ENTERIC PATHOGENIC BACTERIA AND ASSOCIATED RISK FACTORS IN DIARRHOEIC AND DYSENTERIC CHILDREN

TABLE OF CONTENTS

CHAPTER ONE

1.0       INTRODUCTION

1.1       Background

1.2       Statement of research problems:

1.3       Justification of the study

1.4       Aim

1.5       Objectives

CHAPTER TWO

2.0      LITERATURE REVIEW

2.1      Diarrhoeal disease

2.2       Causes of diarrhoeal disease

2.2.1   Congenital or Inherited Causes

2.2.2   Post Surgery/Post Radiotherapy

2.2.3.  Endocrine disorders

2.2.4.  Neoplastic diarrhoea (caused by cancer)

2.2.5. Diarrhoea due to allergic reactions

2.2.6.  Drug-related diarrhoea

2.2.7   Purgative abuse

2.2.8   Mal-absorption of food eaten

2.2.9   Functional (psychogenic or non-specific) cause of diarrhoea

2.2.10 Infectious agents

2.3       Severity of diarrhoeal infection

2.4       Forms of diarrhoea

2.4.1   Infectious diarrhoea

2.4.1.1            Community-acquired infectious diarrhoea

2.4.1.2            Traveler’s diarrhoea

2.4.1.3            Hospital-acquired infectious diarrhoea

2.4.2   Acute diarrhoea

2.4.3   Persistent diarrhoea

2.4.4   Chronic diarrhoea

2.4.5   Secretory Diarrhoea.

2.4.6   Osmotic Diarrhoea.

2.4.7   Childhood diarrhoea

2.5       Aetiologic agents of childhood diarrhoea

2.5.1   Viral diarrhoea

2.5.1.1 Group a rotavirus

2.5.1.2 Enteric adenovirus

2.5.1.3 Norovirus

2.5.1.4 Astrovirus

2.5.1.5 Cytomegalovirus

2.5.1.6 HIV diarrhoea

2.5.2   Bacterial Diarrhoea

2.5.2.1Escherichia coli pathovars

2.5.2.1.1 Enteropathogenic E. coli

2.5.2.1.2 Enterotoxigenic E. coli

2.5.2.1.3 Enteroinvasive E. coli

2.5.2.1.4 Enterohaemorrhagic E. coli

2.5.2.1.5 Enteroaggregative E. coli

2.5.2.1.6 Diffusely adhering E. coli

2.5.2.2 Salmonella

2.5.2.3 Shigella

2.5.2.4 Campylobacter

2.5.2.5 Yersinia

2.5.2.6 Vibrio cholerae

2.5.3   Intestinal parasites

2.5.3.1 Giardiasis

2.5.3.2 Cryptosporidiosis

2.5.3.3 Entamoeba amoebiasis

2.6       Laboratory diagnosis of childhood diarrhoea

2.6.1   Molecular assay for detecting bacterial pathogen causing diarrhoea

2.6.1.2 Polymerase Chain Reaction (PCR)

2.6.1.3 Real-Time PCR

2.7       Malnutrition and diarrhoea

2.8       Poor hygiene and diarrhoea

2.9       Prevention and control of childhood diarrhoea

2.10    Treatment against childhood diarrhoea

CHAPTER THREE

3.0      MATERIALS AND METHODS

3.1       Study Area

3.2       Sampling Design

3.3       Ethical approval

3.4       Sampling Techniques:

3.4.1   Inclusion criteria:

3.4.2   Exclusion criteria:

3.4.3   Sample size:

3.4.5   Sample collection and storage:

3.5       Detection of Enteropathogenic Bacteria from Stool Specimens

3.5.1   Microscopic examination of stool specimens

3.5.2   Stool culture

3.5.3   Identification of Isolates using Biochemical Methods:

3.5.3.1 Gram staining

3.5.3.2 Catalase test

3.5.3.3 Oxidase test

3.5.3.4 Indole test

3.5.3.5 Methyl red test

3.5.3.6 Voges-Proskauer (VP) test

3.5.3.7 Citrate utilization test

3.5.3.8 Urease production test

3.5.3.9 Orthonitrophenyl- -D-Galactopyranoside (ONPG) test

3.5.3.10 Gelatin hydrolysis test

3.5.3.11 Phenylalanine deamination test

3.5.3.12 Decarboxylases test

3.6       Antibiotic Susceptibility Test (Kirby-Bauer Disk Diffusion Method)

3.7       Bacteriological Examination of the Various Source of Water from the Study Areas.

3.7.1   Water sampling

3.7.2   Enumeration of total and faecal coliform from water samples (Membrane Filtration Technique)

3.8       Detection of Enteropathogenic Bacteria Directly from Stool Sample by PCR.

3.8.1   DNA extraction (using feacal DNA MiniPrep kit)

3.8.2   Amplification of target DNA (utilizing Dual Priming Oligonucleotide [DPO])

3.8.3   Detection of bacterial gene on Agarose Gel

3.9       Data analysis

CHAPTER FOUR

4.0       RESULTS

4.1       Prevalence of bacterial diarrhoea among the study population.

4.2       Prevalence of various bacterial agents of diarrhoea in Sokoto state.

4.3       Distribution of enteric pathogens from senatorial zones in Sokoto State by Gender.

4.4       Distribution of diarrhoea among children in Sokoto State by age group

4.5       Occurrences of Campylobacter species among diarrhoeic children in Sokoto state.

4.6       Occurrences of Escherichia coli among diarrhoeic children in Sokoto state.

4.7       Occurrences of Salmonella enterica among diarrhoeic children in Sokoto state.

4.8       Occurrence of Shigella species among diarrhoeic children in Sokoto state.

4.9       Antibiotic susceptibility pattern of Campylobacter species.

4.10    Antibiotic susceptibility pattern of Escherichia coli.

4.11    Antibiotic susceptibility pattern of Salmonella enterica.

4.12    Antibiotic susceptibility pattern of Shigella species.

4.13    Multiple antibiotic resistance (MAR) indices of the isolates

4.14:   Assessment of risk factors associated with diarrhoea in children from Sokoto

4.15:   Enumeration of coliform organism from water samples.

4.16:   Relationship between faecal coliform density and bacterial diarrhoeal disease occurrence

4.17    Prevalence of enteropathogenic bacteria detected by their target genes and product size of PCR.

4.18    PCR results

CHAPTER FIVE

5.0       Discussion

CHAPTER SIX

6.0       Conclusion and Recommendation

6.1       Conclusion

6.2       Recommendation

References

Abstract

Enteric bacterial diarrhoeal and dysenteric illnesses exact a heavy toll on human populations, particularly children. Diarrhoea due to bacterial infections is an important cause of morbility and mortality in infants and young children in most developing countries. Diarrhoea a situation when a person passes more stool than is normal, passing three or more loose or liquid stools per day is caused by a variety of pathogens including bacteria, viruses, and parasites. Among the bacteria, Campylobacter jejuni, Salmonella, Shigella and Yersinia species are known to be the most common and economically important agents but other bacteria, including diarrhoeagenic Escherichia coli (DEC) and Campylobacter coli have also been identified as causes of enteric diarrhoeal disease in children. The aim of this study is to detect enteric bacterial pathogens and associated risk factors in diarrhoeic children in Sokoto state, Nigeria. A total of four hundred and twenty diarrhoeic children five years and less from the three senatorial zones of Sokoto State, Nigeria were screened for the presence of enteropathogenic bacteria namely Escherichia coli, Campylobacter, Salmonella and Shigella species. Stool samples were analyzed by both the conventional culture methods and the standard PCR techniques for the detection of the target enteropathogenic bacterial virulence genes. The antibiogram of the isolates were determined by disk diffusion method. To determine some risk factors associated with incidence of bacterial diarrhoea in children structured questionnaire designed to obtain basic data was used while various water sample from the study area were examine for possible faecal contamination. Of the diarrhoeaic stool specimens analyzed by the culture techniques, Campylobacter species, Escherichia coli, Salmonella enterica and Shigella species were detected in 14 (3.3%), 79 (18.8%), 30 (7%) and 17 (4%) of cases respectively. On the other hand, results of PCR of the stool specimens revealed that, Campylobacter spp., E. coli, Salmonella enterica and Shigella spp. accounted for 34 (8.1%), 87 (20.7%), 35 (8.3%) and 22 (4.8%) of all cases of childhood diarrhoea respectively. E. coli remains the major cause of infantile diarrhoea, followed by Salmonella enterica irrespective of the method of stool analysis. It was observed that, the incidences of diarrhoea among children of 0 to 5 years across the study area is higher (19 - 21 %) among children within the 0 to 3 years age bracket compared with the 13 – 15% recorded for children of 4 - 5 years age bracket. Also, a consistent trend of decreasing incidence with increasing age of the children was noted across the study area. Children of both sexes appear to be equally affected by the disease (51.4% males, 48.6% females). The antibiogram studies on the isolates revealed that most were resistant to Amoxycillin-clavulanic acid, Cotrimaxazole and Erythromycin. However the isolates were found highly susceptible to Ofloxacin, Ciprofloxacin, Ceftriaxone and Cefuraxime. The multiple antibiotics index analysis revealed that all the four isolates had a very high index value (≥0.2%). The percentage frequency of multiple antibiotics resistance (MAR) index value greater than 0.2%, were recorded as 8(57.1%), 44(55.7%), 19(63.3%) and 10(58.8%) isolates for Campylobacter species, Escherichia coli, Salmonella enterica and Shigella species respectively. The risk factors so far identified are lack of portable safe water, improper sewage disposal system and poor hygienic practices. Assessement of various potential risk factors, the incidence of diarrhoea and infection rate indicated that strong association exist and the difference observed was considered statistically significant (P<0.05).

CHAPTER ONE

1.0                       INTRODUCTION

1.1 Background

Diarrhoea is termed as a situation when a person passes more stool than is normal, passing three or more loose or liquid stools per day. Diarrhoea in children can be caused by a variety of pathogens including bacteria, viruses, and parasites. Among the bacteria, Campylobacter jejuni, Salmonella, Shigella and Yersinia Species are known to be the most common and economically important agents (Britton and Veraslovic, 2008) but other bacteria, e.g. enterotoxigenic Escherichia coli (ETEC) and Campylobacter coli have also been identified as causes of enteric disease and diarrhoea in children (Britton and Veraslovic, 2008).

Every year, diarrhoeal diseases contribute to 1.7 million deaths worldwide, mainly in children under the age of 5 years particularly in developing countries (WHO, 2009). Even in industrialized countries, the burden of diarrhoeal diseases is substantial, but in most cases, the etiology is unknown (Richard et al., 2006). Infectious diarrhoea still represents a common public health issue. More than three-quarters of the world population is affected and 1–2 illnesses per person are recorded annually. However, in about 50% of cases, the causative agent remains unknown.

At present, the widespread occurrence of infectious diarrhoea has become one of the major public health problems worldwide. Therefore, a rapid response, which includes identification of the pathogens and prevention of the spread of these pathogens in the community, is crucial for the control of disease outbreak and case investigations (Yuanhai et al., 2008). Transmission of enteric pathogens often occurs through the consumption of contaminated food and water. Although a detailed diagnosis and treatment is usually not required, in outbreak situations, especially in community that share facilities or camps, the causative organism must be identified quickly to prevent further spread. Furthermore, clinicians need an early diagnosis for the effective treatment of the severely ill patients (Guerrant et al., 2001).

Bacterial culture is still the ‘gold standard’ for the identification of enteropathogenic bacteria, but the procedures are time consuming and require 3–5 days (Pawlowski et al., 2009). Most medical microbiology laboratories use conventional diagnostic procedures, such as culture and microscopy, for routine detection of enteric pathogens. These culture procedures include enrichment steps, use of selective culture media, biochemical identification, serotyping, and resistance profiling (Pawlowski et al., 2009). Final results are obtained after 3 to 5 days, making these procedures laborious and time-consuming. Furthermore, the detection of pathogens in stool specimens by culture is complicated. For instance, bacteria belonging to the normal gastrointestinal flora can present with the same colony morphology as enteric pathogens (Cunningham et al., 2010). The resultant misidentification increases hands-on time and delay in reporting of a definite negative result. Other problems are the viable but non-culturable state of some organisms such as Campylobacter jejuni (LaGier et al., 2004) and the

limited viability of shigellae outside the human body. These also compromise the sensitivity of culture methods (Cunningham et al., 2010).

More rapid diagnostic tools are therefore required to circumvent these problems (Weimer et al., 2011). The invention of molecular diagnostics, in particular Polymerase chain reaction (PCR), has allowed rapid identification of microorganisms (Espy et al., 2006). However, in the past, even the PCR methods were laborious and prone to error. But the introduction of hydrolysis and hybridization probes have substantially improved the specificity of the test, and reduced the problem of cross-contamination considerably. Several PCR assays use either gel or hydrolysis probes combining enrichment, DNA extraction, PCR amplification, and detection by hybridization and colour development. The most recent PCR thermo-cyclers allow the simultaneous amplification and detection of several targets (maximum 5) in multiplex PCR (Wilhelm and Pingoud, 2003). So far, only a few attempts have been made to use multiplex PCR for the detection of enteropathogenic bacteria in stool specimens (Linton et al., 1997, Douglas et al., 2003 and Weimer et al., 2011).

1.2 Statement of research problems:

Infectious diarrhoeal disease in children is a leading cause of morbidity and mortality worldwide particularly in developing countries. There are also significant economic and social costs associated in addition to increased morbidity and mortality in Nigeria (Aminu et al., 2007). The prevalence of diarrhoea in Nigeria is 18.8% and is one of the worst in sub-Sahara Africa and above the average of 16% (WHO, 2010). It accounts for over 16% of child deaths in Nigeria. An estimated 150,000 deaths mainly amongst children under five occur annually due to this disease mainly caused by bacterial organisms arising from poor sanitation and hygiene practices (WHO, 2009).

Large proportion of diarrhoeal illnesses in children in developing countries is ascribed to an unknown aetiology because the commonly available methods of diagnosis, such as microscopy and culture, have been associated with low sensitivity (Ajjampur et al., 2007). Conventional culture methods remain the norm for the isolation of bacterial enteric pathogens in clinical laboratories. Diarrhoeal diseases due to known bacterial pathogens require rapid diagnosis of the causative pathogenic bacteria which included Escherichia coli, Vibrio cholerae, Vibrio parahaemolyticus, Salmonella enterica, Campylobacter jejuni, Shigellae, Yersinia enterocolitica, and Listeria monocytogenes (Yuanhai et al., 2008). All these aetiologic agents are often unidentified by traditional methods as both the clinical and analytical sensitivity are low, and require a longer time, as such the diagnostic outputs to allow for earlier treatment, epidemiological investigations and infection control interventions are hindered (Cunningham et al., 2010).

1.3 Justification of the study

Conventional methods of isolation of bacterial strains from faecal samples take 3–5 days to complete and are therefore laborious and require substantial manpower. Besides, very small numbers of viable organisms present in the faeces may fail to grow in artificial laboratory media (Pawlowski et al.,

2009). Traditional methods for detection of the aetiological agents of diarrhoea have ranged from microscopy and bacterial culture to immunoassays, and vary for each enteric pathogen.

This has resulted in a large proportion of samples being ascribed to an unknown aetiology. This study is aimed at decreasing the diagnostic gap in diarrhoeal disease detection by the application of PCR techniques. Identifying the enteric pathogens that contribute significantly to the causes of diarrhoea in children will help to provide an accurate and region-specific estimate of disease burden. Also, the PCR-based methods provide an easy to perform, rapid and uniform method of testing for a wide range of pathogens. This study, therefore, is designed towards assessing the sensitivity of the PCR assays for detecting common enteric pathogens with the view to enhance the identification of aetiological agents of diarrhoea in children. Enteric bacteria such as Campylobacter jejuni, Salmonella, Shigella and Yersinia species are major cause of bacterial gastroenteritis worldwide (Britton and Veraslovic, 2008). A rapid method for their detection in clinical specimens is therefore needed. Multiplex-PCR can obtain results within a shorter time with only a few processing steps, which minimizes processing errors. The cost of PCR is comparatively low and, above all, no infectious waste is produced, and results can be obtained even from inactivated material. The high sensitivity and speed of the test allows clinicians to start early antibiotic treatment in critically ill patients. In the management of outbreak scenarios, the source of infection can be pinned down more quickly, and early action to control the spread can be initiated (Weimer et al., 2011).

1.4 Aim

To detect enteric pathogenic bacteria and determine associated risk factors in diarrhoeic and dysenteric children in Sokoto state, Nigeria.

1.5 Objectives

1.    To determine the prevalence of bacteria in children with diarrhoea using conventional culture method.

2. To determine the antibacterial susceptibility pattern of the enteropathogenic bacteria detected.

3.  To determine some risk factors associated with incidence of bacterial diarrhoea in children.

4.  To screened for total and faecal coliforms in various sources of water from the study areas.

5.  To detect some enteropathogenic bacteria directly from the stool sample by PCR technique.