Mona Mohamed Ali Ahmed

Detection of Salmonella spp. Among School –Age Children at Wad Habouba Rural Hospital, Gezira State, Sudan (2018)

(B.sc in Medical Microbiology Faculty of Medical Laboratory Sciences University of ElzaeimElazhary2005)

Adissertation

Submitted to the University of Gezira in Partial Fulfillment of the Requirements for the Award of the Degree of Master of Science

Medical Microbiology

Faculty of Medical Laboratory Sciences

October 2018

Detection of Salmonella spp. Among School –Age Children at Wad Habouba, Gezira State, Sudan (2018)

Mona Mohammed Ali Ahmed

Supervision Committee:

Name Position Signature

Prof. Adam Dawoud Abakar main supervisor ______

Dr. Sanaa Elfatih Co- supervisor ______

Date Examination:______

Detection of Salmonella spp.Among School –Age Children at WadHabouba, Gezira State, Sudan (2018)

Mona Mohammed Ali Ahmed

Examination Committee:

Name Position Signature

Prof. Adam Dawoud Abakar Chairman ______

______External Examiner ______

______Internal Examiner ______

Declaration

We hereby, declare that this work is original research work; undertakenunder the supervision of Prof. Adam Dawoud Abakar and has not been presentedelsewhere for award of a degree of certificate. All sources have been citedand appropriately acknowledged.

Name:______

Date:______

Signature:______

Dedication

Tothe greatest love in my life,

thewoman whom I loved

and give me the strength,

well and support all the time

Dearest mother…

Tothose whom always on my side

Brothers and Sisters…

To my supporters through my way

Friends…

Acknowledgements

All praiseandthanks to Allah forblessing and for giving me the welling to complete this study,after that I would like to express my appreciation to my supervisor Prof. Adam DawoudAbakar Salim, for his kind help and support,after that I would like to express my appreciation to my supervisor Dr. Sanaa Alfatihalso thanks extended to all staff of the bacteriology laboratory of the medical laboratory of the Gezira University,Also acknowledge with gratitude the assistance,received from my colleges in the Wad Haboubarural hospital.

Finally special thanks to everyone who was beside me to complete this work.

Detection of Salmonella spp.Among School –Age Children at Wad Habouba Rural, Gezira State, Sudan (2018)

Mona Mohammed Ali Ahmed

Abstract

Enteric Fever is an important cause of morbidity and mortality amongchildren indevelopingcountriesincludingSudan.This cross sectional hospital based study aimed to estimate the prevalence rate and risk factor of enteric fever among student attend to wad habouba rural hospital,Blood samples and stools were collected from student aged between (6- 15 years) and suspected to be infected with enteric fever.The study was conducted during the period from August to November 2017 at Wad habouba hospital.Datawascollectedbystructured questionnaire.Specimens were examined by Widal test and stool culture,five ml of blood specimens were collected from student,Out of 100 blood sample only 37 were positive for widal test.stool sample out of 37 sample only 4 were positive in aculture,This study indicate low prevalence rate of enteric fever among the pupils in wad haboubaarea Further studies with large number of patients and more advanced techniques are required to validate the resultsof this study. It is better to perform culture for diagnosing salmonella species more than widal test.

الكشف عن انواع بكتريا السالمونيال وسط اطفال المدارس في منطقة ود حبوبة-والية الجزيرة – السودان

منى محمد علي أحمد

ملخص الدراسة

تعد حمى التيفويدمن المسببات الهامة لالمراضية ومن اهم االسباب تمعظم حاالت الوفاة الطفال الدول النامية ويعد السودان احد الدول النامية، هدفت هذه الدراسة لتقدير معدل انتشار وعوامل خطورة حمي التيفويد من طالب مرحلة االساس من منطقة ود حبوبة، تم جمع مائة عينة دم منالطالب الذين تتراوح اعمارهم بين سن الخامسة والخامسة عشر الذين يشتبه في انهم مصابين بحمي التايفويد في الفترة من اغسطس الي نوفمبر لعام 2017، من مائة عينة دم وجدت 37 عينة موجبة الختبار الفيدال، ومن سبعة وثالثون عينة فسحة وجدت اربعة عينات موجبة في التزريع، وتشير هذه الدراسة الي انخفاض معدل انتشار االصابة بحمي التيفويد بين الطالب في منطقة ود حبوبة والية الجزيرة. اجراء مزيد من الدراسات مع عدد كبير من العينات والمزيد من التقنيات المتقدمة للتحقق من صحة نتائج هذه الدراسة.من األفضل أداء التذريع لتشخيص أنواع السالمونيال أكثر من اختبار الفيدال.

TABLEOF CONTENTS

Serial Title Page

Supervision committee i

Examination Committee Ii

Declaration iii

Dedication iv

Acknowledgements v

English Abstract vi

Arabic Abstract vi

List of Content vii

List of tables ix

List of Figures x

List of Abbreviations xi

CHAPTER ONE: INTRODUCTION

1.1 Introduction 1

1.2 Justification 2

1.3 Topic identification 2

1.4 Objectives 2

1.4.1 General Objective 2

1.4.2. Specific Objectives 2

CHAPTER TWO: LITERATURE REVIEW

2.1 The organism 3

2.1.1 Classification 3

2.2 The disease 3

2.3 Symptoms 3- 4

2.4 Antigenic Structure 5-6

2.5. Epidemiology 6

2.6 Contamination And Transmission 7

2.7 Laboratory Diagnosis 7

2.7.1 Specimens 8

2.7.1.1 Blood 8

2.7.1.2 Stool 8

2.7.1.3 Serum 8

2.7.2 Microbiological Procedure 9

2.7.2.1 Blood Culture 9

2.7.2.2 Stool Culture 9

2.7.3 Colony Characteristics 9- 10

2.7.4 Serology 10-11

2.7.4.1 Widal Test 10-11

2.7.4.2 Haemagglutination 11

2.7.4.3 Counter Immuno Electrophoresis 111

2.9 Prevention 11-13

2.10 Anti-microbial Therapy 13

CHAPTER THREE MATERIALS AND METHODS

3.1 Study Area 14

3.1.1 Study Population 14

3.2 Study design 14

3.3 Inclusion Criteria 14

3.4 Exlusion Criteria 14

3.5 Sample Size 14

3.6 Design 15

3.7 Data Collection 15

3.8 Data Analysis 15

3.9 Ethical consideration 15

3.10 Materials 15-16

3.11 Laboratory methods 16-20

3.11.1 Preparation Of Media 16-17

3.11.2 Collection of specimens 17

3.11.2.1 Collection of blood 17

3.11.2.2 Collection of stool 17

3.11.3 Procedure 17

3.11.4 Culture 17

3.11.4.1 Colonial morphology 17-18

3.11.4.2 Gram smears 18

3.11.4.3 Biochemical tests 18-19

3.11.4.3.1 KIA 18-19

3.11.4.3.2 Indole 19

3.11.4.3.3 Urease 19

3.11.4.3.4 Citrate 19

CHAPTER FOUR

4.1 RESULTS 21-31

CHAPTER FIVE

5.1 Discussion 31

5.2 Conclusions 32

5.3 Recommendations 32

References 33

Appendix 37

List of Tables

Table No. Title Page

Table(4.1.1) Distribution of study population according to residence 22

Table (4.1.2) Distribution of study population according to washing hand 24 before having meal

Table(4.1.3) Distribution of study population according to water source 26

Table (4.1.4) Presenting symptom frequencies

Table(4.1.5) Resultfromdiagnostic test under study 29

Table(4.1.6) Some risk factor associated with infection 30

Table(4.1.7) Frequency and percentage of isolated organism 31

Table(4.1.8) Seroprevalence data together with personal and demographic 31 variables

Listof Figures

FIGURES Title Page

Figure (4.1.1) Distribution of study population according to gender 21

Figure(4.1.2) Distribution of study population according to water source 23

Figure(4.1.3) Washing hand with soap after toilet 25

Figure(4.1.4) Cleaning of dirt at home 27

List of Abbreviations

Abbreviations Expansion

ATP Adenosine triphosphate CIE Counter current immuno electrophoresis DCA Deoxycholate agar ELISA Enzyme linked immunosorbent assay HA Haemagglutination test HAg Flagellar Antigen LPS Lipopoly saccharides LPSHA Lipopolysaccharides heamagglutination MP Macrophage OAg Somatic Antigen RPHA Reverse passive haemagglutantion test SPP Species SS Salmonella Shigella VI Virulent Antigen XLD Xylose-Lysine-deoxycholate Agar

CHAPTER ONE

Introduction

1.1 Background Salmonella spp are important food-borne pathogen associated with many feed stuffs. In order to minimize & control these sources of human infection efforts should be made at all levels of food product supply-chain. Typhoid fever, also known as enteric fever is a bacterial infect caused by a gram -ve bacteria primarily by salmonella enterica, subspecies enteric aserovar typhoid & to lesser extent related serovar's paratyphoid A, B, C.(Chart etal.2007)

Both causes symptoms mild to severe and usually begin six to thirty days after exposure. Often there is gradual onset of fever over several days, weakness, abdominal pain, constipation & headache. Some people develop rash with rose coloured spots. In severe cases there may be confusion. Some people may carry the bacterium without being affected, however they are still spread the disease to others (Prajpati etal.2008).

Poor hygiene habits & public sanitation conditions are main routes the disease spreading so an epidemiological investigation is need to be performed to determine the prevalence rate & subsequently to evaluate appropriate intervention polices, determine control programs. In order to estimate the prevalence of salmonella & to evaluate.

The effectiveness of the intervention accurately as possible, it is necessary to investigate bacteriology & serology method more thoroughly (Prajpati etal 2008).

Serology is one approach for determining salmonella status at the individual or population level & it is performed by widal test, which demonstrates antibodies against salmonella antigen O – somatic & H–Flagellar & It is used extensively in serodiagnosis of typhoid fever (Olopoenia and King 2000). On other hand, stool culture: is more useful in the diagnosis of enteric fever.

1-2 Research Justification 1-Typhoid fever remain an important public health problem in Sudan mainly in rural areas, but its screaning is not done in the study area.

2-Scare information available regarding its prevalence in study area.

3-School age children are frequently presented to health care center and clinics with symptoms resembling typhoid fever.

4- Typhoid fever is usually missed diagnosed as malaria and other related infection.

1-3 Research Identification: The nature of this research topic is to detect prevalence rate & risk factor of Salmonella in order to help in implementation of strong control measures.

1-4 Objectives:

1-4-1General objective: To detect prevalence rate and risk factors of salmonella in children between 6-15 years attended Wad Habouba hospital.

1-4-2 Specific objectives: 1. To compare widal test and culture of stool for detection of salmonella.

2. To determine risk factors associated with disease in the study area.

CHAPTER TWO

Literature Review

2.1 Salmonella

Salmonella spp is genus of rod shaped bacillus .gram negative bacteria of the enterobacteriaeae family .the two species of salmonella are s.enterica and s. bongori .s.entrica is type species and is further divided in to sex sub species (Chiu, 2007) that include over 2500 sero type.

2.1.1 Scientific classification and morphological feature of Salmonella

This classification based on (Fabrega and Vila 2013)

Domain bacteria.

Kingdom: Eubacteria.

Phylum; Proteobacteria

Class; gammaproteobacteria.

Order: Enterobaceriales.

Family; enterobacteriaceae.

Genus: salmonella

Species. S.bongori. S; enterica.

Salmonella species are non-spore forming .motile enterobacteria with cell diameter between about o.7 and 1.5mm, length from 2-5mm, and periterichous flagella (all round the cell body) (Fabrega and Vila 2013) they are chemotrophs obtaining their energy from oxidation and reduction reaction using organic sources. They are also facultative anaerobes capable of generating ATP with oxygen (aerobically) when it is available, using electron acceptors or fermentation (anaerobically).

2.2 The Disease

During an acute infection, S. typhi multiplies in mononuclear phagocytic cells before being released into the bloodstream. After ingestion in food or water, typhoid organisms pass through the pylorus and reach the small intestine. They rapidly penetrate the mucosal epithelium via either microfold cells or enterocytes and arrive in the lamina propria, where they rapidly elicit an influx of macrophages (Mp) that ingest the bacilli but do not generally kill them. Some bacilli remain within Mp of the small intestinal

Lymphoid tissue. Other typhoid bacilli are drained into mesenteric lymph nodes where there is further multiplication and ingestion by Mp. It is believed that typhoid bacilli reach the bloodstream principally by lymph drainage from mesenteric nodes, after which they enter the thoracic duct and then the general circulation. As a result of this silent primary bacteraemia the pathogen reaches an intracellular haven within 24 hours after ingestion throughout the organs of the reticuloendothelial system (spleen, liver, bone marrow, etc.), where it resides during the incubation period, usually of 8 to 14 days. Incubation periods ranging from 3 days to more than 60 days have been reported. Clinical illness is accompanied by a fairly sustained but low level of secondary bacteraemia (~110 bacteria per ml of blood). (Gotuzzo, etal, 1991)

2.3 Symptoms

Clinical presentation of typhoid fever

The course of typhoid fever occurs in phases, which last approximately one week (Edwards, etal 1990). In the course of these phases, patients appear worm out and wasted. The patient has high fever, malaise, leucopenia, headache, bradycardia (faget sign), abdominal pain, Cough, bloody nose, eosinopenia and relative lymphocytosis (Mweu, 2008). Rose spots are visible on the lower chest and on the abdomen. The lung bases have rhonchi. The Abdomen is stretched or becomes swollen and hurts at the right lower quadrant, where Borborygmi can be heard. The patient trots frequently and suffers from hepatosplenomegaly (Prajapati et al., 2008). Many other complications may occur during the third week of infection e.g. congested peyer’s bleeding that results in intestinal he

20 morrhage, intestinal perforation, encephalitis, symptoms of neuropsychiatry, metastatic abscessesy, cholesystitis, endocarditis and osteitis (Kasper, etal 2015). Dehydration ensures and the patient is delirious (typhoid state). Platelet level goes down (Prajapati et al., 2008).

2.4 Antigenic structures of salmonella:

The salmonella spp carry a complex antigenic structure. The antigens which have been detected on these organisms include somatic (O) antigen, flagellar(H) antigen, surface antigens (such as Vi), fimbrial (F) antigen, M and N antigens as well as antigens present on rough strains and designated as R antigen (Madigan etal 2017).

2.4.1 Surface Antigens: The surface antigens which are located on the surface of the salmonella and at times render the bacteria unagglutinable by antibodies against O antigen. these include VI (virulence) antigen is a surface polysaccharide which is formed by most of the strains of Salmonella typhi and some strains of Salmonella paratyphi C, Salmonella Dublin as well as Citrobacter. Freundii;

It is heat labile and destroyed at 100°C within 60 minutes. M and N antigens are responsible for mucoid character and F antigen is carried on fimbriae, as well as R antigen responsible for smooth to rough variation of Salmonella (Madigan etal 2017).

2.4.2. O Antigens: These are heat stable polysaccharide antigens that are part of lipopolysaccharide of the cell wall. These antigens are resistant to boiling up to 2 hours and 30 minutes and alcohol-stable, withstanding treatment with 96% ethanol at 37°C for 4 hours. These also remain unaffected in suspensions having 0.2% formaldehyde. Till date 67 somatic (O) antigens have been described and with their help salmonella have been divided into 46 O serogroups (Coleman and Buxton 1907).

2.4.3. H Antigens: These are flagellar antigens. They are heat and alcohol labile but are well preserved in 0.04 to 0.2% formaldehyde. (Coleman and Buxton 1907).

2.5 Epidemiology of salmonella infection: Typhoid fever is a global health problem. Its real impact is difficult to estimate because the clinical picture is confused with those of many other febrile infections. Additionally, the disease is underestimated because there are no bacteriology laboratories in most areas of developing countries. These factors are believed to result in many cases going undiagnosed. (Edelman and Levine, 1986)) and the incidence of typhoid fever recorded in control groups in large vaccine field trials with good laboratory support it has been estimated that approximately 17 million cases of typhoid fever and 600 000 associated deaths occur annually (Levine, etal.1982). However, the estimates have been biased because study populations have usually been in areas of high incidence. Furthermore, these estimates of burden relate to the clinical syndrome of typhoid fever but not to S. typhi exposure.

In areas of endemicity and in large outbreaks, most cases occur in persons aged between 3 and 19 years. In 1997, for example, this age range was reported during an epidemic of the disease in Tajikistan. Nevertheless, clinically apparent bacteraemic S. typhi infection in children aged less than three years has been described in Bangladesh, India, Jordan, Nigeria, and elsewhere (Sinha, etal, 1999). In Indonesia there is a mean of 900 000 cases per year with over 20 000 deaths. In Indonesia, people aged 3-19 years accounted for 91% of cases of typhoid fever and the attack rate of blood-culture-positive typhoid fever was 1026 per 100 000 per year. A similar situation was reported from Papua New Guinea. When typhoid fever was highly endemic in certain countries in South America the incidence of clinical typhoid fever in children aged less than 3 years was low. In Chile, however, single blood cultures for all children aged less than 24 months who presented at health centres with fever, regardless of other clinical symptoms, showed that 3.5% had unrecognized bacteraemic infections caused by S. typhi or S. paratyphi (Ferreccio, etal.1984).

2.6 Contamination and transmission Humans are the only natural host and reservoir. The infection is transmitted by ingestion of food or water contaminated with faeces. Ice cream is recognized as a significant risk factor for the transmission of typhoid fever. Shellfish taken from contaminated water, and

22 raw fruit and vegetables fertilized with sewage, have been sources of past outbreaks. The highest incidence occurs where water supplies serving large populations are contaminated with faeces. (Ivanoof, etal, 1980).

2.7 Laboratory Diagnosis The definitive diagnosis of typhoid fever depends on the isolation of S. typhi from blood, bone marrow or a specific anatomical lesion. The presence of clinical symptoms characteristic of typhoid fever or the detection of a specific antibody response is suggestive of typhoid fever but not definitive.

Blood culture is the mainstay of the diagnosis of this disease.Although ox bile medium (Oxgall) is recommended for enteric fever pathogens (S. typhi and S. paratyphi), only these pathogens can be grown on it. In a general diagnostic laboratory, therefore, where other pathogens are suspected, a general blood culture medium should be used. More than 80% of patients with typhoid fever have the causative organism in their blood. A failure to isolate the organism may be caused by several factors: (i) the limitations of laboratory media (Colema and Buxton, 1907); (ii) the presence of antibiotics (Guerra- Caceres, etal, 1979); (iii) the volume of the specimen cultured (Wain, etal, 1998); or (iv) the time of collection, patients with a history of fever for 7 to 10 days being more likely than others to have a positive blood culture. Bone marrow aspirate culture is the gold standard for the diagnosis of typhoid fever (Soewandojo,,etal,1998) and is particularly valuable for patients who have been previously treated, who have a long history of illness and for whom there has been a negative blood culture with the recommended volume of blood (Wain, et al, 2001). Duodenal aspirate culture has also proved highly satisfactory as a diagnostic test (Benavente, et al, 1984) but has not found widespread acceptance because of poor tolerance of duodenal aspiration, particularly in children (Katz, 1985).

2.7.1 Specimen. 2.7.1.1 Blood The volume of blood cultured is one of the most important factors in the isolation of S.typhi from typhoid patients: 10-15 ml should be taken from schoolchildren and adults in

23 order to achieve optimal isolation rates; 24 ml are required from toddlers and preschool children (WHO 2003).

2.7.1.2 Stool samples Stools can be collected from acute patients and they are especially useful for the diagnosis of typhoid carriers. Stool specimens should be collected in a sterile wide- mouthed plastic container. Specimens should preferably be processed within two hours after collection. If there is a delay the specimens should be stored in a refrigerator at 4°C or in a cool box with freezer packs, and should be transported to the laboratory in a cool box. Stool culture may increase the yield of culture-positive results by up to 5% in acute typhoid fever. If a stool sample cannot be obtained, rectal swabs inoculated into Carry Blair transport medium can be used but these are less successful. (Wain and Hosoglu 2008).

2.7.1.3 Serum For serological purposes, 13 ml of blood should be inoculated into a tube without anticoagulant. A second sample, if possible, should be collected at the convalescent stage, at least 5 days later. After clotting has occurred the serum should be separated and stored in aliquots of 200 ml at +4°C. Testing can take place immediately or storage can continue for a week without affecting the antibody titer. The serum should be frozen at -20°C if longer-term storage is required. (House, etal.2005)

2.7.2 Microbiological procedures 2.7.2.1 Blood culture A typical blood culture bottle contains 45 ml of tryptic soy broth or brain heart infusion broth. These are inoculated with 5 ml of fresh blood and incubated at 37°C. Negatives should be kept for at least seven days. Because S. typhi is not the only bacterial pathogen found in blood, subculturing is performed on days 1, 2, 3 and 7 on non-selective agar. The best agar is blood agar (horse or sheep blood) as this allows the growth of most bacterial pathogens. If blood agar is not available, nutrient agar can be used in combination with MacKonkey agar.

For suspected typhoid fever, subculture plates should be incubated at 37°C for 18-24 hours in an aerobic incubator. (Popoof 1997)

2.7.2.2 Stool or rectal swab culture

This involves inoculating 1 g of stool into 10 ml of selenite F broth and incubating at 37°C for 18 -48 hours. Because selenite broth is very sensitive to heat the manufacturer’s instructions should be carefully followed during preparation and overheating of the broth during sterilization should be avoided. Once a batch is prepared it should be stored at 4°C. Selenite broth. A subculture of selenite broth on a selective agar is therefore made from the surface of the broth without disturbing the sediment. The choice of agar media includes Mac Conkey agar, desoxycholate citrate agar, and xylose-lysine- desoxycholate agar, and hektoen enteric agar or SS (SalmonellaShigella). The plate is incubated at 37°C for 24 hours. (Kalhan etal.1998)

2.7.3 Colony characteristics

Blood agar: On blood agar, S. typhi and S. paratyphi usually produce non-haemolytic smooth white colonies. MacConkey agar: On MacConkey agar, salmonella produce lactose non-fermenting smooth colonies.

SS agar: On SS agar, salmonella usually produce lactose non-fermenting colonies with black centers (except S. paratyphi A, whose colonies do not have black centers).

Desoxycholate agar: Ondesoxycholate agar, salmonella produce lactose non-fermenting colonies with black centers (except S. paratyphi A, whose colonies do not have black centers).

Xylose-lysine-desoxycholate agar on xylose-desoxycholate agar, salmonella produce transparent red colonies with black centers (except S. paratyphi A, whose colonies do not have black centers).

Hektoen enteric agar, on hektoen enteric agar, salmonella produce transparent green colonies with black centres (except S. paratyphi A, whose colonies do not have black centres).

Bismuth sulfite agar:

On this medium, salmonella produce black colonies. (WHO 2003)

2.7.4 Antibody detection tests (serology)

2.7.4.1 The Widal

The Widal agglutination test, suggested by Widal more than one hundred years ago for the diagnosis of typhoid fever olopoenia and King 2000) a, detects serum antibodies to the O=9, 12 somatic, the H=d flagellar, and the “Vi” capsular antigens of S. Typhi. The interpretation of the Widal test remains problematic to this day, with a great number of articles reporting different cut-offs [Olopoenia,Kingal,2000] and the test has lost some popularity in recent years as antigenic determinants of both typhoid and non-typhoid Salmonella organisms are now characterised. In many places, instead of the standard Widal test, a quantitative slide agglutination test [West, etal 989] is used but this should always be interpreted with reference to clinical data. According to the original papers, a rise in titre over time or a single high test result is diagnostically significant and this is supported by modern studies using ELISA(Wain and Hosoglu 2008). False negative results may occur if the blood is collected too early in the disease; therefore, negative results do not rule out typhoid fever [House, etal, 2005] and may be best used as a baseline for subsequent comparative titrations [House,etal,2001]. False positive results may be associated with a past history of immunization for typhoid fever, cross-reacting antibodies, or a whole host of infections and conditions. The detection of VI antibodies can be used for detection of carriers during specific investigations but is not routinely performed in most diagnostic laboratories and the use of a Vi Widal reagent using tube agglutination has not been well reported. (House, etal 2001)

2.7.4.2 Haemagglutination (HA) Tests

Many researchers have evaluated the usefulness of HA tests in different countries. In a study from India, the anti LPS HA test showed a sensitivity of 60% and specificity of 98.2%. The positive predictive value and negative predictive value were 66.7% and 96.7% respectively. In the same study, the haemagglutination inhibition test targeted Salmonella antigens and was found useful for helping the early detection of S. Typhi in culture [Shulkas, chitins 1997]. In another study, a Reverse Passive Haemagglutination Test (RPHA) was designed for the detection of S. Typhi antigen. The test was found to be 70% sensitive and 92% specific for acute typhoid fever diagnosis [Kalhan, etal .1998]. These studies indicate that the passive HA test is comparable with the Widal test and may be a useful alternative to the Widal test for the serological diagnosis of typhoid fever in busy microbiology laboratories in areas in which the disease is endemic (Coovadia, etal .1986)

2.7.4.3 Countercurrent Immunoelectophoresis (CIE)

This test is based on electrophoresis and the visualization of the precipitin band of antigen-antibody complexes that form. The sensitivity is similar to that of the Widal test and the procedure may be quicker if tests are batched (about one hour for a gel), but bands are often difficult to see, the cost is higher than that of the Widal, and some studies conclude that CIE has a low sensitivity with Vi antigen. A panel of antigens (somatic (O), flagellar (H) and capsular polysaccharide (VI) antigens of Salmonella typhi) is recommended for rapid diagnosis of typhoid fever. (Sharma, etal.1997)

2.8 Prevention of typhoid fever

The major routes of transmission of typhoid fever are through drinking water or eating food contaminated with Salmonella typhi. Prevention is based on ensuring access to safe water and by promoting safe food handling practices. Health education is paramount to raise public awareness and induce behaviour change.

4.1 Safe water

Typhoid fever is a waterborne disease and the main preventive measure is to ensure access to safe water. The water needs to be of good quality and must be sufficient to supply all the community with enough drinking water as well as for all other domestic purposes such as cooking and washing.

During outbreaks the following control measures are of particular interest: In urban areas, control and treatment of the water supply systems must be strengthened from catchment to consumer. Safe drinking water should be made available to the population trough a piped system or from tanker trucks (Acharya, etal 1987).

In rural areas, wells must be checked for pathogens and treated if necessary.

At home, a particular attention must be paid to the disinfection and the storage of the water however safe its source. Drinking-water can be made safe by boiling it for one minute or by adding a chlorine-releasing chemical. Narrow-mouthed pots with covers for storing water are helpful in reducing secondary transmission of typhoid fever. Chlorine is ineffective when water is stored in metallic containers.

In some situations, such as poor rural areas in developing countries or refugee camps, fuel for boiling water and storage containers may have to be supplied.( Acharya, etal 1987)

4.2 Food safety

Contaminated food is another important vehicle for typhoid fever transmission.

Appropriate food handling and processing is paramount and the following basic hygiene measures must be implemented or reinforced during epidemics: washing hands with soap before preparing or eating food; Avoiding raw food, shellfish, ice; Eating only cooked and still hot food or re-heating it

During outbreaks, food safety inspections must be reinforced in restaurants and for street food vendor’s activities.

Typhoid can be transmitted by chronic carriers who do not apply satisfactory food-related hygiene practices. These carriers should be excluded from any activities involving food preparation and serving. They should not resume their duties until they have had three negative stool cultures at least one month apart. (Acharya, etal,1987)

4.3 Sanitation

Proper sanitation contributes to reducing the risk of transmission of all diarrhoeal pathogens including Salmonella typhi. Appropriate facilities for human waste disposal must be available for all the community. In an emergency, pit latrines can be quickly built. Collection and treatment of sewage, especially during the rainy season, must be implemented. In areas where typhoid fever is known to be present, the use of human excreta as fertilisers must be discouraged.

4.4 Health education (Acharya, etal.1987)

2.9 Antimicrobial therapy:

The fluoroquinolones are widely regarded as optimal for the treatment of typhoid fever in adults. They are relatively inexpensive, well tolerated and more rapidly and reliably effective than the former first-line drugs, chloramphenicol, ampicillin, amoxicillin and trimethoprim-sulfamethoxazole. (Arnold, etal, 1993)

CHAPTER THREE

Materials and Methods

3.1 Study Area Wad Habouba Area: it is about three Kilometers from Elhsahisa. Nearly 60 miles (130 Kilometers southern Khartoum. Gezira state. Sudan. This work was done at Wad Habouba teaching hospital.

3.1.1 Study population: Basic – level school aged student who attended to Wad Habouba Rural Hospital during August – November 2017.

3.2 Study design: Descriptive cross sectional hospital based study.

3.3 Inclusion criteria: All students (6 -15) years attending to Basic – level school.

3.4 Exclusion criteria: Student who do not attend to Basic – level school.

3.5 Sample size: One hundred blood sample were collected

3.6 Study design: Random selection, taking random samples from student during the study period.

3.7 Data collection: Structured questionnaire was used to collect data from students after permission of both their parents and the school administrations.

3.8 Data Analysis: Data collected from questionnaire and lab results were analyzed by using SPSS (Statistical package for social science).

3.9 Ethical Concern: This research was submitted to Gezira University, Ministry of health Gezira State of other related authorities for approval of ethical clearance.

3.10 Materials: Instrument: Refrigerator, Autoclave, Microscope, Incubator, Sensitive balance, hot air oven.

Media: XLD, Peptone water, Urea media, Citrate media, KIA, DCA

Reagent and Stain: Kovacs reagent, Normal Saline, Crystal violt, Lougols iodine, 70% Alcohol, Oil immersion.

Glass wares: Slides, petri dishes, Test tubes, bottles, Cover glass

Other material Sterile container, Benzen burner, Wire loop, Racks

Material: serum, slide, Centerfuge, Salmonella Antigen.

3.11 Laboratory methods 3.11.1 Preparation of Media XLD media: 56.68 gram was suspended in 1000 ml distilled water. Heat with frequent agitation until the medium is boils. Do not autoclave or over heat. After cooling pour into sterile petri plates.

DCA media: 70.52 grams was suspended in 1000ml distilled water. Heat with frequent agitation until the medium boils. Do not autoclave or over heat. After cooling pour into sterile petriplates.

Peptone water: 60.4 grams was suspended in 1000ml distilled water. Mix well and dis pense into tubes with or without invreted Durham tubes and sterilize by autoclaving at 151bs pressure ( 121C) for 15 minutes.

KIA Media: 57.52 grams suspended in 1000 ml distilled water. Heat to boil to dissolve the medium completely. Mix well and distribute into tubes. Sterilized by autoclaving at 15 Ibs pressure ( 121C) for 15 minutes.(121C ) for 15 minutes.

Urea Agar: 24 gram was suspended in 950 ml distilled water. Heat to boiling to dissolve the medium completely. Sterilized by autoclaving at 15 Ibs pressure (121oC) for 15 minutes. Cool to 50oC and aseptically add 50 ml of sterile 40% urea solution and mix well. Dispense into sterile tubes and allow to set on slanting position. Do not over heat or reheat the medium.

Simmons Citrate Agar; 24, 28 grams was suspended in 100 ml distilled water. Heat to boiling to dissolve the medium completely. Dispense as desired in tubes or flasks. Sterilze by auto vlaving at 15Ibs pressure (121oC) for 15 minutes.

3.11.2 Sample collection 3.11.2.1 Blood sample; About 3ml of blood was collected under Aseptic condition from pupils in plane plastic tes t tube. By vein puncture then centrifuged at 3000 rpm then collect sera.

3.11.2.2 Stool specimens: The stool specimens were collected, from patients diagnosed clinically to have diarrheal diseases in a clean, dry and leak proof containers and transported to the laboratory as soon as possible.

3.11.3 Procedure Full drop of plasma was taken by automatic pipette in each side of slide then add drop of salmo nella antigen to plasma mix them well wait for minutes then read the reaction.

The stool specimens were investigated by two techniques, culture in suitable medium and biochemical reaction.

3.11.4 Culture: Small portion (3-5mg) of stool specimen was inoculated in peptone cultured in XLD agar incubate overnight at37˚C and culture in DCA.

By using sterile loop apply the inoculum to small area of the plate (the well) of XLD agar. DCA agar media and incubate at 37°C for 24 hours.

3.11.4.1 Colonial morphology: Deoxy cholate agar: On deoxy cholate agar, Salmonella produce lactose non- fermenting colonies with black center.

Shigella produce non lactose fermenting pale coloured, 1-2 mm in diameter.

E.coli donot grow on DCA.

On xylose –deoxycholate agar, salmonella produced transparent red colonies 3-5 mm in diameter with black center.

Shigella produced red pink colonies, 2-4 mm in diameter, without black centers.

E.coli produced yellow colonies.

3.11.4.2 Gram smears: Dried smear was fixed, covered fixed smear with crystal violet stain for 30–60 seconds, rapidly washed off the stain with clean water (When the tap water is not clean, use filtere d water or clean boiled rainwater), all the water were tipped off, and the smear cover with Lugol’siodine for 30–60 seconds, wash off the iodine with clean water, then decolorized rapidly (few seconds) with acetone–alcohol (Appendix 6), then washed immediately with clean water (Acetone–alcohol is highly flammable, therefore use it well a way from an

33 open flame), the smear was covered with safranin stain for 2 minutes, washed off the stain with clean water, the back of the slide clean was wiped, and place it in a draining rack for the smear to air dry, smear was examined microscopically, first with the 40 objective to check the staining and to see the distribution of material, and then with the oil immersion objective to re port the bacteria and cells (Cheesbrough, 2010).

3.11.4.3 Biochemical tests: 3.11.4.3.1 Sugars fermentation, H2S and gas production: This is a multi tests carried out in Kligler Iron Agar (KIA) medium. They performed by inoculating KIA medium. A straight wire was used to inoculate KIA medium, first the butt was stabbed and then the slope was streaked in a zig-zag pattern, after inoculation (make sure the tube tops are left loose). KIA reactions are based on the fermentation of lactose and glucose (dextrose) and the production of hydrogen sulphide. A yellow butt (acid production) and red pink slope indicate the fermentation of glucose only, the slope is pink red due to a reversion of the acid reaction under aerobic conditions, cracks and bubbles in the medium, indicate gas production from glucose fermentation A yellow slope and a yellow butt indicate the fermentation of lactose and possibly glucose, a red- pink slope and butt indicate no fermentation of glucose or lactose and blackening along the stab line or throughout the medium indicates hydrogen sulphide (H2S) production (Cheesbrough, 2010).

3.11.4.3.2 Indole test: The tested organism was inoculated in a bijou bottle containing 3 ml of sterile tryptone water , then incubated at 35–37°C for up to 48 h, indole was tested by adding 0.5 ml of Kovac’s reagent. Then Shaked gently and examined for a red color in the surface layer within 10 minutes. Red surface layer indicate positive indole test and no red surface layer indicate negative indole test (Cheesbrough, 2010).

3.11.4.3.3 Urease test: Inoculate test organism inabijou bottle contain ing 3ml of sterile urea media then the tube was closed and incubated at 35–37°C (preferably in a water bath for a quicker result) for up to 4 hours or overnight (Proteus and M. morganii organism give a positive reaction

34 within 4 hours). Red/purple Color indicates a positive urease test while yellow/orange color indicates a negative urease test (Cheesbrough, 2010).

3.11.4.3.4 Citrate Utilization Test: Prepare slope of the media in abijou bottle using sterile straight wire, first streak the slope with suspension of test organism and then stab the butt Incubate at 35–37°C for 48 hrs Look for bright blue colour in the media

On the second day read the result of biochemical test

S. typhi on KIA, red slope, yellow butt, no gas, hydrogen sulphide weak, indole –ve, citrate –ve, urease –ve.

E. coli KIA produced yellow butt, yellow slope, gas +ve, hydrogen sulphide –ve, indole +ve, citrate –ve, urease –ve.

Shigella on KIA produced red slope, yellow butt, no hydrogen sulphide produced, no gas, citrate –ve, urease –ve, indole –ve.

Chapter Four

Results

4.1 Socio demographic charterization of the study population: Gender: the distribution of study population according togender were 50 (50%) male,50 (50%) female.

Gender

50 50

Female Male

Fiqure (4- 1-1) dustribution of study population according to gender

Residence: The Frequency (Distribution of study population according to residence) and distributed as shown in table (4.1). 15 (15%) from Haj Essayed, 35(35%) from

Dolga, 19(19%) from Ganib, 24(24%) from Alhililiah, 7(7%) fromHelat elsheik

Table (4-1 -1): The frequency distribution of study population according to residence

Frequency Percent Valid Haj Elsayed 15 15.0 Dolga 35 35.0 Ganib 19 19.0 Alhilaliah 24 24.0 HelatElsheikh 7 7.0 Total 100 100.0

Majority of study population according to source of food: Frequency of study population according to source of food 74 (74%) sell from seller, 26 (26%) did not sell from seller.

Is He or She Buy Food From Seller

No Yes

Figure (4-1-2): Frequency distribution of study population according to source of food

Frequency distribution of study population according to washing of hand before having a meal

As shown in table (4-2). 97 (97%) washing hand before having meal, 3(3%) did not washing hand before having meal.

Table (4-1-2): Frequency distribution of study population according to washing of hand before having meal

Frequency Percent

Valid No 3 3.0 Yes 97 97.0 Total 100 100.0

Frequency Distribution of study population according to washing hand by soap after using toilet:

Washing hand 88 (88%), 12 (12%) not washing hand.

Is He or She Washes Hands After Using Toilets

No Yes

Figure (4-1-3): Frequency distribution of study population according to washing and after using toilet

Water source:

Frequency distribution of study population according to water source which distributed as shown in table (4-1-3), tap water 97 (97%), channels 3 (3%).

Table (4-1-3): Frequency Distribution of study population according to water source

Frequency Percent Valid Tab water 97 97.0 Canals 3 3.0 Total 100 100.0

Frequency distribution of study population according to cleaning of dirt at home: Which distributed as shown in figure (4-4) burning 72(72%), burring24 (24%), none of this 4 (4%).

For Elimination of Dirt at Your Home 80 72 70

30 24 20

10 4 0 Deep burning Burning None

Figure (4-1-4) Frequency cleaning of dirt at home

The Presenting Symptoms Frequencies: Distribution of study population according to presenting symptom which distributed as shown in table (4-4), fever 76 (20.9%), headache 88 (24.2), abdominal pain 69 (19.0%), diarrhea or constipation 40 (11.0%), fatigue 89 (24.5%), others 2 (5%).

Table (4- 1-4): The presenting symptom frequency

Responses Percent of Cases N Percent

$The_Presenting_Symptomsa Fever 76 20.9% 76.0% Headache 88 24.2% 88.0% Abdominal Pain 69 19.0% 69.0% Diarrhea or Constipation 40 11.0% 40.0% Fatigue 89 24.5% 89.0% Others 2 .5% 2.0%

Table (4 -1-5) Result from diagnostic test under study

Result Result of widal test Result of stool culture No % No % Positive 37 29.1% 4 4% Negative 63 70.9% 96 96% Total 100 100% 100 100%

Table (4-1-6): Some risk factor associated with infection

Yes 0 96 96 0.002 Cleaning of dirt No 4 0 4 Yes 1 87 88 0.105 Washing hand after using toleit No 3 9 12 Yes 1 96 97 0.022 Washing hand before and after meal No 3 0 3 Yes 4 70 74 0.022 Source of food No 0 26 26 Yes 0 97 97 0.9 Water source No 3 0 3 Yes 2 74 76 0.325 Headache No 2 22 24 Yes 3 66 69 0 Abdominal pain No 1 30 31 Yes 4 36 40 0 Diarrhea or conistipation No 0 60 60 Yes 3 73 76 0.046 Fever No 1 23 24

Table (4- 1-7) Frequency and percentage of isolated organsim

Isolated organism Freguency Percent Salmonella spp 4 10.8 Shigella 7 18.9 E.coli 20 54.1 No groth 6 16.2

Table (4-1-8): Seroprevalence data together with personal and demographic variable

Positive Negative Total P value Age in group 5-10 1 40 41 0.001

/year 11-15 3 56 59

Residence Dolga 2 33 35 0.014 Ganib 0 19 19 Haj Essayed 0 15 15 Helat Sheikh 0 7 7 Alhililah 2 22 24

CHAPTER FIVE

Discussion, Conclusions and recommendations

5.1 Discussion Enteric fever is an infectious infiromental disease especially in areas with poor sanitation if not treated it lead to serious complication and economical loses, therefore it recommended to be screened especially in rural areas .the current study aimed to determine the risk factor and prevalence rate of the disease. Diagnosis is made by widal test and culture of stool. The totals of 100sample s of blood were collected randomly from wad-habouba hospital. The patient categorized into 6-15 years .in this study (37) 29, had positive Widal test.

In Sudan isolation of Salmonella was reported by different investigators (EL Hussein, 2010) isolated four Salmonella isolates from 119 fecal samples (3.4%),(Fath Elrahman, 2008) isolated only 7 (15%) Salmonella typhi Salmonella isolates from 47 blood specimens. (Ahmed, 2008) isolated only 2 (4.3%) paratyphi. A isolates from 46 blood specimens (Mahmood, 2008) isolated only 22 (46.8%) Salmonella paratyphi B isolates from 47blood specimen (Mohammed).Epidemiological investigation of salmonellosis in developing countries like Sudan is difficult because of the very limited scope of the studies and lack of coordinated surveillance systems. Prevalence of Salmonella in this study is10.8unlike the studies conducted in Ethiopia at different times, 4.5% in Addis Ababa (Ashenafi and Gedebou, 1985), 6.4% in Addis Ababa (Mache 2002), 4.5% in Addis Ababa (Ashenafi, 1998), reported in Jimma (15%) (Mache 1997).

Results in the present study found out that Widal test has low sensitivity and specificity. These findings concur with the study carried out by Parry et al. (2009) which found out that Widal test had registered high cases of false positives. The case of false positive in the present study are possibly because the ‘O’ and ‘H’ agglutination usually appear around 8-12 days of infection, hence the probability of them not detected in case of early diagnosis.

The results of the present study found out that both gender were susceptible to infection by typhoid fever. These findings concur with the study carried out by

Youssef et al. (2010) that showed that the attack rates by S. Typhi are similar for both gender. This possibly could be because both genders are involved in outdoor activities exposing them to high risk of infection. In addition, most of the health care facilities lack the equipment and technical expertise to properly diagnose typhoid fever (Pang etal al., 2010). In this study, as significant association was found between some risk factor and infection like water source, washing hand after using toilet, water source.

5.2 Conclusion  Enteric fever is a bacterial disease that may cause serious complication and economical loses if not early diagnosed and treated.  This study is done to a basic level school pupils aged from 6-15 years because they are mostly exposed to risk factor.  All positive cases show typical sign of the disease.  Most of widal test results may be due to other infection like malaria. Brucella, and other infection.  Stool culture is more significance in the diagnosis of enteric fever.

5.3 Recommendations Widal test and stool culture are important for early diagnosis of the disease. 1- Health education about enteric fever plays an important role on it prevention. 2- More research must be done among other age groups to estimate the actual prevalence rate. 3- Those study must be supported by organization and government because their importance and their cost. 4- Avoid dependent on Widal test in the diagnosis of enteric fever as it may cause false results. 5- Application and advanced serological and molecular typing methods to support diagnosis of the disease. 6- It is clear that typhoid fever is still a serious health problem; therefore, it recommended that the public should be made aware of the modes of transmission and the need for proper hygiene and sanitation should be emphasized. 7- Increase the sample size to validate the results of this study.

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Appendix Appendix 1: Questionnaire University of Gezira Faculty of Medical Laboratories Science Questionnaire

1. Age: _____ 2. Gender: Male  Female  3. Residence: ______4. The presenting symptoms: I. Fever  II. Headache  III. Abdominal Pain  IV. Diarrhea or Constipation  V. Fatigue  VI. Others  5. If she or he suffer from the above symptom, go to: I. Private clinic  II. Public hospital  III. Health center  IV. None  6. Types of sample taken from: I. Blood  II. Stool  III. Urine  7. Is she or he buy food from seller: I. Yes  II. No  8. Is she or he wash her/his hands before or after meal: I. Yes  II. No  9. Is she or he wash her/his hands after using toilet: I. Yes  II. No  10. In your area the water supply from: I. Tab water II. Channels III. Other  11. For elimination of dirt at your home: I. Deep burring II. Burning  III. None  12. The lab results: I. Malaria  II. Enteric fever  III. Brucella  IV. Other infection  13. The treatment given: I. Injectable antibiotic 

II. Oral antibiotic (Syrup or tabs) 