AETIOLOGY, MALNUTRITION AND FAECAL LACTOFERRIN LEVELS IN PAEDIATRIC DIARRHOEA By JAPHETH AWULETEY OPINTAN Department o f Microbiology University o f Ghana Medical School, Korle-Bu, Accra. •n University of Ghana http://ugspace.ug.edu.gh AETIOLOGY, MALNUTRITION AND FAECAL LACTOFERRIN LEVELS IN PAEDIATRIC DIARRHOEA Japbeth Awuletey Opintan, MPhil, BSc (Hons), Dip Stat (Ghana) (ID # 10125294) This thesis is submitted to the University of Ghana, Legon in partial fulfillment of the requirement for the award of Doctor of Philosophy (PhD) Microbiology degree Department of Microbiology University o f Ghana Medical School, College o f Health Sciences AUGUST, 2011 University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh ' DECLARATION This thesis has not been presented to any local or international university for the award of any degree. Data was collected from the Princess Marie Louise Children’s Hospital in Accra, Ghana. Initial specimen processing and further molccular analyses were done at the Department of Microbiology, University of Ghana Medical School (UGMS), and the Centre for Global Health, Division of Infectious Diseases and International Health. University of Virginia, USA. The work was jointly supervised by the under-listed Professors. JAPHETH A. OPINTAN (Student) Dare PROFESSOR MERCY J. NEWMAN (Supervisor) Date PROFESSOR MICHAEL D. WILSON (Supervisor) Date .... PROFESSOR RICHARD L.GUERRANT * Date (Supervisor) University of Ghana http://ugspace.ug.edu.gh DEDICATION This PhD thesis is dedicated to; all the children whose specimens and anthropometric data were used in the study, my wife Gladys and daughter Marybeth, and to all the Opintan family. To God is all the glory! Great thing He has done. University of Ghana http://ugspace.ug.edu.gh ACKNOWLEDGEMENTS This PhD research work was jointly sponsored by the University of Ghana Medical School (UGMS), Ghana, and the Center for Global Health/Pfizer Initiative in International Health (CGH/P1IH), University of Virginia, VA, USA. I was blessed to have received support and encouragement from many special people, from the conception of the research to the time o f putting the thesis together. 1 wish therefore to acknowledge the contributions o f these important personalities. I am most grateful to all faculty members. The warm reception and guidance from the Head of the Microbiology Department (UGMS), Dr. Patrick F. Ayeh-Kumi, is very much appreciated. I am very much indebted to Professor Mercy i. Newman, who 1 affectionately call ‘Mummy’. Professor Newman has interest in hard work, and she did not relent to give me all the needed support. 1 am grateful to Professor Michael D. Wilson, from the Noguchi Memorial Institute for Medical Research (NM1MR), who guided me through various stages of my career. I wish to thank Dr Rosina Gepi-Attee, Medical Director and Mr. Raymond Affrim, Head of the Laboratory Unit, of the Princess Marie Louise Children's Hospital, (PML) Accra, Ghana, for their contributions towards the design o f the questionnaire and supervising specimen collection. I am also grateful to Mr. David Quarteng and the nurses at PML for administering questionnaires and collating anthropometric data. Mr. Samuel Obodai from the Geography University of Ghana http://ugspace.ug.edu.gh Department, University of Ghana helped with the plotting of the locations of the study subjects. I am indebted to Professor Iruka N. Okeke from Haverford College, PA, USA, who mentored me in several ways. 1 also received consumables from grants Professor Okeke had from the Branco Weiss Fellowship, Society in Science ETHZ, Zurich, Germany. I wish also to thank Professors Richard L. Guerrant and Michael Scheld, Director of the Center for Global Health (CGH) and Director of the Pfizer Initiative, respectively, for their immense support and supervision of my thesis. I am also indebted to the following special people at CGH, who mentored me in several ways; Professors Cirle Warren and Jim Roche, Doctors Jesus E.A.D Sevilleja and Glynis Rollings. I am also grateful to Ms Leah Barret, April Ballard and Charlotte Martins all of CGH, for making my stay and research in the US a memorable one. I also wish to express my appreciation to Dr Patience Mensah, of the WHO Regional Office for Africa, for the encouragement and constant reminders to prioritize my activities. Last and not the least, I wish to acknowledge the cooperation 1 enjoyed from my wife, Gladys and daughter, Marybeth. I had to leave them alone during difficult moments, spending long hours in the laboratory* and on my work. v University of Ghana http://ugspace.ug.edu.gh TABLE OF CONTENTS TITLE PAGE................................................................................................................. ' DECLARATION.......................................................................................................... •' DEDICATION..............................................................................................................•» ACKNOWLEDGEMENTS.........................................................................................>v TABLE OF CONTENTS.............................................................................................v> LIST OF FIGURES...................................................................................................... LIST OF TABLES...................................................................................................... xii ABBREVIATIONS.................................................................................................. xiii ABSTRACT............................................................................................................... xvi CHAPTER ONE............................................................................ .1 INTRODUCTION..................................................................................... .1 I. I General Introduction........................................................................................... I 1.1.1 Rationale of study....................... 8 1.1.2 Working hypotheses............................................................................. 12 1.1.3 Main objective.......................................................................................13 1.1.3.1 Specific objectives................................................................................13 CHAPTER TWO............................................................................................... 14 LITERATURE REVIEW................................................................................ 14 2.1 The Global Burden of Childhood Diarrhoea.................................................. 14 2.1.1 Definition and forms of acute childhood diarrhoea............................18 2.2 Effect o f pathogen-specific diarrhoeas on nutritional status........................ 20 2.3 Protein-Energy Malnutrition (PEM)............................................................... 24 University of Ghana http://ugspace.ug.edu.gh 2.3.1 Mild and moderate ..................................................................... 26 2.3.2 Epidemiology and causes of PEM..........................................................28 2.4 Aetiological Agents of Infectious Diarrhoea................................................. 30 2.5 Diarrhoeagenic Escherichia coli (DEC)......................................................... 3 i 2.6 Enteroaggregative E. coli (EAEC)................................................................. 33 2.6.1 Virulence profile of EAEC................................................................. 34 2.7 Diagnostic Challenges in the Identification of EAEC...................................37 2.8 Pathogenesis of EAEC infections................................................................... 42 2.9 Inflammatory Diarrhoea and its Assessment................................................. 47 2.9.1 Pathophysiology of Lactoferrin..........................................................49 2.9.2 Lactoferrin (LF) assays for enteric pathogens...................................50 2.10 Cryptosporidiosis: Background and Epidemiology......................................52 2.10.1 Diagnosis and genotyping of Cryptosporidium spp ..........................57 2.11 Dysentery: Background and Epidemiology.................................................. 59 2.11.1 Diagnosis and genotyping of dysentery.............................................. 60 2.12 Giardiasis: Background and Epidemiology.................................................. 61 2.12.1 Diagnosis and genotyping of giardiasis............................................. 62 CHAPTER THREE..................................................................................................63 MATERIALS AND METHODS............................................................................63 3.1 Ethical consideration, Study design, Population and settings.......................63 3.1.1 Interviews and diarrhoea definition....................................................64 3.1.2 Anthropometric data and nutritional status assessment....................65 3.2 Specimen Processing and Microbiological Analysis.....................................66 University of Ghana http://ugspace.ug.edu.gh 3.2.1 Routine stool culture............................................................................. 00 3.2.2 Biochemical and serological identification of isolates......................68 3.2.3 Archiving of bacterial strains.............................................................. 70 3.3 Bacterial DNA Extraction (£. coli Isolates).....................................................71 3.4 Stool DNA Extraction........................................................................................ 73 3.5 Optimization, Sensitivity and Specificity of Real-Time PCR (qPCR) 75 3.6 Detection of Enteric Pathogens from Stool DNA............................................83 3.7 Intestinal Inflammation Assessment................................................................. 86 3.8 Real-Time PCR Detection of EAEC from Bacterial DNA............................ 89 3.9 Genotyping of Amplified Cryptosporidium-positive stools...........................90 3.10 Data Storage and Analysis.................................................................................92 CHAPTER FOUR.................................................................................................... 93 RESULTS ................................................................................................................93 4.1 Study Population and Base-line Characteristics..............................................93 4.2 Nutritional Status and Growth Faltering.......................................................... 96 4.3 Real-Time PCR Detection of Pathogens from Stool DNA.......................... 101 4.4 EAEC Virulence Genes Distribution (Stool DNA)....................................... 103 4.5 Multiple Infection (Stool DNA)......................................................................106 4.6 Conventional Stool Culture Results................................................................ 108 4.7 Faecal Lactoferrin Levels................................................................................ 109 4.8 EAEC’s Dispersin Gene Associates Better with Diarrhoea (p<0.05)......... 113 4.9 Cryptosporidium Genotypes: Preliminary results......................................... 118 4.10 Summaries of Additional Data from Questionnaires..................................... 119 University of Ghana http://ugspace.ug.edu.gh CHAPTER FIVE.........................................................................................................121 DISCUSSION.............................................................................................................. 121 5.1 Nutritional Shortfalls among the Study Population...................................... 121 5.2 PCR for Detection of Pathogens...................................................................... 125 5.3 Co-infection......................................................................................................... 127 5.4 Prevalence of EAEC Virulence-associated Genes in Stool DNA............... 129 5.5 Lactoferrin levels................................................................................................ 132 5.6 Molecular Diagnosis o f EAEC: Stool DNA versus Bacterial DNA 134 5.7 Study Limitations............................................................................................... 137 5.8 Conclusions, Recommendations and Future Research Direction............... 138 REFERENCES......................................... 140 APPENDICES.............................................................................................................184 APPENDIX I: Questionnaires administered in this study................................. 184 APPENDIX II: Media and equipment used.......................................................... 189 APPENDIX III: Cycling conditions of target genes screened.............................195 APPENDIX IV: Sequenced data of Cryptosporidium positive stool DNA 1% APPENDIX V: Abstracts and publication emanating from thesis.................... 207 University of Ghana http://ugspace.ug.edu.gh 315 17 21 23 27 38 67 69 72 74 77 x LIST OF FIGURES The cycle of malnutrition and enteric disease Worldwide distribution of deaths caused by diarrhoea in children < 5 years of age Skewed distribution of the global burden of diarrhoea towards Africa and Asia Diarrhoea linearly ablates catch-up growth Effect of undemutrition on EAEC-challenged weaned C57BL/6 mice An iceberg of Protein Energy Malnutrition (PEM) Adherence pattern of diarrhoeagenic Escherichia coli (DEC) to cultured epithelial cell A flowchart on processing of fresh stool specimen A flowchart on culture of stool specimens for bacteria A flowchart showing how bacterial DNA was extracted A flowchart showing how stool DNA was extracted Optimization curves obtained for real-time PCR for some of the genes screened University of Ghana http://ugspace.ug.edu.gh Figure 13a Figure 13b Figure 14 Figure 15 Figure 16 Figure 17 Figure 18 (i) Figure 18 (ii) Figure 19 Figure 20 Photographs showing some reactions of the lactoferrin 87 assays Standard curve fit of lactoferrin LF) using purified human 88 controls An insert map of Ghana showing settlements of the enrolled 94 children in the study Weight-for-age plot of (a) male and (b) female, infants and 97 young children (<2 years) in the diarrhoea sub-population Weight-for-age plot of (a) male and (b) female, infants and 99 young children (<2 years) in the non-diarrhoea sub­ population Enteric pathogens detected and faecal lactoferrin levels 110 Stool DNA vs bacterial DNA in the molecular diagnosis of 115 EAEC (all stool and bacterial DNAs) Stool DNA vs bacterial DNA in the molecular diagnosis of 116 EAEC (bacterial DNA and their matching stool DNAs) EAEC is better diagnosed in stool DNA as opposed to 117 bacterial DNA HRM data analysis of Cryptosporidium-pos i t i ve DNAs 119 University of Ghana http://ugspace.ug.edu.gh LIST OF TABLES Table 1 Selected EAEC virulence factors and their description 35 Table 2 Summaries of LF assays in relation to pathogen-specific 52 diarrhoeas Table 3 Target genes screened from stool DNA 84 Table 4 Baseline characteristics of study population 95 Table 5 Organisms detected by real-time PCR from stool DNA 102 Table 6 EAEC virulence factor positive in stool DNA 104 Table 7 EAEC’s genes in stool in association with and without 105 malnutrition Table 8 Distribution of pathogens by age from stool DNA 107 Table 9 EAEC genes detected an faecal lactoferrin levels 111 Table 10 EAEC virulence factor-positive in bacterial DNA (E. coli) 114 xii University of Ghana http://ugspace.ug.edu.gh ABBREVIATIONS Hi microlitre HM micro molar AA aggregative adherence AAF aggregative adherence fimbria aap gene encoding dispersin aatA gene encoding dispersin transporter protein aggR gene encoding master regulator AIDS Acquired Immune Deficiency Syndrome bp base pairs CDEC cell detaching E. coli CGH Centre for Global Health cm centimeter Ct melt temperature CVD center for vaccine development DAEC diffusely adherent E. coli DEC diarrhoegenic E. coli EAEC enteroaggregative E. coli EHEC enterohaemorrhagic E. coli El EC enteroinvasive E. coli ELISA enzyme-linked immunosorbent assay EPEC enteropathogenic E. coli University of Ghana http://ugspace.ug.edu.gh ETEC enterotoxigenic E. coli FAO Food and Agriculture Organization h hour HAZ height for age z-score HEp-2 human epithelia cell HRM high resolution melting IBD Inflammatory bowel disease IBS irritable bowel syndrome IgM Immunoglobulin M ipaH invasive plasmid antigen H kb kilo base kg kilogram LEE loci of enterocyte effacement LF lactoferrin MAP membrane-associated protein MDG Millennium Development Goals min minutes ml milliliter ODeoo Optical density at 600 nm wavelength OMP outer-membrane protein ORT Oral rehydration therapy PEM protein energy malnutrition Pet plasmid encoded toxin xiv University of Ghana http://ugspace.ug.edu.gh PML Princess Marie Louise Children Hospital RFLP restriction fragment length polymorphism RFU relative fluorescence unit s seconds spp species UGMS University of Ghana Medical School UNICEF United Nations Children's Fund (formerly United Nations International Children's Emergency Fund) UVa University of Virginia WAZ weight for age z-score WHO World Health Organization WHZ weight for height z-score xv University of Ghana http://ugspace.ug.edu.gh ABSTRACT Diarrhoea is a major public health problem that affects the physical and cognitive development of young children. Anthropometric data was collected from 274 children, 170 with diarrhoea and 104 without diarrhoea. Stool specimens were analyzed by conventional culture, PCR for EAEC, Shigella, Cryptosporidium, Entamoeba, and Giardia species, as well as by ELISA for faecal lactoferrin levels. Additionally, all E. coli recovered from culture were PCR screened for EAEC, and compared with those obtained from the stool DNA. Multiple gene loci (aaiC, aap, attA and aggR) were sought for EAEC. About 50% of the study population was mildly to severely malnourished. Mild to severe malnutrition (WAZ <-l), moderate to severe stunting (HAZ < -2) and moderate to severe wasting (WHZ < -2) were associated with diarrhoea (p = 0.023, 0.026 and 0.062, respectively). In only 1 of 170 diarrhoea stool specimen was Shigella flexneri recovered by culture. EAEC and Cryptosporidium were associated with diarrhoea (p = 0.048 and 0.011, respectively), and malnourished children who had diarrhoea were often co-infected with both Cryptosporidium and EAEC. About 27 % (4/15) C. parvum genotypes were identified by HRM analysis. Faecal lactoferrin levels were higher in children with diarrhoea (p = 0.019). Children who had EAEC infection, with or without diarrhoea had high mean lactoferrin levels irrespective of nutritional status. In conclusion, the current study identified high levels of growth deficits among the children with/without diarrhoea. The use of DNA- biomarkers revealed that EAEC and Cryptosporidium were common intestinal pathogens in Accra, and that elevated faecal lactoferrin was associated with xvi University of Ghana http://ugspace.ug.edu.gh diarrhoea in this group of children. EAEC’s dispersin gene (aap) was significantly associated with diarrhoea in both the faecal and bacterial DNAs, in the children studied (p < 0.05). Publication: Part o f the data presented in this thesis is published as follows: Opintan JA et al. (2010). Pediatric Diarrhea in Southern Ghana: Etiology and Association with Intestinal Inflammation and Malnutrition. Am J. Trop Med Hyg. 83: 936 - 943. xvii University of Ghana http://ugspace.ug.edu.gh CHAPTER ONE INTRODUCTION 1.1 General Introduction Malnutrition in infants and young children has devastating effects on their performance, health and survival (Chang et a l, 2002; Guerrant et a l , 2008; Pelletier and Frongillo, 2003). It is estimated that about 53% of all deaths in young children are attributable to underweight, varying from 45% for deaths due to measles to 61% for deaths due to diarrhoea (Caulfield et a l, 2004). Thus, a specific target goal of the Millennium Development Goals (MDG) is to reduce by 50%, the prevalence of being underweight among children younger than 5 years between 1990 and 2015 (UN, 2002). Three commonly used indicators of under nutrition by the World Health Organization (WHO) are stunting (low height for age), thinness (low body mass index for age) and underweight (low weight for age). Stunting is an indicator of chronic under nutrition, the result of prolonged food deprivation and/or disease or illness. Thinness is an indicator of acute under nutrition, the result of more recent food deprivation or illness. Underweight, which is used as a composite indicator to reflect both acute and chronic under nutrition cannot distinguish between the two indexes (WHO, 1995a). Unfortunately, anthropometric measurements are not routinely performed to identify malnourished children in most clinics and hospitals in Ghana, thereby missing the opportunity for diagnosis and appropriate management (Antwi, 2008). 1 University of Ghana http://ugspace.ug.edu.gh WHO estimated that 1.3 million deaths could be prevented in 42 countries with high rates of mortality, by increased levels of breast feeding among infants (Jones el al., 2003; Lauer el al., 2006). For this reason, exclusive breast feeding up to six months of age is promoted and practiced in many countries including Ghana. However, the appropriate timing of complementary feeding for breastfed infants is often controversial and some studies have shown that growth of such infants appears to falter as early as 2-3 months (Dewey el al., 1992). Sub-optimal breast feeding and malnutrition in younger children has been identified as the most powerful shared risk factor of child death and poses a danger to children’s development. Undernourished children have lowered resistance to infection and are more likely to die from common childhood ailments like diarrhoeal diseases and respiratory infections (Grotto el al., 2003; Osumanu, 2008; Schmidt el al., 2009). The risk of death is often directly correlated with the degree of malnutrition (Chen el al., 1980; Van Den Broeck el al., 1993). A vicious cycle ensues between diarrhoea and malnutrition (Figure 1), and studies have shown that malnutrition with frequent diarrhoeal episodes slow cognitive and physical development of children (Guerrant el al., 2008; Lima el al., 2000). One mechanism for this is that diarrhoeagenic pathogens damage the intestinal epithelium and reduce its absorptive function, leading to nutrient depletion and malnutrition (Guerrant el al., 2008). Malnourished individuals, especially young children living in developing countries, may be unable to repair mucosal damage. University of Ghana http://ugspace.ug.edu.gh The cycle o f malnutrition and enteric disease Pathogen ingestion Enteric infection (± Diarrhea) Exacerbated infectidft severity and/dam age I m pa I re connate and m ucosa l responses J A Intestinal dam age Inflam m ation Malnutrition Fitness im pairm ent C o g n itive im pairm ent Figure 1: The cycle of malnutrition and enteric disease. Enteric infection disrupts the gut’s absorptive function leading to malnutrition, and malnutrition in turn leads to more infection. Source: (rnal-ed, 2010) http://mal-ed.fnih.org/: The interactions of Malnutrition and Enteric Infections: Consequences for child health and development. 3 University of Ghana http://ugspace.ug.edu.gh and thus may become prone to persistent or chronic diarrhoea (Adachi el a l, 2002; Guerrant e! al., 2008; Huppertz et al., 1997; Nataro and Kaper, 1998), The agents capable of causing infectious diarrhoea and the mechanisms responsible for disease pathogenesis are generally known, but the true prevalence of these agents in developing countries is poorly understood (Gomez-Duarte et al., 2009). The present study is particularly interested in the relative importance of EAEC, (an emerging pathogen), as compared to other enteropathogens. In most sub-Saharan African countries including Ghana, microbiological methods for clinical investigation o f diarrhoeal diseases are usually restricted to identifying conventional enteric bacteria such as Salmonella and Shigella. Escherichia coli (£ coli) isolates are often not fully characterized because of the lack of resources. EAEC, a category of the diarrhoegenic E. coli (DEC), is however associated with diarrhoea in several contexts: traveler’s diarrhoea (Adachi et al., 2001; Cabada and White, 2008), paediatric diarrhoea (Cennimo el a l, 2009; Paul et a l, 1994), food borne out-breaks (Kaur et al., 2010; Scavia et a l, 2008). human immunodeficiency virus infection (Samie et al., 2007b), symptomatic and asymptomatic cases (Nataro et a l, 1987; Olesen et al., 2005), acute and persistent diarrhoea (Lima et a l, 2000; Okeke el al., 2003), among others. Additional diarrhoeal pathogens of potential importance include Cryptosporidium spp. Entamoeba histolytica and Giardia lamblia, which are parasitic causes of diarrhoea (Ajjampur et a l, 2008; Gutierrez-Cisneros et a l, 2010; Haque et al., 2003a; Kosek et al., 2001; Ortega and Adam, 1997). Unfortunately, investigation 4 University of Ghana http://ugspace.ug.edu.gh of diarrhoea caused by these parasites in most developing countries largely depends on expert microscopy, where technical competence is necessary (Yakoob etal., 2010). Diarrhoeagenic E. coli (DEC), particularly EAEC, which is not often sought for in developing countries, may be an important unrecognized bacteriological agent associated with acute and persistent diarrhoea in children less than 5 years in the sub-region (Bangar and Mamatha, 2008). A recent study in Accra suggests that asymptomatic carriage of EAEC by adults may place younger children at risk of diarrhoea (Opintan et al., 2010) . The ‘gold standard’ for diagnosis o f EAEC is the human epithelial cell (HEp-2) adherence assay (Nataro et al., 1987). In this assay, EAEC strains form a characteristic ‘stacked brick’ pattern. Unfortunately, the lack of specialized facilities and skills required for this type o f assay, limits many laboratories in Africa to use the adherence assay for identifying EAEC. This obviously is causing a disproportionate investigation and diagnostic insufficiency, particularly in resource-poor countries where diarrhoea is endemic and resources need to be prioritized (Okeke, 2006). A molecular probe, CVD432, was developed as a diagnostic tool and is highly specific for EAEC strains carrying the somewhat variable aggregative adherence (pAA) virulence plasmid (Baudry et al., 1990). While the CVD432 probe made it possible for more investigators to seek EAEC, it varies in sensitivity. As reviewed by Okeke 2009, the sensitivity variation was between 20% to 89%, when 5 University of Ghana http://ugspace.ug.edu.gh comparing to the HEp-2 adherence assay (Okeke, 2009). Far fewer E. coli strains were available from Africa in the earlier tests using the CVD432 probe (Baudry el al.. 1990). It was initially hoped that dispersin, an anti-aggregative protein (aap) secreted by many EAEC strains, could serve as a possible target for diagnosis (Sheikh et al., 2002; Velarde et al., 2007). A recent report however demonstrates that aap is produced by some non-EAEC strains (Monteiro et al., 2009). To increase the chances of recovering EAEC using molecular probes, some authors recommended seeking for multiple gene loci (Monteiro et al., 2009). To date, several dozens of gene probes have been designed and used for screening EAEC, but increasingly, the molecular definition of this pathogen is perplexing. As yet, there is no all-encompassing genetic marker for EAEC, as the organism is often identified in individuals with and without diarrhoea (Okeke, 2009; Opintan et al., 2010; Regua-Mangia et al., 2009). Diarrhoea is both a cause and an effect of malnutrition (Guerrant et a l, 1992b), and this hypothesis has at least been proven in murine models (Coutinho et al., 2008; Roche et al., 2010). These murine models mirror what happens in real human infections, under the condition of malnutrion (Petri et al., 2008). While giardiasis has been consistently associated with nutrient malabsorption and stunting in children, the effects of several bacteria and other protozoan on nutritional status and gastrointestinal morbidity are less clear (Boeke et a l. 2010). Data is required, especially from sub-Saharan Africa, to interrupt at all possible points, the bi-directional nature of diarrhoea and malnutrition (Checkley et a l. 6 University of Ghana http://ugspace.ug.edu.gh 2003; Checkley et a l, 2002; Guerrant el al., 1992b; Mata, 1992). Some authors alude that infection with specific enteric pathogens such as EAEC and Cryptosporidium spp. can affect growth even in the absence of overt diarrhoea (Guerrant et al., 1999a; Huang and Dupont, 2004). Faecal lactoferrin is readily available in the stool, and has the potential of being used routinely in the assessment and management of diarrhoea. Especially in cases of persistent diarrhoea, the lining of the intestional mucosa may be compromised, leading to malnutrition (Guerrant et al., 2008; Guerrant et al., 1992b). In patients with inflammatory bowel disease (IBD), faecal lactoferrin has been shown to be a sensitive and specific marker of intestinal inflammation (Kane et al., 2003; Sugi et al., 1996; Uchida et al., 1994; Vaishnavi et al., 2000). Data on the relationship between pathogen-specific diarrhoea and IBD is scanty, especially in developing countries (Greenberg et al., 2002; Kane et al., 2003; Samie et al., 2007b). In a large cohort of children with/without diarrhoea, the current study also sought to investigate any relationship between specific enteropathogen(s) and intestinal inflammation. 7 University of Ghana http://ugspace.ug.edu.gh 1.1.1 Rationale of study The prevalence of acute malnutrition among children under five years is a sensitive and objective crisis indicator, and reflects the wider situation of at-risk populations (Young and Jaspars, 2006). Additionally, the plight of sub-optimally breast fed and malnourished children is often largely invisible because they are only mildly or moderately undernourished (UNICEF, 2009). Malnutrition and diarrhoeal diseases are linked in a complex, vicious cycle. Under-nutrition contributes to the severity of disease caused by intestinal infections, and infection affects the gut’s capacity to absorb nutrients, thus contributing to further malnutrition [Figure 1]. Important strategies which could be used to break this vicious cycle include the identification of specific enteropathogens capable of inducing intestinal inflammation and malnutrition in young children. This will help prioritize scarce resources in endemic populations. EAEC, Cryptosporidium spp, Entamoeba histolytica and Giardia spp are important enteric pathogens, able to induce repeated and/or prolonged diarrhoea in young children, with potential consequences of growth stunting and reduction of intellectual development (Guerrant et al., 2008; Lima et a!., 2000; Lomtz et al„ 2006). For many of these pathogens, optimal diagnostic testing is unavailable, impractical, or prohibitively expensive for developing countries like Ghana. In most sub-Saharan Africa countries including Ghana, microbiological methods for clinical investigation of diarrhoeal diseases are usually restricted to identifying University of Ghana http://ugspace.ug.edu.gh conventional enteric bacteria such as Salmonella and Shigella. DEC are often not fully characterized because of the lack of resources. Few studies in Ghana which investigated DEC used serological methods (Addy et al., 2004; Agbodaze el al., 1988; Mensah et al., 2002). However, majority of diarrhoea causing strains of E. coli do not fall into standard or even predominant serotypes (Campos et al., 2004). There are geographic variations in the epidemiology of the different DEC pathotypes and their sub-types, and surveillance for DEC remains weak. Although some pathotypes show promise for vaccine development, not enough is known about predominant subtypes to ensure that vaccines will be effective in the places where they are most needed. EAEC is one of the most recently identified DEC and is associated with diarrhoeal disease in different contexts, including paediatric and traveler’s diarrhoea (Huang et al., 2007; Scavia et al., 2008). The major problem today is not the identification of EAEC as a pathotype, but the identification of the true virulent clones especially the ones that are involved in persistent illness. Unfortunatly, the molecular definition of EAEC is confusing, and almost all studies recover EAEC from patients as well as controls without diarrhoea (Ochoa et a l, 2009; Okeke et al., 2003; Opintan et a l, 2010; Samie et al., 2007b). Very little is known about EAEC’s epidemiology in West Africa and there is scanty 9 University of Ghana http://ugspace.ug.edu.gh information from Ghana (Opintan et al., 2010). In particular, the relative importance of EAEC versus other pathogens has not been assessed. Such knowledge would be helpful for prioritizing targeted interventions. As EAEC are often recovered from healthy individuals (without diarrhoea) as well as patients with diarrhoea, it is important to determine whether specific virulence factors are associated with intestinal inflammation, as well as whether any risk factors predispose children to EAEC infection. Asymptomatic carriage of EAEC can result in evidence o f low-level enteritis (Steiner et al., 1998). Several studies have suggested that patients infected with EAEC manifest intestinal inflammation, in which the presence o f faecal lactoferrin and proinflammatory cytokines, notably interleukin (IL)-8, is observed (Greenberg et al., 2002; Jiang et al., 2003). Over the last 20 years, adhesions, enterotoxins and cytotoxins encoded on plasmid and chromosome of EAEC, with well-documented deleterious effects on cells in vivo have been identified. In these studies, a variety of mechanisms of action of these factors at the cellular level have been elucidated (Boisen et al., 2009; Harrington et al., 2006; Harrington et al., 2005). Still unclear and quite challenging to elucidate, however, is which of these EAEC-generated factors drives the most clinically relevant and consequential manifestation in children over time leading to prolonged and recurrent diarrhoea. Human volunteer studies relying on pathogen challenge to define this are ethically difficult and potentially harmful. An alternative approach, less direct but likely to arrive at the same conclusion, is to investigate stool 10 University of Ghana http://ugspace.ug.edu.gh specimens from large cohorts of young children presenting for diarrhoea, with and without growth shortfalls. Such an approach requires strong demographic and clinical data (type of diarrhoea, its duration, height/weight for age, etc.) together with means for quantification of the biomarkers in the stool. The myriad variations of clinical symptoms of EAEC infection has been attributed to factors such as; 1) host genetic susceptibility, 2) host immune response, 3) heterogeneity of virulence among EAEC strains and 4) the amount of bacteria ingested by the infected host (Adachi et al., 2002; Kaur et al., 2010). The current study seeks to identify biomarkers in the stool o f children that will be indicative of enteric infection. Clinical applicability will be enhanced since biomarkers proposed for the study are present in stool, readily available in the field without the need for a blood draw or stool culture. II University of Ghana http://ugspace.ug.edu.gh For this study it is hypothesized that: 1. Enteric infection with specific pathogens leads to malnutrition by causing intestinal inflammation. 2. Under the condition of malnutrition and enteric infection, a biomarker of intestinal inflammation is elevated. 3. The virulence profile of the dominant EAEC strain (amount of virulence factor-positive) in the stools of children less than 5 years initiates clinical disease and growth shortfall. 1.1.2 Working hypotheses 12 University of Ghana http://ugspace.ug.edu.gh The current study seeks to identify biomarkers in the stool of young children that will be clinically useful to determine enteric infection, inflammatory enteropathy, and growth faltering due to specific enteropathogens. 1.1.3.1 Specific objectives 1. To assess the nutritional status of children less than 5 years old with/without diarrhoea, attending a secondary hospital in Ghana. 2. To determine the prevalence of specific enteropathogens in the children, and to assess any association (s) with dianrhoea and nutritional status. 3. To characterize, by genotyping the most prevalent enteropathogen(s) in the children with diarrhoea. 4. To quantify faecal lactoferrin levels in the stools of the children and to determine its association with diarrhoea and nutritional status. 5. To assess the utility of stool DNA and bacterial DNA in the diagnosis of EAEC-associated diarrhoea. 1.1.3 Main objective 13 University of Ghana http://ugspace.ug.edu.gh CHAPTER TWO LITERATURE REVIEW 2.1 The Global Burden of Childhood Diarrhoea Between the 1950’s and the mid I980’s, estimates of global mortality from diarrhoea declined from approximately 4.6 million annual deaths to 2.6 million (Bern et a l, 1992; Rohde and Northrup, 1976; Snyder and Merson, 1982). This decline in mortality is attributable to the promotion and use o f oral rehydration therapy (ORT) for acute childhood diarrhoea (UNICEF. 2002). In the last two decades however, the overall annual incidence has remained relatively stable, between 1.6 - 2.1 million (Bern et al., 1992; Kosek el al., 2003). Despite the different methods and sources of information for these estimates, each successive review of the diarrhoea burden over the last two decades has demonstrated declining mortality but relatively stable morbidity (Bern et al., 1992: Duriey et a l, 2004; Kosek et al., 2003). The incidence of diarrhoeal diseases varies greatly with seasons and the child's age. The youngest children are most vulnerable, and incidence is highest in the first two years of life and declines as the child grows older (Abba et al., 2009; Agbodaze et al., 1988; Albert et a l, 1999; Bry ce et a l, 2005). The global prevalence of diarrhoea is disproportionally skewed towards Africa and South Asia. Africa and South Asia are home to more than 80 per cent of child deaths due to diarrhoea [Figure 2] (Petri et al., 2008; Wardlaw et al., 2010). 14 University of Ghana http://ugspace.ug.edu.gh Figure 2: Worldwide distribution of deaths caused by diarrhoea in children less than five years of age in 2000. in the last 2 decades, global mortality from diarrhoea has remained relatively stable between 1.6-2.1 million per year. Adapted from (Petri et al. , 2008). 15 University of Ghana http://ugspace.ug.edu.gh Just 15 countries account for almost three quarters of all deaths from diarrhoea among children under five years of age annually [Figure 3] (Wardlaw et al., 2010). Globally, diarrhoea closely follows pneumonia as the second leading cause of death among children under five years. Together, pneumonia and diarrhoea account for an estimated 40 per cent of all child deaths globally each year. Nearly one in five child deaths is due to diarrhoea, a loss of about 1.5 million lives each year. The toll is greater than that caused by AIDS, malaria and measles combined (Wardlaw et al., 2010). The causes of diarrhoea are several. Displacement of populations into temporary overcrowded shelters is often associated with contaminated water sources, inadequate sanitation, poor hygiene practices, contaminated food and malnutrition - all of which affect the spread and severity of diarrhoea. The lack o f adequate health services and transport reduce the likelihood of prompt and appropriate treatment of diarrhoea cases. For example, in 1994, between 500,000 and 800,000 Rwandan refugees fled into areas around Goma in what is now the Democratic Republic of the Congo. An estimated 50,000 deaths occurred in the first month alone, with 85 per cent of them attributed to diarrhoea (GEP, 1995). The scarcity of water was cited as the main cause of the outbreak. Malnutrition is also common in emergencies and tends to be heightened when feeding practices are disrupted and sanitation deteriorates (Ezzati et al., 2002; GEP, 1995). 16 University of Ghana http://ugspace.ug.edu.gh RMtK co imm v Tom. m um am or Aim UAL CHILD DEATHS DUE TO QUUWtMQCA 1 In d ia 3 * 6 ,6 0 0 2 N ig e r ia 151*700 3 D e m o c ra tic R e p u b lic o f t h e C o n g o 89.900 4 A fg h a n is ta n 02,100 S E th io p ia 73*700 6 P a k is ta n 53.300 7 B a n g la d e s h 50,800 8 C h in a 40 .000 9 U g a n d a 29*300 i o K e n ya 27.400 11 N ig e r 26.400 12 B u rk in a Faso 24.300 13 U n ite d R e p u b lic o f T a n z a n ia 23*900 14 M a ll 20.900 15 A n g o la 19*700 Figure 3: Skewed distribution of the global diarrhoea burden towards Africa and Asia. More than 80 percent of child deaths due to diarrhoea occur in Africa and South Asia, and nearly three quarters of the deaths occur in just 15 countries. Adapted from (Wardlaw el at., 2010). 17 University of Ghana http://ugspace.ug.edu.gh 2.1.1 Definition and forms of acute childhood diarrhoea Diarrhoea is defined as passage of loose or watery stools at least three times within a 24 h period, or more frequently than normal for an individual (WHO, 1995b). Often, it is usually the patient or the mother/guardian who first diagnoses diarrhoea - noticing that the stool have become more liquid, frequent and different in appearance (Cutting, 1988). Diarrhoea could be infectious or non- infectious. Infectious diarrhoea is the more common cause of diarrhoea worldwide (Casbum-Jones and Farthing, 2004). There are four main types of diarrhoea namely osmotic (malabsorption), secretory, inflammatory and motility related diarrhoeas. The review here will only consider secretory and inflammatory diarrhoea. Most fatal consequences of diarrhoea are due to the loss of water and salts in the stool and therefore medical supervision is usually required to manage diarrhoea in infants and young children. Though most episodes of childhood diarrhoea are mild, acute cases can lead to significant fluid loss and dehydration, which may result in death or other severe consequences if fluid is not replaced at the first sign of diarrhoea. There are three main forms of acute childhood diarrhoea, all of which are potentially life- threatening and require different types of treatment: 1) Acute watery diarrhoea-, is associated with significant fluid loss and rapid dehydration in the infected individual. It usually lasts for several hours or 18 University of Ghana http://ugspace.ug.edu.gh days. The pathogens that generally cause acute watery diarrhoea include V. cholerae or E. coli, as well as rotavirus. 2) Bloody diarrhoea: often referred to as dysentery, is marked by visible blood in the stools. It is associated with intestinal epithelial damage and nutrient losses in an infected individual. The most common cause of bloody diarrhoea is Shigella dysenteriae type la. 3) Persistent diarrhoea: is an episode of diarrhoea, with or without blood, which lasts at least 14 days. Undernourished children and those with other illnesses, such as AIDS, are more likely to develop persistent diarrhoea, and their condition is worsened by malabsorption. 19 University of Ghana http://ugspace.ug.edu.gh 2.2 Effect of pathogen-specific diarrhoeas on nutritional status Diarrhoea resulting from enteric infection may share co-morbidity to several clinical symptoms (Alam and Ashraf, 2003; Aramayo el a l , 2009; Crum el al., 2005; Tormo et al., 2008). The current review focuses on the growth impairment that may result from enteric infection. It is estimated that by the time most children living in imporverished countries attain their second birthday, they would have had about 13 diarrhoea episodes, mainly due to enteric infections (Schorling and Guerrant, 1990; Wardlaw et al., 2010). The vulnerability o f these children to enteric infections is increased soon after weaning, particularly when receiving a protein-poor diet (Woodward, 2001). The diarrhoeas may result in both transient growth deficits and in delayed and cumulative effects resulting in permanent growth faltering later in life (Checkley et al., 2003; Molbak, 2000). Although malnourished children tend to “catch up” if given a chance, those with repeated diarrhoeal episodes as a result of repeated infections with enteric pathogens have this catch-up growth linearly ablated (Checkley et al., 2002; Guerrant et al., 1992b; Petri et a l, 2008; Schorling and Guerrant, 1990) [Figure 4]. It is challenging to design human experiments to establish the relationship between pathogen-specific diarrhoeas and nutritional status. There is therefore only limited knowledge associating pathogen-specific diarrhoea to growth deficits, and available data are mainly observational studies (Checkley et a l. 2008). 20 University of Ghana http://ugspace.ug.edu.gh Percent of days with diarrhea Figure 4: Diarrhoea linearly ablates ‘catch-up’ growth. A plot o f weight gained against percentage days with diarrhoea in an observational study of humans. Adapted from (Petri et al., 2008; Schorling and Guerrant, 1990). University of Ghana http://ugspace.ug.edu.gh A recent murine model however, demonstrated the bi-directional nature of diarrhoea and malnutrition in C57BL/6 mice. The EAEC strains used, EAEC 042 and JM221 can also infect humans (Roche et a l, 2010). Infected mice fed on a low-protein (2%) diet were observed to have minimal weight gain and, in some cases, even weight loss. On the other hand, in infected mice fed a regular diet (20% protein), a progressive weight gain was observed, with no slowing of growth [Figure 5] (Roche et a l, 2010). Additional data from their study revealed important findings which would be usefull in future studies of disease pathophysiology and for preclinical testing of new therapeutics for EAEC. Notably, they found that; i) weaned mice fed a protein-poor diet can be infected with EAEC ii) growth shortfalls occur in neonatal mice challenged with EAEC, iii) stool shedding of organisms accompanies growth shortfalls (Roche et al., 2010). A similar murine model demonstrated that undemutrition with cryptosporidiosis causes mucosal disruption, reduced absorptive surface, and increased proinflammatory cytokine responses, leading to growth impairment (Coutinho et al., 2008). Furthermore, the intensity of the inflammatory response and mucosal disruption parallelled the burden of the C. parvxtm infection (Coutinho et al., 2008). It is postulated that the separate murine models mirror what happens in real human situation under a similar condition o f malnutrition and infection (mal-ed, 2010). 22 University of Ghana http://ugspace.ug.edu.gh Time (day* of lift) Figure 5: Effect of undernutrition on EAEC-challenged weaned C57BLV6 mice. A) Weight was gained progressively in the mice receiving chow having regular amount of protein (RP). The set of mice receiving eucaloric chow (LP) showed minimal weight gain and, in some cases, even weight loss. Growth velocities of nourished and undernourished mice were significantly different on days 23-40 (P < 0 .001). Adapted from (Roche et al., 2010). 23 University of Ghana http://ugspace.ug.edu.gh 2.3 Protein-Energy Malnutrition (PEM) Nutritional disorders stem from imbalance between supply o f protein-energy and the body’s demand for them to ensure optimal growth and function. This imbalance includes both inadequate and excessive nutrient intake; the former leading to malnutrition in the form of wasting, stunting and underweight whilst the latter results in overweight and obesity (Antwi, 2008). Protein-energy malnutrition (PEM) in young children is currently the most important nutritional problem in most countries in Asia, Latin America, the Near East and Africa (Hamer et al., 2004; Joosten and Hulst, 2008; Medhin et al., 2010). The term PEM is used to describe a broad array of clinical conditions ranging from mild to serious malnutrition (WHO, 1995b). At one end of the spectrum, mild PEM manifests itself mainly as poor physical growth in children; at the other end of the spectrum is kwashiorkor and nutritional marasmus. Marasmus represents an adaptive response to starvation whilst kwashiorkor represents a maladaptive response to starvation (Lin el al., 2007; WHO, 2000b). Kwashiorkor and nutritional marasmus are characterized by the presence o f oedema and severe wasting, respectively. It was not until the 1930s that Cicely Williams, working in Ghana, described in detail the condition she termed "kwashiorkor" (using the local Ga word meaning "the disease of the displaced child"). In die 1950s kwashiorkor began to get a great deal of attention. It was often described as the most important form of malnutrition, and it was believed to be caused mainly by protein deficiency (Golden, 1988). 24 University of Ghana http://ugspace.ug.edu.gh The current view is that most PEM is the result of inadequate intake or poor utilization of food and energy, not a deficiency of one nutrient and not usually simply a lack of dietary protein. It has also been increasingly realized that infections contribute importantly to PEM (Checkley et al., 2008). In most populations studied in poor countries, the point prevalence rate for kwashiorkor and nutritional marasmus combined is 1 to 5 percent, whereas 30 to 70 percent of children up to five years of age manifest mild or moderate PEM, diagnosed mainly on the basis of anthropometric measurements. Failure to grow adequately is the first and most important manifestation of PEM. It often results from consuming too little food, especially energy, and is frequently aggravated by infections. A child who manifests growth failure may be shorter in length or height or lighter in weight than expected for a child of his or her age, or may be thinner than expected for height. Three conditions necessary' to prevent malnutrition or growth failure include: adequate food availability and consumption; good health and access to medical care; and adequate care and feeding practices. If any one of these is absent, PEM is a likely outcome (FAO, 2010; WHO, 2000b). PEM or undemutrition remains a major cause of disability and mortality, and is ranked as the top cause of global burden of disease (Ezzati et a/., 2002). Fifty- three percent of deaths in children less than five years of age are attributable to PEM (Bryce et al., 2005; Muller and Becher, 2006). The potential negative 25 University of Ghana http://ugspace.ug.edu.gh impact of child undernutrition goes beyond the individual, affecting society and future generations (Grantham-McGregor el al., 2001\ Victora et al., 2008). 2.3.1 Mild and moderate PEM The prevalence of serious PEM (kwashiorkor, marasmic kwashiorkor and nutritional marasmus) is usually between about 1 and 5 percent, except in famine areas. In contrast, the prevalence of moderate and mild malnutrition in many countries of sub-Saharan Africa and South Asia add up to 30 to 70 percent [Figure 6] (FAO, 2010). PEM may be seen in as many as 15 to 50 percent of young children. Figure 6 illustrates that both energy deficiency and protein deficiency play a part, but that energy deficiency is more important. It suggests that protein deficiency plays a greater part in kwashiorkor and energy deficiency in nutritional marasmus. A method was suggested that distinguished three categories of mild to moderate PEM based on weight and height measurements of children. Subsequently these categories came to be known as follows: a) wasting: acute current, short-duration malnutrition, where weight for age and weight for height are low but height for age is normal; b) stunting: past chronic malnutrition, where weight for age and height for age are low but weight for height is normal; c) wasting and stunting: acute and chronic or current long-duration malnutrition, where weight for age, height for age and weight for height are all low. 26 University of Ghana http://ugspace.ug.edu.gh Nutritional marasmus Kwashiorkor Prevalence (% ) / Severe PEM \ 1-5 J / ^ / Moderate PEM VS \ _3s\ s 10*25/ & & ' \%\ J ► i 20-40 L ' .......... \ \wV 1 No evidence of PEM \ 15-50 Visible, above water Submerged, below water Figure 6: An iceberg of Protein Energy Malnutrition. Only about 20% of severe Protein Energy Malnutrition (kwashiorkor, nutritional marasmus and marasmic kwashiorkor) constitute the top, exposed part of the iceberg: they are easy to be diagnosed from the clinical manifestation. The other 80% is submerged, and can only be diagnosed by the use of anthropometric measurements. Adapted from (FAO, 2010) University of Ghana http://ugspace.ug.edu.gh 2.3.2 Epidemiology and causes of PEM Estimates from WHO suggest that the number of underweight children worldwide rose from 195 million in 1975 to an estimated 200 million at the end of 1994 (de Onis et al., 1993; FAO, 2010). Between 2000 and 2002, it was estimated that 852 million people were undernourished worldwide, with most (about 96%) living in developing countries (FAO, 2004). PEM, unlike the other important nutritional deficiency diseases, is a macronutrient deficiency, not a micronutrient deficiency. Although termed PEM, it is now generally accepted to stem in most cases from energy deficiency, often caused by insufficient food intake. Energy deficiency is more important and more common than protein deficiency. It is very often associated with infections and with micronutrient deficiencies (Dickson et al., 2000; Stoltzfus et a l, 2004). Inadequate care, for example infrequent feeding, may play a part. For satisfactory nutrition, foods and the nutrients they contain must be available to the family in adequate quantity; the correct balance of foods and nutrients must be fed at the right intervals; the individual must have an appetite to consume the food; there must be proper digestion and absorption of the nutrients in the food; the metabolism of the person must be reasonably normal; and there should be no conditions that prevent body cells from utilizing the nutrients or that result in abnormal losses of nutrients. Factors that adversely influence any of these requisites can be causes of malnutrition, particularly PEM. The aetiology, therefore, can be complex. Certain factors that contribute to PEM. particularly in the young child, are related to the host, the agent (the diet) and the environment. The underlying causes could also be categorized as those related to 28 University of Ghana http://ugspace.ug.edu.gh the child's food security, health (including protection from infections and appropriate treatment of illness) and care, including maternal and family practices such as those related to frequency of feeding, breastfeeding and complementary feeding. Some factors enumerated by FAO as involved in the aetiology of PEM include (FAO, 2010): a) the young child's high need for both energy and protein per kilogram relative to those of older family members; b) inappropriate complementary feeding practices; c) staple diets that are often of low energy density (not infrequently bulky and unappetizing), low in protein and fat content and not fed frequently enough to children; d) inadequate or inappropriate child care because of, for example, time constraints for the mother or lack of knowledge regarding the importance of exclusive breastfeeding; e) inadequate availability of food for the family because o f poverty, inequity’ or lack of sufficient arable land, and problems related to intrafamily food distribution; f) infections (viral, bacterial and parasitic) which may cause anorexia, reduce food intake, hinder nutrient absorption and utilization or result in nutrient losses; g) famine resulting from droughts, natural disasters, wars, civil disturbances, etc. 29 University of Ghana http://ugspace.ug.edu.gh 2.4 Aetiologicai Agents of Infectious Diarrhoea A wide array of microbes causes diarrhoea in children, and includes viruses, bacteria and parasites (Brooks et a l, 2006; Gomez-Duarte et al., 2009; Haque et a l, 2003b; Ramani and Kang, 2009). Even in the best of studies no enteric pathogen is identified in one-third of cases, and infections with multiple putative enteric pathogens are observed frequently (Petri et al., 2008). It is most important to ascertain the etiologic agents of diarrhoea in children in developing countries, as this is the predominant group that die from diarrhoea and are at risk for the vicious cycle of diarrhoea and malnutrition (Figure I). Enteric pathogens that are the cause of most severe acute diarrhoea, as assessed by mortality, include rotavirus, Vibrio cholerae, Shigella spp., Salmonella spp., enteropathogenic E. coli (EPEC), and EAEC. Studies linking specific microbes with malnutrition are limited, but currently there is data linking malnutrition and attendant loss of cognitive function to infection with EAEC, enterotoxigenic E. coli (ETEC), Shigella spp., Ascaris lumbricoides, Cryptosporidium spp., Entamoeba histolytica, Giardia lamblia, and Trichuris trichiura (Chang el a l, 2002; Checkley et al., 2003; Mata, 1992; Steiner et a l, 1998). A better understanding of which enteric pathogens are responsible for how much of the burden of diarrhoea morbidity and mortality is required. Although such elucidation would be challenging, it would permit a more informed allocation of resources for the development of treatments and vaccines and should be a research priority (Petri et al., 2008). 30 University of Ghana http://ugspace.ug.edu.gh 2.5 Diarrhoeagenic Escherichia coli (DEC) Escherichia coli are the predominant facultative anaerobe of the human colonic flora. The organism typically colonizes the infant gastrointestinal tract within hours of life, and thereafter E. coli and the host derive mutual benefit for decades (Kaper et al., 2004), As long as these bacteria do not acquire genetic elements encoding virulence factors, they remain commensals in the gut. However, for some reasons that are not so clear, some E coli clones acquire specific virulence factors, which increase their ability to adapt to new niches and allow them to cause a broad spectrum of diseases. E. coli strains that acquire ‘foreign’ DNA encoding enterotoxins, adhesions, or invasion factors become virulent and can cause either a plain, watery diarrhoea or inflammatory dysentery are collectively termed DEC (Weintraub, 2007). DEC have been recognized as intestinal pathogens since the 1940s when Bray hypothesized that E. coli subtypes might account for common infantile diarrhoea of unknown etiology (Bray, 1945). Conclusively, using the Kauffman White scheme of 0:H serotyping, Neter tested the Bray hypothesis and reported that certain ‘enteropathogenic’ serovarieties of E. coli showed close association with infantile diarrhoea (Neter et al., 1955). Their etiologic role in diarrhoea was verified by Levine et al. in volunteer challenge experiments reported in 1978 (Levine et al., 1978). Half a century of microbiology research revealed that most ‘enteropathogenic’ serotypes harboured virulence genes that are absent in non­ pathogens so that by 1998, five categories of DEC that had been unequivocal!)' 31 University of Ghana http://ugspace.ug.edu.gh associated with diarrhoea were known and at least three more categories were under evaluation (Nataro et al., 1998). There are six well-described categories or pathotypes of DEC. Each of these categories has virulence attributes that help them to cause diseases by different mechanisms. The categories include enterotoxigenic E. coli (ETEC), which are characterized by producing heat-stable or heat-labile enterotoxins or both; enterohaemorrhagic E. coli (EHEC), which are characterized by attaching-and- effacing (A/E) lesions and produce shiga-like toxin or verotoxins; enteropathogenic E. coli (EPEC), which elicit characteristic attaching and effacing lesions on the intestinal mucosa; enteroinvasive E. coli (EIEC), which has the ability to invade epithelial cells similar to Shigella and is characterized by the presence of a large invasiveness plasmid; diffusely adherent E. coli (DAEQ demonstrates pattern of diffuse adherence, and enteroaggregative E. coli (EAEC), which demonstrate a characteristic “stacked-brick” aggregative adherence when cultured with HEp-2 cells (Nataro and Kaper, 1998; Nataro et al., 1998). Other categories of DEC have been proposed, such as cell detaching E. coli (CDEC). However, their significance remains uncertain (Abduch Fabrega et a l, 2002). 32 University of Ghana http://ugspace.ug.edu.gh 2,6 Enteroaggregative E. coll (EAEC) Enteroaggregative E. coli (EAEC), is a category of the DEC, and was discovered only in the last two decades (Nataro et al., 1987). EAEC is associated with diarrhoea in several contexts: traveler’s diarrhoea (Adachi et al., 2001; Cabada and White, 2008), paediatric diarrhoea (Paul et al., 1994), food borne out-breaks (Scavia et al., 2008) and human immunodeficiency virus (Samie et al., 2007b; Wanke et al., 1998). The diarrhoea may be symptomatic or asymptomatic (Nataro et al.. 1987), acute or persistent in nature (Lima et al., 2000; Okeke et al., 2003). EAEC is the second most common cause of traveler’s diarrhoea, and is a common cause of acute and persistent diarrhoeal illness in children and adults presenting to emergency departments and inpatient units in the USA (Nataro et al., 2006). A recent study in Ghana indicates that, asymptomatic carriage of EAEC by adults may place infants and younger children at risk (Opintan et a l, 2010). The current review gives priority to EAEC, as an emerging pathogen with new challenges in diagnosis and epidemiology. By definition, EAEC are E. coli lacking the heat-stable (ST) and heat-labile (SL) toxins of ETEC, and adhere to HEp-2 cells in an aggregative or ‘stacked brick’ pattern (Nataro, 2005). Within the EAEC pathotypes (category), two sub-categories have also been described based on the presence or absence of the master (aggR) regulon (Elias et al., 2002a; Gioppo et al., 2000; Itoh et a l, 1997). Typical- and atypical-EAEC refers to EAEC which possess the master regulon, or do not, respectively. 33 University of Ghana http://ugspace.ug.edu.gh 2.6.1 Virulence profile of EAEC A few dozens of putative EAEC virulence genes and virulence factors have been identified to-date. They include the master regulon (Jenkins el al., 2005; Nataro et al., 1995; Nataro el al., 1994; Sheikh el al., 2002), fimbriae and enterotoxins (Bernier et al., 2002; Czeczulin et al., 1997; Nataro et al., 1993; Nataro et al., 1994), outer membrane protein (Monteiro-Neto et al., 2003), dispersin transporter (Ivvashita et al., 2006; Nishi et al., 2003), yersiniabactin iron-scavenging system (Schubert et al., 1998) and lectin (Basu et al., 2004). The clinical implication of these genes and factors however remain elusive (Nataro, 2005). Table 1 shows selected EAEC genes and their description. 34 University of Ghana http://ugspace.ug.edu.gh Table 1. Selected EAEC virulence factor and their description Gene Description/reference Master regulator aggR Secreted proteins aap aaiC pic Dispersin transporter Regulates a package of EAEC plasmid virulence genes, including genes encoding aggregative adherence factors, fimbriae AAF/1 and AAF/II, a dispersin protein (aap), and a large cluster of genes inserted on a pathogenicity island at the PheU locus (Jenkins et al., 2005; Nataro, 2005; Nataro el al., 1994; Sheikh et al., 2002). Is a secreted low-molecular-mass protein (10.2 kDa) that coats the bacterial surface and promotes dispersal of EAEC on the intestinal mucosa, aap lies immediately upstream of aggR in EAEC strain 042 (Grover et al., 2001; Sheikh et al., 2002). Is a chromosomal gene which is regulated by aggR. It encodes some 25 contiguous genes (aaiA-Y), which are localized to a 117 kb pathogenicity island (PAI) inserted at pheU. Many of these genes have homologues in other Gram-negative bacteria and were recently proposed to constitute a type VI secretion system (T6SS). AaiC is a secreted protein that has no apparent homologues within the GenBank (Dudley et al., 2006). The Pic protein has mucinase activity and is capable of causing haemagglutination of erythrocytes (Behrens et al., 2002; Harrington et al., 2009). aalA is one of an autotransport system comprising a 35 University of Ghana http://ugspace.ug.edu.gh aatA Enterotoxins Pet asM/EASTl Fimbriae aggA aafA agg3A cluster of five genes (designated aat-PABCD) on the AA probe. aatA localizes to the outer membrane and facilitates the export of the dispersin (aap) across the outer membrane. It has a homolog with E. coli TolC (lmuta et al., 2008; Iwashita et a l, 2006). Recent gene analyses by some authors suggest that aatA is strongly associated with virulence of E. coli from avian sources (Dai et al., 2010; Li et al., 2010). The plasmid-encoded toxin (Pet) of EAEC is a 104 kDa serine protease autotransporter, produced by the Enterobacteriaceae and other Gram-negative pathogens. After Pet is released into the extracellular medium, it enters intestinal epithelial cells by clathrin-dependent endocytosis. The internalized toxin then escapes the endomembrane system and cleaves the cytosolic actin- binding protein a-fodrin. The resulting disruption to actin architecture and cellular morphology contributes to the pathogen induced mucosal damage (Dautin and Bernstein, 2007; Navarro-Garcia et al., 2007; Navarro- Garcia et al., 2001; Nemec et al., 2010; Scaglione et al., 2008). Encodes the enteroaggregative heat-stable toxin, which has physical and mechanistic similarities to E. coli STa enterotoxin (Harrington et al., 2006; Menard and Dubreuil, 2002). Encodes AAF/I haemagglutination of erythrocytes and adherence adherence to colonic mucosa (Nataro et al., 1993; Nataro et al., 1994). Encodes AAF/II, which mediates adherence to colonic mucosa and haemagglutination of erythrocytes (Czeczulin el al., 1997). Encodes AAF/I 11 haemagglutination of erythrocytes (Bernier el al., 2002). 36 University of Ghana http://ugspace.ug.edu.gh 2.7 Diagnostic Challenges in the Identification of EAEC EAEC was originally identified by the characteristic “stacked-brick” aggregative adherence (Figure 7), when cultured in static Luria broth at 37°C and incubated for 3 hours in HEp-2 cells (Nataro and Kaper, 1998; Nataro et al., 1987). The Hep-2 assay has remained a research tool, and its application has not been translated into the clinical diagnosis of EAEC. Molecular diagnostics have been developed for detection of EAEC as alternatives to the adherence assay to epithelial cells, which is expensive and demands cell culture facilities (Nataro and Kaper, 1998). Among them, a multi-plex PCR detection of three EAEC plasmid borne loci has been proposed (Cema et al., 2003). This assay detects aggR, encoding a transcriptional activator of several EAEC virulence genes (Harrington et al., 2006; Nataro et al., 1994); aatA (formerly known as EAEC or CVD432 probe fragment), encoding an outer membrane protein that is part of a protein transporter system (Baudry et al., 1990; Nishi et al., 2003); and aap, which encodes the anti-aggregation protein or dispersin (Sheikh et al., 2002). These gene markers are present in the high- molecular-weight plasmid (pAA2) of the EAEC prototype strain 042 (Czeczulin etal., 1999). Majority of HEp-2 positive strains are also positive for the anti-aggregation protein transporter gene (aatA) by PCR (Schmidt et al.. 1995). However, in several studies, it has been found that about 10% of EAEC strains verified by the 37 University of Ghana http://ugspace.ug.edu.gh c Figure 7: Adherence pattern of diarrhoeagenic £ coli (DEC) to cultured epithelial cell. A, Localized adherence by enteropathogenic E. coli (EPEC); B, aggregative adherence by enteroaggregative E. coli (EAEC); C, diffuse adherence by Diffusely adherent E. coli (DAEC); and D, non-adherent control strain. Adapted from (Okeke, 2009) 38 University of Ghana http://ugspace.ug.edu.gh HEp-2 assay were negative in the PCR assay (Jenkins el a l, 2006; Menard and Dubreuil, 2002; Weintraub, 2007). This clearly provided evidence to show that it is difficult to provide a genotypic definition for EAEC and to design specific molecular biological assays for detection. The HEp-2 cell adherence assay is currently performed only in research settings, and is labour intensive. Several attempts have been made to develop a molecular biological assay for the identification of EAEC. A cryptic DNA fragment sequence known as “CVD432” or aggregative adherence (AA), from the pAA has been used as an EAEC molecular marker in epidemiological studies and comprises the locus an that encodes an ABC (ATP-binding cassette) transporter system (Baudry et al., 1990; Okeke et a l, 2000a). A transcription activator known as “aggR,” the gene of which lies on pAAs, has been described as the major EAEC virulence regulator for diverse virulence genes (Nataro, 2005). Recently, some epidemiological studies have suggested that CVD432-positive strains, which are predicted to cam the aggR regulon, are the true EAEC pathogens termed “typical EAEC’ (Harrington et a l, 2006; Jenkins et al., 2006). However, AA probe-negative strains share virulence factors with AA probe positive strains, which clearly indicate that additional factors are involved in the AA phenotype in these EAEC strains (Bouzari el al., 2001). A problem with using DNA probes for EAEC demonstrates heterogeneity and no single study has been able to demonstrate a 100% correlation with the HEp-2 cell assay (Sarantuya et a l, 2004). 39 University of Ghana http://ugspace.ug.edu.gh Several assays such as autoagglutination or clump test (Albert et a l , 1993; hvanaga et a l, 2002) and quantitative biofilm test (Wakimoto et a l, 2004) have been devised and at least, evaluated for the screening of EAEC. The clump test may be less useful in the diagnosis of EAEC since several bacteria strains can autoagglutinate. However, the quantitative biofilm assay may be a useful screening tool when a large number of strains are examined in clinical and epidemiologic studies. In the study by Wakimoto et al., all EAEC strains confirmed by the HEp-2 assay demonstrated an OD570 > 0.2, and the incidence of EAEC among the strains with an OD570 > 0.2 was 89.2% (Wakimoto et a l, 2004). Furthermore, the test may be available without the need for a spectrophotometer, since a biofilm demonstrating an OD570 > 0.2 is clearly visible. In addition, this assay may contribute to demonstrating the true incidence of EAEC with and without aggR among clinically isolates o f E. coli. O f the 28 PCR-positive (aggR and EAST) strains screened for biofilm, 25 (89.2%) demonstrated positive results by microtiter plate method (Wakimoto et a l, 2004). Pet is a protease encoded on the pAA plasmid of strain 042 and other EAEC strains (Eslava et a l, 1998). Although this toxin is unique to EAEC, the prevalence of Pet among EAEC isolates varies between 18-44 % (Vila et a l , 2000; Yamazaki et a l, 2000). The emergence of EAEC infection in Brazil (Zamboni et a l, 2004) and the detection complexity of Pet expressing EAEC isolates led to the development of a methodology for Pet detection directly from 40 University of Ghana http://ugspace.ug.edu.gh supernatants of bacterial isolates using a slot blot immunoassay (Taddei et al., 2005). Other proposed diagnostic tests include an enzyme-linked immunosorbent assay (ELISA) for quantitative detection of secretory immunoglobulin A to EAEC (Sutjita et al., 2000) and cytokine response patterns to enteropathogens in which a specific pattern may become a distinguishing pathogen signature (Greenberg et al., 2002). More studies and better diagnostic tools we needed to allow for a better understanding of the true epidemiology of EAEC in children. Serotyping of EAEC is a problem due to their aggregative phenotype, many o f the strains auto-agglutinate and is often described in the literature as nontypable or as O-rough. EAEC from German children demonstrated 14 typable isolates and all belonged to different serotypes (Huppertz et al., 1997). In another study in UK, 97 EAEC strains were serotyped and found to belong to 40 different O-types, In one of the studies, 93 out of 143 EAEC strains could be serotyped and belonged to as many as 47 different serotypes (Jenkins et al., 2006). 41 University of Ghana http://ugspace.ug.edu.gh 2.8 Pathogenesis of EAEC infections The pathogenesis of EAEC is complex (Elias et al., 2002b; Harrington el al., 2005; Nataro. 2005). EAEC is able to bind to jejunal, ileal and colonic epithelium (Huang el al., 2006a). Electron microscopy of infected small and large-intestinal mucosa, from children between 3 and 190 months, infected with several different EAEC strains, reveals bacteria in a thick mucus layer above the intact enterocyte brush border (Hicks el al., 1996). In the colon, EAEC elicits inflammatory mediators and produces cytotoxic effects such as microvillus vesiculation, enlarged crypt openings, and increased epithelial cell extrusion (Harrington et al., 2005). As reviewed by Haung and others, EAEC pathogenesis involves three stages: (1) Adherence to the intestinal mucosa by aggregative adherence fimbriae (AAF) or other adherence factors (Hicks et al., 1996); (2) Production of mucus by bacteria and the host cell forming a biofilm on the surface of the enterocytes; and (3) Release of toxins and elicitation of an inflammatory response, mucosal toxicity and intestinal secretion (Harrington et al., 2005; Huang et al., 2004; Nataro, 2005). EAEC adherence to the intestinal mucosa requires AAF and adherence factors (Moreira et al., 2003). Three AAF structural subunits encoded by agg.4 (AAF/1). aafA (AAF/11) and agg-3 (AAF/111) on the 60-65 MDa pAA plasmid have been described, each EAEC isolate carries only one AAF subtype. aggA encodes 42 University of Ghana http://ugspace.ug.edu.gh AAF/1, and is responsible for the aggregative phenotype and human erythrocyte haemagglutination of some EAEC strains (Nataro et al., 1993). aafA encodes AAF/11, which allows EAEC to adhere to the intestinal mucosa (Czeczulin et a l, 1997). Both aggA and aafA are regulated by the transcriptional activator aggR. AAF/III functions as an adhesin, and is encoded by agg-3, which has a sequence closely related to that of the agg and aaf operon of DAEC (Bernier et al., 2002). Other adherence factors have also been described. Three membrane-associated proteins (MAPs), of 18, 20 and 58 kDa, are believed to play an important role in EAEC adherence to and haemagglutination of animal cells (Spencer et a l, 1998). One study has characterized the outer-membrane protein (OMP) profiles of EAEC strains from children with diarrhoea from Sao Paulo, Brazil, and has observed a heterogeneity in OMP profiles, suggesting that EAEC strains are very heterogeneous (Monteiro-Neto et a l, 2003). AggR regulates the expression o f a secreted low-molecular weight protein known as dispersin (aap) (Sheikh et a l, 2002). Aap lies immediately upstream of aggR in EAEC strain 042. Dispersin is a 10.2 kDa protein that has been identified in 80% of EAEC isolates from one laboratory (Sheikh et a l , 2002). This protein is exported by an ATPbinding cassette (ABC) transporter complex which is encoded by a genetic locus on the EAEC virulence plasmid pAA2 (Nishi et a l, 2003). The locus consists of a cluster of five genes (designated aat-PABCD), including homologues of an inner- membrane permease (AatP), an ABC protein (AatC) and an OMP TolC (AatA). AatA localizes to the outer membrane independently of its ABC partner. Dispersin appears to require the Aat complex for outermembrane translocation. 43 University of Ghana http://ugspace.ug.edu.gh but not for secretion across the inner membrane. In a similar manner to the dispersin gene (aap), transcription of the aat cluster is dependent on AggR, a regulator o f a package of virulence genes in EAEC (Jenkins et al., 2005). Dispersin is responsible for mediating dispersal of EAEC across the intestinal mucosa to allow for efficient adherence and aggregation. This protein neutralizes the negatively charged LPS of the EAEC surface, allowing the positively charged AAF to splay out from the bacterium. In a volunteer challenge study, dispersin has been shown to be highly immunogenic, suggesting that it is a potential vaccine candidate (Nataro et al., 1995). Undoubtedly, other potential AAF and adherence factors exist, and they are currently being investigated. The second stage of EAEC pathogenesis involves production of a mucus layer by the bacteria and the intestinal mucosa. Animal and in vitro culture studies show that EAEC survives within the mucus layer, explaining why individuals infected with EAEC, especially children in developing countries with pre-existent malnutrion, may develop mucoid stools, malnutrition, and persistent colonization with prolonged diarrhoea. One study has identified biofilm production in 48 of 62 (77 %) EAEC strains from Japanese children with diarrhoea, using a quantitative biofilm assay, suggesting that this assay may be a useful and convenient screening tool for EAEC (Wakimoto et al., 2004). Transposon mutagenesis studies suggest that biofilm production by EAEC strain 042 is dependent on two genes. Fis is a chromosomal gene encoding a DNA-binding protein involved in growth-phase- dependent regulation, and yaJK encodes a secreted 28 kDa protein (Sheikh et al.. 44 University of Ghana http://ugspace.ug.edu.gh 2001). Both genes are mediated by AAF and likely reflect its interaction with the intestinal mucosa. Molecular epidemiologic studies are ongoing to determine the clinical impact of infection with EAEC strains that produce biofilm, and to investigate the genetic markers that identify biofilm-producing EAEC. The third stage of EAEC pathogenesis involves release o f EAEC toxins, and elicitation of an inflammatory response, mucosal toxicity and intestinal secretion. Numerous EAEC toxins have been described. Both animal and human studies show that EAEC toxins are destructive to the tips and sides of intestinal villi and enterocytes. Several toxins are part of this process. The three toxins best studied are plasmid encoded toxin (Pet) (Navarro-Garcia et a l , 2001), EAEC heat-stable enterotoxin (EAST1) (Menard and Dubreuil, 2002), and Shigella enterotoxin 1 (ShETl) (Behrens et a l, 2002). Pet is a cytopathic serine protease autotransporter that functions as an enterotoxin and a cytotoxin (Dutta et a l, 2002). Intracellular expression of Pet is accompanied by cleavage of spectrin within the cytoskeleton of intestinal microvilli. In vitro studies show that purified toxin induces cell elongation and rounding, followed by exfoliation of cells from the substratum. These effects are accompanied by loss of actin stress fibres and electrophysiologic changes (Sui et at., 2003). EAST! is encoded by astA and is a heat-stable protein similar to the heat-stable toxin of ETEC. EAST I was originally detected in EAEC strains. 45 University of Ghana http://ugspace.ug.edu.gh However, EAST1 has subsequently been identified in ETEC, EHEC, EPEC and DAEC (Menard and Dubreuil, 2002). The host inflammatory response to EAEC infection is dependent on the host innate immune system and the EAEC strain. The role of putative virulence genes and clinical outcomes is unclear. EAEC carrying ‘virulence’ genes are not always associated with disease; however, virulence factors are associated with increased levels of faecal cytokines and inflammatory markers, such as interleukin 8 (IL-8), interferon (lNF)-y, lactoferrin, faecal leukocytes, and occult blood (Huang et al., 2004; Jiang et al., 2002). IL-8 is an important pro-inflammatory chemokine involved in EAEC pathogenesis. IL-8 is responsible for recruiting neutrophils to the epithelial mucosa without mucosal injury, and facilitates intestinal fluid secretion (Kucharzik et al., 2005; Sansonetti et al., 1999). Travellers to Mexico who developed symptomatic illness due to EAEC infection excreted high concentrations of faecal IL-8 compared to travellers who did not (Jiang et al., 2003). In addition to IL-8, intestinal epithelial cells infected with EAEC 042, the prototype strain, have been shown to upregulate several genes (Huang et al., 2006a). These cellular responses are primarily mediated by flagellin (fliC), a major bacterial surface protein of EAEC (Harrington et al., 2005). Flagellin causes IL-8 release from several epithelial cell lines by binding to Toll­ like receptor 5 (TLR5). TLR5 induce transcription of pro-inflammatory cytokines from epithelial and monocytic cells (Khan et al.. 2004). 46 University of Ghana http://ugspace.ug.edu.gh 2.9 Inflammatory Diarrhoea and its Assessment Diarrhoea can range from a self-limiting, benign condtion to severe, life- threatening illness, the complications of which are often related to infection with pathogens that invade the mucosa to cause inflammation (Guerrant et al., 1999b; Venkataraman et al., 2003). In clinical settings, it is often required to distinguish between inflammatory and irritable bowel syndrome (IBS), a non-inflammatory disease which represents a large part of gastroenterological practice (Tibbie et al., 2000). Usually, clinicians rely on several biochemical studies to help support a diagnosis of either 1BD or gauge a patient’s disease activity during a particular office visit (Walker et a l, 2007). Intestinal inflammation can be assessed by the use of 111 indium neutrophil technique (Saverymuttu et al., 1986). In addition to the high cost (about £300/patient) of this procedure, there are practical problems with obtaining complete faecal collections over 4 days. For these reasons, the use of labeled indium has been limited to only research centers. An alternative non-invasive method to quantify intestinal inflammation is to analyse protease resistant neutrophil derived proteins such as elastase (Adeyemi and Hodgson, 1992) or lactoferrin (Guerrant et al., 1992a; Uchida et al., 1994) in stool specimens. Calprotectin is one such protein, which has also been evaluated in paediatric infections (Bunn et al., 2001a; Bunn et a!., 2001b). 47 University of Ghana http://ugspace.ug.edu.gh Conventional stool culture in all patients presenting with acute diarrhoea is impractical, expensive and time-consuming, and results in a very low yield of positive cultures (Guerrant et al., 1985; Venkataraman el al., 2003). In the investigation of infectious gastro-intestinal disorders and 1BD, several authors used lactoferrin as a marker for neutrophil infiltration (Choi et al., 1996; Fine et al., 1998; Guerrant et a l, 1992a; Kane et al., 2003). In several of these studies however, faecal lactoferin was semi-quantified by the use of latex agglutination (Fine et a l, 1998; Samie et al., 2007b). As both lactoferrin and calprotectin can be measured with ease in faeces, they have the potential to be used in a routine screening procedure as an aid to discriminate between normal and inflammed intestines (Lundberg et a l, 2005; Roseth et al., 1997). Currently, most o f these assays are not used in routine screening of IBD because they are expensive. A 96- well ELISA plate of calprotectin and lactoferrin costs $ 870 and S 835, respectively. 48 University of Ghana http://ugspace.ug.edu.gh Lactoferrin is a glycoprotein consisting of a single polypeptide chain of about 80 kDa with two globular lobes each containing an iron-binding site (Steijns and van Hooijdonk, 2000). Lactoferrin is the second most abundant protein in human milk, and also found in most exocrine secretions including tears, saliva, intestinal mucus and genital secretions (Farnaud and Evans, 2003). Because lactoferrin is found in the specific granules of neutrophils, it serves as a surrogate marker for neutrophil infiltration (Baynes et al., 1988; Farnaud and Evans, 2003). Multiple activities, including anti-microbial, anti-inflammatory, immunomodulatory activity have been described for lactoferrin (Baveye et al., 1999; Brock, 2002; Choi et al., 1996; Kirkpatrick et al., 2002; Levay and Viljoen, 1995). However, the relevance of each of these putative mechanisms in humans remains to be proven (Brock, 2002; Farnaud and Evans, 2003). Human and bovine lactoferrin are well characterized. They consist of 691 and 689 amino acids, respectively; the sequence identity is 69% (Pierce et a l, 1991). The 3-D structures of bovine and human lactoferrin are very similar (Steijns and van Hooijdonk, 2000). The concentration of lactoferrin in human milk is 5.3 ± 1.9 mg/mL in colostrum, and approximately 1 mg/mL after the first month of lactation (Hamosh, 2001). In contrast, the concentration of lactoferrin in bovine milk is very low (1.5 mg/mL in colostral whey and 20-200 |ig/mL in milk) (Steijns and van Hooijdonk, 2000). 2.9.1 Pathophysiology of Lactoferrin 49 University of Ghana http://ugspace.ug.edu.gh Several in vitro studies have demonstrated some inhibitory effects of lactoferrin on enteric pathogens, including bacteria (Bessler et al., 2006), parasites (Leon- Sicairos et al., 2005) and viruses (Seganti et al., 2004). Both recombinant and purified human lactoferrin have been reported to inhibit the AA phenotype of EAEC in tissue culture cells (de Araujo and Giugliano, 2000; Ochoa et al., 2006 ). 2.9.2 Lactoferrin (LF) assays for enteric pathogens Commercially prepared latex beads coated with antibodies against lactoferrin (LEUKO-TEST, TechLab, Blacksburg, VA) provide a semi-quantitative agglutination method for the detection of faecal LF (Guerrant et al., 1992a). It has been used as a screening test for inflammatory diarrhoea (Choi et al., 1996; Huicho et al., 1996; Huicho et al., 1997). This latex agglutination test has been evaluated and found to be useful in the screening of invasive enteropathogens in travellers’ diarrhoea (Scerpella et al., 1994). The latex agglutination test for LF is positive in infection with DEC, Clostridium difficile, Shigella, Campylobacter, E. histolytica and Cryptosporidium spp (Alcantara et al., 2003; Ashraf et al., 2007; Bouckenooghe et al., 2000; Fried et al., 2002; Kohli et al., 2008; Mclver et al., 2001; Miller et al., 1994; Venkataraman et al., 2003). The IBD-SCAN (TechLab, Blacksburg. VA), which is an ELISA method, is able to quantify faecal LF in a gram of stool. Some authors have used the IBD-Scan in 50 University of Ghana http://ugspace.ug.edu.gh assessing IBD, and found that faecal lactoferrin was 90% specific for identifying inflammation in patients with active IBD. Additionally, elevated faecal lactoferrin was 100% specific in ruling out IBS (Kane et al., 2003). Several authors have used the 1BD-SCAN to assess intestinal inflammation caused by C. difficle (Abba et al., 2009; Archbald-Pannone et al., 2010; Vaishnavi el al., 2003; Vaishnavi et al., 2002; van Langenberg et al., 2010). Markers of inflammation, including cytokines and LF was found in stool samples in which EAEC, ETEC, Salmonella or Shigella species were isolated in cases of traveler’s diarrhoea acquired from India and Mexico (Greenberg et al., 2002). In that study, elevated faecal lactoferrin was highest in Shigella-positive cases. Additionally, samples from the S7?/ge//a-positive cases had the highest concentrations of faecal cytokines. A small subset of the patients with EAEC- associated diarrhoea in that study however, failed to develop any inflammatory response (Greenberg et al., 2002). Table 2 summarizes some studies done in both developing and developed countries in relation to pathogen-specific diarrheas and faecal lactoferrin levels. 51 University of Ghana http://ugspace.ug.edu.gh Table 2: Summaries of LF assays in relation to pathogen-specific diarrhoeas Country/year Assay type Summaries and conclusions/references Brazil 2003 semi- quantitative Stool samples of Brazilian children with/without Cryptosporidium associated diarrhoea were analyzed. Only 1/14 volunteers challenged with Cryptosporidium had increased LF. However 12/17 in previous study had mild to moderately elevated LF. The authors concluded that cryptosporidiosis is associated with mild inflammation, especially in children in an endemic area (Alcantara et al., 2003). US 1996 Bangladesh 2007 semi- 55 faecal samples, 46 with and 9 without quantitative diarrhoea were tested. Of 28 samples with salmonellosis, shigellosis or campylobacteriosis, 93% had detectable LF. In 83% of samples with rotavirus or no detectable pathogen, LF was negative at a titre of 1:50. All 9 controls without diarrhoea were negative at 1:50. The authors concluded that the use of LF to screen for inflammatory diarrhoea selects specimens for which stool culture is 5-fold more likely to yield an invasive bacterial pathogen (Choi et al., 1996). semi- 594 patients enrolled under the International quantitative Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR, B) were evaluated. Faecal occult blood test (FOBT) and LF were done in 448/594 patients from whom either a single enteropathogen (53%) or no pathogen (22%) were identified and 146 were excluded for multiple pathogens. Invasive and non-invasive pathogens were isolated from 24% and 76% of the patients, respectively. They concluded that FOBT and LF are not useful in differentiating inflammatory and non-inflammatory diarrhoea in patients in Dhaka (Ashraf et al., 2007). 52 University of Ghana http://ugspace.ug.edu.gh Table 2 (Continuation): Summaries of LF assays in relation to pathogen- specific diarrhoeas Mexico and India 2000 semi- quantitative 45 cases of EAEC diarrhoea were compared to 56 controls with ETEC diarrhoea, and 126 controls without identifiable pathogens. For EAEC, ETEC and the controls without identifiable pathogens, LF was found in 60%, 26.8% and 21.4%, respectively. The authors concluded that EAEC infection is associated with an intestinal inflammatory response (Bouckenooghe et a l, 2000). South Africa 2006 semi- quantitative 244 Stool samples were tested and 44 Cryptosporidium found. 57% o f Cryptosporidium positive samples were diarrhoeic and 26 (59.1%) had elevated LF content (Sarnie et a l , 2006a). South Africa 2007 semi- quantitative Stool samples from 255 patients and 67 primary' school children were analyzed. O f ail pathogens detected, only C. jejuni was significantly associated with diarrhoea and elevated FL (Samie et a l, 2007a). South Africa 2006 semi- quantitative E. histolytica was significantly associated with diarrhoea and with the presence o f LF (Samie et a l, 2006b). US 2010 quantitative 26/46 stools collected had elevated quantitative levels, with only 5 subjects having diarrhoea. Of 2 samples with C. difficle infection, both were liquid and, when compared with all other liquid stools (n=22), the mean LF was statistically higher (134.1 vs 28.8 |ig/ml, p=0.008) (Archbald- Pannone et al., 2010). Korea 2006 semi- quantitative Intestinal inflammation associated with increased faecal LF, important in bacterial enteric infection, was not found in Norovirus-associated gastroenteritis (Ko et a l, 2006). 53 University of Ghana http://ugspace.ug.edu.gh Cryptosporidiosis is a notifiable disease in several countries yet its incidence in humans is probably underestimated in most settings. This is partly because testing for Cryptosporidium in many countries is not included in routine examination of human stool samples, and partly because of insufficient reporting systems (Valentiner-Branth et a l, 2003). Nonetheless, Cryptosporidium species are recognized globally as important causes of diarrhoea in children and adults. Currently, there are at least 20 valid Cryptosporidium species and over 40 unnamed species in mammals, birds and reptiles which only have genotype names (Coupe et a l, 2005; Xiao and Ryan, 2004). Cryptosporidiosis is associated with: a) sporadic, often water-bome, outbreaks of self-limiting diarrhoea in otherwise healthy persons; b) chronic, life-threatening illness in immune-compromised patients, most notably those with human immunodeficiency virus/Acquired immune deficiency syndrome (HiV/AlDS); and c) diarrhoea and malnutrition in young children in developing countries (Mor and Tzipori, 2008). In industrialized nations, improved water-management practices have resulted in a decline in cryptosporidiosis in the general population (Lake, 2007); and antiretrovirals have curbed the incidence and severity in patients with HIV/ AIDS (Maggi et a l, 2000). In the absence of these interventions, the burden of cryptosporidiosis continues to fall heavily on developing regions, where infection is both more ubiquitous and clinically consequential (Mor and Tzipori, 2008). 2.10 Cryptosporidiosis: Background and Epidemiology University of Ghana http://ugspace.ug.edu.gh Cryptosporidiosis occurs worldwide and is responsible for significant morbidity and mortality, especially in HIV-infected patients (Caccio and Pozio, 2006). Most cases of cryptosporidiosis are due to sporadic rather than outbreak-associated infections (Hunter et a l, 2004). However, waterborne and food-borne outbreaks are reported frequently and represent around 10% of all cases of Cryptosporidium infection (Hunter et al., 2004). There is still no curative treatment (Abubakar et al., 2007), making cryptosporidiosis a major public health issue and an economic problem (Corso et al., 2003; Jex et al., 2011; Kothavade, 2011). The prevalence of cryptosporidiosis in children varies considerably across sub- Saharan Africa and within certain subsets of the population. In Ghana for example, Adjei et al reported cryptosporidiosis prevalence of about 29 % in Accra and in Kumasi, Addy et al reported about 12 % (Addy and Aikins-Bekoe. 1986; Adjei et al., 2004). In South Africa, Berkowitz et al reported a prevalence of about 20 % in Johannesburg and Geyer reported 27% in Pretoria (Berkowitz, 1988; Geyer et al., 1993). Although rarely diagnosed in this setting, coinfection with other enteric pathogens occurs frequently, because of common exposure through poor sanitation and hygiene and because of immune predisposition due to HIV infection (Mor and Tzipori, 2008). In most sub-Saharan countries, cryptosporidiosis prevalence peaks among children aged 6-12 months and decreases thereafter. Breast-feeding is speculated to afford some protection, either through conferment of immunoglobin or avoidance of contaminated water (Mor and Tzipori, 2008). This may explain why infection is delayed until after the age 55 University of Ghana http://ugspace.ug.edu.gh of 6 months, an age that is commonly marked by the introduction of complementary foods. Children are likely to experience infection throughout childhood and adolescence, although the clinical significance becomes less apparent with age. This is probably attributable to development of immunity following frequent exposure to oocysts in the contaminated environment. Experimental studies reveal that repeated exposure to Cryptosporidium parvum promotes an IgG response that imparts partial protection against subsequent infection and illness (Chappell et a l, 1999). Cryptosporidiosis was recognised in human beings in 1976, and was prominent as a cause of severe diarrhoeal illness in patients with HIV/AIDS in the 1980s and 1990s. Cryptosporidiosis is now additionally recognised as a major cause of waterborne diarrhoeal illness in developed and developing regions, and as a pathogen with long-term effect on childhood growth and development in impoverished areas (Kosek et al., 2001). Infection and illness caused by Cryptosporidium species is ubiquitous and has been reported in more than 40 countries on six continents (Ethelberg et al., 2009; Fall, 2003; Gay-Andrieu et a l, 2007; Zu etal., 1994). Critical determinants of the epidemiology o f Cryptosporidium infections include its small size, its low infectious dose, its high chlorine resistance, and its zoonotic potential. Cryptosporidia are spread by the ingestion o f oocysts excreted by University of Ghana http://ugspace.ug.edu.gh infected people or animals. Infection can be transmitted through the consumption of faecally contaminated water or food, via direct person-to-person or animal-to- person contact, and contact with contaminated environmental sources (Kosek et al., 2001). 2.10.1 Diagnosis and genotyping of Cryptosporidium spp The pathology and diagnosis of cryptosporidiosis in humans is widely documented for C. hominis and C. parvum (Warren, 2008). Microscopy, employing dyes which stain the oocyst in stool specimens has generally been used in resource poor facilities for the diagnosis of cryptosporidiosis (Addy and Aikins-Bekoe, 1986; Adjei et al., 2004; Morse et al., 2007). There are commercially available enzyme immunoassays (EIAs) for the diagnosis of cryptosporidiosis. These have not only eliminated the rather cumbersome and the time required to perform direct microscopy, but additionally show greater sensitivities and specificities (Garcia et al., 2000; Sharp et al., 2001). Usually, however, the direct microscopy and the E1A methods offer no information on the infecting species and are unhelpful in epidemiological investigations (Fall, 2003). The development of genetic tools has now made possible the detection of Cryptosporidium oocysts by PCR and species identification by sequencing, restriction fragment length polymorphism (RFLP), or the use o f species-specific 57 University of Ghana http://ugspace.ug.edu.gh probes (Caccio and Pozio, 2006; Coupe el al., 2005; Stroup et al., 2006). The molecular probes often target Cryptosporidium oocysts wall protein (COWP) or the 18s rRNA. In several cases, both C. parvum and C. hominis have a relatively conserved target region and further molecular differentiation such as RFLP is required. However, genotyping for specific identification remains restricted to reference or specialized laboratories. An emerging method for characterizing DNA samples is by the use of High Resolution Melting (HRM) (Reja et al., 2010). 58 University of Ghana http://ugspace.ug.edu.gh 2.11 Dysentery: Background and Epidemiology Bacillary dysentery is most commonly caused by microorganisms belonging to the genus Shigella, whereas amebic dysentery is caused by the protozoan parasite Entamoeba histolytica. Approximately, 164.7 million cases of shigellosis are reported worldwide, of which 163.2 million are in developing countries and 1.5 million in industrialized countries (Kotloff et al., 1999). Each year 1.1 million people are estimated to die from Shigella infection and 580,000 cases of shigellosis are reported among travelers from industrialized countries (Kotloff et al., 1999; WHO, 1999). Estimates of E. histolytica infections have primarily been based on examinations of stool for ova and parasites, but these tests are insensitive and cannot differentiate E. histolytica from morphologically identical species that are nonpathogenic, such as E. dispar and E. moshkovskii (Haque et al., 2003a). Specific and sensitive means to detect E. histolytica in stool are now available and include antigen detection and PCR (Haque et al., 2000). The disease is more severe in the very young and old and in patients receiving corticosteroids (Haque et al., 2003a). 59 University of Ghana http://ugspace.ug.edu.gh 2.11.1 Diagnosis and genotyping of dysentery There are four serogroups o f Shigella with varying global distributions. S.flexneri is mostly seen in developing countries (median 60% of isolates), followed by S. sonnei (median 15%), 5. dysenteriae and S. boydii with equal frequency (median 6%). In industrialized countries S. sonnei (median 77%) is mostly seen, followed by 5. flexneri (median 16%), S. boydii (median 2%) and S. dysenteriae (median 1%) (KotlofFet al., 1999). Whilst S. sonnei have no serotype within its serogroup, the others have several serotypes based on their somatic 0 - antigen. Rate of isolation of Shigella by routine stool culture is traditionally low, when the specimens are not cultured promptly or when the specimens are not transported on Cary- Blair transport media. The fastidiousness of the organism may account for low recovery rates (Opintan and Newman, 2007). Molecular methods of diagnosing shigellosis usually target genes coding for the invasive plasmid antigen H (ipaH gene) found in both Shigella spp and EIEC. In addition to the ipaH gene, EIEC also has eae, coding for the protein intimine located on the Locus of enterocyte effacement (LEE). Technically, Shigella, have only ipaH whilst EIEC have both ipaH and eae. Molecular methods such as PCR and pyrosequencing reveal details needed for the differentiation of the Entamoeba complex (Stensvold et al., 2010). Traditionally. E. histolytica is considered the pathogenic form and E. dispar as a commensal. University of Ghana http://ugspace.ug.edu.gh However, current findings employing molecular methods suggest that certain clones of E. dispar can be associated or be potentially responsible for intestinal or liver tissue damage, similar to that observed with E. histolytica (Ximenez et al., 2010). 2.12 Giardiasis: Background and Epidemiology Giardiasis is caused by Giardia duodenal is (synonymous G. lamblia and G. iniestinalis) which is a flagellated protozoan parasite that reproduces in the small intestine. It is a cosmopolitan pathogen with a very wide host range, including domestic and wild animal species, as well as human beings (Plutzer et al., 2010). Giardiasis is found worldwide and is especially common in areas where poor sanitary conditions and insufficient water treatment facilities prevail (Ortega and Adam, 1997). Human-derived Giardia were earlier assigned to a separate species (G. lamblia) and the major lineages have been defined by analysis of human- derived Giardia isolates, designated assemblages A and B (Mayrhofer et a l, 1995). Giardia cysts are transmitted by the faecal-oral route, either direct or indirect. Potential mechanisms of transmission include: person to person, animal to animal, zoonotic (animal to human), waterborne from humans or animals through drinking water or recreational contact such as swimming and food borne from contamination of water used in food preparation and manufacture or from food handlers (Karanis et a l, 2007; Plutzer et a l, 2010; Shields et a l, 2008). 61 University of Ghana http://ugspace.ug.edu.gh Microscopically, giardiasis is diagnosed by the identification of cysts or trophozoites in the feces, using direct mounts as well as concentration procedures. In addition, samples of duodenal fluid (e.g., Enterotest) or duodenal biopsy may demonstrate trophozoites. Despite the value of duodenal biopsy or aspiration for the diagnosis of giardiasis, biopsy only supplements stool examination. Biopsy is less sensitive than stool examination but will identify patients for whom the diagnosis cannot be ascertained by stool examination alone (Ortega and Adam, 1997). Commercial kits for the assay of Giardia spp include antigen detection tests by enzyme immunoassays (EIAs) and detection of parasites by immunofluorescence (Boone et al., 1999; Rosoff et al., 1989; Sharp et al., 2001). Molecular diagnosis of giardiasis has mainly focused on genes coding for the beta giardin of the oocyst or the trophozoite (Calderaro et al., 2010; Guy et al., 2003; Haque et al., 2007). 2.12.1 Diagnosis and genotyping of giardiasis 62 University of Ghana http://ugspace.ug.edu.gh CHAPTER THREE MATERIALS AND METHODS 3.1 Ethical consideration, Study design, Population and settings The study was reviewed and approved by the Institutional Review Board o f the University of Ghana Medical School, Ghana. Participation was voluntary and enrollment was subject to parents/guardians’ approval, through signature or by thumb-printing their names after the purpose of the study was explained to them. This was a prospective cross sectional study, and enrollment was done between August 2007 and May 2008 and involved children < 5 years consulting at the Princess Marie Louise Children’s Hospital (PML), Accra, Ghana. Consecutive children from whom consent was given by their caregivers were included. The diarrhoea sub-population was composed of outpatients brought to the hospital for treatment; and the control children without diarrhoea, were visiting for routine child welfare care. No follow-up was done after the initial recruitment as a part of this study. Sample size for the current study was earlier determined based on achieving a 95% power of detecting major causes or sources of diarrhoea, with significance level set at d= 0.05. The equation n— ; where n= sample size, z= confidence interval, d= significance level, p= proportion in the population with diarrhoea and q= (1-p) = proportion in the population without diarrhoea was used. No reliable 63 University of Ghana http://ugspace.ug.edu.gh record of p was known but based on an earlier study in Korle-Bu (Namboodiri, 2008); p was not expected to exceed 23%. Putting this into the equation above the minimum sample size was 272; HZ = 2.72.! *272 (0 .05 ) 2 3.1.1 Interviews and diarrhoea definition A structured questionnaire was used to obtain information on the children from the parents/guardians. The questionnaire for the two sub-populations was similar. The only difference being that, the questionnaire for the children without diarrhoea had no questions on diarrhoea history. Information that was sought included bio-data, duration of diarrhoea, residence/location, breast feeding status and medication taken before visiting hospital [Appendix I: i, ii and iii]. Diarrhoea was defined as the passage of three or more unformed stools within a 24 h period, according to the WHO’s guidelines on Integrated Management o f Childhood Illness (WHO, 1995b). Diarrhoea lasting < 14 days was defined as acute and those lasting > 14 days, persistent. The control (non-diarrhoea) group consisted of children who did not have diarrhoea at least within the 24 h period prior to enrollment. 64 University of Ghana http://ugspace.ug.edu.gh 3.1.2 Anthropometric data and nutritional status assessment Height or length measurements in centimeter to the nearest one decimal were performed for children above or below two years of age respectively. Weight measurements in kilogram to the nearest one decimal were performed using a 25 kg Salter hanging scale (CMS Weighing equipment, High Holbom, London, UK). The Z-score, weight-for-age (WAZ), height-for-age (HAZ) and weight-for-height (WHZ) were calculated by use of soft-ware designed for nutrition studies (EPINUT, World Health Organization, Geneva; Epi Info version 6.0, Centers for Disease Control and Prevention, Atlanta). These anthropometric Z-scores are a measure of SD above or below the median for the international reference population. Z-score values were used to determine the nutritional status of children based on the following definition: WAZ; well nourished (> -1), mild (-2 to -1), moderate (-3 to -2) and severe (< -3) malnutrition; HAZ, normal height ( > -2), moderate stunting (-3 < -2) and severe stunting (< -3), WHZ; normal weight ( > -2), moderate wasting (-3 < -2) and severe wasting (< -3) (Waterlow et al., 1977). Epi Info graphics was used to draw the growth curves for males and females, for all children less than 2 years of age. 65 University of Ghana http://ugspace.ug.edu.gh 3.2 Specimen Processing and Microbiological Analysis A stool specimen from each participating child was collected into a sterile container and processed within 4 h of collection. A bit of a fresh stool specimen from each child was kept frozen at - 20°C in cryo-vials (deidentified) until it was being sent to the Center for Global Health, University of Virginia, USA, for further analysis. A flow chart of how a stool specimen was processed at the Microbiology Department, UGMS, Ghana and the Centre for Global Health, UVa, USA is shown in Figure 8. 3.2.1 Routine stool culture Bacteria were cultured on MacConkey (MAC), Salmonella-Shigella (SS) and deoxychocolate (DCA) agars (Oxoid, Maryland, USA), using standard techniques (Barrow and Feltham, 1993; WHO, 1987). Selenite F broth was used as enrichment for Salmonella before sub-culturing onto MAC, SS and DCA. A stool specimen was plated onto full plate DCA and SS as primary plates and a loopfull also inoculated into selenite F broth [Figure 9], To obtain discrete colonies, loops were flamed in between plating and aseptic conditions maintained in all cases. Agar plates together with broths were both incubated overnight at 37°C (DAY 1). After 24 hr incubation, primary plates were read and a loop full of broth culture subcultured onto fresh half plate DCA and incubated overnight at 37°C. 66 University of Ghana http://ugspace.ug.edu.gh ;Ns\\>SS' ^V;«- - s*VSK- SS'*'vt:\v £ v-mi»Jm*« 1 :ssas '.w G .5*$*s ::h ; m * Figure 8: A flowchart on processing of a fresh stool specimen. A stool specimen was processed within 4 h of collection, and was cultured for routine enteric bacteria. Genomic DNA was extracted from bacterial isolates (£. coli) and frozen stool specimens by the use of the Wizard genomic kit (Promega, Madison. USA) and the QIAamp stool kit (Qiagen, Valencia, CA), respectively. Faecal lactoferrin was quantified by the use of the 1BD-SCAN (TechLab, Blacksburg. VA). 67 University of Ghana http://ugspace.ug.edu.gh The primary plates were also reincubated overnight at 37°C. After 48 hr incubation the primary plates were further examined, and those without growth were discarded. The half plates were similarly read at 24 hr and 48 hr interval of incubation at 37°C. Latose and non-lactose fermenting colonies were biochemically characterized. 3.2.2 Biochemical and serological identification of isolates The biochemical tests carried out were urea, Triple Sugar Iron (TSI), oxidase, indole and citrate utilization test. A pure colony of suspected isolates on MacConkey plate was used for the biochemical tests, and reactions were read and recorded after a 24 h incubation period. For each test, a suspected isolate was suspended in peptone water and by the use of a straight loop; agar slants of citrate, urea and TSI were inoculated and incubated at 37°C. Indole test was performed by the addition of few drops of Kovac’s reagent to an overnight peptone broth culture. A positive control, indicated by the formation of a pink interphase in the overnight’s culture was set up using E. coli (ATCC 25922). Serogrouping of Shigella was done by the slide agglutination tests with Shigella polyvalent grouping anti-sera (Mast Group Ltd., Merseyside, U.K.). Fresh clean glass slide was used, and a positive and a negative control test were performed alongside each experiment, using test organism and sterile distilled water respectively. 68 University of Ghana http://ugspace.ug.edu.gh J i i l % ! « •.* 'A';,' ■> tx-xswx&\XS\\ wstt* X ssis>'< *5? ■:.%SS*:iIIIm Figure 9: A flowchart on culture of stool specimens for bacteria. In addition to Salmonella and Shigella, other enteropathogens were characterized. * Lfs - lactose fermentors, NLfs - non-iactose fermentors 69 University of Ghana http://ugspace.ug.edu.gh Isolated E. coli and typed Shigella strains were sub-cultured from MacConkey purity plates onto Mueller Hinton (MH) slopes and labeled appropriately. The stoppers o f the slopes were sealed with paraffin material to make them air-tight after overnight’s incubation at 37°C and stored in a dark room for further studies. All information of an identified bacteria strain was recorded on the reverse sheet of the questionnaire and also entered into Microsoft Access data files. 3.2.3 Archiving of bacterial strains 70 University of Ghana http://ugspace.ug.edu.gh 3.3 Bacterial DNA Extraction (£. coli Isolates) A genomic bacterium DNA was extracted from all the E. coli isolates, at the Microbiology Department of the University of Ghana Medical School. Over-night pure growth of bacteria on MacConkey plates were sub-cultured into 3 ml Trypticase Soy Broth (TSB) and incubated aerobically without shaking at 37°C. Bacterial cells were harvested from the over-night culture by centrifugation at 13,000 rpm for 8 min and DNA extracted by the alkaline lyses method using the Wizard Genomic kit (Promega, Madison, USA) with some minor modifications. To lyse cells, bacteria were uniformly suspended in 600 (xl Nuclei Lysis solution and incubated at 80°C for 5 min over a water bath [Figure 10]. After cooling to room temperature, 3 |il RNase was added to the suspension, and mixed by inverting the tube 3-5 times. Suspension was then incubated for 1 h at 37°C. Proteins were precipitated by cold treatment after 200 jil of precipitation solution was added, vortexed and centrifuged at 13,000 rpm for 3 min. 600 |il were transferred into new Eppendolf tubes and DNA precipitated by cold treatment after the addition of 600 (il ethanol. The DNA pellet was purified by ethanol purification and centrifugation and air-dried for about 30 min and kept dry in a cool cupboard for further analysis. DNA pellet was rehydrated in 100 pi of Rehydration solution overnight at 4°C when needed for further analysis and thereafter, rehydrated bacterial DNA was kept frozen at -80 freezer. For purposes of analysis, DNA extracted from the E. coli isolates are referred to as bacterial DNA. 71 University of Ghana http://ugspace.ug.edu.gh Figure 10: A flowchart showing bacterial DNA extraction. DNA was extracted by the alkaline method, using the Wizard Genomic kit (Promega, Madison. USA) 72 University of Ghana http://ugspace.ug.edu.gh 3.4 Stool DNA Extraction The QIAamp stool kit (Qiagen, Valencia, CA) was used to extract genomic DNA from frozen stool specimens with some minor modifications. The modifications included the addition of small and big beads (MO BIO Laboratory Inc., Carlsbad, CA) to weighed stool specimen before the addition of lysis buffer (ASL). The stool-bead-mixture was bead-beated for 2 minutes in a Mini Beadbeater (Biospec Prdt,, Bartlesville, USA) to make a uniform homogeneous mixture with the lysis buffer. Additionally, mixtures were incubated at 80 °C instead of the 70 °C recommended by the manufacture to lyse enteric pathogens, and eluted DNA was further purified in Zymogen columns (MO BIO Laboratory Inc., Carlsbad, CA). Figure 11 shows a flow chart of how stool DNA was extracted and the modifications made in the Qiagen stool kit (modifications highlighted). For each stool aliquot, between 15 - 20 ng or DDe1 of stool was used depending on stool consistency. DNAs were also extracted from pure control strains obtained from the Centre of Global Health, University of Virginia (EAEC 042 and 17-2, Shigella, Cryptosporidium and Giardia oocyst, E. histolytica. The initial steps in the DNA extraction process, which principally removes impurities from stool specimen was avoided, when dealing with pure cells. All DNAs were kept frozen at - 80°C until needed for analysis. 73 University of Ghana http://ugspace.ug.edu.gh Faacal DNA extraction using th« Qlafan kit with modifications Stool (180-200 mol * B «d> (Motlio & 15m g 0.1 ilasi} 1. 1400 f i l ASi + 2 min bead beating 2 . 95°C incubation for 5 min 3. 15 sec vortex * Spin for 1 m in §> 13,600 rpm New Epi tub* with X Tab InbibHEx 1. Vortex continuously to suspend tab 2. Incubate £) room tem p for 1 min 3. Spin for 3 m in @ 13,600 rpm New Epi lube X. Spin for 3 m in© 13,600 rpm New Epi tube ♦ pre 30 pi protie rvase K 1. 400 pi A l + Vortex for 15 sec 2. 70°C incubation for 10 min 3. 400 i d EtOH t 4. Vortex to mix ♦ flash spin 13,600 rpm Labeled QIA amp Column 1. Spin for lm in @ 13,600 rpm New Epi tube ♦ Column from above 1. 500 i l l AW 1 + Spin for lm in $ 13,600 rpm New Epi tu b e + Column from above | 1. 500 f i l AW 2 ♦ Spin for 3min $ 13,600 rpm New labeled Epi tu b e ♦ Column from above 1. 200 i d AE + lm in incubation @ room tem p 2. Spin @ 13,600 rpm for 1 min Purification w ith Zymo Cokimn (Pre prepare columns @ 8,000 for 3 min then e lu te DNA in to new Epi tube @ 800 rpm for 1 min) Figure 11: A flowchart of stool DNA extraction. The QIAamp stool kit (Qiagen. Valencia, CA) was used and modifications are highlighted (red). ASL. AL, AW1. AW2 and AE are stool diluents, lysis, ethanol washing and DNA eluting buffers, respectively. * Epi tube - Eppendolf tube. 74 University of Ghana http://ugspace.ug.edu.gh 3.5 Optimization, Sensitivity and Specificity of Real-Time PCR (qPCR) Real-Time PCR reaction was first optimized using DNA obtained from pure control strains as templates, and annealing temperatures and primer sets described elsewhere (Cema et al., 2003; Czeczulin et al., 1999; Gene ID, 2009; Guy et al., 2003; Haque et al., 2007; Sethabutr et al., 1993). Where necessary, the annealing temperature was modified. A 25 pi reaction volume consisted of 12.5 *il of SYBR-Green -490 (Bio-Rad Laboratories, Madison, USA), I fil of each 6.2 pM primer, 5 pi template and PCR grade water. For the sensitivity o f the PCR reactions, serial dilutions of known quantities (cfu/ml) o f the individual control organisms (cells) were separately suspended and homogenized in 180 pi of phosphate buffer solution (PBS). DNAs were then extracted from each set o f the cell-PBS-mixture, using the Qiagen kit as described earlier. The initial steps in DNA extraction process, which principally removes impurities from stool specimen was avoided, when dealing with pure cells. The eluted DNAs were used to template qPCR to determine the least number of organisms that can reproducibly be detected. For specificity, known quantity (cfu/ml) of each of the control strains (£. coli 042, Cryptosporidium, E. histolytica and Giardia) was used to spike 180 jig of enteric- pathogen-free stool specimen, and again faecal DNA extracted using the methods described above. The faecal DNA obtained was then used to template qPCR for each of the organisms in single-plex reactions to check whether there would be interferences. In large cohorts of experiments, known quantities of all four 75 University of Ghana http://ugspace.ug.edu.gh organisms were also used to spike 180 pg enteric-pathogen-free stool specimen as above, and again faecal DNA extracted and used to template single-plex reactions. Reproducibly, the least amount of organism that was detected in a gram of stool specimen was I03 per a gram of stool. All the qPCR reactions were carried out in a 25 |il final volume as described above. Figures 12 (i-vi) show curves (amplification, standard, negative differentiation of fluorescence unit over temperature) and quantification data table of some of the genes screened. 76 University of Ghana http://ugspace.ug.edu.gh Quantification Data Wei Fluor Content Threshold Cyde( C M ) C(t) Mean C(t) Std. Dev Starting Quantity (SQ) Lofl Starting Quantity SQMean SQ Std Dev Wei Note B07 SYBR Std-2 15.73 15.73 0.000 1.000E+07 7.000 1.00E+07 O.OOE+OO aggR-7 B11 SYBR Std-6 30.61 30.61 0.000 1.000E+03 3.000 1.00E+03 0.00E+00 aggR_3 C09 SYBR Std-4 24.93 24.93 0.000 1.000E+05 5.000 1.00E+05 0.00E+00 aggR-5 005 SYBR Std-1 14.10 14.10 0.000 1.000E+08 8.000 1.00E+08 O.OOE+OO aggR_8 F07 SYBR Std-3 21.39 21.39 0.000 1.000E+06 6.000 1.00E+06 O.OOE+OO aggR_6 F11 SYBR Std-7 34.19 34.19 0.000 1.000E+02 2.000 100E+02 O.OOE+OO aggR_2 Figure 12 (/): EAEC’s aggR gene optimized curves and quantification data. SYBER- Green fluorescence for A) PCR amplification. B) standard curve, C) melt peaks and D) melt curve just before data was captured. 77 University of Ghana http://ugspace.ug.edu.gh Quantification Data Wei Fluor Content Thresh old Cyde( C(t)) C(t) Mean C(t) Std. Dev Starting Quantity (SQ) Log Start n8 Quan tity SQMean SQ Std. Dev We* Note 002 SYBR Std-1 19.45 19.45 0.000 1.000E+07 7.000 1.00E+07 O.OOE+OO aatA10_7 005 SYBR Std-2 23.60 23.60 0.000 1.000E+06 6.000 1.00E+06 O.OOE+OO aatA 10 _6 008 SYBR Std-3 27.88 27.89 0.000 1.000E+05 5.000 1.00E+05 O.OOE+OO aatA10_5 D11 SYBR Std-4 31.28 31.28 0.000 1.000E+04 4.000 1.00E+04 O.OOE+OO aatA10_4 F03 SYBR Std-5 34.81 34.81 0.000 1.000E+03 3.000 1.00E+03 O.OOE+OO aatA 10 _3 F10 SYBR Std-6 37.23 37.23 0.000 1.000E+02 2.000 1.00E+02 O.OOE+OO aatA10_2 Figure 12 (ii): EAEC’s aatA gene optimized curves and quantification data. SYBER- Green fluorescence for A) PCR amplification, B) standard curve, C) melt peaks and D) melt curve just before data was captured. 78 University of Ghana http://ugspace.ug.edu.gh Quantification Data Wefl Fluor Content Threshold Cycle ( C(t)) C(t) Mean C(t) Std. Dev Starting Quantity (SQ) Log Starting Quantity SQ Mean SQ Std Dev Wei Note B02 SYBR Std-1 15.45 15.45 0.000 1.000E+08 8.000 1.00E+08 O.OOE+OO aap10_8 B06 SYBR Std-2 17.54 17.54 0.000 1.000E+07 7.000 1.00E+07 O.OOE+OO aap10_7 BIO SYBR Std-3 20.78 20.78 0.000 1.000E+06 6.000 1.00E+06 O.OOE+OO aap10_6 F02 SYBR Std-4 24.98 24.98 0.000 1.000E+05 5.000 1.00E+05 O.OOE+OO aap10_5 F06 SYBR Std-5 27.97 27.97 0.000 1.000E+04 4.000 1.00E+04 O.OOE+OO aap10_4 F10 SYBR Std-6 29.45 29.45 0.000 1.000E+03 3.000 1.00E+03 O.OOE+OO aap10_3 H04 SYBR Std-7 31.93 31.93 0.000 1.000E+02 2.000 1.00E+02 O.OOE+OO aap10_2 Figure 12 (Hi): EAEC’s aap gene optimized curves and quantification data. SYBER- Green fluorescence for A) PCR amplification, B) standard curve, C) melt peaks and D) melt curve just before data was captured. 79 University of Ghana http://ugspace.ug.edu.gh Quantification Data Wefl Fluor Content Threshold Cyete( C(t>) C(t) Mean C(t) Std. Dev Starting Quantity (SQ) Log Start! ng Quant ity SQ Mean SQ Std. Dev We* Note B06 SYBR Std-1 27.73 27.73 0.000 1.000E+06 6.000 1.00E+06 O.OOE+OO aaiC10_6 C06 SYBR Std-2 32.24 32.24 0.000 1.000E+05 5.000 1.00E+05 O.OOE+OO aaiC10_5 D06 SYBR Std-3 34.77 34.77 0.000 1.000E+04 4.000 1.00E+04 O.OOE+OO aaiC10_4 E06 SYBR Std-4 36.98 36.98 0.000 1.000E+03 3.000 1.00E+03 O.OOE+OO aaiC10_3 F06 SYBR Std-5 N/A 0.00 0.000 1.000E+02 2.000 O.OOE+OO O.OOE+OO aaiC10_2 Figure 12 (iv): EAEC’s aaiC gene optimized curves and quantification data. SYBER- Green fluorescence for A) PCR amplification, B) standard curve, C) melt peaks and D) melt curve just before data was captured. 80 University of Ghana http://ugspace.ug.edu.gh Quantification Data Well Fluor Content Thres hotd Cyde ( C (t )) c(t) Mean C(t) Std. Dev Starting Quantity (SQ) Log Starti ng Quan tfty SQ Mean SQ Std. Dev Wed Note B06 SYBR Std-1 18.95 18.95 0.000 1.000E+06 6.000 1.00E+06 O.OOE+OO Crypt10_6 006 SYBR Std-2 22.20 22.20 0.000 1.000E+05 5.000 1.Q0E+05 O.OOE+OO Crpt10_5 F06 SYBR Std-3 25.89 25.89 0.000 1.000E+03 3.000 1.00E+03 O.OOE+OO Crypt10_3 H06 SYBR Std-4 28.39 28.39 0.000 1.000E+02 2.000 1.00E+02 O.OOE+OO Crypt10_2 Figure 12 (v): Cryptosporidium’s 18s rRNA gene optimized curves and quantification data. SYBER- Green fluorescence for A) PCR amplification, B) standard curve, C) melt peaks and D) melt curve just before data was captured. 81 University of Ghana http://ugspace.ug.edu.gh I - B - »■*» *-»■> 1 Quantification Data Well Fluor Content Thresh oM Cycte( C(t)) c « Mean C(t) Std. Dev Starting Quantity (SQ) Loo Starti ng Quant SQMean SQ Std. Dev Wea Note 806 SYBR Std-1 19.83 19.83 0.000 1.000E+06 6.000 1.00E+06 O.OOE+OO Eh10_6 C06 SYBR Std-2 21.50 21.50 0.000 1.000E+05 5.000 1.00E+05 O.OOE+OO Eh10_5 006 SYBR Std-3 23.17 23.17 0.000 1.000E+04 4.000 1.00E+04 O.OOE+OO Eh10_4 E06 SYBR Std-4 25.71 25.71 0.000 1.000E+03 3.000 1.00E+03 O.OOE+OO Eh10_3 F06 SYBR Std-5 27.50 27.50 0.000 1.000E+02 2.000 1.00E+02 O.OOE+OO Eh10_2 Figure 12 (vi): K histofytica's Eh gene optimized curves and quantification data. SYBER- Green fluorescence for A) PCR amplification. B) standard curve, C) melt peaks and D) melt curve just before data was captured. 82 University of Ghana http://ugspace.ug.edu.gh A single-plex quantitative PCR for each gene pair [Table 3] consisted of template (5 pi faecal), 1 pi of each 6.2 pM primer, 12.5 pi of SYBR-Green -490 (Bio-Rad Laboratories, MD, USA), and PCR grade water to a reaction volume of 25 jil. Reactions for each sample were performed using the Bio-Rad iQCycler Real- Time Detection System in Bio-Rad iCycler 96-well plates, where positive and negative controls were included with each reaction set. The results were analyzed with a user-defined threshold of 200 PCR baseline-subtracted curve-fit relative fluorescence units. Melt curve (ct) data collection and analysis was enabled at cycles 3 and 4, with an increase in set point temperatures after cycle 2 by 0.5°C. Appendix III shows the cycling protocol o f the real-time PCR o f each of the target genes screened. Multiple loci for EAEC (aap, aatA, aggR and aaiC) and single loci for Shigella, Cryptosporidium and Giardia species from faecal DNA were sought. Table 3 shows the target genes, annealing temperatures and the amplification protocol used. Standard cultures with known numbers of E. coli 042 and 17-2, Shigella, Cryptosporidium and Giardia oocysts were used as reference and positive controls. Water and E. coli K-12 were used as negative controls. Melt curve analysis was used to determine positivity of samples using a user defined threshold. 3.6 Detection of Enteric Pathogens from Stool DNA University of Ghana http://ugspace.ug.edu.gh Table 3: Target genes screened from stool DNA Strain Genetarget Location Primer sequence (5 - 3) PCR size (bp) Annealing tempera­ ture (°C) Source/ reference aaiC Chromosome CTTCTGCTCTTAGCAGGGAGTTTG AAGCGTGAAATGCCTGAGGA 123 47.5 Nataro’s Lab EAEC aatA Plasmid CCTRTGTTGATGCTCGAGAGA CKTTCCTCCTCCTCAAGGACAT 118 55 Nataro’s Lab aap Plasmid CTTGGGTATCAGCCTGAATG AACCCATTCGGTTAGAGCAC 310 45 (Cema et al., 2003) aggR Plasmid CTAATTGTACAATCGATGTA ATGAAGTAATTCTTGAAT 308 45 (Czeczulin et al.. 1999) Shigella!El EC ipaH Plasmid GTTCCTTGACCGCCTTTCCGATACCGTC GCCGGTCAGCCACCCTCTGAGAGTAC 619 60.5 (Sethabutr et al.. 1993) 84 University of Ghana http://ugspace.ug.edu.gh Table 3 (Continuation): Target genes screened from stool DNA Crypto­ sporidium 18s rRNA Chromosome CTCCACCAACTAAGAACGGCC TAGAGATTGGAGGTTGTTCCT 213 60 Gene ID cgd7_230 (Gene ID, 2009) E. histolytica Eh Chromosome A AC AGTA AT AGTTTCTTTGGTTAGT AAAA CTTAGAATGTCATTTCTCAATTCAT 134 60 (Haque et al., 2007) Giardia lamblia P241 Chromosome CATCCGCGAGGAGGTCAA GCAGCCATGGTGTCGATCT 74 60 (Guy et a l, 2003) 85 University of Ghana http://ugspace.ug.edu.gh 3.7 Intestinal Inflammation Assessment Intestinal inflammations were quantitatively assessed from frozen stool specimens using the IBD SCAN (TechLab, Blacksburg, VA) according to the manufacturer’s instructions. Stool specimens were allowed to thaw and were serially diluted 10- fold with a diluents supplied by the manufacturer. These were then analyzed by a polyclonal antibody-based ELISA method. Detailed procedure is described elsewhere (Kane et al., 2003), and absorbance of each assay well was measured spectrophotometricaily at 450 and 620 nm (Aj5o/62o)- Faecal lactoferrin concentrations in |ig/ml were determined by comparison with a standard curve using purified human lactoferrin and analyzed by linear regression in Microsoft Excel. The lowest dilution of a specimen with an absorbance at 450/620 nm within the linear portion of the curve was used to determine the lactoferrin concentration. The final lactoferrin concentration was obtained by multiplying the dilution factor by the concentration. A positive control (purified human lactoferrin) and a negative control (washing buffer) was included in each batch of stools analyzed, and linear regression was performed separately for each batch using standard controls supplied by the manufacturer. This assessment was performed only on subjects with adequate (> 60 ml or mg. depending on stool consistency) stool specimens and where necessary, the experiment was repeated. 86 University of Ghana http://ugspace.ug.edu.gh w A * 3' i y E 0 Figure 13a: Photographs showing some reactions of the lactoferrin assay (LF). The IBD-SCAN was used according to the manufacturer’s instruction, a) 200 (j.1 of a stool-diluent suspension was inoculated into a microtitre well, which is coated with immobilized polyclonal antibodies to human LF. b) LF, if present, binds to the antibodies during 30 min incubation at 37°C. After the incubation, polyclonal antibodies are coupled to horseradish peroxidase (conjugate) and allowed to bind to captured LF during 30 min incubation, c) Unbound conjugate was washed, and a substrate (tetra-methyl-benzidene and hydrogen peroxide) is added for colour development, d) After a 15-min substrate incubation, 0.6-N sulfiiric acid solution is added to quench the reaction, and the absorbance of each assay well was measured spectrophotometrically at 450/620 nm (A450/620). 87 University of Ghana http://ugspace.ug.edu.gh Lactoferrin (un/ml) Std. OD 1 0 0 2 Ave. OD 0 0 0 0 6.25 0.03 0.041 0.0355 12.5 0.082 0.11 0.096 25 0.215 0.188 0.2015 a a 50 0.388 0.361 0.3745 1 0 0 0.836 0.766 0.801 ) Figure 13b: Standard curve fit of lactoferrin (LF) using purified human controls. Each batch of assay included purified human lactoferrin of known concentrations, a positive and a negative control, e) Averages of the ELISA readings of each of the known concentrations were calculated, f) A standard curve was generated using the purified human lactoferrin and analyzed by linear regression using Microsoft Excel. The lowest dilution of specimen giving a reading for absorbance at A4S0/620 within the linear portion of the curve was used to determine the lactoferrin concentration. The final concentration was obtained by multiplying the concentration by the dilution factor. Lactoferrin Standard curve y = 0.008x -0 .0 R1 = 0.9987 -Ave . OD -Linear (Ave. 01 University of Ghana http://ugspace.ug.edu.gh 3.8 Real-Time PCR Detection of EAEC from Bacterial DNA The same set of the EAEC genes screened earlier from stool DNA were analyzed in the bacterial DNA, under similar conditions. A single-plex quantitative PCR for the EAEC virulence genes [Table 3] consisted of I pi template, 1 pi of each 6.2 jiM primer, 12.5 pi of SYBR-Green -490 (Bio-Rad Laboratories, MD, USA), and PCR grade water to a reaction volume of 25 |il. Reactions for each sample were performed using the Bio-Rad iQCycIer Real-Time Detection System in Bio- Rad iQCycler 96-well plates, where positive and negative controls were included with each reaction set. The results were analyzed with a user-defined threshold of 200 PCR baseline-subtracted curve-fit relative fluorescence units. Melt curve (ct) data collection and analysis was enabled at cycles 3 and 4, with an increase in set point temperatures after cycle 2 by 0.5°C. 89 University of Ghana http://ugspace.ug.edu.gh 3.9 Genotyping of Amplified Cryptosporidium-positive stools PCR-products from stool DNA that were Cryptosporidium-positive were further analyzed. The amplicons were purified using the PureLink™ PCR Purification Kit (Invitrogen, Carlsbad. CA) according to the manufacturer’s instructions. A minimum volume of 50 nl PCR-product was purified. Basically, the amplified PCR-product was bound onto Spin Columns and washed with buffers, and finally eluted with nuclease free water. Total DNA concentration after purification was measured in jig/mL by a spectrophotometer. Purified PCR-products were then sequenced in both directions using forward and reverse primer sets of Cryptosporidium parvum 18S rRNA (Table 3) at the DNA Sequencing Facility at the Cancer Unit of the University o f Virginia. The forward and reverse sequences (Applied Biosystem) were manually joined and analyzed using BLAST (Basic Local Alignment Search Tool), a public available database (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The Electrogrames and the sequences are documented in Appendix HI. Because the above method was not so helpful in deciphering the genotypes of Cryptosporidium, high resolution melting (HRM) analysis was employed (Herrmann et al., 2006; Reja et al., 2010). The procedure was highly automated using the Rotor-Gene Q 5 plex HRM instrument (QIAGEN, Hilden, German)). The run was done using Eva Green fluorescence dye (Biotium, Hayward, USA), in a 25 nl total volume. The only human involvement was the loading of the 90 University of Ghana http://ugspace.ug.edu.gh master-mix (300 nM each primer), and the amplified DNA templates onto the racks of the machine. Additionally, the concentrations of the amplified DNAs together with a control (C. parvum), previously determined by photospectometry (jig/L) were fed into the software for the purposes of analysis. The system was programmed to compare the melt curves of the positive control (C. parvum) to those of the unknowns and a blank without any DNA. The Qiagen Type-it HRM PCR kit was used in preparing the master-mix according to the manufacturer’s instructions. The basic principle in HRM is that as the PCR products dissociate with increasing temperature, dye is progressively released and fluorescence diminishes. The fluorescence measurements are collected at corresponding temperature increaments and plotted as a ‘melt curve’. Melt curve shape and position are characteristic of each sample allowing them to be compared or discriminated. Even, a single base change between samples can be readily detected and identified (Herrmann et al., 2006; Reja et al., 2010). 91 University of Ghana http://ugspace.ug.edu.gh 3.10 Data Storage and Analysis Data from the questionnaires were stored in Microsoft Access files and later exported to various statistical packages for further analyses. To avoid any experimental biases, stool specimens were coded before testing and only decoded for purposes of analysis. Statistical analyses were performed using SPSS software (version 17.0; SPSS, Chicago) and Epi-lnfo. Z-score calculation and weight for­ age percentile plots were done by the nutritional soft-ware in Epi-lnfo. Statistical tests included x2 for associations of pathogens with age groups, diarrhoea and non-diarrhoea and the paired / test for associations with lactoferrin level. Odds ratio and 95% confidence intervals are reported for all 2 X 2 comparisons. Two-tailed tests were used and p <0.05 was considered statistically significant. 92 University of Ghana http://ugspace.ug.edu.gh CHAPTER FOUR RESULTS 4.1 Study Population and Base-line Characteristics Within the nine-month study period, 287 children < 5 years were recruited with only 13 excluded from analysis because of insufficient data. Figure 14 shows an insert map of Ghana with plots of the locations of the sampled children. Majority of the children enrolled resided in the Greater Accra Metropolis, and a few of them were from the Central (Kasoa) and Eastern (Nsawam) regions o f Ghana. O f the 274 children included in analysis, 170 (62%) were with and 104 (38%) without diarrhoea; there were more males 156 (56.9%) than females. Acute and persistent diarrhoea comprised 85.3% (145) and 7.6% (13) o f total cases respectively. Duration of symptoms in the remaining children with diarrhoea (7.1%) was not recorded. Table 4 shows the baseline characteristics of the study population. 93 University of Ghana http://ugspace.ug.edu.gh LOCATIONS OF SAMPLED CHILDREN Figure 14: Settlements of the enrolled children in the study. Majority of the enrolled children were from the Greater Accra Metropolis. A few of them however came from the Central and the Eastern regions of Ghana, The only Shigella flexneri isolated from stool culture came from a child from the Central region. Sampled children with and without diarrhoea are represented as green dots and red triangles, respectively. The sizes of the shapes are proportional to the number of children enrolled from each locality. University of Ghana http://ugspace.ug.edu.gh Table 4: Baseline characteristics of study population Characteristic Diarrhoea # (%) Non-diarrhoea # (%) Age/months # = 170 # = 104 0 - 6 26(15.3) 35 (33.7) 7 -1 2 54 (31.8) 30 (28.8) 13 -24 77 (45.3) 24 (23.1) 25-60 13(7.6) 15(14.4) Sex N=\70 # = 104 Male 97 (57.1) 73 (42.9) Female 59 (56.7) 45 (43.3) Weight/kg #=170 # = 104 2 .5 -4 .9 10(5.9) 4(3.8) 5 .0 -9 .9 121 (71.2) 75 (72.1) 10-19.9 34 (20.0) 22 (21.2) 20 -86 .0 5 (2.9) 3 (2.9) WAZ #=168 #=101 Normal (> -1) 75 (44.6) 60 (59.4) Mild (-1 to -2) 33 (19.6) 19(18.8) Moderate (-2 to -3) 32(19.0) 12(11.9) Severe (<-3) 28(16.7) 10(9.9) HAZ # = 8 6 # = 8 4 Normal (> -2) 48 (55.8) 61 (72.6) Moderate (-2 to -3) 6(7.0) 11 (13.1) Severe (<-3) 32 (37.2) 12(14.3) WHZ # = 82 # = 7 9 Normal (> -2) 58 (70.3) 66 (83.5) Moderate (-2 to -3) 6(7.3) 8(10.1) Severe (<-3) 18(22.0) 5 (6.3) WAZ, HAZ and WHZ are Z-scores for weight-for-age, height-for-age and weight-for-height, respectively. 95 University of Ghana http://ugspace.ug.edu.gh At least one anthropometric measurement was taken for 269 out of the 274 children analyzed. O f 269 children from whom weight measurements were recorded, 134 (49.8%) showed mild to severe malnutrition (WAZ < -1) [93/168 (55.4%) in children with and 41/101 (40.6%) without diarrhoea, OR=1.82 (95% Cl, 1.102-2.988), p=0.023]. O f 170 children from whom height or length measurements were recorded, 61 (35%) showed moderate to severe stunting (HAZ < -2) [38/86 (44.2%) in children with diarrhoea, and 23/84 (27.4%) without OR=2.10 (95% Cl, 1.110-3.972), p=0.026]. Of 161 children from whom both weight and height measurements were recorded, 37 (22.9%) showed moderate to severe wasting (WHZ < -2) [24/82 (29.3%) in children with diarrhoea, and 13/79 (16.5%) without OR=2.101 (95% Cl, 0.989-4.454), p=0.062]. The mean age, weight and height were; 15.1/14.6 months, 9.8/9.5 kg and 88.0/84.0 cm (diarrhoea/non-diarrhoea, respectively). In the diarrhoea sub-population, growth faltering started as early as 3 months of age in the male sub-group and by the seventh month several (>4) o f the children were observed to fall below the 5% percentile category [Figure 15a, 15b], In the non-diarrhoea sub-population fewer (<9) children were observed to fall below the 5% percentile compared to the diarrhoea sub-population [Figure 16a. 16b]. In the separate sub-populations of diarrhoea and non-diarrhoea, there was no significant statistical association in gender among the children who fell below the 5% percentile curve (p>0.05). 4.2 Nutritional Status and Growth Faltering 96 University of Ghana http://ugspace.ug.edu.gh Age/ months Figure 15a: Weight-for-age plot of male infants and young children (< 2 years of age) in the diarrhoea sub-population. In this sub-category, growth shortfall was observed as early as 3 months, and 37.9% (25/66) of the males fell below the 5% percentile curve. The percentiles (curves) arc measurements that show where a child is compared with a reference population (de Onis and Blossner, 2003). A child with normal growth characteristics is expected to fit above the 5% percentils curve (heavy red curve). University of Ghana http://ugspace.ug.edu.gh b ■ vox Age/months Figure 15b: Weight-for-age plot of female infants and young children (< 2 years of age) in the diarrhoea sub-population. In this sub-category, growth shortfall was observed around 9 months, and 31.1% (18/58) o f the females fell below the 5% percentile curve. The percentiles (curves) are measurements that show where a child is compared with a reference population (de Onis and Blossner, 2003). A child with normal growth characteristics is expected to fit above the 5% percentils curve (heavy red curve). University of Ghana http://ugspace.ug.edu.gh Age/months Figure 16a: Weigbt-for-age plot of infants and young children (< 2 years of age) in the non-diarrhoea sub-population. In this sub-category, fewer male children 20.5% (9/44) fell below the 5% percentile curve. The percentiles (curves) are measurements that show where a child is compared with a reference population (de Onis and Blossner, 2003). A child with normal growth characteristics is expected to fit above the 5% percentils curve (heavy red curve). University of Ghana http://ugspace.ug.edu.gh •" '— w e i g h Age/months Figure 16b: Weight-for-age plot of infants and young children (< 2 years of age) in the non-diarrhoea sub-population. In this sub-category, far fewer female children 9.1% (3/33) fell below the 5% percentile curve. The percentiles (curves) are measurements that show where a child is compared with a reference population (de Onis and Blossner, 2003). A child with normal growth characteristics is expected to fit above the 5% percentils curve (heavy red curve). 100 University of Ghana http://ugspace.ug.edu.gh 4.3 Real-Time PCR Detection of Pathogens from Stool DNA Table 5 shows bacteria and parasitic agents detected from stool DNA by real-time PCR in children with and without diarrhoea. EAEC was defined as positivity for any of the four EAEC virulence genes sought {aap, aatA, aggR and aaiC). Although EAEC was significantly associated with diarrhoea (147/170 vs 80/104, p=0.042), it was also found in high frequencies in both nourished and malnourished children [Table 5]. In six out of 170 diarrhoeal stool specimens, the ipaH gene which is expressed by both Shigella and enteroinvasive E. coli (EIEC) was detected. The ipaH gent was not detected in stool DNA from any of the non-diarrhoea children. The numbers were, however, too small to attain statistical significance (p>0.05). Five out of 6 of the children in whom the ipaH gene was detected were well- nourished (Table 5). Cryptosporidium spp was the most frequently detected protozoan parasite in stool DNA and was associated with diarrhoea (14/170 vs 1/104, /H3.01I). Cryptosporidiosis was also primarily (10 out of 14) detected in children who were malnourished and had diarrhoea (Table 5). E. histolytica was only detected in children with diarrhoea (5 out of 170) and Giardia was not detected in either sub­ populations. 101 University of Ghana http://ugspace.ug.edu.gh Table 5 : Organisms detected by real-time PCR from stool DNA Diarrhoea (n=170) Non-diarrhoea (n=104) No. (%) WN (n=75) MN (n=95) No. (%) WN (n=60) MN (n=41) Odds Ratio [95%CI] p-value Any infection EAEC 147(86.5) 66 79 80 (76.9) 49 30 1.917(1.018-3.612) 0.048 Shigella/EIEC 6(3.5) 5 1 0(0) - - n/a Cryptosporidium spp 14(8.7) 4 10 1(1.0) 1 0 9.244(1.197-71.371) 0.011 E. histolytica 5 (3.0) 2 3 0(0) - - n/a Giardia spp 0(0) - - 0(0) - - - *p-value is between diarrhoeal and non-diarrhoeal stool specimen WN - well nourished (WAZ > 1) MN - malnourished (WAZ < 1) n/a - not applicable 102 University of Ghana http://ugspace.ug.edu.gh Of the four genes associated with EAEC, aatA was the most frequently detected (67.2%) of all stool DNA, followed by aap (59.9%), aggR (42.7%) and aaiC (33.6%) [Table 6]. EAEC’s plasmid gene aap was significantly associated with diarrhoea [OR=2.506 (95% Cl, 1.516-4.144), p <0.001] and the chromosomal gene aaiC was not [OR= 1.639 (95% Cl, 0.0962-2.792), p=0M6] [Table 6], Multiple gene combinations were also observed in EAEC infections in the study population, and the presence of any three genes was associated with diarrhoea [OR=2.I01 (95% Cl, 1.261 -3.502),p=0.006] [Table 6]. None of the EAEC virulence genes was associated with malnutrition (WAZ < -1) (p>0.05) [Table 7], 4.4 EAEC Virulence Genes Distribution (Stool DNA) 103 University of Ghana http://ugspace.ug.edu.gh Table 6: Entcroaggregative E. coli (EAEC) virulence factor-positive in stool DNA Characteristic Diarrhea (n=!70) n (%) Non-diarrhea (n=104) n (%) Total n=274) n (%) Odds Ratio [95% Cl] /rvalue EAEC virulence related gene aaiC 64 (37.6) 2 (26.9) 92 (33.6) 1.639 [0.962 - 2.792] 3.327 0.086 aggR 79 (46.5) 8 36.5 117 (42.7) 1.508 [0.914-2.486] 2.602 0.131 aatA 118(69.4) 66 (63.5) 84 (67.2) 1.307 [0.780-2.188] 1.036 0.354 aap 116(68.2) 48 (46.2) 164 (59.9) 2.506 [1.516-4.144] 13.093 <0.001 EAEC gene combination Any 1 gene 147 (86.5) 80 (76.9) 227 (82.8) 1.917 [1.018-3.612] 4.139 0.048 Any 2 genes 130 (76.5) 73 (70.2) 203 (74.1) 1.380 [0.797-2.391] 1.325 0.259 Any 3 genes 84 (49.4) 33(31.7) 117(42.7) 2.101 [1.261-3.502] 8.224 0.006 All 4 genes 34 (20.0) 13(12.5) 47(17.2) ' "-.u" l 1.750 [0.876-3.496] 2.554 0.110 EAEC - Entcroaggregative Escherichia coli, OR - odds ratio, 95%C! - confidence in terval,/- chi-square, P < 0.05 is significant 104 University of Ghana http://ugspace.ug.edu.gh Table 7 : EAEC's genes in stool in association with and without malnutrition Diarrhoea (n=168) Non-diarrhoea (n=101) *p-valueWN (n=75) MN (n=93) WN (n=60) MN (n=41) EAEC genes aaiC 27 (36.0) 36 (38.7) 21(35.0) 7(17.1) 0.548 aatA 54 (72.0) 62 (66.7) 37 (61.7) 28 (68.3) 0.966 aggR 39 (52.0) 38 (40.9) 25(41.7) 13(31.7) 0.121 aap 53 (70.7) 63 (67.7) 31 (51.7) 16 (39.0) 0.584 *p-value is between diarrhoeal and non-diarrhoeal stool specimen WN - well nourished (WAZ > 1) MN - malnourished (WAZ < 1) 105 University of Ghana http://ugspace.ug.edu.gh 4.5 Multiple Infection (Stool DNA) Co-infections, with two or more pathogens detected in the stool DNA, was found predominantly in children who had diarrhoea. EAEC-Cryptosporidium was the most prevalent (7.6%, 13/170), followed by EAEC-Shigella/EIEC (2.9%, 5/170) and EAEC-£. histolytica (2.4%, 4/170). Cryptosporidium-E. histolytica and Cryptosporidium-ShigellafElEC co-infection each formed 0.6% (1/170), and one child who had diarrhoea was co-infected with EAEC- Cryptosporidium- Shigella/EIEC (0.6%, 1/170). There was no obvious trend in the distribution of pathogens by age in the two sub-populations, especially for EAEC (Table 8). 106 University of Ghana http://ugspace.ug.edu.gh Table 8: Distribution of pathogens by age from stool DNA Diarrhoea (n=170) Non-diarrhoea (n=104) Age category/months Age category/months Any infection 0-6 (n=26) 7-12 (n=54) 13-24 (n=77) 25-60 (n= 13) 0-6 (n=35) 7-12 (n=30) 13-24 (n=24) 25-60 (n=15) EAEC 24 (92.3) 42 (77.7) 69 (89.6) 12 (92.3) 25(71.4) 24(80) 18(75) 13(86.7) Cryptosporidium 3(11.5) 4(7.4) 6(7.8) 1 (7.7) I (4J2) E. histolytica - 2 (3.7) 3 (3.9) - - Shigella/El EC 1 (3.8) 1 (1.8) 3 (3.9) 1 (7.7) - Giardia spp - - - - - 107 University of Ghana http://ugspace.ug.edu.gh In only 1 of 170 diarrhoea stool specimens was Shigella isolated as an enteric bacterial pathogen from culture. This strain was serotyped with Shigella polyvalent anti-sera (Mast Group Ltd., Merseyside, U.K.) and was S. Jlexneri. In the entire study population, E. coli was the predominant commensal recovered fiom culture (79.6%). 4.6 Conventional Stool Culture Results 108 University of Ghana http://ugspace.ug.edu.gh 4.7 Faecal Lactoferrin Levels Figure 17 shows enteric pathogens detected in children with/without diarrhoea and the distribution of faecal lactoferrin levels. Irrespective of the enteric pathogen detected, faecal lactoferrin levels were relatively high (manufacturer’s cut-off value=7.24 |ig/ml). Especially for EAEC infection, both controls and patients had a wide range of lactoferrin levels, irrespective o f whether they were nourished or malnourished (Figure 17). Children with diarrhoea had significantly higher faecal lactoferrin levels (n=143; 1658.9± 204.2 ng/ml) compared to those without diarrhoea (n=84; 935.5± 194.4 pg/ml) (p=0.019). The aatA gene and the presence of any one or two genes of EAEC were also significantly (p<0.05) associated with elevated faecal lactoferrin levels (Table 9). In comparing diarrhoea with non-diarrhoea stool specimens, EAEC’s chromosomal gene aaiC showed the highest fold-increase in faecal lactoferrin level (2.7) followed by the aap gene (2.5). Additionally, detection of multiple virulence gene of EAEC in a stool was associated with an increased fold- rise in mean faecal lactoferrin level between children with and without diarrhoea (Table 9). Among EAEC-infected malnourished children, two stool specimens (out of greater than 200 specimens analyzed) showed lactoferrin values that were 30 - 50 times more than the mean value for their subject group; they were therefore designated as outliers, and not included in the statistical analysis. 109 University of Ghana http://ugspace.ug.edu.gh .((((±. D i ± arhe)Excbtdn .( ± D f ±cs i ± e)Excbtdn vy r/ m ±1s g ±±±±±±±±±±cencIrpdb adre hdodud 9000- • 8000 gg gg l((( F ♦• C R M(((± K 0 • c T± 5000 c T 1 2 U(((± AL # (K H S g g 3000- O Q o Qg O ( g W((( o g F .((( 1 11 )())))2) ~ s~ N Q a0 gcbQQR Q o 5m)OO S gg g.Q.R eOOO a # . -( N MN N MN N MN N MN N MN N MN Crypto Shigella E his EAEC * N * 7 7x03p) Diarrhoea Non-diarrhoea 8c9Exd .l4 *epdxcI 3 rpt )9deb ndpdIpdn ren urdIrh hrIp)udxxce v, 8 s hdodhb Crypto - Cryptosporidium spp. E his - E. histolytica, EAEC - enteroaggrelative Escherichia coli •Breast feeding may cause moderately (15 s 120 ng/ml) increased LF (Lima el al - unpublished observation) 110 University of Ghana http://ugspace.ug.edu.gh Table 9: EAEC genes detected and faecal lactoferrin levels Characteristic Diarrhoeal stool n (mean± S.E) pg/ml Non-diarrhoeal stool n (mean± S.E) pg/ml *Fold- rise P- value EAEC gene aaiC 56 (1021.0±251.6) 25 (821.2±402.1) 1.2 0.667 aggR 66 (1713.9±292.5) 33 (934.1 ±329.2) 1.8 0.104 aatA 98 (1706.1±244.1) 54 (927.1±259.1) 1.8 0.044 aap 101 (1656.7±245.9) 39 (901.2±261.6) 1.8 0.080 EAEC gene combinations Any 1 gene 123(1633.5±215.4) 66 (1175.4±356.8) 1.4 0.029 Any 2 gene 107 (1679.8±231.1) 60 (892.6±234.3) 1.9 0.028 Any 3 gene 73 (1504.9±269.7) 30 (1132.8±394.8) 1.3 0.451 Any 4 gene 30 (1319.5±400.4) 11 (370.5±285.5) 3.6 0.175 All specimens 143 (1658.9±204.2) 84 (935.5±194.4) 1.8 0.019 ♦Ratio o f diarrhoea and non-diarrhoea mean lactoferrin levels 111 University of Ghana http://ugspace.ug.edu.gh In the subset of the children in which bacteria was isolated, faecal lactoferrin levels were further analyzed. In this subset, children with diarrhoea had significantly higher faecal lactoferrin levels (n= 105; 67l.9±341.7 pg/ml) compared to those without diarrhoea (n=75; 946.2±213.1 ng/ml) [95% Cl, - 423.1 to 1261.9, p=0.051]. However, there was no statistical significance between the children who screened positive for EAEC and the non-EAEC [EAEC (n=130); 95% Cl, 14706 ±217.7 vs non-EAEC (n=38); 95% Cl 1051.3 ±258.6,^=0.327] 112 University of Ghana http://ugspace.ug.edu.gh 4.8 EAEC’s Dispersin Gene Associates Better with Diarrhoea (p<0.05) Of the 218 Escherichia coli isolated from culture and DNA extracted, 203 (93.1%) were available for screening by real-time PCR. The aatA was the most frequently detected gene (63.5%) in bacterial DNA, followed by aap (49.3%), aggR (30.5%) and aaiC (20.7%) [Table 10]. Figure 18 (/) and (//) analyzed the entire stool and bacterial DNAs, and bacterial DNA and their matching stool DNAs, respectively. The dispersin gene aap was significantly associated with diarrhoea in the two separate analyzes (p<0.05). The chromosomal aaiC gene attained boarder-line significance in the bacterial DNA and their corresponding matching stool DNAs (p=0.049) [Figure 18 (//")]. In general, the percentage proportion of EAEC genes detected in stool DNA were higher in all cases, compared with those detected from bacterial DNA. Within the diarrhoea sub-populations in the two separate analyses, EAEC’s aaiC and aggR genes were significantly detected in stool DNA (p<0.05) [Figure 18(0 and (//)]. EAEC, defined as positivity for any of the four genes screened was not statistically associated with diarrhoea in the bacterial DNA (p=0.244) [Table 10, Figure 19]. 113 University of Ghana http://ugspace.ug.edu.gh Table 10: Enteroaggregative E. coll (EAEC) virulence factor-positive in bacterial DNA (E . coli) Characteristic Diarrhoea (n=115) n (%) Non-diarrhoea (n=88) n (%) Total n=(203) n (%) Risk estimate: OR [95% cn 7? p-value EAEC virulence related gene aaiC 25 (21.7) 17(19.3) 42 (20.7) 1.160 [0.582-2.313] 0.178 0.673 aggR 32 (27.8) 30 (34.1) 62 (30.5) 0.745 [0.409- 1.359] 0.922 0.359 aatA 77 (67.0) 52 (59.1) 129 (63.5) 1.403 [0.789-2.495] 1.331 0.249 aap 65 (56.5) 35 (39.8) 100(49.3) 1.969 [1.120 - 3.460] 5.595 0.018 EAEC gene combination Any 1 gene 94(81.7) 66 (75.0) 160 (78.8) 1.492 [0.759 - 2.933] 1.356 0.244 Any 2 genes 63 (54.8) 44 (50.0) 107 (52.7) 1.212 [0.695-2.113] 0.457 0.499 Any 3 genes 33(28.7) 21 (23.9) 54 (26.6) 1.841 [0.680-2.423] 0.596 0.440 All 4 genes 9(7.8) 3(3.4) 12(5.9) 2.406 [0.632-9.164] 1.749 0.186 EAEC - Enteroaggregative Escherichia coli, OR - odds ratio, 95%CI - confidence interval, x ' - chi-square, P < 0.05 is significant 114 University of Ghana http://ugspace.ug.edu.gh P<0.001 S_DNA B_DNA S_DNA B_ONA S_DNA B_DNA S_DNA B_DNA S_DNA B_DNA S_ONA B_DNA aa/C aagR aatA aap aaiC aagR aatA aap Diarrhoea Non-Diarrhoea F ig u re 1 8 (/ ): S to o l D N A ve rsu s b a c te r ia l D N A in the m o le c u la r d ia g n o s is o f E A E C (a l l s to o l a n d b a c te r ia l D N A s ) . W ith in the diarrhoea sub -po pu la tio n , the detection o f E A E C 's ch ro m o so m a l gene (a a iC ), and the master plasm id regulon (a g g R ) were significantly detected in s too l D N A , com p are d w ith bacteria D N A (p < 0 .0 5 ). T h e anti-aggregation gene (a a p ) was significantly associated w ith diarrhoea in both the stoo l and bacterial D N A s . s.dna - *iooi dna, b.dna - bacterial dna 90 so 70 60 50 40 30 20 10 0 100 P= 0.004 P-0.002 S_DNA B_DNA S_DNA B_DNA 115 University of Ghana http://ugspace.ug.edu.gh P - 0 . 0 4 0 aa i C aa gR aap aa i C aa gR a a t A aap D i a r rh oe a N o n - D i arrho ea F ig u re 18 (//): S t o o l D N A v er s us b ac t er ia D N A in the m o l e c u l a r d i ag n o s i s of E A E C ( b a c t e r i a D N A a n d thei r c o r r e s p o n d i n g s too l D N A s ) . T h e anti -aggregation gene ( a a p ) was significantly associated with diarrhoea in both the stool and bacteria D N A s . W ithin the diarrhoea sub - popu lat ion , the detection o f E A E C ' s c h r o m o s o m a l g c n e ( a a i C ) . the master plasmid regulon ( a g g R ) and the a a p were significantly detected in stool D N A , c o mp a re d with bacteria D N A ( p < 0 . 0 5 ) . S DNA - 1 10© I DNA, B_D N A • b a c teria DNA 116 University of Ghana http://ugspace.ug.edu.gh p=0.018 S DNA B DNA S_DNA B_DNA Diarrhoea__________ Non-diarrhoea EAEC Figure 19: EAEC is better diagnosed in stool DNAas opposed to bacterial DNA E A E C defined as positivity to any o f the four genes screened foiled to attain statistical signi ficanre in bacterial D N A In the cohort o f children from whom E coli was isolated by culture, EAEC was analysised in both their bacterial and stool DNAs. The association o f EAEC to diarrhoea was lost in the bacterial DM (p=0.244). 117 University of Ghana http://ugspace.ug.edu.gh The amplified region was much conserved, and therefore the BLAST analysis was not helpful in determining the circulating genotypes amplified by PCR. However, the HRM (High Resolution Melting) analysis, interrogating the melt curves of a known C. parvum genotype to the PCR positive unknowns indicated that 26.7% (4/15) of the Cryptosporidium-positive DNAs were C. parvum. The rest were broadly categorized as non-C. parvum [Figure 20]. 4.9 Cryptosporidium Genotypes: Preliminary results 118 University of Ghana http://ugspace.ug.edu.gh VIUUAL W3E* Figure 20: HRM data analysis of Cryptosporidium-positive DNA9. Sample # 1 (type 1) is a positive C. pan'um control. The HRM analysis ‘interogated’ the melt curve of this control with the unknowns as well as a blank, containing only a buffer. ‘Type 1’ and ‘variation’ results generated by the Rotor-gene data file (Qiagen, Germany), represents C. parvum and non- C. parvum genotypes, respectively. Samples analyzed and included in the current study are samples H 2 to 16 (above red horizontal mark). 119 University of Ghana http://ugspace.ug.edu.gh Educational backgrounds of the caregivers of 268 of the children were recorded, ranked and analyzed. No formal education, up to primary, elementary and vocational training was ranked as ‘low’; and middle school leavers, up to Junior and Senior Secondary, and tertiary education, as ‘high’. Low educational background of caregivers was a risk factor for a child presenting with diarrhoea 68.9% (115/167 in the children with, and 49.5% (50/101) without diarrhoea (OR=2.256 95% Cl 1.355-3.754, (p=0.002)]. It was difficult to judge whether caregivers provided true response to the question on how long they exclusively breastfed. Breast feeding is widely promoted in Ghana. It is therefore speculated that caregivers wanted to appear strictly following the message, and it was difficult to include this in analysis. About 43 % (73/170) of the caregivers of the children with diarrhoea responded ‘Yes’ to having taken some form of medication prior to attending hospital, and 50% (85/170) responded ‘No’. No data was recorded for the rest. 4.10 Summaries of Additional Data from Questionnaires 120 University of Ghana http://ugspace.ug.edu.gh CHAPTER FIVE DISCUSSION 5.1 Nutritional Shortfalls among the Study Population The prevalence of early childhood stunting and the number of people living in absolute poverty are good indicators of poor development (Bhutta et al., 2008). These indicators are closely associated with poor cognitive and educational performance in children, and over 200 million children under 5 years, mostly living in south Asia and sub-Saharan Africa are not fulfilling their developmental potential (Muller and Becher, 2006; Pelletier and Frongillo, 2003; Smith, 2000; Victora et al., 2010; Young and Jaspars, 2006). These disadvantaged children are likely to do poorly in school and subsequently have low incomes and provide poor care for their children, thus contributing to the intergenerational transmission of poverty (Grantham-McGregor et al., 2007). In the current study, moderate to severe stunting (HAZ <-2) was observed in 35.9% of the study population; 44.2% and 27.4% in the children with and without diarrhoea, respectively. In Butajira, a rural setting in Ethiopia, the prevalence of stunting increased steadily throughout the first year of life in a cohort o f children studied. At ages 2, 6 and 12 months, the prevalence of stunting was 14.6%, 26.7% and 48.1%, respectively (Medhin et al., 2010). Breastfeeding has already been shown to significantly reduce mortality in infants and young children (Black et al., 2008; Jones et al., 2003; WHO, 2000a), but breast feeding promotions and campaigns only have a small effect on the reduction of stunting (Bhutta et al., 2008). In the current study it was unclear whether the responses the caregivers provided in the 121 University of Ghana http://ugspace.ug.edu.gh questionnaire reflected their true breast feeding practice, since almost everybody responded with either breastfeeding or having exclusively breast fed for at least six month. Elsewhere, exclusive breast feeding practice significantly correlated with factors such as infant birth order, caregivers’ education, occupation and parity (Aghaji, 2002). The current study identified wasting (WHZ <-2) in 21.7% of the children studied, 26.8% and 16.5% in the children with and without diarrhoea, respectively. In surveys conducted in Latin America, Asia and Africa, low birth weight was noted to be a good predictor for wasting in children (Fernandez et al., 2002). The children in the current study were out-patients, and there was no data on their birth weights. Low educational background of caregivers was a risk factor for children presenting with diarrhoea in the current study (p=0.002). This association may be indirectly related to the purchasing powers of these caregivers, in the provision of basic needs for themselves as well as their children. If this argument holds, it was expected that, malnutrition would also be statistically associated with low educational background of the caregivers of the children. However, no significant statistical association was detected between caregivers’ educational background and stunting, wasting and under-weight status of children (p > 0.05). Elsewhere, maternal education and intelligence directly correlated with offspring diet and nutritional status (Hasan et aL. 1991; Wachs etal., 2005). 122 University of Ghana http://ugspace.ug.edu.gh Mild to severe malnutrition (WAZ < -1, which reflects both acute and chronic types of malnutrition) was identified in 49.8% of the study population. Severe growth shortfalls occurred in both children with and without diarrhoea in the entire study population. Growth faltering in infants from developing countries has been reported to occur as early as 2-3 months of age (Davies-Adetugbo, 1997; Dewey et a l , 1992; Sathian et al., 1983) in contrast to developed countries (Dewey el al., 1992), a fact attributable to timing of complementary feeding. For example, in the DARLING (Davis Area Research on Lactation, Infant Nutrition and Growth) study, though breast milk intakes were similar, the amount and nutrient density of food consumed after 6 months were lower in Peru than in the United States (Dewey et a l, 1992). In 2005, Antwi noted a 21.2% incidence of wasting (acute malnutrition) among children seen in Kumasi, Ghana (Antwi, 2008) similar to an overall incidence of 22.9% (37/161) in the current study - both consistent with the 22.1% estimate for Ghana by the World Food Programme (WFP, 2006). In the present study, there were more children who were malnourished (WAZ < -1) and had diarrhoea than those who were malnourished without diarrhoea (93/168 vs 41/101, p=Q.019). Additionally, approximately fourteen percent (38/269) o f the children studied were severely malnourished (WAZ < -3), and. out o f this number. 73.7% had diarrhoea. A self-perpetuating vicious cycle in which malnutrition and diarrhoea are synergistic is suggested, and may explain their effect on cognitive development of children, especially their semantic fluency as documented by others (Guerrant el al., 2008). Data in the present study support this link between 123 University of Ghana http://ugspace.ug.edu.gh malnutrition and diarrhoea, placing these groups of children at a higher risk of morbidity over time. Considerable number of children less than two years of age fell below the 5% percentile curve, and nutritional status appeared to be worsened with increasing age within this sub-population. Elsewhere in India, nutritional status decreased with increasing age, and unhygienic weaning practices were assigned to this (Hasan et al., 1991). 124 University of Ghana http://ugspace.ug.edu.gh PCR methodology is more sensitive in screening for pathogens. In sub-Saharan Africa as in many other similar settings however, the cost involved cannot be passed on to patients and therefore this tool is employed mainly in research facilities. In the present study, routine bacterial culture detected only one stool culture positive for S. flexneri and none for Salmonella spp. PCR however detected six ipaH genes in diarrhoeal stool specimens. Although, the ipaH gene is expressed by both Shigella and enteroinvasive (EIEC), E1EC is not often detected in Ghana (Opintan et al., 2010), and it is speculated that the ipaH gene detected most likely reflects the presence of Shigella spp that may have been missed on culture. The sero-group identified by culture and serology is predominant in Ghana (Opintan and Newman, 2007) as it is in many developing countries (Malakooti et a l, 1997; Subekti et al., 1993). Cryptosporidium spp was not only the most prevalent parasite detected, but it is also significantly associated with diarrhoea (8.7% vs 1.0%, p=0.011). Malnourished children with diarrhoea often had cryptosporidiosis. Prevalence rates of Cryptosporidium spp using presumably less sensitive methods like microscopy and ELISA in Ghana (Addy and Aikins-Bekoe, 1986; Adjei et al., 2004), Liberia (Hojlyng et a l, 1986), Mexico (Miller et al., 1994) and Guinea Bissau (Perch et al.. 2001) reported ranges between 7.7 - 29% in symptomatic children. The current study recorded 8.2% in symptomatic and 1.0% in non-symptomatic patients. 52 PCR for Detection of Pathogens 125 University of Ghana http://ugspace.ug.edu.gh Modem diagnostic tools like PCR are able to discriminate E. histolytica from the non-pathogenic E. dispar (Haque el al., 2007). The present study detected E. histolytica in 2.9 % (5/170) of the children who had diarrhoea. No visible blood was observed in any of the stools from these children. All the E. histolytica detected in the current study were from children who had diarrhoea. In Bangladesh and elsewhere, relatively few numbers of people infected with E. histolytica develop symptomatic disease (Ayeh-Kumi et a l, 2001; Haque et al., 2003a; Verweij et a l , 2003). The present study did not detect any Giardia in the stool specimens screened for the p241 gene o f Giardia spp. The p241 gene target used is well validated (Guy et a l, 2003) and a positive control which was included in each PCR run was amplified whilst the negative control was not. Addy et al in 2004, found a 3.7% prevalence rate of Giardia in Kumasi, Ghana (Addy et a l, 2004). Personal communications from senior scientists indicated that Giardia species used to be the most frequently identified intestinal flagellate seen in routine stool examination in Accra, Ghana. Diarrhoea surviellance screening for major known pathogens and Giardia may indicate whether there is a decline in the incidence of giardiasis in Accra or otherwise. In a 7-year study of diarrhoea caused by parasites in Guinea Bissau, the most prevalent parasite was Giardia lamblia (14.8%) followed by Cryptosporidium (7.7%) (Perch et al., 2001). Though seasonality, study duration or geographical location may influence parasite prevalence, the current study cannot pinpoint additional reasons for the zero prevalence of Giardia. 126 University of Ghana http://ugspace.ug.edu.gh Children who had diarrhoea were often co-infected, with two or more pathogens detected in the stool, and EAEC-Cryptosporidium was the most prevalent (13/170) in the current study. Among HIV infected children in South Africa, Samie et al identified children who were co-infected with as many as six different species of pathogens (Samie et al., 2007b). The present study detected a child who had diarrhoea with EAEC-Cryptosporidium-Shigella/E\EC co-infection. The HIV status of this child is however not known. Relatively, fewer children who are older than 2 years of age were sampled in the present study. However, cryptosporidiosis seemed to be more common in children < 2 years of age, and this is in agreement with an earlier study in Ghana (Adjei et al., 2004). Dozens of Cryptosporidium species have been characterized, and primarily, C. parvum and C. hominis have been associated with cattle and human infections, respectively. Because of the strong bond between humans and animals, C. parvum is also known to be associated with human infection (Samie et al., 2006a; Warren, 2008). Earlier studies in Ghana employed microscopy for the diagnosis of cryptosporidiosis, and therefore did not genotype Cryptosporidium species (Addy and Aikins-Bekoe, 1986; Adjei et al., 2004). Preliminary results in the current study showed that C. parvum genotypes accounts for less than 30% of cryptosporidiosis among young children with diarrhoea in Accra. Though the non-C. panntm could be C. hominis, the lack of an additional control (C. hominis) in the HRM analysis failed to reveal further details. There is little or no data on the epidemiology and genotypes 5.3 Co-infection 127 University of Ghana http://ugspace.ug.edu.gh of cryptosporidiosis in sub-Saharan Africa. Samie et al found more C. hominis (82%) compared to C. parvum (18%) among school children and hospital patients in Venda, South Africa (Samie et al., 2006a). 128 University of Ghana http://ugspace.ug.edu.gh Among the EAEC plasmid genes tested (aap, aatA and aggR), only aap was significantly associated with diarrhoea (/?=0.0003), A recent publication, which compared molecular probes to the ‘gold standard’ (brick-like aggregation in cultured epithelial cells), however, suggested that the aap gene is not restricted to EAEC, but is also detected in diffusely adherent E coli (DAEC) as well as in non pathogenic E. coli (Monteiro et a l, 2009). There was no significant statistical association between any individual EAEC gene (whether plasmid or chromosome borne) and malnutrition (WAZ < -1). In the present study, EAEC virulence genes occurred in both sub-populations (diarrhoea/control, respectively): aap 68.2/46.2%, aatA 69.4/63.5%, aggR 46.5/36.5% and aaiC 37.6/26.9%. Information on the presence of EAEC virulence- associated genes in body fluids of persons in Africa is limited, as is the prevalence and distribution of this organism in Ghana. The aggR is known to regulate its own expression as well as that of several plasmid genes and chromosomal genes of EAEC (Dudley el a l, 2006), including the aggregative adherence fimbriae, a dispersin (aap), a dispersin translocator apparatus called aat and several chromosomal loci including the aaiC (Nataro, 2005). In a different population, Huang et a l failed to show any association between four EAEC virulence-associated genes (aggA, aspU, aafA and aggR) and clinical illness in travelers from the United States to Mexico (Huang et a l, 2003). Further, in that study, aspU (now designated aatA) was the least prevalent gene among the four EAEC virulence genes studied (Huang et a l. 2003), whilst this 5.4 Prevalence of EAEC Virulence-associated Genes in Stool DNA 129 University of Ghana http://ugspace.ug.edu.gh same gene (aatA) was the most prevalent (67.2%) in the current study. In South Africa, Samie et al. found the aap gene to be the predominant among others and also associated with diarrhoea (Samie et al., 2007b). These disparate findings on the relative distribution and importance of EAEC genes in diarrhoeal illnesses suggest that geographical location, type of exposure, and/or host factors may dictate the nature of EAEC infection. Although aatA was the most prevalent gene observed in the present study, it was not associated with diarrhoea (p>0.05). Some studies have shown that the novel protein aatA, which is encoded on EAEC virulence plasmid pAA2, localizes to the outer membrane of this bacterium and facilitates export of the dispersin aap across the outer membrane (Imuta et al., 2008; Sheikh et al., 2002). Results from the current study may support this notion since aatA and aap were detected in greater than 68% of patients with diarrhoea. Further study is required for a firm conclusion. The chromosomal gene aaiC did not show any significant differential association between the diarrhoea and the non-diarrhoea group in the current study (p=0.068). About 12% (13/104) of children without diarrhoea in the current study were positive for all four EAEC genes tested. One explanation for the high frequency of EAEC-associated virulence genes in symptomatic as well as asymptomatic patients and the heterogeneity of the different gene assortments found is that EAEC is endemic in the current study population. In support of this notion, almost all prior studies recover EAEC from controls (Albert et 130 University of Ghana http://ugspace.ug.edu.gh al., 1999; Gascon et al., 2000; Valentiner-Branth et al., 2003) as well as from individuals with diarrhoea (Okeke et al., 2003; Rappelli et al., 2005). Pathogenic and host factors may influence the initiation of a symptomatic phase, determine by and include distinct mechanisms as reviewed by Kaper (Kaper el al., 2004). For example, Huang et al. suggested that a first exposure to EAEC infection ‘primes’ the immune system to prevent a second infection (Huang et al., 2003). Further, in their study of travelers diarrhoea, after the initial EAEC infection, only 4 (11%) of these students had a subsequent symptomatic EAEC infection (Huang et al., 2003). 131 University of Ghana http://ugspace.ug.edu.gh 5.5 Lactoferrin levels Lactoferrin is bactericidal to enteric pathogens, modulates the intestinal immune response, and is released by neutrophils into stool in response to infection (Walker et a l, 2007). In the current study, the mean lactoferrin levels in the children without diarrhoea were high compared to studies done in the developed world (Archbald- Pannone et a l, 2010; Kane et a l, 2003). Several of the available data on enteric infection and intestinal inflammation from developing countries used the latex agglutination method, and therefore lactoferrin levels were only semi-quantified (Alcantara et a l, 2003; Ashraf et a l, 2007; Bouckenooghe el a l, 2000; Samie et a l, 2007a; Samie et a l, 2006b). It is therefore difficult to judge whether mean lactoferrin levels in endemic populations like Ghana are generally high. The current study however observed an association between elevated faecal lactoferrin levels and diarrhoea (p<0.05). In Bangladesh, faecal lactoferrin levels was found not to be useful in differentiating between inflammatory and non-inflammatory diarrhoea, among patients enrolled under the International Center for Diarrhoea Disease Research (ICDDR,B) (Ashraf et al., 2007). In the current study, the mean lactoferrin levels were lower in children who were malnourished and had diarrhoea compared to those without malnutrition who had diarrhoea. It is speculated that the lactoferrin assay may be marginally less sensitive in the setting of malnutrition. Perhaps, the enterocytes are less able to synthesize lactoferrin in children who are malnourished and have diarrhoea. This observation needs further investigation for a firm conclusion. In earlier studies, the mean 132 University of Ghana http://ugspace.ug.edu.gh lactoferrin levels for healthy controls from the US were less than 12.8 (ig/ml (Kane et a l, 2003; Walker et a l, 2007) but healthy controls in the current study had 298.8 Hg/ml. Unpublished observations by Lima et a l suggest that breast milk may contribute to moderately (15 to 120 (ig/ml) increased faecal lactoferrin levels. Some studies have demonstrated an association of EAEC infection with inflammatory cytokines (Cennimo et a l, 2009; Greenberg et a l, 2002), and several others have associated EAEC with elevated lactoferrin levels (Bouckenooghe et a l , 2000; Samie et a l, 2007b). In children with diarrhoea, the current study found a significant statistical association of elevated faecal lactoferrin with the aatA gene, and with the detection of any one or two of the EAEC genes tested (p<0.05). Between diarrhoea and non-diarrhoea stool specimens, EAEC’s chromosomal gene aaiC was associated with the highest fold- rise in faecal lactoferrin level (2.7 fold-rise) followed by the aap gene (2.5 fold-rise). Additionally, an increased number of virulence EAEC genes detected corresponded to a rise in faecal lactoferrin level. The protective function of lactoferrin in infections with enteropathogens have been acknowledged, (Shashiraj et a l, 2006) and colonization/infection, particularly by EAEC in the current study probably contributed to the raised lactoferrin levels. 133 University of Ghana http://ugspace.ug.edu.gh Historically, EAEC was diagnosed by the AA phenotype, when cultured together with HEp-2 cells (Nataro and Kaper, 1998). This procedure requires specialized facilities, and high technical competence. Additionally, the adherence assay requires an initial stool culture - an added cost and takes about three days for results to be ready. Molecular methods eliminate the expensive CO2 facility required for the adherence assay, and has given more scientists in both developing and developed countries the opportunity to investigate EAEC (Aranda et a l, 2004; Dedeic-Ljubovic et a l, 2009; Huang et a l, 2003; Opintan et al., 2010). However, as yet, there is no single-all-encompassing molecular probe for the detection of EAEC and the molecular diagnosis of this pathogen is perplexing (Okeke, 2009). A few dozen investigators have used genomic bacterial DNA or supernatants of boiled bacteria cells to template these PCR reactions (Al-Gallas et al., 2007; Antikainen et al., 2009). Relatively fewer investigators have however, used stool DNAs to template these PCR reactions, and where DNA stool was used, the purpose was generally to screen for multiple enteric pathogens (Antikainen et al., 2009; Samie et a l, 2007b). The current study compared the molecular diagnosis of EAEC using stool DNA as opposed to bacterial DNA. Overall, the detection rates of the genes sought (aaiC, aap, aatA and aggR) were found to be comparable, in both the stool and the bacterial DNAs. In both methods, the aatA was the most detected gene, followed by aap, aggR and aaiC in that order. Though the aatA gene was the most frequently detected, it was not associated with diarrhoea (p>0.05). A recent publication suggests that the aatA is 5.6 Molecular Diagnosis of EAEC: Stool DNA versus Bacterial DNA 134 University of Ghana http://ugspace.ug.edu.gh responsible for virulence in pathogenic E. coli from avian sources (Li et al., 2010). Perhaps some avian clones of E. coli can colonize humans, but are unable to initiate virulence. Only the aap gene was significantly associated with diarrhoea in the two screening methods; stool DNA vs bacterial DNA, respectively (p- <0.001 vs 0.018). The aap gene therefore appeared to be a ‘unique’ gene to screen for EAEC. A recent publication however suggests that the aap is not restricted to EAEC, but could be found in some non-EAECs (Monteiro et al., 2009). With the exception of the chromosomal gene (aaiC), Monteiro et al sought for all the three plasmid genes (aggR, aap and aatA ) investigated in the current study from 252 DEC and non-DEC strains. In that study, 52% (26/50) of the non-DEC strains screened positive by PCR for the aap gene (Monteiro et al., 2009). It is unclear why in the diarrhoea sub­ population, EAEC’s chromosomal (aaiC), the master regulator (aggR), and the dispersin (aap) genes were statistically associated with EAEC’s detection in stool DNA in the current study. This observation requires further investigation. In the literature, association of EAEC with symptomatic and asymptomatic infection is not clear. Factors such as age category, heterogeneity of the strain, inoculum size and polymorphism in the IL8 promoter region have been implicated as factors determining disease course (Flores and Okhuysen, 2009; Huang et al., 2006b: Jiang et al., 2003; Kaur et al., 2010; Okeke et al., 2000b; Usein et al., 2009). Four EAEC’s virulence genes were sought in the current study. For the same set of children, EAEC was only associated with diarrhoea in the stool DNA and not in the bacterial DNA. EAEC was defined as positivity for one or more of the four genes screened in both University of Ghana http://ugspace.ug.edu.gh methods. Using bacterial DNA to template PCRs, some authors have found EAEC to be statistically associated with diarrhoea (Ochoa et al., 2009; Okeke et al., 2003; Opintan et al., 2010). In the current study, EAEC’s association with diarrhoea was lost when bacterial DNA was analyzed from the same children {p>0.05). It is impossible to delineate commensals from pathogenic E. coli on agar plates. Therefore, a possible explanation to the disparity in the current finding may be issues to do with the probability of missing out some pathotypes of E. coli, when fewer bacteria colonies are picked for DEC screening. The current study used the recommendations of Nataro, and picked a minimum of 3 colonies (Nataro et al., 1998). To further eliminate issues with subjectivity as to the number and which bacteria colonies to pick, stool DNA may be a better sample, as demonstrated in the current study. However, in situations where susceptibility may be required to guide therapy, the use of stool DNA may not be too helpful. Secondly, one may be dealing with live as well as dead bacteria when stool DNA is used. 136 University of Ghana http://ugspace.ug.edu.gh The current study had some limitations. Accurate height measurements were the most difficult to obtain on an out-patient basis, and this limited two of the three z-scores of malnutrition (WHZ and HAZ). Knowledge of history of antimicrobial exposure is important; however this information was obtainable in only few of the patients. Some mothers/caregivers do not remember the types of drugs given to their children. Additionally, only selected pathogens were assayed using PCR and thus, other less common pathogens may have been missed. Tests for intestinal parasites other than Cryptosporidium, Giardia and E. histolytica were not carried out. No follow-up o f the children with/without diarrhoea was done as part of the current study. Such a follow-up, especially, for the children without diarrhoea who screened positive for all the four EAEC’s virulence genes may reveal whether they later had diarrhoea or otherwise. 5.7 Study Limitations 137 University of Ghana http://ugspace.ug.edu.gh 5.8 Conclusions, Recommendations and Future Research Direction The current study identified high rates of undernutrition, wasting and stunting, among children from southern Ghana, with the worst forms of these growth shortfalls occurring among children > 6 months. Considerable number of infants with diarrhoea fell below the 5% percentile mark (weight-for-age) when compared with international reference curves. Through the use of specific DNA-biomarkers, the study was able to determine that EAEC and Cryptosporidium were common intestinal pathogens associated with diarrhoea in southern Ghana. HRM analysis indicated that less than 30% of the Cryptosporidium associated diarrhoea were C. parvum genotypes, and none of the four EAEC’s virulence genes investigated was associated with malnutrition (p>0.5). Elevated faecal lactoferrin levels were associated with diarrhoea in this group of children from southern Ghana. A relatively high level o f lactoferrin was observed in EAEC colonization/infection among our study population. Of the individual EAEC’s virulence genes screened in the children with/without diarrhoea, the percentage proportion of each gene found in stool DNA were higher compared to bacterial DNA. The dispersin gene (aap) was significantly associated with diarrhoea in both stool and bacterial DNAs (p<0.5). The use of anthropometric measurements for early identification of at risk children attending health care facilities in Ghana is recommended. Mean faecal lactoferrin levels in the children with/without diarrhoea were relatively high compared to those found in the developed world. Further studies to explore or improve existing diagnostic tools in endemic populations are recommended. Majority of the diarrhoea 138 University of Ghana http://ugspace.ug.edu.gh sub-population in the current study were children who had acute diarrhoea. A prospective study enrolling more children with persistent diarrhoea (duration >14 days) is recommended to help understand EAEC’s colonization/infection. Stool DNA may offer the opportunity for the screening of multiple pathogens. Initial stool culture mid identification of E. coli may still be required for the molecular diagnosis of EAEC, and for purposes of antimicrobial susceptibility. In such a scenario a ‘bacteria sweep’ from a culture plate is recommended for the molecular diagnosis of EAEC. In future, it may be necessary to compare EAEC in HEp-2 assays with markers of inflammation such as lactoferrin levels in stool samples. This will further enhance the knowledge and pathophysiology of diarrhoea caused by EAEC. The ability of the enterocytes to secret proinflammatory cytokines and lactoferrin, need further investigation, especially, under the setting of undemutrition and diarrhoea. 139 University of Ghana http://ugspace.ug.edu.gh REFERENCES 1. Abba, K., Sinfield, R., Hart, C.A., and Garner, P. (2009). Pathogens associated with persistent diarrhoea in children in low and middle income countries: systematic review. BMC Infect Dis 9, 88. Available at: doi: 10.1186/1471-2334-1189-1188. 2. Abduch Fabrega, V.L., Piantino Ferreira, A.J., Reis da Silva Patricio, F., Brinkley, C., and Alfonso Scaletsky, I.C. (2002). Cell-detaching Escherichia coli (CDEC) strains from children with diarrhea: Identification o f a protein with toxigenic activity. 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Seven years' experience with Cryptosporidium par\'um in Guinea- Bissau, West Africa. Ann Trop Paediatr 21, 313-318. 219. Petri, W.A., Jr., Miller, M., Binder, H.J., Levine, M.M., Dillingham, R„ and Guerrant, R.L. (2008). Enteric infections, diarrhea, and their impact on function and development. J Clin Invest 118, 1277-1290. 170 University of Ghana http://ugspace.ug.edu.gh 220. Pierce, A., Colavizza, D., Benaissa, M., Macs, P., Tartar, A., Montreuil, J., and Spik, G. (1991). Molecular cloning and sequence analysis of bovine lactotransferrin. Eur J Biochem 196, 177-184. 221. Plutzer, J., Ongerth, J., and Karanis, P. (2010). Giardia taxonomy, phylogeny and epidemiology: Facts and open questions. Int J Hyg Environ Health 213, 321-333. 222. Ramani, S., and Kang, G. (2009). Viruses causing childhood diarrhoea in the developing world. Curr Opin Infect Dis 22 ,477-482. 223. Rappelli, P., Folgosa, E., Solinas, M.L., Dacosta, J.L., Pisanu, C., Sidat, M., Melo, J., Cappuccinelli, P., and Colombo, M.M. (2005). Pathogenic enteric Escherichia coli in children with and without diarrhea in Maputo, Mozambique. FEMS Immunol Med Microbiol 43 ,67-72. 224. Regua-Mangia, A.H., Gomes, T.A., Vieira, M.A., Irino, K., and Teixeira, L.M. (2009). Molecular typing and virulence of enteroaggregative Escherichia coli strains isolated from children with and without diarrhoea in Rio de Janeiro city, Brazil. J Med Microbiol 58, 414-422. 225. Reja, V., Kwok, A., Stone, G., Yang, L., Missel, A., Menzel, C., and Bassam, B. (2010). ScreenClust: Advanced statistical software for supervised and unsupervised high resolution melting (HRM) analysis. Methods 50, S 10- 14. 226. Roche, J.K., Cabel, A., Sevilleja, J., Nataro, J., and Guerrant, R.L. (2010). 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Am J Trop Med Hyg 51, 1-10. 183 University of Ghana http://ugspace.ug.edu.gh APPENDICES APPENDIX I : Questionnaires administered in this study i) Questionnaire for children with (A) and without (B) diarrhoea QUESTIONNAIRE (A) 01 imi/oii.isnin 1.0 Dale: ______________ 1.1 Project Silo: ______________________ 2.0 B ackg ro und o f p a tien t 2.1 Name: 2.2 Age ; 2 4 .Yroa Residence: 2.5 Who takes care of child most of the time especially week days ’ 2.6 Weight of child: 2.7 I leighl of child: 2.S I’exer? Ycs|| No | | 3.0 H is to ry o f d ia r rh o e a 3.1 Frequency in a day.________________ 3.2 J:or how lone?__ 3.2 Did you lake am anlihiolic drug before visiting clinic? Yes | | 3 J Antibiotic drue name _ 4 .0 IIow Ion® d id y ou exclusively b rea s t feed? 4.1. 6 months [| 4 2 6 months | | 4.3 ollicrs? (please stale)_____________ 4.4 In your estimation, what is the likely cause of the diarrhoea? 5.0 ICdurntloual iKirkground of mother/ guardian (Please tick) 5.1 Up to primary school [ ] 5 21 p to JSS || 5 3 Up to SSS See. sch. |] 5.4 I ’p li* I niv Pol\ tech. | | 5.5 ( >lhers (please stale) ! 3 Sex: No || 184 University of Ghana http://ugspace.ug.edu.gh QUESTIONNAIRE (B) co.vntoi. i J c nuK imoi■: i s n nv 1 II Date: I I Project Site 2.0 Background or pnlic-nt 2 ! Name: 2.2 Age: 2 3 Sex: 24 Aral Resilience: 2.5 Wllo lakes care of child most ofllie lime especially «cek dais ’ 2 .f' W eight of child:______________________ 2.7 lleighl of child:_______________________ 3.0 How long did J °u exclusively breast feed? 3.1. 6 months [ | 3.2 6 months | | 3.3 Others (please slale) 5.0 Educational background of mother/ guardian | Please tick) 5.1 l'p to primary scltool | ] 5.21 >p loJSS || 5.3 I p to SSS Sec. sch. | | 5.4 Up to I nil Poly lech. | | 5.5 Others (please slate) 185 University of Ghana http://ugspace.ug.edu.gh ii) A copy of the consent form used in the study CONSENT FORM (A) PmrrhrHui Study Intruiliuiiun Diarrhoeal diseases remain .1 major cause of childhood death ;ind incapacitation in developing countries. One ol'the main difficulties in ihc management of acutc and persistent diarrhoea is I be inability 10 identify live etiological agents, which could be of viral, parasitic or bacterial source lor many of the pathogens, optimal diagnostic testing is unavailable, impractical, or prohibitively expensive for developing countries like Ghana. In this study we seek to culture for diarrhoeal agents of bacterial origin especially those which arc not routinely cultured for in Ohana. W hat is required from you file client You would be assisted to llll a structured questionnaire after which a stool specimen would he taken. You are please assured that any information >011 provide would be handled in confidentiality and ii will only be used for research purposes Kenefit of study to you the client There is no direct monetary benefit. Indirectly, y ou benefit by not nuking any payment for the laboratory investigations. I11 addition, since sensitivity tests would be carried out on am significant isolate this would positively direct treatment. Consent Ilie study ha> been explained adequately to me and I understand that my participation is purely voluntary. I therefore give my consent and understand that I could withdraw my participation al any time. Thumb print or signature Name Contact Information of Investigator If you have any further questions or lor any reason you wish to redraw your consent you should contact me on the following address: JAIMH ITI A. OPINTAN DI-'PT. O f MICROWOMKJY I NIYI RSITYOI ( il l \NA MI-DIC VI SCIIOOI KORI I-111 . ACC’H \ I n u i l ian li o n i i l l .u i in a l i 'H w r n T el « N lk v ) : t>21 -M-.MiM 186 University of Ghana http://ugspace.ug.edu.gh iii) Completed copies of administered questionnaires 187 University of Ghana http://ugspace.ug.edu.gh oooo University of Ghana http://ugspace.ug.edu.gh APPENDIX II: Media and equipment used 1.1) Preparation o f agar media and broths for culture, identification bacteria The following media, broths and standard solutions were prepared using sterile distilled water. Where appropriated the solutions were either autoclaved at 121°C for 15 min at 1.12 kg cm'2 pressure or filter sterilized with 0.22 or 0.45 nm filter units (MILLEX®-HA, Molsheim, France). For agar plates, dehydrated powders were dissolved in the appropriate volumes of distilled water according to manufacturer’s instructions and autoclaved. When cooled to about 55°C, approximately 25 ml volumes were dispensed into 90 cm sterile Petri Dishes and allowed to set. For broths and slopes, dehydrated powders were dissolved in the appropriate volumes of distilled water and warmed to completely dissolve. Appropriate volumes were then dispensed into appropriate tubes before they were autoclaved. Autoclaved tubes were then slanted at appropriate gradients before they set for agar slopes. Sterility and quality of media after each preparation were controlled by respectively leaving a plate on a bench for the next day and also by inoculating randomly selected media with positive and negative controlled organisms. 189 University of Ghana http://ugspace.ug.edu.gh a) Deoxycholate citrate agar (DCA) per 1 litre i) Composition 5.0 g Beef Extract 5.0 g Peptone 5.4 g Sodium thiosulphate 12.0 g Agar agar 0.02 g Neutral red 1.0 g Ferric citrate 5.0 g Sodium desoxycholate 8.5 g Sodium citrate 10.0 g Lactose pH 7.3 ± 0.2 ii) Preparation Prepared according to the manufacturer’s (BIOTEC) instructions. When cooled to about 55°C, approximately 25 ml volumes were dispensed into 90 cm sterile Petri Dishes and allowed to set. b) MacConkey agar (per 1 litre) i) Composition 20.0 g Peptone 10.0g Lactose 5.0 g Bile salts 5.0 g NaCl 0.075 g Neutral red 12.0 g Agar pH 7.4 ± 0.2 ii) Preparation Prepared according to the manufacturer’s (OXOID) instructions. When cooled to about 55°C, approximately 25 ml volumes were dispensed into 90 cm sterile Petri Dishes and allowed to set. 190 University of Ghana http://ugspace.ug.edu.gh i) Composition 2.0 g Beef infusion solids 17.5 g Acid Hydrolysed Casein 1.5 g Starch 17.0 g Agar No. 1 50-100 mg/L Calcium ions 20-35 mg/L Magnesium ion pH 7.3 ±0.1 ii) Preparation Prepared according to the manufacturer’s (BIOTEC) instructions. When cooled to about 55°C, approximately 25 ml volumes were dispensed into 90 cm sterile Petri Dishes and allowed to set. c) Mueller Hinton agar (per 1 litre) d) Triple Sugar Iron (TSI) per 1 litre i) Composition 3.0 g ‘Lab-Lemco’ powder 3.0 g Yeast extract 20.0 g Peptone 5.0 g NaCl 10.0 g Lactose 1.0 g Glucose 0.03 g Ferric citrate 0.3 g Sodium thiosulphate q.s Phenol red 12.0 g Agar pH 7.4 ± 0.2 ii) Preparation Prepared according to the manufacturer’s (OXOID) instructions. Completely dissolved mixtures were dispensed into tubes before they were autoclaved. Tubes were slanted at appropriate gradient before setting. 191 University of Ghana http://ugspace.ug.edu.gh e) Peptone water (per 1 litre) i) Composition 10.0 g Peptone 5 g NaCl pH 7.1 ± 0 .2 ii) Preparation Prepared according to the manufacturer’s (BIOTEC) instructions. Completely dissolved mixtures were dispensed into tubes before they were autoclaved. f) Urea slope (per 100 ml) i) Composition 1.0 g Peptone 1.0 g glucose 5.0 g NaCl2 1.2 g Di-sodium phosphate 0.8 g Potassium dihydrogen phosphate 0.012 g Phenol red 15 g Agar pH 6.8 ± 0.2 ii) Preparation Prepared according to the manufacturer’s (OXIOD) instructions. Completely dissolved mixtures was autoclaved. When cooled, 5 ml of filter sterilized 40 % urea solution (SR 20) was added aseptically to autoclaved mixtures before they were dispensed into sterile bottles and sloped. 192 University of Ghana http://ugspace.ug.edu.gh g) Selenite F broth per Litre i) Composition 5.0 g Peptone 4.0 g lactose 10.0 g Sodium phosphate pH 7.1 ±0.2 ii) Preparation Prepared according to the manufacturer’s (BIOTEC) instructions. Completely 4 g of sodium biselenite (CAT 3/175) was dissolved in 1 L of distilled water and 19 g of selenite broth base added and warmed for 10 min in a boiling water. Appropriate volumes were then dispensed into appropriate tubes. NB: Under no circumstances was this autoclaved h) Kovac’s indole reagent i) Composition 5.0 g /?-dimethylaminobenzaldehyde 75 ml Amyl alcohol 25 ml Cone. HC1 ii) Preparation The aldehyde was dissolved in the alcohol by gentle warming in a water bath (about 50 - 55 °C). When cooled, the acid was added with care and kept in a brown bottle to protect it from sun light. 193 University of Ghana http://ugspace.ug.edu.gh 1.2) Equipment used in this study Equipment Model Manufacturer -20 Freezer MF 304 Unicef, Denmark -80 Ultra flow freezer MDF-392 Sanyo Electric Co., Ltd., Japan Autoclave Omrou Corporation, Japan Centrifuge Mini Spin Eppendorf, Hamburg, Germany Centrifuge 5804R Eppendorf, Hamburg, Germany Commercial ice system Cornelius Electric pipettor Drummond Sci., Co, Broomall, USA ELISA reader MKII Titerkek Multiskan Plus Icycler iQ Multicolor RT-Time BIO-RAD, USA Incubator IC-62 Yamato Scientific Co., Ltd., Japan Mini Beadbeater 607 Biospec Prdt., Bartlesville, USA PCR/UV Work Station CleanSpot COY Lab. Prds., Michigan, USA Rotor-Gene Q-5 Qiagen, Hilden, Germany Spectrophotometer RS 232C Eppendorf., Hamburg, Germany Water bath 1225 PC VWR Scientific Prdts,. Inc., USA Weighing scale X564 Mettler Toledo, Switzerland 194 University of Ghana http://ugspace.ug.edu.gh APPENDIX III: Cycling conditions of target genes screened Gene target Cycling condition/protocol aaiC 95°C (5 min, 1 cycle); 95 °C, 47.5 °C, and72 °C (20 s at each temperature, 40 cycles); and a final extension step (8 min, 72 °C). aatA 50°C (2 min, 1 cycle); 95 °C (5 min, 1 cycle); 95 °C, 55 °C, and 72 °C (20 s at each temperature, 40 cycles); and a final extension step (10 min, 72 °C). aap 50°C (2 min, 1 cycle); 95 °C (5 min, 1 cycle); 95 °C, 45 °C, and 72 °C (45 s at each temperature, 40 cycles); and a final extension step (8 min, 72 °C). aggR 95°C (5 min, 1 cycle); 95 °C, 45 °C, and 72 °C (10 s at each temperature, 39 cycles); 72 °C (1 min, 39 cycles); and a final extension step (6 min, 72 °C). Shigella/ipaH 95°C (5 min, 1 cycle); 95 °C, 60.5 °C, and72 °C (20 s at each temperature, 40 cycles); and a final extension step (10 min, 72 °C). Crypto 95°C (13.3 min, 1 cycle); 95 °C and 60 °C (15 s, 40 cycle); 72 °C 18sRNA (20 s, 40 cycles); and a final extension step (7 min, 72 °C). Eh 95°C (3 min, 1 cycle); 95 °C, 59 °C, and72 °C (1 min at each temperature, 40 cycles); 72°C (10 min, 1 cycle); and a final extension step (8 min, 72 °C). P241 95°C (10 min, 1 cycle); 95 °C and (15 s , 40 cycle); 60 °C (1 min, 40 cycle); and a final extension step (7 min, 72 °C). 195 University of Ghana http://ugspace.ug.edu.gh APPENDIX IV: Sequenced data of Cryptosporidium positive stool DNA i) Sequence report o f the positive Cryptosporidium >09-22101_012 . ab l C C A A T A C G T T A T G A G A A T C A A A G T C T T T G G G T T C T G G G G G G A G T A T G G T C G C A A G G C T G A A A C T T A A A G G A A T T G A C G G A A G G G C A C C A C C A G G A G T G G A G C C T G C G G C T T A A T T T G A C T C A A C A C G G G A A A A C T C A C C A G G T C C A G A C A T A G G A A G G A T T G A C A G A T T G A T A G C T C T T T C T T G A T T C T A T G G G T G G T G G T G C A T G G C C G T T C T T A G T T G G T G G A G A G C A C C C C C A T T A G T A T T C A G A A G A G C T A T C A A T C T G T C A A T C C T T C C T A T G T C T G G A C C T G G T G A G T T T T C C C G T G T T G A G T C A A A T T A A G C C G C A G G C T C C A C T C C T G G T G G T G C C C T T C C G T C A A T T C C T T T A A G T T T C A G C C T T G C G A C C A T A C T C C C C C C A G A A C C C A A A G A C T T T G A T T T C T C A T A A G G T G C T G A A G G A G T A A G G A A C A A C C T C C A A T C T C T A A A >09-22103_010 . ab l T C C A T A A C T T A T G A G A T C A A G T C T T T G G G T T C T G G G G G G A G T A T G G T C G C A A G G C T G A A A C T T A A A G G A A T T G A C G G A A G G G C A C C A C C A G G A G T G G A G C C T G C G G C T T A A T T T G A C T C A A C A C G G G A A A A C T C A C C A G G T C C A G A C A T A G G A A G G A T T G A C A G A T T G A T A G C T C T T T C T T G A T T C T A T G G G T G G T G G T G C A T G G C C G T T C T T A G T T G G T G G A G A G G G G A A C C C C C A T A G C A T C A G A G A G C T A T C A A T C T G T C A A T C C T T C C T A T G T C T G G A C C T G G T G A G T T T T C C C G T G T T G A G T C A A A T T A A G C C G C A G G C T C C A C T C C T G G T G G T G C C C T T C C G T C A A T T C C T T T A A G T T T C A G C C T T G C G A C C A T A C T C C C C C C A G A A C C C A A A G A C T T T G A T T T C T C A T A A G G T G C T G A A G G A G T A A G G A A C A A C C T C C A A T C T C T A A >09 -22105_024 . ab l A C A G A A T T A T G A G A T C A A G T C T T T G G G T T C T G G G G G G A G T A T G G T C G C A A G G C T G A A A C T T A A A G G A A T T G A C G G A A G G G C A C C A C C A G G A G T G G A G C C T G C G G C T T A A T T T G A C T C A A C A C G G G A A A A C T C A C C A G G T C C A G A C A T A G G A A G G A T T G A C A G A T T G A T A G C T C T T T C T T G A T T C T A T G G G T G G T G G T G C A T G G C C G T T C T T A G T T G G T G G A G A G G G G A T C C A T A G A T C A G A A G A G C T A T C A A T C T G T C A A T C C T T C C T A T G T C T G G A C C T G G T G A G T T T T C C C G T G T T G A G T C A A A T T A A G C C G C A G G C T C C A C T C C T G G T G G T G C C C T T C C G T C A A T T C C T T T A A G T T T C A G C C T T G C G A C C A T A C T C C C C C C A G A A C C C A A A G A C T T T G A T T T C T C A T A A G G T G C T G A A G G A G T A A G G A A C A A C C T C C A A T C T C T A A >09 -22107_022 .ab l C G C A A T G A C A A T C A T G T A G T A T C T G A G T C T A T T G G G T T C T G G G G G G A G T A T G G T C G C A A G G C T G A A A C T T A A A G G A A T T G A C G G A A G G G C A C C A C C A G G A G T G G A G C C T G C G G C T T A A T T T G A C T C A A C A C G G G A A A A C T C A C C A G G T C C A G A C A T A G G A A G G A T T G A C A G A T T G A T A G C T C T T T C T T G A T T C T A T G G G T G G T G G T G C A T G G C C G T T C T T A G T T G G T G G A G G G G G A C A T C A A T A G C A T C A A G A T C T A G C T A T C A A T C T G T C A A T C C T T C C T A T G T C T G G A C C T G G T G A G T T T T C C C G T G T T G A G T C A A A T T A A G C C G C A G G C T C C A G T C C T G G T G G T G C C C T T C C G T C A A T T C C T T T A A G T T T C A G C C T T G C G A C C A T A C T C C C C C C A G A A C C C A A A G A C T T T G A T T T C T C A T A A G G T G C T G University of Ghana http://ugspace.ug.edu.gh A A G G A G T A A G G A A C A A C C T C C A A T C T C T A A >09 -22109_020 . ab l A G C A A T C C C A C T A T G A G A A T C A A A G T C T T T G G G T T C T G G G G G G A G T A T G G T C G C A A G G C T G A A A C T T A A A G G A A T T G A C G G A A G G G C A C C A C C A G G A G T G G A G C C T G C G G C T T A A T T T G A C T C A A C A C G G G A A A A C T C A C C A G G T C C A G A C A T A G G A A G G A T T G A C A G A T T G A T A G C T C T T T C T T G A T T C T A T G G G T G G T G G T G C A T G G C C G T T C T T A A T T G G T G G A G A G G A C T T C C T T A C A T C A G A C G A G C T A T C A A T C T G T C A T C C T T C C T A T G T C T G G A C C T G G T G A G T T T T C C C G T G T T G A G T C A A A T T A A G C C G C A G G C T C C A C T C C T G G T G G T G C C C T T C C G T C A A T T C C T T T A A G T T T C A G C C T T G C G A C C A T A C T C C C C C C A C A A C C C A A A G A C T T T G A T T T C T C A T A A G G T G C T G A A G G A G T A A G G A A C A A C C T C C A A T C T C T A >09 -22111_018 . ab l C C A A T A C G T T A T G A G A A T C A A G T C T T T G G G T T C T G G G G G G A G T A T G G T C G C A A G G C T G A A A C T T A A A G G A A T T G A C G G A A G G G C A C C A C C A G G A G T G G A G C C T G C G G C T T A A T T T G A C T C A A C A C G G G A A A A C T C A C C A G G T C C A G A C A T A G G A A G G A T T G A C A G A T T G A T A G C T C T T T C T T G A T T C T A T G G G T G G T G G T G C A T G G C C G T T C T T A G T T G G T G G A G A G G G A A C C A T A G T A T C A A G A T G A G C T A T C A A T C T G T C A A T C C T T C C T A T G T C T G G A C C T G G T G A G T T T T C C C G T G T T G A G T C A A A T T A A G C C G C A G G C T C C A C T C C T G G T G G T G C C C T T C C G T C A A T T C C T T T A A G T T T C A G C C T T G C G A C C A T A C T C C C C C C A G A A C C C A A A G A C T T T G A T T T C T C A T A A G G T G C T G A A G G A G T A A G G A A C A A C C T C C A A T C T C T A A >09 -22113_024 . ab l A A C A A T A A C G T T A T G A G A A T C A A G T C T T T G G G T T C T G G G G G G A G T A T G G T C G C A A G G C T G A A A C T T A A A G G A A T T G A C G G A A G G G C A C C A C C A G G A G T G G A G C C T G C G G C T T A A T T T G A C T C A A C A C G G G A A A A C T C A C C A G G T C C A G A C A T A G G A A G G A T T G A C A G A T T G A T A G C T C T T T C T T G A T T C T A T G G G T G G T G G T G C A T G G C C G T T C T T A G T T G G T G G A G A G G A T T A C A A A T A C A T C A G A A G A G C T A T C A A T C T G T C A A T C C T T C C T A T G T C T G G A C C T G G T G A G T T T T C C C G T G T T G A G T C A A A T T A A G C C G C A G G C T C C A C T C C T G G T G G T G C C C T T C C G T C A A T T C C T T T A A G T T T C A G C C T T G C G A C C A T A C T C C C C C C A G A A C C C A A A G A C T T T G A T T T C T C A T A A G G T G C T G A A G G A G T A A G G A A C A A C C T C C A A T C T C T A A >09 - 22115_022 . ab l G C A A T T T C G A T T A T C A G A T A G A G G G T C T G T T A C G T T C G T G G G G C T C T A G A G C A T T C T A G G T C C T A T T C T T C C C T T C G A C T T A G T G A A T T G C C G A T G A C G G G G T G G A A C A A A T C A T T T G C C A C C A C T T G T A G T T A T T A T G A C T T C T C A T G G T T T T G G G G G C T G A C T G A A C T C A A C C A G G C C G T T C T T C G A G G A G A G A G T G G T G C A T G G C C G T T C T T A G T T G G T G G A G A A C A T T A C T T T C T C T C T T T A T C T T C G T T G T C A T C G T C A C T C C T T C T G A T T G C T G T T G A T T T G T T C T T T A A G T C A T G T A A C G T G G A C G G C C C C A C A C T C C A A G A A T G G G C C C T G C C C T T A C C T A A A T C C C C A C C T A T T A T C A C A T T T T G T G A C T T G T A A G T T T C C C T T C C C A T C G A G G A A C A A C C T C C A A T C T A T A A G A A G G A G T A A G G A A C A A C C T C C A A T C T C T A A 197 University of Ghana http://ugspace.ug.edu.gh >09 -22117_020 . ab l C C C C A T A C A T T A T G A G A A T C A A G T C T T T G G G T T C T G G G G G G A G T A T G G T C G C A A G G C T G A A A C T T A A A G G A A T T G A C G G A A G G G C A C C A C C A G G A G T G G A G C C T G C G G C T T A A T T T G A C T C A A C A C G G G A A A A C T C A C C A G G T C C A G A C A T A G G A A G G A T T G A C A G A T T G A T A G C T C T T T C T T G A T T C T A T G G G T G G T G G T G C A T G G C C G T T C T T A G T T G G T G G A G G G A A A C C C A T A C A T C A G A A A G A G C T A T C G A T C T G T C A A T C C T T C C T A T G T C T G G A C C T G G T G A G T T T T C C C G T G T T G A G T C A A A T T A A G C C G C A G G C T C C A C T C C T G G T G G T G C C C T T C C G T C A A T T C C T T T A A G T T T C A G C C T T G C G A C C A T A C T C C C C C C A G A A C C C A A A G A C T T T G A T T T C T C A T A A G G T G C T G A A G G A G T A A G G A A C A A C C T C C A A T C T C T A A >09 - 2 2 119_018 . ab l A G G G T C G G T C A T T C T G A C A C G G C T G A A C G G G T C G C T T T A G C C G C T G G C T T A A C T C G G G A G A G C G G T T C A T C C T T G A A C A T G G G C T C G A T G C G G T T G T G T C T C A G A T G G A G A C T G T G A T A G A C C G C T G G T C A T T G A T G A T C A G G G A A G A C A G A T A A A T G G C T A T A T A T T G G G T A T C C T A A C G G C T C G T T A T C G C G G G G C G A A G A C A G T C G T G A C G C C G C T C A G C A C G A C G A G C G C T C T G G A G G A A A G C G G T T G G T T C G A G A A C A T T C A C A G A A C A C G G A T T G G A T C G C C G T A T G T C G T A G C C G A A A T G G C G C G A G C G G T G G C G C A C C C C G T T G T T G G T T T C G A G G C G A A T G G T G G C T T T C T G C T C G G C G A T G A T G T G A C G T T G A A G T C C G G T T T A T T G C G C C G C C T C C C A A C G C G T G A T T C A G T T T T G C C A G C C G T G G C C G T T C T T A G T T G G T G G A G A G C A G G C A A T C A A T C A C C G A T G A C T A G G C G G C G C G G A T G C C G G A A T T G G C C T C G A T C A T C G C C A A G C A A A A A G C C A C C A T T C G A C T C G A A A C C A A G G G G G G G T G C G C C A C C G C T C G C G C G A T T T C G G C T A C G A C A T A C G G C C A T C C A A T C C G T G T T C T G T G A A T G T T C T G C A A C C A A C C G C T T T C C T C A A G A G C G C T C G T C A A G C T G A G C G G C G T C A C G A C T G T T T T C G C A C C G A G A T A T C G A G C C G T T A G G A T A C C C A A T A T A T C G C C A T T T A T C T G T C T T C C C T G A T C A T C A A T G A C C A G A G G T C T A T C A C C G T C T C C A T C T G T C G A C A C A A C T G C A T C G A G C C C A T G T T C T G C G A T A A A T G C G C T C G C C C G A G T T A A G T C A G C G G G G T C A A G C G C T T C T G T A T C A A C C G C G A C G A A C T G T T C C A T C C T G C G A A A A G G A A C A A C C T C C A A T C T C T A A A >09 -22121_032 . ab l A C A G G A C C T T A T G A G A T C A A G T C T T T G G G T T C T G G G G G G A G T A T G G T C G C A A G G C T G A A A C T T A A A G G A A T T G A C G G A A G G G C A C C A C C A G G A G T G G A G C C T G C G G C T T A A T T T G A C T C A A C A C G G G A A A A C T C A C C A G G T C C A G A C A T A G G A A G G A T T G A C A G A T T G A T A G C T C T T T C T T G A T T C T A T G G G T G G T G G T G C A T G G C C G T T C T T A G T T G G T G G A G A G G G A C C C C A A T A G A T C A G A A G A G C T A T C A A T C T G T C A A T C C T T C C T A T G T C T G G A C C T G G T G A G T T T T C C C G T G T T G A G T C A A A T T A A G C C G C A G G C T C C A C T C C T G G T G G T G C C C T T C C G T C A A T T C C T T T A A G T T T C A G C C T T G C G A C C A T A C T C C C C C C A G A A C C C A A A G A C T T T G A T T T C T C A T A A G G T G C T G A A G G A G T A A G G A A C A A C C T C C A A T C T C T A A >09 -22123_030 . ab l A G C G G A G C A G T A T T G A G C A G T C T C A T G A C T T T G T G G T A G C T G G G G G G A G T A T G G T C G C A A G G C T G A A A C T T A A A G G A A T T G A C G G A A G G G C A C C A C C A G G A G T G G A G C C T G C G G C T T A A T T T G A C T C A A C A C G G G A A A A C T C A C C A G G T C C A G A C A T A G G A A G G A T T G A C A G A T T G A T A G C T C T T T C T T G A T T C T A T G G G T G G T G G T G C A T G G C C G T T C T T A G T T G G T G G A G G G G C T A A A C T T A G G A T C G A C A A C G A G C T A T C A T C T A A G 198 University of Ghana http://ugspace.ug.edu.gh >09 - 2 2 117_020 . ab l C C C C A T A C A T T A T G A G A A T C A A G T C T T T G G G T T C T G G G G G G A G T A T G G T C G C A A G G C T G A A A C T T A A A G G A A T T G A C G G A A G G G C A C C A C C A G G A G T G G A G C C T G C G G C T T A A T T T G A C T C A A C A C G G G A A A A C T C A C C A G G T C C A G A C A T A G G A A G G A T T G A C A G A T T G A T A G C T C T T T C T T G A T T C T A T G G G T G G T G G T G C A T G G C C G T T C T T A G T T G G T G G A G G G A A A C C C A T A C A T C A G A A A G A G C T A T C G A T C T G T C A A T C C T T C C T A T G T C T G G A C C T G G T G A G T T T T C C C G T G T T G A G T C A A A T T A A G C C G C A G G C T C C A C T C C T G G T G G T G C C C T T C C G T C A A T T C C T T T A A G T T T C A G C C T T G C G A C C A T A C T C C C C C C A G A A C C C A A A G A C T T T G A T T T C T C A T A A G G T G C T G A A G G A G T A A G G A A C A A C C T C C A A T C T C T A A >09 -22119_01 8 . ab l A G G G T C G G T C A T T C T G A C A C G G C T G A A C G G G T C G C T T T A G C C G C T G G C T T A A C T C G G G A G A G C G G T T C A T C C T T G A A C A T G G G C T C G A T G C G G T T G T G T C T C A G A T G G A G A C T G T G A T A G A C C G C T G G T C A T T G A T G A T C A G G G A A G A C A G A T A A A T G G C T A T A T A T T G G G T A T C C T A A C G G C T C G T T A T C G C G G G G C G A A G A C A G T C G T G A C G C C G C T C A G C A C G A C G A G C G C T C T G G A G G A A A G C G G T T G G T T C G A G A A C A T T C A C A G A A C A C G G A T T G G A T C G C C G T A T G T C G T A G C C G A A A T G G C G C G A G C G G T G G C G C A C C C C G T T G T T G G T T T C G A G G C G A A T G G T G G C T T T C T G C T C G G C G A T G A T G T G A C G T T G A A G T C C G G T T T A T T G C G C C G C C T C C C A A C G C G T G A T T C A G T T T T G C C A G C C G T G G C C G T T C T T A G T T G G T G G A G A G C A G G C A A T C A A T C A C C G A T G A C T A G G C G G C G C G G A T G C C G G A A T T G G C C T C G A T C A T C G C C A A G C A A A A A G C C A C C A T T C G A C T C G A A A C C A A G G G G G G G T G C G C C A C C G C T C G C G C G A T T T C G G C T A C G A C A T A C G G C C A T C C A A T C C G T G T T C T G T G A A T G T T C T G C A A C C A A C C G C T T T C C T C A A G A G C G C T C G T C A A G C T G A G C G G C G T C A C G A C T G T T T T C G C A C C G A G A T A T C G A G C C G T T A G G A T A C C C A A T A T A T C G C C A T T T A T C T G T C T T C C C T G A T C A T C A A T G A C C A G A G G T C T A T C A C C G T C T C C A T C T G T C G A C A C A A C T G C A T C G A G C C C A T G T T C T G C G A T A A A T G C G C T C G C C C G A G T T A A G T C A G C G G G G T C A A G C G C T T C T G T A T C A A C C G C G A C G A A C T G T T C C A T C C T G C G A A A A G G A A C A A C C T C C A A T C T C T A A A >09 -22121_032 . ab l A C A G G A C C T T A T G A G A T C A A G T C T T T G G G T T C T G G G G G G A G T A T G G T C G C A A G G C T G A A A C T T A A A G G A A T T G A C G G A A G G G C A C C A C C A G G A G T G G A G C C T G C G G C T T A A T T T G A C T C A A C A C G G G A A A A C T C A C C A G G T C C A G A C A T A G G A A G G A T T G A C A G A T T G A T A G C T C T T T C T T G A T T C T A T G G G T G G T G G T G C A T G G C C G T T C T T A G T T G G T G G A G A G G G A C C C C A A T A G A T C A G A A G A G C T A T C A A T C T G T C A A T C C T T C C T A T G T C T G G A C C T G G T G A G T T T T C C C G T G T T G A G T C A A A T T A A G C C G C A G G C T C C A C T C C T G G T G G T G C C C T T C C G T C A A T T C C T T T A A G T T T C A G C C T T G C G A C C A T A C T C C C C C C A G A A C C C A A A G A C T T T G A T T T C T C A T A A G G T G C T G A A G G A G T A A G G A A C A A C C T C C A A T C T C T A A >09 -22123_030 . ab l A G C G G A G C A G T A T T G A G C A G T C T C A T G A C T T T G T G G T A G C T G G G G G G A G T A T G G T C G C A A G G C T G A A A C T T A A A G G A A T T G A C G G A A G G G C A C C A C C A G G A G T G G A G C C T G C G G C T T A A T T T G A C T C A A C A C G G G A A A A C T C A C C A G G T C C A G A C A T A G G A A G G A T T G A C A G A T T G A T A G C T C T T T C T T G A T T C T A T G G G T G G T G G T G C A T G G C C G T T C T T A G T T G G T G G A G G G G C T A A A C T T A G G A T C G A C A A C G A G C T A T C A T C T A A G 198 University of Ghana http://ugspace.ug.edu.gh T C A A T C C T T C C T A T G T C T G G A C C T G G T G A G T T T T C C C G T G T T G A G T C A A A T T A A G C C G C A G G C T C C A C T C C T G G T G G T G C C C T T C C G T C A A T T C C T T T A A G T T T C A G C C T T G C G A C C A T A C T C C C C C C A G A A C C C A A A G A C T T T G A T T T C T C A T A A G G T G C T G A A G G A G T A A G G A A C A A C C T C C A A T C T C T A A >09 -22125_02 8 . ab l C A T C A G C G T C A T T T C G T T G C G G C A C C G T G A C G A A G G A T T A A A T C T G G C A G C A T C G G A C G T G C A C T A T C T G T G G C G G A T T C T G C G C G A A T A C T A C C A T C G T G G C C T C T G C G A T C T G C T G G T G G T C A A T T C G C G A C T G G C G C T G G A T C G G G T C T G G A A A G G G G A A C G C T T T A G C T G G T T T A T G A C C C G C C T G C T G C A T G A T T T T C C G G A T C A G A A C G C G T T C G A C G C T A A A A T G C A G G C C G C C G A C C G C C G C T A C T A T C T G G G C T C G C G C G C C G G G C T G A C C A C C A T C G C G G A A A A C T A C G T G G G C T T G C C C A T G G A G C G T G T C G C C T G A A T T A A G A G G A G T C A T C A T G C C G T T C G T T C A A A C C G T G C C C G T A T T T A A G C T G T A T G G C G A A G A G T G C G G C T G G C C G A C G C C G G A C C T G C T G C A C T G T G A G T C G A T T T T A C A G C G C A G C A G C C T G T A T C A G T G G C A T A T T C G C G T C C A T C A G C A T G C G G A A A T G G T C C A G C T T C T C T A T C T G C A T C A G G G G C G G G C G G A G A T C G A A A T T G A A G G C G C C A C C G C G G T G A T G A C G G A G T C C T G C A T C C A G G T G G T T C C C G C C C T C T G T A T T C A C G G G T T T A A T T T T T C A C C C G G G A C G C A A G G G T T T G T G C T C T C T C T G G C G C T G C C G C T G C T G A G C C G G T T T G A A A A A T C A G T T T C G G T C G C C C T C T G G A T G T G C T T T A C G T G C C G C A T G T G T G C C G G T G G G C C C T T T C T T A T T T T G G T A G A G G T G A G G T G T G G T A G G G G A C C A A A G C A A T C G A G A C G G C G A C C G A A C T G A T T T T C A T A C C G G C T C A G C A G C G G C A G C G C C A G A G A G A G C A C A A A C C C T T G C G T C C C G G G T G A A A A A T T A A A C C C G T G A A T A C A G A G G G C G G G A A C C A C C T G G A T G C A G G A C T C C G T C A T C A C C G C G G T G G C G C C T T C A A T T T C G A T C T C C G C C C G C C C C T G A T G C A G A T A G A G A A G C T G G A C C A T T T C C G C A T G C T G A T G G A C G C G A A T A T G C C A C T G A T A C A G G C T G C T G C G C T G T A A A A T C G A C T C A C A G T G C A G C A G G T C C G G C G T C G G C C A G C C G C A C T C T T C G C C A T A C A G C T T A A A T A C G G G C A C G G T T T G A A C G A A C G G C A T G A T G A C T C C T C T T A A T T C A G G C G A C A C G C T C C A T G G G C A A G C C C A C G T A G T T T T C C G C G A T G G T G G T C A G C C C G G C G C G C G A G C C C A G A T A G T A G C G G C G G T C G G C G G C C T G C A T T T T A G C G T C G A A C G C G T T C T G A T C C G G A A A A T C A T G C A G C A G G C G G G T C A T A A A C C A G C T A A A G C G T T C C C C T T T C C A G A C C C G A T C C A G C G C C A G T T G C G A G T A G G C C G C C A G C A G A T C G C T G C G G C C A C G A T G G T A G T A T T C G C G C A G A A T C C G C C A C A G A T A G T T C A C G T C C G A T G C C G C C A G A T T T T A A T C C T T T C G C C C C C G G T T G G C G G A A C G A T A T G C C G C C G C C A T C C C C C C A C C G G G A A A C A A C C T C C A A T C T C T A A A C C C >09 -2 2 127_026 .ab l C C C A A T G A A G T T A T G A G G A A T C A A G T C T T T G G G T T C T G G G G G G A G T A T G G T C G C A A G G C T G A A A C T T A A A G G A A T T G A C G G A A G G G C A C C A C C A G G A G T G G A G C C T G C G G C T T A A T T T G A C T C A A C A C G G G A A A A C T C A C C A G G T C C A G A C A T A G G A A G G A T T G A C A G A T T G A T A G C T C T T T C T T G A T T C T A T G G G T G G T G G T G C A T G G C C G T T C T T A G T T G G T G G A G A G G G G A A T C C C A T A G C A T C A G A A G A G C T A T C A A T C T G T C A A T C C T T C C T A T G T C T G G A C C T G G T G A G T T T T C C C G T G T T G A G T C A A A T T A A G C C G C A G G C T C C A C T C C T G G T G G T G C C C T T C C G T C A A T T C C T T T A A G T T T C A G C C T T G C G A C C A T A C T C C C C C C A G A A C C C A A A G A C T T T G A T T T C T C A T A A G G T G C T G A A G G A G T A A G G A A C A A C C T C C A A T C T C T A A A 199 University of Ghana http://ugspace.ug.edu.gh >09 -22121_032 . ab l A C A G G A C C T T A T G A G A T C A A G T C T T T G G G T T C T G G G G G G A G T A T G G T C G C A A G G C T G A A A C T T A A A G G A A T T G A C G G A A G G G C A C C A C C A G G A G T G G A G C C T G C G G C T T A A T T T G A C T C A A C A C G G G A A A A C T C A C C A G G T C C A G A C A T A G G A A G G A T T G A C A G A T T G A T A G C T C T T T C T T G A T T C T A T G G G T G G T G G T G C A T G G C C G T T C T T A G T T G G T G G A G A G G G A C C C C A A T A G A T C A G A A G A G C T A T C A A T C T G T C A A T C C T T C C T A T G T C T G G A C C T G G T G A G T T T T C C C G T G T T G A G T C A A A T T A A G C C G C A G G C T C C A C T C C T G G T G G T G C C C T T C C G T C A A T T C C T T T A A G T T T C A G C C T T G C G A C C A T A C T C C C C C C A G A A C C C A A A G A C T T T G A T T T C T C A T A A G G T G C T G A A G G A G T A A G G A A C A A C C T C C A A T C T C T A A >09 -22123_030 . ab l A G C G G A G C A G T A T T G A G C A G T C T C A T G A C T T T G T G G T A G C T G G G G G G A G T A T G G T C G C A A G G C T G A A A C T T A A A G G A A T T G A C G G A A G G G C A C C A C C A G G A G T G G A G C C T G C G G C T T A A T T T G A C T C A A C A C G G G A A A A C T C A C C A G G T C C A G A C A T A G G A A G G A T T G A C A G A T T G A T A G C T C T T T C T T G A T T C T A T G G G T G G T G G T G C A T G G C C G T T C T T A G T T G G T G G A G G G G C T A A A C T T A G G A T C G A C A A C G A G C T A T C A T C T A A G T C A A T C C T T C C T A T G T C T G G A C C T G G T G A G T T T T C C C G T G T T G A G T C A A A T T A A G C C G C A G G C T C C A C T C C T G G T G G T G C C C T T C C G T C A A T T C C T T T A A G T T T C A G C C T T G C G A C C A T A C T C C C C C C A G A A C C C A A A G A C T T T G A T T T C T C A T A A G G T G C T G A A G G A G T A A G G A A C A A C C T C C A A T C T C T A A >09 -22 125_028 . ab l C A T C A G C G T C A T T T C G T T G C G G C A C C G T G A C G A A G G A T T A A A T C T G G C A G C A T C G G A C G T G C A C T A T C T G T G G C G G A T T C T G C G C G A A T A C T A C C A T C G T G G C C T C T G C G A T C T G C T G G T G G T C A A T T C G C G A C T G G C G C T G G A T C G G G T C T G G A A A G G G G A A C G C T T T A G C T G G T T T A T G A C C C G C C T G C T G C A T G A T T T T C C G G A T C A G A A C G C G T T C G A C G C T A A A A T G C A G G C C G C C G A C C G C C G C T A C T A T C T G G G C T C G C G C G C C G G G C T G A C C A C C A T C G C G G A A A A C T A C G T G G G C T T G C C C A T G G A G C G T G T C G C C T G A A T T A A G A G G A G T C A T C A T G C C G T T C G T T C A A A C C G T G C C C G T A T T T A A G C T G T A T G G C G A A G A G T G C G G C T G G C C G A C G C C G G A C C T G C T G C A C T G T G A G T C G A T T T T A C A G C G C A G C A G C C T G T A T C A G T G G C A T A T T C G C G T C C A T C A G C A T G C G G A A A T G G T C C A G C T T C T C T A T C T G C A T C A G G G G C G G G C G G A G A T C G A A A T T G A A G G C G C C A C C G C G G T G A T G A C G G A G T C C T G C A T C C A G G T G G T T C C C G C C C T C T G T A T T C A C G G G T T T A A T T T T T C A C C C G G G A C G C A A G G G T T T G T G C T C T C T C T G G C G C T G C C G C T G C T G A G C C G G T T T G A A A A A T C A G T T T C G G T C G C C C T C T G G A T G T G C T T T A C G T G C C G C A T G T G T G C C G G T G G G C C C T T T C T T A T T T T G G T A G A G G T G A G G T G T G G T A G G G G A C C A A A G C A A T C G A G A C G G C G A C C G A A C T G A T T T T C A T A C C G G C T C A G C A G C G G C A G C G C C A G A G A G A G C A C A A A C C C T T G C G T C C C G G G T G A A A A A T T A A A C C C G T G A A T A C A G A G G G C G G G A A C C A C C T G G A T G C A G G A C T C C G T C A T C A C C G C G G T G G C G C C T T C A A T T T C G A T C T C C G C C C G C C C C T G A T G C A G A T A G A G A A G C T G G A C C A T T T C C G C A T G C T G A T G G A C G C G A A T A T G C C A C T G A T A C A G G C T G C T G C G C T G T A A A A T C G A C T C A C A G T G C A G C A G G T C C G G C G T C G G C C A G C C G C A C T C T T C G C C A T A C A G C T T A A A T A C G G G C A C G G T T T G A A C G A A C G G C A T G A T G A C T C C T C T T A A T T C A G G C G A C A C G C T C C A T G G G C A A G C C C A C G T A G T T T T C C G C G A T G G T G G T C A G C C C G G C G C G C G A G C C C A G A T A G T A G C G G C G G T C G G C G G C C T G C A T T T T A G C G T C G A A C G C G T T C T G A T C C G G A A A A T C A T G C A G C A G G 200 University of Ghana http://ugspace.ug.edu.gh C G G G T C A T A A A C C A G C T A A A G C G T T C C C C T T T C C A G A C C C G A T C C A G C G C C A G T T G C G A G T A G G C C G C C A G C A G A T C G C T G C G G C C A C G A T G G T A G T A T T C G C G C A G A A T C C G C C A C A G A T A G T T C A C G T C C G A T G C C G C C A G A T T T T A A T C C T T T C G C C C C C G G T T G G C G G A A C G A T A T G C C G C C G C C A T C C C C C C A C C G G G A A A C A A C C T C C A A T C T C T A A A C C C >09 -22127_026 .ab l C C C A A T G A A G T T A T G A G G A A T C A A G T C T T T G G G T T C T G G G G G G A G T A T G G T C G C A A G G C T G A A A C T T A A A G G A A T T G A C G G A A G G G C A C C A C C A G G A G T G G A G C C T G C G G C T T A A T T T G A C T C A A C A C G G G A A A A C T C A C C A G G T C C A G A C A T A G G A A G G A T T G A C A G A T T G A T A G C T C T T T C T T G A T T C T A T G G G T G G T G G T G C A T G G C C G T T C T T A G T T G G T G G A G A G G G G A A T C C C A T A G C A T C A G A A G A G C T A T C A A T C T G T C A A T C C T T C C T A T G T C T G G A C C T G G T G A G T T T T C C C G T G T T G A G T C A A A T T A A G C C G C A G G C T C C A C T C C T G G T G G T G C C C T T C C G T C A A T T C C T T T A A G T T T C A G C C T T G C G A C C A T A C T C C C C C C A G A A C C C A A A G A C T T T G A T T T C T C A T A A G G T G C T G A A G G A G T A A G G A A C A A C C T C C A A T C T C T A A A 201 University of Ghana http://ugspace.ug.edu.gh 2) Electrogram report o f the positive Cryptosporidium 0* 331 ta_0> I *>t K0 14,0 wcx»m Oil-22107 RB_I730.PO«>7.80t.Jn»to o « o * w r « u c » i f c ^ i m t i n A E n i M N m ^ m u i i i i TI OL GY ,MONMM jCMOCCC OTWQ1 mc*0 A A C W O C T«C «TC T QTCMTCCI T CTCCTOQIOCTtXCCrX *0 50 SO 40 10 to 70 flO K> tOO HO 139 two-j1000-^ A f c S S S X , *0 MI TilTC TMAxyS^i I cnoac antral o * 1 30 jCCTCMTTCCT TTArfCT TTCAOCC" ?GC0*CCATACTCGCCCCAQV0CCAAMACT r7OCTCCTOA*OC*CTA*OI>i*CAACCT OAATC’ CTMA • SO 140 ISO »00 170 100 IK 300 310 220 230 3000— I *100-1 / ( { C l A p p l ie d an2i»_oio«bi a m ( A 4 B io s y s tem s 5 0/ 6 a.ji8.iw.»cT4-i» T 15M C WtT ^ gStf I3Q C»t© W04 P«»K«rr»S»cc*cc*^ACACccAOcr cew&n »acwotyn ocaoi io^ a^qctctttcttq^ ttctatc | *30 I W 140 * » ttO i re I K t*0 SCO 2t0 230 -,f v- «ccroajOCTT*t'r*Ta»cr 10 x » M so *> » « » t « m t » |i*iMUa*TT««r^ TctrrCTT^ TrcT*TORraOTacre»Tmcmr^ t*STTZ3GTOB*t i t io t4o 150 f® iro n o t a » _______________ ts»t c « m W 20*m   C*»T*0 CATOAQ*7 c CC^OOC.TCOC T CC^OOTQO'CKXCT Ai KCCOTCMT TCCt I TMOTT'C*CCCT-aCtW>CCAT«Cl CXCaX^OAMEXAMOACT 1 TOM 1 TC?C*rJUaJTOCJOM OQW M O»Mt*«Xt 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R T 4 M rs45 c*hm ow».ar or TA iO M tw tcA A ora r ra a c r? a oooooanpfl it ± a uattar ±a pt TwoGw«7TQWia>vooam.*i;uigB»w3niBuoa.iiwMTyQw:m I to » » *0 » *0 70 » *> we «r« | « cacocoaaa4ct c * cc ac c t q m o k m AQOAM3CAT n v o o T tg a t a c c t c t TTcrRSirrCtAfQaSTOBracracMOOoCca T c rw c r r e c io o * * . 110 M0 * » 1*0 I TO 1(0 100 WD 219 230 205 University of Ghana http://ugspace.ug.edu.gh o v ? :m .031*1 « « * 0 « f * » a l n ± E l F a ± yf r C f dR h j It t*t OTTO* 3 «* o««*ww iwcnn (pdMf. Kh. o o o o is t a i trrrtflVtftc stwoMwwwiiwiw ■' * Applied Biosystpms a a « * 4 M T «M c « i* ««s « * KB » | !/ W T » C A e* Q . . . . . . , . , keataw i ? c c T T T i^ T iO k < ix u a e « i^ T ^ c 0 x c x M M C C iC M «C M C (T r«* t r tgtcat amxitoc'cuwtn^iMasMCMCK'TceMiC'rcTM 1JC , w |«o 110 1*0 iro WO l » JOB Jl© m J ! A & tiS S U ■ A 3M*.tf? T » * C 20* * * 3« 237 OC**Tr- 0O*TTAX.«C*r *CK«r«T0' W o*»m C * « V* C* A a »* m £ «q i& 'C * s cr (r(ar( v±gTt*AOcrcc?*n crrccef rtcK»-- CMA-’cr^aa>o» ( I M I d o t# a . i 3 » . w j c r « j « » T3«3 CJt^Ji OVS&^S » <90 v» ; c ? fai*cr um» c»cn citAtoo? n x amscttvcr a,Me a uw w o e r t m C*SJ» aw** re ]*»! I ACT ncr Cl cr IITCT t C.0T T'OVkKSTCaOCCT TCTOAI TOCTor tOAtTTOTTCT TT -MO TCAIQIMCOWOACOOCCCC* C^ ra^ CA-irsOTXIXTanX^ (VCCTMH CCOCAOCT>TT*~CACAT’TT m'WCTTOTA*CTT tCCCTTCCCA’ CWOOAAOACCHXMTCT AI»AOb«OWaMOBiMOM » n o « * t » twj m> < » n o 300 s w k b 206 University of Ghana http://ugspace.ug.edu.gh i) Abstracts: Opintan JA, Ayeh-Kumi PF, Assrim R, Gepi-Attee R, Sevilleja JEAD, Roche JK, Nataro JP, Warren CA, Guerrant RL (2009) Cryptosporidium spp and Enteroaggregative E. coli infection and lactoferrin levels in childhood diarrhea in Accra, Ghana. 58th American Society for Tropical Medicine and Hygiene Annual Genera] Meeting Nov 18 - 22, 2009, Washington, DC. Abstract number 84. Opintan JA, Newman MJ, Assrim R, Gepi-Attee R, Sevilleja JEAD, Warren CA, Geurrant RL (2009) Enteroaggregative Escherichia coli virulence genes in association with diarrhoea duration, nutritional status and age in children with and without diarrhoea in Accra, Ghana. 4th Annual Carey, Marshall, Thomer Scholar’s Day April 13 2009, University of Virginia, Charlottesville. ii) Paper. Opintan JA et al (2010). Pediatric Diarrhea in Southern Ghana: Etiology and Association with Intestinal Inflammation and Malnutrition. Am J. Trop Med Hyg. 83; 936-943. APPENDIX V: Abstracts and publication emanating from thesis 207 University of Ghana http://ugspace.ug.edu.gh 4*./ T>vi* Med. Ilyg., 83(4). 2010, pp. 936-943 fofclO.42tMajtmh.201 0.09-0792 Copyright © 2010 by The American Society ofTtopicul Medicine nnd Ilyglcnc Pediatric Diarrhea in Southern Ghana: Etiology and Association with Intestinal Inflammation and Malnutrition Japheth A . Opinion,* Mcrcy J. Newman, Palrick F. Ayeh-Kumi, Raymond Affrim, Rosina Gepi-Allee, Jesus E. A . D. Sevilleja, James K. Roehe, James P. Nataro, Cirlc A . Warren, and Richard L. Guerrant Department o f Microbiology, University of Ghana Medical School, Accra. Ghana: Princess Marie lMiii.se Children's Hospital, Accra. Ghana; Center for Global Health, Division of Infections Diseases and International Health. University of Virginia, Charlottesville, Virginia; Center for Vaccine Development, Department of Pediatrics and Medicine, University of Maryland. Baltimore, Maryland Abstract. Diarrhea is a major public health problem that affects the development of children. Anthropometric data were collected from 274 children with (N = 170) and without (N = 104) diarrhea. Stool specimens were analyzed by con­ ventional culture, polymerase chain reaction for enteroaggregative Escherichia coli ( E A E C ) , Shigella, Cryptosporidium, Entamoeba, and Giardia species, and by enzyme-linked immunosorbent assay for fecal lactoferrin levels. About 50% of the study population was mildly to severely malnourished. Fecal lactoferrin levels were higher in children with diarrhea (P = 0.019). Children who had E A E C infection, with or without diarrhea, had high mean lactoferrin levels regardless of nutritional status.The E A E C and Cryptosporidium were associated with diarrhea (P = 0.048 and 0.011, respectively), and malnourished children who had diarrhea were often co-infecled with both Cryptosporidium and E A E C . In conclusion, the use of DNA-biomarkers revealed that E A E C and Cryptosporidium were common intestinal pathogens in Accra, and that elevated lactoferrin was associated with diarrhea in this group of children. IN T R O D U C T IO N Diarrhea is a principal cause of morbidity and mortality in children < 5 years of age in developing countries, where acute watery diarrhea accounts for nearly two million diarrhea- related deaths annually in this age group.'- In the last decades, however, while mortality caused by diarrhea has been decreas­ ing worldwide mainly because of improved hygiene, morbidity attributable to diarThea remains high.2-1 A vicious cycle ensues between diarrhea and malnutrition, and studies have shown that malnutrition with frequent diarrheal episodes slows cog­ nitive and physical development of children/ '' One mechanism (or this is that diarrheagenic pathogens damage intestinal epi­ thelium and reduce its absorptive function, leading to nutrient depletion and malnutrition.'' Obstacles to recognition of at-risk children are several. The plight of sub-opiimally breast fed and malnourished chil­ dren is often largely invisible because they are only mildly or moderately undernourished.7 Additionally, anthropometric measurements arc not routinely performed to identify mal­ nourished children in most clinics and hospitals in Ghana, thereby missing the opportunity for diagnosis and appropriate management.' The most established method to identify those with malnutrition is by the use of z-scores, with the reference population defined for the study country or from the stan­ dard international reference chart of the National Center for Health and the World Health Organization (W HO).* The agents capable of causing infectious diarrhea and the mechanisms responsible for disease pathogenesis are gener­ ally known, but the true prevalence of these agents in devel­ oping countries is poorly understood.1" For example, in most lub-Saharan African countries including Ghana, microbio­ logical methods for clinical investigation of diarrheal dis­ eases arc usually restricted to identifying conventional enteric * Address correspondence to Japheth A. Opintan. Center for Global Health, Division of Infectious Diseases and International Health, University of Virginia. MK6, C'urtcr-Hnrrison Bldg. Km 27tW, 345 Crispcl Drive. Charlottesville, VA 22903. E-mails: jnph.npinlnnfff jrahoo.com or jyo4g«'virginia,cdii bacteria such as Salmonella and Shigella. Escherichia coli (E . coli) isolates arc often not fully characterized because of the lack of resources. Additional pathogens of potential importance include enteroaggregative E. coli (E A E C ), which is associated with diarrhea in several coniejns: traveler's diar­ rhea,1" 3 pediatric diarrhea.1’ foodborne outbreaks*11 human immunodeficiency virus." symptomatic and asymptomatic cases.1" acute and persistent diarrhea,'1’ among others: and Cryptosporidium, Entamoeba histolytica, and Giardia. which are parasitic causes of diarrhea."t’!° Unfortunately, investiga­ tion of diarrhea caused by these parasites in most developing countries largely depends on expert microscopy, where techni­ cal competence is necessary. The objective of this study was. first, to determine the prev­ alence of E A E C , Shigella spp.. Cryptosporidium spp.. E . his­ tolytica, and Giardia latnblia in children < 5 years of age with and without diarrhea in southern Ghana. Second, this study aims to determine whether these entcropathogens were asso­ ciated with intestinal inflammation in either nourished or mal­ nourished children. M A T E R IA L S A N D M E T H O D S Ethical clearance. The study was reviewed and approved by the Institutional Review Board of the University of Ghana Medical School, Ghana. Participation was voluntary and enroll­ ment was subject to parents/guardians’ approval, through sig­ nature or by thumb printing their names after the purpose of the study was explained to them. Study design, population, and settings. This was a prospec­ tive cross-sectional study carried out between August 2007 and May 200S. of children S 5 years of age consulting at the Princess Marie Louise Children's Hospital (P M L ). Accra, Ghana. Consecutive children from whom consent was given by their caregivers were included in the study. The group of children with diarrhea was recruited from the outpatient clinic anil these children were brought to the hospital for acute health care. The control group of children without diarrhea was also from the outpatient clinic, but these children were visiting for routine child welfare care. No follow-up was done alter the initial recruitment as a part of this study. 936 University of Ghana http://ugspace.ug.edu.gh PEDIATRIC DIARRHEA IN SOUTHERN GHANA 937 Interviews and diarrhea definition. A structured question­ naire was used to obtain informntion on the children from their parents/guardians. Informntion that was obtained included demographic data, duration of diarrhea, residence/location, breast feeding status, and medication taken before visiting the hospital. Diarrhea was defined os the passage of three or more unformed stools within a 24-h period. Diarrhea duration last­ ing < 14 days was defined as acute and those lasting 2 14 days, persistent. The control (non-diarrhea) group consisted of chil­ dren who have not passed three or more unformed stools al least within the 24-h period before enrollment. Anthropometric data and nutritiounl status assessment. Height or length measurements in centimeter to the near­ est one decimal were performed for children above or below 2 years of age. respectively. Weight measurements in kilogram to the nearest one decimal were performed using a 25 kg Salter hanging scale (CM S Weighing equipment. High Holborn, London, U K ). The Z-score. weighl-for-age (W A Z ), height- for-age (H A Z ), and weight-for-height (W H Z ) were calculated by use of software designed for nutrition studies (E P IN U T , World Health Organization. Geneva; Epi Info version 6.0, Centers for Disease Control and Prevention, Atlanta, G A ). These anthropometric Z-scores arc a measure of SD above or below the median for the international reference population. Z-score values were used to determine the nutritional status of children on the basis of the following definition: W A Z , well nourished (> -1 ) . mild ( -2 to -1 ) , moderate ( -3 to -2 ) , and severe (< -3 ) malnutrition; H A Z , normal height (£ -2 ) , mod­ erate stunting ( -3 < -2 ) , and severe stunting (< -3 ) : W H Z , normal weight (2 -2 ) , moderate wasting ( -3 < -2 ) , and severe wasting (< -3 ). Specimen processing and microbiological analysis. A stool specimen from each participating child was collected into a sterile container and processed within 2 h of collec­ tion. Routine enteric bacteria were cultured on MacConkey (MAC).Salnwnclla -Shigella (SS), and deoxvchocolate (D C A ) agars (Oxoid, Columbia. M D ), using standard techniques. Selenite F broth was used as pre-enrichment for Salmonella before sub-culturing onto M A C , SS. and D C A . Bacterial colonies after an overnight incubation period at 37°C were identified by standard biochemical methods and stored on Muellar Hinton slopes for further analysis. No microscopy was performed for the detection of parasites. One aliquot of a fresh stool specimen from each child was kept frozen at -20°C in cryo-vials (deidcntified) until sent to the Center for Global Health, University of Virginia for further analysis. Fecal D N A extraction. We used the Q IAam p stool kit (Oingen, Valencia, C A ) to extract genomic D N A from frozen stool specimens with some minor modifications.The modifica­ tions included the addition of dry beads (M O B IO Laboratory Inc., Carlsbad, C A ) to weighed stool specimen before the addition of lysis buffer (A S L ). The mixture was bead-beated for 2 minutes to make a uniform homogeneous mixture with the lysis buffer. Additionally, we incubated mixtures at 80'C' instead of the 70‘ C recommended by the manufacture to lyse enteric pathogens. For each stool aliquot, between 15 and 20 (Jg or p L of stool was used depending on stool consistency. D N A s were also extracted from appropriate control organisms. A ll D N A s were kept frozen at -8 0 “C until needed for analysis. Quantitative real-time polymerase chain reaction (PCR). A single-plcx quantitative PCR for each gene pair (Table 1) con­ sisted of 5 |jL template, 1 p L of each 6.2 pM primer, 12.5 pL of SYBR-Green -490 (Bio-Rad Laboratories, Beltsville, M D ), and PCR grade water to a reaction volume of 25 p.L. Reactions for each sample were performed using the Bio-Rad iQ C y - cler Real-Time Detection System in Bio-Rad iCycler 96-well plates, where positive and negative controls were included with each reaction set.Table 1 shows the target genes, locations,and annealing temperature for each primer set. The results were analyzed with a user-defined threshold of 200 PCR baseline- subtracted curve-fit relative fluorescence units. Melt curve (ct) data collection and analysis was enabled at cycles 3 and 4, with an increase in set point temperatures after cycle 2 by 0.5’C. We sought multiple loci for E A E C (aap. aatA. aggR. and aaiC ) and single loci for Shigella, Cryptosporidium , and Giardia species. Standard cultures with known numbers of E . coli 042 and 17-2, Shigella, Cryptosporidium , and Giardia oocysts were used as reference and positive controls. Water and E. coli K-12 were used as negative controls. Melt curve analysis was used to determine positivity of samples using a user defined threshhold. Intestinal inflammation assessment. Intestinal inflamma­ tions were quantitatively assessed from frozen stool specimens using the IB D SC A N (TechLab. Blacksburg, V A ) according p a r t m n Target genes screened from fecal DNA* Strain Gene target Location Primer sequence (5 '-? ') PCR si?o (hp) AntKuluYi icm pcm tun : ( C l Socjvc'Tcfeicoce EAEC aaiC Chromosome C TTC TG C TC TTA G C A G G G A G TTTG AA G C G TG A A A TG C C TG A G G A 1 3 4 7 .5 Na taro's Laboratory aatA Plasmid C C TR TG TTG ATG CTCG AG AG A CKTTC C TCC TC C TCA A G G A C AT US 5 5 N a taro's Laboratory aap Plasmid C TTG G G TA TC A G C C TG AA TG AACCCATTCG GTTAGAG CAC 3 1 0 4 5 Cema and others21 Jcc o Plasmid CTA A TTG TAC A ATCG A TG TA ATG A AGTA A T IC IT G A A T 3 0 S 4 5 Czeczulin and others- Shigella/EIEC ipaH Plasmid G TTC C TTG A CC G CC TTTCC G A TA CC G TC GCCGGTCAGCCACCCTCTGAGAGTAC 6 1 9 6 0 .5 Seihabutr and others2' Crypto­ sporidium 18% rRNA Chromosome CTCCACCA ACTA AG A ACG G CO TA G A G A TIG G A G G TTG TTC C T 2 1 3 6 0 Gene ID cgd7_23(P E. histolytica Eh Chromosome A AC AGTA A TA G TT rC T T TG G TTA G TA A A A C T IA G A A TG TC A T ITC TC A A H C A T 13-1 6 0 Haque and others1' Giardia lamblia P24I Chromosome C ATCCGCG AGG AG G TC A A C iCAGCCATGGTGTCGATCT 74 6 0 Guy and others2*  PC R - m: :»   chain reaction: E A E C '» unlcrmtHftrcKflMvc lixcherlctila call-, E U -C - cnlurolnvuKlvc f . colt. University of Ghana http://ugspace.ug.edu.gh 938 OP1NTAN AND OTHERS to the manufacturer's instructions. Stool specimens were allowed to thaw and were serially diluted 10-fold and ana­ lyzed by a polyclonal antibody-based enzyme-linked immu­ nosorbent assay (E L IS A ) method. The detailed procedure is described elsewhere.51 and absorbance of each assay well was measured spectrophotometrically at 450 and 620 nm (A W1W0). Fecal lactoferrin concentrations in jig/mL were determined by comparison with a standard curve using purified human lac­ toferrin and analyzed by linear regression in Microsoft excel (Redmond, W A ). The lowest dilution of a specimen with an absorbance at 450/620 nm within the linear portion of the curve was used to determine the lactoferrin concentration, flie final lactoferrin concentration was obtained by multiply­ ing the dilution factor by the concentration. A positive control (purified human lactoferrin) and a negative control (washing jiu(Ter) was included in each batch of stools analyzed and lin­ ear regression was performed separately for each batch using standard controls.This assessment was performed only on sub­ jects with adequate stool specimens and where necessary, the experiment was repeated. Among EAEC-infected malnour­ ished children, two stool specimens (out of > 200 specimens analyzed) showed lactoferrin values that were 30-50 times more than the mean value for their subject group, were there­ fore designated as outliers, and not included in the statistical analysis. No satisfactory explanation for results on these two specimens was evident, because no diarrhea was present in one. and breast feeding was unlikely in both. Statistical analysis. To avoid any experimental biases, stool specimens were coded before testing and only decoded for purposes of analysis. Statistical analyses were performed using SPSS software (version 17.0. SPSS, Chicago, IL ) and Epi-Info. Statistical tests included x: for associations of pathogens with age groups, diarrhea, and non-diarrhea and the paired r test for associations with lactoferrin level. Odds ratio (O R ) and 95% conGdence intervals (C l ) arc reported for all 2 x 2 compari­ sons Two-tailed tests were used and P < 0.05 was considered statistically significant. R E S U L TS Stndy papulation and nutritional status. Within the 9-month study period, 287 children < 5 years of age were recruited with only 13 excluded from analysis because of insufficient data. Of the274children included for analysis, 170 (62 % ) were with and 104(38%) were without diarrhea; there were more males 156 (56.9%) than females. Acute and persistent diarrhea included 85.3% (145/170) and 7.6% (13/170) of total cases, respectively. Duration of symptoms in the remaining children with diarrhea (7.1%) was not recorded. Al least one anthropometric measurement was taken for 269 out of the 274 children analyzed. O f 269 children from whom weight measurements were recorded, 134 (49.8%) showed mild to severe malnutrition (W A Z < -1 ) (93/168 (55.4%J in children with and 41/101 [40.6%] without diarrhea, OR = 1.82 [95% C l, 1.102-2.988], P = 0.023). O f 170 children Irom whom height or length measurements were recorded, 61 (35%) showed moderate to severe stunting (H A Z < -2 ) (38/86 [44.2%) in children with and 23/84 [27.4%] without diarrhea, O R = 2.10 [95% C l, 1.110-3.972], P = 0.026). Of 161 children from whom both weight and height measure­ ments were recorded, 37 (22.9%) showed moderate to severe wasting (W H Z < -2 ) (24/82 [29.3%] in children with and 13/79 [16.5%] without diarrhea, O R = 2.101 [95% C l, 0.989- 4.454], P = 0.062). 'rile mean age, weight, and height were 15.1/14.6 months, 9.S/9.5 kg, and 88.0/84.0 cm (diarrhea/non­ diarrhea, respectively).Table 2 shows the baseline characteris­ tics of the study population. Microbiological studies. In only I of 170 diarrhea stool spec­ imens was Shige lla recovered as an enteric bacterial pathogen from culture. This strain was scrolypcd with S h ig d la polyva­ lent anti-sera (Mast Group Ltd., Merseyside, U K ) and was Shigella f le xncri. In the entire study population, E . co li was the predominant bacterium obtained from culture (79.6%), fol­ lowed by Klebsiella spp. (5 .1% ). Other commensals included 9.8% ol the total and no bacteria grew in 5.5% of the total stool specimens cultured. Pathogen dclcclion by real-time P CR . Table 3 shows bacte­ rial and parasitic agents delected from fecal D N A by real-time PCR in children with and without diarrhea. The E A E C was defined as posilivily for any of the four E A E C virulence genes sought {aap , a a lA , aggR . and a a iC ). Although E A E C was sig­ nificantly associated with diarrhea (147/170 versus 80/104. O R 1.917 [95% C l = 1.024-3.592], P = 0.048), it was also found in similar frequencies in both nourished and malnourished children. In 6 out of 170 diarrheal stool specimens, the ip a H gene, which is expressed by both Shigella and enleroinvasive £. co li (E IE C ) was delected.The ip a H gene was not detected in fecal D N A from any of the children without diarrhea. The numbers were, however, too small to assess statistical significance. Five out of 6 of the children in whom the ip a H gene was detected were well-nourished (Table 3). C ryp tospo rid iiun spp. was the most frequently delected pro­ tozoan parasite in fecal D N A and was associated with diar­ rhea (14/170 versus 1/104, O R = 9.244 [95% C l 1.197-71371], P = 0.011). Cryptosporidiosis was also primarily (10 out of 14) p a r t m o Baseline characteristics of study population*______ Characteristic Diarrhea >Y ( % ) N oo -d m rtica ;Y ( *5> 1 Age/months N = 170 (V= 104 0-6 26(15.3) 35 ( 33.7) 7-12 54(31.8) 30(28.8) 13-24 77 (45.3) 24(23.1) 25-60 13 (7.6) 15(14.4) Sex N = 170 ,V=104 Male 97(57.1) 73 (42.9) Female 59 (56.7) 45(43J ) Weight/kg A '= 170 nUA LHS 2.5-4.9 10 (5.9) 4 (3.S) 5.0-9.9 121 (712) 75(7X1) 10-19.9 34 (20.0) 22 (21-2) 20-86.0 5 (2.9) 3 (2.9) WAZ 168 -V = 101 Normal (> -1 ) 75 (44.6) 60 (59.4) Mild (-1 to -2 ) 33(19.6) 19(18.8) Moderate (-2 to -3 ) 32(14.0) 12(11.9) Severe(< -3 ) 28(16.7) 10 (9.9) H AZ N = 86 Sr = S4 Normal (2 -2 ) 48 (55.S) 61 (72.6) Moderate (-2 to -3 ) 6(7.0) 11 (13.1) Severe (< -3 ) 32 (37.2) 12(14.3) WHZ N = 82 N = 79 Normal (2 -2 ) 58 (70.3) 66 (S3.5) Moderate (-2 to -3 ) 6 (7.3) S (10.1) Severe (< -3 ) 18(22.0) 5 (6.3) •W AZ - wclghl-for-n^e; H AZ - ho iphi-for-agc;WHZ « wcighi-for-hcipht. University of Ghana http://ugspace.ug.edu.gh PEDIATRIC DIARRHEA IN SOUTHERN GHANA 939 T a h u ! 3 O r g a n i s m s d c l c c t e d b y r C t l l - l i n i c P C R f r o m f c c u l D N A D ln rrl tcn ( N • 170) N on*diarrf)cA ( IV * IM ) O dd» r a l io (9 5% C l ) P v a lu c t No. (% ) WN (N * W ) M N ( /V - 9 5 ) N o . (% ) W N ( X - ftfl) M N ( iV = 4 1 ) A n y i n f e c t i o n E A E C 1 4 7 ( 8 6 .5 ) 6 6 7 9 HO ( 7 6 . 9 ) 4 9 3 0 1 . 9 1 7 ( 1 .0 1 8 - 3 .6 1 2 ) 0 . 0 4 8 S h i g e l l a / E I E C 6 ( 3 . 5 ) 5 1 0 ( 0 ) - - n / a C r y p t o s p o r i d i u m s p p . U ( 8 . 7 ) 4 1 0 1 ( 1 .0 ) 1 0 9 .2 4 4 ( 1 . 1 9 7 - 7 1 .3 7 1 ) 0 . 0 1 1 £ . h i s t o l y t i c a 5 ( 3 .0 ) 2 3 0 ( 0 ) - - n / a G i a r d i a s p p . 0 ( 0 ) - “ 0 ( 0 ) “ - * PC R - |vd\Ti>cr»vc chain reaction; W N - well nourKlied (W A Z > 11: M N » malnou rh tird (W A Z < I ): E A E C «■ ctilcra*BjfrcjwliVc t iu h e r ic h iu n d r . E I E C » crtlcrotnvastvc £ . c o in n/a a nol applicable. t P v a lu e rs b e l^ ’o en du r tt» c .r l a n d n«>n-d iarrheal s lo o l sp e c im en . dclecled in children who were malnourished and had diarrhea (Tabic 3). Enu im oeha h istolytica was only delected in chil­ dren with diarrhea 5 out of 170 (2 .9% ) and G ia rd ia was not detected in either sub-populations. We observed children who were co-infected, with two or more pathogens detected in the stool, predominantly in children who had diarrhea. The E A E C -C ryp to spo r id ium was the most prevalent (7.6% , 13/170). followed bv E A E C - S lugeU a lE lEC (1 9 % , 5/170). and E A E C -f . histolytica (2.4%, 4/170). C ryp io sp o r id iu n i-E . h istolytica and C ryp to spo rid ium - S ltigc lla JE lEC co-infection each formed 0.6% (1/170), and one child who had diarrhea was co-infected with E A E C - C ryp to spo rid ium -S h ige lla fE lEC (0.06%, 1/170). There was no obvious trend in the distribution of pathogens by age in the two sub-populations, especially for E A E C (Table 4). E A E C virulcncc genes distribution. O f the four genes asso­ ciated with E A E C , aatA was the most frequently detected (67,2%) of all fecaJ D N A . followed by aap (59.9%), aggR (42.7%). and aa iC (33 .6% ) (Table 5). The E A E C 's plasmid gene aap was significantly associated with diarrhea (O R = 2306 (95% Ct, 1316-4.144], P < 0.001) and the chromo­ somal gene aa iC was not (O R = 1.639 [95% C l, 0.0962-2.792], P = 0.086) (Table 5). Multiple gene combinations were also observed in E A E C infections in our study population, and the presence of any three genes was associated with diarrhea (O R = 2.101 [95% a, 1.261-3.502],/’ = 0.006). We did nol find any of the E A E C virulence genes associated with malnutrition (W A Z < -1 ) ( P > 0.05). Fecal lactoferrin levels. Figure 1 shows enteric pathogens detected in children with/without diarrhea and the distribu­ tion of fecal lactoferrin levels. Generally, the mean lactoferrin levels were lower in children who had diarrhea and were also malnourished compared with those with diarrhea but were nourished. Regardless of the enteric pathogen detected, fecal lactoferrin levels were relatively high (manufacturer's cut­ off value = 7.24 (ig/mL). Especially for E A E C infection, both controls and patients had a wide range of lactoferrin levels, regardless of whether they were nourished or malnourished (Figure 1). Children with diarrhea had significantly higher fecal lacto­ ferrin levels ( N = 143; 1658.9 ± 204.2 ng/mL) compared with those without diarrhea ( N = 84; 935.5 ± 194.4 (ig/mL) ( P = 0.019). The aatA gene and the presence of any one or two genes of E A E C were also significantly ( P < 0.05) associated with elevated fecal lactoferrin levels (Table 6). In comparing diarrhea with non-diarrhea stool specimens, E A E C s chromo­ somal gene aa iC showed the highest fold-increase in fecal lac- toferrin level (2.7) followed by the aap gene (2.5). Additionally, detection of a multiple virulence gene of E A E C in a stool was associated with an increased fold-rise in the mean fecal lac­ toferrin level between children with and without diarrhea (Table 6). D IS C U S S IO N Nutritional shortfalls among our study population. Mild to severe malnutrition (W A Z < -1 , which reflects both acute and chronic types of malnutrition) was identified in 49.8% of our study population. Severe growth shortfalls occurred in both children with and without diarrhea in the entire study popula­ tion. Growth faltering in infants from developing countries has been reported lo occur as early as 2-3 months of age-’*-'1 in con­ trast to developed countries,25 a fact attributable to timing of complementary'feeding. For example, in the D A R L IN G (Davis Area Research on Lactation, Infant Nutrition and Growth) study, although breast milk intakes were similar, the amount and nutrient density of food consumed after 6 months were lower in Peru than in the United Stales.* In 2005, Antwi* noted a 21.2% incidence of wasting (acute malnutrition) among chil­ dren seen in Kumasi, Ghana, similar to an overall incidence of 22.9% (37/161) in the current study— both consistent with the 22.1 % estimate for Ghana by the World Food Program.*' T a b l e 4 D i s t r i b u t i o n o f p a t h o g e n s b y a g e * A n y inlcclw rti D ia rrh e a ( S o 170) N o n -d ia r rh e a t-Y a HU ) A p e a itc tfo ry /m o n llis A g e c a te g o ry m o n th s 0 - 6 i N - 2ft) 7 -1 2 ( t f - 54) 13—24 ( N * 77 ) 2 5 -6 0 ( N m 13) 0 - 6 (.V * 3 5 ) 7 -1 2 =. 30 ) 13 -24 ( Y ^ 2 4 ) 2 5 -6 0 (A* = 15) E A E C 2 4 ( 9 2 .3 ) 4 2 ( 7 7 .7 ) 6 9 ( 8 9 .6 ) 1 2 ( 9 2 . 3 ) 2 5 ( 7 1 .4 ) 2 4 ( 8 0 ) 1 8 ( 7 5 ) 1 3 ( 8 6 . 7 ) C r y p t o s p o r i d i u m 3 ( 1 1 .5 ) 4 ( 7 .4 ) 6 ( 7 .8 ) 1 3 ( 7 .7 ) - - 1 ( 4 .2 ) E . h i s t o l y t i c a - 2 ( 3 .7 ) 3 ( 3 .9 ) - - _ S h i g e l l a / E l E C 1 ( 3 .8 ) I ( 1 .8 ) 3 ( 3 .9 ) 1 ( 7 .7 ) - - _ _ G i a r d i a s p p . - “ - - - - - - ’ E A E C » cn icro ac rjy cjy illv c l i ir lic r lc h lu co l i , K lliC , - c n lc to in v to iv c li. coll. University of Ghana http://ugspace.ug.edu.gh OPINTAN AND OTHERS p a fMi c EntcronggregativcfeWi.'rfcWa coli (EA EC ) virulence factor-positive in stool samples* CtinrnctcriMjc D iarrhea (.V - 1711) N (%> Nrni-dlorrhan IN - HM> Al <%) T o u t (JVo 274) A l(% ) OR (M% C l| X’ / ’ value EAEC v iru len ce -re la ted ccne aaiC 6 4 ( 3 7 .6 ) 2 8 ( 2 6 ,9 ) 9 2 ( 3 3 .6 ) 1 .6 3 9 | 0 . 9 6 2 - 2 .7 9 2 ) 3 .3 2 7 0.086 aggR 7 9 ( 4 6 .5 ) 3 8 ( 3 6 .5 ) 1 1 7 ( 4 2 .7 ) 1 .5 0 8 ) 0 .9 1 4 - 2 . 4 8 6 ) 2 .6 0 2 0 .1 3 1 aatA 1 1 8 ( 6 9 .4 ) 6 6 ( 6 3 .5 ) 1 84 ( 6 7 .2 ) 1 .3 0 7 1 0 .7 8 0 -2 ,1 8 8 ) 1.036 0.354 M l ’ 116 (68 .2) 48 (46 .2) 164 (59 ,9 ) 2,506 [1.516-4 .144] 13.093 <0.001 EAEC gene com b in a tio n Any 1 gene 147 (86 .5) 8 0 (76.9) 2 2 7 ( 8 2 .8 ) 1 .917(1 .018-3 .612) 4 .1 3 9 0.048 Any 2 genes 130 (76 .5 ) 73 (70 .2) 2 0 3 ( 7 4 , 1 ) I.3K0 (0 .797 -2 .3911 1 .3 2 5 0.259 Any 3 genes 84 (49 .4) 3 3 (3 1 .7 ) 1 1 7 ( 4 2 .7 ) 2.101 ) 1.261-3.502) 8.224 0.006 AIM genes 34 ( 20.0) 1 3 (12 .5 ) 4 7 (1 7 .2 ) 1.750 (0 .876 -3 .4% ) 2.554 0.110 •OR “ odd* C l - onofidcnoc interval; P < 0.05 i* significant. Approximately 14% (38/269) of the children we studied «ere severely malnourished (W A Z < -3 ) , and. oul of this num­ ber. 73.7% had diarrhea. A self-perpetuating vicious cycle in which malnutrition and diarrhea are synergistic is suggested, and may explain their effect on cognitive development of chil­ dren, especially their semantic fluency.1'O u r data supports this link between malnutrition and diarrhea, placing these groups of children at a higher risk of morbidity over time. Bacterial culture versus real-time PCR for detection of pathogens.The PCR methodology is more sensitive in screen­ ing for pathogens. In sub-Saharan Africa as in many settings, however, the cost involved cannot be passed on to patients and therefore this tool is used mainly in research facilities. In this study, routine bacterial culture detected only one stool posi­ tive for S.flexneri and none for Salm one lla spp. However, PCR delected six ip a H genes in diarrheal stool specimens. Although, the ip aH gene is expressed by both Shigella and enteroinva- sive (E l E C ), E IE C is not often detected in Ghana,” and we speculate that the ip a H gene detected most likely reflects the presence of Shigella spp. that was missed on the culture. The sero-group identified by culture and serology is predominant in Ghana31 as it is in many developing countries. C ryp tospo rid ium spp. was not only the most prevalent para­ site detected, but it is also significantly associated with diarrhea (8.7% versus 1.0%, P = 0.011). Children who were malnour­ ished and had diarrhea often had cryptosporidiosis. Prev alence rales of C ryp tospo rid ium spp. using presumably less sensitive methods like microscopy and E L IS A in Ghana, 311 Liberia,3' Mexico,* and Guinea Bissau37 reported ranges between 7.7% and 29% in symptomatic children.The current study recorded 8.2% in symptomatic and 1.0% in non-sympiomatic patients. Entam oeba h istolytica causes amebic colitis, amebic dys­ entery, and liver abscess. Modern diagnostic tools like PCR are able to discriminate E . h isto lytica from the non-pathogenic MN - malnourished ( w a z < -i) N - nourished ( w a z > - d indicates mean levels 8000 E l£ 6000 0 40001 Crypto N MN Shigella MN E his D iorrhoa iL * N MN E A E C -iUU- J S tU E A E C N MN Crypto N on -d lo rrho a F i g u r e 1. Entcric pathogens dctccled and fecal lactoferrin ( L F ) levels. Crypto = Cryptosporidium spp.; E his = E. hiostolytica; EAEC = entero- EscheriMu coll. •Breast-feeding may cause moderately (15 S 120 |ig/mL) increased LF (Lima and others, unpublished observation).aggregative University of Ghana http://ugspace.ug.edu.gh PEDIATRIC D IARRHEA IN SOUTHERN GHANA 941 p a b l eg y EAEC genes detected und fccul Incloferrin levels* Dlnrrlicnl *1001 n (m ean * SIJ.) Im/mL Non-dlnrrhcol Mool n (m ean * SE) Mg/mL P value EAEC gene aaiC 56 (1021.0 ± 251.6) 25 (821.2 ± 402.1) 1.2 0.667 aggR 66(1713.9 1 292.5) 33(934.1 t 329.2) 1.8 0.104 aatA 98(1706.1 ± 244.1) 54 (927.1 ± 259.1) 1.8 0.044 aap 101 (1656.7 ± 245.9) 39 (901.2 ±261.6) 1.8 0.080 EAEC gene combinations Any 1 gene 123 (1633.5 ± 215.4) 66(1175.4 i 356.8) 1.4 0.029 Any 2 gene 107 (1679.8 ±231.1) 60 (892.6 ± 234.3) 1.9 0.028 Any 3 gene 73 (1504.9 ± 269.7) 30(1132.8 ± 394.8) 1.3 0.451 Any 4 gene 30 (1319.5 ± 400.4) 11 (370.5 x 285.5) 3.6 0.175 All specimens 143 (1658.9 ± 204.2) 84 (935.5 ± 194.4) 1.8 0.019 •EA EC ■» Exi-herichia Cttii. t Ratio of dvanrtoca a n d ooa -diarrhea m ean lactoferrin IcvcU. E . dispar.*This sludy detected E .h is to ly tica in 2.9% (5/170) of Ihe children who had diarrhea. No visible blood was observed in any of the stools from these children. A ll Ihe E . h isto lyt­ ica detected in the current study were from children who had diarrhea. In Bangladesh and elsewhere, relatively few num­ bers of people infected with E . histo lytica develop symptom­ atic disease.'*-*1-" This study did not detect any G ia rd ia in (he stool specimens screened for the p241 gene of G ia rd ia spp. The p241 gene tar­ get we used is well validated1" and our positive control, which was included in each PCR run was amplified while the nega­ tive control was nol. Addy and others" in 2004, found a 3.7% prevalence rate of G ia rd ia in Kumasi, Ghana. In a 7-year study of diarrhea caused by parasites in Guinea Bissau, the most prevalent parasite was G ia rd ia Utniblia (14.8%) followed by Cryptosporid ium (7 .7 % ) . t? Although seasonality, study dura­ tion, or geographical location may influence parasite preva­ lence. we cannot pinpoint additional reasons for the zero prevalence of G ia rd ia in the current study. Children who had diarrhea were often co-infecled, wilh two or more pathogens delected in the stool, and E A E C - C ryp tospo rid iw n was the mosl prevalent (13/170) in the currenl study. Among human immunodeficiency virus (H IV ) - infected children in South Africa, Samie and others” iden­ tified children who were co-infected wilh as many as six different species of pathogens. This study detected a child who had diarrhea wilh E A E C -C r y p to s p o r id iu m -S l i ig e lla lE lE C co­ infection. The H IV status of this child is however not known. Relatively fewer children who are > 2 years of age were sam­ pled in this sludy. However, cryptosporidiosis seemed to be more common in children < 2 years of age. and this is in agree­ ment wilh an earlier study in Ghana.11 Prevalence of E A E C virulence-associated genes. Among Ihe E A E C plasmid genes (a ap ,aa tA . and a ggR ) we tested, only aap was significantly associated with diarrhea ( P = 0.0003). A recent publication, which compared molecular probes to the “gold standard" (aggrcgalion of cultured epithelial cells), however, suggested that the aap gene is not restricted to E A E C , but is also detected in diffusely adherent E . co li (D A E C ) and in non- pathogenic E. c o li." We did nol observe any significant statisti­ cal association between any individual E A E C gene (whether plasmid or chromosome borne) and malnutrition (W A Z < - I). Information on the presence of E A E C virulencc-associated genes in body fluids of persons in Africa is limited, as is the prevalence and distribution of this organism in Ghana. The aggR is known to regulate its own expression and that of sev­ eral plasmid genes and chromosomal genes of E A E C c includ­ ing the aggregative adherence fimbriae, a dispersin (aap), a dispersin translocator apparatus called aat, and several chro­ mosomal loci including the aa iC '.** In a different population, Huang and others failed to show any association between four E A E C virulence-associated genes (a g g A .a s p U .a a fA and a g gR ) and clinical illness in travelers from ihe United States to Mexico.'1 Furthermore, in that study, a s p li (now designated a a tA ) was ihe least prevalent gene among the four E A E C vir­ ulence genes studied,*1 whereas this same gene (a a tA ) was the most prevalent (67 .2% ) in the current study. In South Africa, Samie and others1' found the aap gene to be the predominant among others and also associated with diarrhea. These dispa­ rate findings on the relative distribution and importance of E A E C genes in diarrheal illnesses suggest that geographical location, type of exposure, and/or host factors may dictate the nature of E A E C infection. For example, among the risks of contracting traveler's diarrhea, the country of destination was the most important determining factor as reported by Cabada and White.'1 Although aatA was the most prevalent gene observed in this study, it was nol associated with diarrhea ( P > 0.05) (Table 5). Some studies have shown that the novel protein aatA, which is encoded on E A E C virulence plasmid pA A 2 , localizes to the outer membrane of this bacterium and facilitates export of the dispersin aap across the outer membrane.*'* O ur results may support this notion because aa tA and aap were detected in greater than 68% of patients with diarrhea. Further study is required for a firm conclusion. O f interest, the chromosomal gene aa iC did nol show any significant differential association between the diarrhea and the non-diarrhea group in the cur­ rent sludy ( P = 0.068). About 12% (13/104) of children with­ out diarrhea in the current study were positive for all four E A E C genes tested. The presence of 2.3, or 4 genes in stool specimens of study children suggests a strong association. For example, the presence of three of these genes is associated with a higher O R than one gene alone (Table 5). One explanation for the high frequency of EAEC-associated virulence genes in symptomatic and asymptomatic patients and the heterogeneity of Ihe different gene assortments found is that E A E C is endemic in our study population. In support of this notion, almost all prior studies recover E A E C from controls" *" and from individuals wilh diarrhea.17-'0 Pathogenic factors may influence the initiation of a symptomatic phase. University of Ghana http://ugspace.ug.edu.gh 942 OPINTAN AND OTHERS determined by and include distinct mechanisms as reviewed by Kaper and others." For example. Huang and others sug­ gested that a first exposure to E A E C infection “primes” the immune system to prevent a second infection." Furthermore, in their study of traveler's diarrhea, after the initial E A E C infection, only 4 (11 % ) of the subjects had a subsequent symp­ tomatic E A E C infection." Lactoferrin levels. Lactoferrin is bactcricidal to cntcric pathogens, modulates the intestinal immune response, and is released by neutrophils into stool in response to infection.'1 O f interest in the current study, the mean lactoferrin levels were lower in children who were malnourished and had diarrhea compared with those without malnutrition who had diarrhea. We speculate that the lactoferrin assay may be marginally less sensitive in the setting of malnutrition. Perhaps, the entero- cytes are less able to synthesize lactoferrin in children who are malnourished and have diarrhea. This observation needs further investigation for a firm conclusion. In earlier studies, the mean lactoferrin levels for healthy controls were less than 118 jtgftnL:,-c compared with healthy controls (298.8 pg/mL) in the current study. We do not have immediate explanation for this observation. However, unpublished observations by Lima and others suggest that breast milk may contribute to moderately (15 to 120 pg/mL) increased fecal lactoferrin lev­ els. Some studies have demonstrated an association of E A E C infection with inflammatory cytokines"'' and several others have associated E A E C with elevated lactoferrin levels."" In children with diarrhea, we found a significant statistical asso­ ciation of elevated fecal lactoferrin with thenap gene, and with the detection of any one or two of the E A E C genes tested ( P < 0.05). Between diarrhea and non-diarrhea stool speci­ mens. E A E C s chromosomal gene a a iC was associated with the highest fold-rise in fecal lactoferrin level (2.7) followed by the aap gene (23 ). Additionally, more virulence E A E C genes detected corresponded to a rise in the fecal lactoferrin level (Table 6 ).The protective function of lactoferrin on infections with enteropathogens have been acknowledged,* and colo­ nization/infection. particularly by E A E C in the current study probably contributed to the high lactoferrin levels (Table 6). The current study had some limitations. Accurate height measurements were the most difficult to obtain on an outpa­ tient basis.and this limited two of the three z-scores of malnutri­ tion (W H Z and H A Z ). Knowledge of history of antimicrobial exposure is important, however, this information was obtain­ able in only a few of the patients. Only selected pathogens were assayed using PCR and thus, we may have missed other less common pathogens.That is, no stool analyses for intestinal parasites other than C ryp to spo r id ium , G ia rd ia . and E .h is to ly t ­ ica were performed, therefore, missing hclmintic infections. In conclusion, through the use of specific DNA-biomarkers, we were able to determine that E A E C and C ryp tospo rid ium were common intestinal pathogens and that elevated fecal lac- toferrin levels were associated with diarrhea in this group of children from southern Ghana. Rcccivcd December 2Ji, 2009. Accepted for publication July 6 ,2010. Acknowledgments: We thank Dnvid Kwarlcng and the nurses al the Princess Marie Louise Hospital for da ta collection and anth ropom et­ ric measurements. Relanna P inkerton at UVA reviewed all statistical analyses. Financial supporuThis study was supported in pari by the University of Ghana Mcdical School. College of Health Sciences. Accra. Ghana and Pfi/er-supported funds to the C en ter for G lobal H ealth , University o f Virginia, Charlottesville, VA. Authors' addresses: Japheth A. O pintan , Mcrcy J. Newman, and Patrick F. Aych-Kumi, D epartm ent o f M icrobiology, University of G hana Mcdical School, Korle-Bu, Accra. G hana. E-mails: jyo4g@' virginia.edu. newmcrci#yahoo.co.uk, and payehkum iS7 ahoo.com. Raymond Affrim and Rosina G ep i-A ttee . Princess M arie Louise Children 's Hospital, Accra, G hana, E-mails: raffrimV vahixi.com and rgcpLaltce@yahoo.com . Jesus E. A. D. Scvillcja. James K. Roche, Cirle A. Warren, and Richard L. G uerran t. C en te r for G lobal H ealth , Division of Infectious Diseases and International H ealth , University of Virginia. Charlottesville. VA, E-mails: emscvilleja®yahoo.com . jkr7tn(!Pvirginia.edu, ca6lf