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.
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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
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:univen/oai:univendspace.univen.ac.za:11602/1258 |
| Date | 21 September 2018 |
| Creators | Ngobeni, Renay |
| Contributors | Samie, A., Gilchrist, Carol A. |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 1 onlne resource (xxxi, 233 leaves: color illustrations) |
| Rights | University of Venda |
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