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. 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 (estimated trial population: 175,664). 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] versus 3% [0.1 – 27.3]) than at baseline (7% [1.5 – 25.3] versus 5% [0.9 – 22.9]) compatible with a benefit from treatment-as-prevention. Our findings suggest that population mobility patterns are fundamental to HIV transmission dynamics and to the impact of HIV control strategies.Competing Interest StatementThe authors have declared no competing interest.Funding StatementThis 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).Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:The trial was approved by the Botswana Health Research and Development Committee, the institutional review board of the Centers for Disease Control and Prevention and the Harvard School of Public Health Office of Regulatory Affairs and Research Compliance; and was monitored by a data and safety monitoring board and Westat. Written informed consent for enrollment in the trial and viral HIV genotyping was obtained from all participants.All necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable.YesAll relevant data are within the paper and the Supplemental Notes, figures and tables. HIV-1 reads are available on reasonable request through the PANGEA consortium (www.pangea-hiv.org)

We used publicly available data to describe epidemiology, genomic surveillance, and public health and social measures from the first 3 COVID-19 pandemic waves in southern Africa during April 6, 2020-September 19, 2021. South Africa detected regional waves on average 7.2 weeks before other countries. Average testing volume 244 tests/million/day) increased across waves and was highest in upper-middle-income countries. Across the 3 waves, average reported regional incidence increased (17.4, 51.9, 123.3 cases/1 million population/day), as did positivity of diagnostic tests (8.8%, 12.2%, 14.5%); mortality (0.3, 1.5, 2.7 deaths/1 million populaiton/day); and case-fatality ratios (1.9%, 2.1%, 2.5%). Beta variant (B.1.351) drove the second wave and Delta (B.1.617.2) the third. Stringent implementation of safety measures declined across waves. As of September 19, 2021, completed vaccination coverage remained low (8.1% of total population). Our findings highlight opportunities for strengthening surveillance, health systems, and access to realistically available therapeutics, and scaling up risk-based vaccination.

Accurate HIV and CD4 testing are critical in program implementation, with HIV misdiagnosis having serious consequences at both the client and/or community level. We implemented a comprehensive training and Quality Assurance (QA) program to ensure accuracy of point-of-care HIV and CD4 count testing by lay counsellors during the Botswana Combination Prevention Project (BCPP). We compared the performance of field testing by lay counselors to results from an accredited laboratory to ascertain accuracy of testing. All trained lay counselors passed competency assessments and performed satisfactorily in proficiency testing panel evaluations in 2013, 2014, and 2015. There was excellent agreement (99.6%) between field and laboratory-based HIV test results; of the 3002 samples tested, 960 and 2030 were concordantly positive and negative respectively, with 12 misclassifications (kappa score 0.99, p < 0.0001). Of the 149 HIV-positive samples enumerated for CD4 count in the field using PIMA at a threshold of 350 cells/L; there was 86% agreement with laboratory testing, with only 21 misclassified. The mean difference between field and lab CD4 testing was -16.16 cells/L (95% CI -5.4 - 26.9). Overall, there was excellent agreement between field and laboratory results for both HIV rapid test and PIMA CD4 results. A standard training package to train lay counselors to accurately perform HIV and CD4 point-of-care testing in field settings was feasible, with point-of-care results obtained by lay counselors comparable to laboratory-based testing.

BACKGROUND: and Setting: The Botswana Combination Prevention Project (BCPP) demonstrated a 30% reduction in community HIV incidence through expanded HIV testing, enhanced linkage to care, and universal antiretroviral treatment and exceeded the Joint United Nations Programme on HIV/AIDS 90-90-90 targets. We report rates and characteristics of incident HIV infections. METHODS: BCPP was a community-randomized controlled trial conducted in 30 rural/peri-urban Botswana communities from 2013 to 2017. Home-based and mobile HIV-testing campaigns were conducted in 15 intervention communities, with 39% of participants testing at least twice. We assessed the HIV incidence rate (IR; number of new HIV infections per 100 person-years (py) at risk) among repeat testers and risk factors with a Cox proportional hazards regression model. RESULTS: During 27,517py, 195 (women,79%) of 18,597 became HIV-infected (0.71/100py). Women had a higher IR (1.01/100py; 95% CI, 0.99 to 1.02) than men (0.34/100py; 95% CI, 0.33 to 0.35). The highest IRs were among women aged 16-24 years (1.87/100py) and men aged 25-34 years (0.56/100py). The lowest IRs were among those aged 35-64 years (women,0.41/100py; men,0.20/100py). Hazard of incident infection was highest among women aged 16-24 (HR=7.05). Sex and age were significantly associated with incidence (both P<0.0001). CONCLUSIONS: Despite an overall reduction in HIV incidence and approaching the UNAIDS 95-95-95 targets, high HIV incidence was observed in adolescent girls and young women. These findings highlight the need for additional prevention services [pre-exposure prophylaxis (PrEP), DREAMS] to achieve epidemic control in this subpopulation and increased efforts with men with undiagnosed HIV.

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 5,114 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] versus 3% [0.1 - 27.3]) than at baseline (7% [1.5 - 25.3] versus 5% [0.9 - 22.9]) compatible with a benefit from treatment-as-prevention. Conclusion: 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).

Objective: The OraQuick Advance Rapid HIV-1/2 Test is a point-of-care test capable of detecting HIV-specific antibodies in blood and oral fluid. To understand test performance and factors contributing to false-negative (FN) results in longitudinal studies, we examined results of participants enrolled in the TDF2 study, the Bangkok Tenofovir Study, and the Bangkok MSM Cohort Study (BMCS), three separate clinical studies of high-risk, HIV-negative persons conducted in Botswana and Thailand. Design: Retrospective observational analysis. Methods: We compared oral fluid OraQuick (OFOQ) results among participants becoming HIV-infected to results obtained retrospectively using enzyme immunoassay and nucleic acid amplification tests on stored specimens. We categorized negative OFOQ results as true-negative or false-negative relative to NAAT and/or EIA, and determined the delay in OFOQ conversion relative to the estimated time of infection. We used generalized estimating equations to examine the association between FN results and participant, clinical and testing-site factors. Results: Two-hundred and thirty-three FN OFOQ results occurred in 80 of 287 seroconverting individuals. Estimated OFOQ conversion delay ranged from 14.5-547.5 (median 98.5) days. Delayed OFOQ conversion was associated with clinical site and test operator (p<0.05), pre-exposure prophylaxis (p=0.01), low plasma viral load (p<0.02) and time to kit expiration (p<0.01). Participant age, gender, and HIV subtype were not associated with FN results. Long OFOQ conversion delay time was associated with antiretroviral exposure and low PVL. Conclusions: Failure of OFOQ to detect HIV-1 infection was frequent, and multifactorial in origin. In longitudinal trials, negative oral fluid results should be confirmed by testing blood samples.

Successful clinical trials of antiretroviral pre-exposure prophylaxis (PrEP) to prevent HIV infection have been reported in heterosexual men and women, men who have sex with men (MSM), and injection drug users1. A daily oral drug regimen containing nucleoside reverse transcriptase inhibitors (NRTIs) tenofovir disoproxil fumarate (TDF) alone or in combination with emtricitabine (FTC) have been effective when participant adherence is high1. Analysis of PrEP dosing patterns estimate taking at least four TDF doses per week provides 96% protection from HIV infection and at least two doses per week provides 76% protection in MSM and transgender women2, though some estimate more frequent dosing in heterosexual women may be needed3. TDF and FTC require phosphorylation in HIV target cells to tenofovir-diphosphate (TFV-DP) and emtricitabine-triphosphate (FTC-TP) to provide PrEP protection as competitive analogs of naturally occurring deoxyadenosine triphosphate (dATP) and deoxycytidine triphosphate (dCTP), respectively. However, it is unclear if physiological conditions that increase dNTP concentrations can affect pharmacokinetics (PK) and pharmacodynamics (PD) of NRTIs. This may be particularly relevant when cellular deoxynucleoside triphosphate (dNTP) pools in HIV target cells increase in response to immune activation. Decreased ratios of phosphorylated NRTIs to their respective dNTPs have been associated with cell activation in vitro and in nonhuman primate studies4,5, yet this observation remains unexplored in PK and PD studies that measured intracellular drug6-8. The study presented here compared dATP and dCTP concentrations to TFV-DP and FTC-TP in peripheral blood mononuclear cell (PBMCs) of persons using daily oral Truvada™ during a successful HIV PrEP study. We further examined if variations among intracellular drug:dNTP ratios were related to lymphocyte activation.

OBJECTIVE: Among participants of a clinical trial to test the efficacy of tenofovir/emtricitabine in protecting heterosexual men and women living in Botswana from HIV infection, determine 1) if sexual risk behavior, specifically condomless sex acts and number of sex partners, changed over time, 2) factors associated with condomless sex acts and number of sex partners and 3) the effect of participant treatment arm perception on risk behavior to address the possibility of risk compensation. METHODS: A longitudinal modeling of rates of risk behaviors was used to determine if the rate of condomless sex acts (#acts/person) and rate of sex partners (#partners/person) changed over time and which factors were associated with behavior change. RESULTS: 1200 participants were analyzed over 1 year. There was a 25% decrease in the rate of sex partners among participants sexually active in the last 30 days. The rate of reported condomless sex acts was greater for males (RR=1.34, CI=1.07-1.67) and participants whose sexual debut in years was ≤ 15 years of age (RR=1.65, CI=1.14-2.38) and 16-17 (RR=1.68, CI=1.22-2.31) compared to ≥20 years. Rate of reported sex partners was greater for males (RR=3.67, CI=2.86-4.71) and participants whose age at sexual debut in years was ≤15 (RR=2.92, CI=2.01-4.22) and 16-17 (RR=2.34, CI=1.69-3.24) compared to ≥20. There was no effect of participant treatment arm perception on risk behavior. CONCLUSIONS: Our study of PrEP to prevent HIV infection found no evidence of risk compensation which may have been due to participants' motivations to reduce their risk behaviors and risk-reduction counseling.

INTRODUCTION: Accurate clinical laboratory reference values derived from a local or regional population base are required to correctly interpret laboratory results. In Botswana, most reference intervals used to date are not standardized across clinical laboratories and are based on values derived from populations in the United States or Western Europe. METHODS: We measured 14 hematologic and biochemical parameters of healthy young adults screened for participation in the Botswana HIV Pre-exposure Prophylaxis Study using tenofovir disoproxil fumarate and emtricitabine (TDF/FTC) (TDF2 Study). Reference intervals were calculated using standard methods, stratified by gender, and compared with the site-derived reference values used for the TDF2 study (BOTUSA ranges), the Division of AIDS (DAIDS) Grading Table for Adverse Events, the Botswana public health laboratories, and other regional references. RESULTS: Out of 2533 screened participants, 1786 met eligibility criteria for participation in study and were included in the analysis. Our reference values were comparable to those of the Botswana public health system except for amylase, blood urea nitrogen (BUN), phosphate, total and direct bilirubin. Compared to our reference values, BOTUSA reference ranges would have classified participants as out of range for some analytes, with amylase (50.8%) and creatinine (32.0%) producing the highest out of range values. Applying the DAIDS toxicity grading system to the values would have resulted in 45 and 18 participants as having severe or life threatening values for amylase and hemoglobin, respectively. CONCLUSION: Our reference values illustrate the differences in hematological and biochemical analyte ranges between African and Western populations. Thus, the use of western-derived reference laboratory values to screen a group of Batswana adults resulted in many healthy people being classified as having out-of-range blood analytes. The need to establish accurate local or regional reference values is apparent and we hope our results can be used to that end in Botswana.

BACKGROUND: Tenofovir-emtricitabine (TDF-FTC) pre-exposure prophylaxis (PrEP) has been found to be effective for prevention of HIV infection in several clinical trials. Two studies of TDF PrEP among men who have sex with men showed slight bone mineral density (BMD) loss. We investigated the effect of TDF and the interaction of TDF and hormonal contraception on BMD among HIV-uninfected African men and women. METHOD: We evaluated the effects on BMD of using daily oral TDF-FTC compared to placebo among heterosexual men and women aged 18-29 years enrolled in the Botswana TDF2 PrEP study. Participants had BMD measurements at baseline and thereafter at 6-month intervals with dual-energy X-ray absorptiometry (DXA) scans at the hip, spine, and forearm. RESULTS: A total of 220 participants (108 TDF-FTC, 112 placebo) had baseline DXA BMD measurements at three anatomic sites. Fifteen (6.8%) participants had low baseline BMD (z-score of <-2.0 at any anatomic site), including 3/114 women (2.6%) and 12/106 men (11.3%) (p = 0.02). Low baseline BMD was associated with being underweight (p = 0.02), having high blood urea nitrogen (p = 0.02) or high alkaline phosphatase (p = 0.03), and low creatinine clearance (p = 0.04). BMD losses of >3.0% at any anatomic site at any time after baseline were significantly greater for the TDF-FTC treatment group [34/68 (50.0%) TDF-FTC vs. 26/79 (32.9%) placebo; p = 0.04]. There was a small but significant difference in the mean percent change in BMD from baseline for TDF-FTC versus placebo at all three sites at month 30 [forearm -0.84% (p = 0.01), spine -1.62% (p = 0.0002), hip -1.51% (p = 0.003)]. CONCLUSION: Use of TDF-FTC was associated with a small but statistically significant decrease in BMD at the forearm, hip and lumbar spine. A high percentage (6.8%) of healthy Batswana young adults had abnormal baseline BMD. Further evaluation is needed of the longer-term use of TDF in HIV-uninfected persons. TRIAL REGISTRATION: ClinicalTrials.gov NCT00448669.

We examined CD4 cell count and plasma viral load patterns among Botswana TDF/FTC Oral HIV Prophylaxis Trial (TDF2 study) participants who seroconverted, comparing participants assigned to receive tenofovir/emtricitabine with participants assigned to receive placebo. We also evaluated for antiretroviral drug resistance among the breakthrough HIV infections. Among nine seroconverters assigned to tenofovir/emtricitabine and 24 to placebo, there were no significant differences in their CD4 cell count or viral load profiles over time. Of the four participants who seroconverted on-study while receiving tenofovir/emtricitabine, none became infected as a result of drug-resistant HIV; moreover, no resistance mutations emerged following seroconversion.

BACKGROUND: Preexposure prophylaxis with antiretroviral agents has been shown to reduce the transmission of human immunodeficiency virus (HIV) among men who have sex with men; however, the efficacy among heterosexuals is uncertain. METHODS: We randomly assigned HIV-seronegative men and women to receive either tenofovir disoproxil fumarate and emtricitabine (TDF-FTC) or matching placebo once daily. Monthly study visits were scheduled, and participants received a comprehensive package of prevention services, including HIV testing, counseling on adherence to medication, management of sexually transmitted infections, monitoring for adverse events, and individualized counseling on risk reduction; bone mineral density testing was performed semiannually in a subgroup of participants. RESULTS: A total of 1219 men and women underwent randomization (45.7% women) and were followed for 1563 person-years (median, 1.1 years; maximum, 3.7 years). Because of low retention and logistic limitations, we concluded the study early and followed enrolled participants through an orderly study closure rather than expanding enrollment. The TDF-FTC group had higher rates of nausea (18.5% vs. 7.1%, P<0.001), vomiting (11.3% vs. 7.1%, P=0.008), and dizziness (15.1% vs. 11.0%, P=0.03) than the placebo group, but the rates of serious adverse events were similar (P=0.90). Participants who received TDF-FTC, as compared with those who received placebo, had a significant decline in bone mineral density. K65R, M184V, and A62V resistance mutations developed in 1 participant in the TDF-FTC group who had had an unrecognized acute HIV infection at enrollment. In a modified intention-to-treat analysis that included the 33 participants who became infected during the study (9 in the TDF-FTC group and 24 in the placebo group; 1.2 and 3.1 infections per 100 person-years, respectively), the efficacy of TDF-FTC was 62.2% (95% confidence interval, 21.5 to 83.4; P=0.03). CONCLUSIONS: Daily TDF-FTC prophylaxis prevented HIV infection in sexually active heterosexual adults. The long-term safety of daily TDF-FTC prophylaxis, including the effect on bone mineral density, remains unknown. (Funded by the Centers for Disease Control and Prevention and the National Institutes of Health; TDF2 ClinicalTrials.gov number, NCT00448669 .).