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Objective: To identify predictors of HIV acquisition in Botswana., Design: We applied machine learning approaches to identify HIV risk predictors using existing data from a large, well characterized HIV incidence cohort., Methods: We applied machine learning (randomForestSRC) to analyze data from a large population-based HIV incidence cohort enrolled in a cluster-randomized HIV prevention trial in 30 communities across Botswana. We sought to identify the most important risk factors for HIV acquisition, starting with 110 potential predictors., Results: During a median 29-month follow-up of 8551 HIV-negative adults, 147 (1.7%) acquired HIV. Our machine learning analysis found that for females, the most important variables for predicting HIV acquisition were the use of injectable hormonal contraception, frequency of sex in the prior 3 months with the most recent partner and residing in a community with HIV prevalence of 29% or higher. For the small proportion (0.3%) of females who had all three risk factors, their estimated probability of acquiring HIV during 29 months of follow-up was 34% (approximate annual incidence of 14%). For males, nonlong-term relationships with the most recent partner and community HIV prevalence of 34% or higher were the most important HIV risk predictors. The 6% of males who had both risk factors had a 5.1% probability of acquiring HIV during the follow-up period (approximate annual incidence of 2.1%)., Conclusion: Machine learning approaches allowed us to analyze a large number of variables to efficiently identify key factors strongly predictive of HIV risk. These factors could help target HIV prevention interventions in Botswana., Clinical Trials Registration: NCT01965470., (Copyright © 2025 Wolters Kluwer Health, Inc. All rights reserved.)

Background: We 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., Methods: We 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., Results: The 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., Conclusion: The 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Maruapula, Ditshwanelo, Pema, Bareng, Choga, Moraka, Mokgethi, Seatla, Koofhethile, Zuze, Gaolathe, Pretorius-Holme, Lebani, Makhema, Novitsky, Shapiro, Lockman, Moyo and Gaseitsiwe.)

Introduction: Monthly intravenous infusion of broadly neutralizing monoclonal antibodies may be an attractive alternative to daily oral antiretroviral treatment for children living with HIV. However, acceptability among caregivers remains unknown., Methods: We evaluated monthly infusion of dual bNAbs (VRCO1LS and 10-1074) as a treatment alternative to ART among children participating in the Tatelo Study in Botswana. Eligible children aged 2-5 years received 8-32 weeks of bNAbs overlapping with ART, and up to 24 weeks of bNAbs alone as monthly intravenous infusion. Using closed-ended questionnaires, we evaluated caregiver acceptability of each treatment strategy prior to the first bNAb administration visit (pre-intervention) and after the completion of the final bNAb administration visit (post-intervention)., Results: Twenty-five children completed the intervention phase of the study, and acceptability data were available from 24 caregivers at both time points. Responses were provided by the child's mother at both visits (60%), an extended family member at both visits (28%), or a combination of mother and an extended family member (12%). Caregiver acceptance of monthly bNAb infusions was extremely high both pre-and post-intervention, with 21/24 (87.5%) preferring bNAbs to ART pre-intervention, and 21/25 (84%) preferring bNAbs post-intervention. While no caregiver preferred ART pre-intervention, 2/25 preferred it post-intervention. Pre-intervention, 3 (13%) caregivers had no preference between monthly bNAbs or daily ART, and 2 (8%) had no preference post-intervention. Pre-intervention, the most common reasons for preferring bNAbs over ART were the perception that bNAbs were better at suppressing the virus than ART (n = 10) and the fact that infusions were dosed once monthly compared to daily ART (n = 9). Post-intervention, no dominant reason for preferring bNAbs over ART emerged from caregivers., Conclusions: Monthly intravenous bNAb infusions were highly acceptable to caregivers of children with HIV in Botswana and preferred over standard ART by the majority of caregivers., Clinical Trial Number: NCT03707977., Competing Interests: The authors have declared that no competing interest exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Objectives: We sought to determine hepatitis B surface antigen (HBsAg) loss and its predictors among people with chronic hepatitis B (CHB) infections and HIV (PWH) in Botswana., Methods: Archived plasma samples from a cohort of PWH in Botswana (2013-2018) with 3 yearly time-points were used. Samples were screened for HBsAg, immunoglobulin M HBV core antibodies (anti-HBc IgM) and HBV e-antigen (HBeAg) at all time points. HBV deoxyribonucleic acid (DNA) quantification was done at baseline. The Wilcoxon rank-sum was used to compare continuous variables while the chi-squared test and Fishers exact test were used for categorical data wherever appropriate. Logistic regression was used to assess predictors of seroclearance., Results: Of 141 participants with HBsAg-positive serology (HBsAg+) at baseline, 92.2% (131/141) [95% confidence interval (CI) 87.4-96.1] were persistently HBsAg+ at year 1. We report a HBsAg loss of 7.1% (10/141) (95% CI 3.9-12.6) among participants with negative HBeAg and negative IgM serologies. HBsAg loss was 6.3% (7/111) among antiretroviral therapy (ART)-experienced participants and 10.7% (3/28) (95% CI 0.4-5.0) in ART-naive participants. Most participants who had positive anti-HBc IgM serology and did not lose HBsAg were on either lamivudine (3TC)-based therapy or non-tenofovir disoproxil fumarate (TDF)-based therapy, except for one participant. The participants also had varying HBeAg status. HBsAg loss was independent of HIV viral load, CD4 + cell count, age, and sex., Conclusion: We report a HBsAg loss of 6.3% over a 3-year period among ART-experienced CHB participants. Future studies that focus on HBsAg loss in mono-infected patients and the possible correlation between HBeAg status and HBsAg loss are warranted., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

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., Competing Interests: The authors declare no conflict of interest.

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 < 0.05 were considered statistically significant., Results: The study cohort (n = 478) included both HBsAg positive (44 %) and negative (56 %) participants, with a median age of 42 [IQR; 41-43]. Anti-HDV prevalence of (15/211) [7.1 %, 95 % CI: 4.4 - 11.4] was recorded among HBsAg positive participants, all of whom were IgM anti-HBc negative, while 5/6 participants were HBeAg negative. HDV RNA load was detected in 11/12 (92 %) anti-HDV-positive participants. No HDV prevalence was recorded among participants who were HBsAg negative, therefore, the overall HDV prevalence was (15/478) [3.1 %, 95 % CI: 1.9 - 5.1]. HIV viral load suppression was statistically insignificant, irrespective of HDV status., Conclusions: We report high HDV prevalence among HBsAg-positive PWH in Botswana. Most HDV-positive participants had active HDV infection, therefore, we recommend HDV screening in this cohort to guide their clinical care., Competing Interests: Declaration of Competing Interest We have no conflict of interest to declare., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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., (© 2023. Springer Nature Limited.)

Objectives: Pre-existing rilpivirine resistance-associated mutations (RVP-RAMs) have been found to predict HIV-1 virological failure in those switching to long-acting injectable cabotegravir/rilpivirine. We here evaluated the prevalence of archived RPV-RAMs in a cohort of people living with HIV (PWH)., Methods: We analysed near full-length HIV-1 pol sequences from proviral DNA for the presence of RPV-RAMs, which were defined according to the 2022 IAS-USA drug resistance mutation list and Stanford HIV drug resistance database., Results: RPV-RAMs were identified in 757/5805 sequences, giving a prevalence of 13.0% (95% CI 12%-13.9%). Amongst the ART-naive group, 137/1281 (10.7%, 95% CI 9.1%-12.5%) had at least one RPV-RAM. Of the 4524 PWH with viral suppression on ART (VL <400 copies/mL), 620 (13.7%, 95% CI 12.7%-14.7%) had at least one RPV-RAM. E138A was the most prevalent RPV-RAM in the ART-naive group (7.9%) and the ART-suppressed group (9.3%). The rest of the mutations observed (L100I, K101E, E138G, E138K, E138Q, Y181C, H221Y, M230L, A98G, V179D, G190A, G190E and M230I) were below a prevalence of 1%., Conclusions: RPV-RAMs were present in 10.7% of ART-naive and 13.7% of ART-suppressed PWH in Botswana. The most common RPV-RAM in both groups was E138A. Since individuals with the E138A mutation may be more likely to fail cabotegravir/rilpivirine, monitoring RPV-RAMs will be crucial for effective cabotegravir/rilpivirine implementation in this setting., (© The Author(s) 2023. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

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., Competing Interests: Conflict of Interest: None declared

Broadly neutralizing antibodies (bNAbs) may provide an alternative to standard antiretroviral treatment (ART) for controlling HIV-1 replication and may have immunotherapeutic effects against HIV-1 reservoirs. We conducted a prospective clinical trial with two HIV-1 bNAbs (VRC01LS and 10-1074) in children ( n = 25) who had previously initiated small-molecule ART treatment before 7 days of age and who continued treatment for at least 96 weeks. Both bNAbs were dosed intravenously every 4 weeks, overlapping with ART for at least 8 weeks and then continued for up to 24 weeks or until detectable viremia of HIV-1 RNA rose above 400 copies per milliliter in the absence of ART. Eleven (44%) children maintained HIV-1 RNA below 400 copies per milliliter through 24 weeks of bNAb-only treatment; 14 (56%) had detectable viremia above 400 copies per milliliter at a median of 4 weeks. Archived HIV-1 provirus susceptible to 10-1074, lower birth HIV-1 DNA reservoir in peripheral blood mononuclear cells, sustained viral suppression throughout early life, and combined negative qualitative HIV-1 DNA polymerase chain reaction and negative HIV-1 serology at entry were associated with maintaining suppression on bNAbs alone. This proof-of-concept study suggests that bNAbs may represent a promising treatment modality for infants and children living with HIV-1. Future studies using newer bNAb combinations with greater breadth and potency are warranted.

Background: Early antiretroviral treatment (ART) improves outcomes in children, but few studies have comprehensively evaluated the impact of ART started from the first week of life., Methods: Children diagnosed with HIV within 96 hours of life were enrolled into the Early Infant Treatment Study in Botswana and followed on ART for 96 weeks. Nevirapine, zidovudine, and lamivudine were initiated; nevirapine was switched to lopinavir/ritonavir between weeks 2-5 in accordance with gestational age. Clinical and laboratory evaluations occurred at weeks 1, 2, 4, 8, 12, 24, 36, 48, 60, 72, 84, and 96., Findings: Forty children initiated ART at a median of 2 (IQR 2, 3) days of life; 38 (95%) completed follow-up through 96 weeks, and 2 (5%) died between 12 and 24 weeks. ART was well tolerated; 9 children (24%) experienced a grade 3 or 4 hematologic event, and 2 (5%) required treatment modification for anemia. The median 96-week CD4 count was 1625 (IQR 1179, 2493) cells/mm 3 with only 5/38 (13%) having absolute counts <1000 cells/mm 3 . Although 23 (61%) had at least one visit with HIV-1 RNA ≥40 copies/mL at or after 24 weeks, 28 (74%) had HIV-1 RNA <40 copies/mL at the 96-week visit. Median cell-associated HIV-1 DNA at 84/96-week PBMCs was 1.9 (IQR 1.0, 2.6) log 10 copies/10 6 cells. Pre-ART reservoir size at birth was predictive of the viral reservoir at 84/96 weeks., Interpretation: Initiation of ART in the first week of life led to favorable clinical outcomes, preserved CD4 cell counts, and low viral reservoir through 96 weeks of life., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Background: We aimed to determine the prevalence of hepatitis B virus (HBV) infection among people with human immunodeficiency virus (PWH) in rural and periurban communities in Botswana., Methods: PWH from a previous population-based study, the Botswana Prevention Combination Project, which enrolled adults in 30 communities across Botswana (2013-2018), were screened for HBV surface antigen (HBsAg) and HBV core antibody (anti-HBc). HBsAg-positive (HBsAg + ) samples were further screened for HBV core immunoglobulin M antibodies (anti-HBc immunoglobulin M [IgM]) and HBV e antigen (HBeAg). We quantified HBV viral load on participants who tested positive (n = 148) and negative for HBsAg (n = 381)., Results: Of 3304 participants tested, 271 (8% [95% confidence interval {CI}, 7%-9%]) were HBsAg + while 1788 (56% [95% CI, 54%-57%]) of 3218 PWH whom we tested had positive anti-HBc. Approximately 88% of HBsAg + participants were on antiretroviral therapy (ART), 40% and 56% of whom were receiving lamivudine- and tenofovir-containing ART, respectively. Male sex (relative risk ratio [RRR], 1.8 [95% CI, 1.2-2.7]) and the northern geographic region (RRR, 2.5 [95% CI, 1.4-4.7]) were independent predictors of HBV infection (HBsAg + ). Of 381 persons with negative HBsAg who were tested for occult HBV, 126 (33% [95% CI, 29%-38%]) had positive HBV DNA. Eleven participants were highly viremic with high HBV viral load while on a lamivudine- or tenofovir-containing regimen. Ten (91%) of these participants also had positive HBeAg serology, while 4 (36%) had positive anti-HBc IgM serology., Conclusions: The prevalence of HBV was high among PWH in Botswana while on ART regimens with activity against HBV., Competing Interests: Potential conflicts of interest. The authors: No reported conflicts of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

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., Competing Interests: Declaration of Competing Interest None declared., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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 (< 1 year) or chronic (≥ 1 year)., Results: Being male (aOR = 2.23) and younger than 35 years old (aOR = 8.08) was associated with undiagnosed HIV infection (p < 0.001), as was inconsistent condom use (aOR = 1.76). Women were more likely to have undiagnosed infections if they were married, educated, and tested frequently. For men, being divorced increased their risk. The genetic diversity-based algorithm classified most incident infections as recent (75.0%), but almost none of known infections (2.0%). The estimated proportion of recent infections among new diagnoses was 37.0% (p < 0.001)., Conclusion: Our results indicate that those with undiagnosed infections are likely to be young men and women who do not use condoms consistently. Among women, several factors were predictive: being married, educated, and testing frequently increased risk. Men at risk were more difficult to delineate. A sizeable proportion of undiagnosed infections were recent based on a genetic diversity-based classifier. In the era of "test and treat all", pre-exposure prophylaxis may be prioritized towards individuals who self-identify or who can be identified using these predictors in order to halt onward transmission in time., (© 2022. The Author(s).)

Objectives: To assess whether a single instance of low-level viraemia (LLV) is associated with the presence of drug resistance mutations (DRMs) and predicts subsequent virological failure (VF) in adults receiving ART in 30 communities participating in the Botswana Combination Prevention Project., Methods: A total of 6078 HIV-1 C pol sequences were generated and analysed using the Stanford HIV drug resistance database. LLV was defined as plasma VL = 51-999 copies/mL and VF was defined as plasma VL ≥ 1000 copies/mL., Results: Among 6078 people with HIV (PWH), 4443 (73%) were on ART for at least 6 months. Of the 332 persons on ART with VL > 50 copies/mL, 175 (4%) had VL ≥ 1000 copies/mL and 157 (4%) had LLV at baseline. The prevalence of any DRM was 57 (36%) and 78 (45%) in persons with LLV and VL ≥ 1000 copies/mL, respectively. Major DRMs were found in 31 (20%) with LLV and 53 (30%) with VL ≥ 1000 copies/mL (P = 0.04). Among the 135 PWH with at least one DRM, 17% had NRTI-, 35% NNRTI-, 6% PI- and 3% INSTI-associated mutations. Among the 3596 participants who were followed up, 1709 (48%) were on ART for ≥6 months at entry and had at least one subsequent VL measurement (median 29 months), 43 (3%) of whom had LLV. The OR of experiencing VF in persons with LLV at entry was 36-fold higher than in the virally suppressed group., Conclusions: A single LLV measurement while on ART strongly predicted the risk of future VF, suggesting the use of VL > 50 copies/mL as an indication for more intensive adherence support with more frequent VL monitoring., (© The Author(s) 2022. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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)., Competing Interests: LM, YZ, TG, VD, ET, VN, JM, PB, TS, RL, MP, SM, JM, CF, ME No competing interests declared, SL participates in a data safety monitoring board for NIH-funded study of PK of TB drugs and antiretrovirals in children and on a scientific advisory board for observational study of DTG programmatic rollout in Botswana. Is also a member of the Finance Board and a member of the Board of Directors for the Botswana Harvard AIDS Institute Partnership. Receives no financial compensation for these roles, and has no other competing interests to declare, ML is a Reviewing Editor for eLife. Has received consultancy fees from Merck, University of Virginia Miller Center and Janssen, and has performed unpaid consultancy work for Janssen, Pfizer and Astra Zeneca. Has also received payments or honoraria from Sanofi Pasteur and Bristol Myers Squibb. ML has no other competing interests to declare

Objective: To describe the occurrence of HIV drug resistance mutations (DRMs) in both intact and defective HIV-1 cell-associated DNA (HIV-1 CAD) among early-treated infants., Design: The Botswana EIT Study (ClinicalTrials.gov NCT02369406) initiated antiretroviral therapy (ART) in the first week of life and evaluated HIV-1 in plasma and peripheral blood mononuclear cells (PBMCs)., Methodology: We analyzed 257 near-HIV-1 full-length sequences (nFLS) obtained by Illumina next-generation sequencing from infants near birth. Sanger sequencing of pol was performed for mothers at delivery and children with clinical failure through 96 weeks. DRMs were identified using the Stanford HIV Drug Resistance Database., Results: In 27 infants, median PBMC HIV-1 proviral load was 492 copies/ml [IQR: 78-1246 copies/ml] at a median of 2 days (range 1-32); 18 (66.7%) had no DRMs detected; six (22.2%) had DRMs detected in defective DNA only, and three (11.1%) had DRMs in both defective and intact DNA (P = 0.09). A total of 60 of 151 (37.7%) defective sequences had at least one DRM: 31.8% NNRTI, 15.2% NRTI, 5.3% protease inhibitor, and 15.5% INSTI-associated mutations. In intact sequences, 33 of 106 (31.1%) had at least 1 DRM: 29.2% NNRTI, 7.5% NRTI, 0.9% protease inhibitor, and no INSTI-associated mutations. For all three infants with intact sequence DRMs, corresponding DRMs occurred in maternal plasma at delivery. Archived DRMs were detectable at a later clinical rebound on only one occasion., Conclusion: Defective HIV-1 cell-associated DNA sequences may overestimate the prevalence of drug resistance among early-treated children. The impact of DRMs from intact proviruses on long-term treatment outcomes warrants further investigation., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc.)

Introduction: The scale-up of Universal Test and Treat has resulted in reductions in HIV morbidity, mortality and incidence. However, healthcare system and personal challenges have impacted the levels of treatment coverage achieved. We implemented interventions to improve linkage to care, retention, viral load (VL) coverage and service delivery, and describe the HIV care cascade over the course of the Botswana Combination Prevention Project (BCPP) study., Methods: BCPP was designed to evaluate the impact of prevention interventions on HIV incidence in 30 communities in Botswana. We followed a longitudinal cohort of newly identified and known HIV-positive persons not on antiretroviral therapy (ART) identified through community-based testing activities through BCPP and referred with appointments to local HIV clinics in 15 intervention communities. Those who did not keep the first or follow-up appointments were tracked and traced through phone and home contacts. Improvements to service delivery models in the intervention clinics were also implemented., Results: A total of 3,657 newly identified or HIV-positive persons not on ART were identified and referred to their local HIV clinic; 90% (3,282/3,657) linked to care and of those, 93% (3,066/3,282) initiated treatment. Near the end of the study, 221 persons remained >90 days late for appointments or missing. Tracing efforts identified 54/3,066 (2%) persons who initiated treatment but died, and 106/3,066 (3%) persons were located and returned to treatment. At study end, 61/3,066 (2%) persons remained missing and were never reached. Overall, 2,951 (98%) persons living with HIV (PLHIV) who initiated treatment were still alive, retained in care and still receiving ART out of the 3,001 persons alive at the end of the study. Of those on ART, 2,854 (97%) had current VL results and 2,784 (98%) of those were virally suppressed at study end., Conclusions: This study achieved high rates of linkage, treatment initiation, retention and VL coverage and suppression in a cohort of newly identified and known PLHIV not on ART. Tracking and tracing interventions effectively identified those persons who needed more resource intensive follow-up. The interventions implemented to improve service delivery and data quality may have also contributed to high linkage and retention rates. Clinical trial number: NCT01965470., Competing Interests: Authors Lisa Block, Gene Ussery, and Huisheng Wang were employed by Northrup Grumman. There are no patents, products in development or marketed products to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: Phylogenetic mapping of HIV-1 lineages circulating across defined geographical locations is promising for better understanding HIV transmission networks to design optimal prevention interventions., Methods: We obtained near full-length HIV-1 genome sequences from people living with HIV (PLWH), including participants on antiretroviral treatment in the Botswana Combination Prevention Project, conducted in 30 Botswana communities in 2013-2018. Phylogenetic relationships among viral sequences were estimated by maximum likelihood., Results: We obtained 6078 near full-length HIV-1C genome sequences from 6075 PLWH. We identified 984 phylogenetically distinct HIV-1 lineages (molecular HIV clusters) circulating in Botswana by mid-2018, with 2-27 members per cluster. Of these, dyads accounted for 62%, approximately 32% (n = 316) were found in single communities, and 68% (n = 668) were spread across multiple communities. Men in clusters were approximately 3 years older than women (median age 42 years, vs 39 years; P < .0001). In 65% of clusters, men were older than women, while in 35% of clusters women were older than men. The majority of identified viral lineages were spread across multiple communities., Conclusions: A large number of circulating phylogenetically distinct HIV-1C lineages (molecular HIV clusters) suggests highly diversified HIV transmission networks across Botswana communities by 2018., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Background: In settings with high HIV prevalence and treatment coverage, such as Botswana, it is unknown whether uptake of HIV prevention and treatment interventions can be increased further. We sought to determine whether a community-based intervention to identify and rapidly treat people living with HIV, and support male circumcision could increase population levels of HIV diagnosis, treatment, viral suppression, and male circumcision in Botswana., Methods: The Ya Tsie Botswana Combination Prevention Project study was a pair-matched cluster-randomised trial done in 30 communities across Botswana done from Oct 30, 2013, to June 30, 2018. 15 communities were randomly assigned to receive HIV prevention and treatment interventions, including enhanced HIV testing, earlier antiretroviral therapy (ART), and strengthened male circumcision services, and 15 received standard of care. The first primary endpoint of HIV incidence has already been reported. In this Article, we report findings for the second primary endpoint of population uptake of HIV prevention services, as measured by proportion of people known to be HIV-positive or tested HIV-negative in the preceding 12 months; proportion of people living with HIV diagnosed and on ART; proportion of people living with HIV on ART with viral suppression; and proportion of HIV-negative men circumcised. A longitudinal cohort of residents aged 16-64 years from a random, approximately 20% sample of households across the 15 communities was enrolled to assess baseline uptake of study outcomes; we also administered an end-of-study survey to all residents not previously enrolled in the longitudinal cohort to provide study end coverage estimates. Differences in intervention uptake over time by randomisation group were tested via paired Student's t test. The study has been completed and is registered with ClinicalTrials.gov (NCT01965470)., Findings: In the six communities participating in the end-of-study survey, 2625 residents (n=1304 from standard-of-care communities, n=1321 from intervention communities) were enrolled into the 20% longitudinal cohort at baseline from Oct 30, 2013, to Nov 24, 2015. In the same communities, 10 791 (86%) of 12 489 eligible enumerated residents not previously enrolled in the longitudinal cohort participated in the end-of-study survey from March 30, 2017, to Feb 25, 2018 (5896 in intervention and 4895 in standard-of-care communities). At study end, in intervention communities, 1228 people living with HIV (91% of 1353) were on ART; 1166 people living with HIV (88% of 1321 with available viral load) were virally suppressed, and 673 HIV-negative men (40% of 1673) were circumcised in intervention communities. After accounting for baseline differences, at study end the proportion of people living with HIV who were diagnosed was significantly higher in intervention communities (absolute increase of 9% to 93%) compared with standard-of-care communities (absolute increase of 2% to 88%; prevalence ratio [PR] 1·08 [95% CI 1·02-1·14], p=0·032). Population levels of ART, viral suppression, and male circumcision increased from baseline in both groups, with greater increases in intervention communities (ART PR 1·12 [95% CI 1·07-1·17], p=0·018; viral suppression 1·13 [1·09-1·17], p=0·017; male circumcision 1·26 [1·17-1·35], p=0·029)., Interpretation: It is possible to achieve very high population levels of HIV testing and treatment in a high-prevalence setting. Maintaining these coverage levels over the next decade could substantially reduce HIV transmission and potentially eliminate the epidemic in these areas., Funding: US President's Emergency Plan for AIDS Relief through the Centers for Disease Control and Prevention., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: The feasibility of reducing the population-level incidence of human immunodeficiency virus (HIV) infection by increasing community coverage of antiretroviral therapy (ART) and male circumcision is unknown., Methods: We conducted a pair-matched, community-randomized trial in 30 rural or periurban communities in Botswana from 2013 to 2018. Participants in 15 villages in the intervention group received HIV testing and counseling, linkage to care, ART (started at a higher CD4 count than in standard care), and increased access to male circumcision services. The standard-care group also consisted of 15 villages. Universal ART became available in both groups in mid-2016. We enrolled a random sample of participants from approximately 20% of households in each community and measured the incidence of HIV infection through testing performed approximately once per year. The prespecified primary analysis was a permutation test of HIV incidence ratios. Pair-stratified Cox models were used to calculate 95% confidence intervals., Results: Of 12,610 enrollees (81% of eligible household members), 29% were HIV-positive. Of the 8974 HIV-negative persons (4487 per group), 95% were retested for HIV infection over a median of 29 months. A total of 57 participants in the intervention group and 90 participants in the standard-care group acquired HIV infection (annualized HIV incidence, 0.59% and 0.92%, respectively). The unadjusted HIV incidence ratio in the intervention group as compared with the standard-care group was 0.69 (P = 0.09) by permutation test (95% confidence interval [CI], 0.46 to 0.90 by pair-stratified Cox model). An end-of-trial survey in six communities (three per group) showed a significantly greater increase in the percentage of HIV-positive participants with an HIV-1 RNA level of 400 copies per milliliter or less in the intervention group (18 percentage points, from 70% to 88%) than in the standard-care group (8 percentage points, from 75% to 83%) (relative risk, 1.12; 95% CI, 1.09 to 1.16). The percentage of men who underwent circumcision increased by 10 percentage points in the intervention group and 2 percentage points in the standard-care group (relative risk, 1.26; 95% CI, 1.17 to 1.35)., Conclusions: Expanded HIV testing, linkage to care, and ART coverage were associated with increased population viral suppression. (Funded by the President's Emergency Plan for AIDS Relief and others; Ya Tsie ClinicalTrials.gov number, NCT01965470.)., (Copyright © 2019 Massachusetts Medical Society.)

Background: Adolescents and young adults account for more than one-third of incident Human Immunodeficiency Virus (HIV) infections globally. Understanding sexual practices of this high-risk group is critical in designing HIV targeted prevention programming., Objectives: To describe self-reported risky sexual practices of adolescents and young adults aged 16-24 years from 30 Botswana communities., Methods: Cross-sectional, self-reported age at sexual debut; number of sexual partners; condom and alcohol use during sex; intergenerational sex; and transactional sex data were collected. Modified Poisson estimating equations were used to obtain univariate and multivariate-adjusted prevalence ratios (PR) and 95% confidence intervals (CI) comparing engagement in different sexual practices according to gender, accounting for the clustered design of the study., Results: Among the 3380 participants, 2311 reported being sexually active with more females reporting being sexually active compared to males (65% vs. 35%, respectively; p < 0.0001). In univariate analyses, female participants were more likely to report inconsistent condom use (PR 1.61; 95% CI 1.44-1.80), intergenerational sex (PR 9.00; 95% CI 5.84-13.88) and transactional sex (PR 3.46; 95% CI 2.07-5.77) than males, yet less likely to report engaging in sex before age 15 years (PR 0.59; 95% CI: 0.41-0.85), using alcohol around the time of intercourse (PR: 0.59; 95% CI 0.45-0.76) or having ≥ two partners in the last 12 months (PR 0.65; 95% CI 0.57-0.74)., Conclusions: Self-reported risky sexual practices of adolescents and young adults in Botswana differed significantly between males and females. Gender-specific risky sexual practices highlight the importance of developing tailored HIV prevention programming., Competing Interests: The authors declare that this article is not under publication consideration elsewhere and that they have no financial or personal relationships that may have inappropriately influenced them in writing this article.

Background: Scale-up of antiretroviral therapy (ART) and introduction of treat-all strategy necessitates population-level monitoring of acquired HIV drug resistance (ADR) and pretreatment drug resistance (PDR) mutations., Methods: Blood samples were collected from 4973 HIV-positive individuals residing in 30 communities across Botswana who participated in the Botswana Combination Prevention Project (BCPP) in 2013-2018. HIV sequences were obtained by long-range HIV genotyping. Major drug-resistance mutations (DRMs) and surveillance drug resistance mutations (SDRMs) associated with nucleoside reverse transcriptase inhibitors (NRTI) and nonnucleoside reverse transcriptase inhibitors (NNRTI) were analyzed according to the Stanford University HIV Drug Resistance Database. Viral sequences were screened for G-to-A hypermutations. A threshold of 2% was used for hypermutation adjustment. Viral suppression was considered at HIV-1 RNA load ≤400 copies/ml., Results: Among 4973 participants with HIV-1C sequences, ART data were available for 4927 (99%) including 3858 (78%) on ART. Among those on ART, 3435 had viral load data and 3297 (96%) were virologically suppressed. Among 1069 (22%) HIV-infected individuals not on ART, we found NRTI-associated and NNRTI-associated SDRMs were found in 1.5% (95% confidence interval [CI] 1.0-2.5%) and 2.9% (95% CI 2.0-4.2%), respectively. Of the 138 (4%) of individuals who had detectable HIV-1 RNA, we found NRTI-associated and NNRTI-associated drug resistance mutations in 16% (95% CI 10-25%) and 33% (95% CI 25-42%), respectively., Conclusion: We found a low prevalence of NRTI-associated and NNRTI-associated PDR-resistance mutations among residents of rural and peri-urban communities across Botswana. However, individuals on ART with detectable virus had ADR NRTI and NNRTI mutations above 15%.

Background: Botswana is close to reaching the UNAIDS "90-90-90" HIV testing, antiretroviral treatment (ART), and viral suppression goals. We sought to determine HIV incidence in this setting with both high HIV prevalence and high ART coverage., Methods: We used a cross-sectional approach to assessing HIV incidence. A random, population-based sample of adults age 16-64 years was enrolled in 30 rural and peri-urban communities as part of the Botswana Combination Prevention Project (BCPP), from October 2013 -November 2015. Data and samples from the baseline household survey were used to estimate cross-sectional HIV incidence, following an algorithm that combined Limiting-Antigen Avidity Assay (LAg-Avidity EIA), ART status (documented or by testing ARV drugs in plasma) and HIV-1 RNA load. The LAg-Avidity EIA cut-off normalized optical density (ODn) was set at 1.5. The HIV-1 RNA cut-off was set at 400 copies/mL. For estimation purposes, the Mean Duration of Recent Infection was 130 days and the False Recent Rate (FRR) was evaluated at values of either 0 or 0.39%., Results: Among 12,610 individuals participating in the baseline household survey, HIV status was available for 12,570 participants and 3,596 of them were HIV positive. LAg-Avidity EIA data was generated for 3,581 (99.6%) of HIV-positive participants. Of 326 participants with ODn ≤1.5, 278 individuals were receiving ART verified through documentation and were considered to represent longstanding HIV infections. Among the remaining 48 participants who reported no use of ART, 14 had an HIV-1 RNA load ≤400 copies/mL (including 3 participants with ARVs in plasma) and were excluded, as potential elite/viremic controllers or undisclosed ART. Thus, 34 LAg-Avidity-EIA-recent, ARV-naïve individuals with detectable HIV-1 RNA (>400 copies/mL) were classified as individuals with recent HIV infections. The annualized HIV incidence among 16-64 year old adults was estimated at 1.06% (95% CI 0.68-1.45%) with zero FRR, and at 0.64% (95% CI 0.24-1.04%) using a previously defined FRR of 0.39%. Within a subset of younger individuals 16-49 years old, the annualized HIV incidence was estimated at 1.29% (95% CI 0.82-1.77%) with zero FRR, and at 0.90% (95% CI 0.42-1.38%) with FRR set to 0.39%., Conclusions: Using a cross-sectional estimate of HIV incidence from 2013-2015, we found that at the time of near achievement of the UNAIDS 90-90-90 targets, ~1% of adults (age 16-64 years) in Botswana's rural and peri-urban communities became HIV infected annually., Competing Interests: Kara Bennett is employed by Bennett Statistical Consulting, Inc and Jean Leidner was employed by Goodtables Data Consulting. The entities above provided support in the form of salaries for authors Kara Bennett and Jean Leidner, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

: Among 3596 HIV-positive participants enrolled in the Botswana Combination Prevention Project who self-reported no prior antiretroviral (ARV) therapy use and were tested for viral load (n = 951; 27% of all participants), 136 (14%) had HIV-1 RNA less than 400 copies/ml. ARV drugs were detected in 52 (39%) of 134 participants tested. Adjusting for undisclosed ARV use increased the overall estimate of virally suppressed individuals on ARV therapy by 1.4% from 70.2 to 71.6%.