Search

Phylogenetic analysis of pathogens is an increasingly powerful way to reduce the spread of epidemics, including HIV. As a result, phylogenetic approaches are becoming embedded in public health and research programmes, as well as outbreak responses, presenting unique ethical, legal, and social issues that are not adequately addressed by existing bioethics literature. We formed a multidisciplinary working group to explore the ethical issues arising from the design of, conduct in, and use of results from HIV phylogenetic studies, and to propose recommendations to minimise the associated risks to both individuals and groups. We identified eight key ethical domains, within which we highlighted factors that make HIV phylogenetic research unique. In this Review, we endeavoured to provide a framework to assist researchers, public health practitioners, and funding institutions to ensure that HIV phylogenetic studies are designed, done, and disseminated in an ethical manner. Our conclusions also have broader relevance for pathogen phylogenetics.

Background Human immunodeficiency virus (HIV) remains a global challenge and novel measures for transmission disruption are needed. Contact tracing is limited by reluctance or inability of newly diagnosed individuals to name at-risk contacts. Molecular cluster analysis is mostly used for outbreak investigations, and its role in routine public health activities remains uncertain. Methods We conducted a 2-year prospective statewide study in Rhode Island to evaluate integration of HIV cluster analyses into routine contact tracing, by attempting to reinterview all new diagnoses who clustered, notifying them of clustering, and evaluating benefits of this strategy. Clustering was compared between a phylogenetic ensemble versus distance-based HIV-TRACE. Results Of 100 new diagnoses during 2021–2022, 52 individuals clustered, of whom only 31% were reinterviewed. Reinterviewing did not improve contact tracing beyond initial interviews, and the study was stopped early for futility. Clustering concordance within the phylogenetic ensemble was high (88%–89%), but lower (74%) for HIV-TRACE. Despite hypothesis rejection, we established a public health–academic partnership, developed a bioinformatics pipeline enabling near real-time cluster analysis, and identified gaps and unique opportunities for intervention. Conclusions Attempting to reinterview all statewide new HIV diagnoses in molecular clusters showed no evidence of improving contact tracing. However, a strong academic–public health partnership enabled near real-time, longitudinal integration of molecular cluster analysis into routine public health activities, and identified barriers and opportunities tailoring data-driven approaches to unique individual and community characteristics, guiding future work on optimal use of molecular epidemiology to disrupt HIV transmission. [ABSTRACT FROM AUTHOR]

Background: The use of molecular HIV cluster analysis to supplement public health contact tracing has shown promise in addressing HIV outbreaks. However, the potential of HIV cluster analysis as an adjunct to daily, person-by-person HIV prevention efforts remains unknown. We documented lessons learned within a unique public health-academic partnership while guiding workaday HIV prevention efforts with near-real-time molecular cluster analysis. Setting: A public health-academic partnership in the State of Rhode Island, the United States. Methods: We recorded perceptions of our team of academicians and public health practitioners that were encountered in an 18-month study evaluating the integration of molecular cluster analysis with HIV contact tracing for public health benefit. The focus was on monthly conferences where molecular clustering of each new statewide diagnosis was discussed to facilitate targeted interventions and on attempted reinterviews of all newly HIV-diagnosed persons statewide whose HIV sequences clustered to increase partner naming. Results: Three main themes emerged: First, multidisciplinary conferences are substantially beneficial for gleaning actionable inferences from integrating molecular cluster analysis and public health data. Second, universal reinterviews were perceived to potentially have negative consequences but may be selectively beneficial. Third, the translation of cluster analysis into public health action is hampered by jurisdictional surveillance boundaries and within-jurisdictional data silos, across which data sharing is problematic. Conclusions: Insights from a statewide public health-academic partnership support integration of molecular HIV cluster analyses with public health efforts, which can guide public health activities to prevent transmission while identifying substantial barriers to integration, informing continued research. [ABSTRACT FROM AUTHOR]

Justice-involved (JI) populations bear a disproportionate burden of HIV infection and are at risk of poor treatment outcomes. Drug resistance prevalence and emergence, and phylogenetic inference of transmission networks, understudied in vulnerable JI populations, can inform care and prevention interventions, particularly around the critical community reentry period. We analyzed banked blood specimens from CARE+ Corrections study participants in Washington, D.C. (DC) across three time points and conducted HIV drug resistance testing using next-generation sequencing (NGS) at 20% and 5% thresholds to identify prevalent and evolving resistance during community reentry. Phylogenetic analysis was used to identify molecular clusters within participants, and in an extended analysis between participants and publicly available DC sequences. HIV sequence data from 54 participants (99 specimens) were analyzed. The prevalence of transmitted drug resistance was 14% at both thresholds, and acquired drug resistance was 47% at 20%, and 57% at 5% NGS thresholds, respectively. The overall prevalence of drug resistance was 43% at 20%, and 52% at 5% NGS thresholds, respectively. Among 34 participants sampled longitudinally, 21%–35% accumulated 10–17 new resistance mutations during a mean 4.3 months. In phylogenetic analysis within the JI population, 11% were found in three molecular clusters. The extended phylogenetic analysis identified 46% of participants in 22 clusters, of which 21 also included publicly-available DC sequences, and one JI-only unique dyad. This is the first study to identify a high prevalence of HIV drug resistance and its accumulation in a JI population during community reentry and suggests phylogenetic integration of this population into the non-JI DC HIV community. These data support the need for new, effective, and timely interventions to improve HIV treatment during this vulnerable period, and for JI populations to be included in broader surveillance and prevention efforts. [ABSTRACT FROM AUTHOR]

Although there have been many studies of gene variant association with different stages of HIV/AIDS progression in United States and European cohorts, few gene-association studies have assessed genic determinants in sub-Saharan African populations, which have the highest density of HIV infections worldwide. We carried out genome-wide association studies on 766 study participants at risk for HIV-1 subtype C (HIV-1C) infection in Botswana. Three gene associations (AP3B1, PTPRA, and NEO1) were shown to have significant association with HIV-1C acquisition. Each gene association was replicated within Botswana or in the United States-African American or United States-European American AIDS cohorts or in both. Each associated gene has a prior reported influence on HIV/AIDS pathogenesis. Thirteen previously discovered AIDS restriction genes were further replicated in the Botswana cohorts, extending our confidence in these prior AIDS restriction gene reports. This work presents an early step toward the identification of genetic variants associated with and affecting HIV acquisition or AIDS progression in the understudied HIV-1C afflicted Botswana population. [ABSTRACT FROM AUTHOR]

Motivated by the need to jointly model the longitudinal trajectories of HIV viral load levels and CD4 counts during the primary infection stage, we propose a joint penalized spline modeling approach that can be used to model the repeated measurements from multiple biomarkers of various types (eg, continuous, binary) simultaneously. This approach allows for flexible trajectories for each marker, accounts for potentially time‐varying correlation between markers, and is robust to misspecification of knots. Despite its advantages, the application of multivariate penalized spline models, especially when biomarkers may be of different data types, has been limited in part due to its seemingly complexity in implementation. To overcome this, we describe a procedure that transforms the multivariate setting to the univariate one, and then makes use of the generalized linear mixed effect model representation of a penalized spline model to facilitate its implementation with standard statistical software. We performed simulation studies to evaluate the validity and efficiency through joint modeling of correlated biomarkers measured longitudinally compared to the univariate modeling approach. We applied this modeling approach to longitudinal HIV‐1 RNA load and CD4 count data from Southern African cohorts to estimate features of the joint distributions such as the correlation and the proportion of subjects with high viral load levels and high CD4 cell counts over time. [ABSTRACT FROM AUTHOR]

Purpose: CYP2B6 liver enzyme metabolizes the two non-nucleoside reverse transcriptase inhibitors Efavirenz (EFV) and Nevirapine (NVP) used in the antiretroviral therapy (ART) regimens for HIV-infected individuals. Polymorphisms of the CYP2B6 gene influence drug levels in plasma and possibly virological outcomes. The aim of this study was to explore the potential impact of CYP2B6 genotype and haplotype variation on the risk of developing EFV/NVP drug resistance mutations (DRMs) in HIV-1 patients receiving EFV-/NVP-containing regimens in Botswana. Patients and Methods: Participants were a sub-sample of a larger study (Tshepo study) conducted in Gaborone, Botswana, among HIV-infected individuals taking EFV/NVP containing ART. Study samples were retrieved and assigned to cases (with DRMs) and controls (without DRMs). Four single-nucleotide polymorphisms (SNPs) in the CYP2B6 gene (− 82T>C; 516G>T; 785A>G; 983T>C) were genotyped, the haplotypes reconstructed, and the metabolic score assigned. The possible association between drug resistance and several independent factors (baseline characteristics and CYP2B6 genotypes) was assessed by Binary Logistic Regression (BLR) analysis. EFV/NVP resistance status and CYP2B6 haplotypes were also analyzed using Z-test, chi-square and Fisher's exact test statistics. Results: Two hundred and twenty-seven samples were analysed (40 with DRMs, 187 without DRMs). BLR analysis showed an association between EFV/NVP resistance and CYP2B6 516G allele (OR: 2.26; 95% CI: 1.27– 4.01; P=0.005). Moreover, haplotype analysis revealed that the proportion of EFV/NVP-resistant infections was higher among CYP2B6 fast than extensive/slow metabolizers (30.8% vs 16.8%; P= 0.035), with the 516G allele more represented in the haplotypes of fast than extensive/slow metabolizers (100.0% vs 53.8%; P< 0.001). Conclusion: We demonstrated that the CYP2B6 516G allele, and even more when combined in fast metabolic haplotypes, is associated with the presence of EFV/NVP resistance, strengthening the need to assess the CYP2B6 genetic profiles in HIV-infected patients in order to improve the virologic outcomes of NNRTI containing ART. [ABSTRACT FROM AUTHOR]

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/mm3. 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. [ABSTRACT FROM AUTHOR]

Great efforts are devoted to end the HIV epidemic as it continues to have profound public health consequences in the United States and throughout the world, and new interventions and strategies are continuously needed. The use of HIV sequence data to infer transmission networks holds much promise to direct public heath interventions where they are most needed. As these new methods are being implemented, evaluating their benefits is essential. In this paper, we recognize challenges associated with such evaluation, and make the case that overcoming these challenges is key to the use of HIV sequence data in routine public health actions to disrupt HIV transmission networks. [ABSTRACT FROM AUTHOR]

Introduction: Non-citizens often face barriers to HIV care and treatment. Quantifying knowledge of positive HIV status and antiretroviral therapy (ART) coverage among non-citizens in a high HIV-prevalence country like Botswana that is close to achieving UNAIDS “90-90-90” targets may expose important gaps in achieving universal HIV testing and treatment. Methods: The Botswana Combination Prevention Project (BCPP) is a pair-matched cluster-randomized trial evaluating the impact of prevention interventions on HIV incidence in 30 rural or peri-urban communities. Community case finding and HIV testing were conducted in home and mobile venues in 15 intervention communities from October 2013-September 2017. In this secondary analysis, we compared HIV positivity, knowledge of positive HIV-status, and ART status among all citizens and non-citizens assessed at intake in the intervention communities. Results: HIV status was assessed in 57,556 residents in the intervention communities; 4% (n = 2,463) were non-citizens. Five communities accounted for 81% of the total non-citizens assessed. A lower proportion of non-citizens were HIV-positive (15%; n = 369) compared to citizens (21%; n = 11,416) [p = 0.026]; however, a larger proportion of non-citizens did not know their HIV-positive status prior to BCPP testing (75%) as compared to citizens (15%) [p = 0.003]. Among residents with knowledge of their HIV-positive status before BCPP, 79% of the non-citizens (72/91) were on ART compared to 86% (8,267/9,652) of citizens (p = 0.137). Conclusions: Although non-citizens were less likely to know their HIV-positive status compared to citizens, there were no differences in treatment uptake among non-citizens and citizens who knew their status. Designing interventions for non-citizens that provide HIV testing and treatment services commensurate to that of citizens as well as targeting communities with the largest number of non-citizens may help close a meaningful gap in the HIV care cascade and ensure ethical treatment for all HIV-positive persons. Trial registration: ClinicalTrials.gov: (Botswana Combination Prevention Project). [ABSTRACT FROM AUTHOR]

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

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. [ABSTRACT FROM AUTHOR]

Linkage analysis is useful in investigating disease transmission dynamics and the effect of interventions on them, but estimates of probabilities of linkage between infected people from observed data can be biased downward when missingness is informative. We investigate variation in the rates at which subjects' viral genotypes link across groups defined by viral load (low/high) and antiretroviral treatment (ART) status using blood samples from household surveys in the Northeast sector of Mochudi, Botswana. The probability of obtaining a sequence from a sample varies with viral load; samples with low viral load are harder to amplify. Pairwise genetic distances were estimated from aligned nucleotide sequences of HIV-1C env gp120. It is first shown that the probability that randomly selected sequences are linked can be estimated consistently from observed data. This is then used to develop estimates of the probability that a sequence from one group links to at least one sequence from another group under the assumption of independence across pairs. Furthermore, a resampling approach is developed that accounts for the presence of correlation across pairs, with diagnostics for assessing the reliability of the method. Sequences were obtained for 65% of subjects with high viral load (HVL, n = 117), 54% of subjects with low viral load but not on ART (LVL, n = 180), and 45% of subjects on ART (ART, n = 126). The probability of linkage between two individuals is highest if both have HVL, and lowest if one has LVL and the other has LVL or is on ART. Linkage across groups is high for HVL and lower for LVL and ART. Adjustment for missing data increases the group-wise linkage rates by 40–100%, and changes the relative rates between groups. Bias in inferences regarding HIV viral linkage that arise from differential ability to genotype samples can be reduced by appropriate methods for accommodating missing data. [ABSTRACT FROM AUTHOR]

The study estimated the prevalence of HIV-1 intra-subtype recombinant variants among female bar and hotel workers in Tanzania. While intra-subtype recombination occurs in HIV-1, it is generally underestimated. HIV-1 env gp120 V1-C5 quasispecies from 45 subjects were generated by single-genome amplification and sequencing (median (IQR) of 38 (28–50) sequences per subject). Recombination analysis was performed using seven methods implemented within the recombination detection program version 3, RDP3. HIV-1 sequences were considered recombinant if recombination signals were detected by at least three methods with p-values of ≤0.05 after Bonferroni correction for multiple comparisons. HIV-1 in 38 (84%) subjects showed evidence for intra-subtype recombination including 22 with HIV-1 subtype A1, 13 with HIV-1 subtype C, and 3 with HIV-1 subtype D. The distribution of intra-patient recombination breakpoints suggested ongoing recombination and showed selective enrichment of recombinant variants in 23 (60%) subjects. The number of subjects with evidence of intra-subtype recombination increased from 29 (69%) to 36 (82%) over one year of follow-up, although the increase did not reach statistical significance. Adjustment for intra-subtype recombination is important for the analysis of multiplicity of HIV infection. This is the first report of high prevalence of intra-subtype recombination in the HIV/AIDS epidemic in Tanzania, a region where multiple HIV-1 subtypes co-circulate. HIV-1 intra-subtype recombination increases viral diversity and presents additional challenges for HIV-1 vaccine design. [ABSTRACT FROM AUTHOR]

This study analyzed the distribution and prevalence of HIV-1 subtypes, multiplicity of HIV-1 infection, and frequency of inter-subtype recombination among HIV-1-infected female bar and hotel workers in Moshi, Kilimanjaro Region, Tanzania, from 2004 to 2007. The HIV-1 viral sequences spanning the V1-C5 region of HIV-1 env gp120 were analyzed from 50 subjects by single genome amplification and sequencing (SGA/S) technique. A total of 1740 sequences were amplified and sequenced from the HIV-1 proviral DNA template. The median env sequences analyzed per subject per two time points was 38 (IQR 28-50) over one year of HIV infection. In a subset of 14 subjects, a total of 239 sequences were obtained from HIV-1 RNA template at the baseline visit. The most prevalent HIV-1 subtypes were A1 (56%) and C (30%), while HIV-1 subtype D and inter-subtype recombinant viruses were found in 6% and 8% of subjects respectively. Transmission of multiple HIV-1 variants was evident in 27% of the subjects infected with pure HIV-1 subtypes A1, C, or D. The HIV-1 inter-subtype recombinants were found in 8% including HIV-1 C/A, D/A, and complex mosaic recombinants. Multiple viral variants were found in two subjects infected with inter-subtype recombinants. One subject harbored quasispecies of both pure HIV-1 A1 and C/A recombinant. The other subject was infected with two complex mosaic inter-subtype recombinant variants belonging to subtype D. HIV-1 multiple infections and ongoing recombination contribute significantly to the genetic diversity of circulating HIV-1 in Tanzania and have important implications for vaccine design and the development of therapeutic strategies. [ABSTRACT FROM AUTHOR]

Exclusive breastfeeding has been associated with a reduced risk of late vertical HIV transmission as compared to an infant diet composed of breast milk mixed with supplemental foods or liquids. Hypothesized mechanisms include increased infectivity of breast milk from mothers who practice mixed breastfeeding (MBF), or mechanisms such as increased gastrointestinal permeability in the infant caused by mixed feeding. It has been proposed that MBF may result in subclinical mastitis and higher breast milk HIV titers. However, little is known about the relationship between feeding strategy and breast milk viral load. We measured the HIV-1 concentration in breast milk in a sub-cohort of women enrolled in a mother-to-child HIV transmission prevention trial (the 'Mashi' study). We report no observed relationship between MBF and measured breast milk viral RNA load. Our findings suggest that the increased transmission risk associated with higher breast milk HIV-1 RNA during MBF is unlikely. [ABSTRACT FROM AUTHOR]

Abstract: Two analyses of HIV-1 subtype C Gag quasispecies were performed in a prospective cohort of 42 acutely and recently infected individuals by SGA on viral RNA/proviral DNA templates. First, in vivo Gag substitutions were assessed in relation to the HIV-1C consensus sequence, which revealed that 29.3% of detected amino acid substitutions can be classified as reversions to subtype consensus, 61.3% as forward substitutions from subtype consensus, and 9.3% as polymorphisms not associated with the subtype consensus sequence. Second, the proportion, dynamics, and relationships within individual pools of viral quasispecies were analyzed. Among reverse substitutions, 16.1% were minor, 11.0% transient, 13.6% dominant, and 59.2% fixed. In contrast, 31.6% of forward substitutions were minor, 59.3% transient, 3.8% dominant, and 5.3% fixed. The distinct patterns in the spectrum and dynamics of reverse and forward Gag substitutions suggest that these differences should be considered in HIV-1 evolutionary studies and analyses of viral mutational pathways. [Copyright &y& Elsevier]

Risk factors for mother-to-child transmission (MTCT) of human immunodeficiency virus (HIV) via breast-feeding were evaluated in a randomized trial. HIV-infected women and their infants received zidovudine as well as single-dose nevirapine or placebo. Infants were randomized to formula-feed (FF) or breast-feed (BF) in combination with zidovudine prophylaxis. Of 1116 at-risk infants, 6 (1.1%) in the FF group and 7 (1.3%) in the BF group were infected between birth and 1 month (P = .99). Maternal receipt of nevirapine did not predict early MTCT in the BF group (P = .45). Of 547 infants in the BF group at risk for late MTCT, 24 (4.4%) were infected. Maternal HIV-1 RNA levels in plasma (P<.001) and breast milk (P<.001) predicted late MTCT. These findings support the safety of 1 month of breast-feeding in combination with maternal and infant antiretroviral prophylaxis. [ABSTRACT FROM AUTHOR]

The article focuses on the viral dynamics in primary human immunodeficiency virus-1 (HIV-1). It notes that most studies on primary HIV-1 infection does not delve on the evolution of viral parameters in the early phase of HIV-1 subtype C infection. It characterizes the kinetics of viral ribonucleic acid (RNA), proviral deoxyribonucleic acid (DNA), CD4+ T-cell count, and subsets of CD4+ T cells. It yields better understanding of the early phase of subtype C infection.

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

To assess the level of intra-patient diversity and evolution of HIV-1C non-structural genes in primary infection, viral quasispecies obtained by single genome amplification (SGA) at multiple sampling timepoints up to 500 days post-seroconversion (p/s) were analyzed. The mean intra-patient diversity was 0.11% (95% CI; 0.02 to 0.20) for vif, 0.23% (95% CI; 0.08 to 0.38) for vpr, 0.35% (95% CI; −0.05 to 0.75) for vpu, 0.18% (95% CI; 0.01 to 0.35) for tat exon 1 and 0.30% (95% CI; 0.02 to 0.58) for rev exon 1 during the time period 0 to 90 days p/s. The intra-patient diversity increased gradually in all non-structural genes over the first year of HIV-1 infection, which was evident from the vif mean intra-patient diversity of 0.46% (95% CI; 0.28 to 0.64), vpr 0.44% (95% CI; 0.24 to 0.64), vpu 0.84% (95% CI; 0.55 to 1.13), tat exon 1 0.35% (95% CI; 0.14 to 0.56 ) and rev exon 1 0.42% (95% CI; 0.18 to 0.66) during the time period of 181 to 500 days p/s. There was a statistically significant increase in viral diversity for vif (p = 0.013) and vpu (p = 0.002). No associations between levels of viral diversity within the non-structural genes and HIV-1 RNA load during primary infection were found. The study details the dynamics of the non-structural viral genes during the early stages of HIV-1C infection.

To address whether sequences of viral gag and env quasispecies collected during the early post-acute period can be utilized to determine multiplicity of transmitted HIV's, recently developed approaches for analysis of viral evolution in acute HIV-1 infection [1], [2] were applied. Specifically, phylogenetic reconstruction, inter- and intra-patient distribution of maximum and mean genetic distances, analysis of Poisson fitness, shape of highlighter plots, recombination analysis, and estimation of time to the most recent common ancestor (tMRCA) were utilized for resolving multiplicity of HIV-1 transmission in a set of viral quasispecies collected within 50 days post-seroconversion (p/s) in 25 HIV-infected individuals with estimated time of seroconversion. The decision on multiplicity of HIV infection was made based on the model's fit with, or failure to explain, the observed extent of viral sequence heterogeneity. The initial analysis was based on phylogeny, inter-patient distribution of maximum and mean distances, and Poisson fitness, and was able to resolve multiplicity of HIV transmission in 20 of 25 (80%) cases. Additional analysis involved distribution of individual viral distances, highlighter plots, recombination analysis, and estimation of tMRCA, and resolved 4 of the 5 remaining cases. Overall, transmission of a single viral variant was identified in 16 of 25 (64%) cases, and transmission of multiple variants was evident in 8 of 25 (32%) cases. In one case multiplicity of HIV-1 transmission could not be determined. In primary HIV-1 subtype C infection, samples collected within 50 days p/s and analyzed by a single-genome amplification/sequencing technique can provide reliable identification of transmission multiplicity in 24 of 25 (96%) cases. Observed transmission frequency of a single viral variant and multiple viral variants were within the ranges of 64% to 68%, and 32% to 36%, respectively.

Background Understanding drug resistance in perinatally HIV-infected children (PHIC) when viral load (VL) monitoring is limited is critical for life-long antiretroviral use. Resistance data in PHIC in sub-Saharan Africa are limited. Though guidelines recommend PI-based first-line regimens in PHIC, many worldwide remain on NNRTI-based regimens. We examined treatment failure, resistance, and outcomes in Kenyan PHIC on first-line NNRTI-based therapy. Methods PHIC were enrolled in 2010–2013 at the Academic Model Providing Access to Healthcare in Eldoret, Kenya, a large program caring for >160,000 HIV patients; >15,000 PHIC. VL testing, not routinely available then, was done for all, and resistance testing was done in viremic PHIC. Clinical data were derived from medical records. Subtype and resistance interpretation were with Stanford Database tools. Associations between failure (>1,000 copies/mL) or resistance, and demographic, clinical or lab variables were evaluated with Fisher exact and Wilcoxon rank-sum tests. Results Of 482 PHIC enrolled, 52% were female, median age 8.4 years (range 1–15), median CD4% 28 (range 0–53), 79% on zidovudine (AZT)/abacavir (ABC)+lamivudine(3TC)+efavirenz (EFV)/nevirapine (NVP) for median 2.3 years. Treatment failure was seen in 31%, associated with low CD4% and count. Genotypes were available in 124, 47% female, median age 8.3 years (range 2–15), median CD4% 22 (range 0–45), 81% on AZT/ABC+3TC+EFV/NVP for median 2.5 years, median VL 7,515 copies/mL. Subtypes were A 76%, C 3%, D 15%, recombinants 6%. Reverse transcriptase mutations were in 93%; 93%-NNRTIs, median 2/patient, most common Y181C (44%); 89%-NRTIs, median 3/patient, most common M184V (85%); 89%-dual class, median 5/patient. Intermediate-high resistance to potential second-line drugs included 62% etravirine, 66% rilpivirine, and 19% tenofovir. Of 92/124 (74%) PHIC with follow-up data, 27% remained on NNRTI-based first-line (median CD4 count 461), of whom 24% had suppressed VL and 48% died; and 73% switched to PI-based second-line (median CD4 count 591), of whom 72% had suppressed VL and 6% died (P < 0.05 for both). Conclusion PHIC in western Kenya on NNRTI-based first-line regimens had high treatment failure rates and extensive drug resistance with poor clinical outcomes, demanding urgent interventions. Disclosures All Authors: No reported Disclosures. [ABSTRACT FROM AUTHOR]

Background: Mathematical models predict that community-wide access to HIV testing-and-treatment can rapidly and substantially reduce new HIV infections. Yet several large universal test-and-treat HIV prevention trials in high-prevalence epidemics demonstrated variable reduction in population-level incidence. Methods: To elucidate patterns of HIV spread in universal test-and-treat trials, we quantified the contribution of geographic-location, gender, age, and randomized-HIV-intervention to HIV transmissions in the 30-community Ya Tsie trial in Botswana. We sequenced HIV viral whole genomes from 5114 trial participants among the 30 trial communities. Results: Deep-sequence phylogenetic analysis revealed that most inferred HIV transmissions within the trial occurred within the same or between neighboring communities, and between similarly aged partners. Transmissions into intervention communities from control communities were more common than the reverse post-baseline (30% [12.2 - 56.7] vs. 3% [0.1 - 27.3]) than at baseline (7% [1.5 - 25.3] vs. 5% [0.9 - 22.9]) compatible with a benefit from treatment-as-prevention. Conclusions: Our findings suggest that population mobility patterns are fundamental to HIV transmission dynamics and to the impact of HIV control strategies. [ABSTRACT FROM AUTHOR]

Supplemental Digital Content is Available in the Text. Background: Long-term impact of drug resistance in perinatally infected children and adolescents living with HIV (CALWH) is poorly understood. We determined drug resistance and examined its long-term impact on failure and mortality in Kenyan CALWH failing first-line non-nucleoside reverse transcriptase inhibitor–based antiretroviral therapy (ART). Setting: Academic Model Providing Access to Healthcare, western Kenya. Methods: Participants were enrolled in 2010–2013 (timepoint 1) and a subsample re-enrolled after 4–7 years (timepoint 2). Viral load (VL) was performed on timepoint 1 samples, with genotyping of those with detectable VL. Primary endpoints were treatment failure (VL >1000 copies/mL) at and death before timepoint 2. Multinomial regression analysis was used to characterize resistance effect on death, failure, and loss-to-follow-up, adjusting for key variables. Results: The initial cohort (n = 480) was 52% (n = 251) female, median age 8 years, median CD4% 31%, 79% (n = 379) on zidovudine/abacavir + lamivudine + efavirenz/nevirapine for median 2 years. Of these, 31% (n = 149) failed at timepoint 1. Genotypes at timepoint 1, available on n = 128, demonstrated 93% (n = 119) extensive resistance, affecting second line. Of 128, 22 failed at timepoint 2, 17 died, and 32 were lost to follow-up before timepoint 2. Having >5 resistance mutations at timepoint 1 was associated with higher mortality [relative risk ratio (RRR) = 8.7, confidence interval (CI) 2.1 to 36.3] and loss to follow-up (RRR = 3.2, CI 1.1 to 9.2). Switching to second line was associated with lower mortality (RRR <0.05, CI <0.05 to 0.1) and loss to follow-up (RRR = 0.1, CI <0.05 to 0.3). Conclusion: Extensive resistance and limited switch to second line in perinatally infected Kenyan CALWH failing first-line ART were associated with long-term failure and mortality. Findings emphasize urgency for interventions to sustain effective, life-long ART in this vulnerable population. [ABSTRACT FROM AUTHOR]

Abstract: HIV-1 subtype C (HIV-1C) CXCR4-using virus is isolated infrequently and is poorly characterized. Understanding HIV-1C env characteristics has implications for the clinical use of antiretrovirals that target viral entry. A total of 209 env clones derived from 10 samples with mixed CCR5-(R5), CXCR4-using (X4) or dual-tropic HIV-1C were phenotyped for coreceptor usage. Intra-patient X4 and R5 variants generally formed distinct monophyletic phylogenetic clusters. X4 compared to R5 envs had significantly greater amino acid variability and insertions, higher net positive charge, fewer glycosylation sites and increased basic amino acid substitutions in the GPGQ crown. Basic amino acid substitution and/or insertion prior to the crown are highly sensitive characteristics for predicting X4 viruses. Chimeric env functional studies suggest that the V3 loop is necessary but often not sufficient to impart CXCR4 utilization. Our studies provide insights into the unique genotypic characteristics of X4 variants in HIV-1C. [Copyright &y& Elsevier]

The design of antiviral strategies against human immunodeficiency virus type 1 (HIV-1) has been largely derived from studies of subtype B viruses, although they constitute only 12% of infections worldwide. At 50% of all HIV-1 infections worldwide, subtype C viruses are the most predominant. Here, we present evidence that subtype C Nefs display functional Pak2-activating motifs that differ from those found in subtype B and E Nefs. The identification of multiple Pak2-activating structural motifs that singly affect one Nef activity revealed a functional plasticity that has implications for future drug and vaccine design aimed at HIV-1 Nef and its effects on the deregulation of the immune system. [ABSTRACT FROM AUTHOR]

Abstract: Replication-defective adenoviruses have been utilized as candidate HIV vaccine vectors. Few studies have described the international epidemiology of pre-existing immunity to adenoviruses. We enrolled 1904 participants in a cross-sectional serological survey at seven sites in Africa, Brazil, and Thailand to assess neutralizing antibodies (NA) for adenovirus types Ad5, Ad6, Ad26 and Ad36. Clinical trial samples were used to assess NA titers from the US and Europe. The proportions of participants that were negative were 14.8% (Ad5), 31.5% (Ad6); 41.2% (Ad26) and 53.6% (Ad36). Adenovirus NA titers varied by geographic location and were higher in non-US and non-European settings, especially Thailand. In multivariate logistic regression analysis, geographic setting (non-US and non-European settings) was statistically significantly associated with having higher Ad5 titers; participants from Thailand had the highest odds of having high Ad5 titers (adjusted OR=3.53, 95% CI: 2.24, 5.57). Regardless of location, titers of Ad5NA were the highest and Ad36 NA were the lowest. Coincident Ad5/6 titers were lower than either Ad5 or Ad6 titers alone. Understanding pre-existing immunity to candidate vaccine vectors may contribute to the evaluation of vaccines in international populations. [Copyright &y& Elsevier]

Abstract: Human immunodeficiency virus type 1 subtype C (HIV-1C) accounts for about 50% of all HIV infections in the pandemic and is the predominant subtype in the heavily burdened region of southern Africa. HIV-1C possesses unique genetic and phenotypic features that might be associated with biological differences compared to other subtypes. Here, we generated virus isolates from individuals at different stages of HIV-1C infection and investigated the chemokine receptor repertoire that the derived HIV-1C isolates may utilize for entry. Our results show that the R5 phenotype predominates among viruses in Botswana, with a lesser contribution of viruses showing the dualtropic X4R5 phenotype. No viruses of pure X4 phenotype were found, which suggests no discernable evolution of HIV-1C to a monotropic X4 phenotype as the epidemic ages in Botswana. Usage of other coreceptors was rare and apparently insignificant. These results enhance our understanding of HIV-1C biology, with implications for designing and testing therapeutic and prophylactic agents. [Copyright &y& Elsevier]

Globally, human immunodeficiency virus-type 1 (HIV-1) is extraordinarily variable, and this diversity poses a major obstacle to AIDS vaccine development. Currently, candidate vaccines are derived from Isolates, with the hope that they will be sufficiently cross-reactive to protect against circulating viruses. This may be overly optimistic, however, given that HIV-1 envelope proteins can differ in more than 30% of their amino acids. To contend with the diversity, country-specific vaccines are being considered, but evolutionary relationships may be more useful than regional considerations. Consensus or ancestor sequences could be used in vaccine design to minimize the genetic differences between vaccine strains and contemporary isolates, effectively reducing the extent of diversity by half. [ABSTRACT FROM AUTHOR]