PhD (Microbiology) / Department of Microbiology / Background: Diarrheal diseases have a major effect on human health, Globally; it is second only to pneumonia as a leading cause of death among children under five. They are due to a variety of infectious and non-infectious agents; including Entamoeba spp. Entamoeba histolytica is an invasive enteric protozoan parasite that causes amebiasis. Amebiasis is frequent in communities without clean water and poor sanitation, which include low-income South African populations in Giyani and Pretoria. In these populations, the amount of diarrhea caused by Entamoeba histolytica inclusive of all ages, sexes and HIV status is uncertain. Diagnosis of the parasite is usually by microscopy. However, microscopy lacks sensitivity and specificity, therefore it is not reliable. Fortunately, molecular diagnostic tests have been developed to detect different Entamoeba species in humans. It is known that the parasite E. histolytica causes asymptomatic and symptomatic diseases. However, the transition from colonization to disease is still unclear. While parasite and host factors, as well as environmental conditions influence the infection outcome, there is currently no clear explanation of wide variation in the presentation of the disease. This could suggest that there are other factors affecting the disease outcome. A better understanding of these factors as well as their role in disease remains target objectives of modern scientists and it will definitely help in the fight against the disease. In spite of the emerging evidence that the host microbiome, parasite burden and the inflammatory response contribute to the virulence of E. histolytica, their roles have never been defined in developing regions such as Giyani and Pretoria. In addition, the present study hypothesized that co-infections with E. histolytica and secretion of extracellular vesicles/exosomes have a significant impact on the virulence of E. histolytica. Little has been explored or elucidated about responses triggered by other enteropathogens/ameba interplay that could be important in the induction of tissue invasion and disease and also how E. histolytica/enteropathogens interplay in these infections has not been determined. Therefore, the knowledge of this interplay could help in understanding how this modifies disease manifestations by modulating pathogen virulence and the host response. The use of secretion systems is an essential biological process exploited by pathogenic microorganisms to promote survival and spread of the pathogen, which in turn exacerbate the infection. The study of extracellular vesicles (EVs) released by pathogens is a new and exciting field that may realistically contribute to a better understanding of the pathogenic process of E. histolytica and provide alternate control strategies. Aim and objective of the study: The overall aim of the study was to determine the impact of enteric pathogens and secreted extracellular vesicles on amebic virulence and the outcome of infection. This aim was addressed in through a series of six primary objectives, which were: a. To investigate the distribution and prevalence of protozoan parasites in South Africa. b. To investigate novel species of Entamoeba circulating in the South African population. ix c. To elucidate the impact of gut microbiota and immune response during amebic infection. d. To determine the role of Entamoeba histolytica macrophage inhibitory factor (EhMIF) during amebic infection. e. To investigate the impact of co-infections on the outcome of amebiasis. f. To determine the presence of secreted extracellular vesicles/exosomes in Entamoeba histolytica. Brief methodology and results: A modified and validated Taqman qPCR assay (with taqman probes and genus specific primers) was used for amplification and target detection. This assay was used to investigate the distribution and prevalence of protozoan parasites (Cryptosporidium spp and Giardia lamblia) in South Africa, the assay was considered superior for this project because it is more sensitive than conventional PCR and it can be used to detect multiple infection targets. This assay allows fast, accurate, and quantitative detection of a broad spectrum of enteropathogens and is well suited for surveillance or clinical purposes. A total of 484 stool samples collected from diarrheal and non-diarrheal patients from rural and urban communities of South Africa were studied. The overall prevalence of parasites (Giardia lamblia and Cryptosporidium spp) in rural and urban patients were found to be 49% (112/227) and 21% (54/257) respectively (p= < 0.0001). The distribution of specific pathogens in rural areas was Cryptosporidium spp (20%) and Giardia lamblia (14%). Our findings showed no significant difference in parasitic infections between gender and the age of the participants (Chapter 3). The discovery of novel species is of great importance to human health. We have recently discovered stools positive for Entamoeba organisms by microscopy but PCR negative for known Entamoeba species. This led to the hypothesis that novel species of Entamoeba are present in the South African population. A comprehensive assay was used which included probes to identify Entamoeba bangladeshi from diarrheal and non-diarrheal participants. A sensitive qPCR assays and amplicon sequencing was used to detect Entamoeba spp, Prevotella copri and Enterobacteriaceae. Interestingly, E. bangladeshi was identified in the South African population. Entamoeba was present in 27% (E. histolytica 8.5% (41/484), E. dispar 8% (38/484), and E. bangladeshi 4.75% (23/484) E. moshkovskii was not detected in the present study. We were also able to observe changes in the host microbiome and the parasite burden associated with E. histolytica infections in S. African diarrhea cases versus asymptomatic controls but not with E. bangladeshi or E. dispar. In E. histolytica positive samples the level of both parasite and P. copri were lower in non-diarrheal samples (p=0.0034) (Chapter 4). There is accumulating evidence that the inflammatory response contributes to injury. Little is known about the key parasite mediators of host mucosal immunopathology. This study hypothesized that migration inhibitory factor (MIF) mediates the destructive host inflammatory response seen in amebic colitis. To determine the role of EhMIF during amebic infection, we used a genetic approach to test the effect of EhMIF on mucosal inflammation. We found that EhMIF induces IL-8 secretion from intestinal epithelial cells. Mice treated with antibodies that specifically block EhMIF had reduced chemokine expression and neutrophil infiltration in the mucosa. In addition to antibody-mediated neutralization, mice infected with parasites overexpressing EhMIF had increased chemokine expression, neutrophil influx and mucosal damage. We also found that the concentration of EhMIF correlated with the level of intestinal inflammation in persons with intestinal amebiasis. Together, our results reveal a novel parasite mediator of mucosal inflammation and support MIF homologs as potential immunomodulatory targets (Chapter 5). To investigate the impact of co-infections on the outcome of amebiasis, we analyzed the co-occurence of E. histolytica with other enteropathogens known to cause diarrheal infections, such as Shigella/EIEC (IpaH), Campylobacter (cadf), Enterotoxigenic E. coli (STh), Norovirus GII and Adenovirus (Hexon). The results were compared with those obtained with E. histolytica that were not interacted with enteropathogens and with E. histolytica interacted with enteropathogens. The impact of multiple infections on the outcome of the infection was compared between nondiarrheal and diarrheal stool samples. It was found that co-infections with two pathogens were associated with diarrhea compared to single infections. Moreover, Norovirus GII, Campylobacter (Cadf) and co-infections were associated with diarrhea in the study population. This study did not show any significant impact of pathogens co-infecting with E. histolytica on the outcome of amebic infection (Chapter 6). The presence of secreted extracellular vesicles/Exosomes in Entamoeba histolytica was determined by using the Pathogenic ameba strains (HM-1:IMSS or HM-1:IMSS (Sub-strain-US) from petri’s lab to purify exosomes using the commercially available kit to isolate exosomes (total exosomes isolation kit). Our study for the first time revealed that E. histolytica does secrete Evs. This finding increases the appreciation that all organisms are likely to secrete these EVs (Chapter 7). However, the impact of these EVs on the pathogenesis of E. histolytica needs further investigations. Conclusion: This study has contributed significantly to our knowledge on infectious diarrhea and the diversity of Entamoeba species by providing new data on the rate and prevalence of Entamoeba diarrheal infections and their distribution in the South African population. Our study describes for the first time the presence of E. bangladeshi in the South African population. Furthermore, our results reveal a novel parasite mediator of mucosal inflammation and support MIF homologs as potential immunomodulatory targets. This study also, for the first time revealed that E. histolytica does secrete EVs. The results from this work will undoubtedly open an exciting research to establish a deeper understanding of the function and role of these vesicles in amebic infection. We encourage public health interventions like health education programs and improvement of sanitation and hygiene in these populations. Molecular diagnostics should be used for specific diagnostic in clinical settings. / NRF

To determine the relationship between infant-feeding practices and diarrhoeal infections, a descriptive survey was conducted to infants between six to 12 months of age. A guided interview was conducted to 105 mothers of infants who attended the health facilities of Mbabane, Swaziland. The results show that breast-feeding is routinely practiced by the majority of mothers and exclusive breast-feeding is very low, but supplementary feed in the form of formula or solids are introduced by the majority of respondents within the first three months of life. Infants who were given colostrum and breast milk had fewer diarrhoeal attacks. Other factors, for example education and cultural factors influenced the feeding practices and number of diarrhoeal attacks. It is recommended that breast-feeding should be promoted as an important intervention in the control of diarrhoea / Health Studies / M.A. (Nursing Science)

The purpose of this study was to investigate the environmental factors that impact on childhood diarrhoea in children under five years old in Akakikality sub city Addis Ababa, Ethiopia. Objectives of the study were to determine the prevalence of diarrhoea and to determine the environmental factors that impact on diarrhoea in children under five years old. A survey was done and a non-experimental approach was used in this descriptive and analytical quantitative study using a cross sectional study design. The instrument was a self-designed questionnaire. The target population for this study was all mothers or caretakers of children under five years found in the described study context – the sample size was 299. In this study 12.7% of the children had diarrhoea during the survey. Proper utilization of toilets, hand washing and safe storage of water in the households using narrow mouthed water containers were significant predictors of diarrhoea in the children. / Health Studies / M.A. (Public Health)

The purpose of this study was to investigate the environmental factors that impact on childhood diarrhoea in children under five years old in Akakikality sub city Addis Ababa, Ethiopia. Objectives of the study were to determine the prevalence of diarrhoea and to determine the environmental factors that impact on diarrhoea in children under five years old. A survey was done and a non-experimental approach was used in this descriptive and analytical quantitative study using a cross sectional study design. The instrument was a self-designed questionnaire. The target population for this study was all mothers or caretakers of children under five years found in the described study context – the sample size was 299. In this study 12.7% of the children had diarrhoea during the survey. Proper utilization of toilets, hand washing and safe storage of water in the households using narrow mouthed water containers were significant predictors of diarrhoea in the children. / Health Studies / M.A. (Public Health)

To determine the relationship between infant-feeding practices and diarrhoeal infections, a descriptive survey was conducted to infants between six to 12 months of age. A guided interview was conducted to 105 mothers of infants who attended the health facilities of Mbabane, Swaziland. The results show that breast-feeding is routinely practiced by the majority of mothers and exclusive breast-feeding is very low, but supplementary feed in the form of formula or solids are introduced by the majority of respondents within the first three months of life. Infants who were given colostrum and breast milk had fewer diarrhoeal attacks. Other factors, for example education and cultural factors influenced the feeding practices and number of diarrhoeal attacks. It is recommended that breast-feeding should be promoted as an important intervention in the control of diarrhoea / Health Studies / M.A. (Nursing Science)

The aim of this study was to assess and explore the household environmental health factors associated with the occurrence of diarrhoea in under five children in Sebeta town of Ethiopia, in order to develop environmental health strategies for prevention of childhood diarrhoea. A descriptive, quantitative, contextual and cross-sectional study, using stratified random sampling method, was used to conduct the research. The data collection was carried out from November 6 to 28, 2013 using structured interview schedules and a total of 477 households’ mothers/caregivers with under five children had participated in the study. In analyzing data, both bivariate and multivariate analyses were employed using SPSS software. The finding of the study shows that the prevalence of childhood diarrhoea was 9.9%. A number of risk factors including socio-demographic variables, water and hygienic practices, and knowledge risk factors showed significant association with childhood diarrhoea on bivariate analysis using chi-squared and Fischer's exact tests. Multivariate analysis using binary logistic regression was conducted to examine the significance of identified risk factors in bivariate analysis. The results of multivariate regression analysis shows that four variables were associated with risk of childhood diarrhoea; including type of toilet facility (AOR: 0.37; 95% CI 0.16 – 0.87; p=0.023), availability of specific place for handwashing (AOR: 0.40; 95% CI 0.18 – 0.90; p=0.026), availability of handwashing facility (AOR: 0.20; 95% CI 0.06 – 0.70; p=0.012) and mothers’ knowledge on diarrhoea causation (AOR: 3.09; 95% CI 1.24 – 7.68; p=0.015). Although childhood diarrhoea was found to be less prevalent as compared to national and regional prevalence rates, diarrhoea remains one of the causes of morbidity in children of the studied households. The findings of the study concludes that childhood diarrhoea has a number of environmental determinants, notably due to environmental health risk factors associated with lack of improved sanitation and hand-washing facilities and poor knowledge on diarrhoea causation. The study thus recommends that effective measures to curtail prevalence of diarrhoea in urban contexts should be substantially increased by enhancing urban sanitation promotion programmes with emphasis on accelerating universal access to improved sanitation and handwashing facilities, together with efforts in promoting proper hygiene behaviours. / Health Studies / D. Litt. et Phil. (Health Studies)

The aim of this study was to assess and explore the household environmental health factors associated with the occurrence of diarrhoea in under five children in Sebeta town of Ethiopia, in order to develop environmental health strategies for prevention of childhood diarrhoea. A descriptive, quantitative, contextual and cross-sectional study, using stratified random sampling method, was used to conduct the research. The data collection was carried out from November 6 to 28, 2013 using structured interview schedules and a total of 477 households’ mothers/caregivers with under five children had participated in the study. In analyzing data, both bivariate and multivariate analyses were employed using SPSS software. The finding of the study shows that the prevalence of childhood diarrhoea was 9.9%. A number of risk factors including socio-demographic variables, water and hygienic practices, and knowledge risk factors showed significant association with childhood diarrhoea on bivariate analysis using chi-squared and Fischer's exact tests. Multivariate analysis using binary logistic regression was conducted to examine the significance of identified risk factors in bivariate analysis. The results of multivariate regression analysis shows that four variables were associated with risk of childhood diarrhoea; including type of toilet facility (AOR: 0.37; 95% CI 0.16 – 0.87; p=0.023), availability of specific place for handwashing (AOR: 0.40; 95% CI 0.18 – 0.90; p=0.026), availability of handwashing facility (AOR: 0.20; 95% CI 0.06 – 0.70; p=0.012) and mothers’ knowledge on diarrhoea causation (AOR: 3.09; 95% CI 1.24 – 7.68; p=0.015). Although childhood diarrhoea was found to be less prevalent as compared to national and regional prevalence rates, diarrhoea remains one of the causes of morbidity in children of the studied households. The findings of the study concludes that childhood diarrhoea has a number of environmental determinants, notably due to environmental health risk factors associated with lack of improved sanitation and hand-washing facilities and poor knowledge on diarrhoea causation. The study thus recommends that effective measures to curtail prevalence of diarrhoea in urban contexts should be substantially increased by enhancing urban sanitation promotion programmes with emphasis on accelerating universal access to improved sanitation and handwashing facilities, together with efforts in promoting proper hygiene behaviours. / Health Studies / D. Litt. et Phil. (Health Studies)

PhD (Microbiology) / Department of Microbiology / Diarrhoea is still a major problem worldwide. Enteric pathogens such as Enteroaggregative E. coli (EAEC), Enteropathogenic E. coli (EPEC) and Enterotoxigenic E. coli (ETEC) have been reported to cause diarrhoea in children under the age of 5 years. The incidences of these pathogens are due to factors such as poor water quality, sanitation and hygiene practices. Infections with these pathogens result in diarrhoea and have been reported to result in severe disease outcomes more especially in children under 2 years of age. EPEC infections have been well studied using in vitro analyses, with studies highlighting the adherence traits, proteins and virulence genes involved in pathogenesis and inflammatory responses. EPEC is characterized by localized adherence with microcolony formation at the site of infection. In vivo studies have reported on human EPEC infection. However, the current animal models have not been able to replicate clinical outcomes (such as diarrhoea and weigh loss) of EPEC infection similar to humans. Therefore, there is still a need for a suitable small animal model that mimic clinical outcomes of human EPEC infections in vivo. Children living in poor environmental conditions are more susceptible to diarrhoeal pathogens. Furthermore, the incidences of children being exposed to co-infections (more than one pathogen at the same time) is relatively high. The EAEC/EPEC (A/P) and EPEC/ETEC (P/T) co-infections have been increasingly detected in children with and without diarrhoea. It has been suggested that patients infected with these co-infections might result in severe disease outcome than those infected with single pathogens. Pathogens are constantly evolving and the microbe-microbe interaction in the host can result in these pathogens competing for the same niche and thus result in increased virulence. Interaction of co-infections can lead to increased inflammatory responses thus affecting the infected host. The first objective of this study was to develop an EPEC murine model using weaned C57BL/6 mice that have been pretreated with antibiotic cocktail. Mice were orally infected with wild-type (WT) typical EPEC, bfp- and escN mutant strains. The WT had transient weight loss and wet stools with mucous; and the bfp- infected mice also had transient weight loss and bloody stool appearance. Increase in inflammatory biomarkers MPO, LCN-2, CRP, IL-6 and SAA were observed in the WT and bfp- infected mice. The mice infected with escN mutant did not exhibit any weight changes and the stools were similar to the uninfected mice. Furthermore, no inflammatory biomarkers were observed in mice infected with the escN mutant. Metabolic perturbations were observed in WT EPEC infected mice at day 3 post infection with the TCA cycle metabolites (reduced succinate, citrate, fumarate, cis-aconitate) being excreted at lower quantities indicating that the energy production in the infected mice was greatly affected. The second objective of this study was to determine the interaction between the P/T coinfections using in vitro and in vivo analyses. In vitro, human colorectal tumour 8 (HCT-8) cells were infected with single strains of ETEC, EPEC and both the pathogens and incubated for 3 hours. After infection the cells were analysed for bacterial adherence using real-time PCR. The single strains adhered at the same rate similar to the P/T coinfected cells. IL-8, as a marker of inflammatory response, was measured using ELISA. The results indicated that the P/T co-infected cells had a significant increase in IL-8 response higher than the single infections. The P/T co-infections were further analysed in vivo using the EPEC murine model developed in this study. Interestingly, mice infected with P/T co-infections developed severe diarrhoea accompanied with significant increased weight loss and some mice died during the 3-day infection period. The inflammatory responses MPO, LCN-2 and SAA were higher in the co-infected mice indicating a synergistic effect. The bfp and eltA virulence genes were significantly increased in the P/T co-infections. The third objective of this study was to determine the interaction between A/P coinfections using in vitro and in vivo analyses. HeLa cells and HCT-8 cells were infected with EAEC, EPEC and both the pathogens at the same time in order to determine adherence and inflammatory responses. EAEC adherence was higher than EPEC and A/P co-infections adherence. A/P co-infections did not have increased IL-8 response in HCT-8 cells when compared to EAEC alone. The virulence genes involved in EPEC adherence and Type 3 Secretion System (bfp, eae, tir, ler, per, espB and espA) were significantly reduced in A/P co-infected cells. An interesting adherence trait was observed between the A/P co-infections in HeLA cells, EAEC was found to adhere around EPEC altering the localized adherence pattern. The A/P co-infections were further analysed using the EPEC murine model developed in this study. The A/P infected mice had diminished weight changes and EAEC shedding was enhanced when EPEC was present. Faecal inflammatory biomarkers MPO and LCN-2 in A/P infected mice did not have any additive effect. The findings of this study contributed significantly to the knowledge of human EPEC infection in weaned C57BL/6 mice, highlighting clinical outcomes, inflammatory responses and metabolic perturbations. Furthermore, this study also highlighted the interaction of P/T and A/P co-infections using in vitro and in vivo analyses in order to determine the disease severity and outcomes. It was observed in this study that coinfections can result in either synergistic or antagonistic effects. Further studies are therefore, required in order to understand the underlying mechanisms that are involved during co-infections and this can further assist in the development of therapeutic interventions. / NRF

PhD (Microbiology) / Department of Microbiology / Globally, one in ten child deaths before the age of 5 years is due to diarrheal disease, causing almost 800,000 mortalities worldwide, which mostly occur in Sub-Saharan Africa and South Asia. Eighty-eight percent (88%) of diarrheal deaths worldwide are attributable to unsafe water, inadequate sanitation and poor hygiene. Unsanitary environments and poor hygiene practices allow diarrhea causing pathogens including viruses, bacteria and parasites to spread more easily. Norovirus (NoV) are now considered the most common cause of outbreaks of nonbacterial gastroenteritis. However, the factors which control the genetic diversity, the sources of sporadic NoV infections, the transmission and persistence of infection are poorly understood. Limited data are available for NoVs strains in South Africa, especially in rural and peri-urban areas. Despite the excessive burden of diarrhea disease in developing countries, NoVs outbreaks have been to date mostly reported in developed countries. Given that the contribution of the various pathogens to diarrhea may differ substantially between regions depending on local meteorological, geographic, and socio-economic conditions, there is a need to investigate intensively the role of viral agents associated with diarrhea in different settings in Africa continent. How would poor living conditions in rural setting impact the prevalence and genetic characteristics of Norovirus strains circulating Limpopo province is the research question of this study. ix To determine the prevalence and genetic diversity of NoVs strains circulating in the rural communities in the Limpopo Province, South Africa and investigate the genetic relationship between NoVs strains, a cross-sectional study was performed on human stools collected from rural communities. We used qualitative variables of poor living environmental conditions including type of water used at the household of child’s parent or guardian, use of toilet seat, presence of livestock at the household and parent employment status to assess possible environmental risk factors of NoV infection within the study area. Prior to this prospective study, we conducted a systematic review of the PubMed and EMBASE databases for published articles of Human NoVs in Africa between 1990 and 2013 in order to assess the contribution of Human NoVs to diarrhoeal diseases in Africa. This review provides a picture of Human NoVs studies in Africa and reveals that unreported sporadic gastroenteritis cases of Human NoVs are common in Africa. Most are community-associated infections reported from urban settings. Possible environmental transmission routes have been documented. Combined environmental and clinical studies are required for targeted actions to control transmission of Human NoVs in Africa. Between July 2014 and April 2015, outpatient children under 5 years of age from rural communities of Vhembe district, South Africa, were enrolled for the study. A total of 303 stool specimens were collected from those with diarrhea (n=253) and without (n=50) diarrhea. NoVs were identified using real-time one-step RT-PCR. Nucleotide sequencing methods were performed to genotype the strains. Phylogenetic analyses x were performed to compare identified NoVs genotypes to the worldwide circulating strains. One hundred and four (41.1%) NoVs were detected. NoV detection rates in symptomatic and asymptomatic children (OR = 1.24; 95% CI 0.66 – 2.33) were not significantly different. Comparison of the median CT values for NoV in symptomatic and asymptomatic children revealed significant statistical difference of estimated GII viral load from both groups, with a much higher viral burden in symptomatic children to our knowledge this is the first study reporting on the differences in estimated viral load of GII and GI NoV positive cases and controls. The study findings may have implications for the diagnosis of NoV disease and future vaccine development, which may only need to consider GII as the genogroup associated with diarrhea in the South African population. Sequence analyses demonstrated multiple NoV genotypes identified in rural communities of Vhembe district. The most prevalent NoV genotypes were GII.4 Sydney 2012 variants (n=7) among the capsid genotypes, GII.Pe (n=9) among the polymerase genotypes and GII.Pe/GII.4 Sydney 2012 (n=8) putative recombinants among the RdRp/Capsid genotypes. Two unassigned GII.4 variants and an unusual RdRp genotype GII.P15 were found. With note, the rare GII.P15 identified in this study, has a common ancestor with GII.P15 strain from Japan previously reported as GII / untypeable recombinant strain implicated in a gastroenteritis outbreak. To our knowledge this is the first report of this unusual genotype in the African continent. Though not proven predictive of diarrhea disease in this study, the high detection rate of NoV reflects the substantial exposure of children from rural communities to enteric xi pathogens possibly. However in this study no risk factor has been found between NoV positive and qualitative environmental variables of poor living conditions in rural setting. The results also suggest that the difference between asymptomatic and symptomatic children with NoV may be at the level of the viral load of NoV genogroups involved. The findings highlighted NoV genetic diversity and revealed continuous pandemic spread and predominance of GII.Pe/GII.4 Sydney 2012, indicative of increased NoV activity. An unusual RdRp genotype GII.P15 and two unassigned GII.4 variants were also identified from rural settings of the Vhembe district/South Africa. NoV surveillance / NRF