THYPOID FEVER

UPDATE in LABORATORY DIAGNOSTIC

Uleng Bahrun
Manifesto II
Makassar, 11 Maret 2012

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 TYPHOID DESEASE

OVERVIEW

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 What is Typhoid Fever
DEMAM TYPHOID or Enteric Fever or
Tifus abdominalis is a severe and systemic
bacterial infection caused by Salmonella
typhi
 is spread through food and water
contaminated by human waste (stool or
urine) containing Salmonella typhi.

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 Epidemiology of Typhoid Fever
~ endemic in many developing countries >>
South East Asia  pure water supplies, sewage
control and disposal are not always adequate

~ More than 13 million cases occur annually in

Asia alone

~ In Indonesia  a mean of 900.000 cases per

year with over 20.000 deaths.

~ In areas of endemicity and in large outbreaks,

occur in aged between 3 and 19 years
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 Epidemiology
of Typhoid
Fever

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 ETIOLOGY
~ Enteric fever are caused by Salmonella typhi, S.
paratyphi A, S. paratyphi B and S. paratyphi C.

~ Salmonella paratyphi, is very rare ( 10 - 15%),

however it symptoms are very difficult to
differentiated from another major causative
agent- S. typhi
~ Specifically typhoid fever is caused by
infection with Salmonella typhi.
~ The reservoir is person chronically infected by
this
bacteria, except S. paratyphi C which can be
presented at the animals
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The antigenic component of Salmonella Typhi

1. Somatic (0 Ag)

2 Flagellar (H Ag)

3. Capsular (Vi Ag)

4. Outer Membrane
Protein (OMP Ag)
 Pathogenesis
Contaminated food of drinks Gastric acid

Bowel lumen

Mucosal defence

Adhesion to mucose Colonization

Invation to Peyer Patch

Regional Lymphadenitis Thoracic duct

1st systemic bacteriemia

 Pathogenesis
Infection of RE system 2nd Bacteriemia
Liver, Spleen

Lung, Myocard
Gall bladder Kidney, etc

Feces

Reinfection in bowel mucose Systemic manifestation

Hyperplasia Peyer Patch Inflammation, erosion

Bleeding, perforation
 Gambaran Skematis Infeksi S. typhi

Monack DM, 2004

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 Pathogenesis
Antibody Responses to Salmonella typhi

Sources : Prof PL Lim, University of Hongkong

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 DIAGNOSTIC
SITUATION

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 Diagnostic

5%
25%
20%

50%

Sym. only Sym+Sl.Ag

Sym+Sl.Ag+GAL Sym+PCR

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 C LINICAL
DIAGNOSIS &
LIMITATIONS

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 CLINICAL SYMPTOMS

~ long lasting enteric fever, sore throat, mild

to acute gastroenteritis, skin rashes and body
weakness.
~ usually start 1-3 weeks after exposure to
Salmonella typhi and may last up to 2 weeks or
more.
~ Post - infected individuals, who no longer
show clinical symptoms, may still serve as
possible carrier.
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 CLINICAL SYMPTOMS

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 THE DIFFICULTIES TO ESTABLISH A CLINICAL
DIAGNOSIS

Keterlambatan diagnosis merupakan salah satu penyebab

kegagalan pemutusan rantai penularan serta pencegahan
terjadinya komplikasi. Masalahnya tidak jarang ditemui
kesulitan menegakkan diagnosis demam tifoid dengan tepat
dan cepat hanya atas dasar gejala klinis saja.
(Djoko Widodo et al.1999, Perkembangan diagnosis laboratorium demam tifoid. MKI:256-262)

Walaupun gejala klinis jelas tetapi diagnosis demam tifoid

seringkali menjadi masalah karena tidak selalu khas dan
sering sulit dibedakan dengan demam oleh sebab lain.
(Dalima.AW. 2002, Diagnosis Laboratorium Demam Tifoid, PBPK:1-9)

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 L ABORATORY
DIAGNOSIS &
LIMITATIONS

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 DIAGNOSTIC CLASSIFICATION

Classification I : Etiological Agent Isolation

(Salmonella typhi) from all kind
of cinicaly specimen.

Classification II : Serological Test for detecting a

raising titer of antibody level to
S.typhi antigen.
Classification III : Hibridization and PCR for DNA
tracing of specific S.typhi

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 ETIOLOGICAL AGENT ISOLATION

The definitive diagnosis of typhoid fever depends on the

isolations of S.typhi from blood, bone marrow or specific
anatomical lesion.

Blood

Stool
Culture
Bone marrow

Other body fluid

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 ETIOLOGICAL AGENT ISOLATION

~ Bacteremia occurs early in the disease

~ Blood Cultures are positive in

1st week in 90%
2nd week in 75%
3rd week in 60%
4th week and later in 25%

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 A FAILURE & DISADVANTAGE OF ISOLATION

a. A fecal carrier  a false positive in endemic area

b. Low sensitivity (40-60%)
c. Ab, Complemen, or other factor in serum 
inhibiting a basil growth
d. Administration of antibiotics or self medication 
can affecting the growth of isolation.
e. The result may require a days or weeks
f. Not available in each laboratories.

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 SEROLOGICAL TEST
2 groups laboratory assay : determination of S.
typhi specific antibody and S. typhi specific antigen
from serum or body fluid.
O - antigen
(somatic)

Vi - antigen (virulence)
Serological
Salmonella

H - antigen
(flagellar)

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 SEROLOGICAL TEST
Serological test for typhoid fever to be based on
detection the increase of immunologically respon to
Salmonella typhi.

tube agglutinating ab
(felix)
Widal
slide agglutinating ab
Antibody
detection Slide inhibition

Elisa

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 WIDAL vs ELISA
WIDAL / SLIDE ELISA IgM or IgG
AGGLUTINATION

 USES A CRUDE S.TYPHI  USES PURIFIED S.TYPHI

ANTIGEN ANTIGEN (LPS, OMP,
 DETECT WHOLE ANTIBODY FLAGELLA OR CAPSULE/Vi)
 REQUIRES PAIRED SERA  SINGLE SERA
 POOR SENSITIVITY &  DETECT SPECIFIC ANTIBODY
SPECIFICITY  GOOD SENSITIVITY &
SPECIFICITY

 SINGLE STEP  MULTI STEP

 IN MINUTE RESULT (RAPID)  DAILY / WEEKLY RESULT
 REQUIRES NO  REQUIRES WASHER, READER,
INSTRUMENTATION INCUBATORS, ETC
 NO ELECTRICITY  REQUIRES ELECTRICITY
 INEXPENSIVE  REQUIRES INVESTATION &
COSTLY

ADVANTAGE DISADVANTAGE

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 RT- PCR in
Typhoid
PCR amplification for the
detection of pathogens in
biological material is generally
considered a rapid and
informative diagnostic
technique..

Several experimental methods

for PCR methods in progress.

Needs greater validation

Need equipment, electricity,

skill, expensive,
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 DIAGNOSTIC
REQUIREMENT

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 REFLEKSI KEBUTUHAN TES DIAGNOSTIK DEMAM
TIFOID
Diagnosis dini demam tifoid sangat berperan dalam pencegahan
komplikasi dan penularan, sementara uji widal tunggal (spesimen
tunggal) yang banyak digunakan ternyata kurang bermakna,
teknik DNA probe dan PCR yang memiliki nilai diagnostik tinggi saat
ini hanya bisa dimanfaatkan untuk kepentingan penelitian. Untuk
itu dibutuhkan pemeriksaan penunjang laboratorium yang sensitif,
spesifik, praktis dan tidak mahal bilamana gejala klinis tidak khas.
(Djoko Widodo, Irsan Hasan. Perkembangan diagnosis laboratorium
demam tifoid. Maj Kedokt Indon 1999, Vol; 49: 256-262)

Melihat pengalaman selama ini banyak sekali kasus

infeksi yang dikirim ke rumah sakit dengan diagnosa
Demam Tifoid dan sudah mulai diberikan obat
antibiotika, yang kemudian ternyata suatu Demam
karena Virus – antara lain Dengue
(Iskandar Zulkarnain. Perkembangan diagnosis dan penatalaksanaan beberapa penyakit infeksi, khusus
beberapa segi yang memprihatinkan yang perlu mendapat perhatian, 2004. Pidato Upacara Pengukuh
Tetap – Ilmu Penyakit Dalam FKUI.)
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 REFLEKSI KEBUTUHAN
TES DIAGNOSTIK DEMAM TIFOID

Despite the fact that is not reliable, the Widal test continues
to be used. A reason for this is the simplicity of the test,
inexpensive, requires no instrumentation and a single step is
involved, whereas multiple step are required by the ELISA,
enzyme conjugates and electronic reader are also costly
necessities. An ideal test is one that is not only reliable but,
first and foremost, simple and affordable for the countries
which need it most.
(Lim PL, etal, 1998. One step 2-minutes test to detect typhoid specific antibodies based on particle
separation in Tubes. J Clin Microbiol,36,2271-2278)

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 REFLEKSI KEBUTUHAN
TES DIAGNOSTIK DEMAM TIFOID

Isolation of serotype Typhi from blood, urine, or stool is the

most reliable means of confirming an infection. However,
this requires laboratory equipment and technical training
that are beyond the means of most primary health care
facilities in the developing world. Most serotype Typhi
infections are diagnosed purely on clinical grounds and
treated presumptively. As a result, the diagnosis may be
delayed or missed while other febrile illnesses are
considered, and patient without typhoid fever may receive
unnecessary and inappropriate antimicrobial therapy.

(Sonja J. Olsen et al. Evaluation of Rapid Diagnostic Tests for Typhoid Fever. JOURNAL
OF CLINICAL MICROBIOLOGY, May 2004, p. 1885-1889.)

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 KEBUTUHAN UNTUK TES
DIAGNOSTIK DEMAM TIFOID
Mampu mendeteksi demam sedini mungkin  erat kaitannya
dengan derajat infeksi yang masih ringan

Lebih cepat & lebih sensitive

Mampu membedakan Demam Tifoid akut secara signifikan

terhadap infeksi lain dengan gejala yang sangat mirip, terutama
di daerah endemis

Effektif mendeteksi secara akurat hanya dengan spesimen tunggal

Prosedur kerja sepraktis WIDAL dan Akurasi sebagaimana ELISA

Hasil pengujian konsisten dan tidak timbul variasi hasil antar

Laboratorium.

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 ALTERNATIVE SOLUTION

A practical, inexpensive but yet sensitive

Here I am !
and specific laboratory test for Typhoid
fever has been coming

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 ALTERNATIVE SOLUTION
- has the advantages
of the Widal test, and
the specificity ELISA

- utilize purified
antigen for detection.

-consist of one step

R - result is read
visually based on the
appearance of the
reactant colors.
(Lim PL, etal, 1998. One step 2-minutes test to detect typhoid specific antibodies
based on particle separation in Tubes. J Clin Microbiol,36,2271-2278)
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 REAGENT COMPOSITION

BROWN Magnetic particle coated with S.typhi LPS -

REAGENT O9 Antigen by passive adsorption

BLUE Blue carboxylated latex particle coated with

REAGENT anti 09 Mab by using carbodiimide & added
with BSA-coated red latexparticles

NEGATIVE Protein stabilized buffer

CONTROL

POSITIVE Control Ab in protein stabilized

CONTROL buffer
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 PRODUCT PROFILE

Immunodominant & Robust

Immunogenic in infants
O-9 Antigen Potent B cell mitogen
Very rare in nature

Only in group D Salmonella

(WHO, 2003. Background document : The diagnosis, treatment and prevention of typhoid fever)

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 Epitope site monoclonal IgM used

TUBEX
used

Antibody
reacting

Antibody
not
reacting

JOHN M. C. LUK AND ALF A. LINDBERG. JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1991, p. 2424-2433
 TEST PRINCIPLE

negative positive

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 TEST PROCEDURE

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 INTERPRETASI HASIL
Separate on TUBEX
Magnetic Color Scale

5 min
0 2 4 6 8 10

Magnetic Color Interpretation Result

Scale 1 2 3

0 8 4

Neg Control Pos Control Serum Sample Pos

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 INTERPRETATION

Score Interpretation Guide

< 2 NEGATIVE - Does NOT indicate current typhoid fever

infection

Borderline, inconclusive score.

3 Repeat analysis. If still inconclusive, repeat sampling at a
later date.
4-5 POSITIVE - Indication of current typhoid fever infection
> 6 POSITIVE - Strong indication of current typhoid infection
No clear score obtained due to :
1) Poor adherence to assay protocol. Repeat
INDETERMINATE analysis.
2) Poor specimen quality. Repeat sampling and analysis

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 Special features

As the only semi-quantitative serum test

based on ELISA inhibition for typhoid fever
which requires no instrumentation

As the only test with magnetic binding

inhibiting format to separate reacted
X
from unreacted indicator particles which
is able and effective to detects specific
IgM anti-Salmonella O-9 antigen.

As the only test for detecting typhoid fever

by the development of a special tube
which allows efficient reaction to take
place
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 (Pak-Leong Lim,et al,, 1998. One-Step 2-Minute Test to Detect Typhoid-Specific Ab Based on Particle
Separation in Tubes. Journal of Clinical Microbiology, August 1998, p. 2271-2278, Vol. 36, No. 8)

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 Sensitivity of TUBEX TF vs Antibody
respond & Days after infection

Sources : Prof PL Lim, University of Hongkong

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 TUBEX-TF di berbagai penelitian

No Peneliti Sensitifitas Spesifisitas

(negara) % %
1 Lim PL (Malaysia) 100 100

2 Oracz (Polandia) 92,6 94,8

3 Olsen (Vietnam) 78 94

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 IDL Biotech

Semudah Widal
Dan
Secanggih ELISA

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Simple hand hygiene and washing can reduce
several cases of Typhoid
 SALMONELLA

The salmonellae that cause typhoid fever and paratyphoid

fever have the following antigenic compositions and belong
to the serogroups indicated.

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