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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.

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.)

Immune mechanisms that modulate human immunodeficiency virus-1 (HIV-1) reservoir size in neonates are poorly understood. Using samples from neonates who initiated antiretroviral therapy shortly after birth, we demonstrate that interleukin-8-secreting CD4 T cells, which are selectively expanded in early infancy, are more resistant to HIV-1 infection and inversely correlated with the frequency of intact proviruses at birth. Moreover, newborns with HIV-1 infection displayed a distinct B-cell profile at birth, with reduction of memory B cells and expansion of plasmablasts and transitional B cells; however, B-cell immune perturbations were unrelated to HIV-1 reservoir size and normalized after initiation of antiretroviral therapy. Clinical Trials Registration. NCT02369406., Competing Interests: Potential conflicts of interest. D. R. K. has received consulting honoraria and/or research support from Gilead, Merck, and ViiV. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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.)

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 χ 2 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 (χ 2 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.

Objectives: We compared the patterns of HIV-1 drug resistance mutations between the CSF and plasma of individuals with HIV-associated cryptococcal meningitis., Methods: This is a cross-sectional study of archived CSF and plasma samples collected from ART-exposed participants recruited in the Phase 3 AmBisome Therapy Induction Optimisation randomized controlled trial (ISRCTN72509687) conducted in Botswana between 2018 and 2021. HIV-1 RT and protease genes were genotyped using next-generation sequencing and HIV-1 drug resistance mutations were compared between the CSF and plasma compartments stratified by thresholds of ≥20% and <20%., Results: Overall, 66.7% (16/24) of participants had at least one HIV-1 drug resistance mutation in the CSF and/or plasma. A total of 15/22 (68.2%) participants had HIV-1 drug resistance mutations at ≥20% threshold in the plasma and of those, 11 (73.3%) had been on ART longer than 6 months. HIV-1 drug resistance mutations were highly concordant between the CSF and plasma at ≥20% threshold despite a substantial number of individuals experiencing CSF viral escape and with only 54.5% with CSF WBC count ≥20 cells/mm3. Minority HIV-1 drug resistance mutations were detected in 20.8% (5/24) of participants. There were no mutations in the CSF that were not detected in the plasma., Conclusions: There was high concordance in HIV-1 drug resistance mutations in the CSF and plasma, suggesting intercompartmental mixing and possibly a lack of compartmentalization. Some individuals harboured minority HIV-1 drug resistance mutations, demonstrating the need to employ more sensitive genotyping methods such as next-generation sequencing for the detection of low-abundance mutations., (© 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.)

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: Broadly neutralizing monoclonal antibodies (bNAbs) suppress HIV-1 RNA and may deplete residual viral reservoirs. We evaluated the safety and pharmacokinetics (PK) of dual intravenous VRC01LS and 10-1074 in very early-treated children with HIV-1 on suppressive antiretroviral treatment (ART)., Setting: Botswana., Methods: Children with HIV-1 (median age 3.1 years) on ART from <7 days old were enrolled. In phase A, 6 children received 10-1074 (30 mg/kg at day 0, 28, and 56) and 6 children received VRC01LS (30 mg/kg at day 0, 10 mg/kg at days 28 and 56) by intravenous infusion. In phase B, 6 children received the 2 bNAbs combined (with higher VRC01LS maintenance dose, 15 mg/kg) every 4 weeks for 32 weeks with PK evaluations over 8 weeks. Population PK models were developed to predict steady-state concentrations., Results: BNAb infusions were well tolerated. There were no infusion reactions nor any bNAb-related grade 3 or 4 events. The median (range) first dose Cmax and trough (day 28) combined from both phases were 1405 (876-1999) μg/mL and 133 (84-319) μg/mL for 10-1074 and 776 (559-846) μg/mL and 230 (158-294) μg/mL for VRC01LS. No large differences in bNAb clearances were observed when given in combination. The estimated VRC01LS half-life was shorter than in adults. Predicted steady-state troughs [median (90% prediction interval)] were 261 (95-565) and 266 (191-366) μg/mL for 10-1074 and VRC01LS, respectively, when given in combination., Conclusions: 10-1074 and VRC01LS were safe and well-tolerated among children receiving ART. Troughs exceeded minimal targets with every 4-week administration of 10-1074 at 30 mg/kg and VRC01LS at 15 mg/kg., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2022 Wolters Kluwer Health, Inc. 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).)

Initiation of antiretroviral therapy (ART) in infected neonates within hours after birth limits viral reservoir seeding but does not prevent long-term HIV-1 persistence. Here, we report parallel assessments of HIV-1 reservoir cells and innate antiviral immune responses in a unique cohort of 37 infected neonates from Botswana who started ART extremely early, frequently within hours after birth. Decline of genome-intact HIV-1 proviruses occurs rapidly after initiation of ART and is associated with an increase in natural killer (NK) cell populations expressing the cytotoxicity marker CD57 and with a decrease in NK cell subsets expressing the inhibitory marker NKG2A. Immune perturbations in innate lymphoid cells, myeloid dendritic cells, and monocytes detected at birth normalize after rapid institution of antiretroviral therapy but do not notably influence HIV-1 reservoir cell dynamics. These results suggest that HIV-1 reservoir cell seeding and evolution in early-treated neonates is markedly influenced by antiviral NK cell immune responses., Competing Interests: Declaration of interests D.R.K. has received consulting honoraria and/or research support from Gilead, Merck, and ViiV. M.L. has received speaking and consulting honoraria from Merck., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Individuals living with human immunodeficiency virus (HIV) who experience virological failure (VF) after combination antiretroviral therapy (cART) initiation may have had low-frequency drug resistance mutations (DRMs) at cART initiation. There are no data on low-frequency DRMs among cART-naïve HIV-positive individuals in Botswana., Methods: We evaluated the prevalence of low-frequency DRMs among cART-naïve individuals previously sequenced using Sanger sequencing. The generated pol amplicons were sequenced by next-generation sequencing., Results: We observed low-frequency DRMs (detected at <20% in 33/103 (32%) of the successfully sequenced individuals, of whom four also had mutations detected at >20%. K65R was the most common low-frequency DRM detected in 8 individuals. Eighty-two of the 103 individuals had follow-up viral load data while on cART. Twenty-seven of the 82 individuals harbored low-frequency DRMs. Only 12 of 82 individuals experienced VF. The following low-frequency DRMs were observed in four individuals experiencing VF: K65R, K103N, V108I, and Y188C. No statistically significant difference was observed in the prevalence of low-frequency DRMs between individuals experiencing VF (4/12) and those not experiencing VF (23/70) (P = .97). However, individuals with non-nucleoside reverse transcriptase inhibitors-associated low-frequency DRMs were 2.68 times more likely to experience VF (odds ratio, 2.68; 95% confidential interval, 0.4-13.9) compared with those without (P = .22)., Conclusion: Next-generation sequencing was able to detect low-frequency DRMs in this cohort in Botswana, but these DRMs did not contribute significantly to VF., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2022 the Author(s). Published by Wolters Kluwer Health, Inc.)

Background: Oral pre-exposure prophylaxis has been introduced in more than 70 countries, including many in sub-Saharan Africa, but women experience considerable barriers to daily pill-taking, such as stigma, judgement, and the fear of violence. Safe and effective long-acting agents for HIV prevention are needed for women. We aimed to evaluate the safety and efficacy of injectable cabotegravir compared with daily oral tenofovir diphosphate plus emtricitabine (TDF-FTC) for HIV prevention in HIV-uninfected women., Methods: HPTN 084 was a phase 3, randomised, double-blind, double-dummy, active-controlled, superiority trial in 20 clinical research sites in seven countries in sub-Saharan Africa. Participants were eligible for enrolment if they were assigned female sex at birth, were aged 18-45 years, reported at least two episodes of vaginal intercourse in the previous 30 days, were at risk of HIV infection based on an HIV risk score, and agreed to use a long-acting reversible contraceptive method. Participants were randomly assigned (1:1) to either active cabotegravir with TDF-FTC placebo (cabotegravir group) or active TDF-FTC with cabotegravir placebo (TDF-FTC group). Study staff and participants were masked to study group allocation, with the exception of the site pharmacist who was responsible for study product preparation. Participants were prescribed 5 weeks of daily oral product followed by intramuscular injections every 8 weeks after an initial 4-week interval load, alongside daily oral pills. Participants who discontinued injections were offered open-label daily TDF-FTC for 48 weeks. The primary endpoints of the study were incident HIV infection in the intention-to-treat population, and clinical and laboratory events that were grade 2 or higher in all women who had received at least one dose of study product. This study is registered with ClinicalTrials.gov, NCT03164564., Findings: From Nov 27, 2017, to Nov 4, 2020, we enrolled 3224 participants (1614 in the cabotegravir group and 1610 in the TDF-FTC group). Median age was 25 years (IQR 22-30); 1755 (54·7%) of 3209 had two or more partners in the preceding month. 40 incident infections were observed over 3898 person-years (HIV incidence 1·0% [95% CI 0·73-1·40]); four in the cabotegravir group (HIV incidence 0·2 cases per 100 person-years [0·06-0·52]) and 36 in the TDF-FTC group (1·85 cases per 100 person-years [1·3-2·57]; hazard ratio 0·12 [0·05-0·31]; p<0·0001; risk difference -1·6% [-1·0% to -2·3%]. In a random subset of 405 TDF-FTC participants, 812 (42·1%) of 1929 plasma samples had tenofovir concentrations consistent with daily use. Injection coverage was 93% of the total number of person-years. Adverse event rates were similar across both groups, apart from injection site reactions, which were more frequent in the cabotegravir group than in the TDF-FTC group (577 [38·0%] of 1519 vs 162 [10·7%] of 1516]) but did not result in injection discontinuation. Confirmed pregnancy incidence was 1·3 per 100 person-years (0·9-1·7); no congenital birth anomalies were reported., Interpretation: Although both products for HIV prevention were generally safe, well tolerated, and effective, cabotegravir was superior to TDF-FTC in preventing HIV infection in women., Funding: National Institute of Allergy and Infectious Diseases, ViiV Healthcare, and the Bill & Melinda Gates Foundation. Additional support was provided through the National Institute of Mental Health, the National Institute on Drug Abuse, and the Eunice Kennedy Shriver National Institute of Child Health and Human Development. ViiV Healthcare and Gilead Sciences provided pharmaceutical support., Competing Interests: Declaration of interests PLA has received fees from Merck, ViiV Healthcare, and Gilead Sciences, and research support from Gilead Sciences paid to his institution. AR, WRS, and KS are employees of ViiV Healthcare. JFR is an employee of Gilead Sciences. All other authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

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.)

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.

Sensitive and reproducible diagnostics are fundamental to containing the spread of existing and emerging pathogens. Despite the reliance of clinical virology on qPCR, technical challenges persist that compromise their reliability for sustainable epidemic containment as sequence instability in probe-binding regions produces false-negative results. We systematically violated canonical qPCR design principles to develop a Pan-Degenerate Amplification and Adaptation (PANDAA), a point mutation assay that mitigates the impact of sequence variation on probe-based qPCR performance. Using HIV-1 as a model system, we optimized and validated PANDAA to detect HIV drug resistance mutations (DRMs). Ultra-degenerate primers with 3' termini overlapping the probe-binding site adapt the target through site-directed mutagenesis during qPCR to replace DRM-proximal sequence variation. PANDAA-quantified DRMs present at frequency ≥5% (2 h from nucleic acid to result) with a sensitivity and specificity of 96.9% and 97.5%, respectively. PANDAA is an innovative advancement with applicability to any pathogen where target-proximal genetic variability hinders diagnostic development.

Background: Phylogenetic analysis can be used to assess human immunodeficiency virus (HIV) transmission in populations. We inferred the direction of HIV transmission using whole-genome HIV sequences from couples with known linked infection and known transmission direction., Methods: Complete next-generation sequencing (NGS) data were obtained for 105 unique index-partner sample pairs from 32 couples enrolled in the HIV Prevention Trials Network (HPTN) 052 study (up to 2 samples/person). Index samples were obtained up to 5.5 years before partner infection; partner samples were obtained near the time of seroconversion. The bioinformatics method, phyloscanner, was used to infer transmission direction. Analyses were performed using samples from individual sample pairs, samples from all couples (1 sample/person; group analysis), and all available samples (multisample group analysis). Analysis was also performed using NGS data from defined regions of the HIV genome (gag, pol, env)., Results: Using whole-genome NGS data, transmission direction was inferred correctly (index to partner) for 98 of 105 (93.3%) of the individual sample pairs, 99 of 105 (94.3%) sample pairs using group analysis, and 31 of the 32 couples (96.9%) using multisample group analysis. There were no cases where the incorrect transmission direction (partner to index) was inferred. The accuracy of the method was higher with greater time between index and partner sample collection. Pol region sequences performed better than env or gag sequences for inferring transmission direction., Conclusions: We demonstrate the potential of a phylogenetic method to infer the direction of HIV transmission between 2 individuals using whole-genome and pol NGS data., (© 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.)

Although antiretroviral therapy (ART) effectively suppresses HIV replication, the latent reservoir remains the barrier to HIV eradication. It remains unknown whether long-term ART impacts levels of inducible replication-competent provirus. To address this knowledge gap, we assessed the proviral reservoir in HIV-1 perinatally infected adolescents having received ART for >13 years. We recruited 15 vertically infected adolescents living with HIV in Botswana. Historical viral load, CD4 + T cell count, and treatment data were retrieved from their outpatient medical records. Inducible replication-competent proviruses from cryopreserved peripheral blood mononuclear cells were quantified using a TZM-bl based assay (TZA). Total proviral DNA copies were quantified using droplet digital PCR. The mean age of study participants was 16 years (standard deviation = 0.7) and median CD4 + T cell count at enrollment was 784 [interquartile range (IQR) = 728.8-1,288] cells/mm 3 . Median age at ART initiation was 8 (IQR = 6-12) months. Fourteen (93%) participants had HIV-1 RNA <400 copies/mL at the time of enrollment in the study. A median of 19 (IQR = 18-27) HIV-1 RNA measurements were available per participant. Six (40%) participants displayed viral suppression at all clinic visits since initiating ART, whereas the remaining 9 (60%) had one or more clinic visits with detectable HIV-1 RNA. The median inducible replication-competent provirus count was 7.4 infectious units per million cells (IQR = 6.7-19.2), and did not differ significantly by either complete or incomplete viral suppression (7.2 vs. 7.4, p  = .86), or by age at ART initiation (7.4 if <12 months, 11.2 if >12 months, p  = .85). The median total HIV DNA count was 129.1 copies per million cells (IQR = 18.9-212.3). Our data suggest that long-term ART initiated within the 1st year in perinatally infected infants did not eliminate proviral DNA or inducible replication-competent proviruses.

To determine effects of cryptococcal meningitis (CM) on human immunodeficiency virus (HIV)-1C cerebrospinal fluid (CSF) viral escape, CSF/plasma viral discordance, and drug resistance mutation (DRM) discordance between CSF and plasma compartments, we compared CSF and plasma viral load (VL) and DRMs in individuals with HIV-associated CM in Botswana.This cross-sectional study utilized 45 paired CSF/plasma samples from participants in a CM treatment trial (2014-2016). HIV-1 VL was determined and HIV-1 protease and reverse transcriptase genotyping performed. DRMs were determined using the Stanford HIV database. CSF viral escape was defined as HIV-1 ribonucleic acid ≥0.5 log10 higher in CSF than plasma and VL discordance as CSF VL > plasma VL.HIV-1 VL was successfully measured in 39/45 pairs, with insufficient sample volume in 6; 34/39 (87.2%) participants had detectable HIV-1 in plasma and CSF, median 5.1 (interquartile range: 4.7-5.7) and 4.6 (interquartile range:3.7-4.9) log10 copies/mL, respectively (P≤.001). CSF viral escape was present in 1/34 (2.9%) and VL discordance in 6/34 (17.6%). Discordance was not associated with CD4 count, antiretroviral status, fungal burden, CSF lymphocyte percentage nor mental status. Twenty-six of 45 (57.8%) CSF/plasma pairs were successfully sequenced. HIV-1 DRM discordance was found in 3/26 (11.5%); 1 had I84IT and another had M46MI in CSF only. The third had K101E in plasma and V106 M in CSF.Our findings suggest that HIV-1 escape and DRM discordance may occur at lower rates in participants with advanced HIV-disease and CM compared to those with HIV associated neurocognitive impairment.

Background: Risk for nondiagnostic and false-positive HIV testing has not been quantified for neonates., Methods: From April 2015 to July 2018, we screened HIV-exposed infants in Botswana less than 96 h from birth by qualitative DNA PCR. Repeat blood draws for DNA and RNA PCR testing occurred for initial positive and indeterminate results to establish final diagnosis. We compared screening DNA PCR cycle threshold values with final HIV status of the child., Results: Of 10 622 HIV-exposed infants, 10 549 (99.3%) had no HIV DNA detected (negative), 42 (0.4%) had HIV DNA detected (positive), and 31 (0.3%) tested indeterminate at first HIV screen. Repeat testing identified 2 (5.0%) of 40 positive screens (2 declined additional testing) as false positives and confirmed 2 (6.5%) of 31 indeterminate screens as infected. Median cycle threshold value at screening was 28.1 (IQR 19.8--34.8) for children with final positive status, and 35.5 (IQR 32.8--41.4) for indeterminates who were ultimately negative. Six (15%) of 40 infants with final positive status had cycle threshold value greater than 33 at first screen, whereas 3 (9.7%) of 31 indeterminates with final negative status had cycle threshold value 33 or less at first screen. This threshold resulted in a negative predictive value of 82% and a positive predictive value of 92% for a single screen., Conclusion: Although a DNA PCR cycle threshold value of 33 was predictive of the final HIV status in newborns, overlap occurred for true positives, false positives, and initial indeterminates. Testing additional samples should be standard practice for positive and indeterminate HIV DNA PCR tests in the first week of life.

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: Roll-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., Methods: We 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., Results: Among 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., Conclusions: The 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., Competing Interests: The authors have declared that no competing interests exist.

Rationale: Early initiation of antiretroviral therapy (ART) leads to long-term viral suppression, reduces proviral reservoir size, and prolongs time to rebound. Since human immunodeficiency virus (HIV) is a lifelong disease, diagnostic monitoring after confirmed infection is typically not performed; therefore, little is known about the impact of early initiation and long-term ART on the sensitivity of assays that detect HIV antibodies and viral nucleic acid in children and adolescents., Patient Concerns: Here we report 1 case of diagnosed and confirmed perinatal HIV-1C infection with longstanding viral suppression, who subsequently had a negative HIV-1 deoxyribonucleic acid (DNA) test, undetectable antibodies to HIV-1, and high CD4+ T cell count after 14 years of ART., Diagnosis: The patient was diagnosed with HIV in 2002 at 1 and 2 months of age using DNA polymerase chain reaction. At 8 months old, his viral load was 1210 HIV ribonucleic acid (RNA) copies/mL and CD4 T cell count was 3768 cells/mm., Intervention: At the age of 9 months, highly active antiretroviral therapy comprising of zidovudine, nevirapine, and lamivudine was initiated. The patient remained on this treatment for 14 years 11 months and was virally suppressed., Outcomes: At the age of 14 years 4 months, the participant decided to visit a local voluntary HIV testing center, where a rapid HIV test came out negative and the viral load was undetectable (<400 HIV-1 RNA copies/mL). These results led to termination of ART which led to viral rebound within 9 months., Lessons: As more people with early HIV infection initiate early ART in the context of "Test and Treat all" recommendations, aspects of this report may become more commonplace, with both clinical and public health implications. If the possibility of functional cure (or false-positive diagnosis) is being considered, decisions to terminate ART should be made cautiously and with expert guidance, and may benefit from highly sensitive quantification of the proviral reservoir.

Differences among hosts, resulting from genetic variation in the immune system or heterogeneity in drug treatment, can impact within-host pathogen evolution. Genetic association studies can potentially identify such interactions. However, extensive and correlated genetic population structure in hosts and pathogens presents a substantial risk of confounding analyses. Moreover, the multiple testing burden of interaction scanning can potentially limit power. We present a Bayesian approach for detecting host influences on pathogen evolution that exploits vast existing data sets of pathogen diversity to improve power and control for stratification. The approach models key processes, including recombination and selection, and identifies regions of the pathogen genome affected by host factors. Our simulations and empirical analysis of drug-induced selection on the HIV-1 genome show that the method recovers known associations and has superior precision-recall characteristics compared to other approaches. We build a high-resolution map of HLA-induced selection in the HIV-1 genome, identifying novel epitope-allele combinations.

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: We evaluated the association between maternal cytomegalovirus (CMV) viremia during pregnancy and adverse birth and infant health outcomes in HIV-infected mothers and their HIV-exposed uninfected infants., Methods: HIV-positive women and their infants were followed prospectively from pregnancy through 2 years postpartum in the "Tshipidi" study in Botswana. We analyzed the association between detectable CMV DNA in maternal blood at delivery and adverse birth outcomes (stillbirth, preterm delivery, small for gestational age, or birth defect), as well as infant hospitalization and mortality through 24 months., Results: We measured CMV DNA in blood samples from 350 (77.1%) of 454 HIV-positive women from the Tshipidi study. The median maternal CD4 count was 422 cells/mL, and median HIV-1 RNA at entry was 3.2 log10 copies/mL. Fifty-one (14.6%) women had detectable CMV DNA. In unadjusted analyses, detectable CMV DNA was associated with higher maternal HIV-1 RNA [odds ratio (OR) 1.4, 95% confidence interval (CI): 1.1 to 1.9], presence of a birth defect (OR 9.8, 95% CI: 1.6 to 60.3), and occurrence of any adverse birth outcome (OR 2.0, 95% CI: 1.04 to 3.95). In multivariable analysis, we observed a trend toward association between detectable maternal CMV DNA and occurrence of any adverse birth outcome (adjusted OR 1.9, 95% CI: 0.96 to 3.8). Maternal CMV viremia was not associated with infant hospitalization and/or death by 24 months., Conclusions: Approximately 1 in 6 HIV-positive women in Botswana had detectable CMV DNA in blood at delivery. The presence of maternal CMV viremia had a borderline association with adverse birth outcomes but not with 24-month morbidity or mortality in HIV-exposed uninfected children.

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.

: There are limited data on the effectiveness of dolutegravir (DTG)-based combination antiretroviral therapy (ART) in real-life settings in southern Africa where HIV-1 subtype C predominates. We report a patient infected with HIV-1 subtype C on DTG-based ART previously exposed to raltegravir who developed multidrug resistance mutations to four antiretroviral classes. There is need for drug resistance monitoring and clinical vigilance to ensure effectiveness of HIV treatment programs even in the era of DTG-based ART.

Rilpivirine (RPV) and Etravirine (ETR) are approved second-generation non-nucleoside reverse transcriptase inhibitors (NNRTIs) for HIV treatment. There is a cross-resistance HIV mutation profile between first- and second-generation NNRTI drugs. We determined the prevalence of HIV-1 drug resistance mutations (DRMs) to RPV and ETR in Botswana. A total of 168 HIV-1 polymerase gene sequences from participants failing nevirapine (NVP)- or efavirenz (EFV)-containing regimens were analyzed for DRMs using the Stanford University HIV drug resistance database. Forty-one sequences were from an adult antiretroviral therapy (ART) study, the Tshepo study, and 127 from a prevention of mother-to-child transmission (PMTCT) study, the Mashi study, all conducted in Botswana. Prevalence of RPV and ETR highest DRM in the adult ART study (n = 41) were K101E (26.2%), E138A (23.8%), and Y181C (26.2%). The PMTCT cohort's (n = 127) high prevalence mutations were Y181C (15.7%), E138A (15%), and K101E (11%). A total of 42.9% and 3.2% of patients in the adult ART study and PMTCT study, respectively, had three or more NNRTI mutations at virologic failure. We identified HIV-1 mutations conferring resistance to RPV and ETR even though they have not been used in Botswana. Of concern was the high proportion of sequences from the adult ART study that displayed multiple DRMs; as the number of NNRTI mutations increases, the level of cross-resistance increases. It is plausible that patients displaying such profiles maybe at increased risk of failing second-generation NNRTI drugs, hence, calls for genotyping in patients with prior NVP or efavirenz exposure before prescription of RPV- or ETR-containing cART.

: 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%.

Intrahost human immunodeficiency virus (HIV)-1 diversity increases linearly over time. We assessed the extent to which mean pairwise distances and the time to the most recent common ancestor (tMRCA) inferred from intrahost HIV-1C env sequences were associated with the estimated time of HIV infection. Data from a primary HIV-1C infection study in Botswana were used for this analysis (N = 42). A total of 2540 HIV-1C env gp120 variable loop region 1 to conserved region 5 (V1C5) of the HIV-1 envelope gp120 viral sequences were generated by single genome amplification and sequencing, with an average of 61 viral sequences per participant and 11 sequences per time point per participant. Raw pairwise distances were calculated for each time point and participant using the ape package in R software. The tMRCA was estimated using phylogenetic inference implemented in Bayesian Evolutionary Analysis by Sampling Trees v1.8.2. Pairwise distances and tMRCA were significantly associated with the estimated time since HIV infection (both P < 0.001). Taking into account multiplicity of HIV infection strengthened these associations. HIV-1C env-based pairwise distances and tMRCA can be used as potential markers for HIV recency. However, the tMRCA estimates demonstrated no advantage over the pairwise distances estimates.

Routine monitoring of HIV-1 RNA or viral load (VL) in patients on antiretroviral therapy (ART) is important, but there are multiple impediments to VL testing in resource-constrained settings. An accurate point-of-care (POC) HIV-1 VL test could alleviate many of these challenges. We compared the performance of the Cepheid Xpert HIV-1 VL assay against the laboratory-based Abbott m2000sp/m2000rt assay (Abbott assay). ART-naive individuals participating in the Botswana Combination Prevention Project in 20 communities provided EDTA-blood specimens during household surveys. Both the POC Xpert HIV-1 VL and Abbott assays were performed on specimens sampled from 277 individuals. We found a high correlation between the Xpert HIV-1 VL and Abbott assay results (r 2 = 0.92; P < 0.001). The overall mean difference in the HIV-1 RNA values obtained by Xpert HIV-1 VL assay and Abbott assay was 0.34 log 10 copies/ml (95% confidence interval [CI], 0.26 to 0.40 log 10 copies/ml) (P < 0.001). Using a clinically relevant level of 1,000 copies/ml as a threshold, agreement was 90.6% (95% CI, 87.9 to 93.1%), with a sensitivity of 98.6% (95% CI, 97.2 to 100%). The two methods agreed on their detectability of HIV-1 RNA (>40 copies/ml) at 97.1% (95% CI, 95.5 to 98.7%), with a sensitivity of 99.6% (95% CI, 97.2 to 100%). The POC Cepheid Xpert HIV-1 VL assay showed high agreement and accuracy with a laboratory-based method of HIV-1 RNA testing. The POC Xpert HIV-1 VL assay tended to overestimate HIV-1 VL, although the difference was below a clinically relevant threshold of 0.5 log 10 copies/ml. The POC Cepheid Xpert HIV-1 VL assay is a promising tool for monitoring patients on ART in southern Africa., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Background: A single viral variant is transmitted in the majority of HIV infections. However, about 20% of heterosexually transmitted HIV infections are caused by multiple viral variants. Detection of transmitted HIV variants is not trivial, as it involves analysis of multiple viral sequences representing intra-host HIV-1 quasispecies., Methodology: We distinguish two types of multiple virus transmission in HIV infection: (1) HIV transmission from the same source, and (2) transmission from different sources. Viral sequences representing intra-host quasispecies in a longitudinally sampled cohort of 42 individuals with primary HIV-1C infection in Botswana were generated by single-genome amplification and sequencing and spanned the V1C5 region of HIV-1C env gp120. The Maximum Likelihood phylogeny and distribution of pairwise raw distances were assessed at each sampling time point (n = 217; 42 patients; median 5 (IQR: 4-6) time points per patient, range 2-12 time points per patient)., Results: Transmission of multiple viral variants from the same source (likely from the partner with established HIV infection) was found in 9 out of 42 individuals (21%; 95 CI 10-37%). HIV super-infection was identified in 2 patients (5%; 95% CI 1-17%) with an estimated rate of 3.9 per 100 person-years. Transmission of multiple viruses combined with HIV super-infection at a later time point was observed in one individual., Conclusions: Multiple HIV lineages transmitted from the same source produce a monophyletic clade in the inferred phylogenetic tree. Such a clade has transiently distinct sub-clusters in the early stage of HIV infection, and follows a predictable evolutionary pathway. Over time, the gap between initially distinct viral lineages fills in and initially distinct sub-clusters converge. Identification of cases with transmission of multiple viral lineages from the same source needs to be taken into account in cross-sectional estimation of HIV recency in epidemiological and population studies., Competing Interests: The authors have declared that no competing interests exist.

Background: Cross-sectional, biomarker methods to determine HIV infection recency present a promising and cost-effective alternative to the repeated testing of uninfected individuals. We evaluate a viral-based assay that uses a measure of pairwise distances (PwD) to identify HIV infection recency, and compare its performance with two serologic incidence assays, BED and LAg. In addition, we assess whether combination BED plus PwD or LAg plus PwD screening can improve predictive accuracy by reducing the likelihood of a false-recent result., Methods: The data comes from 854 time-points and 42 participants enrolled in a primary HIV-1C infection study in Botswana. Time points after treatment initiation or with evidence of multiplicity of infection were excluded from the final analysis. PwD was calculated from quasispecies generated using single genome amplification and sequencing. We evaluated the ability of PwD to correctly classify HIV infection recency within <130, <180 and <360 days post-seroconversion using Receiver Operator Characteristics (ROC) methods. Following a secondary PwD screening, we quantified the reduction in the relative false-recency rate (rFRR) of the BED and LAg assays while maintaining a sensitivity of either 75, 80, 85 or 90%., Results: The final analytic sample consisted of 758 time-points from 40 participants. The PwD assay was more accurate in classifying infection recency for the 130 and 180-day cut-offs when compared with the recommended LAg and BED thresholds. A higher AUC statistic confirmed the superior predictive performance of the PwD assay for the three cut-offs. When used for combination screening, the PwD assay reduced the rFRR of the LAg assay by 52% and the BED assay by 57.8% while maintaining a 90% sensitivity for the 130 and 180-day cut-offs respectively., Conclusion: PwD can accurately determine HIV infection recency. A secondary PwD screening reduces misclassification and increases the accuracy of serologic-based assays., Competing Interests: The authors have declared that no competing interests exist.

The development of biomedical interventions to reduce acquisition of HIV-1 infection remains a global priority, however their potential effectiveness is challenged by very high HIV-1 envelope diversity. Two large prophylactic trials in high incidence, clade C epidemic regions in southern Africa are imminent; passive administration of the monoclonal antibody VRC01, and active immunization with a clade C modified RV144-like vaccines. We have created a large representative panel of C clade viruses to enable assessment of antibody responses to vaccines and natural infection in Southern Africa, and we investigated the genotypic and neutralization properties of recently transmitted clade C viruses to determine how viral diversity impacted antibody recognition. We further explore the implications of these findings for the potential effectiveness of these trials. A panel of 200 HIV-1 Envelope pseudoviruses was constructed from clade C viruses collected within the first 100 days following infection. Viruses collected pre-seroconversion were significantly more resistant to serum neutralization compared to post-seroconversion viruses (p = 0.001). Over 13 years of the study as the epidemic matured, HIV-1 diversified (p = 0.0009) and became more neutralization resistant to monoclonal antibodies VRC01, PG9 and 4E10. When tested at therapeutic levels (10ug/ml), VRC01 only neutralized 80% of viruses in the panel, although it did exhibit potent neutralization activity against sensitive viruses (IC50 titres of 0.42 μg/ml). The Gp120 amino acid similarity between the clade C panel and candidate C-clade vaccine protein boosts (Ce1086 and TV1) was 77%, which is 8% more distant than between CRF01&#95;AE viruses and the RV144 CRF01&#95;AE immunogen. Furthermore, two vaccine signature sites, K169 in V2 and I307 in V3, associated with reduced infection risk in RV144, occurred less frequently in clade C panel viruses than in CRF01&#95;AE viruses from Thailand. Increased resistance of pre-seroconversion viruses and evidence of antigenic drift highlights the value of using panels of very recently transmitted viruses and suggests that interventions may need to be modified over time to track the changing epidemic. Furthermore, high divergence such as that observed in the older clade C epidemic in southern Africa may impact vaccine efficacy, although the correlates of infection risk are yet to be defined in the clade C setting. Findings from this study of acute/early clade C viruses will aid vaccine development, and enable identification of new broad and potent antibodies to combat the HIV-1 C-clade epidemic in southern Africa.

Background: Hepatitis B virus (HBV) is a major global health problem especially in sub-Saharan Africa and in East Asia. Ten hepatitis B virus genotypes have been described that differ by geographic distribution, disease progression, and response to treatment. Escape mutations within the surface open reading frame (ORF) affect HBV antigenicity leading to failures in diagnosis, vaccine and hepatitis B immunoglobulin therapy. However, the molecular characteristics of HBV in Botswana, a highly endemic country, are unknown. We describe the molecular characteristics of HBV and prevalence of escape mutants among HIV/HBV coinfected individuals Botswana., Methods: DNA was extracted from archived plasma samples from 81 HIV/HBV co-infected participants from various clinical studies at the Botswana Harvard AIDS Institute Partnership. A 415 base pair (bp) fragment of the polymerase gene was amplified by semi-nested PCR. In a subset of samples, a 2100 bp fragment was amplified. The PCR product was genotyped using Big Dye sequencing chemistry and the sequences were analysed for genotypes and mutations., Results: Of the 81 samples included, 70 (86 %) samples were successfully genotyped. Genotype A was found in 56 (80 %) participants, D in 13 (18.6 %), and 1 (1.4 %) was genotype E. Escape mutations previously linked with failure of diagnosis or escaping active vaccination and passive immunoglobulin therapy were detected in 12 (17.1 %) participants at positions 100, 119, 122, 123, 124, 126, 129, 130, 133, 134 and 140 of the S ORF. Genotypes and escape mutations were not significantly associated with aspartate aminotransferase (AST), alanine aminotransferase (ALT) and AST platelet ratio index (APRI)., Conclusion: Genotypes A, D and E were found in this cohort of HIV coinfected patients in Botswana, consistent with the findings from the sub-Saharan Africa region. Some escape mutations which have previously been associated with diagnosis failure, escaping vaccine and immunoglobulin therapy were also observed and are important in guiding future policies related to vaccine implementation, therapeutic guidelines, and diagnostic guidelines. They are also important for identifying patients who are at an increased risk of disease progression and to choose optimal therapy. Future research should focus on determining the clinical significance of the different HBV genotypes and mutations found in this population.

The goal of the study was to improve the methodology of HIV genotyping for analysis of HIV drug resistance and HIV clustering. Using the protocol of Gall et al. (A. Gall, B. Ferns, C. Morris, S. Watson, M. Cotten, M. Robinson, N. Berry, D. Pillay, and P. Kellam, J Clin Microbiol 50:3838-3844, 2012, doi:10.1128/JCM.01516-12), we developed a robust methodology for amplification of two large fragments of viral genome covering about 80% of the unique HIV-1 genome sequence. Importantly, this method can be applied to both viral RNA and proviral DNA amplification templates, allowing genotyping in HIV-infected subjects with suppressed viral loads (e.g., subjects on antiretroviral therapy [ART]). The two amplicons cover critical regions across the HIV-1 genome (including pol and env), allowing analysis of mutations associated with resistance to protease inhibitors, reverse transcriptase inhibitors (nucleoside reverse transcriptase inhibitors [NRTIs] and nonnucleoside reverse transcriptase inhibitors [NNRTIs]), integrase strand transfer inhibitors, and virus entry inhibitors. The two amplicons generated span 7,124 bp, providing substantial sequence length and numbers of informative sites for comprehensive phylogenic analysis and greater refinement of viral linkage analyses in HIV prevention studies. The long-range HIV genotyping from proviral DNA was successful in about 90% of 212 targeted blood specimens collected in a cohort where the majority of patients had suppressed viral loads, including 65% of patients with undetectable levels of HIV-1 RNA loads. The generated amplicons could be sequenced by different methods, such as population Sanger sequencing, single-genome sequencing, or next-generation ultradeep sequencing. The developed method is cost-effective-the cost of the long-range HIV genotyping is under $140 per subject (by Sanger sequencing)-and has the potential to enable the scale up of public health HIV prevention interventions., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

There is progressive concern about the evolving burden of morbidity and mortality caused by coinfection with HIV-1 and hepatitis B virus (HBV) in sub-Saharan Africa, but the epidemiology and impact of this problem are not well defined. We therefore set out to assimilate more information about the nature of HBV/HIV coinfection in this region by undertaking a retrospective observational study of southern African adult women. We used samples from previously recruited HIV-1 positive women attending antenatal clinics in three settings in South Africa and Botswana (n = 950) and added a small cohort of HIV-negative antenatal South African women for comparison (n = 72). We tested for HBsAg and followed up HBsAg-positive samples by testing for HBeAg, HBV DNA, HBV genotype, presence of drug-resistance associated mutations (RAMs) and HDV. We identified HBsAg in 72 individuals (7% of the whole cohort), of whom 27% were HBeAg-positive, and the majority HBV genotypes A1 and A2. We did not detect any HDV coinfection. HBV prevalence was significantly different between geographically distinct cohorts, but did not differ according to HIV status. Among adults from South Africa, HBV/HIV coinfected patients had lower CD4+ T cell counts compared to those with HIV-monoinfection (p = 0.02), but this finding was not replicated in the cohort from Botswana. Overall, these data provide a snapshot of the coinfection problem at the heart of the HIV/HBV co-epidemic, and are important to inform public health policy, resource allocation, education, surveillance and clinical care.

HLA class I polymorphism has a major influence on adult HIV disease progression. An important mechanism mediating this effect is the impact on viral replicative capacity (VRC) of the escape mutations selected in response to HLA-restricted CD8+ T-cell responses. Factors that contribute to slow progression in pediatric HIV infection are less well understood. We here investigate the relationship between VRC and disease progression in pediatric infection, and the effect of HLA on VRC and on disease outcome in adult and pediatric infection. Studying a South African cohort of >350 ART-naïve, HIV-infected children and their mothers, we first observed that pediatric disease progression is significantly correlated with VRC. As expected, VRCs in mother-child pairs were strongly correlated (p = 0.004). The impact of the protective HLA alleles, HLA-B*57, HLA-B*58:01 and HLA-B*81:01, resulted in significantly lower VRCs in adults (p<0.0001), but not in children. Similarly, in adults, but not in children, VRCs were significantly higher in subjects expressing the disease-susceptible alleles HLA-B*18:01/45:01/58:02 (p = 0.007). Irrespective of the subject, VRCs were strongly correlated with the number of Gag CD8+ T-cell escape mutants driven by HLA-B*57/58:01/81:01 present in each virus (p = 0.0002). In contrast to the impact of VRC common to progression in adults and children, the HLA effects on disease outcome, that are substantial in adults, are small and statistically insignificant in infected children. These data further highlight the important role that VRC plays both in adult and pediatric progression, and demonstrate that HLA-independent factors, yet to be fully defined, are predominantly responsible for pediatric non-progression.

Introduction: It would be useful to understand which populations are not reached by home-based HIV-1 testing and counselling (HTC) to improve strategies aimed at linking these individuals to care and reducing rates of onward HIV transmission., Methods: We present the results of a baseline home-based HTC (HBHTC) campaign aimed at counselling and testing residents aged 16 to 64 for HIV in the north-eastern sector of Mochudi, a community in Botswana with about 44,000 inhabitants. Collected data were compared with population references for Botswana, the United Nations (UN) estimates based on the National Census data and the Botswana AIDS Impact Survey IV (BAIS-IV). Analyzed data and references were stratified by age and gender., Results: A total of 6238 age-eligible residents were tested for HIV-1; 1247 (20.0%; 95% CI 19.0 to 21.0%) were found to be HIV positive (23.7% of women vs. 13.4% of men). HIV-1 prevalence peaked at 44% in 35- to 39-year-old women and 32% in 40- to 44-year-old men. A lower HIV prevalence rate, 10.9% (95% CI 9.5 to 12.5%), was found among individuals tested for the first time. A significant gender gap was evident in all analyzed subsets. The existing HIV transmission network was analyzed by combining phylogenetic mapping and household structure. Between 62.4 and 71.8% of all HIV-positive individuals had detectable virus. When compared with the UN and BAIS-IV estimates, the proportion of men missed by the testing campaign (48.5%; 95% CI 47.0 to 50.0%) was significantly higher than the proportion of missed women (14.2%; 95% CI 13.2 to 15.3%; p<0.0001). The estimated proportion of missed men peaked at about 60% in the age group 30 to 39 years old. The proportions of missed women were substantially smaller, at approximately 28% within the age groups 30 to 34 and 45 to 49 years old., Conclusions: The HBHTC campaign seems to be an efficient tool for reaching individuals who have never been tested previously in southern African communities. However, about half of men from 16 to 64 years old were not reached by the HBHTC, including about 60% of men between 30 and 40 years old. Alternative HTC strategies should be developed to bring these men to care, which will contribute to reduction of HIV incidence in communities.

Background: Regional and subtype-specific mutational patterns of HIV-1 transmitted drug resistance (TDR) are essential for informing first-line antiretroviral (ARV) therapy guidelines and designing diagnostic assays for use in regions where standard genotypic resistance testing is not affordable. We sought to understand the molecular epidemiology of TDR and to identify the HIV-1 drug-resistance mutations responsible for TDR in different regions and virus subtypes., Methods and Findings: We reviewed all GenBank submissions of HIV-1 reverse transcriptase sequences with or without protease and identified 287 studies published between March 1, 2000, and December 31, 2013, with more than 25 recently or chronically infected ARV-naïve individuals. These studies comprised 50,870 individuals from 111 countries. Each set of study sequences was analyzed for phylogenetic clustering and the presence of 93 surveillance drug-resistance mutations (SDRMs). The median overall TDR prevalence in sub-Saharan Africa (SSA), south/southeast Asia (SSEA), upper-income Asian countries, Latin America/Caribbean, Europe, and North America was 2.8%, 2.9%, 5.6%, 7.6%, 9.4%, and 11.5%, respectively. In SSA, there was a yearly 1.09-fold (95% CI: 1.05-1.14) increase in odds of TDR since national ARV scale-up attributable to an increase in non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance. The odds of NNRTI-associated TDR also increased in Latin America/Caribbean (odds ratio [OR] = 1.16; 95% CI: 1.06-1.25), North America (OR = 1.19; 95% CI: 1.12-1.26), Europe (OR = 1.07; 95% CI: 1.01-1.13), and upper-income Asian countries (OR = 1.33; 95% CI: 1.12-1.55). In SSEA, there was no significant change in the odds of TDR since national ARV scale-up (OR = 0.97; 95% CI: 0.92-1.02). An analysis limited to sequences with mixtures at less than 0.5% of their nucleotide positions—a proxy for recent infection—yielded trends comparable to those obtained using the complete dataset. Four NNRTI SDRMs—K101E, K103N, Y181C, and G190A—accounted for >80% of NNRTI-associated TDR in all regions and subtypes. Sixteen nucleoside reverse transcriptase inhibitor (NRTI) SDRMs accounted for >69% of NRTI-associated TDR in all regions and subtypes. In SSA and SSEA, 89% of NNRTI SDRMs were associated with high-level resistance to nevirapine or efavirenz, whereas only 27% of NRTI SDRMs were associated with high-level resistance to zidovudine, lamivudine, tenofovir, or abacavir. Of 763 viruses with TDR in SSA and SSEA, 725 (95%) were genetically dissimilar; 38 (5%) formed 19 sequence pairs. Inherent limitations of this study are that some cohorts may not represent the broader regional population and that studies were heterogeneous with respect to duration of infection prior to sampling., Conclusions: Most TDR strains in SSA and SSEA arose independently, suggesting that ARV regimens with a high genetic barrier to resistance combined with improved patient adherence may mitigate TDR increases by reducing the generation of new ARV-resistant strains. A small number of NNRTI-resistance mutations were responsible for most cases of high-level resistance, suggesting that inexpensive point-mutation assays to detect these mutations may be useful for pre-therapy screening in regions with high levels of TDR. In the context of a public health approach to ARV therapy, a reliable point-of-care genotypic resistance test could identify which patients should receive standard first-line therapy and which should receive a protease-inhibitor-containing regimen.

Laboratory cross-sectional assays are useful for the estimation of HIV incidence, but are known to misclassify individuals with long-standing infection as recently infected. The false recent rate (FRR) varies widely across geographic areas; therefore, accurate estimates of HIV incidence require a locally defined FRR. We determined FRR for Botswana, where HIV-1 subtype C infection is predominant, using the BED capture enzyme immunoassay (BED), a Bio-Rad Avidity Index (BAI) assay (a modification of the Bio-Rad HIV1/2+O EIA), and two multiassay algorithms (MAA) that included clinical data. To estimate FRR, stored blood samples from 512 antiretroviral (ARV)-naive HIV-1 subtype C-infected individuals from a prospective cohort in Botswana were tested at 18-24 months postenrollment. The following FRR mean (95% CI) values were obtained: BED 6.05% (4.15-8.48), BAI 5.57% (3.70-8.0), BED-BAI 2.25% (1.13-4.0), and a combination of BED-BAI with CD4 (>200) and viral load (>400) threshold 1.43% (0.58-2.93). The interassay agreement between BED and BAI was 92.8% (95% CI, 90.1-94.5) for recent/long-term classification. Misclassification was associated with viral suppression for BED [adjusted OR (aOR) 10.31; p=0.008], BAI [aOR 9.72; p=0.019], and MAA1 [aOR 16.6; p=0.006]. Employing MAA can reduce FRR to <2%. A local FRR can improve cross-sectional HIV incidence estimates.

Background: Determining patterns of HIV transmission is increasingly important for the most efficient use of modern prevention interventions. HIV phylogeny can provide a better understanding of the mechanisms underlying HIV transmission networks in communities., Methods: To reconstruct the structure and dynamics of a local HIV/AIDS epidemic, the phylogenetic relatedness of HIV-1 subtype C env sequences obtained from 785 HIV-infected community residents in the northeastern sector of Mochudi, Botswana, during 2010-2013 was estimated. The genotyping coverage was estimated at 44%. Clusters were defined based on relatedness of HIV-1C env sequences and bootstrap support of splits., Results: The overall proportion of clustered HIV-1C env sequences was 19.1% (95% CI 17.5% to 20.8%). The proportion of clustered sequences from Mochudi was significantly higher than the proportion of non-Mochudi sequences that clustered, 27.0% vs. 14.7% (p = 5.8E-12; Fisher exact test). The majority of clustered Mochudi sequences (90.1%; 95% CI 85.1% to 93.6%) were found in the Mochudi-unique clusters. None of the sequences from Mochudi clustered with any of the 1,244 non-Botswana HIV-1C sequences. At least 83 distinct HIV-1C variants, or chains of HIV transmission, in Mochudi were enumerated, and their sequence signatures were reconstructed. Seven of 20 genotyped seroconverters were found in 7 distinct clusters., Conclusions: The study provides essential characteristics of the HIV transmission network in a community in Botswana, suggests the importance of high sampling coverage, and highlights the need for broad HIV genotyping to determine the spread of community-unique and community-mixed viral variants circulating in local epidemics. The proposed methodology of cluster analysis enumerates circulating HIV variants and can work well for surveillance of HIV transmission networks. HIV genotyping at the community level can help to optimize and balance HIV prevention strategies in trials and combined intervention packages.

Risk of developing drug resistance after stopping antiretroviral regimens to prevent mother-to-child HIV-1 transmission is unknown. The Mma Bana Study randomized treatment-naive pregnant women with CD4 ≥200 cells per cubic millimeter to receive either abacavir/zidovudine/lamivudine [triple nucleoside reverse transcriptase inhibitor (NRTI) arm] or lopinavir/ritonavir/zidovudine/lamivudine [protease inhibitor (PI) arm]. Drugs were discontinued after 6 months of breastfeeding. One month after discontinuation, 29 NRTI arm samples and 25 PI arm samples were successfully genotyped. No clinically significant antiretroviral resistance mutations were detected. Eight minor resistance mutations were found among 11 (20%) women (3 from NRTI arm and 8 from PI arm), occurring at similar frequencies to those reported in HIV-1 subtype C treatment-naive cohorts.

To detect and characterize polymerase gene (pol) polymorphisms and mutation patterns in HIV-1C-infected Batswana patients treated with reverse transcriptase inhibitors, samples from AIDS patients treated with highly active antiretroviral therapy (HAART) were sequenced for the region encompassing the entire HIV-1 protease (PR) and the first 335 amino acids of reverse transcriptase (RT). Amongst the 16 patients treated with antiretroviral (ARV) drugs, eight started HAART regimens containing didanosine, stavudine and nevirapine (ddI/d4T/NVP) or efavirenz (EFV) (arm A) while the others started with zidovudine (AZT) and lamivudine (3TC) given together as combivir (CBV) with either NVP or EFV as arm B. Arm B is the first line regimen currently provided by the Botswana ARV national programme. Greater efficacy, in terms of treatment duration, was observed in patients in arm B (14 months) as compared with patients in arm A (9 months); P<0.05, n=8. Appearance of the M184V mutation in the arm B patients coincided with a rebound of viral load (VL) (4.3 +/-0.1 log10 RNA copies/ml) and a significantly improved immunological parameter (deltaCD4=207.0+/-48.1 cells/microl; P<0.05). Interestingly, patients developing the M184V mutation preferentially harboured polymorphisms Q174K and/or I178L located in close proximity to pol position 184. The M184V mutation occurred following a clear clinical benefit consisting of increased CD4 cell counts and lower plasma viral loads. Primary mutations known to be associated with NNRTI and NRTI resistance for HIV-1B were observed in 10 of the 16 treated patients.