Evaluation of a Rapid Serological Test for Diagnosis of Typhoid Fever Detecting Anti-O9 Antibodies

Thesis

Submitted for Partial Fulfillment of Master Degree in

Medical Microbiology & Immunology

Presented by Ahmad Mohammad Al-Shanawany

M.B.B.CH

Faculty of Medicine Tanta University

Under Supervision of

Prof. Dr. Tahany Ahmad Abdel-

Raouf

Professor of Medical Microbiology & Immunology

Faculty of Medicine

Ain-Shams University

Dr. Rasha Ahmad Reda Nasr

Assistant Professor of Medical Microbiology & Immunology

Faculty of Medicine

Ain-Shams University

Faculty of Medicine

Ain Shams University

Faculty of Medicine

Medical Microbiology & Immunology Dep.

Evaluation of a Rapid Serological Test for Evaluation of a Rapid Serological Test for Evaluation of a Rapid Serological Test for Evaluation of a Rapid Serological Test for

Diagnosis of Typhoid Fever Detecting Diagnosis of Typhoid Fever Detecting Diagnosis of Typhoid Fever Detecting Diagnosis of Typhoid Fever Detecting

AntiAntiAntiAnti----OOOO9999 Antibodies Antibodies Antibodies Antibodies

Protocol of ThesisProtocol of ThesisProtocol of ThesisProtocol of Thesis

Submitted for Partial Fulfillment of Master

Degree in Medical Microbiology& Immunology

Presented byPresented byPresented byPresented by Ahmad Mohammad Al-Shanawany

M.B.B.M.B.B.M.B.B.M.B.B.CHCHCHCH

Faculty of Medicine Faculty of Medicine Faculty of Medicine Faculty of Medicine TantaTantaTantaTanta University University University University

Supervised bySupervised bySupervised bySupervised by

Prof. Dr. Tahany Prof. Dr. Tahany Prof. Dr. Tahany Prof. Dr. Tahany AhmAhmAhmAhmaaaad d d d AbdelAbdelAbdelAbdel----RaoufRaoufRaoufRaouf Professor of Professor of Professor of Professor of Medical Medical Medical Medical Microbiology & ImmunologyMicrobiology & ImmunologyMicrobiology & ImmunologyMicrobiology & Immunology

Faculty of Medicine Faculty of Medicine Faculty of Medicine Faculty of Medicine

AinAinAinAin----Shams UniversityShams UniversityShams UniversityShams University

Dr. Rasha AhmDr. Rasha AhmDr. Rasha AhmDr. Rasha Ahmaaaad Reda Nasrd Reda Nasrd Reda Nasrd Reda Nasr

Assistant professor of Assistant professor of Assistant professor of Assistant professor of Medical Medical Medical Medical Microbiology & ImMicrobiology & ImMicrobiology & ImMicrobiology & Immunologymunologymunologymunology

Faculty of Medicine Faculty of Medicine Faculty of Medicine Faculty of Medicine

AinAinAinAin----Shams UniversityShams UniversityShams UniversityShams University

Faculty of Medicine

Ain-Shams University

2008

Introduction

Typhoid fever is a febrile and often serious systemic illness caused

by Salmonella enterica typhi (S.typhi). It remains a global threat with an

estimated 21 million illnesses and more than 200 000 deaths annually

(Crump et al., 2004). Rapid accurate diagnosis and early treatment with

suitable antimicrobials is essential for rapid recovery and for prevention of

complications and deaths due to this disease and also for the control of

transmission (WHO, 2003).

Because the clinical features of typhoid fever are nonspecific, the

isolation of the causative organism from blood, bone marrow, or stool is

required to confirm diagnosis (Duta et al., 2006). However, the problem

with culture is that the method is laborious and time consuming (taking

days) and even stool cultures are positive in only 30% of patient with acute

illness (Parry et al., 2002).

Serology based on antibody detection has been widely used to

diagnose typhoid fever since the introduction of the Widal test (Widal,

1896). This test has remained the mainstay of serological test in many of

the endemic countries despite numerous accounts of its shortcomings

(Quiroga et al., 1992, Parry et al., 1999 and Nuseba et al., 2002). The

main reason for its continued use is the fact that it is simple-requiring just a

single step with no instrumentation and is affordable by the affected

countries (Tam et al., 2008).

Several new serological tests for typhoid fever have been introduced

which detect antibodies to various purified antigens of S. typhi (Rahman et

al., 2007). Of these tests is the TUBEX O9 antibody test. In this test, when

magnetic particles coated with S. typhi LPS are mixed with indicator

(colored) particles coated with a monoclonal antibody specific for the O9

antigen in S. typhi lipopolysaccharide (LPS), the two types of particles will

bind together and sediment by use of magnet leaving a clear supernatant. If

a patient’s anti-O9 antibodies are present in the reaction mixture, they will

inhibit the binding of the colored particles to the magnetic particles.

Consequently, the supernatant remains colored, the results are graded from

0 (pink, most negative) to 10 (blue, more positive) according to the color

chart provided (Oracz et al., 2003).

Aim of the Work

The aim of this study is to evaluate a rapid serological test in

diagnosis of typhoid fever by detecting anti-O9 antibodies of S.typhi in

comparison with isolation of S.typhi and Widal test.

Patients and Methods

Patients

This study will include 30 patients selected from Armed Forces

Tropical Hospital in Almaza, Cairo, presenting by clinical suspicion of

typhoid fever. All patients will be subjected to full history taking and

clinical examination.

Methods

A- Blood sample will be taken from each patient to do the following:

� Blood culture for isolation & identification of S. typhi.

� Widal test for detection of salmonella antibodies in the patients' serum.

� Detection of IgM antibodies specific for S. typhi O9 LPS by TUBEX O9

antibody test (IDL Biotech, Sweden).

B- Stool samples will be collected for isolation and identification of

S. typhi.

References

• Crump JA, Luby SP and Mintz ED. (2004): The global burden of

typhoid fever. Bull WHO 82: 346-352

• Dutta J, Sur D, Manna B, Sen B, Deb AK, Deen JL, Wain J, Seidlein

LV, Ochiai L, Clemens JD and Bhattachary SK. (2006):

Evaluation of new-generation serologic tests for the diagnosis of

typhoid fever. Diag. Microbiol. Infect. Dis. 56: 359-365.

• Oracz G, Feleszko W, Golika D, Maksymiuk J, Klonowska A and

Szajewska H. (2003): Rapid diagnosis of acute Salmonella

gastrointestinal infection. Clin. Infect. Dis. 36: 112-115.

• Parry CM, Hien TT, Dougan G, White NJ and Farrea J. (2002):

Typhoid Fever. N Engl. J Med. 347: 1770-1782.

• Rahman M, Siddique AK, Tam FCH, Sharmin S, Rashid H,

Iqubal A, Ahmed S, Nair GB, Chaignat CL and Lim PL. (2007):

Rapid detection of early typhoid fever in endemic community

children by the TUBEX O9-antibody test. Diag. Microbiol Infect.

Dis. 58: 275-281.

• Tam FCH, Ling YKW, Wong KT, Leung DTM Chan RCY and Lim PL.

(2008): The TUBEX test detects not only typhoid-specific

antibodies but also soluble antigens and whole bacteria. J Med.

Microbiol. 57:316-323.

• WHO (2003): Background Document: The diagnosis, Treatment and

Prevention of typhoid fever, WHO document: Geneva WHO/V and

B/03.07.

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تنـتج عـن و الحمى التيفية من الأمراض الحمية الى تصيب أجهزة الجسم المختلفة إن

تؤدى لمـا مليون مصاب و ٢١لميا يهدد قرابة هى تمثل خطرا عا و. الإصابة بالسلمونيلة التيفية

ب يعـد العلاج المناسسنويا لذلك فان التشخيص السريع والدقيق و ألف حالة وفاة ٢٠٠يزيد عن

الوفيات الناتجة عن هذا المرض وايضا من أجل ضروريا من أجل الشفاء وتجنب المضاعفات و

.انتشارهاو السيطرة على نقل العدوى

ونتيجة لأن العلامات الاكلينيكية للحمى التيفية تتشابه مع أمراض أخرى فـان فـصل

يا لتأكيد التشخيص از يعد ضرور الكائن المسبب للمرض من الدم أو من نخاع العظم أو من البر

أيـضا فـان ة تستغرق وقت طويل يستمر لأيـام و لكن تبقى مشكلة أن المزارعة وسيلة مرهق و

.فقط من الحالات الحادة% ٣٠مزارع البراز تعطى نتائج ايجابية فى

لذلك فان التشخيص السيرولوجى المعتمد على اكتشاف الأضداد قد أصـبح مـستخدما

لقد ظل هذا الاختبار أساسيا فـى و ١٨٩٦ور اختبار فيدال عام لتشخيص الحمى التيفية منذ ظه

يعـد رغم من أوجه القصور العديـدة بـه و كثير من الدول التى يتوطن بها هذا المرض على ال

السبب الأساسى لاستمراره كونه سهل الأداء لا يتطلب الا خطوة واحدة ولا يتطلب أى أجهزة

. يمكن لهذه الدول أن تتحمل تكلفتهو

التـى تتعـرف علـى رات السيرولوجية للحمى التيفية و وقد تم تقديم العديد من الاختبا

فيـه و) TUBEX O9(منها اختبار ة المختلفة للسلمونيلة التيفية والأضداد للمستضدات النوعي

الخاصـة ) LPS( ات الممغنطة المغلفة بالـدهون عديـدة الـسكريات عندما يتم مزج الجزيئ

المغلفة بالأضداد الأحادية الخاصـة بالمستـضد ) الملونة(بالسلمونيلة التيفية مع الجزيئات الدالة

)O9 ( الموجود بالدهون عديدة السكريات الخاصة بالسلمونيلة التيفية فسوف يتم حدوث ارتبـاط

يئات ويتم ترسيبها باستخدام مغناطيس تاركا السائل الطـافى رائقـا بين هذين النوعين من الجز

فى المزيج فسوف يمنـع ارتبـاط ) O9(وفى حالة وجود الأضداد الخاصة بالمريض للمستضد

وردى الأكثر (نتائج من يتم تقدير ال والجزئ الملون بالجزئ الممغنط وبالتالى يظل الطافى ملونا

.تدريج الملون المصحب للاختباروفقا ) أزرق أكثر ايجابية (١٠لى إ) سلبية

ovfÖ]<àÚ<Í‚�]ovfÖ]<àÚ<Í‚�]ovfÖ]<àÚ<Í‚�]ovfÖ]<àÚ<Í‚�]< << << << <

سريع فى تـشخيص الحمـى التيفيـة تقييم اختبار سيرولوجى الىتهدف هذه الدراسة

اختبار بفصل السلمونيلة التيفية و مقارنة للسلمونيلة التيفية ) O9(ـلكتشاف الأضداد الخاصة با إب

.فيدال

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مريض يتم اختيارهم من مستشفى القوات المـسلحة ٣٠سوف تشتمل هذه الدراسة على

جميع المرضى سيتم أخذ التاريخ المرضـى . بالقاهرة مصابين بالحمى التيفية للحميات بألماظة

.إكلينيكيا كاملا كما سيتم فحصهم منهم

:ذ عينات من دم المرضى لعمل الاتىسيتم أخ •

.تحديد السلمونيلة التيفيةومزرعة دم لفصل .١

.اختبار فيدال للكشف عن الاضداد الخاصة بالسلمونيلة التيفية فى مصل المريض .٢

باســتخدامللــسلمونيلة التيفيــة) O9(ل الخــاص بــا) IgM(اكتــشاف الأضــداد .٣

. )TUBEX O9(اختبار

. براز لفصل السلمونيلة التيفية والتعرف عليهاسيتم جمع عينات •

Acknowledgement

First, I thank AALLLLAAHH for granting me the power to

proceed and to accomplish this work.

I would like to express my deep appreciation and sincere

gratitude to Prof. Dr. Tahany Ahmad Abdel-Raouf,

Professor of Medical Microbiology & Immunology, Faculty of

Medicine Ain-Shams University, for her active cooperation, deep

concern and enthusiastic encouragement throughout this study.

I am also grateful and owe special thanks to Dr. Rasha

Ahmad Reda Nasr, Assistant professor of Medical

Microbiology & Immunology, Faculty of Medicine Ain-Shams

University, for supervising the work, valuable guidance and

continuous advice and support throughout this study.

I would like to express sincere thanks to all Staff members

of Medical Microbiology & Immunology department, Faculty of

Medicine Ain-Shams University, for their continuous support

and precious advice.

Contents

Item Page

Introduction ……..………….…..………………….. 1

Aim of the Work …...…………………….………… 3

Review of Literature…..

………………………….. 4

(A)

Salmonella……..………………………………….……

…………

4

Background 4

Morphology and Cultural Characters 5

Biochemical Activity 7

Antigenic Structure 9

Typing 18

Habitat 24 Susceptibility to antimicrobial agents 25

Antibiotic resistance 25

(B) Typhoid Fever ……………………………………….…………

32

Background 32

Transmission 33

Pathophysiology 34

Risk factors 35

Mortality/Morbidity 36

Clinical Presentation 37

Laboratory Diagnosis 40

Treatment 44

Epidemiology and control 50

Patients and Methods

……..……………………… 53

Results ……………..…..

………………………….... 58

Discussion ……………………………………………. 66

Summary and Conclusion ………….……………… 73

Recommendations ………………………………… 75

References ………………………………………….. 76

Arabic Summary …………………………………... -

List of Abbreviations

aPTT : Activated partial thromboplastin time

AUC : Area under curve

CDC : Centers for disease control

CFTR : Cystic fibrosis transmembrane conductance receptor

DCA : Deoxycholate citrate agar

DIC : Disseminated intravascular coagulation

DT : Definitive phage types

EGF : Epidermal growth factor

ESR : Erythrocyte sedimentation rate

KUB : Kidneys, ureters, and bladder

LPS : Lipopolysaccharide

PAMPs : Pathogen-associated molecular patterns

PCR : Polymerase chain reaction

PFGE : Pulsed field gel electrophoresis

PT : Prothrombin time

ROC : Receiver operating characteristic

SD : Standard deviation

SMZ : Sulfamethoxazole

SPSS : Statistical program for social science

SSPs : Serotype-specific plasmids

TAB : Typhi, Paratyphi A and Paratyphi B

TLR : Toll-like receptor

TMP : Trimethoprim

TSI : Triple sugar iron

WHO : World Health Organization