Comparison of different test methods including

polymerase chain reaction for early and reliable

diagnosis of typhoid fever

Dr. Safia Sultana MBBS

Department of Microbiology Mymensingh Medical College

Mymensingh, Bangladesh January 2012

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TO WHOM IT MAY CONCERN

This is to certify that Dr. Safia Sultana, a student of thesis part of M Phil (Microbiology) has

completed the thesis entitled �Comparison of different test methods including polymerase

chain reaction for early and reliable diagnosis of typhoid fever� in the Department of

Microbiology, Mymensingh Medical College under my guidance and supervision and this is

up to my satisfaction. Her protocol was approved by protocol approval committee of the

Department of Microbiology and Ethical review committee of Mymensingh Medical College.

Mymensingh December 05, 2011

Professor Dr. Md. Akram Hossain Head of the Department of Microbiology

Mymensingh Medical College Mymensingh, Bangladesh

Declaration

I hereby declare that the whole work submitted as a thesis entitled �Comparison of different

test methods including polymerase chain reaction for early and reliable diagnosis of

typhoid fever� in the department of the microbiology, Mymensingh Medical College, Dhaka

University, from July 2010 to June 2011 for the Degree of Master of philosophy is the result

of my own investigation and was carried out under the supervision of Professor Dr. Md.

Akram Hossain.

I, further declare this thesis or part thereof has not been concurrently submitted for the award

of any Degree or Diploma anywhere.

Dr. Safia Sultana Signature of the Candidate

ACKNOWLEDGEMENT

All praises belongs to Almighty Allah, the most merciful, the most beneficent and the most

kind for giving me the opportunity, courage and enough energy to carry out and complete the

entire thesis work.

I am very grateful and deeply indebted to my honourable teacher and guide Professor Dr. Md.

Akram Hossain, Head of the Department of Microbiology, Mymensingh Medical College. It

is my great pleasure to express my deepest regards and whole hearted indebtedness to him for

his inspiring encouragement, continuous guidance, active cooperation, constant supervision,

valuable suggestions, constructive criticism and help in carrying out this work successfully.

I am grateful and express thanks to the honorable members of the Ethical Review Committee

for giving kind approval to my research protocol. I am obliged to Professor Md. Aminul

Haque Principal of Mymensingh Medical College, Mymensingh for his kind permission to

conduct the thesis.

I would like to express my deepest regards and gratitude to my respected teacher Dr. Md.

Ashraful Alam, Assistant Professor, Department of Microbiology, Mymensingh Medical

College, Mymensingh, for his constructive criticism in correcting this thesis with his wise

advice and active cooperation in correcting the manuscript.

I owe my gratitude to respected teacher Md Chand Mahamud, Assistant Professor,

Department of Microbiology, Mymensingh Medical College, Mymensingh for valuable

advice and cordial cooperation.

I am cordially expressing my respect and complements to Dr. Shaymol Kumar Paul,

Assistant Professor, Department of Microbiology, Mymensingh Medical College,

Mymensingh, for his cordial cooperation, and thoughtful suggestions in my thesis work.

I also grateful to Professor Nobumichi Kobayashi, Sapporo Medical University of Japan for

providing with us primers of PCR and technical assistance for this study and also greatful to

Professor Jalaluddin Ashraful Hoque, Professor of BIRDEM and Ibrahim medical college for

sponsoring blood culture tube.

I owe my gratitude to Professor Abu Ahmed Saleh, Professor of microbiology, BSMMU, for

supplying reference strain of Salmonella typhi.

I like to express gratitude to all other teachers, M.Phil students, laboratory technologist and

other staffs of Microbiology department, Mymensingh Medical College, Mymensingh for

their constant help and sincere cooperation during the entire study period.

I also grateful to Alamin, for his active help in computer typing, data entry and sincere

cooperation during the entire study period. I like to express gratitude to Dr. Aklima Sultana,

medical officer of pathology, BSMMU for kind help in my study period.

I express my gratitude to my parent, who always inspired me for higher studies and is my

constant source of inspiration and encouragement in every step of my life.

I remained incomplete if I do not express whole hearted thanks and gratitude to my

husband Dr. Mohammed Abdoullah Al Maruf for sparing me so much time in this job from

all sorts of social and family responsibilities and sharing my pain and pleasure. I would like

to express my heartiest affection for my sons, Mikail Abid and Yousuf Aryan who had been

deprived of my care and attention during the thesis work.

I remained incomplete if I do not express wholehearted thanks and gratitude to my beloved

mother in law Jahanara Begum her blessings, affectionate support and spearing me so much

time in this job from all sorts of social and familial responsibilities.

Lastly I am indebted to all those persons from whom I have collected samples. May Allah

give them better rewards. Thanks again.

Mymensingh, January, 2012 Dr. Safia sultana

CONTENTS

Page No.

LIST OF TABLES VI

LIST OF FIGURES VII

LIST OF ABBREVIATIONS VIII

SUMMARY X

INTRODUCTION 1

CHAPTER 1: HYPOTHESIS 5

OBJECTIVES 6

CHAPTER 2: REVIEW OF LITERATURE 7

CHAPTER 3: MATERIALS AND METHODS 54

CHAPTER 4: RESULTS 68

CHAPTER 5: DISCUSSION 90

CHAPTER 6: CONCLUSION AND RECOMMENDATIONS 98

CHAPTER 7: LIMITATION 99

CHAPTER 8: LIST OF REFERENCES 100

CHAPTER 9: PHOTOGRAPH i

CHAPTER 10: APPENDICES xii

LIST OF TABLES

Table No. Title Page No.

1 Age group distribution of suspected cases of typhoid fever. 71

2 The socio-demographic characteristics of study population 73

3 Rate of isolation of S. typhi among study cases 76

4 Relationship of blood culture positivity with duration of fever

of the cases

77

5 Antibiotic usage by the blood culture-negative suspected

cases of typhoid fever

78

6 Results of ICT for identification of IgM antibody of S. typhi 81

7 Sensitivity and specificity of ICT for diagnosis of typhoid

fever.

83

8 Results of polymerase chain reaction (PCR) for identification

of flagellin gene of S. typhi

84

9 Comparison of culture and PCR methods for identification of

S. typhi

85

10 Sensitivity and specificity of blood culture in study population 88

11 Diagnostic accuracy of PCR for diagnosis of typhoid fever 89

LIST OF FIGURES

Figure No. Title Page No.

1 Presenting complaints by the suspected cases of typhoid

fever.

69

2 Age group distribution of study population 70

3 Sex pattern of study population. 72

4 Different test performed for diagnosis of typhoid fever. 75

5 Antimicrobial susceptibility pattern of the Salmonella

typhi isolates

79

6 Antibody detection among suspected cases by ICT

method.

82

7 Comparison of positivity of Blood culture and PCR for

diagnosis of typhoid fever.

86

LIST OF ABBREVIATIONS

ATCC American Type Culture Collection

BMAC Bone-marrow-aspirate culture

BP Base pair

CIE Counter current Immunoelectophoresis

CLSI Clinical and Laboratory Standards Institute

CMIR Cell-mediated immune responses

DDW Deionized distilled water

DNA Deoxyribonuclic acid

DSCC Duodenal string-capsule culture

DTH Delayed-type hypersensitivity response

EDTA Ethylene diamine tetraacetic acid

EIA Enzyme Immunoassay

ELISA Enzyme-linked immunosorbent assay

et al. et alia ( and others )

FAE Follicle-associated epithelium

GALT Gut-associated lymphoid tissues

HLA Human leucocyte antigen

ICDDRB International Center for Diarrrhoeal Diseses and Research Bangladesh,

ICT Immunochromatography test

IROMPs Iron-regulated outer-membrane protein

Kb Kilobyte

Kda Kilo dalton

LDC Lysine decarboxylase

LMI Leucocyte migration inhibition test

MDR Multidrug resistant

MDRTF Multi Drug Resistant typhoid fever

MHC Major Histocompatibility Complex

OMPs Outer membrane proteins

PCR Polymerase chain reaction

PPs Peyer's patch

rEPA Recombinant Pseudomonas aeruginosa exotoxin

RPHA Reverse Passive Haemagglutination assay

Rpm Rotation per minute

SIADH Syndrome of inappropriate release of antidiuretic hormone

SPI Salmonella pathogenecity island

SPS Sodium polyanethol sulfonate

SPV Salmonella plasmid virulence

Taq Thermus aquaticus

TBE Tris/Borate/EDTA

TTSSs Type III Secretion Systems

USDA The United States Department of Agriculture

VICPS Capsular polysaccharide vi antigens

SUMMARY

Background

Typhoid fever, caused by Salmonella typhi, is an important cause of morbidity and mortality

among all age groups in many developing countries including Bangladesh. A rapid and

reliable method for the detection of S. typhi is essential for early diagnosis. The blood culture

though less sensitive and technically demanding is the gold standard method for diagnosis of

typhoid fever. In addition, the disease is diagnosed serologically by the Widal test and other

methods, which have limitations of sensitivity and specificity. The most promising recently

published method is polymerase chain reaction (PCR) based amplification of DNA from the

blood samples of typhoid fever patients.

Objective

Keeping in mind the above considerations, the study was designed- (i) to detect IgM antibody

to S. typhi specific antigen by Immunochromatographic (ICT) method, (ii) to isolate the S.

typhi by lytic centrifugation method of blood culture and (iii) to detect flagellin gene of S.

typhi using appropriate primers by a nested PCR directly from blood sample.

Methodology

The study was carried out in the department of Microbiology, Mymensingh Medical College,

Mymensingh between July, 2010 and June, 2011 including 200 individuals of different age

and sex. Of them, 150 were clinically suspected cases of typhoid fever and 25 were febrile

non-typhoid controls, and remaining 25 were apparently healthy controls. Specimens of

whole blood were collected from each of the cases and controls following universal safety

precautions. The collected samples were tested by culture, ICT and PCR and then results

were analyzed using appropriate statistical methods.

Results

Among the 150 suspected typhoid fever cases, PCR showed maximum positive result 133

(88.7%) followed by ICT for IgM 106(70.7%) and blood culture 23(15.3%). All culture

positive cases were positive by PCR and among 127 culture negative cases, 110(86.5%) were

also positive by PCR. Neither of the controls was positive by PCR or blood culture. At first

week of illness, out of 23 culture positive samples, 14 were positive by the ICT (IgM). Out of

127 cultures negative cases 92 were positive by ICT (IgM). The sensitivity, specificity of

PCR (100%, 100%) using blood culture as gold standard and ICT (77.4%, 82.3%) using PCR

as a gold standard was calculated.

Conclusion

In the present study, the PCR appears to be highly sensitive and specific and superior to

blood culture. So it is recommended to take necessary steps for setup and start PCR at least in

the tertiary care hospitals. The ICT remains one of the easily accessible, cheap and simple

method with moderate sensitivity for the diagnosis of typhoid fever, especially in the resource

poor countries like Bangladesh. Analyzing the findings of the study it is concluded that ICT

and PCR are the suitable methods for diagnosis of typhoid fever.

Introduction

Typhoid fever, caused by Salmonella typhi, is widely recognized as a major public health

problem in many developing countries. The disease emerged as an important infectious

disease in the early 19th century. It is endemic in the Indian subcontinent including

Bangladesh, South-East and Far- East Asia, the middle East, Africa, central and South

America (Jenkins and Gillespie 2009). It is a systemic infection and is transmitted through

the faecal oral route by the consumption of contaminated water and food, particularly raw or

undercooked meat, poultry, eggs and milk. The consumption may occur either directly from

person-to-person or by ingestion of food or water contaminated with faeces or urine carrier as

well as through flies (Singh 2001). The disease may occur in all ages, with the highest

incidence found particularly in children (Anggraini, Handoyo and Aryati 2004). In addition

infection is most common in young children and elderly with peak incidence in summer and

fall (Jerrold and Turner 2010). It is therefore, presumed that typhoid fever is a major health

problem in all those parts of the world where safe drinking water and sanitation is inadequate.

There was an estimation that typhoid fever caused more than 21 million illnesses and more

than 216,000 deaths in the world during 2000 (Crump, Luby and Mintz 2004). A previous

study from Pakistan in 2006 revealed an incidence rate of 170/100,000 (using blood culture)

whereas a serology based incidence rate was 710/100,000 (using Typhidot) (Siddiqui et al.

2006). The incidence of cases of bacteraemic typhoid fever in Bangladesh per year was found

390/100,000 in 2001. In the same investigation, the incidence of typhoid fever among the

children per year was 210/100,000 children of >5 years of age and 1870/100,000 of <5 years

of age (Brooks et al. 2005).

Enteric fevers including typhoid fever occur only in the humans, which may turn into a

severe infection progressing to complications and death. Persons with typhoid fever carry the

causative agent S. typhi in their bloodstream and intestinal tract. S. typhi is uniquely adapted

to humans and carriers represent the sole source of these organisms for a short period of time

(convalescent carrier) or chronic carriers who shed the organism for longer than 1 year

(Zwadyk 1992).

Since all the signs and symptoms of typhoid fever are nonspecific, a definitive diagnosis of

the disease depending on the clinical presentation alone is very difficult. Therefore,

laboratory-based investigations are essential for supporting the diagnosis of typhoid fever.

Several different techniques are used for the diagnosis of the disease. The �gold standard� for

diagnosis of typhoid fever is the isolation of S. typhi from appropriate samples including

blood, bone marrow aspirates, stool, urine and rose spots (Gasem et al. 1995; Wain et al.

2001). A lytic centrifugation technique of blood culture has some added advantage over

conventional method to reduce the isolation time (Old 2006; Betly et al. 2010). Serologic

diagnostic tests for typhoid fever by immunochromatographic test (ICT) are good alternatives

(Bhutta and Mansurali 1999) and PCR identification of the S. typhi specific gene (e,g-

flagellin gene) are the better techniques.

Drug resistance in S. typhi is of considerable importance to both clinicians and

microbiologists and poses a major problem for public health authorities. The emergence of

antibiotic resistant strains of the bacteria is closely linked to the irrational use of antibiotic in

treating human infections; especially ciprofloxacin resistance to commonly used antibiotics

such as chloramphenicol, ampicillin and cotrimoxazole has been reported from different parts

of world including India (Gautam et al. 2002). A study in Bangladesh reported showed a

gradual change in resistance to ampicillin and cotrimoxazole among S. typhi. In the same

study, the rate of resistance to cotrimoxazole, ampicillin and chloramphenicol decreased from

59.6 to 5.6% of the organisms over a 3 years period (Asna and Haq 2000). For this reason,

antimicrobial susceptibility test is essential to see the changing trend of antibiogram of

circulating strains in the community.

In the perspective of Bangladesh, it is presumed that the diagnosis of typhoid fever is usually

based on clinical presentation as well as Widal test, both of which are associated with

numerous limitations. The diagnosis of typhoid fever on clinical presentations alone is

difficult, as the presenting symptoms are diverse and similar to those observed with other

febrile illnesses, especially during the first weeks of the infection (Jenkins and Gillespie

2009). On the other hand, ICT method for detection of S. typhi antibodies is a simple and

rapid diagnostic test. The test simultaneously detects and differentiates the IgG and IgM

antibodies to S. typhi specific-antigen in whole blood (Ismail, Kader and Ong 1991). The

detection of IgM reveals acute typhoid fever in the early phase of infection, the IgG detection

reveals late phase as well as the carriage of infection, while the detection of both IgG and

IgM suggests acute typhoid in the middle phase of the infection (Saha et al. 1999; Choo et al.

1999).

The development of molecular methods for diagnosis of infectious diseases, including

typhoid fever has improved the sensitivity and specificity of diagnosis. One of the molecular

methods, Polymerase chain reaction (PCR) is the most sensitive and rapid method to detect

microbial pathogens in clinical specimens (Massi et al. 2003). In particular, when the specific

pathogen is difficult to culture in vitro or requires a long cultivation period, the diagnostic

value of PCR appears very significant (Kumar et al. 2002). Among the various PCR

techniques, nested PCR assay was used in the early diagnosis of typhoid fever (Prakash et al.

2005). The nested PCR has good potential to be a rapid tool for the definitive diagnosis of

typhoid fever and is superior to conventional methods of PCR (Ali et al. 2009). The nested

PCR had greatest diagnostic value for detection of S. typhi among all the diagnostic tests used

and also had higher efficacy in detecting the disease than other methods like the Widal test,

blood and urine cultures (Ambati, Nath and Das 2007).

The present study was designed to identify the cases of typhoid fever employing the

techniques of blood culture, serology and molecular methods keeping in mind the commonly

occurring problems and to overcome them. For this reason, blood culture was done by lytic

centrifugation method to cut short the length of isolation of S. typhi. Antimicrobial

susceptibility test was done in the present study to see the susceptibility pattern of circulating

strains in Bangladesh. The ICT method has been shown to be cheap, less time-consuming,

applicable for field use, easy to perform and highly sensitive and specific for detection of

antibodies in patients with typhoid fever. Considering this, the ICT method was applied for

the detection of S. typhi specific IgM antibodies in blood samples of the present study. The

nested PCR method was included in the present study as a tool for diagnosis of typhoid fever

and was compared to blood culture and serology.

Hypothesis

Polymerase chain reaction is highly sensitive, specific and superior to other available

methods for diagnosis of typhoid fever.

Objectives

General Objective:

To compare different test methods including polymerase chain reaction (PCR) for early and

reliable diagnosis of typhoid fever.

Specific objectives:

1. To isolate S. typhi by lytic centrifugation method of blood culture and to test

antimicrobial susceptibility of S. typhi isolates by disk diffusion method.

22.. To detect IgM and IgG antibodies to S. typhi-specific antigen by

immunochromatographic test (ICT).

3. To detect flagellin gene of S. typhi by a nested polymerase chain reaction from blood

samples.

4. To compare results of the test methods used for diagnosis of typhoid fever.

Review of literature

Typhoid fever caused by Salmonella typhi is a disease of global distribution. It is

characterized by insidious onset of sustained fever, headache, malaise, anorexia, relative

bradycardia, constipation or diarrhoea, and nonproductive cough. Epidemics are more

common in spring and summer; sporadic in other seasons (CDC 1997; Crum 2003). It is

continues to be a health major problem in many developing countries where there is poor

standard of personal hygiene and prevalence of contaminated food, safe drinking water and

sanitation is inadequate (Lifshitz 1996; House et al. 2001).

Background and history of typhoid fever

Until the first quarter of the 19th century, typhoid fever was not recognized as a separate

clinical entity and was often confused with other prolonged febrile illness of typhus fever of

rickettsial origin. �Typhos in Greek means smoke and typhus fever got its name from smoke

that was believed to cause it. Typhoid means typhus like and thus the name was given to this

disease. Although typhoid fever was first discovered by Willis in 1643 (cited in collier�s

Encyclopedia 1989), it was mistakenly understood to be typhus fever for a long time. Gerhard

in 1837 (cited in collier�s Encyclopedia 1989), distinguished the two illnesses and invented

the name typhoid fever which means typhus like fever. The causative organism was

visualized in tissue sections from Peyer�s patches and spleen of infected patients by Ebertt in

1880 and named it as Salmonella typhosum and was grown in pure culture by Gaffky in 1884

(Topley and Wilson 1990).

The significance of water contamination in the spread of the disease was first recognized by

Budd in 1856. As a young practitioner in North Devon (is a local government district in

Devon, England) his observations provided one of the greatest milestones in the development

of hygiene. He for the first time proved that the disease is infectious and spread through

patients� faeces. He further discovered that milk and water played an important role in the

transmission of typhoid fever (Topley and Wilson 1990).

Mary Mallon also known as Typhoid Mary was the first person in the United States to be

identified as a healthy carrier of typhoid fever. She became the first American carrier to be

identified and traced. She was a cook in New York. Some believed she was the source of

infection for several hundred people. Over the course of her career as a cook, she is known to

have infected 53 people, three of whom died from the disease. Public health authorities told

Mary to give up working as a cook or have her gall bladder removed; Mary quit her job but

returned later under a false name. She was detained and quarantined after another typhoid

outbreak. Mallon spent the rest of her life in quarantine. Six years before her death, she was

paralyzed by a stroke. On November 11, 1938, aged 69, she died of pneumonia. She was still

infectious on the day of her death. An autopsy found evidence of live typhoid bacteria in her

gallbladder. Her body was cremated; the ashes were buried at Saint Raymond's Cemetery in

the Bronx (Wikipedia 2011).

Epidemiology

Mode of transmission

Typhoid fever is a systemic infection caused primarily by S. typhi and is transmitted through

the fecal oral route by the consumption of contaminated meat, poultry, eggs and milk.

Infection is most common in young children and elderly with peak incidence in summer and

fall (rainy season) (Jerrold and turner 2010). The infection is transmitted by ingestion of food

or water contaminated with faeces. The disease is transmitted by either directly through hands

soiled with faeces or urine of cases or carriers or indirectly by ingestion of contaminated

water, milk, food or through flies. Contaminated ice, ice-cream and milk products are rich

sources of infection (Singh 2001).

Two hospital based case-control studies from Vietnam found that risk of infection was related

to recent contact with a typhoid infected person, lack of education and drinking untreated

water (Luxemburger et al. 2001; Tran et al. 2005).

Depending on age, 1% - 5% of patients become chronic carriers, harbouring S. typhi in the

gall bladder (WHO 2003). Chronic typhoid carrier status may be responsible for the

endemicity and outbreaks of the disease in the region. High prevalence of typhoid carriers

occurs in patients with biliary, gastrointestinal and other related disease (Vaishnovi et al.

2005).

World wide distribution

Typhoid fever caused by S. typhi is widely recognized as a major public health problem in

many developing countries. The disease is endemic in the Indian subcontinent including

Bangladesh, South-East and Fareast Asia, the Middle East, Africa, Central and South

America (Jenkins and Gillespie 2009).

Crump et al. estimated the global burden of typhoid fever and showed that 21.6 million cases

of illnesses and 0.2 million cases of death were by typhoid fever during 2000 (Crump, Luby

and Mintz 2004). They also identified the regions with high incidence of typhoid fever

(>100/100000 caser per year) including South-East and South-central Asia. Other regions of

medium incidence (10-100/100000 cases/per year) included rest of Asia, Africa, Latin

America, the Caribbean and Oceania (except Australia and New Zealand) and region low

incidence (<10/100000 cases/year) of typhoid fever included Europe, North America and

the rest of the developed world. In contrast to that seen in rich countries, typhoid fever

remains an important cause of illness in developing world where annual incidences in Papua

New Guinea and Indonesia may reach 1200/100,000 population (Crump, Luby and Mintz

2004).

It is commonly observed that the majority of patients, 60% - 90% are treated as out patients

and therefore, hospital based studies was underestimate true incidence. The annual typhoid

incidence (per 100,000 person years) among 5-15 year-olds age group varied from 24.2 and

29.3 in sites in Viet Nam and China, respectively, to 180.3 in the site in Indonesia; and to

412.9 and 493.5 in sites in Pakistan and India, respectively (Ochiai et al. 2008). In a study

during 2000-2001 among children in an urban slum Bangladesh, the overall incidence of

typhoid fever was 390 cases per 100,000 populations per year. The incidence among >5 years

was 210 per 100,000 per year and among children <5 years the rate was 1870 per 100,000 per

year (Brooks et al. 2005).

Another study conducted by Sinha et al. 63 culture-positive typhoid fever cases were

detected. The incidence rate of typhoid per 1000 person-years was 27.3 at age under 5 years,

11.7 at 5-19 years, and 1.1 between 19 and 40 years. The difference in the incidence of

typhoid fever between those under 5 years and those aged 5-19 years was significant (Sinha

et al. 1999).

The organism Salmonella typhi

Historical background

The genus Salmonella was named after Daniel Elmer Salmon, an American veterinary

pathologist. Although Theobald Smith was the actual discoverer of the bacterium (Salmonella

enterica Var. cholerasuis) in 1885. As Dr. Salmon was the administrator of the United States

drug administration (USDA) research program, and thus the organism was named after him.

Smith and Salmon had been searching for the cause of common �hog cholera� and proposed

this organism as the causal agent. Later research, however, showed that this organism (now

known as Salmonella enterica) rarely causes enteric symptoms in pigs and was thus not the

agent they were seeking (Which was eventually shown to be a virus). However related

bacteria in the genus Salmonella were eventually shown to cause other important infectious

diseases (Wikipedia 2011).

Salmonella Nomenclature:

Salmonella nomenclature is complicated. Initially each Salmonella species was named

according to clinical consideration e,g. S. typhimurium (mouse typhoid fever), S. Cholerasuis

(hog cholera) (Kauffmann 1941)(Cited in Wikipedia 2011). Later, molecular findings led to

the hypothesis that Salmonella consisted of only one species (Leminor and Popoff 1987).

The terminology introduced by White and modified by Kauffman accorded specific rank to

each antigenically distinguishable Salmonella type and the convention was established that

each new type should be named after the place in which it was first isolated. The first

published table contained some 20 serotypes. The current number in about more than 2500

serotypes (Moreno et al. 2009).

The antigenic formulae of Salmonella serotype are defined and maintained by the World

Health Organization (WHO) collaborating centre for Reference and Research of Salmonella

at the Pasteur institute, Paris, France, and new serotype have been listed in annual updates of

the Kauffman�White scheme (Popoff, Bockemüh and Brenner 2000).

Morphology

These are Gram negative, motile, non spore forming, non capsulated bacilli measuring 2-4 x

0.6 µm. Most strain are motile due to presence of peritrichous flagella except S. gallinarum

and S. pullorum which are non motile. They may possess fimbriae (Chacraborty 2003). Most

strains of most serotypes form type 1 fimbriae (Mannose � Sensitive, haemagglutinating); S.

gallinarum, S. pullorum and a few strains of other serotypes form type2 fimbriae (non-

haemagglutinating) and most S. paratyphi A strains are non-fimbriate. S. typhi also

synthesizes type IV pili and such pili are important in adherence to or invasion of human

intestinal cells (Zhang et al. 2000).

Antigenic structure of Salmonella typhi

The O and H antigens are the major antigens used to serotype the Salmonella. The O antigens

are Similar to the O antigens of other Enterobacteriaceae but H antigens are different in that

they are diphasic. i.e, the H antigens can exist in either of two major antigenic phases. Phase

1 (Specific phase) and phase 2 (non specific phase). Phase 1 antigens are shared by only a

few organisms and react only with homologous antigens but phase 2 antigens are shared by

many organisms and react with heterologous antisera (Zwadyk 1992).

O antigens or somatic antigens

The O antigens are the most surface exposed lipopolysaccharide (LPS) component and

displays enormous structural variability, resulting in a large variety of serotypes (Reeves

1993). These somatic antigens represent the side-chains of repeating sugar units projecting

outwards from the lipopolysaccharide layer on the surface of the bacterial cell wall.

The O antigens are heat stable being unaffected by heating for 2.5 hour at 1000c and alcohol

stable. The O antigens are unaffected by suspension of the bacteria in 0.2% formaldehyde

(Old 2006). Antibodies to O antigens are predominantly IgM and tend to agglutinate O

antigens in granular masses. The presence and proper Chain length distribution of the O

antigens polysaccharide are essential for serum resistance of S. typhi but not for invasion of

epithelial cells (Hoare et al. 2006).

H (Flagellar) Antigens

These antigens represent determinant groups on the flagellar protein. They are heat�labile

and alcohol labile, but are well preserved in 0.04 � 0.2 % formaldehyde. Heating at

temperature above 600C detaches the flagella from the bacteria and detachment of all flagella

is achieved by heating for 30 minute at 100°c. The deflagellated bacteria are inagglutinable

by H antibodies but the detached flagella remain immunogenic and suspensions of bacteria to

be used for the production of O antisera should be freed from detached flagella by

centrifugation and washing or by inactivation by heating for 2.5 hour at 1000C (Old 2006).

Vi � antigen

S. typhi also produces a group 1 exopolysaccharide known as the vi antigen; which is made of

a homopolymer of high molecular mass (Virlogeux and Popoff 1996) and form a capsular

structure. The vi antigen is found in virtually all clinical isolates from patients with acute

typhoid infection. It protect S. typhi against complement mediates lysis as well as

phagoaytosis (Kossack et al. 1981).

M antigens

It is a loose extracellular polysaccharide slime consisting of colanic acid. It occurs in a

serologically similar form in various unrelated enterobacteria, including serotypes of S. typhi

and many stains of E. coli and resembles the vi antigen in preventing agglutination by O

antibodies (Old 2006).

R antigens

In S-R mutation the O antigens are lost and new R antigens revealed at the bacterial surface.

The R variant bacteria tend to out grow the parenteral S bacteria during serial culture in the

laboratory. They form rough colonies, and are autoagglutinable in saline and sensitive to

killing by complement because they are autoagglutinable and lack serotype specificity, they

are unsuitable for serologic tests (Old 2006).

Fimbrial antigens

The type 1 fimbriae formed by most strains of S. typhi bear antigens that determine

agglutination by sera containing anti-fimbrial antibodies. Fimbriae are not found in young (6-

24 hours old) broth cultures, but can be found in 24-48 hours old broth cultures (Old 2006).

Determinants of pathogenecity

Whether an infection with S. typhi leads to a disease largely depends on the virulence of the

strain and the constitution of the host. The virulence of the strain is determined by So-called

virulence factors. S. typhi is a complex organism that produces a variety of virulence factors.

These include surface antigens, factors contributing to invasiveness, endotoxin, cytotoxin and

enterotoxin (Zwadyk 1992).

Whereas a number of virulence factors of S. typhi have been identified only recently, others

have been studied for decades. These latter virulence factors i, e, virulence plasmids, toxin,

fimbriae and flagella are therefore referred to as classic virulence factors (Asten and Dijk

2005).

Surface Antigen

a) O antigen

The ability of S. typhi to attach to host receptors cells and to survive intracellularly may be

due to O antigenic side chain. The O antigen of S. typhi apparently is important in

determining the susceptibility of some serotypes to complement, to host cationic proteins and

to an interaction with host macrophages. Organisms with intact O antigens are more resistant

to the complement mediated killing of normal serum. The resistance to killing by normal

serum probably is due to the shielding of the complement activating lipid A and LPS core

polysaccharides by the polysaccharides of the O antigen (Zwadyk 1992).

b) Vi antigen

The capsular polysaccharide vi antigens (VICPS) is an essential virulence factor and also a

protective antigen of S. typhi (Tang et al. 2003). The vi antigen is found in virtually all

clinical isolates from patients with acute typhoid infection. It protects S. typhi against

complement mediate lysis as well as phagocytosis (Kossack et al. 1981).

c) Fimbrial antigen

S. typhi synthesizes type IV pilli and such pilli are important in adherence to or invasion of

human intestinal cells (Zhang et al. 2000).

d) Outer membrane protein (OMP)

Act as virulence factor such as 55 KDa outer membrane protein from short chain fatty acids

exposed to S. typhi induces apoptosis in macrophages (Chander et al. 2006).

Invasive ness

Unlike most bacteria, which rely on receptor mediated endocytosis to invade a target cell, S.

typhi utilizes a complex process known as bacterial mediated endocytosis, where bacterial

proteins enter the host cell and manipulate signaling cascades that control cytoskeletal

architecture membrane trafficking and gene expression, all of which force the host to

endocytose S. typhi (Ohi and Miller 2001).

The target cell for S. typhi is the macrophage. The ability of S. typhi to survive in

macrophages is due to the production of bacterial proteins that enable the organism to

withstrand both the oxygen-dependent and the non oxygen-dependent killing mechanisms of

these professional phagocytic cells. The oxygen-dependent mechanisms include the

production of hydrogen peroxide and super oxide. The oxygen independent mechanisms

include the production of antibacterial, cationic proteins known as defensins (Zwadyk 1992).

Endotoxin

Endotoxin may play a role in the pathogenesis of S. typhi infections, especially during the

bacteremic stages of typhoid fever. The fever is produced by the endotoxin acting directly

and indirectly through the release of endogenous pyrogens from leukocytes. Endotoxin

activation of the chemotactic properties of the complement system may cause the localization

of leukocytes in the classic lesions of typhoid fever (Chakraborty 2003).

Virulence plasmid

Certain Salmonella carry a large, low copy number plasmid that contains virulence genes.

Virulence plasmids are required to trigger systemic disease; their involvement in the enteric

stage of the infection is unclear. Salmonella virulence plasmids are heterogeneous in size (50-

90kb), but all share a 7.8 kb region, SPV, required for bacterial multiplication in the

reticuloendothelaial system (Rotger and Casadesus 1999).

Cultural Characteristics

Salmonellae are aerobic and facultatively anaerobic. They grow on simple laboratory media

in temperature range 150c - 450c, optimally at 370c, and require enrichment of the minimal

medium with one or more amino acids or vitamins e.g, cystin or Nicotinamide; most S. typhi

strains require tryptophan (Old 2006).

On Nutrient agar

The colonies of most strains are moderately large 2-3 mm in diameter after 24 hours at 370c.

They are grey white, Moist Circular discs with a smooth convex surface and entire edge (Old

2006).

On Blood agar

S. typhi produce grey white 2-3 mm in diametre colonies. Some strain produce mucoid

colonies (Cheesbrough 2010).

Peptone water and Nutrient broth

In liquid media most strains give abundant growth with uniform turbidity. A thin surface

pellicle usually forms on prolonged incubation, Rough (R) variants, which have a

hydrophobic a granular deposit and a thick pellicle (Old 2006).

Mac Conkey�s agar

After 18-24 hour at 370c the colonies are pale yellow or nearly colour less, 1-3 mm in

diameter and easily distinguished from the pink red calories of lactose fermenting commensal

coliform bacilli e.g. � Escherichia coli (Richard and Thompson 2007; Cheesbrough 2010).

Salmonella � Shigella agar (SS agar)

SS agar is highly selective medium formulated to inhibit the growth of most coli form

organisms and permit the growth of species of Salmonella and shigella from environmental

and clinical specimens. The high bile salts concentration and sodium citrate inhibit all Gram

positive bacteria and many Gram negative organisms, including coliforms (Old 2006).

Enrichment media

These are liquid media used to assist the isolation of S. typhi from faeces, sewage, food stuffs

and other materials containing a mixed bacterial flora. The enriched culture is placed on

selective and/or differential media, usually after 24 hours of incubation (Richard and

Thompson 2007; Cheesbrough 2010).

Tetrathionate broth

Enriches S. typhi and sometimes shigellae but permits the growth of proteus species.

Selenite F broth

It is the most commonly used enrichment medium for specimens that may contain either

salmonella or shigellae. It is excellent for S. typhi and S. Dublin. It is not suitable particularly

for isolation of S. paratyphi A and S. choleraesuis (Old 2006; Richard and Thompson 2007).

Immune response

The nature of protective immunity in typhoid fever in man is not well understood. The

development of the humoral immune response to O, H, and Vi antigens of S. typhi has been

regularly demonstrated during and after typhoid fever as well as after TAB vaccination

(Kumer et al. 1774).

The development of specific humoral antibody response as well as CMIR in patients with

typhoid fever at various stages of their illness. These immune responses are correlated with

the clinical picture and specific CMIR give protection against typhoid fever (Sarma et al.

1977).

Cell mediated immune response (CMIR)

The cell-mediated immune response in typhoid fever develops almost invariably during the

second week of illness in uncomplicated cases while it was often negative in complicated

cases (Sarma et al. 1977).

Cell-mediated immunity was assessed by the leucocyte migration inhibition tests (LMI), and

developed in all cases with typhoid fever. Positive LMI was evident in the first week of the

illness and was maintained during the evolution of disease and in some patients was still

present after a year. It also developed at the end of 3 weeks in TAB vaccinated subjects

(Dham and Thompson 1982). Positive LMT is associated with good clinical recovery (Sarma

et al. 1977).

A studies have shown that, iron-regulated outer-membrane protein (IROMPs) expressed by

S.typhi induce a cellular immune response against infection through Th1 and Th2 type cells

(Sood et al. 2005). The cellular immune response induced by IROMPs resulted in an

enhanced DTH (delayed-type hypersensitivity response) and exhibited a significant increase

the ratio of CD4+/CD8+ cells and increased production of interleukin (IL-2) and interferon

(IFN) in early period and in the late period of the study, increased production of IL-

4_producing cells. The increase in the lymphocytes in Peyer�s patches (PPs) might have

caused the increase in the secretory immunoglobulin A (sIgA). Therefore, it is speculated that

immunization with IROMPs may evoke peripheral as well as mucosal immunity against S.

typhi infection (Sood et al. 2005). The uncomplicated cases of typhoid fever were found to

have and intact CMIR as compared to the complicated cases (Rajagoplan, Kumar and

Malaviya 1982).

Humoral immune response

The humoral response to S. typhi is important for protective immunity against typhoid fever,

as indicated by the protection obtained with killed cell vaccines and component vaccines

(outer membrane proteins, Vi antigen) in animals and human beings (Aron et al. 1993).

Although antibodies to S. typhi O, H, and Vi antigen appear to be involved in protection

against S. typhi infection, it is unknown whether such antibodies mediate protection, act in

conjunction with other adaptive responses, or serve as a surrogate for the presence of other,

more dominant protective immune responses e.g. cell-mediated immunity (Sztein 2007).

Anti-O-polysaccharide chain antibody titres are lower at the first week and increase up to the

third week of the infection. On the other hand, antilipid A antibody levels, which are already

higher at the beginning of the disease, progressively augment during the following weeks

(Mastroianni et al. 1989).

The antibodies and cellular reactively developed almost simultaneously but there was no

correlation between the agglutination titres and LMI positivity. Typhoid patients also showed

significantly raised serum IgM and IgA levels and increased concentrations of secretory IgA

in their saliva (Dham and Thompson 1982). The antibodies appeared after the 1st week of

illness and the titres gradually increased during the following days. Chloramphenicol therapy

did not interfere with antibody production and antibody titres did not correlate with the

severity of typhoid fever (Sarma et al. 1977).

S. typhi IROMP have also been observed to have immunogenic potential and are able to

stimulate antibody-mediated protection at systemic and mucosal levels (Sood et al. 2005). For

protection against Salmonella spp. both antibody and cell-mediated immune (CMI) responses

are considered to be important. The O antigen (O9, 12 serotype) is most relevant to protection

against typhoid fever; other antigens include the virulence capsule antigen and some outer

membrane proteins (Viret et al. 1999).

Classification of Salmonella:

Kauffman � White classification

Based on their O and H antigen composition, more than 2500 Salmonella serotypes (species)

are described in the Kauffman- White scheme. Salmonella are placed in groups by their O

antigens (A, B, C. etc.) and subdivided by their H (phase 1 and 2) antigens. The grouping and

antigenic composition of some Salmonella are shown in the following table (Cheesbrough

2010).

Group, Serogroup Serotype

O Antigen H Antigens Phase1 Phase 2

Group A, Serogroup 2

S. paratyphi A

1,2,12

a -

Group B, Serogroup 4

S. paratyphi B

S. derby

S. typhimurium

1,4,5,12

1,4,5,12

1,4,5,12

b 1,2

f.g (1,2)*

i 1,2

Group C, Serogroup 7

S. choleraesuis

S. paratyphi C

6,7

6,7,(Vi)*

c 1,5

c 1,5

Group D, serogroup, 9

S. typhi

S. enteritidis

S. pullorum-gallinarum

9,12,(Vi)*

1,9,12

1,9,12

d -

g.m -

- -

Group E, Serogroup 3,10

S. weltevreden

S. anatum

3,10

3,10

r z6

e,h 1,6

Group G, serogroup

13,22

13,22

z 1,6

S. Poona

S. cubana

1,13,23 z29 -

Brackets indicate that the antigen may present or absent. Note:The O Antigen in bold type is common to all members of the group.

Pathogenesis

Natural infection of typhoid fever occurs by ingestion of contaminated food followed by

penetration of salmonella typhi through the intestinal mucosa. Disease production is

dependent on several factors: i) number of organisms swallowed, ii) state of gastric acidity

and iii) possession of vi antigen by the organisms (Jenkins and Gillespie 2009). Studies

involving human volunteer showed that only 25% of people become infected on ingestion of

105 organisms, with the infection rate increasing to 95% when the infecting dose increases to

109 viable organisms (Zwadyk 1992).

Many factors influence the infective dose. The vehicle of ingestion also matters. Organisms

in water and other drinks may be carried through the stomach relatively rapidly, and thus

escape the effect of the gastric acid. Similarly, the administration of antacids, or the effects of

gastric resection, reduces the infective dose (Lewis 1997).

After ingestion, organisms enter the lumen of intestine. They are able to multiply. Some of

bacteria attach to the microvilli of the ileal mucosa by means of adhesions on the bacterial

surface, which adhere specifically to mannose-containing receptors on the epithelium. S.

typhi crosses the intestinal mucosal barrier after attachment to the microvilli by a complex

mechanism involving membrane ruffling, acting rearrangement, and internalization in an

intracellular vacuole (Bhutta 2008). Attachment is followed by degeneration of the microvilli

to form breaches in the cell membrane through which S. typhi enter the cell (Lewis 1997).

Gastric acidity is an important defense against enteric infections and gastric hypo acidity

from any cause will allow a greater number of organisms to enter the small intestine (Jones,

Ghori and Falkaw 1994).

From the submucosa, invading bacteria are taken up by macrophages and the organisms

travel to mesenteric lymph nodes. After a brief period of multiplication in the lymph nodes,

the organisms enter blood stream via thoracic duct causing transient primary bacteraemia.

The organisms are then transported to the liver and spleen. After a period of further

multiplication in these organs, huge numbers of organisms enter the blood stream and onset

of clinical illness due to Secondary bacteraemia. During this secondary bacteraemia, which

continues for the greater part of illness and involvement of gall bladder Peyers patches in the

lower small intestine, have important clinical significance (Jenkins and Gillespie 2009).

Invasion of the Peyers patches occurs either during the primary intestinal infection or during

the secondary bacteraemia and further seeding occurs through infected bile. The Peyers

patches become hyperplasic with infiltration of chronic inflammatory cells. Later necrosis of

the superficial layer leads to formation of irregular, ovoid ulcers along the long axis of the

gut, so that stricture does not occur after healing. When an ulcer erodes into a blood vessel,

severe haemorrhage results and transmural perforation leads to peritonitis (Jenkins and

Gillespie 2009).

The clinical syndrome of fever and systemic symptoms is produced by a release of

proinflammatory cytokines (IL-6, IL-1ß, and TNF-Ü) from infected cells. In addition to the

virulence of the infecting organisms, host factors and immunity may also play an important

role in predisposition to infection (Bhutta 2008).

Molecular basis of pathogenesis

To be effective pathogens, salmonellae must be able to invade epithelial cells for organisms

to cause enteric fever they have to be adapted to survive inside cells of the reticulo-

endothelial system.

Epithelial invasion

Membranous or microfold cells, commonly referred to as M cells, are specialized epithelial

cells of the gut-associated lymphoid tissues (GALT). M cells form part of the follicle-

associated epithelium (FAE) which overlies the Peyers Patches and other lymphoid

aggregates (Miller et al. 2007).

The target of S. typhi invasion is the M cell but must cross epithelial later to achieve this

(Jones, Ghori and Falkaw 1994). S. typhi invade the intestinal epithelial cells by a complex

mechanism which includes triggering active rearrangements, formation of pseudopodia and

phagocytosis of the bacterium into the cells. Membrane ruffling then returns to normal after

the bacterium has invaded. The ruffling- internalization process is controlled by a type III

secretion system encoded by genes found in the inv locus (Collazo and Galan 1997). These

genes are located on a pathogenecity island SPI -1(Salmonella pathogenecity island 1) which

encodes all of the genes necessary for the invasion of intestinal epithelial cells and induction

of intestinal secretory and inflammatory responses (Galan 2001).

Intracellular survival

S. typhi causing typhoid fever must be able to survive and replicate within the host

macrophage system. Once inside these locations they are shielded from the effect of

immunity, but to do this they must overcome the nutrient poor environment within the

macrophage and defeat its bactericidal mechanisms. The PhoP-PhoQ two-component

regulatory system is required for the virulence of S. typhi in humans. PhoP-PhoQ plays a

more significant role in resistance of the organisms to deoxycholic and chenodeoxycholic

acids than in resistance to other bile acids or detergents (VanVelkinburgh and Gunn 1999).

Salmonella genes necessary for survival inside macrophages are constituents of a two

component response regulator termed phoP/ phoQ. Genes activated by this phoP/ phoQ are

known as pag genes of which pag A �C have been characterized. The pag genes are

expressed within the macrophage phagosome and are required for survival within it (Behlau

and Miller 1993; Pegues et al. 1995).

Complication: Intestinal perforation and bleeding

Typhoid perforation of the intestinal wall is an important complication of typhoid fever. It is

seen rarely, but shows a high mortality and morbidity. It usually occurs when sloughs

overlying the Peyer�s patches are separated during the late second or early third week of the

illness (Atamanalp et al. 2007; Jenkins and Gillespie 2009).

Typhoid fever leads to hyperplasia in the reticuloendothelial system. In addition necrosis and

ulceration may limited to the Peyer�s patches (Hosoglu et al. 2004; Saxena, Basu and Sharma

2007). While typhoid fever often affects the terminal ileum, in rare cases, the jejunum and

caecum may also be involved (Eggleston, Santoshi and Singh 1979).

Typhoid encephalopathy

Typhoid encephalopathy, often accompanied by shock, is associated with a high mortality.

Patients may display the �typhoid� facies, a thin, flushed face with a staring, apathetic

expression. Mental apathy may progress to an agitated delirium, frequently accompanied by

tremor of the hands, tremulous speech and gait ataxia, and then muttering delirium,

twitchings of the fingers and wrists (subsultus tendinum), agitated plucking at the bedclothes

(carphology), and a staring, unrousable stupor (coma vigil) (Parry et al. 2002).

The mechanisms responsible for the neurological manifestations of typhoid fever have been

variously described. Possible mechanisms implicated are hyperpyrexia (>43°C), fluid and

electrolyte disturbances, typhoid neurotoxin, vasculitis with peri-vascular cuffing,

autoimmune mechanism, pressure effect on blood vessels resulting in cerebral infarction and

acute disseminated encephalomyelitis (Vidyasagar et al. 2004).

Hepatobiliary manifestation of typhoid fever

Mild jaundice may occur in typhoid fever due to hepatitis, cholangitis, cholecystitis or

haemolysis. Biochemical changes indicative of hepatitis have been observed during the acute

stage (Khosla 1990). The spectrum of hepatic injury in typhoid fever has been studied in

children aged below 18 years. Among 100 children with confirmed typhoid fever, 29 had

moderate hepatitis (Balasubramanian et al. 2010).

Typhoid fever in pregnancy

Typhoid fever in pregnancy may be complicated by miscarriage; although antimicrobial

treatment has made this less common (Seoud et al. 1988). Pregnancy is a risk factor for and

affects typhoid disease expression, typhoid fever does not appear to affect pregnancy

outcome (Sulaiman and Sarwari 2007). A study described by Hasbun and others found that

the hepatic dysfunction occurred in 10 cases out of 32 women with typhoid fever during

pregnancy. This was associated with late diagnosis and maternal and perinatal complications.

Hepatic dysfunction in typhoid fever during pregnancy must be interpreted as a severe

damage of cell function with potential progress to maternal multisystemic failure and

perinatal death (Hasbun, Osorio and Hasbun 2006).

Complications of typhoid fever in children

Bacteraemia by Salmonella typhi in younger children can have serious consequences and

potentially fatal outcomes. Typhoid fever in children can lead to intracranial infections

(meningitis, focal brain abscesses) and osteomyelitis with sickle cell disease. Reactive

arthritis may follow Salmonella gastroenteritis in children with HLA �B27 antigen.

Complications of typhoid fever in children include anicteric hepatitis, bone marrow

suppression, paralytic ileus, myocarditis, psychosis, cholecystitis, osteomyelitis, peritonitis,

pneumonia and syndrome of inappropriate release of antidiuretic hormone (SIADH) (Malik

2002).

Intestinal haemorrhage and perforation is infrequent among children. Other reported

complications include fatal bone marrow necrosis, disseminated intravascular coagulation,

haemolytic uraemic syndrome, pyelonephritis, nephritic syndrome, meningitis, endocarditis,

parotitis, orchitis, and suppurative lymphadenitis (Bhutta 2008).

Relapse and Carriers

Two types of relapse in case of typhoid fever have been reported such as early and late

relapse. An early relapse occurs in 5 to 10 percent of patients, usually two to three weeks

after the resolution of fever. Reinfection may also occur and can be distinguished from

relapse by molecular typing of the causative agent (Wain et al. 1999; Parry et al. 2002).

Late relapse of typhoid fever may occur in some patients (10-20%), several weeks later or

after apparent recovery. There is usually an afebrile period between the first and second

episode of fever which may be a few days to a few weeks. Clinical manifestations and course

of the disease are usually milder and shorter than the primary attack (Joshi 2001).

There are two types of carrier have been identified. Carrier of S. typhi are either convalescent

carriers who secrete the organism for a limited period of time after apparent clinical cure, or

chronic carriers in whom persistent excretion of S. typhi in stool or urine can be detected a

year after clinical illness. Chronic faecal carriers occur more commonly than do chronic

urinary ones (Singh 2001).

It is reported that 1% - 5% of those infected become chronic carriers (WHO 2003) and carrier

status persists throughout the life of a person. High prevalence of typhoid carriers occur in

patients with biliary, gastrointestinal and other related disease (Vaishnovi et al. 2005). Apart

from this, it may be responsible for deaths due to hepato biliary cancer. Faecal carriage is

more frequent individual with gall bladder disease and is most common in women over 40

(Vaishnavi et al. 2005). Chronic Carriage lead to an increased risk of carcinoma of the gall

bladder, Pancreas and Large bowel (Parry et al. 2002).

Laboratory diagnosis

Laboratory diagnosis of typhoid fever is based on isolation and identification of Salmonella

typhi from a suitable clinical specimen such as blood, stool, urine, bone marrow, and

duodenal aspirate by culture, detection of S. typhi-specific antibodies by serological test and

antigen by immunological test and identification of nucleic acid by Polymerase chain reaction

(Pearson and Guerrant 1995).

Isolation of the organism

Culture isolation of the S. typhi remains the most effective diagnostic procedure in suspected

typhoid fever. Where culture is available, typhoid fever may account for two thirds of cases

of community acquired septicaemia admitted to hospital (Hoa et al. 1998). Blood has been

the mainstay of culture for S. typhi since 1900. S. typhi maximally isolated from blood in the

first week of disease; from faeces in the second and subsequent weeks and urine in the third

and fourth weeks (Old 2006).

The various culture methods available are: 1. Blood culture 2. Clot cultures 3.Faeces culture

4. Bone marrow culture 5. Urine culture 6. Bile culture 7. Duodenal aspirate culture.

Blood Culture

Blood culture is the gold standard diagnostic method for diagnosis of typhoid fever (Parry et

al. 2002). The sensitivity of blood culture is highest in the first week of the illness and

reduces with advancing illnesses (Ananthanarayan and Panikar 1999). The organisms may be

recovered from bloodstream at any stage of the illness, but are most commonly found during

the first 7-10 days and during relapses (Lewis 1997).

Blood culture is the method of choice and has the great advantage over culture from the

faeces, urine or bile. It is showing not only that patient is infected with the bacillus but that

the infection is active (Parker 1990). Though it is gold standard, the yield of blood culture is

quite variable. In the untreated patient, blood culture is usually positive in about 80% during

first week and declining 20% - 30% later in the course of the disease (Jenkins and Gillespie

2009). Sensitivity of cultures can be affected by antibiotic treatment of the patient, inadequate

sampling, type of culture medium, lengths of incubation, and variations of bacteraemia in the

patients. In addition, Salmonella cultures take 4-7 days for isolation and identification of the

organisms (Miller and Pegues 2000).

Adequate volumes of medium should be used in blood culture system to avoid negative

results. A study finding suggested that 50 ml of medium was adequate for 8 ml of blood,

presumably because of very low degrees of bacteraemia in some patients (Watson 1978).

If whole blood is to be cultured, it is essential to prevent bactericidal effects of serum either

by adequate dilution of the sample in an adequate medium volume or by inhibition of serum

bactericidal factors. Sodium polyanethol sulfonate (SPS) and bile salt inhibit this bactericidal

effect (Parker 1990).

The SPS in concentration of 0.025% to 0.03% is the best anticoagulant for blood. It is also

anticomplementary and antiphagocytic, and interferes with the activity of some anti microbial

agents, notably amino glycosides (Betly et al. 2010). A study was reported that SPS aids in

early recovery of S. typhi and S. Paratyphi A from blood cultures (Escamilla et al. 1985).

Taking samples of blood on several occasions may improve the results of culture (Le and

Hoffman 1999).

Three types of blood cultures have been in use such as i) traditional or conventional blood

culture ii) lysis centrifugation iii) automated blood culture (Collee and Marr 2006).

Traditional or conventional technique

Tryptone soya broth, bile broth or glucose broth, brain heart infusion broth are usually used

for conventional methods of blood culture.

The media is incubated aerobically at 37°c. Subculture should be done on Mac conkey�s agar,

blood agar media daily for 1 week and checked for turbidity, gas formation and other

evidence of growth after 1, 2, 3 and 7 days. For days 1, 2 and 3 only bottles showing signs of

positive growth are cultured on agar plates. On day 7 all bottles should be sub-cultured before

being discarded as negative (Watson 1978).

Automated technique

Modern blood culture techniques (automated) permit the bacteriological confirmation of

typhoid fever in a higher proportion of cases. These systems employ equipment that

automatically detects an early sign of bacterial growth in a special blood culture bottle

(Collee and Marr 2006). An isolation rate of 92% of blood culture with the Bactec 460

Radiometric system using a blood: broth ratio of 1:6 was found in a study (Duthie and French

1990).

Lysis centrifugation

Lysis centrifugation system consists of a tube containing anticoagulant (SPS), EDTA, and

saponin. After the tube is filled with blood during phlebotomy, the contents are mixed and

centrifuged and the resulting pellet is inoculated onto agar media taking all aseptic measures.

The system effectively recovers S. typhi from blood specimen (Richard and Thompson 2007).

The benefit of this system include; i) the more rapid and greater recovery of the organism; ii)

the presence of actual colonies for direct identification and susceptibility testing after initial

incubation; iii) the ability to quantify the colony forming units present in the blood; iv) rapid

detection of polymicrobial bacteraemia; and v) possible recovery of intracellular

microorganisms caused by lysis of host cells. Limitation of the method is high rate of

possible contamination (Betly et al, 2010).

Clot culture

The blood clot culture was found to be much more sensitive for S. typhi than whole blood

culture. Bacterial growth was significantly faster in cultures of blood clot compared to whole

blood. The rapid confirmation of the aetiological agent would facilitate an early institution of

appropriate antimicrobial therapy, thereby reducing clinical morbidity, especially in an

endemic population (Mantur et al. 2007).

Blood clot from which serum has been removed often gives a positive result when a similar

volume of whole blood yields no growth (Parker 1990). A method of clot culture with

streptokinase has been recommended (Watson 1956). An amount of 8 ml quantities of venous

blood is taken from patient and allowed to clot in sterile screw capped universal containers.

The separated serum is removed. The medium used consists of a Wilson and Blair agar slope

in a 120 ml bottle to which is added 15 ml of streptokinase bile salt broth. The streptokinase

causes rapid clot lysis with release of bacteria trapped in the clot. The cultures are then

incubated and positive results may be obtained in less than 24 hours (Watson 1956). Clot

culture is more sensitive than blood cultures with isolation rate of 92% and the clot technique

has many advantages over conventional whole blood culture, both in reliability and in cost

(Watson 1978).

Culture of the mononuclear cell �Platelet Fraction of Blood

The moderate or low sensitivity of blood culture is probably due to low concentration of S.

typhi (<10 bacteria per ml) in cells of the blood of patients with typhoid fever. Virtually all

intracellular S. typhi are found within only mononuclear cells (MNC) and platelets. By the

method of culture of mononuclear cells (MNC) and platelets fraction of blood from typhoid

patients is subjected to density gradient centrifugation to isolate the mononuclear cells.

Colonies of S. typhi were present in all mononuclear cell�platelet layer�positive cultures

within 18 hours of plating and were identified within an additional 10 minutes by a co-

agglutination technique. In contrast, identification of all positive culture by conventional

blood culture required 3 days (Rubin et al. 1990).

Bone marrow culture

Salmonella typhi is an intracellular pathogen in the reticuloendothelial cells of the body

including the bone marrow. The overall sensitivity of bone marrow cultures ranges from 80-

95% and is good even in the late phase of the disease and despite prior antibiotic therapy

(Parry et al. 2002).

Bone marrow aspirates are know to yield a higher rate of positive cultures than peripheral

blood in typhoid fever cases (Gilman et al. 1975; Farooqui et al. 1991) Bone marrow culture

may give a positive result when blood culture fails, particularly in patients admitted to

hospital while on antibiotic treatment. As a result unlike blood culture bone marrow culture is

highly (90%) sensitive (Lesser and Miller 2005). Another study reported that the

concentration of S. typhi in the bone marrow was found considerably higher than in

peripheral blood (Wain et al. 2001). In the bone marrow there were over 10 times more

bacteria than in peripheral blood. it seems likely to the positively rate of a 1 ml bone marrow

culture is equivalent to the result of 10 ml of peripheral blood (Wain et al. 2001). The

invasive nature of bone marrow aspiration discourage from its use as a first line investigation

for diagnosis of typhoid fever (Kundu 2006).

Stool Culture

In typhoid fever, stool cultures are usually positive from the second week of the infection.

Stool is usually plated on desoxycholate- citrate agar and also inoculated into fluid

enrichment media such as tetrathionate or selenite broth. The limitation of liquid of medium

is that the growth of fluid enrichment medium is subcultured appropriate medium for proper

identification. Suspicious colonies from culture plates are tested directly for the presence of

salmonella O antigens by slide agglutination and subcultured to peptone water for

determination of H antigen structure and for further biochemical analysis (Lewis 1997).

Urine Culture

Urine cultures are not recommended for diagnosis in view of poor sensitivity (Parry et al.

2002) (Gilman et al. 1975). Bacteria are not excreted continuously and therefore, several

specimens may need to be cultured before organisms can be isolated (Chessbrough 2010).

In typhoid fever, urine cultures are usually positive from the third week of the infection. The

centrifuged urine deposit is plated on desoxycholate- citrate agar and is also inoculated into

fluid enrichment media such as tetrathionate or selenite broth. The growth of fluid enrichment

medium is subcultured appropriate medium for proper identification (Lewis 1997).

Duodenal string � Capsule Culture

Duodenal string test was found to be a simple, non-invasive and a reliable test which when

used in combination with blood culture could identify almost all cases of typhoid fever

irrespective of duration of fever and prior use of antibiotics (Antony et al. 1993).

Duodenal content cultures have been proved to be more sensitive (86%) in diagnosis than

bone marrow (75%) and more effective than blood (42%) and stool (26%) cultures in

recovery of S. typhi. The sensitivity of duodenal content cultures was found not modified by

the duration of illness at admission or by previous antibacterial therapy (Benavente et al.

1984).

Culture of duodenal aspirate is important in the detection of typhoid carriage. Individuals can

excrete S. typhi in the bile and yet be undetected by stool culture (Madanagopalan et al.

1975). Because of patient�s discomfort and the time required for tube placement, duodenal

aspiration has not been widely used (Gilman and Rechard 1976).

Antibody detection tests (serology)

Widal test

The information regarding Widal test has been noted in Britannica encyclopaedia. The Widal

agglutination test was introduced as a serologic technique to aid in diagnosis of typhoid fever.

The test was named after Georges Fernand Isidore Widal, a French physician and

bacteriologist. In 1896, Widal developed a procedure for diagnosing typhoid fever based on

the fact that antibodies in the blood of an infected individual cause the bacteria to bind

together into clumps (the Widal reaction) (Encyclopaedia Britannica 2011).

The test was based on demonstrating the presence of agglutinin (antibody) in the serum of an

infected individual, against the H (flagellar) and O (somatic) antigens of Salmonella typhi

(Jenkins and Gillespie 2009).

The �O� antigen is the somatic antigen of S. typhi and is shared by S. paratyphi A, S.

paratyphi B, other Salmonella species and other members of the Enterobacteriaceae family

(Rodrigues 2003). Antibodies against the O antigen are predominantly IgM, rise early (appear

on day 6-8) in the illness and disappear early (Rodrigues 2003). The H antigens are flagellar

antigens of S. typhi, paratyphi A and paratyphi B. Antibodies to H antigens are both IgM and

IgG, rise late (on days 10-12) in the illness and persist for a longer time (Olopoenia and King

2000; Rodrigues 2003).

Serological diagnosis relies classically on the demonstration of a rising titre of antibodies in

paired samples at an interval of 10�14 days (Parry et al. 1999). In typhoid fever, however, a

four- fold rise after 2 weeks in not always demonstrable, even in blood culture confirmed

cases. This situation may occur when the acute phase sample is obtained late in the natural

history of the disease, because of high levels of probable background antibodies in an

endemic region, or because in some individuals the antibody response is blunted by the early

administration of an antibiotic (Schroeder 1968).

There is a controversy about the predictive value of O and H antibodies for diagnosis of

enteric fever. Some authorities claim that O antibodies have superior specificity and positive

predictive value (PPV) because these antibodies decline early after an acute infection

(Schroeder 1968).

It can be negative in up to 30% of culture- proven cases of typhoid fever. The purity and

standardization of antigens used for the Widal test is a major problem and often results in

poor specificity and poor reproducibility of test results (Olopoenia and King 2000).

Followings are the causes of a positive Widal agglutination test: i) the patient being tested has

typhoid fever; ii) previous immunization with Salmonella antigen; iii) cross-reaction with

non-typhoidal Salmonella; iv) infection with other enterobacteriaceae ; v) other diseases such

as dengue, schistosomal infection, chronic liver disease associated with raised globulin

levels; and vi) disorders such as rheumatic fever, rheumatoid arthritis, multiple myeloma,

nephritic syndrome and ulcerative colitis (Cheesbrough 2010). False negativity is one of the

obstructive features of the Widal test. Hosoglu et al conducted a study to evaluate the

associated factors with Widal test negativity in an endemic area. Widal test negativity was

retrospectively analyzed by them among culture-proven typhoid fever cases. The potential

features including age, gender, previous antibiotic usage, duration of symptoms, leucopoenia,

haematocrit value, and erythrocyte sedimentation rate (ESR) were evaluated for association

with Widal test negativity (Hosoglu et al. 2008).

It has been shown that the antibody response to the O antigen of S. typhi was markedly

reduced in acute episodes of malaria compared to controls and that humoral immunity is

transiently impaired (Greenwood et al. 1972). In a recent study, subjects with dual infection

of malaria and typhoid fever had significantly higher rates of nausea, vomiting, abdominal

pain, and diarrhoea�the common features of enteric fever (Khan et al. 2005). In the last two

decades, this relationship between the two diseases has been reported in studies from Africa

and India (Ammah et al. 1999; Ohanu et al. 2003; Kanjilal et al. 2006).

A study conducted in Cameron found that the number of fever cases diagnosed as malaria-

typhoid fever co-infection were actually overestimated (Ammah et al. 1999).

Immunochromatographic method

ICT has been studied in many countries and they found significantly higher sensitivity and

specificity (Jesudason, Esther and Mathai 2002; Pastoor et al. 2008; Anusha, Ganesh and

Lalitha 2011). An evaluation of ICT (Typhidot) in India was found to be 100% sensitive and

80% specific compared to a blood culture as �gold standard� (Jesudason, Esther and Mathai

2002).

Haemagglutination (HA) Tests

Many researchers have evaluated the usefulness of HA tests in different countries. In a study

from India, the anti-LPS HA test showed a sensitivity of 60% and specificity of 98.2%. The

positive predictive value and negative predictive value were 66.7% and 96.7% respectively.

In the same study, the haemagglutination inhibition test targeted Salmonella antigens and was

found useful for helping the early detection of S. typhi in culture (Shukla, Patel and Chitinnis

1997). In another study, a Reverse Passive Haemagglutination Test (RPHA) was designed for

the detection of S. typhi antigen. The test was found to be 70% sensitive and 92% specific for

acute typhoid fever diagnosis (Kalhan et al. 1998).

Countercurrent Immunoelectophoresis (CIE)

This test is based on electrophoresis and the visualization of the precipitin band of antigen-

antibody complexes that form. The sensitivity is similar to that of the Widal test and the

procedure may be quicker if tests are batched (about one hour for a gel), but bands are often

difficult to see, the cost is higher than that of the Widal, and some studies conclude that CIE

has a low sensitivity with Vi antigen. A panel of antigens (somatic (O), flagellar (H) and

capsular polysaccharide (Vi) antigens of S. typhi is recommended for rapid diagnosis of

typhoid fever (Sharma et al. 1979).

Other serological test

In view of the limitations of the Widal test and need for a cheap and rapid diagnostic method,

several attempts to develop alternative serologic tests have been made. These include rapid

dipstick assays, dot enzyme immuno-assays and agglutination inhibition tests.

Antibody detection:

Dot Enzyme Immunoassay (EIA) test

A dot enzyme immunoassay that detects IgG and IgM antibodies against a 50 KD outer

membrane protein, distinct from the somatic (O), flagellar (H) or capsular (Vi) antigen of

Salmonella typhi is commercially available as Typhidot (Gasem et al. 2002). commercially it

is available in two different properties, i) The Typhidot M that detects only IgM antibodies of

S. typhi has been reported to be slightly more specific in a couple of studies (Choo et al.

1999; Hatta et al. 2002).

ii) Typhidot® test detects specific IgM and IgG antibodies to S. typhi. It has undergone full-

scale multinational clinical evaluation of its diagnostic value (Ismail, Kader and Ong 1991).

In areas of high endemicity, where the rate of S. typhi transmission is high, the detection of

specific IgG increases. The IgG can persist for more than two years after typhoid fever

Infection (Choo et al. 1997; Bhutta and Mansurali 1999). The detection of specific IgG can

not differentiate between acute and convalescent cases (Choo et al. 1999). Furthermore, false

positive results attributable to previous infection may occur. On the other hand, IgG positivity

may also occur in the event of current reinfection. In cases of reinfection there is a secondary

immune response with a significant boosting of IgG over IgM, such that the later can not be

detected and its effect is masked. A possible strategy for solving this problem is to enable the

detection of IgM by ensuring that it is unmasked (Bhutta 1996).

IgM dipstick test

A rapid dipstick assay for the detection of S. typhi-specific IgM antibodies in serum and

whole blood samples was previously reported and the sensitivity and specificity was

evaluated (Gasem et al. 2002; Hatta et al. 2002; House et al. 2005).

The dipstick assay may thus also be useful for the serodiagnosis of culture-negative patients

with clinical signs and symptoms consistent with typhoid fever. The advantages of the

dipstick assay are that the result can be obtained on the same day allowing a prompt

treatment, that only a small volume of serum is needed, and that no special laboratory

equipment is needed to perform the assay. The stability of the reagents of the dipstick and the

simplicity of the assay allows its use in places that lack laboratory facilities (Hatta et al.

2002).

Antigen detection tests

There is clearly a demand for a simple diagnostic test for typhoid fever. An ideal test is

reliable, simple, and affordable for the countries where the need is the greatest. Many of the

affected countries are poor, and some places do not have electricity. The antigen detection,

rather than antibody detection, could provide such a test (Wain and Hosoglu 2008).

Protein and Vi antigens

Enzyme immuno-assay, counterimmune electrophoresis and co-agglutination tests to detect

serum or urinary somatic/flagellar/Vi antigens of S. typhi have been evaluated (Fadeel et al.

2004; Kalhan et al. 1999). Sensitivity of Vi antigen had been found to be superior than

somatic and flagellar antigen, had been reported as ranging from 50-100% in different studies

(Fadeel et al. 2004; Kalhan et al. 1999; Rao et al. 1999). Similarly, specificity estimates have

been reported to vary from 25% -90%. The suboptimal and variable sensitivity and specificity

estimates, inability to detect S. paratyphi infection and Vi antigen negative strains of S. typhi

are serious limitations of the Vi antigen detection tests. S. typhi antigen can be detected in the

urine of some typhoid patients by co-agglutination test (West, Richens and Howard 1989). A

monoclonal antibody specific for group D Salmonellae antigen 9 was used in an indirect

enzyme-linked immunosorbent assay (ELISA) for detecting the antigen in urine specimens

collected from patients with clinical typhoid fever in Jakarta, Indonesia. The ELISA had a

sensitivity of 95% in identifying patients in whom S. typhi was isolated from blood cultures,

73% in patients in whom S. typhi was isolated from stool specimens, and 40% in patients in

whom the organism was isolated from bone marrow cultures, but specificity varies from 25-

90% (Chaicumpa et al. 1992).

Molecular methods

The molecular method for diagnosis of typhoid fever has been evolved to overcome the

limitations of cultures and serologic tests. Many authors have explored the use of polymerase

chain reaction (PCR) for detecting specific DNA sequence of the organisms present in

clinical specimens. The PCR as a diagnostic modality for typhoid fever was first evaluated in

1993 when Song et al successfully amplified the flagellin gene of S. typhi in all cases of

culture proven typhoid fever and from none of the healthy controls. By using two pairs of

primers evaluated in the study by Song et al, amplification of the flagellin gene of S. typhi

confirmed the presence of the organism in the patient�s blood (Song et al. 1993).

The PCR has been used to enable diagnosis of typhoid fever within few hours and more

specific and sensitive than blood cultures.In addition nested PCR has been shown promising

results. These results show that the nested PCR has good potential to be a rapid tool for the

definitive, differential diagnosis of typhoid and is superior to conventional methods (Ali et al.

2009).

The nested PCR resulted in amplified fragments that were visible after agarose gel

electrophoresis. The whole procedure to identify S. typhi DNA in the blood by agarose gel

electrophoresis took only 16 hours, demonstrating the PCR to be a specific and rapid method

for the early diagnosis of typhoid fever (Song et al. 1993). Frankel et al. amplified flagellin

gene sequences specifically from S. typhi (Frankel et al. 1989).

Massi in the year 2005 conducted a study to establish the nested PCR for DNA detection of S.

typhi in the urine of patients with suspected typhoid fever. This research was used 107 urine

samples from patients suspected with typhoid fever which were examined with nested PCR

using two primer pairs with the final amplification result of 343 basepair (bp). This study

reported that 64 (59%) urine samples were positive with S. typhi DNA. This research

concluded that nested PCR specifically from urine specimen can be used as an alternative

method in diagnosis and management of typhoid fever (Massi et al. 2005).

Kumar et al used blood samples from 40 clinically suspected cases of typhoid fever, and

found 20 of 20 culture positive and 12 of 20 culture negative cases to be positive by PCR in

Delhi, India (Kumar et al. 2002). Using single primer in South Sulawesi, Indonesia, 46 of 73

(63.0%) blood samples collected from patients with clinically suspected typhoid fever were

positive by PCR compared to 13.7% positive by blood culture (Massi et al. 2003). In

Varnassi, India, nested PCR was again better 53 of 57 (73.0%) were positive than blood

culture 17 of 53 (32%) were positive on specimens from 63 clinical typhoid fever cases

(Prakash et al. 2005).

A large, well-designed study in Indonesia investigated 131 patients with a clinical diagnosis

of typhoid fever and diagnosed the cases by blood culture and PCR from blood (84.5%) and

urine samples (69.3%). The PCR diagnosis was found more sensitive than the blood culture

(61.8%) (Hatta and Smith 2007).

A study from Nepal on specimens from 71 children with suspected typhoid fever reports

82.7% positivity for PCR from blood and urine, showing similar results for each specimen

and PCR results were much higher than blood culture (26.9%). In Pakistan, 55 cases of

suspected typhoid fever and a control group of 20 healthy persons were diagnosed by PCR

from blood samples and blood culture. The PCR and blood culture gave 58.2% and 14.5%

positivity, respectively showing significantly better results by PCR (Haque et al. 2001).

Again in Pakistan, a multiplex PCR targeting five different genes for differential diagnosis of

typhoidal pathogens has been developed for use directly on clinical blood samples. Of 42

multiplex PCR-positive blood samples, 35 were positive for S. typhi and two for S. Paratyphi

A and interestingly remaining 5 patients were found to have mixed infection (Ali et al. 2009).

Moreover, some patients with culture negative typhoid fever were PCR positive suggesting

that PCR diagnosis of typhoid fever may have superior sensitivity than cultures. Over the

next 10 years, a handful of studies have reported PCR methods targeting the flagellin gene,

somatic gene, Vi antigen gene, 5S-23S spacer region of the ribosomal RNA gene, invA gene

and hilA gene of S. typhi for diagnosis of typhoid fever (Prakash et al. 2005; Jimenez and

Castro 2004; Haque et al. 2001; Cocolin et al. 1998). These studies have reported excellent

sensitivity and specificity when compared to positive (blood culture proven) and healthy

controls. The time required for diagnosis has been less than 24 hours (Kundo et al. 2006).

Chaudhry et al. developed standardization of PCR for the detection of S. typhi in typhoid

fever. The PCR technique was used for detection of S typhi in blood or clot cultures from 84

patients clinically suspected of having typhoid fever, and from 20 healthy controls. Twenty

five of 84 samples from clinically suspected cases were positive by PCR; four of which were

culture negative. No amplification was seen in samples from patients who were culture

positive for organisms other than S. typhi or from controls. The time taken for each sample

for PCR analysis was less than 48 hours, compared with three to five days for blood or clot

culture. The PCR was found to appear to be a promising diagnostic test for typhoid fever

(Chaudhry et al. 1997).

Multiplex PCR has been successfully applied for differential diagnosis of many diseases

caused by viruses, bacteria, fungi, and parasites (Rithidech, Dunn, and Gordon 1997; Hirose

et al. 2002). The multiplex PCR technique was evaluated by Ali et al directly on blood

samples of 60 clinically suspected cases of typhoid fever. The PCR detected typhoidal

species in 42 cases, only 17 of which were culture positive. The presence of Salmonella in

these samples was confirmed with genus-specific primers (Kidgell et al. 2002). Quick

multiplex PCR-based detection method was developed for early diagnosis of typhoid fever,

using specific genetic markers of S. typhi. Primers of tyv gene, flag gene, viaB gene and ratA

gene confirmed the specificity and sensitivity of the PCR. The serum samples of the

suspected typhoid patients were taken directly for PCR without culturing the organism and

extracting genomic DNA. Overall diagnosis required 2 hours which is the least time ever

reported for a PCR based method. The sensitivity of the method was up to 5 famtogram (fg)

genomic DNA. The genetic markers were specific and the four pairs of primers give selective

amplification and differentiate S. typhi from closely related S. typhimurium (Kumar et al.

2010).

Antimicrobial resistance of S. typhi

Most drug resistance is the result of a genetic change in the organism caused either by a

chromosomal mutation or the acquisition of a plasmid or transposon (Winstaniey and Hart

2001; Levinson 2010). Plasmid-mediated resistance is more common in multidrug resistant

(MDR) typhoid fever and occurs with a high frequent rate (Levinson 2010). In 1948,

chloramphenicol became the standard antibiotic for treating typhoid fever (Woodward et al.

1948; Kidgell et al. 2002). Emergence of S. typhi strains resistant to chloramphenicol was

reported in 1970 cited in Rahman et al (Rahman et al. 2006) then subsequently resistance to

ampicillin and co-trimoxazole emerged soon (Morshed et al. 1986; Parry and Threlfall 2008).

Toward the end of the 1980s and the 1990s, S. typhi developed resistance simultaneously to

all the drugs that were then used as first-line treatment (chloramphenicol, trimethoprim,

sulfamethoxazole, and ampicillin (Mirza, Beeching and Hart 1996). The MDR S. typhi being

resistant to three of the first-line antimicrobials emerged sporadically (Goldstein et al. 1986).

First documented outbreak of multidrug resistance occurred in Malaysia in 1984 (Sharma et

al. 1979). Since then the MDR S. typhi spread throughout south-east Asia and China, where

they became endemic (Ling and Chang 1984). Outbreaks of infections with these strains

occurred in India (Threlfal et al. 1992; Shanahan et al. 1998), Pakistan (Bhutta 1996;

Shanahan et al. 2000), Bangladesh (Saha et al. 1996), Vietnam (Hoa et al. 1998), the Middle

East (Lancet 1990), and Africa (Kariuki et al. 2000) . In most cases, resistance to

chloramphenicol, ampicillin and cotrimoxazole was transferable on plasmid, either

individually or all together (Goldstein et al. 1986; Mirza, Beeching and Hart 1996; Mirza et

al. 2000; Connerton et al. 2000; Wain et al. 2001). Plasmids are extrachromosomal

supercoiled loops of DNA that were probably originally derived from bacterophages (Sinha

et al. 1999; Levinson 2010). When greater than 40 kbp in size the plasmids are able to

transfer the carrying genes from host bacterium to others by conjugation.

The plasmids found in S. typhi are of two major types. First as pH CM2 cryptic plasmid

which can carry gene encoding mechanisms of DNA metabolism and replication and

virulence are found in Asia but not in Africa. Second, type approximately 140-180 kbp self

transferable plasmid. Large resistant plasmids are built up by the addition of resistant gene

encoded on integrons and transposons (Levinson 2010). These large plasmids can then be

transferred to and from enteric gram negative bacterium such as Echerichia coli, Klebsiella

pneumoniae and S. enterica, especially when antimicrobials are being administered

(Levinson 2010).

Chomosomally acquired quinolone resistance in S. typhi has been found in different parts of

Asia (Park 1999; Rahman et al. 2006; Levinson 2010) which may be the consequence of wide

spread indiscriminate use of these drugs. Resistance rarely emerges during the course of

treatment (Schwalbe et al. 1990). There have been sporadic reports of high-level resistance to

ceftriaxone (minimal inhibitory concentration [MIC], 64 mg per liter) in S. typhi and S.

paratyphi A (Saha et al. 1999; Bhutta, Farooqui and Sturm 1992), although these strains are

very rare. S. typhi strains with reduced susceptibility to fluoroquinolones have become a

major problem in Asia (Wain et al. 1997; Brown et al. 1997; Threlfall and Ward 2001a). An

outbreak of typhoid with such strains in Tajikistan in 1997, caused illness in 8000 people in a

six-month period and 150 deaths. Although they were reported to be susceptible to

fluoroquinolones, by disk testing with the use of recommended break points, these organisms

were resistant to nalidixic acid and the MIC of fluoroquinolones for these strains was 10

times that for fully susceptible strains. This reduction in susceptibility results in a poor

clinical response to treatment (Wain et al. 1997; Threlfall et al. 1999).

Quinolone resistance is frequently mediated by single point mutations in the quinolone-

resistance� determining region of the gyrA gene, characteristically occurring at position 83 of

the DNA gyrase enzyme (changing serine to phenylalanine) and position 87 (changing

aspartate to tyrosine or glycine) (Wain et al. 1997; Brown et al. 1999). In other

Enterobacteriaciae, higher levels of quinolone resistance have been associated with additional

mutations in the gyrA gene, mutations in other Topoisomerase genes, or alterations in

fluoroquinolone uptake. No such mutations have been reported yet in S. typhi, although there

are sporadic reports of fully fluoroquinolone-resistant isolates (Mehta, Randhawa and

Mohapatra 2001). Because the clinical response to fluoroquinolones in patients infected with

nalidixic acid�resistant strains is greatly inferior to the response in those infected with

nalidixic acid�susceptible strains (Threlfall, Skinner and Ward 2001b).

Treatment of multidrug resistant (MDR) S. typhi infection

The MDR S. typhi infections are those caused by the organism resistant to chloramphenicol,

ampicillin, and cotrimoxazole (trimethoprim�sulphamethoxazole). Emergence of drug

resistance is a major challenge in the treatment of typhoid fever (Bhutta 2008).

The MDR cases began to appear around 1990 (Rahman et al. 2007). In 1992, 40% of the

isolates were MDR (Hoque et al. 1992). Studies from Institute of child and maternal health

(ICMH) in Dhaka (1994) and BSMMU (1992) revealed similar pattern, while isolates were

equally sensitive to ciprofloxacin and ceftriaxone (Saha 1994).

WHO recommends in the year 2003 ciprofloxacin and ofloxacin for MDR cases and

azithromycin, third generation cephalosporin and high dose older generation fluroquinolones

in nalidixic acid resistant cases (WHO 2003). Resistance to azithromycin and ceftriaxone is

rarely reported and this is if they can be used as empirical therapy in enteric fever (Islam et

al. 2007; William et al. 2001). Azithromycin is a macrolide azolide antibiotic with relatively

poor in vitro activity against S. typhi. However, the antibiotic is concentrated 50-100 fold

inside cells such as macrophages, where the S. typhi reside usually. It also has a long half life

(68 hours) and is thus given as a once-daily regimen. In a study in Bangladesh, 94% cure rate

with azithromycin was observed. An Indian study showed 88% response with once daily

azithromycin for 7 days, where 100% of the cases were disease free after 14 days therapy

(Butler, Sridhar and Daga 1999).

The MDR S.typhi isolates with reduced susceptibility to fluoroquinolones indicated by

resistance to nalidixic acid have caused epidemics and become endemic in southern Viet

Nam during the 1990s. Short courses of ofloxacin have proved acceptable for treating MDR

or nalidixic acid sensitive isolates of S. Typhi (Parry et al. 2002).

Prevention of typhoid fever

Typhoid fever is a food and waterborne disease and the main preventive measure is to ensure

access to safe water and water. Adequate water treatment, waste disposal, and protection of

food supply from contamination are important public health measures. Typhoid can be

transmitted by chronic carriers who do not apply satisfactory food � related hygiene practices.

These carriers should be excluded from any activities involving food preparation and serving

(Bhutta 2008; Zwadyk 1992).

Vaccination

Vaccination in high-risk areas is a potential control strategy recommended by WHO for the

short-to-intermediate term. Two safe and efficacious typhoid vaccines, the injectable Vi

polysaccharide and the oral Ty21a, have been licensed; and new, improved candidate

vaccines are currently being tested. This vaccine is licensed in 56 countries in Africa, Asia,

Europe; South America, and the USA However; typhoid vaccination has not been

implemented as a routine public health measure in most typhoid-endemic countries (WHO

2003).

Inactivated whole-cell typhoid vaccine

Vaccines of this type were introduced in 1896 (WHO 2005). Their efficacy was established

only in 1960 in controlled trials in the Cochrane countries of Yugoslavia, the Soviet Union,

Poland, and Guyana. The 1998 version of the vaccine demonstrated that two doses of this

type of vaccine resulted in 73% efficacy over three years .Different methods of inactivating

cells of S. typhi have been used to prepare these vaccines: acetone-inactivated, alcohol-

inactivated, or heat inactivated and phenol preserved. In field trials, the vaccine has been

associated with fever and systemic reactions in 9% to 34% of the recipients, and with short

absences from work or school in 2% to 17% of the cases (WHO 2000). Therefore, the

inactivated whole cell typhoid vaccine is considered unsuitable for use as a public health

vaccine and although licensed, it is no longer available for use. The old parenteral killed

whole cell vaccine was effective but produced strong side effects because of

lipopolysacharide (LPS) (Garmory, Brown and Titball 2002).

Vi polysaccharide vaccine

The Vi polysaccharide vaccine is given as a single parenteral dose. Protection was found to

begin seven days after injection and maximum protection reached 28 days after injection

when the highest antibody concentration was obtained (Garmory, Brown and Titball 2002).

The vaccine is approved for persons aged over two years. Revaccination is recommended

every three years. Mild local adverse reactions was found to occur including 17% of adult

vaccinees and 86.7% of child vaccines experience local pain at the injection site, although

these reactions are usually transient and mild (Garmory, Brown and Titball 2002).

In field trials conducted in Nepal and South Africa, where the disease was endemic and attack

rates reached 900/100000, the protective efficacy was 72%, one and half years after

vaccination (Acharya et al. 1987) and was found 55% three years after a single dose

(Klugman et al. 1996). In another field trial in South Africa, 10 years after immunization,

58% of the vaccines still had protective level of antibodies of over 1 ìg/ml. (Keddy et al.

1999). In efficacy trials conducted in Chiang Su and Guangxi, China, in 1995 and 1997

respectively, with a locally produced Vi vaccine, 72% protection was obtained by vaccination

(Yang et al. 2001). A protective efficacy of 70% was reported in a population vaccinated

before or during an outbreak situation in the same country (Yang and Kilgore 2001). It is

mainly used by travelers, visiting areas at high risk of typhoid fever because of the presence

of multidrug-resistant strains (Jegathesan 1983).

Ty2la vaccine

The live oral vaccine Ty21a is available as enteric coated capsule or liquid formulation. It

elicits protection as from 10-14 days after the third dose. It is approved for use in children

aged at least 5 years or older and adults. A booster dose is needed every 5 years for people

who remain at risk. Travelers should be revaccinated annually, and those living in disease

endemic areas, every three years (Khan 2005; Black et al. 1990).

In randomized, controlled field trials in Area Norte and Area Occidente of Santiago, Chile, 2

(Norte) or 3 (Occidente) doses of live oral typhoid vaccine Ty21a in enteric-coated capsules

conferred protection against confirmed S. typhi disease (53% efficacy in Norte; 67% efficacy

in Occidente) during 3 years of follow-up. There was also a trend in each trial showing

protection against S. paratyphi B disease (56% efficacy in Norte; 42% efficacy in Occidente)

(Levine et al. 2007). The use of vaccines in the control of typhoid fever has had mixed

results. The most promising was an oral vaccine of an attenuated typhoid bacillus, TY21a. In

a field trial in Alexandria, Egypt, involving 32,000 children, the infection rate was 0/100,000

in the vaccinated group as compared to 126/100,000 in the placebo group and 133/100,000 of

non-vaccinated group (Zwadyk 1992).

Future vaccines (Vi-rEPA vaccine)

A new modified Vi vaccine conjugated to a nontoxic recombinant Pseudomonas aeruginosa

exotoxin A (rEPA) has also been evaluated in a randomized controlled trial among children

aged two to five years. This vaccine has the potential of being immunogenic in infants under

the age of two (Parry et al. 2002). Research continues to develop more effective typhoid

vaccines. It is concentrated on two main efforts. The first is development of new parenteral

Vi polysaccharide protein conjugate vaccines, which are expected to produce higher antibody

titres following initial and booster immunizations. The second effort is to genetically

attenuate strains of S. typhi to achieve high immunogenicity, while at the same time rendering

the strain nonpathogenic (Garmory, Brown and Titball 2002). Vi-rEPA vaccine has enhanced

immunogenecity in adults and in children aged 5-14 years and has induced a booster response

in children aged 2-4 years (Kossaczka et al. 1999). In a double-blind randomized control trial,

11091 Vietnamese children aged 2-5 years, were given two injections of Vi- rEPA separated

by six weeks (Lin et al. 2001). No serious side effects observed. The efficacy after 27 months

of active surveillance was 91.2%. Passive surveillance in the 16 months since the study ended

showed 88% efficacy.

Materials and methods

Type of study: The study was designed as Descriptive type of comparative study.

Place of study:

The study was carried out in the Department of Microbiology Mymensingh Medical College,

Mymensingh.

Period of study:

The study was carried out from 1st July 2010 to 30th June 2011.

Sampling technique:

Non probability purposive type of sampling.

Data collection, recording and analysis:

A set of questionnaire was used for each of the cases and controls. All the relevant

information (history, clinical and laboratory findings) of every cases were systematically

recorded in a pre-designed data sheet (Appendix I). The data were analyzed in computer

using appropriate program.

Study population:

One hundred and fifty (150) cases and fifty (50) age sex matched controls were studied.

Sample size determination:

In this study, the sample size was calculated by the following formula:

2

2

d

pqzn

In this study, sample size was taken 150, due to limitation of budget, resource and time.

Subjects/Cases:

All febrile patients suspected of typhoid fever attending at out patient departments (OPD) of

Medicine and Paediatrics unit of MMCH were included in this study. The suspicion was

made by characteristics presentation of typhoid fever by the medical officer working at the

relevant OPD�s.

Inclusion criteria for cases:

1. Fever, characteristics for typhoid fever for 3 days or more, irrespective of antibiotic

treatment.

2. Any other additional presentation suggestive of typhoid fever like headache, anorexia,

nausea, vomiting, abdominal discomfort with diarrhea, soft enlarged spleen, coated

tongue, toxic look and relative bradycardia.

3. Individuals of both sexes representing all ages.

Exclusion criteria of the cases:

1. Fever with any obvious focus for other infection such as urinary tract infection, otitis

media etc.

2. History of immunization with typhoid vaccines.

The control group:

A total of 50 age and sex- matched controls of which 25 were healthy control individuals

with no recent history of fever and 25 were sick suffering from non-typhoidal febrile illness.

Consent taking:

Informed written consent was taken from each patient or from guardian of the minor patients

before his/her entry into the study (Appendix II).

Socioeconomic condition:

Socio-economic conditions of the study population were ascertained on the basis of their

monthly income of the patients or their guardians from all possible sources. The cases and

controls were classified into three groups:

i. Low income group (Up to 15,000 TK. /month.)

ii. Middle income group (15,000-50,000 TK. /month.)

iii. Upper income group (>50,000 TK. /month.)

Laboratory procedures

Specimen collection and storage:

Whole blood 10 ml for adults and 6 ml for children.

Venous blood was collected aseptically following universal safety precaution. For this

purpose:

i) Wearing a sterile disposable glove, the puncture area was washed with iodine and

70% alcohol.

ii) With a sterile disposable syringe, 10 ml of blood in case of adult and 6 ml in case of

children were collected.

iii) Of the collected sample, 8 ml for adults and 4 ml for children were transferred in

heparinized tube for blood culture.

iv) From the remaining blood sample, 1 ml of was transferred in a tube with EDTA for

PCR and immediately would be refrigerated at -200C for molecular study.

v) Remaining blood was transferred in another clean tube for ICT.

Blood culture by lytic centrifugation method:

The aseptically collected blood sample in an amount of 8 ml in case of adult and 4 ml in case

of children were collected in heparinized tube containing lytic solution which was made in

our laboratory (Appendix III) for initial processing. Blood sample were mixed well by

inverting the tube 3-5 times to mix blood with lytic solution and mixture were shaked,

vortexed and centrifuged at 3000 rpm for 30 minutes. After centrifugation, the supernatant

were discarded and 1ml of the deposit containing the pathogen is vigorously vortexed and

entire sediment were directly inoculated onto blood agar and Mac Conkey agar media

(Appendix IV). The inoculated culture plates were immediately placed in an incubator at

370C for 24 - 48 hours. After proper incubation colonies if any as Salmonella typhi were

identified by colony morphology, Gram staining, motility test and relevant method of

biochemical tests (Richard and Thompson 2007; Cheesbrough 2010).

Colony morphology:

MacConkey agar: Produce non lactose fermenting colonies of 1-3 mm in diameter, colonies

are pale yellow or nearly colour less.

Blood agar: The colonies of S. typhi were moderately large 2-3 mm in diameter after 24 hours

at 370C. They were non haemolytic, grey-white, moist circular discs with a smooth convex

surface and entire edge.

Salmonella � Shigella agar (SS agar): In SS agar medium, Colonies of S. typhi were colorless

with or without black centers.

Gram�s staining

Gram�s staining was done for morphological identification of S. typhi. A drop of distilled

water was taken on the middle of a clear glass slide. Then a colony of S. typhi (young culture)

was transferred with a sterilized inoculating wire loop on the drop of water. Then a very thin

smear was prepared on the slide by spreading specimen uniformly. The smear was fixed by

passing it over the flame for two or three times. Then Gram�s staining was done (Appendix-

V) and S. typhi was found to be Gram negative short bacilli.

Motility test:

It was done by hanging drop preparation and by using Motility indole urease (MIU) medium.

In MIU medium, S. typhi were motile and Urease and indole negative. In hanging drop

preparation, S. typhi were motile.

Biochemical tests:

S. typhi were presumptively identified using triple sugar iron (TSI) medium, citrate utilization

test and oxidase test.

On Triple sugar iron agar medium (TSI)

The subculture of S. typhi in TSI medium was inoculated by using a sterile straight wire to

stab into the butt first and then use the same wire to streak slope in a zig-zag pattern. Then the

medium were incubated at 370C for 24 hours.

S. typhi in TSI medium was identified by following findings:

- Pink-red (alkaline) slope and yellow (acid) butt, indicating fermentation of glucose

but not lactose.

- No cracks in the medium (if serotype produces gas from glucose fermentation, it is (S.

Para typhi A)

- Blackening in the medium due to H2S production (only a small amount of blackening

is seen with S. typhi).

Citrate utilization test:

By using a sterile straight wire, first streak the slope with a saline suspension of S. typhi and

then stab the butt. Then the medium were incubated at 350C for 48 hours. S. typhi were citrate

negative, it was identified that there was no color change in the medium.

Oxidase test (Cheesbrough, 2010)

Objective: To distinguish S. typhi from Pseudomonas species.

A piece of filter was placed in a clean petridish and 2 to 3 drops of 1% freshly prepared

oxidase reagent (tetramethyl-p-phenylene diamine dihydrocloride) was added. With a sterile

glass rod 1 or 2 colonies of the test organism was taken and rubbed on the filter paper.

Development of blue- purple colours within 10 seconds indicates positive test. Pseudomonas

aeruginosa was used as positive control and Escherichia coli as negative control.

Antimicrobial susceptibility test by disk diffusion method

All the isolated S. typhi were put into antibiotic susceptibility test by Kirby-Bauer disk

diffusion technique as per recommendation of the Clinical and Laboratory Standards Institute

(CLSI 2010). Panel of antibiotics were used � namely chloramphenicol, cotrimoxazole,

amoxycillin, ciprofloxacin, cephradine, gentamicin, ceftriaxone, azithromycin and nalidixic

acid. All tests were performed on Muller-Hinton agar (Appendix VI) media. The surface was

lightly and uniformly inoculated by sterile cotton swab stick. Prior to inoculation, the swab

stick was dipped into bacterial suspension having visually equivalent turbidity to 0.5

McFarland standards (Appendix VII). The swab stick then took out and squeezed on the wall

of the test tube to discard extra suspension. Inoculated plates were incubated at 35 to 370C for

24 hours. On the next day, plates were read by taking measurement of zone of inhibition.

Inhibition zones were measured in millimeter (mm) by using a ruler over the surface of the

plate with the lid open. The plates were hold a few inches above a black, non-reflecting

background and illuminated with reflected light. Results were recorded and graded as

Resistant (R) and Sensitive (S) according to the reference zone of inhibition for particular

antibiotic (CLSI 2010).

Standardization of the disk

In order to standardize the disk potency, a representative disk was tested against the reference

strains of S. typhi ATCC 19430. The zone of inhibition was compared with standard value as

recommended by CLSI (2010).

Serological tests:

Detection of Salmonella typhi antibody

Immunochromatographic test (Typhoid IgG/IgM combo rapid test ® USA) is a rapid assay

for the detection of S. typhi antibody in whole blood/serum specimen.

Principle of the test: Detection of S. typhi antibody by ICT is a qualitative test. The ICT

utilizes a unique combination of monoclonal antibody/colloidal gold dye conjugate and a

polyclonal antibody immobilized on the solid phase. This was selectively identifying S. typhi

antibody associated S. typhi infection with a high degree of sensitivity and specificity.

As the specimen follows through the absorbent pad in the sample well and through the

antigen/colloidal gold complex any S. typhi antibody present in the sample binds to the

conjugate forming an antigen/antibody complex. The sample and dye complex continue to

migrate along the membrane to the immobilized polyclonal antibody. In the presence of S.

typhi, the polyclonal antibody captures the complex. This forms a visible pink/purple band in

the test area of the card. If no antibody is present, there is no line in the area. The remaining

complex continues to migrates to another immobilized antibody on the membrane in the

control area of the card and is captured which then forms a band indicating proper

performance of the test.

The test cassette consists of � A colored conjugate pad containing recombinant S. typhi H

antigen and O antigen conjugated with colloid gold (Typhoid conjugates) and rabbit IgG gold

conjugates.

A nitrocellulose membrane strip containing two test bands (T1 and T2 bands) and a control

band. The T1 band is pre-coated with monoclonal anti human IgM for the detection of IgM

anti- S. typhi, T2 band is pre coated with reagents for the detection of IgG anti S. typhi and

the C band is pre-coated with goat anti rabbit IgG.

Reagent

1. Test strip

2. Sample Diluent

3. Positive Control

Procedural note:

All specimens or reagents were brought to room temperature (18-250) prior to testing and Foil

pouch was not opened until ready to perform the test.

Assay procedure

Step 1: Place the test device on a clean, flat surface.

Step 2: Be sure to label the device with specimen�s ID number.

Step 3: For whole blood test

One drop of whole blood (about 40-50 µl) was put to the sample well of the test unit, then

add 1 drop (about 35-50 ìl) of sample diluent immediately. The test unit was kept for 15

minutes. Results were read after 15 minutes. Result was not interpreted after more than 15

minutes. Positive results can be visible in as short as 1 minute.

Interpretation of ICT was given in (Appendix �VIII) (Photograph-12)

Polymerase chain reaction for detection of S. typhi directly from blood

sample:

Processing of blood samples and PCR amplification:

Three major steps:

Extraction of DNA from blood samples.

Amplification of DNA in thermal cycler.

Electrophoresis and then documentation under UV light.

DNA extraction from blood:

Extraction of DNA from blood samples (1ml) was carried out by modified lysis buffer

method. One ml EDTA containing blood was centrifuged by micro-centrifuge at 13000 rpm

for 5 minutes. Supernatant was discarded. Then 1 ml 0.2% Triton X-100 (Appendix-IX) was

added to the pellet. The mixture was vortexed, incubated at room temperature for 10 minutes

and centrifuged at 13000 rpm for 10 minutes. Supernatant was decanted. One ml 0.2% Triton

X-100 was added to the pellet again, vortexed and centrifuged at 13000 rpm for 10 minutes.

Then washed with 1 ml nuclease free water, centrifuged for 3 minutes and supernatant

discarded. The pellet was resuspended in 30 µl nuclease free water. Boiled for 10 minutes at

990C then centrifuged for 3 minutes. Supernatant was used as template for PCR (Jimenez and

Castro 2004; Nagarajan et al. 2009).

Preparation of reaction mixture

Sterile micro-centrifuge tubes (1.5ml) were taken and the tubes were labeled with date and

identification number. A total volume of 50 µl master mixture including 1 µl sample in which

included 10 x buffers, primer mixture, deoxy nucleoside triphosphate, taq polymerase and

PCR water.

Preparation of master mixture for each reaction (50 µl) contained:

10 x buffers - 5µl

2.5 micro mol deoxy nucleoside triphosphate - 4µl

25 Pico mol of each primer mix- 1µl (STI =0.5 µl, ST2 =0.5 µl)

2units of Taq DNA polymerase - 0.5µl

DDW/PCR water - 38.5µl

DNA sample - 1µl

Polymerase chain reaction for detection of S. typhi directly from blood sample DNA

amplification was carried out as described by Nagarajan et al. 2009 with some modification.

A mixture consisting of 5µl of 10X Ex Taq buffer (Takara, Japan), 4µl 2.5 mM dNTP mix

(2.5 mM each of dATP, dCTP, dGTP and dTTP, Takara, BIO INC, Japan), 38.5 µl of DDW

was taken in a 1.5 ml of micro centrifuge tube. Then 0.5 µl (25 pmol) each of gene specific

forward (STI) and reverse (ST2) primers were added. Afterwards 1µl of extracted DNA was

added and finally 0.5 µl Taq polymerase (Takara, BIO INC, Japan) was added and contents

of tube mixed well by pipetting. The tubes were placed in a thermal cycler and allowed to run

by the following protocol by Massi and others, Ambati and others with some modification

(Massi et al. 2003; Ambati, Nath and Das 2007).

Protocol of thermal cycles:

Denaturation at 940C for 30 seconds.

Primer annealing at 540C for 30 seconds.

Extension at 72 0C for 1 minute.

Final extension at 720C for 5 minutes.

Above step were repeated for 30 cycles in an automated DNA thermal cycler (Eppendorf,

Germany). Final extension was done at 720C for 5 minutes. The PCR products were run in

1% agarose gels stained with ethidium bromide and visualized under the UV transilluminator.

In nested PCR, the amplified product of the first PCR is used as template. In the second

amplification (nested PCR), a second set (ST3 and ST4) of primer was added to the reaction

mixture (50 µl). The tube was kept in a thermal cycler and allowed to run for 30 cycles as

well as the same protocol of the first round PCR.

Primer used for PCR:

30 cycles

For nested PCR oligonucleotide primers as first described by Song et al. 1993 and used by

Massi et al. 2003 and Ambati and others 2007. The sequences from 5' to 3' ends of these

oligonucleotide primers were as follows:

Designation- sequence (5' to 3') Target gene Amplicon size

ST1- 5'-TAT GCC GCT ACA TAT GAT GAG-3' Flagellin gene 495 bp

ST2 - 5'-TTA ACG CAG TAA AGA GAG-3'

Above primers were used for first round PCR to amplify 495 bp sequence of flagellin gene.

Designation- sequence (5' to 3') Target gene Amplicon size

ST3- 5'-ACT GCT AAA ACC ACT ACT-3' Flagellin gene 364 bp

ST4 - 5`-TGG AGA CTT CGG TCG CGT AG-3'

Above pairs of primers were used for second round PCR to amplify a 364 bp sequence of

flagellin gene.

Agarose gel electrophoresis:

The PCR product was analyzed in 1% agarose gel by electrophoresis for 35 minutes to detect

specific band.

Preparation of agarose gel:

One percent agarose gel was prepared by melting 3 gm agarose in 200 ml of diluted TBE

buffer (1:4) using microwave woven. The agarose was allowed to cool about 500C and was

poured into gel electrophoresis unit with spacers and comb. After solidification of the gel, the

comb was removed and wells were formed. During electrophoresis, gel was placed in a

solution containing Tris- EDTA- borate buffer (TBE buffer) and ethidium bromide

(0.5µg/ml) in the horizontal electrophoresis chamber.

Loading and Running of the sample:

Five µl of amplified product was mixed with 2.0 µl gel loading dye. The mixture was slowly

loading into the well using disposable micropipette tips. Marker DNA of known size (100 bp

ladder) was loaded in one well to determine the size of amplified PCR products.

Electrophoresis was carried out at 100 volts for 35 minutes in 1% agarose gel, prestained with

ethidium bromide in a submarine gel apparatus (Mupid 2 plus submarine electrophoresis,

Japan).

Visualization /documentation under Transilluminator

Following electrophoresis, amplified product, the bands (495 bp in first round and 364 bp in

second round of nested PCR) of the sample of the study was visualized by

transilluminator.The amplicon size was determined by comparing position of amplicon in

respect to that of a 100 bp ladder that was loaded in adjacent well and simultaneously

electrophoresed. And the gel was photographed by a digital camera and was transferred data

to computer for further documentation.

Interpretation

PCR positive: Samples were scored as positive when a PCR product of 495 bp could be

detected in first round PCR.

In nested PCR, samples were scored as positive when a PCR product of 364 bp could be

detected.

Results

The present study was conducted on a total 200 individuals of different age and sex,

including 150 cases of clinically suspected typhoid fever and 50 controls of 25 non-typhoidal

illnesses and 25 healthy persons. Among the 150 suspected cases of typhoid fever, almost all

of the cases complained of fever (148, 98.7%) followed by headache (52, 34.7%), anorexia,

nausea and vomiting (39, 25.8 %), abdominal discomfort with diarrhoea (34, 22.6 %) and

constipation (29, 19.3%) for 3-10 days (Figure-1)

Age range of the study population was from 2 years to 60 years, mean age was 18.6 years.

Age distribution of 150 suspected cases and 50 controls showed that the majority of the

respondents of the cases (43, 28.6%) belonged to the age group of 1 to 5 years followed by

(34, 22.7%) to the age group of 16 -20 years and (32, 21.3%) to the age group of >30

years(Table-I) (Figure-2).

Distributing the sex of study population showed that total male were 81(54%) and female

69(46%) in the cases group and corresponding values for controls were 26 (52%) and

24(48%) respectively. Male to female ratio of cases were 1.2:1(Figure-3).

The socio-demographic characteristics of suspected cases of typhoid fever showed that the

majority of the respondents were pre-school children (53, 35.3%), belonged to lower class

(121, 80.7%), lived in tinshed (89, 59.3%), used open latrine (79, 52.7%) and drinking from

tubewell (112, 74.7%) (Table-II).

Presenting complaints by the suspected cases of typhoid fever

148

5139 34 29

98.7

34.725.8 22.6 19.3

0

20

40

60

80

100

120

140

160

Fever Headache Anorexia, Nausea,vomiting

Abdominaldiscomfort with

Diarrhoea

Constipation

Total no. of cases

Percentatge

Figure-1: Presenting complaints by the suspected cases of typhoid fever.

Age group distribution of study population

0%

10%

20%

30%

40%

50%

60%

1-5 ye

ars

6-10

years

11-15y

ears

16-20y

ears

20-25y

ears

26-30y

ears

>30y

ears

Control

Case

Figure 2: Age group distribution of study population

Table I: Age group distribution of suspected cases of typhoid fever

Age in years No of cases (%)

1 - 5 43 (28.6%)

6 - 10 14 (9%)

11 - 15 8 (5.3%)

16 - 20 34 (22.7%)

21 - 25 12 (7.7%)

26 - 30 7 (4.7%)

>30 33 (21.3%)

Male female ratio 1:1.2

Sex pattern of suspected cases of typhoid fever

54%

46% Male

Female

Sex pattern of control group

52%

48%Male

Female

Figure-3: Sex pattern of study population.

Table-II: The socio-demographic characteristics of study population (n=150)

Factors Number of cases (%)

Illiterate 26 (17.3)

Pre-school 53 (35.3)

Primary level 31 (20.7) Education status

Secondary level or higher 36 (24)

Lower class (Up to Tk. 15,000/=) 121(80)

Middle class (Tk. 15,000-50,000/=) 26(17.3) Socio-economic class as per monthly income

Upper class (Tk. >50,000/=) 03(2.0)

Tin shed 89 (59.3)

Mud house 35 (23.3)

Tin shed building 20 (13.3) Housing condition

Building 06 (4)

Tube well 112 (74.7)

Municipal Supply 35 (23.3) Source of drinking water

Ponds water 3 (2)

Figures in parenthesis indicate percentage.

Among the three investigations performed for the diagnosis of typhoid patients, polymerase

chain reaction showed maximum 133(88.7%) positive result followed by

Immunochromatography (ICT) test 106 (70.7%) and blood culture 23(15.3%) (Figure-4).

Among 150 blood samples from the suspected cases, 23(15.3%) were positive for Salmonella

typhi by blood culture. None of the healthy controls was positive by blood culture. The

highest rate of blood culture positivity for S. typhi 09(39.1%) was found among 1 to 5 years

of age group followed by 5(21.8%) among 6 to 10 years (Table-III).

By observing the relationship of blood culture positivity and duration of illness, it was found

that all of the samples yielding positive result were collected from the cases complaining of

3-7 days of fever (Table-IV).

Considering the relationship of antibiotic usage with the results of blood culture of the

suspected cases of typhoid fever, it was found that none of the blood culture positive cases

taken any antibiotic during the episode and majority of the blood culture negative cases gave

history of taking different antibiotics for variable periods (Table-V).

Antimicrobial susceptibility pattern of S. typhi isolates (n=23) showed that the highest

number (73.9%) of isolates were sensitive to chloramphenicol, followed by cotrimoxazole

(69.6%), ceftriaxone (60.9%), gentamicin (52.2%), ceftazidime (47.8%) and azithromycin

(43.5%) (Figure-5).

Results of three tests performed typhoid fever

15.30%

70.70%

88.70%84.70%

29.30%

11.30%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Blood culture ICT PCR

Positive

Negative

Figure-4: Different test performed for diagnosis of typhoid fever.

Table-III: Rate of isolation of Salmonella typhi among study cases

Age in years Blood culture Positive ( n= 23)

1-5 09(39.1)

6 - 10 05(21.8)

11-15 02(8.7)

16-20 04(17.4)

>20 03(13.04)

Total = 23 23(100)

Figures in parenthesis indicate percentage.

Table-IV: Relationship of blood culture positivity with duration of fever of the cases

No. (%) of patients and duration of illness in days Result of blood culture 1-3 4-7 8-10 11-14

Positive 1(0.7%)* 22(14.7%) 0(0%) 0(0%)

Negative 01(0.7%) 96(64%) 26 (17.3%) 2 (1.3%)

*The case was presenting with fever for 3 days.

Table-V: Antibiotic usage by the blood culture-negative suspected cases of typhoid fever

Antibiotic Dosage schedule Duration of intake

(days) No. (%) of cases

Ciprofloxacin 100-500 mg twice

daily 2-7 23 (25.9%)

Cotrimoxazole 960 mg twice

daily 2-5 14 (22.2%)

Tetracycline 250 mg twice

daily 2-7 9(5.6%)

Azithromycin 500 mg single 3 18 (14.8%)

Amoxycillin 250-500mg thrice

daily 3-5 13(13.0%)

Levofloxacin 500 mg single 3-7 17 (5.6%)

Cephradin 250 mg thrice

daily 6 16 (1.9%).

*17 patients taken some drugs but cannot mentioned the name properly.

Antimicrobial susceptibility pattern of the Salmonella typhiisolates (n-23)

1716

14

1211

109

76

7

9

1112

1314

16

0

2

4

6

8

10

12

14

16

18

Chloram

phen

icol

Cotrimoxa

zole

Ceftriax

one

Genta

micine

Ceftazid

ime

Azithr

omycine

Ampic

illin

Cipro

floxa

cine

Sensitive

Resistant

Figure-5: Antimicrobial susceptibility pattern of the Salmonella typhi isolates (n=23).

By ICT, among 150 blood samples from the suspected cases, 106(70.7%) were positive for

IgM of S. typhi. Whereas, among 50 controls 5 were positive for IgM of S. typhi. (Table VI).

Among the ICT positive cases, it was found that 82 (71.9%) cases had IgM antibody,

24(21.05%) cases had both IgM and IgG antibody and only 8(7.01%) cases positive for IgG

antibody (Figure-6).

By observing the relationship between ICT and PCR, it was found that 103 cases were both

ICT (IgM) and PCR positive (Table�VII), 3 samples (clotted blood) were ICT positive but

PCR negative.

The ICT (IgM) was positive in 14(60.9%) out of 23 culture positive typhoid cases. Sensitivity

and specificity of ICT was 77.4% and 82.3% respectively by considering PCR as gold

standard (Table VII).

Among 150 blood samples from the suspected cases, subjected to PCR, 133(88.7%) showed

positive results for flagellin gene of S. typhi, and all of the 50 blood samples from the

controls were negative by PCR (Table IX). The samples of the cases showing negative

results with blood culture (n=127) were also tested by PCR and 110 (86.5%) of them were

positive by the PCR (Table X). The rate of positivity by PCR (88.7%) was found to be much

higher than blood culture (15.3%) method (Figure- 7).

Table -VI: Results of ICT for identification of IgM antibody of S. typhi

ICT results Study population

Total number of samples tested Positive Negative

Suspected cases 150 106 (70.7%) 44 (29.3%)

Controls 50 05 (10%) 45 (90%)

Antibody detection among suspected cases by ICT method

71.90%

21.09%

7.01%

IgM

Both IgM and IgG

IgG

Figure-6: Antibody detection among suspected cases by ICT method.

Table-VII -: Sensitivity and Specificity of ICT

Disease status Test result

PCR positive PCR negative Total Sensitivity Specificity

ICT positive 103 (a) 3( b) 106

ICT negative 30 (c) 14(d) 44

Total 133 17 150

77.4% 82.3%

N.B: Sensitivity and specificity was calculated by the following formula considering PCR as

gold standard

Sensitivity= 100 x a/a+c, Specificity = 100 x d/b+d.

Positive predictive value = a x 100 / a+b = 97.2%

Negative predictive value = d x 100 / c+d =31.8%

a = True positive, b = False positive, c = False negative and d = True negative.

PPV = Positive predictive value; NPV = Negative predictive value

Table-VIII: Results of polymerase chain reaction (PCR) for identification of flagellin

gene of S. typhi

PCR results Study population

Total number of samples tested Positive Negative

Suspected cases 150 133 (88.7) 17 (11.3)

Controls 50 00 (00) 50 (100)

Figures in parenthesis indicate percentage.

Table-IX: Comparison of culture and PCR methods for identification of Salmonella

typhi

No(%) of cases showing culture results PCR results

Positive Negative Total

Positive 23(100) 110(86.7) 133(88.7)

Negative 0(0) 17(13.3) 17(11.3)

Total 23(100) 127(100) 150(100)

Figures in parenthesis indicate percentage.

2315.3

133

88.7

0

20

40

60

80

100

120

140

Blood culture PCR

Test method

Comparison of Blood culture and PCR

Total no. of cases

Percentatge

Figure 7: Comparison of positivity by blood culture and PCR for diagnosis of typhoid

fever.

Sensitivity, specificity, positive predictive value and negative predictive value of blood

culture by lytic centrifugation method into consideration of clinical suspicion as disease

positive for a case, it was found that blood culture showed a sensitivity, specificity, positive

predictive value and negative predictive value were 15.3%, 100%, 100% and 28.3%

respectively (Table-X).

On calculating diagnostic accuracy of PCR method taking blood culture as gold standard, it

was found that PCR showed a sensitivity of 100%, specificity 100%, (Table XI)

Table-X: Sensitivity and specificity of blood culture in study population:

Disease status of the cases Blood culture

results Positive (n=150) Negative (n=50) Total Sensitivity Specificity

Positive 23 0 23

Negative 127 50 177

Total 150 50 200

15.3% 100%

N.B: Sensitivity and specificity was calculated by the following formula

Sensitivity= 100 x a/a+c, Specificity = 100 x d/b+d.

a = True positive, b = False positive, c = False negative and d = True negative.

PPV = Positive predictive value; NPV = Negative predictive value

Positive predictive value = a x 100 / a+b = 100%

Negative predictive value = d x 100 / c+d =28.3%.

Table-XI: Diagnostic accuracy of PCR for diagnosis of typhoid fever

PCR results No(%) of cases showing culture results Total

Positive Negative

Positive 23(100%) 110(86.7%) 133(88.7%)

Negative 0(0%) 17(13.3%) 17(11.3%)

Total 23(100%) 127(100%) 150(100%)

Sensitivity and specificity of PCR was calculated by considering blood culture as a gold

standard method.

Sensitivity= 100%

Specificity= 100%

N.B: Sensitivity and specificity was calculated by the following formula

True Positive

Sensitivity = True Positive + False Negative

×100

True Negative

Specificity = True Negative + False Positive

×100

Discussion

Typhoid fever is one of the most common infectious disease in developing countries

including Bangladesh. The disease is present especially in areas where healthcare facilities

are limited and peoples are illiterate, living in unhygienic surroundings, drink raw-water from

tube-wells and not habitual of hand- washing after toilet by soap. Symptoms and signs of the

disease are non-specific and laboratory tests are essential for diagnosis. Early and reliable

diagnosis of the disease is not only important in relieving patients� suffering, but also

necessary to avoiding life threatening complications including perforation of the intestine.

In the present study, 200 purposefully selected individuals were investigated including 150

clinically suspected cases of typhoid fever and 50 controls (25 non-typhoid febrile illnesses

and 25 healthy individuals). The disease affected all ages, however most of the cases (28%)

of the study were in the age group of 1�5 years (Table I). This findings correlates with the

observation made by Saha and associates who found that children between 2-3 years of age

are most susceptible age group (35.6%) (Saha et al. 2003). Almost similar study done by

Brooks and others showed that the prevalence of typhoid fever in children of under 5 years

were much higher than other age group (Brooks et al. 2005). The child aged group < 5 years

were more prone to infection, which may be due to a lack of immunity transferred by

mothers� milk or the non consumption of potable drinking water as is the common practice in

rural areas (Saha et al. 2003).

In present study, among 150 clinically suspected typhoid cases 54% were male and 46% were

female. Another study done by Butler and others also showed similar result that infection rate

was slightly higher in male (Butler et al. 1991). Butler expressed his opinion that greater

exposure of male to contaminated food and water out side the home might be region of higher

rate of infection among this population.

The socio-demographic characteristics of suspected cases of typhoid fever in this present

study showed that the majority of the respondents were pre-school children (53, 35.3%) and

(26, 17.3%) were illiterate, belonged to lower class (121, 80.7%), used open latrine (79,

52.7%) and drinking from tubewell (112, 74.7%) (Table II). Similar findings also have been

reported by Sur and others showing that the illiteracy rates were highest in the cases of

typhoid fever. In the same study unhygienic latrines were the main sources of spreading

typhoid diseases and the sanitation condition of low income areas was remarkably poor (Sur

et al. 2007). Lack of safe drinking water and unhygienic sanitation are believed to contribute

the transmission of typhoid fever (Jenkins and Gillespie 2009). It has been observed that the

water and sewage pipelines lie close together in the slum areas of Bangladesh and they are

prone to leakage and cross-contamination. In addition most of the people cannot afford the

sanitary latrines in Bangladesh due to poverty which further carries the risk of exposure to

Salmonella typhi.

In the present study, blood culture, ICT and nested PCR were studied. The nested PCR

showed maximum (133 out of 150, 88.7%) positive result followed by ICT (106, 70.7%) and

blood culture (23, 15.3%). All of the culture positive cases were positive by PCR and among

culture negative cases 86.5% were positive by PCR. Almost similar study was done in Delhi,

India, and used blood samples from 40 clinically suspected cases of typhoid fever, and found

100% of culture positive (n=20) and 75% (15/20) of culture negative cases positive by PCR

(Kumar et al. 2002).

In the present study, among 150 clinically diagnosed typhoid fever cases 23(15.3%) showed

positive blood culture for S. typhi (Table III). Similar finding was also reported by Begum

and associates in Bangladesh in 2007, where they found an isolation rate of S. typhi was 14%

(Begum et al. 2009). Saha and others from Kolkata in 2003 found an isolation rate of S. typhi

was 21.1% (Saha et al. 2003). Hossain (2001) from Bangladesh reported an isolation rate of

S. typhi was 16.67% (Hossain 2001). The relative low sensitivity of the blood culture in

diagnosing typhoid fever is a common phenomenon. It may be due to indiscriminate use of

antibiotics and difficulties in obtaining adequate volume of blood for culture from children

(Bhutta and Mansurali 1999; Mohanty and Ramana 2007). Blood culture has the promise of

diagnosis in the first week and is very specific, but its sensitivity is poor due to various

factors. Sensitivity of cultures can be affected by antibiotic treatment, type of culture

medium, length of incubation and variations of bacteraemia (Gilman et al. 1975; Miller and

Pegues 2000).

In this study, the highest rate of positive blood culture for S. typhi 09(39.1%) was found

among 1 to 5 years of age group followed by 5(21.8%) among 6 to 10 years (Table-III).

Another similar study by Saha and others from Bangladesh showed that the majority (54.5%;

213 of 391) of S. typhi isolates were from children who were younger than 5 years, and 27%

(105 of 391) were from children in the first 2 years of life. They also mentioned that the

isolation rate was highest (17.4%, 68 of 486) in the second year of life (Saha et al. 2001).

In present study, majority of the blood culture negative cases gave history of taking different

antibiotics for variable periods. Antibiotic treatment prior to collection of sample inhibits the

growth on blood cultures (Gasem et al. 1995; Cheesebrough 2010). Similar finding were

found by Lin and others, where they showed that the S. typhi was recovered from 5.3% of

patients with prior antibiotic intake versus 5.8% without prior antibiotics (Lin, Ho and Bay

2000).

In this study, the majority (73.9%) of isolates of S. typhi were sensitive to chloramphenicol,

followed by cotrimoxazole (69.6%). There have been some reports of the reemergence of the

sensitivity of S. typhi to chloramphenicol and other first line drugs. Bhatia and others reported

that the highest (96%) sensitivity of Salmonella typhi to chloramphenicol in India (Bhatia,

Mathur and Arora 2007). Another study by Yashavanth and Vidyalakshmi found that the re-

emergence of chloramphenicol (97.4%) sensitivity among the strain of S. typhi pathogens in

Mangalore in 2007 (Yashavanth and Vidyalakshmi 2010).

In present study, some isolates of S. typhi were found resistant to ciprofloxacin and nalidixic

acid. Nalidixic acid-resistant (NAR) S. typhi with decreased susceptibility to ciprofloxacin is

now endemic in India and neighbouring countries, constituting a threat to global health

(Chandel et al. 2000; Threlfall and Ward 2001a; Hakanen et al. 2001).

Another study from Bangladesh showed that the decreased susceptibility to ciprofloxacin was

detected in 24 (18·2%) out of 132 randomly selected strains during 1990�2002 (Rahman et al.

2006). Fluoroquinolones, especially ciprofloxacin, have been in use for more than 18 years

and have remained an important weapon against S. typhi. In spite of this, in recent years,

several reports have appeared worldwide concerning reduced activity of ciprofloxacin against

S. typhi (Wain et al. 1997; Chandel et al. 2000; Threlfall and Ward 2001a; Hakanen et al.

2001). In perspective of Bangladesh, ciprofloxacin is commonly used in case of typhoid fever

may be due to ciprofloxacin continues to be the mainstay in the treatment of typhoid fever as

it is orally effective and economical.

In this study, 40% of isolates of S. typhi were found resistant to ceftriaxone. Third generation

cephalosporins have been recommended as an alternative to quinolone treatment in enteric

fever and several physicians have claimed good results with them, particularly with

ceftriaxone (Girgis et al.1995a). As consequences of extensive use of ceftriaxone and other

third generation cephalosporin, resistance is being reported with increasing frequency all over

the world. Another study by Saha and others reported about the highly ceftriaxone resistant

strain of S. typhi in Bangladesh (Saha et al. 1999).

ICT has been studied in many countries and they found significantly higher sensitivity and

specificity (Jesudason, Esther and Mathai 2002; Pastoor et al. 2008; Anusha, Ganesh and

Lalitha 2011). An evaluation of ICT (Typhidot) in India was found to be 100% sensitive and

80% specific compared to a blood culture as �gold standard� (Jesudason, Esther and Mathai

2002). In present study, sensitivity and specificity of ICT in suspected typhoid cases were

found 77.4% and 82.3% respectively. In another study from India by Nakhla and others

showed almost similar type of result that the sensitivity and specificity of ICT (IgM) was

80% and 71.4% respectively (Nakhla et al. 2011). Abdoel and associates showed that the

sensitivity of ICT improved with the duration of illness from 30.8% for samples collected

during the first 4-5 days of illness to 45.5% for samples collected between days 7 and 9, and

to 84.6% for the samples collected more than 9 days after the onset of illness (Abdoel et al.

2007).

In this study, out of 127 cultures negative typhoid cases 92 (72.5%) were positive by ICT

method. Almost similar types of results were also reported by Anggraini and others, where

they showed that ICT (IgM) positive 66% among culture negative but clinically diagnosed

typhoid fever (Anggraini Handoyo and Aryati 2004). In another study from Pakistan showed

that only 28% (7 out of 25) of culture negative typhoid fever cases were ICT (IgM) positive

(Bhutta and Mansurali 1999). The lower detection rate in that study may be explained by the

reason that the study was conducted on random selection rather than the clinically diagnosed

typhoid fever cases.

In this study, out of 23 culture positive cases, 9 (39.1%) were negative for ICT (IgM).

Although these patients may have had antibodies at a lower titre. It is well recognized that

patients with blood culture-confirmed typhoid fever may have a negative antibody response

throughout the course of their illness. This lack of antibody response among such patients has

been attributed to short duration of illness (3-7 days, Table-IV). Seropositivity increased with

duration of illness and seroconversion was observed in the majority of typhoid patients with a

negative IgM result for the first collected serum sample. Seropositivity in the IgM increased

from less than 30% for samples collected during the first week of illness to over 95% for

samples collected from patients ill for more than 9 days (Hatta et al. 2002).

Among 106 ICT (IgM) positive cases, 3 cases showed negative result by PCR due to clotting

of blood because of delayed processing. Thirty PCR positive cases showed negative result by

ICT (Table-VII). The false negative ICT in this case was probably due to the failure of ICT to

detect antibodies the because antibody titre did not yet reach the detectable level in these

patients.

In the present study the rate of diagnosis of typhoid fever by the PCR was 88.7% compared

with blood culture, where it was 15.3%. The rate of positivity of the nested PCR using 1 ml

of blood appears to be higher than that of blood culture performed with 8 ml blood samples.

The results show that nested PCR method can be an alternative tool to confirm clinical

diagnosis of typhoid fever. The finding of the PCR results is consistent with the previous

observations, which showed that the same nested PCR, as well as other PCRs for typhoid

fever, had higher sensitivities than blood culture (Haque et al. 2001; Kumar et al. 2002; Massi

et al. 2003; Prakash et al. 2005; Hatta and Smit 2007). In Varnassi, India, Prakash and others

used the nested PCR with a different DNA extraction method and reported nested PCR was

again better (53 positive of 57 samples, 73.0%) than blood culture (17 positive of 53 samples,

32.0%) (Prakash et al. 2005). In another study, in Pakistan, 55 cases of suspected typhoid

fever were diagnosed by the PCR and blood culture, gave 58.2% and 14.5% positivity,

respectively, showing significantly better results by PCR (Hoque et al. 2001). A large, well-

designed study in Indonesia by Hatta and Smith reported a sensitivity of 61.8% by blood

culture and 84.5% by PCR from blood samples (Hatta and Smit 2007). A study from Nepal

reported sensitivity of PCR 82.7% which is much higher than blood culture (26.9%) (Ambati

et al. 2007). Another study in South Sulawesi, Indonesia, showed 63.0% were positive by

PCR compared to 13.7% positive by blood culture (Massi et al. 2003).

In this study, nested PCR was used to yield 364 bp amplicon specific for flagellin gene of S.

typhi. This was preferred because hypervariable region VI of the flagellin gene is unique for

S. typhi and its amplification was found to provide 100% specificity (Frankel, 1994; Song et

al. 1993). In an alternative, targetting Vi gene, gives false-positive results, because of the

presence of the gene in another salmonella namely S. paratyphi C as well (Hashimoto et al.

1995).

Among the suspected typhoid cases in the present study, PCR was able to detect S. typhi in

88.70% of the blood samples and none of the healthy controls was positive. The rate of

detection by PCR was significantly higher (p<0.001 each) than blood culture. The low rate of

detection by blood culture is probably due to antibiotics taken by the patients before sample

collection. Antibiotics are freely available in Bangladesh over the counter without

prescription and many of the patients reported to have antibiotics before collection of

samples.

It is, therefore, concluded that the PCR method was much superior to blood cultures yielding

very high sensitivity and specificity. Although the PCR method requires extensive

infrastructure and specialized skilled personnel, and cannot be made available everywhere,

especially in developing countries, it can be made available to the reference centres for

utilizations by other healthcare facilities following referral system. In fact, due to the rapid

and definitive diagnosis, hospital admission of the patient can be avoided, reduce patients�

suffering, save working days and unnecessary expenditure on unrelated and misdirected

treatment which may be many times more than the cost of PCR.

Conclusion and recommendations Analyzing the findings of the present study, it can be concluded that typhoid fever remain to

be as an endemic disease in this locality. All the signs and symptoms of the disease are

nonspecific common with other acute febrile illnesses; a definitive diagnosis of the disease is

required for treatment and to prevent transmission. The ICT and PCR can be the suitable

method for rapid diagnosis of typhoid fever. Detection of antibody (IgM) from whole blood

by ICT method is more easy, non-invasive and highly sensitive and specific method. Thus,

Detection rate of antibody by ICT method is quite satisfactory, so this test can be applicable

for field level use. So, efforts should be made to establish antibody (IgM) detection from

whole blood by ICT method at field level, especially in the endemic areas of Bangladesh.

It is, therefore, concluded that the PCR method was much superior to other method yielding

very high sensitivity and specificity. Although the PCR method requires extensive

infrastructure and specialized skilled personnel, and cannot be made available everywhere,

especially in developing countries, it can be made available to the reference centres for

utilizations by other healthcare facilities following referral system. So it is strongly

recommended to take necessary steps for setup and start PCR at least in the tertiary care

hospitals.

Comparison of different test methods including polymerase

chain reaction for early and reliable diagnosis of typhoid

fever

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Photograph-1: Steps of preparing and processing of sample for Nested PCR

One ml EDTA containing blood

in a appendorf tube

Centrifuge at 13000 rpm for 5

minutes.

Supernatant

discarded

One ml 0.2%

triton X-100

was added to

the pellete

Boiled at 990C for 10 minutes

Preparation of master mixture in PCR tubes

Placing the PCR tube in thermal cycler

Gel electrophoresis of amplified

products

Visualization of the amplified products by UV light trans-

illuminator.

The mixture was vortexed.

Flagellin gene of Salmonella typhi (495 bp)

L 1 2 3 4 5 6 7 8 9 10 11 12 13 No Ladder - - - + - - - - + + + ladder

Photograph-2: Flagellin gene of Salmonella typhi after first round of nested PCR.

L 1 2 3 4 5 6 7 8 9 10 11 12 13

No ladder + + + + + + + + + + + ladder

Photograph-3: Flagellin gene of Salmonella typhi after second round of nested PCR

Flagellin gene of S. typhi (364

bp)

Photograph-4: Heparinized tube containing lytic solution

Photograph-5: Initial processing of blood for culture by lytic centrifugation method

.

Photograph-6: Growth of S. typhi after 24 hours of incubation by lytic centrifugation method. Photograph-7: Growth of non lactose fermenting colony Salmonella typhi on Mac conkey�s

agar media.

Photograph-8: Growth of non lactose fermenting colony Salmonella typhi on Salmonella - shigella agar media. .

Photograph-9: Growth of Salmonella typhi on blood agar.

Photograph-10: Gram negative short bacilli of Salmonella typhi

Photograph-11: Citrate utilization test showing citrate negative S. typhi

Negative Control

known S. typhi

Test Positive Control

Klebsiella

Photograph-12: Photograph of results of Immunochromatographic method

ICT Negative

IgG Positive

IgM Positive

Both IgM and IgG Positive

Photograph-13: Photograph of micro centrifuge

Photograph-14: Photograph of Thermal cycler

Photograph-15: Photograph of Documentation chamber with digital camera.

Photograph-16: Photograph of Horizontal Electrophoresis.

Appendix-I

DATA COLLECTION SHEET

Title of the thesis: Comparison of different test methods including polymerase chain reaction

for early and reliable diagnosis of typhoid fever.

ID no. �������..

Date of enrollment: ����/����./������

Particulars of the patient

1. Name: �������������.Age: ����. Sex: ����

2. Address: Vill: ���������. PO: ����..

Thana: �������. District: ���

5. Education: Yes/ No If Yes, level of education:������

6. Income per month of the patient/ guardian: Tk��������./-

7. Living conditions:

a. Living area : Rural/ City/ Slum

b. Housing condition : Kancha (mud house)/ Tinshed/ Tinshed Building/

Buiding

c. Type of latrine using : Sanitary / Open

d. Source of drinking water : Municipal supply/ Tube well/ Pond/ River

8. Presenting complaints:

a) Fever for ����days; Temperature at maximum rise: ��.. 0C

b) ��������c)������������d)��������

9. On examination

a) Physical condition: alert/ toxic b) Temperature: ���..0C

b) Pulse: ����. / min c) Coated tongue

d) Rose spot e) Abdominal tenderness

f) Caecal gurgling g) Liver

h) Spleen

10. Previous history

a) Antibiotic intake: Yes/ No

If Yes, name of antibiotic���������

Dose & route of administration����� Duration: for���..days

b) History of contact from similar illness in last 1 moth: Yes/ No

c) History of vaccination: Yes/ No If Yes, date of vaccination: ���

Laboratory findings:

1) ICT: Positive/ Negative If Positive: IgG/ IgM/ both

2) PCR: Flagellin gene positive/ negative

3) Blood culture (lytic centrifugation method) result:

Shows growth of �����������������after 24/48 hrs

Identification tests done:

Date Test name Findings comments

Motility by Hanging

drop

MIU

TSI

Citrate Utilization test

Sensitivity test result

Antibiotic IZD

(mm)

Result Antibiotic IZD

(mm)

Result

Appendix - II For Patients

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Appendix III

Preparation of lytic solution

Composition of lytic solution:

For adult:

Sodium polyanethol sulfonate (SPS) ----1.53 gram

Saponin ----- 2.80 gram

Distilled water -----100ml

For Infant:

Sodium polyanethol sulfonate (SPS) ---- 0.19 gram

Saponin ---- 0.80 gram

Distilled water ----- 20ml

Sterilization of lytic solution:

By autoclaving at a temparature of 121ºc, pressure of 15ib for 15 minutes.

With all aseptic precaution by the help of sterile pipette and sterile tips lytic solution

dispensed into Heparinized tubes within a biosafety cabinet.

For adult 1 ml and for infant 125µå of lytic solution in each heparinized tube.

Appendix IV

The composition and methods of preparation of different media used in this study are given

bellow:

Blood agar media:

Dehydrated blood agar base : 40 grams.

Distilled water : 1000.0ml

PH : 6.8

Autoclave at 1210 C for 15 minutes under 15 Ibs. Pressure, cooled down at 500 C, 7% sheep

blood (defibrinated, aseptically colleted) was added, mixed well and poured into sterile Petri

dishes.

MacCokey�s agar media:

Dehydrated MacConkey�s agar base : 51.5 gm

Distilled water : 1000.0ml

Media was dissolved and PH was adjusted to 7.2 to 7.6 at 250 C and autoclave at 1210 C for

15 minutes. When it was cooled to 550 C, poured into sterile Petri dish.

Nutrient agar media:

Dehydrated blood agar base : 40 gm

Distilled water : 1000.0ml

PH 7.3 ± 0.2

Autoclave at 1210 C for 15 minutes. When it was cooled to 550 C, poured into sterile Petri

dish.

Triple sugar iron (TSI) agar media:

Dehydrated TSI agar base : 65.0 gm.

Distilled water : 1000.0 ml

PH : 7.2-7.6

The ingredients were dissolved by boiling and the PH was checked, distributed into test tube

in 5 ml amounts and sterilized by autoclaving at 121oC for 15 minutes under 15 Ibs. Pressure.

Allow the medium to solidify in a sloped position to give a butt 25-30 mm and a slope 20-25

mm long. Store in a cool dark place or at 2- 80 C.

Simmon�s citrate media:

Dehydrated media : 24.2 gm.

Distilled water : 1000.0 ml

PH : 6.8

Ingredient dissolved, dispensed into test tubes and autoclave at 1210 C for 15 minutes and

allowed to set as slopes.

Appendix - V

Gram staining technique (Cheesbrough 2010)

Required

Crystal violet stain, Lugol�s iodine, Acetone-alcohol decolourizer, Neutral red, 1 g/l (0.1%

w/v).

Before Gram staining smear of positive control of known S. typhi in one side of a slide, a

negative control of gram positive cocci in another side of slide and test in the middle of slide

were prepared.

Method:

1. Fix the dried smear by gentle heat.

2. Cover the fixed smear with crystal violet stain for 30-60 seconds.

3. Rapidly wash off the stain with clean water.

4. Tip of all the water, and cover the smear with Lugol�s iodine for 30-60 seconds.

5. Wash of the iodine with clean water.

6. Decolourize rapidly (few seconds) with acetone-alcohol. Wash immediately with clean

water.

7. Cover the smear with neutral red stain for 2 minutes.

8. Wash off the stain with clean water.

9. Wipe the back of the slideclean, and place it in a draining rack for the smear to air dry.

10. Examine the smear microscopically, first with the 40X objectives to check the staining

and to see the distribution of material, and then with the oil immersion objectives to report

the bacteria and cells.

Appendix - VI

Muller Hinton agar medium

Composition

Ingredients gram/liter

Beef dehytrated infusion 300

Casein hydrolysate 17.50

Starch agar 1.5

Agar 17.00

Thirty-eight grams of dehydrated Mueller Hinton agar medium was suspended in 1000 ml

cold distilled water and boiled to dissolve the medium completely. The solution was then

sterilized by autoclaving at 1210C and 15 lbs pressure for 15 minutes. The autoclaved media

was stored at 40C

Appendix �VII

McFarland Turbidity Standard No. 0.5 (Cheesbrough 2010)

McFarland Standard 0.5 are used as a reference to adjust the turbidity of bacterial

suspensions so that the number of bacteria will be within a given range. A 0.5 McFarland

standard is equivalent to a bacterial suspension containing between 1 x 108 and 2 x

108 CFU/ml of E. coli.

Original McFarland standards were mixing specified amounts of barium chloride and sulfuric

acid together. Mixing the two compounds forms a barium sulfate precipitate, which causes

turbidity in the solution. A 0.5 McFarland standard is prepared by mixing 0.05 mL of 1.175%

barium chloride dihydrate (BaCl2�2H2O), with 9.95 mL of 1% sulfuric acid (H2SO4).

Reagents:

McFarland Turbidity Standard No. 0.5

Approximate Formula Per 100 mL Purified Water

Sulfuric Acid, 0.18 M 99.5 mL

Barium Chloride, 0.048 M 0.5 mL

Preparation of 1 % (V/V) solution of Sulphuric acid by adding 1 ml of concentrated sulphuric

acid to 99 ml of water and 1% (W/V) solution of barium chloride was prepared by dissolving

0.5g of dehydrated barium chloride (BaCl2�2H2O) in 50 ml of distilled water in two separate

sterile flasks. Then 99.5 ml of sulphuric acid and o.5 ml of barium chloride were added to the

clean screw capped test tube, mixed well and 5- 10 ml was distributed in sterile capped test

tubes and sealed.

Appendix �VIII Interpretation of ICT

Positive result:

Both presence of C band and T1 band were indicate for presence of anti S. typhi IgM, The

result was positive.

Both presence of C band and T2 band were indicate for presence of anti S. typhi IgG, The

result was positive.

Presence of C band both T1and T2 band were indicate for presence of anti S. typhi IgG and

IgM, The result was also positive.

Negative results:

If only presence of the C band and absence of any color in the both T (T1 and T2) bands

indicate the result was negative.

Invalid result:

If no C band was developed indicate the assay was invalid.

Appendix �IX

Preparation of 0.2% Triton X 100

To make 100 ml 0.2% Triton X 100 required:

Distilled water���������.100ml.

Concentrated Triton X������0.2ml.

Triton X 100 was mixed well with distilled water by inverting the tube 3-5 times and then

stored at room temperature.

Appendix �X

Maintenance and preservation of culture strains

Organisms grown in appropriate medium for 24 hours were preserved in a nutrient agar slant

at 2-80C in a refrigerator and this culture was used for three month. For long-term

preservation selected and identified strains were stored in brain heart infusion broth with 20%

glycerol and stored frozen without significant loss of viability at �200C until further study

(Cheesbrough 2010).

Appendix �XI

Formula for sensitivity

True Positive

Sensitivity = True Positive + False Negative

×100

Formula for Specificity

True Negative

Specificity = True Negative + False Positive

×100

Formula for calculation of sample size:

56.365 0025.0

91391664.0

2)05(.

61.039.02)96.1(

2d

pq2Zn

n= Desired sample size,

p= Prevalence of the disease / problem in

community (incidence of typhoid fever is

3.9/1000),

q= (1-p),

d= Degree of accuracy (it is .05),

z = Confidence interval (usually we take 95%

CI, in which z = 1.96).