wherein the scissile amide bond is replaced
polyprotein substrate. A mutagenesis study by an electrophile which reacts with the
with the dengue virus NS2B co-factor had catalytic serine residue.
revealed that substitutions of the -
residues (Leu75 and Ile79 in NS2B of The NS3 protease reacts with small
dengue virus type 2) by alanine resulted in model substrates for serine proteases such as
preponderant effects on the catalytic activity N--benzoyl-L-arginine-p-nitroanilide (BAPA)
of the NS3 protease rather than on substrate and activity of the unliganded NS3 protease
binding [P. Niyomrattanakit, unpublished towards this substrate is higher than that of
data]. A single residue in the N-terminal the NS2B-NS3 co-complex[24] , a finding
region of NS2B, Trp62, was critical for which suggests that substrate recognition in
protease activation and replacement of this the complex requires additional interactions
residue yielded a NS3 protease which was extending beyond the P1 side for optimal
catalytically inactive in autoproteolysis and activity. A number of fluorogenic tripeptides
containing dibasic residues at the P1 and P2

positions are cleaved by NS2B(H)-NS3pro 10-fold better than the best commercially
protease and the best substrate identified in available substrate tested so far, GRR-AMC
these experiments was GRR-AMC, which [J. Wikberg, unpublished data]. In the near
had a Km value of 180 M, a kcat value of future, these investigations will likely lead to
0.031 s-1 and a catalytic efficiency expressed the identification of NS3 substrates with
as kcat / Km of 172 s-1 M-1 [24] . optimized sequence length and improved
binding affinities, which can be applied as
Chromogenic p-nitroanilide peptides
sensitive probes for enzyme activity in high-
representing hexameric sequences of the
throughput inhibitor screenings.
native polyprotein cleavage junctions were
tested in a photometric assay and the most
efficiently cleaved substrate derived from Perspectives for inhibitor
the NS4B/NS5 site (Ac-TTSTRR-pNA) had a
Km of 346 M, kcat of 0.095 s-1 and a kcat / Km development
of 275 s-1 M-1 [26] . Principally, every step of viral
Recently, we have shown by a HPLC- morphogenesis, from cell-entry, uncoating,
based assay with fluorometric detection that replication and assembly of new virus
the NS2B-NS3pro protease incorporating a particles, is a potential target for antiviral
full-length NS2B co-factor could cleave N- inhibitors. However, the molecular events in
terminally dansylated 12mer peptides the infectious cycle of flaviviruses such as
mimicking native polyprotein junctions in dengue virus are characterized only to a
the absence of microsomal membranes[28] . very limited extent, making the design of
However, this protein was completely specific inhibitors an adventurous task. In
inactive in autocleavage and the efficiency contrast, proteases and their inhibitors have
of this recombinant protease with the been intensively studied because of their
peptide substrate was markedly reduced potential for the development of selective
when compared to constructs incorporating antiviral compounds. Although tremendous
the 40-residue NS2B core sequence, likely progress in the field is indicated by the
due to structural distortions induced by the design and clinical use of antiviral drugs
flanking regions of NS2B and inefficient against HIV (AIDS) and hepatitis C virus
activation of the protease. proteases, the potential of dengue and
related flaviviral proteases for inhibitor
Currently, a detailed study on substrate discovery is largely unexploited. In response
specificity of the dengue virus NS3 protease to the global problem of the dengue virus
is in progress, which uses combinatorial epidemics, considerable efforts are now
libraries of internally quenched fluorogenic being devoted to the development of drugs
peptides labelled with the aminobenzoyl / which will eventually be suitable for a
p-nitrotyrosine reporter pair. For a substrate chemotherapeutic intervention in acute
peptide based on the NS3/NS4A cleavage dengue diseases, not only by academic
site, Abz-AAGRK?SLTLY(NO2)R-NH2 (? institutions but also by pharmaceutical
denotes the cleavage site), a Km value of 141 companies (for example see
M, a kcat of 0.18 s-1 and a kcat / Km of 1262 http://www.nitd.novartis.com).
s-1 M-1 was found, which is approximately

In a first step towards a rational inhibitor suggest the existence of a high-affinity

design for the dengue virus NS3 serine binding site in the non-prime region of the
protease, inhibition by synthetic peptides enzyme and offer the prospect of
mimicking uncleavable transition state developing effective inhibitors against the
isosteres of the P6-P2 residues of the native dengue virus protease by combinatorial
polyprotein sites, was demonstrated[26] . The optimization based on the structure(s) of
peptides with an -keto amide in place of native polyprotein cleavage site peptides.
the scissile amide bond acted as competitive However, in general, peptide-based
inhibitors of the NS3 protease with Ki - inhibitors exhibit poor pharmacokinetic
values in the micromolar range. properties and usually the conversion of
Replacement of the P1-P2 residues by a these structures into less peptide-like
carboxyl-terminal aldehyde in the compounds (peptidomimetics) is required
NS3/NS4A-derived peptide (Ac-FAAGRR- to generate drug-like entities.
CHO) yielded a competitive inhibitor with a
Inhibitor discovery for the dengue virus
Ki of 16 M. For the dengue virus protease,
NS3 protease is currently limited by the lack
the hexapeptides displayed Ki - values which
of a 3-dimensional structure for the NS2B-
were only 2- to 6-fold lower than the Km -
NS3 co-complex; however, it can be
value for the corresponding substrate, a
expected that crystallographic studies and
feature which discriminates dengue virus
NMR-experiments will provide more insight
NS3 from the HCV protease, where product
into the structure and catalytic mechanism of
inhibitors had binding affinities which were
the enzyme in the near future. In addition,
one order of magnitude lower than those for
powerful computer modelling approaches
the substrates[33,34] .
exist which may help to obtain information
Product inhibition of the HCV NS3 required for a rational drug design even in
protease by cleavage-site derived peptides the absence of crystallographic structures.
led to the discovery of very potent inhibitors
Proteochemometric modelling (PCM) is
of this enzyme with IC50 values in the
currently explored as a tool to analyse the
nanomolar range by cyclic optimization of
structural and physicochemical properties
the inhibitor structures[33,34] . Recently, we
which are necessary for the interaction of
have shown that peptides representing non- potential inhibitors with the dengue virus
prime-side residues of the dengue virus NS3
NS3 protease target structure [35] . Preliminary
protease act as competitive inhibitors of the data obtained by this approach suggest that
enzyme, whereas prime-side peptides
the proteochemometric models are valid
appeared to have negligible effects on and useful for the accelerated design of
enzyme inhibition at concentrations >1.0
novel inhibitors. This approach does not
mM. (S. Chanprapaph, unpublished data). Ki only circumvent the traditional erratic dug
- values for hexapeptides derived from all 4
discovery process with its high attrition rates,
dengue virus cleavage sites were in the low but also allows to incorporate potential
micromolar range and the best inhibitor was
resistance of the target and the development
based on the NS2A/NS2B site (Ac-RTSKKR- of drug resistance resistant compounds as
CONH2) and gave a Ki of 12 M. In analogy an initial consideration in the design process.
to the HCV NS3 protease, these findings The existence of large conformational

ensembles is particularly challenging in the biomedical targets in the dengue virus

case of rapidly mutating viral enzymes, polyprotein would even make a therapy
where a design against a moveable target feasible, which uses combinations of
would require large sets of corresponding different inhibitors and therefore could
inhibitors. In the future, these problems are minimize the risk of rapid resistance
likely to be addressed by shotgun development.
approaches to the structure of enzyme-
It can also be expected that progress for
inhibitor complexes and the identification of
inhibitors against the dengue virus protease
hot spots of interaction flexibility by the
will be of large benefit for drug design
use of fast, high-resolution methods such as
against related human pathogens of the
NMR. Detailed accounts on this strategy are
flavivirus complex such as yellow fever virus,
given in Reference 36.
Japanese encephalitis virus and West Nile
virus.
Conclusions However, in order to bring an effective
anti-dengue drug from the bench to the
Substantial progress has been made over the
past few years in the biochemical bedside, several questions and limitations
need to be addressed. These include the
characterization of the dengue virus NS2B-
evaluation of prognostic markers for disease
NS3 two-component protease. The data,
severity, the pathobiochemistry of dengue
which are available now, make the dengue
haemorrhagic fever and shock syndrome,
virus NS3 protease a valid molecular target
the problem of selectivity against
for the development of antiviral compounds.
pharmacologically relevant human proteases
A large repertoire of powerful methods for
such as furin and the risk of adverse effects.
inhibitor development and evaluation exists
Potential complications may also arise from
which includes state-of-the-art technology in
the presence of four related dengue
organic synthesis and computer-aided serotypes in the case that their NS3
molecular design. Although there is no proteases show marked differences in their
suitable animal model available for dengue inhibition profiles. Intensive efforts and
virus diseases, initial screening of potential sustained multidisciplinary research is
antiviral compounds would be facilitated by required in the future to cope with the
well-established insect and mammalian cell challenging task of a causative treatment for
culture systems which are useful to monitor dengue virus diseases.
the effects of anti-NS3 inhibitors on the
propagation of the virus.
Acknowledgements
Moreover, alternative drug targets
which are present on the dengue virus NS3 We thank Dr J. Wikberg, Department of
protein can be exploited for inhibitor Pharmaceutical Biosciences, Uppsala
development. These include the binding site University, Sweden, for intensive discussions
of the NS2B co-factor to the NS3 protease, and access to data prior to publication. This
the NS3 NTPase / helicase portion and the work was supported by grants from the
interaction surface of NS3 with the NS5 Thailand Research Fund (TRF).
replicase. The presence of multiple

References
[1] Chambers TJ, Hahn CS, Galler R and Rice [9] Preugschat F, Yao CW and Strauss JH. In
CM. Flavivirus genome organization, vitro processing of dengue virus type 2
expression and replication. Annu Rev nonstructural proteins NS2A, NS2B and NS3.
Microbiol, 1990, 44: 649-688. J Virol, 1990, 64: 4364-4374.
[2] Ryan MD, Monaghan S and Flint M. Virus- [10] Falgout B, Pethel M, Zhang YM and Lai CJ.
encoded proteinases of the Flaviviridae. J Both nonstructural proteins NS2B and NS3
Gen Virol, 1998, 79: 947-959. are required for the proteolytic processing of
[3] Zhang L, Mohan PM and Padmanabhan R. dengue virus nonstructural proteins. J Virol,
Processing and localization of dengue virus 1991, 65: 2467-2475.
type 2 polyprotein precursor NS3-NS4A- [11] Arias CF, Preugschat F and Strauss JH. Dengue
NS4B-NS5. J Virol, 1992, 66: 7549-7554. virus NS2B and NS3 form a stable complex
that can cleave NS3 within the helicase
[4] Nowak T, Farber PM, Wengler G and
Wengler G. Analyses of the terminal domain. Virology, 1993, 193: 888-899.
sequences of West Nile virus structural [12] Lobigs M. Flavivirus premembrane protein
proteins and of the in vitro translation of cleavage and spike heterodimer secretion
these proteins allow the proposal of a require the function of the viral proteinase
complete scheme of the proteolytic NS3. Proc Natl Acad Sci USA, 1993, 90:
cleavages involved in their synthesis. 6218-6222.
Virology, 1989, 169: 365-376. [13] Teo KF and Wright PJ. Internal proteolysis of
[5] Stadler K, Allison SL, Schalich J and Heinz the NS3 protein specified by dengue virus 2.
FX. Proteolytic activation of tick-borne J Virol, 1997, 72: 624-632.
encephalitis virus by furin. J Virol, 1997, 71: [14] Gorbalenya AE, Donchenko AP, Koonin EV
8475-8481. and Blinov VM. N-terminal domains of
[6] Falgout B and Markoff L. Evidence that putative helicases of flavi- and pestiviruses
flavivirus NS1-NS2A cleavage is mediated by may be serine proteases. Nucleic Acids Res,
a membrane-bound host protease in the 1989, 17: 3889-3897.
endoplasmic reticulum. J Virol, 1995, 69: [15] Amberg SM and Rice CM. Flavivirin. In:
7232-7243. Barrett AJ, Rawlings ND, Woessner JF (Eds)
[7] Lin C, Amberg SM, Chambers TJ and Rice Handbook of proteolytic enzymes.
CM. Cleavage at a novel site in the NS4A Academic Press, London, 1998: 268-272.
region by the yellow fever virus NS2B-3 [16] Bazan JF, Fletterick RJ and Rice CM. Evidence
proteinase is a prerequisite for processing at that the N-terminal domain of nonstructural
the downstream 4A/4B signalase site. J Virol, protein NS3 from yellow fever virus is a serine
1993, 67: 2327-2335. protease responsible for site-specific
[8] Chambers TJ, Weir RC, Grakoui A, McCourt cleavages in the viral polyprotein. Proc Natl
DW, Bazan JF, Fletterick RJ and Rice CM. Acad Sci USA, 1990, 87: 8898-8902.
Evidence that the N-terminal domain of [17] Li H, Clum S, You S, Ebner KE and
nonstructural protein NS3 from yellow fever Padmanabhan R. The serine protease and
virus is a serine protease responsible for site- RNA-stimulated nucleoside triphosphatase
specific cleavages in the viral polyprotein. and RNA helicase functional domains of
Proc Natl Acad Sci USA, 1990, 87: 8898- dengue virus type 2 NS3 converge within a
8902. region of 20 amino acids. J Virol, 1999, 73:
3108-3116.

[18] Gorbalenya AE, Koonin EV, Donchenko AP protein NS2B: identification of a domain
and Blinov VM. Two related superfamilies of required for NS2B-NS3 protease activity. J
putative helicases involved in replication, Virol, 1993, 67: 2034-2042.
recombination, repair and expression of
[26] Leung D, Schroeder K, White H, Fang NX,
DNA and RNA genomes. Nucleic Acids Res,
Stoermer MJ, Abbenante G, Martin JL,
1989, 17: 4713-4730.
Young PR and Fairlie DP. Activity of
[19] Valle RPC and Falgout B. Mutagenesis of the recombinant dengue virus NS3 protease in
NS3 protease of dengue virus type 2. J Virol, the presence of a truncated NS2B cofactor,
1998, 72: 624-632. small peptide substrates and inhibitors. J Biol
Chem, 2001, 276: 45762-45771.
[20] Chambers TJ, Nestorowicz A, Amberg SM
and Rice CM. Mutagenesis of the yellow [27] Khumthong R, Angsuthanasombat C,
fever virus NS2B protein: effects on Panyim S and Katzenmeier G. In vitro
proteolytic processing, NS2B-NS3 complex determination of dengue virus type 2 NS2B-
formation, and viral replication. J Virol, 1993, NS3 protease activity with fluorescent
67: 6797-6807. peptide substrates. J Biochem Mol Biol,
2002, 35: 206-212.
[21] Kapoor M, Zhang L, Ramachandra M,
Kusukawa J, Ebner KE and Padmanabhan R. [28] Khumthong R, Niyomrattanakit P,
Association between NS3 and NS5 proteins Chanprapaph S, Angsuthanasombat C,
of dengue virus type 2 in the putative RNA Panyim S and Katzenmeier G. Steady-state
replicase is linked to differential cleavage kinetics for dengue virus type 2
phosphorylation of NS5. J Biol Chem, 1995, NS2B-NS3(pro) serine protease with
270: 19100-19106. synthetic peptides. Prot Pept Lett, 2003, 10:
19-26.
[22] Krishna Murthy HM, Clum S and
Padmanabhan R. Dengue virus NS3 serine [29] Butkiewicz N, Yao N, Zhong W, Wright-
protease: crystal structure and insights into Minogue J, Ingravallo P, Zhang R, Durkin J,
interaction of the active site with substrates Standring DN, Baroudy BM, Sangar DV,
by molecular modeling and structural Lemon SM, Lau JY and Hong Z. Virus-
analysis of mutational effects. J Biol Chem, specific cofactor requirement and chimeric
1999, 274: 5573-5580. hepatitis c virus/GB virus B nonstructural
proteins. J Virol, 2000, 74: 4291-4301.
[23] Kim JL, Morgenstern KA, Lin C, Fox T,
Dwyer MD, Landro JA, Chambers SP, [30] Yan Y, Li Y, Munshi S, Sardana V, Cole JL,
Markland W, Lepre CA, OMalley ET, Sardana M, Steinkuehler C, Tomei L, De
Harbeson SL, Rice CM, Murcko MA, Caron Francesco R, Kuo LC and Chen Z. Complex
PR and Thomson JA. Crystal structure of the of NS3 protease and NS4a peptide of BK
hepatitis C virus NS3 protease domain strain hepatitis C virus: a 2.2 A resolution
complexed with a synthetic NS4A cofactor structure in a hexagonal crystal form. Protein
peptide. Cell, 1996, 87: 343-355. Sci, 1998, 7: 837-847.
[24] Yusof R, Clum S, Wetzel M, Krishna Murthy [31] Barbato G, Cicero DO, Cordier F, Narjes F,
HM and Padmanabhan R. Purified Gerlach B, Sambucini S, Grzesiek S, Matassa
NS2B/NS3 serine protease of dengue virus VG, De Francesco R and Bazzo R. The
type 2 exhibits cofactor NS2B dependence solution structure of the N-terminal protease
for cleavage of substrates with dibasic amino domain of the hepatitis c virus (HCV) NS3
acids. J Biol Chem, 2000, 275: 9963-9969. protein provides new insights into its
activation and catalytic mechanism. EMBO J,
[25] Falgout B, Miller RH and Lai CJ. Deletion
2000, 19: 1195-1206.
analysis of dengue virus type 4 nonstructural

[32] Bianchi E, Orru S, Dal Piaz F, Ingenito R, [35] Wikberg JES, Lapinsh M and Prusi P.
Casbarra A, Biasiol G, Koch U, Pucci P and Proteochemometrics a tool for modelling
Pessi A. Conformational changes in human the molecular interaction space. In: Mueller
hepatitis C virus NS3 protease upon binding G, Kubinyi H (Eds) Chemogenomics.
of product-based inhibitors. Biochemistry, Methods and Principles in Medicinal
1999, 38: 13844-13852. Chemistry. Wiley-VCH, Weinheim, 2004, in
press.
[33] Ingallinella P, Altamura S, Bianchi E, Taliani
M, Ingenito R, Cortese R, De Francesco R, [36] Bianchi E and Pessi A. Inhibiting viral
Steinkuehler C and Pessi A. Potent peptide proteases: challenges and opportunities.
inhibitors of the human hepatitis C virus Biopolymers, 2002, 66: 101-114.
NS3 protease are obtained by optimizing
the cleavage products. Biochemistry, 1998,
37: 8906-8914.
[34] Ingallinella P, Fattori D, Altamura S,
Steinkuehler C, Koch U, Cicero D, Bazzo R,
Cortese R, Bianchi E and Pessi A. Prime site
binding inhibitors of a serine protease:
NS3/4A of hepatitis C virus. Biochemistry,
2002, 41: 5483-5492.

Prognostic Factors of Clinical Outcome in
Non-Paediatric Patients with
Dengue Haemorrhagic Fever/Dengue Shock Syndrome
Jaime R. Torres*#, Jos M. Torres-Viera**, Hiplito Garca**, Jos R. Silva**,
Yasmn Baddour**, Angel Bajares** and Julio Castro M.*
*Tropical Medicine Institute, Infectious Diseases Section, Universidad Central de Venezuela

**Clnica Santa Sofa, Caracas, Venezuela
Abstract
A total of 112 adults with dengue haemorrhagic fever (DHF) admitted at Clnica Santa Sofa, Caracas,
Venezuela, were studied during June 1998June 2001. Capillary leakage (CL) occurred in 28.8% cases,
21.6% experienced bleeding, 9.2% developed pleural effusion (PE) and 9.2% developed acute
acalculous cholecystitis (AAC). High correlation was noticed between the length of illness prior to
admission (IPA) and length of hospitalization (LH) and levels of Hct, Hb and leukocytes. Significant
differences were seen in the length of IPA, LH and level of platelets for patients with or without bleeding
(P<0.05) or CL (P<0.005), and in LH for patients with or without PE (P<0.005), or CL (P<0.05).
Patients with AAC reached higher leukocyte counts (P<0.05). ANOVA showed an association between
IPA and LH, between either of them and levels of Hb, Hct or leukocytes, and between platelets levels
and PE, CL or bleeding. The length of IPA and degree of alteration of Hb, Hct, leukocytes and platelets
predicted a more severe course in adults with DHF.
Keywords: Dengue, dengue haemorrhagic fever, adults, prognostic factors.
Introduction been reported in at least 25 countries in the

Americas [4] . Venezuela has recorded large
Over the last decade, dengue fever has numbers of DHF cases every year, and, in
dramatically spread in virtually all Latin 1995, the country reported the largest
American and Caribbean countries which outbreak in the region with almost 30,000
are infested with Aedes aegypti. During this dengue cases and 5,000 DHF cases.
period, the number of cases reported every Although DEN-1, DEN -2 and DEN -4 had
year in these countries jumped from been isolated during this epidemic, DEN-2
250,000 to more than 750,000[1] . was the predominant serotype [5] .
Furthermore, recent serological surveys
suggest the occurrence of millions of such In contrast with observations made in
infections[2] . After its emergence in Cuba in Asian countries, where DHF is almost
1981[3] , epidemics or sporadic cases of completely restricted to young children, in
dengue haemorrhagic fever (DHF) have the Americas, older age groups are widely
involved[6- 8] .
#
E-mail: torresj@iname.com; Fax: + (58-212) 9876590

Prognostic Factors in Adult Patients with Haemorrhagic Dengue
The hosts immune response appears to <105 per L, and evidence of plasma
be a major factor influencing the type and extravasation, such as haemoconcentration
severity of disease, as sequential infection (20% increase over base Hct, or 20%
with different dengue virus serotypes in the decrease after rehydration), polyserositis, or
presence of non-neutralizing antibodies has hypoproteinemia, with or without signs of
been strongly incriminated in the circulatory failure, including narrowing of
occurrence of DHF/DSS[8-10] , and cases of pulse pressure ( 20 mm Hg), hypotension,
DHF/DSS are seldom documented in or shock[15] . The level of severity of the
patients with primary infection[11-13] . condition was established according to the
Individual factors, such as age, sex, genetic following scale:
background and underlying diseases, may
(i) Grade I, fever + nonspecific
also play a role[14] .
constitutional symptoms + positive
Since most of the currently available tourniquet test + evidence of
clinical and epidemiological data on haemoconcentration and thrombo-
DHF/DSS derives from observations on cytopenia;
infected children, information is sparse
(ii) Grade II, all of the above +
regarding prognostic factors of poor spontaneous bleeding, usually
evolution among adult patients. In an
restricted to the skin other sites;
attempt to identify potential prognostic
factors in this specific setting, a retrospective (iii) Grade III, all of the above +
study was carried out among non-paediatric circulatory failure manifested by
inpatients with DHF/DSS followed at a rapid and weak pulse, narrowing of
single South American private medical pulse pressure (20 mm Hg or less),
institution. or hypotension with the presence
of cold, clammy skin, and
restlessness or agitation, and
Materials and methods (iv) Grade IV, all of the above +
A total of 112 non-paediatric patients profound shock with undetectable
(male/female ratio: 64/48; age range: 15-92 blood pressure and pulse[15] .
years; median 36 years) admitted to Clnica Clinical evidence of gross capillary
Santa Sofa, Caracas, Venezuela, during a leakage (CL) was defined as the occurrence
36-month period from June 1998 to June of polyserositis, expressed by any of the
2001, who were attended to by the same following: symptomatic pleural effusions,
team of physicians, were included in the ascytis, pericardial effusions, gallbladder wall
study. The endemic dengue transmission edema and/or acute acalculous cholecystitis.
season in the country typically extends from
May to October, matching the yearly cycle Acute acalculous cholecystitis (AAC) was
of rains. diagnosed according to the following
criteria: fever; persistent abdominal pain;
All cases fulfilled the diagnostic criteria nausea and vomiting. On physical
of DHF/DSS, according to WHO and PAHO examination, occurrence of tenderness or
definitions: acute febrile illness with muscle rigidity in the right upper abdominal
evidence of bleeding, thrombocytopenia quadrant, epigastrium, or both, and

Murphy's sign. Additional relevant findings bleeding, and 14 (12.5%) developed pleural
were a palpable mass in the region of the effusion on plain chest X-ray films. AAC
gall-bladder, jaundice, and mild elevations ensued in 14 (12.5%) cases. No deaths
in the serum levels of bilirubin, alkaline occurred (Table 1).
phosphatase, and/or transaminases. On
ultrasound, demonstration of an enlarged
Table 1. Degree of severity of DHF and
gall-bladder with thickened wall ( 6 mm) clinical complications in 112 non-paediatric
and pericholecystic fluid appearing as a halo,
Venezuelan patients
or the presence of a diffuse, homogeneous,
non-shadowing, medium level echogenicity Severity of
within the gall-bladder lumen, were all disease and Number Percentage
considered positive findings[16] . clinical of cases (%)
complication
Either specific IgM or IgG
seroconversion over a period of 15 days was DHF grade I 71 63.4.9
documented by means of a rapid
DHF grade II 23 20.5
commercial qualitative immuno-
chromatographic test (PanBio Dengue , DHF grade III 18 16.1
Windsor, Australia). or IV
Statistical analysis was performed using Bleeding 25 22.3
a StatSoft, Inc. (1995), STATISTICA for
Windows, Computer programme manual, Pleural effusion 14 12.5
Tulsa, OK. Either Students t-test for the Acalculous 14 12.5
comparison of means from independent cholecystitis
samples of unknown variances, Pearsons
correlation analysis, univariate and Pearsons correlation analysis results
multivariate logistic regression analysis was according to clinical complications are
performed with CAA, vascular leakage or depicted in Table 2. A significant correlation
bleeding as main outcomes, by means of a was found between the levels of Hb and
forward stepwise independent variable entry Hct (r=0.762; P<0.0001), and between the
in the final model. All calculations were level of platelets and Hb (r=-0.280;
two-tailed, and 0.05 significant criteria were P<0.01). Correlation was also observed
used. ANOVA analysis of variables was between the number of days of illness prior
performed as required. to admission (IPA) and the length of
hospitalization (r=-0.233; P<0.05), as well
as between the length of hospitalization
Results (LH) and the degree of alteration in platelets
Out of 112 patients, 71 (63.4%) developed level (r=-0.198; P<0.05). A higher
DHF grade I, 23 (20.5%) had DHF grade II, correlation was seen between the length of
and 18 (16.1%) had either DHF grade III or hospitalization and the number of days of
grade IV. In 37 patients (33%), clinical signs IPA (r=-0.426; P<0.005) for patients with
of vascular leakage were evident, 25 dengue grade II or higher, or those with
(22.3%) experienced moderate to severe bleeding (r=-0.427, P<0.005).

Significant differences were seen in the longer (mean 4.35 days vs. 3.26 days;
length of IPA (mean 4.32 days vs. 3.60 days; P<0.05), the length of hospitalization was
P<0.05), and the minimum level of platelets longer (mean 4.85 days vs. 3.61 days;
(mean 43,000 per L vs. 65,816 per L; P<0.001), the serum levels of alkaline
P<0.05) for patients with or without clinical phosphatase were higher (mean 183 /L vs.
bleeding, as well as those with or without 91 /L; P<0.05), and the minimum level of
dengue type 2 (mean 4.43 vs. 3.56 days, leukocytes was higher (mean 12,890 per L
and 48,870 vs. 70,800 per L; P<0.05, vs. 5,026 per L; P<0.001) in patients
respectively). Significant differences were developing vascular leakage. Patients with
also found in the length of hospitalization AAC exhibited a significantly longer hospital
(mean 5.77 days vs 3.61 days; P<0.005) for stay (4.71 days vs. 3.71 days; P<0.05), and
patients with or without pleural effusion, as a higher mean level of peripheral blood
well as for those with or without clinical leukocytes (15,986 x mm3 vs. 5,071 x mm3;
vascular leakage (mean 4.85 days vs. 3.53 P<0.001).
days; P<0.005). The length of IPA was
Table 2. Significant differences in disease outcome in 112 non-paediatric Venezuelan patients

with dengue haemorrhagic fever according to type of clinical complication
Clinical complication Variable Mean value Significance
Bleeding Yes Illness prior to admission 4.66 days P<0.05

No 3.70 days
Yes Minimum platelet level 43,619 per L P<0.05
No 61,128 per L
Pleural Yes Length of hospitalization 5.0 days P<0.005
effusion No 3.6 days
Signs of Yes Length of hospitalization 4.24 days P<0.05

capillary No 3.53 days
leakage
Yes Illness prior to admission 4.71 days P<0.001
No 3.56 days
Yes Minimum platelet level 45,178 per L P<0.01

No 68,784 per L
AAC Yes Peripheral blood leukocytes 11,300 per L P<0.05
No 3,418 per L
Both one-way and multivariate logistic bleeding were illness severity on admission
regression analysis revealed that the only according to WHO scale (OR=5.3;
variables significantly associated with P<0.001), and length of IPA (OR=1.7;

P<0.05). Vascular leakage was also Table 4. Logistic regression analysis of

associated with illness severity on admission clinical variables associated significantly with
in the multivariate regression analysis bleeding in 112 Venezuelan patients with
(OR=14.3; P<0.005), as well as with the dengue haemorrhagic fever
length of hospitalization (OR=1.79;
Univariate Multivariate
P=0.001), illness severity on admission Variable
(OR=26.5; P=0.001), leukocytes level OR P OR P
(OR=1.33; P=0.001), and a prolonged
Degree of severity 5.85 0.001 5.3 0.01
aPTTA (OR=18.9; P=0.001), in the
univariate regression analysis. Of note was Length of illness 1.37 0.032 1.7 0.13
prior to admission
the finding that whereas patients with
bleeding did not remain hospitalized longer, Platelets (nadir) 0.99 0.015 >0.5
those with vascular leakage did (OR=1.79; Platelets <50.000 3.83 0.005 >0.05
P=0.001). Statistically significant results for
Pleural effusion 6.3 0.02 >0.05
the one-way and multivariate logistic
OR = odds ratio
regression analysis are summarized in Tables
Blank cells indicate values that should not be included
3, 4 and 5. since they are not significant
Table 3. Logistic regression analysis of Table 5. Logistic regression analysis of

clinical variables associated significantly clinical variables significantly associated with
with capillary leakage in 112 non-paediatric acute achalculous cholecystitis (AAC) in 112
Venezuelan patients with dengue non-paediatric Venezuelan patients with
haemorrhagic fever dengue haemorrhagic fever
Univariate Multivariate Univariate Multivariate

Variable Variable
OR P OR P OR P OR P
Degree of severity 50.5 0.001 >0.05

Degree of severity 26.5 0.001 14.3 0.002
Blood leucocytes 1.58 0.001 1.34 0.04
Length of 1.79 0.001 >0.05 level
hospitalization
Abnormal PTT 1.81 0.001 11.3 0.01
Blood leucocytes 1.33 0.001 >0.05
Pleural effusion 9.75 0.001 >0.05
level
OR = odds ratio
Abnormal PTT 18.9 0.001 >0.05 Blank cells indicate values that should not be included
since they are not significant
Bleeding 3.51 0.015 >0.05
OR = odds ratio
Blank cells indicate values that should not be included Discussion
since they are not significant
The age distribution for DHF cases in the
Americas differs from that observed in
South-East Asia[1,3,6,7,8,17,18] , where young

Activated partial thromboplastin time

children continue to be the age group patients show severe bleeding of the
almost exclusively affected. In contrast, an gastrointestinal (GI), or of other sites,
age range of 31 to 45 years has been preceding the shock, which may be severe
reported for Brazilian patients with enough to cause death[20-21,25] .
DHF/DSS[6] , while in Puerto Rico, the mean
Relevant laboratory findings in DHF
age of the patients reported in 1990-91 was
cases include thrombocytopenia, haemo-
38 years [7] . Furthermore, during the
concentration and hypoproteinemia[15] . A
outbreaks in Cuba in 1981 and in
drop in platelet count to below 100,000 per
Venezuela in 1989, about one third of the
L and an increase of 20% or more in the
deaths were among patients older than 14
haematocrit, both resulting from increased
years [8,19] , and in the 1997 Cuban outbreak,
vascular permeability, are consistent findings.
all registered deaths were seen among
Other signs of plasma leakage include pleural
adults[8] .
effusion, ascites and hypoproteinemia.
The main pathogenic feature of dengue Leukopenia and leukocytosis are common,
is an increase in vascular permeability and aminotransferases are usually elevated.
leading to loss of plasma from blood vessels, Thrombin, prothrombin and partial
which causes haemoconcentration, low thromboplastin times are often prolonged.
blood pressure and shock. This may also be Fibrinogen levels decrease and fibrin
accompanied by haemostatic abnormalities degradation products may increase. In
such as thrombocytopenia, vascular changes patients with DSS, the severity of laboratory
and coagulopathy[15] . abnormalities described for DHF tends to be
worse. Dilutional hyponatremia and
The clinical spectrum of dengue virus
hypoproteinemia correlate with disease
infection may range from an asymptomatic
severity[1] .
infection to a severe and rapidly fatal
disease[15,20-24] . The most severe end of the In the current series, patients with
spectrum of dengue virus infection in longer length of hospitalization exhibited
children is represented by dengue shock significantly lower levels of platelets, as well
syndrome (DSS) [15] . Adults seem less likely as a higher tendency to severe capillary
than children to suffer from DSS. Indeed, in vascular leakage (CVL) . Overall, a shorter
a retrospective study of 108 adult duration of IPA associated surprisingly with
Malaysians with DHF, the overall morbidity a longer length of hospitalization, probably
was significant (29.4%) but the case-fatality reflecting the fact that more severely ill
rate remained low (2.0%)[25] . patients tended to seek medical attention
earlier. Nevertheless, patients with dengue
Haemorrhagic manifestations in DHF
type II, as well as those with evidence of
usually consist of petechiae, ecchymoses,
CVL, exhibited significantly more protracted
easy bruising and bleeding from
IPA. The occurrence of the latter two
venipuncture sites. Epistaxis, gum bleeding
conditions most likely reflect the delay in
and gastrointestinal haemorrhage are less
common[23-24] . If improperly treated, shock
leads to metabolic acidosis, severe
CVL may induce many other clinical manifestations
generalized bleeding and, eventually,
and complications besides clinical bleeding, such as
death[15] . Unlike children, many adult pleural effusion, ascitis, joint swelling, etc.

initiating a proper and adequate fluid- Viral, serological and genetic factors
replacement treatment, which is the key to may influence virulence. Molecular studies
treating DHF in order to compensate for the have identified genetic variation among all 4
loss of plasma from blood vessels due to dengue virus serotypes[31-34] . Of note here is
increased vascular permeability[1,15] . the finding that DEN-2 strains associated
Of note is the finding that a with grade II DHF or DSS grow to higher
considerable percentage of the cases titers in peripheral blood leukocytes than do
(12.5%) developed AAC. Recent data DEN-2 strains isolated from mildly ill
suggest that in children with DHF, a gall- patients[35,36] . Although characterization of
the viral serotypes involved in these patients
bladder wall thickening =5 mm on
was not performed, DEN -1, 2 and 4 all
ultrasonography correlates with a higher risk
of hypovolemic shock[26] . However, despite circulated in Venezuela during the period
a few scattered reports of AAC complicating when the cases occurred, DEN-2 being the
adult DHF patients[27- 30] , little information most prevalent one [5] .
exists in medical literature on the In conclusion, the number of days of
pathological and clinical implications of this IPA, the length of hospitalization and the
newly recognized condition. It is worth degree of alteration in the level of Hb, Hct,
mentioning that while our nine patients with leukocytes and platelets were all predictors
AAC exhibited a significantly increased level of a more severe and complicated course in
of peripheral blood leukocytes during adult patients with DHF/DSS. The onset of
hospitalization, their clinical outcome in leukocytosis must suggest the occurrence of
terms of IPA, length of hospitalization or inflammatory complications such as AAC.
occurrence of other life-threatening DHF/DSS in the Americas continues to
complications, did not differ from that of occur in a significant number of adults, but
patients without AAC. Details of the clinical it is not clear whether this relates with the
aspects and imaging techniques findings for genetic background of the populations, the
this set of patients will be discussed epidemiological events or, else, with other
elsewhere. unknown factors.
References
[1] Istriz R, Gubler D and Brea del Castillo J. epidemic. Bull Pan Am Health Organ, 1986,
Dengue and dengue haemorrhagic fever in 20: 24-30.
Latin America and the Caribbean. Infect Dis [4] Gubler DJ and Trent DW. Emergence of
Clin North Am, 2000, 14: 121-140. epidemic dengue/dengue haemorrhagic fever
[2] Pinheiro FP and Corber SJ. Global situation as a public health problem in the Americas.
of dengue and dengue haemorrhagic fever Infect Agents Dis, 1994, 2: 383-385.
and its emergence in the Americas. World [5] Salas RA, Tovar D, Barreto A, de Miller E,
Health Stat Q, 1997, 50: 161-169. Leitmeyer K and Rico-Hesse R. Serotypes
and genotypes of dengue virus circulating in
[3] Kouri G, Guzman MG and Bravo J.
Venezuela, 1990-1997. Acta Cient Venez,
Haemorrhagic dengue in Cuba: history of an
1998, 49 (Suppl 1): 33-37.

[6] Zagne SM, Alves VG, Nogueira RM, [16] Nahrwold DL. Acute Cholecystitis. In:
Miagostovich MP, Lampe E and Tavares W. Sabistons Textbook of Surgery, 15 th ed.,
Dengue haemorrhagic fever in the state of 1997. W.B. Saunders Company, Editors,
Rio de Janeiro, Brazil: a study of 56 1127-1132.
confirmed cases. Trans R Soc Trop Med Hyg, [17] Sumarmo, Wulur H, Jahja E, Gubler DJ,
1994, 88: 677-679. Suharyono W and Sorensen K. Clinical
[7] Rigau-Perez JG. Clinical manifestations of observations on virologically confirmed fatal
dengue haemorrhagic fever in Puerto Rico, dengue infections in Jakarta, Indonesia. Bull
1990-1991. Puerto Rico Association of World Health Organ, 1983, 61: 693-701.
Epidemiologists. Rev Panam Salud Publica, [18] Thongcharoen P and Jatanasen S.
1997, 1: 381-388. Monograph on dengue/dengue haemorrhagic
[8] Kouri G, Guzman MG and Bravo J. Why fever. New Delhi: WHO Regional
dengue haemorrhagic fever in Cuba? 2: An Publications, South-East Asia Series, No. 22,
integral analysis. Trans R Soc Trop Med Hyg, 1993, 1-8.
1987, 81: 821-823. [19] Torres JR and Torres-Viera CG. Dengue in
[9] Dengue and dengue haemorrhagic fever in Latin America a unique situation. Dengue
the Americas: Guidelines for prevention and Bull, 2002, 26: 62-69.
control. Pan American Health Organization, [20] Funahara Y, Sumarmo and Wirawan R.
Scientific Publication No. 548, 1994. Features of DIC in dengue haemorrhagic
[10] Halstead SB. Pathogenesis of dengue: fever. Bibliotheca haematologica, 1983, 49:
challenges to molecular biology. Science, 201-211.
1988, 239: 476-481 [21] Tsai CJ, Kuo CH, Chen PC and Changcheng
[11] Gubler DJ. Dengue. In Monath TP (Ed.), The CS. Upper gastrointestinal bleeding in
arboviruses: epidemiology and ecology, Vol. dengue fever. Am J Gastroenterol, 1991, 86:
II. Boca Raton, CRC Press Inc, 1988: 223- 33-35.
260. [22] Nimmannitya S, Thisyakorn U and
[12] Scott RM, Nimmannitya S, Bancroft WH Hemsrichart V. Dengue haemorrhagic fever
and Mansuwan P. Shock syndrome in with unusual manifestations. Southeast Asian J
primary dengue infections. Am J Trop Med Trop Med Public Health, 1987, 18: 398-403.
Hyg, 1976, 25: 866-874. [23] Bhamarapravati N, Tuchinda P and
[13] Rosen L. The emperor's new clothes Boonyapaknavik V. Pathology of Thailand
revisited, or reflections on the pathogenesis haemorrhagic fever: a study of 100 autopsy
of dengue haemorrhagic fever. Am J Trop cases. Ann Trop Med Parasitol, 1967, 61:
Med Hyg, 1977, 26: 337-343. 500-510.
[14] Bravo JR, Guzman MG and Kouri GP. Why [24] Pinheiro FP. Dengue in the Americas, 1980-
dengue haemorrhagic fever in Cuba? 1: 1987. Epidemiol Bull, 1989, 10: 1-8.
Individual risk factors for dengue [25] Ibrahim NM and Cheong I. Adult dengue
haemorrhagic fever/dengue shock syndrome haemorrhagic fever at Kuala Lumpur
(DHF/DSS). Trans R Soc Trop Med Hyg, Hospital: retrospective study of 102 cases.
1987, 81: 816-820. Br J Clin Pract, 1995, 49: 189-191.
[15] Anonymous. Dengue haemorrhagic fever: [26] Setiawan MW, Samsi TK, Wulur H, Sugianto
diagnosis, treatment, prevention and control, D and Pool TN. Dengue haemorrhagic
ed. 2. Geneva, Switzerland, World Health fever: ultrasound as an aid to predict the
Organization, 1997. severity of the disease. Pediatr Radio, 1998,
28: 1-4.

[27] Van Troys H, Gras C, Coton T, Deparis X, [33] Lewis JG, Chang G-J, Lanciotti RS, Kinney
Tolou H and Durand JP. Imported dengue RM, Mayer LW and Trent DW. Phylogenetic
haemorrhagic fever: aprops of 1 case relationships of dengue-2 viruses:
presenting with signs of acute alithiasic correlation with epidemiology. Virology,
cholecystitis. Med Trop (Mars), 2000, 60: 1993, 197: 216-224.
278-280. [34] Lanciotti RS, Gubler DJ and Trent DW.
[28] Sood A, Midha V, Sood N and Kaushal V. Molecular evolution and phylogeny of
Acalculous cholecystitis as an atypical dengue-4 viruses. J Gen Virol, 1997, 78:
presentation of dengue fever. Am J 2279-2286.
Gastroenterol, 2000, 95: 3316-3317. [35] Morens DM, Marchette NJ, Chu MC and
[29] Coton T, Debonne JM, Molinier S, Chaudier Halstead SB. Growth of dengue type 2 virus
B, Gras C, Carre D and Raillat A. Alithiasic isolates in human peripheral blood
cholecystitis and haemorrhagic dengue. leukocytes correlates with severe and mild
Gastroenterol Clin Biol, 1999, 23: 789-790. dengue disease. Am J Trop Med Hyg, 1991,
[30] Wu KL, Changchien CS, Kuo CM, Chuah SK, 45: 644-651.
Lu SN, Eng HL and Kuo CH. Dengue fever [36] Vaughn DW, Green S, Kalayanarooj S, Innis
with acute acalculous cholecystitis. Am J BL, Nimmannitya S, Suntayakorn S, Endy TP.
Trop Med Hyg, 2003, 68: 657-660. Dengue viremia titer, antibody response
[31] Rico-Hesse R. Molecular evolution and pattern, and virus serotype correlate with
distribution of dengue viruses types 1 and 2 disease severity. J Infect Dis, 2000, 181: 2 -9.
in nature. Virology, 1990, 174: 479-493.
[32] Lanciotti RS, Lewis JG, Gubler DJ and Trent
DW. Molecular evolution and epidemiology
of dengue-3 viruses. J Gen Virol, 1994, 75:
65-75.

Dengue Haemorrhagic Fever with
Encephalopathy/Fatality at Petchabun Hospital:
A three-year Prospective Study (1999-2002)
Prasonk Witayathawornwong#
Department of Paediatrics, Petchabun Hospital, Petchabun Province, Thailand
Abstract
During the three-year period from 1 May 1999 to 30 April 2002, there were 1,465 cases of dengue
haemorrhagic fever (DHF) admitted to the Department of Paediatrics, Petchabun Hospital. The male to
female ratio was 722:743 (1:1.03). Their ages ranged from 80 days to 15 years with a median of 9 years.
Thirty-two patients (2.2%) were under one year of age with a median of 8 months, and all except two
had primary dengue infection.
There were 34 DHF patients with encephalopathy (2.3% of all DHF cases). The male to female ratio
was 17:17 (1:1). The median age was 8 years and 11 months (range 10 months to 13 years). Thirty
patients (88.2%) were older than 5 years. Thirty and four patients respectively developed
encephalopathy in shock and convalescent stages. All 17 fatal cases (1.16% of the total DHF cases,
male:female = 8:9) had both prolonged shock and massive gastrointestinal haemorrhage since
admission. About 64.7%, 76.4% and 58.8% of the seventeen non-fatal cases (male:female = 9:8) had
gastrointestinal haemorrhage, shock state and massive fluid overload since admission respectively. The
risk factors for encephalopathy included prolonged shock, severe gastrointestinal haemorrhage, severe
hepatic dysfunction and prior fluid overload.
Keywords: Dengue haemorrhagic fever, encephalopathy, fatality, gastrointestinal haemorrhage, shock, fluid overload,
hepatic dysfunction.
Introduction 0.88-5.4%[7-11] first reported in 1976[12] .

Although dengue encephalitis existed as
The major pathophysiological hallmarks in evident from the direct dengue viral
dengue haemorrhagic fever (DHF) are invasion[13-17] , the more common conditions
leakage and abnormal haemostasis that were encephalopathy secondary to fluid
leads to hypovolemic shock and/or extravasation, cerebral oedema,
haemorrhage. Generally, vital organs are not hypoperfusion, haemorrhage, hyponatremia,
primarily involved in DHF but unusual liver failure and renal failure [2,18-19] . The
manifestations, mainly the involvement of treatment of DHF with CNS involvement is
the central nervous system (CNS) and severe supportive and symptomatic. Early detection
hepatic dysfunction, are increasingly being and proper fluid management of DHF
detected[1-6] . The incidence of CNS should be done to prevent any risk factors.
involvement in dengue infection was about
#
E-mail: prasonkw@thaimail.com

DHF with Encephalopathy/Fatality at Petchabun Hospital, Thailand (1999-2002)
Materials and methods Results

There were 1,465 cases of DHF admitted to Of the 1,465 cases of DHF admitted to the
the Department of Paediatrics, Petchabun Department of Paediatrics, Petchabun
Hospital, Petchabun province, Thailand, Hospital, from 1 May 1999 to 30 April 2002.
from 1 May 1999 to 30 April 2002. The The male to female ratio was 722:743
diagnosis methods used followed the WHO (1:1.03). The patients age ranged from 80
criteria[20] and about 82% of cases were days to 15 years with the median of 9 years.
serologically confirmed using either enzyme- The highest incidence was in the 5-10-year
linked immunosorbent assay or age group (57%) and the second highest was
haemagglutination inhibition tests. The in the 10-15-year age group (26.2%). Thirty-
treatment consisted of general measures two patients (2.2%) were under one year of
(closely observed vital signs, general age with the median of 8 months, and all of
appearance and serially recorded them except two had primary dengue
haematocrit in 24-48 hours after fever, no infection. Eighty-seven per cent of all cases
medication except antacid for moderate to were found in the rainy season during May-
severe abdominal pain) and fluid therapy October. The patients were from all 11
(minimal amount to normalize vital signs districts in the Petchabun province, 84%
and haematocrit: 5% dextrose saline for from the central district alone. There were
DHF patients from all villages (178) of all the
infants, 5% dextrose Ringer acetate for older
subdistricts (17) of the central district.
patients, Dextran-40 in normal saline for
impending or fluid overload and fresh whole There were 34 DHF patients with
blood or packed red cells for significant encephalopathy (2.3%). The male to female
bleeding, platelet concentrate and plasma ratio was 17:17 (1:1). The median age was
not used). 8 years 11 months (range 10 months to 13
years). Thirty patients (88.2%) were older
Patients with encephalopathy (drowsy,
than 5 years. Thirty and four patients
stuporous, comatose and convulsion) were
developed encephalopathy in the shock and
closely observed for blood sugar or
convalescent stages respectively. There were
dextrostix every six hours. Serum
7, 18 and 9 patients with encephalopathy
transaminase was done once a day until
stage II (drowsy), stage III (stuporous) and
recovery. Vitamin K 1 and 10% calcium
stage IV (comatose) respectively. Twenty-
gluconate were administered for three days. eight patients (82.3%) were referred from
Lumbar puncture was performed cautiously district hospitals and 17 patients (50%) had
if there was no other risk. a massive fluid overload. Lumbar puncture
The statistical analysis included the was performed in 2 non-fatal cases with
percentage, mean, standard deviation and normal findings. Their serology indicated
range for demographic data and Students t secondary dengue response.
and chi-square tests for comparing non- About 17 fatal cases (1.6% of total DHF
categorized and categorized variables, cases, male:female = 8:9 , median age 8
respectively. years and 4 months, range 10 months to 12

years) had the median duration of 13 hours 9 (52.9%) and 7 (41.1%) cases respectively.
for deaths (range 1-26 hours, meanSD = There was no history of taking
13.48.4 hours). Sixteen of the 17 cases acetaminophen more than 60 mg per kg/day.
(94.1%) were dead within 24 hours. Aspirin and non-steroidal anti-inflammatory
Profound shock and massive gastrointestinal drug (NSAID), Ibuprofen, were taken by each
haemorrhage since admission were detected patient for many days during the febrile stage.
in all patients. Hepatic failure, comatose Convulsion was detected in 4 cases (Tables 1
stage and massive fluid overload were and 2).
detected since admission in 12 (70.6%),
Table 1. Clinical manifestations of 17 fatal cases
Age Weight L/S DHF Haemorrhage Encephalopathy Fluid Associated

Patients Sex TDF/DI Referral*
(years) (kg) (cm) grade manifestations signs/onset overload diseases
1NW 1.3 F 81 3/3 3/- 4 GIH Comatose/s + Diarrhea +

convulsion
2SS 11.7 F 42 6/7 2/- 4 GIH Comatose/s -
3TR 13 M 37 4/3 2/- 4 GIH Stuporous/s + ASA +
4KB 6 F 132 3/3 10/4 4 GIH Comatose/s Thalasemia +
convulsion
5SM 7 M 20 5/6 3/- 4 GIH Stuporous/s Pneumonia +
G6PDdef
6KC 7 F 191 4/4 2/- 4 GIH Comatose/s + +
7UA 2.11 F 21 7/7 3/- 4 GIH Comatose/s + NSAID +
convulsion
8WT 10 M 50 5/5 2/- 4 GIH Stuporous/s + +
9KF 6-36.3 M 40 5/6 3/- 4 GIH Stuporous/s
10RM 0.10 F 71 4/4 4/- 4 GIH Stuporous/s +
11JP 10 F 31 4/5 3/- 4 GIH Stuporous/s + ARDS, DIC +
12DP 9.0 M 17 5/6 3/- 4 GIH Comatose/s + +
13BP 9.5 F 231 4/4 5/- 4 GIH Comatose/s +
14NO 8.4 F 24 6/7 3/- 4 GIH Comatose/s
1
15TW 8.10 M 20 5/6 3/- 4 GIH Drowsy/s Hypoglycemia +
convulsion
16WK 10 M 35 4/4 3/- 4 GIH Comatose/s +
17PL 12 M 45 5/6 2/- 4 GIH Stuporous/s
TDF = total duration of fever; DI = duration of illness

GIH = gastrointestinal haemorrhage; S = shock stage; kg = kilograms
1,2
= first, second degree protein energy malnutrition
* or prior medications especially excessive fluid replacement
ARDS = A dult respiratory distress syndrome
DIC = Disseminated intravascular coagulation
NSAID = Non-steroidal anti inflammatory drug
L/S = liver/spleen

Table 2. Laboratory investigations of 17 fatal cases
Platelets/ AST/ALT PT/PTT* Sodium DHF Duration

Patients
Hct (max-min) (IU/L) (sec) (mEq/L) serology in hospital
1NW 23,00 (37-) 481/161 20.2/96.6 129 SDI 6h
2SS 27,000 (50-34) 10,081/2,410 20.9/97.9 135 SDI 24 h
3TR 98,000 (39-21) 1,880/636 29.0/>180 130 SDI 26 h

4KB 35,000 (20-12) 1,346/420 137.0/58.0 130 SDI 7.5 h
5SM 15,000 (28-8) 50/20 26.2/42.0 138 SDI 2h
6KC 33,000 (47-27) 9,729/4,821 24.0/170 134.9 SDI 21 h
7UA 24,000 (48-37) 10,043/3,734 19.7/171 129.6 SDI 13 h
8WT 17,000 (49-34) 3,537/1,599 20.1/>180 134 SDI 8.5 h
9KF 18,000 (57-47) 600/1,524 13.8/77.7 136 SDI 17.5 h

10RM 22,000 (29-27) 2,582/671 23.1/119.6 128 SDI 6h
11JP 46,000 (42-6) 3,298/1,274 43.9/472 136.7 SDI 16 h
12DP 12,000 (48-31) 1,452/797 17.1/84.8 137 SDI 24 h
13BP 25,000 (42-34) 10,215/5,015 43.1/172 135 SDI 21 h
14NO 10,675 (35-26) 1,486/631 35.6/>180 138 SDI 1 h 10 m
15TW 2,000 (48-28) 3,940/1,592 35.3/>180 134.0 SDI 12 h
16WK 2,000 (56-32) 234/99 12.6/41.1 136 SDI 20 h

17PL 89,000 (41- ) 17,950/10,446 45.2/160.8 135 SDI 2 h15 m
* normal (10-14)/(23-35) sec

h = hours, m = minutes
SDI = secondary dengue infection
There were 17 non-fatal cases district hospitals. Haemodialysis and

(male:female = 9:8, median age = 9 years, plasmapheresis were done in 3 renal failure
range = 3-12 years). Massive gastrointestinal cases and 2 cases with only supportive
haemorrhage, shock and massive fluid treatment. All 17 cases recovered
overload were detected since admission in completely without neurological sequelae.
11 (64.7%), 13 (grade 3-9, grade 4-4, The average (mean SD) hospitalization
76.4%) and 10 (58.8%) cases respectively. duration and recovery period from
There were 5 cases with acute renal failure encephalopathy were 8.3 5.31 days and
and one with liver failure since admission. 5.1 3.7 days respectively (Tables 3, 4).
Fifteen of the 17 cases were referred from

Table 3. Clinical manifestations of 17 non-fatal encephalopathy cases

Fluid
Age Weight L/S DHF Hemato Enceph Associated Referral
Patients Sex TDI/DI over-
(y.m) (kg) (cm) grade manifes signs/onset diseases
load
1YB 9 M 25 4/4 3/- 4 GIH Stuporous/s + ARF +
2PS 4.4 F 121 5/3 3/- 4 Petichiae Stuporous/s Pneumonia
3ST 10.3 F 29 6/6 3/- 3 GIH Stuporous/s + - +

4SU 6 F 151 7/7 3/- 3 GIH Stuporous/s + Pneumonia, +
Tracheitis
5CP 5.9 M 17 7/7 1/- 2 GIH Stuporous/s - +
6WD 9.9 F 43 5/5 JP 3 GIH Stuporous/s + ARF +
7WB 6.6 M 181 3/3 3/- 3 Petichiae Stuporous/s + - +
8SSr 9 F 41.5 5/5 2/- 3 GIH Stuporous/s Hypoglycemia, +
ARF
9SN 11 F 301 4/7 4/- 2 GIH Stuporous/s ARF +
10PP 11 M 34 5/5 2/- 4 GIH Stuporous/s + - +
11Sma 9.11 M 28 5/5 5/- 3 GIH Drowsiness/s + - +
12WL 6.11 F 19 5/5 8/6 3 GIH Stuporous/s + Thalasemia, +
ARDS,
pneumonia
13ThP 7.9 M 22 5/5 4/- 2 GIH Stuporous/s - +
14SL 5.9 M 251 5/6 5/- 3 Petichiae Stuporous/s + ARF +
15KN 11.11 M 232 5/5 1/- 4 Petichiae Drowsiness/s - +

16MK 10.4 M 49 6/6 2/- 3 Petichiae Drowsiness/s -
17WTh 3 F 13.5 5/7 2/- 2 Petichiae Drowsiness/s + Rhinitis +
1,2
= first, second degree protein energy malnutrition
ARF = acute renal failure; ARDS = Adult respiratory distress syndrome
GIH = gastrointestinal haemorrhage; JP = just palpable
Table 4. Laboratory investigations of 17 non-fatal encephalopathy cases

Platelets/ AST/ALT PT/PTT * Sodium DHF
Patients Duration **
Hct (max-min) (IU/L) (sec) (mEq/L) serology
1YB 62,000(51-30) 9,420/2,239 23.4/>120 128 SDI 13/7
2PS 17,000(50-26) 2,023/458 14.4/59.9 135 SDI 9/5
3ST 16,000(50-38) 1,098/452 14.7/121.6 130 SDI 4/2
4SU 44,000(46-34) 255/148 16.8/48.5 135 SDI 17/10
5CP 90,000(52-30) 168/110 18.4/64.5 140. 3 SDI 13/10
6WD 36,000(41-30) 13,895/5,200 24.6/77.6 127 SDI 11/7
7WB 4,000(58-34) 2,128/918 15.1/67.8 128 SDI 6/3
8SSr 20,000(48-32) 14,580/5,852 16.4/56.0 134 SDI 10/7
9SN 68,000(49-27) 6,987/3,789 32.5/57.2 138 SDI 7/3
10PP 21,000(42-27) 3,810/1,935 15.3/52.5 129 SDI 4/2
11Sma 34,000(48-33) 2,250/448 14.9/78.9 128 SDI 6/3
12WL 20,000(42-27) 3,436/1,841 15.0/89.3 143 SDI 21/15
13ThP 56,000(42-30) 1,315/549 12.4/61.5 134 SDI 2/2
14SL 45,000(56-33) 4,746/1,470 15.9/1,133 131.6 SDI 2/2
15KN 40,000(49-32) 8,150/4,450 16/72.2 137 SDI 7/4
16MK 3,000(51-35) 2,996/1,482 13.8/64.0 132.8 SDI 5/3
17WTh 60,000(32-30) 275/599 15.4/66.7 128 SDI 4/3
* normal: (10-14 )/(23-35 ) sec
** duration in hospital/consciousness change (days)

A comparison between the clinical manifestations and encephalopathy cases

manifestations and laboratory investigations are shown in Tables 5, 6 and 7 respectively.
of the fatal and non-fatal cases, usual
Table 5. Comparison between clinical manifestations and laboratory investigations

of fatal and non-fatal cases
Clinical manifestations or laboratory Fatal cases
Non-fatal cases P-value
investigations* (n=17)
Shock (grade IV) 17 4 <0.001
Internal bleeding 17 11 0.007
Onset of encephalopathy in shock stage 17 13 0.035
Coma since admission 9 0 <0.001
Platelets (/cubic millimeters)** 29,33.8 (26,662.1) 37,411.7 (24,153.3) 0.362
AST (IU/L) 4,641.4 (5082.7) 4,560.7 (4,545.8) 0.961
ALT (IU/L) 2,108.8 (2,655.1) 1,878.8 (1,838.6) 0.771
PT in sec 25.5 (11.1) 17.3 (5.0) 0.011
PTT in sec 146.1 (98.8) 75.9 (25.5) 0.011
Serum sodium (mEq/L) 133.9 (3.3) 132.8 (4.8) 0.471
* mean (standard deviation, SD)
** minimum value
PT=Prothrombin time
PTT=Partial thromboplastin time
Table 6. Comparison between clinical manifestations of usual manifestations

and encephalopathy cases
Usual manifestations Encephalopathy
Clinical data P-value
(n=1431) (n=34)
Age <1 yr 31 (2.1%) 1 (2.9%) 0.775
1 4 yr 210 (14.6%) 4 (11.7%) 0.661
5 10 yr 812 (56.7%) 23 (67.6%) 0.219
10 15 yr 378 (26.4%) 6 (17.6%) 0.252
Female 726 (50.7) 17 (50.0%) >0.993
Shock 332 (23.2) 30 (88.2%) <0.001
(grade 3=330, 4=2) (grade 3=9, 4=21)
Referal or prior medications 266 (18.6%) 28 (82.3%) <0.001
Internal bleeding 130 (9.1%) 28 (82.3%) <0.001
Malnutrition[21] 716 (50.0%) 12 (35.3%) 0.091
(1=511, 2=192, 3=13) (1=10, 2=2)
Death 17 (50%) <0.001
Duration in hospital (days):mean 3.4 (1.38) 83 (5.31)* 0.002
(standard deviation)
* only non fatal cases

Table 7. Comparison between laboratory investigations of usual and encephalopathy cases
Usual manifestations Encephalopathy

Laboratory data* P-value
(n=1431) (n=34)
AST (IU/L) 257.3 (250.6) 4601.0 (4748.3) <0.001
ALT (IU/L) 119.5 (138.1) 1993.8 (2251.8) <0.001

Platelets (minimum) 70697.9 (2975.7) 33372.8 (2538315) <0.001
PT (10-14 sec) 11.2 (1.1) 21.4 (9.4) <0.001

PTT (23-35 sec) 51.9 (13.4) 111.0 (79.5) <0.001
* mean (standard deviation, SD)
Discussion DHF/DSS patients were well-nourished but

patients with CNS involvement were more
DHF patients from all the 11 districts in the undernourished without significant
Petchabun province were included in this difference[24,26] . In this study, patients with
study. The minors were referred from 10 usual manifestations and encephalopathy
other districts. The adults were from the 178 were underweight[21] by about 50% and
villages in all 17 subdistricts of the central 35.3% respectively. Patients with
district. This epidemic was similar to the encephalopathy were mainly in stage
previous dengue epidemic in 1997[22] . This III [7,8,18,27] (88.2%) initially developed in
data implied that the dengue virus had shock stage (52.9%), more than in febrile or
spread nationwide. The percentage of DHF convalescent stage[7,9,28] . Seizure in DHF
patients with CNS involvement in this study with encephalopathy had been reported in
was 2.3%, the same as in the previous about 18.8-100%[7- 9,13,18,24,27,29 ,30] . In this
study[22] as well as in other studies[7-11] . study seizure was detected in 4 out of 34
Female patients were reported to be more patients (11.7%). Two patients (1NW, 7UA),
severely affected and accounted for more under 5 years, developed seizure in afebrile
fatalities than male patients but without any stage, indicating that the seizure might have
significant difference[18,23-25] . In this study, had a specific primary cause[10] . However,
the number of both sexes was equal and some children had possible confounding
both had usual manifestations and factors such as hyponatremia (3 patients,
encephalopathy cases. Young patients, 1NW, 4KB, 7UA), hypoglycemia (15TW),
especially those less than 1 year of age, had and liver failure (3 patients, 1NW, 7UA,
the tendency to be more severely and fatally 15TW). Other factors could be a history of
affected[18,23-25] . Age itself was not a risk previous febrile seizure, co-infection[10] and
factor of disease severity in this study. drug ingestion.
Although there was one fatal case aged 10
The causes or factors contributing to
months (10 RM), 85% of those who died
CNS manifestations included the following:
were older than 5 years. Most of the
direct CNS infection a rare entity, CNS

bleeding[6,18] and severe hepatic dysfunction. occur in severe cases with prolonged shock,
Lumbar puncture was done in only two DIC and also hepatorenal syndrome[37] .
patients (5CP, 15KN) in the convalescent There were five patients with ARF in this
stage with normal findings. Direct CNS study. All of them recovered completely,
infection could not be evaluated in this three (6WD, 8SSr, 14SL) with haemodialysis
study. There was massive gastrointestinal and plasmapheresis and two (1YB, 9SN)
haemorrhage in 28 out of 34 cases (82.3%). with only supportive and symptomatic
Acute liver failure could be the direct or therapy. Hyponatremia was a factor in CNS
indirect cause of encephalopathy[31,32] and involvement. Serum sodium of
an important cause of death in DHF[33] . encephalopathy patients in this study was
Twelve of the 17 fatal cases (70.5%) had this slightly low. It was due to excessive
severe condition. Severe hepatic hypotonic solution replacement. The risk
dysfunction could be due to profound shock, factors of encephalopathy in this study were
massive gastrointestinal haemorrhage or profound shock, massive gastrointestinal
immune complex mechanism[34] . The liver haemorrhage, excessive fluid overload and
pathology in profound shock stage might be severe hepatic dysfunction. Among the fatal
centrilobular hepatocellular necrosis [35] or and non-fatal encephalopathy cases,
extensive necrosis of hepatocytes usually in laboratory investigations except for
a massive or submassive distribution[33] . coagulogram, were not significantly different.
Massive gastrointestinal haemorrhage might But fatal cases had more severe shock
cause hepatic dysfunction and vice versa. conditions, gastrointestinal haemorrhage
Immune complexes, detected in 80% of and onset and depth of encephalopathy
DHF patients[36] , might be deposited in (Table 5).
hepatocytes and then destroyed as in
hepatitis B virus infection[34] . Excessive use
of hepatotoxic drugs such as acetaminophen, Conclusion
an antiemetic drug, might interfere with In conclusion, encephalopathy in DHF was
liver function. There was no history of a severe complication with high mortality,
excessive use of such drugs in this study. although neurological sequelae in recovered
Two fatal cases with liver failure and massive patients were rare. Prevention should be
gastrointestinal haemorrhage had taken attempted by early diagnosis and proper
aspirin (3TR) and non-steroidal anti- management of fluid therapy.
inflammatory drug, Ibuprofen (7UA), for
many days during the febrile stage. Both
drugs aggravated the gastrointestinal Acknowledgements
haemorrhage and aspirin could have
The author thanks officials of the Regional
induced Reye syndrome[37] .
Medical Science Centre, Phitsanuloke,
Thalassemia, of which two cases with a Thailand, for DHF serology testing and all
large liver and spleen were included in this nursing staff and workers of the Paediatrics
study (4KB, 12WL), was a risk factor for Department of Petchabun Hospital for
hepatic failure [18] . Acute renal failure (ARF) taking good care of the patients.
was a rare complication of DHF but could

References
[1] Nogueira RMR, Filippis AMB, Coelho JMO, [11] Attavinijtrakarn P. Hepatic dysfunction in
Segueira PC, Schatmayr HG, Paiva FG, dengue haemorrhagic fever in
Ramos AMO and Miagostovich MP. Dengue Paholpolpayuhasaena Hospital. Thai J Pediatr,
virus infection of the central nervous system 2000, 39: 265-276.
(CNS): a case report from Brazil. Southeast [12] Sanguansermsri T, Poneprasert B and
Asian J Trop Med Public Health, 2002, 33: Pornphutkul B. Acute encephalopathy
68-71. associated with dengue infection.
[2] Jimerez DR, Santana JL and Ramirez-Ronda Conference on dengue haemorrhagic fever,
CH. Neurological disorders associated with current knowledge. SEAMEO TROPMED,
dengue infection. Bol Assoc Med PR, 1988, 1976, 10-11.
80: 208-211. [13] Lum LCS, Lam SK, Choy YS, George R and
[3] Ramos C, Sanchez G, Pando RH, Baquera J, Harun F. Dengue encephalitis: a true entity?
Hernandez D, Mota J, Ramos J, Flores A and Am Trop Med Hyg, 1996, 54: 256-259.
Llausas E. Dengue virus in the brain of a fatal [14] Thisyakorn U, Limpitikul W and Nisalak A.
case of haemorrhagic dengue fever. J Dengue infection with central nervous
Neurovirol, 1998, 4: 465-468. system manifestations. Proceedings of the
4th international symposium on dengue
[4] Hendarto SK and Hadinegoro SR. Dengue
fever, Tahiti, 1997: 47.
encephalopathy. Acta Paediatr Jpn, 1992,
34: 350-357. [15] Miagostovich MP, Ramos RG, Nicol AF,
Nogueira RMR, Cuzzi-Maya T, Oliveira AV,
[5] Chauhan GS, Khangaro DK, Shah PK and
Marchevsky RS, Mesquita RP and
Rodrigues FM. Encephalitis as a Schatzmayr HG. Retrospective on dengue
manifestation or co-incidence in case of fatal cases. Clin Neuropathol, 1997, 16:
classical dengue fever. J Assoc Physicians 204-208.
India, 1987, 35: 658-659.
[16] Kankirawatana P, Chokepaibulkit K,
[6] Patey O, Ollivaud L, Breuil J and Lafaix C. Puthavathana P, Yoksan S, Apintanapong S
Unusual neurological manifestations and Pongthapisit V. Dengue infection
occurring during dengue fever infection. Am presenting with central nervous system
J Trop Med Hyg, 1993, 48: 793-802. manifestation. J Child Neurol, 2000, 15:
[7] Pongrithsukda V. Dengue haemorrhagic 544-547.
fever and hepatic encephalopathy. [17] Solomon T, Dung NM, Vaughn DW, Kneen
Ramathibodi Med J, 1986, 9: 11-18. R, Thao LTT, Rangsakulrach B, Loan HT,
Day NPJ, Farrar J, Myint KSA, Warrell MJ,
[8] Sirisanthana V. Dengue haemorrhagic fever James WS, Nisalak A and White NJ.
at the Department of Pediatrics Chiangmai Neurological manifestations of dengue
University Hospital in 1987. Thai J Pediatr, infection. Lancet, 2000, 355: 1053-1059.
1988, 27: 36-43.
[18] Nimmannitya S, Thisyakorn U and
[9] Vasanawathana S. Encephalopathy in DHF Hemsrichart V. Dengue haemorrhagic fever
in Khon Kaen Hospital. Khon Kaen Med J, with unusual manifestations. Southeast Asian J
1992, 16: 91-103. Trop Med Public Health, 1987, 18: 398-406.
[10] Pancharoen C and Thisyakorn U. [19] Lum L, Lam SK, George R and Devi S.
Neurological manifestations in dengue Fulminant hepatitis in dengue infection.
patients. Southeast Asian J Trop Med Public Southeast Asian J Trop Med Public Health,
Health, 2001, 32: 341-345. 1993, 24: 467-471.

[20] World Health Organization. Dengue [29] Sumarmo, Wulur H, Jahja E, Gubler DJ,
haemorrhagic fever: diagnosis, treatment Sutomenggolo TS and Saroso JS.
and control, 2nd ed. Geneva, WHO, 1997. Encephalopathy associated with dengue
infection. Lancet, 1978, 1: 449-450.
[21] Thailand, Ministry of Public Health,
Department of Health. Reference scales of [30] Tin U, Aye M, Swe TN, Khin MM and Rosen
weight, height and indicators of nutritional L. Dengue haemorrhagic fever with
status of Thai population - 1 day to 19 years encephalitic symptoms. SEAMEO TROPMED
old. Bangkok, 1999. conference, Bangkok, 1976: 29-30.
[22] Witayathawornwong P. Dengue haemorrhagic [31] [31] Hoyumpa AM Jr, Desmond PV and
fever patients with encephalopathy at Avan GR. Hepatic encephalopathy.
Petchabun hospital: A prospective study for Gastroenterology, 1979, 76: 184.
two years during May 1997 to April
[32] Rueff B and Benhamou JP. Acute hepatic
1999.Thai Pediatr J, 2001, 8: 65-73.
necrosis and fulminant hepatic failure. Gut,
[23] Nimmannitya S. Dengue haemorrhagic fever 1973, 14: 805-815.
in Thailand. Southeast Asian J Trop Med
[33] Innis BL, Myint KSA, Nisalak A, Ishak KG,
Public Health, 1987, 18: 281-284.
Nimmannitya S, Laohapand T,
[24] Thisyakorn U and Thisyakorn C. Dengue Tanprasertsuk S, Pongritsukda V and
infection with unusual manifestations. J Med Thisyakorn U. Acute liver failure is one
Assoc Thai, 1994, 77: 410-413. important cause of fatal dengue infection.
Southeast Asian J Trop Med Public Health,
[25] Kalayanarooj S, Nimmannitya S and
1990, 21: 695-696.
Eaksangsri P. Fatal cases of dengue
haemorrhagic fever at children's hospital [34] Woolf I, El Sheilk N and Cullens H.
1987. Bull Dept Med Serv, 1989, 14: 771- Enhanced HBsAb production in
778. pathogenesis of fulminant hepatitis type B.
Br Med J, 1976, 2: 669-671.
[26] Thisyakorn U and Nimmannitya S.
Nutritional status of children with dengue [35] Nunes G, Blasdell FW and Margaretten W.
haemorrhagic fever. Clin Infect Dis, 1993, Mechanism of hepatic dysfunction following
16: 295-297. shock and trauma. Arch Surg, 1970, 100:
546-566.
[27] Kho LK, Sumarmo, Wulur H, Jahja E and
Gubler DJ. Dengue haemorrhagic fever [36] Ruangjirachuporn W, Boonpucknavig S and
accompanied by encephalopathy in Jakarta. Nimmannitya S. Circulating immune
Southeast Asian J Trop Med Public Health, complexes in serum from patients with
1981, 12: 83-86. dengue haemorrhagic fever. Clin Exp
Immunol, 1979, 36: 46-53.
[28] Suvattee V, Vajaradule C and Laohapand T.
Liver failure and hepatic encephalopathy in [37] Committee on infectious diseases: Aspirin
dengue haemorrhagic fever/dengue shock and Reye syndrome. Pediatrics, 1982, 3: 321.
syndrome: a co-relation study with
acetaminophen usage. Southeast Asian J
Trop Med Public Health, 1990, 21: 694-695.

A New Tool for the Diagnosis and Molecular
Surveillance of Dengue Infections in Clinical Samples
C. Domingo* #, G. Palacios**, M. Niedrig***, M. Cabrerizo*, O. Jabado**,
N. Reyes*, W.I. Lipkin** and A. Tenorio*
*Laboratorio de Arbovirus y Enfermedades Vricas Importadas, Centro Nacional de Microbiologa,
Instituto de Salud Carlos III, Carretera de Pozuelo Km 2 (28220 Majadahonda), Madrid, Spain
**Jerome L and Dawn Greene Infectious Disease Laboratory, Columbia University, New York, USA
***Robert Koch Institute, Nordufer 20, 13353 Berlin, Germany
Abstract
Dengue fever and dengue haemorrhagic fever are amongst the most important challenges in tropical
diseases due to their expanding geographical distribution, increasing outbreak frequency,
hyperendemicity and evolution of virulence.
Here, the use of a RT-nested PCR for both the diagnosis and genetic characterization of dengue
infections in clinical samples is described.
Keywords: Dengue, dengue haemorrhagic fever, diagnosis, molecular epidemiology, surveillance, glycoprotein E
gene, NS1 gene.
Introduction infrastructure in most endemic areas are the

likely factors contributing to the surge in new
Dengue fever (DF), dengue haemorrhagic cases[4] . A major concern is the potential
fever (DHF) and dengue shock syndrome spread of dengue fever into the United States
(DSS) are considered to be the most of America and Europe due to climate
important arthropod-borne viral diseases due warming and the spread of its vector.
to the high rates of morbidity and mortality
Travellers to areas where dengue is
caused by them. Over 2.5 billion people are
endemic are a potential source of the spread.
at risk of the infection and more than 100
Most infections manifest as a mild febrile
countries are home to endemic dengue
illness during travel that coincides with the
transmission with an increasing incidence of
peak of viral shedding and risk for
DHF cases[1-3], making dengue an archetypal
transmission. Imported dengue virus
emerging disease. International travel, infections have been reported in several non-
urbanization, overpopulation, crowding, endemic countries; and dengue virus
poverty and a weak public health infection is one of the most frequent causes
#
E-mail: cdomingo@isciii.es; Phone: (+34) 918223954, Fax: (+34) 915097966

A New Tool for the Diagnosis and Molecular Surveillance of Dengue Infections in Clinical Samples
of febrile illness in tourists and people Materials and methods

working in dengue-endemic areas [5,6,7,8] . As
early symptoms of DF mimic those of other Virus isolates and clinical samples
diseases such as malaria or leptospirosis,
Viral RNAs were provided by the National
rapid laboratory diagnosis is important for
Collection of Pathogenic Viruses (Porton
proper patient care. Dengue fever can be
Down, Salisbury, UK): serotype 1 dengue
diagnosed by virus isolation, genome and
virus (DEN-1; strain Hawaii), serotype 2
antigen detection, or serological studies.
dengue virus (DEN-2; strain New Guinea C),
Samples obtained for the detection of
serotype 3 dengue virus (DEN-3; strain H87),
dengue virus by cell-culture require proper
and serotype 4 dengue virus (DEN-4; strain
handling of the sample for viral viability. H241); the RNAs from prototype strains of
Serology, even for anti-dengue IgM Japanese encephalitis (JEV), yellow fever
antibodies, is feasible only after 5 days (YFV), tick-borne encephalitis (TBEV),
following the onset of the symptoms. Thus, Murray Valley encephalitis (MVEV) and St.
molecular techniques fulfil an important role Louis encephalitis (SLEV) viruses were used
in the diagnosis of dengue infection during its to check the specificity of the dengue virus
early stages. assay. Serial dilutions of this genome material
The characterization of circulating were prepared to obtain the standards to
dengue virus serotypes is important in assess the sensitivity of the assay.
surveillance, since the introduction of a new Viremic human sera samples were
variant to areas affected by pre-existing obtained from patients with a clinical
serotypes constitutes a risk factor for diagnosis of dengue infection (Sera
DHF/DSS[9] . By defining intra-serotypic 1794F02; 13VI02; 366VI03; 438VI03).
genetic variation, the global distribution and These were travellers who presented at the
spread of virus strains can be mapped and Spanish Tropical Medicine Units with
followed up[10-13] , and the genetic differences dengue-compatible symptomatology on
associated with disease severity can be their return from the Dominican Republic,
identified[10,14-16] . Moreover, recent India, Indonesia and Nicaragua, respectively,
epidemiological analyses suggest that the and suffered from classical DF as defined by
more virulent genotypes are now displacing the WHO criteria[19] .
those of lower epidemiological impact[17] ,
resulting in dengue outbreaks [18]. In this
Selection and synthesis of
context, a methodology for real-time,
worldwide surveillance is needed to track oligonucleotide primers
dengue strains and help anticipate changes in A RT-nested PCR protocol was developed
the epidemiology of the infection. for the detection of the four serotypes of
Here we report the amplification and dengue virus in clinical samples. Dengue
analysis of a genomic interval spanning the virus primers for amplification and/or
E/NS1 junction of the dengue genome for sequencing (Table) were designed based on
the detection and typing of all four dengue dengue virus sequences in the public
virus serotypes in clinical specimens. This sequence databases, using a computer-
assisted analysis (MACAW version 32
sensitive, specific and rapid alternative assay
software, 1995, NCBI, Maryland) to
requires only a single acute phase serum
sample. determine consensus sequences. The Table

shows the sequences and the respective S2176DEN2, S2176DEN3, S2176DEN4,

primer positions in the prototype strains of AS2504DEN) and 2.5 U of DNA Taq
the four dengue serotypes. To address the Polymerase (Perkin-Elmer). The samples were
natural variability of dengue viruses, mixtures subjected to a denaturation step (94 C, 2
of degenerated primers were used to enable minutes) followed by 40 cycles of
hybridization with all known serotypes. denaturation (94 C, 30 seconds), primer
annealing (57 C, 4 minutes), and primer
RT-nested PCR extension (72 C, 30 seconds) and a further
extension step at 72 C for 5 minutes.
Using purified dengue virus RNA as a
template, relevant aspects of the RT-PCR and Dengue virus sequence database
nested PCR assay (Mg2+ concentration,
primers, RT temperature, number of cycles, A dengue sequence database was
annealing temperatures) were initially constructed by extracting sequences from
optimized to achieve the greatest sensitivity. the NCBI GenBank. Each sequence was
A PTC-200 Peltier thermal cycler (MJ identified by name, place, date and
Research) was used throughout. 5 l of viral serotype. Previously described genotypes
RNA solution were added to 45 l of a were taken from the references listed:
medium compatible with both the reverse dengue strains genotypes were noted as
transcription and PCR amplification steps described by Rico-Hesse for DEN-1, 3 and
(QIAGEN OneStep RT-PCR kit). The RT- 4[17] and by Twiddy et al. for dengue virus
PCR mix contained 1OneStep RT-PCR type 2[20] . A manual search was employed
buffer, 400 mM of each dNTP, 20 pmol of for all the sequences in GenBank
each sense or antisense degenerated primer encompassing the targets of selected primers.
(S1871DEN1, 1871DEN2, 1871DEN3, Next, we used BUSSUB, a new tool
1871DEN4, AS2622DEN1, AS2622DEN2, developed at the Bioinformatics Unit of the
AS2622DEN3, AS2622DEN4) and an Institute of Health Carlos III[21] . This software
optimized combination of Omniscript and simplifies and boosts the process of retrieving
Sensiscript reverse transcriptases and HotStar sequences contained between two given
Taq DNA polymerase. The RT-PCR reactions flanking regions, improving the final results of
were carried out using an initial reverse a search. Genetic characterization was
transcription step at 41 C for 45 minutes performed on a total data set of 113 DEN-1,
followed by a denaturation and Hot Star Taq 191 DEN-2, 102 DEN-3 and 153 DEN -4
polymerase activation step (94 C, 15 sequences.
minutes) and 40 cycles of denaturation
(94 C, 30 seconds), primer annealing Sequence analysis of amplified
(55 C, 1 minute), and primer extension products
(72 C, 30 seconds). A final incubation was
Original sequence data were first analysed by
carried out at 72 C for 5 minutes. A second
the CHROMAS software (version 1.3,
amplification reaction (nested PCR) was
McCarthy 1996; School of Biomolecular and
seeded with 1 l of the initial amplification
Biomedical Science, Faculty of Science and
product. The reaction mixture contained 1
Technology, Griffith University, Brisbane,
buffer B (60 mM Tris-HCl pH 8.5, 2 mM
Queensland, Australia); forward and reverse
MgCl2, 15 mM (NH4) 2SO 4, 40 pmol of each
sequence data of each sample were aligned
sense and antisense primer (S2176DEN1,
using the programme EDITSEQ (DNASTAR

Inc. Software, Madison, Wisconsin, USA). Scores were calculated to test the significance
The consensus sequence was compared and of each pair-wise alignment by Monte Carlo
aligned to other samples or DNA database simulation on the shuffled sequences.
sequences using the programme CLUSTAL X, Statistical analysis was conducted with the
version 1.83[22]. Programmes from the MEGA SPSS statistical package (SPSS Software,
package[23] were used to produce Chicago, IL).
phylogenetic trees using NJ as the method to
reconstruct the phylogeny and Kimura-2p as
nucleotide substitution calculation method. Results
The statistical significance of a particular tree
topology was evaluated by bootstrap re- Design of the primers
sampling of the sequences 1,000 times. The E/NS1 region of the genome was
Published sequences used in the chosen for the development of a RT-nested
comparisons were obtained from the PCR. The primers selected specifically
GenBank databases. Pair-wise comparisons amplify the four dengue viruses with no
of the dengue virus database were done by cross reactivity to other members of the
global alignment using the Needleman flavivirus family. A mix of degenerate
Wunsch[24] algorithm using the primers representing each serotype was
implementation from EMBOSS, the European used to ensure coverage for the highly
Molecular Biology Open Software Suite[25] . Z- variable dengue serotypes (Table).
Table. Primers used in RT-nested PCR assays and sequencing
Primer* Sequence Genome position PCR

S1871DEN1 5-TGGCTGAGACCCARCATGGNAC-3 1869 to 1890 RT-PCR
S1871DEN2 5-TAGCAGAAACACARCATGGNAC-3 1871 to 1889
S1871DEN3 5-TCTCCGAAACGCARCATGGNAC-3 1863 to 1884
S1871DEN4 5-TGGCAGAAACACARCAYGGNAC-3 1873 to 1894
AS2622DEN1 5-CAATTCATTTGATATTTGYTTCCAC-3 2620 to 2644 RT-PCR
AS2622DEN2 5-CAATTCTGGTGTTATTTGYTTCCAC-3 2622 to 2646
AS2622DEN3 5-CAGTTCATTRGCTATTTGYTTCCAC-3 2614 to 2638
AS2622DEN4 5-TAGCTCGTTGGTTATTTGYTTCCAC-3 2624 to 2648
S2176DEN1 5-ATCCTGGGAGACACYGCNTGGG-3 2174 to 2195 Nested
S2176DEN2 5-ATTTTRGGTGACACAGCNTGGG-3 2176 to 2197
S2176DEN3 5-ATCTTGGGAGACACAGCNTGGG-3 2168 to 2189
S2176DEN4 5-ATTCTAGGTGAAACAGCNTGGG-3 2178 to 2199
AS2504DEN 5-TGRAAYTTRTAYTGYTCTGTCC-3 2506 to 2527 DEN-1 Nested
2504 to 2525 DEN-2
2496 to 2517 DEN-3
2506 to 2527 DEN-4
*Primers name s beginning with S indicate a genome (plus)-sense orientation; names beginning with AS indicate a
complementary sense orientation.
The genome positions are given according to each dengue virus serotype prototype strain (DEN-1; strain Mochizuki,
DEN-2; strain Jamaica N-109, DEN-3; strain H87, DEN-4; strain 814669)
Degenerate positions: N:A/C/g/T, R:A/g, Y:T/C

Dengue virus RT-nested One hundred and sixty-four serum

samples from cases of febrile illness
PCR specificity
associated with travel were tested with the
The specificity of the RT-nested PCR was assay. Thirty-seven cases were diagnosed as
determined by analysing serial dilutions of of classical dengue fever by the WHO
RNA from related flavivirus (JEV, MVEV, criteria[19] . Sixteen of these cases were found
SLEV, TBEV, WNV, YFV) and no positive by using our E/NS1 assay.
amplification was obtained (data not shown). Convalescent sera were available for 13 of
The amplification was successful with these cases; all were later confirmed to be
both commercial RNA and serum samples seroconvert. All serum samples found
for all dengue virus serotypes as shown positive by RT-nested PCR were collected in
(Figure 1), yielding a distinct DNA product the first week after the onset of the
of the expected size (328-pb) in agarose gels. symptoms.
Figure 1. Amplification products obtained Sequence analysis results

through PCR analysis of sera from subjects
The phylogenetic trees obtained by the
with acute dengue virus infection
analysis of the representative strains of the
[Arrow indicates 328 bp E/NS1 products. 1% four serotypes and unknown sample
agarose gel. MWM: Molecular weight markers; sequences allowed rapid differentiation of
438VI03 (DEN-1); 13VI02 (DEN-2); 1794F02 the corresponding serotype (Figure 2).
(DEN-3); 366VI03 (DEN-4)]
Pair-wise sequence analysis using
Needleman Wunsch global alignment was
carried out on the 220bp sequence where a
1794F02
438VI03
366VI03
higher amount of sequences were available

13VI02
MWM
for comparison. As expected, comparisons

between serotypes showed a low sequence
similarity and could be easily grouped. An
all-against-all sequence comparison was
done within each serotype to evaluate the
possibility of using sequence similarity to
396
344 classify genotypes. Significant sequence
298 similarity was observed when comparing
sequences within the same genotype. This
was evaluated by an analysis of variance
between groups (ANOVA), comparing the
scores of sequence comparisons within
genotypes to comparisons between
genotypes. Each group was significant to the
P<0.001 level. Genotypes with only one
member sequence were excluded from the
analysis.

Figure 2. Phylogenetic tree constructed with the E/NS1 fragment which identifies
the four dengue serotypes
[Phylogenetic analysis was performed using the Kimura-two parameter model as a model of nucleotide
substitution and using the neighbor joining method to reconstruct the phylogenetic tree (MEGA version
2.1 software package)]
Den1-Peru9615-00-PERU
Den1-FGA89-FRENCHGUYANA1989
Den1-Peru9581-00-PERU
Den1-BR90-BRASIL1990
Den1-Br01MR-BRASIL2001
Den1-233-BRASIL1997
Den1-111-BRASIL1997
Den1-409-BRASIL1997
Den1-Peru5198-97 DENGUE SEROTYPE 1
Den1-SingaporeS275-90-SINGAPORE1990
Den1-CAMBODIA-CAMBODIA1998
Den1-GZ80-CHINA1980
Den1-DJIBOUTI1998
Den1-MOCHIZUKI-JAPAN1953
Den1-16007-THAYLAND1964
Den1-A88-INDONESIA1988
Den1-clonePDK27
Den1-WestPac-NAURU1974
Den3-80-2-CHINA1980
Den3-H87-PHILIPPINES1956 DENGUE SEROTYPE 3
Den3-11069
Den2-IQT2913-PERU1996
Den2-539-96-PERU1996
Den2-IQT1797-PERU1995
Den2-131-MEXICO1992
Den2-VEN2-VENEZUELA1987
Den2-44-CHINA1989
Den2-NewGuineaC-NEWGUINEA1944
Den2-43-CHINA1987
Den2-04-CHINA1985
Den2-Mara4-VENEZUELA1990 DENGUE SEROTYPE 2
Den2-N.1409-JAMAICA1983
Den2-CookIslands-AUSTRALIA1997
Den2-16681-THAYLAND1964
Den2-ThNH54-93-THAYLAND1993
Den2-ThNHp36-93-THAYLAND1993
Den2-CO371-THAYLAND1995
Den2-K0008-THAYLAND1994
Den2-K0010-THAYLAND1994
Den4-TRI94-TRINIDAD1994
Den4-HON91-HONDURAS1991
Den4-MON94A-MONTSERRAT1994
Den4-TRI99-TRINIDAD1999
Den4-BAH98A-BAHAMAS1998
Den4-MON94B-MONTSERRAT1994
Den4-SUR94A-SURINAM1994
Den4-BDS93A-BARBADOS1993 DENGUE SEROTYPE 4
Den4-MEX91-MEXICO1991
Den4-JAM83-JAMAICA1983
Den4-TRI82A-TRINIDAD1982
Den4-814669-DOMINICA1981
Den4-SUR82D-SURINAM1982
Den4-JAMAICA1981
Den4-TRI84A-TRINIDAD1984
YellowFever
WestNile
JVE
0.1
Sequences that had no known genotype generate a full taxonomy tree, but did fully
were classified with respect to the group to differentiate the genotypes (data not shown).
which they were most similar. To verify the Even with this simple method, all unknowns
utility of this method, a phylogenetic tree were classified correctly into their genotypic
was built in parallel with the unknown and group (Figure 3 illustrates one example
characterized sequences. Bootstrap values result for each serotype, compared to
in the 220bp region were too low to known sequences).

Figure 3. Pair-wise analysis of four dengue strains detected by PCR amplification of 328 bp
E/NS1 products from patient sera. Samples are (a) 438VI03, DEN-1 AMERICAN-AFRICAN
genotype, (b) 13VI02, DEN-2 INDIAN genotype, (c) 1794F02, DEN-3 COSMOPOLITAN
genotype, and (d) 366VI03, INDONESIAN DEN-4 genotype
a) Dengue 1 - 438VI03 Nicaragua 2003
(a) d) Dengue 2 - 13VI02 India 2002
(b)
1100
1100
1000
1000
NW Score
NW Score
900 900
800 800
700
700
COS ASIAN ASIAN AMERICAN ASIAN AFRICAN
AMAF ASIAN MAL SP THAI
GENOTYPE 2 AMERICAN GENOTYPE 1
Genotype
Genotype
b) Dengue 3 - 1794F02 Dominican Republic 2002

(c) c) Dengue 4 - 366VI03 Indonesia 2003
(d)
1200 1200
1100 1100
1000
NW Score
1000
NW Score
900 900
800 800
700 700
SE Asia/SP THAILAND INDIAN AMERICAS INDONESIA SE ASIA MALAYSIA
SUBCONTINENT
Genotype
Genotype
Discussion governments and healthcare workers in

planning for potential outbreak situations.
The efficient worldwide control of dengue The advent of a simple and accurate
virus requires the definition of sources of method for diagnosis and surveillance could
epidemic viruses and the precise improve the establishment of these
identification of virus genotypes. A key programmes in developing countries
objective of DF and DHF surveillance affected by the disease, and in non-endemic
programmes is early detection of outbreaks areas where dengue is a travel-acquired
to permit the implementation of control infection.
measures. DHF outbreaks can be
The RT-nested PCR described here
anticipated by monitoring the emergence of
allows rapid direct diagnosis of acute
new genotypes in a region. The need for
dengue infection in laboratories without
surveillance is warranted, since air travellers
BSL-3 (bio-safety level 3) facilities.
can quickly move viruses from an endemic
area to a receptive area. Dengue virus Pair-wise comparison to classify
surveillance should be implemented in sequences has been used for enteroviruses
endemic and non-endemic areas to aid and potyvirus [26-28] . Multiple alignment and

rigorous phylogenetic methods are preferable Acknowledgements

to establish exact lineages of sequence strains
and discover recombination events. Pair-wise This investigation received financial support
comparisons can substitute if only a high level from the Instituto de Salud Carlos III (ISCIII)
of taxonomic classification is desired. Our through research project grants (MPY
method allows classification of dengue 1194/02 and C03/04). G. Palacios and WI
genotypes using the sequence of the 220bp Lipkin were supported by the Ellison Medical
region amplified by the PCR assay. The Foundation and the National Institutes of
advantage of pair-wise comparison for Health (AI 51292 and U54 AI57158-Lipkin).
classification is its speed, simplicity and C. Domingo was contracted by an agreement
availability. The database and classification between the Public Health Division of the
scheme provides a repository for sequences, Spanish Ministry of Health (DGSP-MSC) and
complementing efforts in tracking dengue the Instituto de Salud Carlos III (ISCIII) for the
genotype distribution. A website could be development of the Haemorrhagic Viral
deployed wherein clinical laboratories post Fevers Surveillance and Control Programme.
their sequences, location and circumstances The authors thank Dr J. Gascn, Dr
of isolation. This would allow rapid R. Lpez-Vlez and Dr S. Puente and the
centralized analysis detailing the genotype, many scientists who contributed dengue-
date and location of the most similar
infected patient samples for this work. The
sequence isolate in the database. New
authors are grateful to Dr J.E. Mejia for
genotypes could be rapidly identified by
assisting in manuscript preparation.
failure to relate them to a described group.
References
[1] Gubler DJ. Epidemic dengue/dengue [7] Lopez-Velez R, Perez-Casas C, Vorndam AV
haemorrhagic fever: a global public health and Rigau J. Dengue in Spanish travelers
problem in the 21st century. Dengue Bulletin, returning from the tropics. Eur J Clin
1997, 21. Microbiol Infect Dis, 1996, 15: 823-826.
[2] World Health Organization. Dengue and [8] Trofa AF, DeFraites RF, Smoak BL, Kanesa-
dengue haemorrhagic fever. Fact sheet, thasan N, King AD, Burrous JM, MacArthy
2002, No. 117. PO, Rossi C and Hoke CH, Jr. Dengue fever
in US military personnel in Haiti. JAMA,
[3] Clarke T. Dengue virus: break-bone fever. 1997, 277: 1546-1548.
Nature, 2002, 416: 672-674.
[9] Halstead SB. Pathogenesis of dengue:
[4] Lifson AR. Mosquitoes, models, and dengue. challenges to molecular biology. Science,
Lancet, 1996, 347: 1201-1202. 1988, 239: 476-481.
[5] Jelinek T. Dengue fever in international [10] Lanciotti RS, Lewis JG, Gubler DJ and Trent
travelers. Clin Infect Dis, 2000, 31: 144-147. DW. Molecular evolution and epidemiology
of dengue-3 viruses. J Gen Virol, 1994, 75
[6] Jelinek T, Dobler G, Holscher M, Loscher T
(Pt 1): 65-75.
and Nothdurft HD. Prevalence of infection
with dengue virus among international [11] Lanciotti RS, Gubler DJ and Trent DW.
travelers. Arch Intern Med, 1997, 157: Molecular evolution and phylogeny of
2367-2370. dengue-4 viruses. J Gen Virol, 1997, 78 (Pt
9): 2279-2284.

[12] Rico-Hesse R. Molecular evolution and [21] Snchez-Marino JPL-C, G.; Spiteri, I.;
distribution of dengue viruses type 1 and 2 Martn-Snchez, F. BUSSUB: A virtual
in nature. Virology, 1990, 174: 479-493. amplicon retrieval software. ISMB/ECCB
2004. Glasgow, 2004.
[13] Chungue E, Deubel V, Cassar O, Laille M
and Martin PM. Molecular epidemiology of [22] Thompson JD, Gibson TJ, Plewniak F,
dengue 3 viruses and genetic relatedness Jeanmougin F and Higgins DG. The
among dengue 3 strains isolated from CLUSTAL_X windows interface: flexible
patients with mild or severe form of dengue strategies for multiple sequence alignment
fever in French Polynesia. J Gen Virol, 1993, aided by quality analysis tools. Nucleic Acids
74 (Pt 12): 2765-2770. Res, 1997, 25: 4876-4882.
[14] Leitmeyer KC, Vaughn DW, Watts DM, [23] Kumar S, Tamura K, Jakobsen IB and Nei M.
Salas R, Villalobos I, de C, Ramos C and MEGA2: molecular evolutionary genetics
Rico-Hesse R. Dengue virus structural analysis software. Bioinformatics, 2001, 17:
differences that correlate with pathogenesis. 1244-1245.
J Virol, 1999, 73: 4738-4747.
[24] Needleman SB and Wunsch CD. A general
[15] Pandey BD, Morita K, Hasebe F, Parquet method applicable to the search for
MC and Igarashi A. Molecular evolution, similarities in the amino acid sequence of
distribution and genetic relationship among two proteins. J Mol Biol, 1970, 48: 443-453.
the dengue 2 viruses isolated from different
[25] Rice P, Longden I and Bleasby A. EMBOSS:
clinical severity. Southeast Asian J Trop Med
the European Molecular Biology Open
Public Health, 2000, 31: 266-272.
Software Suite. Trends Genet, 2000, 16:
[16] Rico-Hesse R, Harrison LM, Salas RA, Tovar 276-277.
D, Nisalak A, Ramos C, Boshell J, de Mesa
[26] Palacios G, Casas I, Tenorio A and Freire C.
MT, Nogueira RM and da Rosa AT. Origins
Molecular identification of enterovirus by
of dengue type 2 viruses associated with
analyzing a partial VP1 genomic region with
increased pathogenicity in the Americas.
different methods. J Clin Microbiol, 2002,
Virology, 1997, 230: 244-251.
40: 182-192.
[17] Rico-Hesse R. Microevolution and virulence
[27] Oberste MS, Maher K, Kilpatrick DR,
of dengue viruses. Adv Virus Res, 2003, 59:
Flemister MR, Brown BA and Pallansch MA.
315-341.
Typing of human enteroviruses by partial
[18] Gubler DJ, Suharyono W, Lubis I, Eram S sequencing of VP1. J Clin Microbiol, 1999,
and Gunarso S. Epidemic dengue 3 in 37: 1288-1293.
central Java, associated with low viremia in
[28] Ward CW, McKern NM, Frenkel MJ and
man. Am J Trop Med Hyg, 1981, 30: 1094-
Shukla DD. Sequence data as the major
1099.
criterion for potyvirus classification. Arch
[19] World Health Organization. Clinical Virol Suppl, 1992, 5: 283-297.
diagnosis. 2 nd edition. Geneva: WHO, 1997.
[20] Twiddy SS, Farrar JJ, Vinh Chau N, Wills B,
Gould EA, Gritsun T, Lloyd G and Holmes
EC. Phylogenetic relationships and
differential selection pressures among
genotypes of dengue-2 virus. Virology, 2002,
298: 63-72.

Clinical and Laboratory Observations Associated with
the 2000 Dengue Outbreak in Dhaka, Bangladesh
Monira Pervin* #, Shahina Tabassum**, Md. Mobarak Ali***,
Kazi Zulfiquer Mamun* and Md. Nazrul Islam**
*Department of Virology, Dhaka Medical College, Dhaka, Bangladesh

**Department of Virology, Bangabandhu Sheikh Mujib Medical University, Shahbag, Dhaka, Bangladesh
***Shahid Suhrawardi Hospital, Dhaka Bangladesh
Abstract
A large outbreak of dengue fever (DF)/dengue haemorrhagic fever (DHF) occurred in Dhaka city,
Bangladesh, in 2000. The present study was conducted on 105 clinically-suspected cases of DF to
confirm the diagnosis, determine the major clinical manifestations and correlate the haemorrhagic
manifestations with different dengue serotypes circulating during the outbreak. A total of 97 cases were
positive for anti-dengue IgM and were considered as recent dengue infection; 52.6% patients had
secondary and 47.4% had primary dengue infection. According to WHO case-definition, 79 cases were
classified as DF, 17 as DHF and 1 as DSS. Among the 18 DHF/DSS cases, 14 had secondary and 4 had
primary type of antibody response. The mean age of the dengue patients was 29.212.9 years and most
of them (37.1%) belonged to the 20-29-year age group. All the clinically-suspected patients had fever
ranging from 100-104 F, but the secondary dengue fever patients had higher (101.61.4 F) mean
body temperature. Common complaints included myalgia (84.5%), headache (82.5%), arthralgia (68.0%),
lethargy (80.4%) and retro-orbital pain (49.5%). Rash, especially maculopapular type, was significantly
higher in primary infection (P<0.01), while hepatomegaly was higher in secondary infections (P<0.01).
Haemorrhagic manifestations were observed both in primary and secondary dengue patients and were
mostly associated with serotypes DEN-3. In 22.7% of cases, the platelet count was <1x105/mm3 and was
associated more with secondary infection. Haematocrit more than 45% was found in 16.5% patients and
a significantly higher association was detected among the secondary dengue fever patients (P=0.02).
Although all 4 dengue serotypes were prevalent during the outbreak, DEN-3 was the predominant
serotype (70.5%) and was associated with more severe clinical manifestations.
Keywords: Primary dengue fever, secondary dengue fever, dengue haemorrhagic fever, serotypes, Bangladesh.
#
E-mail: msakib@dhaka.net

Clinical and Laboratory Observations during the 2000 Dengue Outbreak in Dhaka
Introduction Materials and methods

Dengue was first reported in Bangladesh in The study was conducted on clinically
1964 and the outbreak came to be known suspected cases of dengue fever attending
as Dacca Fever [1] . For a long period after the inpatient and outpatient departments of
that, dengue cases remained undetected. A Medicine and Paediatrics, Bangabandhu
few cases were reported in 1999 before a Sheikh Mujib Medical University (BSMMU)
large outbreak occurred in 2000, during Hospital, and patients admitted at the Dhaka
Medical College Hospital during June
which 5,551 cases and 93 dengue-related
December 2000. Some patients seeking
deaths were reported[2,3] . Cases reported
diagnostic facilities at the Department of
during the outbreaks were mostly diagnosed
Virology, BSMMU, were also included in the
clinically, except for a few serologically study.
diagnosed cases[4] .
According to specific inclusion criteria,
Several studies have observed that 105 clinically suspected patients with fever
sequential or secondary dengue infections (presenting within 5 days of onset with body
are more likely to produce a severe form of temperature above 100 F at the time of
the disease. DHF and DSS occur mostly in blood sample collection) and fulfilling the
persons with pre-existing dengue antibodies case-definition criteria of dengue fever (DF)
acquired actively or passively[5-7] . and dengue haemorrhagic fever (DHF) of
WHO[10] were enrolled in the study. The
Detection of anti-dengue IgM indicates
exclusion criteria defined cases of febrile
the diagnosis of recent dengue infection in illness of more than 5 days and/or with
both primary and secondary cases. Anti- definite sources of infection, chronic
dengue IgM develops earlier than IgG in illnesses like tuberculosis, bronchial asthma,
primary infection and is usually detectable congenital heart disease, renal failure,
by day 5 of illness and wanes after 1-2 history of bleeding tendency since birth and
months[8] . The ratio of IgM and IgG patients who refused to give two blood
antibodies determined by ELISA is useful for samples.
distinguishing primary and secondary
Clinical data were collected through
infections[8,9] . In primary infection, the
interviewing the patients or their attendants
IgM/IgG ratio generally exceeds 1.8 OD
and meticulous physical examination of the
units in acute or convalescent sera[8] . patients conducted by a doctor. The
Detection of an early and excess of IgG tourniquet test was performed in all patients
characterizes secondary infection. In the by conventional method[11] . Hepatomegaly
present study, dengue patients were and ascites were ascertained by physical
classified into primary and secondary cases examination and on reports of
on the basis of this concept and it was ultrasonography and X-rays. 5 ml of venous
carried out to confirm the clinical diagnosis, blood was collected aseptically from all
correlate the clinical manifestations with patients during both the early and
laboratory findings and establish the convalescent stages of fever irrespective of
association of haemorrhagic manifestations age and sex. This was processed and stored
with different dengue virus serotypes appropriately for virus isolation, antibody
responsible for the outbreak of 2000. assay, platelet count and haematocrit

estimation. Detection of IgM and IgG anti- anti-dengue IgM or IgM and IgG antibodies
dengue antibody, isolation of dengue viruses and 38 (36.2%) patients developed only
by mosquito inoculation technique and anti-dengue IgG in acute stage by ELISA.
serotyping of the isolated viruses were done When these patients were tested in the
as described previously[12,13] . Patients were convalescent stage, 21 (20%) were positive
classified into DF and DHF or DSS for only IgM and 76 (72.4%) were positive
according to WHO, 1997[10] . for both IgM and IgG antibodies. Eight
patients did not develop anti-dengue IgM
Statistical analysis and IgG in either of the specimens (Table 1)
The numerical data obtained from the study and were also negative on virus isolation.
were analysed and the significance of the Thus, a total of 97 (92.4%) cases were
difference was estimated by using statistical diagnosed serologically as current dengue
methods. The data were expressed in infection, while 8 (9.8%) cases were
frequency, percentage, mean and standard considered as non-dengue febrile illness and
deviation as applicable. The comparison were excluded from further analysis. Of the
between groups was done by Students t 97 dengue patients, 46 (47.4%) had primary
test and Chi square and Z test as and 51 (52.6%) had secondary infection
applicable. All data were analysed by using depending on anti-dengue IgM and IgG
the computer-based SPSS programme. antibody level of acute and convalescent
Probability less than 0.05 was considered as stage sera (Table 2). Among them, 17
significant. patients developed DHF and one developed
DSS. Of the 18 DHF/DSS patients, 14 had
Results secondary type of infection and 4 had
primary infection.
Of the 105 clinically suspected dengue
patients, 39 (37.1%) were positive for either
Table 1. IgM and IgG dengue antibody positive cases by ELISA
Only IgM Only IgG Both IgM and IgM and IgG
Time of serum antibody antibody IgG antibody antibody Total
collection positive cases positive cases positive cases negative cases n (%)
n (%) n (%) n (%) n (%)
Acute stage serum 21 (20.0)* 38 (36.2) 18 (17.1)* 28 (26.7) 105 (100)
(within 5 days of
fever)
Convalescence 21 (20.0)** 00 (0.0) 76 (72.4)** 8 (7.6)*** 105 (100)
stage serum
(within 14-21
days of fever)
* Total number of dengue infection cases detected in acute stage serum (21+18) = 39 (37.1%).
**Total number of dengue infected cases detected in convalescence serum (21+76) = 97 (92.4%).
*** Non-dengue febrile illness cases = 8 (7.6%).
Figures in parenthesis indicate percentage

Table 2. Age distribution of dengue patients by primary and secondary dengue infection
Primary infection Secondary infection Total

Age in years
n (%) n (%) n
<10 3 (42.9) 4 (57.1) 7
10-19 4 (44.4) 5 (55.6) 9
20-29 20 (55.6) 16 (44.4) 36
30-39 11 (45.8) 13 (54.2) 24
40-49 7 (53.8) 6 (46.2) 13
>50 1 (12.5) 7 (87.5) 8
Total 46 (47.4) 51 (52.6) 97 (100.0)
*MeanSD 27.511.0 30.714.4 29.212.9
P value 0.214 (unpaired students t test)
* Mean of age
The involvement of all age groups, Dengue viruses were isolated from 44
especially an adult predominance, was of the 97 dengue patients. The rate of
observed. The mean age of the dengue dengue virus isolation was significantly
patients was 29.212.9 years and most higher (68.2% vs 31.2%) among primary
belonged to the 20-29-year age group. The than secondary infection patients (P=0.018).
mean age of primary dengue fever patients The isolation rate decreased gradually with
was 27.511.7 years and that of secondary the increasing duration of fever (see Figure).
patients was 30.714.4 years.
Figure. Isolation rate of dengue virus between primary and secondary dengue patients by
day of fever
4/4
100 9/10
5/6
Virus isolation rate (%)
8/11
80
3/5
60
40 1/3
5/18 4/14 4/15
20
1/1
0
1st 2nd 3rd 4th 5th
Days of fever
Primary Secondary
(P=0.018)
P value reached from Z test

The distribution of clinical manifestations The most common presenting sign was rash,
in dengue cases is given in Table 3. The especially maculopapular type, and its
mean body temperature of the dengue association was significantly higher with
patients was 101.51.4 F but there was no primary DF cases (P=0.016). Ascitis was
significant difference in the mean body observed in 5 (9.8%) cases; all had
temperature between primary and secondary secondary DF. Hepatomegaly was present in
DF patients. Other common symptoms 13 (13.4%) patients and its association was
included myalgia (84.5%), headache (82.5%), significantly higher (P=0.002) in secondary
arthralgia (68%), lethargy (80.4%) and retro- DF patients. Abdominal pain (6.2%) and
orbital pain (49.5%). Patients with primary lymphadenopathy (4.1%) was less frequent
dengue infection presented more commonly among our study patients though abdominal
with headache, arthralgia and retro-orbital pain was more common (7.8%) in secondary
pain, whereas lethargy was commonly DF patients.
associated with secondary dengue infection.
Table 3. Distribution of clinical manifestations in dengue patients
Types of dengue
Secondary Total
Clinical characteristics Primary infection P-value
infection n=97
n=46
n=51
Mean temperature (F) 101.41.4 101.61.4 101.51.4
Headache 41 (89.1) 39 (76.5) 80 (82.5)
Arthralgia 34 (73.9) 32 (62.7) 66 (68.0)
Retro-orbital pain 25 (54.3) 23 (45.1) 48 (49.5)
Myalgia 39 (84.8) 43(84.3) 82 (84.5)
Lethargy 36 (78.3) 42 (82.4) 78 (80.4)
Rash 19 (41.3) 13 (25.5) 32 (32.9)
Maculopapular 17 (36.9) 8 (15.7) 25 (25.8) 0.016S
Petechial 2 (4.3) 5 (9.8) 7 (7.2)
Anorexia, nausea and 16 (34.8) 19 (37.3) 35 (36.1)
vomiting
Abdominal pain 2 (4.3) 4 (7.8) 6 (6.2)
Ascitis 0 (0.0) 5 (9.8) 5 (5.1)
Enlarged lymph node 2 (4.3) 2 (3.9) 4 (4.1)
Hepatomegally 1 (2.2) 12 (23.5) 13 (13.4) 0.002S
P-value reached from chi-square analysis

Haemorrhagic manifestations in primary (12.4%), gum bleeding 12 (12.4%) and

and secondary dengue infections are haematemasis/malaena 11 (11.3%) were the
indicated in Table 4. The most common other bleeding manifestations. Besides these,
sign of bleeding manifestation, i.e. a positive conjunctival bleeding, haematuria and per
tourniquet test, was observed in 18 (18.6%) rectal bleeding also occurred in a small
patients. Petechiae 15 (15.5%), purpura 12 number of patients.
Table 4. Haemorrhagic manifestations among primary and secondary dengue cases
Types of dengue
Secondary Total
Clinical characteristics Primary infection
infection n=97
n=46
n=51
Positive tourniquet test 7 (15.2) 11 (21.6) 18 (18.6)
Petechiae 8 (17.4) 7 (13.7) 15 (15.5)
Purpura 5 (10.9) 7 (13.7) 12(12.4)
Epistaxis 1 (2.2) 2 (3.9) 3 (3.1)
Haematemesis/melaena 8 (17.4) 3 (5.9) 11 (11.3)
Gum bleeding 5 (10.9) 7 (13.7) 12 (12.4)
Per vaginal bleeding 2 (4.3) 1 (2.0) 3 (3.1)
Conjunctival bleeding 1 (2.2) 1 (1.0) 2 (2.0)
Haematuria 1 (2.2) 0 (0.0) 1 (1.0)
Per rectal bleeding 0 (0.0) 2 (3.9) 2 (2.0)

The haematological features in primary association of >45% haematocrit level was

and secondary dengue infections are given detected among secondary DF patients
in Table 5. Platelet counts of <1x10 5/mm3 (P=0.02).
was detected in 22 (22.7%) patients and
Haemorrhagic manifestations were
was more frequently 16 (31.4%) associated
mostly associated with DEN-3 and DEN-4
with secondary DF (Table 5). Haematocrit infections; only one patient with DEN-1
value of >45% was observed in 16 (16.5%) infection had per rectal bleeding (Table 6).
patients and a significantly higher

Table 5. Haematological features in primary and secondary dengue infection
Types of dengue infection

Secondary Total
Haematology Primary infection P-value
infection n=97
n=46
n=51
Platelets/cu mm
<1105 6(13.1) 16(31.4) 22(22.7) 0.059
>1105 40(86.9) 35(68.6) 75(77.3)
Haematocrit
>45% 3(6.5) 13(25.5) 16(16.5) 0.020S
<45% 43(93.5) 38(74.5) 81(83.5)
P-value reached from chi-square analysis
Table 6. Haemorrhagic manifestations in different serotypes of dengue virus infections
Serotypes of dengue viruses

Clinical characteristics DEN-1 DEN-2 DEN-3 DEN-4
(n=6) (n=3) (n=31) (n=4)
Positive tourniquet test 0 (0.0) 0 (0.0) 6 (19.4) 3 (75.0)
Petechiae 0 (0.0) 0 (0.0) 1 (3.2) 1 (25.0)
Purpura 0 (0.0) 0 (0.0) 3 (9.7) 1 (25.0)
Epistaxis 0 (0.0) 0 (0.0) 1 (3.2) 0 (0.0)
Haematemesis/melaena 0 (0.0) 0 (0.0) 4 (12.9) 1 (25.0)
Gum bleeding 0 (0.0) 1 (33.3) 4 (12.9) 1 (25.0)
Haematuria 0 (0.0) 0 (0.0) 1 (3.2) 1 (25.0)
Per rectal bleeding 1 (16.7) 0 (0.0) 0 (0.0) 1 (25.0)
Discussion dengue infection. The presence of

secondary cases indicated that DF was
The secondary dengue infection is usually present in the area and perhaps a low-grade
associated with more severe manifestations transmission was continuing during the
than the primary infection. In our study, out previous years. Thus, it may be speculated
of the 97 dengue cases, 46 (47.4%) had that a considerable proportion of cases,
primary and 51 (52.6%) had secondary which were diagnosed as viral flu in the

past, may have been due to dengue virus DHF in Cambodian[20] and Thai children[21] ,
infection. On serological test in acute stage, while it was less frequent in patients in our
only 39 (37.1%) cases were found to be study. In the present study, statistically
positive for dengue antibody. However, significant (P=0.002) hepatomegaly (13.4%)
when the serum from the patients was in secondary dengue infection was noted.
tested again in the convalescence stage, 97 Hepatomegaly was also a common clinical
(92.4%) cases were found to be positive, finding in several other studies[22-24] . Acute
indicating that they had recent dengue virus abdominal pain, which is considered as an
infection (Table 1). Thus, the detection of a early sign of shock in DF/DHF[10] , was
significant number of cases could have been present in only 6 (6.2%) patients in the
missed if the tests were not done again at present study. This is similar to a study from
the convalescence stage. A second test Lucknow, India, where abdominal pain was
should therefore be considered at the found in only 5% of cases in an epidemic of
convalescence stage to delineate the actual DHF[19] . In some studies, acute abdominal
numbers of infection due to dengue. pain was strongly correlated with DHF and
Our study indicates the involvement of DSS[10,24,25] . In the present study, ascitis, a
all age groups of the population with adult sign of plasma leakage, was present in only
predominance. This finding is not consistent 5 (9.8%) secondary infection cases and was
with the epidemiological data from other in agreement with the findings of other
endemic countries in Asia where young studies[25,26] .
children were affected[14,15] . However, adults The positive tourniquet test, which
may also be the major victims of DHF as reflects capillary fragility and is used as a
reported in different epidemics where guiding test for detecting dengue illness[27] ,
dengue was endemic[16,17,18] . No significant was found in only 18.6% cases in the
age or sex difference among patients was present study. There was no statistically
observed between primary and secondary significant difference in test positivity
infections in the present study. Similar between primary and secondary DF patients
observations were also reported from an although it was more frequently positive in
outbreak in Fiji[11] . secondary DF. In most studies, comparing
Anorexia, nausea, vomiting, abdominal DHF with classic DF, either a higher
pain and ascitis were associated more with incidence of tourniquet test positivity in
secondary than primary dengue infections DHF[23,26,28] or no difference between the
(Table 3). In the present study, a statistically two groups [24,29] , was reported. These
significant (P=0.01) association of rash variable findings are probably because the
(32.9%) among primary dengue infection pathogenesis of tourniquet positivity and
cases was demonstrated. The predominant other bleeding manifestations in dengue
type of rash in primary infection was cases are different. Moreover, the defects
macular or maculopapular whereas which lead to increase in capillary fragility
petechial rash was found frequently in may also predispose to the development of
secondary infection. Other studies also shock, thus indicating a better correlation of
reported similar association of rash in the test to shock rather than to haemorrhage.
DF[11,19] . Petechiae were frequently found in

In our study, platelet counts of there is a wide variation in the presence of

<1105/mm3 were observed among 22 various symptoms, which may misguide the
patients and were associated more with physician regarding the severity of the
secondary than primary infections (Table 4). disease. Therefore, the use of clinical case-
Lack of correlation between a moderately definition results in inaccuracies. Thus, in
low platelet count and bleeding order to identify the cases accurately, one
manifestation in dengue patients has been should take the help of a simple and cheap
noted by other investigators [8]. The bleeding diagnostic test that is able to diagnose
in dengue is probably due to other factors accurately in the laboratory. In this context
with or without the additive effect of ELISA is clearly very promising for the
thrombocytopenia. Haemoconcentration, the confirmation of clinical diagnosis and to
evidence of plasma leakage and shock, was differentiate between primary and
observed in 16 patients with secondary secondary infections. This will guide the
infection and the association was statistically clinician to take prompt and meticulous
significant (P=0.02). Increased haematocrit clinical management and early hospitalization
was found in 95.5% patients with shock in of severe cases. Indeed, it is hoped that
contrast to 31.7% cases without shock[21] . accurate laboratory diagnosis will not only
reduce the morbidity and mortality but will
In the present study, the virus isolation
also reduce the economic burden of the
rate was significantly higher in patients with
patient and the government.
primary (68.2%) than secondary (31.2%)
infections (P=0.018). Similar results have
been reported in a study from Fiji[11] . In Acknowledgements
Bantal, Indonesia, the isolation rate was
100% among patients with primary dengue We express our sincere thanks to
infection and 57% with secondary dengue Dr Vorndam Vance, CDC Dengue Lab, San
infection[30] . However, a study from Juan, Puerto Rico, USA, and Dr Ichiro
Thailand reported a high rate of virus Kurane, Arbovirus Research Laboratory,
isolation in both primary (98%) as well as NIID, Japan, for their kind support in
secondary (93%) infections[31] . The reason providing cell line and anti-dengue
for a higher isolation rate in secondary monoclonal antibodies. We are also grateful
infection was probably because blood to the patients who participated in the study.
samples were collected at an early febrile We would like to thank Mr Tauhid Uddin
phase (within 72 hours). In our study, the Ahmed, Ex-PSO, IEDCR, Bangladesh, and
isolation rate was also high in both primary Noor-e -Jannat for providing mosquito
(100%) and secondary (68%) infections in colony and rearing techniques. We would
early febrile period, i.e. on day 1 of fever. also like to thank Dr Bijon Kumar Sil, Ex-
Thereafter, the rate of isolation was always Senior Scientific Officer, Bangladesh
higher in primary than in secondary dengue Livestock Research Institute, Savar, Dhaka,
infection in the subsequent days of fever Bangladesh. We also express our gratitude
(Figure). to the Department of Medicine and
Paediatrics, BSMMU, and Dhaka Medical
In conclusion, the clinical diagnosis is College, Bangladesh, for their support in the
not very reliable in dengue infection as study.

References
[1] Aziz MA, Gorham JR and Gregg MB. Dacca [9] Kuno G, Gomez I and Gubler DJ. An ELISA
Fever- An outbreak of dengue. Pakistan procedure for the diagnosis of dengue
Journal of Medical Research, 1967, 6: 83-92. infections. Journal of Virological Methods,
1991, 33: 101-113.
[2] Amin MMM, Hussain AMZ, Nahar K,
Chowdhury IA, Murshed M and Chowdhury [10] World Health Organization. Dengue
SA. Sero-diagnosis of dengue infections in haemorrhagic fever diagnosis, treatment and
four metropolitan cities of Bangladesh. control, Geneva: WHO, 1997.
Dengue Bulletin, 2000, 24: 34-41.
[11] Kuberski T, Rosen L, Reed D and Mataika J.
[3] Yunus EB, Bangali AM, Mahmood MAH, Clinical and laboratory observations on
Rahman MM, Chowdhury AR and Talukder patients with primary and secondary dengue
RK. Dengue outbreak 2000 in Bangladesh: type I infections with haemorrhagic
From speculation to reality and exercise. manifestations in Fiji. The American Journal
Dengue Bulletin, 2001, 25: 15-20. of Tropical Medicine and Hygiene, 1977,
26(4): 775-783.
[4] Ahmed FU, Mahmood CB, Sharma JD,
Hoque SM, Zaman R and Hasan MH. [12] Pervin M, Tabassum S, Sil BK and Islam MN.
Dengue and dengue haemorrhagic fever in Isolation and serotyping of dengue viruses
children during the 2000 outbreak in by mosquito inoculation and cell culture
Chittagong, Bangladesh. Dengue Bulletin, technique: An experience in Bangladesh.
2001, 25: 33-39. Dengue Bulletin, 2003, 27: 81-90.
[5] World Health Organization. Comprehensive [13] Pervin M, Tabassum S and Islam MN.
guidelines for prevention and control of Isolation and serotyping of dengue viruses
dengue and dengue haemorrhagic fever. by mosquito inoculation technique from
New Delhi: WHO/SEARO Regional clinically suspected cases of dengue fever.
Publication, No. 29, 1999. Bangladesh Medical Research Bulletin, 2002,
28(3): 104-111.
[6] Sangkawibha S, Rojanasuphot S, Ahandrik S,
Viriyapongse S, Jatanasen S, Salitul V, [14] Guzman MG, Kouri G, Vazqez S, Rasario D,
Panthumachinda B and Halstead SB. Risk Bravo J and Valdes L. DHF epidemics in
factors in dengue shock syndrome. A Cuba, 1981 and 1997: some interesting
prospective epidemiologic study in Rayong, observations. Dengue Bulletin, 1999, 23:
Thailand. The American Journal of 39-43.
Epidemiology, 1984, 120: 653-669.
[15] Rojanapithayakorn W. Dengue haemorrhagic
[7] Halstead SB. Dengue haemorrhagic fever fever in Thailand. Dengue Bulletin, 1998,
a public health problem and a field of 22: 60-68.
research. Bulletin of the World Health [16] Lam SK. Epidemiology of DF/DHF in
Organization, 1980, 58(1): 1-21.
Malaysia-status report, 1995. Dengue
[8] Innis BL, Nisalak A, Nimmannitya S, Bulletin, 1996, 20: 62-65.
Kusalerdchariya S, Chongswasdi V, [17] Goh KT. Changing epidemiology of dengue
Suntayakorn S, Puttisri P and Hoke CH. An in Singapore. The Lancet, 1995, 346: 1098.
enzyme-linked immunosorbent assay to
characterize dengue infections where [18] Vitarana T, Jayakuru WS and Withane N.
dengue and Japanese encephalitis co- Historical account of dengue haemorrhgic
circulate. The American Journal of Tropical fever in Sri Lanka. Dengue Bulletin, 1997,
Medicine and Hygiene, 1989, 40: 418-427. 21: 117-118.

[19] Agarwal R, Kapoor S, Nagar R, Misra A, [26] Kabra SK, Jain Y, Pandey RM, Madhulika,
Tandon R, Mathur A, Misra AK, Srivastava Singhal T, Tripathi P, Broor S, Seth P and
KL and Chaturvedi UC. A clinical study of Seth V. Dengue haemorrhagic fever in
the patients with dengue haemorrhagic fever children in the 1996 Delhi epidemic, Trans
during the epidemic of 1996 at Lucknow, Royal Soc Trop Med and Hyg, 1999, 93:
India. South-East Asian Journal of Tropical 294-298.
Medicine and Public Health, 1999, 30(4):
[27] Kalayanarooj S, Nimmannitya S,
735-740.
Suntayakorn S, Vaughn DW, Nisalak A,
[20] Lan TN, Hung TN, Ha QD, Phuong MTB, Green S, Chansiriwongs V, Rothman A and
Lien LB, Tuan LA, Huong VTQ, Hieu LTM, Ennis FA. Can doctors make an accurate
Tran TN, Cam LT and Taun NA. Treatments diagnosis of dengue infections at an early
of dengue haemorrhagic fever at childrens stage? Dengue Bulletin, 1999, 23: 1-9.
hospital no. 1, Ho Chi Minh City, Viet Nam
[28] Hayes CG, Manaloto CR, Gonzales A and
1991-1996. Dengue Bulletin, 1998, 22: 99-
Ranoa CP. Dengue infections in the
105.
Philippines: clinical and virological findings
[21] Cohen SN and Halstead SB. Shock in 517 hospitalized patients. American
associated with dengue infection I. Clinical Journal of Tropical Medicine and Hygiene,
and physiological manifestations of dengue 1988, 39: 110-116.
haemorrhagic fever in Thailand, 1964.
[29] Kalayanarooj S, Vaughn DW, Nimmannitya
Journal of Pediatrics, 1966, 68: 448-456.
S, Green S, Suntayakorn S, Kunentrasai N,
[22] Ming KC, Thein S, Thaung U, Oo T, Myint Viramitrachai W, Ratanachu-eke S,
KS, Shwe TN, Halstead SB and Diwan AR. Kiatpolpoj S, Innis BL, Rothman AL, Nisalak
Clinical and laboratory studies on A and Ennis FA. Early clinical and laboratory
haemorrhagic fever in Burma 1970-72. indicators of acute dengue illness. The
Bulletin of the World Health Organization, Journal of Infectious Diseases, 1997, 176:
1974, 51: 227-235. 313-321.
[23] Nimmannitya S, Halstead SB, Cohen SN and [30] Gubler DJ, Suharyono S, Lubis I, Eram S and
Margiotta MR. Dengue and Chikungunya Gunarso S. Epidemic dengue 3 in Central
virus infection in man in Thailand, 1962- Java, associated with low viremia in man.
1964. The American Journal of Tropical American Journal of Tropical Medicine and
Medicine and Hygiene, 1969, 18(6): 954- Hygiene, 1981, 30(5): 1094-1099.
971.
[31] Vaughn DW, Green S, Kalayanarooj S, Innis
[24] Aggarwal A, Chandra J, Aneja S, Patwari AK BL, Nimmannitya S, Suntayakorn S,
and Dutta AK. An epidemic of dengue Rothman AL, Ennis FA and Nisalak A.
haemorrhagic fever and dengue shock Dengue in early febrile phase: viremia and
syndrome in children in Delhi. Indian antibody responses. The Journal of
Pediatrics, 1998, 35: 727-732. Infectious Diseases, 1997, 176: 322-330.
[25] Guzman MG, Kouri G, Morior L and
Fernandez A. A study of fatal haemorrhagic
dengue cases in Cuba 1981. Bulletin of Pan
American Health Organization, 1984, 18:
213-220.

Current Status of Dengue Diagnosis at the
Center for Disease Control, Taiwan
Pei-Yun Shu, Shu-Fen Chang, Yi-Yun Yueh, Ling Chow, Li-Jung Chien,
Yu-Chung Kuo, Chien-Lin Su, Tsai-Ling Liao, Ting-Hsiang Lin
and Jyh-Hsiung Huang#
Center for Research and Diagnostics, Center for Disease Control, Department of Health,
161, Kun-Yang Street, Taipei, Taiwan
Abstract
A national-level diagnostic laboratory has been set up in Taiwan for routine diagnosis of reported cases of
dengue fever (DF)/dengue haemorrhagic fever (DHF), Japanese encephalitis (JE) and yellow fever (YF).
The facilities include serological diagnosis, virus isolation by cell culture, molecular diagnosis and
molecular tools for epidemiological investigations. To detect and differentiate dengue, JE and YF virus
infections, a differential diagnostic system has been developed. For acute-phase sera, virus isolation by
cell culture and real-time one-step reverse transcription-polymerase chain reaction (RT-PCR) has been
established. For all of the serum samples reported, serological diagnosis of specific antibodies based on
envelope and membrane (E/M)-specific capture IgM and IgG enzyme-linked immunosorbent assay
(ELISA) are performed. In this report, a case study from Taiwan has been presented with the analysis of
959 serum samples (including some paired sera) collected between day 1-30 of illness from 799
confirmed dengue cases reported in 2002. The results demonstrated that 94.5% of acute-phase serum
samples of confirmed dengue cases could be identified as positive or probable with the combined use of
real-time one-step RT-PCR and E/M-specific capture IgM and IgG ELISA. Furthermore, a nonstructural
protein NS1 serotype-specific indirect IgG ELISA has been developed and used to analyse dengue NS1-
specific IgG antibodies. Both E/M-specific capture IgM and IgG ELISA and the NS1 serotype-specific
indirect IgG ELISA have been used to detect and differentiate primary and secondary dengue virus
infections. In addition, the NS1 serotype-specific indirect IgG ELISA has the potential of replacing the
plaque-reduction neutralization test (PRNT) and is being used for a large-scale seroepidemiological study.
Keywords: Dengue virus, virus isolation, real-time one-step RT-PCR, E/M-specific capture IgM and IgG ELISA, NS1
serotype-specific indirect IgG ELISA.
#
E-mail: jhhuang@cdc.gov.tw; Tel.: 886-2-26531374, Fax: 886-2-27883992

Dengue Diagnosis in CDC, Taiwan
Introduction haemagglutination inhibition (HI) test and

E/M-specific capture IgM and IgG ELISA[8]
The dengue viruses cause a broad spectrum are the two most commonly used serological
of illness ranging from inapparent infection, techniques for the routine diagnosis of
mild undifferentiated fever and classic flavivirus infection. The serodiagnosis of
dengue fever to a more severe form, dengue flavivirus is rather complicated due to the
haemorrhagic fever/dengue shock syndrome high cross-reactivity of IgG antibodies to
(DHF/DSS), resulting in high morbidity and homologous and heterologous viruses. We
mortality. The diagnosis of dengue virus have attempted to set up an ELISA system
infection based on clinical syndromes is not that can be easily and reliably used to detect
reliable, and a confirmation of the infection and differentiate various flavivirus infections.
should rely on laboratory diagnosis with the To accomplish this goal, three different
detection of the specific virus, viral antigen, forms of ELISA were developed including:
genomic sequence and/or antibodies. (i) E/M-specific capture IgM and IgG ELISA;
A rapid, simple, sensitive and specific (ii) E/M-specific antigen-coated indirect IgM
assay system to detect the virus in the acute- and IgG ELISA; and (iii) NS1 serotype-
phase serum is essential to improve the specific indirect IgG ELISA[9-11] .
clinical treatment, etiological investigation
and disease control of dengue virus Case study of 2002 outbreak of
infection. Among the various assays for virus DEN-2 in southern Taiwan
detection, virus isolation by cell culture and
We present here a case study of the analysis
dengue virus antigen detection by ELISA[1,2]
of biological material obtained during a
suffer from some disadvantages while the
DEN-2 outbreak in 2002 in southern
former needs a longer time, the latter has
Taiwan, by utilizing the facilities available in
low sensitivity. However, recent advances in
the national diagnostic laboratory.
molecular diagnosis have demonstrated that
various RT-PCR protocols can be reliably A major DEN-2 epidemic occurred in
used to detect the viral genomic sequence southern Taiwan, affecting Kaohsiung city,
with high sensitivity and specificity. More Kaohsiung county and Pingtung county
recently, several investigators have reported between October 2001 and December 2002,
real-time RT-PCR assays for the detection of with more than 5,000 confirmed cases.
dengue virus in acute-phase serum Among these, 227 cases were classified as of
samples[3-7]. The real-time RT-PCR assay has DHF with 21 deaths. This outbreak was a
many advantages over the conventional RT- repeat of the 1987-1988 DEN-1 epidemic in
PCR methods, which include rapidity, many aspects[11,12] . In this report, we present
quantitative measurement, lower the results of a total of 959 acute- and
contamination rate, higher sensitivity, higher convalescent-phase sera collected from 799
specificity and easy standardization. confirmed dengue patients reported to the
Kun-Yang office of the Center for Disease
For convalescent sera, detection of
Control (CDC), Taiwan, 2002.
specific IgM and IgG antibodies based on

Materials and methods performed as previously described[7,9] . The

culture supernatants were used as the
source of E/M and NS1 antigens for ELISA.
Human serum samples The control antigen was prepared by the
The serum samples used in this study were same procedure from Vero cells culture
collected from the confirmed cases of without viral infection.
dengue patients reported to the Arbovirus
Laboratory in the Kun-Yang office, CDC, One-Step SYBR Green I Real-Time
Department of Health, 2002. A total of 959 RT-PCR
acute- and convalescent-phase sera
collected from 799 confirmed dengue One-step SYBR Green I real-time RT-PCR
patients were analysed. Most of these serum for dengue virus was performed in the
samples were from the major DEN-2 Mx4000 TM quantitative PCR system
outbreak, together with a few serum (Stratagene) as recently described[7] . Briefly,
samples from imported cases contracted a set of flavivirus- (in the NS5 gene region),
during travel to the neighbouring South-East dengue- and serotype-specific primer pairs
Asian countries. (in the core gene region) was selected and
used for analysis. To assure the specificity of
Case definitions amplicons produced from SYBR Green I
real-time RT-PCR in daily routine screening,
A confirmed case of dengue virus infection both flavivirus- and dengue -specific primer
was defined as febrile illness associated pairs were used for each of the serum
with: (i) the isolation of dengue virus; samples tested. Serum samples found
(ii) positive test of real-time one-step RT- positive for initial screening were then
PCR; (iii) positive seroconversion or four- tested for serotype by each of the four
fold increase in dengue-specific IgM or IgG serotype-specific primer pairs.
antibody from appropriately timed paired
serum; or (iv) high-titer dengue-specific IgM ELISA
and IgG antibody in a single serum
specimen where cross-reaction to Japanese E/M-specific capture IgM and
encephalitis (JE) had been excluded. Sera IgG ELISA
collected during day 1-7 after the onset of
symptoms are referred to as acute-phase A modified E/M-specific capture IgM and
sera. Early and late convalescent sera refer IgG ELISA was performed to measure the
to the specimens collected during day 8-13 dengue-specific IgM and IgG antibodies as
and day 14-30, respectively. recently described[13] . Briefly, each
microtiter 8 wells strip was coated with
Virus isolation by cell culture and 5 g/ml, 100 l/well of affinity purified goat
anti-human IgM (-specific) or IgG (-
virus antigen preparation
specific) antibodies, followed by incubation
The isolation of dengue virus by cell culture with 1:100 diluted serum, incubation with
and virus antigen preparation from culture cocktail contained 1:3 diluted pooled virus
supernatants of DEN-1, DEN-2, DEN -3, antigens from culture supernatants of DEN-1,
DEN-4 or JE-virus infected Vero cells were DEN-2, DEN-3 or DEN -4 infected Vero cells

and 1 g/ml mAb D56.3, incubation with with goat anti-human IgG conjugated to
1:1,000 diluted alkaline phosphatase- alkaline phosphatase. The enzyme activity
conjugated goat anti-mouse IgG (-specific). was developed and OD was taken 30
The enzyme activity was developed with the minutes later.
substrate p-nitrophenyl-phosphate and
optical density (OD) was taken 30 minutes Data analysis
later. For routine screening, culture
supernatant from JE virus-infected Vero cells For E/M-specific capture IgM and IgG ELISA,
was used as negative control antigen due to primary dengue virus infection was defined
the limited cross-reactivity between dengue- if the IgM:IgG OD ratio was 1.2, or
and JE-specific IgM antibodies measured by secondary if the OD ratio was <1.2. For
E/M-specific capture IgM ELISA. This was in those sera with positive NS1-specific IgG
contrast to the high cross-reactivity of antibody response, NS1 serotyping was
dengue- and JE-specific IgG antibodies calculated by the ratio of the highest OD
among dengue patients with secondary value and the second highest OD value read
infection. from the four dengue serotypes. Positive
serotype-specificity is defined if the OD
E/M-specific antigen-coated indirect ratio is 1.2 and negative serotype-
specificity is defined if the OD ratio is <1.2.
IgM and IgG ELISA
Based on NS1 serotype-specific indirect IgG
E/M-specific antigen-coated indirect IgM ELISA, primary dengue virus infection was
and IgG ELISA were performed as previously defined if: (i) negative NS1-specific IgG
described[10] . Two-fold serial dilutions of antibody response was found for sera
appropriately timed paired sera diluted from collected between day 1 and 14 of illness,
1:100 to 1:12,800 were analysed to or (ii) positive serotype-specificity for sera
determine whether four fold increase of collected 9 days of illness. Secondary
dengue-specific IgM or IgG antibody could dengue virus infection was defined if:
be found. This assay has the advantage of (i) positive NS1-specific IgG antibody
better sensitivity in the detection of IgM and response was found for sera collected
IgG antibody increase than E/M-specific between day 1 and 8 of illness, or
capture IgM and IgG ELISA. (ii) positive NS1-specific IgG antibody
response and negative serotype-specificity
NS1 serotype-specific indirect was found any time after the onset of
IgG ELISA infection.
NS1 serotype-specific indirect IgG ELISA

was performed as previously described[9,13] . Results
Briefly, each microtiter 8 wells strip was
coated with 5 g/ml, 100 l/well of mAbs
Dengue surveillance system and
D2/8-1, followed by incubation with 1:3
diluted NS1-containing culture supernatants laboratory diagnosis in Taiwan
of DEN-1, DEN -2, DEN-3, DEN-4 or JE Taiwan has an integrated programme for
viruses-infected Vero cells, incubation of dengue surveillance and control. The
serum samples at a 1:50 dilution, incubation dengue prevention and control centre is a

mission-oriented structure jointly sponsored Pingtung county due to the large samples
by the Department of Health and the generated by the DEN-2 outbreak. For
Environment Protection Administration routine diagnosis, serum samples from the
responsible for the planning and execution reported cases were sent to the laboratory
of dengue control. To assure the on a daily basis and tested according to the
effectiveness of dengue surveillance, three flow chart shown in Figure 1. The periods of
report systems are currently associated with time required to complete these tests were
the dengue surveillance programme 7 days, 6 hours and 4 hours for virus
including: (i) hospital-based passive report isolation, real-time one-step RT-PCR and
system; (ii) syndrome report system (under E/M-specific capture IgM and IgG ELISA,
the classification of viral haemorrhagic respectively. The results were reported as
fever); and (iii) active surveillance system. positive, negative or probable cases. The
The Arbovirus Laboratory in Kun-Yang office, probable case was referred to ELISA result
Taipei City, CDC, Department of Health, is with only IgM or IgG antibody positive. For
responsible for the diagnosis of various negative and probable cases, the
flavivirus. In addition, a second dengue convalescent serum samples collected after
diagnostic laboratory was set up in the day 14 of the illness were demanded and
Fourth Branch, Kaohsiung City, CDC, in July tested for the presence of or increase in IgM
2002 to provide prompt service to and/or IgG antibodies.
Kaohsiung city, Kaohsiung county and
Figure 1. Flow chart of laboratory diagnosis of dengue virus infection
1-7 days
Virus isolation (+) Result
Serum
Real-Time Probable or
1-30 days RT-PCR (-) Result
E/M-specific
Current
Capture
dengue
IgM/IgG ELISA (+) Result
infection
1:100 dilution
>=14 days serum

E/M-specific
Capture Final
Probable or (-) Result Result
IgM/IgG ELISA
1:100 dilution

Representative results of routine provided a good example of the dynamic

change of dengue virus and specific IgM and
diagnosis measured by virus
IgG antibodies in the acute- and
isolation, real-time one-step convalescence-phase sera from patients with
RT-PCR, and E/M-specific capture primary or secondary dengue virus infection
IgM and IgG ELISA covering all four serotypes. As shown in
Figure 1, all of the three assays were
Table 1 shows the representative results of
performed for acute-phase sera, whereas
serum samples analysed by virus isolation,
only E/M-specific capture IgM and IgG
real-time one-step RT-PCR and E/M-specific
ELISA was tested for convalescence-phase
capture IgM and IgG ELISA. The results
sera.
Table 1. Representative results of routine diagnosis of serum samples from reported dengue cases
E/M-specific capture IgM and

Real-time one-step RT-PCR
IgG ELISA
Dengue Dengue Onset Virus Ct value
OD 405 nm
infection serotype days isolation
Flavi- Dengue- Serotype- Dengue- JE-IgM Dengue- JE-IgG
specific specific specific IgM IgG
Primary DEN-1 3 + 28 25 25 0.526 0.237 0.200 0.330
infection 16 2.791 0.951 1.687 1.453
DEN-2 2 + 26 33 29 0.209 0.192 0.182 0.174
15 3.484 0.245 1.165 0.708
DEN-3 3 + 25 31 32 0.478 0.104 0.148 0.228
16 3.074 0.329 2.535 1.704
DEN-4 3 26 35 35 0.094 0.082 0.057 0.063
15 2.225 0.148 1.301 0.096
Secondary DEN-1 1 + 27 28 24 0.166 0.151 0.226 0.176
infection 16 1.949 0.469 3.606 1.699
DEN-2 2 + 17 18 20 0.267 0.217 0.222 0.164
18 0.496 0.157 3.482 1.069
DEN-3 1 + 18 23 24 0.175 0.142 0.180 0.216
7 3.631 0.691 3.494 3.474
DEN-4 7 + 20 28 29 1.298 0.105 2.883 0.273
12 1.468 0.108 3.424 0.637
+ = positive
= negative
Due to the long time needed to isolate reliable and universal RT-PCR protocol that
virus using the cell culture method, it has can be used to systemically detect and
limited value in rapid diagnosis. The isolated differentiate various flavivirus. To assure the
virus, however, is the key material for the specificity of amplicons produced from
later studies of molecular epidemiology and SYBR Green I real-time RT-PCR in routine
pathogenesis. The real-time one-step SYBR screening, both flavivirus- and dengue-
Green I RT-PCR we developed is a simple, specific primer pairs were run (Table 1).

Those serum samples positive for initial Therefore, a positive dengue-specific IgM
screening were then tested for serotype by and IgG antibody response can be easily
each of the four serotype-specific primer used to detect and differentiate primary and
pairs. The analysis of acute-phase serum secondary dengue virus infections.
samples demonstrated that the one-step
SYBR Green RT-PCR was more sensitive to E/M-specific antigen-coated
the virus isolation method and could detect indirect IgM and IgG ELISA
two-times more the acute-phase sera with
positive dengue-specific IgM and/or IgG for the detection of dengue
antibodies[7] . virus infection
The E/M-specific capture IgM and IgG Occasionally, there were acute-phase sera
ELISA has several advantages in the which tested positive with E/M-specific
detection of dengue-specific IgM and IgG capture IgM or IgG antibody response, but
antibodies including: (i) high sensitivity; did not show an apparent increase in
(ii) high specificity (only for IgM antibody); antibody titers in convalescent sera. Due to
(iii) analysis of isotype-specific antibody the higher sensitivity of E/M-specific indirect
responses; (iv) easy automation to test large IgM and IgG ELISA (especially for IgG
amount of serum samples; and antibody), it can be reliably used to
(v) differentiation of primary and secondary determine whether four-fold increase of
dengue infections. The results shown in dengue-specific IgM or IgG antibody were
Table 1 demonstrated the low cross- presented. Figure 2 shows an example
reactivity between dengue - and JE-specific where significant dengue-specific IgG
IgM antibody and inverse pattern of IgM:IgG antibody increase in a convalescent serum.
OD ratio of primary and secondary infection.
Figure 2. E/M-specific antigen-coated indirect IgM and IgG ELISA to detect the increase of
dengue-specific IgM and/or IgG antibodies in the pair sera. The serum samples showed a
= four-fold increase in dengue-specific IgG antibody titer in the convalescent sera
3.0
2.5
2.0
OD 405nm
S9100837A-day 5 IgM
1.5 S9100837B-day 15 IgM
S9100837A-day 5 IgG
S9100837B-day 15 IgG
1.0
0.5
0.0
50x 100x 200x 400x 800x 1600x 3200x 6400x
Patient serum dilution (S9100837)

Statistical analysis of results of respectively. The positive rate for E/M-

specific capture IgM and/or IgG ELISA was
serum samples from confirmed
31.6%, 32.6%, 30%, 39%, 52.6%, 87.5%
dengue cases reported in 2002 and 80% for day 1-7 of illness, respectively.
Table 2 shows the comprehensive analysis Thus, the combined results of real-time RT-
of the results of serum samples from PCR and E/M-specific capture IgM and IgG
confirmed dengue cases reported to Kun- ELISA (IgM and/or IgG positive) could detect
Yang office in 2002. The results of virus an average 94.5% (89.7% to 97.5%) of
isolation, real-time RT-PCR and E/M-specific acute-phase serum samples of confirmed
capture IgM and IgG ELISA were analysed dengue cases. The results also showed that
separately or in combination from the sera the real-time RT-PCR was more sensitive
samples collected on 1-30 day of illness. than virus isolation although very few sera,
The positive rate for real-time RT-PCR was which were virus-isolation positive, were
74.7%, 69.5%, 72.3%, 76.6%, 57.7%, missed by real-time RT-PCR.
36.3% and 22.2% for day 1-7 of illness,
Table 2. Statistical analysis of results of serum samples from confirmed dengue cases
reported in 2002
Days after onset

Assays
1 2 3 4 5 6 7 8 9 10 11 12 13 14-30 Total
Total serum no. 95 95 130 128 97 80 45 58 27 24 9 16 7 148 959
tested
V.I.+ (Virus 53 52 64 58 32 9 2 270
isolation)
% of V.I.+ 55.8 54.7 49.2 45.3 33.0 11.3 4.4
RT-PCR+ and 52 50 60 57 31 8 2 260
V.I.+
RT-PCR+ or 72 68 98 99 57 30 10 434
V.I.+
RT-PCR+ (Real- 71 66 94 98 56 29 10 424
time)
% of RT-PCR+ 74.7 69.5 72.3 76.6 57.7 36.3 22.2
-
RT-PCR (Real - 24 29 36 30 41 51 35 246
time)
ELISA + (E/M- 20 25 21 27 29 41 27 45 19 23 8 12 7 140 444
specific capture
IgM+IgG+)
Probable (ELISA 10 6 18 23 22 29 9 9 8 1 1 3 0 8 147
IgM+ or IgG+)
% of IgM+ 31.6 32.6 30.0 39.1 52.6 87.5 80.0 93.1 100 100 100 93.8 100 100
and/or IgG+
RT-PCR -, V.I.-, 3 3 5 3 9 1 3 4 0 0 0 1 0 0 32
ELISA IgM-IgG-

Days after onset

Assays
1 2 3 4 5 6 7 8 9 10 11 12 13 14-30 Total
+
RT-PCR and 3 2 4 5 6 7 2 29
IgM+IgG+
RT-PCR+ and 6 2 4 7 9 13 1 42
IgM+IgG-
RT-PCR+ and 1 3 4 12 5 1 1 27
IgM-IgG+
RT-PCR+ and 61 59 82 74 36 8 6 326
IgM-IgG-
RT-PCR - and 17 23 17 22 23 34 25 45 19 23 8 12 7 140 415
IgM+IgG+
RT-PCR - and 3 0 8 3 4 14 6 8 7 0 0 3 0 6 62
IgM+IgG-
RT-PCR - and 0 1 2 1 4 1 1 1 1 1 1 0 0 2 16
IgM-IgG+
RT-PCR - and 4 5 9 4 10 2 3 4 0 0 0 1 0 0 42
IgM-IgG-
RT-PCR+ or 88 89 111 120 79 63 35 45 19 23 8 12 7 140 839
ELISA +
% (RT-PCR +or 92.6 93.7 85.4 93.8 81.4 78.8 77.8 77.6 70.4 95.8 88.9 75.0 100 94.6
ELISA +) / Total
serum no.
tested
RT-PCR+ or 91 90 121 124 87 78 42 54 27 24 9 15 7 148 917
ELISA IgM+
and/or IgG+
% (RT-PCR +or 95.8 94.7 93.1 96.9 89.7 97.5 93.3 93.1 100 100 100 93.8 100 100
ELISA IgM+
and/or IgG+) /
Total serum no.
tested
NS1 serotype-specific indirect IgG specific capture IgM and IgG ELISA and NS1
ELISA in the differentiation of JE, serotype-specific indirect IgG ELISA showed
good correlation with 95.90% agreement[13] .
primary and secondary dengue Most importantly, retrospective sero-
virus infections and for the DEN epidemiological studies on serum samples
serotyping of primary infection collected from Liuchiu Hsiang, Pingtung
county and Tainan city in southern Taiwan,
More recently, we have developed a NS1 demonstrated that NS1 serotype-specific
serotype-specific indirect IgG ELISA in the indirect IgG ELISA could replace plaque-
detection and differentiation of primary and reduction neutralization test (PRNT) for
secondary infections. Comparisons of E/M- seroepidemiological study to differentiate JE,

primary and secondary dengue virus evaluated the real-time RT-PCR method for
infections and for the DEN serotyping of rapid detection of dengue virus in the acute-
primary infection[11] . phase sera. In this report, we have
presented a detailed analysis of a total of
959 acute- and convalescent-phase sera
Discussion collected from 799 confirmed dengue
Recent advances in molecular and patients reported to the Kun-Yang office of
serological assays have revolutionized the CDC, Taiwan, in 2002. The results
laboratory diagnosis of flavivirus demonstrated that 94.5% of acute-phase
infection[7,13,14]. Rapid diagnosis of dengue serum samples of confirmed dengue cases
virus infection in the acute-phase sera, could be identified as positive or probable
which is important for disease control with the combined use of real-time one-step
measures and potential treatment, will RT-PCR and E/M-specific capture IgM and
require very sensitive and specific assays. IgG ELISA. The results are very encouraging
With the maturation of real-time RT-PCR and suggest that these two assays are well-
technique, its routine application to clinical suited for routine tests for the early diagnosis
and laboratory diagnosis has now become a of dengue virus infection.
reality. For serodiagnosis, E/M-specific
capture IgM and IgG ELISA has become the
new standard assay for the detection and Conclusion
differentiation of flavivirus infection.
The real-time RT-PCR assay has many
The large DEN -2 epidemic in southern advantages over conventional RT-PCR
Taiwan, was uncontrolled despite vigorous methods, which include rapidity, quantitative
attempts to contain it by the central and measurement, lower contamination rate,
local health governments during October higher sensitivity, higher specificity and easy
2001 December 2002. Although standardization. Therefore, real-time
insecticide -resistance was blamed as an quantitative assay might eventually replace
important factor for this disaster, other virus isolation and conventional RT-PCR as
elements including, political, social, the new gold standard for the rapid diagnosis
environmental, community and human of virus infection in the acute-phase serum
factors were also responsible for this setback. samples.
This epidemic was a strong warning to us
and suggested that more effective measures
should be sought and applied. There is an Acknowledgements
urgent need to improve the surveillance
system and laboratory diagnosis which We wish to thank Hsiu-Ling Pan, Yun-Yih
would help to identify confirmed cases in Chang and Chih-Heng Chen for their expert
the acute-phase sera and respond promptly technical assistance. This work was in part
and effectively to control the transmission supported by grants DOH91-DC -2007 and
chain. DOH91-DC -2016 from the Center for
Disease Control, Department of Health,
Along with the progress of the DEN-2
Taiwan.
outbreak in 2002, we have developed and

References
[1] Alcon S, Talarmin A, Debruyne M, Falconar A, [8] Innis BL, Nisalak A, Nimmannitya S,
Deubel V and Flamand M. Enzyme-linked Kusalerdchariya S, Chongswasdi V,
immunosorbent assay to dengue virus type 1 Suntayakorn S, Puttisri P and Hoke CH. An
nonstructural protein NS1 reveals circulation enzyme-linked immunosorbent assay to
of the antigen in the blood during the acute characterize dengue infections where dengue
phase of disease in patients experiencing and Japanese encephalitis co-circulate. Am J
primary or secondary infections. J Clin Trop Med Hyg, 1989, 40: 418-427.
Microbiol, 2002, 40: 376-381. [9] Shu PY, Chen LK, Chang SF, Yueh YY, Chow
[2] World Health Organization. Dengue L, Chien LJ, Chin C, Lin TH and Huang JH.
haemorrhagic fever: diagnosis, treatment and Dengue NS1-specific antibody responses:
control (second edition). Geneva, WHO, 1997. isotype distribution and serotyping in patients
with dengue fever and dengue hemorrhagic
[3] Callahan JD, Wu SJ, Dion-Schultz A, fever. J Med Virol, 2000, 62: 224-232.
Mangold BE, Peruski LF, Watts DM, Porter
KR, Murphy GR, Suharyono W, King CC, [10] Shu PY, Chen LK, Chang SF, Yueh YY, Chow
Hayes CG and Temenak JJ. Development L, Chien LJ, Chin C, Lin TH and Huang JH.
and evaluation of serotype- and group- Antibody to the nonstructural protein NS1 of
specific fluorogenic reverse transcriptase Japanese encephalitis virus: potential
PCR (TaqMan) assays for dengue virus. J Clin application of mAb-based indirect ELISA to
Microbiol, 2001, 39: 4119-4124. differentiate infection from vaccination.
Vaccine, 2001, 19: 1753-1763.
[4] Drosten C, Gottig S, Schilling S, Asper M,
[11] Shu PY, Chen LK, Chang SF, Yueh YY, Chow
Panning M, Schmitz H and Gunther S.
L, Chien LJ, Chin C, Yang HH, Lin TH and
Rapid detection and quantitation of RNA of
Huang JH. Potential application of
Ebola and Marburg viruses, Lassa virus,
nonstructural protein NS1 serotype-specific
Crimean-Congo hemorrhagic fever virus, Rift
immunoglobulin G enzyme-linked
valley fever virus, dengue virus and yellow
immunosorbent assay in the
fever virus by real-time reverse transcription-
seroepidemiologic study of dengue virus
PCR. J Clin Microbio, 2002, 40: 2323-2330.
infection: correlation of results with those of
[5] Houng HH, Hritz D and Kanesa-thasan N. the plaque reduction neutralization test. J
Quantitative detection of dengue 2 virus Clin Microbiol, 2002, 40: 1840-1844.
using fluorogenic RT-PCR based on 3'- [12] Ko YC, Chen MJ and Yeh SM. The
noncoding sequence. J Virol Methods, 2000, predisposing and protective factors against
86: 1-11. dengue virus transmission by mosquito
[6] Laue T, Emmerich P and Schmitz H. vector. Am J Epidemiol, 1992, 136: 214-220.
Detection of dengue virus RNA in patients [13] Shu PY, Chen LK, Chang SF, Yueh YY, Chow
after primary or secondary dengue infection L, Chien LJ, Chin C, Lin TH and Huang JH.
by using the TaqMan automated Comparison of capture immunoglobulin M
amplification system. J Clin Microbio, 1999, (IgM) and IgG enzyme-linked
37: 2543-2547. immunosorbent assay (ELISA) and
[7] Shu PY, Chang SF, Kuo YC, Yueh YY, Chien nonstructural protein NS1 serotype-specific
LJ, Sue CL, Lin TH and Huang JH. IgG ELISA for differentiation of primary and
Development of group- and serotype- secondary dengue virus infections. Clin
specific one-step SYBR Green I-based real- Diagnos Lab Immunol, 2003, 10: 622-630.
time reverse transcription-PCR assay for [14] Shu PY and Huang JH. Mini Review: Current
dengue virus. J Clin Microbiol, 2003, 41: advances in dengue diagnosis. Clin Diagnos
2408-2416. Lab Immunol, 2004, 11: 642-650.

Detection of Dengue Virus Serotype-specific IgM
by IgM Capture ELISA in the Presence of
Sodium thiocyanate (NaSCN)
Masaru Nawa* #, Tomohiko Takasaki**, Mikako Ito**, Ichiro Kurane**
and Toshitaka Akatsuka*
*Department of Microbiology, Saitama Medical School, 38, Moroyama, Saitama 350-0495, Japan
**Division of Vector-Borne Viruses, Department of Virology 1, National Institute of Infectious Diseases,
1-23-1, Toyama, Shinjuku-ku, Tokyo 162-8640, Japan
Abstract
Dengue virus serotype-specific IgM was detected by IgM-capture enzyme-linked immunosorbent assay
(IgM-ELISA) in the presence of a chaotropic agent, sodium thiocyanate (NaSCN). NaSCN did not affect the
reactions between anti-human IgM and patients IgM, and between dengue viral antigens and detecting
antibody, peroxidase-conjugated flavivirus-specific monoclonal antibody D1-4G2 IgG. Among 18 dengue-
confirmed cases, highest IgM responses were detected to infecting serotypes in 14 cases in the presence of
0.5 M of NaSCN. The results indicate that: (i) the protein-denaturing agent, NaSCN, affects antigen-
antibody reaction in IgM-ELISA, and enables the differentiation of serotype-specific IgM from cross-reactive
IgM; and (ii) IgM responses against the infecting serotypes are higher than those against the other three
serotypes in most primary dengue virus infection. In conclusion, the addition of NaSCN to IgM-capture
ELISA is useful for highlighting serotype-specific IgM responses in primary dengue virus infections.
Keywords: Dengue, IgM-capture ELISA, serotype-specific IgM response, sodium thiocyanate, NaSCN.
Introduction tropical and subtropical areas and nearly two

million people visit Japan from these areas.
Dengue is currently one of the most Therefore, dengue fever (DF) and dengue
important arboviral disease in humans. haemorrhagic fever (DHF) has become an
Dengue viruses, belonging to the family infectious disease of significance and worthy
Flaviviridae, are comprised of four of more attention from the medical
antigenically cross-reactive serotypes and are community in Japan.
responsible for epidemics in tropical and
We have earlier reported the laboratory
subtropical countries. Since the dengue
outbreaks in Osaka, Kobe, Hiroshima and diagnosis of dengue by reverse transcription
Nagasaki from 1942 to 1945, dengue has not polymerase chain reaction (RT-PCR) and
occurred in an epidemic form in Japan[1]. IgM-ELISA[3-5]. We demonstrated that IgM-
However, imported dengue cases have been ELISA was a reliable diagnostic method and
that IgM responses were generally serotype
reported[2]. Approximately five million
Japanese people annually visit countries in cross-reactive but often highest against
#
E-mail: mnawa@saitama-med.ac.jp; Tel.: +81-49-276-1438; Fax: +81-49-295-9107

Detection of Dengue Virus Serotype-specific IgM by IgM Capture ELISA in the Presence of NaSCN
infecting virus serotype in most Japanese Japan from 2000 to 2002 and sent to the
cases[4]. The serotype specificity of IgM Department of Virology 1, National Institute
responses in dengue patients has been of Infectious Diseases, Tokyo, Japan.
controversial[6-8]. Burke had reported that
Prototype dengue viruses were
serotype-specific IgM responses
propagated in the Aedes albopictus
corresponding to the isolated virus type were
mosquito cell clone C6/36, and the infected
detected in primary dengue virus infection[6].
Gubler had reported that in dengue infection, cell culture supernatants were inactivated by
frequent monotypic IgM responses were not incubation with beta-propiolactone at a final
correlated with the virus serotype isolated concentration of 0.2% for 30 minutes at
from patients[7]. In 1984, Inouye et al. [9] 37 C as previously reported[10]. Tetravalent
demonstrated a new technique for the and four monovalent dengue viral antigens
differentiation of antibody avidity after virus were prepared by the method as previously
infection, i.e. rubella, rota and Japanese reported[4,5].
encephalitis viruses. They estimated the The IgM capture ELISA was carried out
antibody avidity to viral antigen using a low according to the method described
concentration of a protein-denaturing agent, previously[3-5]. Anti-human IgM ( chain
guanidine hydrochloride, in the diluent of specific) goat serum and peroxidase-
antibody in the ELISA. They concluded that conjugated anti-human IgM were purchased
the stringent immunosorption technique from Sigma-Aldrich, Inc, USA. The IgG
was useful for investigating the antigenic fraction of the flavivirus-specific monoclonal
relationship among closely-related viruses. antibody, D1-4G2, was prepared by the
In the present study, we examined Protein G affinity chromatography kit
serotype-specific IgM responses under (ImmunoPure G IgG Purification kit, PIERCE,
stringent conditions in the presence of a USA), and then conjugated with horseradish
chaotropic agent, sodium thiocyanate peroxidase by a commercial kit (EZ-Link Plus
(NaSCN), in the reaction mixture of dengue Activated Peroxidase kit, PIERCE, USA).
viral antigens and patients sera. The In order to detect the serotype-specific
development of a simple method to reaction between dengue viral antigen and
distinguish serotype-specific reaction from dengue virus-specific IgM antibody, patient
cross-reaction will be useful not only for serum was treated with the Protein G affinity
laboratory diagnosis but also for chromatography kit described above. The
seroepidemiological studies. IgG fraction in the serum specimen was
removed by adding Protein G beads
Materials and methods according to the manufacturers instruction.
Unless otherwise stated, data were presented
Twenty-eight serum specimens from 18 as the mean of independent two-to-three
confirmed Japanese dengue cases were used assays.
in the study. Serum samples from 22
Japanese subjects with other illnesses, who
had never been to areas where dengue was Results and discussion
epidemic-prone or endemic, were used as One of the authors has previously reported
the control. These sera were obtained for
that the addition of NaSCN to the reaction
diagnostic purposes in clinics and hospitals in
mixture of ELISA highlights serotype-specific

reaction between crude dengue viral antigen reactive one. According to these results, we
and anti-dengue hyperimmunized mouse decided to use NaSCN in IgM-capture ELISA
sera[11]. The antigen-antibody reaction was at the following conditions: (i) NaSCN was
affected not only by the concentration of included at a final concentration of 0.5 M in
NaSCN but also by the procedures of the 10% normal calf serum-PBS; (ii) peroxidase-
NaSCN treatment. A concentration higher conjugated flavivirus- specific monoclonal
than 0.7 M inhibited the reaction non- antibody D1-4G2 (D1-4G2) was diluted in
specifically, while a concentration lower than 0.5 M NaSCN; and (iii) viral antigens
0.3 M had no effect on the discrimination of captured by patients IgM were detected by
serotype-specific reaction from the cross- the detection antibody in 0.5 M NaSCN.
Figure 1. The effects of NaSCN treatment on antigen-antibody reaction

(a)
1.5
Control
NaSCN
1
ELISA O.D.
0.5
0
50 60 70 80 90100 200 300 400
Serum dilution
(b)
2
Control
NaSCN
1.5
ELISA O.D.
0.5
0
1 2 3 4 5 6 7 8
Antigen dilution
(a) Human serum was serially diluted from 1:50 to 1:400 with 10% CS-PBS containing 0.5 M NaSCN, and IgM was
captured on anti-human IgM goat serum-coated wells. IgM was detected with peroxidase-conjugated anti-human
IgM goat serum. (b) Serially diluted tetravalent dengue viral antigen was captured on the solid phase sensitized
with D1-4G2 IgG, and then detected with peroxidase-conjugated D1-4G2 IgG in the presence of 0.5 M NaSCN.

Figure 1 shows the reactions between with the Protein G beads (UltraLink
anti-human IgM and patients IgM (a), and Immobilized Protein G Plus, capacity: ~25
dengue viral antigen and the detection mg IgG/ml of the gel, PIERCE). The levels of
antibody (b), in the presence of 0.5 M cross-reaction between dengue viral antigens
NaSCN in the ELISA. These two reactions and patients IgM antibody (a) were
were not affected by 0.5 M NaSCN, decreased after the treatment with 0.5 M
suggesting that IgM capture and detection of NaSCN in the ELISA (b): The level of reaction
dengue viral antigens were not affected in with homologous dengue-1 antigen was less
IgM-capture ELISA.
affected. The results suggested that the
Figure 2 shows anti-dengue IgM titration addition of 0.5 M NaSCN to the reaction
curves in the presence or absence of 0.5 M mixture of ELISA may highlight a serotype-
NaSCN in the ELISA. The IgG fraction in the specific reaction between crude dengue viral
serum specimen was removed by adsorption antigen and anti-dengue IgM antibody.
Figure 2. Titration curves of dengue virus specific IgM antibody in the presence or
absence of 0.5 M NaSCN in the ELISA
(a)
2
v.s.D1 Ag
v.s.D2 Ag
v.s.D3 Ag
v.s.D4 Ag
1.5
0.5
0
100 1000
Serum dilution
(b)
2
1.5
0.5
0
100 1000
Serum dilution
A serum specimen obtained from the dengue virus type 1-infected patient was used in the study. The IgG fraction
was removed according to the method described in the Materials and Methods section. Protein G-treated serum
specimen was serially diluted from 1:20 to 1:2560 in the absence (a) and in presence of 0.5 M NaSCN (b) in 10%
CS-PBS, and then reacted on the plates coated with each of 4 dengue viral antigens. Dengue virus-specific IgM
antibody was detected with 1:500 diluted peroxidase-conjugated anti-human IgM goat serum.

We examined the IgM levels by ELISA all the tested cases, the serotype-specific
with the antigen of 4 dengue virus serotypes. IgM levels were the highest against the
The table below shows the results of 28 infecting dengue virus serotype than against
serum samples from 18 Japanese dengue three other serotypes in the presence of
patients. The infecting dengue virus NaSCN. The serotype-specific IgM
serotypes were determined by RT-PCR. The responses were more highlighted in the
data were presented as the index value presence of NaSCN than in its absence.
according to the method described These data suggest that IgM responses to the
previously[4] . We defined index values of infected dengue virus serotype determined
2.28 or greater and 26.60 or greater as by RT-PCR in primary dengue infection
positive, respectively, in the absence and were the highest. These results agreed with
presence of 0.5 M NaSCN in the ELISA. In the report by Burke [6] .
Table. Serodiagnosis of dengue by IgM-ELISA with and without 0.5M NaSCN
Index values*
Patient RT-PCR Disease day Without NaSCN (cut off = 2.28) With 0.5M NaSCN (cut off = 26.60)
D1 D2 D3 D4 D1 D2 D3 D4
1 D3 4 1.20 1.47 2.29 1.42 5.33 3.67 45.67 5.67
9 8.58 7.73 16.40 10.46 110.30 47.67 471.67 273.00

2 D2 7 4.89 52.18 7.96 4.49 128.00 1,246.00 670.00 239.00
14 5.27 37.93 8.87 5.42 68.00 395.00 243.70 179.00
3 D3 6 5.34 3.34 9.60 2.35 50.25 15.00 283.00 39.50
17 6.79 7.07 12.38 4.00 78.00 46.25 413.25 108.50
4 D3 8 5.61 2.84 9.20 2.63 67.25 12.75 305.50 77.00
10 4.77 3.16 9.10 3.45 54.00 14.00 274.20 97.20

5 D2 6 1.13 4.77 1.71 0.81 2.78 9.36 8.00 0.00
13 2.65 12.31 4.49 2.15 10.27 46.27 28.73 14.45
6 D3 6 3.07 1.75 6.55 0.89 11.83 4.00 140.33 2.50
8 4.06 2.40 9.23 1.34 21.00 6.83 198.33 11.83
7 D1 5 2.76 1.46 6.33 1.39 7.86 0.29 20.71 0.00
7 13.51 6.65 11.49 3.67 127.14 72.86 11.43 25.71
8 16.91 9.53 13.28 4.43 143.37 100.00 20.37 42.00

14 24.51 11.49 9.86 3.63 214.50 68.25 34.00 51.13

Index values*
Patient RT-PCR Disease day Without NaSCN (cut off = 2.28) With 0.5M NaSCN (cut off = 26.60)
D1 D2 D3 D4 D1 D2 D3 D4
8 D1 7 1.73 3.89 1.66 1.45 5.54 6.38 6.00 1.62
11 6.95 10.19 2.27 1.76 26.42 20.25 19.00 10.17
9 D2 5 1.93 3.02 1.28 0.72 1.80 4.70 3.90 0.00
10 D2 ND 0.75 2.98 0.92 0.28 2.13 4.63 3.75 0.00
11 D1 5 1.25 1.28 1.32 0.69 4.00 1.57 10.71 0.00

12 D1 ND 20.35 15.44 15.02 8.00 161.28 36.71 39.90 97.71
13 D1 19 18.37 10.69 10.02 12.75 244.43 22.86 138.14 135.00
14 D1 8 7.55 6.78 6.41 3.37 50.25 11.67 27.92 19.92
15 D4 19 1.12 1.44 1.19 2.41 2.56 3.44 7.22 35.44
16 D1 5 1.36 1.13 1.27 0.27 6.27 1.45 2.45 0.00
17 D1 6 4.74 2.01 5.20 0.98 58.67 3.71 17.58 0.00
18 D1 15 14.18 8.39 8.52 3.58 137.38 33.27 31.00 49.88
D1 to D4, dengue virus type 1 to 4, respectively. ND, not determined.

* index values were calculated by the formula A492 with the viral antigen/A492 with uninfected control antigen.
The chaotropic agent, NaSCN, is known represent the envelope glycoprotein and NS1,
to denature protein structure and inhibit the respectively[11]. Trent et al.[15,16] observed
formation of immune complexes as was three antigenic determinants on the envelope
exploited for immunoaffinity glycoprotein: (i) flavivirus group- reactive;
chromatography[12,13]. There are multiple (ii) complex-specific; and (iii) serotype-
factors which affect NaSCNs ability to specific. Henchal et al.[17] characterized four
highlight serotype-specific reaction in antigenic determinants on the envelope
comparison with cross-reaction in the ELISA. glycoprotein: (i) flavivirus group-reactive;
One of the factors is a characteristic of (ii) dengue complex-specific; (iii) dengue
dengue viral antigens, which are prepared subcomplex-specific; and (iv) dengue
from the infected mosquito cell culture. It serotype-specific.
was reported that a rapidly sedimenting
Brandt et al. [14] reported that the DEN-2
haemagglutinin (RHA) was cross-reactive and
SCF antigen extracted from infected mouse
labile, but the soluble complement-fixing
brains was resistant to the treatment with
antigen (SCF) was serotype-specific and
protein denaturing agents. Falconar and
relatively stable against the NaSCN treatment
Young[18] reported serotype-specific epitopes
in the ELISA[11,14]. The predominant
on NS1. These results suggest that
polypeptides in RHA and SCF fractions

heterologous antigenic determinants on reactive IgM. This procedure may be useful

dengue viral antigens contribute to the cross- for determining infecting dengue virus
reactivity among the four dengue serotypes, serotypes, especially in primary dengue virus
and that the presence of a chaotropic agent infection.
in the reaction mixture induces changes of
viral antigens and may decrease the cross-
reactive antigenicity. Acknowledgement
In conclusion, we demonstrated that the This work was supported by a grant for
addition of NaSCN to IgM-capture ELISA research on emerging and re-emerging
highlighted the detection of serotype-specific infectious diseases from the Ministry of
IgM in comparison with serotype cross- Health, Labour and Welfare, Japan.
References
[1] Hotta S. Twenty years of laboratory [6] Burke DS. 1983. Rapid methods in the
experience with dengue virus. In Saunders laboratory diagnosis of dengue virus
M and Lennnet EH (Eds.), 1965, Medical infections. In T Pang and R Pathmananathan
and Applied Virology. Green, St Louis: 228- (Eds.), Proceedings of the International
256. Conference on Dengue/Dengue
Hemorrhagic Fever. University of Malaya,
[2] Yamada K, Takasaki T, Nawa M, Nakayama
Kuala Lumpur, Malaysia, pp. 72-84.
M, Arai TY, Yabe S and Kurane I. The
features of imported dengue fever cases [7] Gubler DJ. Dengue and dengue
from 1996 to 1999. Jpn J Infect Dis, 1999, hemorrhagic fever. Clin Microbiol Rev, 1998,
52: 257-259. 11: 480-496.
[3] Yamada K, Nawa M, Takasaki T, Yabe S and [8] Innis BL. Antibody responses to dengue virus
Kurane I. Laboratory diagnosis of dengue infection. In DJ Gubler and G Kuno (Eds.),
virus infection by reverse transcriptase Dengue and dengue hemorrhagic fever,
polymerase chain reaction (RT-PCR) and Wallingford: CAB International, 1997: 221-
IgM-cpature enzyme-linked immunosorbent 243.
assay (ELISA). Jpn J Infect Dis, 1999, 52:
[9] Inouye S, Hasegawa A, Matsuno S and
150-155.
Katow S. Changes in antibody avidity after
[4] Nawa M, Yamada K, Takasaki T, Akatsuka T virus infections: detection by an
and Kurane I. Serotype-cross-reactive immunosorbent assay in which a mild
immunoglobulin M responses in dengue protein-denaturing agent is employed. J Clin
virus infections determined by enzyme- Microbiol, 1984, 20: 525-529.
linked immunosorbent assay. Clin Diag Labo
[10] Nawa M, Takasaki T, Yamada K, Kurane I
Immunol, 2000, 7: 774-777.
and Akatsuka T. Development of IgM-
[5] Nawa M, Takasaki T, Yamada K, Akatsuka T cpature enzyme-linked immunosorbent
and Kurane I. Development of dengue IgM- assay for serodiagnosis of dengue using beta-
cpature enzyme-linked immunosorbent propiolactone-inactivated dengue viral
assay with higher sensitivity using antigens. Dengue Bulletin, 2003, 27: 95-99.
monoclonal detection antibody. J Virol
Methods, 2001, 92: 65-70.

[11] Nawa M. An enzyme-linked immunosorbent [16] Trent DW. Antigenic characterization of

assay using a chaotropic agent (sodium flavivirus structural proteins separated by
thiocyanate) for serotype specific reaction isoelectoric focusing. J Virol, 1977, 22: 608-
between cude dengue viral antigen and anti- 618.
dengue mouse antibody. Microbiol
[17] Henchal EA, Gentry MK, McCown JM and
Immunol, 1992, 36: 721-730.
Brandt WE. Dengue virus-specific and
[12] Dandlinker WB and de Saussure VA. flavivirus group determinants identified with
Antibody purification at neutral pH utilizing monoclonal antibodies by indirect
immunospecific adsorbents. Immunochemistry, immunofluorescence. Am J Trop Med Hyg,
1968, 5: 357-365. 1982, 31: 830-836.
[13] Avrameas S and Ternyck T. Use of iodide [18] Falconar AKI and Young PR. Production of
salts in the isolation of antibodies and the dimer-specific and dengue virus group cross-
dissolution of specific immune precipitates. reactive mouse monoclonal antibodies to
Biochem J, 1976, 102: 37c-39c. the dengue 2 virus non-structural
glycoprotein NS1. J Gen Virol, 1991, 72:
[14] Brandt WE, Cardiff RD and Russell PK.
961-965.
Dengue virions and antigens in brain and
serum of infected mice. J Virol, 1970, 6:
500-506.
[15] Trent DW, Harvey CL, Qureshi A and
Lestourgeon D. Solid-phase radioimmunoassay
for antibodies to flavivirus structural and
nonstructural proteins. Infect Immunol,
1976, 13: 1325-1333.

Genetic Influences on Dengue Virus Infections
J.F.P. Wagenaar #, A.T.A. Mairuhu and E.C.M. van Gorp
Department of Internal Medicine, Slotervaart Hospital, Louwesweg 6, 1066 EC Amsterdam,

The Netherlands
Abstract
Dengue virus infections are an important cause of morbidity and mortality in the tropics, with 100
million people infected annually and an estimated 2.5 billion people at risk. Human infections can be
asymptomatic or can manifest as the self-limited febrile dengue fever, or the more severe and life-
threatening dengue haemorrhagic fever (DHF). There are several possible reasons why some infected
individuals might develop a more severe form of the disease than others. Antibody enhancement and
viral virulence have been implicated in the pathogenesis of DHF but host genetic factors may also be
relevant and predispose some individuals to DHF. This review discusses the possible involvement of a
variety of genetic polymorphisms on the course of dengue virus infections. It has been shown that several
common genetic polymorphisms can influence the susceptibility to dengue haemorrhagic fever. Gene
polymorphisms concerning human leucocyte antigens, antibody receptors, inflammatory mediators and
other factors with immunoregulatory effects are described. The study of genetic polymorphisms might
provide important insights into the pathogenesis of a more severe disease and could have an impact on
the design of future vaccines.
Keywords: Dengue haemorrhagic fever, genetic polymorphisms.
Introduction headache and muscle and joint pain.

Occasionally, dengue virus infections
Dengue has become one of the most progress to dengue haemorrhagic fever
important arthropod-borne diseases in (DHF) and dengue shock syndrome (DSS).
tropical and subtropical regions of the world. These are potentially life-threatening
Approximately 100 million cases of dengue illnesses characterized by haemorrhagic
infections occur annually, and an estimated manifestations and the loss of plasma from
2.5 to 5 billion people are at risk of dengue the vascular compartment, which may give
virus infection[1] . The four serotypes of rise to shock in severe cases.
dengue virus (DEN-1, 2, 3 and 4) are
Central in the pathogenesis of DHF and
transmitted to humans through the bite of
an infective female Aedes mosquito and DSS is the loss of endothelial integrity that is
believed to be the result of an abnormal
may result in dengue fever (DF), an acute
viral infection characterized by fever, rash, immune response and a disturbance in
#
E-mail: jfpwagenaar@hotmail.com; Tel.: + 31-(0)20-5124591; Fax: + 31-(0)20-5124783

immune regulation. Elevated levels of inflammatory response is related to viral

several cytokines and chemical mediators, virulence. Several studies have found that
which cause capillary leakage and may lead infection with DEN-2 caused more severe
to shock, have been found in those suffering disease than other serotypes, suggesting that
from DHF and DSS. Replication of dengue the virus phenotype influences the
viruses occurs primarily in mononuclear outcome[7,8,11] . In addition, genetic variations
phagocytes, which are a major source of within a specific serotype may also account
tumor necrosis factor (TNF)- and other for differences in disease severity, although
vasoactive inflammatory mediators. Several reports remain scanty[14].
studies have demonstrated that TNF- and Hyperendemic transmission of multiple
other cytokines that are produced
DEN serotypes in a Haitian population and
downstream of TNF- in the inflammatory the apparent absence of DHF and DSS, in
cascade, e.g. IL-1, IL-6 and IL-8, are addition to the observation that black people
related with disease severity[2-4] . Other were hospitalized less frequently with DHF
inflammatory mediators, like IL-2 and and DSS than the whites during epidemics in
interferon (IFN)-, are released from T Cuba, led to the hypothesis that human
lymphocytes that are activated during genetic factors, e.g. gene mutations and gene
dengue virus infections. The levels of these polymorphisms, may contribute to variable
cytokines are significantly higher in DHF susceptibility[15-17] . Genetic polymorphisms
and DSS patients than in DF patients[5,6] . are stable gene variants that usually have
There are several possible reasons why minor effects on the regulation or function of
some infected individuals might produce a proteins. These subtle chances might very
greater inflammatory response than others. well have important consequences for
The most favoured hypothesis concerns the susceptibility to the disease[18] . Several studies
antibody-dependent enhancement theory. have confirmed that some genetic
Several epidemiological studies demonstrate polymorphisms may protect or predispose an
that prior infection with a different viral individual to DHF and DSS. Understanding
serotype constitutes the largest risk factor for the molecular basis for these differences in
DHF[7-11] . In vitro studies demonstrated that susceptibility should provide useful insight in
the presence of anti-dengue antibodies at the pathogenesis of DHF and DSS and aid in
sub-neutralizing concentrations augment the development of effective therapies and
dengue virus infection of Fc receptor- vaccines. This review attempts to describe
positive cells, such as monocytes[12,13] . Based the current knowledge of the role of genetic
on these epidemiological and laboratory influences on dengue virus infections.
observations, it has been hypothesized that
dengue cross-reactive antibodies may
increase the number of dengue virus- Human leucocyte antigen
infected monocytes during secondary genes
infections, and lysis of these dengue virus-
The function of the human leukocyte
infected monocytes may lead to DHF and
antigens (HLAs), encoded by the major
DSS. Another possibility why some infected
histocompatibility complex (MHC) and
individuals might produce a greater
whose genes are on chromosome 6, are to

display antigen-proteins to antigen receptors HLA gene regions influence peptide epitope
of host T-lymphocytes in order to activate binding[18] . A number of studies have looked
cellular host immune responses. HLA genes at the variation in HLA genes and found
show great variability and it could well be some of them to be associated with the
that specific polymorphisms seen in human severity of dengue virus infections (Table).
Table. Effect of HLA and non-HLA alleles on the severity of dengue virus infections
Alleles Class Effect Population Reference
HLA alleles Class I

A1 Susceptibility Cubans 20
A2 Susceptibility Thai 19, 22
A*0203 Protective Thai 22
A*0207 Susceptibility Thai 22
A24 Susceptibility Vietnamese 21
A29 Protective Cubans 20
A33 Protective Vietnamese 21
B blank Susceptibility Cubans/Thai 19, 20
B13 Protective Thai 19
B14 Protective Cubans 20
B46 Susceptibility Thai 22
B51 Susceptibility Thai 22
Class II
DRB1*04 Resistance Mexicans 29
Non-HLA alleles Fc gamma-receptor Resistance Vietnamese 30
Vitamin D receptor Resistance Vietnamese 30
HLA class I Polymorphisms in this class I region gene

were found to be associated with DHF
HLA class I alleles consist of HLA-A, -B, and - disease susceptibility. Chiewslip and Paradoa
C; its products have a wide distribution and were the first to report an association
are present on the surface of all nucleated between HLA class I and the severity of
cells and on platelets. Antigens associated dengue virus infection[19,20] . In two ethnically
with HLA class I products will interact with and geographically distinct populations
CD8 cells during an immune response. evidence was presented suggesting that HLA-

A1, HLA-A2 and HLA-B blank increased in haemorrhagic fever[23] . HLA-B52 showed a
frequency in DHF patients. A negative strong association with less severe DF. The
relationship was found for HLA-B13, HLA- reduced frequency of the HLA-B15 group of
B14 and HLA-A29. However, these studies serotypes, including HLA-B62, B76 and B77,
had a small sample size and additional in patients with secondary infections, suggests
studies with a larger number of patients were that they may be protective against
needed. Subsequently, a large case control developing clinical disease in
study in 560 study subjects was performed, immunologically primed individuals. By
which mainly confirmed the observations contrast, HLA-B46 that also belongs to the
made by the two previous studies that HLA HLA-B15 group of serotypes, was increased
class I was important[21] . The data in DHF patients with secondary infections.
demonstrated that polymorphisms in the Since HLA-B46 is in strong equilibrium with
HLA class I region, particularly of the HLA-A HLA-A*0207, it is believed that the effect of
gene, were significantly associated with B46 was likely to be an adjunct to that of
susceptibility to DHF. Of the 15 alleles A*0207. Finally, HLA-B44 appeared also to
studied, two particular alleles were relevant: be protective against the development of
patients with HLA-A33 were less likely to severe disease in patients with secondary
develop DHF (odds ratio 0.56; 95% dengue virus infections.
confidence interval 0.34-0.39), whereas
those with HLA-A24 were at an increased HLA class II
risk to develop DHF (odds ratio 1.54; 95%
confidence interval 1.05-2.25). The HLA-B Class II HLA products consist of HLA-D, -DR,
alleles were not associated with DHF disease -DP, and -DQ; they have a more limited
susceptibility. distribution on B-cells, macrophages,
dendritic cells, Langerhans cells and activated
Another case control study, in a Thai T cells. HLA class II alleles have shown to
population, also demonstrated that the HLA- play a role in mycobacterial diseases, and
A2 locus serotype was associated with their association with hepatitis clearance is
disease susceptibility[22]. HLA-A*0203 was also established[24-26] . HLA-DRB1, which is
associated with the less severe DF, whereas one of the most polymorphic loci of the HLA
HLA-A*0207 was associated with complex in the Mexican population[27,28] , was
susceptibility to the more severe DHF. studied in Mexican patients suffering from a
Interestingly, these associations were only dengue virus infection[29] . Although the
found in immunologically primed persons, sample size was relatively small, the
but not in immunologically nave patients investigators found that the frequency of
with primary infection. Dengue virus-specific HLA-DRB1*04 was lower in DHF patients.
associations were also observed within the Persons homozygous for DRB1*04 were less
HLA-B5 group of related alleles, whereby likely to develop DHF than persons who
molecularly-determined HLA-B51 alleles were DRB1*04 negative (odds ratio 0.28;
were associated with the development of 95% confidence interval 0.12-0.66),
DHF after secondary infections. HLA-B51- suggesting a protective effect. The envelope
restricted CTL responses to a variety of protein (E) of the virus is responsible for viral
viruses have been described, including entry into target cells. The immunological
Hantaan virus which also causes a determinants of protein E are probably

processed and presented by HLA class II gene has been associated with
antigens. The HLA-DRB1*04 molecule may meningococcal disease and recurrent
present these viral antigens to CD4+ respiratory tract infections[33,34] . It chances the
lymphocytes leading to an effective immune IgG binding affinity of the receptor with
response and therefore protection from DHF. reduced opsonisation of IgG2 antibodies
These findings are in contrast to the findings causally associated with the arginine variant.
of Loke et al., who studied polymorphisms in Loke et al. found that homozygotes for the
the HLA-DRB1 gene but did not find an arginine variant at position 131 of the FcRIIA
association[21] . gene may be less susceptible to DHF[30] .
HLA class III Vitamin D receptor (VDR)

Genes in the class III region encode a This gene mediates the immuno-regulatory
number of proteins, including complement effects of 1.25-dihydroxyvitamin D3, which
proteins (C4A, C4B, C2 and Bf), TNF- and include activating monocytes, stimulating
TNF-[18] . Loke and colleagues studied cellular immune responses and suppressing
promotor polymorphisms in the TNF- gene immunoglobulin production and lymphocyte
but did not find an association[21] . No other proliferation[35] . Recently the tt genotype of a
studies are reported to have studied HLA single nucleotide polymorphism at position
class III polymorphisms. 352 of the VDR gene has been associated
with tuberculoid leprosy, enhanced
clearance of HBV infection and resistance to
Non-HLA host genetic factors pulmonary tuberculosis [36,37] . Expression of
The number of studies on polymorphisms VDR may affect susceptibility to DHF since
within non-HLA genes remains low. Loke activated B and T lymphocytes express VDR
and colleagues investigated the association and 1,25D3 affects monocytes, the main sites
between susceptibility to DHF and of dengue virus infection and replication[12] .
polymorphic non-HLA alleles: vitamin D The t allele at position 352 of the vitamin D
receptor (VDR), Fc receptor II (FcRII), receptor (VDR) gene was associated with
Interleukin-4 (IL-4), Interleukin-1 repeat resistance to severe dengue, although the
alleles (IL-1RA), and mannose-binding lectin exact mechanism needs to be explored.
(MBL) [30] . Two of the five genes assessed
showed evidence of association with altered Interleukin-4 (IL-4)
risk of severe dengue.
IL-4, primarily produced by Th2 subset of
CD4+ T-cells, regulates B-cell growth, IgG
Fc receptor class switching and suppresses Th1-type
The Fc receptor is a widely distributed responses as well[38,39] . Since this gene affects
receptor for all subclasses of IgG and is able both antibody responses and inflammatory
to mediate antibody dependent responses during disease, IL-4 promotor
enhancement in vitro by binding to virus-IgG polymorphisms were studied in order to find
complexes[31,32] . An arginine to histidine a relationship in susceptibility to DHF.
substitution at position 131 of the FcRIIA However, no associations were found in this
context[30] .

Interleukin-1 repeat allele (IL-1RA) infections but also of viral infections in

general. The finding of a protective
IL-1RA was thought to be a good candidate association with particular HLA or non-HLA-
gene as well because IL-1RA is involved in types may encourage the design of future
the regulation of IL-1-mediated inflammatory vaccines, whereas polymorphisms associated
responses by competitive binding to IL- with the susceptibility to develop a more
receptors [40] . But no significant difference severe disease may help to identify certain
could be found in the DHF group in addition risk groups in a population. It is therefore of
to the controls[30] . great importance to stimulate the study of the
interaction of single and multiple
Mannose-binding lectin (MBL) polymorphisms in severe dengue virus
Several mutations in the MBL gene, which infections.
encodes for a protein involved in the The few studies performed thus far have
activation of the classical complement demonstrated that host genetic factors can be
pathway[41,42] , have been associated with a important in susceptibility to DHF. It is most
marked reduction in serum MBL levels and likely that classical HLA class I and class II
MBL-mediated complement activation[43,44] . gene products play a crucial role in
Polymorphisms in this gene were not proved determining the severity of dengue virus
to have any effect on the susceptibility to infections. Two polymorphic non-HLA alleles,
DHF. However, this variant allele was the FcRII receptor and VDR, could also play
relatively low in the observed population, an important role in susceptibility to DHF.
which limits the statistical power of the Some polymorphic HLA alleles were
analysis [30] . observed in several studies, e.g. HLA-A2 in a
Thai and Vietnamese population, but
differences in susceptibility to DHF were
Discussion and future observed[19,21,22] . An explanation for the
perspective observed difference may be that a genetic
polymorphism is more frequent in a
The number of candidate susceptibility and
population whereas another is relatively
protective genes is expanding rapidly, but
infrequent. Overall, such disease associations
what is the use of studying these genes in
warrant further analysis, but also emphasize
relation to DHF? Studying host genetic factors
the need to expand the scope of investigation
will clearly contribute to our understanding of
to other candidate genes within and outside
the pathophysiology of dengue virus
of the HLA region.
References
[1] Rigau-Perez JG, Clark GG, Gubler DJ, Reiter [2] Suharti C, van Gorp EC, Dolmans WM,
P, Sanders EJ and Vorndam AV. Dengue and Setiati TE, Hack CE, Djokomoeljanto R and
dengue haemorrhagic fever. Lancet, 1998, van der Meer JW. Cytokine patterns during
352: 971-977. dengue shock syndrome. European Cytokine
Network, 2003, 14: 172-177.

[3] Bethell DB, Flobbe K, Cao XT, Day NP, [10] Russell PK, Yuill TM, Nisalak A, Udomsakdi
Pham TP, Buurman WA, Cardosa MJ, White S, Gould DJ and Winter PE. An insular
NJ and Kwiatkowski D. Pathophysiologic outbreak of dengue hemorrhagic fever. II.
and prognostic role of cytokines in dengue Virologic and serologic studies. American
hemorrhagic fever. The Journal of Infectious Journal of Tropical Medicine and Hygiene,
Diseases, 1998, 177: 778-782. 1968, 17: 600-608.
[4] Hober D, Poli L, Roblin B, Gestas P, [11] Sangkawibha N, Rojanasuphot S, Ahandrik S,
Chungue E, Granic G, Imbert P, Pecarere JL, Viriyapongse S, Jatanasen S, Salitul V,
Vergez-Pascal R, Wattre P et al. Serum levels Phanthumachinda B and Halstead SB. Risk
of tumor necrosis factor-alpha (TNF-alpha), factors in dengue shock syndrome: a
interleukin-6 (IL-6), and interleukin-1 beta prospective epidemiologic study in Rayong,
(IL-1 beta) in dengue-infected patients. The Thailand. I. The 1980 outbreak. American
American Journal of Tropical Medicine and Journal of Epidemiology, 1984, 120: 653-
Hygiene, 1993, 48: 324-331.
669.
[5] Green S, Vaughn DW, Kalayanarooj S,
[12] Halstead SB and O'Rourke EJ. Dengue viruses
Nimmannitya S, Suntayakorn S, Nisalak A, Lew
and mononuclear phagocytes. II. Identity of
R, Innis BL, Kurane I, Rothman AL and Ennis
blood and tissue leukocytes supporting in
FA. Early immune activation in acute dengue
vitro infection. The Journal of Experimental
illness is related to development of plasma
Medicine, 1977, 146: 218-229.
leakage and disease severity. The Journal of
Infectious Diseases, 1999, 179: 755-762. [13] Halstead SB. In vivo enhancement of
[6] Kurane I, Innis BL, Nimmannitya S, Nisalak dengue virus infection in rhesus monkeys by
A, Meager A, Janus J and Ennis FA. passively transferred antibody. The Journal
Activation of T lymphocytes in dengue virus of Infectious Diseases, 1979, 140: 527-533.
infections. High levels of soluble interleukin [14] Rico-Hesse R. Molecular evolution and
2 receptor, soluble CD4, soluble CD8, distribution of dengue viruses type 1 and 2
interleukin 2, and interferon-gamma in sera in nature. Virology, 1990, 174: 479-493.
of children with dengue. The Journal of
Clinical Investigation, 1991, 88: 1473-1480. [15] Halstead SB, Streit TG, Lafontant JG,
Putvatana R, Russell K, Sun W, Kanesa-
[7] Vaughn DW, Green S, Kalayanarooj S, Innis Thasan N, Hayes CG and Watts DM. Haiti:
BL, Nimmannitya S, Suntayakorn S, Endy TP,
absence of dengue hemorrhagic fever
Raengsakulrach B, Rothman AL, Ennis FA
despite hyperendemic dengue virus
and Nisalak A. Dengue viremia titer,
transmission. The American Journal of
antibody response pattern, and virus
Tropical Medicine and Hygiene, 2001, 65:
serotype correlate with disease severity. The
180-183.
Journal of Infectious Diseases, 2000, 181: 2 -9.
[8] Burke DS, Nisalak A, Johnson DE and Scott [16] Kouri GP, Guzman MG, Bravo JR and Triana
RM. A prospective study of dengue C. Dengue haemorrhagic fever/dengue
infections in Bangkok. American Journal of shock syndrome: lessons from the Cuban
Tropical Medicine and Hygiene, 1988, 38: epidemic, 1981.Bulletin of the World
172-180. Health Organization, 1989, 67: 375-380.
[9] Halstead SB, Nimmannitya S and Cohen SN. [17] Kouri G, Guzman MG, Valdes L, Carbonel I,
Observations related to pathogenesis of del Rosario D, Vazquez S, Laferte J, Delgado
dengue hemorrhagic fever. IV. Relation of J and Cabrera MV. Reemergence of dengue
disease severity to antibody response and in Cuba: a 1997 epidemic in Santiago de
virus recovered. The Yale Journal of Biology Cuba. Emerging Infectious Diseases, 1998,
and Medicine, 1970, 42: 311-328. 4: 89-92.

[18] Cooke GS and Hill AV. Genetics of [27] Vargas-Alarcon G, Gamboa R, Zuniga J,
susceptibility to human infectious disease. Hernandez-Pacheco G, Ramos-Kuri M,
Nature Reviews Genetics, 2001, 2: 967-977. Castillo E, Gomez-Casado E, Martinez-Laso J,
Arnaiz-Villena A and Granados J. HLA-DR4
[19] Chiewsilp P, Scott RM and Bhamarapravati
allele frequencies on Indian and Mestizo
N. Histocompatibility antigens and dengue
population from Mexico. Human
hemorrhagic fever. The American Journal of
Immunology, 2000, 61: 341-344.
Tropical Medicine and Hygiene, 1981, 30:
1100-1105. [28] Vargas-Alarcon G, Hernandez-Pacheco G,
Gamboa R, Zuniga J, Flores C, Gomez-
[20] Paradoa Perez ML, Trujillo Y and Basanta P.
Casado E, Martinez-Laso J, Granados J and
Association of dengue hemorrhagic fever
Arnaiz-Villena A. Polymorphism and
with the HLA system. Haematologia, 1987,
distribution of HLA-DR2 alleles in Mexican
20: 83-87.
populations. Human Immunology, 2001,
[21] Loke H, Bethell DB, Phuong CX, Dung M, 62: 286-291.
Schneider J, White NJ, Day NP, Farrar J and
[29] LaFleur C, Granados J, Vargas-Alarcon G,
Hill AV. Strong HLA class I--restricted T cell
Ruiz-Morales J, Villarreal-Garza C, Higuera L,
responses in dengue hemorrhagic fever: a
Hernandez-Pacheco G, Cutino-Moguel T,
double-edged sword? The Journal of
Rangel H, Figueroa R, Acosta M, Lazcano E
and Ramos C. HLA-DR antigen frequencies
[22] Stephens HA, Klaythong R, Sirikong M, in Mexican patients with dengue virus
Vaughn DW, Green S, Kalayanarooj S, Endy infection: HLA-DR4 as a possible genetic
TP, Libraty DH, Nisalak A, Innis BL, resistance factor for dengue hemorrhagic
Rothman AL, Ennis FA and fever. Human Immunology, 2002, 63:
Chandanayingyong D. HLA-A and -B allele 1039-1044.
associations with secondary dengue virus
[30] Loke H, Bethell D, Phuong CX, Day N,
infections correlate with disease severity and
White N, Farrar J and Hill A. Susceptibility to
the infecting viral serotype in ethnic Thais.
dengue hemorrhagic fever in Vietnam:
Tissue Antigens, 2002, 60: 309-318.
evidence of an association with variation in
[23] Van Epps HL, Schmaljohn CS and Ennis FA. the vitamin d receptor and Fc gamma
Human memory cytotoxic T-lymphocyte receptor IIa genes. American Journal of
(CTL) responses to Hantaan virus infection: Tropical Medicine and Hygiene, 2002, 67:
identification of virus-specific and cross- 102-106.
reactive CD8(+) CTL epitopes on
[31] van de Winkel JG and Capel PJ. Human IgG
nucleocapsid protein. Journal of Virology,
Fc receptor heterogeneity: molecular aspects
1999, 73: 5301-5308.
and clinical implications. Immunology Today,
[24] Abel L and Dessein AJ. Genetic epidemiology 1993, 14: 215-221.
of infectious diseases in humans: design of
[32] Littaua R, Kurane I and Ennis FA. Human
population-based studies. Emerging
IgG Fc receptor II mediates antibody-
dependent enhancement of dengue virus
[25] McNicoll J. Host genes and infectious infection. Journal of Immunology, 1990,
diseases. Emerging Infectious Diseases, 1998, 144: 3183-3186.
4: 423-426.
[33] Fijen CA, Bredius RG and Kuijper EJ.
[26] Thursz M. MHC and the viral hepatitides. Polymorphism of IgG Fc receptors in
The Quarterly Journal of Medicine, 2001, meningococcal disease. Annals of Internal
94: 287-291. Medicine, 1993, 119: 636.

[34] Sanders LA, van de Winkel JG, Rijkers GT, [40] Dinarello CA and Thompson RC. Blocking
Voorhorst-Ogink MM, de Haas M, Capel PJ IL-1: interleukin 1 receptor antagonist in
and Zegers BJ. Fc gamma receptor IIa vivo and in vitro. Immunology Today, 1991,
(CD32) heterogeneity in patients with 12: 404-410.
recurrent bacterial respiratory tract
[41] Ohta M, Okada M, Yamashina I and
infections. The Journal of Infectious Diseases,
Kawasaki T. The mechanism of
1994,170: 854-861.
carbohydrate-mediated complement
[35] MacDonald PN, Dowd DR and Haussler MR. activation by the serum mannan-binding
New insight into the structure and functions protein. The Journal of Biological Chemistry,
of the vitamin D receptor. Seminars in 1990, 265: 1980-1984.
Nephrology, 1994,14: 101-118.
[42] Lipscombe RJ, Sumiya M, Summerfield JA
[36] Roy S, Frodsham A, Saha B, Hazra SK, and Turner MW. Distinct physicochemical
Mascie-Taylor CG and Hill AV. Association characteristics of human mannose binding
of vitamin D receptor genotype with leprosy protein expressed by individuals of differing
type. The Journal of Infectious Diseases, genotype. Immunology, 1995, 85: 660-667.
1999, 179: 187-191.
[43] Madsen HO, Garred P, Kurtzhals JA, Lamm
[37] Bellamy R, Ruwende C, Corrah T, McAdam LU, Ryder LP, Thiel S and Svejgaard A. A
KP, Thursz M, Whittle HC and Hill AV. new frequent allele is the missing link in the
Tuberculosis and chronic hepatitis B virus structural polymorphism of the human
infection in Africans and variation in the mannan-binding protein. Immunogenetics,
vitamin D receptor gene. The Journal of 1994, 40: 37-44.
[44] Super M, Gillies SD, Foley S, Sastry K,
[38] Paul WE and Seder RA. Lymphocyte Schweinle JE, Silverman VJ and Ezekowitz
responses and cytokines. Cell. 1994, 76: RA. Distinct and overlapping functions of
241-251. allelic forms of human mannose binding
protein. Nature Genetics, 1992, 2: 50-55.
[39] Swain SL. T-cell subsets. Who does the
polarizing? Current Biology, 1995, 5: 849-
851.

Identification and Phylogenetic Analysis of DEN-1 Virus
Isolated in Guangzhou, China, in 2002
Jun-lei Zhang, Rui Jian, Ying-jie Wan, Tao Peng and Jing An#
Department of Microbiology, College of Medicine, Third Military Medical University,

Chongqing, 400038, Peoples Republic of China
Abstract
Virus was isolated and identified from the serum samples of patients with suspected classical dengue
fever (DF) in Guangzhou province, China, in 2002. The serum was incubated with the Aedes albopictus
cell line, C6/36, for isolation and 5 of 20 serum samples caused cytopathologic effects on C6/36 cells. A
539-nucleotide (nt) fragment in the NS1 region of the isolated virus genome was amplified using
universal primers of 4 serotypes of DEN viruses. By sequencing the primer-extension production and
blasting in the GenBank, the isolated strain was the closest to rDEN-1 dalte30 (AY145123), which was an
attenuation strain of DEN-1 virus. A 593nt fragment from the Envelope-Nonstructural protein 1 (E/NS1)
of these isolates was also sequenced to compare it with published sequences of other DEN-1 viruses. The
240nt from the E/NS1 region of this DEN-1 virus genome was the closest to DEN-1/T14 strain (M32931),
which was genotype IV. The phylogenetic analysis of the NS1 (480nt) and E/NS1 (240nt) regions of the
gene nucleotide sequences was performed using neighbour-joining methods: 11 strains of DEN-1 virus
were divided into three genotypes: I, IV and V, as defined by Rico-Hesse, or Asia, South Pacific and
Americas/Africa, as defined by Goncalvez. Based on the sequence information and the definition, the
isolated virus (named DEN-1/GZ 2002) strain belonged to DEN-1 and IV or South Pacific genotypes. The
suckling mice died on days 10 to 11 after intracerebral inoculation with the isolated virus. TCID50 of the
virus was 4.37 log pfu/0.2ml. Small plaques with an unclear edge were seen on Vero cells on days 8 to 9
after infection. Combined with clinical data that thousands of patients only showed DF manifestations,
the results suggested that DEN-1/GZ 2002 might be a low-virulence strain.
Keywords: Dengue virus, DEN-1/GZ2002, virulence, phylogenetic analysis.
Introduction RNA, approximately 11,000 nucleotides

long, encoding 10 distinct proteins. The
Dengue (DEN) viruses belong to the genus gene order is 5-C-prM(M)-E-NS1-NS2A-
Flavivirus. There are four closely related, but NS2B-NS3-NS4A-NS4B-NS5-3, expressed
antigenically distinct, virus serotypes (DEN-1, as a single polyprotein that is cleaved by
DEN-2, DEN -3 and DEN-4). The viral both viral and cellular proteases to form the
genome is a positive-sense single-stranded viral polypeptides. The three 5 proteins are
#
E-mail: anjing60@yahoo.com.cn; Tel.: +86-23-68752774; Fax: +86-23-65463259

Identification and Phylogenetic Analysis of Dengue-1 Virus
three structural proteins: capsid (C), I) America, Africa and South-East Asia;
membrane (M) and envelope (E), and the II) Sri Lanka; III) Japan; IV) South-East Asia,
remaining seven are nonstructural (NS) the South Pacific, Australia and Mexico; and
proteins[1]. The DEN viruses cause classical V) Taiwan and Thailand[14]. Recently,
dengue fever (DF) and dengue Goncalvez set up the phylogeny of 36 DEN -
haemorrhagic fever/dengue shock syndrome 1 viruses according full E gene sequences.
(DHF/DSS). DF is a non-specific viral febrile Statistical analyses of the validity of
illness while DHF/DSS is a severe and fatal branching patterns (bootstrap) also
haemorrhagic disease. Up to 100 million suggested five classifications: 1) from Asia;
cases of DF occur annually in the world, 2) from Thailand; 3) from sylvatic/Malaysia;
with some 2 billion people at risk of the 4) from South Pacific; and 5) from the
infection in tropical and subtropical regions Americas/Africa, in which three genotypes
of Africa, Asia and the Americas [2,3]. indicated as 1), 4) and 5) were consistent to
I), IV) and V) mentioned by Rico-Hesse[15] .
The pathogenesis of the DEN virus
Aviles-G and collaborators sequenced
infection is not clear. Although many factors
Capsid-pre Membrane (C/prM) and E/NS1
are proposed to be involved in the DEN
regions of 24 recent isolates of DEN-1 from
virus epidemiology and occurrence of
South America, suggesting that the recent
DHF/DSS, the evolution of the DEN viral
epidemics in Argentina and Paraguay were
virulence and the spread of DEN viruses in
due to the re-emergence of a previously
different geographical areas are two
circulated strain[ 16] . This indicated that small
important features in the epidemiology and
gene fragment sequences are available for
pathogenesis of the disease. Studies have
analysis of viral genetic characterization.
suggested that specific viral structures might
contribute to the replication capability in Current methods for obtaining DEN
the target cells and to their transmission[4] . virus sequences no longer require a viable
Co-circulation of novel DEN genotype, virus isolate. In fact, the entire genome
another genotype of the same serotype or sequences can be obtained by enzymatic
different serotypes of DEN viruses in the amplification of viral RNA template in the
same area, might lead to the displacement patients blood sample. Unfortunately, there
of the native genotype by a new genotype are numerous sequences that contain errors,
and thus to the occurrence of DHF/DSS[5] . which have led to serious mistakes in their
interpretation. Therefore, it is wise to obtain
Several studies have shown that the
virus isolates for further genetic
phylogenetic trees obtained from small gene
characterization[17]. In this study, a viral
fragment sequences are congruent with the
strain that circulated in Guangzhou, China,
trees obtained from an entire gene
in 2002 was isolated first, then identified by
sequence, although there might be minor
amplifying and sequencing 539 nucleotide
rearrangements in the terminal branches[6-13] .
(nt) in NS1 region of DEN virus genome
Using a partial sequence from Envelope-
using universal primers. Also, E/NS1 regions
Nonstructural protein 1 (E/NS1) junction
of the isolated virus were sequenced to
region, Rico-Hesse defined five genotypes
determine their origin.
for DEN-1 viruses isolated worldwide:

Materials and methods primer sequences are: 5'GTG CAC ACA

TGG ACA GAA CA 3'(forward) and 5'CTT
TCT ATC CAA TAA CCC AT 3'(reverse).
Collection of blood samples Their combination generated a 539nt
Twenty patients with suspected DEN virus fragment. Briefly, 5 l of the extracted RNA
infection admitted to the Eighth Peoples was mixed with 50 pmol (2l) of reverse
Hospital of Guangzhou were enrolled 12 primer at 70 C for 5 minutes and cooled
males and 8 females aged between 23-57 down on ice. Then 13 l of RT mix
years. The blood samples were collected on containing 4l of 5X RT XL buffer, 0.5 l of
day 1 to 11 after the onset of fever. The sera 10mM dNTP, 6.0 l of sterile UHQ water,
were separated by centrifugation and stored 0.5l RNase inhibitor and 20 U of AMV
at 70 C until use. reverse transcriptase XL (TaKaRa, Japan)
were added. The mixture was incubated at
Viral isolation 42 C for 1 hour for RT reaction, then
heated to 95 C to inactivate AMV and
Aedes albopictus mosquito cell line, C6/36, cooled down on ice. PCR was subsequently
was cultured in RPMI 1640( pH6.8~ 7.0) carried out by adding 24 l of PCR mix
containing 10% fetal bovine serum (FBS) containing 2.5 l of 10X Taq Polymerase
and 2 mM glutamine with 5% CO 2 at 28 C. buffer (PROMEGA, USA), 2.5 l of 25mM
30 l of the serum sample was diluted with MgCl2, 12.5 pmol each of sense and reverse
1 ml RPMI 1640 containing 2% FBS and primers, 0.5 l of 10mM dNTP, 2U of Taq
was incubated with overnight C6/36 cells for DNA Polymerase (PROMEGA, USA) and
1 hour at 28 C. After removing the serum, 17.5 l of sterile UHQ water to the 1st
the cells were cultured in RPMI 1640 strand synthesis tube containing 1 l of
( pH6.8 ~ 7.0 ) containing 2% FBS at cDNA. PCR with denaturation at 94 C for
28 C. The cells were observed daily for 30 seconds; annealing at 53 C for 30
cytopathic effect (CPE) [18] . If CPE was not seconds; and extension at 72 C for 1
seen initially, the cells were passaged several minute 30 seconds for 30 cycles. The RT-
times to confirm whether there was virus in PCR conditions for the amplification of
the serum samples or not. nucleotides from positions 2107~2701
coding for the E/NS1 fragment were similar
RNA extraction to those used for NS1 fragment, but with an
annealing temperature of 58 C instead of
Viral RNA was extracted from the culture 53 C.
supernatants of infected C6/36 with
ISOGEN (Nippon Gene, Toyama, Japan)
DNA purification and sequencing
according to the manufacturers instructions.
PCR-amplified DNA products were
electrophoresed in 1% agarose gels and
RT-PCR amplification
stained with 1g/ml ethidium bromide. The
Nucleotides from positions 2503~3041 bands of predicted size (539nt for the NS1
coding a fragment of the NS1 gene were fragment and 593nt for E/NS1 fragment)
amplified using RT-PCR. The universal were excised from the gel and purified using

a Golden Beads Product Purification Kit NS1 region and the E/NS1 junction of our
(Songon, CN) according to the isolates were carried out with the neighbor-
manufacturers instructions. Purified PCR joining method (NJ)[20] , calculating bootstrap
products were cloned on to pMD18-T confidence intervals of 1,000 replicates.
Vector (TaKaRa, Japan) and sent to Co. Character state tree-building algorithms
Bioasia, Shanghai, CN, in 15% Glycerol to (PHYLIP package) were also tested. A strict
perform sequencing using an ABI Prism 377 consensus bootstrap tree was obtained by
Genetic Analyzer. Amplifying primers were using the following programmes:
M13-48. (i) SEQBOOT to generate 100 replicas,
(ii) DNADIST and NEIGHBOR to acquire
Computer analysis the tree of each reiterated data, and
(iii) CONSENSE to build a strict consensus
The multiple sequence alignment bootstrap tree. Phylogenetic trees were
programme Clustalx, version 1.8[19] , was drawn using TreeView[21] . Published
used to obtain an optimal nucleotide sequences used in the analysis are listed in
sequence alignment file. Phylogenetic Table 1.
analysis of nucleotide sequences from the
Table 1. Dengue viral sequences from GenBank used in the phylogenetic analysis
Virus Strains Origin Year isolated Accession number
DEN-1 FGA/89 French Guiana 1989 AF226687
DEN-1 BR/90 Brazil 1990 AF226685
DEN-1 S275/90 Singapore 1990 M87512
DEN-1 Abidjan Ivory Coast 1999 AF298807
DEN-1 WestPac/74 Nauru 1974 U88535
DEN-1 A88 Indonesia 1988 AB074761
DEN-1 GD05/99 Guangdong, CN 1999 AY376738
DEN-1 GD23/95 Guangdong, CN 1995 AY373427
DEN-1 GZ/80 Guangzhou, CN 1980 AF350498
DEN-1 Mochizuki Japan 1943 AB074706
DEN-1 Djibouti Djibouti 1998 AF298808
DEN-2 TR1751* Trinidad 1954 M32969

* The full-length gene segment sequence of this viral strain is unavailable in GenBank and the NS1 region in the
phylogenetic analysis was sequenced by us

The resulting unrooted trees were out- Figure 1. Cultured C6/36 cells:
grouped to the sequence of DEN-2 (TR1751), (a) C6/36 cells without DEN-1 infection;
which was kindly provided by Dr Oya A (b) Cell-cell fusion and syncytia on C6/36
(National Institute of Infectious Diseases, cells was seen on day 6 after infection
Japan) and isolated from a DF patient[22,23] . with DEN-1/GZ2002
The RT-PCR amplification of the NS1 region
of this virus strain using universal primers and
sequencing of amplified products was done
as described above.
Biological character detection

The virus isolated from the serum samples
was propagated in C6/36 cells (MOI = 1)
and the titer was determined by the plaque
assay using Vero cells monolayer culture Figure 1 (a)
under methylcellulose overlay medium.
TCID50 of the virus was also measured by
the plaque assay and calculated using Reed-
Muench method[24] . Eighteen suckling mice
were intracerebrally (ic) inoculated with
104 pfu/mouse of the virus for observing
clinical signs and their survival time.
Results
Figure 1 (b)
General observation
After two passages, 5 of the 20 serum Figure 2. Plaque formation on Vero cells
specimens caused CPE on C6/36 cells. was detected on day 8 after infection
Infected C6/36 cells became syncytia and A. Blank control; B. Vero cells were
cell-cell fusion. Some cells showed necrosis infected with DEN-2/Tr1751; C and D. Vero
and got detached from the plate at a late cells were infected with DEN-1/GZ2002
stage of the infection (Figure 1). The virus
was propagated in C6/36 cells and the
highest titer was 5105 pfu/ml at day 6 post-
infection (pi). Using the Reed-Muench
method, TCID50 of the virus was 4.37 log
pfu/0.2ml. Plaque formation on Vero cells
was detected on day 8 or 9 after infection
and they were small and unclear (Figure 2).
After ic inoculation with isolated virus, all of
the suckling mice showed kyphoscoliosis
and paralysis of hind legs and died on days
10 to 11 after infection.

Comparison of nucleotide which was an attenuation strain of DEN-1

sequences virus. According to the full-length gene
sequences of rDEN-1 dalte30 (AY145123),
As shown in Figure 3, a 539nt fragment we designed the primers to amplify the
from the NS1 region (positions 2503 to nucleotides of a fragment from the E/NS1
3041) was amplified using the universal region (positions 2107 to 2701). The 240nt
primer by RT-PCR and the amplified from the E/NS1 region (position 2309 to
products were sequenced to determine the 2548) of this DEN-1 virus genome is the
relationships among the 5 isolates and their closest to DEN-1/T14 strain from Australia
origin. Comparisons of the NS1 region of isolated in 1981 (M32931), which was
the 5 isolates virus strains showed little genotype IV, their divergence was 2%.
divergence, so we chose one as the Based on sequence information and
representative strain to compare with the definition by Rico-Hesse, our isolates
sequences published in GenBank. The belonged to DEN-1 and genotype IV,
nucleotide sequence of this strain was the named DEN -1/GZ2002.
closest to rDEN-1 dalte30 (AY145123),
Figure 3. Agarose gel analysis of the cDNA products from RT-PCR of RNA samples isolated
from the supernatant of the infected C6/36 cells
(a) After amplification with the universal primer, a band of 539nt for the NS1 fragment
was seen. Lanes 2-6: PCR amplified DNA products from different samples respectively;
lane 7: blank control; lanes 1 and 8: 1Kb DNA Ladder marker (GeneRulerTM )
(b) After amplification with a primer for 593nt fragment of E/NS1 region, an expected
band about 593nt was seen (lane 1), Lane 2: 1Kb DNA Ladder marker (GeneRulerTM )
Figure 3 (a)
Figure 3 (b)

Phylogenetic analysis branches (Figure 4). From the NJ trees in our

study, 11 strains of the DEN-1 virus were
We compared 480nt from the NS1 region divided into three genotypes coinciding with
(position 2516 to 2995) and 240nt from the I, IV and V as defined by Rico-Hesse or Asia,
E/NS1 region (position 2309 to 2548) of this South Pacific and Americas/Africa, as
DEN-1 virus genome with sequences of defined by Goncalvez. Our isolated DEN -
other published DEN -1 viruses. The 1/GZ2002 strain belonged to genotype IV or
phylogenetic analysis of the NS1 (480nt) South Pacific genotype. In addition, it was
and E/NS1 (240nt) regions of the gene found that an arrangement of the S275/90
nucleotide sequences was performed using strain showed a major difference between
NJ methods respectively. Two NJ trees were the two trees, which supports the previous
set up and they showed a similar result, hypothesis that the S275/90 strain is a
except for a little difference in terminal recombined strain[25] .
Figure 4. Phylogenetic relationship of DEN-1/GZ2002 to previously characterized DEN-1

viruses. Two phylogenetic trees were set up using 240nt from the E/NS1 region, position
2309 to 2548 (a) and using 480nt from the NS1 region, position 2516 to 2995 (b) of these
DEN-1 virus genomes. The resulting unrooted trees were out-grouped to the sequence of
DEN-2 (TR1751)
Discussion vector and the environment. DEN virus

evolution is also determined by many
It is evident that the DEN virus complex interactions. Some genetic changes
epidemiology is determined by many factors, that occur during the natural transmission
including those in the host, the virus, the cycles of the DEN virus might affect its

virulence and cause the disease. Therefore, in DEN virus evolution and the NS1
understanding DEN virus variation is sequences were a highly conserved region.
especially important to clarify the In our study, after sequencing E/NS1 (240nt)
pathogenesis of DEN virus infection. and the NS1 (480nt) gene region, we
Although current methods of obtaining DEN analysed the phylogenetic relationship of 11
virus sequences no longer require viable DEN-1 virus representatives of three
virus isolates, virus isolates are necessary for genotypes coincidence with I, IV, and V as
further studies of the biological and genetic referred to by Rico-Hesse (1990), or Asia,
characteristics[17]. In this study, we isolated South Pacific and Americas/Africa, as
DEN-1 virus that circulated in Guangzhou in defined by Goncalvez. This indicated that
2002 and analysed its possible origin and the isolated virus belonged to genotype
partial biological features. IV/South Pacific.
In an In vitro experiment, the virus Tolou completed the genome sequence
caused typical CPE on C6/36 cells such as analysis to demonstrate the likelihood of
syncytia and cell-cell fusion, which was recombination between different strains of
continuously shown when the virus DEN-1 virus. The region of 1295~2592nt
passaged several times. Their titer was considered to be a hot spot for the
maintained a level about 5 105 pfu/ml recombination[25] . We sequenced one
during passages. On Vero cells, a small and region outside (2516~2995nt) and one
unclear plaque, a biological marker of region inside (2309~2548nt) the possible
attenuation, was seen. The suckling mice recombination site. No recombination event
survived for 11 days after ic inoculation. occurred in our strains, at least in the
Interestingly, the sequences of 539nt from regions analysed. However, no virus isolates
the NS1 were very close to rDEN-1 dalte30 meeting the stringent criteria for
(AY145123), which was an attenuation recombination have yet been described and
strain of DEN-1 virus. Combined with it remains to be determined whether and at
clinical data where thousands of patients what frequency DEN viruses undergo
only showed DF clinical signs, our results recombination in nature [17].
indicated that the isolated virus might be a
The evolution of DEN viruses has had a
low-virulence strain.
major impact on their virulence for humans
As is known, a comparison of full-length and on the epidemiology of the DEN virus
gene segment sequences is impractical, around the world. It is necessary that a more
especially when looking at a large number complete and systematic survey of DEN-1
of samples. Recently, several studies have samples be undertaken before a link
shown that the phylogenetic trees obtained between specific genotypes and the
from small gene fragment sequences are virulence of these viruses can be established.
highly congruent with those obtained from
an entire gene sequence, except for a minor
rearrangement in the terminal branches, Acknowledgment
suggesting thereby that small gene fragment This work was partially supported by grants
sequences were effective for the study of nos. 30170848 and 30300303 from the
viral genetic characteristics[6-13] . The E/NS1 National Science Foundation of China
junction sequences were a useful indicator (NSFC).

References
[1] Trent DW, Manske CL, Fox GE, Chu MC, [9] Morzunov SP, Feldmann H, Spiropoulou CF,
Kliks SC and Monath TP. The molecular Semenova VA, Rollin PE, Ksiazek TG, Peters
epidemiology of dengue viruses: Genetic CJ and Nichol ST. A newly recognized virus
variation and microevolution. Applied associated with a fatal case of hantavirus
Virology Research, 1990, 2: 293-315. pulmonary syndrome in Louisiana. Journal
[2] Monath TP and Heinz FX. Flaviviruses. In: of Virology, 1995, 69: 1980-1983.
Fields BN, Knipe DM and Howley PM (Eds.), [10] Nichol ST, Spiropoulou CF, Morzunov S,
Fields Virology. Philadelphia: Lippincott Rollin PE, Ksiazek TG, Feldmann H, Sanchez
Raven, 1996, 9611034. A, Childs J, Zaki S and Peters CJ. Genetic
identification of a hantavirus associated with
[3] Monath TP. Dengue: the risk to developed an outbreak of acute respiratory illness.
and developing countries. Proceedings of Science, 1993, 262: 914-917.
the National Academy of Sciences of the [11] Ravkov EV, Rollin PE, Ksiazek TG, Peters CJ
United States of America, 1994, 91: 2395- and Nichol ST. Genetic and serologic
2400. analysis of Black Creek Canal virus and its
[4] Leitmeyer KC, Vaughn DW, Watts DM, association with human disease and
Salas R, Villalobos I, de Chacon, Ramos C Sigmodon hispidus infection. Virology, 1995,
and Rico-Hesse R. Dengue virus structural 210: 482-489.
differences that correlate with pathogenesis. [12] Spiropoulou CF, Morzunov S, Feldmann H,
Journal of Virology, 1999, 73: 4738-4747. Sanchez A, Peters CJ and Nichol ST.
[5] Rico-Hesse R, Harrison LM, Salas RA, Tovar Genome structure and variability of a virus
D, Nisalak A, Ramos C, Boshell J, de Mesa causing hantavirus pulmonary syndrome.
MT, Nogueira RM and da Rosa AT. Origins Virology, 1994, 200: 715-723.
of dengue type 2 viruses associated with [13] Xiao SY, Leduc JW, Chu YK and Schmaljohn
increased pathogenicity in the Americas. CS. Phylogenetic analyses of virus isolates in
Virology, 1997, 230: 244-251. the genus Hantavirus, family Bunyaviridae.
[6] Hjelle B, Krolikowski J, Torrez Martinez N, Virology, 1994, 198: 205-217.
Chavez-Giles F, Vanner C and Laposata E. [14] Rico-Hesse R. Molecular evolution and
Phylogenetically distinct hantavirus distribution of dengue viruses type 1 and 2
implicated in a case of hantavirus pulmonary in nature. Virology, 1990, 174: 479-493.
syndrome in the northeastern United States.
[15] Goncalvez AP, Escalante AA, Pujol FH,
Journal of Medical Virology, 1995, 46: 21-
Ludert JE, Tovar D, Salas RA and Liprandi F.
27.
Diversity and evolution of the envelope
[7] Khan AS, Spiropoulou CF, Morzunov S, Zaki gene of dengue virus type 1. Virology, 2002,
SR, Kohn MA, Nawas SR, McFarland L and 303: 110119.
Nichol ST. Fatal illness associated with a [16] Aviles G, Rowe J, Meissner J, Manzur
new hantavirus in Louisiana. Journal of Caffarena JC, Enria D and St Jeor S.
Medical Virology, 1995, 46: 281-286. Phylogenetic relationships of dengue-1
[8] Liang M, Li D, Xiao SY, Hang C, Rossi CA viruses from Argentina and Paraguay.
and Schmaljohn CS. Antigenic and Archives of Virology, 2002, 147: 2075-2087.
molecular characterization of hantavirus [17] Rico-Hesse R. Microevolution and virulence
isolates from China. Virus Research, 1994, of dengue viruses. Advances in Virus
31: 219-233. Research, 2003, 59: 315-341.

[18] Gubler DJ, Kuno G, Sather GE, Velez M and [22] Igarashi A. Isolation of a Singh's Aedes
Oliver A. Mosquito cell cultures and specific albopictus cell clone sensitive to dengue and
monoclonal antibodies in surveillance for Chikungunya viruses. The Journal of General
dengue viruses. The American Journal of Virology, 1978, 40: 531-544.
Tropical Medicine and Hygiene, 1984, 33: [23] An J, Kimura Kuroda J, Hirabayashi Y and
158-165. Yasui K. Development of a novel mouse
[19] Thompson JD, Higgins DG, Gibson TJ and model for dengue virus infection. Virology,
Clustal W. Improving the sensitivity of 1999, 263: 70-77.
progressive multiple sequence alignment [24] Reed LJ and Muench H. A simple method of
through sequence weighting, position estimating fifty percent endpoints. American
specific gap penalties and weight matrix Journal of Hygiene, 1938, 27:493-497.
choice. Nucleic Acids Research. 1994, 22,
4673-4680. [25] Tolou HJ, Couissinier Paris P, Durand JP,
Mercier V, de Pina JJ, de Micco P, Billoir F,
[20] Saitou N and Nei M. The neighbor-joining Charrel RN and de Lamballerie X. Evidence
method: a new method for reconstructing for recombination in natural populations of
phylogenetic trees. Molecular Biology and dengue virus type 1 based on the analysis of
Evolution, 1987, 4: 406-425. complete genome sequences. The Journal of
[21] Page RD. TREEVIEW: An application to General Virology, 2001, 82: 1283-1290.
display phylogenetic trees on personal
computers. Computer Applications in the
Biosciences: CABIOS, 1996, 12: 357-358.

Induction of Cytotoxic T Lymphocytes by Immunization
with Dengue Virus Derived, Modified Epitope Peptide,
Using Dendritic Cells as a Peptide Delivery System
Yoshiki Fujii*, Hideyuki Masaki** #, Takanori Tomura**, Kiyohiro Irimajiri*
and Ichiro Kurane***
*Department of Pharmacotherapy, Kinki University School of Pharmaceutical Sciences,
Higashi-Osaka, Osaka, Japan
** First Department of Biochemistry, Kinki University School of Medicine, Osaka-Sayama,
Osaka 589-8511, Japan
***Department of Virology 1, National Institute of Infectious Diseases, Shinjuku, Tokyo, Japan
Abstract
A single 9-amino acid peptide of the defined murine cytotoxic T lymphocyte (CTL) epitope (named
peptide-1), which corresponds to the amino acid residues 298-306 (GYISTRVEM) of NS3 of dengue virus
serotypes DEN-2 and 4, was examined for induction of specific CTLs. Immunization of BALB/c mice
subcutaneously with the peptide-1 emulsified with complete Freund adjuvant (CFA) did not induce
specific CTLs. The peptide-2 (GYISTRVEL), in which the residue (M) at 9th position of the peptide-1 was
substituted for L, was prepared. The peptide-2 possessed the complete H-2Kd-binding motif. Intravenous
immunization with 5x10 5 dendritic cells (DCs) pulsed with the peptide-2-induced specific CTLs.
Furthermore, subcutaneous immunization with the peptide-2 emulsified with CFA-induced CTLs which
lysed peptide-1-pulsed target cells as well as peptide-2-pulsed ones. These results indicate that
immunization with dengue virus-derived CTL epitope peptide induces specific CTLs, and that DC can be
used as a vehicle for the modified epitope peptide.
Keywords: Dengue virus, cytotoxic T lymphocyte, dendritic cell, epitope, peptide, binding motif.
Introduction have CTL epitope expressed in host cells by

infection with live viruses, or by
Major histocompatibility complex (MHC) administration of an expression plasmid
class I restricted, CD8+ cytotoxic T vector (i.e. DNA vaccine) in which the
lymphocytes (CTLs) are known to play an epitope gene is incorporated. The other is
essential role in the recovery from viral immunization with a defined CTL epitope
infection by lysing virus-infected cells[1] . peptide. The former strategy is more
There are two major strategies to induce physiological; however, the preparation of
CTL-mediated protective immunity. One is to immunogen is often difficult and there is a
#
E-mail: masaki@med.kindai.ac.jp; Tel.: +81-72-366-0221, Ext.3152; Fax: +81-72-366-0206

Induction of Cytotoxic T Lymphocytes by Immunization with Dengue Virus
potential risk that the immunogen may be the Animal Facility of Kinki University School
pathogenic. Immunization with a CTL of Medicine under conventional conditions.
epitope peptide is relatively easy; however, Mice were used at the age of 6 to 12 weeks.
the epitope varies depending on T cell
receptor repertoires and MHC class I
Cells
haplotypes. Furthermore, the peptide Murine mastcytoma line, P815 (H-2d), was
administrated into the body may be used as target cells in CTL assays. The cells
degraded or washed away soon. Thus, the were maintained in RPMI 1640 medium
peptide is usually less immunogenic for CTL (Sigma, St. Louis, MO) with 5x10-5M 2-
induction, and an appropriate delivery mercaptoethanol (2-ME), 100U penicillin,
system is necessary for induction of CTLs [2,3]. 100g/ml streptomycin, 10mM HEPES, and
10% heat-inactivated fetal calf serum
Dendritic cells (DCs), which are potent
(Complete medium) at 37 C in 5% CO2.
antigen-presenting cells, are postulated to be
one of the peptide delivery systems for
inducing CTLs [2,4] . It has been reported that Peptides
intravenous immunization with DCs pulsed The peptide-1 (GYISTRVEM), which
with virus-derived peptides, or tumour- corresponds to the amino acid residues 298-
derived peptides, elicits specific CTLs [5-7] . 306 of NS3 of dengue virus types 2 and 4,
Dengue viruses cause dengue fever and and the peptide-2 (GYISTRVEL) were
dengue haemorrahgic fever/dengue shock synthesized with 9-fluorenylmethoxycarbonyl
syndrome (DHF/DSS). Vaccine development chemistry by Sigma Genosis, Japan. The
against dengue virus infection has not been purity was determined to be 95.0% for the
accomplished yet. It is important to analyse peptide-1 and 96.2% for the peptide-2 by
CTL responses elicited by a single epitope in reverse phase HPLC.
order to understand the role of CTLs in
dengue virus infection. In the present study, Induction of dendritic cells
we employed a single 9-amino acid (a.a.)
BALB/c mouse bone marrow cells (9x105)
peptide, in which C-terminal residue was
were cultured in 1ml of AIM-V medium
replaced to provide the complete H-2K d-
(Invitrogen, Carlsbad, CA) supplemented with
binding motif. This peptide was a derivative
20ng/ml mouse GM-CSF (R&D Systems,
of the defined H-2K d-restricted 9-a.a. CTL
Minneapolis, MN) in 24-well plate at 37 C
epitope peptide that corresponds to the
in 5% CO2. On days 4 and 6, 50 to 75%
residues 298-306 of NS3 of dengue virus
volume of the culture medium was changed
types 2 and 4[8] . We examined whether
with the fresh one supplemented with the
intravenous immunization with bone
same amount of GM-CSF. On day 7, cells
marrow-derived DCs pulsed with this peptide
were harvested, subjected to flow cytometry
elicited specific CTL response.
analysis and used as DCs for immunization.
Materials and methods Flow cytometry analysis

Bone marrow-derived cells (4x105) were
Mice incubated with 1g of FITC-conjugated anti-
Female BALB/cAJcl mice were purchased mouse I-Ad/I-Ed antibody (BD PharMingen,
from Clea, Japan, and were maintained in San Diego, CA), PE-conjugated anti-mouse

CD86 antibody (BD PharMingen), and and 10 U recombinant mouse IL-2 was
biotinylated anti-mouse CD11c (BD added. On day 7, the cells were harvested
PharMingen) in 100l of phosphate buffered and used as CTL (cytotoxic T lymphocyte)
saline (PBS) containing 0.02% NaN 3 effector cells. Mice were also immunized by
(PBS/NaN3) at 4 C for 20 minutes. Same subcutaneous injection with 1 n mole of the
amounts of FITC-conjugated rat IgG2a, (BD peptide emulsified with complete Freund
PharMingen), PE-conjugated rat IgG2a, (BD adjuvant (CFA) into two-foot pads. In this
PharMingen) and biotinylated hamster IgG immunization protocol, draining lymph node
group 1, (BD PharMingen) were used as (popliteal lymph nodes) cells were used as
isotype controls. The cells were washed three the effector cells after stimulation in vitro with
times with PBS/NaN 3 at 4 C, and then the peptide as described above. When mice
incubated with 0.1g of streptoavidin- were immunized with the peptide-1, the cells
conjugated Cy-ChromeTM (BD PharMingen) were stimulated in vitro with peptide-1-
in 100l of PBS/NaN 3 at 4 C for 20 minutes. pulsed spleen cells.
The cells were washed three times, fixed with
1ml of PBS containing 1% paraformaldehyde, Cytotoxicity assays
and analysed by a FACS Calibur (Becton
P815 cells (1x106) were pulsed with the
Dickinson, San Jose, CA) and CELL QuestTM
version 3.3 software. peptide at a concentration of 10M in
complete medium at 37 C for 3 hours. The
cells were labelled with 100 Ci of
Immunization and CTL induction Na251CrO 4 (NEN Life Science Products,
Bone marrow cells (10x106) stimulated with Boston, MA) for one hour, then washed three
GM-CSF for 7 days were incubated in the times and suspended in complete medium.
presence of 10M peptide-2 in 1ml of AIM-V Peptide-pulsed, 51Cr-labelled cells were
at 37 C for 2 hours. The cells were washed seeded in 96-well V-bottom plate at 1.5x103
two times with RPMI-1640. Peptide-2-pulsed cells in 100l of complete medium per well.
cells (2x106) were injected intravenously into Effector cells were added to the plate to
BALB/c mice. Four weeks later, the spleens make various effector/target ratios (E/T ratios)
were collected, minced into single cell in a total volume of 0.2ml per well, and the
suspension, erythrocyte-lysed, and treated plate was incubated at 37 C in 5% CO2 for
with anti-CD4 antibody (BD PharMingen) at four hours. The supernatant fluids were
the rate of 1g/1x107 cells and 10% baby harvested with a Supernatant Collecting
rabbit complement (Cederlane, Hornby, Ont, System (Skatoron, Lier, Norway), and 51Cr
Canada) to deplete CD4-positive cells. Five content was measured by a gamma counter
million cells were then co-cultured with the (Aloka model ARC-300). Maximum 51Cr
same number of peptide-2-pulsed, 33Gy X- release was determined by adding 0.1%
ray-irradiated syngeneic spleen cells in 2ml of Triton X, and spontaneous 51Cr release was
EHAA medium (Sigma) supplemented with determined with the wells that contained
100g/ml nucleic acid precursors, 2mM L- target cells and medium only. Assays were
glutamine, 5x10-5M 2-ME, 100U penicillin, performed in triplicate, and the mean value
100g/ml streptomycin, 10mM HEPES, and was used to calculate percent-specific lysis
10% fetal calf serum in 24-well plate at with the following formula: % specific lysis =
37 C in 5% CO2. On day 4, half volume of 100 x [(release with effector cells
the medium was replaced with fresh one, spontaneous release) / (maximum release

spontaneous release)]. Spontaneous release were cultured with GM-CSF for seven days,
did not exceed 28.9% of the maximum by flow cytometry three colour analysis, and
release. evaluated the purity of DC. As shown in
Figure 1a, 25.07% of the bone marrow cells
strongly expressed both I-Ad/I-Ed and CD86.
Results and discussion The percentage of CD11c-positive cells in
these double positive cells was 95.81%
It is known that mature murine DCs strongly
(Figures 1b and 1c). In contrast, freshly
express major histocompatibility complex
isolated bone marrow cells did not express
(MHC) class II antigen, co-stimulatory
these surface molecules (data not shown).
molecules CD80 and CD86, and CD11c, the
These results suggest that bone marrow cells
chain of p150/95 2-integrin[9,10] . We
were differentiated into DCs during the
examined the expression of MHC class II
culture with GM-CSF for seven days and that
antigen I-Ad/I-Ed, CD86, and CD11c on
DCs accounted for one-fourth of the entire
BALB/c mouse bone marrow cells, which
population.
Figure 1. Expression of the dendritic cell markers on bone marrow cells after culture
in the presence of GM-CSF for 7 days
(a) (b) (c)
R1
CD86 CD11c CD11c
I-Ad/I-E d I-Ad/I-E d CD86

[BALB/c mouse bone marrow cells were cultured for 7 days in AIM-V medium with 20 ng/ml of
murine GM-CSF. The cells were stained with anti I-Ad/I-Ed FITC, anti-CD86 PE, anti-CD11c
biotin, and streptoavidin Cy-Chrome TM. (a); The cells in the region (R1) strongly expressed I-Ad/I-Ed
and CD86, and accounted for 25.07% of the entire cultured bone marrow cells. (b) & (c): The cells
in the region (R1) were examined for the expression of CD11c. 95.81% expressed CD11c.]
We first attempted to induce specific induce specific CTLs might partly be due
CTLs by two foot pad immunizations with to the low binding affinity to H-2K d MHC
the peptide-1 (GYISTRVEM) emulsified class I molecule, because the peptide-1
with CFA. Specific CTL activity was not possesses only one anchor residue (Y) for
detected in draining lymph node cells. binding to H-2K d molecule. We, therefore,
(Table, Experiment no. 1). We speculated prepared the peptide-2 (GYISTRVEL) in
that the inability of the peptide-1 to which the last residue M of the peptide-1

was substituted for L in order to provide were DCs and it was reported that
the complete H-2K d-binding motif [11]. intravenous immunization with 1x105 to
5x10 5 purified DCs pulsed with peptides
It was reported that immunization
induced antiviral immunity[5] . Spleen cells
with virus epitope peptidepulsed DCs
from the mice immunized with peptide-2-
efficiently induced virus -specific CD8+
pulsed DCs lysed peptide-2-pulsed P815
CTLs and protective immunity[5,6] . We
cells in a dose dependent fashion after
attempted to induce specific CTLs by
stimulation in vitro with X-ray-irradiated,
immunization with peptide-2-pulsed DCs.
peptide-2-pulsed syngeneic spleen cells in
We intravenously injected 2x106 bone
the presence of recombinant IL-2 for
marrow cells, which were stimulated with
seven days (Table, Experiment no. 2). This
GM-CSF for seven days and pulsed with
result demonstrates that intravenous
the peptide-2, into BALB/c mice. One-
immunization with peptide-2-pulsed DCs
fourth of the cultured bone marrow cells
induced peptide-2-specific CTLs.
Table. Induction of specific CTLs by immunization with peptide-2-pulsed dendritic cells

and the peptide-2 emulsified with complete Freund adjuvant
Experi- % Specific lysis*

ment Immunization E/T ratio Peptide-1- Peptide-2-
no. Non-pulsed
pulsed pulsed
1. Peptide-1/CFA 20 5.4 7.9 Not done
100 22.9 21.9 Not done
2. Peptide-2/CFA 10 6.5 Not done 36.8
20 8.4 Not done 55.2
40 9.9 Not done 65.4
Peptide-2-pulsed DC 10 20.9 Not done 24.5
20 18.7 Not done 35.8
40 24.0 Not done 44.2
3. Peptide-2/CFA 5 12.7 23.0 23.9
10 20.8 30.4 34.2
20 28.7 46.3 48.3
* 51Crlabeled P815 mastcytoma (H-2d) pulsed with the peptide (10M, 3hours) were used as target cells.
Specific CTL activity was observed in pulsed ones. (Table, Experiment nos. 2 and
the draining lymph node cells after 3). These results suggest that the peptide-2
immunization with peptide-2/CFA. which has the complete binding motif to H-
Interestingly, CTLs induced by peptide- 2kd molecule can induce specific CTLs
2/CFA demonstrated lower levels of non- when used with CFA, and that peptide-2-
specific cytotoxic activity to P815 cells than specific CTLs also lyse original peptide-1-
those induced by peptide -2-pulsed DCs. pulsed target cells. Induction of higher levels
Peptide-2/CFA-induced CTLs lysed peptide- of non-specific cytotoxicity by immunization
1-pulsed target cells as well as peptide-2- with DCs may be due to the high antigen

presentation ability of DCs to prime various activiity. The data, however, only suggest
repertoires of T cells. The other possibility is how efficiently measurable specific CTLs
the difference in the source of lymphocytes. can be induced in vitro. It is plausible that
We observed that spleen-derived intravenous immunization with peptide-
lymphocytes tended to show higher levels of pulsed DCs induces higher levels of specific
non-specific lysis than lymph node -derived CTLs in vivo and protective immunity
cells (data not shown). It seems that the against viral infections. Moreover, CFA is not
draining lymph node cells from mice accepted for human use. Thus,
immunized with the peptide and CFA are a immunization strategy using peptide-pulsed
better source of CTLs than the spleen cells DCs is still worth investigating for induction
from those immunized with peptide-pulsed of CTL-mediated anti-dengue virus
DCs because of low non-specific cytotoxic immunity.
References
[1] Gotch F, Gallimore A and McMichael A. syngeneic irradiated HIV-1 envelope derived
Cytotoxic T cells protection from disease peptide-pulsed dendritic cells. Int Immunol,
progression protection from infection. 1993, 5: 849-857.
Immunol Lett, 1996, 51: 125-128. [7] Porgador A, Snyder D and Gilboa E.
[2] Toes RE, van der Voort EI, Schoenberger SP, Induction of antitumor immunity using bone
Drijfhout JW, van Bloois L, Storm G, Kast marrow-generated dendritic cells. J Immunol,
WM, Offringa R and Melief CJ. 1996, 156: 2918-2926.
Enhancement of tumor outgrowth through [8] Spaulding AC, Kurane I, Ennis FA and
CTL tolerization after peptide vaccination is Rothman AL. Analysis of murine CD8+ T-cell
avoided by peptide presentation on clones specific for the dengue virus NS3
dendritic cells. J Immunol, 1998, 160: 4449- protein: flavivirus cross-reactivity and
4456. influence of infecting serotype. J Virol, 1999,
[3] BenMohamed L, Belkaid Y, Loing E, Brahimi 73: 398-403.
K, Gras-Mass H and Druihe P. Systemic [9] Maraskovsky E, Brasel K, Teepe M, Roux ER,
immune responses induced by mucosal Lyman SD, Shortman K and McKenna HJ.
administration of lipopeptides without Dramatic increase in the numbers of
adjuvant. Eur J Immunol, 2002, 32: 2274- functionally mature dendritic cells in Flt3
2281. ligand-treated mice: multiple dendritic cell
[4] Steinman RM. The dendritic cell system and subpopulations identified. J Exp Med, 1996,
its role in immunogenicity. Annu Rev 184: 1953-1962.
Immunol, 1991, 9: 271-296. [10] Son YI, Egawa S, Tatsumi T, Redlinger RE Jr,
[5] Ludewig B, Ehl S, Karrer U, Odermatt B, Kalinski P and Kanto T. A novel bulk-culture
Hengartner H and Zinkernagel RM. method for generating mature dendritic cells
Dendritic cells efficiently induce protective from mouse bone marrow cells. J Immunol
antiviral immunity. J Virol, 1998, 72: 3812- Methods, 2002, 262: 145-157.
3818. [11] Romero P, Corradin G, Luescher IF and
[6] Takahashi H, Nakagawa Y, Yokomuro K and Maryanski. H-2Kd-restricted antigenic
Berzofsky JA. Induction of CD8+ cytotoxic T peptides share a simple binding motif. J Exp
lymphocytes by immunization with Med, 1991, 174: 603-612.

Molecular Characterization of Brazilian Dengue Viruses
Marize Pereira Miagostovich#, Flvia Barreto dos Santos
and Rita Maria Ribeiro Nogueira
Laboratory of Flavivirus, Department of Virology, Oswaldo Cruz Institute, Oswaldo Cruz Foundation,
Fiocruz Avenida Brasil 4365 Manguinhos, Rio de Janeiro 21040-360, RJ, Brazil
Abstract
Many countries in Central and South America as well as Brazil have been characterized by a rise in
dengue endemicity. Since 1986, dengue infection has gained endemicity in these countries and more
than 3 million dengue cases have been reported along with the emergence also of the severe forms of
the disease. Once intratypic variations among dengue virus (DEN) serotypes have been associated with
the disease severity, the molecular characterization of DEN becomes an indispensable tool for the
laboratories performing virological surveillance programmes. In countries endemic for DEN, as in Brazil,
the monitoring of DEN activity should be an ongoing programme to detect the eventual introduction of
new serotypes/genotypes to curb the impact of the circulating strains. Here, the molecular
epidemiological studies performed on Brazilian DEN strains are presented in order to contribute to a
better understanding of the dengue epidemiology in the country.
Keywords: Dengue viruses, molecular epidemiology, genotypes, Brazil.
Introduction frame (ORF) of over 10,000 nucleotides that

encodes a polyprotein precursor of about
The dengue (DEN) virus belongs to the 3,400 amino acid residues which is co- and
Flaviviridae family, genus Flavivirus, and it has post-translationally processed by the host cell
four distinct antigenic serotypes (1 to 4) that and virus-specific protease to yield structural
cause a spectrum of diseases ranging from and nonstructural proteins. The coding
asymptomatic, mild, undifferentiated fever protein region starts with the sequence for
and classic dengue fever (DF) to more severe the core (C), precursor of membrane (prM/M)
forms known as dengue haemorrhagic fever and the envelope (E) structural proteins,
(DHF) and dengue shock syndrome (DSS) [1,2]. followed by a series of seven nonstructural
The DEN virus is a spherical particle of (NS) proteins ordered as follows: NS1, NS2A,
approximately 500 in diameter, lipid NS2B, NS3, NS4A, NS4B and NS5. The ORF
enveloped that includes one segment of a is flanked by two untranslated regions (5 and
single-stranded positive sense RNA with 3 UTR) and has a type I cap at its 5 end
~11,000 nucleotides in length. The genomic (m7GpppAmp) and appears to lack a 3 -
RNA contains a single long open reading terminal poly A tract[3,4].
#
E-mail: marizepm@ioc.fiocruz.br; Fax: 55-21-2598-4373

Electron micrographs show that the characterization of DEN strains isolated in

virion is characterized by an electron dense Brazil during the last 18 years since dengue
core that consists of an isometric became endemic and surveillance
nucleocapsid, made up of a single C (100 programmes were implemented in the
amino acids) protein, surrounded by a country.
double lipid layer, whereas both E (495
amino acids) and M (75 amino acids)
proteins are associated[5] . E-glycoprotein is DEN-1
the major surface protein and as showed by The first molecular analysis for DEN Brazilian
crystallography, the flat elongated dimmer strains characterization was performed by an
extends parallel to the viral membrane [6] . The analysis on genome fragments from DEN-1
E protein is associated with a number of by using restriction endonuclease (RE)
biological activities, being the most important enzymes. In this study, restriction fragment
antigen with regard to virus biology and heterogeneity by Hae III digestion of cDNA
immunity[7] . products was used to map the distribution of
The intratypic variation of DEN was DEN-1 topotype found in the American
demonstrated by the fingerprinting method region. The strains isolated in the state of Rio
that determined genetic variants within each de Janeiro from human serum specimens
serotype and employed the term topotype to from 1986 to 1994[23,24] were grouped in the
define variants representing samples from the American (Caribbean) topotype, recognized
same geographical region[8] . From 1990 as the only one circulating in the
onwards, the molecular analysis by the partial Americas [8,25] . The percentage of the similarity
sequencing of the DEN genome gathered the observed among DEN-1 Brazilian strains
topotypes in genomic groups (genotypes) and ranged from 60% to 94%, showing the
became an important tool to determine their evolution of those samples since its
genetic variation and to identify risk factors introduction in the state in 1986.
associated with the transmission of particular By the sequencing of 240-nucleotides
strains[9] . (nts) spanning the E/NS1 junction (111 nts
The E gene has been the most from the 3 end of E gene and 129 nts from
commonly surveyed gene in dengue the 5 end of NS1 gene), the DEN-1 Brazilian
molecular epidemiology[10-15] , although genes strains were classified as belonging to
that encode nonstructural proteins and non- genotype I which comprises of strains from
coding regions have also been used in the the Americas, Africa and South-East Asia[9] .
phylogeny studies[16-22]. The DEN-1 Brazilian strains were also
Once intratypic variations among analysed by using one-step amplification with
different serotypes were associated with the four primers that target regions spanning
disease severity, technologies for the polymorphic endonuclease restriction
molecular characterization of DEN became specific sites (RSS-PCR) and all of them were
an indispensable tool for laboratories grouped into subtype C, which corresponds
performing virological surveillance to the largest genotypic group of DEN-1
programmes. This paper presents the most described as genotype 1[9,26] . The RSS-PCR
relevant results of the molecular has become an alternative tool routinely used,

which has allowed the characterization of DEN-2 from Brazil, Colombia, Mexico and
DEN strains for molecular epidemiological Venezuela have a common progenitor with
studies performed in endemic countries, those from South-East Asia[36] .
providing rapid identification of viruses
In Brazil, the first molecular
currently circulating[27-30] .
characterization of DEN-2, introduced in the
Another study performed with the DEN- country in 1990[37,38] , was performed by RE
1 Brazilian strains compared the complete analysis and showed a similarity of 80% with
sequences of three strains isolated in 1990, the 1981 Jamaica isolate, suggesting the
1997 and 2001. The genome analysis of spread of those viruses from the Caribbean
those strains revealed a remarkable region to South America[25] .
conservation of the structural proteins and 27
The geographical origin of DEN-2
amino acids substitutions in the nonstructural
Brazilian strains was also established by the
genes, and 12 of them in the NS4B-NS5 and
direct sequencing of cDNA fragments
nine specific to strains BR/97 and BR/01.
amplified by the polymerase chain reaction
Those findings also suggested that
of a fragment encoding amino acids 29 to 94
recombinant events might have occurred,
in the E gene. Considering a divergence of
since some amino acids substitutions were
6% between the nucleotide sequences as a
previously identified in DEN-1 strains
cut-off for genotype classification, it was
sequenced so far[31]. The evidence that the
demonstrated that the Brazilian strains
genetic diversity of DEN might be generated
belonged to the South-East Asian/American
by recombination among those viruses has
genotype. The comparison of the three DEN-
been described[32-34].
2 strains isolated in Rio de Janeiro, two of
them obtained from classic dengue cases and
DEN-2 one from a fatal case, did not identify the
markers for virulence in the region studied[10] .
The DEN-2 fingerprinting analysis of the
American strains showed that this serotype The analysis of DEN-2 samples isolated
exists in the American continent as two in the states of Rio de Janeiro, Cear, Bahia,
topotypes representing strains from the and Alagoas between 1990 and 1995 was
Caribbean region (Puerto Rico) and from the performed by the partial sequencing of nts
Americas, India and South Pacific[8]. By the 1685 and 2504 encompassing the E gene
sequencing of the E/NS1 junction those and demonstrated the spread of this serotype
topotypes could be related to genotype I from Rio de Janeiro to other states[39] .
(Native American) and to genotype III (South- All the characteristics observed in the
East Asian/American), respectively. This latter Brazilian DEN-2 genotype were confirmed by
genotype was introduced into the Americas the full-length analysis of the nucleotide and
in 1981, and was responsible for the first amino acids sequence (GenBank access #
DHF/DSS epidemic that occurred in the AF489932) [40] . The Asian-specific non-
continent and spread throughout the region conserved amino acid differences, previously
over the next two decades[35,36] . The direction described by Leitmeyr et al.[41] as well as
of the transmission from South-East Asia to additional differences specific to the Brazilian
the Americas was demonstrated as well, since strain were found in E, NS3, and NS5

genes[40]. Changes in the E protein could strains presented the same pattern,
affect the immunogenicity or cell presenting consistent and reproducible
entry/tropism, whereas changes in NS3 amplicons of 582bp and 100bp and,
(helicase/protease) and NS5 (RNA-dependent occasionally, extra amplicons of 676 bp or
RNA polymerase) could affect replication 150 bp[29] . The DEN-2 Brazilian RSS-PCR
efficiency. In addition, differences in the pattern was consistent; however, it did not
predicted secondary structure of the 5 and match any of the RSS-PCR patterns
3 untranslated regions were found between previously described by Harris et al.[46] Once
the South-East Asian/American and native the method was developed with DEN-2
American genotypes; in these regions, the isolates obtained from 1964-1986, the
Brazilian isolate was identical to the South- ongoing evolution of those viruses over the
East Asian/American strains in sequence and last 15 years could explain the genetic
consequently in the predicted secondary diversity observed. Despite those
structures[40]. These similarities with the observations, the sequence of the E/NS1
South-East Asian/American genotype were gene junction (GenBank Access # AF529064
also reported recently for the Martinique to AF529078) showed that the Brazilian
703/98 strain after a complete analysis of the DENV-2 strains still belonged to the South-
genome[42] . East Asian/American genotype [29].
In Brazil, this DEN-2 genotype was
responsible for some clinical features, mainly DEN-3
related to the severity of the disease. In
regions where DEN-2 accounted for primary Different from the DEN-3 genotype IV
infections, as in the states of Bahia and (topotype Caribbean) responsible for
Espirito Santo[43] , the most common clinical epidemics in the 60s and 70s, the DEN-3
feature consisted of classic fever, with re-introduced in the American continent after
frequent exanthema, pruritus and a few an absence of 17 years belonged to genotype
severe cases. However, in other states where III (topotype Sri Lanka), represented by
DEN-2 circulated after extensive epidemics strains from Sri Lanka and India, which are
caused by DEN-1, as in Rio de Janeiro, Cear, associated with DHF/DSS cases in those
Pernambuco and Rio Grande do Norte, an countries[13,47] .
increase in the number of severe cases was By the time of DEN-3 isolation in
observed. The first DHF/DSS case was Brazil[48] , RSS-PCR was extremely valuable
reported in Rio de Janeiro after the which once allowed the rapid
introduction of DEN-2 in 1990, and it was characterization of the first strain as subtype
accompanied by an increasing number of C, confirming the introduction and direction
hospital admissions resulting from DEN-2 of transmission of those viruses from Central
secondary infections[44,45] . to South America[28,46] .
By using RSS-PCR we were able to After the RSS-PCR analysis, th e nucleic
analyse geographically and temporally acid sequencing from positions 278 to 2550
distinct Brazilian DEN-2 strains encompassing of DEN genome was performed (Access
ten years (1990 to 2000). The analysis of the number AY038605) and the parsimony
RSS-PCR products showed that all Brazilian analysis generated a phylogram assigning a

Brazilian DEN-3 strain to genotype III, The severity of the disease and the
reconfirming those data[13,28] . The similarity occurrence of deaths resulting from primary
rate of a Brazilian DEN-3 strain to others infections during the DEN-3 epidemic in the
represented by the same subtype III ranged state of Rio de Janeiro in 2002 could be
from 96% to 98% and 98% to 99% for explained partially by the virulence of this
nucleic acid and deduced amino acid particular genotype [28,54] . Fatal cases, resulting
sequences, respectively. The comparison of from primary dengue infections, were
the Brazilian DEN-3 with strains isolated in previously described[55] before the DEN-3
Guatemala showed a total of 14 nucleic acid genotype III introduction in Brazil. However,
substitutions, with one of them resulting in an the highest number of DHF/DSS cases that
amino acid change from histidine to occurred in the state were due to secondary
arginine [28] . infection by the South-East Asian/American
DEN-2 genotype [45] . Those findings
As a result of several DEN-3 epidemics in
corroborated the previous observations that
Latin American countries, a large number of
some DEN strains can be more virulent than
DEN-3 genome sequences have been
others, representing an important risk factor
recently deposited in the GenBank[49-51] . A
for DHF/DSS and that antibody-dependent
phylogenetic study compromising DEN-3
enhancement (ADE) itself does not explain all
strains isolated in Sri Lanka, East Africa and
cases of severe disease [51,56-58] . Recently, it
Latin America confirmed the establishment of
was suggested that the more virulent
the new DEN-3 genotype[51] . According to
genotypes were now replacing those that had
the author, there are two separate lineages
a lower epidemiological impact throughout
formed within genotype III: Group A
the world[59] .
consisting of isolates from 1981 to 1989 in Sri
Lanka and Group B which was expanded in
three distinct clades including isolates from Conclusion
1989 to 1998 in Sri Lanka, strains isolated in
East Africa from 1985 to 1993 and isolates In the last few decades, dengue has spread as
from 1994 in Latin America. The a pandemic in the American continent,
phylogenetic analysis suggested that genotype starting in the Caribbean islands and
III was introduced from the Indian expanding to North, Central and South
subcontinent into East Africa in the 1980s America[60] . In this context, the dengue
and from Africa into Latin America in 1994, epidemiological profile in Brazil has changed
showing a single genotype introduction in the from a non-endemic to a hyperendemic one.
continent and its subsequent diversification[52] . Since the 1980s when the first DEN strains
In the same year, Peyrefitte et al.[53] showed a were isolated, more than 3 million dengue
high similarity between the DEN-3 cases and nearly 2,090 DHF/DSS cases have
Martinique and the Brazilian strains. been reported in the country
Furthermore, the complete genome (www.funasa.gov.br) [61,62] .
characterization of the Brazilian DEN-3 The endemicity of dengue in 25 out of
sequence (AY679147) strain confirmed an the 27 federative units, the remarkable
insertion of 11 nts in the 5 non-coding virulence of the DEN-2 and DEN-3
region of the genome as previously described genotypes and the risk of the introduction of
for the Martinique strain[53] .

DEN-4 in the country highlight the alarming disease, not because of the virulence-
dengue epidemiological picture in Brazil. In inherited properties but because antibodies
this scenario, use of rapid methods for DEN from a primary infection may enhance
identification and molecular characterization infection with one genotype while
are indispensable tools in the virological neutralizing infection with a distinct one[52,65] .
surveillance laboratories, mainly due to an
Given the limited options available for
obvious need to characterize dengue
dengue control, active surveillance
genotypes before a major outbreak
programmes with continuous monitoring of
occurs [26,46,63] . The partial sequencing of DEN
dengue infection in communities is still one
strains genome has also been used routinely
of the strategies available to detect the
for DEN molecular epidemiological studies,
introduction of new serotypes/genotypes, and,
and it recently characterized the co-infecting
consequently, to prevent the occurrence of
genotypes of DEN-1 and DEN-2 in a patient
epidemics, thus minimizing the impact of the
presenting classic dengue fever in So
circulating strains.
Paulo[64] .
The knowledge of the virus genotype
circulating in a particular region has also Acknowledgements
implications for the potential introduction of To Conselho Nacional de Desenvolvimento
vaccines, allowing the evaluation of the Cientfico e Tecnolgico (CNPq), Fundao
genomic relations between the viruses used de Amparo a Pesquisa do Estado do Rio de
in vaccine development and the circulating Janeiro (FAPERJ), Fundao Oswaldo Cruz
strains. The ability of pre-existing dengue (FIOCRUZ), PAPES III FIOCRUZ. Thanks
antibodies to neutralize better certain DEN are also due to the staff of the Laboratory of
variants than others has been demonstrated. Flavivirus for their technical assistance.
Some strains may produce a more severe
References
[1] Westaway EG, Brinton MA, Gaidamovich SY, [5] Kuhn RJ, Zhang W, Rossmann MG, Pletnev
Horzinek MC, Igarashi A, Kaariainen L, Lvov SV, Corver J, Lenches E, Jones CT,
DK, Porterfield JE, Russell PK and Trent D W. Mukhopadhyay S, Chipman PR, Strauss EG,
Flaviviridae. Intervirol, 1985, 24: 183-192. Baker TS and Strauss JH. Structure of
[2] World Health Organization. Dengue dengue virus: implications for flavivirus
haemorrhagic fever: diagnosis, treatment organization, maturation, and fusion. Cell,
and control. Geneva: WHO, 1997. 2002, 108: 717-725.
[3] Chambers TJ, Hahn CS, Galler R and Rice [6] Rey FA, Heinz FX, Mandl C, Kunz C and
CM. Flavivirus genome organization, Harrison C. The envelope glycoprotein from
expression, and replication. Ann Rev tick-borne ecephalitis virus at 2 A resolution.
Microbiol, 1990, 44: 649-688. Nature, 1995, 375: 291-298.
[4] Heinz FZ and Roehrig JT. Flaviviruses. In:
[7] Roehrig JT. Immunochemistry of dengue
MHV Van Regenmorte, AR Neurath.
viruses. In: DJ Gubler, G Kuno (Eds).
Immunochemistry of viruses II. The basis for
Dengue and dengue haemorrhagic fever.
serodiagnosis and vaccines. Elsevier Science
New York: CAB International, 1997: 23-44.
Publisher, BV, 1990, 289-305.

[8] Trent DW, Manske CL, Fox GE, Chu MC, [16] Chow VTK, Seah CLK and Chan YC. Use of
Kliks SC and Monath TP. The molecular NS3 consesus primers for the polymerase
epidemiology of dengue viruses: genetic chain reaction amplification and sequencing
variation and microevolution. In: Applied of dengue viruses and other flavivirus. Arch
Virology Research, Vol 2. Virus Variation Virol, 1993 133: 157-170.
and Epidemiology (E Kurstak, RG Marusky,
[17] Deubel V, Kinney RM and Trent DW.
MHV Regenmortel, Eds.), New York:
Nucleotide sequence and deduced amino
Plenum Publishing Corporation, 1990: 293-
acid sequence of the non-structural proteins
315.
of dengue type 2 virus, Jamaica genotype:
[9] Rico-Hesse R. Molecular evolution and comparative analysis of the full length
distribution of dengue viruses type 1 and 2 genome. Virology, 1988, 165: 234-244.
in nature. Virol, 1990, 174: 479-493.
[18] Blok J, Gibbs AJ, McWilliam SM and Vitaran
[10] Deubel, V, Nogueira, RMR, Drouet MT, UT. NS1 gene sequences from eight
Zeller M, Reynes J and Ma DQ. Direct dengue-2 viruses and their evolutionary
sequencing of genomic cDNA fragment relationships with other dengue-2 viruses.
amplified by the polymerase chain reaction Arch Virol, 1991, 118: 209-233.
for molecular epidemiology of dengue 2
[19] Kuno G. Phylogeny of the genus Flavivirus. J
viruses. Arch Virol, 1993, 129: 197-210.
Virol, 1998, 72: 73-83.
[11] Chungue E, Deubel V, Cassar O, Laille M
[20] Batista WC, Kashima S, Marques AC and
and Martin PMV. Molecular epidemiology of
Figueiredo LTM. Phylogenetic analysis of
dengue 3 viruses and genetic relatedness
Brazilian flavivrus using nucleotide
among dengue 3 strains isolated from
sequences of parts of NS5 gene and 3 non-
patients with mild or severe form of dengue
coding regions. Virus Res, 2001, 75: 35-42.
fever in French Polynesia. J Gen Virol, 1993,
74: 2765-2770. [21] Shurtleff AC, Beasley DWC, Chen JJY, Ni H,
Suderman MT, Wang H, Xu R, Wang E,
[12] Chungue E, Cassar O, Drouet MT, Guzman
Weaver SC, Watts DM, Russell KL and
MG, Laille M, Rosen L and Deubel V.
Barrett ADT. Genetic variation in the 3' non-
Molecular epidemiology of dengue-1 and
coding region of dengue viruses. Virol, 2001,
dengue-4 viruses. J Gen Virol, 1995, 76:
281: 75-87.
1877-1884.
[22] Baleottti FG, Moreli ML and Figueiredo LTM.
[13] Lanciotti RS, Lewis JG, Gubler DJ and Trent
Brazilian flavivirus phylogeny based on NS5.
DW. Molecular evolution and epidemiology
Mem Inst Oswaldo Cruz, 2003, 98(3): 379-
of dengue-3 viruses. J Gen Virol, 1994, 75:
382.
65-75.
[23] Schatzmayr HG, Nogueira RMR and
[14] Lanciotti RS, Gubler DJ and Trent DW.
Travassos Da Rosa APA. An outbreak of
Molecular evolution and phylogeny of
dengue virus at Rio de Janeiro 1986. Mem
dengue-4 viruses. J Gen Virol, 1997, 78:
Inst Oswaldo Cruz, 1986, 81: 245-246.
2279-2286.
[24] Miagostovich MP, Nogueira RMR,
[15] Goncalves AP, Escalante AA, Pujol FH,
Cavalcante SMB, Marzochi KBF and
Ludert JE, Tovar D, Salas RA and Liprandi F.
Schatzmayr HG. Dengue epidemic in the
Diversity and evolution of the envelope
state of Rio de Janeiro, Brazil: virological
gene of dengue virus type 1. Virol, 2002,
and epidemiological aspects. Rev Inst Med
3003: 110-119.
Trop So Paulo, 1993, 35: 149-154.

[25] Vorndam V, Nogueira RMR and Trent DW. [33] Tolou H, Couissinier-Paris P, Durand JP, de
Restriction enzyme analysis of American Pina JJ, De Micco P, Billoir F, Charrel RN
region dengue viruses. Arch Virol, 1994, and De Lamballerie X. Evidence for
136: 191-196. recombination in natural populations of
dengue virus type 1 based on the analysis of
[26] Miagostovich MP, Santos FB, Gutierrez CM,
complete genome sequences. J Gen Virol,
Riley LW and Harris E. Rapid subtyping of
2001, 82, 1283-1290.
dengue virus subtypes 1 and 4 by restriction
site specific PCR, J Clin Microbiol, 2000, [34] Twiddy SS and Holmes EC. The extent of
38(3), 1286-1289. homologous recombination in members of
the genus Flavivirus. J Gen Virol, 2003, 84(Pt
[27] Balmaseda A, Sandoval E, Perez L, Gutierrez
2): 429-440.
CM and Harris E. Application of molecular
typing techniques in the 1998 dengue [35] Guzman Tirado MG, Kouri GP, Bravo J,
epidemic in Nicaragua. Am J Trop Med Hyg, Soler M, Vazquez S, Santos M, Villa Esclusa
1999, 61: 893-897. R, Basanta P, Indan G and Ballester JM.
Dengue hemorrhagic fever in Cuba. II
[28] Miagostovich MP, Santos FB, De Simone TS,
clinical investigations. Trans R Soc Trop Med
Costa EV, Filippis AMB, Schatzmayr HG and
Hyg, 1984, 78: 239-241.
Nogueira RMR. Genetic characterization of
dengue virus type 3 isolates in the state of [36] Rico-Hesse R, Harrison LM, Salas RA, Tovar
Rio de Janeiro, 2001. Brazilian J Med Biol D, Nisalak A, Ramos C, Boshell J, Mesa MTR,
Res, 2002, 35: 1-4. Nogueira RMR and Travassos Da Rosa A.
Origins of dengue type 2 viruses associated
[29] Miagostovich MP, Sequeira PC, Santos FB,
with increased pathogenicity in the Americas.
Maia A, Nogueira RMR, Schatzmayr HG,
Virol, 1997, 230: 652-658.
Harris E and Riley LW. Molecular typing of
dengue virus type 2 in Brazil. Rev Inst Med [37] Nogueira RMR, Miagostovich MP, Lampe E
Trop So Paulo, 2003, 45: 17-21. and Schatzmayr HG. Isolation of dengue
virus type 2 in Rio de Janeiro. Mem Inst
[30] De Simone TS, Nogueira RMR, Arajo ESM,
Oswaldo Cruz, 1990, 85: 253.
Guimares FR, Santos FB, Schatzmayr HG,
Souza RV, Teixeira Filho G and [38] Nogueira RMR, Miagostovich MP, Lampe E,
Miagostovich MP. Dengue virus surveillance: Souza RW, Zagne SMO and Schatzmayr HG.
the co-circulation of DENV-1, 2 and 3 in the Dengue epidemic in the state of Rio de
state of Rio De Janeiro, Brazil. Trans R Soc Janeiro, Brazil, 1990-1991: co-circulation of
Trop Med Hyg, 2004, 98(9): 553-562. dengue 1 and dengue 2. Epidemiol Infect,
1993, 111: 163-170.
[31] Duarte dos Santos CN, Rocha CFS, Cordeiro
M, Fragoso SP, Rey F, Deubel V and [39] Miagostovich MP, Nogueira RMR,
Desprs P. Genome analysis of dengue type- Schatzmayr HG and Lanciotti RS. Molecular
1 virus isolates between 1990 and 2001 in epidemiology of DEN-2 virus in Brazil. Mem
Brazil reveals a remarkable conservation of Inst Oswaldo Cruz, 1998, 93: 625-626.
the structural proteins but amino acid
[40] Santos FB, Miagostovich MP, Nogueira RMR,
differences in the nonstructural proteins.
Edgil D, Schatzmayr HG, Riley LW and
Virus Res, 2002, 90(1-2): 197-205.
Harris E. Complete nucleotide sequence
[32] Holmes EC and Burch SS. The causes and analysis of a Brazilian dengue virus type 2
consequences of genetic variation in dengue strain. Mem Inst Oswaldo Cruz, 2002,
virus. Trends Microbiol, 2000, 8: 74-77. 97(7): 991-995.

[41] Leitmeyer KC, Vaughn DW, Watts DM, [49] Usuku S, Castillo L, Sugimoto C, Noguchi Y,
Salas R, Villalobos de Chacon I, Ramos C Yogo Y and Kobayashi N. Phylogenetic
and Rico-Hesse R. Dengue virus structural analysis of dengue-3 viruses prevalent in
differences that correlate with pathogenesis. Guatemala during 1996-1998. Arch Virol,
J Virol, 1999, 73: 4738-4747. 2001, 146: 1381-1390.
[42] Tolou H, Couissinier-Paris P, Mercier V, [50] Uzcategui NY, Comach G, Camacho D,

Pisano MR, De Lamballerie X, De Micco P Salcedo M, Cabello de Quintana M,
Jimnez M, Siera G, Cuello de Uzcategui R,
and Durand JP. Complete nucleotide
James WS, Turner S, Holmes EC and Gould
sequence of dengue type 2 virus from the
EA. Molecular epidemiology of dengue virus
French West Indies. Biochem Biophys Res
type 3 in Venezuela. J Gen Virology, 2003,
Commun, 2000, 277: 89-92.
84: 1569-1575.
[43] Nogueira RMR, Miagostovich MP, [51] Messer WB, Vitarana UT, Sivananthan K,
Schatzmayr HG, Moraes GC, Cardoso FMA, Elvtigala J, Preethimala LD, Ramesh R,
Ferreira J, Cerqueira V and Pereira M. Withana N, Gubler DJ and de Silva AM.
Dengue type 2 outbreak in the south of the Epidemiolopgy of dengue in Sri Lanka
state of Bahia, Brazil: laboratorial and before and after the emergence of epidemic
epidemiological studies. Rev Inst Med Trop dengue hemorrhagic fever. Am J Trop Med
So Paulo, 1995, 37: 507-510. Hyg, 2002, 66(6): 765-773.
[44] Nogueira RMR, Zagne SMO, Martins ISM, [52] Messer WB, Gubler DJ, Harris E,
Lampe E, Miagostovich MP and Schatzmayr Sivananthan K and de Silva AM. Emergence
HG. Dengue haemorrhagic fever/dengue and global spread of a dengue serotype 3,
shock syndrome (DHF/DSS) caused by subtype III virus. Emerg Infect Dis, 2003,
serotype 2 in Brazil. Mem Inst Oswaldo 9(7): 800-808.
Cruz, 1991, 86(2): 269. [53] Peyrefitte CN, Couissinier-Paris P, Mercier-
[45] Zagne SMO, Alves VGF, Nogueira RMR, Perennee V, Bessaud M, Martial J, Kenane N,
Miagostovich MP, Lampe E and Tavares W. Durand J-P A and Tolou HJ. Genetic
Dengue haemorrhagic fever in the state of characterization of newly reintroduced
dengue virus type 3 in Martinique (French
Rio de Janeiro, Brazil: a study of 56
West Indies). J Clin Microbiol, 2003, 41(11):
confirmed cases. Trans R S Trop Med Hyg,
1994, 88: 677-679. 5195-5198.
[54] Nogueira RMR. Personal communication.
[46] Harris E, Sandoval E, Johnson M, Xet-Mull
AM and Riley LW. Rapid subtyping of [55] Nogueira RMR, Schatzmayr HG, Cunha RV,
dengue viruses by restriction site-specific Zagne SMO, Gomes FP and Miagostovich
(RSS)-PCR. Virology, 1999, 253: 86-95. MP. Dengue fatal cases in primary infections
in Brazil. Trans R Soc Trop Med Hyg, 1999,
[47] Anonymous. Dengue 3 in Central America. 93: 418.
Dengue surveillance summary. Dengue
[56] Rosen L. The emperor's new clothes
Branch, San Juan, Puerto Rico. Division of
revisited, or reflections on the pathogenesis
Vector-Borne Infectious Diseases, 1995, 70: 4.
of dengue hemorrhagic fever. Am J Trop
[48] Nogueira RMR, Miagostovich MP, Filippis Med Hyg, 1977, 26: 337-343.
AMB, Pereira MAS and Schatzmayr HG. [57] Halstead SB. Dengue haemorrhagic fever, a
Dengue virus type 3 in Rio de Janeiro, Brazil. public health problem and a field for
Mem Inst Oswaldo Cruz, 2001, 96: 925- researches. Bull World Health Organ, 1980,
926. 58: 1-21.

[58] Watts DM, Porter KR, Putvatana P, Vazquez [62] Nogueira RMR, Miagostovich MP and
B, Calampa C, Hayes CG and Halstead SB. Schatzmayr HG. Dengue virus in Brazil.
Failure of secondary infection with American Dengue Bulletin, 2002, 26: 77-83.
genotype dengue 2 to cause dengue [63] Lanciotti RS, Calisher CH, Gubler DJ and
haemorrhagic fever. Lancet, 1999, 354: Vorndam V. Rapid detection and typing of
1431-1434. dengue viruses from clinical samples by using
[59] Rico-Hesse R. Microevolution and virulence reverse transcriptase-polymerase chain
of dengue viruses. Adv Virus Res, 2003, 59: reaction. J Clin Microbiol, 1992, 30: 545-551.
315-341. [64] Santos CLS, Bastos MAA, Sallum MAM and
[60] Gubler DJ. Epidemic dengue/dengue Rocco IM. Molecular characterization of
hemorrhagic fever as a public health, social dengue viruses type 1 and 2 isolated from
and economic problem in the 21st century. concurrent human infection. Rev Inst Med
Trends Microbiol, 2002, 10: 100-103. Trop So Paulo, 2003, 45(1): 11-16.
[61] Da Silva Jnior JB, Siqueira Jnior JB, Coelho [65] Kochel TJ, Watts DM, Halstead SB, Hayes
GE, Vilarinhos PTR and Pimenta Jnior FG. CG, Espinoza A and Felices V. Effect of
Dengue in Brazil: current situation and dengue-1 antibodies on American dengue-2
prevention and control activities. Epidem viral infection and dengue haemorrhagic
Bulletin, 2002, 23(1): 1-7. fever. Lancet, 2002, 360: 310-312.

Unusual Emergence of Guate98-like Molecular Subtype
of DEN-3 during 2003 Dengue Outbreak in Delhi
Manoj Kumar*#, S.T. Pasha*, Veena Mittal*, D.S. Rawat*, Subhash Chandra Arya**,
Nirmala Agarwal**, Depesh Bhattacharya*, Shiv Lal* and Arvind Rai*
*National Institute of Communicable Diseases, 22 Shamnath Marg, Delhi 110 054, India
**Sant Parmanand Hospital, 18 Shamnath Marg, Delhi 110 054, India
Abstract
With a view to identifying the molecular subtype of the circulating dengue virus responsible for a major
outbreak of dengue fever (DF) / dengue haemorrhagic fever (DHF) in and around Delhi during the post-
monsoon period in 2003, 32 serum samples were collected from clinically suspected cases. These were
subjected to reverse transcription/polymerase chain reaction (RT/PCR) for amplification of 511 bp C-
PreM gene region of the dengue virus. Seven specimens, yielding a satisfactory quantum of viral RNA,
were subsequently processed for automated nucleotide sequencing. Five of the seven analysed isolates
showed close DNA sequence homology with Guate96-98 strains of DEN-3 virus, whereas two turned out
to be genotype IV of DEN-2. Earlier, DEN-2 (genotype IV) had been identified as the etiological agent
during a major DF/DHF outbreak in Delhi in 1996 and also in 2000. Though DEN-2 continues to prevail,
DEN-3, having a close sequence homology with Guate96-98 strains, seems to have entered India for the
first time in late 2003, resulting in a major DF/DHF outbreak. How the Guate96-98 strain of DEN-3
entered India remains to be linked epidemiologically.
Keywords: Dengue outbreak, molecular typing, CpreM gene, DEN-3, Delhi.
Introduction alone, a number of outbreaks of dengue

virus infection were recorded in 1967, 1970,
Dengue fever/dengue haemorrhagic fever 1982, 1988 and 1990[9-13] . Again in 1996, a
(DF/DHF) is caused by one or more of the major DHF outbreak, resulting in 10,252
four antigenically-related dengue virus cases with 432 deaths, occurred in and
serotypes DEN-1 to DEN-4. It is widespread around Delhi. DEN -2 genotype IV was the
in tropical and subtropical countries in the predominant etiological agent[14,15] .
world and is a serious cause of morbidity
Delhi and its adjoining areas were again
and mortality, threatening about one third
of the total human population[1-3] . Many struck by a major outbreak of DF/DHF
between September and December 2003.
outbreaks and epidemics of DF/DHF have
The present study was undertaken to unveil
been reported in different parts of India
the predominant molecular subtype of the
during the past four decades[4-8] . In Delhi
dengue virus involved in this outbreak.
#
E-mail: manojkumardelhi@yahoo.co.in; Tel./Fax: 91-11-23912960

Emergence of DEN-3 in Delhi in 2003
Materials and methods Virus RNA isolation

Thirty-two serum samples were subjected to
Clinical specimens
dengue viral RNA isolation. 140l sera
A total of 32 serum samples from clinically sample was processed for RNA isolation using
suspected acute cases of DF/DHF were QIAamp viral RNA Kit (QIAGEN, Germany)
collected from different hospitals in Delhi using standard kit protocol. Finally, viral RNA
between September and December 2003. was eluted in 30l nuclease-free water.
Most of the serum samples were collected
within the first five days of the clinical onset. Reverse transcription/polymerase
Samples were transported to the laboratory chain reaction (RT/PCR)
within six hours of collection and stored at
RT-PCR was carried out using previously
70 C until processed. Thirty-two sera are reported D1 and D2 primers that were M13
considered adequate to establish the tailed for the convenience of nucleotide
predominant molecular subtype of dengue sequencing. This primer set is capable of
virus. amplifying all the four types of dengue viruses
(DEN-1 to DEN-4).
Primer D1 (with M13F tail)

(5-TGTAAAACGACGGCCAGTTCAATATGCTGAAACGCGCGAGAAACCG-3)
(M13 forward primer sequence underlined)
Primer D2 (with M13R tail)
(5-CAGGAAACAGCTATGACCTTGCACCAACAGTCAATGTCTTCAGGTTC-3)
(M13 reverse primer sequence underlined)
Complementary DNA (cDNA) synthesis followed by 40 cycles of 95 C for 30

and gene amplification of 511bp CpreM seconds, 55 C for 30 seconds, 72 C for 1
gene region of the dengue virus was minute and final extension for 72 C for 10
performed using one step GeneAmp RNA minutes and hold at 4 C. Appropriate
Gold RT PCR Kit (Applied Biosystems, USA) positive and negative controls were used in
with D1 and D2 primers for detection and RT/PCR. PCR products were
typing of all the four types of dengue virus [16] . electrophoresed on 1.5% agarose gel along
Briefly, 50l reaction mix containing final with 100bp DNA ladder marker (MBI
concentration of 1X of 5X RT buffer, 1.5mM Fermentas, USA) and were visualized on gel
MgCl2, 200M dNTPs, 5mM dithiotheratol documentation system (Biometra, Germany).
and 10pmol of D1 and D2 primers, 10U
RNAase inhibitor, 15U of MuLV MultiScribe Gene sequencing and phylogenetic
reverse transcriptase, 2.5U of Amplitaq Gold analysis
DNA polymerase and 5 l of extracted viral
RNA. RT was performed at 42 C for 20 PCR amplicons were purified using
minutes on GeneAmp 9700 PCR System. Centricon-100 columns (Millipore, USA)
Then pre-hold at 95 C for 10 minutes and subjected to automated dideoxy chain

termination nucleotide cycle-sequencing from 18,000 to 250,000. Most of the

using commercial ABI PRISMTM Big Dye samples collected within five days from the
Terminator Cycle Sequencing Kit with onset of the fever were selected for RT-PCR
Amplitaq DNA Polymerase FS, following the testing.
manufacturers protocol and run on ABI
Out of the 32 serum samples subjected
PRISM TM 310 Genetic Analyser (Applied
to RT-PCR, only seven yielded amplification
Biosystems, USA). Nucleotide sequences
of 511bp C-PreM gene region of dengue
were edited and aligned using Sequence
virus. Lane 1-14 in the Figure included
Navigator Software. Subsequently, blast
clinical samples, 100bp DNA ladder marker
search (http://www.ncbi.nlm.nih.gov/blast)
in lane M and negative control in lane Neg.
and phylogenetic analysis using DNA Star
Automated nucleotide sequencing of these
software, were done to reveal the dengue
seven RT/PCR products revealed two groups
virus molecular subtype.
of sequences, the first group had five almost
identical sequences, while the second group
Result and discussion had two similar sequences. All samples were
subjected to blast search, which revealed
During the study, a majority of the subject that five isolates had a very close sequence
patients had clinical symptoms of DF and homology (=98%) with Guate98 AB038478
only sporadic cases presented symptoms of strain of dengue type 3 (DEN -3), while two
DHF/DSS {DF: 24 (75%), DHF: 7 (21.9%) turned out to be DEN-2 and showed =99%
and DSS: 1 (3.1%)}. The samples belonged homology with Delhi96 AF047394 strain
to all age groups ranging from 5 to 50 years. Delhi 2000 strains (Table). These Cpre M
The male-female ratio was 18:14. The mean gene sequences were submitted in the Gene
platelet count was 77,120, which ranged Bank wide accession AY 706094-AY706099.
Figure. PCR products visualization on 1.5% agarose gel

Table. Clinical and molecular analysis data of seven samples, yielding RT-PCR positivity for
511bp CpreM gene of dengue virus
Assigned
S. Sample Serum Clinical Platelet RT-PCR genotype
No. ID* collection state Age/Sex count 511bp on blast
search
1 04DEL03 22.09.03 DF 30/F 89,000 +ve DEN-3
2 06DEL03 24.09.03 DF 20/M 79,000 +ve DEN-2

3 10DEL03 07.10.03 DHF 15/M 39,000 +ve DEN-3
4 11DEL03 08.10.03 DF 14/F NA +ve DEN-3

5 16DEL03 01.10.03 DF 34/F 18,000 +ve DEN-2
6 19DEL03 04.10.03 DHF 40/M 38,000 +ve DEN-3

7 26DEL03 13.10.03 DHF 16/M 36,000 +ve DEN-3
Out of 32 samples subjected to RT-PCR for 511bp Cpre M gene of dengue virus, only 7 turned positive, while 25 did
not show any amplification.
All five DEN-3 strains had =98% nucleotide sequence homology with Guate98 strains.
Both DEN-2 strains had =99% nucleotide sequence homology with already circulating Delhi96 and Delhi2000
strains.
NA: Information not available
Several studies have shown that dengue although cases of DEN-1 were also
virus infection has been endemic in detected[19-21] .
different parts of India, as documented for
During the present study, the majority
over four decades[17,18] , and almost all the
of the patients had clinical symptoms of DF
four known serotypes of dengue virus (DEN -
and only sporadic cases presented with
1 to DEN-4) have been reported. The
symptoms of DHF or DSS. The samples
metropolitan city of Delhi witnessed several
referred to our laboratory for molecular
outbreaks of DF/DHF in 1967, 1970, 1982,
characterization were accompanied by IgM
1988,1996 and 2000. DEN -1 and DEN-3
serology results. When cross-checked, we
viruses were associated with the 1970
found that five RT/PCR-positive samples
epidemic, DEN-1 and DEN -2 with the 1988
were IgM-negative, while two RT/PCR-
epidemic, while genotype IV of DEN-2 was
positive samples were dengue IgM-positive.
responsible for the major DHF outbreak in
Previous studies had also shown that most,
1996[15] . Our previous findings revealed
but not all, RT/PCR-positive samples had
genotype IV of DEN-2 as the predominant
negative IgM serology. The reason for this is
type circulating from 1996 onwards, based
attributed to the fact that the virus had not
on RT-PCR and C-PreM gene sequencing,
been recovered from most of the DF/DHF
patients beyond the 5th day of the onset of

the symptoms, while detectable levels of Americas had also been reported[25] . The
dengue-specific IgM antibodies appear after first epidemic of DEN -3 in Jamaica and
the 4th or the 5th day. Our findings re- Puerto Rico was witnessed in 1963, which
established that the ideal time for the was followed by another epidemic of DEN -
collection of samples for molecular test was 3 in Colombia and Puerto Rico in the mid-
between days 1-5. 1970s, and in the Pacific islands in early
1980s [26] . In 1994, a new strain of DEN-3
The nucleotide sequence alignment and
was introduced in the Americas, causing a
blast search of the seven RT/PCR-positive
major epidemic of DF/DHF in Nicaragua
samples in the present study, when
and an outbreak of DF in Panama[27] . But
compared with those of earlier ones,
this DEN-3 was genetically different from
revealed that only ~29% (2/7) belonged to
the DEN-3 strains which circulated in the
the already prevalent genotype IV of DEN-
Americas earlier. Interestingly, in 1994, this
2; whereas the majority ~71% (5/7) showed
DEN-3 genotype was reported to have a
close genomic homology (=98%) with
close identity with those strains which
GUATE98 AB038478 strain of dengue type
caused DHF epidemic in some of the South-
3 (DEN-3) which caused a widespread
East Asian countries around the same
dengue outbreak in Guatemala in the late
period[28] . This DEN -3 strain subsequently
Nineties[22] . A similar strain is also reported
spread from Asia to Central America and
to have been reintroduced in Marlinique
Mexico in 1995 and caused major
(French West Indies)[23] and in Rio de
epidemics. A classic example of the
Janerio, Brazil (unpublished data vide Gene
replacement of one type by the other is
Bank Accession No. AY679147). The first
evident from the fact that, in 1971, DEN-2
reported evidence of DEN-3 in Delhi was in
was introduced into the Pacific areas
1970, based on serotyping, but no genomic
followed by a new strain of DEN-1 in 1975
data of the 1970 strain of DEN -3 is available.
and DEN-4 by 1979, and in early 1980s by
It is difficult to ascertain, after a long gap of
yet another new strain of DEN -3[29] . These
33 years, whether the old 1970 strain of
reports support our current findings that,
DEN-3 had re-emerged during the current
although DEN -2 (genotype IV) has been
outbreak; or Guate98 DEN -3 strain
predominant in northern India over the past
(prevalent in South American countries) had
few years, Guate96-98-like DEN-3 strain
been introduced for the first time in India.
dominated during the major outbreak of DF
The changing epidemiology of different in Delhi in 2003.
subtypes of dengue virus and their co-
existence and/or replacement of one type
by the other is well documented[24] . During Acknowledgements
the present outbreak, we found DEN-3 as We thank Ms Priyanka, Ms Kamini Singh
the predominant type, but it did not seem and Ms Seema George for their technical
to completely replace the previously assistance. The secretarial assistance of
circulating DEN -2. Prior to 1977, co- Mr A.K. Manchanda, Ms Kiran Bhatt and
existence of DEN-2 and DEN -3 in the Ms Sarita Kumar is acknowledged.

References
[1] Gubler DJ and Clark GG. Dengue/dengue disease. Epidemiol Infect, 2000, 125(1):
haemorrhagic fever and emergence of a 195-200.
global health problem. Emerg Infect Dis,
[9] Balaya S, Paul SD, DLima LV and Parvi KM.
1995, 1(2): 55-57. Investigations on an outbreak of dengue in
[2] Monath TP. Dengue: the risk to developed Delhi in 1967. Indian J Med Res, 1969, 57:
and developing countries. Proc Natl Acad 767-774.
Sci, USA, 1994, 91: 2395-2400.
[10] Diesh P, Pattanayak S, Singha P, Arora DD,
[3] World Health Organization: Dengue Mathur PS, Ghosh TK, Mondal MM and
haemorrhagic fever: diagnosis, treatment Raghavan NGS. An outbreak of dengue
and control. Geneva: WHO, 1997: 12-23. fever in Delhi 1970. J Commun Dis, 1972,
4: 13-18.
[4] Rodrigues FM. Patnakar MR, Banerjee K,
Bhatt PN, Goverdhan MK, Pavri KM and [11] Rao CVRM, Bagchi SK, Pinto BD, Ilkal MA,
Vittal M. Etiology of the 1965 epidemic of Bhardwaj M, Shaikh BH, Dhanda V, Dutta
febrile illness in Nagpur city, Maharashtra M and Pavri KM. The 1982 epidemic of
state, India, Bull World Health Organ, 1972, dengue fever in Delhi. Indian J Med Res,
46: 173-179. 1985, 82: 271-275.
[5] Myers RM, Carey DE, De Ranitz CM, [12] Kabra SK, Verma IC, Arora NK, Jain Y and
Reuben R and Bennet B. Virological Kalra V. Dengue haemorrhagic fever in
investigations of the 1966 outbreak of children in Delhi. Bull World Health Organ,
dengue type 3 in Vellore, Southern India. 1992, 70: 105-108.
Indian J Med Res, 1969, 57(8): 1392-1401.
[13] Srivastava VK, Suri S, Bhasin A, Srivastava L
[6] Chaturvedi UC, Kapoor AK. Mathur A, and Bhardwaj M. An epidemic of dengue
Chandra D, Khan AM and Mehrotra RM. A haemorrhagic fever and dengue shock
clinical and epidemiological study of an syndrome in Delhi: a clinical study. Ann
epidemic of febrile illness with Trop Paediatr, 1990, 10: 329-334.
haemorrhagic manifestations, which
[14] Broor S, Dar L, Sengupta S, Chakraborty M,
occurred at Kanpur, India in 1968. Bull
Wali JP, Biswas A, Kabra SK, Jain Y and Seth
World Health Organ, 1970, 43(2): 281-287. P. Recent dengue epidemic in Delhi, India.
[7] Padbidri VS, Dandawate CN, Goverdhan In: Saluzzo JF and Dodet B (Eds.): Factors in
MK, Bhat UK, Rodrigues FM, DLima LV, the emergence of arbovirus diseases.
Kaul HN, Guru PY, Sharma R and Gupta NP. Amsterdam: Elsevier, 1996: 123-128.
An investigation of the aetiology of the 1971
[15] Singh UB, Maitra A, Broor S, Rai A, Pasha ST
outbreak of febrile illness in Jaipur city, India. and Seth P. Partial nucleotide sequencing
Indian J Med Res, 1973, 61(12): 1737-1743. and molecular evolution of epidemic
[8] Singh J. Balakrishnan N, Bhardwaj M, causing dengue 2 strains. J Infect Dis, 1999,
Amuthadvi P, George EG, Subramani K, 180: 959-965.
Soundararajan K, Appavoo NC, Jain DC,
[16] Lanciotti RS, Calisher CH, Gubler DJ, Chang
Ichhpujani RL, Bhatia R and Sokhey J. Silent G and Vorndam V. Rapid detection and
spread of dengue and dengue haemorrhagic typing of dengue viruses from clinical
fever to Coimbatore and Erode districts in
samples by using reverse transcriptase
Tamil Nadu, India. Need for effective
polymerase chain reaction. J Clin Microbiol,
surveillance to monitor and control the
1992, 30: 545-551.

[17] Kalra NL, Kaul SM and Rastogi RM. [23] Peyrfitte CN, Paris PC, Perennec M, Bessaud
Prevalence of Aedes aegypti and Aedes M, Martial J, Kenane, Durand JP and Tolou
albopictus vectors of dengue and dengue HJ. Genetic characterization of newly
haemorrhagic fever in North, North-East and reintroduced dengue virus type 3 in
Central India. Dengue Bulletin, 1997, 21: Martinique (French West Indies). J Clin
84-92. Microbiol. 2003, 41(11): 5195-5198.
[18] Mathew T, Nayar M, Gupta JP, Suri NK, [24] Gubler DJ. Epidemic dengue/dengue
Bhola SR, Ghosh TK, Suri JC, Talwar V and haemorrhagic fever: A global public health
Pattanayak S. Serological investigations on problem in the 21st century. Dengue Bulletin,
arbovirus activity in and around Delhi, a 1997, 21: 1-15.
five-year study. Indian J Med Res, 1979, 69: [25] Gubler DJ and Trent DW. Emergence of
557-566. epidemic dengue/dengue haemorrhagic
[19] Kumar M, Rai A, Pasha ST and Datta KK. fever as a public health problem in the
Molecular detection and typing of dengue Americas. Infect Agents Dis, 1994, 2: 383-
viruses in clinical specimen from dengue 393.
outbreak in Delhi in 2000. The Proceedings [26] Gubler DJ and Casta-Valez A. A programme
of the International Conference of Indian for prevention and control of epidemic
Public Health Congress, New Delhi, 14-16 dengue and dengue haemorrhagic fever in
April 2001. Puerto Rico and the US Virgin Islands. Bull
[20] Singh UB and Pradeep Seth. Use of PAHO, 1991, 25: 237-247.
nucleotide sequencing of the genomic [27] Centers for Disease Control. Imported
cDNA fragments of the capsid/pre dengue-United States, 1993-1994. MMWR,
membrane junction region for molecular 1995, 44: 353-356.
epidemiology of dengue type 2 viruses.
Southeast Asian J Trop Med Public Health, [28] Lanciotti RS, Lewis JG, Gubler DJ, and Trent
2001, 32(2): 326-335. DW. Molecular evolution and epidemiology
at dengue-3 viruses. J Gen Virol, 1994, 75:
[21] Das PK, Parida MM, Saxena P, Kumar M,
65-75.
Rai A, Pasha ST and Jana AM. Emergence
and continued circulation of dengue-2 [29] Gubler DJ. Dengue and dengue
(genotype IV) virus strains in northern India. haemorrhagic fever. Its history and
Journal of Medical Virology, 2004, 74(2): resurgence as a global public health problem.
In DJ Gubler and G Kuno (Eds.), Dengue
314-322.
and dengue haemorrhagic fever, CAB
[22] Usuku S, Castillo L, Sugimoto C, Noguchi Y, International, Wallingford, Oxon OX108DE,
Yogo Y and Kobayashi N. Phylogenetic UK, 1997: 1-23.
analysis of dengue-3 viruses prevalent in
Guatemala during 1996-1998. Arch Virol,
2001, 146(7): 1381-1390.

The Animal Models for Dengue Virus Infection
Tao Peng, Junlei Zhang and Jing An #
Department of Microbiology, College of Medicine, Third Military Medical University, Chongqing, 400038,
Peoples Republic of China
Abstract
Currently, the mechanisms involved in the pathogenesis of DHF/DSS remain poorly understood and
there is no effective vaccine available to prevent infection with DEN virus. The lack of a reliable small
animal model that mimics dengue disease is a major obstacle. In this paper, the development of small
animal models such as mice for dengue virus infections is reviewed.
Keywords: Dengue virus, animal model, mice.
Introduction However, the mechanisms involved in the

pathogenesis of DHF/DSS remain poorly
Dengue (DEN) viruses are mosquito-borne understood and there is no effective vaccine
RNA viruses, which belong to the genus available to prevent infection with any of
Flavivirus (family Flaviviridae), and are the four serotypes of DEN virus. A major
grouped into four antigenically distinct types technical barrier is the absence of a suitable
(DEN-1, DEN-2, DEN-3, and DEN-4). Every animal model that mimics DEN disease,
year, they infect millions of people and can including DHF/DSS. So far, there are only
cause a mild-to-debilitating febrile illness three known hosts for DEN virus infections:
(classical dengue fever, DF) or life- mosquitoes, humans and lower primates[2] .
threatening syndrome (dengue Although these lower primates infected with
haemorrhagic fever/dengue shock syndrome, wild type DEN viruses develop viremia, they
DHF/DSS). In recent years, the geographical generally manifest only very mild or no
range of dengue in tropical and subtropical clinical signs of disease[3] . Since the
regions of the world has extended and appearance of the severe combined
DHF/DSS is occurring in new areas and with immunodeficiency (SCID) mice in 1983[4] ,
increased incidence[1] . Cardinal signs of efforts have been made to develop new
DHF/DSS include haemorrhage, abrupt small animal models that may be useful for
onset of vascular leakage and shock, the development of a future DEN virus
accompanied by severe thrombocytopenia vaccine and for studying the pathogenesis of
and massive complement activation. DEN virus infections.
#
E-mail: anjing60@yahoo.com.cn; Tel.: +86-23-68752774; Fax: +86-23-65463259

Animal models based on virus titers were detected in the peripheral

blood and the brain tissues, indicating that
SCID mice DEN virus had replicated in the infected
The SCID mice, which do not produce K562-SCID mice. Other serotypes of DEN
functional T and B cells and lack detectable viruses were also used to infect the K562-
immunoglobulin (Ig), can support DEN - SCID mice, and the mortality rates of the
susceptible human cell lines xenografts, and infected mice varied with different challenge
this system has been employed to study strains, suggesting that this animal system
DEN virus infection in vivo. SCID mice might potentially be utilized to define the
reconstituted with human peripheral blood virulence of various human DEN isolates
lymphocytes (hu-PBL-SCID) have been used and to characterize the molecular
for studies on the pathogenesis of infection determinants for such viral virulence. K562-
with the human immunodeficiency virus SCID mice were also challenged with DEN -
(HIV)[5,6] and for research on treatment of 2 virus and received antibody administration
HIV infection[7] . at the same time or one day earlier, and the
results revealed that these mice exhibited a
The hu-PBL-SCID mice were firstly reduction in mortality and a delay of
evaluated as an animal model for DEN viral paralysis onset after DEN virus infection.
infection in 1995[8] . SCID mice were These results indicated that an in vitro
injected intraperitoneally (ip) with hu-PBL neutralizing antibody also defended K562-
for reconstitution and successful SCID mice against DEN-2 virus infection.
engraftment was demonstrated by the
presence of a high serum level of human Target cells and organs for DEN virus
IgG. Hu-PBL-SCID mice were ip-infected replication in humans remain unclear.
with DEN-1 virus. Unfortunately, only 5 of Unusual clinical manifestations, mostly
19 hu-PBL-SCID mice showed sensitivity to cerebral and hepatic symptoms, have
DEN-1 virus. become more common in patients with
DEN virus infection in recent years [10,11] . The
It was suggested that the main reason involvement of liver cells in the
for the low DEN infection rate was a scanty pathogenesis of DEN virus infection has
number of appropriate human target cells in been indicated by abnormal liver function,
the reconstituted mice. Thus, investigators pathological findings and detection of viral
searched for more DEN-susceptible human antigen in hepatocytes and Kupffers cells at
cell lines to improve the infection rate of the biopsies[12] . It was reported that DEN virus
SCID mouse model. One promising could replicate in a human heptocarcinoma
candidate was K562 cell, an cell line, HepG2, and infectious particles
erythroleukemia cell line. SCID mice were were released into the culture medium[13,14] .
engrafted ip with K562 cells (K562-SCID Therefore, HepG2 cells were transplanted
mice)[9] . After intratumor injection into the into SCID mice to develop an animal model
peritoneal tumor masses of DEN-2 virus, for studying the pathogenesis of DEN virus
K562-SCID mice showed neurological signs infection[15] . The replication of HepG2 cells
of paralysis and died at approximately 2 in host mice was confirmed by an increase
weeks post-infection (pi). In addition to of serum human albumin and propagation
being detected in the tumour masses, high of HepG2 cells in the liver. At 7-8 weeks

after transplantation, HepG2-grafted SCID such as arching of the back, ruffling of the
mice were ip-infected with DEN-2 virus. A fur and slowing of activity appeared at end
high titer of the virus was detected in the of day 4 pi. The presence of DEN-2 virus in
liver and serum but not in the brain in the the blood was confirmed on day 2 pi by
early stage of the infection. When the mice reverse transcriptase-polymerase chain
showed paralysis, the highest titer of virus reaction (RT-PCR). The development of the
was detected in the serum and brain. DEN - experimental DEN -2 virus infection in
2 antigens were also found in HepG2 cells mouse model was accompanied by the virus
of the liver in the early stage and some reproduction in all animals. Within 5 and 6
neurons of the brain in the late stage. Upon days pi, all mice showed severe sickness
clinical examination, thrombocytopenia, with anorexia and weight loss ending in
prolonged partial thromboplastin time, limb paralysis and 100% mortality rate was
increased haematocrit, blood urea nitrogen noted at 7 days pi. The most impressive
and tumour necrosis factor a (TNF a) were changes were seen with TNF-a, which
seen in the paralyzed mice. Moreover, mild abruptly and steeply increased 24 h before
haemorrhages in the liver and tarry stool in death. Serum levels of interleukin (IL)-1, IL-
the small intestine were observed in some 6, IL-10, IL-1 receptor antagonist and
mice. soluble TNF receptor I continuously
increased during the time of infection.
All of above animal models based on
Treating animals with anti-TNF-a serum
SCID mice with transplanted DEN -
reduced the mortality rate down to 40%.
susceptible human cells mimic some of the
This model supports the view that the
aspects of human disease, which may be
activation of innate immune response is at
helpful for studying DEN virus infection,
least partially responsible for mortality in
especially in the areas of viral pathogenesis,
DEN-2 virus infection, and in line with this
virus-host interaction and vaccine
concept, anti-TNF treatment significantly
development against DEN infections.
reduces the mortality rates. Therefore,
However, it is generally agreed that
inbred 4-week-old BALB/c mice are useful
DHF/DSS is an immune-mediated disease,
models to research the immune activation
and since SCID mice are unable to produce
of host in DEN-2 virus infection.
the innate immune response, this may
impose some limitations on the use of these
animal models to extrapolate the situation in Gene knockout mouse
human DHF/DSS.
(AG129) model
There is evidence that alpha and beta
BALB/c mouse model interferons (IFN-a/) and gamma IFN (IFN-?)
Inbred four-week-old BALB/c mice were might be involved in human DEN virus
found sensitive (haplotype H-2d) to the infection[17,18] . In addition, exogenously
challenge with dengue virus type 2 (strain administered IFN appears to protect mice
P23085) [16] . Mice were ip-infected with a from DEN virus challenge[19] . This
dose of 5 LD 50 of the mouse-adapted DEN - information suggested that mice defective in
2 virus, and the first clinical manifestations their IFN response might provide a suitable

model for DEN virus infection. paraplegia after virus infection, they
Intraperitoneally administered mouse- recovered after one month. However, there
adapted DEN -2 virus was uniformly lethal in was transient thrombocytopenia at 10-13
AG129 mice[20] , which lack IFN-a/ and days pi. When the mice were re-infected
IFN-? receptor genes, regardless of age. The with the same DEN-2 virus two months later,
mice showed neurological abnormalities, thrombocytopenia was manifested again at
including hind-leg paralysis and blindness at 10 days after infection. Anti-platelet
7 days pi, and died at 12 days after infection. antibody was also generated after injection.
The immunized mice were protected from And there was strain variation in DEN-2
virus challenge, and the survival time virus infection; the A/J strain was more
increased following passive transfer of anti- sensitive than BALB/c or B6 mice. These
DEN polyclonal antibody. To determine results show that this DEN-2 virus-infected
which aspect of the IFN response was mouse system accompanied by thrombo-
critical in protecting these mice from DEN cytopenia and anti-platelet antibody may be
virus infection, animals individually deficient a suitable model to study the pathogenicity,
in either IFN-a/ (A129) or IFN-? (GKO) especially immune activation in DEN virus
functions as well as BALB/c controls were infection. On the other hand, A/J mice had
subjected to a similar DEN virus challenge. to be inoculated with a large quantity of
None of these mice exhibited any overt DEN-2 virus; a dose of less than 110 8 pfu
symptoms of illness, indicating that for DEN per mouse was not effective in causing
virus infection, IFN-a, -, and -? paraplegia. Furthermore, viremia was low
abnormalities in combination were and transient in A/J mice compared with
necessary for the mouse-adapted virus to be that in SCID or IFN-deficient AG129 mice.
lethal when the ip challenge route was used. DEN-2 virus could not be isolated from the
These results demonstrated that AG129 blood of infected mice; it could only be
mice were a promising small animal model detected by sensitive RT-PCR in A/J mice.
for DEN virus vaccine trials.
Conclusion
A/J mouse model Although the above-mentioned new small
DEN virus infection causes DF and animal models, which mimic some of the
DHF/DSS. No animal model is available that aspects of human DEN virus disease, may
mimics this clinical manifestation. The facilitate not only the study of DEN
immunocompetent mouse (A/J strain) was pathogenesis but also the evaluation of anti-
reported as a mouse model for DEN virus DEN virus as well as vaccine development,
infection that resembles the thrombo- there are still drawbacks in each model,
cytopenia manifestation[21] . Intravenous especially in mimicing DHF/DSS. Presently,
injection of DEN-2 virus into A/J mice the molecular mechanisms underlying the
induced paraplegia at 2-3 weeks, while the pathogenesis of DHF/DSS remain unknown,
mock-infected controls were normal. such as the receptors of DEN virus which
Viremia detected by RT-PCR was found are still not clear. Even though the use of
transiently at two days but at no other time transgenic animals has been proposed in the
after infection. Although A/J mice developed quest for an animal model, it is apparent

that one needs to know more about the Acknowledgment

mechanisms involved in the pathogenesis of
DHF/DSS at the molecular level before one This work was partially supported by grants
can construct a transgenic animal to serve as nos. 30170848 and 30300303 from the
a model for use in research on the National Science Foundation of China
pathogenesis, vaccine development and (NSFC).
therapy for DHF/DSS.
References
[1] Rico-Hesse R, Harrison LM, Salas RA, Tovar combined immunodeficient (SCID) mouse
D, Nisalak A, Ramos C, Boshell J, de Mesa as an animal model for dengue viral
MT, Nogueira RM and da Rosa AT. Origins infection. The American Journal of Tropical
of dengue type 2 viruses associated with Medicine and Hygiene, 1995, 52: 468-476.
increased pathogenicity in the Americas.
[9] Lin YL, Liao CL, Chen LK, Yeh CT, Liu CI,
Virology, 1997, 230: 244-251.
Ma SH, Huang YY, Huang YL, Kao CL and
[2] Gubler DJ. Dengue viruses. In R.G. Webster King CC. Study of dengue virus infection in
and A. Granoff (Eds.), Encyclopedia of SCID mice engrafted with human K562 cells.
virology. Academic Press, Inc., San Diego, Journal of Virology, 1998, 72: 9729-9737.
California, 1994: 324-331.
[10] Rajajee S and Mukundan D. Neurological
[3] Schlesinger RW. Dengue virus. New York, manifestations in dengue hemorrhagic fever.
Spriger-Verlag, 1977: 72-73. Indian Pediatrics, 1994, 31: 688-690.
[4] Bosma GC, RP Custer and MJ Bosma. A [11] Thisyakorn U and Thisyakorn C. Dengue
severe combined immunodeficiency infection with unusual manifestations.
mutation in the mouse. Nature, 1983, 301: Journal of the Medical Association of
527-530. Thailand, 1994, 77: 410-413.
[5] Mosier DE. Immunodeficient mice [12] Kuo CH, Tai DI, Chien CSC, Lan CK, Chiou
xenografted with human lymphoid cells: SS and Liaw YF. Liver biochemical tests and
new models for in vivo studies of human dengue fever. The American Journal of
immunobiology and infectious diseases. Tropical Medicine and Hygiene, 1992, 47:
Journal of Clinical Immunology, 1990, 10: 265-270.
185-191.
[13] Marianneau P, Megret F, Olivier R, Morens
[6] Mosier DE. Viral pathogenesis in hu-PBL- DM and Deubel V. Dengue 1 virus binding
SCID mice. Seminars in Immunology, 1996, to human hepatoma HepG2 and simian
8: 255-262. Vero cell surface differs. Journal of General
Virology, 1996, 77: 2547-2554.
[7] McCune JM, Namikawa R, Shih CC, Rabin L
and Kaneshima H. Suppression of HIV [14] Marianneau P, Cardona A, Edelman L,
infection in AZT-treated SCID-hu mice. Deubel V and Despres P. Dengue virus
Science, 1990, 247: 564-566. replication in human hepatoma cells
activates NF-kB which in turn reduces
[8] Wu S-JL, Hayes CG, Dubois DR,
apoptotic cell death. Journal of Virology,
Windheuser MG, Kang Y-H, Watts DM and
1997, 71: 3244-3249.
Sieckmann DG. Evaluation of the severe

[15] An J, Kimura-Kuroda J, Hirabayashi Y and [19] Cole GA and Wisseman CL Jr. Pathogenesis
Yasui K. Development of a novel mouse of type 1 dengue virus infection in suckling,
model for dengue virus infection. Virology, weanling and adult mice. 1. The relation of
1999, 263: 70-77. virus replication to interferon and antibody
[16] Atrasheuskaya A, Petzelbauer P, Fredeking formation. American Journal of
Epidemiology, 1969, 89: 669-680.
TM and Ignatyev G. Anti-TNF antibody
treatment reduces mortality in experimental [20] Johnson AJ and Roehrig JT. New mouse
dengue virus infection. FEMS Immunology model for dengue virus vaccine testing.
and Medical Microbiology, 2003, 35: 33-42. Journal of Virology, 1999, 73: 783-786.
[17] Kurane I, Innis BL, Nimmannitya S, Nisalak [21] Huang K-J, Li S-YJ, Chen S-C, Liu H-S, Lin
A, Meager A, Janus J and Ennis FA. YS, Yeh T-M, Liu C-C and Lei H-Y.
Activation of T lymphocytes in dengue virus Manifestation of thrombocytopenia in
infections. High levels of soluble interleukin dengue-2-virus-infected mice. Journal of
2 receptor, soluble CD4, soluble CD8, General Virology, 2000, 81: 21772182.
interleukin 2, and interferon-gamma in the
sera of children with dengue. Journal of
Clinical Investigation, 1991, 88: 1473-1480.
[18] Kurane I, Innis BL, Nimmannitya S, Nisalak
A, Meager A and Ennis FA. High levels of
interferon alpha in the sera of children with
dengue virus infection. The American
Journal of Tropical Medicine and Hygiene,
1993, 48: 222-229.

Philippine Species of Mesocyclops (Crustacea:
Copepoda) as a Biological Control Agent of
Aedes aegypti (Linnaeus)
Cecilia Mejica Panogadia-Reyes*#, Estrella Irlandez Cruz**
and Soledad Lopez Bautista***
*Department of Biology, Emilio Aguinaldo College, Ermita, Manila, MM, Philippines
**Research Institute for Tropical Medicine, Alabang, Muntinlupa, MM, Philippines
***Department of Medical Technology, Emilio Aguinaldo College, Ermita, Manila, MM, Philippines
Abstract
The predatory capacity of two local populations of Mesocyclops aspericornis (Daday) and Mesocyclops
ogunnus species were evaluated, for the first time in the Philippines, as a biological control agent for
Aedes aegypti (L) mosquitoes. Under laboratory conditions, Mesocyclops attacked the mosquito first
instar larvae by the tail, side and head. The mean of first instar larvae consumed by M. aspericornis and
M. ogunnus were 23.96 and 15.00, respectively. An analysis of the variance showed that there was a
highly significant difference between the mean number of first instar mosquito larvae consumed by
M. aspericornis and by M. ogunnus, which indicated that the former is a more efficient predator of
dengue mosquito larvae.
The results of the small-scale field trials showed that the mean number of surviving larvae in
experimental drums was 63.10 and in control drums was 202.95. The Student t-test of means indicated
that there was a significant difference between the mean number of surviving larvae in the drums with
and without M. aspericornis. The findings indicated that M. aspericornis females were good biological
control agents, for they destroyed/consumed about two-thirds of the wild dengue mosquito larvae
population.
Keywords: Mesocyclops aspericornis, Mesocyclops ogunnus, biological control agent, Aedes aegypti, Aedes albopictus,
Philippines.
Introduction aquatic animals of their own size including

small fishes and mosquito larvae. Some
Copepods feed on paramecium (ciliates adults are able to tear pieces out of the
protozoans) while naupli feed on body of their victims with their strong
Chilomonas spp. The adult planktonic mandibles. In the Philippines, around 14
copepods utilize diatoms as the principal species of Cyclops have been recorded, of
food, while predacious adult copepods which Mesocyclops aspericornis and
feed on protozoa, rotifers and several M. ogunnus are more common[1,2] .
#
E-mail: cpreyes@eac.edu.ph

Mesocyclops as a Biological Control Agent of Aedes aegypti
In Brazil, a study[3] reported that under Laboratory trials

laboratory conditions, four different strains
of M. aspericornis showed the potential as In a 600 ml beaker, 500 ml filtered tap
biological control agents of Aedes aegypti water with pH 7 was poured. Then 50
larvae. In Viet Nam, under laboratory Aedes aegypti L1 obtained from laboratory
conditions, M. aspericornis consumed a culture and a female M. aspericornis were
mean 23.75 L1 and killed a mean 13.43 added at the same time. The same
L1, or a total of 37.18 L1 within 24 hours. procedure was done for M. ogunnus. The
M. ogunnus, on the other hand, consumed control group did not receive Mesocyclops.
a mean 8.48 L1 and killed a mean 7.54 L1 The experiment was replicated six times
or a total of 16.02 L1 within 24 hours [4] . In and was observed every day for five
Australia, six species of Mesocyclops were consecutive days, with daily replacements
evaluated as biological control agents of of new L1. The daily number of L1
Aedes aegypti[5] . Of these, M. aspericornis destroyed or consumed by M. aspericornis
was found to be the most effective and M. ogunnus and those that died in the
predator. This study attempted to evaluate, control group were determined.
for the first time, the potential of the local Copepods feeding behaviour was
population of two species of Mesocyclops observed.
as biological control agents of Aedes
aegypti (L) under laboratory and field Field trials
conditions in the Philippines. Permission to conduct the study in Estero
de Tanque located at P Nieto Street,
Barangay 674, Zone 73, Paco, Manila, was
Methodology secured from the community health
officials and Barangay chairperson.
Mesocyclops culture
Members of the households were
Mesocyclops aspericornis and M. ogunnus informed regarding the procedures to be
were raised in laboratory following the undertaken and the possible benefit they
techniques adopted by Marten and could derive from it. Consent from the
Thomson[6] . To ensure the establishment of caretakers of the sample households was
a single species culture, a female with egg also secured.
sacs was captured and placed in a petri
A preliminary study was conducted for
dish for examination under a dissecting
15 days. Twenty houses were chosen as
microscope before it was transferred to a
study sites. Two drums per household or a
wide-mouthed beaker containing 100 ml
total of 40 drums were emptied and filled
of mixed culture of Paramecium caudatum
almost to the brim with tap water, pH 7.2.
and Chilomonas sp. (food of Mesocyclops).
A litre of water as sample from each drum
Populations of Chilomonas sp. and
container was collected in sterile bottles
P. caudatum in the culture bottle were
and brought to the laboratory to exclude
maintained using sterile wheat seeds.
the presence of fungi, bacteria and
Sample specimens from copepods culture
indigenous copepods. Mosquito eggs that
were sent to Maria Holynska, Museum
hatched into larvae in drums were
and Institute of Zoology, Warsaw, Poland,
monitored and collected daily for
for identification.

recording and identification. Water pH, Results and discussion

water temperature and ambient
temperature were taken daily. Drums Laboratory trials
were checked daily for the presence of
mosquito eggs and larvae with the aid of a The results of the predatory capacity of
magnifying glass. Drums with mosquito M. aspericornis and M. ogunnus as
larvae were marked and female copepods evaluated under laboratory conditions and
were introduced in experimental drums analysis of variance are presented in
using the ratio one Mesocyclops Tables 1 and 2. The mean L1 consumed
aspericornis per 50 Aedes aegypti first by M. aspericornis was 23.96 while that of
instar larvae (L1). No copepods were M. ogunnus was 15.00. Control means
introduced in the 20 control drums. were 0.63 L1 and 0.60 L1, respectively.
Observation was made daily for 15 The findings showed that there was a
consecutive days. Surviving larvae were highly significant difference between the
collected from all drums and brought to mean number of Ae. aegypti L1 consumed
the laboratory for recording and species by M. aspericornis and that of M. ogunnus
identification. and the control group.
Table 1. Predatory capacity of female Mesocyclops aspericornis vs Aedes aegypti first instar
larvae in 500 ml filtered water under laboratory conditions
Statistical
Treatment Mean F value Tabular f
significance
Experimental group 23.96 338.83 7.12 Highly significant
Control group 0.63
Table 2. Predatory capacity of female Mesocyclops ogunnus vs Aedes aegypti first instar
larvae in 500 ml filtered water under laboratory conditions
Statistical
significance
Experimental group 15.00 319.18 7.12 Highly significant
Control group 0.60
To determine if there was a difference difference between the mean number of

between the mean number of L1 L1 consumed by M. aspericornis and by
consumed by M. aspericornis and M. ogunnus. The study showed that
M. ogunnus, the analysis of variance was M. aspericornis was a more efficient
carried out (Table 3). The findings showed predator of Aedes aegypti larvae.
that there was a highly significant

Table 3. Comparison of the predatory capacity of M. aspericornis and M. ogunnus

under laboratory conditions
Statistical
significance
M. aspericornis 23.96 36.11 7.12 Highly significant
M. ogunnus 15.00
Table 4. Predatory capacity of Mesocyclops aspericornis vs Aedes larvae in small-scale

field trials using drum containers
Statistical
Treatments # Replicates Mean + SD P value
significance
Experimental group 20 63.10 + 59.43 0.0002 Significant

Control group 20 202.95 + 140.43
Field trials laboratory and field conditions. These

copepods could be effectively used for the
The results of the field trials are presented in control of Aedes breeding in non-removable
Table 4. containers, viz. drums and used tyres.
The mean L1 in experimental drums was
63.10 and 202.95 L1 in control drums. To
determine the difference between the mean Acknowledgements
number of surviving larvae in the drums with The World Health Organization and the
M. aspericornis and in the drums without M. Yaman Lahi Foundation, Inc. Emilio
aspericornis, a Student t-test was used. The Aguinaldo College provided financial
findings indicated that there was a significant support for this project. We acknowledge
difference between the mean number of the kind assistance of Dr Kevin Palmer of
surviving larvae in the drums with and the WHO Western Pacific Region; Dr Jose
without Mesocyclops. This significant Paulo E. Campos of Yaman Lahi Foundation,
difference suggests that M. aspericornis is a Inc. Emilio Aguinaldo College (YLFI-EAC),
good biological control agent, for it Philippines; Dr Remigio M. Olveda of the
consumed about two-thirds of the wild, Department of Health Research Institute
dengue mosquito larvae population. for Tropical Medicine (DOH-RITM),
Philippines; Dr Maria Holynska of Museum
and Institute of Zoology Polish Academy
Conclusion of Science (MIZ-PAS), Poland; Dr Vu Sinh
The results of the study showed that Nam of the National Institute of Hygiene
Mesocyclops aspericornis was an efficient and Epidemiology (NIHE), Viet Nam; and
predator of Aedes aegypti larvae both in Dr Brian H. Kay of the Queensland Institute
of Medical Research (QIMR), Australia.

References
[1] Mamaril AC and Fernando CH. Freshwater [4] Nam VS, Tien TV, Huan TQ, Yen NT, Kay B,
zooplankton of the Philippines (Rotifera, Marchand R, Marten G, Holynska M and
Cladocera, and Copepoda). Natural and Reid J. Mesocyclops of Vietnam Part I -
Applied Science Bulletin, 1978, 30(4): 109- laboratory evaluation as biological agent for
221. control of Aedes aegypti. Dengue Bulletin,
1999, 23: 89-93.
[2] Tuyor JB and Baay MO. Contribution to the
knowledge of freshwater copepods of the [5] Brown MD, Kay BH and Hendrikz JK.
Philippines. Asia Life Sciences, 2001, 10(1): Evaluation of Australian Mesocyclops
35-43. (Cyclopoida: Cyclopidae) for mosquito
control. J Med Entomol, 1991, 28(5): 618-
[3] Kay BH, Cabral CP, Sleigh AC, Brown MD,
623.
Ribeiro ZM and Vasconcelos AW.
Laboratory evaluation of Brazilian [6] Marten G and Thompson G. Copepod
Mesocyclops (Copepoda: Cyclopidae) for production and application for control
mosquito control. J Med Entomol, 1992, procedure for New Orleans Mosquito Board,
29(4): 599-602. City of New Orleans, USA, 1997.

Susceptibility of Aedes aegypti to Insecticides
in Viet Nam
Vu Duc Huong#, Nguyen Thi Bach Ngoc, Do Thi Hien and Nguyen Thi Bich Lien
Department of Entomology, National Institute of Malariology, Parasitology and Entomology,
B.C. 10 200 Tu Liem, Hanoi, Viet Nam
Abstract
During 2000-2002, studies on the susceptibility of Aedes aegypti to insecticides were conducted at 22
places in 11 provinces and cities in four different regions of Viet Nam. Aedes aegypti was found
susceptible to malathion, but resistant to DDT in almost all the study sites. It continues to be susceptible
to the pyrethroid group of insecticides (permethrin, lambda-cyhalothrin, deltamethrin and alpha-
cypermethrin) in many places in the North and Centre regions, but is resistant to these insecticides in
many places in the South and Central Highlands in Viet Nam. However, the species was found highly
and widely resistant to etofenprox.
Keywords: Aedes aegypti, pyrethroids, insecticides, Viet Nam.
Introduction Both malaria and DHF were endemic in

many mountainous, forested and coastal
Insecticidal measures, especially in the plain areas of Viet Nam where house spray
outbreak-risk areas, are the most important and bednet treatment were applied for
for the control of Aedes aegypti, the main years. DDT was widely used before 1990
vector of DHF. Many insecticides of the and later lambda-cyhalothrin, permethrin
group organochlorine (DDT), organo- and deltamethrin[5] were introduced. In
phosphorous (fenthion, malathion and 1999, Aedes aegypti was found resistant to
temephos) and pyrethroid (permethrin, DDT and some insecticides of the
deltamethrin, lambda-cyhalothrin, etc.) have pyrethroid group in many places in Nam Bo
been used for the malaria control programme (the South), Central Highlands, but not yet
and for Aedes aegypti. Aedes aegypti has to malathion[6] . This study provides more
data on the susceptibility of Aedes aegypti to
been resistant to DDT since the early 1960s
insecticides in different regions of Viet Nam.
and cross-resistant to many insecticides of the
pyrethroid and temephos groups in many
countries, but is not yet resistant to Materials and methods
malathion[1-3] . When this species is resistant
to the insecticides of the pyrethroid group, Time and study regions
the organophosphorous ones could take their During 2000-2002, studies were conducted
place[4] . at 22 places (located in 11 provinces and
#
E-mail: entomology@fpt.vn

Susceptibility of Aedes aegypti to Insecticides in Viet Nam
cities) six places in the North, six in the The susceptibility to the insecticides was
Centre, six in the South and four in the evaluated on the following criteria:
Central Highlands.
Mortality 98-100%: Susceptible to
insecticide.
Methods Mortality 80-97%: Possibility of
The WHO standard bioassay tests (1998) [7] resistance to insecticide.
were followed and the papers treated with Mortality <80%: Resistant to insecticide.
DDT 4%, the control paper with OC
(organochlorine control), malathion 5% and
the control paper with OP (organophosphate Results
control) and 5 insecticides of the pyrethroid
group (permethrin 0.75%, lambda- North
cyhalothrin 0.05%, deltamethrin 0.05%,
Of the six study places, Aedes aegypti was
alpha-cypermethrin 30mg/m2 and etofenprox
found resistant to DDT at five places and
0.5%) with the same control paper PY
possibly resistant to DDT at one place; it
(pyrethroid control).
was susceptible to malathion at five places
The tests were done at a temperature of and possibly resistant to this insecticide at
25 C 2 C and humidity of 75-85%. one place. It was susceptible to all the tested
The unfed, F1 mosquitoes, one or two days insecticides of the pyrethroid group such as
old at least 150 mosquitoes for each permethrin, lambda-cyhalothrin, deltamethrin
insecticide, 100 for the test and 50 for the and alpha-cypermethrin in at five places and
control, were used. Twenty-five mosquitoes the possibility of resistance to these four
were put in each test tube and the per cent insecticides existed at one place. Aedes
mortality count was done 24 hours after the aegypti was resistant to etofenprox at four
exposure. The mosquitoes in the resting places and possibly resistant to etofenprox at
tubes were then fed with glucose 10% in two places (Table 1).
soaked cotton.
Table 1. Results of suceptibility tests on Aedes aegypti to insecticides

in the North and Centre, Viet Nam
% Mortality
S. No. Places Lambda - Alpha-

Permethrin Deltamethrin Etofenprox DDT Malathion
cyhalothrin cypermethrin
0.75% 0.05% 2 0.05% 4% 5%
0.05% 30mg/m
1. Thi Cau (Co) 100 100 100 100 68 57 100
Bac Ninh (T)
Bac Ninh (P)
2. Phu Lang (Co) 100 100 100 100 92 94 100
Que Vo (D)
Bac Ninh (P)
3. Cat Ba (S) 100 100 100 100 42 14 100
Cat Hai (D)
Hai Phong (C)

% Mortality
S. No. Places Lambda - Alpha-

0.75% 0.05% 2 0.05% 4% 5%
0.05% 30mg/m
4. Niem Nghia (Co) 91 92 94 95 92 21 100
Le Chan (D)
Hai Phong (C)
5. Ly Thai To (Co) 99 96 (200) 100 100 18 21 97.33
Hoan Kiem (D) (150)
Ha Noi (C)
6. Thinh Liet (Co) 100 99 98 98 37 60 98
Thanh Tri (D)
Ha Noi (C)
7. Thach Phu (Co) 100 100 100 100 100 63 100
HaTinh (T)
Ha Tinh (P)
8. Duc Tho (S) 100 100 100 100 93 37.39 100
Duc Tho (D) (115)
Ha Tinh (P)
9. Song Cau (S) 100 100 100 98 51 4 100
Song Cau (D)
Phu Yen (P)
10. No. 6 (Co) 100 100 100 91 76 2 100
Tuy Hoa (T)
Phu Yen (P)
11. Dong Luong (Co) 94 96 97 95 16 2 100
Dong Ha (T)
Quang Tri (P)
12. Trieu Do (Co) 100 100 100 100 86 11 100
Trieu Phong (D)
Quang Tri (P)
No. 1 - 6 in the North
No. 7 - 12 in the Centre
Co: Commune; S: Small town; D: District; T: Town; P: Province; C: City
Figures in parenthesis indicate the number of mosquitoes tested
Central possibly resistant and susceptible to

etofenprox at two and one places,
Aedes aegypti was found resistant to DDT respectively (Table 1).
but was susceptible to malathion in all six
places. It was susceptible to four insecticides
South
of the pyrethroid group such as permethrin,
lambda-cyhalothrin, deltamethrin and Aedes aegypti was resistant to DDT at all six
alpha-cypermethrin in five places and places; but susceptible to malathion at four
possibly resistant to them in one place, and places and possibility of resistance to
resistant to etofenprox in three places, and malathion at two places; it was resistant to

permethrin and lambda-cyhalothrin at four resistance to alpha-cypermethrin at two

places and the possibility of resistance to places and possibility of resistance to alpha-
permethrin and lambda-cyhalothrin at twor cypermethrin at four places as well as
places. It also showed resistance to resistance to etofenprox at all six places
deltamethrin at one place and possibility of (Table 2).
resistance to deltamethrin at five places;
Table 2. Results of susceptibility tests on Aedes aegypti to insecticides

in the South and Central Highlands, Viet Nam
% Mortality
No. Places Lambda - Alpha-

0.75% 0.05% 2 0.05% 4% 5%
0.05% 30mg/m
1. No. 6 (Co) 62 67 90 82 4 2 81
Ben Tre (T)
Ben Tre (D)
2. BinhThuan (Co) 94 90 97 92 53 20 100
Binh Dai (D)
Ben Tre (P)
3. Binh Khanh (Co) 18 24 84 75 1 0 99
Can Gio (D)
Ho Chi Minh (C)
4. Binh Trung Tay (Co) 96 93 97 96 20 11 95
No. 2 (D)
Ho Chi Minh (C)
5. An Loc (S) 79 56 84 96 1 0.8 90
Binh Long (D) (125)
Binh Phuoc (P)
6. Tan Xuan (Co) 8 28 19.33 25 7 2 99
Dong Xoai (T) (150)
Binh Phuoc (P)
7. Plei Can (S) 36 32 51 28 1 1 100
Ngoc Hoi (D)
Kon Tum (P)
8. Quyet Thang (Co) 57 61 66 47 1 1 100
Kon Tum (T)
Kon Tum (P)
9. Buon Trap (Co) 5 11 41 40 0 6 97
Krong Ana (D)
Dak Lak (P)
10. Thang Loi (Co) 24 36 63 34 23 0 98
Buon Me Thuot (C)
Dak Lak (P)
No. 1 - 6 in the South
No. 7 - 10 in the Central highlands
Co: Commune; S: Small town; D: District; T: Town; P: Province; C: City
Figures in parenthesis indicate the number of mosquitoes tested

Central Highlands were not completely comparable with the

observations made by Reiter and Gubler
Aedes aegypti was resistant to DDT, (1997) [3] , and Vu Duc Huong and Nguyen
permethrin, lambda-cyhalothrin, deltamethrin, Thi Bach Ngoc (1999) [6] . Aedes aegypti was
alpha-cypermethrin and etofenprox at all possibly resistant to malathion in some
four places in this region and susceptible to places. This discrepancy in different regions
malathion at three places and showed was possibly due to the longer and more
possibility of resistance to malathion at one extended use of the insecticides of the
place (Table 2). pyrethroid group in malaria and dengue
haemorrhagic fever control programmes and
in agriculture in the Southern and Central
Discussion Highlands. It is therefore suggested that the
Aedes aegypti was susceptible to malathion susceptibility tests should be conducted on
and resistant to DDT at almost all study all insecticides before use. Moreover, the
places in Viet Nam. It was still susceptible to cross-resistance of Aedes aegypti to
the four insecticides of the pyrethroid group insecticides belonging to the pyrethroid
(permethrin, lambda-cyhalothrin, deltamethrin group should also be checked. Aedes
and alpha-cypermethrin) in many places in aegypti was highly and widely resistant to
North and Centre Viet Nam, but resistant to etofenprox and further studies should be
these insecticides in many places in the conducted in this context.
South and Central Highlands. These results
References
[1] Yap HH, Cheng NL, Foo AES and Lee CY. [5] Phan VT. Malaria epidemiology and malaria
Dengue vector control: present status and control in Vietnam. Medical Publishing
future prospects. Kaoshiung J Med Sci, House, Hanoi, 1996: 218-241 (in
1994,10: 102-108. Vietnamese).
[2] World Health Organization. Dengue [6] Huong VD and Bach Ngoc NT.
haemorrhagic fever: diagnosis, treatment, Susceptibility of Aedes aegypti in south
prevention and control. 2nd edn. Geneva: Vietnam. Dengue Bulletin, 1999, 23: 85-88.
WHO, 1997: 48-59.
[7] World Health Organization. Report of the
[3] Reiter P and Gubler DG. Surveillance and WHO informal consultation. Test
control of urban dengue vectors. Dengue procedures for insecticide resistance
and dengue haemorrhagic fever. Colorado: monitoring in malaria vector, bio-efficacy
CAB International, 1997: 425-454. and persistence of insecticides on treated
surfaces. Geneva: WHO, 1998. Document
[4] Kantachuvesiri A. Dengue haemorrhagic WHO/CDC/CPC/MAL/98.12: 1-43.
fever in Thai society. Southeast Asian J Trop
Med Public Heath, 2002, 33(1): 56-67.

Ovipositioning Behaviour of Aedes aegypti in
Different Concentrations of Latex of Calotropis procera:
Studies on Refractory Behaviour and its Sustenance
across Gonotrophic Cycles
Manju Singhi, Vinod Joshi#, R.C. Sharma and Keerti Sharma
Desert Medicine Research Centre (Indian Council of Medical Research), New Pali Road,
Jodhpur 342 005, India
Abstract
Dengue fever associated with dengue haemorrhagic fever is gaining endemicity in India. Due to lack
of any chemotherapy against this arboviral infection, the control of the disease depends largely on
preventive measures against Aedes mosquito vectors. A wild shrub, Calotropis procera, commonly
growing in the desert areas of Rajasthan has shown a remarkable effect as a larvicide against Aedes
aegypti. However, different water concentrations of this biocide have also brought forward very
important observations on the ovipositioning behaviour of Aedes aegypti. At 0.7% concentration of
latex, the ovipositiong was avoided by the gravid female mosquitoes and this behaviour continued till
three gonotrophic cycles. However, at lower concentrations (0.2% and 0.1%) of the larvicidal latex,
the refractory behaviour of ovipositioning could not be retained up to the third gonotrophic cycle. The
concentration of latex such as 0.7% and 0.2% were observed as ovicidal also and this effect continued
across all the gonotrophic cycles. The behavioural observations reported in the present study may
serve as significant information on choosing bio-larvicides for vector control against dengue.
Keywords: Dengue, Aedes aegypti, Calotropis procera, ovipositioning, refractory behaviour, gonotrophic cycle.
Introduction have been reported[2-4] . It has also been

established that dengue virus undergoes
Dengue fever and dengue haemorrhagic transovarial transmission across generations
fever (DF/DHF) is gaining endemicity in of Aedes aegypti under natural as well as
many states in India[1] . In the absence of experimental conditions[5,6] . Latex of
chemotherapy and vaccines, vector control, Calotropis procera, a milky weed plant
largely based on larval control, is the only growing all across the desert areas in the
option available. In Rajasthan, a number of state has shown promising larvicidal
epidemics of dengue associated with DHF properties in a series of laboratory
#
E-mail: vinodjoshi@dmrcjodhpur.org

Ovipositioning Behaviour of Aedes aegypti in Different Concentrations of Latex of Calotropis procera
experiments carried out against Aedes The experiments were conducted at 25-
aegypti (unpublished data, Desert Medicine 30 C (room) temperature and at relative
Research Centre). In an attempt to assess humidity of about 60-70%. The eggs of each
the value of this agent as an oviposition generation were counted under a dissecting
attractant for use in ovitraps, it was microscope in the control as well as
discovered that the agent showed experimental sets. All the eggs were
oviposition refractoriness instead. The immersed in plain water to observe whether
present communication incorporates the exposure to larvicide had any ovicidal effect
findings of these studies. on them.
Materials and methods Results and discussion

Six experimental sets were designed for the Table 1 shows relative observations on
study. Cages of 30 cm3 size, with wooden different concentrations of latex of
frame and iron mesh with muslin cloth on Calotropis procera on the ovipositioning
one side, were used as units of present behaviour of Aedes aegypti. In experimental
experiments. In each cage, 16 gravid cage A, while in the control set, 65 eggs
females of Aedes aegypti were released. The were laid, in the corresponding lethal
concentration of latex in water, which concentration of 0.7% of the same cage, no
showed the highest (0.7%), moderate (0.2%) eggs were laid. In this cage all the
and no mortality (0.1%) effect in the mosquitoes showed persistent refractiveness
larvicidal efficacy experiments, were of ovipositioning across all gonotrophic
prepared and put in beakers in the cages A, cycles from G1-G3. In cage B where 0.2%
B, C, D and E while cage F was kept without concentration was offered along with
latex solution and contained only water. A control, refractive behaviour up to two
beaker containing plain water was also cycles only was observed while in the third
placed in each of the six cages to serve as cycle (G3), 127 eggs were laid. Similar
control. While in cage D all the observations were made in cage C. It was
experimental concentrations were placed interesting to note that in cage D where all
along with the control, in cage E all the the experimental concentrations were
concentrations were placed without choice offered to mosquitoes along with choice of
of a control. In the sixth cage (cage F) two control, even in G1 cycle no refractiveness
beakers with plain water were placed was shown. In experiment E where choice
without any experimental lethal of control (plain water) was not offered,
concentration. The eggs laid by female maximum preference for ovipositioning was
Aedes aegypti were counted after 48 hours shown in lowest concentration (0.1%). The
in each of the experimental cages. Second different preference among different
and third blood meals were provided to concentrations continued across all the
facilitate G2 and G3 (gonotrophic cycles) to gonotrophic cycle (Table 1).
mosquitoes.

Table 1. Ovipositing preference of Aedes aegypti in different larvicidal concentrations of latex of

Calotropis procera
Latex concentration (%) in experimental cages

Cage
A B C D E F
Eggs laid C 0.7 C 0.2 C 0.1 C 0.1 0.2 0.7 0.1 0.2 0.7 C C
within 48
hrs
G1 65 0 156 0 180 0 109 18 18 6 101 51 11 102 110
G2 93 0 156 1 149 0 100 0 0 0 33 10 7 96 83
G3 275 0 230 127 275 52 270 98 31 0 215 150 20 233 197
C: Control
G1-G3: Gonotropic cycles
Table 2. Effect of latex on the percentage viability of eggs of Aedes aegypti
Experimental cages
Cage
A B C D E F
Latex (%) C 0.7 C 0.2 C 0.1 C 0.1 0.2 0.7 0.1 0.2 0.7 C C
Eggs 65 0 156 0 180 0 109 18 18 6 101 51 11 102 110
immersed
Eggs 55 0 78 0 126 0 78 0 0 0 6 2 0 96 103
hatched
% eggs 84.6 0.0 50 0.0 70 0.0 73 0.0 0.0 0.0 5.9 4.0 0.0 93.1 93.6
hatched
C: Control
Table 2 shows the results of the Dengue fever associated with DHF has
experiment carried out to study the viability become a problem of public health
of eggs that were exposed to different importance. Available evidence shows that
larvicidal concentrations and to control the virus undergoes vertical transmission
groups. The eggs laid in experiments A, B, C across generations of mosquitoes[6] . Larval
and D showed no hatching, while in control, therefore, is the most effective
experimental cage E where no control had approach to restrict the vector and virus
been kept, the eggs laid up to a sustenance in nature. The wildly grown
concentration 0.2% showed some viability plant, Calotropis procera, has shown very
and in cage F where only controls were kept encouraging results in its different lethal
the laid eggs showed 93% viability. concentrations. However, if this plant

species is to be used as a material of choice, domestic mosquito fauna in such premises

its other aspects such as the ovipositioning will lay the eggs but they will lose their
behaviour of gravid females towards the viability to hatch into larvae (Table 2).
larvicide can also become known.
The role of Calotropis procera as a
The significance of the reported larvicide has been reported by other workers
observations is that the refractiveness from India[7] . However, contrary results have
developed by the species is sustained across been reported by some workers[8] where
two gonotrophic cycles when one larvicidal insect growth regulators actually enhanced
concentration was offered with a ovipositioning.
corresponding control (Table 1). However,
The observations reported by us not only
when all the concentrations viz. 0.1 %,
present the results of the ovipositioning
0.2 % and 0.7% were of fered with one
behaviour induced by a bio-larvicide, but
control, such avoidance was not shown.
also add the information on relative
Similarly, in the experimental set E where all
preference in ovipositioning and its
concentrations of latex were made available
sustenance across gonotrophic cycles when
without any control, the maximum egg
different larvicidal concentrations were
laying was preferred in 0.1 % of latex. The
offered.
data suggest fine chemo-sensation in Aedes
aegypti where the ovipositioning female
could distinguish to choose the least Acknowledgements
larvicidal concentration for its egg laying.
The authors gratefully acknowledge the
The observation showed that if in the guidance and inspiration received from
various types of domestic containers where Mr N.L. Kalra, Member, Scientific Advisory
water is stored a non-lethal concentration of Committee of the Desert Medicine Research
latex (0.1%) is used, the refractiveness of Centre, Jodhpur, India.
ovipositioning will not be there and the
References
[1] Dengue alert in South-East Asia Region. outbreak of febrile illness in Jaipur city, India.
New Delhi: World Health Organization, Indian Journal of Medical Research, 1973,
Regional Office for South-East Asia, 2004 61(12): 1737-1743.
(http://w3.whosea.org/index.htm, accessed [4] Chouhan GS, Rodrigues FM, Shaikh BH,
25 August 2004). Ilkal MA, Khangaro SS, Mathur KN, Joshi KR
[2] Ghosh SN and Sheikh BH. Investigations on and Vaidhye NK. Clinical and virological
the outbreak of dengue fever in Ajmer city, study of dengue fever outbreak in Jalore city,
Rajasthan. Part II: Results of serological tests. Rajasthan, 1985, Indian Journal of Medical
Indian Journal of Medical Research, 1974, Research, 1990, 91: 414-418.
62: 523-533. [5] Joshi V, Singhi M and Chaudhary RC.
[3] Padbidri VS, Dandawate CN, Goverdhan Transovarial transmission of dengue 3 virus
MK, Bhat UK, Rodrigues FM, D'Lima LV, by Aedes aegypti. Transaction of Royal
Kaul HN, Guru PY, Sharma R and Gupta NP. Society of Tropical Medicine and Hygiene,
An investigation of the etiology of the 1971 1996, 90: 643-644.

[6] Joshi V, Mourya D and Sharma RC. [7] Girdhar G, Deval K, Mittal PK and
Persistence of vertical transmission of Vasudevan P. Mosquito control by
dengue-3 virus through vertical transmission Calotropis latex. Pesticides, 1984, 26-29.
passage in successive generations of Aedes
[8] Moore CG. Insecticide avoidance by
aegypti mosquitoes. American Journal of
ovipositioning Aedes aegypti. Mosquito
Tropical Medicine and Hygiene, 2002,
News, 1977, 37: 291-293.
67(2): 158-161.

Community-based Assessment of Dengue-related
Knowledge among Caregivers
Khynn Than Win* #, Sian Za Nang** and Aye Min***
*Health Systems Research Division, Department of Medical Research (Lower Myanmar), Myanmar
**Health Education Bureau, Department of Health Planning, Myanmar
***Vector Borne Disease Control Programme, Department of Health, Myanmar
Abstract
The study was conducted in Thaketa township in Myanmar involving 405 respondents aged 18 years and
above. It was aimed: (i) to explore the extent of dengue-related knowledge among caregivers; (ii) to
identify the exposure of community members to the existing IEC materials; and (iii) to find out the factors
related to high knowledge scores. The findings were triangulated by results from personal interviews,
focus group discussions and observational checklist. The difference of mean scores among males and
females was not statistically significant. Knowledge scores of the caregivers were not statistically different
whether there was a primary DHF case at home or not. Almost 60% of the interviewees had received
information on DHF by watching television and they observed that television was the most effective
medium. Females with more than six years of schooling, persons who had access to pamphlets/posters,
television, newspapers and journals got higher scores than the unexposed group. Less than 15% were not
exposed to any of the IEC materials. Aedes aegypti larvae were found in 67% of water storage tanks and
15.9% of flower vases when using observational checklist. Focus group discussions were held for drafting
IEC materials. Community members were more interested in the mode of DHF transmission to children
rather than in the elimination of the Aedes mosquitoes. A low practice score was observed in those with
high knowledge level, which means that high knowledge does not necessarily lead to high practice. Less
than half of the respondents had seen posters and pamphlets. IEC materials need to be improved so that
they present the message most effectively and they should be extensively distributed in the community.
Keywords: Dengue, caregivers, community, IEC material, knowledge score, Myanmar.
Introduction termed DHF as one of the diseases under

national surveillance. DHF is also listed as
Dengue haemorrhagic fever (DHF) is the 17th priority disease in Myanmar. One of
endemo-epidemic in 12 out of 14 states and the strategies devised in NHP for the
divisions in Myanmar and is transmitted by prevention and control of DHF is
Aedes aegypti. Most of the reported cases production of guidelines for basic health staff
are under 15 years of age[1] . The Myanmar (BHS) as part of the information, education
National Health Plan (NHP) (1996-2001) and communication (IEC) programme.
#
E-mail: thaint@mail4u.com.mm

Assessment of Dengue-related KAP among Caregivers
IEC initiatives are based on the concepts materials; and (iii) to find out the factors
of prevention and primary health care. They responsible for high knowledge scores.
create awareness, increase knowledge,
change attitudes and motivate people to
adopt new ideas [2] . Methods and materials
Communication participation appears This community-based cross-sectional study
to be one of the most promising innovative was based on multistage sampling to identify
means to prevent and control DHF. Simple 405 caregivers in Thaketa township, Yangon.
elimination of vector-breeding water This area was one of the dengue endemic
collections or source reduction is the regions in the Yangon division and the case-
possible answer to the problem. Community fatality rate (CFR) was 1.65% in 2002. Both
activities are identified mainly as reduction quantitative and qualitative data collection
of non-essential water containers, protection methods, including observation checklist,
of water containers from larvae breeding, were used in the study. The study sample
larviciding and release of larvivorous fish. included household members aged 18 years
Community participation needs to be and above. The households with children
sustained by dissemination of health were selected randomly. In these
messages through various channels[3] . households, we chose one subject from
each household, regardless of sex. The
Existing IEC materials in Myanmar respondent was a relative of the child
included health talks routinely carried out in (mother/father/grandfather/grandmother/
schools and in the community. Health brother/sister/uncle/aunt). Mothers were the
messages were distributed through radio, key persons to be interviewed after taking
television, newspapers and journals before their consent. The questionnaire was pilot-
and during the epidemic season. Pamphlets tested for clarity and validity; all questions
were developed locally in states and were reviewed by epidemiologists, public
divisions. However, it is necessary to find health experts, health educators and by
out the most appropriate IEC materials and investigators experienced in conducting
means that would be relevant for various community-based surveys in DHF. In order
communities. to ensure the accuracy and completeness of
This study attempted to improve the data, our surveyors were trained before and
existing IEC materials on DHF control based after pre-testing.
on the knowledge and practice of child-care Ten sessions of Focus Group
providers in the Thaketa township of the Discussions (FGDs) were performed among
Yangon division in Myanmar. DHF control basic health staff (BHS), general practitioners,
will be more effective in the future by Maternal and Child Welfare Association
strengthening community participation on (MCWA) members, other volunteers
case information and source reduction. including Ward Law and Order Restoration
This research aimed to: (i) explore the Council members, voluntary fire brigade
members, etc., for recommendation of
extent of dengue-related knowledge among
existing IEC activities (including television,
caregivers; (ii) identify the exposure of
radio, newspapers, local journals and
community members to the existing IEC
pamphlets). In every FGD, the moderator

explained thoroughly the purpose of Table 1. Dengue-related knowledge

conducting the discussions. The Township responses of caregivers
Health Centre, Ward Law and Order
Restoration Council offices and homes were Responses Frequency Percent
chosen for holding FGDs. DHF is common in 254 44.4
children 3-8 years of age
Caregivers were those who took care of
children at home, or supervised at the DHF is transmitted by 331 81.7
the mosquito
health centre or clinic, or advised parents on
home care of a child with fever. Mosquito species of 204 61.6
DHF vector is Aedes
Data checking, cleaning and validation
Biting time of mosquitoes 266 80.4
were performed using Epi-info 6.0, and data
is at daytime
analysis was conducted using SPSS 10.0.
P<0.05 was used as the definition of Aedes breed in
statistical significance. The study period clear water 135 33.3
lasted one year starting in June 2002. polluted water 166 41.0
The Aedes mosquito 253 62.5
breeds inside the house 144 35.6
Results flower vases 96 23.7
ant traps
About respondents
The Aedes mosquito 292 72.1
The sex ratio of the respondents was 7:1 for breeds outside the house
females and males. The mean age was water containers 178 61.0
35.910.3. The majority of the respondents old tyres, broken pots, 75 25.7
were aged 18-35 years, literate and and coconut shells
dependants. About 8.6% of them had blocked gutters 9 2.2
received 12 years school education. DHF transmission is 348 85.9
Nearly 40% lived in their own wooden highest in the rainy
houses. Most of the households (77.8%) had season
1 to 2 children under 15 years of age. Only
DHF may be fatal 382 94.3
34 (8.4%) of the households that
participated in the interview had a child There is a vaccine for 243 60.0
prevention of DHF
with history of DHF.
A previously infected 293 72.3
The difference of mean score among child may get DHF again.
males and females was not statistically
If the child is febrile, 262 64.7
significant (P=0.271).
DHF should be
observed
Dengue-related knowledge
responses The total knowledge scores were
categorized as low (0-19) and high (20-39).
The responses of caregivers are included in The percentage of the high score group was
Table 1. more than that of the low score group

(68.6% vs 31.4%). Signs and symptoms of Existing exposure to IEC material in

DHF were fever (57.3%), vomiting (51.6%), the community
purpura (36.3%), drowsiness (28.1%), cold
extremities (17%), etc. Table 2 contains the responses of the
communities to IEC materials.
Knowledge score of respondents
with and without history Table 2. Exposure to IEC materials in the
community
of DHF case in their homes
Community members exposed
Knowledge scores with and without past (n) Percent
to IEC materials
history of primary DHF cases at home are
given in the Figure. had seen pamphlets 197 48.6
listened to radio 137 33.8
watched on television 246 60.7
Figure. Past history of primary DHF cases
read in newspapers/journals 133 32.8
at home and knowledge scores
Percent exposed to any type 352 87.0
Low High of IEC
300 Percent not exposed to any 53 13.0
258
IEC
250 Percent exposed to all types 87 21.5
of IEC
200
Knowledge scores
Are the facts easily recognized?

150 Yes 317 78.3
113
No 23 5.7
100
The percentage of people watching
50 20 television was the highest as compared to
14
other types of exposure. Nearly half of the
0
Present Absent
respondents had seen the pamphlets about
DHF (48.6%). Exposure to radio talks and
Past history of primary DHF cases at home
information in newspapers and journals was
very low. The extent of respondents
The figure illustrates that the knowledge exposed to any type of IEC was 87%. The
scores of the caregivers were not statistically facts in those IEC materials were concise
and easily recognized (78.3%) (Table 2).
different according to the presence of
primary DHF case at home (P=0.137). Almost 60% of the interviewees felt that
television was the most effective medium for
Prevention of mosquito bites dissemination of knowledge on DHF in the
community.
To prevent and protect children from
mosquito bites, the following measures were The logistic regression model of
taken: use of mosquito nets (47.9%), use of knowledge score by the respondents
repellants (47.2%), wearing of long sleeves characteristics and exposure to health
(12.6%), others (8.6%), and none (5.2%). education media is given in Table 3.

Assessment of Dengue-related KAP Among Caregivers
Table 3. Logistic regression model of knowledge scores, by respondents characteristics and

exposure to health education materials
Odds ratio
Variables (n) Percent
(95% confidence interval)
Respondent's characteristics
Sex
Male 54 13.3 1
Female 351 86.7 0.379 (0.198-0.727)**
Years of formal schooling
0 to 5 (r) 133 32.8 1
6 to 20 272 67.2 0.588 (0.364-0.950)*
Mean years of schooling 7.63.6
Exposure to health education media

Pamphlets/posters
Not seen 197 48.6 1
Seen 208 51.4 0.478 (0.291-0.784)**
Television
Not watched (r) 159 39.3 1
Watched 246 60.7 0.353 (0.218-0.571)***
Newspapers/journals
Not read (r) 272 67.2 1
Read 133 32.8 0.443 (0.245-0.803)**
* Significant at P<0.05; ** P<0.01; *** P<0.001; (r) = Reference category
An analysis of the findings suggested Cross-check by observation

that females with over six years of schooling checklist
were significantly related to total knowledge
scores. Survey respondents with 6-20 years The researchers used an observation
of schooling were more likely to obtain high checklist to support the findings. Nearly
scores than those with 0-5 years of 55% of the households had two to three
water containers. Although 46.5% of the
schooling. Respondents who were exposed
water containers had lids, only 19% were
to health education media such as
covered tightly. Larvae were found in 67%
pamphlets/posters, television, newspapers
of the water storage tanks and 15.9% of the
and journals obtained higher scores than the flower vases at the time of interviews. Gutter
unexposed group. The respondents who blockage was observed in 3.2% of cases.
watched television were more likely to score Old tyres, coconut shells and tins were
higher than those who did not (Table 3). found in the compounds and larvae existed
in half of these solid wastes.

Approaches considered for vector breeding places. The subjects knew

little of blocked gutters as possible breeding
improvement of IEC activities
sites. Nearly 80% thought that DHF could
Many recommendations were extracted be prevented by immunization. A majority
from FGDs for the improvement of existing of the interviewees used measures to
IEC materials. It was suggested that messages prevent children from being bitten by
should be short and clear. Pamphlets should mosquitoes. A very small percentage did not
be widely distributed among the community, use any measure. Some points that require
especially in schools. The same messages emphasis for community awareness include:
could be published in newspapers and
Aedes is the main vector for DHF.
journals once a week before and during the
rainy season. Health magazines and Aedes only breeds in clear water, not in
magazines on astrology were the most polluted water.
preferred media. They also pointed out the
The biting time of Aedes is daytime.
most suitable times for telecast and broadcast
of health messages. Regular clearing of Blocked gutters should be mentioned as
gutters should be mentioned in the health possible breeding sites for Aedes in
message as well as in the book, Facts for existing IEC materials.
Life. The fact that DHF may attack again a
There is no vaccine available for the
previously-infected child was important to
prevention of DHF.
remind mothers of the danger. Cartoons and
art competitions and exhibitions on DHF The chance to get high scores was
would be the most effective media for better in females with high education.
schoolchildren and mothers. Caregivers with low educational levels
should be targeted for health education. The
level of knowledge scores was not related to
Discussion the history of a previously infected child at
The mean scores of male and female home. Knowledge scores may be changed
respondents were not significantly different through exposure to various media. Also,
in the community. Community members sufficient numbers of IEC materials should
knew a lot (more than 80%) about be distributed in the community. Only a
transmission of DHF by mosquito bite, the small percentage of the interviewees were
biting habit of Aedes and the resultant not exposed to any type of existing IEC. Ways
fatality. Nevertheless, only 61.6% correctly and means should be found to improve
identified the main vector of DHF. Nearly peoples exposure by further discussions with
20% answered that Aedes usually bit at community members and health workers
nighttime. Some people were still confused using participatory approaches.
that polluted water was also a breeding Pamphlets/posters, television, newspapers
place. Most of the interviewees responded and journals are still popular media for the
that the common breeding sites of Aedes public. Television is the most effective
were water containers and flower vases. Not medium and various types of programmes,
more than 30% identified ant traps, broken such as songs, comedies, short movies with
pots, tins, old tyres and coconut shells as the famous actors, actresses, etc., apart from

discussions and health talks can be telecast. with volunteers and local NGOs should also
However, radio is still a valuable tool in the be strengthened.
health education process because of its easy
availability ad popular use in semi-urban
and rural areas. The effectiveness of various Acknowledgement
existing IEC materials should be reviewed for
We express our gratitude to the Township
further improvement.
Medical Officer and basic health staff in
Many water containers were not Thaketa township for their support. We are
covered tightly to prevent larval breeding. grateful to volunteers, NGO members and
Social mobilization for sustainability of the study area residents for their active
larvae control activities should be participation in the study.
implemented. Coordination and cooperation
References
[1] Tha NO. A study on the larval control [3] Yoon SY. Community participation in the
operations for DHF prevention in control and prevention of DF/DHF: Is it
Sanchaung township, Yangon, Myanmar, possible? Dengue Newsletter, 1987, (13): 7-
2000-2001 (Unpublished report). 14.
[2] World Health Organization. Information,
education and communication. Lessons
from the past: perspectives for the future.
Department of Reproductive Health and
Research, World Health Organization,
Geneva, 2001.

Students Perceptions about Mosquito Larval Control
in a Dengue-Endemic Philippine City
Jeffrey L. Lennon #
Foundation University, School of Education, Dumaguete City, Negros Oriental, The Philippines
Abstract
A study was carried out among university students to find out their perceptions about mosquito larval
control in a dengue-endemic Philippine city. This formative research was conducted to obtain
information for formulating future school-related dengue control strategies. The study was carried out in-
class through a semi-structured, open-ended question format. The study yielded information on students
perceptions about the most important measures for mosquito larval control and perceived reasons why
people did not implement them. The study also explored the opinions of the students by gender. The
study yielded students knowledge on the types of mosquito larval habitats. Little was expressed about
specific indoor mosquito larval control nor about the frequency of conducting source-reduction activities.
Perceived barriers to constructing mosquito larval control centred on themes such as apathy, laziness and
lack of time. Further studies are necessary to follow-up on these themes in depth.
Keywords: Dengue, formative research, mosquito larval control, students, open-ended questionnaire, Philippines.
Introduction Consequently, it has become endemic in

Dumaguete city, Philippines[12].
The control of Aedes aegypti mosquito
Aedes control is largely based on source
larvae is essential for the control of dengue
fever (DF) and dengue haemorrhagic fever reduction. Therefore, knowledge of the
(DHF) [1] . The need to know the perceptions types of mosquito breeding sites is a
prerequisite for health personnel,
of key informants is necessary in order to
schoolteachers and children and the
better address the dengue-related control
community at large for the control of
issues in a specific area or community[2-5].
dengue. Various types of containers have
Schools are potential mosquito breeding
sites[6-8] . Also, primary, secondary and been identified as potential mosquito
tertiary school-age students are principal breeding sites. These include plastic and
targets of the Aedes mosquitoes[9-11]. metal containers, animal-feeding dishes,
tyres, flower vases, coconut shells and water
Dengue has become a steadily increasing storage drums[3,4,13,14] .
health problem in the Philippines[9] .
#
E-mail: jeffchona2@yahoo.com

Students Perceptions about Mosquito Larval Control in a Dengue Endemic Philippine City
The knowledge about the types of College of Education, Dumaguete city,

breeding containers alone is not enough to Philippines. There were 36 female and 7
achieve mosquito control. Attitudes and male subjects. All subjects were Filipinos.
beliefs impact a persons knowledge about The participants were students in a college-
mosquito control. For example, the belief based personal and community hygiene
that dengue is not a fatal or serious problem course. This study took place prior to any
impairs a person from carrying out adequate course coverage or discussions about
mosquito control practices. Some people dengue fever or any of the mosquito-borne
believe that mosquitoes within the home diseases. The participation was voluntary
and outside are different. So it is believed and no student in the class declined it. The
that mosquitoes inside the house do not results and data were confidentially held. All
carry disease[15] . results were tabulated and reconciled so
Gender-related responsibilities for that the responses would be anonymous.
control of certain mosquito breeding
containers might also exist. There may be Procedure
different responsibilities for one gender
Two open-ended semi-structured questions
inside the residence compared to the
were administered to the subjects on
outside surroundings. Or, there may be a
January 16, 2003. The students instruction
distribution of clean-up activities depending
upon ownership of specific items such as was the facilitation of the questionnaire.
tyres[15] . The two questions were as follows:
One study in Mexico indicates that (i) What is the best way to control
informants devalued the use of screens in mosquito larvae?
dengue prevention and believed that the
(ii) Why dont some people use the
screens were not effective in keeping out
measure you suggest for question
mosquitoes[2] .
No. 1?
Since dengue is already endemic in this
study city in the Philippines, it was The questions were given verbally. The
presupposed that there would already be a students were also told that they were
high level of awareness about dengue in allowed to give more than one response per
general. So, this study sought to explore the question. The students gave written
more specific topic related to dengue responses to the questions. The responses
control, through source reduction. were classified by gender. There were no
Therefore, this objective of the study was to predetermined categories. Categories of
explore the opinions of Philippine university responses were created from post-survey
education students about the mosquito results. As categories emerged, sub-
larvae control in a dengue endemic city. categories were created according to their
relationship to the broader response
Materials and methods categories. The time allotted for the whole
process of issuing instructions to the
Subjects students and completion of response-writing
The subjects consisted of 43 university was approximately 15 minutes.
major students at Foundation University,

Results activities, activities not specified as inside or

outside, activities related to specific types of
On the responses to Question 1, the containers, use of insecticide, unrelated
principal categories that emerged activities, and not familiar with mosquito
concerning mosquito larvae control activities larvae (Table 1).
were as follows: outside activities, inside
Table 1. Responses to Question 1
Category Male Female Total
I. Outside activities
A. General outside activities
A.1. Clean surroundings or yard 5 22 27
A.2. Clean surroundings daily 1 1 2
B. Specific water-containing items outside
B.1. Turn over coconuts 1 0 1
B.2. Dispose of water in coconut shells 0 1 1
B.3. Put tyres in a safe place 0 2 2
B.4. Dispose of tyres 1 0 1
B.5. Dispose of stagnant water in drums 0 1 1
C. Other environmental activities
C.1. Clean canals 1 5 6
C.2. Burn grass 0 1 1
C.3. Put trash cans in a safe place (to avoid rain-water entry) 0 1 1
C.4. Throw garbage properly 0 5 5
D. Activities involving other participants
D.1. Clean-up drive participation 0 1 1
II. Inside activities
A. Clean house (No specific activities mentioned) 0 4 4
III. Activities not specified as inside or outside
A. Non-specific cleaning
A.1. Clean things with stagnant water 1 0 1
A.2. Clean or eliminate stagnant water 1 5 6
A.3. Empty all stagnant water 0 2 2
A.4. Avoid or prevent stagnant water 0 4 4
A.5. Dispose of anything where mosquito eggs are laid 0 1 1
A.6. Kill mosquito larvae in stagnant water (not specified) 0 2 2
A.7. Clean things where mosquito larvae are laid 0 2 2
(non-specific item)

B. Activities related to non-specific types of containers

B.1. Prevent water from entering open containers 0 1 1
B.2. Cover any water container 0 8 8
B.3. Clean water containers 0 1 1
B.4. Dry water containers 0 1 1
B.5. Empty containers 0 1 1
C. Clothes-hanging arrangement 0 1 1
IV. Activities related to specific types of containers

A. Avoid open cans 0 1 1
B. Cover water containers such as gallon containers 0 1 1
C. Cover water barrels and tanks 0 2 2
D. Put open cans in a safe place 0 3 3
E. Dispose of cans 1 0 1
F. Cover jars 0 1 1
G. Cover basins 0 1 1
H. Clean bottles 0 1 1
I. Replace flower vase water (no time mentioned) 1 0 1
J. Replace flower vase water daily 1 1 2
K. Replace flower vase water weekly 0 1 1
L. Cover garbage 0 2 2
M. Put garbage in a can (No mention of placing over) 0 1 1
V. Other control-related activities

A. Spraying insecticide 0 4 4
VI. Unrelated activity

A. Remove an injured person 0 1 1
VII. Not familiar with mosquito larvae 0 1 1
Total 14 94 108
n 7 36 43
No. of students with multiple responses 5 29 34

Table 2. Responses to Question 2

I. Knowledge -related categories
A. Lack of education 1 1 2
B. Lack of knowledge 0 5 5
C. Lack of consciousness of surroundings or environment 0 3 3
D. Lack of consciousness of health 0 3 3
E. Specific responses related to knowledge
E.1. Do not know that the suggested method of larval control 1 1 2
is effective
E.2. Do not know the cause and effect of the mosquito to 0 1 1
dengue
E.3. They think that they know everything about mosquito 1 0 1
control already
E.4. Not aware of the consequences 0 2 2
E.5. Do not know that the mosquito is dangerous 0 2 2
II. Attitude-related categories
A. Dont care apathy 1 9 10
B. Dont care because they have enough money to pay 0 1 1
hospital bill
C. Lazy 3 7 10
D. Busy 2 6 8
E. Since people are busy, they only use mosquito sprays to 1 0 1
kill adult mosquitoes instead of mosquito larvae
F.1. No time 0 6 6
F.2. Insufficient time or little time 0 1 1
G. No money especially to pay for insecticide spray 0 1 1
H. Tired 1 1 2
III. Practice-related categories
A. Lack of cooperation with others 0 2 2
B. They just use insecticide spray instead of source 0 1 1
restriction environment control
IV. Invalid or unrelated responses
A. Misunderstood the question (Answered in the affirmative) 1 8 9
B. Either answered Question 1 incorrectly or unfamiliar 0 2 2
with the term mosquito larvae
V. No response 1 0 1
Total 13 63 76
n 7 36 43
Students with multiple responses 4 20 24

For the responses to Question 2, the Female subjects responded to all but
principal categories that emerged one response, activities not specified as
concerning reasons why people do not inside or outside (Table 1, Category III).
undertake mosquito larvae control were as Perhaps, the male respondents were not
follows: knowledge-related categories, carrying out these activities. The male
attitude-related categories, practice-related response emphasis on general response
categories, invalid or unrelated responses items Table 1 A, General outside activities
and no response (Table 2). may suggest their lack of specificity in the
details necessary to conduct proper dengue-
related mosquito control. The largest
Discussion response in Table 1, Category III, was on the
In response to Question 1, both female and section cover any water container
male students rated clean surroundings (Category III B.2). This practice is consistent
with recommendations throughout the
(Table 1, Category I A.2) with the largest
literature [4,6,7,13,14] . However, these responses
number of responses. This reflects the need
in Table 1, Category III B.2, are less than
for emphasis on specific mosquito control
one-third the number of the responses given
efforts while developing training and health
in Table 1, Category I A.1.
education strategies[5,14] .
The training of students in
The higher rating of outside activities
environmental surveys and in the
compared to inside activities reflects the
environmental action plan known as 4
perceived importance of outside clean-up,
oclock habit, has previously been
and conversely, the relative lack of
successful[6] . It is suggested that this be
importance of inside clean-up and control
projected on a larger scale to include other
of inside mosquito breeding sites. This may
members of the community.
follow the perception as recorded in
literature that house mosquitoes do not The responses in activities related to
transmit dengue [2,5] . Perhaps students specific types of containers Table 1,
believe that the Aedes mosquitoes do not Category IV, were scattered across the item
live inside houses or other living quarters. sub-categories by the respondents. Many of
these response items could be used as
No male subject gave any response inside containers. Not only is it necessary to
related to indoor mosquito control measures cover, empty, and clean containers, but also
(Table 1, Category II). Besides the to indicate the frequency of the activity in
perception that the indoors lack importance order to conduct adequate mosquito control.
in mosquito control, the males may have Only the responses in Table 1, Category IV J,
deemed the indoors as the domain of and Category IV K, both dealing with
females. The role of women as homemakers flower-water replacement, and Category I
needs to be considered in vector control A.2 clean surroundings daily mentioned
programmes. This was reviewed in a the frequency and timing of activities. This
number of international settings[15] . amounted to four responses out of a total of
108 responses in Table 1. Perhaps,

programmes need to stress health education mosquito larvae. However, only a minority
on the mosquito life-cycle and include gave specific details either in types of
emphasis on the time interval to conduct control measures or in the timing of control
successive mosquito control clean-ups. The activities.
lack of responses on the frequency of
For Question 2, the greatest number of
environmental clean-up is in agreement
responses among the knowledge-related
with previous literature on the incomplete
category was the general category, Lack of
understanding about the mosquito life-cycle,
knowledge, Table 2, Category I B. There
and thus, the need to include this topic in
was no concentration of specific knowledge-
future health education programmes[2,5] .
related responses. There were nearly twice
Only four female respondents and no as many attitude -related responses as
male respondent in Table 1, Category V A, knowledge-related responses. Attitude
indicated that insecticide spraying was the responses were greater than knowledge
most important mosquito control measure. responses for both male and female subjects
The overwhelming majority of the (Table 2). Since dengue is endemic in
respondents discussed general or specific Dumaguete City[12] , and various health
environmental clean-up activities. Source education programmes have continued for
reduction of mosquito breeding sites years, it is reasonable to agree that there is a
without the use of adult insecticides has high general awareness about dengue.
been stressed in dengue-related health However, knowledge alone is generally not
education programmes in Honduras [3] , sufficient to change attitudes and
Mexico[4] , and the Philippines[6,13,16] . Since behaviour[17] . Health education to increase
larvicides are not currently being used or only knowledge without addressing health
promoted in this Philippine study site, the behaviours has also been ineffective in the
respondents most likely were referring to the dengue control experience[5] .
use of adult insecticides. In contrast, dengue
The greatest numbers of responses were
control programmes in Puerto Rico utilized
in the attitude-related categories of Dont
both source reduction of mosquito breeding
care apathy, Table 2, Category II A, and
sites and insecticide use[14] . However, for
Lazy, Table 2, Category II C. The
developing countries, regular adult aerosol
responses of apathy or laziness could have
insecticides were deemed to be too
been a result of a weak belief in the
expensive for regular home use, and thus
effectiveness of the proposed measures from
not recommended for routine control[13] .
Table 1 to control mosquitoes and dengue.
Only one respondent as seen in Table 1, The health belief model may help to explain
Category VI, gave a response to an activity these responses. Two key components of
unrelated to mosquito control. Also, there the health belief model are perceived
was only one respondent as seen in Table 1, benefits and perceived barriers. A lower
Category VII, who responded to the perception of benefits coupled with
category, Not familiar with mosquito elevated barriers may result in a lower
larvae. These responses validated that the possibility for change[18] . Likewise, people
respondents in general had awareness about having low self-efficacy (a construct of the

Social Cognitive Theory and also the Health the collective efficacy for the desired
Belief Model), or, in other words, behavioural change activities. High levels of
confidence in doing something[18,19] could perceived collective efficacy may increase
have resulted in their lack of interest to carry the likelihood of a group carrying out
out the suggested mosquito control tas ks. desired behavioural change activities[21].
Therefore, promoting strategies to increase
The large number of responses found in
collective efficacy may enhance the
Table 2, Categories II D, II E, II F1 and II F2,
likelihood that community mosquito control
related to the factors of insufficient time to
activities will be carried out and sustained.
perform clean-up tasks is also suggested in
the literature [15] . While people may know Asking the questions orally, rather than
about various individual mosquito breeding in a written form, had some limitations. This
sites source reduction tasks, they may lack may have contributed to the nine
the self-confidence necessary to perform a respondents who misunderstood Question 2.
regular, comprehensive environmental They answered Question 2 by explaining
clean-up task. Thus, skill development on their reason of choice for the best mosquito
how to conduct the steps of an larval control, rather than explain why
environmental action plan for dengue people were not using the best mosquito
control should be emphasized in order to larval control method. See these responses
increase self-efficacy and mosquito control in Table 2, Category IV A.
behaviours. This promotion of skill
This form of questioning was also
development may, in turn, increase
limited in the lack of in-depth follow-up.
personal efficiency to perform source
The procedure in this study did not allow for
reduction tasks and thus, decrease the
follow-up of such responses as the reason
perception of time as a limitation to perform
for apathy as a response to Question 2.
mosquito control activities.
Unlike other studies that used interviews[2-
5,14]
There were only two responses to Table or focus groups [4,5] , the facilitator of the
2, Category III A, Lack of cooperation with questions did not interact with the
others. This low number may have been respondents , nor probe for follow-up
due to a high value placed on the important responses.
relational imperative or supportive Filipino
The method and procedure also had its
norm of bayanihan or cooperation[20] . Yet,
strengths. In spite of utilizing open-ended
there were still responses to perceived lack
questions, the procedure was very efficient
of cooperation as a cause for lower rate of
in time and in organization. Unlike a
mosquito larval control activities. Addressing
previous study where interviews lasted for
this norm through various communications
an hour per respondent, and at home[5] ,
and other health promotion means may
respondents in this study were able to
help in increasing mosquito larvae control
complete the questions in a matter of
activity.
minutes, and in one sitting.
Increasing the self-efficacy of individuals
All questions were completed at the
in the community may help in increasing
same time, reducing potential biases that

could result from interaction with others in Efforts are needed to create awareness
the outside environment. Also, having the regarding frequency of mosquito
students complete the questions individually, activities.
without interaction with fellow students and
There should be an in-depth
reduced biased or blended responses.
exploration of the reasons behind the
The procedure also demonstrated the perceived causes of non-participation in
strength of producing a large variety of mosquito larval control. Especially, the
response categories, multiple responses and reasons behind apathy and laziness
overall total responses in a very short period as perceived causes for non-
of time. This was evident for both sexes and participation in mosquito larval control
in response to both questions. should be further explored.
Activities such as an environmental
control action plan and creation of a
Summary and
mosquito control checklist and
recommendations implementation for houses and schools
The study is suggestive that there was an as a means to increase self-efficacy
understanding by most students of the should be promoted.
term larvae, and its general The determinants of collective efficacy
relationship to mosquito and dengue in mosquito larval control and dengue
control. This was exemplified by the control should be explored.
low number of incorrect responses to
Teachers play an important role in
content unrelated to mosquito larval
facilitating of health promotion in dengue
control as indicated in Table 1,
endemic areas. Students and teachers
Categories VI and VII, and Table 2,
should be properly oriented to carry out
Categories III B, IV B and V.
personal, school and community mosquito
There were no male responses for and dengue control measures. Antecedent
indoor control measures to Question 1. to this is an understanding of students
Also, there were almost no specific perceptions about mosquito-related dengue
mosquito control measures mentioned control. The in-class semi-structured
by the male respondents. Further question method is one tool to carry out this
studies should explore the possible type of formative research.
gender relationship to mosquito control
practices.
Acknowledgement
The majority of male and female
respondents did not mention indoor The author is associated with the
mosquito larval control. Therefore, International Technical Assistance Group
future mosquito control programmes (ITAG), Seattle, WA, USA. ITAGs support is
should stress the importance of indoor gratefully acknowledged. Also, thanks to
mosquito larval control measures. Chona F. Lennon and Fernando N.
Florendo for their assistance.

References
[1] Gubler DJ. Aedes aegypti and Aedes aegypti- [9] Custodio GGV. (Ed.). National objectives for
borne disease control in 1990s: top down or health, Philippines, 1999-2004: Kalusugan
bottom up. American Journal of Tropical Para sa Masa. HSRA Monograph Series
Medicine and Hygiene, 1989, 40: 571-578. (Series No. 1), 1999 (On-line Serial).
[2] Kendall C, Hudelson P, Leontsini E, Winch P, Retrieved on 1 November 2000, from
Lloyd L and Cruz F. Urbanization, dengue http/www.doh.gov.ph/noh/default.shtm.
and the health transition: anthropological [10] Gubler DJ. Dengue and dengue
contributions to international health. haemorahagic fever. Clinical Microbiology
Medical Anthropology Quarterly, 1991, 5: Reviews, 1998, 11: 480-496.
257-268.
[11] Pinheiro FP and Corber SJ. Global situation
[3] Leontsini E, Gil E, Kendall C and Clark GG. of dengue and dengue haemorrhagic fever
Effect of a community-based Aedes aegypti and its emergence in the Americas. World
control programme on mosquito larval Health Statistics Quarterly, 1995, 50: 161-
production sites in El Progreso, Honduras. 169.
Transactions of the Royal Society of Tropical
Medicine and Hygiene, 1993, 17: 267-271. [12] Dengue cases by Barangay, Dumaguete City,
Philippines, City Health Office, 2003.
[4] Lloyd LS, Winch P, Ortega-Canto J and
Kendall C. The design of a community- [13] Advocacy and Health Promotion Service.
based health education intervention for the Best way to prevent dengue haemorrhagic
control of Aedes aegypti. American Journal fever. Cebu City, Philippines, Department of
of Tropical Medicine and Hygiene, 1994, Health.
50: 401-411. [14] Winch PJ, Leontsini E, Rigau-Perez JG, Clark
[5] Winch P, Lloyd L, Godas MD and Kendall C. GG and Gubler DJ. Community-based
Beliefs about the prevention of dengue and dengue prevention programmes in Puerto
other febrile illnesses in Merida, Mexico. Rico: impact on knowledge, behavior and
Journal of Tropical Medicine and Hygiene, residential mosquito infestation. American
1991, 94: 377-387. Journal of Tropical Medicine and Hygiene,
[6] Lennon JL. Knowledge of dengue 2002, 67: 363-370.
haemorrhagic fever by Filipino University [15] Winch PJ, Lloyd LS, Hoemeke L and
students. Dengue Bulletin, 1996, 20: 82-86. Leontsini E. Vector control at the household
[7] Swaddiwudhipong W, Chaovakiratipong C, level: An analysis of its impact on women.
Nguntra P, Koonchote S, Khumklan P and Acta Tropica, 1994, 56: 327-339.
Lerdlukanavonge P. Effect of health
[16] Lennon JL and Gonzales MJCP. The
education on community participation in
utilization of college of education students in
control of dengue hemorrhagic fever in an
a dengue health education campaign.
urban area of Thailand. Southeast Asian
Philippine Journal of Education, 1995, 73:
Journal of Tropical Medicine and Public 393, 421, 425-426.
Health, 1992, 20: 200-206.
[17] Green LW and Kreuter MW. Health
[8] Strickman D and Kittayapong P. Dengue
promotion planning: an educational and
and its vectors in Thailand: introduction to
environmental approach, Second Edition.
the study and seasonal distribution of Aedes
larvae. American Journal of Tropical Mountain View, CA, USA: Mayfield
Publishing, 1991: 155.
Medicine and Hygiene, 2002, 67: 247-259.

[18] Strecher VJ and Rosenstock IM. The health [20] Andres JD and Ilada-Andres PB.
belief model. In: K Glanz, FM Frances and Understanding the Filipino. Quezon City,
BK Rimer (Eds). Health behavior and health Philippines: New Day Pub, 1987: 55-56.
education: Theory, research and practice,
[21] Bandura, A. Exercise of human agency
Second Edition, San Francisco, CA, USA:
through collective efficacy. Current
Jossey-Bass Pub, 1997: 41-59.
Directions in Psychological Research, 2000,
[19] Bandura A. Self-efficacy: the exercise of 9: 75-78.
control. New York: WM Freeman Pub. 1997.

Short Note 1
Sero-surveillance in Delhi, India An Early Warning

Signal for Timely Detection of Dengue Outbreaks
D. Bhattacharya*, Veena Mittal* #, M. Bhardwaj*, Mala Chhabra*,
R.L. Ichhpujani** and Shiv Lal*
*National Institute of Communicable Diseases, 22-Sham Nath Marg, Delhi 110 054, India
**Directorate General of Health Services, Nirman Bhavan, New Delhi 110 011, India
In India the first major outbreak of dengue These samples were tested for dengue by
fever (DF) accompanied with dengue haemagglutination inhibition (HI) test[3] or
haemorrhagic fever (DHF) was reported in IgM Capture ELISA Test[4] . A titre of
Kolkata (Calcutta) in 1963[1] . More than 60 1:1280 in HI test in acute phase serum is
outbreaks have been reported since 1956 to considered a presumptive diagnosis of a
date[2] . Of these two major outbreaks of current dengue infection[5] . Samples positive
DF/DHF occurred in 1996 and 2003 in for IgM antibodies against dengue virus
Delhi and its adjoining states. Surveillance is indicate recent infection with dengue virus.
the most cost-effective approach for
The results of the sentinel sero-
prevention and control of dengue. A strong
surveillance from 1996 to 2003 are
surveillance system will help in detecting
summarized in the Table. The analysis of
early warning signals of an outbreak,
data over the period of eight years shows that
instituting timely and appropriate control
dengue strikes Delhi every year. The
measures, assessing the impact of
positivity ranges from approximately 13%-
intervention measures and early containment
33%, except in 1996 and 2003 when dengue
of the outbreak. Considering the above facts,
fever occurred in epidemic proportions along
the arbovirus laboratory at the National
with DHF. The positivity in these two years
Institute of Communicable Diseases, Delhi,
was 53.4% and 57.8% respectively.
has started sero-surveillance and monitoring
of dengue fever in Delhi since 1996 as an The month-wise distribution of samples
ongoing activity. It was intended to develop tested from 1996-2003 shows that the
an early warning signal for timely detection of positivity for dengue starts appearing in the
an impending outbreak and institution of month of August and reaches a peak in
preventive and control measures in high-risk October and continues till mid-November
areas. and then a decline starts and the last cases
are reported upto 2nd week of December.
Sera samples of clinically suspected
The data for 2003 also shows a similar trend
cases of DF and/or DHF are received from
(Figure).
various hospitals of Delhi round the year.
#
E-mail: veena_m12@yahoo.com

Table: Dengue serology during the years 1996 2003 in Delhi
208
1996 1997 1998 1999 2000 2001 2002 2003
Year/
Month
Tested +ve Tested +ve Tested +ve Tested +ve Tested +ve Tested +ve Tested +ve Tested +ve
JAN 1 0 5 0 21 1 5 0 11 0 19 0 18 0 7 0
FEB 4 0 6 0 11 0 11 0 8 0 21 0 19 0 5 0
MAR 3 0 4 0 24 0 12 0 9 0 11 0 19 0 13 0
APR 0 0 39 1 28 0 7 0 8 0 10 0 34 0 8 0
MAY 3 0 33 0 67 0 11 1 26 0 13 0 6 0 10 0
JUN 0 0 8 0 27 0 12 0 9 0 22 0 12 0 11 5
JUL 0 0 15 0 31 1 11 0 18 0 22 1 23 0 11 0
AUG 3 0 20 3 48 0 27 0 32 1 43 6 17 0 43 16
SEP 159 73 169 65 70 5 71 17 56 13 127 44 31 1 701 433
OCT 643 343 369 159 201 38 145 35 188 74 304 129 55 12 2,351 1,438
NOV 104 74 81 26 293 140 130 35 254 112 225 94 60 18 877 450
Sero-surveillance in Delhi An Early Warning Signal for Timely Detection of Dengue Outbreaks
DEC 11 3 15 1 47 21 40 5 77 13 35 9 34 11 35 14
Total 931 493 764 255 868 206 482 93 696 213 852 283 328 42 4,072 2,356
(53.4%) (33.3%) (23.7%) (19.2%) (30.6%) (33.2%) (12.8%) (57.8%)
Dengue Bulletin Vol 28, 2004

Sero-surveillance in Delhi An Early Warning Signal for Timely Detection of Dengue Outbreaks
Figure. Month-wise samples tested/positive for dengue antibodies in Delhi

during the year 2003
2,500
2,000
1,500
1,000
500
September
April
November
December
June
October
May
July
August
March
January
February
Tested Positive
The studies for the estimation of the to dengue virus can act as an early warning
House Index (HI) of mosquitoes also shows signal for an impending outbreak. The
that the house index for Aedes aegypti, the above observations show that serological
vector of dengue fever, starts building-up surveillance throughout the year, especially
during the rainy season, i.e. from July and during the outbreak-prone period, can play
reaches a peak in August-September[6]. an important role in the detection of early
cases.
A regular monitoring of suspected
dengue cases by detection of IgM antibodies
References
[1] Ramakrishnan SP, Gelfand HM, Bose PN, [4] Lam SK, Devi S and Pang T. Detection of
Sehgal PN and Mukherjee RN. The specific IgM dengue infection. Southeast
epidemic of acute haemorrhagic fever, Asian J Trop Med Public Health, 1987, 18:
Calcutta. Ind J Med Res, 1964, 52: 633-650. 532-538.
[2] Epidemic preparedness dengue fever, [5] Gubler DJ. Serological diagnosis of
DHF and DSS. CD Alert, Monthly dengue/dengue haemorrhagic fever. Dengue
Newsletter, National Institute of Bulletin, 1996, 20: 20-23.
Communicable Diseases, Delhi, June 2001, [6] Katyal R, Singh K and Kumar K. Seasonal
Volume 5 (16). variation in Aedes aegypti population in
[3] Clark DH and Casals J. Techniques for Delhi, India, Dengue Bulletin, 1996, 20: 78-
haemagglutination and haemagglutination 81.
inhibition with arthropod borne viruses. Am
J Trop Med, 1958, 7: 561-573

Short Note 2
Detection of Dengue Virus in Wild Caught Aedes

albopictus (Skuse) around Kozhikode Airport,
Malappuram District, Kerala, India
B.P. Das*, L. Kabilan**, S.N. Sharma*, S. Lal*, K. Regu*** and V.K. Saxena*
*National Institute of Communicable Diseases, 22 Sham Nath Marg, Delhi 110 054, India
**Centre for Research in Medical Entomology, 4 Sarojini Street, Chinna Chokkikulam,
Madurai 625 002, India
***National Institute of Communicable Diseases, Kozhikode Branch, Kerala, India
Introduction in and around the airport area. The survey

was carried out in May 2004.
In India, outbreaks of dengue fever
(DF)/dengue haemorrhagic fever (DHF) have
been reported in various parts of the Materials and methods
country during the past four decades[1] .
The Kozhikode airport is situated at 11 .15'
Aedes aegypti is the only vector that has so
N latitude and 75 .49' E longitude, in a hilly
far been implicated in dengue
area of Malappuram district, Kerala. It
transmission[1,2] , even though Aedes
became functional as an international
albopictus is known to be present in some
airport in 1988. A larval survey was carried
of the peri-urban and rural areas [2] . Recently,
out in various types of water-holding
a survey was carried out in the Kozhikode
containers to detect the breeding of Aedes
(earlier known as Calicut) airport area of
(Stegomyia) mosquitoes, both inside the
Malappuram district, Kerala. During 2002
airport premises and its periphery up to
and 2003 (up to July), 75 and 150 clinical
about 600 metres. The larvae were
dengue fever cases, respectively, were
identified as per the method described
reported from the district[3] . Earlier, reports
earlier[6,7] . Adults of Aedes (Stegomyia)
of Aedes survey in Kerala had shown the
mosquitoes were collected while landing on
presence of Aedes albopictus in rubber
human baits by aspirator tube in a forested
plantation areas [4] and in plastic cups [5] .
residential area about 600 metres away
This communication presents the results from the airport.
of the detection of dengue virus from the
The wild Aedes albopictus females
wild and dry preserved, adult females of
caught from outside the Kozhikode airport,
Aedes albopictus and their breeding indices
the adults reared from larval collections

Detection of Dengue Virus in Wild Caught Ae. albopictus (Skuse) around Kozhikode Airport
from inside the Kozhikode airport and the positive for Aedes albopictus and two for
city, and the adults of Aedes aegypti reared Aedes vittatus (container index 15.1% and
from larval collection from the 0.7% respectively). The most preferred
Thiruvananthapuram international airport containers for Aedes albopictus breeding
were separated sex-wise, pooled by species were discarded tyres, coconut shells and
(about 15 adults per pool) and transported plastic containers. The average landing rate
to the Centre for Research in Medical of Aedes albopictus on humans was 20
Entomology (CRME), Madurai, Tamil Nadu, females/human bait/hour.
in a dry state, for detection of dengue virus.
The methodology followed was similar to Dengue virus detection in
that used for the detection of JE virus and
Aedes albopictus
based on the protocol developed and
standardized by CRME[8] . However, the Of the three pools of Aedes albopictus
antibody (D3-5C9-1) was diluted at 1:5000 tested for dengue virus infection following
as being followed by CRME. antigen-capture enzyme immunoassay (EIA),
one pool was found positive for dengue
virus (OD-0.32), thereby indicating dengue
Results and discussion viral activity in this mosquito species. The
mosquitoes in the positive mosquito pool
Aedes survey were collected as landing collection around
the Kozhikode airport on 28 May 2004, and
The survey for larval infestation in 52
transported as dry specimens to the CRME
houses/premises around the airport area
laboratory and processed on 8 June 2004
revealed 16 premises as positive for Aedes
(Table). Earlier dengue virus was also
albopictus breeding (house index 30.7%). A
isolated from Aedes albopictus collected in
total of 272 wet containers searched for
a village in Vellore district of Tamil Nadu[9] .
Aedes breeding revealed 41 containers as
Table. Aedes mosquito pools tested for dengue virus infection by ELISA
No. of pools tested/No. of adults/No. of

Collected on pools positive
Mosquito
Locality (Processed
species Wild caught adults Reared adults
on)
(landing) (immature)
Kozhikode airport 27/05/04 1/15/0
(08/06/04)
Residential area 28/05/04 1/10/1*
Aedes
around the airport (08/06/04)
albopictus
City area 28/05/04 1/20/0
(2 kms from (08/06/04)
airport)
* Positive pool

Detection of Dengue Virus in Wild Caught Ae. albopictus (Skuse) around Kozhikode Airport
The present study confirms that antigen- Centre for Research in Medical Entomology,
capture enzyme immunoassay is a useful Madurai, Tamil Nadu, is gratefully
surveillance tool for monitoring dengue virus acknowledged. The authors are thankful to
infection in mosquitoes. Mrs V. Thenmozhi and Mr S. Venkatesan,
CRME, for providing laboratory assistance.
Acknowledgements
The provision of laboratory facilities and the
permission for the study by the Director,
References
[1] Yadava RL and Narasimham MVVL. [6] Das BP. A simple modified method for
Dengue/dengue hemorrhagic fever and its mounting mosquito larvae. J Commun Dis,
control in India. Dengue Newsletter, 1992, 1986, 18: 63-64.
17: 3-8. [7] Das BP and Kaul SM. Pictorial key to the
[2] Das BP, Sharma SK and Datta KK. common Indian species of Aedes
Prevalence of Aedes aegypti at the (Stegomyia) mosquitoes. J Commun Dis,
International port and airport, Kolkata (West 1998, 30: 123-127.
Bengal), India. Dengue Bulletin, 2000, 24: [8] Tewari SC, Thenmozhi V, Rajendran R,
124-26. Appavoo NC and Gajanana A. Detection of
[3] Anonymous. Annual Report of the Centre Japanese encephalitis virus antigen in
for Research in Medical Entomology, Indian desiccated mosquitoes: an improved
Council of Medical Research, 2002-2003: surveillance system. Trans Roy Soc Trop
82. Med Hyg, 1999, 93: 525-26.
[4] Sumodan PK. Potential of rubber plantation [9] Tewari SC, Thenmozhi V, Katholi CR,
as breeding source for Aedes albopictus in Manavalan R, Munirathinam A and
Kerala, India. Dengue Bulletin, 2003, 27: Gajanana A. Dengue vector prevalence and
197-98. virus infection in a rural area in south India.
[5] Hiriyan J, Tewari SC and Tyagi BK. Aedes Trop Med Int Health, 2004, 9(4): 499-507.
albopictus (Skuse) breeding in plastic cups
around tea-vendor spots in Ernakulam city,
Kerala state, India. Dengue Bulletin, 2003,
27: 195-96.

Short Note 3
Entomological Investigations for DF/DHF in

Alwar District, Rajasthan, India
Kalpana Baruah #, Avdhesh Kumar and V.R. Meena
National Institute of Communicable Diseases, 22 Shamnath Marg, Delhi-110 054
Introduction is usually of a short duration (July-August),

the average rainfall being 61.16 cm. The
During 2001, small outbreaks of DF/DHF highest temperature during June goes up
were reported in many districts in to 47 C whereas the lowest may go down
Rajasthan, including the capital city of to freezing point.
Jaipur and the industrial city of Alwar.
There was a total of 1,820 laboratory- Alwar town has a number of industrial
confirmed cases (based on serology using units. Migration of labour thus poses an
kits for IgG and IgM antibodies) with 30 increased threat for malaria and other
deaths (CFR: 1.65%)[1] . The present vector-borne diseases, including DF/DHF.
communication deals with the The city has irregular piped water supply
entomological investigations carried out by resulting in water storage practices for
the National Institute of Communicable household purposes. In rural areas no such
Diseases, Delhi, during the outbreak piped water supply system exists; therefore,
period in a few urban and rural areas of water from wells, bore-wells and natural
Alwar district that were affected. streams is used for household purposes
with minimal storage practices.
Study area
Larval survey
Alwar district is situated in the north-
eastern part of Rajasthan between 274 An Aedes survey, as per WHO guidelines[2] ,
and 284 north latitude and 767 and was carried out in four localities out of 10
7713 longitude. The central part of the in urban areas and in five localities under
district is occupied by the Aravali hills. The four primary health centres (PHCs) in rural
population of the district is 29,90,862 areas, all reporting fever cases. The results
(2001 census), of which 85% is of the Aedes survey are given in Table 1.
predominantly rural. The monsoon season
#
E-mail: kalpana_baruah@yahoo.co.in

Entomological Investigations for DF/DHF in Alwar District of Rajasthan, India
Table 1. Aedes aegypti larval indices in the urban and rural areas, Alwar, Rajasthan
Name of Total Houses

House Containers Containers Container Breteau
the locality houses found
Index searched positive Index Index
(or villages) searched positive
Urban areas
Sonwa 30 10 33.3 62 17 27.4 56.7
Karaulikund 30 9 30.0 57 13 22.8 43.3
Arya Nagar 20 4 20.0 47 4 8.5 20.0
Kalakuan 25 9 36.0 111 15 13.5 60.0
Rural areas
Indok 25 0 47 0
Madhogarh 20 0 32 0
Malakhera 25 0 34 0
(Kalachara)
Bhartahari 25* 5 20.0 20 5 25.0 20.0
Tiraha
Kushalgarh 10* 1 10.0 15 1 6.7 10.0
* Shops and nearby houses just outside the villages
Results and discussion Container Index 6.7% to 25.0% and Breteau

Index from 10.0 to 20.0 only. The shops
used earthen pots for storing drinking water
Larval surveys wherein co-breeding of Aedes aegypti and
In urban areas, the House, Container and Aedes albopictus was detected. The
Breteau indices ranged from 20.0% to dwelling houses along the shops were also
36.0%, 8.5% to 27.4% and 20.0 to 60.0, found positive, as Aedes breeding was
respectively. Mixed breeding of Aedes detected in cement containers used for
aegypti and Anopheles stephensi was also providing drinking water to cattle.
detected in cement tanks in some areas.
The area-wise infestation by containers
In comparison, the House Index was nil is given in Table 2. In the urban areas, out
in rural residential areas as no mosquito of the total of 49 positive containers,
breeding could be detected; however, 67.35% were domestic water-storing
shops in the marketplaces near the villages containers (like cement tanks, clay pots and
and their adjacent houses were found to be overhead tanks), followed by 30.61%
positive. The House/Premise Index in these evaporation coolers and the remaining
localities ranged from 10.0% to 20.0%, 2.04% trash.

Entomological Investigations for DF/DHF in Alwar District of Rajasthan, India
Table 2. Area-wise infestation of Aedes aegypti in the urban areas, Alwar district, Rajasthan
Type of container
Evaporation Cement Overhead
Clay pots Others
Area coolers tanks tanks
% % % % %
S S S S S
+ve +ve +ve +ve +ve
Sonwa 27 18.5 16 56.3 12 16.7 7 0
Karaulikund 19 21.1 21 38.1 17 5.9

Arya Nagar 24 8.3 9 11.1 14 7.1
Kalakuan 37 10.8 27 25.9 41 4.9 4 25.0 2 50.0
Total 107 14.0 73 34.2 84 8.3 11 9.1 2 50.0
S=Searched
Adult surveys (Rajasthan), where people from the

periphery picked up the infection during
Landing collections were also undertaken in their day visit to the city area where Aedes
the same urban and rural areas where larval aegypti breeding indices were very high.
surveys were carried out. In urban areas the
adult density of Aedes aegypti ranged from
2.0 to 7.0 per man-hour. The houses with Acknowledgments
poor ventilation and light yielded higher
numbers. The authors are grateful to the Director,
National Institute of Communicable
In rural areas both Aedes aegypti and Diseases, Delhi, for providing the
Aedes albopictus were detected in shopping opportunity and necessary facilities to
areas only. The cases reported from these undertake this investigation. The
rural areas could be attributed to population cooperation extended by the staff of the
movement from rural to urban areas (city) Chief Medical Officers office, Alwar district,
during daytime for earning their livelihood and the technical support of the staff of the
or shopping purposes. A similar observation NICD, Alwar branch, is gratefully
was made earlier by Kalra et al.[3] in Ajmer acknowledged.
References
[1] Anonymous. Report of State Health [3] Kalra NL, Ghosh TK, Pattanayak S and
Authority, Jaipur, 2001 (unpublished). Wattal BL. Epidemiological and
entomological study of an outbreak of
[2] World Health Organization. Prevention and
dengue fever at Ajmer, Rajasthan (1969). J
control of dengue and dengue haemorrhagic
fever comprehensive guidelines, 1999, Com Dis, 1976, 8(4): 261-279.
WHO Regional Publication, SEARO 29.

Short Note 4
Essentiality of Source Reduction in both Key and

Amplification Breeding Containers of Aedes aegypti
for Control of DF/DHF in Delhi, India
B.N. Nagpal, Aruna Srivastava #, M.A. Ansari and A.P. Dash
Malaria Research Centre, 20 Madhuban, Delhi - 110 092
Delhi, the Capital of India, reported the city Municipal Corporation of Delhi (MCD).
first-ever outbreak of dengue fever in The MCD organized a campaign aimed at
1967[1] . Since then the city has experienced source reduction, which was backed by
cyclic epidemics every 2-3 years. During vehicle-mounted thermal fogging for the
1996, a large epidemic swept the city when control of the outbreak. However, the
10,252 cases were hospitalized and 423 reduction in the DF incidence was not
deaths were recorded[2] . The disease has found to be commensurate with the inputs
now become endemic and the yearly made into the campaign. At the request of
incidence has varied between 160 and 300 the MCD, a team of experts from the
cases of DF/DHF, with a couple of deaths[3] . Malaria Research Centre undertook a
Aedes aegypti has been invariably found to comprehensive survey in five affected
be associated with these outbreaks in Delhi. localities, viz. Dayanand Colony (Lajpat
Overhead tanks (OHTs) and ground level Nagar Phase IV), Lajpat Nagar (Phases I and
tanks (GLTs) as key containers, and II), Kotla Mubarakpur, Dayalpur Extension
evaporation coolers have repeatedly been and Harsh Vihar Tulsi Niketan, to identify
reported as seasonal amplification sites[4,5] . both the key containers (OHTs and GLTs)
The key containers maintain mosquito and the amplification containers
breeding throughout the year, whereas (evaporation coolers, earthen pots, flower
coolers amplify the A edes population from vases, household ornamental fountains, etc.)
May to November, and thereafter they go during November 2003 as per WHO
dry[6] . techniques. The results of the study are
included in this communication.
During 2003, DF/DHF resurged in some
localities in Delhi and a total of 2,604 The results of the larval survey of Aedes
DF/DHF cases and 33 deaths were aegypti in five dengue-affected localities in
registered up to 9 November 2003 by the Delhi as mentioned above are given in the
#
E-mail: srivastavaa@icmr.org.in, aruna@del3.vsnl.net.in

Source Reduction for DF/DHF Control in India
Table. The results revealed that among the also interact with householders and
key containers, out of 243 OHTs and 20 impress upon them to remove small
GLTs, 64 OHTs (26.34%) and 8 GLTs (40%) water collections indoors/outdoors,
were found positive for breeding of Aedes specially water evaporation coolers.
aegypti. On the other hand, out of the 533
During the survey it was found that
evaporation coolers, which are identified as
fogging generally lacked a pre-fogging
the major amplification-breeding site,
public information campaign requiring the
checked, none was found positive for
public to keep the house doors/windows
breeding of Aedes aegypti. Ornamental
open to permit the entry of fog. Health
fountains inside the drawing rooms and
workers, while interacting with
mud-pots containing water for birds were
householders, invariably talked about the
supporting the heavy breeding (>1,000
removal of breeding from coolers and tanks
larvae in each site) of Aedes aegypti. The
but did not point out other sites of breeding.
maximum positivity of OHTs was found in
The crosschecking teams of the MCD also
Kotla Mubarakpur (39.39%), followed by
laid emphasis on inspection of evaporation
Phases I and II of Lajpat Nagar (30%) and
coolers as focal points and did not verify
Dayanand Colony (20.69%). Two OHTs
OHTs/GLTs and trash materials, as they
located on the roofs of the market area were
found it a time-consuming affair. In urban
found supporting the breeding of Aedes
areas, for security reasons or houses being
albopictus. In a childrens home for boys
locked, access by health workers into the
belonging to the Social Welfare Department
houses was also a constraint.
of the Delhi Government, five out of 12
OHTs were found positive for Aedes aegypti In view of the aforesaid, it can be
while two were found dry. It would thus concluded that: (i) source reduction should
appear that during the MCD campaign, cover both the key containers as well as the
source reduction efforts had concentrated amplification breeding sites, viz. evaporation
on evaporation coolers, and key containers coolers and other trash articles breeding the
and other trash materials breeding the vector species; (ii) the fogging operation
species remained intact in domestic habitats. should be preceded by a pre-fogging
information campaign in order to seek full
cooperation of communities to derive
IEC Activities of MCD optimal benefits from fogging; (iii) the IEC
The IEC activities of the MCD generally campaign based on KAP studies should be
covered the following aspects: prepared with particular emphasis on
community participation; (iv) special efforts
(1) Creating awareness through media and should be focused on behavioural change in
spreading vocal messages through the accordance with the guidelines laid down
use of loudspeakers in DF-affected by WHO[7] ; and (v) health staff of the MCD
localities, distribution of pamphlets, should be trained in entomological
etc.; surveys/techniques related to Aedes
(2) MCD workers who visit houses for breeding, prevention and its control.
physical verification of breeding sites

Table. Breeding of Aedes aegypti in key and amplifier containers in dengue-affected localities of Delhi during November 2003
218
Key containers Amplification containers
Ground level tanks

Locality Overhead tanks (OHTs) Evaporation coolers Other domestic containers
(GLTs)
Examined +ve % Examined +ve % Examined +ve % Examined +ve %
Dayanand 58 12 20.69 13 5 38.46 147 0 0 1 1 100

Colony
(Lajpat Nagar
Phase IV)
Lajpat Nagar 80 24 30.00 138 0 0 2 2 100

(Phases I and II)
Kotla 66 26 39.39 7 3 42.86 134 0 0 2 2 100

Mubarakpur
Dayalpur 5 0 0.0 69 0 0
Extension
Harsh Vihar 34 2 5.88 45 0 0

Tulsi Niketan
Total 243 64 26.34 20 8 40.00 533 0 0 5 5 100
* Ornamental fountain and trash
Dengue Bulletin Vol 28, 2004

: NIL
Acknowledgement former Deputy Director, National Vector-

Borne Diseases Control Programme, for his
The authors are thankful to Dr K.N. Tiwari, valuable suggestions and guidance. We
Municipal Health Officer-cum-Director, acknowledge the efforts of the field staff for
Health Services, Municipal Corporation of assisting in carrying out the survey. Thanks
Delhi, for providing assistance in the are also due to Mr Sanjeev Gupta for his
conduct of the study, and to Mr N.L. Kalra, help in various ways.
References
[1] Balaya S, Paul SD, D'lima LV and Pavri KM. [5] Ansari MA and Razdan RK. Seasonal
Investigations of an outbreak of dengue in prevalence of Aedes aegypti in five
Delhi in 1967. Indian Journal of Medical localities of Delhi, India. Dengue Bulletin,
Research, 1969, 57: 767-774. 1998, 22: 28-32.
[2] Sharma S, Sharma SK, Mohan A, Wadhwa [6] Katyal R, Gill KS and Kumar K. Seasonal
J and Dar L. Clinical profile of dengue variation in Aedes aegypti population in
haemorrhagic fever in adults during the Delhi, India. Dengue Bulletin, 1996, 20:
1996 outbreak in Delhi, India. Dengue 78-81.
Bulletin, 1998, 22: 20-27.
[7] Parks W and Lloyd L: Planning social
[3] Katyal R, Kumar K, Gill KS and Sharma RS. mobilization and communication for
Impact of intervention measures on dengue fever prevention and control. A
DF/DHF cases and Aedes aegypti indices step-by-step guide. WHO, Geneva, 2004.
in Delhi, India: an update 2001. Dengue
Bulletin, 2003, 27: 163-167.
[4] Krishnamurthy BS, Kalra NL, Joshi GC and
Singh NN. Reconaissance survey of Aedes
mosquito in Delhi, Bull Ind Soc Mal Com
Dis, 1965, 2: 56-67.

Short Note 5
Breeding of Dengue Vector Aedes aegypti (Linnaeus) in

Rural Thar Desert, North-western Rajasthan, India
B.K. Tyagi# and J. Hiriyan
Centre for Research in Medical Entomology, Indian Council of Medical Research, 4 Sarojini Street,
Chinna Chokkikulam, Madurai 625 002, India
Aedes aegypti, the vector of dengue fever, is considered worthwhile to highlight the
widely present in India[1,2] , including the association of Aedes aegypti and tankas in
Thar desert in north-western Rajasthan. sustaining the vector population under
Jalore town in the Thar desert experienced extreme xeric conditions.
the first-ever epidemic of dengue fever in
A total of 33 villages in the three
1985. Entomological studies carried out in
districts of Jodhpur, Jaisalmer and Sri
Jalore during 1990 and subsequently in
Ganganagar (now incorporated partly in the
1996 observed extensive breeding of Aedes
newly created Hanumangarh district) were
aegypti[3,4] . Recently, dengue fever again
surveyed for the presence of tankas (Table
struck the Thar, this time in Jodhpur district,
1). Compared to those of Jodhpur and
warranting a serious review of the vector
Jaisalmer, most villages in Sri Ganganagar
ecology in the region, particularly in view of
are highly irrigated and adequately supplied
the prodigious breeding of Aedes aegypti in
with the conduit-water system. Four major
discarded household and community-based
types of water storing facilities were
underground water reservoirs called tankas,
identified which supported the breeding of
of which thousands are formed in different
Aedes aegypti. About 13.6% of the tankas,
forms in the Thar[3] . These tankas, which
which constituted 77.1% of the four water
originally attracted only the malaria vector
bodies, were found to be breeding Aedes
Anopheles stephensi as long as the water
aegypti. It is noteworthy that the typical
remained potable, started breeding Aedes
century-old traditional earthen-type tanka
aegypti only after being discarded by local
was almost invariably present in most
populations in the wake of the recent
villages of Jodhpur (88.8%) and Jaisalmer
availability of conduit-based water supply
(85.7%) but less so in Sri Ganganagar (17%)
under Indira Gandhi Nahar Pariyojana
where canal-based water storage sources
(IGNP canal project). It is, therefore,
#
E-mail: bk_tyagi@sify.com

Aedes aegypti Breeding in Tankas in the Thar Desert
had greatly reduced the existence of the while the fourth instar larvae (7.8%) was
tankas. As many as 11 (24.4%) out of a total present the least, followed by pupae (2.5%).
of 45 community tankas existing This situation clearly indicated a sustained
peripherally in Kanasar village were breeding of Aedes aegypti in the tankas.
abandoned for want of proper water storage Cement tanks, invariably constructed in
facility and rendered uncared for by the close vicinity of bore-wells, for cattle
villagers. All such tankas, wherein the water drinking bred Aedes aegypti as soon as the
turned turbid in course of time including water there turned turbid due to prolonged
vegetation growth, Aedes aegypti was use by cattle, sometimes with Anopheles
invariably found to breed, replacing in the subpictus. None of the beris, another type of
process its earlier and original occupant, the earthen reservoir in the desert environment,
malaria vector Anopheles stephensi. The first supported the breeding of Aedes aegypti.
instar larvae abounded the most (52.3%),
Table 1. Distribution of different kinds of tankas and beris in various villages in three districts
currently under varying degrees of irrigation and/or conduit water supply from canals
No. of villages with different types of tankas

Cement
Typical intra- Metallic Central Peripheral Beris
No. of tanks
District domestic mobile community village tankas
villages
earthen tankas tankas tankas assembly
+ ++ + ++ + ++ + ++ + ++ + ++
Sri 17 9 5 3 4 13 0 0 17 0 17 0 0 17 0 0 2 12 3
Ganganagar
Jodhpur 9 1 5 3 0 8 1 0 9 0 0 8 1 8 1 0 0 7 2
Jaisalmer 7 1 6 0 0 7 0 0 7 0 0 6 1 6 1 0 0 6 1
Total 33 11 16 6 4 28 1 0 33 0 17 14 2 31 2 0 2 25 6
= Absence of breeding habitat

+ = Presence of breeding habitat, with potable water breeding Anopheles stephensi
++ = Presence of abandoned breeding habitat positive for Aedes aegypti breeding
Conclusion the tankas in the rural areas of the Thar

desert is considered to be a rather recent
The dengue vector, Aedes aegypti, has so far phenomenon, possibly due to the easy
been collected from the Thar desert only accessibility of newly provided conduit-
from townships and/or desert fringe areas in water supply to villages, which has led to
the vicinity of urban environment, breeding the abandoning of the traditional water
mostly in household pitchers and cement reservoirs.
tanks [2,4,5] . The breeding of Aedes aegypti in

Aedes aegypti Breeding in Tankas in the Thar Desert
References
[1] Kalra NL, Wattal BL and Raghvan NGS. [4] Joshi V, Mathur ML, Dixit AK and Singhi M.
Distribution pattern of Aedes aegypti in India Entomological studies in a dengue endemic
and some ecological considerations. Bull Ind area Jalore, Rajasthan. Indian J Med Res,
Soc Mal Comm Dis, 1968, 5: 307-334. 1996, 104: 161-165.
[2] Kalra NL, Kaul SM and Rastogi RM. [5] Tyagi BK. A note on the breeding of vector
Prevalence of Aedes aegypti and Aedes mosquitoes in cement tanks and pit latrines.
albopictus vectors of DF/DHF in north, J Appl Zool Res, 1994, 5: 149-151.
north-east and central India. Dengue
Bulletin, 1997, 21: 84-92.
[3] Chouhan GS, Rodrigues FM, Sheikh BH,
Ilkal MA, Khangaro SS, Mathur KN, Joshi KR
and Vaidhye NK. Clinical and virological
study of dengue fever outbreak in Jalore city,
Rajasthan, 1985. Indian J Med Res, 1990,
91: 414-418.

Book Review 1
A Review of Entomological Sampling Methods and

Indicators for Dengue Vectors
Dana A. Focks
Infectious Disease Analysis, Gainsville, Florida, USA
TDR / IDE / Den / 03.1
This review was developed in response to a abundance, temperature, and sero-

recommendation of the WHO Informal conversion rates in the human population.
Consultation on Strengthening
The document reviews and critiques
Implementation of the Global Strategy for
current methods, focusing especially on
Dengue Fever/ Dengue Haemorrhagic Fever
sampling methods that provide information
Prevention and Control, held in October of
on (1) the risk of transmission as a function
1999, urging the refinement of existing
of vector abundance, and (2) the relative or
entomological indicators and/or the
absolute importance of the various types of
development of new indicators that better
containers in the environment. This second
reflect transmission potential. The
aspect is essential when considering a
Consultation recommended that such
suppression strategy designed to minimize
indicators should provide clear, meaningful
costs or to improve sustainability by
information for communities as well as for
targeting only a subset of the breeding
programme managers and policy-makers.
containers for control or elimination
Whereas the traditional Stegomyia indices
specifically those container types that are
(the House, Container, and Breteau indices,
responsible for the majority of adult
and various related derivations) are of some
production. In reviewing current and
operational value for measuring the
generally-used sampling methods, each is
entomological impact of larval control
discussed with respect to transmission risk
interventions against the mosquito vectors of
assessment and evaluated in terms of being
dengue virus, they are not proxies for adult
useful for either research or special studies
vector abundance. Neither are they useful
or as a practical operational tool providing
for assessing transmission risk because they
useful information for planning and
do not take into consideration the
management of vector control programmes.
epidemiologically important variables,
including adult vector and human

Book Review 2
DengueNet in India
Weekly Epidemiological Record, 2004, 79(21): 201-203
Epidemic dengue fever (DF) and dengue implementation in South-East Asia and the
haemorrhagic fever (DHF) have emerged as Western Pacific was held in Kuala Lumpur
a global public health problem in recent on 11-13 December 2003 2 . The objective
decades. In fact, the problem has become of the meeting was to expand the pilot
hyperendemic in many urban, periurban project to these two regions, building upon
and rural areas, with frequent epidemics. the lessons learned from the pilot project in
The South-East Asia Region is one of the the Americas. Based on the
regions at highest risk of DF/DHF, recommendations of this meeting, two
accounting for 52% of the global risk. country workshops were organized in India
Dengue outbreaks now occur in India, as in in March 2004, supported by the
other high-burden countries in the Region, WHO/CSR and USAID project to strengthen
such as Indonesia, Myanmar and Thailand. surveillance in India. The first took place in
New Delhi on 11-12 March 2004 with the
Strengthening epidemiological and
northern states and the second in Bangalore
laboratory surveillance of dengue and
on 16-17 March 2004, with the southern
dengue haemorrhagic fever including, the
states. WHO collaborating centres attended
implementation of DengueNet 1 , is one of
both meetings. The proceedings and
the priorities of the global and regional
recommendations from the New Delhi
strategies for dengue prevention and control.
meeting were discussed at the Bangalore
DengueNet is WHOs global data
meeting to ensure that the consensus
management system created on the Internet
recommendations addressed national issues,
to collect and analyse standardized
needs and priorities. Experts from health
epidemiological and laboratory surveillance
service departments of all states and the
data with the objective to improve capacity
Delhi City Corporation, the National
for effective national and international
Institute for Communicable Diseases (NICD),
planning for the prevention and control of
the National Institute of Virology (NIV) in
epidemic dengue and DHF.
Pune, the WHO Department of
Following the pilot use of DengueNet in Communicable Disease Surveillance and
the Americas, a joint WHO Response, WHO Representative for India
HQ/SEARO/WPRO meeting on DengueNet and SEARO participated.
1 2
1 See No. 36, 2002, pp. 300-304. See No. 6, 2004, pp. 57-62.

DengueNet in India
The main objective of the workshops significantly improved its surveillance system,
was to strengthen disease surveillance and including strengthening of laboratory
response to vector-borne diseases using services. Uttaranchal, although lacking a
DengueNet as an entry point. Work focused good network of laboratories, was included
specifically on assessing current surveillance because of its close proximity to Delhi.
practices (including the use of case
It was also decided to designate two
definitions, reporting formats and
WHO laboratory collaborating centres
mechanisms for flow of information),
(WHO CC) for northern and southern states
laboratory facilities and tests for DHF, on
to ensure practical use of these facilities.
identifying and strengthening regional
Accordingly, NIV-Pune will continue as
collaborative laboratories and on establishing
WHO CC for the south, and it is
a framework for participation in DengueNet.
recommended that WHO designate NICD
Experiences both from India and from to serve as WHO CC for the northern states
the region on surveillance and control were (following a request from NICD). The two
discussed. The need for an integrated centres would be responsible for training,
approach to surveillance of vector-borne quality control, use of standard procedures
diseases and application of lessons and and networking at national and international
experiences from malaria and other vector- level.
borne diseases was identified. The
Based on the recommendations of the
consensus was to implement DengueNet in
workshops, a follow-up meeting has been
accordance with the Integrated Disease
planned to develop an activity plan for 2004
Surveillance Programme (IDSP) that is
focusing on laboratory strengthening,
starting in India. This would require capacity
training, disease and vector surveillance,
building for disease surveillance and
networking, information sharing and
response at national, state and district levels,
reporting to DengueNet. NICD will take the
through training of health workers and
lead role in this follow-up activity, which is
programme managers and strengthening of
scheduled for the end of May 2004,
laboratory services.
working closely with all major stakeholders 3.
Given the vastness of the country, the
heterogeneity of the disease burden and the
organizational network, the group
recommended piloting the participation of
selected states in global DengueNet through 3
Major stakeholders include the Indian Council for
focal points at state and national level. Medical Research, IDSP Cell, Delhi City Corporation,
NICD WHO CC for Training and Epidemiology,
States in which implementation would be
WHO CC for Rabies Epidemiology, WHO Regional
piloted included Delhi, Karnataka, Reference Laboratory for Polio Surveillance, National
Maharashtra, Tamil Nadu and Uttaranchal. Reference Laboratory for SARS and Avian Influenza,
Maharashtra and Tamil Nadu have a well state nodal officers of the pilot states and states that
were not represented in the earlier meetings, national -
developed disease surveillance system and a
level institutes such as the Central Bureau of Health
network of public health laboratories at both Intelligence, Vector Control and Research Centre
district and state level, with strong linkages WHO CC for Filariasis and Vector Control, National
to national-level laboratories. Delhi, Vector Borne Disease Control Programme and NIV
following the recent dengue outbreak, has WHO CC for Arboviral Disease Diagnosis and Research
and National Influenza Surveillance Centre.

Book Review 3
WHO/WPRO/SEARO Meeting on DengueNet

Implementation in South-East Asia and the
Western Pacific, Kuala Lumpur, 11-13 December 2003
Weekly Epidemiological Record, 2004, 79(6): 57-62
Dengue/DHF Global public Rationale for DengueNet1

health problem DengueNet, WHOs global surveillance
system for dengue fever and DHF, has been
Epidemic dengue fever and dengue created as a web-based central data
haemorrhagic fever (DHF) have emerged as a management system to collect and analyse
global public health problem in recent standardized epidemiological and virological
decades, with the development of data in a timely manner and to present
hyperendemicity in urban and peri-urban epidemiological trends as soon as new data
centres of many tropical and subtropical are entered. Strengthening epidemiological
countries. Asia-Pacific countries have more and virological surveillance of dengue and
than 70 % of the disease burden; in several DHF, including implementation of
of them, DHF has become a leading cause of DengueNet, for early detection, planning and
hospitalization and death among children. response is one of the four main priorities of
Latin America and the Caribbean appear to WHOs global prevention and control
be following the same DHF epidemic trend, strategy, adopted in resolution WHA55.17 in
with the disease affecting all ages and case- May 2002. DengueNet, when fully
fatality rates as high as 10-15 % in areas with implemented, will facilitate WHOs global
limited health service infrastructure. The outbreak and response activities and support
African and Eastern Mediterranean regions the GOARN2.
are much less affected. Air travel is also Epidemiological and laboratory-based
facilitating the rapid global movement of surveillance is required to monitor and guide
dengue viruses and increasing the risk of dengue/DHF prevention and control
DHF epidemics through the introduction of programmes whether these are based on
new serotypes. Globally, 2.5 billion people vector control or possible future vaccination
live in areas where dengue viruses can be with a safe, effective and affordable vaccine.
transmitted: an estimated 50 million dengue Recent and encouraging research
infections occur each year, with 500,000 developments have made it likely that a
cases of DHF and at least 22,000 deaths, dengue vaccine will become available. As a
mainly among children. Although dengue is a consequence, the public health community
notifiable disease in many endemic countries,
only a small proportion of cases are reported 1
See No. 36, 2002, pp. 300-304.
to WHO. 2
Global Outbreak Alert and Response Network.

DengueNet Implementation in South-East Asia and the Western Pacific
needs to define the burden of dengue for representing 20 island countries in the
society, so that adequate cost-benefit Americas, and after changes to the
analyses can be presented to government supporting computer hardware, software,
leaders before they decide to use the vaccine. and routines, a second meeting was
Standardized global dengue surveillance data, convened jointly with WHO/WPRO/SEARO
one of the principal results expected from the and the WHO collaborating centre for
establishment of DengueNet, have become dengue/DHF at the University of Malaya in
critical. Kuala Lumpur, 11-13 December 2003. The
objective was to expand the pilot to South-
East Asia and the Western Pacific, building on
Phased implementation of the lessons learned from the pilot conducted
DengueNet in the Americas. About 70 participants
included national epidemiologists, laboratory
First meeting in San Juan, specialists, and clinicians from 19 Asia-Pacific
Puerto Rico, July 2002 countries, three countries in the Americas, six
WHO collaborating centres, and WHO HQ,
The first DengueNet meeting was held jointly
regional and country staff4.
with WHO/PAHO and the WHO
collaborating centre for dengue/DHF at the The plenary presentations and
Centers for Disease Control and Prevention discussions focused on: (1) the challenges
(CDC Dengue branch; San Juan, Puerto Rico). and need for standardized global
Its objective was to describe and demonstrate epidemiological and laboratory surveillance
DengueNet to prospective users and to of dengue and DHF; (2) the activities of the
launch a pilot, building on the existing Pediatric Dengue Vaccine Initiative (PDVI);
reporting systems and network of dengue (3) the national surveillance and reporting
laboratories in the Americas. Epidemiologists systems in the participating countries in
and virologists from eight countries in the South-East Asia and the Western Pacific;
Americas, three countries in Asia and five (4) the activities of the participating WHO
WHO collaborating centres provided collaborating centres; (5) the WHO global
recommendations for the administrative and
technical procedures involved in making
DengueNet operational. 4
Participants included:
South-East Asian and Western Pacific regions:
Second meeting in Kuala Lumpur, national programmes from Bangladesh,
Cambodia, China, Fiji, French Polynesia, India,
Malaysia, December 20033 Indonesia, Lao Peoples Democratic Republic,
After pilot use of DengueNet by four Maldives, Malaysia, Myanmar, Nepal, Philippines,
Sri Lanka, Singapore, Thailand, Viet Nam; the bi-
Member States and one network
regional Mekong Basin Disease Surveillance
Network; WHO collaborating centres and
3
research institutes in Australia, India, Japan,
This meeting was organized by the WHO Department Malaysia, Thailand.
of Communicable Disease Surveillance and Response,
Global Alert and Response, jointly with the WHO Americas: DengueNet pilot country Brazil; WHO
Regional Offices for South-East Asia and the Western collaborating centres in Cuba and USA; interim
Pacific, and the WHO Collaborating Centre for director of the Dengue Pediatric Vaccine Initiative
dengue/DHF at the University of Malaya in Kuala (PDVI).
Lumpur, Malaysia, with technical and financial support WHO: HQ; regional offices (PAHO, SEARO,
from the US Centers for Disease Prevention and Control. WPRO); country offices (India, Malaysia).

strategy and regional programmes; to provide early warning of potential

(6) WHOs global outbreak and response outbreaks of dengue disease or of the
activities and GOARN; (7) the DengueNet introduction of dengue viruses into
pilot and lessons learned; (8) presentation of epidemiologically silent areas, for the
DengueNet and a hands on session with purpose of implementing timely control
the new prototype web site in Global Atlas. measures and notifying decision-makers
Two working groups were convened. The in institutions whose occupations or
first reviewed and defined the epidemiological livelihood may be affected;
data and reporting requirements for
to strengthen and standardize
DengueNet, modifications needed to the epidemiological surveillance of DF and
present format, identification of countries
DHF;
for expanding the DengueNet pilot to Asia-
Pacific regions, and roles and responsibilities to promote the use of standardized
of national and international partners. A clinical case definitions and reporting
subgroup also reviewed and defined the criteria for dengue illnesses, permitting
objectives of DengueNet. The second comparisons between countries and
working group reviewed the existing over time;
laboratory capacity in South-East Asian and to strengthen the network of
Western Pacific countries in relation to collaborating centres and national
DengueNet, identifying the current needs laboratories for serotype determination
(and gaps) for laboratory standards, quality and strain characterization;
control, and dengue serological diagnosis
and virus isolation, as well as for reporting to promote improvement in the quality
and information exchange. The group made of laboratory data reported at national
recommendations that focused on and international levels;
strengthening regional dengue laboratory to provide a standardized database for
diagnosis capacity, so that laboratories epidemiological research and analysis;
participating in DengueNet are able to
report data of the highest quality possible to provide data useful for estimating the
within their working environment. burden of disease (including the social
and economic burden) on a national,
regional, or global scale;
Meeting outcomes to support the improvement in national
and international alert and response
Objectives of DengueNet capacity for dengue/DHF outbreaks;
The participants agreed that the overall and
objective of DengueNet is to improve to promote the free and timely exchange
capacity for effective national and of epidemiological information between
international planning for the prevention affected countries, their neighbours, and
and control of dengue and that the specific other stakeholders in order to facilitate
objectives for implementing this global and promote dengue control activities
surveillance system are: within the region.

Recommendations of the In collaboration with WHO country and

regional offices, WHO collaborating
laboratory working group
centres should provide reference
To strengthen the regional dengue reagents to national laboratories
laboratory diagnosis capacity, the standard inactivated antigens,
participants of this group made the monoclonal antibodies, standard sera,
following recommendations for national cell lines for virus isolation, and
laboratories, WHO collaborating centres, prototype dengue virus strains.
WHO, and government health authorities.
Laboratory training
Quality control
WHO should organize regional training
Quality control/proficiency testing courses on laboratory diagnosis of
should be undertaken by the national dengue and other flaviviruses.
laboratory/WHO collaborating centre
WHO should develop a laboratory
for other laboratories in the country
concerned. manual for dengue diagnosis.
WHO HQ should establish a global
A reference centre should be
established at the WHO Collaborating technical advisory group, including
representatives from collaborating
Centre for Tropical Viral Diseases,
Nagasaki, Japan, to undertake centres, to meet annually to advance
laboratory training, capacity building,
coordination of quality assurance/
control for other WHO collaborating reagents, quality issues, and DengueNet.
centers and designated national
laboratories in the two Regions. Reporting and information exchange
The Nagasaki reference centre should With regard to collection of laboratory data
coordinate, organize, and distribute a and information transfer, the group
WHO panel of reference sera for identified a strong need for government
validation of tests/kits/rapid tests by health authorities to develop a reporting
WHO, national laboratories, and WHO system to collect, centralize, and
collaborating centres. disseminate these data, identify key
laboratories to participate in this system, and
designate a focal point for DengueNet.
Reference services
The group recommended that WHO
Countries that do not have facilities for
virus isolation should send appropriate support national health ministries to assess
current laboratory status in Asia-Pacific
samples to a WHO collaborating centre
of their choice after consultation with countries and to plan mechanisms to
strengthen laboratories for DengueNet. A
that centre.
draft DengueNet laboratory assessment tool
WHO should recommend capacity- is available for review and use.
building for virus isolation to the
ministries of health of countries that The group expressed appreciation of
the efforts made to develop DengueNet and
lack facilities.

recommended that WHO work with - Countries should provide annual

partners to develop strategies for raising epidemiological data by sex and
crucial resources. age groups.
Recommendations of the Rate calculations

epidemiology working group - Both incidence and mortality rates
The group reviewed currently available data should be expressed per 100 000
and reporting practices in the Asia-Pacific mid-year population; countries
countries in relation to DengueNet. The should provide updates to
discussion was organized around the DengueNet in the event of
principal epidemiological variables of time, significant change.
place and personal characteristics, plus - The system should not show
information about the virus. The group incidence and mortality rates for
made recommendations on the countries that report data only from
modifications to be made to the present sentinel sites.
format of the DengueNet prototype in
Global Atlas, on strengthening Virus serotype data all available
epidemiological surveillance, and on a
framework for implementation of the - Data should be entered (when
DengueNet in Asia-Pacific regions with provided by the laboratory) as the
emphasis on the quality of available data cumulative number of isolations of
and the active participation of national each serotype in the country from 1
programmes. January.
Data collection General recommendations
Epidemiological data For countries

- To accommodate currently available Countries should promote implementation
case classification reporting practices, of the WHOrecommended surveillance
countries should provide three standards for dengue. (Participants were
categories DF cases, DHF/DSS provided with copies of these standards.)
(dengue shock syndrome) cases,
total dengue cases (DF/DHF/DSS). For DengueNet
These data should be provided
monthly, at state/department level A country information page should be
by large countries and at island provided on the DengueNet web site for all
level by island nations. country-specific information, definitions,
and methods used (e.g. sentinel site
- Countries should provide data,
information, reporting by time of onset or
monthly when available, on
notification, case classification other than
dengue deaths (probable or
according to the WHO definition, etc.).
confirmed).

WHO-recommended surveillance Country participation

standards should be made available on the
DengueNet web site. A major outcome of the meeting was that
representatives of all participating countries
showed interest in collaborating with
Roles and responsibilities of DengueNet and agreed to present the
meetings recommendations to their health
the partners in this network ministries. Country participation will require
Countries will collect, validate, and provide a letter of request from WHO to the
epidemiological and laboratory data. They ministry of health; ministry authorization for
will designate the participating centres and participation and designation of a national
focal points, and WHO country offices will DengueNet focal point; and, for some
facilitate the process. WHO collaborating countries, an external budget.
centres will provide laboratory support,
The DengueNet pilot will be expanded
proficiency panels, and training to national
to countries in American, South-East Asian,
laboratories. WHO regional offices will
and Western Pacific regions in 2004 after
implement the country support activities,
modifications to the system have been
and WHO HQ will maintain and moderate
made in Global Atlas. The lessons learned
the DengueNet web site. WHO regional
from the pilot will be used to develop a
offices and HQ will seek financial support
consensus framework for DengueNet
for dengue surveillance activities.
implementation for standardized global
surveillance of dengue and DHF.

Instructions for Contributors
The Dengue Bulletin welcomes all original research papers, short notes, review articles, letters
to the Editor and book reviews which have a direct or indirect bearing on dengue
fever/dengue haemorrhagic fever prevention and control, including case management. Papers
should not contain any political statement or reference.
Manuscripts should be typewritten in English in double space on one side of white A4

size paper, with a margin of at least one inch on either side of the text and should not exceed
15 pages. The title should be as short as possible. The name of the author(s) should appear
after the title, followed by the name of institution and complete address. E-mail address of the
corresponding author should also be included.
References to published works should be listed on a separate page at the end of the
paper. References to periodicals should include the following elements: name and initials of
author(s); title of paper or book in its original language; complete name of the journal,
publishing house, or institution concerned; volume and issue number, relevant pages and date
of publication, and place of publication (city and country). References should appear in the
text in the same numerical order (Arabic numbers in parenthesis) as at the end of the article.
For example:
(1) Nimmannitaya S. Clinical spectrum and management of dengue haemorrhagic

fever. The Proceedings of the International Conference on Dengue Haemorrhagic
Fever, Kuala Lumpur, September 1-3, 1983:16-26.
(2) Gubler DJ. Dengue and dengue haemorrhagic fever: Its history and resurgence as
a global public health problem. In: Gubler DJ, Kuno G (ed.), Dengue and dengue
haemorrhagic fever. CAB International, New York, NY, 1997, 1-22.
(3) Nguyen Trong Lan, Nguyen Thanh Hung, Do Quang Ha, Bui Thi Mai Phuong, Le
Bich Lien, Luong Anh Tuan, Vu Thi Que Huong, Lu Thi Minh Hieu, Tieu Ngoc
Tran, Le Thi Cam and Nguyen Anh Tuan. Treatment of dengue haemorrhagic
fever at Children's Hospital N.1, Ho Chi Minh City, 1991-1996. Dengue Bulletin,
1997, 22: 150-161.
Figures and tables (Arabic numerals), with appropriate captions and titles, should be
included on separate pages, numbered consecutively, and included at the end of the text with
instructions as to where they belong. Abbreviations should be avoided or explained. Graphs or
figures should be clearly drawn and properly labeled, preferably using MS Excel, and all data
clearly identified.

Instructions for Contributors
Articles should include a self-explanatory abstract at the beginning of the paper of not
more than 300 words explaining the need/gap in knowledge and stating very briefly the area
and period of study. The outcome of the research should be complete, concise and focused
conveying the conclusions in totality. Appropriate keywords and a running title should also be
provided.
Articles submitted for publication should be accompanied by a statement that they

have not already been published, and, if accepted for publication in the Bulletin, will not be
submitted for publication elsewhere without the agreement of' WHO, and that the right of
republication in any form is reserved by the WHO Regional Offices for South-East Asia
(SEARO) and the Western Pacific (WPRO).
One hard copy, original and clear figures/tables and a computer diskette indicating
the name of the software, of the manuscript should be submitted to:
The Editor
Dengue Bulletin
WHO Regional Office for South-East Asia
Mahatma Gandhi Road
New Delhi 110002
India
Telephone: 91-11-23370804
Fax: 91-11-23379507, 23370972
E-mail: dengue@whosea.org
Manuscripts received for publication are subjected to in-house review by a

professional expert and are peer-reviewed by experts in the respective disciplines. Papers are
accepted on the understanding that they are subject to editorial revision, including, where
necessary, condensation of the text and omission of tabular and illustrative material.
Original copies of articles will not be returned. The principal author will receive 10
reprints of his article published in the Bulletin. A PDF file can be supplied on request.

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D ENGUE

WORLD HEALTH ORGANIZATION

World Health Organization

Indexation: Dengue Bulletin is being indexed by BIOSIS and

3. Oon Chong Teik

Dengue Bulletin Vol 28, 2004 iii

iv Dengue Bulletin Vol 28, 2004

DengueNet, the WHO Global Surveillance System for management of epidemiological

A supplement, featuring experiences from different countries in social mobilization and

1. Annual Changes of Predominant Dengue Virus Serotypes in Six Regional 1

2. A Retrospective Study of the 1996 DEN-1 Epidemic in Trinidad: 7

3. Ecological Study of Rio de Janeiro City DEN -3 Epidemic, 2001-2002 20

4. Sero-epidemiological and Virological Investigation of Dengue Infection 28

5. Spatial and Temporal Dynamics of Dengue Haemorrhagic Fever 35

6. Sporadic Prevalence of DF/DHF in the Nilgiri and Cardamom Hills of 44

7. Autoimmunity in Dengue Virus Infection 51

8. Inhibition of the NS2B-NS3 Protease Towards a Causative Therapy for 58

Dengue Bulletin Vol 28, 2004 v

9. Prognostic Factors of Clinical Outcome in Non-Paediatric Patients with 68

10. Dengue Haemorrhagic Fever with Encephalopathy/Fatality at Petchabun 77

15. Genetic Influences on Dengue Virus Infections 126

16. Identification and Phylogenetic Analysis of DEN-1 Virus Isolated in 135

vi Dengue Bulletin Vol 28, 2004

17. Induction of Cytotoxic T Lymphocytes by Immunization with Dengue 145

18. Molecular Characterization of Brazilian Dengue Viruses 151

19. Unusual Emergence of Guate98-like Molecular Subtype of DEN-3 161

20. The Animal Models for Dengue Virus Infection 168

21. Philippine Species of Mesocyclops (Crustacea: Copepoda) as a Biological 174

22. Susceptibility of Aedes aegypti to Insecticides in Viet Nam 179

23. Ovipositioning Behaviour of Aedes aegypti in Different 184

24. Community-based Assessment of Dengue-related Knowledge 189

25. Students Perceptions about Mosquito Larval Control in a Dengue- 196

Dengue Bulletin Vol 28, 2004 vii

1. Sero-surveillance in Delhi, India An Early Warning Signal for 207

2. Detection of Dengue Virus in Wild Caught Aedes albopictus (Skuse) 210

3. Entomological Investigations for DF/DHF in Alwar District, Rajasthan, India 213

4. Essentiality of Source Reduction in both Key and Amplification Breeding 216

1. A Review of Entomological Sampling Methods and Indicators for 223

2. DengueNet in India 224

3. WHO/WPRO/SEARO Meeting on DengueNet Implementation in South- 226

Instructions to Contributors 232

viii Dengue Bulletin Vol 28, 2004

Introduction occurred in a pattern of 2-year cycle, and

Dengue Bulletin Vol 28, 2004 1

Materials and methods

2 Dengue Bulletin Vol 28, 2004

stained by IFA, and dengue virus serotypes Results and discussion

Table 1. Number of samples used for the analysis

Convalescent Virus isolation

1999 Lampang 128 108 74

2001 Lampang 262 238 150

2002 Lampang 339 236 95

Dengue Bulletin Vol 28, 2004 3

4 Dengue Bulletin Vol 28, 2004

Acknowledgements Dr Vitaya Jiwariyaves and Ms Vanna

Dengue Bulletin Vol 28, 2004 5

6 Dengue Bulletin Vol 28, 2004

Introduction and potentially fatal form of dengue

Dengue Bulletin Vol 28, 2004 7

8 Dengue Bulletin Vol 28, 2004

Dengue Bulletin Vol 28, 2004 9

Dengue cases Rainfall

10 Dengue Bulletin Vol 28, 2004