Department of Biochemistry and Microbiology
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Item Open Access Detection of Cryptosporidium species in stools of HIV/AIDS patients in Bela-Bela, South Africa(2010-06) Makuwa, Stenly Modupi; Bessong, P. O.; Samie, A.; Potgieter, N.Item Open Access Drug Resistance Mutations in Naive HIV-1 South African Patients, and Construction of Molecular Clones to Phenotype Putative Resistance Mutations(2009-03) Mavhandu, Lufuno Grace; Bessong, P. O.; Rekosh, David; Hammarskjold, Marie-LouiseIn countries such as South Africa where access to therapy is progressing data is required on patterns of resistance and evolution of resistance. Thirty protease (PR) and 31 reverse transcriptase (RT) amino acid sequences of HIV primary isolates from drug naNe patients from rural settings in South Africa were examined for resistance mutations. Samples were collected between May and August 2007. Phylogenetic analysis showed that all the sequences were HIV-1 subtype C in both the protease and reverse transcriptase genes. The mean genetic distances among the sequences were 0.0170-0.0786 for the protease, and 0.0045-0.0890 for the reverse transcriptase. However, it was noted that 3 pairs of samples 07VGNF5ZA and 07VGNF6ZA, 07VGNF7ZA and 07VGNF8ZA, 07VGNF10ZA and 07VGNF13ZA did not show any genetic variability among their protease sequences. No major resistance mutation was observed among the protease sequences. However, the following minor resistance mutations were noted: L101N (3/30), A71T (1/30), and T74S (2/30). Examination of the reverse transcriptase gene for resistance mutations reveal the presence of V118I (1/30), V179D (1/30), K103N (2/30). Most of the RT sequences were wild-type, although V118I (3.3%) and k103N (6.7%) associated with resistance to lamivudine and nevirapine, respectively, were observed. In summary, this study has shown that most of the viruses in Limpopo Province, representing the northeastern part of South Africa are HIV-1 subtype C, and that the prevalence of resistant mutations among the drug na"fve patients is still low. Although combination antiretroviral therapy has resulted in a considerable improvement in the treatment of human immunodeficiency virus type 1 (HIV-1) infection, the emergence of resistant virus is a significant obstacle to the effective management of HIV infection and AIDS. Systems to be used in the testing of phenotypic drug resistance and susceptibility are being developed. These may intimately be used in guiding therapy to improve long term suppression of HIV replication. Two proviral chimeric clones making use of pMJ4 and pNL4-3, and two vector plasmids which deletions of sequences encoding HIV-1 protease or reverse transcriptase were constructed for cloning of HIV-1 PCR products. Growth of constructs was monitored by p24 antigen production. Susceptibility to protease and reverse transcriptase inhibitors was measured by using resistance test vectors that contain a Luciferase indicator gene. Cells were co-transfected with packaging plasmids, pluc, and pEnv, resulting in the production of virus particles that were used to infect target cells. Luciferase activity was measured following a single round of replication. The chimeric constructs MJ4 carrying the NL4-3 Apal-Hpal cassette (MJ4/NL4-3) and NL4-3 carrying the MJ4 Apal-Hpal cassette (NL4-3/MJ4) were successfully developed as shown by restriction digestion analysis. Considering growth of the constructed chimeras NL4-3/MJ4 was better than MJ4/NL4-3 although not robustly. Good p24 production was obtained from all four gap-pol plasmids. MJ4/NL4-3 worked better in delivering luciferase to the target cells while NL4-3/ML4 appeared totally devoid of any infectivity. The vectors pCMVGagPol(MJ4)-RREr and pCMVGagPol(NL4.3)-RREr were created and both expressed the viral gag-pol protein. Viral inhibition test showed that the vectors can be inhibited by NRTI, NNRTI and Pl. Inhibition was seen in all drugs in different concentrations indicating that the system works. The results showed that vector systems constructed can be used to evaluate putative drug resistant mutations, coding for resistance to protease and reverse transcriptase inhibitors, detecte� in patient viruses. In addition, the system can also be used to evaluate candidate drugs and assist in the development of new drugs that are active against resistant HIV-1 virus.Item Open Access HIV co-infections with cytomegalovirus, hepatitis c virus and human papillomavirus in northern South Africa(2014-11-03) Rikhotso, Mikateko; Bessong, P. O.; Tebit, Denis.Item Open Access Immunodulation of inflammation in a murine pnemococcal sepsis model(2013-10-01) Musie, Mbulaheni Edgar; Bessong, P. O.; Scheld, M. W.Mortality from pneumococcal infections remains high, despite the development of potent antibiotics. Antibiotic resistance among pathogens including S. pneumoniae calls for new therapeutics. lmmunomodulation represents a novel approach to antimicrobial therapy that depends on bolstering host immunity, rather than direct antimicrobial activity. The use of innate immune stimulation to improve survival has previously been described for Gram negative pathogens. The effect of TLR4 stimulation on survival in mice during lethal S. pneumoniae, serotype 2 infections was examined. C57BL/6, BALB/c, CBA/CaHN-Btkxid'/J, A/J, Rag-1 KO, IL-10 KO , C3H/HeN and C3H/HeJ mice were inoculated intravenously with a lethal dose of 106 cfu of S. pneumoniae serotype 2 48 hours after treatment with five doses of 10ug highly purified LPS or vehicle (PBS) at 12 hours interval. Another group of LPS or PBS treated C57bl/6 received 25 mg/kg ceftriaxone at 6 hours post infection. Survival was monitored for 5 days. Blood samples were collected at different time points (6h, 12h and 24h) after bacterial challenge for bacteriological examinations, serum cytokine measurements, and biochemical assays for liver function. Spleens were harvested for flow cytometric analysis of splenic lymphocytes or NK activation. Innate stimulation with LPS reduced systemic bacteremia by at least four logs in LPS- pretreated C57BL/6 (104 v.s 108 cfu/ml) mice compared with controls during the recorded course of infection. Death in experimental controls occurred within 48 hours. Reduced bacteremia corresponded with improved survival in all 3 strains. Survival for LPS-treated C57bl/6, Balb/c and C3H/HeN was 90% (N=29; p=0.001), 50% (N=14; p = 0.017) and 60% (N=8; p=0.009), respectively, and mortality for controls was 100% for all the strains. Mortality for ceftriaxone-treated C57bl/6 was 75% (N=12, p=0.0018). Survival for LPS-treated and non-treated mice was 80% (n=10) and O % (n= 6) in CBA/CaHN-Btkx,dJ/J mice and 85% (n=14) and O % (n=9) in A/J mice which suggests that deficiency in complement and antibodies did not ablate survival benefits. Lack of B and T cells ablated survival benefits of LPS pretreatment in RAG-1 KO mice even when mice received a high dose of LPS. These results suggest that T cells are responsible for protection and B cells are partially involved in tolerance. The level of TNF-a, IFN-g, IL-12 (p40/70), IL-6, IL-1a/b, IL-10, Eotaxin, MCP-1, and MlP-1a/b were attenuated in C57BL/6 LPS-treated mice 12 hours after infection compared to untreated group. Pretreatment with a low dose of LPS also prevented hypoglycemia (glucose level was 149 vs. 45) and liver failure (reduced the AST [139 vs. 445, p<0.025] and ALT [27 vs. 129, p<0.03] to near baseline) induced by S. pneumoniae infection. LPS pretreatment restored the splenic NK population and decrease their activation demonstrated by lower mean percentage of CD69 expression of 35.3 vs 97.3 (p<0.05) than of control infected mice. This study demonstrates a survival benefit from TLR4 stimulation prior to infection with S. pneumoniae. It provides evidence that induction of profound LPS tolerance, despite reducing cytokine production, improves host defense against infection with virulent strain of S. pneumoniae. TLR4 agonist activity can be potentially exploited to provide short-term resistance to infectious challenge such as might occur in the setting of exposure to bio-threat agents or epidemic infections. These observations have implications for prophylactic treatment after an index case is identified or as adjunctive therapies with antibiotics. It is plausible that compounds capable of stimulating early host defense and microbial clearance, but not the latter phases of inflammatory tissue injury associated with sepsis, may be advantageous. The effect of LPS postinfection, as adjunctive therapy, in a lethal murine model of systemic Streptococcus pneumoniae was investigated. Mice were inoculated intravenously with a lethal dose of 107 cfu of S. pneumoniae serotype 2; and treated with a combination of 25 mg/kg Ceftriaxone (i.p) and 10ug of LPS (i.v) at 6 hours post infection or ceftriaxone alone. The survival of mice treated with the combination of ceftrioxone and LPS was 80% in C57BL/c (n=20), compared to 40% of mice receiving ceftrioxone only (n=20) and 0% of vehicle controls (n=10) (p=0.0001). Treatment with a non-lethal dose of LPS beginning at 3 hours after infection reduced mortality by 50% in C57BL/6 mice (n=7) (p=0.03) and no survival benefits (100 % mortality) were observed when LPS alone was administered 6h after bacterial challenge (n=5) (p=0.07). Administration of LPS in combination with ceftriaxone resulted in a significant reduction of bacteremia at 7h (2.5 ± 1.6 vs 6.6 ± 1.6 log10 CFU/ml, p=0.002 and 12h (0.5 ± 1.0 vs 4.9 ±1.6 log10 CFU/ml, p=0001) after infection, compared with animals treated with ceftriaxone alone and also decreased significantly the level of TNF-alpha, IFN-gamma, IL-12p70, MIP-1 alpha, IL-1 beta, and RANTES 12h after bacterial challenge and elevated levels of IL-2, IL-3, IL4, and KC were elevated 12h after bacterial challenge compared to those treated with ceftriaxone alone (p<0.05). In this mouse model, stimulation of TLR4 by highly purified LPS as an additional therapy to the antibiotic Ceftrioxone achieved a survival benefit in the reduction of mortality in severe S. pneumoniae infections. These results show the potential for innate immune stimulation as adjunctive therapy in the treatment of pneumococcal infections and these results warrant further studies. The treatment of pneumococcal infections- such as meningitis, pneumonia, and bacteremia- with 13-lactam antibiotics can result in the paradoxical enhancement of inflammation as a result of the release of proinflammatory bacterial cell components, such as lipoteichoic acid, peptidoglycan, and bacterial toxin pneumolysin. Adenosine has strong immunosuppressive and anti-inflammatory effects mediated by A2A receptor (A2AR) expressed on immune cells. We investigated the effect of A2AR agonist, ATL313, as adjunctive therapy in a lethal C57bl/6 mouse model of systemic Streptococcus pneumoniae. Female C57bl/6 mice were inoculated intravenously with 107 colony forming units (CFU) of S. pneumoniae and treated with the A2A AR agonist ATL313 (5ug/kg or 25ug/kg), or PBS, at t=1, 6hrs and then every 24 hours after bacterial challenging spanning 48 hours. To test whether the effect of ATL313 was through functional A2A adenosine receptors and if hematopoietic cells are important in the protective effect of A2AAR, C57BL/6 A2A-receptor-deficient mice, and chimera were used and groups of C57BL/6 mice were co-injected intraperitoneally with ATL313 and ZM243185 (antagonist ligand selective for the adenosine A2Areceptor). Survival was monitored for 7 days. Blood samples were collected at 7h and 12h after bacterial challenge for bacterial counts and cytokine measurements. ATL 313 (2.5- 25ug/kg), had no survival benefits (100% mortality) when administered without antibiotic ceftriaxone. But when administered in combination with antibiotic ceftriaxone at 6h after infection, 25ug/kg ATL313 increased survival (89%, n=26) of mice compared to ceftriaxone alone treated mice (23%, n=31). Survival benefit was reversed after treatment with ZM241385 (25%; n=8/group) and after ATL313 treatment in A2A AR KO and chimeric mice (17%; N=6/group) compared to ATL 313 treated C57BL/6 mice (89%; p<0.001). Treatment with ATL313 plus ceftriaxone reduced bacteremia by 2.1 log10 and 3.3 log10 fewer bacteria at t=12h and 24h, respectively, than those of mice treated with ceftriaxone alone (p<0.05); and also decreased level of TNF-alpha (p=0.010), IL-6 (p=0.027) , IFN-gamma (p=0.001), IL- 12p70 (p=0.031), MIP-1 alpha/ beta (p=0.005), IL-10 (p=0.022), IL-1 beta (p=0.0071), IL-5 (p=0.014), and Eotaxin (p=0.023) at 12h after bacterial challenge compared to those treated with ceftriaxone alone (p<0.05). In this study, ATL313 in association with ceftriaxone reduced both the magnitude and the duration of inflammation and increased survival in a lethal murine model of systemic Streptococcus pneumoniae. The anti -inflammatory effects of A2A AR agonist ATL313 may be particularly useful for the treatment in bacterial diseases when antibiotics have been administered. Further investigations are warranted to assess the therapeutic benefit of A2A receptor agonists as an adjunctive agent to antibiotics in the treatment of pneumococcal infection and also to further understand the mechanism of protection involved in the ATL 313 protection. The mechanism of protection was investigated by characterizing the effects of A2A R activation on the NK cell activity in pneumococcal sepsis model. Natural killer (NK) cells are potent mediators of the innate immune response and their effect is attributed to both direct cytotoxiciy and indirect stimulation of macrophages by cytokine signaling. A2A adenosine receptor signaling has been implicated in adenosine-mediated inhibition of cytokine production and cytotoxic activity by activated natural killer (NK) cells, although the process of NK cell granule exocytosis is apparently suppressed via a distinct and as yet uncharacterized adenosine receptor. There is evidence that NK cells can be activated during septic events and may contribute to the pathogenesis of this condition via the secretion of IFN-y, which acts primarily to augment macrophage function. Whether modulation of NK activity during pneumococcal sepsis could improve pneumococcal sepsis outcomes and the . ability of an A2A AR agonist to modulate the NK response to improve sepsis has not been tested or available data is scanty and controversial. In this study, mice were inoculated intravenously (i.v) with a lethal dose (107 cfu) of S. pneumoniae serotype 2 and treated with a combination of ceftriaxone and 25ug/kg ATL313 or ceftriaxone alone. To assess the importance of NK cells in pneumococcal sepsis, C57BU6 and Ja281 mice were treated with a single i.p. injection of 200µg anti-NK1.1 (PK136) antibodies 2 days before bacterial challenge. Survival was monitored for seven days. Spleens were harvested and processed to assess NK cell activation (expression of markers CD69 and NK1.1, perforin and intracellular interferon gamma) using flow cytometry. Treatment with the combination of ATL and ceftriaxone (n=4) showed a high level of NK cell populations (2.063 ± 0.277) compared with ceftriaxone alone treated group (1.648 ± 0.566) (p=0.535); down regulated CD69 expression, mean percentage 32.78 ± 6.327 vs 70.03 ± 8.163) (p=0.011); reduced the release of perforin (3,4% vs 0.21% )(p<0.05); and also reduced the level of intracellular and plasma IFN-y (0.55% compared to 12.1%) (p=0.02). Blockade of NK cell activation with the treatment with PK136 and antibiotics resulted in 80% survival compared with 40% of mice that received ceftriaxone alone (p=0.01) in pneumococcal animal sepsis model. The results of the study implicate NK cells as one of the mediator of inflammation sensitive to regulation by A2AR activation. Furthermore, profound protection is imparted when this early event in the inflammatory cascade is inhibited by A2AR activation. Therapeutic agents that inhibit the activity of NK and NKT cells may therefore hold promise in the treatment of pneumococcal infection i.e. it is possible to reduce cytokine levels more substantially by targeting an upstream event in the cascade (NK cell activation). A further study to improve understanding on the negative impact of ATL313 on NK cell function during sepsis is needed. These results suggest that analysis of NK cell activation during the septic process may yield insights into the interactions that occur between NK cells and the monocytes or macrophages compartment during the course of severe bacterial infections. However, the precise molecular and functional mechanism by which the negative impact of NK cells occurs is not known and an improved understanding of NK cell function during sepsis is needed. To investigate the role of decreased NK cell activation by A2AAR treatment in the improved outcomes from experimental sepsis. We need to study the impact of A2AAR agonist on NK cell trafficking during sepsis i.e. study how NK cells impact the pathophysiology of sepsis and determine where NK cells traffic during experimental sepsis and where and to what extent they proliferate. This will permit clarity on whether organ damage corresponds to areas where NK cells traffic. Furthermore, the understanding gained in this process will potentially provide tools with which we may in turn control the immune system when it runs awry in sepsis. Growing antibiotic resistance and paradoxical enhancement of lethality as a result of the release of proinflammatory cell components after antibiotic treatment prompted our study of Etanercept (a tumor necrosis factor alpha neutralizing agent) as an adjunctive therapy against systemic S. pneumoniae. TNF is a major therapeutic target since it appears early and is related to disease severity. Balb/c mice were inoculated intravenously with a lethal dose (107 cfu) of S. pneumoniae serotype 2 and then treated with 25mg/kg ceftriaxone with/out 100ug of Etanercept (i.v) at 4 hours after bacterial challenge. Survival was monitored for several days. Serum samples from mice were analyzed for inflammatory cytokines with bead-based multi-analyte flow cytometry at 24 hours after challenge. Etanercept as adjunctive therapy significantly improved bacterial clearance and survival (70%, n=16) compared with 25% (n=20) survival of ceftriaxone alone treated mice (p=0.001). Inflammatory cytokines, TNF-alpha (787 v/s 5801 pg/ml), IFN- gamma (504 v/s 2642 pg/ml), interleukin-6 (IL-6) (255 v/s 2806 pg/ml), were reduced in Etanercept treated mice compared with ceftriaxone treated mice (p<0.05). In conclusion, this study indicates that early TNF-alpha is a critical component of antibacterial host defense and late neutralization of that same endogenous TNF alpha provides survival benefits when combined with ceftriaxone. Moreover, the current wave of enthusiasm regarding the treatment of patients with anti- TNF antibodies or soluble receptors must be tempered by the awareness of potential infectious complications that may occur as a result of this specific therapy. The study outlines the potential usefulness of Etanercept as an adjunctive therapy for pneumococcal infection and underlines the need for further research in the field. Therefore, more investigations are warranted, to further assess the therapeutic benefit of etanercept as an adjunctive agent to antibiotics in the treatment of pneumococcal infections in a clinical set-up.Item Embargo Molecular characterization of entamoeba species and impact of host genetic polymorphisms on amoebiasis(2022-11-10) Davhana, Ndivhudzannyi Caroline; Samie, A.; Bessong, P. O.; ElBakri, A. M. K.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.Item Open Access Molecular detection of norovirus GI ang GII genotypes in children less than two years of age and impact on child growth(2014-11-03) Moloro, Glenton Thabo; Samie, A.; Bessong, P. O.Background: Noroviruses (NoVs) are, after Rotaviruses, the second most common causative agents of acute gastroenteritis in young children. In South Africa, NoV was first reported in 1993 in gastroenteritis associated with the consumption of salad. NoV antibody prevalence was later reported in both urban and rural South African populations, and Norwalk and Hawaii strains were detected. However very few studies have been conducted to identify strains of oV in the country and their impact on child growth is not well understood. This study aims to identify common NoV genogroups and the strains that are more prevalent in diarrhoeal and non-diarrhoeal stool samples of children less than 2 years of age in rural areas in Vhembe district, South Africa, using reverse transcriptase Real Time PCR. Moreover, an inve tigation of the impact of different genotypes on child group was determined. Methodology: In the present study, 185 children were recruited of whom 88 were males and 97 females. Of all the children 141 had experienced diarrhoea at least once while 44 never had diarrhoea. Samples were treated with Sodium Chloride (NaCl), and RNA was purified from them using the QiaAmp viral RNA purification kit and stored at -20°C. Following RNA purification, samples were subjected to One step reverse transcriptase real-time PCR to detect oV genotypes. Positive samples were further run in reverse transcriptase PCR using specific primers that amplify genogroup-specific sequences of the N-terminal and shell (N/S) region of the NoV VPI gene, and the cDNA synthesized was run in a conventional PCR. uccessfully amplified conventional PCR products for NoV GI and NoV GII were sequenced and the sequences aligned and compared with the existing sequences in the GeneBank, in order to determine the genetic relationship and variability of strains of NoV in Vhembe district, South Africa. Results: In the study, 708 samples were tested, of which 256 (36.2%) were diarrhoeal samples and 453 (63.8%) were non-diarrhoeal samples. Norovirus GIi was the most common genogroup detected in the present study. Among the 256 diarrhoeal samples, 34 (24.1%) were GI, and 93 (66.0%) were GIi. Five (13.2%) of the children who were born stunted presented with NoV GI, whilst 22 (57.9%) of them presented with NoV GIi at 12 months. The number of infection increased with the children's age. About 12 (61.7%) of the children who were stunted presented with NoV GI and 29 (61.7%) of the children who were stunted presented with NoV GIi. Norovirus GIi infections showed to be the highest in June (48.1%), October (48.0%) and November (50.6%) whilst infection with Norovirus GI was the highest in October (20.0%). Analysis of sequences showed that the NoV strains differed in their sequences up to 40%. Comparison of study strains with reference has shown the difference in the sequences of the strains, indicating a high mutation rate among Norovirus strains. Discussion: The present study shows that infection with GIi is the most common variant among children and contracted easily during the winter months of the year. Reverse-transcriptase RT-PCR can be recommended as a rapid and sensitive method that can be used to detect NoV, in order to give a quick response to eliminate and prevent infection. Though sequences detected are of similar strains, their nucleotide alignment varies due to the high mutation rate of NoV. Mutations in NoV can cause a problem with vaccine development or an alteration to confer vaccination in order to prevent infection, and spread ofNoV. Conclusion: The present study shows how NoV affects the children's health and has an effect on the child's nutritional state and growth. It also shows that Norovirus strains differ from one country to another, this also include seasonal variation of Norovirus genogroups, which calls for a lot of attention on vaccine development. Norovirus has shown to be more active during winter season and has NoV GII as a predominant strain of which other studies also agree with these results.Item Open Access Molecular diagnosis and characterization of clinical isolates of entamoeba histolytica, giadia lamblia and cyptosporidium species from the United Arab Emirates and South Africa(2014-11-03) ElBakri, Ali Mohammed Kamal; Bessong, P. O.; Potgieter, N.; AbuOdeh, R. OItem Metadata only Screening of herbal preparation (Pheko) for anti HIV-1 replication properties(2015-01-14) Matume, Nontokozo Daphney; Bessong, P. O.; Sekhoacha, M.Item Open Access Serologic markers and molecular pidemiology of HBV in an HIV infected cohort from Cameroon(2016-05) Magoro, Tshifhiwa; Bessong, P. O.; Mavhandu, Lufuno G.; Masebe, Tracy M.See the attached abstract below