dc.description.abstract |
Background: Enteric infections constitute a serious public health problem globally,
especially in low and middle-income countries, particularly in areas of poor sanitation, low
socio-economic conditions, inadequate water supply and poor hygiene practices. South
Africa is one of the developing countries that has been significantly impacted by diarrheal
infections, many of which are due to Entamoeba species. It is still not clear why some
individuals once infected with E. histolytica develop clinical amoebiasis while others do
not show any symptoms. It has been suggested that both the parasite and host factors
play a significant role in the outcome of E. histolytica infection. However, this does not
explain why only few infected individuals develop symptomatic diseases. This suggests
that there are other factors to explain this transition.
Tumor necrosis factor-α (TNF-α) is a multifunctional pro-inflammation cytokine which has
been considered as one of the pathogenic factors for various diseases. Several studies
have reported an association between TNF-α polymorphisms and inflammatory diseases.
However, no study has been carried out on the association between polymorphisms in
the promoter of the TNF-α gene and high risk of E. histolytica infections.
Aim and objective of the study: The overall aim of the study was to determine the
molecular characteristics of Entamoeba species in relation to the occurrence of diarrhea
among children and determine the impact of host genetic polymorphism on the
occurrence of amoebiasis. This aim was addressed by the following primary objectives:
a. To investigate the prevalence, distribution and genetic diversity of Entamoeba species
and other parasites circulating among children in the Northern part of South Africa.
b. To investigate the genetic polymorphism of TNF-α gene promoter region in a cohort of
children in Northern South Africa.
c. To identify any association between TNF-α promoter gene polymorphism with diarrhea,
vomiting, fever, acute lower respiratory infection, gender and malnutrition.
d. To identify any potential association that may exist between TNF-α promoter gene
polymorphism and parasitic infections.
Brief methodology and results: This study was nested within the Madi project and
Malnutrition and Enteric Diseases project (MAL-ED) South Africa and received approval
from the Health, Safety and Research Ethics Committee of the University of Venda. The
stool samples were from Madi project and the blood samples were from the MAL-ED
project. A total of 394 stool samples were collected in selected household with children
under the age of 5 years who were randomized to receive a silver-impregnated ceramic
water, a silver-impregnated ceramic disk, a safe-storage water container, or no
intervention, and were followed quarterly for two years from Dzimauli rural communities
of South Africa in Limpopo province. All the stool samples were observed under a light
microscope for the presence of intestinal parasites. In order to accurately detect
Entamoeba species in all faecal samples, polymerase chain reaction (PCR) protocols
were performed using genus-specific PCR primers based on small-subunit rRNA gene
sequences for E. polecki, E. chattoni, E. dispar, E. histolytica, E. hartmanni and E. coli.
Real-time PCR protocol was also used to detect and identify the specific Entamoeba
species such as E. histolytica, E. dispar, E. bangladeshi and E. moshkovskii in order to
gain information on their accurate prevalence, distribution and genetic diversity in the
community.
The present study reported an overall prevalence of intestinal parasites including
Entamoeba species, Strongyloides stercoralis, Cystoisospora belli and Ascaris
lumbricoides as determined by microscopy to be 140 (35.5%), 138 (35.0%), 114 (28.9%)
and 78 (19.8%), respectively. The genus-specific PCR was positive for Entamoeba spp.
in 140 (35.5%) samples. Real time PCR detected E. histolytica in about 4% of the samples
while E. moshkovskii occurred in about 9% of the samples. Results identified by qPCR
showed that children in silver-impregnated ceramic water filter group at month 12 had
higher E. moshkovskii infection 6 (13.0%), while those in intervention group had lower E.
moshkovskii infection 3 (6.8%). None of the participants were infected with Entamoeba
bangladeshi. Sequence analysis showed a wide variety of Entamoeba species with E.
coli and E. polecki appearing to be the most common organisms followed by E.
moshkovskii and E. dispar. Further studies using next generation sequencing
technologies are needed to understand the real importance of each of these organisms
in the community in terms of the pathogenesis of amoebiasis (Chapter 3).
TNF-α is a multi-functional pro-inflammatory cytokine and a primary mediator involved in
the early phase of the cytokine cascade and plays an important role in the initiation and
regulation of the immune response. The present study aimed to investigate the genetic
polymorphism of tumor necrosis factor-α (TNF-α) gene promoter region in a cohort of
children in Northern South Africa. A total of 199 blood samples were evaluated from
children who were part of the MAL-ED study cohort. The TNF-α gene at positions
‑1031T/C and ‑308G/A were genotyped by polymerase chain reaction–restriction
fragment length polymorphism (PCR–RFLP) assay and confirmed by DNA sequencing. Out
of these 199 participants, 94(47.2%) were males and 105(52.8%) were females. Of all
the children, 23(11.6%) had low birth weight. A strong association was noted between the
CC homozygous genotype at position -1031 and children with diarrhea (P=0.043,
OR=4.167, 95% CT=0.942-18.43); whereas TC heterozygous genotype was significantly
common in healthy children with no diarrhea (P=0.019, OR=0.446, 95% CT=0.226-
0.882). The T-allele was significantly common in children with diarrhea (P=0.043,
OR=0.240, 95% CT=0.054-1.062). A strong association was also noted between the TT
homozygous genotype at position -1031 and children with dehydration (P=0.014, 95%
CI= 1.224-12.443). A strong association was noted between the GA heterozygous
genotype at position -308 and children with diarrhea (P=0.040, OR=2.579, 95%
CT=1.019-6.528); while AA homozygous genotype was significantly common in healthy
children with no diarrhea (P=0.012, OR=0.3420, 95% CT=143-0.815). Heterozygous GA
genotype was more common among healthy children with no dehydration and the result
was statistically insignificant (P = 0.894, X2 = 0.018, OR = 1.095, 95% CT = 0.288-4.168);
while a strong association was also noted between the heterozygous GA genotype at
position -308 and children with vomiting (P=0.019, OR=2.694, 95% CT=1.160-6.256).
The G allele was significantly common in children with vomiting (P=0.010, OR=2.816,
95% CT=1.263-6.279). Our study has for the first time revealed that the -1031(T/C)
polymorphism of TNF-α promotor gene is associated with diarrhea, dehydration and
acute lower respiratory infection among children at five years of age, while the -308(G/A)
polymorphism was associated with diarrhea and vomiting among these children (Chapter
4).
Intestinal parasitic diseases are common in developing countries including South Africa
and have been documented to be the most common in children under the age of five. The
present study aimed to identify any potential association that may exist between TNF-α
promoter gene polymorphism and parasitic infections. A total of 199 blood samples was
evaluated from children who were part of the MAL-ED study cohort. The DNA was used
to investigate polymorphism in the promoter region of the TNF-α gene at position
1031T/C. The polymorphisms were detected by polymerase chain reaction–restriction
fragment length polymorphism (PCR–RFLP) assay. The TC genotype at position -1031
was significantly higher in healthy controls children than in children who were infected
with Entamoeba species (59.9% vs 29.4%, P = 0.015) and Entamoeba coli (59.1% vs
30.8%, P = 0.046), indicating that TC genotype may be protective against Entamoeba
infections and Entamoeba coli infections. The CC genotype at position -1031 was more
common among children with parasite and diarrhea and the results was statistically
significant (P = 0.04). This study has revealed that the CC genotype may be is a risk
factor for symptomatic parasitic infections, while the TC genotype might be protective of
Entamoeba infections among children in Dzimauli community (Chapter 5).
Overall conclusion: The present study demonstrated new data on the prevalence,
distribution and genetic diversity of Entamoeba species and other parasites in Northern
South Africa. Several Entamoeba species are circulating in the region and the importance
of E. moshkovskii in the pathogenesis of amoebiasis seems to be underestimated. This
study also revealed for the first time that the -1031(T/C) polymorphism of TNF-α promotor
gene is associated with diarrhea and acute lower respiratory infection, Entamoeba
species and Cyclospora infections among the children in Dzimauli community, while -
308(G/A) was associated with vomiting and overall illness. Information generated from
this study will be useful for understanding the transmission, source of infection and clinical
outcome of infection with parasitic infections. However, larger studies need to be
conducted in order to confirm these findings. |
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