1

Comparison between Widal and Immunochromatography

Tests in Detecting of Salmonella Gezira state, Sudan

Rowida Yousif Mohamed Ahmed

B.Sc Medical Laboratories science, University of Science and Technology

(2007)

Postgraduate Diploma in Medical Laboratory, University of Khartoum

(2008)

A Dissertation

Submitted to the University of Gezira in Partial Fulfillment of the

Requirements for the Award of the Degree of Master of Sciences

in

Biosciences and Biotechnology(Biotechnology)

Center of Bioscience and Biotechnology

Faculty of Engineering and Biotechnology

University of Gezira

March 2018

2

Comparison between Widal and Immunochromatography Tests in

Detecting of Salmonella Gezira state, Sudan

Rowida Yousif Mohammed Ahmed

Supervision Committee:

Name Position Signature

Dr : Mutaman Ali Abd Elgadir main-Supervisor .………..

Dr : Attalla Mohammed AttallaCo-supervisor …………

Date :March, 2018

3

Comparison between Widal and Immunochromatography Tests in

Detecting of Salmonella Gezira state, Sudan

Rowida Yousif Mohammed Ahmed

Examination Committee:

Name Position Signature

Dr:Mutaman Ali Abd Elgadir Chairperson ………………

Dr:Salma Osman Mohamed External examiner ………………

Dr:Yasir Mohamed Abdelrehim Internal examiner ………………

Date :22 / 3 / 2018.

4

DECLERATION

Authorized that my dissertation “Comparison Between Widal Test and

Immunochromatography Test in Detection of Salmonella, Gezira state,

Sudan”

By me, under the supervision of Dr. Mutaman Ali Abd Elgadir for the partial

fulfillment for the award of Master degree in Medical Laboratory Sciences in

Microbiology. University of Gezira Faculty of Medical Laboratory Sciences

Department of Microbiology; Wad-Madani, Sudan and this is original and it

was not submitted in part or in full, in any printed or electronic means, and is

not being considered elsewhere for publication .

5

DEDICATION

To my parents with thankful

To my husband and

Children’s

with love

6

ACKNOWLEDAGMENTS

First and foremost, I would like to thanks my advisor Doctor Mutaman Ali

Abd Elgadir, for his guidance, teaching, insightful ideas and long time work.

For all his help and monitoring, I 'm very grateful. I would like to extend my

thanks to my advisor

I am deepest gratitude goes to my family for their unflagging love and support

throughout my life.

I'm indebted my father for his care and love. He had never complying in spite

of all the hardships in his life. I cannot ask to my mother, as she is simply

perfect. I have no suitable words that can fully describe her everlasting love

for me.

I have to give special mention for the unlimited support given

by my husband my kids.

During this work I have collaborated with many colleagues for whom may

have great regard, and I wish to extend warmest thanks to all whose have

helped, support and assisted me, I would like to express my apologies that I

could not mentioned one by one.

7

Comparison between Widal and Immunochromatography Tests in

Detecting of Salmonella Gezira state, Sudan

Rowida Yousif Mohammed Ahmed

M.Sc in Biosciences and Biotechnology (Biotechnology),March (2018)

Abstract

Typhoid fever, caused by Salmonella typhi O and typhi BOis 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. The objective of this study was compare

between widal test and immunochromatography test(ICT) in estimating of Salmonella

species .50 samples were collected from Patients at range 20-80 years old from different

sex . This study was conducted in Gezira state, Wad Madani Great locality . Blood from

patients was collected and tested for widal test to detect the agglutination and titration for

both typhi O and typhi BO antibodies , Beside immunochromatography test(ICT) S.typhi

and S.paratyphi antigens to detect the positive and negative test results for salmonella from

stool sample . The result obtained by widal test typhi O Antibody was 18% suggestive ,

54% positive and 28% negative . Beside the results obtained by typhi BO Antibody was

13% suggestive, 39% positive, 44% negative and 4% doubtful . In ICT test method

according to S.typhi antigen 2% were positive and 98% were negative . Beside in ICT

S.paratyphi antigen 22% were positive and 78% were negative . Comparison between

immunochromatogrphy( ICT) S.typhi antigen and widal test typhi O antibody by chi-

squire statistical analysis showed no significant difference (P. Value 0.651), most of result

obtained by ICT were negative in comparing with that obtained by widal test . But

comparison between immunochromatogrphy( ICT) S.paratyphi antigen and widal test

typhi BO antibody by chi-squire statistical analysis showed positive difference (P. value

0.003) . The study recommend more studies with large sample size and advanced

techniques like culture and PCR should be done .

8

و الاستشراب المناعي اللوني في الكشف عن السالمونيلا مقارنه بين إختبار الفيدال

رويدا يوسف محمد أحمد

2018ماجستير العلوم في العلوم والتقنية البيولوجية )تقنية بيولوجية(, فبراير

ملخص الدراسة

حمى التيفويد الناجمة عن السالمونيلا هي مشكلة صحية رئيسية في كثير من البلدان النامية. وقد ظهر المرض كمرض

معدي في أوائل القرن التاسع عشر الهدف من هذه الدراسة مقارنة بين اختبار فيدال واختبارالاستشراب المناعي اللوني

سنة من أجناس مختلفة . أجريت هذه الدراسة 80-20ضيأعمارهم بين عينة من المر 50فيتشخيص السالمونيلا. تم جمع

في محلية ودمدني الكبري ,ولاية الجزيرة.تم جمع عينات الدم من المرضى واختباره من أجل اختبار فيدال للكشف عن

بالاستشراب ، وعينات البراز و اختبارهاBO والتايفويد Oالتراص والمعايرة لكل من الاجسام المضادة التايفويد

للكشف عن نتائج الاختبار الإيجابية والسلبية للسالمونيلا.كانت BOوالتايفويد Oالمناعي اللوني للمستضدات التايفويد

٪ 28٪ موجبة و 54٪ محتملة ، O 18النتيجة التي تم الحصول عليها من قبل اختبار فيدال للأجسام المضادة التايفويد

٪ 44٪ موجبة، 39٪ محتمله ، 13كانت BOحصل عليها للأجسام المضادة التايفويد سالبة. إلى جانب النتائج التي

٪ كانت سلبية. 98٪ كانت إيجابية و O 2٪ مشكوك فيها. في اختبار الاستشراب المناعي اللوني وفقاللمستضد 4سالبة و

٪ كانت سلبية. أظهرت المقارنة بين اختبارالاستشراب 78٪ كانت إيجابية و BO 22إلى جانب الأجسام المستضد

التايفويد من خلال التحليل الإحصائي عدم O واختبار فيدال في الأجسام المضادةOالمناعي اللوني للمستضد التايفويد

المناعياللوني سلبية ، وكانت معظم النتائج التي حصل عليها من خلال الاستشراب (0.651) وجود فرق معنوي

بالمقارنة مع تلك التي تم الحصول عليها عن طريق الاختبار فيدال . ولكن المقارنة بين الاستشراب المناعي اللوني

بواسطة التحليل الإحصائي أظهرت فرقا كبيرا BOواختبار فيدال في الأجسام المضادة التايفويد BOللمستضد التايفويد

دراسة بإجراء المزيد من الدراسات مع زيادة حجم العينة و استخدام تقنيات متطورة كالتزريع كما توصي ال (0.003 .) .

وسلسلة تفاعل البوليمريز .

9

TABLES OF CONTENT

Page No Content

III Declaration

IV Dedication

V Acknowledgements

VI Abstract English

VII Abstract Arabic

VIII Table of Contents

X List of Tables

XI List of Abbreviations

CHAPTER ONE : INTRODUCTION

1 1.1 General introduction

2 1.2 Justification

2 1.3 Objective

2 1.3.1 General objective

2 1.3.2 Specific objective

CHAPTER TWO: LITERATURE REVIEW

3 2.1 Typhoid Fever

3 2.2 Cause

3 2.2.1 Transmission

4 2.2.2 Bacteria

4 2.3 Signs and symptoms

5 2.4 Diagnosis

5 2.4.1 Rabid Diagnostic Test

6 2.4.2 Widal test

6 2.4.3 Nucleic acid amplification tests

7 2.4.4 Blood Culture

7 2.5 Treatment

8 2.6 Prevention

8 2.7 Vaccination

10

8 2.8 Previous studies

CHAPTER THREE :MATERIALS AND METHODS

10 3.1 Study design

10 3.2 Study area and duration

10 3.3 Study population

10 3.4 Sample size

10 3.4.1 Inclusion criteria

10 3.4.2 Exclusion criteria

10 3.5 Data collection

10 3.6Data analysis

11 3.7 Ethical consideration

11 3.8Laboratory diagnosis

11 3.8.1. Widal test (Salmonella Ab)

11 3.8.1.1 Materials

11 3.8.1.2 Principle

11 3.8.1.3 Procedure

12 3.8.1.4 Interpretation of results

12 3.8.2 Rapid Test Device

12 3.8.2.1 Materials

13 3.8.2.2 Principle

13 3.8.2.3 Procedure

13 3.8.2.4 Interpretation of result

CHAPTER FOUR: RESULTS AND DISCUSSION

15 4.1Results

19 4.2 Discussion

CHAPTER FIVE:CONCLUSION AND RECOMMENDATIONS

20 6.1 Conclusion

20 6.2 Recommendations

21 REFERENCE

11

LIST OF TABLES

ABBREVATIONS

Abs Antibodies

Ag Antigen

ICT Immuno chromatographic test

PCR Polymerase Chain Reaction

BO Salmonella Paratyphi

O Salmonella Typhi

RDTs Rapid Diagnostic tests

Table

No.

TITLE Page

4.1 Distribution of study population according to Gender 15

4.2 Distribution of participant according to Age 15

4.3 Distribution of participant according to Widal O Ab 16

4.4 Distribution of participant according to Widal Bo Ab 16

4.5 Distribution of study population according to ICT O Ab 17

4.6 Distribution of study population according to ICT Bo Ab 17

4.7 ICT O Ab * Widal O Ab Crosstabulation 18

4.8 ICT Bo Ab * Widal Bo Ab Crosstabulation 18

12

W T Widal test

NAATs Nucleic acid amplification tests

SPS Sodium polyanethol sulfonate

CHAPTER ONE

INTRODUCTION

1.1 General Introduction

Typhoid and paratyphoid (enteric) fever are diseases caused by Salmonella

enterica serovar Typhi and Paratyphi A, respectively. Typhoid, the more common infection, is an

important infectious disease in low- and middle-income countries (LMICs) with over 22 million

new cases worldwide and an estimated 200,000 deaths annually (WHO, 2003). South and South-

East Asia are the most affected areas of the world, with an estimated annual incidence in some

areas of greater than 100 people per 100,000 population (Crump et al, 2004). Enteric fever is

common in areas with inadequate sanitation and hygiene, particularly regarding food, water, and

disposal of human excrement, and only to this extent are these diseases tropical (Gill et al, 2011).

Despite advances in technology and public health strategies, enteric fever remains a major cause

of morbidity in the developing world (Bhutta, 2006). Urbanization, global warming, and

traditional methods of waterside living have created even greater demands for clean water in

developing countries (UNICEF 2006). The causative organisms are Gram-negative bacilli that

are transmitted by the faecal-oral route when a person ingests food or water that is contaminated

with infected human faeces. The most important reservoirs of infection are short-term

convalescents or chronic human carriers. Food handlers who are carriers are a particularly

important source of transmission (Gill et al, 2011; Andrews and Ryan, 2015).

The clinical presentation of enteric fever varies from a mild illness with a low-grade

fever, malaise, and slight dry cough to a severe clinical illness with multiple complications

including intestinal perforation (Ismail, 2006). Toxic apathy, blanching 'rose spots' on the trunk,

abdominal organomegaly, and diarrhoea are also associated with enteric fever, but the clinical

picture is highly variable between geographical location and age groups. Enteric fever can

present in many different and non-specific ways, thus posing a diagnostic challenge for the health

professional. Enteric fever is usually diagnosed on clinical grounds and treated presumptively.

13

The diagnosis may be delayed or missed, while other febrile illnesses are being considered (Parry

et al, 2002).

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). Serologic diagnostic tests for

typhoid fever by immuochromatography 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.

There is antimicrobial resistance to S. enterica serovar Typhi and Paratyphi A worldwide

(Kariuki et al, 2015). Health professionals in the tropics overprescribe antimicrobials for many

reasons, including cultural factors and patient expectation (Okeke et al, 2005). The purchase of

drugs such as antimicrobials from untrained vendors and unlicensed pharmacists is common

place in the developing world (Larsson et al, 2008). A major challenge is the inability to confirm

diagnoses in resource-limited settings where traditional laboratory methods of diagnosing enteric

fever are unavailable. Healthcare workers are therefore reliant on their clinical skills to make an

educated guess of the cause of illness or to prescribe an antimicrobial that targets several bacteria,

or both (Shetty, 2008). This over treatment has contributed to increasing resistance to

fluoroquinolones (for example, ciprofloxacin) and multiple drug resistance (resistance to

chloramphenicol, ampicillin, and co-trimoxazole) in S. enterica serovar Typhi and Paratyphi A in

endemic Asian countries (Chuang et al, 2009).

1.2 Justification

Typhoid fever estimated in over 22 million new cases worldwide and cause 200,000

deaths annually (WHO, 2003). Differentiating the common causes of the febrile patient by

clinical criteria is challenging without the laboratory support for blood films, serology, or blood

cultures (Bhutta, 2006). A diagnostic test in such a setting must be cheap, simple to perform, and

able to quickly deliver a result. Such a test should correctly identify true enteric fever cases

among febrile patients, ensuring prompt and specific treatment, allowing the avoidance of broad-

spectrum medication that cover all common causes of fever (Larsson et al, 2008).

1.3 Objectives

1.3.1 General objective

14

To compare between widal test and rabid diagnostic test in estimating of salmonella species .

1.3.2 Specific objective

To perform widal test to detect Salmonella typhi and Salmonella paratyphi .

To perform immuochromatography test to detect Salmonella typhi Salmonella paratyphi .

To compare between the two methods .

To determine the effectiveness of salmonella infection on age groups .

CHAPTER TWO

LITERATURE REVIEW

2.1 Typhoid Fever

In 2000, typhoid fever caused an estimated 21.7 million illnesses and 217,000 deaths

(Crump and Mintz, 2010). It occurs most often in children and young adults between 5 and 19

years old (WHO, 2011). In 2013 it resulted in about 161,000 deaths – down from 181,000 in

1990 (Abubakar et al, 2015). Infants, children, and adolescents in south-central and Southeast

Asia experience the greatest burden of illness (Crump and Mintz, 2010). Outbreaks of typhoid

fever are also frequently reported from sub-Saharan Africa and countries in Southeast Asia

(Muyembe-Tamfum et al, 2009)(Baddam et al, 2012;Yap et al, 2012). Historically, in the pre-

antibiotic era, the case fatality rate of typhoid fever was 10–20%. Today, with prompt treatment,

it is less than 1% (Heymann, 2004). However, about 3–5% of individuals who are infected will

develop a chronic infection in the gall bladder (Levine et al, 1982). Since S. typhi is human-

restricted, these chronic carriers become the crucial reservoir, which can persist for decades for

further spread of the disease, further complicating the identification and treatment of the disease

(Gonzalez-Escobedo et al, 2011). Lately, the study of Typhi associated with a large outbreak and

a carrier at the genome level provides new insights into the pathogenesis of the pathogen (Yap et

al, 2012;Yap et al, 2014).

In industrialized nations, water sanitation and food handling improvements have reduced the

number of cases (Crump et al, 2015). Developing nations, such as those found in parts of Asia

and Africa, have the highest rates of typhoid fever. These areas have a lack of access to clean

water, proper sanitation systems, and proper health care facilities. For these areas, such access to

basic public health needs is not in the near future (Khan et al, 2015).

15

2.2 Cause

2.2.1 Transmission

The bacterium that causes typhoid fever may be spread through poor hygiene habits and

public sanitation conditions, and sometimes also by flying insects feeding on feces. Public

education campaigns encouraging people to wash their hands after defecating and before

handling food are an important component in controlling spread of the disease. According to

statistics from the United States Centers for Disease Control and Prevention (CDC),

the chlorination of drinking water has led to dramatic decreases in the transmission of typhoid

fever in the United States (Marriott and Robertson, 1997).

2.2.2 Bacteria

The cause is the bacterium Salmonella typhi, also known as Salmonella enterica serotype Typhi

(Kishore and Swathi, 2017) .

There are two main types of Typhi namely the ST1 and ST2 based on MLST sub typing scheme,

which are currently widespread globally (Yap et al, 2016) .

2.3 Signs and symptoms

Classically, the course of untreated typhoid fever is divided into four distinct stages, each lasting

about a week. Over the course of these stages, the patient becomes exhausted and emaciated

(Kumar and Kumar, 2016).

In the first week, the body temperature rises slowly, and fever fluctuations are seen with

relative bradycardia (Faget sign), malaise, headache, and cough. A bloody nose (epistaxis) is seen

in a quarter of cases, and abdominal pain is also possible. A decrease in the number of circulating

white blood cells (leukopenia) occurs with eosinopenia and relative lymphocytosis; blood

cultures are positive for Salmonella typhi or S. paratyphi. The Widal test is usually negative in

the first week .

In the second week, the person is often too tired to get up, with high fever in plateau around

40 °C (104 °F) and bradycardia (sphygmothermic dissociation or Faget sign), classically with

a dicrotic pulse wave. Delirium is frequent, often calm, but sometimes agitated. This delirium

gives to typhoid the nickname of "nervous fever". Rose spots appear on the lower chest and

abdomen in around a third of patients. Rhonchi are heard in lung bases.

16

The abdomen is distended and painful in the right lower quadrant, where borborygmi can be

heard. Diarrhea can occur in this stage: six to eight stools in a day, green, comparable to pea soup,

with a characteristic smell. However, constipation is also frequent. The spleen and liver are

enlarged (hepatosplenomegaly) and tender, and liver transaminases are elevated. The Widal test

is strongly positive, with antiO and antiH antibodies. Blood cultures are sometimes still positive

at this stage.

(The major symptom of this fever is that the fever usually rises in the afternoon up to the first and

second week.)

In the third week of typhoid fever, a number of complications can occur:

Intestinal hemorrhage due to bleeding in congested Peyer's patches; this can be very serious, but

is usually not fatal.

Intestinal perforation in the distal ileum: this is a very serious complication and is frequently fatal.

It may occur without alarming symptoms until septicemia or diffuse peritonitis sets in.

Encephalitis

Respiratory diseases such as pneumonia and acute bronchitis

Neuropsychiatric symptoms (described as "muttering delirium" or "coma vigil"), with picking at

bedclothes or imaginary objects.

Metastatic abscesses, cholecystitis, endocarditis, and osteitis

The fever is still very high and oscillates very little over 24 hours. Dehydration ensues, and the

patient is delirious (typhoid state). One-third of affected individuals develop a macular rash on

the trunk.

Platelet count goes down slowly and risk of bleeding rises.

By the end of third week, the fever starts subsiding (Kishore and Swathi, 2017) .

2.4 Diagnosis

Diagnosis is made by any blood, bone marrow or stool cultures and with the Widal test

(demonstration of antibodies against Salmonella antigens O-somatic and H-flagellar). In

epidemics and less wealthy countries, after excluding malaria, dysentery, or pneumonia, a

therapeutic trial time with chloramphenicol is generally undertaken while awaiting the results of

the Widal test and cultures of the blood and stool (Ryan and Ray, 2004) .

2.4.1 Rabid Diagnostic Test

17

Current enteric fever rapid diagnostic tests (RDTs) include a variety of different methods

and formats. RDTs can be applied to blood or urine samples, with blood RDTs (using either

venous or capillary samples, or both) most common. Test formats are based on lateral flow, flow-

through, agglutination, or solid phase methods (Pastoor et al, 2008). RDTs may detect antigens

(components of the causative Salmonella organism) or antibodies (markers of the person's

immune response to the antigen). The type of antibody class or immunoglobulin detected could

be either immunoglobulin-M (IgM), which may be indicative of recent exposure, or

immunoglobulin-G (IgG), which can indicate recent or previous exposure (Baker et al,

2010;Thriemer et al, 2013). Future RDTs are also likely to take a serological approach, although

the identification of novel antigens that are free of cross-reacting epitopes is a major challenge

(Baker et al, 2010).

2.4.2 Widal test

The Widal test (WT) is a serological test that detects agglutinating antibodies to LPS (O

antigen) and flagella (H antigen). The WT is the principal alternative test and is widely used but

is neither sensitive nor specific (Olopoenia and King, 2000). In its original format the WT

required both acute and convalescent-phase serum samples taken approximately 10 days apart.

The test has also been evaluated as a single, acute-phase serum sample (Saha et al, 1996). In

people with enteric fever, titres often rise before the clinical onset, making it very difficult to

demonstrate the diagnostic four-fold rise between initial and subsequent samples (Gill et al,

2011).

The role of the WT is controversial because the sensitivity, specificity, and predictive values

vary considerably between geographical areas (Parry et al, 2002). Test results need to be

interpreted carefully in the light of previous history of enteric fever and vaccination.

Interpretation of the result is also greatly helped by knowledge of the background levels of

antibodies in the local healthy population (House et al, 2001). The increasing use of enteric fever

vaccines and the occurrence of infection with other Salmonella enterica serovars lower the

specificity of the WT (Waddington et al, 2014). Infection with non-Salmonella organisms (for

example, malaria, dengue, brucellosis) also leads to cross-reactivity in the WT in enteric fever-

endemic regions (Olopoenia and King, 2000). There is considerable variation in agglutinin levels

among non-infected populations. These levels are susceptible to change over time and depend on

the degree of endemicity (Parry et al, 2002). Despite these shortcomings of both sensitivity and

specificity, because the WT is simple and inexpensive, it is still widely used as a diagnostic test

(Fadeel et al, 2004).

18

2.4.3 Nucleic acid amplification tests

Nucleic acid amplification tests (NAATs) for enteric fever diagnosis, such as polymerase

chain reaction (PCR), and real-time PCR are being explored. Theoretically, NAATs could

amplify DNA from dead or unculturable bacteria, thus addressing the concern of poor culture

positivity because of pre-treatment with antimicrobials (Wain et al, 2001). One study found that a

novel three-colour real-time PCR technique had the same limitations in test sensitivity as culture

and deemed it an unsuitable methodology for the routine diagnosis of enteric fever (Nga et al,

2010). Methods that combine culture and PCR methods have been also been tested (Zhou and

Polard, 2010). The use of NAATs in developing countries will most likely be limited in the

medium-term because of high cost and the lack of laboratory infrastructure (Olsen et al, 2004).

2.4.4 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).

2.5 Treatment

19

The rediscovery of oral rehydration therapy in the 1960s provided a simple way to

prevent many of the deaths of diarrheal diseases in general.Where resistance is

uncommon, the treatment of choice is a fluoroquinolone such as ciprofloxacin (Parry

and Beeching, 2009;Thaver et al, 2008) Otherwise, a third-generation cephalosporin

such as ceftriaxone or cefotaxime is the first choice (Soe and overturf, 1987;Wallace et

al, 1993) Cefixime is a suitable oral alternative (Bhutta et al, 1994;Cao et al, 1999).

Typhoid fever, when properly treated, is not fatal in most

cases. Antibiotics,suchas ampicillin, chloramphenicol, trimethoprimsulfamethoxazole,

amoxicillin, and ciprofloxacin, have been commonly used to treat typhoid fever in

microbiology (Baron, 1996).

Without treatment, some patients develop sustained fever, bradycardia, hepatosplenomegaly,

abdominal symptoms and, occasionally, pneumonia. In white-skinned patients, pink spots, which

fade on pressure, appear on the skin of the trunk in up to 20% of cases. In the third week,

untreated cases may develop gastrointestinal and cerebral complications, which may prove fatal

in up to 10–20% of cases. The highest case fatality rates are reported in children under 4 years.

Around 2–5% of those who contract typhoid fever become chronic carriers, as bacteria persist in

the biliary tract after symptoms have resolved (WHO, 2017).

2.6 Prevention

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).

2.7 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).

20

2.8 Previous studies

Study to Differentiating both typhoid (Salmonella typhi) and paratyphoid

(Salmonella paratyphi A) infection from other causes of fever in endemic areas. Study found that

thirty-seven studies met the inclusion criteria and included a total of 5080 participants (range 50

to 1732). Enteric fever prevalence rates in the study populations ranged from 1% to 75% (median

prevalence 24%, interquartile range (IQR) 11% to 46%) (Wijedoru et al, 2017).

Study to Comparison of different test methods including polymerase chain reaction for early

and reliable diagnosis of typhoid fever . Study found that 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 ICT (77.4%, 82.3%) using PCR as a gold standard was

calculated (Sultana, 2012).

21

CHAPTER THREE

MATERIALS AND METHODS

3.1 Study Design

Cross sectional and comparative study aimed to detect salmonella species .

3.2 Study area and duration

This study was conducted in wad Madani from May to November 2012.

3.3 Study population

The study were conducted in patient attended hospital for enteritis disease between the ages of

20-80 years were collected the data in the questionnaire .

3.4 Sample size

A total 50 participants were enrolled in this study.

3.4.1 Inclusion criteria

Any patient have symptoms of typhoid fever .

3.4.2 Exclusion criteria

- Asymptomatic individuals .

22

- Individuals under treatment .

- Patient who gave one sample (Blood or stool) .

3.5 Data collection

The data was collected by a structural interview

- questionnaire" age, gender, and result of ICT and Widal test were recorded.

.

3.6 Ethical consideration

Ethical consideration obtained from Ministry of Health, Gezira State.

Permission obtained from wad Madani administration .

Written consent from participant .

Ethical clearance was obtained from the faculty of Medical Laboratory Sciences

Ethical Committee Gezira University in May 2012 .

3.7 Laboratory diagnosis

3.7.1. Widal test (Salmonella Ab)

3.7.1.1 Materials

S.typhi O antigen suspension .

S.paratyphi BO Antigen suspension

Slide test method : stop watch , variable micropipette, mixing sticks, microscope and centrifuge .

3.7.1.2 Principle

Enteric fever occur when pathogenic micro-organism like S.typhi, S.paratyphi A, S.paratyphi

B, S.paratyphi C infect the human body . During the course of disease the body respond to the

antigenic stimulus by producing antibodies whose titer rise slowly in early stages , to a maximum

and then slowly falls till it is undetectable . Antibodies to salmonella organisms may be detected

in the patient serum from the second week after onset of infection . Information regarded the titer

and whether or not they are rising or falling can be obtained by performing serological tests using

23

spectrum salmonella antigen suspension . Usually tube titers of 1:80 and above are taken as

diagnostically significant , however for endemic areas cut-offs may need to be establish .

3.7.1.3 Procedure

A. Reagent were brought to room temperature before using .

B. Antigens were shaken and mixed well before dispensing .

Slide screening method

1. One drop were placed of positive control onto a reaction circle of the glass slide .

2. One drop were placed (50 ul) of isotonic saline onto the next reaction circle of the glass slide .

3. One drop were placed (50 ul) of patient serum to be tested onto each of the required number of

reaction circle .

4. One drop were added of appropriate spectrum salmonella antigen suspension to the reaction

circle containing positive control and isotonic saline .

5. One drop were added of appropriate spectrum salmonella antigen suspension to the reaction

circle containing patient serum .

6. Contents were mixed of each circle uniformly over the entire circle with separate mixing sticks .

7. The slide were rocked gently back and forth , and observe for agglutination macroscopically at

one minute .

Slide semi-quantitative method

1. placed 80ul, 40ul, 20ul, 10ul and 5ul of patient serum was tested on 5 different reaction circle on

the glass slide . The corresponding titers obtained were 1:20, 1:40, 1:80, 1:160, 1:320

respectively .

2. Steps Number 5-7 of slide screen method were followed .

3.7.1.4 Interpretation of results

Slide screening method

Agglutination is a positive test result and indicate presence of the corresponding antibody in the

patient serum .

No agglutination is a negative test result and indicate absence of the corresponding antibody in

the patient serum .

Slide semi-quantitative

24

Agglutination is a positive test result . The titer of the patient serum correspond to the viable

agglutination in the test circle with the smallest amount of serum sample (Cruickshank,

1984;Felix 1942).

3.7.2 Rapid Test Device

3.7.2.1 Materials

Specimen collection container .

Micropipette .

Timer .

3.7.2.2 Principle

The S.typhi and S.paratyphi rapid test device (feces) has been designed to detect S.typhiand

/or S.paratyphi through visual interpretation of color development in internal strip. Anti S.typhi

and Anti S.paratyphi monoclonal antibodies are immobilizedon the respective of the

nitrocellulose (NC) membrane. A fecal sample is added to the sample diluted buffer which is

optimized to extract the S.typhi and S.paratyphi antigen from specimen , During testing,the

extracted antigen , if present will bind to anti S.typhi and /or S.paratyphi antibodies conjugated

to colored particles on the sample pad. As the specimen migrates along the strip by capillary

action and interact with reagent on the NC membrane ,the complex will be captured by anti

S.typhiand /or S.paratyphi antibodies at the detection zone. The presence of a colored band

indicates a positive result, while it absence indicate a negative result. EeA red band at the control

region (C) serves a procedural control, indicating that the proper volume of specimen has been

added and membrane wicking is working.

3.8.2.3 Procedure

Bring tests, specimens, buffer and/or control at room temperature (15-30°C) before use.

1. The test device removed from its sealed pouch, and placed on a clean, level surface. The device

were labeled with patient or control identification. For best results, the assay should be perform

within one hour .

2. The provided disposable pipette were using to transferd small amount of stool (approximately

25ul) and 2 drop of buffer to the sample, then start the timer .

Avoid trapping air bubble in the specimen well , and do not add any solution to the result area.

25

At the best being to wark, color was migrate across the membrane .

3. The result were readed in 15 minutes. The result should not interpret after 20 minutes .

3.7.2.4 Interpretation of result

IgG+IgM positive: One red band appears in the control region (C), and two others red band

appear in both IgG region and IgM region. The shade of color may vary from pink to purple, but

it indicates a positive result even with a faint line.

IgG Positive: One red band appears in the control region (C), and another red band in the IgG

region. The shade of color may vary from pink to purple, but it indicates a positive results even

with a faint line.

IgM Positive: One red band appears in the control region (C), and another red band in the IgM

region. The shade of color may vary from pink to purple, but it indicate a positive result even

with a flint line .

Negative: Only one red brand appears in the control region (C), and no band appears either on

the IgG region or IgM region.

Invalid: No red brand appears in the control region (C), whether a test band(s) is present or not.

Repeat invalid test with a new sample, new test device and reagent. Insufficient sample volume,

inaccurate operating procedure or expired tests may yield an invalid result. Contact your local

distributor if the problem continues (Christie, 1969).

3.8 Data analysis

Data were analyzed by statistical package for social sciences (SPSS) version 22

26

.CHAPTER FOUR

RESULTS AND DISCUSSION

4.1 Results

The study population found that 21(45.5%) were males and 25(54.3%) were females (Table 4.1).

The study population found that 31(67.4%) age between 20 – 40Year , 11(23.9%) between 41 –

60Year and 4(8.7%) between 61 – 80Year . These result reflect that prevelance high in young

people(Table 4.2) .

The study population found that 8(17.4%) suggestive for enterica (titer 1/160) , 25(54.3%)

significant for enterica (titer 1/320) and 13(28.3%) were negative(Table 4.3).

The study population according to BO Ab found that 6(13%) suggestive for enterica (titer

1/160) , 18(39.1%) significant for enterica (titer 1/320) , 20(43.5%) were negative and 2(4.3%)

Doubtful (titer 1/80) (Table 4.4).

27

Table (4.1) : Distribution of study population according to Gender

Gender Frequency Percent% Valid Percent% Cumulative Percent

Valid Male 21 45.7 45.7 45.7

Female 25 54.3 54.3 100.0

Total 46 100.0 100.0

28

Table (4.2) : Distribution of study population according to age

Age group Frequency Percent% Valid Percent% Cumulative Percent

Valid 20 – 40 Year 31 67.4 67.4 67.4

41 – 60 Year 11 23.9 23.9 91.3

61 – 80 Year 4 8.7 8.7 100.0

Total 46 100.0 100.0

29

Table (4.3) : Distribution of study population according to Widal O Ab :

Widal O Ab Frequency Percent% Valid Percent% Cumulative Percent

Valid Suggestive 8 17.4 17.4 17.4

Significant 25 54.3 54.3 71.7

Negative 13 28.3 28.3 100.0

Total 46 100.0 100.0

30

Table (4.4) : Distribution of study population according to Widal BO Ab

Widal Bo Ab Frequency Percent% Valid Percent% Cumulative Percent

Valid Suggestive 6 13.0 13.0 13.0

Significant 18 39.1 39.1 52.2

Negative 20 43.5 43.5 95.7

Doubtful 2 4.3 4.3 100.0

Total 46 100.0 100.0

31

The study population according to ICT O Ag found that 1(2.2%) positive , 45(97.8%) negative

for enteric (Table 4.5).

The study population according to BO Ag found that 10(21.7%) were positive for enterica ,

36(78.3%) were negative for enteric(Table4.6) .

The study population according to O Ab by widal test in correlation with O Ag by ICT showed

that most of patient negative by ICT, were positive by widal test(Table 4.7) .

The study population according to BO Ab by widal test in correlation with BO Ag by ICT

showed that most of patient negative by ICT, were positive by widal test(Table 4.8) .

32

Table (4.5) : Distribution of study population according to ICT O Ag .

ICT O Ab Frequency Percent% Valid Percent% Cumulative Percent

Valid Positive 1 2.2 2.2 2.2

Negative 45 97.8 97.8 100.0

Total 46 100.0 100.0

33

Table (4.6) : Distribution of study population according to ICT BO Ag

ICT Bo Ag Frequency Percent% Valid Percent% Cumulative Percent

Valid Positive 10 21.7 21.7 21.7

Negative 36 78.3 78.3 100.0

Total 46 100.0 100.0

34

Table (4.7) : ICT O Ag Widal O Ab Crosstabulation

Widal O Ab

Total P. Value Suggestive Significant Negative

ICT O Ag Positive 0 1 0 1

.651 Negative 8 24 13 45

Total 8 25 13 46

35

Table (4.8) : ICT BO Ag Widal BO Ab Crosstabulation

Widal Bo Ab

Total P. Value Suggestive Significant Negative Doubtful

ICT BO Ag Positive 2 8 0 0 10

.007 Negative 4 10 20 2 36

Total 6 18 20 2 46

36

4.2 Discussion

Typhoid fever is one of the most common infectious disease in developing

countriesincluding 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

study50 individual 25(50%) were Males and 25(50%) were Females, their age range from 20-

80years with mean 50year : 33(66%) 20-40year, 12(24%) 41-60year and 5(10%) 61-80year . The

result obtained by widal test O Antibody was 20% suggestive , 50% significant and 30%

negative . Beside the results obtained by BO Antibody was 16% suggestive, 40% significant, 40%

negative and 4% doubtful . In ICT test method according to O antigen 2% were positive and 98%

were negative . Beside in ICT BO antigen 24% were positive and 76% were negative.

Comparison between ICT O antigen and widal test O antibody by chi-squire statistical

analysis showed no significant difference (P. Value 0.458), most of result obtained by ICT were

negative in comparing with that obtained by widal test . But comparison between ICT BO antigen

and widal test BO antibody by chi-squire statistical analysis showed significant difference (P.

value 0.002) and this study was similar to study done by Sultana, 2012 who found thatout of 23

culture positive samples, 14 were positive by the ICT (IgM). Out of 127 cultures negative cases

92 were positive by ICT (IgM).ICT has been studied in many countries and they found

significantly higher sensitivity and specificity (Jesudason, Esther and Mathai 2002; Pastooret al.

37

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 Mathai2002).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.

CHAPTER FIVE

CONCLUSIONS AND RECOMMENDATIONS

.

5.1 Conclusions

These study concluded that there were no difference in the detection of salmonella O antibody

using Widal test and ICT, but there were statistically different in detection of salmonella BO

antibody by widal test and ICT.

5.2 Recommendations

Focus on using a antigen detection method rather than antibody based techniques in salmonella

diagnosis.

Health education to improve personal hygiene in order to reduce salmonella prevalence.

More studies with large sample size.

Advanced techniques like culture and PCR should be done.

38

REFERENCE

Abubakar, I.I., Tillmann, T. and Banerjee, A., (2015). Global, regional, and national

age-sex specific all-cause and cause-specific mortality for 240 causes of death,

a systematic analysis for the Global Burden of Disease Study

2013. Lancet,Vol. 385 (9963):117-171.

Ananthanarayan, R and Panikar, CKJ (1999), in Textbook of Microbiology, Chennai,

Orient Longman, pp. 244-249.

Andrews, J.R. and Ryan, E.T., (2015). Diagnostics for invasive Salmonella infections:

current challenges and future directions. Vaccine, 33:8-15.

Baddam, R., Kumar, N., Thong, K.L., Ngoi, S.T., Teh, C.S.J., Yap, K.P., Chai, L.C.,

Avasthi, T.S. and Ahmed, N., (2012). Genetic fine structure of a Salmonella

enterica serovar Typhi strain associated with the 2005 outbreak of typhoid fever

in Kelantan, Malaysia. Journal of bacteriology,Vol. 194(13):3565-3566.

Baker, S., Favorov, M. and Dougan, G., (2010). Searching for the elusive typhoid

diagnostic. BMC infectious diseases, Vol.10 (1):45.

Baron, S., 1996. Epidemiology--Medical Microbiology. University of Texas Medical

Branch at Galveston.

Bhutta, Z.A., (2006). Current concepts in the diagnosis and treatment of typhoid

fever. BMJ: British Medical Journal,Vol. 333(7558):78.

39

Bhutta, Z.A., Khan, I.A. and Molla, A.M., (1994). Therapy of multidrug-resistant

typhoid fever with oral cefixime vs. intravenous ceftriaxone. The Pediatric

infectious disease journal,Vol. 13(11) :990-993.

Bhutta, ZA (2008), Salmonella in Nelson Textbook of peadiatric, vol.1, 18th edition,

Kliegman, Behrman, Jenson, Stanton editors, India, Saunders, Elsevier,

pp.1182-1191.

Bhutta, ZA and Mansurali, N (1999), Rapid serologic diagnosis of paediatric typhoid

fever in an endemic area: a prospective comparative evaluation of two dot-

enzyme immunoassays and the Widal test, American Journal of Tropical

Medicine and Hygiene, 61: 654-657.

Cao, X.T., Kneen, R., Nguyen, T.A., Truong, D.L., White, N.J. and Parry, C.M.,

(1999). A comparative study of ofloxacin and cefixime for treatment of typhoid

fever in children. The Dong Nai Pediatric Center Typhoid Study Group. The

Pediatric infectious disease journal,Vol. 18(3):245-248.

Christie, A.B., (1969). Infectious diseases: epidemiology and clinical practice. Infectious

diseases: epidemiology and clinical practice.

Chuang, C.H., Su, L.H., Perera, J., Carlos, C., Tan, B.H., Kumarasinghe, G., So, T.,

Van, P.H., Chongthaleong, A., Hsueh, P.R. and Liu, J.W., (2009).

Surveillance of antimicrobial resistance of Salmonella enterica serotype Typhi

in seven Asian countries. Epidemiology & Infection,Vol. 137(2):266-269.

Cruickshank, J.G., (1984). The investigation of salmonella outbreaks in hospitals. Journal

of Hospital Infection,Vol. 5(3):241-243.

Crump, J.A. and Mintz, E.D., (2010). Global trends in typhoid and paratyphoid

fever. Clinical Infectious Diseases,Vol. 50(2):241-246.

Crump, J.A., Luby, S.P. and Mintz, E.D., (2004). The global burden of typhoid

fever. Bulletin of the World Health Organization,Vol. 82(5):346-353.

Crump, J.A., Sjölund-Karlsson, M., Gordon, M.A. and Parry, C.M., (2015).

Epidemiology, clinical presentation, laboratory diagnosis, antimicrobial

resistance, and antimicrobial management of invasive Salmonella

infections. Clinical microbiology reviews, Vol.28(4):901-937.

Fadeel, M.A., Crump, J.A., Mahoney, F.J., Nakhla, I.A., Mansour, A.M., Reyad, B.,

El Melegi, D., Sultan, Y., Mintz, E.D. and Bibb, W.F., (2004). Rapid

40

diagnosis of typhoid fever by enzyme-linked immunosorbent assay detection of

Salmonella serotype typhi antigens in urine. The American journal of tropical

medicine and hygiene,Vol. 70(3):323-328.

Felix, A., (1944). Technique and interpretation of the Weil-Felix test in typhus

fever. Transactions of the Royal Society of Tropical Medicine and

Hygiene,Vol. 37(5):321-341.

Fraser, A., Goldberg, E., Acosta, C.J., Paul, M. and Leibovici, L., (2007). Vaccines for

preventing typhoid fever. Cochrane Database Syst Rev, Vol.3(3).

Gasem, MH, Dolmans, WM, Isbandrio, BB, Wahyono, Keuter, HM, Djokomoeljanto,

R et al. (1995), Culture of Salmonella typhi and Salmonella paratyphi from

blood and bone marrow in suspected typhoid fever, Tropical Geographical

Medicine, 47: 164-167.

Gill, G.V. and Beeching, N. eds., (2011). Lecture Notes: Tropical Medicine (Vol. 67).

John Wiley & Sons.

Gill, N., Wlodarska, M. and Finlay, B.B., (2011). Roadblocks in the gut: barriers to

enteric infection. Cellular microbiology,Vol. 13 (5):660-669.

Gonzalez-Escobedo, G., Marshall, J.M. and Gunn, J.S., (2011). Chronic and acute

infection of the gall bladder by Salmonella Typhi: understanding the carrier

state. Nature Reviews Microbiology,Vol. 9(1):9-14.

Heymann, D.L., (2004). Control of communicable diseases manual 18th Edition.

House, D., Wain, J., Ho, V.A., Diep, T.S., Chinh, N.T., Bay, P.V., Vinh, H., Duc, M.,

Parry, C.M., Dougan, G. and White, N.J., (2001). Serology of typhoid fever

in an area of endemicity and its relevance to diagnosis. Journal of clinical

microbiology, Vol.39(3):1002-1007.

Ismail, TF, (2006). Rapid Diagnosis of Typhoid Fever. Indian Journal of Medical

Research ;Vol.123(4):513-6.

Jenkins, C and Gillespie, SH (2009), Salmonella Infections in Gordon, C, Cook and

Zumla, AI editors, in Manson.s Tropical Diseases, 22nd edition, China,

Saunders, Elsevier, pp. 931-952.

Kariuki, S., Gordon, M.A., Feasey, N. and Parry, C.M., (2015). Antimicrobial

resistance and management of invasive Salmonella disease. Vaccine, 33:21-29.

Khan, M.I., Pach, A., Khan, G.M., Bajracharya, D., Sahastrabuddhe, S., Bhutta, W.,

Tahir, R., Soofi, S., Thapa, C.B., Joshi, N. and Puri, M.K., (2015). Typhoid

41

vaccine introduction: An evidence-based pilot implementation project in Nepal

and Pakistan. Vaccine, 33:62-67.

Kishore, P. and Swathi, T. (2017), International Journal of Current Research In Health

And Biological Sciences.

Kumar P, Kumar R, (2016). "Enteric Fever". Indian Journal of

Pediatrics.doi:10.1007/s12098-016-2246-4.

Larsson, M., Kronvall, G., Chuc, N., Karlsson, I., Lager, F., Hanh, H., Tomson, G.

and Falkenberg, T., (2000). Antibiotic medication and bacterial resistance to

antibiotics: a survey of children in a Vietnamese community. Tropical Medicine

& International Health,Vol. 5(10):711-721.

Levine, M.M., Black, R.E. and Lanata, C., (1982). Precise estimation of the numbers of

chronic carriers of Salmonella typhi in Santiago, Chile, an endemic area. The

Journal of infectious diseases, Vol.146(6):724-726.

Lewis, MJ (1997), Salmonella, in Medical Microbiology, Greenwood, D, Slack, R and

Pebtherer, J, editors, 15th edition, ELST, USA, pp. 252-261.

Marathe, S.A., Lahiri, A., Negi, V.D. and Chakravortty, D., (2012). Typhoid fever &

vaccine development: a partially answered question. The Indian journal of

medical research, Vol.135(2):161.

McKinnon, L.R. and Karim, Q.A., (2014). Host-pathogen Interactions: Honing in on

enteric fever. eLife, 3:35-45.

Miller, IS and Pegues, DA (2000), Salmonella species, including Salmonella typhi, in

Mandell, Douglar and Bennetts principles and practice of infectious disease,

Mandell, GL, Bennett, JE, Dolin, R, editors, 5th edition, Philadelphia, Churchill

Livingstone, pp. 2344-2263.

Muyembe-Tamfum, J.J., Veyi, J., Kaswa, M., Lunguya, O., Verhaegen, J. and

Boelaert, M., (2009). An outbreak of peritonitis caused by multidrug-resistant

Salmonella Typhi in Kinshasa, Democratic Republic of Congo. Travel medicine

and infectious disease, Vol.7(1):40-43.

Nga, T.V.T., Karkey, A., Dongol, S., Thuy, H.N., Dunstan, S., Holt, K., Campbell, J.I.,

Chau, T.T., Chau, N.V.V., Arjyal, A. and Koirala, S., (2010). The sensitivity

of real-time PCR amplification targeting invasive Salmonella serovars in

biological specimens. BMC infectious diseases,Vol. 10(1):125.

Okeke, I.N., Laxminarayan, R., Bhutta, Z.A., Duse, A.G., Jenkins, P., O'Brien, T.F.,

Pablos-Mendez, A. and Klugman, K.P., (2005). Antimicrobial resistance in

42

developing countries. Part I: recent trends and current status. The Lancet

infectious diseases,Vol. 5(8):481-493.

Olopoenia, L.A. and King, A.L., (2000). Widal agglutination test− 100 years later: still

plagued by controversy. Postgraduate medical journal,Vol. 76(892):80-84.

Olsen, S.J., Pruckler, J., Bibb, W., Thanh, N.T.M., Trinh, T.M., Minh, N.T.,

Sivapalasingam, S., Gupta, A., Phuong, P.T., Chinh, N.T. and Chau, N.V.,

(2004). Evaluation of rapid diagnostic tests for typhoid fever. Journal of clinical

microbiology,Vol. 42(5):1885-1889.

Parker, MT (1990), enteric infections: typhoid and paratyphoid fever, in Topley and

Wilson.s Microbiology and Microbial infections, 8th edition. Collies, L, Balows,

A, Susman, M. Arnold, editors, London, Vol. 3, pp. 423-446.

Parry CM, Hien TT, Dougan G, White NJ, Farrar JJ, (2002). Typhoid Fever. New

England Journal of Medicine;Vol.347(22):1770-1782.

Pastoor, R., Hatta, M., Abdoel, T.H. and Smits, H.L., (2008). Simple, rapid, and

affordable point-of-care test for the serodiagnosis of typhoid fever. Diagnostic

microbiology and infectious disease,Vol. 61(2):129-134.

Saha, S.K., Ruhulamin, M., Hanif, M., Islam, M. and Khan, W.A., (1996).

Interpretation of the Widal test in the diagnosis of typhoid fever in Bangladeshi

children. Annals of tropical paediatrics,Vol. 16(1):75-78.

Shetty P. (2008) .Antibiotic resistance: frequently asked questions.

www.scidev.net/global/health/feature/antibiotic-resistance-frequently-asked-

questions.

Soe, G.B. and Overturf, G.D., (1987). Treatment of typhoid fever and other systemic

salmonelloses with cefotaxime, ceftriaxone, cefoperazone, and other newer

cephalosporins. Reviews of infectious diseases, Vol.9(4):719-736.

Sultana, S., (2012). Comparison of different test methods including polymerase chain

reaction for early and reliable diagnosis of typhoid fever (Doctoral dissertation,

M. Phil.

Thaver, D., Zaidi, A.K., Critchley, J.A., Azmatullah, A., Madni, S.A. and Bhutta, Z.A.,

(2008). Fluoroquinolones for treating typhoid and paratyphoid fever (enteric

fever). Cochrane Database Syst Rev, 4.

Thriemer, K., Ley, B., Menten, J., Jacobs, J. and van den Ende, J., (2013). A

systematic review and meta-analysis of the performance of two point of care

43

typhoid fever tests, Tubex TF and Typhidot, in endemic countries. PLoS

One,Vol. 8(12):81263

Waddington, C.S., Darton, T.C. and Pollard, A.J., (2014). The challenge of enteric

fever. Journal of Infection,Vol. 68:S38-S50.

Wain, J., Bay, P.V.B., Vinh, H., Duong, N.M., Diep, T.S., Walsh, A.L., Parry, C.M.,

Hasserjian, R.P., Ho, V.A., Hien, T.T. and Farrar, J., (2001). Quantitation of

bacteria in bone marrow from patients with typhoid fever: relationship between

counts and clinical features. Journal of clinical microbiology,Vol. 39(4):1571-

1576.

Wallace, M.R., Yousif, A.A., Mahroos, G.A., Mapes, T., Threlfall, E.J., Rowe, B. and

Hyams, K.C., (1993). Ciprofloxacin versus ceftriaxone in the treatment of

multiresistant typhoid fever. European Journal of Clinical Microbiology &

Infectious Diseases, Vol.12(12):907-910.

Watson, KC (1978), Laboratory and Clinical Investigation of Recovery of Salmonella

Typhi from Blood, Journal of Clinical Microbiology, Vol. 7 ( 2):122-126.

World Health Organization (2003). Background document: the diagnosis, treatment and

prevention of typhoid fever (WHO/V&B/03.07). Geneva, Switzerland: World

Health Organization.

World Health Organization, (2011) . the diagnosis, treatment and prevention of typhoid

fever (WHO/V&B/03.07) . Geneva, Switzerland: World Health Organization.

World Health Organization, (2017) . treatment of typhoid fever

(WHO/V&B/03.07) . Geneva, Switzerland: World Health Organization.

Yap, K.P., Gan, H.M., Teh, C.S.J., Chai, L.C. and Thong, K.L., (2014). Comparative

genomics of closely related Salmonella enterica serovar Typhi strains reveals

genome dynamics and the acquisition of novel pathogenic elements. BMC

genomics,Vol. 15(1):1007.

Yap, K.P., Ho, W.S., Gan, H.M., Chai, L.C. and Thong, K.L., (2016). Global MLST of

Salmonella Typhi revisited in post-genomic era: genetic conservation,

population structure, and comparative genomics of rare sequence

types. Frontiers in microbiology, 7.

Yap, K.P., Teh, C.S.J., Baddam, R., Chai, L.C., Kumar, N., Avasthi, T.S., Ahmed, N.

and Thong, K.L., (2012). Insights from the genome sequence of a Salmonella

enterica serovar Typhi strain associated with a sporadic case of typhoid fever in

Malaysia. Journal of bacteriology, Vol.194(18):5124-5125.

44

Zhou, L. and Pollard, A.J., (2010). A fast and highly sensitive blood culture PCR method

for clinical detection of Salmonella enterica serovar Typhi. Annals of clinical

microbiology and antimicrobials,Vol. 9(1):14.

Zwadyk, P (1992), Enterobacteriaceae: Salmonella and Shigella, Intestinal pathogens, in

Joklik, WK, Willet, HP, Amos, B and Wilfert, CM editors, Zinsser

Microbiology, 20th editon, USA, Appleton and Lange, pp. 556-565.

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Questionnaire of salmonella study

Rowida Yousif Mohammed Ahmed

ID:…………………………………………………

Patient name: ………………………………………………………………………………………

Age: ……………………………………….

Locality : ……………………………………..

Phone :………………………………………

Onset of symptoms :………………………..

Duration of symptoms :…………….

Other chronic disease : …………………………..

Endoscope : yes no

Biopsy : yes no

Had salmonella before : yes no

Blood sample :

Stool sample :

Other sample mention :………………………….