Department of Biochemistry and Microbiology

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Browsing Department of Biochemistry and Microbiology by Author "Bessong, Pascal Obong"

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    Burden of infection and genetic characterization of human herpes virus type 8 in HIV infected individuals in Northern South Africa
    (2019-05-16) Etta, Elizabeth Mashu; Bessong, Pascal Obong; Mavhandu - Ramarumo, Lufuno Grace
    Human herpes virus type 8 (HHV-8), also known as Kaposi’s sarcoma associated herpes virus (KSHV), is the etiologic agent of Kaposi’s sarcoma (KS), and AIDS related Kaposi’s sarcoma (AIDS-KS). HHV-8 which is a member of the Herpesviridae family, exhibits extensive genetic diversity globally. In endemic regions, infection with HHV-8 occurs very early on in life, which is an indication of both environmental and vertical routes of transmission. The advent of HIV leads to the classification of an AIDS-KS defining condition in HIV infections. This suggests that in regions where HIV and HHV-8 are endemic, KS may become common in a mature HIV epidemic. Just like the prevalence of HIV in Northern South Africa is generally high as in most regions of the country, as the HIV epidemic matures in South Africa, it is important to understand the burden and distribution of HHV-8 infection, and the likely genotypes infecting the population. The main objective of the thesis was to establish the epidemiology and infecting genotypes of HHV-8 in Northern South Africa (Limpopo Province), where no data exists. First, a systematic review of the literature was carried out for the entire African continent to determine the seroprevalence and genotype distribution of HHV-8 in all African countries (n=53). In this review, Sudan and South Sudan were considered as one country. Articles were searched using the PRISMA guideline and exported using an article grid. More than two-thirds (64%) of the studies reported on seroprevalence, 29.3% on genotypes; and 9.5% were on both seroprevalence and genotypes. About 45% (24/53) of the African countries had data on HHV-8 seroprevalence exclusively, and more than half (53%) had data on either seroprevalence or genotypes. Almost half (47%) of the countries had no data on HHV-8 infection. There was high heterogeneity in the types of tests and interpretation algorithms used in determining HHV-8 seropositivity across the different studies. Generally, seroprevalence ranged from 2.0% in a group of young children in Eritrea to 100% in a small group of individuals with KS in the Central Africa Republic and a larger group of KS in individuals in Morocco. Approximately, 16% of all the studies reported on children. The difference in seroprevalence across the African region was not significant (95% CI, X2 =0.86; p =0.35), although specifically, a relatively significant ETTA MASHU ELIZABETH, PHD IN MICROBIOLOGY|UNIVERSITY OF VENDA, 2019|VIII level of infection was observed in HIV-infected children. About 38% of the countries had data on K1 genotypes A, A5, B, C, F and Z which occurred at frequencies of 5.3%, 26.3%, 42.1%, 18.4%, 5.3% and 2.6% respectively. Twenty-three percent of the countries had data for K15 genotypes, whereas genotypes P, M and N occurred at frequencies of 52.2%, 39.1% and 8.7% respectively. Data on HHV-8 inter-genotype recombinant is scanty. Our finding suggests that HHV-8 is endemic on the entire African continent, and in HIV endemic regions, but there is need for a harmonized testing protocol for better understanding of HHV-8 seropositivity. HHV-8 genotype A5 and B for K1 gene and genotype P and M for K15 gene are the most predominant genotypes in Africa. The review, for the first time, has provided information on HHV-8 burden on the entire African continent, and suggests that vaccine development efforts for Africa should focus on genotypes B and P. The second component of the investigation focused on the burden of HHV-8 in an HIV population in Northern South Africa (Limpopo Province). Plasma from 3501 HIV infected individuals from 5 districts in Limpopo Province were assessed for antibodies to both the lytic antigen (ORFK8.1) and the latent antigen (ORF73). The distribution of infection was analyzed based on demographic, socioeconomic, and immunological parameters. Statistical inferences for significant differences were determined by Chisquare at a confidence interval of 95%. P-values less than 0.05 were considered significant. About 19.0% of the study population was positive for antibodies to either the lytic or latent antigens or both. Prevalence of antibodies to the lytic antigen was significantly higher than prevalence of antibodies to the latent antigen (17.3% vs 4.1%; p=0.0001). Significant differences were observed for age groups, racial population groups, districts and year of sample collection (p=<0.0001, p=<0.0001, p=<0.0001 and p=0.0385) respectively. Associations were found between both antigens in comparison to the different variables such as age group, racial population groups and districts (R2 value ranging between 0.886 and 1.0). The burden of HHV-8 has now been established for the first time in Northern South Africa. The third aspect of the investigation was a meta-analysis of HHV-8 seroprevalence in Southern Africa in order to understand the impact of geographical location (urban vs rural) on infection. The analysis revealed a significant association between urban settings and HHV-8 infection (p=0.0001). ETTA MASHU ELIZABETH, PHD IN MICROBIOLOGY|UNIVERSITY OF VENDA, 2019|IX The fourth component of the thesis examined the detection of HHV-8 antigen through polymerase chain reaction (PCR) in 534 participants in HIV infected and HIV noninfected populations. A selection of mouthwash DNA samples were subjected to Next Generation Sequencing (NGS) for subsequent genotype inference. Mouth wash samples were obtained from each consenting individual before eating or smoking, and their DNA was purified. A 233bp fragment of the ORF26 gene of HHV-8 was amplified by PCR. HHV-8 was detected in 150 of the 534 participants (28.1%). A significant difference in detection was observed for gender, HIV status, district and the level of education (p=0,0003; p=0.0094; p=0.0002 and p=0.0095) respectively. Consensus sequences were derived from NGS reads for 13 samples. The genotyping results revealed that genotype Q, B, E and N are the genotypes predominant in the study population. As such no mixed infections were detected. Therefore, from the investigations foregoing have demonstrated for the first time the following: (1) HHV-8 is endemic in the entire African continent, which suggest a coendemicity in regions already endemic for HIV; (2) HHV-8 is endemic in Northern South Africa; (3) Urban settings in Southern Africa are associated with high HHV-8 infection; (4) HHV-8 genotypes Q, B, E and N may be predominant in Northern South Africa, with B and P common on the entire African continent. Hence, studies should focus on the generation of full length HHV-8 genomes of the common genotypes to support the selection of genes for vaccine design and development.
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    Characterization of cervicovaginal HPV virome and bacteriome in HIV -i infected women in Northern South Africa
    (2023-05-19) Ratshilindela, Mpho; Bessong, Pascal Obong; Tebit, Denis
    BACKGROUND: The presence of a highly diverse bacterial species in the cervicovaginal niche is linked to a higher risk of contracting human immunodeficiency virus (HIV), persistent infection with human papillomavirus (HPV) and consequently cervical cancer. It is unknown how cervicovaginal bacterial species associate and interact with other viruses. This study hypothesized that HIV/HPV co-infected women have an increased diversity of cervicovaginal virome and bacteriome. OBJECTIVES: The study’s primary objective was to characterize cervicovaginal HPV virome and bacteriome in HIV-infected women from selected health care facilities in Northern South Africa. Specific objectives were to determine the presence of HPV virome in HIV-infected women and HIV-noninfected women, to determine the presence of bacteriome in HIV-infected women and HIV-noninfected women, and to determine the relational occurrence of virome and bacteriome according to HIV status. METHODOLOGY: Cervical swabs from 50 HIV/HPV co-infected women; 50 HIV-infected, HPV-noninfected women; and from 50 HPV-infected, HIV-noninfected women were used to extract total deoxyribonucleic acid (DNA). To determine HPV virome, total DNA was enriched by rolling circle amplification (RCA) using an illustra TempliPhi amplification kit. To determine bacteriome, a two-round polymerase chain reaction (PCR) was employed targeting a bacterial 16S rRNA gene. Amplification products obtained from RCA and PCR were purified and sequenced by Next Generation Sequencing (NGS) using a MiniSeq platform (Illumina). The quality of the sequences was validated using the FastQC program. Sequences of good quality were assigned to viral family and genera using the Dragen metagenomics online tool with BaseSpace Sequence Hub. Bacterial sequences were assigned and categorised into vaginal community state types (CSTs) using the Dragen metagenomics online tool with BaseSpace Sequence Hub. The relational occurrence of virome and bacteriome according to HIV status was assessed through Chi-square statistical analysis available in GraphPad Prism version 9.3.1. Differences in occurrence were expressed as probability (P)-values. RESULTS: A diverse group of viral families was observed among HIV/HPV co-infected women. Papillomaviridae (14/34; 41%) was the most prevalent (P<0.0001), followed by Herpesviridae and Phycodnaviridae (12/34; 35%) each, Poxviridae (10/34; 29%), Mimiviridae (7/34; 20%), Maiseilleviridae (3/34; 9%) and Anelloviridae (2/34; 6%) in order of decreasing prevalence. A highly diverse group of bacteriophages was observed, with Myoviridae (31/34, 91%) being the most prevalent (P<0.0001). Among HPV-infected HIV-noninfected women, v Papillomaviridae (8/26; 31%) was the most prevalent (P<0.0001), followed by Anelloviridae (4/26; 15%), Phycodnaviridae (4/26; 15%), Poxviridae and Herpesviridae (2/26; 8%) each in order of decreasing prevalence. Myoviridae (6/26; 23%) was the most prevalent bacteriophage family detected (P=0.0005). Among HIV-infected HPV-noninfected women, a small group of viral families was observed, with Myoviridae (3/11,27%) and Siphoviridae (3/11, 27%) being the most prevalent viral families detected (P=0.0005 each). HPV 16 was the most common high risk (hr) HPV type in HIV-infected women and HIV-noninfected women of this study. This genotype co-existed with other hrHPV or probable hr types including HPV 54, HPV 53, and HPV 26. Overall, hrHPV types were more prevalent in HIV-infected women than in HIV-noninfected women, although this difference was not statistically significant (P=0.2832). When the occurrence of hrHPV types were considered individually, HPV 54 occurred more significantly in HIV-noninfected women than in HIV-infected women (P<0.0003). Bacteriome revealed the presence of community state types (CSTs) one, two, three, four and five among study participants. Among HIV/HPV co-infected women, CST one (L. crispatus), CST three (L. iners) and CST four (high bacterial diversity) were observed, with CST four being the most prevalent. Among HPV-infected, HIV-noninfected women, CST one, three, four and five (L. jensenni) were observed, with CST four, also being the most prevalent. Among HIV-infected, HPV noninfected women, CST three and four were observed. CST three and four were associated with viral infections. Gardnerella vaginalis (75%), Pretovella spp (54%), Atopobium spp (50%), Bifidobacterium spp (27%), Porphyromonas spp (53%), Pseudomonas spp (50%), Faecalibacterium prausnitzii (47%) and Bacteroides spp (35%) were the most prevalent anaerobic bacterial species detected in CST four. A diverse group of bacterial families occurred more significantly among HIV-infected women as compared to HIV-noninfected women for NGS data of RCA products (P<0.0001) and NGS data of 16S amplification products (P<0.0001). CONCLUSION: In conclusion, this study showed a higher diversity of cervicovaginal virome and bacteriome in women who were HIV/HPV co-infected than in those singly infected with HIV or HPV. The relationship between viral infections and a high diversity of bacterial species (CST four) observed herein may be a useful indicator of the individual’s disease state, indicating the likelihood of developing cervical lesions and cervical cancer. As a result, additional research is necessary to uncover the association between viral infections and CST four with disease state. Vaccine development and antiviral research should also target compounds that boost the cervicovaginal environment and maintain vaginal homeostasis.
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    Characterization of HIV-1 drug resistance mutations from plasma and peripheral mononuclear cells in patients failing antiretroviral treatment in Bela-Bela, South Africa
    (2015-09-16) Etta, Elisabeth Mashu; Bessong, Pascal Obong; Tebit Denis Manga
    BACKGROUND: The current expansion of antiretroviral treatment (ART) in developing countries, such as South Africa, which lacks routine virological monitoring raises concerns on the outcome of the strategy in terms of virological success and the drug resistant burden. With an estimated 3.7% of the patients failing first-line treatment after 2 years and 17.9% after 4 years on treatment, there is qualified need for practical and routine drug resistance testing to provide data to clinicians in order to improve the lives of these patients. Thus, this study was conducted to characterize and compare HIV-1 drug resistance mutations in peripheral blood mononuclear cells (PBMCs) and in the plasma of patients whose therapeutic regimen is failing. METHOD: The study was nested within the Bela-Bela Wellness clinic, Limpopo Province South Africa. Approval for the study was obtained from the Health, Safety and Research Ethics Committee of the University of Venda. Blood specimens were collected from 23 HIV-infected drug experienced, individuals between July 2013 and October 2014 who met the following criteria: (1) two consecutive viral loads on treatment greater than 1000 copies/ml after previous suppression; (2) one viral load greater than 1000 copies/ml after previous suppression followed by a change in treatment; and (3) one viral load greater than 1000 copies/ml after 180 days on ART without suppression. The protease (PR) and reverse transcriptase (RT) genes of the 23 treatment exposed HIV infected patients failing therapy were PCR amplified, sequenced, subtyped and analysed for the presence of drug resistance mutations. RESULTS: Twenty one (91%), out of 23 specimens were successfully amplified. Comparison of the amino acid sequence of the PR and RT genes in the cell-associated variants of HIV-1 with that of plasma revealed that 17(81%), out of the 21 specimens tested, exhibited major drug resistance both in PBMCs and plasma. However, the greatest number of mutations were found in plasma (D67N, K103N, V106M, Y181C and M184V) occurred most frequently. On the other hand, V106M, K103N and M184V were observed most often in PBMCs. The prevalence of predicted resistance mutations in our study corresponding to respective ART usage were thus: NRTI, (n=11), NNRTI, (n=13) and Pl, (n=4). The most common mutations for RT were M184V>K103N>V106M>D67N. It is noteworthy that M230I an NNRTI was found in PBMCs only. Major mutations for PR were M46I> D30N>V82A. Seventeen of the 21 viruses (81%) were HIV- 1 subtype C in the polymerase gene; one virus was subtype B (4.8%) and one was a C/B recombinant (4.8%). One virus (4.8%) could not be conclusively typed. In conclusion, this study firstly, confirmed previous findings that the cellular compartment of blood may contain an archive of drug resistant variants making an interesting compartment for analysing the evolution of drug resistance in a given patient. Secondly, the prevalence of drug resistance mutations observed in Bela-Bela drug experienced individuals is very fairly high; and finally, there appears to be a continual presence of recombinant viruses circulating in the study region.
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    Drug resistance genotyping and phylogenetic analysis of HIV in chronically infected antiretroviral naive patients
    (2019-05-18) Baloyi, Tlangelani; Bessong, Pascal Obong; Traore, Afsatou Ndama
    Background: Antiretroviral treatment (ART) has grown to be one of the most effective tool in the fight to control HIV/AIDS morbidity and mortality worldwide. However, due to the emergence of drug resistant HIV, ART efficacy can be jeopardized. Drug resistant HIV strain has a potential of becoming a major public threat, as its limit treatment options on people living with HIV. With several findings worldwide reporting drug resistant HIV to be currently being transmitted to ART-naïve persons, measures have been taken to genotype drug resistant HIV prior to treatment initiation. However, in resource limited countries such measures are not executed especially in public sectors due to the costs associated with the required assays for genotyping. Objective: The objectives of the study was to establish a deep sequencing protocol (Next Generation Sequencing-NGS) using an Illumina MiniSeq Platform and subsequently apply it to genotype HIV in chronically infected drug naïve persons for resistance mutations and viral genotypes Methods: HIV positive Individuals without any exposure to ART (Treatment-naive) were recruited. Partial pol fragment (complete protease and ~1104bp reverse transcriptase) were amplified and purified. Libraries were prepared using Nextera XT library preparation kit, fragmented, tagmented, pooled and denatured then sequenced with Illumina MiniSeq instrument. Consensus sequences were derived, aligned and phylogenetically analysed. The Stanford HIV Drug Resistance Algorithm was used to infer the presence of drug resistant mutants, at the viral minority and majority population levels. Results and discussion: An NGS protocol to generate nucleotide sequences for drug resistance inference was established. No major drug resistance mutations were detected against protease, reverse transcriptase inhibitors in the study subjects investigated. Nevertheless, V179D change was observed in one patient (8.3%). V179D has been shown to impact a low-level resistance to NNRTI. On the other hand, several secondary and unusual mutations at known drug sites were detected even at minority threshold level of <20%. Conclusion: No major drug resistance mutations was detected in the drug naïve study population. This finding suggests that there is no risk of treatment failure to the investigated subjects, however it is important to assess the potential phenotypic v | P a g e significance of the identified secondary resistance mutations in the context of HIV-1 subtype C. The established NGS protocol should be applied in subsequent HIV drug resistance studies.
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    Evaluation of adherence to antiretroviral therapy using efarivenz as a marker
    (2019-09-20) Tambe, Lisa Arrah Mbang; Mavhandu-Ramarumo, Lufuno Grace; Bessong, Pascal Obong; Keterere, David R.
    Background: Patients on antiretroviral (ART) are expected to be at least 95% adherent to their treatment, as this will increase their chances of achieving treatment success (maximum and durable suppression of HIV-1 viral load); non-adherence may lead to the development of HIV drug resistance, which may lead to virologic failure and treatment failure. Therapeutic drug monitoring (TDM) has been reported to be the most efficient method to assess treatment adherence in HIV individuals, since it quantifies the concentration of ARTs in biological matrices. This is very effective when using a robust technique such as liquid chromatography tandem mass spectrometry (LCMS/MS), which has played a significant role in the evaluation and interpretation of bioavailability, bioequivalence and pharmacokinetic data. Even with patient adherence, various intra-individual factors have an influence on the expression and function of the genes responsible for the transport (MDR1) and metabolism (CYP2B6) of Efavirenz (EFV). This may lead to single nucleotide polymorphisms (SNPs) in these genes, and this may affect the way antiretrovirals (ARVs) are metabolized. The aim of this study was to evaluate the EFV concentration in plasma to assess patient adherence to treatment and correlate this with genomic occurrences in human and viral genes. Hypothesis: The concentration of ARVs in patient plasma can be used to estimate adherence to treatment; while ARVs’ transport and metabolism can affect bioavailability in a patient’s system. Research Question: Can EFV concentration in plasma be used to estimate patient adherence to treatment? Can transport and metabolism of EFV affect their bioavailability in the patient’s system? Objectives: To determine EFV concentration in plasma to assess patient adherence to treatment and correlate this with genomic occurrences in human genes and viral genes. Methodology: Twenty blood samples were collected from HIV positive individuals before treatment initiation (baseline) and between six to twelve months following treatment initiation (follow-up). The concentration of EFV in patient plasma was measured by LC-MS/MS technique. To infer other factors influencing patient pharmacokinetics output, drug resistance and human genetic characteristics were analyzed. A 1.65kb fragment of the HIV-1 Pol gene was amplified and sequenced to determine drug resistant mutations; while 363bp and 289bp of the MDR-1 and CYP2B6 human genes respectively, were also amplified and sequenced to determine polymorphisms in the transport and metabolism genes. Obtained sequences were manually edited and analyzed using Geneious Version 11.1.5 software. The Stanford HIV Drug Resistance database was used for drug resistant mutation (DRMs) analysis and MDR1 and CYP2B6 test sequences were compared with variant reference sequences to detect the presence of any SNPs. Results: The plasma EFV concentration at baseline and follow-up range was as follows: 0 – 1183ng/ml and below limits of quantification (BLQ) to 15,670ng/ml, respectively. At baseline, 0ng/ml is the expected plasma EFV concentration for patients about to commence treatment; however, two out of twenty patients had 769.9 and 1,183ng/ml drug levels in their system. Post treatment, plasma EFV levels in patients are expected to range from 1,000 – 4,000ng/ml, however, of the twenty patients, two had <1,000ng/ml, and three patients had >4,000ng/ml in their plasma. For Pol amplification, 35% (7/20) were positively amplified at baseline and 25% (5/20) were positively amplified from the follow-ups; 100% (20/20) samples were amplified for both CYP2B6 and MDR1 genes. Detection of drug resistance in the baseline Pol sequences revealed the absence of major mutations in both NRTI and NNRTI drug classes. The G516T polymorphism was present in 15% of the study participants while the homozygous GG and heterozygous GT genotype was present in 25% and 40% of the study participants, respectively. Allele determination was impossible in 20% of the samples, due to the poor nature of the sequence. The homozygous TT variant polymorphism at position 3435 was absent in the entire population, however, the CC and CT genotype was present in 15% and 85% of the study participants respectively. Analysis of EFV concentration in close proximity with the human genetic characteristics reveals that the presence of a Single Nucleotide Polymorphism affects the pharmacokinetic output observed. Discussion and Conclusion: Post treatment, 90% of the study participants indicate adherence to treatment, with only 10% of them having lower than expected EFV concentrations, implying they were non-adherent to their treatment. However, because plasma drug concentrations only reflect a patient’s adherence pattern for a few hours to at most two days, the adherence patterns of these individuals cannot be concluded with certainty. Using plasma EFV as a biomarker to evaluate adherence to treatment in HIV seropositive individuals is a feasible technique, however, its application in non-research settings is still a drawback due to the cost of the method. Characterizing patient inter-individual differences should be taken into consideration, especially since any polymorphism in their transporter and metabolizing genes may influence their overall treatment success.
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    Expression of TN5 transposase for next generation sequencing protocol of HIV ad selected oncoviruses
    (2021-03) Mathobo, Phindulo; Bessong, Pascal Obong; Mavhandu-Ramarumo, Lufuno Grace
    Objective: The development of HIV drug resistance is a significant challenge in maintaining suppressed viral load in the management of patients. Next generation sequencing (NGS) is a more sensitive approach to determine the burden of HIV drug resistance. We aimed to describe the uptake of NGS in HIV drug resistance studies in Africa. Methodology: Electronic search was done for studies published between 2005 and 2019, from three databases: Pubmed, Web of Science and Google scholar. The search approach was carried out according to PRISMA guidelines. Studies included in the analysis were those that reported the use of NGS on HIV drug resistance using samples from Africa or in which the studies were done in Africa. Only articles published in English were included in the analysis. Results: Four thousand one hundred and eighty articles were identified according to the search criteria. Out of these, 238 studies met the inclusion criteria and were included in the analysis. Thirty studies (12.6%) used NGS, 194 (81.5%) used Sanger sequencing, and 14 (5.9%) used both techniques. Evidence of in-country application of NGS was observed in six studies (13.6%), all from South Africa. In other studies, NGS was either done outside of Africa using samples obtained from Africa or the country in which NGS was done was not indicated. From 2005 to 2012, only one study was reported on HIV drug resistance using NGS; however, 44 studies were published by 2019. Out of the 54 African countries, investigators from 13 countries (24.1%) published data on HIV drug resistance using NGS, as at end of 2019. Conclusion: There is an uptake in the application of NGS in HIV drug resistance studies in Africa, albeit in a slower pace, with investigators from about a quarter of African countries applying the technology for this purpose.
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    Genetic analysis of HIV-1 integrase sequences from treatment naive individuals in Northeastern South Africa
    (Molecular Diversity Preservation International (MDPI), 2013-03-01) Bessong, Pascal Obong; Nwobegahay, Julius
    Raltegravir, an integrase inhibitor, is not a component of the current South African antiretroviral treatment guidelines, but it could be introduced in the near future as cases of virological failures from current treatment regimens begin to occur. The aim of this study was to analyze the complete HIV integrase gene obtained from individuals at two treatment sites in northeastern South Africa for the presence of Raltegravir associated drug resistant mutations and viral subtypes based on the integrase gene. Examination for mutations against other integrase inhibitors, such as Elvitegravir and Dolutegravir, was also done. Viruses from 127 treatment naive individuals were analyzed. Genetic drug resistance mutations were determined using the Stanford HIV Drug Resistance Interpretation program and the International AIDS society-USA guidelines. Viral subtyping was done by phylogenetic analysis, and recombinants were determined using the REGA, jpHMM and RIP tools. No major resistance mutations were detected. However, 7% of the sequences had minor mutations and polymorphisms. The majority (99%) of the viruses were HIV-1 C. Recombination analysis showed that the polymerase gene of one virus was likely composed of HIV-1 subtype A1 and C sequences. The present study indicates that Raltegravir, Elvitegravir and Dolutegravir resistant mutations may be absent in the study communities and further indicates the presence of recombinant viruses in northeastern South Africa.
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    Genetic analysis of human papillomavirus in a cohort of women in routine care in Northern South Africa
    (2019-05-18) Rikhotso, Rixongile Rhenny; Bessong, Pascal Obong; Mavhandu - Ramarumo, Lufuno Grace
    BACKGROUND: Human papillomavirus (HPV) is a common sexually transmitted virus known to be a causative agent of cervical cancer (CC), one of the most frequent cancers in women worldwide. HPV is a double stranded DNA virus of approximately 7,900 bp; belonging to Papillomaviridae family. To date, about 202 low risk (LR) and high risk (HR) HPV genotypes have been identified. However, available vaccines against HPV infection are designed based on the most common known genotypes. Therefore, it is critical to understand the scope and diversity of HPV genotypes in all geographical locations which can help to inform the design and development of future vaccines. OBJECTIVE: The objective of this study was to describe the burden and diversity of HPV genotypes in a cohort of women in routine care in northern South Africa. METHODS: Eighty seven women consented to participate in the study and each provided a specimen for analysis. With the help of qualified health care practitioners, Aptima Cervical Specimen Collection and Transport Kit (Hologic, San Diego, CA) was used to collect cervical specimens from each study participant following the manufacturer’s procedure. Total DNA was purified from the cervical pellet using QIAamp DNA mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The purified DNA was then subjected to a single round conventional PCR in a reaction volume of 100 μl to amplify HPV L1 gene comprising of approximately 450 bp. A portion of each PCR amplicon from each participant was denatured, hybridized and genotyped using the Linear Array HPV genotyping Test Kit (Roche Molecular Systems, Inc. Branchburg, NJ USA). The kit is designed to detect 37 HPV genotypes (genotypes 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 64, 66, 67, 68, 69, 70, 71, 72, 73, 81, 82, 83, 84, IS39 and CP6108). To detect the HPV genotypes, the Linear Array (LA) reference guide was used for results interpretation following the manufacturer’s instructions. The other portion of each of the amplicons was subjected to next generation sequencing (NGS) using the Illumina MiniSeq platform. Using the Nextera XT DNA Library preparation kit, an initial input of 1ng genomic DNA was tagmented, cleaned up, normalized and pooled. The pooled library was then denatured with 0.1 N NaOH and diluted into a final volume of 500 μl at 1.8 pM then sequenced using the Local Run Manager option following the manufacturer’s instructions. The generated sequence data was downloaded into fastaQ format and analysed using Genious 11.0.5 software. RESULTS: Of the 87 participants, the overall proportion of women harbouring HPV DNA by linear array (LA) PCR was 23% (n=20). Of the 20, 16 (80%) were living with HIV. However, this difference was not significant (p=0.077). Genotyping data generated by Roche LA method was successful for all the 20 positive amplicons. In this study, 27 (73%) of the 37 HPV genotypes incorporated in the Roche Linear Array method were detected. The detected genotypes include: types 84, 83, 81, 73, 72, 71, 70, 69, 68, 66, 62, 61, 59, 54, 53, 52, 51, 45, 42, 39, 35, 26, 18, 16, 6, IS39 and CP6108. Most women (15/20;75%) harboured multiple infections compared to single infection. In terms of genotypes distribution, the most frequent genotypes detected LR HPV types in increasing order of frequency included HPV type 61 and 83 (12%), 62 (36%) and 81 (43%). On the other hand, HPV type 66, 53, 52, 51, 18 and 16 were the most common genotypes detected HR HPV types. In contrast, although genotyping data was successfully generated from 15 of 20 women (75%), NGS technology was seen to be more sensitive compared to Roche LA method. Nearly all the detected genotypes identified by the commercial kit were detected by NGS. In addition, NGS detected 10 namely: HPV types 11, 31, 33, 40, 55, 56, 58, 64, 67, and 82 that were not detected by the LA yet incorporated in the kit. Moreover, it was observed that NGS identified additional 6 HPV types including HPV types 2, 27, 30, 35, 85 and 102 not incorporated in the Roche LA kit. A similar distribution of HPV multiple infections was observed in the study population, however, high frequency of 93% (14 of 15) was detected by NGS. The proportion of women harbouring one or more of the 22 LR HPV types was 100% (n=15).The most frequent LR genotypes in increasing order of frequency was HPV type 62 and 70 (27%), 6 (40%) and 11 (47%). HPV types 40, 42, 54, 72, 64, and 81 were the least detected genotypes with n=1 (7%) each. Furthermore, the common combination observed among the participants was type 6 and 11. In contrast, the most frequent detected genotypes in the study population by NGS under the HR HPV types in increasing order of frequency include type 35 (21%), 39, 56 and 82 (29%), 68 (36%) and 51 (50%). In addition, HPV types 26, 31, 45, 53, 56, 58 and 66 were the least detected genotypes n=1 (7%) in the study population. HPV 39 and 68 were observed as the common combination detected under HR HPV types. Following genotyping by LA and NGS, the demographic and clinical data of all the 20 positive subjects by PCR were subjected to statistical analysis to determine the association between HPV positive DNA status and associated risk factors. Smoking status (p=0.000), age at first sexual intercourse (p=0.011), vaccination status (p=0.000), gender of sexual partner (p=0.000), highest level of education (p=0.004), marital status (p=0.008) and number of sexual partners (p=0.000) were found to be having a positive statistical association. CONCLUSION: Amplification of targeted HPV DNA from cervical specimens demonstrated the presence of HPV infection in the study cohort, with a proportion of 23%. The findings illustrate that there is a diversity of HPV genotypes prevalent in the study population as shown by Roche LA and NGS methods. However, the NGS method was observed to be more sensitive than Roche LA in detecting HPV genotypes. Furthermore, NGS identified 6 additional HPV types not incorporated in the Roche LA. Thus, there are genotypes that may be present in the study population that the Roche commercial kit may fail to detect. Therefore, is it imperative to use both genotyping methods to confirm HPV genotypes.
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    Genetic characterization of human immunodeficiency virus from Northern South Africa
    (2012-12-19) Iweriebor, Benson Chuks; Bessong, Pascal Obong; Mphahlele, Jeffrey; Moyo, Sylvester Rodgers
    Globally, human immunodeficiency virus type 1 (HIV-1) is extraordinarily variable, and this diversity poses a major obstacle to AIDS vaccine development, diagnosis and therapy. Since HIV-1 M group began its expansion in humans roughly 70 years ago it has diversified rapidly now comprising a number of different subtypes and circulating recombinant forms (CRFs). Currently, strains belonging to the same subtype can differ by up to 20% in their envelope gene, and between subtype distances can soar to 35%. Moreover this diversity is continually growing. Although the scale of the HIV-1 pandemic makes action imperative, there is still much to learn about the extent and immunological implications of HIV-1 sequence diversity. As with other infectious agents, effective public health surveillance is essential to track the epidemic, guide research, and direct prevention activities. Significant challenges were highlighted by recent finding that some of the more divergent HIV-1 strains were not reliably detected by all antibody screening tests in current use. HIV-1 subtype C is responsible for the vast majority of the estimated 5 million infected South Africans. Thus, while subtype C viruses dominate the epidemic, the exceptional high prevalence rates in South Africa could provide significant opportunities for the spread of new genetic subtypes and/or evolution of current subtypes. Furthermore, analysis should therefore focus on areas where little information is known, such as the Limpopo Province, and where the opportunity for the introduction of new variants exists, and where prevalence rates are relatively high. The strategic location of the Province makes it imperative for regular diversity studies to be carried out. The implications of HIV-1 diversity in diagnostics and vaccine development; and the formulation of treatment regimens necessitated this study. Thus, this study therefore identified and characterized circulating HIV-1 genetic variants in sere-positive, drug na"fve populations from Limpopo Province where there is an estimated prevalence rate of about 19%. The molecular epidemiology of HIV-1 in two highly endemic areas in Limpopo Province of South Africa; mutations associated with drug resistance to protease inhibitors (Pis), nucleotide reverse transcriptase inhibitors (NRTls) and non nucleoside reverse transcriptase inhibitor (NNRTs), co-receptor usage and epitope mapping of HIV-1 isolates from drug naive individuals were investigated. Subtyping was done by phylogenetic analysis making use of ClustalX2 software while drug resistance mutation analysis, co-receptor usage prediction epitope mapping and substitution of the functional motifs were all determined by interpretation algorithms. Phylogenetic analysis of the test isolates revealed that subtype C is predominantly driving the epidemic in northern South Africa (Limpopo Province). Co-receptor usage prediction showed that majority of the isolates were R5 viruses as all had their tetrapeptide GPGQ motif characteristic of subtype C utilizing CCR5 viruses conserved. Genetic drug resistance mutation analysis of the 35 PR gene sequences did not reveal any major mutation associated with resistance but a high degree of minor mutations and polymorphisms were observed. Examination of the 44 RT genes showed a K103N substitution in two isolates. K103N change causes high level resistance to nevirapine. Epitope mapping of the gag p17 and p24 consensus sequences of the test isolates did not reveal any difference between them and the subtype C consensus sequences. They all had the same dissociation constant for the epitopes recognized by the HLA they were mapped against. Also, all the functional motifs in the PR and RT genes were conserved in majority of the test isolates. Molecular characterization of the test isolates has helped to update the baseline data on the circulating strains of HIV -1 in northern South Africa. Since all the isolates are subtype C as in other regions of South Africa and result of epitope mapping compares very well with those of subtype C consensus sequences, vaccine based on subtype C viruses could be designed and evaluated in the Province. Also, it has shown that entry inhibitor- the new class of antiretroviral drug could be of significance should the current NRTls and NNRTls begins to fail as majority of the isolates had their GPGQ motifs conserved. Since no important resistance mutation to the Pis and RTls was found among the test isolates, usage of these classes of drugs will continue to have positive impact in reducing morbidity and mortality due to AIDS in the studied area. The second element of this study focused on the amplification and analysis of a unique recombinant form composed of subtypes A1 and C subgenomic regions. Recombination plays a key role in HIV-1 genetic diversity which on the long run has a grave implication on diagnosis, therapy and vaccine development. The URF recombinant that was analysed was isolated from a female patient residing at Bela Bela which has a high HIV-1 prevalence. The amplified near-full length genome was sequenced by the 454 Genome Sequencer FLX system. The sequence generated was delineated into respective gene by the sequence locator- a web­ based online tool. Analysis of the recombinant virus was carried out in order to determine the subtypes that constituted the mosaic genome making use of jpHMM and REGA subtyping analytical tools. The results obtained revealed that the mosaic genome is composed of A1/C with seven breakpoints of recombination partitioning the genome into eight segments alternating between sub-subtypes A1 and C viruses. Analysis of the near-full length genome by subtyping tools showed discordant assignment of some gene regions by the different tools. Further analysis of the accessory genes did not reveal any major changes like premature termination or loss of functional motifs but deletions and insertions were observed in the tat, rev and net genes respectively. The isolation of a recombinant virus in a region where subtype C is the dominant variant shows the dynamic nature of this virus and calls for regular monitoring of the HIV-1 genetic landscape of the region. The third component of this study also involved the analysis of a recombinant virus isolated from a female patient at Mankweng Hospital near Polokwane the provincial capital of Limpopo Province. Partial fragment of 5665 nucleotides was generated, sequenced and analysed by various subtype analytical tools. Presence of drug resistance mutations in the PR, RT and IN genes was determined as well as prediction of co-receptor usage. Results revealed a mosaic recombination between subtype C and CRF11_cpx only at the RT gene of the isolate. All other gene regions analysed phylogenetically belong to pure subtype C virus. This is a novel strain as there is no known variant that has this genomic recombination in the HIV database. The epidemiological implication of this strain in Limpopo Province is not known. The frequency and pattern of polymorphisms among HIV-1 subtypes associated with resistance or resulting to a faster emergence of drug resistance once under drug pressure has been evaluated extensively on subtype B and little information exist about other subtypes. Subtype C variants are responsible for more than 50% of the global epidemic, and it is the subtype that is driving the epidemic in Southern Africa, the region with the highest HIV prevalence in the world. Antiretroviral scale up in the Limpopo region is high and therefore, it is necessary to determine whether genetic subtype differences will influence therapy outcome. The investigative theme of chapter five of this thesis is on the implications of nucleotide polymorphisms on the genetic barrier to evolution of drug resistance mutations in subtype C viruses when compared to the global subtype B consensus sequence. The protease and reverse transcriptase nucleotide sequences generated in this study were compared with the global subtype B consensus sequence at codons known to code for drug resistance mutations according to the Stanford drug resistance algorithm. The results revealed a reduced genetic barrier in subtype C viruses at codon V106M (GTA to GTG) and an increased barrier at codon L21OW (TTA/CTG/CTA to TGG) when compared to subtype B global consensus sequence. From this analysis, these are the only codons where significant differences exist between the subtypes. Apparently, there are, no major genetic barriers existing between subtypes B and C at known positions that code for drug resistance mutations. In conclusion, HIV-1 subtype C viruses is the predominant circulating variant in Limpopo Province as phylogenetic analyses of the partial gag, pol, and env C2- C3 gene fragments from HIV chronically infected patients showed that majority of the viruses are HIV-1 subtype C. The circulating HIV-1 viruses will be susceptible to the currently available protease inhibitors and reverse transcriptase inhibitors as drug resistance mutations in the naive population are very low (4.7%). Two unique recombinant forms HIV-1 A1/C and HIV-1 C/CRF11_cpx were each detected from two different individuals in the Waterberg and Capricorn districts respectively. There is no immunological difference between the HIV-1 subtype C viruses from Limpopo Province and the global consensus of subtype C viruses as epitope mapping using the consensus generated from test isolates had the same dissociation constant as the subtype C consensus sequence. Also, there is apparently no significant difference on the impact of nucleotide polymorphisms on the genetic barrier to antiretroviral drug resistance between subtype C viruses and subtype B viruses.
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    Genetic diversity of Human Herpesvirus Type 8 in Northern South Africa
    (2024-09-06) Raphalalani, Mulalo; Bessong, Pascal Obong; Mavhandu-Ramarumo, Lufuno Grae
    Background: Human herpesvirus type 8 (HHV-8), is an oncogenic virus responsible for causing all forms of Kaposi`s sarcoma (KS). HHV-8 prevalence varies globally, however, it is more prevalent in African countries, with South Africa having over 50% of HHV-8 infections. HHV-8 encodes a highly diverse open reading frame (ORF) K1 gene, which has led to the identification of seven major genotypes (A-F and Z) that are heterogeneously distributed across the world. The viral genetic landscape of any geographical area is of paramount importance in vaccine development and diagnostics. However, data on HHV-8 genotypes is scarce in northern South Africa. Therefore, this study will provide genetic diversity of HHV-8 in northern South Africa, and this may aid in the selection of genes for vaccine development. Objective: The main objective of the study was to describe the genetic diversity of human herpesvirus type 8 in northern South Africa. Methodology: Deoxyribonucleic acid (DNA) was extracted from 115 archived mouthwash samples collected from five healthcare facilities in northern South Africa. The partial open reading frame (ORF) K1 gene (~840bp) was amplified in a two round conventional PCR using JumpStart REDTaq master mix. The band of interest was extracted by phenol-freeze protocol and enriched using conventional PCR. Enriched amplicons were purified and sequenced in an Illumina MiniSeq platform. K1 genotypes were inferred using an online BioAfrica HHV-8 subtyping tool and confirmed by computing a phylogenetic tree. Intra-genetic diversity among HHV-8 genotypes was described by aligning study sequences with their respective prototype strains. Synonymous and nonsynonymous mutation rates were computed by the online SNAP tool. Results: K1 gene was successfully amplified in 61.7% (71/115) samples, along with unspecified DNA bands. The band of interest was successfully recovered in 67 amplicons (94.4%). Sixty-five gel extracted products (65/67; 97%) were successfully enriched and purified using magnetic beads. Of the 65 purified samples, 63 were sequenced using Illumina MiniSeq platform. Thirty-seven sequences had an acceptable nucleotide base call. The prevalence of HHV-8 in the study sequences was 94% (35/37) and majority of the sequences (24/35;68%) had sequence reads that span partial or complete K1 gene. Two major genotypes were detected (A and B); genotype B (19/24;79%) had a higher prevalence than genotype A (5/24; 21%). All sequences which grouped with genotype A were further classified as subtype A5. Interestingly, all sequences that were classified as genotype B did not cluster to any of the B subtypes. A higher genetic drift was observed among the study sequences reaching up to 33.7% at the amino acid level. Genotypes A and B exhibited 16.67% and 7.41% intra-genetic diversity at the amino acid level, respectively. Several amino acid polymorphisms were observed at the ITAM region of genotype A sequences (OUHC 013 and ODF 029), while the ITAM region of the B sequence was conserved. Conclusion: In this study, a predominance of HHV-8 genotype B was observed in northern South Africa. Additionally, there was a high degree of evolutionary divergence among the studied sequences. A higher frequency of nonsynonymous mutations was detected at the ITAM region of A5 sequences and these mutations may potentially affect the functionality of ITAM.
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    Genetic divesity of Human Herpesvirus Type 8 in Northern South Africa
    (2024-09-06) Raphalalani, Mulalo; Bessong, Pascal Obong; Mavhandu-Ramarumo, Lufuno Grace
    Background: Human herpesvirus type 8 (HHV-8), is an oncogenic virus responsible for causing all forms of Kaposi`s sarcoma (KS). HHV-8 prevalence varies globally, however, it is more prevalent in African countries, with South Africa having over 50% of HHV-8 infections. HHV-8 encodes a highly diverse open reading frame (ORF) K1 gene, which has led to the identification of seven major genotypes (A-F and Z) that are heterogeneously distributed across the world. The viral genetic landscape of any geographical area is of paramount importance in vaccine development and diagnostics. However, data on HHV-8 genotypes is scarce in northern South Africa. Therefore, this study will provide genetic diversity of HHV-8 in northern South Africa, and this may aid in the selection of genes for vaccine development. Objective: The main objective of the study was to describe the genetic diversity of human herpesvirus type 8 in northern South Africa. Methodology: Deoxyribonucleic acid (DNA) was extracted from 115 archived mouthwash samples collected from five healthcare facilities in northern South Africa. The partial open reading frame (ORF) K1 gene (~840bp) was amplified in a two round conventional PCR using JumpStart REDTaq master mix. The band of interest was extracted by phenol-freeze protocol and enriched using conventional PCR. Enriched amplicons were purified and sequenced in an Illumina MiniSeq platform. K1 genotypes were inferred using an online BioAfrica HHV-8 subtyping tool and confirmed by computing a phylogenetic tree. Intra-genetic diversity among HHV-8 genotypes was described by aligning study sequences with their respective prototype strains. Synonymous and nonsynonymous mutation rates were computed by the online SNAP tool. Results: K1 gene was successfully amplified in 61.7% (71/115) samples, along with unspecified DNA bands. The band of interest was successfully recovered in 67 amplicons (94.4%). Sixty-five gel extracted products (65/67; 97%) were successfully enriched and purified using magnetic beads. Of the 65 purified samples, 63 were sequenced using Illumina MiniSeq platform. Thirty-seven sequences had an acceptable nucleotide base call. The prevalence of HHV-8 in the study sequences was 94% (35/37) and majority of the sequences (24/35;68%) had sequence reads that span partial or complete K1 gene. Two major genotypes were detected (A and B); genotype B (19/24;79%) had a higher prevalence than genotype A (5/24; 21%). All sequences which grouped with genotype A were further classified as subtype A5. Interestingly, all sequences that were classified as genotype B did not cluster to any of the B subtypes. A higher genetic drift was observed among the study sequences reaching up to 33.7% at the amino acid level. Genotypes A and B exhibited 16.67% and 7.41% intra-genetic diversity at the amino acid level, respectively. Several amino acid polymorphisms were observed at the ITAM region of genotype A sequences (OUHC 013 and ODF 029), while the ITAM region of the B sequence was conserved. Conclusion: In this study, a predominance of HHV-8 genotype B was observed in northern South Africa. Additionally, there was a high degree of evolutionary divergence among the studied sequences. A higher frequency of nonsynonymous mutations was detected at the ITAM region of A5 sequences and these mutations may potentially affect the functionality of ITAM.
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    Identification of human papilloma virus, hepatitis B virus and human herpes virus type 8 in plasma of benign prostatic hyperplasia and prostate cancer patients in South Africa
    (2017-05) Munzhedzi, Mukhethwa; Bessong, Pascal Obong; Borman, Riana
    Background: Prostate cancer (PCA) is a major health concern in males, particularly those above 40 years old. It is the most common form of cancer in males worldwide, including South Africa. In South Africa, the rate of histologically diagnosed prostate cancer is 40 per 100 000 in whites and 14 per 100 000 in blacks, and 1 in 8 men will develop PCA in their lifetime. Several reports have suggested the association of viruses in the pathogenesis of prostate cancer. Objectives: This study was aimed at identifying Hepatitis B virus (HBV), human papilloma virus (HPV) and human herpes virus type 8 (HHV-8), implicated in other forms of cancer, in a cohort of South African patients with either PCA or benign prostatic hyperplasia (BPH); and to seek possible associations thereof. Methods: The study group comprised 187 male patients recruited from Polokwane Hospital presenting with either PCA (staged by Gleason scores) or BPH. Enzyme-linked immunosorbent assay was used to detect antibodies to HHV-8 and HPV; and to detect hepatitis B surface antigen (HBsAg) in the plasma of the study subjects. Total DNA was extracted from plasma and targeted for the identification of HBV and HHV-8 DNA by nested PCR protocols. The HBV nested PCR protocol amplifies a 336bp fragment of the overlapping surface polymerase gene of HBV. The HHV-8 nested protocol amplifies a 233bp fragment of the ORF 26 gene of HHV-8. Amplified DNA products were purified, sequenced by the Sanger protocol and phylogenetically analysed for viral genotypes. The Chi-square test was used to infer statistically significant differences in the level of detection of viruses and the stage of prostate cancer development. Results: Of the 187 participants, a seroprevalence of 4.8% (9/187, HBsAg), 5.3% (10/187, HPV IgG antibody) and 27% (33/124, HHV-8 IgG antibody) were observed. HBsAg was detected more in individuals with BPH than those without and this was statistically significant at ( 2=6.0, p< 0.05). HHV-8 DNA was detected more in individuals in the 60-79 years age range and this was statistically significant at ( 2=61.1, p< 0.05). Occult HBV infection (that is the presence of HBV DNA in the absence of HBsAg) was detected in 23/178 (12.9%) of patients. Taking into account occult HBV infection, the overall prevalence of HBV was 17.7%. HBV genotype E was more prevalent (86.7%) followed by genotype A (13.3%). HHV-8 genotypes K and R were inferred. Apparently, this is the first report on the identification of HHV-8 genotypes K and R from South Africa. Conclusion: The current study has demonstrated for the first time, the presence of genotypes K and R of HHV-8 in South Africa. This study also suggests that there is a high level of occult genotype E HBV infection. Future studies will explore the virome in prostate cancer biopsies.
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    Indentification and molecular characterization of human papillomavirus infection among women living with and without HIV in selected health facilities in Limpopo Province, South Africa
    (2025-09-05) Rikhotso, Rixongile Rhenny; Bessong, Pascal Obong; Mitchell, Emma McKim
    Background: Co-infection of human papillomavirus (HPV) and human immunodeficiency virus (HIV) has been well established. Both viruses are sexually transmitted with paramount public health implications due to their interaction with cervical cancer (CC). Globally, the estimated prevalence of HPV is 11.7%, with high-risk (hr-) HPV 16 and HPV 18 attributing for most cases of CC. Due to the high genetic diversity of HPV, it is important to study the virus in different geographic locations, people, and in persons living with HIV, to inform the selection of genes for vaccine improvements and potential vaccine trial sites. Consequently, it is important to describe the prevalence, genotype distribution and genomes of HPV in Limpopo Province, where there is limited data. The general objective of this study was to therefore to identify and characterize HPV DNA among women living with and without HIV in selected health facilities in Limpopo Province, South Africa. The specific objectives were: (1) To provide a narrative literature review on the prevalence and distribution of selected cervical HPV genotypes among women living with and without HIV in South Africa; (2) To determine the prevalence of cervical HPV infection among women living with and without HIV in selected health facilities in Limpopo Province, South Africa; (3) To describe HPV genotypes among women living with and without HIV in selected health facilities in Limpopo Province, South Africa; and (4) To generate and characterize HPV full genomes among women living with and without HIV in selected health facilities in Limpopo Province, South Africa. Methods: A systematic review of the available literature was conducted in the quest to understand and determine the prevalence and distribution of cervical HPV among women living with HIV (WLWH) and those without HIV in South Africa, following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) workflow. Records were retrieved from PubMed and Web of Science databases. An analytic cross-sectional study on HPV prevalence and genotype distribution among 450 women who were living with and without HIV from selected health facilities in Limpopo Province was done. In this regard, total DNA was purified from cervical specimens and amplified using a double-nested polymerase chain reaction (PCR) strategy, targeting the L1 gene. A sample that shows an amplification product of 450 bp in the first nested PCR or a product of 150 bp in the second nested reaction was taken as positive for the presence of HPV DNA. Statistical analysis, using models in R-statistical package, was done to determine comparative inferences of the presence of HPV DNA and associated risk factors. HPV genotypes were determined through deep sequencing of the 450 bp PCR products on an Illumina MiniSeq platform. The generated sequences were quality assured and analyzed for viral genotypes. Rolling circle amplification (RCA) was used for enriching DNA in samples that tested positive for HPV DNA to generate and characterize near-full length HPV genomes. The enriched DNA was purified with AMPure XP beads and quantified with Qubit, followed by DNA library preparation, and then sequenced on an Illumina MiniSeq platform following the manufacturer’s protocols. Results Prevalence and distribution of selected cervical HPV genotypes in South Africa A total of sixty-nine (69) articles met the inclusion criteria for determining the prevalence and distribution of cervical HPV genotypes among South African women who were either living with HIV or not. These articles were on studies done in 5 of the 9 South African Provinces. The studies were conducted between 1989-2021. Sequencing was the least genotyping tool used in these studies, while most studies utilized PCR-based hybridization commercial kits. Compared to other investigated genotypes, HPV 16, HPV 18, and HPV 35 were predominant in the study population regardless of the HIV status. Prevalence of HPV DNA and genotype distribution in selected health facilities in Limpopo Province, South Africa The HPV DNA was detected in cervical specimens from 147 of 450 women (32.7%), significantly higher at 52.21% (p = 0.00) among WLWH compared to women living without HIV. Forty-eight samples were of acceptable quality and analyzed for viral diversity. The study detected 41 HPV genotypes, all of which belong to the Alphapapillomavirus genus of the Papillomaviridae family. In general, HPV 45 (16.7%) was the predominant genotype. However, HPV 81 (18.8%) and HPV 56 (25.0%) were the most common genotypes among women living with and without HIV, respectively. Multiple infections and high-risk HPV genotypes were more common among WLWH. Characterization of whole genomes of cervical human papillomavirus Six full genomes of HPV genotypes 34 (n=1), 35 (n=1), 42 (n=1), 45 (n=2), and 90 (n=1) are reported herein. The study further identified variant sub-lineages, including the A1 sub-lineages of hr-HPV 35 and hr-HPV 45. The genome sequences are available at the National Center for Biotechnology Information (NCBI) in the Sequence Read Archive (SRA) database. Interpretations and conclusions Prevalence and distribution of selected cervical HPV genotypes in South Africa The narrative review of the literature indicated that there are limited studies on cervical HPV prevalence and genotype distribution in South Africa. Therefore, further research is required to identify HPV genotypes, including the use of sensitive approaches such as next generation sequencing. This will enhance our understanding of HPV geo-diversity patterns in various provinces across South Africa, and subsequently inform decisions on screening strategies, and selection of geographically relevant genes for vaccine improvement and development. The high prevalence of hr-HPV 16, HPV 18 and HPV 35 regardless of HIV status in the country is a public health concern, considering their significant contribution to the burden of CC. Consequently, routine HPV screening and prevention measures such as HPV vaccination for all individuals remain critical. Because current vaccines exclude HPV 35 as a target, there is a need to consider this genotype in future vaccine development efforts. Prevalence of HPV DNA and genotype distribution in selected health facilities in Limpopo Province, South Africa The study reported a relatively high prevalence of HPV infection among the study participants, which signified the burden of HPV in the province. The prevalence was significantly higher among WLWH, including more HPV genotypes and hr-HPV genotypes as compared to women living without HIV, potentially due to immunosuppression. Considering the high rate of HPV-HIV co-infection observed in this study and other studies in South Africa, prioritizing women living with HIV for cervical HPV screening and HPV vaccination is recommended. Strengthening integrated HPV and HIV screening programs and ensuring regular monitoring of cervical health among WLWH remains a public health imperative. Moreover, the study also showed that women living with and without HIV may have different profiling in terms of HPV infections which is important in intervention strategies, example development of targeted HPV screening strategies. The current study also fills a significant gap of limited data by providing baseline information on the prevalence and genotype distribution of cervical HPV infection among women living with and without HIV in the study area. The study findings further encourage national-level surveillance with the inclusion of expanded regional representation. Characterization of whole genomes of cervical HPV Near full-length genomes of six HPV genotypes were successfully characterized in the study population. These sequences could serve as a valuable resource for further research and vaccine development studies. The identified variant sub-lineages may underscore the need to strengthen HPV vaccine coverage, routine cervical screening, and genomic monitoring in underrepresented areas such as Limpopo Province. Moreover, this data supports the development of risk stratification models aimed at identifying women at high-risk for persistent infection and progression to CC, thereby enabling early, tailored interventions to reduce the HPV related disease burden.
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    Serologic and genotypic characterization of hepatitis B virus in HIV-1 infected patients from South West and Littoral Regions of Cameroon
    (BioMed Central, 2016) Magoro, Tshifhiwa; Gachara, George; Mavhandu, Lufuno; Lum, Emmaculate; Kimbi, Helen K.; Ndip, Roland N; Bessong, Pascal Obong
    Background: HBV and HIV share similar transmission routes. Concurrent infection with the two viruses usually results in more severe and progressive liver disease, and a higher incidence of cirrhosis, liver cancer and mortality. Further, this co-infection may lead to cross-resistance between HIV and HBV drugs and increased liver injury, either due to direct hepatotoxicity or drug-related immune-reconstitution hepatitis. These challenges necessitate continuous surveillance for HBV among HIV infected individuals to guide patient management. We conducted this study to understand the serologic and genotypic characteristics of HBV among HIV/HBV infected patients in South West and Littoral Regions of Cameroon. Methods: Plasma samples were screened for HBsAg, HBeAg, Anti-HBs and anti-HBc using ELISA followed by DNA extraction from all HBsAg positive samples. A 366 bp region covering the overlapping surface/polymerase gene was amplified by a nested PCR and the product sequenced using Big Dye sequencing chemistry. The resulting sequences were then analyzed for genotypes and both escape and drug resistance mutations. Results: Of the 455 samples in this study, 25.5 % (n = 116) were HBsAg positive and 46 of these had their DNA successfully amplified. Genotype E was found in 32 samples (69.6 %) and genotype A in the rest of the samples. Escape mutations associated with failure of diagnosis (Y100C, R122K and Q129H) and with vaccine escape (Q129R and T131N) were detected in varying frequencies in the population. Polymerase mutations implicated in resistance to lamivudine and other ʟ-nucleoside analogues were detected in seven patients (15.2 %), while all the samples lacked mutations associated with resistance to adefovir and tenofovir. Conclusions: These findings suggest the endemicity of HBV and the predominance of genotypes A and E in the study population. Also, drug resistance findings support the use of tenofovir based ART regimens among HIV/HBV co-infected persons. There is need for continuous HBV screening and monitoring in HIV infected individuals in these regions
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    Studies on severe acute respiratory syndrome coronavirus type -2 in Northern South Africa
    (2025-05-16) Tambe, Lisa Arrah Mbang; Mavhandu-Ramarumo, Lufuno Grace; Tebit, Denis Manga; Bessong, Pascal Obong
    Background: The last three decades have been characterized by the re-emergence of the Coronaviridae family into the human population, causing severe respiratory disease with increased morbidity rates. A dearth of information exists on human coronavirus (HCoV) molecular epidemiology, and circulation in different populations in Africa. As the COVID-19 pandemic progressed across the globe, wastewater-based epidemiology (WBE) was proposed as an alternative tool for assessing and monitoring the occurrence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the community level. Additionally, through wastewater-based genomic surveillance of SARS-CoV-2, the evolutionary patterns and distribution of viral types at the population level can be comprehensively characterized. This study systematically reviewed literature published prior to the SARS-CoV-2 outbreak to investigate the prevalence and molecular epidemiology of HCoVs circulating in Africa. Secondly, this study established a wastewater-based surveillance (WBS) system to track the trends of SARS-CoV-2, investigate SARS-CoV-2 variants of concern (VOC) circulating in the population, and determine the prevalence of people infected in the Vhembe and Mopani districts. Thirdly, through WBS, to describe the molecular epidemiology of SARS-CoV-2 and document the respiratory viruses occurring in the Vhembe and Mopani districts. Methodology: A systematic literature review was conducted according to the PRISMA guidelines, to understand the prevalence and molecular epidemiology of HCoVs in Africa. For the second and third objectives, wastewater influents from seven wastewater treatment plants (WWTPs) and one waste sedimentation pond (WSP) were collected weekly from January 2021 to June 2022. Out of a total of 487 samples collected, about 75% (365/487) were positive for SARS-CoV-2 RNA by qRT-PCR. Of these, 80 met the criteria for allele-specific genotyping (ASG). Positive SARS-CoV-2 RNA detected throughout the surveillance period were compared to 7-day moving average (7D-MA) of clinical cases reported per sub-district. Next, SARS-CoV-2 RNA detected during the surveillance period was normalized using the flow rate and population size. The Spearman’s correlation coefficient was used to determine the relationship between non-normalized, and normalized SARS-CoV-2 RNA data when compared to the reported clinical cases. Finally, positive SARS-CoV-2 RNA copies with a standard deviation of less than one (SD <1) were used to predict the prevalence of people infected in the study sites using the Monte Carlo simulation model. This predicted prevalence was also compared to the 7D-MA clinical cases reported per sub-district, and the correlation between them was determined. Subsequently, samples positive for SARS-CoV-2 were subjected to whole genome sequences (WGS) using the ATOPlex next-generation sequencing method and analyzed for lineage and clade assignment using the Pangolin and Nextclade tool. Relatedness of identified sequences was determined by phylogenetic analysis. VOC was analyzed for prevalence and geographical distribution. Concordance for VOC between ASG and WGS analyses was determined. Results: Findings from the systematic literature review showed that thirteen out of 54 (24%) African countries had published data on HCoV prevalence and/or genomic epidemiology, from hospitals, clinics, homes, community gatherings, farms, and individuals at airports. The first published data on HCoV was from South Africa in 2008. There was heterogeneity in the type of tests used in determining HCoV prevalence. Two studies reported that risk factors for HCoV include exposure to infected animals or humans. The second objective, establishing a wastewater-based surveillance system was achieved. Briefly, SARS-CoV-2 viral load was detected in wastewater one week prior to increased infection cases reported at the district level during the third and fourth waves, thus serving as an early warning system. Of interest, towards the end of the surveillance period, increased SARS-CoV-2 viral load detected in wastewater were not reflected in the reported clinical cases. Comparing the reported number of cases per district to the predicted prevalence revealed more cases in the Vhembe District than in the Mopani District. Third, SARS-CoV-2 molecular epidemiology and the distribution of respiratory virus were described. A total of 60 SARS-CoV-2 full genomes were analyzed. Delta and Omicron variants were detected as early as January and February 2021, respectively, while the Beta variant was detected in July 2021. Delta variant was significantly predominant at a prevalence of 45%, followed by Omicron (32%), and Beta (5%). Eighteen percent (11/60) of the sequences were assigned a lineage by Pangolin tool, but not a specific WHO variant name. Upon phylogenetic analysis, some of these sequences were seen clustering with the Alpha (2/11) and Delta (2/11) variants, while the remaining sequences clustered with each other. Mutations in the receptor-binding domain (RBD) of the Spike protein (S-protein) were investigated, with some peculiarities observed such as mutation E484K absent in all Beta variant study sequences. Three previously undescribed mutations (A631S, V327I, D427Y) were detected in Delta variant sequences. Concordance in variant assignments between allele-specific genotyping and WGS was seen in 51.2% of the study sequences. Respiratory virus surveillance revealed year-round circulation of human Adenoviruses (HAdVs), while HCoVs, influenza viruses and human parainfluenza viruses (HPIVs) were mostly detected in winter. Influenza A and B viruses (IAV and IBV) detected in the study site in 2021 were remarkably different from those reported in circulation nationwide by the NICD. Specifically, IAV (H5N1)/Guandong, a highly pathogenic influenza virus, was detected, although at a low frequency. Discussion and Conclusion: The systematic review revealed that despite the outbreaks of SARS in Southeast Asia in 2002 and MERS in 2012 in the Middle East, the quantum of virologic investigations on HCoV on the African continent was scanty. Pandemic preparedness requires cognizance of disease outbreaks in other continents, establishment of test and surveillance protocols, and infrastructure for eventualities. Regarding the establishment of a wastewater-based surveillance system for SARS-CoV-2 monitoring, this study demonstrates effective surveillance over an extended period in rural settings. This is important because most reports about the application of WBE for monitoring SARS-CoV-2 and circulating variants are predominantly from more urbanized regions in South Africa and other parts of the world. Thus, it reveals applicability of monitoring pathogens in rural areas, despite challenges encountered such as poor or non-existent sewerage systems. Such challenges are common in the African continent, highlighting the need for more of such investigations to strengthen pandemic preparedness measures. The presence of Delta and Omicron VOCs observed prior to other reports in South Africa highlights the importance of population-based approaches in genomic surveillance over approaches that rely on individual samples. Again, it also emphasizes the need for pandemic preparedness efforts to be extended to all geographic regions. Wastewater is known to potentially capture more viral diversity, including SARS-CoV-2 genetic diversity, and could reveal new viruses and VOCs in circulation before they emerge in the wider human population. Thus, continuous surveillance is necessary for documentation of cryptic lineages, which may contribute towards improving vaccine.