Recommendations on data sharing in HIV drug resistance research.

• Human immunodeficiency virus (HIV) drug resistance has implications for antiretroviral treatment strategies and for containing the HIV pandemic because the development of HIV drug resistance leads to the requirement for antiretroviral drugs that may be less effective, less well-tolerated, and more expensive than those used in first-line regimens. • HIV drug resistance studies are designed to determine which HIV mutations are selected by antiretroviral drugs and, in turn, how these mutations affect antiretroviral drug susceptibility and response to future antiretroviral treatment regimens. • Such studies collectively form a vital knowledge base essential for monitoring global HIV drug resistance trends, interpreting HIV genotypic tests, and updating HIV treatment guidelines. • Although HIV drug resistance data are collected in many studies, such data are often not publicly shared, prompting the need to recommend best practices to encourage and standardize HIV drug resistance data sharing. • In contrast to other viruses, sharing HIV sequences from phylogenetic studies of transmission dynamics requires additional precautions as HIV transmission is criminalized in many countries and regions. • Our recommendations are designed to ensure that the data that contribute to HIV drug resistance knowledge will be available without undue hardship to those publishing HIV drug resistance studies and without risk to people living with HIV.

Novel Criteria for Diagnosing Acute and Early Human Immunodeficiency Virus Infection in a Multinational Study of Early Antiretroviral Therapy Initiation.

BACKGROUND: Antiretroviral therapy (ART) initiation during acute and early human immunodeficiency virus infection (AEHI) limits HIV reservoir formation and may facilitate post-ART control but is logistically challenging. We evaluated the performance of AEHI diagnostic criteria from a prospective study of early ART initiation. METHODS: AIDS Clinical Trials Group A 5354 enrolled adults at 30 sites in the Americas, Africa, and Asia who met any 1 of 6 criteria based on combinations of results of HIV RNA, HIV antibody, Western blot or Geenius assay, and/or the signal-to-cutoff (S/CO) ratio of the ARCHITECT HIV Ag/Ab Combo or GS HIV Combo Ag/Ab EIA. HIV status and Fiebig stage were confirmed by centralized testing. RESULTS: From 2017 through 2019, 195 participants were enrolled with median age of 27 years (interquartile range, 23-39). Thirty (15.4%) were female. ART was started by 171 (87.7%) on the day of enrollment and 24 (12.3%) the next day. AEHI was confirmed in 188 (96.4%) participants after centralized testing, 4 (2.0%) participants were found to have chronic infection, and 3 (1.5%) found not to have HIV discontinued ART and were withdrawn. Retrospectively, a nonreactive or indeterminate HIV antibody on the Geenius assay combined with ARCHITECT S/CO ≥10 correctly identified 99 of 122 (81.2%) Fiebig II-IV AEHI cases with no false-positive results. CONCLUSIONS: Novel AEHI criteria that incorporate ARCHITECT S/CO facilitated rapid and efficient ART initiation without waiting for an HIV RNA result. These criteria may facilitate AEHI diagnosis, staging, and immediate ART initiation in future research studies and clinical practice. CLINICAL TRIALS REGISTRATION: NCT02859558.

HIVIntact: a python-based tool for HIV-1 genome intactness inference.

The characterisation of the HIV-1 reservoir, which consists of replication-competent integrated proviruses that persist on antiretroviral therapy (ART), is made difficult by the rarity of intact proviruses relative to those that are defective. While the only conclusive test for the replication-competence of HIV-1 proviruses is carried out in cell culture, genetic characterization of genomes by near full-length (NFL) PCR and sequencing can be used to determine whether particular proviruses have insertions, deletions, or substitutions that render them defective. Proviruses that are not excluded by having such defects can be classified as genetically intact and, possibly, replication competent. Identifying and quantifying proviruses that are potentially replication-competent is important for the development of strategies towards a functional cure. However, to date, there are no programs that can be incorporated into deep-sequencing pipelines for the automated characterization and annotation of HIV genomes. Existing programs that perform this work require manual intervention, cannot be widely installed, and do not have easily adjustable settings. Here, we present HIVIntact, a python-based software tool that characterises genomic defects in NFL HIV-1 sequences, allowing putative intact genomes to be identified in-silico. Unlike other applications that assess the genetic intactness of HIV genomes, this tool can be incorporated into existing sequence-analysis pipelines and applied to large next-generation sequencing datasets.

Intact HIV Proviruses Persist in Children Seven to Nine Years after Initiation of Antiretroviral Therapy in the First Year of Life.

In adults starting antiretroviral therapy (ART) during acute infection, 2% of proviruses that persist on ART are genetically intact by sequence analysis. In contrast, a recent report in children treated early failed to detect sequence-intact proviruses. In another cohort of children treated early, we sought to detect and characterize proviral sequences after 6 to 9 years on suppressive ART. Peripheral blood mononuclear cells (PBMC) from perinatally infected children from the Children with HIV Early antiRetroviral (CHER) study were analyzed. Nearly full-length proviral amplification and sequencing (NFL-PAS) were performed at one time point after 6 to 9 years on ART. Amplicons with large internal deletions were excluded (<9 kb). All amplicons of ≥9 kb were sequenced and analyzed through a bioinformatic pipeline to detect indels, frameshifts, or hypermutations that would render them defective. In eight children who started ART at a median age of 5.4 months (range, 2.0 to 11.1 months), 733 single NFL-PAS amplicons were generated. Of these, 534 (72.9%) had large internal deletions, 174 (23.7%) had hypermutations, 15 (1.4%) had small internal deletions, 3 (1.0%) had deletions in the packaging signal/major splice donor site, and 7 (1.0%) were sequence intact. These 7 intact sequences were from three children who initiated ART after 2.3 months of age, one of whom had two identical intact sequences, suggestive of a cell clone harboring a replication-competent provirus. No intact proviruses were detected in four children who initiated ART before 2.3 months of age. Rare, intact proviruses can be detected in children who initiate ART after 2.3 months of age and are probably, as in adults, maintained by clonal expansion of cells infected before ART initiation.IMPORTANCE There are limited data about the proviral landscape in children exhibiting long-term suppression after early treatment, particularly in Sub-Saharan Africa where HIV-1 subtype C predominates. Investigating the sequence-intact reservoir could provide insight on the mechanisms by which intact proviruses persist and inform ongoing cure efforts. Through nearly full-length proviral amplification and sequencing (NFL-PAS), we generated 733 NFL-PAS amplicons from eight children. We showed that rare, genetically intact proviruses could be detected in children who initiated ART after 2.3 months of age. The frequency of intact proviruses was lower (P < 0.05) than that reported for HIV subtype B-infected adults treated during early HIV infection. We show that cells harboring genetically intact HIV proviruses are rare in children exhibiting long-term suppression after early treatment and may require the processing of a large number of cells to assess reservoir size. This points to the need for efficient methods to accurately quantify latent reservoirs, particularly in pediatric studies where sample availability is limited.

Pharmacogenetics and pharmacokinetics of CNS penetration of efavirenz and its metabolites.

BACKGROUND: There are limited data on the pharmacogenetics and pharmacokinetics of the CNS penetration of efavirenz. OBJECTIVES: We investigated genetic polymorphisms associated with CSF concentrations of efavirenz and its metabolites and explored the relationships with neurocognitive performance. METHODS: We included 47 HIV-infected South African black adults with and without HIV-associated neurocognitive disorder on efavirenz/tenofovir/emtricitabine and collected paired plasma-CSF samples. We considered 2049 SNPs, including SNPs known to affect plasma efavirenz exposure, from potentially relevant genes (ABCC5, ABCG2, ABCB1, SLCO2B1, SCLO1A2, ABCC4, CYP2B6 and CYP2A6) and 880 met a linkage disequilibrium (LD)-pruning threshold. RESULTS: We identified 9 slow, 21 intermediate and 17 extensive metabolizers. The CYP2B6 983 genotype in multivariate analyses predicted log10-transformed concentrations of plasma efavirenz (ß = 0.38, P = 2.7 × 10-03), plasma 7-hydroxy-efavirenz (ß = 0.59, P = 3.7 × 10-03), plasma 8-hydroxy-efavirenz:efavirenz ratio (ß = -0.31, P = 1.8 × 10-04) and CSF efavirenz (ß = 0.36, P = 0.01). Lower plasma 7-hydroxy-efavirenz concentrations were independently associated with CYP2A6 rs10853742 (ß = -0.55, P = 3.5 × 10-05), ABCB1 rs115780656 (ß = -0.65, P = 4.1 × 10-05) and CYP2A6 -48A→C (ß = -0.59, P = 0.01). CYP2A6 -48A→C was independently associated with higher CSF 8-hydroxy-efavirenz:efavirenz ratio (ß = 0.54, P = 0.048). CYP2B6 rs2279345 polymorphism was associated with lower plasma 7-hydroxy-efavirenz:efavirenz ratio in multivariate analyses (P < 0.05). No polymorphisms were associated with CSF:plasma ratios of efavirenz, plasma or CSF concentrations of 8-hydroxy-efavirenz or neurocognitive performance. CONCLUSIONS: We identified novel genetic associations with plasma efavirenz, plasma 7-hydroxy-efavirenz, plasma 7-hydroxy-efavirenz:efavirenz ratio, plasma 8-hydroxy-efavirenz:efavirenz ratio, CSF efavirenz and CSF 8-hydroxy-efavirenz:efavirenz ratio.

Pooled PCR testing of dried blood spots for infant HIV diagnosis is cost efficient and accurate.

BACKGROUND: Access to qualitative HIV PCRs for early infant diagnosis (EID) is restricted in resource-limited settings due to cost. We hypothesised that pooling of dried blood spots (DBS), defined as combining multiple patient samples in a single test with subsequent individual testing of positive pools, would be cost saving while retaining clinical accuracy compared to individual patient testing. METHODS: Cost savings: A model was developed to simulate reagent and consumable cost saving of pooled compared to individual sample testing. Daily sample/result data of a public health laboratory in South Africa were used to illustrate outputs from the model. Samples were randomly allocated to pools and the process was repeated 1000 times to measure variation in estimates due to this stochasticity. Clinical accuracy: 1170 patient samples were tested using the Roche CAP/CTM Qual assay in pools of five 50 µl DBS. Negative pools comprised DBS previously tested in single reactions; positive pools included 1 positive sample. RESULTS: Pooling would have saved 64% of laboratory costs in 2015. The model is published as an R-based web tool, into which the user enters sample/positivity estimates and workflow management parameters to obtain cost saving estimates at an optimal pool size. Sensitivity of pooled testing was 98.8% overall; 100% for strongly reactive pools. One pool tested false positive which would not impact clinical specificity as individual patient testing is performed prior to reporting. CONCLUSIONS: Pooled PCR testing for EID remains accurate and dramatically reduces costs in settings with moderate to low prevalence rates and sufficient sample numbers.

Paper-based detection of HIV-1 drug resistance using isothermal amplification and an oligonucleotide ligation assay.

Regular HIV-1 viral load monitoring is the standard of care to assess antiretroviral therapy effectiveness in resource-rich settings. Persistently elevated viral loads indicate virologic failure (VF), which warrants HIV drug resistance testing (HIVDRT) to allow individualized regimen switches. However, in settings lacking access to HIVDRT, clinical decisions are often made based on symptoms, leading to unnecessary therapy switches and increased costs of care. This work presents a proof-of-concept assay to detect M184V, the most common drug resistance mutation after first-line antiretroviral therapy failure, in a paper format. The first step isothermally amplifies a section of HIV-1 reverse transcriptase containing M184V using a recombinase polymerase amplification (RPA) assay. Then, an oligonucleotide ligation assay (OLA) is used to selectively label the mutant and wild type amplified sequences. Finally, a lateral flow enzyme-linked immunosorbent assay (ELISA) differentiates between OLA-labeled products with or without M184V. Our method shows 100% specificity and 100% sensitivity when tested with samples that contained 200 copies of mutant DNA and 800 copies of wild type DNA prior to amplification. When integrated with sample preparation, this method may detect HIV-1 drug resistance at a low cost and at a rural hospital laboratory.

CD4 count-based failure criteria combined with viral load monitoring may trigger worse switch decisions than viral load monitoring alone.

OBJECTIVE: CD4 count decline often triggers antiretroviral regimen switches in resource-limited settings, even when viral load testing is available. We therefore compared CD4 failure and CD4 trends in patients with viraemia with or without antiretroviral resistance. METHODS: Retrospective cohort study investigating the association of HIV drug resistance with CD4 failure or CD4 trends in patients on first-line antiretroviral regimens during viraemia. Patients with viraemia (HIV RNA >1000 copies/ml) from two HIV treatment programmes in South Africa (n = 350) were included. We investigated the association of M184V and NNRTI resistance with WHO immunological failure criteria and CD4 count trends, using chi-square tests and linear mixed models. RESULTS: Fewer patients with the M184V mutation reached immunologic failure criteria than those without: 51 of 151(34%) vs. 90 of 199 (45%) (P = 0.03). Similarly, 79 of 220 (36%) patients, who had major NNRTI resistance, had immunological failure, whereas 62 of 130 (48%) without (chi-square P = 0.03) did. The CD4 count decline among patients with the M184V mutation was 2.5 cells/mm(3) /year, whereas in those without M184V it was 14 cells/mm(3) /year (P = 0.1), but the difference in CD4 count decline with and without NNRTI resistance was marginal. CONCLUSION: Our data suggest that CD4 count monitoring may lead to inappropriate delayed therapy switches for patients with HIV drug resistance. Conversely, patients with viraemia but no drug resistance are more likely to have a CD4 count decline and thus may be more likely to be switched to a second-line regimen.

Early Antiretroviral Therapy in South African Children Reduces HIV-1-Infected Cells and Cell-Associated HIV-1 RNA in Blood Mononuclear Cells.

We measured cell-associated human immunodeficiency virus (HIV)-1 DNA (CAD) and RNA (CAR) and plasma HIV-1 RNA in blood samples from 20 children in the Children with HIV Early Antiretroviral (CHER) cohort after 7-8 years of suppressive combination antiretroviral therapy (cART). Children who initiated cART early (<2 months; n = 12) had lower HIV-1 CAD (median, 48 vs 216; P < .01) and CAR (median, 5 vs 436; P < .01) per million peripheral blood mononuclear cells than children who started later (≥ 2 months; n = 8). Plasma HIV-1 RNA levels were not significantly lower in early-treated children (0.5 vs 1.2 copies/mL; P = .16). Early treatment at <2 months of age reduces the number of HIV-infected cells and HIV CAR.

Irreproducible positive results on the Cobas AmpliPrep/Cobas TaqMan HIV-1 Qual test are different qualitatively from confirmed positive results.

Criteria that define low positive results on the COBAS® AmpliPrep/COBAS® TaqMan (CAP/CTM) HIV-1 Qual test as inconclusive have been adopted by all academic centres in South Africa that conduct infant HIV PCR, following previous investigations that showed poor specificity of these results. Retesting all inconclusive specimens has considerable cost implications. Therefore, it was attempted to characterise such inconclusive results, by comparing those that prove to be either negative or positive on follow-up testing. This retrospective, laboratory-based study found that 193 of 211 (91.5%) patients with previous inconclusive results (defined as reported positive by CAP/CTM but with cycle threshold [Ct ] values of >32 and/or fluorescence intensity [FI] values of <5) tested negative and only 18 (8.5%) tested positive using independently obtained follow-up samples after a median of 28 days. The only significant independent predictor of a later positive result was a higher FI value (3.326 vs. 0.495, P < 0.0001), whereas Ct values were not predictive independently. Specimens from patients negative on follow-up testing differed qualitatively from specimens that proved to be true positives. As the lower FI values in false-positive compared to true-positive results probably are indicative of a non-specific signal, the incorporation of stringent amplification slope criteria in the assay's test definition file may improve correct classification and thus reduce the need for repeat testing of a large number of inconclusive specimens.

Nucleoside reverse transcriptase inhibitor resistance mutations associated with first-line stavudine-containing antiretroviral therapy: programmatic implications for countries phasing out stavudine.

BACKGROUND: The World Health Organization Antiretroviral Treatment Guidelines recommend phasing-out stavudine because of its risk of long-term toxicity. There are two mutational pathways of stavudine resistance with different implications for zidovudine and tenofovir cross-resistance, the primary candidates for replacing stavudine. However, because resistance testing is rarely available in resource-limited settings, it is critical to identify the cross-resistance patterns associated with first-line stavudine failure. METHODS: We analyzed HIV-1 resistance mutations following first-line stavudine failure from 35 publications comprising 1,825 individuals. We also assessed the influence of concomitant nevirapine vs. efavirenz, therapy duration, and HIV-1 subtype on the proportions of mutations associated with zidovudine vs. tenofovir cross-resistance. RESULTS: Mutations with preferential zidovudine activity, K65R or K70E, occurred in 5.3% of individuals. Mutations with preferential tenofovir activity, ≥ two thymidine analog mutations (TAMs) or Q151M, occurred in 22% of individuals. Nevirapine increased the risk of TAMs, K65R, and Q151M. Longer therapy increased the risk of TAMs and Q151M but not K65R. Subtype C and CRF01_AE increased the risk of K65R, but only CRF01_AE increased the risk of K65R without Q151M. CONCLUSIONS: Regardless of concomitant nevirapine vs. efavirenz, therapy duration, or subtype, tenofovir was more likely than zidovudine to retain antiviral activity following first-line d4T therapy.

Impact of antiretroviral therapy during acute or early HIV infection on virologic and immunologic outcomes: results from a multinational clinical trial.

OBJECTIVE: To assess how antiretroviral therapy (ART) initiation during acute or early HIV infection (AEHI) affects the viral reservoir and host immune responses. DESIGN: Single-arm trial of ART initiation during AEHI at 30 sites in the Americas, Africa, and Asia. METHODS: HIV DNA was measured at week 48 of ART in 5 million CD4 + T cells by sensitive qPCR assays targeting HIV gag and pol . Peripheral blood mononuclear cells were stimulated with potential HIV T cell epitope peptide pools consisting of env , gag , nef, and pol peptides and stained for expression of CD3, CD4, CD8, and intracellular cytokines/chemokines. RESULTS: From 2017 to 2019, 188 participants initiated ART during Fiebig stages I ( n  = 6), II ( n  = 43), III ( n  = 56), IV ( n  = 23), and V ( n  = 60). Median age was 27 years (interquartile range 23-38), 27 (14%) participants were female, and 180 (97%) cisgender. Among 154 virally suppressed participants at week 48, 100% had detectable HIV gag or pol DNA. Participants treated during Fiebig I had the lowest HIV DNA levels ( P  < 0.001). Week 48 HIV DNA mostly did not correlate with concurrent CD4 + or CD8 + T cell HIV-specific immune responses (rho range -0.11 to +0.19, all P  > 0.025). At week 48, the magnitude, but not polyfunctionality, of HIV-specific T cell responses was moderately reduced among participants who initiated ART earliest. CONCLUSION: Earlier ART initiation during AEHI reduced but did not eliminate the persistence of HIV-infected cells in blood. These findings explain the rapid viral rebound observed after ART cessation in early-treated individuals with undetectable HIV DNA by less sensitive methods.

Deep sequencing reveals minor protease resistance mutations in patients failing a protease inhibitor regimen.

Standard genotypic antiretroviral resistance testing, performed by bulk sequencing, does not readily detect variants that comprise <20% of the circulating HIV-1 RNA population. Nevertheless, it is valuable in selecting an antiretroviral regimen after antiretroviral failure. In patients with poor adherence, resistant variants may not reach this threshold. Therefore, deep sequencing would be potentially valuable for detecting minority resistant variants. We compared bulk sequencing and deep sequencing to detect HIV-1 drug resistance at the time of a second-line protease inhibitor (PI)-based antiretroviral regimen failure. Eligibility criteria were virologic failure (HIV-1 RNA load of >500 copies/ml) of a first-line nonnucleoside reverse transcriptase inhibitor-based regimen, with at least the M184V mutation (lamivudine resistance), and second-line failure of a lopinavir/ritonavir (LPV/r)-based regimen. An amplicon-sequencing approach on the Roche 454 system was used. Six patients with viral loads of >90,000 copies/ml and one patient with a viral load of 520 copies/ml were included. Mutations not detectable by bulk sequencing during first- and second-line failure were detected by deep sequencing during second-line failure. Low-frequency variants (>0.5% of the sequence population) harboring major protease inhibitor resistance mutations were found in 5 of 7 patients despite poor adherence to the LPV/r-based regimen. In patients with intermittent adherence to a boosted PI regimen, deep sequencing may detect minority PI-resistant variants, which likely represent early events in resistance selection. In patients with poor or intermittent adherence, there may be low evolutionary impetus for such variants to reach fixation, explaining the low prevalence of PI resistance.

The largest HIV-1-infected T cell clones in children on long-term combination antiretroviral therapy contain solo LTRs.

Combination antiretroviral therapy (cART) suppresses viral replication but does not cure HIV infection because a reservoir of infectious (intact) HIV proviruses persists in long-lived CD4+T cells. However, a large majority (>95%) of HIV-infected cells that persist on effective cART carry defective (non-infectious) proviruses. Defective proviruses consisting of only a single LTR (solo long terminal repeat) are commonly found as endogenous retroviruses in many animal species, but the frequency of solo-LTR HIV proviruses has not been well defined. Here we show that, in five pediatric donors whose viremia was suppressed on cART for at least 5 years, the proviruses in the nine largest clones of HIV-infected cells were solo LTRs. The sizes of five of these clones were assayed longitudinally by integration site-specific quantitative PCR. Minor waxing and waning of the clones was observed, suggesting that these clones are generally stable over time. Our findings show that solo LTRs comprise a large fraction of the proviruses in infected cell clones that persist in children on long-term cART. IMPORTANCE This work highlights that severely deleted HIV-1 proviruses comprise a significant proportion of the proviral landscape and are often overlooked.

Advancing HIV Drug Resistance Technologies and Strategies: Insights from South Africa's Experience and Future Directions for Resource-Limited Settings.

Monitoring of HIV drug resistance (HIVDR) remains critical for ensuring countries attain and sustain the global goals for ending HIV as a public health threat by 2030. On an individual patient level, drug resistance results assist in ensuring unnecessary treatment switches are avoided and subsequent regimens are tailored on a case-by-case basis, should resistance be detected. Although there is a disparity in access to HIVDR testing in high-income countries compared to low- and middle-income countries (LMICS), more LMICs have now included HIVDR testing for individual patient management in some groups of patients. In this review, we describe different strategies for surveillance as well as where HIVDR testing can be implemented for individual patient management. In addition, we briefly review available technologies for HIVDR testing in LMICs, including Sanger sequencing, next-generation sequencing, and some point-of-care options. Finally, we describe how South Africa has implemented HIVDR testing in the public sector.

HIV drug resistance in various body compartments.

PURPOSE OF REVIEW: HIV drug resistance testing using blood plasma or dried blood spots forms part of international guidelines. However, as the clinical utility of assessing drug resistance in other body compartments is less well established, we review this for blood cells and samples from other body compartments. RECENT EVIDENCE: Although clinical benefit is not clear, drug resistance testing in blood cells is often performed when patients with suppressed plasma viral loads require a treatment substitution. In patients with HIV neurocognitive disease, cerebral spinal fluid (CSF) drug resistance is rarely discordant with plasma but has nevertheless been used to guide antiretroviral drug substitutions. Cases with HIV drug resistance in genital fluids have been documented but this does not appear to indicate transmission risk when blood plasma viral loads are suppressed. SUMMARY: Drug-resistant variants, which may be selected in tissues under conditions of variable adherence and drug penetration, appear to disseminate quickly, and become detectable in blood. This may explain why drug resistance discordance between plasma and these compartments is rarely found. Partial compartmentalization of HIV populations is well established for the CSF and the genital tract but other than blood plasma, evidence is lacking to support drug resistance testing in body compartments.

Antiretroviral resistance patterns and factors associated with resistance in adult patients failing NNRTI-based regimens in the Western Cape, South Africa.

Antiretroviral drug resistance in patients failing non-nucleoside reverse transcriptase inhibitor (NNRTI)-based first-line combination antiretroviral treatment (ART) is influenced by: regimen choice, HIV-1 subtype, detection of and response to therapy failure. In order to describe resistance patterns by genotypic testing, at the time of first-line ART failure and to describe associations with having M184I/V, K65R, three or more thymidine analog mutations (TAMs) and etravirine (ETV) resistance, the prevalence of antiretroviral drug resistance associated mutations in a cross-sectional study, at two South African public health clinic settings, at the time of virologic failure (HIV-1 RNA load >400 copies/ml) are described. Also reported are associations of therapy choice, prolonged virologic failure, and concurrent HIV viral load and CD4 count with the presence of M184I/V, TAMs, K65R, and resistance to ETV. Of 167 adult patients with virologic failure on first-line ART, 28 (17%) had no resistance, 137 (82%) had NNRTI resistance, 101 (60%) M184I/V, 20 (12%) TAMs, of which 4 had 3 or more TAMs, and 7 (4%) had K65R, of which 6 were on D4T and one on AZT. A prolonged estimated period of failure was associated with having ≥3 TAMs. Patients treated with nevirapine (NVP) were more likely to have ETV resistance than those treated with efavirenz (EFV). Major protease inhibitor mutations were not detected. A delayed response to ART failure may risk accumulation of TAMs in patients on an NNRTI-based regimen. The use of NVP rather than EFV was associated with ETV resistance.

HIV-1 Persistence in Children during Suppressive ART.

There is a growing number of perinatally HIV-1-infected children worldwide who must maintain life-long ART. In early life, HIV-1 infection is established in an immunologically inexperienced environment in which maternal ART and immune dynamics during pregnancy play a role in reservoir establishment. Children that initiated early antiretroviral therapy (ART) and maintained long-term suppression of viremia have smaller and less diverse HIV reservoirs than adults, although their proviral landscape during ART is reported to be similar to that of adults. The ability of these early infected cells to persist long-term through clonal expansion poses a major barrier to finding a cure. Furthermore, the effects of life-long HIV persistence and ART are yet to be understood, but growing evidence suggests that these individuals are at an increased risk for developing non-AIDS-related comorbidities, which underscores the need for an HIV cure.

HIV false positive screening serology due to sample contamination reduced by a dedicated sample and platform in a high prevalence environment.

Automated testing of HIV serology on clinical chemistry analysers has become common. High sample throughput, high HIV prevalence and instrument design could all contribute to sample cross-contamination by microscopic droplet carry-over from seropositive samples to seronegative samples resulting in false positive low-reactive results. Following installation of an automated shared platform at our public health laboratory, we noted an increase in low reactive and false positive results. Subsequently, we investigated HIV serology screening test results for a period of 21 months. Of 485 initially low positive or equivocal samples 411 (85%) tested negative when retested using an independently collected sample. As creatinine is commonly requested with HIV screening, we used it as a proxy for concomitant clinical chemistry testing, indicating that a sample had likely been tested on a shared high-throughput instrument. The contamination risk was stratified between samples passing the clinical chemistry module first versus samples bypassing it. The odds ratio for a false positive HIV serology result was 4.1 (95% CI: 1.69-9.97) when creatinine level was determined first, versus not, on the same sample, suggesting contamination on the chemistry analyser. We subsequently issued a notice to obtain dedicated samples for HIV serology and added a suffix to the specimen identifier which restricted testing to a dedicated instrument. Low positive and false positive rates were determined before and after these interventions. Based on measured rates in low positive samples we estimate that before the intervention, of 44 117 HIV screening serology samples, 753 (1.71%) were false positive, declining to 48 of 7 072 samples (0.68%) post-intervention (p<0.01). Our findings showed that automated high throughput shared diagnostic platforms are at risk of generating false-positive HIV test results, due to sample contamination and that measures are required to address this. Restricting HIV serology samples to a dedicated platform resolved this problem.

NanoHIV: A Bioinformatics Pipeline for Producing Accurate, Near Full-Length HIV Proviral Genomes Sequenced Using the Oxford Nanopore Technology.

HIV-1 proviral single-genome sequencing by limiting-dilution polymerase chain reaction (PCR) amplification is important for differentiating the sequence-intact from defective proviruses that persist during antiretroviral therapy (ART). Intact proviruses may rebound if ART is interrupted and are the barrier to an HIV cure. Oxford Nanopore Technologies (ONT) sequencing offers a promising, cost-effective approach to the sequencing of long amplicons such as near full-length HIV-1 proviruses, but the high diversity of HIV-1 and the ONT sequencing error render analysis of the generated data difficult. NanoHIV is a new tool that uses an iterative consensus generation approach to construct accurate, near full-length HIV-1 proviral single-genome sequences from ONT data. To validate the approach, single-genome sequences generated using NanoHIV consensus building were compared to Illumina® consensus building of the same nine single-genome near full-length amplicons and an average agreement of 99.4% was found between the two sequencing approaches.