Comparison of Widal Test In Diagnosis of Typhoid Fever ...

1

Comparison of Widal Test In Diagnosis of Typhoid Fever with

Blood Culture and Immuno chromatographic Test among

Patients in Wad Medani Teaching Hospital, Gezira State, Sudan

(2012-2017)

Tayallah Ali Tayallah Yousif

B. Sc. Faculty of Environmental and Health Sciences- University of Khartoum (1985)

M.Sc. in Environmental Health (Malaria - Epidemiology) Faculty of Medicine-

University of Gezira- (2004)

A Thesis

Submitted to the University of Gezira in Fulfillment of the

Requirement for the award of the Degree of Doctor of

Philosophy

In

Epidemiology (Epidemiology of Communicable Diseases)

Department of Epidemiology

Faculty of Health and Environmental Sciences

Feb 2018

2




(2012-2017)


Supervision Committee:

Date: 13 / 2 / 2018

Name Position Signature

Dr. Imadeldin Eljack Suleiman Main Supervisor

…………….……………………..………

Prof. Bakri Yousif Mohamed Nour Co. Supervisor …………….………………...……………

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(2012-2017)


Examination Committee:

Date of Examination: 13 / 2 /2018

Name Position Signature

Dr. Imadeldin Eljack Suleiman Chairperson …………….……………………..………

Prof. External Examine …………….………………...……………

Prof. Internal Examiner …………….………………………………

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Dedication

To my mother

My father

My family

My brothers and my sisters

5

Acknowledgement

I would like to convey my cordial thanks to my main supervisor Dr.

Imadeldin Eljack Suleiman, and my Co-supervisor Prof. Bakri Yousif

Mohamed Nour, whom paid much attention and careful consideration in

supervising my study.

I would like to extend my thanks and gratefulness to Miss Rehab, laboratory

staff from Blue Nile National Institute for Communicable Diseases and the staff

of the main laboratory at Wad – Medani Teaching Hospital, Fatimah, Rimaz,

and suhair.

I would like to thank Fatimah Osman Elkagm and Osman sheikh Idrees and

all staff of the epidemiology department Ministry of Health Gezira State.

I wish to thanks all those whom assist me by any means until I Should

completed my study successfully.

I wish them all good health.

6




(2012-2017)

Tayallah -Ali Tayallah Yousif

Abstract

Typhoid fever is a major health problem in developing countries and its diagnosis on clinical

ground is difficult .In developing countries including Sudan, the diagnosis mostly done by

Widal test. However, the value of the test has been debated. Hence, evaluating the result of

this test is necessary for correct interpretation of the result; the main objective of this study

was to compare the result of Widal test with blood culture test and immuno chromato graphic

test (ICT) in the diagnosis of typhoid fever in febrile patients. Blood samples were collected

from 237 febrile patients with symptoms clinically similar to typhoid fever and visiting Wad-

Medani Teaching Hospital from mid November 2012 to mid December 2012.Blood culture

test was used to isolate S. typhi, slide agglutination test and tube agglutination test were used

for the determination of antibody titer. An antibody titere of 1:320 was taken as a cut off

value to indicate infection of typhoid fever. Immune chromate graphic test (ICT) also was

used to confirm the infection of Typhoid fever by positive IgG and IgM. The total

participants in the study were 237, of which females were 126 (53.2%) while males were 111

(46.8%).Data were analyzed by using statistical package for social science (SPSS), and the

results revealed that, the Positive cases of Salmonella Typhi identified by hospital Widal test

slide method were 47 (19.8 %) and the Negative cases were 190 (80.2 %). The Positive cases

of Salmonella Typhi identified by study Widal test slide method were 132 (55.7 %) and the

Negative cases were 105 (44.3 %). The Positive cases of Salmonella Typhi identified by

study Widal test tube method were 62 (26.2 %) and the Negative cases were 175 (73.8). The

Positive cases of Salmonella Typhi identified by study ICT were 58 (24.5 %) and the

Negative cases were 179 (75.5 %). All cases (237) diagnosed by study Culture test were

Negative for Salmonella Typhi.Widal test has a low sensitivity, (26.5%, 19.4%), for Widal

test slide and Widal test tube, respectively, and low prevalence rate 55.7%,26.2%

respectively, and high specificity 88.6%,80% respectively. The blood culture test was the best

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test for diagnosis of typhoid fever rather than Widal test. Widal agglutination test should not

be used for diagnostic method for typhoid fever.

8

مناعة د مع تزريع الدم واختبار يحمي التيفوئ تشخيص ( فيWidal test) فيدلاختبار مقارنهالسودان ,ولاية الجزيرة , التعليمي بمستشفى مدني المرضى( وسط ICT ) اللون التخطيطي

2012-2017 تاي الله علي تاي الله يوسف

ملخص الدراسة

لبصد ا الامميدح ويةدتش تلهيةدرم فدييييما دي البصد ا الامميدح وماردم الفدو ا حمي التيفوئد يدي ملد صح ةدحيح يئيفديح دي ا

( تمد مامشلدترم وتتتبدي مرمدح widalعصدي ييدح حدم ييميدح الاهتبدمي ) يد ) ولايح الجزيية ( يدت تلهيةدرم لملبدما بمهتبدمي

ع اهتبددمي تزييددع الدد والفحدد الفددييع لتففدديي الاتيجددح ل والردد ي اليئيفددي لردد ه ال يافددح يددو مصمياددح اتيجددح حدد الددو ا مدد

(ICT لحمددا التيفوئدد وفددض المي ددا و. الحمددا ل تدد جمددع عدد )عياددح مدد المي ددا الدد ي ح دديوا لمفتلددفي 237

، 2012حتدا ماتةدي يفدمبي 2012و م اي ويعيا ر تضمبق يعياض حمي التيفوئد دي الفتدية مد ماتةدي ادو مبي

الفددملموايت تددميفي واهتبددمي تدديا اللددييحح واهتبددمي تدديا اتابددوش يفددتتم لتح يدد عيددمي اهتبددمي تزييددع الدد يفددتم لتددز

يدد عصدددي ا ةدددمبح بحمددا التيفوئددد ، اهتبدددمي مامعددح الصدددو التهضيضدددي 320: 1الجفدد الم دددم ، عيددمي الجفددد الم دددم

مددار ا اددم عدد و 237ال يافددح وبصددع عدد الملددمي ي ددي IgMو IgGيفددتتم لتي يدد التدد و بحمددا التيفوئدد بميجمبيددح

الحزمددح الاحةددمئيح لصتصدددو تدد تحصيدد البيماددم بمفددته ا بياددمم %( 8ل46) 111الدد وي بيامددم عدد %( 2ل53) 123

( ويظري الاتدمئ ن الحدملا الايجمبيدح لصفدملموايت التميفيدح التدي تد عزلردم بمهتبدمي تديا اللدييحح ) SPSS)الاجتممعيح

%( ل والحملا الايجمبيح لصفملموايت التميفيدح التدي تد 2ل80) 190%( والحملا الفملبح 8ل19) 47( ي بمفتلفي م اي

%( ل والحددملا 3ل44) 105%( والحددملا الفددملبح يددي 7ل55) 132) ال يافددح ( يددي عزلرددم بمهتبددمي تدديا اللددييحح

175%( والحددددملا الفدددددملبح 2ل26) 62ابدددددوش يددددي الايجمبيددددح لصفددددملموايت التميفيدددددح التددددي تدددد عزلردددددم بمهتبددددمي تدددديا ات

58يددي ICT)%( ل الحددملا الايجمبيددح لصفددملموايت التميفيددح التددي تدد عزلرددم بمهتبددمي مامعددح الصددو التهضيضددي )8ل73)

( التدددي تددد حةدددرم بمهتبدددمي التزييدددع ماددد فدددملبح 237%( ل ددد الحدددملا )5ل75) 179%( والحدددملا الفدددملبح 5ل24)

ضييصددح اهتبددمي اللدديائ يدد ا % ، لاهتبددمي 4ل19% ، 5ل26ت التميفيددح ل اهتبددمي و ا ل يددي حفمفدديح ماهف ددح لصفددملمواي

% عصدددي التدددوالي و شدددح بافدددبح 2ل26% ، 7ل55عصدددي التدددوالي ، ومتددد ااتلدددمي مددداهفض يددد ا وضييصدددح اهتبدددمي يابدددوش

v

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يد حمدا التميفويد مد اهتبدمي اهتبمي تزييع الد يدو ات د دي حد % عصي التوالي ل80% , 6ل88

ل ال . يجش الا يفتتم

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List of Contents

Page No. Contents

i Supervision Committee ……………………………………………………………………

ii Examination Committee …………………………………………………………………..

iii Dedication…………………………..………………………………….………….…………

iv Acknowledgement…………………………..……………………………………….………

v English Abstract……………..……………………..……………………..………….……

v Arabic Abstract……………..……………..……………………..………………………

vii List of content……………..……………………..……………………..……………………

xi List of Tables……………..……………………..……………………..………..………….

xiii List of Figures ……………………………..…………………..…………………..………

xiv List of Abbreviations ……………………………..……………………..…………………..

Chapter One: Introduction

1

1.1 General Introduction…………………………..……………………………………..

5 1.2 Rationale…………………………..……………………………………..……………

6 1.3 Research Objectives…………………………..……………………………………..…

6 1.3.1 General Objectives…………………………..……………………………………..…

6 1.3,2 Specific Objectives…………………………..…………………………………….

Chapter Two: Literature Review

7 Literature Review

7 2.1 Epidemiology of Typhoid Fever

8 2.2 Diagnosis of Typhoid fever

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8 2.2.1 Culture

Page No. Content

9 2.2.2 Widal test

10 2.2.3 Immuno Chromatographic Test

10 2.3 Previous Studies of Typhoid Fever

13 2.4 Treatment of Typhoid Fever 13 2.4.1 Treatment at Home

14 2.4.2 Staying off Work or School

14 2.4.3 Hospital Treatment

14 2.4.4 Relapses

14 2.4.5 Long-term Carriers

15 2.4.6 Antibiotics

15 2.5 Typhoid Fever Prevention

16 2.5.1 Vaccine

16 2.5.2 Personal Prevention

17 2.6 Incubation Period

17 2.6.1 Public Health Significance and occurrence

17 2.6.2 Reservoir

17 2.6.3 Communicability Period

17 2.6.4 Susceptibility

17 2.6.5 Sensitivity and Specificity

Chapter Three

18 Materials and Methods

18 3.1 Study area and Period

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Page No. Content

18 3.2 Study Design

19 3.3 Study Population

19 3.4 Sample Size

20 3.5 Sample Technique

20 3.6 Data Collection

21 3.7 Data Analysis

21 3.8 Research Ethics

21 3.9 Quality Controls

23 3.2 Materials and Methods

23 3.2.1 Sterilization Procedure

23 3.2.1.1 Hot Air Oven

23 3.2.1.2 Autoclaving

23 3.2.1.3 Ultra-Violet Light

23 3.2.2 Disinfection

23 3.2.3 Solutions

23 3.2.3.1 Thioglycolate broth

23 3.2.3.2 Normal Saline

23 3.2.3.3 Peptone Water

24 3.2.4 Identification of the Isolates

24 3.2.4.1 Microscopic Examination

24 3.2.4.2 Gram’s Stain

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Page No. Content

24 3.2.4.3 Biochemical Test

24 3.2.4.3.1 Indol production Test

24 3.2.4.3.2 Oxidase Test

24 3.2.4.3.3 UreaseTest

24 3.2.4.3.4 Citrate Utilization Test

24 3.2.4.3.5 Voges -Proskauer

25 3.2.4.3.6 Methyl- Red (MR) Test

25 3.2.4.3.7 Kilglers Iron Agar(KIA)

25 3.2.5 Widal Test

25 3.2.5.1 Widal Test Slide Method

25 3.2.5.2 Widal Test Tube Method

25 3.2.6 Immuno chromatographic Test (ICT)

26 3.2.7 Blood Culture

26 3-2-7-1 Morphological and Biochemical Tests

Chapter four

27 Results and Discussions

Chapter Five

58 5.1 Conclusions

58 5.2 Recommendations

59 References

Appendices

63 Appendix (1)

67 Appendix (2)

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List of Tables Page No. Title Table No.

3 Distribution of cases of typhoid fever in Gezira State -Sudan in the years 2007- 2011 1.1

7 Prevalence rate of typhoid fever in Sudan per 1000000 populations in the years 2011-

2014

2.1

7

Prevalence rate of typhoid fever in Gezira State per 1000000 populations in the years

2012-2014

2.2

11 Distribution of Salmonella serotypes among schistosomiasis patients In an endemic area

in Sudan

2.3

11 Comparison between Culture method and Widal test in diagnosis of typhoid fever 2.4

31 Distribution of the participants by food hygiene At Wad-Medani Teaching Hospital -

Gezira state - Sudan -year 2012.

4.1

31 Distribution of the participants by Sewage system -Gezira state - Sudan -year 2012. 4.2

32 Distribution of participants by refuse collection -Gezira state - Sudan -year 2012. 4.3

33 Distribution of participants by symptoms At Wad-Medani Teaching Hospital-Gezira

state - Sudan -year 2012.

4.4

34 Relationship between the sex and the tests among the participants At Wad-Medani

Teaching Hospital-Gezira state - Sudan -year 2012

4.5

35 Relationship between the education and the tests among the participants At Wad-Medani

Teaching Hospital-Gezira state - Sudan -year 2012.

4.6

36 Relationship between the occupation and the tests among the participants At Wad-

Medani Teaching Hospital-Gezira state - Sudan -year 2012.

4.7

37 Relationship between the water supply and the tests among the participants At Wad-


4.8

38 Relationship between the daily meals at home and the tests among the participants At

Wad-Medani Teaching Hospital-Gezira state - Sudan -year 2012

4.9

39 Relationship between the daily meals at market and the tests among the participants At

Wad-Medani Teaching Hospital-Gezira state - Sudan -year 2012.

4.10

40 Relationship between the Sustained fever 39°-40 °and the tests among the participants At

Wad-Medani Teaching Hospital-Gezira state - Sudan -year 2012.

4.11

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43 Relationship between the headache and the tests among the participants At Wad-Medani


4.14

44 Relationship between the Loss of appetite and the tests among the participants At Wad-


4.15

45 Relationship between the rash of flat and the tests among the participants At Wad-


4.16

50 Comparison between the study widal test tube method and the hospital Widal test slide

method at Wad Medani Teaching Hospital – Gezira state - Sudan -year 2012.

4.17

51 Comparison between ICT and study Widal test slide method at Wad Medani Teaching

Hospital – Gezira state - Sudan -year 2012.

4.18

52 Comparison between the ICT and study Widal test tube Method at Wad Medani

Teaching Hospital – Gezira state - Sudan -year

4.19

53 Comparison between the study Widal test slide method and the hospital Widal test slide

method in Wad Medani Teaching Hospital – Gezira state – Sudan-year 2012

4.20

54 Comparison between the study widal test tube method and The hospital Widal test slide

method at Wad Medani Teaching Hospital –, Gezira state - Sudan - year 2012.

4.21

55 Comparison between ICT and hospital Widal test slide method at Wad Medani Teaching

Hospital – in Wad -Medani Teaching Hospital, Gezira state - Sudan - year 2012.

4.22

56 Distribution of cases by sex at Wad - Medani Teaching Hospital - Gezira state - Sudan –

year

4.23

.

Page No. Title Table No.

41

Relationship between the feel weak and the tests among the participants At Wad-Medani


4.12

42 Relationship between the stomach pains and the tests among the participants At Wad-


4.13

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List of Figure

Page No. Title Figure No.

27 Distribution of participants by sex in Wad - Medani Teaching Hospital, Gezira state -

Sudan - year 2012

4.1

27 Distribution of participants by age group in Wad -Medani Teaching Hospital, Gezira

state - Sudan - year 2012

4.2

28 Distribution of the participants by resident in Wad - Medani Teaching Hospital,

Gezira state - Sudan - year 2012

4.3

28 Distribution of participants by education level in Wad -Medani Teaching Hospital,


4.4

29 Marital status of participants in Wad -Medani Teaching Hospital, Gezira state -

Sudan - year 2012

4.5

29 Distribution of participants by occupation In Wad -Medani Teaching Hospital, Gezira


4.6

30 Distribution of participants by Income/month S.P in Wad -Medani Teaching

Hospital, Gezira state - Sudan - year 2012

4.7

30 Distribution of participants by Water supply In Wad -Medani Teaching Hospital,


4.8

46 Results of Study Widal test Slide method in Wad Medani Teaching Hospital, Gezira

state - Sudan –- year 2012

4.9

46 Results of Study Widal test Tube method in Wad -Medani Teaching Hospital, Gezira


4.10

47 Results of Study ICT Results in Wad -Medani Teaching Hospital, Gezira state -

Sudan - year 2012

4.11

47 Results of Study Culture test in Wad -Medani Teaching Hospital, Gezira state -

Sudan - - - year 2012

4.12

48 Results of widal, ICT, and culture tests in Wad -Medani Teaching Hospital, Gezira

state - Sudan - -year 2012

4.13

48 Results of Hospital Widal test slide method in Wad Medani Teaching Hospital,

Gezira state - Sudan – year 2012

4.14

49 Results of widal, ICT, and culture tests in Wad -Medani Teaching Hospital, Gezira


4.15

17

List of Abbreviations

April Apr.

Degree of Freedom D.F

Ethylene Diamine Tetra-acetic Acid EDTA

Hour h.

Immuno chromatographic Test ICT

Immunoglobulin Gama IgG

Immunoglobulin Moue IgM

Kilglers Iron Agar KIA

Sample Size N

Number No.

October Oct.

Plasmodium P

Salmonella S

September Sep.

Sudanese Pounds SP.

Statistical Package for Social Science SPSS

True Negative Rate TNR

True Positive Rate TPR

United State US

World Health Organization WHO

Negative -ve

Positive +ve

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CHAPTER ONE

INTRODUCTION

1.1General Introduction

History of Typhoid fever:-

Typhoid fever known as enteric fever ,caused by Salmonella enterica S.typhi was related to

Greek typhus or smoke ,cloud that believed to cause disease , (Rehman , 2015)

Emperor Augustus 2000 year ago was a case of typhoid fever, treated with cold baths by

Antonius Musa, a Roman physician .First description of typhoid fever in 1659 by Thomas

Willis. The name “typhoid fever proposed by Pierre Charl Alexander Louis who was French

physician.

The clear differentiation between typhus fever and typhoid was firstly discovered by William

Wood Gerhard in1837. The typhoid bacillus discovered by Carl Joseph Eberth in 1880. The

Widal agglutination reaction of the blood was described by Georges Widal in 1896. The

famous typhoid carrier was “Typhoid Mary”, her name was Mary Mallon, she was a cooker

in Oyster Bay in New York in 1906, she was infecting 53 people, and five of them died

(Deepika etal, 2004).

Various agglutination tests have been developed & which the Widal method is the oldest &

remains the most widely used. The test was first introduced by F. Widal in 1896.

Laboratories in industrialized countries have stopped performing the assay. In Africa the

Widal test is still widely used, because typhoid fever is perceived to be endemic in the area

and the Widal test is the only rapid diagnostic assay that is available & affordable. The Widal

test is commonly performed when children and adults present with fever to treatment centers,

as few centers have the capacity to perform micro-bacterial culture. Despite this wide-spread

use, little has been published on its performance in Africa (BanooS, 2006).

Definition of Typhoid Fever:-

Typhoid fever is a bacterial infection caused by drinking or eating contaminated water or

food with bacterium Salmonella Typhi, it is a common disease in the developing countries

with poor sanitation, sewage system and low standard of hygiene. (Kumar etal, 2009). `

19

It is a contagious disease caused by the bacteria Salmonella typhi. It is known as enteric fever

or typhoid fever. It is difficult to differentiate clinically between Typhoid fever and

paratyphoid fever, jointly called enteric fever. Usually spread via contaminated food and

water supplies and close contact with infected cases. The disease is described by very high

fever, sweating, gastroenteritis, and diarrhea. Although typhoid is very rare in the developed

world, it is a serious public health problem, threat the developing world. Typhoid is treatable

disease. (Altman etal, 1994).

Typhoid fever is a bacterial infection caused by Salmonella typhi, which cause salmonella

food poisoning (Bayram etal, 2014).

Typhoid fever was one of the infectious diseases, it occurred usually in summer season,

characterized by inflammation of the intestines and ulceration that caused by Salmonella

Typhi, which enter the intestines through contaminated food and/or water (Houghton, 2005).

It is an acute infectious disease caused by Salmonella typhi and characterized by a continued

fever, physical and mental depression, rose-colored spots on the chest and abdomen, and

diarrhea. Also called enteric fever. (Parent etal, 2016).

Infectious Agent and Transmission:-

Salmonella typhi was the causative agent of typhoid fever which infects humans only.

Paratyphoid and enteric fevers are caused by other species of Salmonella, which infect

domestic animals as well as humans. The transmission of typhoid bacillus is occurred by

consumption of contaminated food or water. Occasionally, direct fecal–oral transmission may

occur. Contaminated areas are an important source of infection, transmission also occurs

through eating raw fruit and vegetables fertilized by human excreta and through ingestion of

contaminated milk and milk products. Flies may cause human infection through transfer of

the infectious agents to foods. Contamination of water sources by S.typhi may cause

epidemics of typhoid fever when large numbers of people use the same source of drinking-

water. Higher risk of typhoid fever occurs in countries or areas with low standards of hygiene

and shortage of potable- water supply facilities for human consumsion. (WHO, 2017).

S. typhi is spread through contaminated food, drink, or water. If a person eat or drink

something that is contaminated with the bacteria, the bacteria enter the intestines, and then

into the blood. The bacteria pass via the blood to your lymph nodes, gallbladder, liver, spleen,

and other parts of the body.

20

The carriers of S. typhi carry on to emission the bacteria in their stools spreading the

disease for years; Typhoid fever is common in developing countries.

Symptoms or clinical presentation

The symptoms of typhoid fever start with, fever (39.5º or 103º F), feeling-ill, abdominal pain,

sever diarrhea happened when the disease become desirable. In some cases a rash appeared

on the abdomen and the chest, called rose spots. There is an other symptoms include.

Abdominal tenderness, agitation, bloody stools, Chills, confusion, nosebleeds, severs fatigue,

weakness, these symptoms occurred after one to three weeks from the exposure to the

causative agent. (Harris etal, 2015).

Later Stage:-

Untreated cases of typhoid may become delirious, lie motionless, exhausted with eyes half-

closed which is known as the typhoid state, the complications that were threatening the life

often developed at this stage. (CDC, 2015).

Burden of Disease:-

Table NO. (1.1) distribution of the cases of typhoid fever in Gezira State -Sudan in the

years 2007- 2011.

Year Cases Deaths Prevalence rate

2007 10518 9 303/100000 (Epidemiology,2007)

2008 14485 3 405/100000 (Epidemiology,2008)

2009 21123 4 573/100000 (Epidemiology,2009)

2010 27013 7 712/100000 (Epidemiology,2010)

2011 41498 7 1161/100000 (Epidemiology,2011)

The estimated global burden of typhoid fever was 20.6million cases, typhoid fever remains

one of the most serious public health problems in Gezira State causing high morbidity,

approximately 42000 cases per year (Epidemiology,2012-2014), and it was endemic

21

throughout Sudan (Epidemiology report Sudan,2010-2014). And 223000 deaths in low-

income and middle-income countries (Danish, 2005)).

Salmonella was an aerobic bacterium, gram negative bacilli. (Danish,2005).Typhoid fever

one of the water-borne diseases (WHO, 2014). Typhoid fever it was an acute illness caused

by infection with the bacterium Salmonella Typhi, characterized by fever, headache,

constipation, malaise, chills and muscles pain, and diarrhea is uncommon and un sever

vomiting. Death may occur in sever cases. The disease is transmitted through contaminated

drinking water or food. Epidemics related to fecal contamination of water supplies or foods

sold on the streets. (John , 2017).

Salmonella serotype Typhi (S. typhi), the causative agent of typhoid fever, was calculated to

have caused approximately 200000 deaths globally in 2000. The clinical picture of typhoid

fever is nonspecific; confirmed diagnosis through blood culture requires expensive and

laboratory intensive isolation and identification of the organism, which may take up to seven

days. A cheap and rapid alternative laboratory test is desirable, especially for developing

countries settings where typhoid fever is major public health burden (Ley etal, 2010).

Typhoid fever is the one of the infectious disease caused by salmonella typhi, it is known as

enteric fever and commonly typhoid, clinically it is indistinguishable disease; it is spread by

contaminated food, water and close contact with the infected persons (WHO, 2015).

There were 22 studies identified Regions according to the incidence of typhoid fever.

Regions with high incidence (>100/100000 cases/year) include south-central Asia and south-

east Asia. Medium incidence (10-100/100000 cases/year) includes the rest of Asia, Africa,

Latin America, Caribbean and Oceania. The rest of the developed World have low incidence

(<10/100000 cases/year).

1) Typhoid fever caused approximately 21650974 cases and 216510 deaths during the year

2000 (WHO, 2004). The burden of Typhoid fever in low and middle income countries in

the year 2010 after adjusting of water was 11.9 million cases and 129000 deaths, by

comparison with unadjusting water were cases was 20.6 million and 223000 deaths

(Bayram etal, 2014).

2) In the year 2010 there were 13.5 million cases of typhoid fever (Bayram etal, 2014).

22

World wide each year occur 22 million cases of typhoid fever and 200000 deaths, and 6

million cases of paratyphoid fever (Anna etal, 2016).

Typhoid fever is a bacterial infection caused by drinking or eating contaminated water or

food with bacterium Salmonella Typhi, it is a common disease in the developing countries

with poor sanitation, sewage system and low standard of hygiene. (Kumar etal,2009).

It is a contagious disease caused by the bacteria Salmonella typhi. It is known as enteric fever

or typhoid fever. It is difficult to differentiate clinically between Typhoid fever and

paratyphoid fever, jointly called enteric fever. Usually spread via contaminated food and

water supplies and close contact with infected cases. The disease is described by very high

fever, sweating, gastroenteritis, and diarrhea. Although typhoid is very rare in the developed

world, it is a serious public health problem, threat the developing world. Typhoid is treatable

disease. (Altman etal, 1994).

Typhoid fever is a bacterial infection caused by Salmonella typhi, which cause salmonella

food poisoning (Bayram etal, 2014).

Originally the diagnosis of typhoid fever (enteric fever) is clinically. The reported

sensitivities of tests for S. typhi vary greatly in the literature, even among the most recent

articles and respected journals. (John etal, 2017)

1.2 Rationale:

• Typhoid Fever is a major health problem in developing countries including Sudan

(Gezira State).

• The diagnosis on clinical ground is difficult.

• In developing countries the diagnosis mostly done by Widal test.

• The value of the Widal test has been debated.

• Hence, evaluating the result of this test is necessary for correction the interpretation of

the result and diseases surveillance.

Justification:

Typhoid fever is the one of the most prevalence diseases in Gezira state in the last five years,

2007-2011 and till now, the prevalence of Typhoid fever in the year 2007 was 303 cases per

100000 populations (303/100000), and increase until reaches 1161 cases per 100000

populations in the year 2011, and the prevalence in the years 2012, 2013, 2014, was 1078,

1013and1050 cases per 100000 populations respectively. (State ministry, 2007-2014)

23

The prevalence of Typhoid fever could not be precisely calculated because of different unit’s

registration system and the method used for diagnosis is a single Widal test rather than

culture, some cases it is not notified to the state Ministry of Health. So all the cases of

Typhoid fever notified to the state ministry of health consider as suspected cases, most of the

health units (governmental and non governmental) not notified to the state ministry of health,

in addition to this, there is a loss or misuse of drugs. However similar studies of endemic

diseases have done, so we can make a scientific calculation of the level of the prevalence. For

malaria it was estimated in the study (Testing the sensitivity and specificity of the

fluorescence microscope (Cyscope) for malaria diagnosis) (Ley etal, 2010).

as being 20%, it is not likely to be very much different from such estimate, but we can make

an absolute prevalence 20± 1%, this given arrange of 18-22 and by taking the geometric

mean of the range we have the prevalence of Typhoid fever as 19%.

The diagnosis of typhoid fever is confirmed by culture of salmonella enteric Serotype Typhi

(S. typhi).

According to the last knowledge there was no similar study conducted in the same area to

measure related finding.

The study can help in choosing better diagnosis for typhoid fever

However, a more rapid, simple, and cheaper diagnostic method would be very useful

especially in developing countries. The Widal test is widely used in Africa but little

information exists about its reliability.

1 -3 Research objectives:

1.3.1 General objective:

To assess the reliability of the Widal test compared to blood culture, ICT among adults in

Wad Medani teaching hospital.

1.3.2 Specific objectives:

1- To estimate and compare the prevalence of typhoid fever among adults using Widal test,

ICT and blood culture.

2- To estimate the sensitivity and specificity of the Widal test among adults in Wad Medani

teaching hospital.

Research Hypotheses:-

The blood culture test is the best diagnostic method for typhoid fever rather than Widal and

ICT test.

24

CHAPTER TWO

Literature Review

2.1 Epidemiology of Typhoid Fever

Throughout Africa and Asia the disease is endemic and continues in the Middle East, a few

Southern and Eastern European countries and central and South America. A recent study

estimated that approximately 22 million cases of typhoid fever occurred each year with at

least 200000 deaths. However, the real magnitude is difficult to measure, because clinical

picture is confused with many other febrile diseases, and most typhoid fever endemic areas

had lack facilities to confirm the diagnosis.( Pietro etal , 2005).

Table NO. (2.1) Prevalence rate of typhoid fever in Sudan per 1000000 populations in

the years 2011-2014.

(Federal, 2014)

Table NO. (2.2) Prevalence rate of typhoid fever in Gezira State per 100000 populations

in the years 2012-2014.

Year No of cases No of deaths Total

population

Prevalence

Rate

2012 42055 0 3900440 1078

2013 40408 0 3986250 1013

2014 42806 0 4073948 1050

(State, (2012-2014)

Year No. of cases No. of

deaths

Total

population

Prevalence

Rate

2011 154481 3 35720328 432

2012 149842 7 36825080 406

2013 137920 29 37964000 363

2014 143678 7 39102920 367

25

2.2 Diagnosis of Typhoid Fever:-

2.2.1 Culture: - Originally the diagnosis of typhoid fever (enteric fever) is clinically. The

reported sensitivities of tests for S. typhi vary greatly in the literature, even among the most

recent articles and respected journals.

The principle diagnosis of typhoid fever has long been culture isolation of the organism.

Cultures are widely considered 100% specific.

Blood, intestinal secretions (vomitus or duodenal aspirate), and stool culture results are

positive for S. typhi in approximately 85%-90% of patients with typhoid fever who present

within the first week of onset. They decline to 20%-30% later in the disease course. In

particular, stool culture may be positive for S. typhi several days after ingestion of the

bacteria secondary to inflammation of the intraluminal dendritic cells. Later in the illness,

stool culture results are positive because of bacteria shed through the gallbladder. (John etal,

2017)

The diagnosis of typhoid fever based on isolation of Salmonella from a patients, generally by

blood culture ,which is not available in the areas where the disease is endemic. The Tube

Widal agglutination test is the alternative, which demonstrates the presence of somatic (O)

and flagella (H) agglutinins to Salmonella in the patient’s serum using O and H antigen

suspension; it is commonly used for diagnostic of typhoid fever in developing countries. In

the recent past, slide Widal test was developed and now is the most commonly used

technique in local laboratories because of its convenience, 300 blood samples of patients

were diagnosed by tube Widal test and slide Widal test using the available Salmonella

antigen test kits. From the 300 samples studied, 17(5.7%) serum samples were positive by

Widal slide test method, while 25(8.7%) were positive by Tube test method. The study

shows, little difference in the results of the two methods. As a conclusion, the Tube Widal

test is more sensitive and specific than slide Widal test, but the slide method is good

alternative test, because less time consuming, easy to perform, affordable cost and can be

applied in poor areas. ( Sood etal, 2014).

26

2.2.2 Widal test

The Widal test is a hypothetical serological test for enteric fever or undulant fever, the

bacteria causing typhoid fever are mixed with serum containing specific antibodies obtained

from an infected individual. In case of Salmonella infections, it is an evidence of the presence

of O-somatic false-positive result. Test results need to be explained carefully according to the

past history of enteric fever, typhoid vaccination, and the general level of antibodies in the

populations in endemic areas of the world. Typhidot is the other test used to discover the

diagnosis of typhoid fever. As with all serological tests, the rise in antibody levels needed to

perform the diagnosis takes 7–14 days, which limits it applicability in early diagnosis. Other

means of diagnosing Salmonella Typhi (and ParaTyphi) include cultures of blood, urine and

faeces, these organisms produce H2S from thiosulfate and can be easily identified on

differential media such as, Bismuth sulfite agar.

Often 2-mercaptoethanol is added to the Widal test. This agent more easily denatures the

IgM class of antibodies, so if a decrease in the titer is seen after using this agent, it means

that the contribution of IgM has been removed leaving the IgG component. This

differentiation of antibody classes is important; as it allows for the distinction of a recent

(IgM) from an old infection (IgG).

The Widal test is positive if TO antigen titer is more than 1:160 in an active infection, or if

TH antigen titer is more than 1:160 in past infection or in immunized persons. A single

Widal test is of little clinical relevance due to the number of cross reacting infections,

including malaria. If no other tests (either bacteriologic culture or more specific serology)

are available, a fourfold increase in the titer (e.g., from 1:40 to 1:160) in the course of the

infection, or a conversion from an IgM reaction to an IgG reaction of at least the same titer,

would be consistent with a typhoid infection.

A new serological test is called the Tubex test is not superior than the Widal test and even

the Widal test gives better performance results, so the Tubex test is not recommended for

diagnosis of typhoid fever.( Bakr etal,2017).

27

2.2.3 Immuno chromatographic Test:-

The test was done to detect IgM, IgG or combined IgM/IgG in serum in first week of fever

for the diagnosis of typhoid fever. ICT can be done for diagnosis of typhoid fever by a single

blood sample in the first week of illness.ICT is a more sensitive and specific test which is

easy to perform and more reliable as compared to the Widal test and it is useful in early

therapy (Hassan etal, 2013).

2.3 Previous Studies:-

The Widal test was conduct in Gezira Sate in Sudan, for 114 normal individuals.

Salmonella typhi O agglutinins were found at a titer of 1:320 in 12(10.5%) of them. None

of these individuals had a history of typhoid fever or vaccination. The following points

was emerged:-

1- Normal healthy people in the Sudan have a high antibody titer of Salmonella typhi.

2- The Widal test in the Sudan should be interpreted against this background.

3 - Diagnostic titer of 1:160 for Salmonella typhi results in high false Positive results.

4) A titer above 1:320 is suggested as diagnostic for Salmonella typhi. (El-shafie ,1991)

A study was conducted in Northwest Ethiopia for Malaria, Typhoid Fever and their co-

infection among febrile patients at Rural Health Center. It is across- sectional studies on

200 Health Center, blood sample were collected for blood culture, Widal test, and blood

film preparation. Results; Malaria prevalence was 36.5 %( n =73).Among these

32(43.8%) were positive for P.falciparm, 30 (41.1%) were positive for P.vivax, and

11(15.1%) were mixed infection. The seroprevalence of typhoid fever was 38(19%), but 1

(0.5%) with blood culture. Malaria typhoid fever co infection was 13 (6.5%).Poor hand

washing habit were significantly associated with typhoid infection (P<0.05). As a

conclusions the prevalence malaria and Typhoid fever was found high. (Meseret, 2014).

A cross sectional descriptive study was conducted between November 2005 and May

2006 in El Managil Locality, Gezira State, Sudan, the goal of the study to determine the

presence of typhoid and paratyphi Salmonella Among schistosomiasis patients. A study

samples was 203, samples of urine, stool, and blood were collected and examined based

on standard methods. Of the 203 samples, 50 (24.6%) were diagnosed with schist soma,

where 42(20.7%) with Schist soma hematobium and 8 (3.9%) with Schist soma mansoni

infection.

28

One of these, Salmonella Species were detected in 30 (60%) cases, of which Salmonella typhi

represented by 63.3%, Salmonella Paratyphi A (16.7%), Salmonella Paratyphi B 16.7% and

Salmonella Paratyphi C 3.3%. Based on the culture results 30 cases were positive for enteric

fever, Widal test was positive in 12 cases, with a sensitivity of 40% and specificity of 75%.

Of the widal positive cases, titers of 1:160, 1:320, and 1:640 were detected in 58.3, 33.3 and

8.3% of samples, respectively. In areas endemic with schistosomiasis, enteric fever was

linked with schistosomiasis, which requires investigation of both infections concomitantly

Table NO. (2.3) Distribution of Salmonella serotypes among schistosomiasis patients In

an endemic area in Sudan.

Type of infection Number of

Salmonella

Isolate

Salmonella Serotype

S. typhi S. typhi A S. Typhi

B

S. typhi C

schist soma

haematobium (n = 42) 23 16 3 4 0

schist soma Mansoni

(n=8) 7 3 2 1 1

Total 30 19

(63.3%)

5

(16.7% )

5

(16.7% )

1

(3.3 %)

Table NO. (2.4) Comparison between Culture method and Widal test in diagnosis of

typhoid fever.

Number of cases Culture Widal test

15 -ve -ve

18 +ve -ve

12 +ve +ve

5 -ve +ve

Total (n=50) 30/50 (60%) 17/50 (34%)

(AJMR, 2015).

29

A study conducted in South-East Asia to evaluate the prevalence of malaria and

Typhoid.A503 samples of blood were collected from patients suspected for Malaria and

Typhoid, A microscopic examination were done , the results, 158 patients male (69%) were

found positive ,followed by 71 patients female (31%).The prevalence rate was 45.5%,the

prevalence was high.(Bisht etal , 2015).

A comparative study conducted in Ethiopia one of the developing countries, the value of the

Widal test has been debated, for correct interpretation the test must be evaluated. Blood

samples for 270 were collected and examined for typhoid fever in St.Pauls General

Specialized Hospital from mid December 2010 to March 2011.Blood culture was done to

isolate Salmonella Typhi, slide and tube agglutination tests were used for the determination of

antibody titer. An antibody titer of ≥ 1:180 for anti TO and ≥ 1:160 for antiTH were taken as

a cut off value to indicate recent infection of typhoid fever.168 of the participants (68.9%)

were females and 84(31.1%) were males.7 (2.6%) cases of Salmonella Typhi were identified

with the total prevalence of typhoid fever 4.1%.The sensitivity and specificity of Widal test

were 71.4%, 68.4% respectively. As a conclusion, Widal test has a low sensitivity and

specificity but it was good in indication for the absence of the disease. (Gizachew etal, 2014)

A study of Typhoid fever was conducted in five Asian countries, China, India, Indonesia,

Pakistan and Viet Nam, study sites where Typhoid was considered as a health problem by

local authorities, A total of 21874 febrile person of fever were detected, Salmonella typhi was

isolated from 475(2%) by blood cultures, the annual incidence of typhoid fever varied from

site to an other, being high in India and Pakistan, intermediate in Indonesia, and low in China

and Viet Nam.The burden of disease in force policy decisions to take action to control this

disease. (WHO, 2008).

A study was conducted in India to determine the baseline titer of antibodies among healthy

persons and to define the cutoff titer. A total of 500 blood samples were diagnosed, among

those 260 samples were positive for agglutinins. 141 and 163 samples were positive for O

and H agglutinins of Salmonella typhi respectively. Among the 141samples the baseline titer

for 30 samples was 1:20, 100 and 25 samples the baseline titer was 1:40, 1:80 respectively.

Among 163 samples the baseline titer for 18, 120, and 21 samples was 1:20, 1:40 and 1:80

respectively. As a conclusion, the baseline titer for Salmonella typhi must be ≥ 1:160. (Jey,

2017).

30

A study conduct in Nigeria about Widal agglutination test at Tertiary Hospital. Using Widal

Agglutination test, a blood sample of 271 febrile persons were diagnosed, 124(45.8%) were

positive, 60(22.1%) blood samples grew Salmonella on blood culture, and 55(20.3%) blood

samples showed a co – infection of typhoid fever and malaria, a sensitivity of 35%,

specificity of 51% were observed for Widal test. As a conclusion Widal agglutination test is

not a valid diagnostic method for typhoid fever while co-infection with malaria parasite is

confirmed in typhoid fever infection. Malaria parasitemia is associated with positive titers on

Widal test. (Osahon etal, 2016)

2.4 Treatment of Typhoid fever:-

Typhoid fever can be treated with a course of antibiotic, most cases can be treated at home

but if the condition is sever, admition may be required.

2.4.1 Treatment At Home:-

If it diagnosed in its early stages, a course of antibiotic tablets may be needed for a patient,

most people need to take these for 7 to 14 days.

In South East Asia some strains of Salmonella Typhi have developed a resistance to one or

more types of antibiotics. Any blood, stool or urine samples have taken for the diagnosis

must be tested in a laboratory to determine the type of the strain, for appropriate treatment

with antibiotics. ( Rajive , 2012).

Symptoms should begin to improve within two to three days of taking antibiotics. However,

it is very important to finish the course, so to make sure the bacteria are completely removed

from your body. ( Rajive , 2012).

Make sure you rest, drink plenty of fluids and eat regular meals. You may find it easier to eat

smaller meals more frequently, rather than three larger meals a day.

You should also maintain good standards of personal hygiene, such as regularly washing your

hands with soap and warm water, to reduce the risk of spreading the infection to others.

In a small number of cases, the symptoms or infection may reoccur. This is known as a

relapse. (Rajive , 2012).

31

2.4.2 Staying Off Work or School:-

Many cases being treated for typhoid fever can return to work or school as soon as they start

to feel better.

The exclusions of these cases who work with food and vulnerable people, such as children

under five, the elderly and those in poor health.

In these cases, you or your child should only return to work or nursery after tests on three

stool samples taken at 48-hour intervals have shown that the bacteria are no longer present.

2.4.3 Hospital Treatment:-

Hospital admission is usually recommended if you have severe symptoms of typhoid fever,

such as persistent vomiting, severe diarrhea or a swollen stomach. As a precaution, young

children who develop typhoid fever may be admitted to hospital. In hospital, you will

have antibiotic injections and you may also be given fluids and nutrients directly into a

vein through an intravenous drip. (Rajive , 2012).

2.4.4 Relapses:-

Some treated cases of typhoid fever experience a relapse, in which the symptoms usually

return around a week after antibiotic treatment has finished. The symptoms are usually

milder and last for a short time than the original illness, but further treatment with antibiotics

is usually recommended. (Rajive, 2012).

2.4.5 Long-term carriers:-

After the symptoms disappeared, you should have another stool test to check if there are still

Salmonella typhi bacteria in your faeces. If there are, you may have become a carrier of the

typhoid infection, and you may need to have a further 28-day course of antibiotics to "flush

out" the bacteria.

Until test results show that you are free of bacteria, avoid handling or preparing food. It is

also very important that you wash your hands thoroughly after going to the toilet (Rajive,

2012).

http://www.nhs.uk/Conditions/Typhoid-fever/Pages/Symptoms.aspx

32

2.4.6 Antibiotics:

Antibiotics are medications are used to treat and in some cases prevent bacterial infections

(GOV, 2015). Several antibiotics are effective for the treatment of typhoid fever.

Chloramphenicol was the original drug of choice for many years. Because of rare serious

side effects, chloramphenicol has been replaced by other effective antibiotics. The choice of

antibiotics needs to be guided by identifying the geographic region where the organism was

acquired and the results of cultures once available. (Certain strains from South America

show a significant resistance to some antibiotics.) Ciprofloxacin (Cipro) is the most

frequently used drug in the U.S. for no pregnant patients. Ceftriaxone (Rocephin), an

intramuscular injection medication, is an alternative for pregnant patients. Ampicillin

(Omnipen, Polycillin, Principen ) and trimethoprim-sulfamethoxazole (Bacterium, Septra)

are frequently prescribed antibiotics although resistance has been reported in recent years. If

relapses occur, patients are retreated with antibiotics. (Rajive , 2012).

2.5 Typhoid Fever Prevention:-

1) Prevention within the hospital setting, infected people are cared for in isolation. Proper

hand hygiene is the most important way of preventing further spread in hospital. Stool

samples are also taken from members of the patient's family to identify any 'healthy'

carriers. (Charlie, 2013).

Typhoid fever is an infection caused by Salmonella enterica serotype Typhi (S. typhis). The

disease remains an important public health problem in developing countries. In 2000, it was

estimated that over 2.16 million cases of typhoid occurred worldwide, resulting in 216 000

deaths, and that more than 90% of this morbidity and mortality occurred in Asia. Although

improved water quality and sanitation constitute ultimate solutions to this problem,

vaccination in high-risk areas is a potential control strategy recommended by WHO for the

short-to-intermediate term. (Sally, 2015).

Typhoid fever is a life threatening bacterial infection that affects some organs of the body.

The infection causes by Salmonella Typhi .Untreated typhoid cases may lead to sever

complications and even death. Typhoid fever is affecting about 21.5 million people every

year worldwide. Infected person with typhoid fever carry the bacteria in their intestinal tract

http://www.nhs.uk/conditions/Antibiotics-penicillins/Pages/Introduction.aspx

http://www.drugs.com/article/antibiotics.html

33

and blood stream. The bacteria spread through the feces and sometimes in the urine. Typhoid

fever infection spread through the contaminated water and food with Salmonella Typhi.

2.5.1 Vaccine:-

For the prevention of typhoid fever there are two vaccines approved by the World Health

Organization, these are:-

1- Ty21-one capsule is taken orally for four days, children received this vaccine at six years

of age and then require boosters’ dose every five years.

2- ViCPS- this is a single dose injection given to children at two years of age, and given

booster dose every two years. The two vaccines can not give a complete protection

against typhoid fever.

2.5.2 Personal Prevention:-

1-Wash hands with soap and hot water before eating or preparing food and after using toilet.

2-Avert drinking contaminated water unless you are boiled or treated it.

3- Wash your teeth by using potable water.

4- Avoid using ice that made of unsafe water.

5- Eat thoroughly cooked food and avoid that stored at room temperature.

6- Eat only peeled vegetables.

6-Evade food and drink being sold by street vendors. How to prevent others:-

7-Complete the treatment by taking full course of antibiotics.

8-During the contagious period avoid preparing food for others.

9-Thoroughly hand washing with soap and hot water and scrubbed hands for at least thirty

seconds. (CDC, 2015).

34

2.6 Incubation period:-

The incubation period for typhoid fever is usually 8-14 days but this depends on the infective

dose and can vary from three to one month, paratyphoid fever is usually one to ten days.

2.6.1 Public health significance and occurrence:-

Typhoid infections occur throughout the world, Outbreaks usually occur in areas with poor

sanitation and inadequate sewerage systems.

2.6.2 Reservoir:

Reservoir for typhoid are humans (gallbladder) and rarely human urinary carriers and for

paratyphoid fever Humans and rarely domestic animals.

2.6.3 Communicability Period:-

Communicability continues as long as bacilli are present in excreta. Some patients become

permanent carriers.

2.6.4 Susceptibility Period:-

The humans can be susceptible to infection, immunization is not sufficient to protect from

large dose of infection (GOV, 2015).

2.6.5 Sensitivity and specificity:-

Sensitivity and specificity are statistical measures of the performance of the diagnostic tests,

also known as classification function.

Sensitivity also known as true positive rate or recall , it measures the proportion of positive

that are correctly identified as such , ( the percentage of sick people who are correctly

identified as having the disease ) .

Specificity, also known as true negative rate (TNR) it measure the proportion of negative

that are correctly identified as such . Thus Sensitivity quantifies the avoiding of false

negatives, specificity to avoid false positives.

Sensitivity described as 100% Sensitive when all sick are identified as sick and 100%

specific when no healthy are identified as sick .( Harris etal,2015).

35

CHAPTER THREE

MATERIALS AND METHODS

3-1. Study area and period:-

Gezira state is located at the middle of Sudan, between Latitudes 13 – 15 north, and

Longitudes 34 -32.5 east. The area of the state is about 27549 square kilometers while the

total population is about 3734320 according to 2008 census. The state is boarding by

Khartoum state from the North and Sinner state to the south, White Nile state to the West and

Gedarif state to the East.

Administratively, the state was divided into eight Localities namely: Wad Madani alkubra,

Alkamleen, Elhasaheisa, Um Algora, East Algezira, Alqurashi, South Elgezira and El

Managil. The state had benefited from the the Gezira Scheme economically and on the other

hand suffered from the cultivation that makes the state exposed to water associated diseases

like Malaria, schistosomiasis and water borne diseases like Typhoid, Acute watery diarrhea

particularly during and after the rainy season where the environment became suitable for the

prevalence of these diseases. Most of the population works in agriculture while others hered

livestock .79.2% of the population using improved join king water source, the coverage is

54% - 96% in urban and 60% - 90% in rural areas. (Gezira, 2014).

The study was conducted in Wad Medani Teaching hospital from 2012 to 2017. This hospital

was one of the largest public hospitals in Sudan, located in Wad Madani city it has 282 beds,

with an estimated 418 health staffs, provide care to more than 82557 people each year.

(Annual, 2016)

3-2 Study Design:

Across sectional study was conducted in which patients were screened for typhoid fever and

suspected patients were enrolled in the study, then blood sample were collected and tested for

confirmation of the disease. Patients were screened by their physician for the clinical

symptoms of typhoid fever which is fever of two or more days before admission

accompanied by other clinical symptoms of typhoid fever in the absence of any other known

febrile illnesses. Febrile patients whose presumptive clinical diagnosis were typhoid fever

and sent to the laboratory by their physician for Widal test were included in the study.

However, those febrile patients who had received antibiotic treatment for their symptoms

36

within two weeks before coming to the hospital and those who are diagnosed for other known

febrile illness and outside of the state were not included in this study. By using these

inclusion and exclusion criteria, about 237 suspected febrile patients were recruited for this

study ,then data and blood samples were collected from these 237 patients for Widal test ICT

and Culture.. The samples were transported immediately to the laboratory for bacteriological

investigation. Cultivation Bacteriological tests were done according to Barrow and

Feltham1993. All specimens were inoculated on culture media including, thioglycolate broth,

and KIA, and incubated in aerobic atmosphere at optimum temperature 35-37°C and pH

range from 7 -7.2.

3-3 Study population:

The study population were those who were come to the Wad-Medani Teaching Hospital, as

febrile patients of Typhoid fever during the study period mid November 2012 – mid

December 2012.

3.4 Sample size:

The prevalence of Typhoid fever could not be precisely calculated because of different unit’s

registration system and the method used for diagnosis is a single Widal test rather than

culture.

However similar studies of endemic diseases have done, For malaria it was estimated in the

study (Testing the sensitivity and specificity of the fluorescence microscope (Cyscope) for

malaria diagnosis) as being 20%, so we can make a scientific calculation of the level of the

prevalence. (Ley etal, 2010).

it is not likely to be very much different from such estimation, but we can make an absolute

prevalence 20± 1%, this given arrange of 18-22 and by taking the geometric mean of the

range we have the prevalence of Typhoid fever as 19%.

For the calculation of the sample size, we used the following equation:

= n = (Z + 1.28)2 P (1 – P) / (P – P0)

2

= n = (1.96 + 1.28)2 P (1 – P) / (P – P0)

2

37

Where:-

N= sample size, the suspected sensitivity (P) is 0.90 and the minimal acceptable sensitivity

(P0) is 0.75, Z= 1.96.

This translate into N= 42. Among the patients in the study, the prevalence of Typhoid is

expected to be not less than 19%. Therefore, to adjust the sample size for this factor, the

calculated sample size (42) will be divided by 19 and multiplied by 100]42/19(100) [ to get a

sample size of 221.

3-5 Sample Technique:-

237 Samples were collected from patients directed to the laboratories for Widal test by

attending physicians from Wad Medani Teaching Hospital. The blood was collected

aseptically by vein puncture technique 10ml of blood collected from each person were tested

for Salmonella typhi O and H antibodies, Rabid Diagnostic Test to detect IgG and IgM also

cultured for Salmonella typhi.8ml was put in thioglycolate broth media and incubated in

aerobic atmosphere at optimum temperature 35-37°C and pH range from 7 -7.2., 2ml of

blood were put in Ethylene Diamine Tetra acetic Acid (EDTA) to be used for Widal and ICT

test.

3-6 Data collection:

Data will be collected through:

3-6-1 Blood samples: Widal test, ICT, culture.

3-6-2 Questionnaire variables :

- Socioeconomic data (age, sex, residence, marital status, education, occupation,

employees, income and water supply.

- Complaints data.

- Sewage data

- Investigation data.

Questionnaire:-

A questionnaire was fulfilled from the febrile suspected patients of typhoid fever according to

the sample size (237).

3-7 Data Analysis:-

Statistical software package of social science (SPSS Version 16) was used for the analysis of

the data .Sensitivity, specificity were calculated for Widal test, ICT, and Culture.

38

3-8 Research Ethics:-

Ethical review and clearance of the research protocol were obtained from the Ethical Review

Committee of the Gazira State Ministry of Health. Samples were collected from Wad–

Medani Teaching Hospital with the permission of the hospital authorities. The patients’

consent was obtained prior to sampling by filling the Patient Consent Form. All subjects who

were accepted were provided their bio-data. Patients who were already on antibiotics or

outside the State were excluded from this study.

3-9 Quality Controls:-

Standard operational procedures were followed during processing of each sample and all

the instruments used for sample processing were checked every morning for proper

functioning.

3.9-1 Inclusion criteria:

All patients from Gezira state who come to Wad Medani teaching hospital suspected for

Typhoid fever.

3.9-2 Exclusion criteria:

- All patients from outside Gezira state.

- Patients complain from other conditions (diseases).

- Patients receive antibiotics within two weeks before coming to the hospital.

3.9-3 Limitation of the study:

Lack of financial support.

39

(Gezira State Emergency Profile, 2014)

40

3-2 Materials and Methods:-

3-2-1. Sterilization procedures:-

3-2-1-1. Hot air oven:-

Glass wares (flasks, test tubes, pipettes and Petri-dishes) were sterilized in hot air oven at

160ºC for 1 hour.

3-2-1-2 Autoclaving:-

Autoclaving was used to sterilize culture media and solutions at121ºC for 15 minutes.

3-2-1-3 Ultra –Violet Light:-

It was used to sterilize the vacuums, benches and walls of media pouring room.

3-2-2 Disinfection:-

Alcohol (70%) was used to disinfect the surfaces of working benches before and after use.

3-2-3 Solutions:

3-2-3-1. Thioglycolate broth:-

Thioglycollate broth is a medium contains sodium thioglycollate, which reacts with

molecular oxygen keeping free oxygen levels low. The sodium thioglycollate in the broth

creates a redox potential in the tube, with higher levels of oxygen at the top of the tube, and a

complete absence of oxygen at the bottom of the tube. Fluid thioglycollate broth also

typically contains a redox potential indicator such resazurin, which produces a pink color in

an oxidized environment

3-2-3-2 Normal Saline.

This solution was prepared by dissolving 8.5 grams of sodium chloride in one liter of distilled

water; the solution distributed into test tubes as 10 ml per tube and will be sterilized by

autoclaving at121ºC for 15 minutes.

3-2-3-3. Peptone water:

As a tryptophan-containing medium used for detecting indole production (an important

character of E. coli. (Cowan etal, 2003).

41

3-2-4 Identification of the isolates:

3-2-4-1 Microscopic examination:

3-2-4-2 Gram’s stain:

Smear was prepared from the pure culture of each isolate by emulsifying a portion of a

colony in a drop of sterile normal saline on a glass slide, dried and fixed by heating. Then the

slide flooded by crystal violet for one minute and then washed with tap water. Iodine solution

applies for one minute and then the slide washed with tap water. Then the smear decolorized

with few drops of acetone for seconds and wash immediately with water. Then the smear

flooded with diluted carbol fuchsin for 30 seconds and washed with tap water. Slide blotted

dry with filter paper and examine under oil immersion lens.

3-2-4-3 Biochemical Tests:

3-2-4-3-1 Indole production test:

This test demonstrates the ability of certain bacteria to decompose amino acid tryptophan to

indole which accumulates in the medium, about 0.5ml of kovac’s reagent was added to

culture of the test organism grown on suitable tryptophan rich medium, mainly peptone

water.

3-2-4-3-2 Oxidase test:

This test depends on the presence of oxidase enzyme in certain bacteria, that was catalyzed

the transport of electrons between electron donors in the bacteria and redox dye (tetramrthyl-

p-phenylene Diamine dihydrochloride).

3-2-4-3-3 Urease test:

This test was run to detect urease enzyme activity, which is important in recognizing enter

bacteria. By inoculating slopes of Christensen’s medium, which contain urea.

3-2-4-3-4 Citrate utilization test:

This test based on the ability of an organism to use citrate as its only source of carbon and

ammonia also as its only source of nitrogen. By inoculation of Simmons’s citrate medium

with a broth culture of test organism.

3-2-4-3-5 Voges-Proskauer (V-P) Test:

Many bacteria fermented carbohydrates with the production of acetyl methyl carbinol

(acetone) by inoculating the test organism in glucose phosphate peptone water and added

small amount of sodium hydroxide.

42

3-2-4-3-6 Methyl Red (MR) Test:

This test is implemented to detect the production of sufficient amount of acid during the

fermentation of glucose. By inoculating the test organism in 5ml of sterile glucose phosphate

peptone water, a drop of methyl red was added.

3-2-4-3-7 Kilgler, s Iron Agar (KIA):

The test organism was sub cultured into KIA by stabbing the agar, which is significantly used

for the detection of lactose and glucose fermentation, evolution of gas during fermentation

and production of hydrogen sulphide. (Hassan , 2013).

3-2-5 Widal test:

Widal test is an agglutination test which detects the presence of serum agglutinins (H and O)

in patient’s serum with typhoid and paratyphoid fever. It was developed by Georges

Ferdinand Widal in 1896

Blood samples collection (237) venous blood samples were collected from individuals 3-5

mL of blood sample was collected into a sterile test tube and centrifuged for 5 minutes to

separate the serum from the blood.

3-2-5-1 Slide test Method:-

A drop of the serum (0.08mL) was pipette and dropped on a sterile slide in four different

parts for Salmonella typhi O and another for Salmonella paratyphi H antigens. Antigens O

and H were shaken and dropped into the serum accordingly. It was then mixed and rocked

gently for 2 seconds. The results were recorded as thus: depending on the gravity of the

agglutination, 1/20 is negative while 1/80 – 1/360 is positive.

3-2-5-2 Tube test Method:-

Serial dilutions of sera starting at a dilution of 1:10 were made with 0.9% saline. Tubes

containing O and H antigens and sera were incubated at 37°C for 18 h, and examined for

visible agglutination. Appropriate positive and negative control sera were included. the titer

of the patient serum using Widal test antigen suspensions is the highest dilution of the serum

sample that gives a visible agglutination.

3-2-6 ICT Test:-

Typhoid rapid antibody tests were carried out according to manufacturer’s instructions. The

typhoid test becomes positive within 2-3 days of infection and separately identifies IgM and

IgG antibodies. The test is based on the presence of specific IgM and IgG antibodies to a

specific antigen, which is impregnated on nitrocellulose strips.

43

3-2-7 Blood Culture:-

The organism may be recovered from the bloodstream at any stage of the illness but are most

commonly found during the first 7-10 days and during relapses. Incubated the samples on

thioglycolate broth and at 37°C for an initial period of 48 hours and sub-cultured on Blood

and Macconky Agar Salmonella typhi were identified on the basis of standard culture,

microscopic and biochemical characteristics. Inoculated blood culture medium was discarded

as negative if there is no growth after 7-10 days.

3-2-7-1 Morphological and Biochemical Tests:-

Morphological and Biochemical tests were carried out on blood culture isolates of each

sample to confirm the presence of Salmonella. The tests include gram reaction, motility test

and biochemical test such as catalase, coagulase, lactose fermentation, glucose fermentation

test, citrate utilization test etc. The procedures for the test are according to Cheesbrough

(.Hassan, 2013).

44

CHAPTER FOUR

Results and Discussion

Participants from Gezira State, female (53.2%) more than male (46.8%),the

highest age group was 15-24 years, most were urban (57%)with a potable water

and low income per month.

A study samples was two hundred and thirty seven, samples of blood were collected from

febrile patients and examined based on standards methods, at Wad- Medani teaching hospital

from 13 November 2012 up to 13 December 2012, and analyzed statistically based on

standards methods, and the results comes as follows:-

Figure (4.1) Distribution of participants by sex.

N = 237

The majority were Females (53.2%)

%

Figure (4.2) Distribution of participants by age group.

N = 237

Most of the participants (57%) in the age group (15 -34).

45

Figure (4.3) Distribution of the participants by resident.

N = 237

The study targeted the participants in Gezira state. Urban = 135 (57%), rural 102 (43%).

Figure (4.4) Shows distribution of participants by education level .

N = 237

The primary school is highest (29.1%).

46

Figure (4.5) Marital status of participants .

N = 237

The majority were married (54%).

Figure (4.6): Distribution of participants by occupation.

N = 237

The majority were House wife (34.6%).

47

Figure (4.7) Distribution of participants by Income/month S.P.

N = 237

The majority of participants in group 600-1000 (48.5%) SP.

Figure (4.8): Distribution of participants by Water supply source

In Wad -Medani Teaching Hospital, Gezira state - Sudan - year 2012.

N = 237

84.4% 0f the participants were used ground water.

48

Table (4.1) Distribution of the participants by food hygiene.

Food hygiene

Frequency Frequency

Total Yes No

the food was covered after cooking 228

( 96.2% )

9

( 13.8% )

237

(100%)

the food stored in the refrigerator) 216

( 91.1% )

21

( 8.9% )

237

(100%)

daily meals at home 178

( 75.1% )

59

( 24.9% )

237

(100%)

daily meals at market (outside) 73

( 30.8% )

164

( 69.2% )

237

(100%)

N = 237

Most of the food stored and covered in a hygienic way.

Table (4.2) Distribution of the participants by Sewage system.

N = 237

The majority used pit latrines (70.9%).

Sewage system Frequency Percent

Buckets system 5 2.1

open places 8 3.4

pit latrines 168 70.9

siphon latrines 21 8.9

water close latrines 35 14.8

Total 237 100

49

Table (4.3) Distribution of participants by refuse collection.

Semi solid west Frequency Percent

Collection by car from house to house 129 54.4

Collection in certain place (kosha) 108 45.6

Collection in the roads 0 0

Collection outside the residence area 0 0

Total 237 100

N = 237

The majority used collection by car (54.4%).

50

Table (4.4) Distribution of participants by symptoms.

At Wad-Medani Teaching Hospital-Gezira state - Sudan -year 2012.

Symptoms

Frequency Frequency

Total

Yes No

sustained fever 39°-40 187

(78.9%)

50

(21.1%)

237

(100%)

feel weak 44

(18.6%)

193

(81.4%)

237

(100%)

stomach pains

58

(24.5%)

179

(75.5%)

237

(100%)

headache 106

(44.7%)

131

(55.3%)

237

(100%)

loss of appetite

7

(2.9%)

230

(97.1%)

237

(100%)

rash of flat, rose-colored

spots

2

(0.8%)

235

(99.2%)

237

(100%)

others (mentioned)

2

(0.8%)

235

(99.2%)

237

(100%)

N = 237

The majority of the participants complains from sustained fever 39-40(78.9%).

51

Table (4.5 ) Relationship between the sex and the tests among the participants.

As shown in table ( 4.5) chi-square is 0.133; d.f is 2 and P.value 0.936, the result shows that

the relationship between the sex and the widal test slide method it was not significant at

P.value 0.936 (P≥0.05). According to this result, the sex did not affect the Widal test slide

method.

Also shows that the chi-square is 1.646; d.f is 2 and P.value 0.439, the result shows that the

relationship between the sex and the widal test Tube method it was not significant at P.value

0.439 (P≥0.05). According to this result, the sex did not affect the Widal test Tube method.

Also shows that the chi-square is 0.654; d.f is 2 and P.value 0.721, the result shows that the

relationship between the sex and the ICT method it was not significant at P.value 0.721

(P≥0.05). According to this result, the sex did not affect the ICT method.

Test

Sex

Total Chi-Square

Tests df P.Value

female male

Widal Slide

method

Negative 57 48 105

0.133(a) 2

0.936

Non sig.

Positive 69 63 132

Total 126 111 237

Widal Tube

method

Negative 96 79 175

1.646(a) 2

0.439

Non sig.

Positive 30 32 62

Total 126 111 237

ICT Results

Negative 95 84 179

0.654(a) 2

0.721

Non sig.

Positive 31 27 58

Total 126 111 237

52

Table (4.6) Relationship between the education and the tests among the participants.

Test

Education Level

Total Value df

P.

Value higher

school

khalw

a

Postg

radu

ate

primar

y

school

secon

dary

schoo

l

under

gradu

ate

unive

rsity

Widal

Slide

method

Negati

ve 20 24 0 32 6 1 22 105

6.145

(a)

7

0.523

Non sig.

Positiv

e 34 22 1 35 9 0 31 132

Total 54 46 1 67 15 1 53 237

Widal

Tube

method

Negativ

e 33 41 1 49 12 1 38 175

12.261

(a) 7

0.092

Non sig. Positive 21 5 0 18 3 0 15 62

Total 54 46 1 67 15 1 53 237

ICT

Results

Negativ

e 35 39 1 50 11 1 42 179

10.288

(a) 7

0.173

Non sig. Positive 19 7 0 17 4 0 11 58

Total 54 46 1 67 15 1 53 237

As shown in table (4.7)chi-square is 6.145; d.f is 7 and P.value 0.523, the result shows that

the relationship between the education and the Widal test slide method it was not significant

at P.value 0.523 (P≥0.05). According to this result, the education did not affect the Widal test

slide method.

53

Also shows that the chi-square is 12.261, d.f is 7 and P.value 0.092, the result shows that the

relationship between the education and the Widal test Tube method it was not significant at

P.value 0.092 (P≥0.05). According to this result, the education will not affect the Widal test

Tube method.

Also shows that the chi-square is 10.288, df is 7 and P.value 0.173, the result shows that the

relationship between the education and the ICT method it was not significant at P.value 0.173

(P≥0.05). According to this result, the education will not affect the ICT method.

Table ( 4.7) Relationship between the occupation and the tests among the participants

At Wad-Medani Teaching Hospital-Gezira state - Sudan -year 2012.

As shown in table (4.7 ) chi-square is 2.391, df is 6 and P.value 0.88-0, the result shows that

the relationship between the occupation and the Widal test slide method it was not significant

Test

Occupation

Tota

l Value df

P.

Value farmer free

trade

house

wife

lab-

tech student teacher

Widal

Slide

method

Negative 9 34 37 1 24 0 105

2.391

(a)

6 0.880

Non sig.

Positive 8 43 45 1 33 2 132

Total 17 77 82 2 57 2 237

Widal

Tube

method

Negative 7 50 57 1 38 2 175

6.314

(a) 6

0.389

Non sig.

Positive 0 20 16 1 09 0 62

Total 17 77 82 2 57 2 237

ICT

Results

Negative 17 59 58 2 41 2 179

5.521

(a) 6

0.479

Non sig.

Positive 0 18 24 0 16 0 58

Total 17 77 82 2 57 2 237

54

at P.value 0.880 (P≥0.05). According to this result, the occupation will not affect the Widal

test slide method.


relationship between the occupation and the Widal test slide method it was not significant at

P.value 0.880 (P≥0.05). According to this result, the occupation will not affect the Widal test

tube method.


relationship between the occupation and the ICT method it was not significant at P.value

0.479 (P≥0.05). According to this result, the occupation will not affect the ICT method.

Table (4.8) Relationship between the water supply and the tests among the participants.

Test

Water Supply

Total

Chi-

Square

Tests

Df P.Value

. general

net

ground

water

hand

pump

surface

water

Widal Slide

method

Negative 4 15 83 1 2 105

4.209(a) 4

0.378

Non sig. Positive 3 11 116 0 2 132

Total 7 26 199 1 4 237

Widal

Tube

method

Negative 6 21 143 1 4 1751

7 4

0.378

Non sig. Positive 1 5 56 0 0 62

Total 7 26 199 1 4 237

ICT

Results

Negative 6 20 149 1 3 179

0.791

(a)

4

0.940

Non sig.

Positive 1 6 50 0 1 58

Total 7 26 199 1 4 237

As shown in table.( 4.8) chi-square is 4.209, df is 4 and P.value 0.378, the result shows that

the relationship between the water supply and the Widal test slide method it was not

55

significant at P.value 0.378 (P≥0.05). According to this result, the water supply will not affect

the Widal test slide method in the current study.

Also shows that the chi-square is.791, df is 4 and P.value 0.940, the result shows that the

relationship between the water supply and the ICT method it was not significant at P.value

0.940 (P≥0.05). According to this result, the water supply will not affect the ICT method in

the current study. Also shows that the chi-square is 3.333, df is 4 and P.value 0.504, the result

shows that the relationship between the water supply and the Widal test tube method it was

not significant at P.value 0.504 (P≥0.05). According to this result, the water supply will not

affect the Widal test tube method in the current study

Table (4.9) Relationship between the daily meals at home and the tests among the

participants.

As shown in table (4.9 ) chi-square is 0.073, df is 1 and P.value 0.788, the result shows that

the relationship between the daily meals at home and the Widal test slide method it was not

significant at P.value 0.788 (P≥0.05). According to this result, the daily meals at home will

not affect the Widal test slide method in the current study.

Test

Daily meals at home

Total

Chi-

Square

Tests

df P.Value

Yes No

Widal Slide

method

Negative 77 27 104

0.073

1

0.788

Non sig. Positive 99 32 131

Total 176 59 235

Widal Tube

method

Negative 134 39 173

2.291

1

0.130


Total 176 59 235

ICT Results

Negative 133 44 177

0.023

1

0.878


Total 176 59 235

56


relationship between the daily meals at home and the Widal test tube method it was not


not affect the Widal test tube method in the current study.

Also shows that the chi-square is 0.023, df is 1 and P.value 0.878, the result shows

that the relationship between the daily meals at home and the ICT method it was not


not affect the ICT method in the current study

.

Table (4.10) Relationship between the daily meals at market and the tests among the

participants.

Test

Daily meals at

market (outside) Total

Chi-

Square

Tests

df P.Value

Yes No

Widal Slide

method

Negative 34 70 104

0.370

(b) 1

0.543


Total 72 163 235

Widal Tube

method

Negative 47 126 173

3.717

(b) 1

0.054


Total 72 163 235

ICT Results

Negative 52 125 177

0.536

(b) 1

0.464

Non sig.

.464

Positive 20 38 58

Total 72 163 235

As shown in table ( 4.10) chi-square is 0.370, df is 1 and P.value 0.543, the result

shows that the relationship between the daily meals at market and the Widal test slide method

it was not significant at P.value 0.543 (P≥0.05). According to this result, the daily meals at

market will not affect the Widal test slide method in the current study.


relationship between the daily meals at market and the Widal test Tube method it was

57

significant at P.value 0.054 (P≤ 0.05). According to this result, the daily meals at market will

affect the Widal test Tube method in the current study.


relationship between the daily meals at market and the ICT method it was not significant at

P.value 0.464 (P≥0.05). According to this result, the daily meals at market will not affect the

ICT method in the current study.

Table (4.11 ) Relationship between the Sustained fever 39°-40 °and the tests among the

participants.

As shown in table ( 4.11) chi-square is 0.754, df is 1 and P.value 0.385, the result shows that

the relationship between the symptom sustained fever 39°-40 ° and the Widal test slide

method it was not significant at P.value 0.385 (P≥0.05). According to this result, the

sustained fever 39°-40 ° will not affect the Widal test slide method in the current study.


relationship between the symptom sustained fever 39°-40 ° and the Widal test tube method it

Test

Sustained fever 39°-40 °

Total

Chi-

Square

Tests

df P.Value

Yes No

Widal Slide

method

Negative 85 19 104

0.754

(b) 1

0.385


Total 186 49 235

Widal Tube

method

Negative 139 34 173

0.570

(b) 1

0.450


Total 186 49 235

ICT Results

Negative 139 38 177

0.166

(b) 1

0.684


Total 186 49 235

58

was not significant at P.value 0.450 (P≥0.05). According to this result, the sustained fever

39°-40 ° will not affect the Widal test tube method in the current study.


relationship between the symptom sustained fever 39°-40 ° and the ICT method it was not

significant at P.value 0.684 (P≥0.05). According to this result, the sustained fever 39°-40 °

will not affect the ICT method in the current study.

Table (4.12 ) Relationship between the feel weak and the tests among the participants.

As shown in table (4.12 ) chi-square is 4.113, df is 1 and P.value 0.043, the result

shows that the relationship between the symptom feel weak and the Widal test slide method it

was significant at P.value 0.043 (P≤ 0.05). According to this result, the feel weak will affect

the Widal test slide method in the current study.


relationship between the symptom feel weak and the Widal test tube method it was not

significant at P.value 0.096 (P≥0.05). According to this result, the symptom feel weak will

not affect the Widal test tube method in the current study

Test

Feel weak

Total

Chi-

Square

Tests

df P.Value

Yes No

Widal Slide

method

Negative 25 79 104

4.113

(b) 1

0.043

Sig. Positive 18 113 131

Total 43 192 235

Widal Tube

method

Negative 36 137 173

2.766

(b) 1

0.096


Total 43 192 235

ICT Results

Negative 34 143 177

0.398

(b) 1

0.528


Total 43 192 235

59


relationship between the symptom feel weak and the ICT method it was not significant at

P.value 0.528 (P≥0.05). According to this result, the symptom feel weak was not affect the


Table (4.13) Relationship between the stomach pains and the tests among the

participants.


the relationship between the symptom stomach pain and the Widal test slide\ method it was

not significant at P.value 0.085 (P≥0.05). According to this result, the symptom stomach pain

will not affect the Widal test slide method in the current study.


relationship between the symptom stomach pain and the Widal test tube\ method it was not

significant at P.value 0.841 (P≥0.05). According to this result, the symptom stomach pain

will not affect the Widal test tube method in the current study.

Test

Stomach pains

Total

Chi-

Square

Tests

df P.Value

Yes No

Widal Slide

method

Negative 30 74 104

2.971

(b) 1

0.085


Total 55 179 234

Widal Tube

method

Negative 41 131 172

0.040

(b) 1

0.841


Total 55 179 234

ICT Results

Negative 43 133 176

0.340

(b) 1

0.560


Total 55 179 234

60


relationship between the symptom stomach pain and the ICT method it was not significant at

P.value 0.560 (P≥0.05). According to this result, the symptom stomach pain will not affect

the ICT method in the current study

Table (4.14) Relationship between the headache and the tests among the participants.


the relationship between the symptom headache and the Widal test tube method it was not

significant at P.value 0.612 (P≥0.05). According to this result, the symptom headache will not

affect the Widal test tube method in the current study.


relationship between the symptom headache and the ICT method it was not significant at

P.value 0.072 (P≥0.05). According to this result, the symptom headache will not affect the


Test

Headache

Total

Chi-

Square

Tests

df P.Value

Yes No

Widal Slide

method

Negative 52 52 104

2.136

(b) 1

0.144


Total 105 130 235

Widal Tube

method

Negative 79 94 173

0.257

(b) 1

0.612


Total 105 130 235

ICT Results

Negative 85 92 177

0.257

(b) 1

0.612


Total 105 130 235

61


relationship between the symptom loss of appetite and the Widal test slide\ method it was not

significant at P.value 0.774 (P≥0.05). According to this result, the symptom loss of appetite

will not affect the Widal test slide method in the current study.

Table (4.15) Relationship between the Loss of appetite and the tests among the

participants.


the relationship between the symptom loss of appetite and the Widal test slide\ method it was

not significant at P.value 0.774 (P≥0.05). According to this result, the symptom loss of

appetite will not affect the Widal test slide method in the current study.


relationship between the symptom loss of appetite and the Widal test tube method it was not

significant at P.value 0.696 (P≥0.05). According to this result, the symptom loss of appetite

will not affect the Widal test tube method in the current study

Test

Loss of appetite

Total

Chi-

Square

Tests

df P.Value

Yes No

Widal Slide

method

Negative 3 101 104

0.082

(b) 1

0.774


Total 6 229 235

Widal Tube

method

Negative 4 169 173

0.153

(b) 1

0.696


Total 6 229 235

ICT Results

Negative 6 171 177

2.018

(b) 1

0.155


Total 6 229 235

62


relationship between the symptom loss of appetite and the ICT method it was not significant

at P.value 0.155 (P≥0.05). According to this result, the symptom loss of appetite will not

affect the ICT method in the current study.

Table (4.16) Relationship between the rash of flat and the tests among the participants.


the relationship between the symptom rash of flat( rose-colored spots) and the Widal test

slide\ method it was not significant at P.value 0.261 (P≥0.05). According to this result, the

symptom rash of flat (rose-colored spots) will not affect the Widal test slide method in the

current study.


relationship between the symptom rash of flat( rose-colored spots) and the Widal test tube\

method it was not significant at P.value 0.549 (P≥0.05). According to this result, the

symptom rash of flat (rose-colored spots) will not affect the Widal test tube method in the

current study.

Test

Rash of flat, rose-

colored spots Total

Chi-

Square

Tests

df P.Value

Yes No

Widal Slide

method

Negative 1 103 104

1.265

(b) 1

0.261


Total 1 234 235

Widal Tube

method

Negative 1 172 173

0.360

(b) 1

0.549


Total 1 234 235

ICT Results

Negative 1 176 177

0.329

(b) 1

0.566


Total 1 234 235

63


relationship between the symptom rash of flat( rose-colored spots) and the ICT method it was

not significant at P.value 0.566 (P≥0.05). According to this result, the symptom rash of flat

(rose-colored spots) will not affect the ICT in the current study

Figure (4.9) shows the results of Study Widal test Slide method in Wad Medani

Teaching Hospital, Gezira state - Sudan –- year 2012-2017

N =237

Only 55.7% were positive.

Figure (4.10) shows the results of Study Widal test Tube method in Wad -Medani

Teaching Hospital, Gezira state - Sudan - year 2012-2017.

N =237


64

Figure (4.11) show the results of Study ICT Results in Wad -Medani Teaching

Hospital, Gezira state - Sudan - year 2012-2017

N =237


Figure (4.12) shows the results of Study Culture test in Wad -Medani Teaching

Hospital, Gezira state - Sudan - - - year 2012.

N =237

65

The higher growth was gram positive cocci 24.5%.

Figure (4.13) Shows the results of widal, ICT, and culture tests in Wad -Medani

Teaching Hospital, Gezira state - Sudan - -year 2012-2017.

Figure (4.14) shows the results of Hospital Widal test slide method in Wad Medani

Teaching Hospital , Gezira state - Sudan – year 2012-2017.

N =237

Only 19.8 % were positive.

+ve

55.7

+ve

24.5

-ve

44.3

-ve

73.8

-ve

75.5

-ve

100

-+ve

26.2

66

Figure (4.15) Results of widal, ICT, and culture tests in Wad -Medani Teaching

Hospital, Gezira state - Sudan - year 2012-2017.

N =237

Salmonella typhi 100 % negative by culture.

Table (4.17) Comparison between widal test tube method and

Widal test slide method.

Study Widal Test Slide

method

Study Widal Test Tube method

+ve -ve Total

+Ve 61 71 132

-Ve 1 104 105

Total 62 175 237

Chi-square Value = 62.016, DF = 1, P.Value = 0.000 , significant

- As shown in table (4.17 there is a relationship between the Study Widal Test slide

method and study Widal test tube method, thus the relationship is highly significant.

Since chi-square is 62.016; d.f is 1 and P.value 0.000. (P<0.05). That means the study

Widal test tube method was better than the study Widal test slide method.

+ve

19.8

+ve

55.7

+ve

24.5

+ve

26.2

-ve

80.2

-ve

44.3

-ve

73.8

-ve

75.5

-ve

100

+ve

0.0

67

- The sensitivity of the study widal test tube method in comparison with study widal

test slide method = 61/62*100 = 98.4%

- The specificity of the study widal test tube method in comparison with study widal


- The prevalence = 62/237*100 = 26.2 %

The total positive result by the study Widal test slide method was 132(55.7%),61(25.7%) of

them were positive by Widal test tube method and 71 (30%) of them were negative , the

sensitivity, specificity and the prevalence of Widal test tube method were 98.4%,59.4% and

26.2% respectively. Similar study was conducted in North Ethiopia in April 2013 for 200

febrile patients, the prevalence of Typhoid fever was 38(19%), but 1(0.5%) patient was

positive by blood culture, (Meseret, 2014). The reported sensitivities of tests for S. typhi vary

greatly in the literature, even among the most recent articles and respected journals, (John

etal, 2017), and this was similar to the results of the current study.

Table (4.18) Comparison between ICT and Widal test slide method.

Study Widal Test Slide

method

ICT Results

+ve -ve Total

+Ve 55 77 132

-Ve 3 102 105

Total 58 179 237

Chi-square Value = 47.655, D.F = 1, P.Value = 0.000, significant

- As shown in table (4.18) there is a relationship between the study Widal test slide

method and ICT Results, Thus the relationship is highly significant. Since chi-square

is 47.655; d.f is 1 and P.value 0.000 (P<0.05).

- That means the ICT test was better than the study Widal test slide method, but the

blood culture was the best test for typhoid fever as mentioned by (John etal, 2017).

- The sensitivity of the ICT in comparison with hospital widal test slide method =

55/58*100 = 94.9%

68

- The specificity of the ICT in comparison with hospital widal test slide method =

102/179*100 = 57.0%

- The prevalence = 58/237*100 = 24.5 %

The total positive result by the study Widal test slide method was 132(55.7%), 55(23.2%) of

them were positive by ICT test and 77 (32.5%) of them were negative, this matching what

was mentioned by (Hassan, 2013). ICT test was better than Widal test slide method, the

sensitivity; specificity and the prevalence of ICT method were 94.9%, 57% and 24.5%

respectively.

Table (4.19) Comparison between ICT and Widal test tube Method.

Study Widal Test Tube

method

ICT Results

+ve -ve Total

+Ve 51 11 62

-Ve 7 168 175

Total 58 179 237

Chi-square Value = 151.689, D.F = 1, P.Value = 0.000 , significant

- As shown in table (4.19) there is a relationship between the study Widal test tube

method and ICT Results, Thus the relationship is highly significant. Since chi-square

is 151.689; d.f is 1 and P.value 0.000 (P<0.05).

- That means the study ICT test was better than the study Widal test tube method, and

the culture was the best test for typhoid fever as mentioned by (John etal, 2017).

- The sensitivity of the ICT in comparison with study widal test tube method =

51/58*100 = 87.9%

69

- The specificity of the ICT in comparison with study widal test tube method =

168/179*100 = 93.9%

- The prevalence = 58/237*100 = 24.5 %

The total positive result by the study Widal test tube method was 62(26.2%),51(21.5%) of

them were positive by study ICT test and 11 (4.6%) of them were negative, the sensitivity,

specificity and the prevalence of study ICT test were 87.9%,93.9% and 24.5% respectively.

Table (4.20) Comparison between modified Widal test slide method and Widal test slide

method.

Hospital Widal test slide

method

Study Widal Test Slide method

+Ve -Ve Total

+Ve 35 12 47

-Ve 97 93 190

Total 132 105 237

Chi-square Value = 8.372, D.F = 1, P.Value = 0.004, significant.

As shown in table (4.20) there is a relationship between the study Widal test slide method and

hospital Widal test slide method, thus the relationship is highly significant. Since chi-square

is 8.372; d.f is 1 and P.value 0.004 (P<0.05). The result shows that the relationship between

the hospital Widal test slide method and the study Widal test slide method was highly

significant at P.value 0.004. That means the study Widal test slide method was better than the

hospital Widal test slide method, and the culture was the best test for typhoid fever as

mentioned by (John etal, 2017).

- The sensitivity of the study widal test slide method in comparison with hospital

widal test slide method = 35/132*100 = 26.5%

- The specificity of the study widal test slide method in comparison with hospital

widal test side method = 93/105*100 = 88.6%

- The prevalence = 132/237*100 = 55.7 %

70

The total positive result by the hospital Widal test slide method was 47(19.8%),35 of them

were positive by study Widal test slide method and 12 (25.5%) of them were negative, the

sensitivity, specificity and the prevalence of study Widal test slide method were 26.5%,88.6%

and 55.7% respectively.

Table (4.21) Comparison between Widal test tube method and hospital Widal test slide

method.


method

Study Widal Test Tube method

`+ve -ve Total

+Ve 12 35 47

-Ve 50 140 190

Total 62 175 237

Chi-square Value = 0.012, D.F = 1, P.Value = 0.913 , not significant

As shown in table (4.21) there is no relationship between the study Widal test tube method

and hospital Widal test slide method. The relationship was not significant. Since chi-square is

0.012; d.f is 1 and P.value 0.913 (P>0.05). That means the study Widal test tube method and

the hospital Widal test slide method were the same, and the culture was the best test for

typhoid fever as mentioned by (John etal, 2017).

The total positive result by the hospital Widal test slide method was 47(19.8%),12(5%) of

them were positive by study Widal test tube method and 35(14.8%) of them were negative,

that difference in the results between the two test was due to that, the typhoid fever was

indistinguishable disease (WHO, 2015).

There was a little difference between the Widal test slide method and the study Widal test

tube method, as mentioned in the previous study by (Sood etal, 2014).

- The sensitivity of the study widal test tube method in comparison with hospital widal


- The specificity of the study widal test tube method in comparison with hospital

widal test slide method = 140/175*100 = 80.0%

- The prevalence = 62/237*100 = 26.2 %

- The sensitivity, specificity and the prevalence of study Widal test tube method were

19.4%, 80% and 26.2% respectively.

71

Table (4.22) Comparison between ICT and hospital Widal test slide method.


method

ICT Results

+ve -ve Total

+Ve 11 36 47

-Ve 47 143 190

Total 58 179 237

Chi-square Value = 0.036, D.F = 1, P.Value = 0.849 , not significant.

As shown in table (4.22) there is no relationship between the ICT results and Hospital Widal

test slide method. The relationship it was not significant. Since chi-square is 0.036; d.f is 1

and P.value 0.849 (P>0.05). That means the ICT method and the hospital Widal test slide

method were the same, but with little difference as mentioned in reference (Jain , 2014), the

culture was the best test for typhoid fever as mentioned in reference (John etal , 2017).

- The sensitivity of the ICT in comparison with hospital widal test slide method =

11/58*100 = 19.0%

- The specificity of the ICT in comparison with hospital widal test slide method =

143/179*100 = 79.9%

- The prevalence = 58/237*100 = 24.5 %

The total positive result by the hospital Widal test slide method was 47(19.8%),11of them

were positive by study ICT method and 36 (15.2%) of them were negative, the sensitivity,

specificity and the prevalence of study ICT method were 19%,79.9% and 24.5% respectively

Table (4.23) Distribution of cases by sex.

Sex Hospital Widal test slide method Study Widal Test Slide method

+ve -ve Total -ve +ve Total

Female 88 38 126 56 70 126

Male 102 9 111 49 62 111

Total 190 47 237 105 132 237

Chi-square Value = 18.132, D.F = 2,

P.Value = 0.000 , significant

Chi-square Value = 0.133, D.F = 2,

P.Value = 0.936, not significant

72

As shown in table (4.23) chi-square is 18.132; d.f is 2 and P.value 0.000, the result showed

that the relationship between the sex and the hospital widal test slide method it was highly

significant at P.value 0.000 (P<0.05). According to this result, the sex will affect the

Hospital Widal test slide method. Also the result showed that the relationship between the

sex and the study Widal test slide method it was not significant (P>0.05). Since chi-square is

0.133; DF is 2 and P.value 0.936.

The total of the participants were 237, the female were 126 (53.2%) and the male

were 111 (46.8%), the daily meals at market were have a statistically significance

relationship (table NO.4.10) in the study widal test tube method, this will match what was

mentioned by (WHO, 2014) and (John etal, 2017), that means contamination of meals by S.

typhi cause typhoid fever.

The symptoms of typhoid fever in the participants has no statistically significance

relationship, except the symptoms ,feel weak in the study widal test slide method ( table

4.12), that means all the positive cases were not matching the case definition of typhoid fever

, thus all the participants (237) were negative for typhoid fever by culture test,

This matched what was mentioned by (John etal, 2017) that is to say cultures are

widely considered 100%specific.

A similar study conduct in Gezira State –Sudan, by El-Shafie (1991), for 114 normal

individuals. Salmonella typhyi O agglutinins were found in 12(10.5%) of them at a titer of

1:320. In the current study a samples of 237 febrile persons were examined. Salmonella typhi

were found in 132(44.3%) at a titer of 1:320.The two studies exclude a titer of 1:160 as a cut

off value for diagnosis of Salmonella typhi .

A study conducted in Northwest Ethiopia by) Meseret, 2014(, a 200 febrile patients

suspected for malaria and/or typhoid fever, among those 73(36.5%) were positive for malaria,

11(15.1%) were mixed infection. The prevalence of Typhoid and Malaria were 38(19%),

73(36.5%) respectively, the prevalence of Typhoid fever (19%) it was similar to that in

Gezira State by which we calculate the sample size of the current study, among the positive

cases(38) of Typhoid fever, only 1(0.5%) case was positive by Culture method and the other

cases were positive by Widal test, that means Culture method is better than Widal test, this

matching the results of the current study. Across-sectional study was conducted(AJMR,2015)

in Elmanagil Locality- Gezira- State-Sudan, a blood samples were collected from 203 febrile

patients and examined for Typhoid fever based on standard methods,30 cases were positives

by culture method,12 cases were positive by Widal test slide method, with a sensitivity of

73

40% and75% specificity, this low sensitivity match the results(26.5%) obtained from the

current study and with high specificity 88.6%,50 samples were examined ,30(60%)were

positive by culture method and 17 (34%)were positive by Widal test. A study was conducted

in Ethiopia (Gizachew etal, 2014), the conclusion, Widal test has a low sensitivity (71.45%)

and specificity (68.4%) but it was good in negative cases.

A study about Typhoid fever (WHO, 2008) was conducted in five Asian countries ,China,

India, Indonesia, Pakistan, Viet Nam.Atotal of 21874 febrile persons of fever were examined

by blood culture method, only 475(2%) were positive.

In a study was conducted in India (Jey etal, 2017) to determine the cut off value of

titer for positive Salmonella typhi, and as a result the bases line titer for Salmonella typhi

must be ≥1:160, and in the current study this value was excluded, and the cut of value was

1:320 for positive titer. In a study conducted in Nigeria (Osahon etal, 2016) about Widal

agglutination test, a blood samples of 271febrile persons were collected and examined on

standard base,124(45.8%) were positive for Salmonella typhi by Widal test,60(22.1%) blood

samples grew Salmonella on blood culture, thus, the False positive cases by Widal test were

64(23.6%), however blood culture method remains the best test for diagnosis of typhoid

fever.55(20.3%) blood samples showed a co-infection of Typhoid fever and Malaria. As a

conclusion Widal agglutination test is not a valid diagnostic method for typhoid fever, while

co-infection with malaria parasite confirmed in typhoid fever infection. Malaria parasite is

associated with positive titers on Widal test.

74

Chapter Five

Conclusions and Recommendations

5-1 Conclusions:-

The results that were obtained in the present study were discussed and summarized , modified

Widal test slide method was better than the hospital Widal test slide method at P value

0.004(P<0.05,table 4.20). There is a relationship between the Study Widal Test slide method

and study Widal test tube method, thus the relationship is highly significant. P.value 0.000.

(P < 0. 05). That means the study Widal test tube method was better than the study Widal test

slide method.

There is a highly significant relationship between the study Widal test slide, tube methods

and ICT at P.value 0.000 (P<0.05). ICT test was better than the study Widal test slide and

tube methods.

- Culture was the best test that performed adequate results in diagnosis of typhoid

fever.

- The current techniques used for diagnosis of typhoid fever is not performed

adequately, hence obtained high false positive results, and high prevalence rate.

- Widal test has a low sensitivity and high specificity.

5-2 Recommendations

1-Widal agglutination test should not be used for diagnostic method for typhoid fever.

2- Culture test was the best diagnostic method for typhoid fever, if it was available and

affordable.

3-Immuno chromatographic test (ICT) should be used as an alternative of Widal test in

identifying typhoid fever.

4-To raise the awareness of the decisions makers to supply hospitals with equipments and

materials recommended for diagnosis of typhoid fever.

5- Additional research is recommended in an other area to elucidate more results

75

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80

Appendix (1)

Questionnaire about typhoid fever

1. Name: …………………………………………………………………………… No.

2. Sex:

2.1 male 2.2 female

3. Age: 3.1 15-30

3.2 31-45

3.3 46-60

3.4 + 60

4. Residence:

4.1 State …………………………

4.2 locality ………………………

4.3 City …………………………. Hai ……………….. Square ……………

4.4 Village ………………………………..

4.5 Tel. No: …………………………………

4.6 Email: …………………………………………………………………….

5. Marital status:

5.1 Marriage

5.2 Single

5.3 Divorce

5.4 Widow

81

6. Education:

6.1 khalwa

6.2 primary school

6.3 Secondary school

6.4 Higher school

6.5 University

6.6 Undergraduate

6.7 Postgraduate

7. Occupation:

7.1 house wife

7.2 farmers

7.3 free trade

7.4 employees:

7.4.1 Public sector

7.4.2 Private sector

7.4.3 Governmental sector

7.4.4 Organization

7.4.5 Others

8. Income/ month S.P

8.1 250-500

8.2 600-1000

8.3 1100-2000

8.4 2000<

82

9. Water Supply:

9.1 surface water

9.2 Ground water (wells)

9.3 General net

9.4 hand pump

9.5 Others (mentioned)

10. Food hygiene:

10.1 The food was covered after cooking

yes No

10.2 The food store in the refrigerator

yes No

10.3 Daily meals at home

yes No

10.4 Daily meals at market (outside)

yes No

11. Sewage system:

11.1 Open places

11.2 Bukets system

11.3 Pit latrines

11.4 Water close latrine.

11.5 Siphone latrines (sewage system)

83

12. Semi solid waste:

12.1 Collection by car from house to house

12.1 Collection in certain place (koosha)

12.3 Collection in the roads

12.4 Collection outside the residence area

13. Complaints:

13.1 Sustained fever 39 o to 40 o

13.2 Feel week

13.3 Stomach pains

13.4 Headache

13.5 Loss of appetite

13.6 Rash of flat, rose-colored spots

13.7 Others (mention)

14. Typhoid fever spread by:

14.1 Contaminated food by S.typhi

14.2 Drinking of contaminated water by S. typhi

15. Investigation results:

15.1 Widal test.

15.2 Culture test.

15.3 ICT test.

84

Appendix (2)

ICT positive For IgG -IgM

Picture (5) ICT at

Wad medani Teaching hospital - Gezira State –Sudan - 2012.

85

ICT Negative

Picture (4) ICT at


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Picture (3) Widal test Tube method


87

Picture (2) Widal test Slide method wad medani Teaching hospital - Gezira State –Sudan - 2012.

88

Picture (1)

Central laboratory of wad medani Teaching hospital - Gezira State –Sudan - 2012.

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Picture (6) Laboratory works at


(The Co-Supervisor and the researcher)

90



91



92

Picture (9) Blood culture test


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