Doctoral Degrees (Medical Virology)

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Analysis of HIV-1 diversity and inflammatory markers in HIV-associated neurocognitive disorders (HAND).
(Stellenbosch : Stellenbosch University, 2022-04) Ruhanya, Vurayai; Engelbrecht, Susan; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology.
ENGLISH SUMMARY: HIV-associated neurocognitive disorders (HAND), which involve impairment or disruption of neurocognitive functioning have become one of the most frequent complications in adult HIV-1 infections with global estimates ranging from 42% to 45%. The screening and diagnosis for HAND relies on multiple clinical and neuro-psychometric methods. However, these methods have a low reliability because they are not precise as most of possess inadequate psychometric properties and diagnostic accuracy. Therefore, this study aimed to describe and characterise viral and immunological determinants of HAND and evaluated their relationship with specific clinical, neuromedical and neuropsychological data to identify putative easy-to-measure biological markers for diagnosis of the condition. This study demonstrated that higher peripheral blood lymphocyte-derived HIV-1 proviral DNA is a predictor of global and domain-specific neurocognitive impairment among individuals infected with HIV-1 subtype C. The study also determined proviral load cut-off /threshold value for neurocognitive impairment and associated diagnostic accuracy. It also identified IP-10 and RANTES as a plasma chemokine bio-signature for HIV-associated neurocognitive impairment with diagnostic accuracy comparable to standard psychometric tests used to screen and describe severity of HAND. In addition, the study identified 3 viral genetic signatures for cognitive impairment, namely Lysine at codon 24, (24K) and Arginine at codon 29 (29R) on Tat protein and Tyrosine (Y) at position 45 (45Y) of Vpr. These three signature amino acids were related to classical markers for monitoring HIV infection. Finally, we identified 4 conserved fragment sequences, PEDQGPQREPYNEWTLE (5 to 21), LGQYIY (42 to 47), TYGDTW (49 to 54), PEDQGPQREPYNEW (5 to 18) on viral protein R, that were associated with higher plasma viral load and proviral load. The study has identified novel cytokine/chemokine and viral biomarker signatures for HIV associated neurocognitive impairment with low to moderate diagnostic accuracy. The findings demonstrated a need for interdisciplinary approach to elucidate possible molecular interactions between the peripheral blood immune markers, viral signatures and the CNS that are linked to observed clinical outcomes of neurodegradation in HIV infection. The identified biomarkers can be further investigated for use as screening tools and treatment endpoints for HAND.
Blood dendritic cells in chronically HIV-1 infected individuals in South Africa: Phenotype, function and immune modulation
(Stellenbosch : Stellenbosch University, 2016-12) De Swardt, Dalene; Glashoff, Richard H.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology
ENGLISH ABSTRACT :HIV-1 infection detrimentally affects CD4 T lymphocytes as well as the blood plasmacytoid (pDC) and myeloid dendritic cell (mDC) compartment. DCs act as innate sensors and as initiators and directors of antigen-specific immune responses. Whereas, mDCs have the unique ability to prime naïve T-lymphocytes and activate adaptive immune responses, pDCs are primary producers of type 1 interferons (IFNs), playing a pivotal role in anti-viral immunity. In the current study both pDCs and mDCs from chronically HIV-1 infected South African individuals (on or naïve for ARV therapy) as well as with and without concurrent TB disease, were compared to matched uninfected controls. Similar to CD4 T lymphocytes, bothpDCs and mDCs, were significantly depleted during HIV-1 infection, (reduction of pDC, mDC and CD4 T lymphocyte was 63% (p 0.001), 80% (p 0.001) and 31% (p 0.01), respectively). In parallel, significantly higher levels of generalised immune activation and exhaustion (CD38+CD8+, PD-1+CD8+ and CD38+PD-1+CD8+ T lymphocytes) were observed. ARV treatment ( 1 year) did not result in DC number recovery despite a significant increase of CD4 T lymphocytes numbers (CD4 T lymphocyte number gain of 89% (p 0.01), it fell short of full recovery).TB co-infection did not exacerbate number loss. Phenotypic characterisation of DC populations in circulation during HIV-1 infection may indicate the underlying reasons for the loss from circulation. Phenotypic profiling by multiparameter flow cytometry included: markers of activation (CD86, CD80 and CD62L), maturation (CD83), apoptosis (TNF-R2, FAS, FASL and TRAIL R1-R4) and chemotaxis (CCR5, CCR7, CCR9 and CXCR6). HIV-infection was associated with a significantly higher percentage of CD86+mDCs which may be indicative of early maturation or transition to secondary lymphoid tissue. The frequency of the CD86+mDCs subset normalised upon ARV therapy. Also, HIV-1 infection influenced the distribution of TNF-R2+pDCs and TNF-R2+mDCs. Increased TNF-R2 expression in both subsets, may attest to enhanced survival function. Functionally, DCs of HIV-1 infected individuals were reactive to TLR-L stimulation and in some cases showed enhanced responses compared to uninfected individuals. A significantly higher frequency of TNF-R2+pDCs, IFN- +pDCs, and TNF +mDCs was observed in whole blood TLR cultures of HIV-1 infected individuals (TNF-R2+pDCs: LPS (p = 0.002) and R848 (p = 0.01), IFN- +pDCs: R848 (p = 0.04), TNF +mDCs: LPS (p = 0.003))s.In addition, whole blood TLR cultures of the ARV treated study group generally showed normalisation of the responses, however; in certain cases ARV therapy reduced responsiveness to levels significantly lower than the control study group (i.e.TNF-R2+pDCs and TNF-R2+mDCs in CpG ODN stimulation). In contrast, a significantly lower frequency of IL12p40+mDCs was observed during HIV-1 infection (p = 0.02). TLR-L cultures of the ARV treated study groups showed normalisation of IL12p40+mDCsfrequency. Notably, treatment with the immunomodulating peptide VIP induced a decline in IL12p40+mDCs to levels lower than the control study group.The frequency of TNF +pDCs in TLR-L whole blood cultures was similar between the healthy, untreated and treated HIV-1 infected study groups, however, significantly reduced frequencies were observed in these study groups upon VIP treatment. These data indicate unique phenotypic and functional changes in DC subsets in chronic HIV- 1 infection which may provide potential targets for immunotherapeutic intervention.
Characterization of the persisting HIV-1 reservoir in early treated children on long-term suppressive ARV regimens
(Stellenbosch : Stellenbosch University, 2019-12) Katusiime, Mary Grace Kato; Van Zyl, Gert Uves; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Medical Virology.
Introduction: The major barrier to curing HIV-1 infection is a latent reservoir of long-lived, replication-competent proviruses that persists despite suppressive antiretroviral therapy (ART). Initiating ART during acute infection limits the development of phylogenetically diverse reservoirs. Novel approaches for reservoir elimination are emerging, providing hope for a cure. Perinatally infected, early-treated children, are likely good candidates for cure interventions as they have low immune activation states and a low proportion of central memory T-cells. We studied the post-CHER cohort of perinatally infected children, who initiated ART during acute infection and are in long-term follow-up. To inform cure interventions, we aimed to: (i) quantify latently infected cells and describe longitudinal genetic diversity, (ii) describe mechanisms that enable long-term reservoir persistence, (iii) describe the extent to which early therapy shapes the proviral landscape. Methods: In Aim I, we used a sensitive quantitative PCR assay for HIV-1 cell associated DNA (iCAD), followed by cell-associated DNA-single genome sequencing (CAD-SGS) of 1200bp in HIV-1 gag-pol to investigate genetic evolution in the reservoir during long-term ART. We performed 3 tests for evolution: (i) average pairwise distance (APD) for intra-patient viral population diversity, (ii) panmixia for probability of shifts in viral population structure, (iii) maximum likelihood (ML) root-to-tip distances to detect emergence of new viral populations. In Aim II, we performed integration site analysis (ISA) on samples from close to therapy initiation (baseline) and after 6-9 years on ART to investigate clonal expansion as a mechanism for reservoir persistence despite early, suppressive therapy. In Aim III, near full length- proviral amplification and sequencing (NFL-PAS) was performed to determine the proportion of genetically intact vs defective proviruses after 6-9 years on ART. Findings: We found low iCAD levels (median iCAD:22.45cp/106) in 16 children who initiated ART within the first 18 months of life. No significant changes in intra-patient proviral diversity, shifts in viral population structure or emergence of new viral populations were detected in children who were fully suppressed on ART, suggesting that ART prevents ongoing replication that replenishes the reservoir. ISA detected expanded clones in baseline samples of 6 children treated as early as 2 months of age, suggesting that infected cells begin clonally expanding before ART. Furthermore, there was a significant increase in the proportion of expanded clones after several years. A total of 738 NFL amplicons were generated from 9 children. Of these, 72.9% had large internal deletions, 23.7% were hypermutated, 1.4% had small internal deletions, and 1% had deletions in the gag-leader region. Intact proviruses were detected at a frequency of 1%. This study showed that early therapy and long-term suppression in children leads to limited reservoir size and genetic diversity, factors that are favourable for cure interventions. The reservoir appears to be maintained by clonal expansion that begins before therapy is initiated. Although a large proportion of proviral DNA in long-term suppressed children is defective, genetically intact variants persist and likely form part of expanded clones. This suggests the need for novel approaches that target HIV reservoirs by reducing proliferation of cells that harbour replication-competent proviruses.
Diversity and Ecology of Astroviruses in South African Bats
(Stellenbosch : Stellenbosch University, 2020-03) Barnard, Karlien; Preiser, Wolfgang; Schultz-Cherry, Stacey; Ithete, Ndapewa Laudika; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Medical Virology.
ENGLISH ABSTRACT: Emerging infectious diseases are mostly zoonotic in origin and defined as “infections that have newly emerged in a population or have existed but are rapidly increasing in incidence or geographic range". Zoonotic viruses are directly (e.g. bite from a rabid bat) or indirectly (via an intermediate host or vector) transmitted from animals to humans. Bats have received increasing attention as potential hostsfor zoonotic diseases. Bats belong to the order Chiroptera, which consists of two suborders: Yinpterochiroptera and Yangochiroptera. More than 1 300 species have been described globally, occurring on almost all continents excluding Antarctica. Specific physiological and ecological characteristics make bats extraordinary evolutionary vessels to carry numerous infectious agents including pathogens. Astroviruses (AstVs) are amongst the vast array of viruses that have been detected in bats. AstVs are single stranded, positive sense, RNA viruses that are transmitted via the faecal-oral route. Infection with AstVs causes acute diarrhoea, however, more serious clinical presentations such as neurological deficits, stunted growth and encephalitis have also been documented. Bats on the other hand, seem to be asymptomatically infected with AstVs. Little attention has been given to the evolution, phylogenetic relationship, ecology and diversity of AstVs in South African bats. In 2013 the first study in South Africa screening for a variety of viruses in small mammals, including SAn bats, found that bats were frequently co-infected with AstVs and coronaviruses. The overall aims of the current study were to describe the prevalence, diversity and ecology of AstVs in South African bats, to determine the potential threat to environmental and animal health at wastewater treatment works (WWTW) through testing water and bat samples for the presence of AstVs, to monitor AstV and CoV co-infection in a Neoromicia capensis colony over time and to isolate and propagate a bat AstV in vitro. The results will be used to determine the potential One Health implications of AstVs in a South African setting. Sample collection was done via non-invasive capture and release methods by collaborating zoologists. Morphological and ecological data of each bat were recorded. Bat faecal samples (n=500) were screened for AstVs using the hemi-nested screening assay that targets the RNA-dependent RNA polymerase (RdRP) gene of the virus. Plasmid positive controls were generated to ensure an optimal AstV screening PCR assay. The One Health concept emphasizes the interlinkage between human, animal and environmental health. To determine the impact that potential exposure to human AstVs at WWTW might have on animal and environmental health, water samples upstream and downstream of two WWTW were also collected and screened for AstVs. The overall detection rate of AstVs across bat species was 13%, but it differed significantly between species (Miniopterus natalensis, 55%; Rhinolophus capensis, 39%; and R. clivosus, 17%). Positive samples were further analysed to try and amplify the capsid protein gene (ORF2), which is highly variable and only one ORF2 gene fragment was obtained. Twenty-five novel AstV RdRp sequences and one ORF2 sequence were identified, bringing the total RdRp sequences available for South African bat AstVs to forty-four. Maximum likelihood analyses of the RdRp gene fragments suggest that South African bat AstVs are not restricted by host species identity or geographical location. Interestingly, the maximum likelihood analyses of the ORF2 sequence suggest that the South African bat AstVs might be more similar to human AstVs from Japan compared to any bat AstVs. The water samples collected from the WWTW tested negative for the presence of AstVs and only one bat sample collected at the WWTW tested positive for AstV. Two real-time PCR assays were designed to monitor AstVs and coronaviruses in a N. capensis colony over time, as these two viruses regularly co-infect bats. The results indicated that both these viruses had a single amplification peak that was associated with colony formation after migration. Interestingly the peak in viral loads did not correlate with the pupping season of the bats, as was found by another study conducted on these two viruses in Germany. Statistical analyses of ecological and individual bat factors suggest that being a sexually active adult male bat, species identity and occurrence in the Succulent Karoo biome could contribute to AstV positivity. The current study was the first ever to successfully isolate and propagate a Miniopterus bat derived AstV in vitro. During the isolation attempts three different cell lines were used, human adenocarcinoma, Neoromicia capensis kidney and baby hamster kidney cells. Isolation and propagation was only successful in the baby hamster kidney cells. The refined protocol for isolation and propagation of bat AstVs in cell culture will enable future studies to successfully isolate bat AstVs as well as enable genomic and functional studies. The results also gave insight into the potential zoonotic risk of bat AstVs. The findings of the current study indicated that bat AstVs are diverse and relatively prevalent in South African bats. Phylogenetic analyses of the 24 novel RdRp and one ORF2 genes from this study indicated that the virus was not limited by species identity or host geographical range. Furthermore, the phylogenetic analyses of the bat AstV ORF2 gene would suggest that the bat AstV is more similar to human AstVs, which could imply that South African bat AstVs have zoonotic potential. The results of current study gave some potential insights into the One Health implications of AstVs in the SA setting.
The diversity of coronaviruses in Southern African bat populations
(Stellenbosch : Stellenbosch University, 2017-12) Cronje, Nadine; Preiser, Wolfgang; Schoeman, Corrie; Ithete, Ndapewa; Stellenbosch University. Faculty of Health Sciences. Dept. of Pathology. Medical Virology.
ENGLISH ABSTRACT: Coronaviruses are RNA viruses encompassing four genera. The alpha- and betacoronaviruses have commonly been associated with mild disease in humans. However, outbreaks of severe respiratory disease in 2002 and 2012 led to the identification of novel highly pathogenic human coronaviruses, SARS- and MERS-CoV, respectively. Bats, order Chiroptera, are believed to be the reservoir host from which all mammalian coronaviruses have emerged. To date, few studies have been published on coronaviruses in South African bats. With little known about the diversity and prevalence of bat coronaviruses in this region; this study aimed to describe the existing coronavirus diversity within South African bat populations as well as factors that might influence bat-coronavirus ecology. It detected nine different coronavirus species, eight alphacoronaviruses and one betacoronavirus, from ten different bat species. The study not only demonstrated that diverse coronaviruses can be found in different bat species of Southern Africa but lends additional support to an ongoing circulation of MERS-related betacoronaviruses in South African bats, with divergent variants detected in two different vespertilionid bat species. A species-specific surveillance of Neoromicia capensis (Cape serotine) bats detected three different bat coronavirus species and revealed genetic diversity across different geographic regions. Several instances of coinfection with two different coronaviruses were detected, demonstrating the potential for recombination that could lead to the emergence of a new coronavirus that might have zoonotic potential. This study demonstrated that both host and environmental factors may influence CoV ecology. Female Neoromicia capensis bats trapped at low altitude sites within the Forest biome had the highest likelihood of being coronavirus positive. Discrepancies between detection rates obtained with different screening assays led to the adoption of an improved approach and recommendations for future bat coronavirus surveillance studies were made.
From bedside to bench and back : future options for antiretroviral drugs in non-B HIV-1 subtypes
(Stellenbosch : Stellenbosch University, 2020-12) Njenda, Duncan Tazvinzwa; Engelbrecht, Susan; Jacobs, Graeme Brendon; Neogi, Ujjwal; Sonnerborg, Anders; Spetz, Anna-Lena; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Medical Virology.
Introduction: HIV-1 drug resistance remains a burden in low- and middle-income countries (LMIC). Regardless of the advances in antiretroviral (ARV) therapy, there is an increase in the trend of acquired and pre-treatment drug resistance mutations (DRM) in LMIC affected by HIV-1 non-B subtypes. The overall aim of this thesis is to understand the potential subtype-specific differences in viral fitness and drug susceptibility against the drugs that target different stages of viral replication that includes, protease-mediated cleavage and maturation (Paper I), reverse transcription (Paper II) and integration (Paper III). Methods:The thesis presents cross-sectional drug resistance data from predominantly HIV-1 non-B subtypes. It also includes virological drug sensitivity and enzyme-based in vitro assay results of recombinant viruses derived from predominantly HIV-1 non-B and few HIV-1B infected patients. The following areas were investigated: a) the role that HIV-1 subtype C (HIV-1C) gag-p6 gene could play in response to proteaseInhibitors PIs in the absence of known PI primary mutations (Paper I). b) Ex vivo potency of the newer drug 4’-Ethynyl-2’-Fluoro-2’ deoxyadenosine (EFdA),first and second generation non-nucleoside reverse transcriptase inhibitors (NNRTIs)and Tenofovir alafenamide(TAF) against diverse HIV-1 subtypes (Paper III). c)Ex vivo potency of Integrase strand transfer inhibitors (INSTIs) against diverse HIV-1subtypes as well as genotypic resistance data comparing INSTI naïve and INSTI-experienced patients. Results: In paper I, the study showed an increase in viral fitness for HIV-1C viruses carrying the PYxE insertion in gag-p6 when compared to the wild-type (WT) HIV-1C viruses. Furthermore, some PYxE-carrying viruses had low sensitivity to LPV and (TAF) when tested in drug sensitivity assays. Clinical data analysis showed a higher pre-therapy viral load and a decrease in CD4+ T-cell counts for patients harboring PYxE-carrying viruses when compared to WT. Paper II. demonstrated that EFdA has a high inhibitory potential independent of HIV-1 subtype and high antiviral activity against resistant viruses. However, HIV-1C viruses had a significantly reduced susceptibility to NNRTIs, specifically Rilpivirine and Etravirine. In paper III, the drug susceptibility of INSTIs results indicated that INSTIs such as Dolutegravir (DRV), Bictegravir (BIC) and Cabotegravir (CAB) inhibited non-B subtypes significantly as compared to HIV-1B subtypes. Conclusion: Inferences can be made from the results to suggests that subtype-specific differences play an essential role in influencing the ARV susceptibility which could further impact the treatment efficacy in sub-optimal adherence. To reduce the trend of increasing DRMs in non-B HIV-1 subtypes which are mainly dominating in LMICs, adherence support and viral load monitoring should be prioritized. A rapid adaptation of INSTIs and newer drugs that have long-acting potential is encouraged. However, pre-clinical studies and clinical trials that are mainly restricted to the HIV-1B enrolled patients, should be inclusive of non-HIV-1B infected patients before the massive roll-out of INSTIs and newer drugs continues in non-HIV-1B dominated settings.
Genetic aspects of HIV-1 risk in an African setting
(Stellenbosch : Stellenbosch University, 2006-12) Petersen, Desiree C.; Hayes, Vanessa M.; Dean, Michael; Janse van Rensburg, Estrelita; Glashoff, Richard H.; Stellenbosch University. Faculty of Health Sciences. Dept. of Pathology. Medical Virology.
Host susceptibility to human immunodeficiency virus-1 (HIV-1) infection and disease progression to acquired immunodeficiency syndrome (AIDS) varies widely amongst individuals. This observation led to the identification of host genetic factors playing a vital role in HIV-1 pathogenesis. Previous studies mainly focusing on Caucasian-based populations have indicated possible associations between genetic variants and host susceptibility to HIV-1/AIDS. The limited studies performed on African-based populations have emphasised the need for extensive investigation of both previously reported and particularly novel genetic variants within the older and genetically diverse Sub-Saharan African populations. In this study, the case-control samples were represented by African individuals of Xhosa descent, all residing in the Western Cape Province of South Africa. This included 257 HIV-1 seropositive patients and 110 population-matched HIV-1 seronegative controls. Mutational screening was performed in a subset of individuals for the entire coding regions of the CC chemokine receptor 5 (CCR5) and CC chemokine receptor 2 (CCR2) genes, and the 3’ untranslated region of the CXC chemokine ligand (CXCL12) gene, as previously reported (Petersen, 2002). Further analysis of these genes in a larger study sample involved the genotyping of previously identified mutations and single nucleotide polymorphisms (SNPs), which forms part of the present study. In addition, mutational screening was performed for the entire coding region of the CXC chemokine receptor 4 (CXCR4) gene, partial coding region of the mannose binding lectin (MBL) gene, and the promoter regions of interleukin 4 (IL4), interleukin 10 (IL10) and the solute carrier 11A1 (SLC11A1) genes. This was followed by genotyping of SNPs occurring in CCR5, CCR2, CXCL12, MBL, IL4, IL10, CX3C chemokine receptor 1 (CX3CR1), CC chemokine ligand 5 (CCL5) and tumour necrosis factor alpha (TNFα) genes. Significant associations were observed with HIV-1 susceptibility in the Xhosa population of South Africa. These included the CCR5-2733A>G, CX3CR1V249I, IL10-819C>T and IL10-592C>A SNPs being associated with a reduced risk for HIV-1 infection, while the CCR5-2135C>T and SDF1-3’G>A (CXCL12-3’G>A) SNPs were associated with increased susceptibility to HIV-1 infection. Furthermore, certain haplotypes for IL4 and IL10 showed association with reduced risk for HIV-1 infection. This included the identification of a novel IL4 haplotype restricted to the HIV-1 seronegative control group. This study emphasises the importance of considering genetic diversity across all populations, as certain HIV-1/AIDS associations appear to be restricted to specific ethnic groups. These findings have also provided an understanding for further elucidating the functional roles of genetic variants in determining HIV-1/AIDS susceptibility. Ultimately, such genetic association studies will contribute to establishing HIV-1/AIDS risk profiles for African-based populations from pandemic-stricken Sub-Saharan Africa.
Hepatitis B virus mother-to-child transmission in Namibia: transmission dynamics and possibilities for elimination
(Stellenbosch : Stellenbosch University, 2019-12) Tamandjou Tchuem, Cynthia Raissa; Andersson, Monique I.; Preiser, Wolfgang; Wiysonge, Charles S.; Jacobs, Graeme Brendon; Stellenbosch University. Faculty of Health Sciences. Dept. of Pathology. Medical Virology.
ENGLISH ABSTRACT: Introduction: Hepatitis B virus (HBV) remains endemic in sub-Saharan Africa (SSA). While the roll-out of pediatric HBV immunization from six weeks of age has had an impact on horizontal transmission of the virus, mother-to-child transmission (MTCT) has been identified as the driver of the current HBV epidemic in the region. Given the high likelihood of developing chronic HBV infection (CHB) if the infection is acquired during infancy, preventing HBV MTCT in SSA is vital. Where MTCT occurs, it is essential to identify the HBV-infected children for appropriate management, especially in the context of HIV. Current antiretroviral therapy (ART) for the management of HIV infection includes tenofovir for children ≥ 10 years old. Children below the age of 10 years are treated with lamivudine. However, many of the HIV/HBV co-infected children are left on lamivudine treatment for more than ten years and are at risk of developing HBV drug resistance and uncontrolled HBV infection. Uncontrolled HBV infection is a known factor for increased risk of severe liver damage. Aim: This research project aimed to (1) assess the molecular character of HBV and the liver health of HIV/HBV co-infected children who have been on long-term lamivudine treatment, (2) to determine the feasibility of a screen-treat-vaccinate intervention to prevent HBV MTCT, and (3) to measure the costs and health outcomes of combined prophylactic measures against HBV MTCT, in Namibia. Methodology: Three sub-studies were conducted as part of this research project, to answer each of its aims. The first sub-study involved HIV/HBV co-infected children and adolescents below the age of 18 years old, and who have been exposed to lamivudine. Venous blood samples were collected from these children for HBV serological testing (HBsAg, HBeAg, anti-HBe and anti-HBc total) using Murex ELISA assays. Dried blood spots (DBS) samples were used for HBV DNA levels measurement and genotyping. HBV DNA measurements were completed using the automated AmpliPrep/COBAS TaqMan HBV test V2.0. Genotyping and mutation analyses were performed using online tools. Liver health was assessed through AST platelet ratio index (APRI). An APRI score > 0.5 was considered a sign of liver fibrosis. Mothers attending with these children and adolescents were also enrolled in the study, to determine the role of HBV MTCT in these pediatric HBV infections. DBS were collected from these mothers for HBV molecular characterization as well. The second sub-study focused on pregnant women attending antenatal clinics (ANCs) in Windhoek. These women were recruited following informed consent and screened for HBV using the Alere DetermineTM HBsAg rapid test. HBsAg positive pregnant women were tested for further HBV serological markers (HBeAg, anti-HBe, anti-HBc IgM), and HBV viral loads were measured to determine the risk of MTCT. Positive mothers at high risk of MTCT were reviewed for antiviral prophylaxis and offered treatment where necessary. HBV-exposed babies were immunized as per Namibian guidelines, and followed-up to determine the rate of MTCT. The feasibility of offering routine antenatal HBV rapid testing was assessed quantitatively and qualitatively. The former involved determining the diagnostic accuracy of the rapid test used for HBsAg screening, and the latter focused on the perceptions of this antenatal care service by healthcare workers (HCWs). In the third sub-study, the costs and health outcomes of four interventions against HBV MTCT were assessed through a cost-effectiveness analysis. The interventions included: (1) universal birth dose (BD) vaccination, (2) BD vaccination and HBIG, (3) BD vaccination, HBIG, and maternal antiviral prophylaxis informed by sequential HBV viral load testing, and (4) BD vaccination, HBIG, and maternal antiviral prophylaxis informed by sequential HBeAg testing. All resources including consumables, HCW’s time, building space and facilities (and their quantity) were measured and valued to determine the unit costs of HBsAg screening at the ANCs, providing antenatal treatment and administering pediatric immunoprophylaxis. Health outcomes were measured in terms of the number of pediatric HBV infections averted. The incremental cost-effectiveness ratios (ICERs) of these interventions were calculated and were used to compare each intervention to the previous less expensive one. Results: Fifteen HIV/HBV co-infected children/adolescents and six mothers attending with the children were enrolled in the first sub-study. Ten serum samples obtained from Windhoek were further tested for HBV serological markers; seven were HBeAg positive/anti-HBe negative (7/10; 70%), three were HBeAg negative (3/10; 30%), and all were reactive for anti-HBc (total) (10/10; 100%). Among HBeAg negatives, one was anti-HBe negative and two were anti-HBe positive. Eight of the fifteen children (8/15; 53.3%) were HBV DNA positive. The viral strains were grouped with genotype E (6/8; 75%) and genotype D3 (2/8; 25%) and harbored lamivudine drug-associated resistance variants and immune escape mutants. Liver health was assessed in nine children: five with detectable levels of HBV DNA and four with undetectable levels of HBV DNA. An abnormal APRI score of 0.713, was detected in one HBV DNA positive child (1/9; 11.1%). In the second sub-study an HBsAg seroprevalence of 5.4% was observed among 515 (28/515) pregnant women enrolled at ANCs in Windhoek. Three pregnant women (3/28; 10.7%) were positive for HBeAg; of whom one was HIV/HBV co-infected and the other two were HBV mono-infected. The two (2/28; 7.14%) HBV mono-infected/HBeAg-positive patients presented with viral load > 105 IU/ml, the study cut-off for antenatal treatment to prevent HBV MTCT; one received antiviral prophylaxis with tenofovir, the other was offered prophylaxis but did not receive it. Postpartum, 25 of the 28 HBV-exposed babies (25/28; 89.3%) were traced and followed-up to determine their HBV status and the rate of HBV MTCT. Fourteen (14/25; 56%) were males, and eleven were females (11/25; 44%). All babies had been vaccinated against HBV at birth, and 15 (15/25; 60%) had received hepatitis B immunoglobulin (HBIG). The 25 babies were tested for HBsAg at a median age of seven weeks (Range: 5.57 weeks – 20.29 weeks). All were non-reactive for HBsAg, including both babies born to the highly viremic women. With regards to the feasibility of HBV rapid testing as part of antenatal care services, the DetermineTM HBsAg rapid test had a 100% diagnostic sensitivity and specificity. HCWs found the test simple to use and showed a preference for rapid testing over laboratory testing for routine antenatal screening of HBV. They believed that this method would improve early diagnosis and treatment of HBV of pregnant women. In the cost-effectiveness analysis conducted in the third sub-study, a preventive strategy with universal BD vaccination alone was the cheapest option but was less effective. Adding HBIG to BD vaccination, and providing maternal antiviral prophylaxis was the most effective and the most costly strategy. The strategy that includes antiviral prophylaxis with sequential HBeAg testing added to BD vaccination and HBIG had an ICER of US$6 262.42 per infection averted, in comparison to the strategy including BD vaccination and HBIG only. The BD vaccination and HBIG strategy had an ICER of US$4 550.34/ pediatric HBV infection averted, in comparison to BD vaccination alone. These ICERs were highly sensitive to the prevalence of highly infectious pregnant women, the cost of the HBeAg test, and the effectiveness of each strategy for preventing MTCT in highly infectious pregnant women. Conclusion: Results from this research project reemphasized the issue of pediatric CHB, especially in HIV/HBV co-infected children. The data described in this study also showed that elimination of MTCT of HBV in Namibia is achievable, through routine antenatal HBsAg screening, treating pregnant women at high risk of MTCT, and providing HBV vaccination from birth. Screening using rapid testing was found cheap, and a feasible alternative for detecting HBV infection in pregnant women. The costs and health benefits of implementing antenatal antiviral prophylaxis and HBIG presented in the study provide background data for further assessment of the value for money of these interventions in SSA, and to explore alternatives excluding HBIG for HBV PMTCT.
Hepatitis B virus-associated hepatocellular carcinoma in South Africa: epidemiology and impact of HIV-1 co-infection and immune dysregulation
(Stellenbosch : Stellenbosch University, 2016-12) Maponga, Tongai Gibson; Andersson, Monique I.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Medical Virology
ENGLISH ABSTRACT : Co-infection with the human immunodeficiency virus (HIV) negatively impacts the natural progression of hepatitis B virus (HBV) infection, including causing rapid progression to liver fibrosis and hepatocellular carcinoma (HCC). In sub-Saharan Africa the overlap between high HIV and HBV prevalence may increase the incidence of HCC. The aim of this study was to investigate the effect of HIV co-infection on presentation of HCC among HBV-infected patients. Since HCC is thought to be driven by ongoing severe inflammation, the study also evaluated the association between the expression of markers of immune activation/exhaustion and liver inflammation in patients with chronic hepatitis B (CHB) to determine if the risk of hepatofibrosis is increased by exposure to gut microbial products and compared HIV-infected patients with HBV-infected and HIV-HBV co-infected patients. Ethical approval was obtained to conduct two sub-studies. The first sub-study (HCC Epidemiology Study) involved recruitment of patients diagnosed with HCC at oncology units of selected teaching hospitals in South Africa. A total of 107 HCC cases were recruited between December 2012 and October 2015. Demographic, laboratory and clinical data together with blood specimens were collected. Patients were tested for HBV, hepatitis C virus (HCV) and HIV. Molecular characterization of HBV and HCV was also performed. For the second sub-study (Liver Fibrosis and Immune Markers Study), 46 HBV/HIV co-infected; 47 HBV monoinfected; 39 HIV monoinfected and; 39 HIV-/HBV-uninfected controls were recruited following informed consent. All HIV-infected patients had been on highly active antiretroviral therapy (HAART) for ≥3 months. Liver stiffness measurements were taken using the Fibroscan 402. Cell-based immunomarkers of activation/exhaustion were measured using flow cytometry of fresh whole blood. Soluble serum/plasma immunomarkers were measured using ELISA and Luminex. HIV and HBV viral loads and genotyping of HBV were performed. Of 107 cases in the HCC Epidemiology study, 83 (78%) were male and 68/106 (64%, 95% CI: 59-77) were positive for HBsAg. HIV seropositivity was seen in 22/100 (22%, 95% CI: 14-30) of all HCC cases. Among HBsAg-positive HCC cases, 19/66 (29%, 95% CI: 18-40) were HIV-infected compared to only 3/34 (9%) among those that were HBsAg-negative, p=0.04. The proportion of females among the HBV/HIV co-infected HCC cases 6/18 (33%, 95% CI: 11-55) was significantly higher compared to those that were HBV-mono-infected 6/47 (13%, 95% CI: 3-23), p=0.005. HIV/HBV co-infected females presented younger, at mean age 36.8 years (95% CI: 32.2-41.5) compared to 50.5 years (95% CI: 30.2-70.8) in HBV-mono-infected women, p=0.09. Males continue to be disproportionally affected with HCC. There is a trend towards younger age at diagnosis of HCC among HIV-positive compared to HIV-negative women. The Liver Fibrosis and Immune Markers Study showed a high percentage of CD8+ T lymphocytes from co-infected subjects expressing HLA-DR/CD38 and PD-1 (p<0.05). Soluble CD14 and IP-10 were also significantly elevated in plasma of co-infected patients. Co-infected subjects exhibited delayed immune recovery with lower CD4/CD8 T cell ratio; CD4 cell counts and frequent HIV viremia compared to HIV mono-infected participants (p<0.05). The HBV mono-infected group had the highest proportion of participants with moderate/advanced liver fibrosis measured by Fibroscan, together with highest plasma concentrations of most of the cytokines measured. The results showed positive correlation between HIV and HBV viral replication and liver fibrosis. The results suggest that there is persistent T-lymphocyte dysregulation and delayed immune recovery in ART-experienced HBV/HIV co-infected patients. However this does not appear to be associated with severity of liver fibrosis in this cohort. HAART used in HIV is also effective against HBV and may therefore have led to control of viral replication leading to better fibrosis scores compared to the HBV mono-infected patients. Moderate/advanced liver fibrosis in HBV-mono-infection may well be an indicator of poor access to HBV screening and treatment.
HIV-1C dynamics and evolutionary trends in Botswana
(Stellenbosch : Stellenbosch University, 2016-12-09) Moyo, Sikhulile; Engelbrecht, Susan; De Oliveira, Tulio; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology
ENGLISH ABSTRACT : Introduction: HIV incidence estimates are critical for monitoring HIV transmission dynamics, and for design and evaluation of the impact of interventions. Biomarkers and assays for cross-sectional surveillance of HIV incidence are greatly needed because of the high costs and time needed to maintain prospective cohorts to determine HIV incidence. New cross-sectional assays for estimation of HIV incidence are attractive due to their improved performance and cost-effectiveness. In this dissertation, methods for identification and characterization of recency of HIV infection are described. An in-depth review of HIV recency determination methods, including novel cross-sectional application of molecular methods, is given in “From serological assays to genomics.” Multi-assay approaches were evaluated in order to increase the sensitivity and specificity of the commercial incidence assays in the context of high treatment coverage and stable but high HIV prevalence in Botswana. A novel biomarker based on HIV viral diversity was investigated as a complementary or standalone tool to characterize HIV recency. In this thesis, an innovative use of pairwise diversity and the time to the most recent common ancestor (tMRCA) in a heterosexual HIV-1 subtype C (HIV-1C) epidemic were introduced as novel approaches for HIV incidence estimation. We evaluated the properties of the new potential tools for estimating time since infection, including their specificity and predictive performance in the context of the HIV-1C epidemic in Botswana. Methods: Characterization of HIV recency and novel biomarkers for estimation of HIV infection incidence is based on application of immunologic and molecular methods: a) Evaluation of the long-term specificity (false recent classification rates) of serological tests for recent infection, and algorithms for estimating HIV-1C incidence utilizing samples from patients with known long-standing HIV infection. b) Application of within-host viral diversity for estimation of HIV-1C recency in Botswana using samples collected from patients with known time since seroconversion in the primary HIV-1C infection cohort. c) Investigation of intra-host viral pairwise diversity and the time to the most common recent ancestor (tMRCA), as potential markers for HIV infection recency. Results: We estimated for the first time false recency rates (FRR) of the commercially available BED and Limiting Antigen (LAg) assays in Botswana. We demonstrated that combined algorithms reduce FRR to the recommended < 2%. Including viral load in the assay algorithm resulted in an FRR of 0.4% for LAg. Analysis of the within-host viral pairwise diversity provided more accurate estimation of HIV recency, as compared with the recommended LAg and BED using the receiver operator characteristic analysis (ROC). We demonstrated that intra-host viral pairwise distances reduce misclassification and increase the accuracy of serologic assays. tMRCA and intra-host viral pairwise distances correlated with time since HIV infection provide additional novel tools for reliable estimation of HIV recency. Conclusion: HIV infection recency can be determined cross-sectionally using a combination of serological and molecular biomarkers. Including viral load and an assessment of prior exposure to ARVs is critical for accurate estimation of HIV incidence. Intra-host pairwise diversity and tMRCA are able to predict time since HIV infection and can be used to improve accuracy in estimation of HIV infection recency.
Identification and characterisation of paramyxoviruses in species-rich small mammals from South Africa
(Stellenbosch : Stellenbosch University, 2022-12) Kleinhans, Bronwyn; Preiser, Wolfgang; Ithete, Ndapewa Laudika; Drexler, Jan Felix; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology.
ENGLISH ABSTRACT: Paramyxoviruses are negative-sense RNA viruses and include a substantial collection of ubiquitous viruses, comprising one of the most important viral groups with numerous pathogens historically impacting on public and veterinary health. The Paramyxoviridae family consists of four subfamilies including Avulavirinae (genera: Metaavulavirus, Orthoavulavirus and Paraavulavirus), Metaparamyxovirinae (genus: Synodonvirus), Orthoparamyxovirinae (genera: Aquaparamyxovirus, Ferlavirus, Henipavirus, Jeilongvirus, Morbillivirus, Narmovirus, Respirovirus and Salemvirus) and Rubulavirinae (genera: Orthorubulavirus and Pararubulavirus). Over the past three decades, an increasing number of novel paramyxoviruses of especially the Orthoparamyxovirinae and Rubulavirinae subfamilies have emerged from small mammal reservoir hosts, some of which, especially the deadly henipaviruses, Hendra- and Nipah virus, have demonstrated a propensity to spillover from their natural reservoir hosts into human and domestic animal populations. Although some small mammals have been implicated as potential hosts for novel paramyxoviruses within southern Africa, little to no data exists on such discovery in insectivorous bats, rodents and particularly shrews and sengis in South Africa. This study identified an additional 23 previously unimplicated species representing four different orders (Chiroptera, Eulipotyphla, Macroscelidea and Rodentia), greatly expanding on the known host and geographic range of these putative paramyxoviruses, reiterating this group of virus’ ubiquitous nature and high diversity. The presumptive paramyxoviruses discovered in this study demonstrated phylogenetic relatedness to at least five of the known genera including: Henipavirus, Jeilongvirus, Morbillivirus, Narmovirus and Rubulavirus as well as to previously discovered viral sequences clustering within the Orthoparamyxovirinae subfamily but that could not be assigned to any of the currently known genera. Unique to this study and of particular value in the South African context was the discovery that: Cape horseshoe bats (Rhinolophus capensis) harbour different variants / strains of a paramyxovirus displaying multiple nonsynonymous mutations resulting in amino acid changes, raising concerns over these putative paramyxoviruses’ zoonotic potential; the widely distributed and populous Rhabdomys (R. bechuanae, R. dilectus, R. intermedius and R. pumilio), endemic to southern Africa, harbour diverse and abundant (overall prevalence of 19.41% [106/546]) paramyxoviruses, displaying diversity on the individual, species and population levels; at least four different shrew species commonly found in South Africa were implicated as hosts for putative henipaviruses; the sustained presence of one of these novel viruses in greater red musk shrews (Crocidura flavescens) implicated this particular species as the reservoir host thereof, of which the near full-length genome sequence further revealed a close phylogenetic relationship to the rat-borne henipavirus, Mòjiāng virus. Given the abundance and diversity of novel potential paramyxoviruses discovered herein and the implication of a multitude of previously unimplicated species as potential reservoir hosts, this study reaffirms the importance and need for ongoing surveillance efforts of especially small mammals in South Africa. This study further highlights the importance of not only the identification of novel paramyxoviruses but also their characterisation, especially those demonstrating close relation to pathogenic members within the family. Further investigation into host-pathogen dynamics at the wildlife – domestic animal – human interface is however warranted to establish the potential for these viruses’ ability to spillover into humans and/or their domestic animals.
Investigating pathogens of the gastrointestinal tract in sudden and unexpected death in infancy cases at the Tygerberg medico-legal mortuary, compared to an age-matched healthy control group.
(Stellenbosch : Stellenbosch University, 2023-07) Cupido, Danielle Tiffany; De Beer, Corena; Whitelaw, Andrew; Dempers, Johan; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology.
ENGLISH ABSTRACT: Background: Sudden and unexpected deaths in infancy (SUDI) includes infants under the age of one year that die suddenly and without apparent cause. Childhood diarrhoea is one of the leading causes of death for children under five, with around 1.7 billion cases worldwide each year and is often reported prior to death in SUDI cases. Poor socioeconomic conditions and inadequate water supplies in developing countries contribute to diarrhoea, and diarrhoeagenic Escherichia coli (DEC) were detected in 30-40% of these cases, while acute viral gastroenteritis causes ± 70% of all episodes. The microbiome influences host immunity, infectious susceptibility, and health, disease, and death outcomes. Limited information is available on the gastrointestinal tract (GIT) pathology, as well as the GIT microbiome as contributory factors to SUDI in South Africa. This study aims to investigate the bacterial and viral pathogens and colonisation of the GIT in SUDI cases admitted to the Tygerberg Medico-Legal Mortuary in the Western Cape in the process of determining the cause of death. Finally, the SUDI microbiome was compared to age-matched, apparently healthy infants. Methods: Swabs of the GIT and stool samples were collected from SUDI cases at Tygerberg Medico-legal Mortuary between June 2017 and May 2018. To serve as controls, stool samples were collected from the nappies of 45 healthy and age-matched infants. In stool and swab samples positive for Escherichia coli, DEC were detected using the AllplexTM GI-Bacteria (II) Assay and gastrointestinal viruses were detected in stool samples using the Allplex™ GI-Viral Assay. Positive rotavirus samples were genotyped and the intestinal microbiome was characterised by full-length 16S rRNA sequencing, on the PacBio Sequel IIe System platform. Results: This study included 186 SUDI cases (107 males and 79 females) and 45 controls (24 males and 21 females). Several known demographic factors increase the risk for SUDI, including age between 2-4 months, male sex, cold season, bedsharing, prone and side sleeping positions, as well as informal housing. Enteroaggregative Escherichia coli (EAEC) ) were detected in 87.3% of cases and enteropathogenic Escherichia coli (EPEC) were detected in 78.2% of cases. Co-infections between DEC pathotypes were observed in 85.2% of cases. Rotavirus was detected in 38.6%, of cases followed by norovirus GI and GII (30.0%), whereas norovirus GII was more prevalent in the controls (36.7%). Forty-eight cases had enteric virus co-detections. The association between most viruses and seasons was highly significant. Among the rotavirus genotypes, combinations of the G type and the P type, G1P[8] had the highest prevalence (40%), followed by G2P[4] (30%), while G9P[8] (20%) and G8P[4] (10%) genotypes had the lowest prevalence. Firmicutes, Bacteroidota, Proteobacteria, and Actinobacteria were found to be the most common organisms in the GIT. Significant differences were observed in alpha diversity and beta diversity between cases and controls, as well as the different final diagnoses. Conclusion: This study demonstrated that autopsy sampling procedures should include other sampling sites, e.g., GIT, as these pathogens may contribute to death, particularly with virus and bacterium co-infections. Determining the cause of death based on GIT pathogens, may decrease the number of Sudden Infant Death Syndrome (SIDS) cases reported in the future.
Investigating the structural impact of hiv-1 integrase natural occurring polymorphisms and novel mutations identified among group m subtypes circulating in sub-Saharan Africa
(Stellenbosch : Stellenbosch University, 2020-12) Mikasi, Sello Given; Jacobs, Graeme Brendon; Cloete, Ruben; Van Zyl, Gert Uves; Engelbrecht, Susan; Ikomey, George Mondinde; Kasang, Christa; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Medical Virology.
Introduction HIV/AIDS remains a major health concern worldwide, with sub-Saharan Africa (SSA) carrying the largest burden.HIV is characterised by extremely high genetic diversity, with all the major groups and subtypes circulating in SSA. Combination antiretroviral therapy (cART) have substantially reduced HIV related deaths, but this is counteracted by the development of HIVdrug resistance, caused by certain drug resistance-associated mutations (RAMS). Integrase (IN) strand transferase inhibitors (INSTIs), the newest class of antiretroviral drugs,has a high genetic barrier and can be used in individuals that previously exhibited resistance to other classes of drugs. The World Health Organisation (WHO) approved the use of Dolutegravir (DTG) as part of first-line cART. Methods This is a descriptive experimental design study, which aimed to identify IN natural occurring polymorphisms (NOP) among different HIV-1 group M subtypes and Drug resistance mutations within the HIV-1 pol gene fragment of INSTI naïve patients from South Africa (SA) and Cameroon (CR), using the Stanford University genotypic resistance interpretation algorithm. Structural computational methods that included; homology modelling, molecular docking, molecular dynamics simulations and interaction analysis was performed to understand the structural impact of mutations from diverse HIV-1 subtypes on DTG drug binding. ResultsWe observed low-level RAMs against INSTIs in SA (2.2%) and CR sequences (5.4%). Through Fisher’sexact test we noted that the two NOPs occurred: VI72I and R269K, with p-values ≤0. 05, were statistically enriched. The impact of having these mutations are yet to be fully understood. Through molecular modelling and stability predictions, we observed a destabilizing effect of the known G140S mutant on the HIV-1C IN protein structure and simulation analysis showed that it affected structural stability and flexibility of the protein structure. Interactions analysis of different drug binding conformations to different HIV-1 IN subtypes reported differences in the number of binding interactions to different HIV-1 IN subtypes, but we did not observe any significant differences in binding affinity for each INSTIs. This implies no significant alteration to the binding site in the wild type IN, which may not prevent INSTIs drug binding. In addition, all accessory mutations that resulted in a change in the number of interactions encompassing residues were found within the stable alpha-helix secondary structure element and not in close proximity to the drug active site.ConclusionThe study data indicate that RAMS against INSTIs remain low both in SA and in CR.Subtype C in SA and CRF02_AG in CR continues to be the driving force ofthe epidemic. We further reported on the impact of various NOPs on drug susceptibility. The analyses suggested that NOPs does not have an impact on IN protein structure and stability,and does not affect drug binding in the WT IN, but the known mutation G140S affect DTG binding. The study support recommendations made by the WHO to use DTG as part of salvage therapy in patients with RAM’s and accessory mutations. Data obtained from thisstudy can help to tailor effective treatment strategies in the African population, where diverse HIV subtypes circulate.
The investigation of genotypic antiretroviral drug resistance in the context of the South African national antiretroviral roll-out programme
(Stellenbosch : Stellenbosch University, 2012-03) Van Zyl, Gert Uves; Preiser, Wolfgang; Nachega, Jean; Stellenbosch University. Faculty of Health Sciences. Dept. of Pathology.
ENGLISH ABSTRACT: Introduction: Since the South African public sector antiretroviral roll-out programme started in 2004, the success of antiretroviral combination therapy (cART) has been experienced in terms of survival, prevention of mother-to-child transmission (PMTCT) and quality of life. However, as the programme matures, viral resistance to the constituent drugs will increase. Monitoring antiretroviral drug resistance (ARVDR) should therefore be a priority in the public health approach to HIV treatment. Methods: A cross-sectional investigation of genotypic antiretroviral drug resistance in: a) HIV-infected mothers who were exposed to a PMTCT regimen of short course azidothymidine (AZT) with single dose nevirapine (NVP) during labour. b) HIV-infected adults and children who were cART-naïve (transmitted or initial resistance). c) HIV-infected adults and children who were failing cART (drug-induced or acquired resistance). In case of adults, this includes patients on a first-line, non-nucleoside reverse transcriptase (NNRTI)-based regimen, or on a second-line, protease inhibitor (PI)-based regimen, and in case of children, this includes patients on a first-line PI-based regimen. Results: In mothers who received a PMTCT-regimen that combined AZT and NVP the prevalence of NNRTI resistance mutations was 17.1% (95% CI: 8.7-25.6%). The prevalence of transmitted ARVDR in adults was low, as was initial ARVDR in young children (mostly PMTCT-exposed), except for NNRTI resistance in children who had received NVP as part of PMTCT. Drug-induced resistance was found in adults failing first-line NNRTI-based cART, with 83% having resistance to ≥1 drug. In contrast, adult patients failing second-line PI-based cART had a low prevalence of PI resistance; the predominant reason for failure was poor drug exposure, as detected by measuring lopinavir concentrations in blood plasma and hair samples. In contrast, PI resistance in children was not rare, largely due to historic exposure to un-boosted PIs. This resulted in extensive resistance to PIs and reverse transcriptase inhibitors (RTI) in some children. Conclusions: A combined regimen of short course AZT with intrapartum NVP for PMTCT may, in addition to reducing the risk of neonatal infection, also reduce the risk of NVP resistance in the mothers compared to a regimen of NVP only. In South Africa, the prevalence of transmitted ARVDR remains low relative to industrialised countries, probably as comparatively little time has elapsed since the scale-up of cART. Adults failing first-line cART are likely to respond to second-line cART, without failure due to resistance. However some children with PI and RTI resistance cannot be adequately treated with drugs currently available through the roll-out programme. This emphasizes the urgent need for a rational and science-based approach to managing cART-experienced children, including access to additional drugs to form a third-line paediatric cART regimen.
Investigation of small mammal-borne viruses with zoonotic potential in South Africa
(Stellenbosch : Stellenbosch University, 2013-12) Ithete, Ndapewa Laudika; Preiser, Wolfgang; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology, Division of Medical Virology.
ENGLISH ABSTRACT: The emergence and re-emergence of viral human pathogens from wildlife sources in the recent past has led to increased studies and surveillance of wildlife for potentially zoonotic agents in order to gain a better understanding of the pathogens, their sources as well as events that may lead to viral emergence. Of the >1407 known human pathogens, 13% are classified as emerging or re-emerging, and 58% as zoonotic; 37% of the (re-)emerging and 19% of the zoonotic pathogens are RNA viruses, accounting for the majority of recently emerged infectious diseases with a zoonotic origin, such as HIV, Ebola, Hendra, Nipah, Influenza and SARS. This study focusses on potentially zoonotic viruses hosted by rodents (Muridae family), shrews (order previously known as Insectivora/Soricomorpha, now reclassified as Eulipotyphla) and bats (order Chiroptera). Rodents and bats represent the largest (~40%) and second largest (~25%) mammalian orders and both occur on every continent except Antarctica. Together, the three mammalian orders investigated represent the most relevant potential sources of new zoonoses. In this study I investigated the occurrence of astroviruses, arenaviruses, coronaviruses and hantaviruses in South African small mammal species belonging to the orders mentioned above. These viruses have either been implicated in recent emerging zoonotic events or are considered to have the potential to cause cross-species transmissions resulting in a zoonotic event. In the first part of the study specimens collected from various bat, rodent and shrew species were screened for viral sequences by broadly reactive PCRs; positive samples were characterised by sequencing and sequence analysis. A separate part of the study focussed on hantavirus disease in humans: a seroprevalance survey was conducted to determine the presence of hantavirus antibodies in the local population. Additionally, acutely ill patients with potential hantavirus disease were tested in an attempt to identify possible acute infections and define clinical hantavirus disease in South Africa. Screening of rodent and shrew specimens resulted in the identification of eight novel arenavirus sequences. Seven of the sequences are related to Merino Walk virus, a recently identified South African arenavirus, and the eighth sequence represents a novel lineage of Old World arenaviruses. Screening of bat specimens resulted in the identification of highly diverse novel astrovirus and coronavirus sequences in various South African bat species, including the identification of a viral sequence closely related to the recently emerged Middle East Respiratory Syndrome coronavirus. While the study did not identify hantavirus infections in any of the acutely ill patients, it found seroprevalences similar to those observed in Europe and West Africa. The results obtained highlight the importance of small mammals in the emergence of potential zoonoses and further reinforce the importance of viral surveillance of relevant wildlife species. Further in-depth studies of naturally infected reservoir host populations are required in order to gain a better understanding of virus-host dynamics and the events that lead to virus emergence.
A Longitudinal Perspective on the Impact of Immune Status on the HIV-1 Latent Reservoir and Neurocognitive Outcomes in Virologically Suppressed Children
(Stellenbosch : Stellenbosch University, 2022-04) Naidoo, Shalena; Glashoff, Richard; De Beer, Corena; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology.
ENGLISH SUMMARY: Background: Children remain the most vulnerable population affected by the Human Immunodeficiency Virus-1 (HIV-1) pandemic whose reliance on lifelong therapy is accompanied by several immune abnormalities. In the absence of an effective vaccine, there is currently a strong emphasis on HIV-1 “cure” and although cART leads to viral suppression the virus is still able to establish latent reservoirs within host cells and this is considered the major barrier to achieving cure. Immune factors are considered critically important for the establishment and maintenance of HIV-1 latent reservoirs, but these factors are not well characterised, particularly in children. Delineating and understanding the immunological mechanisms that drive HIV-1 persistence and other chronic diseases such as metabolic, cardiovascular and neurodegenerative diseases is important in children approaching adolescence. Longitudinal studies evaluating the relationship between immune status, the HIV-1 latent reservoir and neurocognitive outcomes are limited, and inadequately studied, specifically within the South African subtype C context. The aim of this study was to longitudinally investigate and characterise host immune status before and after early initiated, delayed and interrupted cART in relation to HIV-1 latent reservoir size and neurocognitive outcomes in perinatally HIV-1 infected children. Methods: Study participants originated from the well-characterised Children with HIV Early AntiRetroviral Therapy (CHER) randomised controlled trial. This was a descriptive study that employed both a longitudinal (birth to 8 years of age) and cross-sectional study design and analysed samples collected retrospectively and prospectively. For the extensive longitudinal evaluation of immunological biomarker (cytokine, chemokine and receptor antagonist) profiling, we utilised Luminex® Multiplex Assays as well as Enzyme Linked ImmunoSorbent Assays (ELISAs). Multiparameter flow cytometry was utilised for the analysis of monocyte subset distribution. The quantitative viral outgrowth assay (QVOA) was implemented on a subset of participants for measuring of the HIV-1 replication-competent viral reservoir in conjunction with a novel highly sensitive RT-qPCR single copy assay targeted at HIV-1 integrase (iSCA). In addition, HIV-1 cell-associated DNA, specific for integrase (iCAD), were measured longitudinally as a molecular biomarker of HIV-1 persistence and correlated to immune and neurocognitive parameters. Longitudinal neurocognitive assessments were completed independently and correlated to immune, virological and clinical parameters. Age and demographically matched HIV exposed uninfected (HEU) and HIV unexposed uninfected (HUU) were included at the last time point (8 years of age). Key Findings: HIV-1 infected (HIV+) children showed significantly higher levels of biomarkers associated with generalised chronic inflammation, particularly those associated with myeloid cell activation compared to HEU and HUU controls at 8 years of age. These included hsCRP (p=0.01), MIP-1β (p=0.03), IL-1α (p<0.001), INF-α (p<0.001), CD40L (p<0.001), sCD14, sCD163, IL-18, IL-17F (p<0.001) and PDGF-BB (p<0.00001). We also observed a significant elevation of soluble calcium binding alarmin protein involved in the regulation of the inflammatory process and immune response, s100A8/A9, in the HIV+ group compared to the two study control groups (p<0.001). Within the HIV+ group, significant elevation of biomarkers associated with gut epithelial damage was observed at 8 years of age. IL-18 was the only immune biomarker that significantly correlated with HIV-1 CAD at baseline (r = +0.35; p=0.04) and at the 8-year follow-up (r = +0.38; p=0.02) and associated to the innate IL-1 family of immune biomarkers including IL-1RA (r = +0.33; p<0.01), IL-1α (r = +0.22; p<0.01), IL-1β (r = +0.26; p<0.01), and IL-1RA (r = +0.32; p<0.01). IL-18 also significantly correlated with biomarkers of monocyte/macrophage activation and gut damage, including: sCD14 (r = +0.50; p<0.01), sCD163 (r = +0.40; p<0.01), LBP (r = +0.26; p<0.01) and MIP-1β (r = +0.19; p=0.02). IL-18 showed significant negative associations (p<0.01) with CD4 % at baseline, longitudinal CD4 count and CD4:CD8 ratio at 8 years (r = +0.35; p=0.04). For longitudinal cytokine analysis, principal component analysis indicated that about 36% of the soluble biomarkers measured including: IL-15, TNFβ, GCSF, IL-1RA, IL-5, IL-4, IL-8, IL-10 and RANTES contributed to the largest parameter change over time across all treatment groups. The key early clinical predictors of plasma biomarker expression over time include time to therapy initiation, time to viral suppression, longitudinal CD4% and absolute count and CD4 and CD8% and absolute counts at birth. Neurocognitive outcomes can be predicted by early immunological, virological and clinical parameters. Conclusion: By investigating and profiling participants from the CHER randomised control trial cohort, we were able to provide important insights into immunological mechanisms that contribute to driving HIV-1 persistence during long-term suppressive therapy. The findings reported in this thesis have highlighted some key features of immune abnormalities that persists after early initiated long-term ART in paediatric populations. The evaluation of the persistence of any of the key associations through and beyond adolescence will aid in building a lifelong profile of immune changes in the MTCT-infected children. This research also provides important knowledge to further exploring PHIV children as potential “cure” and remission candidates.
Multidisciplinary viral analyses in people living with HIV-1C and receiving second-line combination antiretroviral therapy (cART) in South Africa
(Stellenbosch : Stellenbosch University, 2019-12) Obasa, Adetayo Emmanuel; Jacobs, Graeme Brendon; Neogi, Ujjwal; Kamalendra, Singh; Cloete, Ruben; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Medical Virology
ENGLISH ABSTRACT: The use of combination Antiretroviral Therapy (cART) has grown since its first introduction into the South African public sector. cART has significantly reduced the mortality rate caused by human immunodeficiency virus (HIV) in both high- and low-to-middle-income countries. The development of drug resistance has challenged the outcome of cART. This has led to the introduction of Integrase (IN) strand transfer inhibitors (InSTIs) as part of the first-line cART regimen. Due to their superior efficacy and high genetic barrier, this class of drugs was previously reserved as salvage therapy. The World Health Organization (WHO) supports InSTIs as first-line regimen non-nucleoside reverse transcriptase inhibitors (NNRTIs) particularly in regions where pre-treatment drug resistance to NNRTIs reaches 10%. Therefore, this study aimed to (i) to investigate the prevalence of InSTI mutations in treatment-naïve and treatment-experienced PLHIV using genotypic assays, which included Sanger sequencing, next-generation sequencing (NGS) and molecular modelling; (ii) analysed Long Terminal Repeats (LTR) to identify transcription factor binding sites. Chapter 2: Ninety-one (n = 91) treatment-naïve patients were obtained before the start of antiretroviral treatment in South Africa. Furthermore, we included 314 South African patient sequences obtained from the Los Alamos National Library database (www.lanl.gov). The IN gene ~ 900 base pairs [bps] was amplified and sequenced using conventional DNA Sanger sequencing. Homology structure was generated using the cryoEM structure of HIV-1B IN intasome (PDB file 5U1C) using ‘Prime’ of Schrodinger Suit. Chapter 3: Ninety-six (n = 96) treatment-experienced patients receiving boosted protease inhibitors (bPIs) as part of their cART treatment regimen were obtained for further analyses. We performed conventional DNA Sanger sequencing to analyse the complete pol gene (~3011bps) and sequences were analysed using the Stanford HIV drug resistance database to assess genotypic resistance associated mutations (RAMs). Chapter 4: Fifty-six (n = 56) treatment-experienced patients receiving boosted protease inhibitors (bPIs) as part of their cART treatment regimen were obtained. We performed a high-throughput (HT) sequence analyses on the complete pol gene using Illumina HiSeq2500, followed by bioinformatics analysis to quantify the RAMs according to the Stanford HIV drug resistance database. Chapter 5 and 6: We performed in-silico analyses on diverse HIV-1 subtypes based on 8114 sequences. These included treatment naïve and downloaded sequences from the HIV Los Alamos National Library Database (www.lanl.gov). Homology derived molecular models of HIV-1 IN tetramers from different subtypes were generated using cryoEM structure of the HIV-1B IN intasome. Chapter 7: Fifty-six (n = 56) treatment-experienced patients receiving boosted protease inhibitors (bPIs) as part of their cART treatment regimen were obtained. We performed Sanger sequencing to analyse the LTR gene (~ 474 bps) followed by bioinformatics analyses to identify transcription factor binding sites.The data indicates that in South Africa, the prevalence of RAMs against InSTIs is low and InSTIs can be used as a potential viable salvage therapy option and/or first-line regimen. Molecular modelling was done for IN structural analyses, which revealed how naturally occurring polymorphisms might affect structural stabilities, viral DNA binding and drug-binding propensity. This study represents a true baseline InSTI resistance rate as the treatment-naïve patients were obtained before the cART introduction. We propose GRT for people living with HIV (PLHIV) before treatment initiation and we recommend continued InSTIs drug resistance monitoring when introduced on a larger scale in South African.
Mutagenesis and functional studies of the HIV-1 vpr gene and Vpr protein obtained from South African virus strains
(Stellenbosch : University of Stellenbosch, 2011-03) Romani, Bizhan; Engelbrecht, Susan; Glashoff, Richard H.; University of Stellenbosch. Faculty of Health Sciences. Dept. of Pathology. Medical Virology
ENGLISH ABSTRACT: Background: Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) is an accessory protein that interacts with a number of host cellular and other viral proteins. Vpr exerts several functions such as induction of apoptosis, induction of cell cycle G2 arrest, modulation of gene expression, and suppression of immune activation. The functionality of subtype C Vpr, especially South African strains, has not been studied. The aim of this study was to describe the diversity of South African HIV-1 subtype C vpr genes and to investigate selected functions of these Vpr proteins. Methodology: The HIV-1 vpr region of 58 strains was amplified, sequenced, and subtyped using phylogenetic analysis. Fragments containing natural mutations were cloned in mammalian expression vectors. A consensus subtype C vpr gene was constructed and site-directed mutagenesis was used to induce mutations in postions in which no natural mutations have been described. The functionality of all constructs was compared with the wild-type subtype B Vpr, by transfecting human 293T cell line to investigate subcellular localization, induction of apoptosis and cell cycle G2 arrest. The modulation of genes expressed in the induction of apoptosis using TaqMan Low density arrays (TLDA) was also investigated. Results: Phylogenetic analysis characterized 54 strains as HIV-1 subtype C and 4 strains as HIV-1 subtype B. The overall amino acid sequence of Vpr was conserved including motifs FPRPWL and TYGDTW, but the C-terminal was more variable. The following mutations were constructed using site-directed mutagenesis: P14I, W18C, Y47N, Q65H and Q88S. Subtype B and all natural mutants of subtype C Vpr localized to the nucleus but the W18C mutation disturbed the nuclear localization of Vpr. The cell cycle G2 arrest activity of all the mutants, as well as consensus-C, was lower than that of subtype B Vpr. All the natural mutants of subtype C Vpr induced cell cycle G2 arrest in 54.0-66.3% of the cells, while subtype B Vpr induced cell cycle G2 arrest in 71.5% of the cells. Subtype B and the natural mutant Vpr proteins induced apoptosis in a similar manner, ranging from 95.3-98.6% of transfected cells. However, an artificially designed Vpr protein containing the consensus sequences of subtype C Vpr indicated a reduced ability to induce apoptosis. While consensus-C Vpr induced apoptosis in only 82.0% of the transfected cells, the artificial mutants of Vpr induced apoptosis in 88.4 to 96.2% of the cells. The induction of apoptosis associated gene expression was similar for all constructs, indicated that apoptosis was efficiently induced through the intrinsic pathway by the mutants. Conclusion: This study indicated that both HIV-1 subtype B and C Vpr display a similar ability for nuclear localization and apoptosis induction. The induction of cell cycle G2 arrest by HIV-1 subtype B Vpr may be more robust than many subtype C Vpr proteins. The natural mutations studied in the isolates did not disturb the functions of subtype C Vpr and in some cases even potentiated the protein to induce apoptosis. Naturally occurring mutations in HIV-1 Vpr cannot be regarded as defective, since enhanced functionality would be more indicative of an adaptive role. The increased potency of the mutated Vpr proteins suggests that Vpr may increase the pathogenicity of HIV-1 by adapting apoptotic enhancing mutations.
Origin and phylodynamics of HIV-1 subtype C in South Africa
(Stellenbosch : Stellenbosch University, 2013-12) Wilkinson, Eduan; Engelbrecht, Susan; De Oliveira, Tulio; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology, Division of Medical Virology.
ENGLISH ABSTRACT: The HIV epidemic in the past couple of decades has spread at an alarming rate throughout Southern Africa. Today the region accounts for roughly one third of all HIV infections, while prevalence rates in other areas of sub-Saharan Africa remain low. In the following study, sampled sequences from Cape Town, spanning over a 21-year period were used to investigate the epidemic history of HIV, which was compared to epidemic trends across Southern Africa. Longitudinal sequence data sets were generated from stored patient samples from Cape Town through standard molecular techniques. Firstly, these sequences were used to estimate the date of origin of the HIV epidemic in Cape Town and to reconstruct a demographic history of the epidemic with advanced Bayesian inference methods. These analyses placed the estimated date of origin of the Cape Town epidemic around the mid 1960‟s with periods of strong epidemic growth observed during the mid 1980‟s and 1990‟s. Secondly, reference strains of HIV from Southern African countries were used to estimate the date of origin of the epidemic in the Southern African region. These analyses placed the date of origin of the epidemic in the Southern African region around the mid 1950‟s roughly ten years before the start of the epidemic in Cape Town/South Africa. These sequences were also used for the reconstruction of the demographic history of the epidemic in the region. A two phased growth in the HIV epidemic in the Southern African region was observed with exponential growth occurring in the mid 1980‟s and 1990‟s. Such findings are also supported by HIV prevalence estimates made by some of the leading HIV research centres and government health departments. Thirdly, a large number of homologous reference strains were used to establish the evolutionary relationship of HIV isolates from Cape Town with those from around the world. A close genetic relationship between Cape Town isolates with other South African and other Southern African isolates was observed in these analyses. Finally, large monophyletic clusters of Cape Town isolates, which was observed during the evolutionary inference, were further investigated. After detailed analyses it appears that these transmission clusters of HIV-1 have been in circulation amongst the infected population of Cape Town for several years or decades.
Population structure, host cell interactions and pathogenesis of Staphylococcus aureus strains isolated at Tygerberg hospital, South Africa
(Stellenbosch : Stellenbosch University, 2013-12) Oosthuysen, Wilhelm Frederick; Wasserman, Elizabeth; Orth, Heidi; Sinha, Bhanu; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology.
ENGLISH ABSTRACT: Numerous studies conducted internationally have identified and described several endemic methicillin-susceptible Staphylococcus aureus (MSSA) clones. However, only some of these clones are associated with methicillin resistance (CC5, CC8, CC22, CC30 and CC45). To date, studies reporting on the population structure of S. aureus isolated in South Africa represent limited demographic areas, focus on methicillin-resistant S. aureus (MRSA) only and have displayed little emphasis on virulence. This study was undertaken to elucidate the population structure of S. aureus isolated from specific clinical sources at Tygerberg hospital, and to investigate specific host-pathogen interactions of representative isolates. Consecutive non-repetitive clinical S. aureus isolates were collected over one year (September 2009/2010) with patient demographics and limited clinical information. Strains were typed by PFGE and molecular markers (spa, multi-locus sequence typing (MLST), agr, Staphylococcal Chromosome Cassette mec and Panton-Valentine leukocidin (PVL)). Representative isolates were selected and investigated for the presence of virulence genes, adherence (to immobilised fibronectin [Fn], fibrinogen [Fg], collagens IV [CnIV] and VI [CnVI]), cellular invasion and cell death induction. Statistical association were determined between all in vitro results and methicillin-resistance, clonality, patient HIV status and bacterial PVL status. Fifteen percent of the isolates (n = 367) were MRSA. Forty four present of isolates were PVL+. agr I-IV and SCCmec I-V were identified. The MSSA population was diverse: ST22 (dominant), ST1865 and ST121 were PVL+. ST45, ST1863 and ST15 were PVL-. PVL- MRSA were diverse: ST612-MRSA-IV (dominant), ST5-MRSA-I, ST239-MRSA-III, ST36-MRSA-II and ST22-MRSA-IV. The genes fnbA/B (fibronectin-binding protein A/B), clfA/B (clumping factor A/B), eap (extracellular adherence protein), nuc (nuclease), coa (coagulase) and hld (delta toxin) were detected in all representative isolates. The CC8 and CC6 isolates adhered strongly to all ligands (100-700% of control, ligand dependent), while isolates of CC45, CC22 and CC88 adhered strongly only to Fg and Fn. The CC30, CC15, and CC12 isolates adhered extremely strongly to CnIV (>300%) and CC8, CC15, and C6 to CnVI (>200%). Isolates from CC30, CC8, CC15, CC6, CC12, CC97, CC88 and CC45 were highly invasive (>100%). ST121 was non-invasive (>50%). Isolates of CC5, CC30 and CC121 were non-cytotoxic (<50%), while isolates of CC22, CC8, CC15, CC45 and CC88 were very cytotoxic (>70%). No significant difference was observed in adherence or cell death induction of MRSA vs. MSSA clones or between isolates from HIV+ vs. HIV- persons. PVL- isolates displayed higher cellular invasiveness than PVL+ isolates. The presence of ST612-MRSA-IV, ST22-MRSA-V and ST8-MRSA-V points to local SCCmec acquisition, as we found MSSA isolates with the same spa types. Numerous MSSA clones were prevalent, but do not appear to have a major common genetic background with MRSA. PVL was highly prevalent among MSSA, indicating acquisition of PVL genes independently of SCCmec. The abilities to adhere to specific immobilised ligands in vitro were diverse and grouped with the genetic background, while the vast majority of isolates were invasive and induced significant cell death. We can conclude that the population of S. aureus at Tygerberg hospital is composed of a vast number of MSSA and MRSA clones, which display varying patters of adherence to selected ligands and of which, the majority clones are invasive and cytotoxic.

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