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Abstract Background Cervical cancer remains a public health problem despite heavy global investment in health systems especially in low-and-middle-income countries (LMIC). Prophylactic vaccines against the most commonly detected human papillomavirus (HPV) types in cervical cancers are available and decisions on the selection of vaccine design depends on the prevalence of high-risk (hr) HPV genotypes for a particular region. In 2015, Botswana adopted the use of a quadrivalent HPV vaccine as a primary prevention strategy. Secondary prevention includes cervical smear screening whose uptake remains notably low among indigenous and marginalized communities despite efforts to improve access. Aim To determine the prevalence of hrHPV genotypes and cervical lesions’ burden in women from the indigenous and marginalized communities of Botswana. Methods This prospective survey enrolled 171 non-HPV vaccinated women aged 21 years and older. Face-to-face interviews, Pap smear screening, hr-HPV and Human Immuno-deficiency virus (HIV) testing were carried out. Conventional Papanicolau smears were analyzed and cervical brushes were preserved for hrHPV testing using the Ampfire Multiplex HR-HPV protocol which detects the following genotypes: HPV 16, 18, 31, 35, 39, 45, 51, 52, 53, 56, 58, 59 and 68. Results In this study, 168/171 (98.6%) of the women consented to HIV testing; 53/171 (31%) were living with HIV and self-reported enrolment on antiretroviral therapy. Among the women examined, 23/171 (13.5%) had cervical dysplasia with most presenting with Atypical Squamous Cells of Undetermined Significance 8/23 (35%), Low-Grade Squamous Intraepithelial Lesions 8/23 (35%), Atypical Squamous Cells-High Grade 4/23 (17%), Atypical Endocervical Cells 2/23 (9%) and Atypical Endocervical Cell favoring neoplasia 1/23(4%). However, no High-Grade Squamous Intraepithelial Lesions (HSIL) or squamous cell carcinoma (SCC) were detected. Overall hrHPV prevalence in this study was at 56/171 (32.7%). The most commonly detected hrHPV genotypes in women with cervical dysplasia were HPV39 (6.25%), HPV51 (14.5%), HPV52 (12.5%) and HPV56 (4%). Notably, HPV 16 and 18 were not found in women with cervical dysplasia. Conclusions Our study provides valuable insights into the prevalence and distribution of hrHPV genotypes in indigenous and marginalized communities in Botswana, and the need for further investigation of their potential role in cervical carcinogenesis in this population. These results may also serve as baseline data to facilitate future evaluation of the HPV vaccine needs.

BackgroundWe evaluated the prevalence of archived proviral drug resistance mutations (DRMs) associated with resistance to integrase strand transfer inhibitors (INSTIs) shortly before Botswana transitioned in 2016 to using dolutegravir (DTG)-based antiretroviral treatment in first-line regimens.MethodsWe used the Stanford University HIV drug resistance database to analyze INSTI-resistance associated mutations (RAMs) in a large representative population-based cohort of adults recruited in 30 geographically dispersed communities as part of the Botswana Combination Prevention Project (BCPP) cohort from 2013 to 2018. A total of 5,144 HIV-1 proviral DNA sequences were included in our analysis; 1,281 sequences were from antiretroviral therapy (ART)-naïve individuals and 3,863 sequences were from non-nucleoside reverse transcriptase inhibitor (NNRTI) ART-experienced individuals. None of the sequences were from DTG-ART experienced participants.ResultsThe overall prevalence of major INSTIs DRMs was 1.11% (95% CI 0.82–1.39%). The prevalence of INSTI DRMs in ART-naïve individuals was 1.64% (21/1,281) and 0.93% (36/3,863) in ART-experienced individuals. Major INSTI-RAMs detected in ART-naïve individuals were E138K (2/1,281; 0.16%), G140R (8/1,281;0.62%), E92G (2/1,281;0.16%), R263K (5/1,281; 0.4%), N155H (1/1,281; 0.08%), P145S (1/1,281;0.008%). Among the ART-experienced individuals, major INSTI RAMs detected were E138K (4/3,863; 0.10%), G140R (25/3,863;0.65%), G118R (2/3,863, 0.05%), R263K (4/3,863, 0.10%), T66I (1/3,863;0.03%), E138K + G140R (1/3,863, 0.03%|), G140R + R263K (1/3,863, 0.03%). High-level resistance to cabotegravir (CAB), elvitegravir (EVG), and raltegravir (RAL) was detected in 0.70, 0.16 and 0.06% of the individuals, respectively. Notably, bictegravir (BIC) and dolutegravir (DTG) showed no high-level resistance.ConclusionThe overall prevalence of archived INSTI RAMs in Botswana was low prior to transitioning to first-line DTG-based ART regimens, and did not differ between ART-naïve and ART-experienced individuals. Ongoing surveillance of INSTI DRMs in Botswana will allow for re-assessment of INSTI resistance risk following nationwide DTG rollout.

We investigated intra-host genetic evolution using two SARS-CoV-2 isolates from a fully vaccinated (primary schedule x2 doses of AstraZeneca plus a booster of Pfizer), >70-year-old woman with a history of lymphoma and hypertension who presented a SARS-CoV-2 infection for 3 weeks prior to death due to COVID-19. Two full genome sequences were determined from samples taken 13 days apart with both belonging to Pango lineage FL.2: the first detection of this Omicron sub-variant in Botswana. FL.2 is a sub-lineage of XBB.1.9.1. The repertoire of mutations and minority variants in the Spike protein differed between the two time points. Notably, we also observed deletions within the ORF1a and Membrane proteins; both regions are associated with high T-cell epitope density. The internal milieu of immune-suppressed individuals may accelerate SARS-CoV-2 evolution; hence, close monitoring is warranted.

Hepatitis delta virus (HDV) co-infections more often result in severe hepatitis compared to hepatitis B virus (HBV) infections alone. Despite a high HDV prevalence (7.1%), information regarding circulating HDV clades is very limited in Botswana. We extracted total nucleic acid from confirmed HDV-positive samples and quantified their viral load. We then sequenced the large hepatitis delta antigen (L-HDAg) using Oxford Nanopore Technology (ONT). Genotyping was performed using the HDV Database, and HDV mutation profiling was performed on AliView. All participants with HBV genotypic information belonged to sub-genotype A1, and 80% (4/5) of them had a higher HDV viral load and a lower HBV viral load. We sequenced 75% (9/12) of the HDV-positive samples, which belonged to HDV clade 8. A total of 54 mutations were discovered, with the most prevalent being Q148R (16%), D149P (16%) and G151D (16%). Known mutations such as S117A, K131R, R139K and G151D were detected, while the other mutations were novel. Our results reveal that HDV clade 8 is the predominant clade in Botswana. The significance of all mutations remains unclear. Future studies with a larger sample size to detect other HDV clades that might be circulating in Botswana and functionally characterize the detected mutations are warranted.

HIV-1 drug resistance (HIVDR) impedes treatment and control of HIV-1, especially in high-prevalence settings such as KwaZulu-Natal (KZN) province, South Africa. This study merged routine HIV-1 genotypic resistance test (GRT) data with Geographic Information Systems coordinates to assess patterns and geographic distribution of HIVDR in KZN, among ART-experienced adults with virological failure. We curated 3133 GRT records generated between 1 January 2018 and 30 June 2022, which includes the early phase of dolutegravir (DTG) rollout, of which 2735 (87.30%) had HIVDR. Of the 2735, major protease, nucleoside, and non-nucleoside reverse transcriptase inhibitor mutations were detected in 41.24%, 84.97% and 88.08% of GRTs, respectively. Additional genotyping of HIV-1 integrase for 41/3133 (1.31%) GRTs showed that 17/41 (41.46%) had integrase strand transfer inhibitor resistance. Notably, of 26 patients on DTG with integrase genotyping, 9 (34.62%) had DTG-associated resistance mutations. Dual- or triple-class resistance was observed in four of every five GRTs. The odds of HIVDR increased significantly with age, with ≥60 years having 5 times higher odds of HIVDR compared to 18–29 years (p = 0.001). We identified geospatial differences in the burden of HIVDR, providing proof of concept that this could be used for data-driven public health decision making. Ongoing real-time HIVDR surveillance is essential for evaluating the outcomes of the updated South African HIV treatment programme.

AimThis study aimed to determine the kinetics of occult hepatitis B virus infections (OBI) among people with HIV (PWH).MethodsThe study used archived plasma samples from longitudinal HIV natural history studies. We identified new OBI cases and assessed risk factors for OBI using Cox proportional hazards regression analysis.ResultsAt baseline, 8 of 382 [(2.1%) (95% CI: 1.06–4.1)] samples tested positive for hepatitis B surface antigen (HBsAg+). Of the 374 HBsAg-negative samples, 76 had sufficient sample volume for HBV DNA screening. OBI positivity (OBI+) at baseline was reported in 11 of 76 [14.7 95% CI (8.3–24.1)] HBsAg-negative (HBsAg−) participants. Baseline HBsAg-negative samples with sufficient follow-up samples (n = 90) were used for analysis of newly identified OBI cases. Participants contributed 129.74 person-years to the study and were followed for a median of 1.02 years (IQR: 1.00–2.00). Cumulatively, there were 34 newly identified OBI cases from the 90 participants, at the rate of 26.2/100 person-years (95% CI: 18.7–36.7). Newly identified OBI cases were more common among men than women (61.1% vs. 31.9%) and among participants with CD4+ T-cell counts ≤450 cells/mL (p-value = 0.02). Most of the newly identified OBI cases [55.9% (19/34)] were possible reactivations as they were previously HBV core antibody positive.ConclusionThere was a high rate of newly identified OBI among young PWH in Botswana, especially in men and in participants with lower CD4+ T-cell counts. OBI screening in PWH should be considered because of the risk of transmission, possible reactivation, and risk factors for the development of chronic liver disease, including hepatocellular carcinoma.

BackgroundThe COVID-19 pandemic brought challenges requiring timely health data sharing to inform accurate decision-making at national levels. In Botswana, we adapted and integrated the Research Electronic Data Capture (REDCap) and the District Health Information System version 2 (DHIS2) platforms to support timely collection and reporting of COVID-19 cases. We focused on establishing an effective COVID-19 data flow at the national public health laboratory, being guided by the needs of health care professionals at the National Health Laboratory (NHL). This integration contributed to automated centralized reporting of COVID-19 results at the Ministry of Health (MOH). ObjectiveThis paper reports the experiences, challenges, and lessons learned while designing, adapting, and implementing the REDCap and DHIS2 platforms to support COVID-19 data management at the NHL in Botswana. MethodsA participatory design approach was adopted to guide the design, customization, and implementation of the REDCap platform in support of COVID-19 data management at the NHL. Study participants included 29 NHL and 4 MOH personnel, and the study was conducted from March 2, 2020, to June 30, 2020. Participants’ requirements for an ideal COVID-19 data management system were established. NVivo 11 software supported thematic analysis of the challenges and resolutions identified during this study. These were categorized according to the 4 themes of infrastructure, capacity development, platform constraints, and interoperability. ResultsOverall, REDCap supported the majority of perceived technical and nontechnical requirements for an ideal COVID-19 data management system at the NHL. Although some implementation challenges were identified, each had mitigation strategies such as procurement of mobile Internet routers, engagement of senior management to resolve conflicting policies, continuous REDCap training, and the development of a third-party web application to enhance REDCap’s capabilities. Lessons learned informed next steps and further refinement of the REDCap platform. ConclusionsImplementation of REDCap at the NHL to streamline COVID-19 data collection and integration with the DHIS2 platform was feasible despite the urgency of implementation during the pandemic. By implementing the REDCap platform at the NHL, we demonstrated the possibility of achieving a centralized reporting system of COVID-19 cases, hence enabling timely and informed decision-making at a national level. Challenges faced presented lessons learned to inform sustainable implementation of digital health innovations in Botswana and similar resource-limited countries.

AbstractOpportunistic infections due to Cryptococcus neoformans and C. gattii species complexes continue to rise unabated among HIV/AIDS patients, despite improved antifungal therapies. Here, we collected a total of 20 environmental and 25 presumptive clinical cryptococcal isolates from cerebrospinal fluid (CSF) samples of 175 patients enrolled in an ongoing clinical trial Ambition 1 Project (Botswana-Harvard Partnership). Identity confirmation of the isolates was done using MALDI-TOF MS and PCR. We describe the diversity of the isolates by PCR fingerprinting and sequencing (Oxford Nanopore Technology) of the intergenic spacer region. Mating types of the isolates were determined by amplification of the MAT locus. We report an unusual prevalence of 42.1% of C. neoformans x C. deneoformans hybrids Serotype AD (n = 16), followed by 39.5% of C. neoformans Serotype A (n = 15), 5.3% of C. deneoformans, Serotype D (n = 2), 7.9% of C. gattii (n = 3), and 5.3% of C. tetragattii (n = 2) in 38 representative isolates that have been characterized. Mating type-specific PCR performed on 38 representative environmental and clinical isolates revealed that 16 (42.1%) were MATa/MATα hybrids, 17 (44.7%) were MATα, and five (13.2%) possessed MATa mating type. We used conventional and NGS platforms to demonstrate a potential link between environmental and clinical isolates and lay a foundation to further describe mating patterns/history in Botswana.

Background: Approximately 15–20 million people worldwide are infected with hepatitis delta virus (HDV), which is approximately 5 % of people with chronic hepatitis B virus (HBV). Sub-Saharan Africa has high HDV prevalence, leading to worse clinical outcomes among people who are HIV/HBV/HDV tri-infected. There are limited data on HDV prevalence among people with HIV (PWH) who are HBV-infected and uninfected in Botswana. We, therefore, determined HDV prevalence among PWH in Botswana. Methods: This was a retrospective cross-sectional study utilizing archived plasma samples from PWH with results for HBV markers such as hepatitis B surface antigen (HBsAg), hepatitis B core antibody (anti-HBc), immunoglobulin M antibody to hepatitis B core antigen (IgM anti-HBc) and hepatitis B e antigen (HBeAg). Samples were categorized according to their HBsAg status and screened for anti-HDV antibodies. Total nucleic acid was extracted from samples with a single positive anti-HDV result, and HDV ribonucleic acid (RNA) load was quantified using the Altona Diagnostic RealStar® HDV RT-PCR kit. Statistical analysis was performed using STATA version 14.0 where p-values

Abstract We used HIV-1C sequences to predict (in silico) resistance to 33 known broadly neutralizing antibodies (bnAbs) and evaluate the different HIV-1 Env characteristics that may affect virus neutralization. We analyzed proviral sequences from adults with documented HIV-1 seroconversion (N = 140) in Botswana (2013–2018). HIV-1 env sequences were used to predict bnAb resistance using bNAb-ReP, to determine the number of potential N-linked glycosylation sites (PNGS) and evaluate Env variable region characteristics (VC). We also assessed the presence of signature mutations that may affect bnAb sensitivity in vitro. We observe varied results for predicted bnAb resistance among our cohort. 3BNC117 showed high predicted resistance (72%) compared to intermediate levels of resistance to VRC01 (57%). We predict low resistance to PGDM100 and 10-1074 and no resistance to 4E10. No difference was observed in the frequency of PNGS by bNAb susceptibility patterns except for higher number of PNGs in V3 bnAb resistant strains. Associations of VC were observed for V1, V4 and V5 loop length and net charge. We also observed few mutations that have been reported to confer bnAb resistance in vitro. Our results support use of sequence data and machine learning tools to predict the best bnAbs to use within populations.

(1) Background: Hepatitis B virus (HBV) sequencing data are important for monitoring HBV evolution. We aimed to molecularly characterize HBV sequences from participants with HBV surface antigen-positive (HBsAg+) serology and occult hepatitis B infection (OBI+). (2) Methods: We utilized archived plasma samples from people living with human immunodeficiency virus (PLWH) in Botswana. HBV DNA was sequenced, genotyped and analyzed for mutations. We compared mutations from study sequences to those from previously generated HBV sequences in Botswana. The impact of OBI-associated mutations on protein function was assessed using the Protein Variation Effect Analyzer. (3) Results: Sequencing success was higher in HBsAg+ than in OBI+ samples [86/128 (67.2%) vs. 21/71 (29.2%)]. Overall, 93.5% (100/107) of sequences were genotype A1, 2.8% (3/107) were D3 and 3.7% (4/107) were E. We identified 13 escape mutations in 18/90 (20%) sequences with HBsAg coverage, with K122R having the highest frequency. The mutational profile of current sequences differed from previous Botswana HBV sequences, suggesting possible mutational changes over time. Mutations deemed to have an impact on protein function were tpQ6H, surfaceV194A and preCW28L. (4) Conclusions: We characterized HBV sequences from PLWH in Botswana. Escape mutations were prevalent and were not associated with OBI. Longitudinal HBV studies are needed to investigate HBV natural evolution.

(1) Background: we determined the prevalence of the hepatitis B virus (HBV) amongst people without human immunodeficiency virus (HIV) in rural and peri-urban areas in Botswana. (2) Methods: We screened for the hepatitis B surface antigen (HBsAg) from archived plasma samples of people without HIV (n = 2135) randomly selected from the Botswana Combination Prevention Program (BCPP) (2013–2018). We sequenced 415 bp of the surface region using BigDye sequencing chemistry. (3) Results: The median age of participants was 31 (IQR: 24–46) and 64% (1360/2135) were female. HBV prevalence was 4.0% (86/2135) [95% CI: 3.3–4.9]) and ranged between 0–9.2%. Older participants (>35 years) had increased odds of HBV positivity (OR: 1.94; 95% CI: [1.32–2.86]; p = 0.001). Thirteen samples were sequenced and seven (53.8%) were genotype A, three (23.1%) were genotype D and genotype E each. Clinically significant mutations were identified in the surface region, but no classic drug resistance mutations were identified. (4) Conclusions: We report an HBV prevalence of 4.0% (95% CI 3.3–4.9) among people without HIV in rural and peri-urban communities in Botswana with varying rates in different communities. A comprehensive national HBV program is required in Botswana to guide HBV prevention, testing and management.

Abstract We sought to investigate whether SARS‐CoV‐2 was present, and to perform full‐length genomic sequencing, in a 5‐year‐old male crossbreed dog from Gaborone, Botswana that presented overt clinical signs (flu‐like symptoms, dry hacking cough and mild dyspnoea). It was only sampled a posteriori, because three adult owners were diagnosed with SARS‐CoV‐2 infection. Next‐generation sequencing based on Oxford Nanopore Technology (ONT) was performed on amplicons that were generated using a reverse transcriptase real‐time polymerase chain reaction (RT‐qPCR) of confirmed positive SARS‐CoV‐2 nasopharyngeal and buccal swabs, as well as a bronchoalveolar lavage with mean real cycle threshold (qCt) value of 36 based on the Nucleocapsid (N) gene. Descriptive comparisons to known sequences in Botswana and internationally were made using mutation profiling analysis and phylogenetic inferences. Human samples were not available. A near‐full length SARS‐CoV‐2 genome (∼90% coverage) was successfully genotyped and classified under clade 20 O and Pango‐Lineage AY.43 (Pango v.4.0.6 PLEARN‐v1.3; 2022‐04‐21), which is a sublineage of the Delta variant of concern (VOC) (formerly called B.1.617.2, first detected in India). We did not identify novel mutations that may be used to distinguish SARS‐CoV‐2 isolates from the dog and humans. In addition to Spike (S) region mutation profiling, we performed phylogenetic analysis including 30 Delta sequences publicly available reference also isolated from dogs. In addition, we performed another exploratory analysis to investigate the phylogenetic relatedness of sequence isolated from dog with those from humans in Botswana (n = 1303) as of 31 March 2022 and of same sublineage. Expectedly, the sequence formed a cluster with Delta sublineages – AY.43, AY.116 and B.1.617.2 – circulating in same time frame. This is the first documented report of human‐associated SARS‐CoV‐2 infection in a dog in Botswana. Although the direction of transmission remains unknown, this study further affirms the need for monitoring pets during different COVID‐19 waves for possible clinically relevant SARS‐CoV‐2 transmissions between species.

BackgroundApproximately 30,000 non-citizens are living with HIV in Botswana, all of whom as of 2020 are eligible to receive free antiretroviral treatment (ART) within the country. We assessed the prevalence of HIV-1 mutational profiles [pre-treatment drug resistance (PDR) and acquired drug resistance (ADR)] among treatment-experienced (TE) and treatment-naïve (TN) non-citizens living with HIV in Botswana.MethodsA total of 152 non-citizens living with HIV were enrolled from a migrant HIV clinic at Independence Surgery, a private practice in Botswana from 2019–2021. Viral RNA isolated from plasma samples were genotyped for HIV drug resistance (HIVDR) using Sanger sequencing. Major known HIV drug resistance mutations (DRMs) in the pol region were determined using the Stanford HIV Drug Resistance Database. The proportions of HIV DRMs amongst TE and TN non-citizens were estimated with 95% confidence intervals (95% CI) and compared between the two groups.ResultsA total of 60/152 (39.5%) participants had a detectable viral load (VL) >40 copies/mL and these were included in the subsequent analyses. The median age at enrollment was 43 years (Q1, Q3: 38–48). Among individuals with VL > 40 copies/mL, 60% (36/60) were treatment-experienced with 53% (19/36) of them on Atripla. Genotyping had a 62% (37/60) success rate – 24 were TE, and 13 were TN. A total of 29 participants (78.4, 95% CI: 0.12–0.35) had major HIV DRMs, including at least one non-nucleoside reverse transcriptase inhibitor (NNRTI) associated DRM. In TE individuals, ADR to any antiretroviral drug was 83.3% (20/24), while for PDR was 69.2% (9/13). The most frequent DRMs were nucleoside reverse transcriptase inhibitors (NRTIs) M184V (62.1%, 18/29), NNRTIs V106M (41.4%, 12/29), and K103N (34.4%, 10/29). No integrase strand transfer inhibitor-associated DRMs were reported.ConclusionWe report high rates of PDR and ADR in ART-experienced and ART-naïve non-citizens, respectively, in Botswana. Given the uncertainty of time of HIV acquisition and treatment adherence levels in this population, routine HIV-1C VL monitoring coupled with HIVDR genotyping is crucial for long-term ART success.

ABSTRACT There are limited data on the prevalence of fostemsavir (FTR) resistance-associated polymorphisms in people with HIV in Botswana. We sought to determine the prevalence of FTR resistance-associated polymorphisms in HIV-1 subtype C sequences from antiretroviral therapy (ART)-naïve and ART-experienced individuals in Botswana. Previously reported FTR resistance-associated polymorphisms from literature were surveyed from HIV-1C proviral sequences generated from a large HIV-1 population-based cohort enrolled between 2013 and 2018. Sequences from ART-naïve and ART-experienced individuals were included in the analysis. A total of 6,078 participants with gp120 sequences were included in this study, and 6,030 (99.2%) had known ART status; 1,282 (21.3%) were from ART-naïve individuals, while 4,748 (78.7%) were from individuals on ART at study enrollment. Viral load (VL) data were available for 4,739 (99.8%) ART-experienced individuals, of whom 4,526 (95.5%) were suppressed and 213 (4.5%) had virologic failure (VF) (VL >400). Among those with VF and ART naive, the overall prevalence of FTR resistance was 13.3% (95% CI 11.6%–15.1%) and did not differ between ART-experienced with VF individuals (29/213, 13.6%) and ART-naïve individuals (170/1,282, 13.3%) (P-value = 0.9). The most predominant mutations (prevalence ≥1.0%) were M434I (9.8%), M475I (5.9%), and M426L (1.1%). The overall prevalence of FTR polymorphisms was similar in both ART-naïve and ART-experienced individuals with VF in a setting with no prior FTR exposure. We recommend further studies on the phenotypic impact of these polymorphisms to guide how to monitor for FTR resistance. (This study has been registered at ClinicalTrials.gov under registration no. NCT01965470.) IMPORTANCE Fostemsavir (FTR) is a newly licensed antiretroviral drug that has been shown to have activity against HIV-1. The mechanism of action of FTR is different from all currently available antiretrovirals (ARVs), and as such, it offers hope for HIV-1 suppression in those people with HIV (PWH) who harbor HIV-1 variants with drug resistance mutations to currently used ARVs. Using 6,030 HIV-1 sequences covering the HIV-1 envelope from PWH in Botswana who are antiretroviral therapy (ART) naïve as well as those who are failing ART, we explored the sequences for FTR resistance-associated polymorphisms. We found the prevalence of FTR polymorphisms to be similar in both ART-naïve and ART-experienced individuals with VF in this setting, with no prior FTR exposure. Further studies on the phenotypic impact of these polymorphisms are warranted to guide how to monitor for FTR resistance.

Abstract Background Low- and middle-income countries (LMICs) account for nearly 85% of the global cervical cancer burden, yet have the least access to high-performance screening. International guidelines recommend human papillomavirus testing (HPV) as primary screening, yet implementation is inhibited by the cost of HPV testing. Atila AmpFire® HPV Assay (AmpFire) is both affordable and easy to use, and offers individual genotyping. The objective of this study was to compare the performance of the AmpFire HPV assay to the Xpert® HPV assay in detection of both HPV and clinically significant cervical disease. Methods We utilized stored cervical specimens from a prospective cohort study of women living with human immunodeficiency virus (HIV) in Botswana conducted from May to July 2018. Positive and negative percent agreement was calculated for the AmpFire and Xpert assays, as was detection of high-grade cervical dysplasia. Results 63 stored cervical specimens had detectable DNA after thawing and were included in the analysis. The positive percent agreement was 91.2% (95%CI 76.3–98.1) and negative percent agreement was 79.3% (95% CI 60.3–92.0). Six cases positive by AmpFire but negative by Xpert were HPV genotypes 35, 52 (n = 2), 58, 68, and co-infection with HPV 45 and 68. Both Xpert and AmpFire assays detected HPV in all 10 samples of women who had high-grade cervical dysplasia. Conclusions The AmpFire HPV assay demonstrated excellent analytic performance in both detection of HPV and clinically significant cervical disease. AmpFire HPV is a promising option to increase access to affordable, type-specific HPV screening for cervical cancer in LMICs.

We evaluated subsequent virologic outcomes in individuals experiencing low-level virem ia (LLV) on dolutegravir (DTG)-based first-line antiretroviral therapy (ART) in Botswana. We used a national dataset from 50,742 adults who initiated on DTG-based first-line ART from June 2016–December 2022. Individuals with at least two viral load (VL) measurements post three months on DTG-based first-line ART were evaluated for first and subsequent episodes of LLV (VL:51–999 copies/mL). LLV was sub-categorized as low-LLV (51–200 copies/mL), medium-LLV (201–400 copies/mL) and high-LLV (401–999 copies/mL). The study outcome was virologic failure (VF) (VL ≥ 1000 copies/mL): virologic non-suppression defined as single-VF and confirmed-VF defined as two-consecutive VF measurements after an initial VL < 1000 copies/mL. Cox regression analysis identified predictive factors of subsequent VF. The prevalence of LLV was only statistically different at timepoints >6–12 (2.8%) and >12–24 (3.9%) (p-value < 0.01). LLV was strongly associated with both virologic non-suppression (adjusted hazards ratio [aHR] = 2.6; 95% CI: 2.2–3.3, p-value ≤ 0.001) and confirmed VF (aHR = 2.5; 95% CI: 2.4–2.7, p-value ≤ 0.001) compared to initially virally suppressed PLWH. High-LLV (HR = 3.3; 95% CI: 2.9–3.6) and persistent-LLV (HR = 6.6; 95% CI: 4.9–8.9) were associated with an increased hazard for virologic non-suppression than low-LLV and a single-LLV episode, respectively. In a national cohort of PLWH on DTG-based first-line ART, LLV > 400 copies/mL and persistent-LLV had a stronger association with VF. Frequent VL testing and adherence support are warranted for individuals with VL > 50 copies/mL.

Cytomegalovirus (CMV) has been linked with increased cardiovascular risk and monocyte activation in people living with HIV (PLWH). This cross-sectional study aimed to compare CMV immunoglobulin G (IgG) levels between combined antiretroviral therapy (cART)-treated PLWH versus ART-naïve PLWH and those without HIV, and to investigate their associations with biomarkers of endothelial injury and carotid atherosclerosis, in Gaborone, Botswana. All participants were between 30 and 50 years old. Carotid intimal media thickness (cIMT) and biomarkers of endothelial injury and monocyte activation were also assessed. The association between quantitative CMV IgG and cardiovascular disease risk was assessed in multivariate logistic regression analysis. The results showed that the mean CMV IgG level among ART-naïve participants was significantly higher than both the cART group and controls. However, CMV IgG levels did not differ significantly between the controls and cART groups. Among PLWH, CMV IgG levels were associated with ICAM-1 levels and cIMT. Increases in CMV IgG among ART-naïve participants were significantly associated with increases in log VCAM-1. In conclusion, CMV IgG levels are elevated among PLWH in sub-Saharan Africa, and higher levels are associated with biomarkers of endothelial injury and cIMT. Future research should investigate the long-term impact of elevated CMV IgG among PLWH.

(1) Background: We aimed to determine the prevalence of hepatitis B virus (HBV) resistance-associated mutations (RAMs) in people with HBV and human immunodeficiency virus (HBV/HIV) in Botswana. (2) Methods: We sequenced HBV deoxyribonucleic acid (DNA) from participants with HBV/HIV from the Botswana Combination Prevention Project study (2013–2018) using the Oxford Nanopore GridION platform. Consensus sequences were analyzed for genotypic and mutational profiles. (3) Results: Overall, 98 HBV sequences had evaluable reverse transcriptase region coverage. The median participant age was 43 years (IQR: 37, 49) and 66/98 (67.4%) were female. Most participants, i.e., 86/98 (87.8%) had suppressed HIV viral load (VL). HBV RAMs were identified in 61/98 (62.2%) participants. Most RAMs were in positions 204 (60.3%), 180 (50.5%), and 173 (33.3%), mostly associated with lamivudine resistance. The triple mutations rtM204V/L180M/V173L were the most predominant (17/61 [27.9%]). Most participants (96.7%) with RAMs were on antiretroviral therapy for a median duration of 7.5 years (IQR: 4.8, 10.5). Approximately 27.9% (17/61) of participants with RAMs had undetectable HBV VL, 50.8% (31/61) had VL < 2000 IU/mL, and 13/61 (21.3%) had VL ≥ 2000 IU/mL. (4) Conclusions: The high prevalence of lamivudine RAMs discourages the use of ART regimens with 3TC as the only HBV-active drug in people with HIV/HBV.

ABSTRACT: Objectives: There are limited data on the prevalence of doravirine (DOR)-associated drug resistance mutations in people with HIV (PWH) in Botswana. This cross-sectional, retrospective study aimed to explore the prevalence of DOR-associated resistance mutations among ART-naïve and -experienced PWH in Botswana enrolled in the population-based Botswana Combination Prevention Project (BCPP). Methods: A total of 6078 HIV-1C pol sequences were analysed for DOR-associated resistance mutations using the Stanford HIV drug resistance database, and their levels were predicted according to the Stanford DRM penalty scores and resistance interpretation. Virologic failure was defined as HIV-1 RNA load (VL) >400 copies/mL. Results: Among 6078 PWH, 5999 (99%) had known ART status, and 4529/5999 (79%) were on ART at time of sampling. The suppression rate among ART-experienced was 4517/4729 (96%). The overall prevalence of any DOR-associated resistance mutations was 181/1473 (12.3% [95% confidence interval {CI}: 10.7–14.1]); by ART status: 42/212 (19.8% [95% CI: 14.7–25.4]) among ART-failing individuals (VL ≥400 copies/mL) and 139/1261 (11.0% [95% CI: 9.3–12.9]) among ART-naïve individuals (P < 0.01). Intermediate DOR-associated resistance mutations were observed in 106/1261 (7.8% [95% CI: 6.9–10.1]) in ART-naïve individuals and 29/212 (13.7% [95% CI: 9.4–8.5]) among ART-experienced participants (P < 0.01). High-level DOR-associated resistance mutations were observed in 33/1261 (2.6% [95% CI: 1.8–3.7]) among ART-naïve and 13/212 (6.1% [95% CI: 3.6–10.8]) among ART-failing PWH (P < 0.01). PWH failing ART with at least one EFV/NVP-associated resistance mutation had high prevalence 13/67 (19.4%) of high-level DOR-associated resistance mutations. Conclusion: DOR-associated mutations were rare (11.0%) among ART-naive PWH but present in 62.7% of Botswana individuals who failed NNRTI-based ART with at least one EFV/NVP-associated resistance mutation. Testing for HIV drug resistance should underpin the use of DOR in PWH who have taken first-generation NNRTIs.

Abstract Background HIV-1 is endemic in Botswana. The country’s primary challenge is identifying people living with HIV who are unaware of their status. We evaluated factors associated with undiagnosed HIV infection using HIV-1 phylogenetic, behavioural, and demographic data. Methods As part of the Botswana Combination Prevention Project, 20% of households in 30 villages were tested for HIV and followed from 2013 to 2018. A total of 12,610 participants were enrolled, 3596 tested HIV-positive at enrolment, and 147 participants acquired HIV during the trial. Extensive socio-demographic and behavioural data were collected from participants and next-generation sequences were generated for HIV-positive cases. We compared three groups of participants: (1) those previously known to be HIV-positive at enrolment (n = 2995); (2) those newly diagnosed at enrolment (n = 601) and (3) those who tested HIV-negative at enrolment but tested HIV-positive during follow-up (n = 147). We searched for differences in demographic and behavioural factors between known and newly diagnosed group using logistic regression. We also compared the topology of each group in HIV-1 phylogenies and used a genetic diversity-based algorithm to classify infections as recent (

Abstract Background The variation of human papillomavirus (HPV) genotypes shapes the risks of cervical cancer and these variations are not well defined in Africa. Nucleotide changes within the L1 gene, nucleotide variability, and phylogeny were explored in relation to HIV in samples from Botswana and Kenya. Methods A total of 98 HPV-positive cervical samples were sequenced to identify different HPV variants. Phylogenetic inferences were used to determine HPV genotypes and investigate the clustering of sequences between women living with HIV (WLWHIV) and -women not living with HIV (WNLWHIV). Results Out of 98 generated sequences, 83.7% (82/98) participants had high-risk (HR) HPV genotypes while 16.3% (16/98) had low-risk (LR) HPV genotypes. Among participants with HR-HPV genotypes, 47.6% (39/82) were coinfected with HIV. The prevalence of HR-HPV genotypes was statistically higher in the Botswana population compared to Kenya (p-value

[This corrects the article DOI: 10.1371/journal.pone.0288971.].

BackgroundWomen living with HIV-1 (WLHIV) are at higher risk of having an adverse birth outcome, but the underlying mechanism(s) are unknown. We hypothesized that HIV-associated endothelial activation could adversely impact placental function and lead to impaired fetal growth or stillbirth.MethodsWe used stored samples from WLHIV and HIV-negative women who had enrolled during pregnancy in the observational Botswana Tshipidi cohort. Written informed consent was obtained from the participants. We measured plasma levels of markers of endothelial activation (soluble vascular adhesion molecule 1 [VCAM-1], intercellular adhesion molecule 1 [ICAM-1] and E-selectin) from samples taken during pregnancy. We compared log10 biomarker levels by maternal HIV status and by the timing of ART initiation (ART prior to conception vs. during pregnancy; ART prior to sample collection vs. no ART prior to sampling) using t-tests and the Kruskal-Wallis rank test. We evaluated the association between these biomarkers and adverse birth outcomes (composite of stillbirth or small for gestational age [SGA]) using univariate and multivariate log-binomial regression controlling for maternal age (continuous) and timing of ART start. We also used generalized linear models (GLM) to evaluate the association between continuous birthweight (in grams) and gestational age (in weeks) and markers of endothelial dysfunction, adjusting for maternal age (continuous) and timing of ART relative to sample collection.ResultsSpecimens collected before delivery were available for 414 women (372 WLHIV and 42 HIV-negative women), with a median age of 28 years and median gestational age at sample collection of 30 weeks (range 26, 35 weeks). WLHIV had significantly higher median VCAM1 (p = 0.002) than HIV-negative women, but HIV-negative women had higher median ICAM1 (p = 0.01); e-Selectin levels did not differ by maternal HIV status. Women starting ART during pregnancy had higher log10 VCAM1 levels than those on ART before conception, regardless of whether the sample was collected before (p = 0.02) or after (p = 0.03) ART initiation. However, ICAM1 and e-Selectin did not differ significantly by ART status or ART timing. Ninety-eight women (91 WLHIV and 7 HIV-negative), or 9 (2%) and 89 (22%) included in this study, had a stillborn or SGA baby respectively. Univariate and adjusted analyses did not show significant associations between levels of any of the biomarkers with these adverse birth outcomes. However, lower birthweight (p = 0.03) and lower gestational age at delivery were associated e-Selectin and ICAM (p = 0.008), respectively.ConclusionMaternal HIV infection and lack of ART (or recent ART initiation) were associated with one marker of greater endothelial activation (VCAM-1), but not with other markers (ICAM-1 nor E-selectin) in pregnancy. e-Selectin was associated with lower birthweight and every unit increase in log ICAM-1 at delivery was associated with lower gestation age at delivery.

Abstract Background Botswana updated its antiretroviral treatment (ART) guidelines in May 2016 to support breastfeeding for women living with HIV (WLHIV) on ART who have documented HIV RNA suppression during pregnancy. Methods From September 2016 to March 2019, we evaluated feeding method at discharge among WLHIV at eight government maternity wards in Botswana within the Tsepamo Study. We validated the recorded feeding method on the obstetric record using the prevention of mother-to-child transmission of HIV (PMTCT) counsellor report, infant formula dispensing log or through direct observation. Available HIV RNA results were recorded from the obstetric record, and from outpatient HIV records (starting February 2018). In a subset of participants, we used electronic laboratory records to verify whether an HIV RNA test had occurred. Univariable and multivariable logistic regression analyses were performed to identify factors associated with infant feeding choice. Results Among 13,354 WLHIV who had a validated feeding method at discharge, 5303 (39.7%) chose to breastfeed and 8051 (60.3%) chose to formula feed. Women who had a documented HIV RNA result in the obstetric record available to healthcare providers at delivery were more likely to breastfeed (50.8%) compared to women who did not have a documented HIV RNA result (35.4%) (aOR 0.59; 95% CI 0.54, 0.65). Among women with documented HIV RNA, 2711 (94.6%) were virally suppressed ( 400 copies/mL, and 134 (27.4%) of 489 women with no reported ART use. A sub-analysis of electronic laboratory records among 150 women without a recorded result on the obstetric record revealed that 93 (62%) women had an HIV RNA test during pregnancy. Conclusions In a setting of long-standing use of suppressive ART, with majority of WLHIV on ART from the time of conception, requiring documentation of HIV RNA suppression in the obstetric record to inform infant feeding decisions is a barrier to breastfeeding but unlikely to prevent a substantial amount of HIV transmission.

(1) Background: Hepatitis B core antibodies (anti-HBc) are a marker of hepatitis B virus (HBV) exposure; hence, a normal HBV serology profile is characterized by HBV surface antigen (HBsAg) and anti-HBc positivity. However, atypical HBV serologies occur, and we aimed to determine the prevalence of an atypical profile (HBsAg+/anti-HBc-) in a cohort of people with HIV-1 (PWH) in Botswana. (2) Methods: Plasma samples from an HIV-1 cohort in Botswana (2013–2018) were used. The samples were screened for HBsAg and anti-HBc. Next-generation sequencing was performed using the GridION platform. The Wilcoxon rank-sum test and Chi-squared tests were used for the comparison of continuous and categorical variables, respectively. (3) Results: HBsAg+/anti-HBc- prevalence was 13.7% (95% CI 10.1–18.4) (36/263). HBsAg+/anti-HBc- participants were significantly younger (p < 0.001), female (p = 0.02) and ART-naïve (p = 0.04) and had a detectable HIV viral load (p = 0.02). There was no statistically significant difference in the number of mutations observed in participants with HBsAg+/anti-HBc- vs. those with HBsAg+/anti-HBc+ serology. (4) Conclusions: We report a high HBsAg+/anti-HBc- atypical serology profile prevalence among PWH in Botswana. We caution against HBV-testing algorithms that consider only anti-HBc+ samples for HBsAg testing, as they are likely to underestimate HBV prevalence. Studies to elucidate the mechanisms and implications of this profile are warranted.

Sapoviruses are an important cause of acute viral gastroenteritis in children. The objective of this study was to determine the burden and genetic diversity of sapoviruses in children hospitalized with acute gastroenteritis in Botswana between 2016 and 2018. Samples were screened using the BioFire® Filmarray® GI Panel assay and for positive sapovirus samples, conventional RT-PCR was used to amplify the partial VP1 gene. Strain characterization was performed using both the Human Calicivirus Typing tool and the NCBI GenBank BLAST. Phylogenetic and evolutionary analysis was inferred using BEAST V1.10.4. General time-reversible was used as a nucleotide substitution model. Sapovirus was detected in 6.5% of participants and two different sapovirus genotypes GI.1 (5/7) and GI.6 (2/7) were identified from the seven samples. This study provides the first data on sapovirus infections and their genetic diversity in a clinical setting in Botswana, contributing to the global knowledge of this virus.

Objectives:Coronavirus disease 2019 was declared a global pandemic in March 2020 with correct and early detection of cases using laboratory testing central to the response. Hence, the establishment of quality management systems and monitoring their implementation are critical. This study describes the experience of implementing the COVID-19 Laboratory Testing and Certification Program (CoLTeP) in Africa.MethodsPrivate and public laboratories conducting SARS-CoV-2 testing using polymerase chain reaction were enrolled and assessed for quality and safety using the CoLTeP checklists.ResultsA total of 84 laboratories from 7 countries were assessed between April 2021 to December 2021 with 52% of these from the private sector. Among them, 64% attained 5 stars and were certified. Section 4 had the highest average score of 92% and the lowest of 78% in Section 3. Also, 82% of non-conformities (NCs) were related to sample collection, transportation, and risk assessments. Non-availability, inconsistency in performing, recording, instituting corrective actions for failed internal and external quality controls were among major NCs reported.ConclusionsLaboratories identified for SARS-CoV-2 testing by public and private institutions mostly met the requirements for quality and safe testing as measured by the CoLTeP checklist.

HIV is known to accumulate escape mutations in the gag gene in response to the immune response from cytotoxic T lymphocytes (CTLs). These mutations can occur within an individual as well as at a population level. The population of Botswana exhibits a high prevalence of HLA*B57 and HLA*B58, which are associated with effective immune control of HIV. In this retrospective cross-sectional investigation, HIV-1 gag gene sequences were analyzed from recently infected participants across two time periods which were 10 years apart: the early time point (ETP) and late time point (LTP). The prevalence of CTL escape mutations was relatively similar between the two time points—ETP (10.6%) and LTP (9.7%). The P17 protein had the most mutations (9.4%) out of the 36 mutations that were identified. Three mutations (A83T, K18R, Y79H) in P17 and T190A in P24 were unique to the ETP sequences at a prevalence of 2.4%, 4.9%, 7.3%, and 5%, respectively. Mutations unique to the LTP sequences were all in the P24 protein, including T190V (3%), E177D (6%), R264K (3%), G248D (1%), and M228L (11%). Mutation K331R was statistically higher in the ETP (10%) compared to the LTP (1%) sequences (p < 0.01), while H219Q was higher in the LTP (21%) compared to the ETP (5%) (p < 0.01). Phylogenetically, the gag sequences clustered dependently on the time points. We observed a slower adaptation of HIV-1C to CTL immune pressure at a population level in Botswana. These insights into the genetic diversity and sequence clustering of HIV-1C can aid in the design of future vaccine strategies.

Abstract. The two non-nucleoside reverse transcriptase inhibitors (NNRTIs), efavirenz (EFV) and nevirapine (NVP), are currently the core antiretroviral drugs for treatment of HIV in sub-Saharan Africa including Botswana. The drugs are metabolized by Cytochrome P450 2B6 (CYP2B6) liver enzyme. The CYP2B6 gene that encodes for metabolism of these drugs is known to be highly polymorphic. One of the polymorphism in the CYP2B6 gene, 516G>T, particularly the 516T allele, is known to confer poor metabolism of EFV and NVP. This may lead to high levels of plasma drug concentrations and development of treatment toxicities, like central nervous system toxicities, and cutaneous and hepatic toxicities, for EFV and NVP, respectively. The CYP2B6 516G allele on the other hand is associated with an extensive metabolism of the two NNRTIs drugs. We sought to establish association between possible developments of NNRTIs toxicities with CYP2B6 516G>T variation in Botswana. A total of 316 peripheral blood mononuclear cells samples were used in a retrospective view. All the samples were from participants on EFV/NVP-containing regimen with known toxicity output. TaqMan Real-Time PCR approach was applied for assessing CYP2B6 516 allele variation in cases with treatment toxicity and those without. Analysis was performed by chi-square statistics and logistic regression analysis. The rate of poor metabolizers among participants with toxicity and those without toxicity was 18.4% and 15.1%, respectively. The CYP2B6 516 genotype distribution comparisons between the participants with toxicity and those without were not statistically different (chi-square = .326; P = .568). CYP2B6 516 variation was not associated with NNRTI toxicity. No other factors were associated with toxicity when considering age, baseline body mass index, baseline CD4, baseline HIV viral load and adherence. The results were discussed in the context of all the studies done in Botswana to date.

Background: Mathematical models predict that community-wide access to HIV testing-and-treatment can rapidly and substantially reduce new HIV infections. Yet several large universal test-and-treat HIV prevention trials in high-prevalence epidemics demonstrated variable reduction in population-level incidence. Methods: To elucidate patterns of HIV spread in universal test-and-treat trials, we quantified the contribution of geographic-location, gender, age, and randomized-HIV-intervention to HIV transmissions in the 30-community Ya Tsie trial in Botswana. We sequenced HIV viral whole genomes from 5114 trial participants among the 30 trial communities. Results: Deep-sequence phylogenetic analysis revealed that most inferred HIV transmissions within the trial occurred within the same or between neighboring communities, and between similarly aged partners. Transmissions into intervention communities from control communities were more common than the reverse post-baseline (30% [12.2 – 56.7] vs. 3% [0.1 – 27.3]) than at baseline (7% [1.5 – 25.3] vs. 5% [0.9 – 22.9]) compatible with a benefit from treatment-as-prevention. Conclusions: Our findings suggest that population mobility patterns are fundamental to HIV transmission dynamics and to the impact of HIV control strategies. Funding: This study was supported by the National Institute of General Medical Sciences (U54GM088558), the Fogarty International Center (FIC) of the U.S. National Institutes of Health (D43 TW009610), and the President’s Emergency Plan for AIDS Relief through the Centers for Disease Control and Prevention (CDC) (Cooperative agreements U01 GH000447 and U2G GH001911).

Background: The novel coronavirus disease 2019 (COVID-19), declared a pandemic by the World Health Organization (WHO) in March 2020, has taught us about the importance of epidemic preparedness. Objective: We analysed the pre-COVID-19 preparedness of sub-Saharan African countries and how this may have influenced the trajectory of COVID-19 cases. Methods: The WHO Joint External Evaluation (JEE) tool and the Global Health Security (GHS) Index were used to determine the epidemic preparedness of countries in the WHO African Region. The relationship between pre-COVID-19 preparedness and the reported number of cases per million people was evaluated over the first 120 days of the first reported case in each country, between February 2020 and September 2020. Results: The overall performance of the 42 countries was 40% in the 19 JEE core capacities and 32% in the six GHS Index indicators. At Day 1, the mean number of cases per million population was significantly higher among countries rated as ‘prepared’ in the JEE legislation, policy and finance (p = 0.03), ports of entry (p = 0.001), and international health regulation coordination, communication and advocacy (p = 0.03) categories. At Day 90, countries rated as ‘prepared’ in the national laboratory systems (p = 0.05) and real-time surveillance (p = 0.04) JEE categories had statistically significantly fewer cases per million population. Conclusion: This analysis highlights the importance of building capacity for pandemic preparedness in Africa. The WHO African Region was not adequately prepared for the COVID-19 pandemic as measured by the WHO JEE tool and the GHS Index.

It remains unknown whether the C-C motif chemokine receptor type 5 (CCR5) coreceptor is still the predominant coreceptor used by Human Immunodeficiency Virus-1 (HIV-1) in Botswana, where the HIV-1 subtype C predominates. We sought to determine HIV-1C tropism in Botswana using genotypic tools, taking into account the effect of antiretroviral treatment (ART) and virologic suppression. HIV-1 gp120 V3 loop sequences from 5602 participants were analyzed for viral tropism using three coreceptor use predicting algorithms/tools: Geno2pheno, HIV-1C Web Position-Specific Score Matrices (WebPSSM) and the 11/25 charge rule. We then compared the demographic and clinical characteristics of people living with HIV (PLWH) harboring R5- versus X4-tropic viruses using χ2 and Wilcoxon rank sum tests for categorical and continuous data analysis, respectively. The three tools congruently predicted 64% of viruses as either R5-tropic or X4-tropic. Geno2pheno and the 11/25 charge rule had the highest concordance at 89%. We observed a significant difference in ART status between participants harboring X4- versus R5-tropic viruses. X4-tropic viruses were more frequent among PLWH receiving ART (χ2 test, p = 0.03). CCR5 is the predominant coreceptor used by HIV-1C strains circulating in Botswana, underlining the strong potential for CCR5 inhibitor use, even in PLWH with drug resistance. We suggest that the tools for coreceptor prediction should be used in combination.

Background: Viral hepatitis is a major global health problem. There is a paucity of data from Botswana on the seroprevalence of markers of hepatitis. The objective of the study was to determine the seroprevalence and risk factors of hepatitis B virus (HBV), hepatitis D virus (HDV) and hepatitis C virus (HCV) infections in patients with clinical features of hepatitis and/or altered liver function tests. Method: This cross-sectional study was done at Princess Marina Hospital (PMH) in Gaborone, Botswana, from February 2015 to July 2016. It involved 328 adult patients with any of the following: jaundice, history of liver disease and/or increased serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and serum bilirubin of 2 times the upper limit of normal (ULN). Results: Active or chronic active hepatitis (hepatitis B surface antigen [HBsAg] positive) was identified in 46.7% of patients. Antibodies to HDV infection were detected in 4.6% of the HBsAg-positive patients and antibodies to HCV infection in 4.3% of the study patients. Immunity against HBV infection was noted in 34.5% of patients. Human immunodeficiency virus (HIV) co-infection was self-reported by 42.7% of HBsAg-positive patients with known HIV status. Conclusion: High prevalence rate of HBV, HCV, HDV infection and HIV co-infection was observed in patients with liver disease attending PMH.

Abstract Background Hepatitis C virus (HCV) infection is a major cause of chronic liver disease globally. Direct acting antivirals (DAAs) have proven effective in curing HCV. However, the current standard of care (SOC) in Botswana remains PEGylated interferon-α (IFN-α) with ribavirin. Several mutations have been reported to confer resistance to interferon-based treatments. Therefore, there is a need to determine HCV genotypes in Botswana, as these data will guide new treatment guidelines and understanding of HCV epidemiology in Botswana. Methods This was a retrospective cross-sectional pilot study utilizing plasma obtained from 55 participants from Princess Marina Hospital in Gaborone, Botswana. The partial core region of HCV was amplified, and genotypes were determined using phylogenetic analysis. Results Four genotype 5a and two genotype 4v sequences were identified. Two significant mutations – K10Q and R70Q – were observed in genotype 5a sequences and have been associated with increased risk of hepatocellular carcinoma (HCC), while R70Q confers resistance to interferon-based treatments. Conclusion Genotypes 5a and 4v are circulating in Botswana. The presence of mutations in genotype 5 suggests that some patients may not respond to IFN-based regimens. The information obtained in this study, in addition to the World health organization (WHO) recommendations, can be utilized by policy makers to implement DAAs as the new SOC for HCV treatment in Botswana.

Cerebrospinal fluid (CSF) viral escape has been poorly described among people with HIV-associated cryptococcal meningitis. We determined the prevalence of CSF viral escape and HIV-1 viral load (VL) trajectories in individuals treated for HIV-associated cryptococcal meningitis. A retrospective longitudinal study was performed using paired CSF and plasma collected prior to and during the antifungal treatment of 83 participants recruited at the Botswana site of the phase-3 AMBITION-cm trial (2018–2021). HIV-1 RNA levels were quantified then CSF viral escape (CSF HIV-1 RNA ≥ 0.5 log10 higher than plasma) and HIV-1 VL trajectories were assessed. CSF viral escape occurred in 20/62 (32.3%; 95% confidence interval [CI]: 21.9–44.6%), 13/52 (25.0%; 95% CI: 15.2–38.2%) and 1/33 (3.0%; 95% CI: 0.16–15.3%) participants at days 1, 7 and 14 respectively. CSF viral escape was significantly lower on day 14 compared to days 1 and 7, p = 0.003 and p = 0.02, respectively. HIV-1 VL decreased significantly from day 1 to day 14 post antifungal therapy in the CSF but not in the plasma (β = −0.47; 95% CI: −0.69 to −0.25; p < 0.001). CSF viral escape is high among individuals presenting with HIV-associated cryptococcal meningitis; however, antifungal therapy may reverse this, highlighting the importance of rapid initiation of antifungal therapy in these patients.

Background: HIV-1 drug resistance poses a major threat to the success of antiretroviral therapy. The high costs of available HIV drug resistance assays prohibit their routine usage in resource-limited settings. Pan-degenerate amplification and adaptation (PANDAA), a focused genotyping approach based on quantitative PCR (qPCR), promises a fast and cost-effective way to detect HIV drug resistance mutations (HIVDRMs). Given the high cost of current genotyping methods, we sought to use PANDAA for screening key HIVDRMs in antiretroviral-naïve individuals at codons 103, 106 and 184 of the HIV-1 reverse transcriptase gene. Mutations selected at these positions have been shown to be the most common driver mutations in treatment failure. Methods: A total of 103 samples from antiretroviral-naïve individuals previously genotyped by Sanger population sequencing were used to assess and verify the performance of PANDAA. PANDAA samples were run on the ABI 7500 Sequence Detection System to genotype the K103N, V106M and M184V HIVDRMs. In addition, the cost per sample and reaction times were compared. Results: Sanger population sequencing and PANDAA detected K103N mutation in three (2.9%) out of 103 participants. There was no evidence of baseline V106M and M184V mutations observed in our study. To genotype the six HIVDRMs it costs approximately 40 USD using PANDAA, while the reagents cost per test for Sanger population sequencing is approximately 100 USD per sample. PANDAA was performed quicker compared to Sanger sequencing, 2 hours for PANDAA versus 15 hours for Sanger sequencing. Conclusion: The performance of PANDAA and Sanger population sequencing demonstrated complete concordance. PANDAA could improve patient management by providing quick and relatively cheap access to drug-resistance information.

Background: Epidemics of human immunodeficiency virus (HIV) and cervical cancer are interconnected. DNA hypermethylation of host genes' promoter in cervical lesions has also been recognized as a contributor to cervical cancer progression.Methods: For this purpose we analyzed promoter methylation of four tumor suppressor genes (RARB, CADM1, DAPK1 and PAX1) and explored their possible association with cervical cancer in Botswana among women of known HIV status. Overall, 228 cervical specimens (128 cervical cancers and 100 non-cancer subjects) were used. Yates-corrected chi-square test and Fisher's exact test were used to explore the association of promoter methylation for each host gene and cancer status. Subsequently, a logistic regression analysis was performed to find which factors, HIV status, high risk-HPV genotypes, patient's age and promoter methylation, were associated with the following dependent variables: cancer status, cervical cancer stage and promoter methylation rate.Results: In patients with cervical cancer the rate of promoter methylation observed was greater than 64% in all the genes studied. Analysis also showed a higher risk of cervical cancer according to the increased number of methylated promoter genes (OR = 6.20; 95% CI: 3.66–10.51; P < 0.001). RARB methylation showed the strongest association with cervical cancer compared to other genes (OR = 15.25; 95% CI: 6.06–40.0; P < 0.001). Cervical cancer and promoter methylation of RARB and DAPK1 genes were associated with increasing age (OR = 1.12; 95% CI: 1.01-1.26; P = 0.037 and OR = 1.05; 95% CI: 1.00-1.10; P = 0.040). The presence of epigenetic changes at those genes appeared to be independent of HIV status among subjects with cervical cancer. Moreover, we found that cervical cancer stage was influenced by RARB (χ2= 7.32; P = 0.002) and CADM1 (χ2=12.68; P = 0.013) hypermethylation, and HIV status (χ2= 19.93; P = 0.001).Conclusion: This study confirms the association between invasive cervical cancer and promoter gene methylation of tumor suppressing genes at the site of cancer. HIV infection did not show any association to methylation changes in this group of cervical cancer patients from Botswana. Further studies are needed to better understand the role of HIV in methylation of host genes among cancer subjects leading to cervical cancer progression.

Background Oral HIV self-testing (HIVST) may be useful for increasing testing in persons at elevated risk of acquiring HIV. Methods We conducted a pilot study to evaluate the feasibility (defined by uptake) of HIVST among FSW in Gaborone, Botswana. FSW age 18 years and above were recruited through a non-governmental organization serving FSW. FSW with unknown or negative HIV status at screening performed HIVST in the study clinic following brief training. FSW testing HIV-negative were each given two test kits to take home: one kit to perform unassisted HIVST and another to share with others. Feasibility (use) of HIVST (and sharing of test kits with others) was assessed in these women at a study visit four months later. Results Two hundred FSW were screened. Their average age was 34 years (range 18–59), and 115 (58%) were HIV-positive. Eighty-five (42%) tested HIV-negative at entry and were eligible to take part in the HIVST pilot study. All 85 (100%) agreed to take home HIVST kits. Sixty-nine (81%) of these 85 participants had a follow-up visit, 56 (81%) of whom reported performing HIVST at a mean of three and half months after the initial visit. All 56 participants who performed HIVST reported negative HIVST results. Fifty (73%) of the 69 participants who took HIVST kits home shared them with others. Of the 50 women sharing HIVST kits, 25 (50%) shared with their non-client partners, 15 with a family member, 8 with friends, and 3 with a client. One participant did not test herself but shared both her test kits. Most participants 53/56 (95%) found oral HIVST very easy to use whilst 3/56 (5%) felt it was fairly easy. Conclusion Oral HIVST is feasible among FSW in Gaborone, Botswana. The majority of FSW used the HIVST kits themselves and also shared extra HIVST kits with other individuals.

BackgroundHIV prevalence among female sex workers (FSW) in sub-Saharan Africa is much higher than in the general population. HIV self-testing (HIVST) may be useful for increasing testing rates in FSW.MethodsWe conducted semi-structured in-depth interviews among FSW, nurses and lay counsellors providing services to FSWs in Botswana. We aimed to gain understanding of perceived acceptability, anticipated barriers, and preferred approaches to HIVST among FSW. Interviews were audio-recorded, transcribed and translated. Transcripts were reviewed and coded independently by two investigators; high inter-coder agreement was achieved (Kappa = 0.80).ResultsWe interviewed five care providers whose average age was 40 years (SD = 2,64, range = 37-43); three nurses and two counsellors. Thirty FSW were interviewed, with mean age 34 years (range = 20-52). Most (27; 90%) FSW expressed great interest in using HIVST kits. Facilitators of HIVST were: awareness of own risky sexual behaviours, desire to stay healthy, and perceived autonomy over one's healthcare decisions. Perceived advantages of HIVST included convenience, privacy, and perception of decreased stigma. Identified barriers to HIVST included lack of knowledge about the HIVST kit, fear of testing due to anticipated stigma, mistrust of the test's accuracy, doubt of self-competency to perform HIVST, and concerns about not linking to care. Assisting someone to test was noted as good for providing emotional support, but there were concerns about confidentiality breaches. Providers expressed concerns over low literacy among FSWs which could affect comprehension of testing instructions, and competency to perform testing and interpret results. Participants' recommendations for implementation of HIVST included: ensuring wide dissemination of information on HIVST, engaging peers in information-sharing and education, making test kits accessible in FSW-friendly centres, and having clear instructions for linkage to healthcare and support.ConclusionHIVST shows high acceptability among FSWs in Gaborone Botswana, with providers expressing some concerns. Implementation should be peer-driven with healthcare provider oversight.

BackgroundBotswana introduced the HBV vaccine at birth for all newborns in 2000. To the best of our knowledge, since the introduction of HBV vaccination, there have been limited data for vaccine response to HBV and its impact on early childhood HBV infections among children HIV exposed but uninfected in Botswana.AimsTo determine the prevalence of hepatitis B surface antigen (HBsAg) and HBV vaccine response in 18 months old children HIV exposed but uninfected in Botswana.MethodsStored plasma samples from 304 children at 18 months of age and 287 mothers from delivery were tested for HBsAg. Mothers with positive HBsAg had HBV DNA level tested, and their HBV genotypes were determined by amplifying a 415-base pair (bp) region of the surface gene. Plasma samples from children exposed to HIV were tested for hepatitis B surface antibody (anti-HBs) titers.ResultsNo children (0 of 304) were positive for HBsAg at 18 months while 5 (1.74%) of 287 HIV-positive mothers were HBsAg positive. Four of the HBsAg positive mothers were infected with genotype A1, while 1 was infected with genotype E. The median anti-HBs titer in children was 174 mIU/mL [QR: 70, 457]. Three (1.1%) of 269 children had an inadequate vaccine response (ConclusionNo HBsAg positivity was identified in a cohort of children HIV exposed but uninfected. The absence of HBsAg positives was associated with good HBV vaccine responses and low maternal HBsAg prevalence in Botswana.

Abstract Background Norovirus is a leading cause of viral gastroenteritis worldwide with a peak of disease seen in children. The epidemiological analysis regarding the virus strains in Africa is limited. The first report of norovirus in Botswana was in 2010 and currently, the prevalence and circulating genotypes of norovirus are unknown, as the country has no systems to report the norovirus cases. This study investigated the prevalence, patterns and molecular characteristics of norovirus infections among children ≤5 years of age admitted with acute gastroenteritis at four hospitals in Botswana. Methods A total of 484 faecal samples were collected from children who were admitted with acute gastroenteritis during the rotavirus vaccine impact survey between July 2013 and December 2015. Norovirus was detected using real-time RT-PCR. Positive samples were genotyped using conventional RT-PCR followed by partial sequencing of the capsid and RdRp genes. Norovirus strains were determined by nucleotide sequence analysis using the online Norovirus Genotyping Tool Version 1.0, and confirmed using maximum likelihood tree construction as implemented in MEGA 6.0. Results The prevalence of norovirus was 9.3% (95% CI 6.7–11.9). The genotype diversity was dominated by the GII.4 strain at 69.7%. This was followed by GII.2, GII.12 each at 9.1%, GI.9 at 6.6% and GII.6, GII.10 each at 3.0%. The most common combined RdRp/Capsid genotype was the GII.Pe/GII.4 Sydney 2012. Norovirus was detected during most part of the year; however, there was a preponderance of cases in the wet season (December to March). Conclusion The study showed a possible decline of norovirus infections in the last 10 years since the first report. An upward trend seen between 2013 and 2015 may be attributable to the success of rotavirus vaccine introductions in 2012. Knowledge of circulating genotypes, seasonal trends and overall prevalence is critical for prevention programming and possible future vaccine design implications.

Archived Formalin-Fixed Paraffin-Embedded (FFPE) tissue specimens can be a valuable source of human papillomavirus (HPV) nucleic acids for molecular biological analyses in retrospective studies. Although successful amplification with polymerase chain reaction (PCR) is essential for analysis of HPV DNA extracted from cervical FFPE specimens, extensive DNA damage due to cross-linking and fragmentation results in poor yield. Therefore, techniques to improve the diagnostic rate and sensitivity from FFPE tissues through PCR is highly desired and of wider interest. To overcome this, a highly sensitive double-nested PCR methodology was designed and optimized for limited-resource laboratories coupled with an organic extraction of DNA. This method allows the detection of a broad range of HPV genotypes and also allowing the sequencing of the final amplicon. Validation of the new approach developed was done with an automated DNA extraction coupled with Real Time PCR. Results showed that the proposed method achieves 96.3% of HPV detection as compared to 100% Abbott m2000rt used as ‘gold standard’. Moreover, the concordance rate between the two methods was equal for detecting HPV -16 or -18 genotypes. Nevertheless, the newly introduced assay has an advantage of: • Simultaneously identifying broad range of HPV genotypes besides HPV-16 and -18 from clinical samples. • It is an easy and cost-effective method that can be beneficial in resource-limited setting and can be employed for various molecular applications. • The method is indicated for highly degraded FFPE samples. Method name: Double-nested PCR for detecting and genotyping HPV, Keywords: HPV, FFPE, DNA, Double-nested PCR, RFLP, DNA sequencing

Dolutegravir (DTG) is a potent anti-HIV drug that is used to treat HIV globally. There have been reports of mutations in the HIV-1 3′-polypurine tract (3′PPT) of the nef gene, contributing to DTG failure; however, there are limited ‘real-world’ data on this. In addition, there is a knowledge gap on the variability of 3′PPT residues in patients receiving combination antiretroviral therapy (cART) with and without viral load (VL) suppression. HIV-1 subtype C (HIV-1C) whole-genome sequences from cART naïve and experienced individuals were generated using next-generation sequencing. The nef gene sequences were trimmed from the generated whole-genome sequences using standard bioinformatics tools. In addition, we generated separate integrase and nef gene sequences by Sanger sequencing of plasma samples from individuals with virologic failure (VF) while on a DTG/raltegravir (RAL)-based cART. Analysis of 3′PPT residues was performed, and comparison of proportions computed using Pearson’s chi-square test with p-values < 0.05 was considered statistically significant. A total of 6009 HIV-1C full genome sequences were generated and had a median log10 HIV-1 VL (Q1, Q3) copies/mL of 1.60 (1.60, 2.60). A total of 12 matching integrase and nef gene sequences from therapy-experienced participants failing DTG/ RAL-based cART were generated. HIV-1C 3′PPT nef gene sequences from therapy-experienced patients failing DTG cART (n = 12), cART naïve individuals (n = 1263), and individuals on cART with and without virological suppression (n = 4696) all had a highly conserved 3′PPT motif with no statistically significant differences identified. Our study confirms the high conservation of the HIV-1 nef gene 3′PPT motif in ‘real-world’ patients and showed no differences in the motif according to VL suppression or INSTI-based cART failure. Future studies should explore other HIV-1 regions outside of the pol gene for associations with DTG failure.

Abstract Background Human papillomavirus (HPV) associated malignancies are the leading cause of cancer death in Botswana. We sought to determine causative HPV types in patients with anogenital malignancies in Botswana to inform vaccine strategy. Methods We used formalin-fixed and paraffin-embedded (FFPE) tissue blocks from patients diagnosed with anal, penile and vulvar squamous cell carcinomas between the years, 2014 and 2016. Presence of HPV 16, 18, or other high-risk (HR) types was detected using Abbott m2000 real-time PCR platform. Tissues with other high-risk types were subsequently analysed using a multiplex qPCR assay that includes 15 validated fluorophore probes. Results A total of 126 tissue specimens, comprising of 21 anal (9 males, 12 females), 31 penile and 74 vulvar were studied. Ninety-three (73.8%) patients had their HIV status documented in the records while the rest did not. Eighty-three (83) out of 93 were HIV positive, a prevalence of 89.4% (95% CI: 81–94). HPV was detected in 68/126 (54%) tissues, of which 69% (95% CI: 54–79) had HPV 16 only, 28% (95% CI: 19–40) had other hr.-HPV types and 2.9% (95% CI, 3.5–10.1) were co-infected with HPV 16 and other hr.-types. Other high-risk types detected included HPV 26, 31, 33, 35, 39, 45, 51, 52, 66 and 68. HPV 18 was not detected. Multiple-type HPV infection was detected in 44 of 47 (93.6%) HIV positive participants co-infected with HPV. In HIV-negative individuals, only HPV 16 was detected. Conclusion In our study, anogenital carcinomas were associated with HPV 16 and other hr.-HPV types besides HPV 16 and 18. HIV co-infected patients had multiple hr.-HPV types detected whereas in HIV-negative patients only HPV 16 was detected. Our study suggests that multivalent vaccines may be more suitable in this setting, especially for HIV-infected individuals.

BACKGROUND:High-risk human papillomavirus (HR-HPV) has been demonstrated to be the necessary cause of cervical carcinoma. High-risk HPV detection has a prognostic significance for the women who are at increased risk of disease progression. HPV genotyping in cervical cancer precursor lesions is crucial for prevention and management of cervical cancer. This study was designed to investigate the distribution of HR-HPV genotypes among a group of patients with high-grade squamous intraepithelial lesions and higher, of the cervix, in Botswana. MATERIALS AND METHODS:185-archived residual formalin-fixed paraffin-embedded cervical biopsies collected between the years, 2006 and 2008 were studied. These tissues were diagnosed with HSIL (n = 146) and squamous cell carcinoma (n = 39). DNA was extracted using the Abbott m2000 analyser (Abbott Laboratories, Illinois) using reagents provided by the manufacturer. HPV genotyping was done using the Abbott RealTime HR-HPV PCR, which qualitatively detects 14 HR-HPV (reported as HPV 16, 18 & Other HR-HPV). RESULTS:DNA was successfully extracted from 162/185 (87.6%) tissues as indicated by a positive β-globin test. 132/162 (82%) tested positive for HR-HPV The HPV 16 prevalence was 50% (66/132), HPV 18 at 15.2% (20/132) and other Group 1 HR-HPV plus HPV 66 and 68 had a prevalence of 56.1% (74/132). Other HR-HPV types were common in HSIL than in carcinoma, while HPV 16 was more prevalent in carcinomas than other HR-HPV genotypes. CONCLUSION:In this study, HPV 16 and other HR-HPV genotypes were commonly associated with HSIL but HPV 18 was uncommon among Botswana women. Our data highlights the need for multivalent HPV vaccines with cross coverage for other high risk HPV other than HPV 16 and 18.

BackgroundMolecular typing of Mycobacterium tuberculosis (M.tb) isolates can inform Tuberculosis (TB) control programs on the relative proportion of transmission driving the TB epidemic. There is limited data on the M. tb genotypes that are circulating in Botswana. The aim of this study was to generate baseline data on the genetic diversity of M.tb isolates circulating in the country.MethodsA total of 461 M.tb isolates received at the Botswana National Tuberculosis Reference Laboratory between March 2012 and October 2013 were included in this study. Drug susceptibility testing was conducted using the BD BACTEC MGIT 960 System. M.tb strains were genotyped using spoligotyping and spoligotype patterns were compared with existing patterns in the SITVIT Web database. A subset of drug resistant isolates which formed spoligo clusters (n = 65) was additionally genotyped with 12-loci MIRU. Factors associated with drug resistance and clustering were evaluated using logistic regression.ResultsOf the 461 isolates genotyped, 458 showed 108 distinct spoligotype patterns. The predominant M.tb lineages were Lineage 4 (81.9%), Lineage 2 (9%) and Lineage 1 (7.2%). The predominant spoligotype families within Lineage 4 were LAM (33%), S (14%), T (16%), X (16%). Three hundred and ninety-two (86%) isolates could be grouped into 44 clusters (2-46 isolates per cluster); giving a clustering rate of 76%. We identified 173 (37.8%) drug resistant isolates, 48 (10.5%) of these were multi-drug resistant. MIRU typing of the drug resistant isolates allowed grouping of 46 isolates into 14 clusters, giving a clustering rate of 49.2%. There was no association between age, sex, treatment category, region and clustering.ConclusionsThis study highlights the complexity of the TB epidemic in Botswana with multiple strains contributing to disease and provides baseline data on the population structure of M.tb strains in Botswana.

BackgroundRoll-out of Integrase Strand Transfer Inhibitors (INSTIs) such as dolutegravir for HIV combination antiretroviral therapy (cART) in sub-Saharan Africa necessitates the development of affordable HIV drug resistance (HIVDR) assays targeting the Integrase gene. We optimised and evaluated an in-house integrase HIV-1 drug resistance assay (IH-Int) and compared it to a commercially available assay, ViroSeq™ Integrase Genotyping kit (VS-Int) amongst HIV-1 clade C infected individuals.MethodsWe used 54 plasma samples from treatment naïve participants and one plasma sample from a patient failing INSTI based cART. Specimens were genotyped using both the VS-Int and IH-Int assays. Stanford HIV drug resistance database were used for integrase resistance interpretation. We compared the major and minor resistance mutations, pairwise nucleotide and amino-acid identity, costs and assay time.ResultsAmong 55 specimens tested with IH-Int, 53 (96.4%) successfully amplified compared to 45/55 (81.8%) for the VS-Int assay. The mean nucleotide and amino acid similarity from 33 paired sequences was 99.8% (SD ± 0.30) and 99.8% (SD ± 0.39) for the IH-Int and VS-Int assay respectively. The reagent cost/sample were 32 USD and 147 USD for IH-Int and VS-Int assay, respectively. All sequenced samples were confirmed as HIV-1 subtype C.ConclusionsThe IH-Int assay had a high amplification success rate and high concordance with the commercial assay. It is significantly cheaper compared to the commercial assay. Our assay has the needed specifications for routine monitoring of participants on Dolutegravir based regimens in Botswana.

There are limited real-world mutational and virological outcomes data of treatment-experienced persons diagnosed with HIV-1 subtype C (HIV-1 C) who are failing Integrase Strand Transfer Inhibitor-based regimens. Requisition forms sent for HIV-1 genotypic resistance testing (GRT) between May 2015 and September 2019 were reviewed and participants experiencing virologic failure while on dolutegravir (DTG) or raltegravir (RAL) cART at sampling recruited. Sanger sequencing of the HIV-1 Pol gene was performed from residual plasma samples and drug resistance mutational (DRM) analysis performed using the Stanford University HIV drug resistance database. 40 HIV-1C integrase sequences were generated from 34 individuals, 24 of whom were on DTG cART, three on RAL cART and seven on an unknown (DTG or RAL)-anchored cART at time of GRT. 11/34 (32%) individuals had DRMs to DTG and other integrase inhibitors. 7/11 (64%) patients had exposure to a RAL-based cART at the time of sampling. Out of the 11 individuals with DRMs, one (9%) had 2-class, 6 (55%) had 3-class, and 4 (36%) had 4-class multidrug-resistant HIV-1C. 7/11 individuals (64%) are currently virologically suppressed. Of the four individuals not virologically suppressed, three had extensive DRMs involving 4-classes of ARV drugs and one individual has demised. Resistance to DTG occurs more often in patients exposed to RAL cART. Individuals with 4-class DRMs plus integrase T97 and E157Q mutations appear to have worse outcomes. There is a need for frequent VL monitoring and GRT amongst treatment-experienced HIV-1C diagnosed individuals.

HIV-1 compartmentalization in reservoir sites remains a barrier to complete HIV eradication. It is unclear whether there is variation in HIV-1 env and gag between cerebrospinal fluid (CSF) and plasma of individuals with HIV-associated cryptococcal meningitis (CM). We compared HIV-1 env characteristics and the gag cytotoxic T-lymphocyte (CTL) escape mutations from CSF and plasma samples. Employing population-based Sanger sequencing, we sequenced HIV-1 env from CSF of 25 patients and plasma of 26 patients. For gag, 15 CSF and 21 plasma samples were successfully sequenced. Of these, 18 and 9 were paired env and gag CSF/plasma samples, respectively. There was no statistically significant difference in the proportion of CCR5-using strains in the CSF and plasma, (p = 0.50). Discordant CSF/plasma virus co-receptor use was found in 2/18 pairs (11.1%). The polymorphisms in the HIV-1 V3 loop were concordant between the two compartments. From the HIV-1 gag sequences, three pairs had discordant CTL escape mutations in three different epitopes of the nine analyzed. These findings suggest little variation in the HIV-1 env between plasma and CSF and that the CCR5-using strains predominate in both compartments. HIV-1 gag CTL escape mutations also displayed little variation in CSF and plasma suggesting similar CTL selective pressure.