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Background Multi-assay algorithms (MAAs) are used to estimate population-level HIV incidence and identify individuals with recent infection. Many MAAs use low viral load (VL) as a biomarker for long-term infection. This could impact incidence estimates in settings with high rates of early HIV treatment initiation. We evaluated the performance of two MAAs that do not include VL. Methods Samples were collected from 219 seroconverters (infected 1 year) in the HPTN 071 (PopART) trial; 28.8% of seroconverter samples and 73.2% of non-seroconverter samples had VLs ≤ 400 copies/mL. Samples were tested with the Limiting Antigen Avidity assay (LAg) and JHU BioRad-Avidity assays. Antibody reactivity to two HIV peptides was measured using the MSD U-PLEX assay. Two MAAs were evaluated that do not include VL: a MAA that includes the LAg-Avidity assay and BioRad-Avidity assay (LAg + BR) and a MAA that includes the LAg-Avidity assay and two peptide biomarkers (LAg + PepPair). Performance of these MAAs was compared to a widely used MAA that includes LAg and VL (LAg + VL). Results The incidence estimate for LAg + VL (1.29%, 95% CI: 0.97–1.62) was close to the observed longitudinal incidence (1.34% 95% CI: 1.17–1.53). The incidence estimates for the other two MAAs were higher (LAg + BR: 2.56%, 95% CI 2.01–3.11; LAg + PepPair: 2.84%, 95% CI: 1.36–4.32). LAg + BR and LAg + PepPair also misclassified more individuals infected > 2 years as recently infected than LAg + VL (1.2% [42/3483 and 1.5% [51/3483], respectively, vs. 0.2% [6/3483]). LAg + BR classified more seroconverters as recently infected than LAg + VL or LAg + PepPair (80 vs. 58 and 50, respectively) and identified ~ 25% of virally suppressed seroconverters as recently infected. Conclusions The LAg + VL MAA produced a cross-sectional incidence estimate that was closer to the longitudinal estimate than two MAAs that did not include VL. The LAg + BR MAA classified the greatest number of individual seroconverters as recently infected but had a higher false recent rate.

IntroductionLow HIV viral load is associated with delayed disease progression and reduced HIV transmission. HIV controllers suppress viral load to low levels in the absence of antiretroviral treatment (ART). We used an antibody profiling system, VirScan, to compare antibody reactivity and specificity in HIV controllers, non-controllers with treatment-induced viral suppression, and viremic non-controllers.MethodsThe VirScan library contains 3,384 phage-displayed peptides spanning the HIV proteome. Antibody reactivity to these peptides was measured in plasma from a Discovery Cohort that included 13 elite controllers, 27 viremic controllers, 12 viremic non-controllers, and 21 non-controllers who were virally suppressed on ART. Antibody reactivity to selected peptides was also assessed in an independent cohort of 29 elite controllers and 37 non-controllers who were virally suppressed on ART (Validation Cohort) and in a longitudinal cohort of non-controllers.ResultsIn the Discovery Cohort, 62 peptides were preferentially targeted in HIV controllers compared to non-controllers who were virally suppressed on ART. These specificities were not significantly different when comparing controllers versus viremic non-controllers. Aggregate reactivity to these peptides was also high in elite controllers from the independent Validation Cohort. The 62 peptides formed seven clusters of homologous epitopes in env, gag, integrase, and vpu. Reactivity to one of these clusters located in gag p17 was inversely correlated with viral load set point in an independent cohort of non-controllers.ConclusionsAntibody reactivity was low in non-controllers suppressed on ART, but remained high in viremic controllers despite viral suppression. Antibodies in controllers and viremic non-controllers were directed against epitopes in diverse HIV proteins; higher reactivity against p17 peptides was associated with lower viral load set point. Further studies are needed to determine if these antibodies play a role in regulation of HIV viral load.

We combined behavioral survey data from the human immunodeficiency virus (HIV) Prevention Trials Network 068 study with phylogenetic information to determine if cluster membership was associated with characteristics of young women and their partners. Clusters were more likely to involve young women from specific villages and schools, indicating some localized transmission.Supplemental digital content is available in the text.

BACKGROUND: Antiretroviral (ARV) drugs are used for HIV treatment and prevention. We analyzed ARV drug use and HIV drug resistance in a cohort of young women in rural South Africa enrolled in the HPTN 068 study, which evaluated use of a cash transfer conditional on school attendance to reduce HIV incidence. METHODS: ARV drug testing was performed using plasma samples from 2,526 young women. This included 2,526 enrollment samples (80 HIV-infected, 2,446 HIV-uninfected) and 162 seroconversion samples (first HIV-positive study visit). Testing was performed using a qualitative assay that detects 20 ARV drugs from five drug classes. HIV drug resistance testing was performed with the ViroSeq HIV-1 Genotyping System for samples that had HIV viral loads ≥400 copies/mL. RESULTS: At enrollment, ARV drugs were detected in 10 (12.5%) of 80 HIV-infected young women. None of 2,446 HIV-uninfected young women had ARV drugs detected at enrollment. ARV drugs were also detected in 16 (9.9%) of 162 seroconverters. At enrollment, nine (13.4%) of 67 young women with genotyping results had HIV drug resistance; resistance was also detected in nine (6.9%) of 131 seroconverters with genotyping results. CONCLUSIONS: Most of the HIV-infected young women in this cohort from rural South Africa were not taking ARV drugs, suggesting they were unaware of their HIV status or were not in care. HIV drug resistance was detected in young women with both prevalent and new HIV infection.

HPTN 067/ADAPT evaluated tenofovir disoproxil fumarate/emtricitabine (TDF/FTC) pre-exposure prophylaxis (PrEP) in women (South Africa) and men who have sex with men (Thailand, US). Participants received once-weekly directly observed TDF/FTC (DOT), and were then randomized to daily, time-driven, or event-driven PrEP. This report describes characterization of 12 HIV seroconversion events in this trial. HIV rapid testing was performed at study sites. Retrospective testing included: 4th generation assays; HIV RNA testing; Western blot; an HIV-1/2 discriminatory assay; resistance testing; and antiretroviral (ARV) drug testing. Six of the 12 seroconverters received TDF/FTC in the DOT phase, but were not randomized (3 were acutely infected at enrollment; 2 were infected during the DOT phase; one was not randomized due to pregnancy). One of the six randomized participants had acute infection at randomization but was not diagnosed for 3-4 months because HIV rapid tests were non-reactive; continued daily PrEP use was associated with false-negative antibody tests and low HIV RNA levels. The five participants infected after randomization included four with low adherence to the PrEP regimen, and one who reported a 7-day period without dosing prior to infection. Three participants had TDF/FTC resistance (M184I, K65R), including two who received only four once-weekly TDF/FTC doses; most TDF/FTC mutations were detected by next generation sequencing only. In HPTN 067/ADAPT, participants who acquired HIV infection had infrequent PrEP dosing or low/suboptimal adherence. Sensitive assays improved detection of HIV infection and drug resistance. Drug resistance was observed with limited PrEP exposure. This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial License 4.0 (CCBY-NC), where it is permissible to download, share, remix, transform, and buildup the work provided it is properly cited. The work cannot be used commercially without permission from the journal.

It has been postulated that the direction of HIV transmission between two individuals can be determined by phylogenetic analysis of HIV sequences. This approach may be problematic, since HIV sequences from newly infected individuals are often more similar to index sequences from samples collected years before transmission, compared to those from samples collected at the time of transmission. We evaluated the accuracy of phylogenetic methods for determining the direction of HIV transmission by analyzing next-generation sequencing (NGS) data from index–partner pairs enrolled in the HIV Prevention Trials Network (HPTN) 052 trial. HIV-infected index and HIV-uninfected partner participants were enrolled as serodiscordant couples; samples were analyzed from couples with index-to-partner HIV transmission that was confirmed by genetic linkage studies. NGS for HIV gp41 (HXB2 coordinates: 7691–8374) was performed using plasma samples from thirty-nine index–partner pairs (seventy-eight samples collected within 3 months of partner seroconversion). Maximum likelihood trees were generated using the entire dataset using FastTree v.2. Topological patterns of HIV from each index–partner pair were analyzed. The analysis included 9,368 consensus sequences and 521,145 total sequence reads for the seventy-eight samples analyzed. In 10 per cent (four out of thirty-nine) of couples, the phylogeny was inconsistent with the known direction of transmission. In 26 per cent (ten out of thirty-nine) of couples, the phylogeny results could not discern directionality. In 64 per cent (twenty-five out of thirty-nine) of couples, the results correctly indicated index-to-partner transmission; in two of these twenty-five cases, only one index sequence was closest to the most recent common ancestor. Phylogenetic analysis of NGS data obtained from samples collected within 3 months of transmission correctly determined the direction of transmission in 64 per cent of the cases analyzed. In 36 per cent of the cases, the phylogenetic topology did not support the known direction of infection, and in one-third of these cases the observed topology was opposite to the known direction of transmission. This demonstrates that phylogenetic topology alone may not be sufficient to accurately determine the direction of HIV transmission.

BackgroundAn interim analysis of data from the HIV Prevention Trials Network (HPTN) 052 trial showed that antiretroviral therapy (ART) prevented more than 96% of genetically linked infections caused by human immunodeficiency virus type 1 (HIV-1) in serodiscordant couples. ART was then offered to all patients with HIV-1 infection (index participants). The study included more than 5 years of follow-up to assess the durability of such therapy for the prevention of HIV-1 transmission.MethodsWe randomly assigned 1763 index participants to receive either early or delayed ART. In the early-ART group, 886 participants started therapy at enrollment (CD4+ count, 350 to 550 cells per cubic millimeter). In the delayed-ART group, 877 participants started therapy after two consecutive CD4+ counts fell below 250 cells per cubic millimeter or if an illness indicative of the acquired immunodeficiency syndrome (i.e., an AIDS-defining illness) developed. The primary study end point was the diagnosis of genetically linked HIV-1 infection in the previously HIV-1-negative partner in an intention-to-treat analysis.ResultsIndex participants were followed for 10,031 person-years; partners were followed for 8509 person-years. Among partners, 78 HIV-1 infections were observed during the trial (annual incidence, 0.9%; 95% confidence interval [CI], 0.7 to 1.1). Viral-linkage status was determined for 72 (92%) of the partner infections. Of these infections, 46 were linked (3 in the early-ART group and 43 in the delayed-ART group; incidence, 0.5%; 95% CI, 0.4 to 0.7) and 26 were unlinked (14 in the early-ART group and 12 in the delayed-ART group; incidence, 0.3%; 95% CI, 0.2 to 0.4). Early ART was associated with a 93% lower risk of linked partner infection than was delayed ART (hazard ratio, 0.07; 95% CI, 0.02 to 0.22). No linked infections were observed when HIV-1 infection was stably suppressed by ART in the index participant.ConclusionsThe early initiation of ART led to a sustained decrease in genetically linked HIV-1 infections in sexual partners. (Funded by the National Institute of Allergy and Infectious Diseases; HPTN 052 ClinicalTrials.gov number, NCT00074581 .).

BackgroundWe evaluated use of phylogenetic methods to predict the direction of human immunodeficiency virus (HIV) transmission.MethodsFor 33 pairs of HIV-infected patients (hereafter, “index patients”) and their partners who acquired genetically linked HIV infection during the study, samples were collected from partners and index patients close to the time when the partner seroconverted (hereafter, “SC samples”); for 31 pairs, samples collected from the index patient at an earlier time point (hereafter, “early index samples”) were also available. Phylogenies were inferred using env next-generation sequences (1 tree per pair/subtype). The direction of transmission (DoT) predicted from each tree was classified as correct or incorrect on the basis of which sequences (those from the index patient or the partner) were closest to the root. DoT was also assessed using maximum parsimony to infer ancestral node states for 100 bootstrap trees.ResultsDoT was predicted correctly for both single-pair and subtype-specific trees in 22 pairs (67%) by using SC samples and in 23 pairs (74%) by using early index samples. DoT was predicted incorrectly for 4 pairs (15%) by using SC or early index samples. In the bootstrap analysis, DoT was predicted correctly for 18 pairs (55%) by using SC samples and for 24 pairs (73%) by using early index samples. DoT was predicted incorrectly for 7 pairs (21%) by using SC samples and for 4 pairs (13%) by using early index samples.ConclusionsPhylogenetic methods based solely on the tree topology of HIV env sequences, particularly without consideration of phylogenetic uncertainty, may be insufficient for determining DoT.

Evaluation of pretreatment HIV genotyping is needed globally to guide treatment programs. We examined the association of pretreatment (baseline) drug resistance and subtype with virologic failure in a multinational, randomized clinical trial that evaluated 3 antiretroviral treatment (ART) regimens and included resource-limited setting sites.Pol genotyping was performed in a nested case-cohort study including 270 randomly sampled participants (subcohort), and 218 additional participants failing ART (case group). Failure was defined as confirmed viral load (VL)1000 copies/mL. Cox proportional hazards models estimated resistance-failure association.In the representative subcohort (261/270 participants with genotypes; 44% women; median age, 35 years; median CD4 cell count, 151 cells/µL; median VL, 5.0 log10 copies/mL; 58% non-B subtypes), baseline resistance occurred in 4.2%, evenly distributed among treatment arms and subtypes. In the subcohort and case groups combined (466/488 participants with genotypes), used to examine the association between resistance and treatment failure, baseline resistance occurred in 7.1% (9.4% with failure, 4.3% without). Baseline resistance was significantly associated with shorter time to virologic failure (hazard ratio [HR], 2.03; P = .035), and after adjusting for sex, treatment arm, sex-treatment arm interaction, pretreatment CD4 cell count, baseline VL, and subtype, was still independently associated (HR, 2.1; P = .05). Compared with subtype B, subtype C infection was associated with higher failure risk (HR, 1.57; 95% confidence interval [CI], 1.04-2.35), whereas non-B/C subtype infection was associated with longer time to failure (HR, 0.47; 95% CI, .22-.98).In this global clinical trial, pretreatment resistance and HIV-1 subtype were independently associated with virologic failure. Pretreatment genotyping should be considered whenever feasible.NCT00084136.

Introduction: We evaluated HIV drug resistance in adults who received early vs. delayed antiretroviral therapy (ART) in a multinational trial [HIV Prevention Trials Network (HPTN) 052, enrollment 2005-2010]. In HPTN 052, 1763 index participants were randomized to start ART at a CD4 cell count of 350-550 cells/mm 3 (early ART arm) or 1000 copies/mL >24 weeks after ART initiation. Drug resistance testing was performed for pretreatment (baseline) and failure samples from participants with virologic failure. Results: HIV genotyping results were obtained for 211/249 participants (128 early ART arm and 83 delayed ART arm) with virologic failure. Drug resistance was detected in 4.7% of participants at baseline; 35.5% had new resistance at failure. In univariate analysis, the frequency of new resistance at failure was lower among participants in the early ART arm (compared with delayed ART arm, P = 0.06; compared with delayed ART arm with ART initiation before May 2011, P = 0.032). In multivariate analysis, higher baseline viral load (P = 0.0008) and ART regimen (efavirenz/lamivudine/zidovudine compared with other regimens, P = 0.024) were independently associated with higher risk of new resistance at failure. Conclusions: In HPTN 052, the frequency of new drug resistance at virologic failure was lower in adults with early ART initiation. The main factor associated with reduced drug resistance with early ART was lower baseline viral load.

Background South Africa has one of the highest rates of HIV-1 (HIV) infection world-wide, with the highest rates among young women. We analyzed the molecular epidemiology and evolutionary history of HIV in young women attending high school in rural South Africa. Methods Samples were obtained from the HPTN 068 randomized controlled trial, which evaluated the effect of cash transfers for school attendance on HIV incidence in women aged 13–20 years (Mpumalanga province, 2011–2015). Plasma samples from HIV-infected participants were analyzed using the ViroSeq HIV-1 Genotyping assay. Phylogenetic analysis was performed using 200 pol gene study sequences and 2,294 subtype C reference sequences from South Africa. Transmission clusters were identified using Cluster Picker and HIV-TRACE, and were characterized using demographic and other epidemiological data. Phylodynamic analyses were performed using the BEAST software. Results The study enrolled 2,533 young women who were followed through their expected high school graduation date (main study); some participants had a post-study assessment (follow-up study). Two-hundred-twelve of 2,533 enrolled young women had HIV infection. HIV pol sequences were obtained for 94% (n = 201/212) of the HIV-infected participants. All but one of the sequences were HIV-1 subtype C; the non-C subtype sequence was excluded from further analysis. Median pairwise genetic distance between the subtype C sequences was 6.4% (IQR: 5.6–7.2). Overall, 26% of study sequences fell into 21 phylogenetic clusters with 2–6 women per cluster. Thirteen (62%) clusters included women who were HIV-infected at enrollment. Clustering was not associated with study arm, demographic or other epidemiological factors. The estimated date of origin of HIV subtype C in the study population was 1958 (95% highest posterior density [HPD]: 1931–1980), and the median estimated substitution rate among study pol sequences was 1.98x10-3 (95% HPD: 1.15x10-3–2.81x10-3) per site per year. Conclusions Phylogenetic analysis suggests that multiple HIV subtype C sublineages circulate among school age girls in South Africa. There were no substantive differences in the molecular epidemiology of HIV between control and intervention arms in the HPTN 068 trial.

HIV Prevention Trials Network (HPTN) 052 demonstrated that antiretroviral therapy (ART) prevents HIV transmission in serodiscordant couples. HIV from index-partner pairs was analyzed to determine the genetic linkage status of partner infections. Forty-six infections were classified as linked, indicating that the index was the likely source of the partner’s infection. Lack of viral suppression and higher index viral load were associated with linked infection. Eight linked infections were diagnosed after the index started ART: four near the time of ART initiation and four after ART failure. Linked infections were not observed when the index participant was stably suppressed on ART.

Early initiation of antiretroviral treatment (ART) reduces HIV transmission and has health benefits. HIV drug resistance can limit treatment options and compromise use of ART for HIV prevention. We evaluated drug resistance in 85 participants in the HIV Prevention Trials Network 052 trial who started ART at CD4 counts of 350-550 cells per cubic millimeter and failed ART by May 2011; 8.2% had baseline resistance and 35.3% had resistance at ART failure. High baseline viral load and less education were associated with emergence of resistance at ART failure. Resistance at ART failure was observed in 7 of 8 (87.5%) participants who started ART at lower CD4 cell counts.

We analyzed antiretroviral drug susceptibility in HIV-infected adults failing first- and second-line antiretroviral treatment (ART) in Rakai, Uganda. Samples obtained from participants at baseline (pretreatment) and at the time of failure on first-line ART and second-line ART were analyzed using genotypic and phenotypic assays for antiretroviral drug resistance. Test results were obtained from 73 samples from 38 individuals (31 baseline samples, 36 first-line failure samples, and six second-line failure samples). Four (13%) of the 31 baseline samples had mutations associated with resistance to nucleoside or nonnucleoside reverse transcriptase inhibitors (NRTIs and NNRTIs, respectively). Among the 36 first-line failure samples, 31 (86%) had NNRTI resistance mutations and 29 (81%) had lamivudine resistance mutations; only eight (22%) had other NRTI resistance mutations. None of the six individuals failing a second-line protease inhibitor (PI)-based regimen had PI resistance mutations. Six (16%) of the participants had discordant genotypic and phenotypic test results. Genotypic resistance to drugs included in first-line ART regimens was detected prior to treatment and among participants failing first-line ART. PI resistance was not detected in individuals failing second-line ART. Surveillance for transmitted and acquired drug resistance remains a priority for scale-up of ART.

OBJECTIVE: Single-dose nevirapine (NVP) (sdNVP) can reduce the risk of HIV vertical transmission. We assessed risk factors for NVP resistance in plasma and breast milk from sdNVP-exposed Ugandan women. METHODS: Samples were analyzed using the Roche AMPLICOR HIV-1 Monitor Test Kit version 1.5 and the ViroSeq HIV-1 Genotyping System. NVP concentrations were determined by liquid chromatography with tandem mass spectroscopy. RESULTS: HIV genotypes (plasma and breast milk) were obtained for 30 women 4 weeks after sdNVP (HIV subtypes: 15A 1C 12D two recombinant). NVP resistance was detected in 12 (40%) of 30 breast milk samples. There was a nonsignificant trend between detection of NVP resistance in breast milk and plasma (P = 0.06). There was no association of HIV resistance in breast milk with median maternal pre-NVP viral load or CD4 cell count median breast milk viral load at 4 weeks breast milk sodium more than 10 mmol/l HIV subtype or concentration of NVP in breast milk or plasma. CONCLUSION: NVP resistance was frequently detected in breast milk 4 weeks after sdNVP exposure. In this study we were unable to identify specific factors associated with breast milk NVP resistance.

HIV-infected patients receiving antiretroviral (ARV) therapy (ART) in India are not all adequately virally suppressed. We analyzed ARV drug resistance in adults receiving ART in three private clinics in Mumbai, India. HIV viral load was measured in 200 patients with the Roche AMPLICOR HIV-1 Monitor Test, v1.5. HIV genotyping was performed with the ViroSeq HIV-1 Genotyping System for 61 participants who had HIV-1 RNA >1000 copies/ml. Genotyping results were obtained for 51 samples. The participants with resistance results were on ART for a median of 24 months and were on their current regimen for a median of 12 months (median CD4 cell count: 217 cells/mm3; median HIV viral load: 28,200 copies/ml). ARV regimens included nonnucleoside reverse transcriptase inhibitor (NNRTI)-based regimens (n = 27), dual nucleoside reverse transcriptase inhibitors (NRTIs, n = 19), protease inhibitor (PI)-based regimens (n = 3), and other regimens (n = 2). Twenty-six participants (51.0%) were on their first ARV regimen and 24 (47%) reported >95% adherence. Forty-nine participants (96.1%) had resistance to at least one ARV drug; 47 (92.2%) had NRTI resistance, 32 (62.7%) had NNRTI resistance, and four (7.8%) had PI resistance. Thirty (58.8%) had two-class resistance and three (5.9%) had three-class resistance. Four (8%) had three or more resistance mutations associated with etravirine resistance and two (4%) had two mutations associated with reduced darunavir susceptibility. Almost all patients with HIV-1 RNA >1000 copies/ml had NRTI resistance and nearly two-thirds had NNRTI resistance; PI resistance was uncommon. Nearly 60% and 6% had two- and three-class resistance, respectively. This emphasizes the need for greater viral load and resistance monitoring, use of optimal ART combinations, and increased availability of second- and third-line agents for patients with ARV resistance.

We compared the ability of the LigAmp assay and an ASPCR assay to detect and quantify K103N-containing HIV variants in samples from 63 women who received single-dose nevirapine in a clinical trial. Samples were first analyzed with the ViroSeq HIV Genotyping system, and ViroSeq PCR products were used as templates for the LigAmp and ASPCR assays. A cutoff of 0.5% K103N for detection of K103N was used for both assays. Results for the percentage K103N were similar for the two assays (R(2) = 0.92). Forty-six samples (73.0%) were positive for K103N by both assays and 13 samples (20.6%) were negative by both assays. Four samples (6.3%) were positive by ASPCR only. No samples were positive by LigAmp only. Eight discordant samples were analyzed in more detail. Sequence polymorphisms near oligonucleotide binding sites provided a possible explanation for the discordance in four of eight samples. The percentage K103N was also determined by analyzing 40 HIV clones from each of these eight samples, using a combined amplification/sequencing method (AmpliSeq). The percentage K103N determined by clonal analysis was consistent with the LigAmp result for five of eight samples, and was consistent with the ASPCR result for three of eight samples. Among 320 clones analyzed, we identified eight different codons at position 103 (mean = 3.8 codons/sample), which encoded six different amino acids, illustrating the extensive genetic diversity in HIV. Further studies are needed to compare performance of assays for detection and quantification of HIV drug resistance mutations in clinical samples.

K103N is frequently detected in HIV-infected women after single dose (SD) nevirapine (NVP). K103N-containing variants were detected more frequently by the ViroSeq HIV-1 Genotyping System in women with subtype C (69.2%) than subtypes A (19.4%, p < 0.0001) or D (36.1%, p < 0.0001). K103N-containing variants were also detected more frequently and at higher levels in women with subtype C by the LigAmp assay. In this report, we analyzed samples collected prior to or within hours after SD NVP administration from antiretroviral drug-naive African women with subtypes A, C, and D. Only 1/254 samples had an NVP resistance mutation detected with the ViroSeq system, and only 4/236 samples had K103N detected at < 0.5% with the LigAmp assay [2/110 (1.8%) with subtype A, 1/46 (2.2%) with subtype C, and 1/80 (1.3%) with subtype D] (p = 0.92). We did not detect significant differences in the pre-NVP frequency of NVP resistance mutations or the pre-NVP levels of K103N-containing variants in women with subtypes A, C, and D ...

Objective: Antiretroviral drug treatment may be complicated in individuals infected with antiretroviral drug-resistant or non-subtype B HIV-1 strains. HIV-1 tropism may also affect disease progression. We analyzed antiretroviral drug resistance, HIV-1 subtype, and HIV-1 tropism among 195 men who have sex with men from six major cities in the United States, using samples collected within 6 months of HIV-1 seroconversion (1999-2003). Methods: HIV-1 genotyping was performed using the ViroSeq HIV-1 Genotyping System. HIV-1 tropism was determined using a commercial assay. HIV-1 subtyping was performed by phylogenetic analysis of pol region sequences. Results: Thirty-one (15.9%) of the men had evidence of antiretroviral drug resistance. Seven (3.6%) men had multi-class resistance, including three (1.5%) with resistance to all three antiretroviral drug classes. We found no statistically significant association of antiretroviral drug resistance with demographic factors, sexual practices, self-reported sexually transmitted infections, use of recreational drugs, or use of antiretroviral drug post-exposure prophylaxis. All samples were HIV-1 subtype B. Four men had CXCR4-using HIV-1 strains. One man with a CXCR4-using strain also had antiretroviral drug resistance. Conclusions: Antiretroviral drug resistance is relatively common among recently infected men who have sex with men in the United States. CXCR4-using strains were detected in a small number of these infections, which were all subtype B HIV-1.

Replication capacity and transmission of human immunodeficiency virus (HIV)-1 in antiretroviral drug-naive Malawian women who had subtype C infection were investigated. Infant children of these women received either 1 dose of nevirapine or 1 dose of nevirapine plus 1 week of daily doses of zidovudine. PhenoSense HIV was used to determine replication capacity in 49 women whose infants were infected with HIV-1 and in 47 women whose infants were uninfected by 6-8 weeks of age. Mean replication capacity was higher in transmitters than in nontransmitters (P=.01). In a multivariate model, higher replication capacity was associated with transmission (odds ratio, 1.45 for each 10% increase in replication capacity [95% confidence interval, 1.11-1.90]; P = .0063), after adjustment for maternal HIV-1 load and other factors.

Objective. In the Human Immunodeficiency Virus (HIV) Network for Prevention Trials (HIVNET) 012 trial in Uganda, 6-8 weeks after single-dose nevirapine (SD-NVP), NVP resistance mutations were detected at a higher rate in women with HIV-1 subtype D than in women with subtype A. Here, we evaluate the rate of NVP resistance mutations in women with subtype C. Methods. NVP resistance mutations were detected using the ViroSeq HIV-1 Genotyping System. Results. The portion of women with any NVP resistance mutation was higher in those with subtype C (45/ 65 [69.2%] in the NVP and zidovudine trial, Malawi) than in those in the HIVNET 012 trial with either subtype A (28/144 [19.4%]; P

The HIVNET 012 trial in Uganda demonstrated that a regimen of single-dose nevirapine (NVP) can prevent HIV-1 mother-to-child transmission. Previous studies show that HIV-1 with one or more NVP resistance (NVPR) mutations can be selected in many women as early as 7 days after single-dose NVP. We evaluated the genetic linkage of NVPR mutations in plasma from women in HIVNET 012 collected 7 days after single-dose NVP administration. The HIV-1 pol region was amplified and cloned from 20 plasma samples (16 with NVPR mutations detected by population sequencing and 4 with no NVPR mutations detected), and 10 clones from each sample were sequenced. Up to five different NVPR mutations were detected in clones from a single sample. K103N and Y181C were the most common mutations detected. Clones with two genetically linked mutations were detected in four samples. Different combinations of NVPR mutations were linked in individual clones, but none of the clones contained both K103N and Y181C. Further studies are needed to evaluate whether selection of minority variants with one or more NVPR mutations after single-dose NVP is clinically relevant.

A prototype assay was used to genotype integrase (IN) from 120 HIV-1- infected IN inhibitor-naive adults from Argentina, Brazil, Cameroon, South Africa, Thailand, and Uganda. Subtype designations based on analysis of pol IN sequences were A (14), B (15), C (12), D (11), F (12), G (7), H (1), CRF01_AE (9), CRF02_AG (34), CRF22_01A1 (4), and CRF37_cpx (1). Ten (8.3%) of 120 samples had mutations associated with reduced susceptibility to the IN inhibitors, raltegravir and elvitegravir. Two samples had E92Q (both subtype B) and eight had E157Q (2A, 1C, 1D, 1F, 3 CRF02_AG). Some samples had other mutations selected by these drugs including T97A, and some had amino acid polymorphisms at positions associated with raltegravir and elvitegravir resistance. Mutations associated with other investigational HIV IN inhibitors were also identified. This suggests that HIV strains may vary in their natural susceptibility to HIV IN inhibitors.

The administration of single-dose nevirapine to women in labor and their infants can prevent HIV-1 mother-to-child transmission. We examined nevirapine resistance in infants who were HIV-1 infected despite single-dose nevirapine prophylaxis, including 18 Ugandan infants (HIVNET 012 trial, nine subtype A and nine subtype D) and 23 Malawian infants (NVAZ trial, all subtype C). Nevirapine resistance was more frequent in infants with subtype C than with subtypes A and D (87 versus 50%, P = 0.016).

Background Data describing the pharmacokinetics and safety of tenofovir in neonates are lacking. Methods The HIV Prevention Trials Network 057 protocol was a phase 1, open-label study of the pharmacokinetics and safety of tenofovir disoproxil fumarate (TDF) in HIV-infected women during labor and their infants during the first week of life with 4 dosing cohorts: maternal 600 mg doses/no infant dosing; no maternal dosing/infant 4 mg/kg doses on days 0, 3, and 5; maternal 900 mg doses/infant 6 mg/kg doses on days 0, 3, and 5; maternal 600 mg doses/infant 6 mg/kg daily for 7 doses. Pharmacokinetic sampling was performed on cohort 1 and 3 mothers and all infants. Plasma, amniotic fluid, and breast milk tenofovir concentrations were determined by liquid chromatographic-tandem mass spectrometric assay. The pharmacokinetic target was for infant tenofovir concentration throughout the first week of life to exceed 50 ng/mL, the median trough tenofovir concentration in adults receiving standard chronic TDF dosing. Results One hundred twenty-two mother-infant pairs from Malawi and Brazil were studied. Tenofovir exposure in mothers receiving 600 and 900 mg exceeded that in nonpregnant adults receiving standard 300 mg doses. Tenofovir elimination in the infants was equivalent to that in older children and adults, and trough tenofovir plasma concentrations exceeded 50 ng/mL in 74%-97% of infants receiving daily dosing. Conclusions A TDF dosing regimen of 600 mg during labor and daily infant doses of 6 mg/kg maintains infant tenofovir plasma concentration above 50 ng/mL throughout the first week of life and should be used in the studies of TDF efficacy for HIV prevention of mother-to-child transmission and early infant treatment.

Background. The HIV Prevention Trials Network (HPTN) 052 trial demonstrated that early initiation of antiretroviral therapy (ART) reduces human immunodeficiency virus (HIV) transmission from HIV-infected adults (index participants) to their HIV-uninfected sexual partners. We analyzed HIV from 38 index-partner pairs and 80 unrelated index participants (controls) to assess the linkage of seroconversion events. Methods. Linkage was assessed using phylogenetic analysis of HIV pol sequences and Bayesian analysis of genetic distances between pol sequences from index-partner pairs and controls. Selected samples were also analyzed using next-generation sequencing (env region). Results. In 29 of the 38 (76.3%) cases analyzed, the index was the likely source of the partner’s HIV infection (linked). In 7 cases (18.4%), the partner was most likely infected from a source other than the index participant (unlinked). In 2 cases (5.3%), linkage status could not be definitively established. Conclusions. Nearly one-fifth of the seroconversion events in HPTN 052 were unlinked. The association of early ART and reduced HIV transmission was stronger when the analysis included only linked events. This underscores the importance of assessing the genetic linkage of HIV seroconversion events in HIV prevention studies involving serodiscordant couples.

Background: Cross-sectional assessment of HIV incidence relies on laboratory methods to discriminate between recent and non-recent HIV infection. Because HIV diversifies over time in infected individuals, HIV diversity may serve as a biomarker for assessing HIV incidence. We used a high resolution melting (HRM) diversity assay to compare HIV diversity in adults with different stages of HIV infection. This assay provides a single numeric HRM score that reflects the level of genetic diversity of HIV in a sample from an infected individual. Methods: HIV diversity was measured in 203 adults: 20 with acute HIV infection (RNA positive, antibody negative), 116 with recent HIV infection (tested a median of 189 days after a previous negative HIV test, range 14–540 days), and 67 with nonrecent HIV infection (HIV infected .2 years). HRM scores were generated for two regions in gag, one region in pol, and three regions in env. Results: Median HRM scores were higher in non-recent infection than in recent infection for all six regions tested. In multivariate models, higher HRM scores in three of the six regions were independently associated with non-recent HIV infection. Conclusions: The HRM diversity assay provides a simple, scalable method for measuring HIV diversity. HRM scores, which reflect the genetic diversity in a viral population, may be useful biomarkers for evaluation of HIV incidence, particularly if multiple regions of the HIV genome are examined.

We analyzed HIV gp41 from 195 men in the United States who were HIV-1 infected between 1999 and 2002, before enfuvirtide (ENF) was approved for clinical use in the United States. gp41 genotyping results were obtained for 175 samples. None of the samples had major ENF resistance mutations. Six (3.4%) samples had minor ENF resistance mutations in the HR1 region (V38G, N43K, L44M, L45M). Twenty-eight (16%) samples had the N42S polymorphism, which is associated with ENF hypersusceptibility. Accessory mutations in the HR2 region were identified in some samples (E137K, S138A). Five of the six samples with HR1 resistance mutations were analyzed with a phenotypic assay; one sample had reduced ENF susceptibility (a sample with N42S + L44M + E137K). Prior to the availability of ENF, some men in the United States were infected with HIV that contained mutations associated with ENF resistance or hypersusceptibility. However, most of the mutations were not associated with phenotypic ENF resistance.

The HIV fusion inhibitor enfuvirtide (ENF/Fuzeon) targets the env gp41 transmembrane domain. Mutations in gp41 are associated with ENF resistance. We developed a prototype assay to genotype a 676-bp region spanning the heptad repeat domains (HR1 and HR2) of HIV-1 gp41. Plasma samples were collected from 126 HIV-1-infected blood donors in Cameroon, Brazil, Uganda, South Africa, Thailand, and Argentina. Based on analysis of gag p24, pol integrase, and env gp41 genes, the panel was composed of subtypes A/A2 (18), B (11), C (14), D (10), F/F2 (9), G (7), CRF01_AE (9), CRF02_AG (33), and recombinant strains (15). Genotyping was successful for 119 of the 126 samples (94.4%). Although numerous amino acid polymorphisms were detected in some samples, none had primary mutations associated with ENF resistance. The gp41 HIV-1 research reagents developed by Celera are useful tools for genotyping analysis of the gp41 region in diverse HIV-1 strains.

K103N is frequently detected in HIV-infected women after single dose (SD) nevirapine (NVP). K103N-containing variants were detected more frequently by the ViroSeq HIV-1 Genotyping System in women with subtype C (69.2%) than subtypes A (19.4%, p0.0001) or D (36.1%, p0.0001). K103N-containing variants were also detected more frequently and at higher levels in women with subtype C by the LigAmp assay. In this report, we analyzed samples collected prior to or within hours after SD NVP administration from antiretroviral drug-naive African women with subtypes A, C, and D. Only 1/254 samples had an NVP resistance mutation detected with the ViroSeq system, and only 4/236 samples had K103N detected at0.5% with the LigAmp assay [2/110 (1.8%) with subtype A, 1/46 (2.2%) with subtype C, and 1/80 (1.3%) with subtype D] (p = 0.92). We did not detect significant differences in the pre-NVP frequency of NVP resistance mutations or the pre-NVP levels of K103N-containing variants in women with subtypes A, C, and D that explain the dramatic subtype-based differences in emergence of HIV-1 variants with these mutations after SD NVP exposure.

We analyzed the development of nevirapine (NVP) resistance in human immunodeficiency virus type 1 (HIV-1)-infected Malawian infants who received regimens containing single-dose NVP (SD-NVP) for the prevention of mother-to-child transmission (MTCT) of HIV-1. All infants received SD-NVP, and some randomly received zidovudine (ZDV) as well. Mothers did or did not receive SD-NVP on the basis of when they arrived at the hospital for delivery. In infants 6-8 weeks of age, NVP resistance was less frequent when infants had received SD-NVP plus ZDV and mothers had not received SD-NVP than when infants had received SD-NVP alone and mothers had received SD-NVP (4/15 [27%] vs. 20/23 [87%]; P < .001). The risk of MTCT of HIV-1 was comparable with these regimens. Infant-only prophylaxis also eliminates the development of NVP resistance in mothers.

India has the second largest burden of HIV-1-infected persons worldwide. Access to antiretroviral drugs in India is increasing. We analyzed HIV-1 protease and reverse transcriptase sequences in 12 acute seroconverters from Pune, India, and evaluated HIV-1 evolution in these individuals over time. HIV-1 genotyping was performed with the ViroSeq HIV-1 Genotyping System. Baseline samples, collected between 1999 and 2001, had viral loads from 3,523 to 8,556,280 copies/ml. All subjects had subtype C HIV-1. None of the samples had primary drug resistance mutations. The sequence identity between baseline and 1-year samples ranged from 99.7% to 99.9%, and between baseline and 2-year samples ranged from 99.4% to 100%. Most of the nucleotide changes were silent (synonymous). Amino acid substitutions were rare, and varied from subject to subject. In this cohort, drug resistance was not observed and evolution in the pol region was very limited during the first 2 years of infection.

In the HPTN 052 study, transmission between HIV-discordant couples was reduced by 96% when the HIV-infected partner received suppressive antiretroviral therapy (ART). We examined two transmission events where the newly infected partner was diagnosed after the HIV-infected partner (index) initiated therapy. We evaluated the sequence complexity of the viral populations and antibody reactivity in the newly infected partner to estimate the dates of transmission to the newly infected partners. In both cases, transmission most likely occurred significantly before HIV-1 diagnosis of the newly infected partner, and either just before the initiation of therapy or before viral replication was adequately suppressed by therapy of the index. This study further strengthens the conclusion about the efficacy of blocking transmission by treating the infected partner of discordant couples. However, this study does not rule out the potential for HIV-1 transmission to occur shortly after initiation of ART, and this should be recognized when antiretroviral therapy is used for HIV-1 prevention.