HIV-1 Reverse Transcriptase Genes Research Articles

Identification of integrase inhibitor-related drug resistance mutations in newly diagnosed ART-naïve HIV patients

BackgroundIn China, the recommended treatment regimens for HIV-infected individuals were tenofovir in combination with lamivudine or emtricitabine as NRTIs, efavirenz or rilpivirine as NNRTIs, lopinavir/ritonavir as protease inhibitors, and raltegravir or dolutegravir as INSTIs. The development of drug resistance increases the risk of viral rebound, opportunistic infections, and ultimately treatment failure such that the early detection of resistance is ideal. This study was developed to explore primary drug resistance characteristics and genotypic distributions in newly diagnosed antiretroviral therapy (ART)-naïve HIV-1 patients in Nanjing with the goal of establishing a basis for their individualized treatment in the clinic. MethodsSamples of serum were collected from newly diagnosed ART-naïve HIV patients from the Second Hospital of Nanjing between May 2021 and May 2022. The HIV-1 integrase (IN), protease (PR), and reverse transcriptase (RT) gene coding sequences were amplified from these samples, sequenced, and assessed for drug resistance-related mutations. ResultsMajor integrase resistance-related mutations were detected in 4/360 amplified samples, with 5 other patient samples exhibiting accessory resistance mutations. The overall prevalence of PR and RT inhibitor-related transmitted drug resistance mutations (TDRMs) in this patient population was 16.99% (61/359). The most common mutations were non-nucleoside reverse transcriptase inhibitor-related mutations (51/359; 14.21%), followed by those associated with nucleoside reverse transcriptase inhibitors (7/359; 1.95%) and protease inhibitors (7/359; 1.95%). Dual-resistant strains were also observed in a subset of patients. ConclusionsIn summary, this study is the first to have surveyed the prevalence of integrase inhibitor resistance-related mutations and other drug resistance-related mutations among newly diagnosed ART-naïve HIV-positive patients in Nanjing, China. These results highlight the need for further molecular surveillance-based monitoring of the HIV epidemic in Nanjing.

Affordable drug resistance genotyping of HIV-1 reverse transcriptase, protease and integrase genes, for resource limited settings

BackgroundAs use of dolutegravir (DTG) becomes more common in resource limited settings (RLS), the demand for integrase resistance testing is increasing. Affordable methods for genotyping all relevant HIV-1 pol genes (i.e., protease (PR), reverse transcriptase (RT) and integrase (IN)) are required to guide choice of future antiretroviral therapy (ART). We designed an in-house HIV-1 drug resistance (HIVDR) genotyping method that is affordable and suitable for use in RLS.MethodsWe obtained remnant plasma samples from CAPRISA 103 study and amplified HIV-1 PR, RT and IN genes, using an innovative PCR assay. We validated the assay using remnant plasma samples from an external quality assessment (EQA) programme. We genotyped samples by Sanger sequencing and assessed HIVDR mutations using the Stanford HIV drug resistance database. We compared drug resistance mutations with previous genotypes and calculated method cost-estimates.ResultsFrom 96 samples processed, we obtained sequence data for 78 (81%), of which 75 (96%) had a least one HIVDR mutation, with no major-IN mutations observed. Only one sample had an E157Q INSTI-accessory mutation. When compared to previous genotypes, 18/78 (23%) had at least one discordant mutation, but only 2/78 (3%) resulted in different phenotypic predictions that could affect choice of subsequent regimen. All CAPRISA 103 study sequences were HIV-1C as confirmed by phylogenetic analysis. Of the 7 EQA samples, 4 were HIV-1C, 2 were HIV-1D, and 1 was HIV-1A. Genotypic resistance data generated using the IDR method were 100% concordant with EQA panel results. Overall genotyping cost per sample was estimated at ~ US$43–$US49, with a processing time of ~ 2 working days.ConclusionsWe successfully designed an in-house HIVDR method that is suitable for genotyping HIV-1 PR, RT and IN genes, at an affordable cost and shorter turnaround time. This HIVDR genotyping method accommodates changes in ART regimens and will help to guide HIV-1 treatment decisions in RLS.

Genomic signatures of protease and reverse transcriptase genes from HIV-1 subtype C isolated from first-line ART patients in India.

Genomic signatures of the protease and reverse transcriptase gene of HIV-1 from HIV infected North Indian patients who were under ART from 1 to ≤ 7 years were analyzed. The DNA from plasma samples of 9 patients and RNA from 57 patients were isolated and subjected to amplification for the protease and reverse transcriptase gene of HIV-1 subtype C. Then sequencing was carried out following the WHO dried blood spot protocol. The drug resistance mutation patterns were analyzed using the HIV Drug Resistance Database, Stanford University, USA. Lamivudine-associated drug-resistance mutations such as M184V/M184I, nevirapine-associated drug resistance mutations Y181C and H221Y, and efavirenz-associated drug resistance mutations M230I were observed in reverse transcriptase gene of archived DNA of two HIV-1 infected patients. No mutation was observed in the remaining 7 patients. Various computational tools and websites like viral epidemiological signature pattern analysis (VESPA), hyper mutation, SNAP version 2.1.1, and entropy were utilized for the analysis of the signature pattern of amino acids, hyper mutation, selection pressure, and Shannon entropy in the protease and reverse transcriptase gene sequences of the 9 archived DNA, 56 protease gene and 51 reverse transcriptase gene from the HIV-1 DNA amplified sequences of RNA. The HIV-1 Subtype-C (Gene bank accession number: AB023804) and first isolate HXB2 (Gene bank accession number: K03455.1) was taken as reference sequence. The signature amino acid sequences were identified in the protease and reverse transcriptase gene, no hyper mutation, highest entropy was marked in the amino acid positions and synonymous to non-synonymous nucleotide ratio was calculated in the protease and reverse transcriptase gene of 9 archived DNA sequences, 56 protease and 51 reverse transcriptase gene sequences of HIV-1 Subtype C isolates.

The frequency of HIV-1 infection and surveillance drug-resistant mutations determination among Iranians with high-risk behaviors, during 2014 to 2020.

Background and Objectives:Human immunodeficiency virus (HIV) has various transmission routes. Instant antiretroviral therapy (ART) is the recommended treatment for HIV infection. Highly active antiretroviral therapy (HAART) significantly decreases the acquired immunodeficiency syndrome (AIDS) and AIDS-related co-morbidities. Notwithstanding the suitability of HAART, the antiretrovirals (ARVs) have adverse effects and antiretroviral drug resistance mutations are reported among those who receive ARVs. In this survey, the abundance of HIV-1 infection in Iranians with high-risk behaviors, and detection of the surveillance drug-resistant mutations (SDRMs) were evaluated.Materials and Methods:This cross-sectional study was conducted on 250 individuals with high-risk behaviors from September 2014 to February 2020. HIV-1 Ag/Ab in plasma samples was detected using enzyme immunoassay (EIA) kits. The conserved region of HIV-1 was detected in the plasma samples by real-time polymerase chain reaction (PCR) assay. Furthermore, in individuals with positive HIV-1 RNA, HIV-1 viral load testing was performed. After amplification and sequencing of the HIV-1 protease, reverse transcriptase, and integrase genes, surveillance drug resistance mutation (SDRM) and phylogenetic analysis were determined.Results:Out of the 250 participants with high-risk behaviors, six (2.4%) were infected with HIV-1. According to the phylogenetic analysis, the CRF35_AD (83.3% or 5/6) was the dominant subtype, followed by CRF01_AE (16.7% or 1/6). In this research, in none of the HIV-1 infected patients, SDRM for protease inhibitors (PIs), nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), and integrase inhibitors (INs) were observed. Nevertheless, in one of the patients, V179L mutation was detected which is a rare non-polymorphic mutation and is listed as a rilpivirine (RPV) -associated resistance mutation.Conclusion:The results of the current survey revealed that 2.4% of people with high-risk behaviors are infected with HIV and the level of drug resistance mutations (DRMs) in these people is very low.

Acquired HIV drug resistance and virologic monitoring in a HIV hyper-endemic setting in KwaZulu-Natal Province, South Africa

BackgroundIntroduction of tenofovir (TDF) plus lamivudine (3TC) and dolutegravir (DTG) in first- and second-line HIV treatment regimens in South Africa warrants characterization of acquired HIV-1 drug resistance (ADR) mutations that could impact DTG-based antiretroviral therapy (ART). In this study, we sought to determine prevalence of ADR mutations and their potential impact on susceptibility to drugs used in combination with DTG among HIV-positive adults (≥ 18 years) accessing routine care at a selected ART facility in KwaZulu-Natal, South Africa.MethodsWe enrolled adult participants in a cross-sectional study between May and September 2019. Eligible participants had a most recent documented viral load (VL) ≥ 1000 copies/mL after at least 6 months on ART. We genotyped HIV-1 reverse transcriptase and protease genes by Sanger sequencing and assessed ADR. We characterized the effect of ADR mutations on the predicted susceptibility to drugs used in combination with DTG.ResultsFrom 143 participants enrolled, we obtained sequence data for 115 (80%), and 92.2% (95% CI 85.7–96.4) had ADR. The proportion with ADR was similar for participants on first-line ART (65/70, 92.9%, 95% CI 84.1–97.6) and those on second-line ART (40/44, 90.9%, 95% CI 78.3–97.5), and was present for the single participant on third-line ART. Approximately 89% (62/70) of those on first-line ART had dual class NRTI and NNRTI resistance and only six (13.6%) of those on second-line ART had major PI mutations. Most participants (82%) with first-line viraemia maintained susceptibility to Zidovudine (AZT), and the majority of them had lost susceptibility to TDF (71%) and 3TC (84%). Approximately two in every five TDF-treated individuals had thymidine analogue mutations (TAMs).ConclusionsSusceptibility to AZT among most participants with first-line viraemia suggests that a new second-line regimen of AZT + 3TC + DTG could be effective. However, atypical occurrence of TAMs in TDF-treated individuals suggests a less effective AZT + 3TC + DTG regimen in a subpopulation of patients. As most patients with first-line viraemia had at least low-level resistance to TDF and 3TC, identifying viraemia before switch to TDF + 3TC + DTG is important to avoid DTG functional monotherapy. These findings highlight a need for close monitoring of outcomes on new standardized treatment regimens.

Use of a mutation-specific genotyping method to assess for HIV-1 drug resistance in antiretroviral-naïve HIV-1 Subtype C-infected patients in Botswana.

Background: HIV-1 drug resistance poses a major threat to the success of antiretroviral therapy. The high costs of available HIV drug resistance assays prohibit their routine usage in resource-limited settings. Pan-degenerate amplification and adaptation (PANDAA), a focused genotyping approach based on quantitative PCR (qPCR), promises a fast and cost-effective way to detect HIV drug resistance mutations (HIVDRMs). Given the high cost of current genotyping methods, we sought to use PANDAA for screening key HIVDRMs in antiretroviral-naïve individuals at codons 103, 106 and 184 of the HIV-1 reverse transcriptase gene. Mutations selected at these positions have been shown to be the most common driver mutations in treatment failure. Methods: A total of 103 samples from antiretroviral-naïve individuals previously genotyped by Sanger population sequencing were used to assess and verify the performance of PANDAA. PANDAA samples were run on the ABI 7500 Sequence Detection System to genotype the K103N, V106M and M184V HIVDRMs. In addition, the cost per sample and reaction times were compared. Results: Sanger population sequencing and PANDAA detected K103N mutation in three (2.9%) out of 103 participants. There was no evidence of baseline V106M and M184V mutations observed in our study. To genotype the six HIVDRMs it costs approximately 40 USD using PANDAA, while the reagents cost per test for Sanger population sequencing is approximately 100 USD per sample. PANDAA was performed quicker compared to Sanger sequencing, 2 hours for PANDAA versus 15 hours for Sanger sequencing. Conclusion: The performance of PANDAA and Sanger population sequencing demonstrated complete concordance. PANDAA could improve patient management by providing quick and relatively cheap access to drug-resistance information.

Use of a mutation-specific genotyping method to assess for HIV-1 drug resistance in antiretroviral-naïve HIV-1 Subtype C-infected patients in Botswana

Background: HIV-1 drug resistance poses a major threat to the success of antiretroviral therapy. The high costs of available HIV drug resistance assays prohibit their routine usage in resource-limited settings. Pan-degenerate amplification and adaptation (PANDAA), a focused genotyping approach based on quantitative PCR (qPCR), promises a fast and cost-effective way to detect HIV drug resistance mutations (HIVDRMs).  Given the high cost of current genotyping methods, we sought to use PANDAA for screening key HIVDRMs in antiretroviral-naïve individuals at codons 103, 106 and 184 of the HIV-1 reverse transcriptase gene. Mutations selected at these positions have been shown to be the most common driver mutations in treatment failure.  Methods: A total of 103 samples from antiretroviral-naïve individuals previously genotyped by Sanger population sequencing were used to assess and verify the performance of PANDAA. PANDAA samples were run on the ABI 7500 Sequence Detection System to genotype the K103N, V106M and M184V HIVDRMs. In addition, the cost per sample and reaction times were compared. Results: Sanger population sequencing and PANDAA detected K103N mutation in three (2.9%) out of 103 participants.  There was no evidence of baseline V106M and M184V mutations observed in our study. To genotype the six HIVDRMs it costs approximately 40 USD using PANDAA, while the reagents cost per test for Sanger population sequencing is approximately 100 USD per sample. PANDAA was performed quicker compared to Sanger sequencing, 2 hours for PANDAA versus 15 hours for Sanger sequencing. Conclusion: The performance of PANDAA and Sanger population sequencing demonstrated complete concordance. PANDAA could improve patient management by providing quick and relatively cheap access to drug-resistance information.

Primary resistance to antiretroviral drugs of HIV strains in Chad: a retrospective investigation by analysis of frozen dried blood spot samples.

No data concerning antiretroviral drug's (ARV) primary resistance mutation rates in Chad are available. We retrospectively analysed frozen-stored dried blood spot samples that were collected from 48 Chadian human immunodeficiency virus (HIV)-1 seropositive patients naïve of ARV. HIV-1 protease and reverse transcriptase genes were successfully sequenced for 24 (60.0%) of the 40 patients displaying a viral load > 1000 copies/ml. Seven (29.2%) displayed mutations conferring resistance against one or more classes of ARV. We evidenced high levels of primary ARV resistance mutations in Chad, but lower than those observed in patients with failure to first-line ARV.

Human immunodeficiency virus infection and syphilis among homeless people in a large city of Central-Western Brazil: prevalence, risk factors, human immunodeficiency virus-1 genetic diversity, and drug resistance mutations

Homeless people are at high risk for sexually transmitted infections (STIs), such as human immunodeficiency virus (HIV) infection and syphilis. We investigated the epidemiology of HIV-1 infection and syphilis among homeless individuals in a large city in Central-Western Brazil. In this cross-sectional study, we interviewed and tested 355 individuals from September 2014 to August 2015. Rapid test samples positive for syphilis were retested using the Venereal Disease Research Laboratory (VDRL) test. Blood samples from HIV-infected participants were collected for POL sequencing using HIV-1 RNA extracted from plasma, reverse transcription, and nested polymerase chain reaction. Anti-HIV-1-positive samples were subtyped by sequencing the nucleotides of HIV-1 protease and part of the HIV-1 reverse transcriptase genes. Transmitted and acquired drug resistance mutations and susceptibility to antiretroviral drugs were also analyzed. Anti-HIV was positive in 14 patients (3.9%; 95% confidence interval [CI]: 2.3–6.4). HIV-1 RNA was detected in 8 of the 14 samples. Two of the eight (25%) isolates showed HIV-1 drug resistance mutations. Furthermore, 78 (22%; 95% CI: 17.9–26.5) and 29 (8.2%; 95% CI: 5.6–11.4) homeless individuals tested positive for syphilis using the rapid test and VDRL test, respectively. Two individuals were anti-HIV-1 and VDRL test positive. Daily alcohol use (adjusted odds ratio [AOR]: 3.2, 95% CI: 1.0–10.4), sex with people living with HIV (PLWH) infection (AOR: 6.8, 95% CI: 1.9–25.0), and sex with people of the same sex (AOR: 5.4, 95% CI: 1.7–17.5) were predictors of HIV infection. Age ≤35 years (AOR: 3.8, 95% CI: 1.4–10.8), previous syphilis testing (AOR: 3.5, 95% CI: 1.4–8.4), history of genital lesions (AOR: 4.9, 95% CI: 1.3–19.1), and crack use in the last six months (AOR: 3.1, 95% CI: 1.3–7.6) were predictors of syphilis. Our findings highlight the importance of STI prevention and control strategies among the homeless.

Patterns of acquired HIV-1 drug resistance mutations and predictors of virological failure in Moshi, Northern Tanzania.

Emergence of HIV drug resistance poses a serious risk of inactivity to all currently approved antiretroviral drugs. Profiles of HIV drug resistance mutations (HIVDRM) and virological failure (VF) are not extensively studied in Tanzania. This study aimed to determine HIVDRM and predictors of VF in HIV-infected individuals failing first-line HIV drugs in Moshi, Northern Tanzania. A case-control study was conducted at Kilimanjaro Christian Medical Centre, Mawenzi, Pasua and Majengo health facilities with HIV-care and treatment clinics from October, 2017 to August, 2018. Cases and controls were HIV-infected individuals with VF and viral suppression (VS) respectively. HIV-1 reverse transcriptase and protease genes were amplified and sequenced. Stanford University’s HIV drug resistance database and REGA subtyping tool 3.0 determined HIVDRM and HIV-1 subtypes respectively. Odds ratios (OR) with 95% confidence interval (95% CI) investigated predictors of VF. P-value < 5% was considered statistically significant. A total of 124 participants were recruited, of whom 63 (50.8%) had VF, 61 (49.2%) had VS and 82 (66.1%) were females. Median [IQR] age and duration on ART were 45 [35–52] years and 72 [48–104] months respectively. Twenty-five out of 26 selected samples from cases were successfully sequenced. Twenty-four samples (96%) had at least one major mutation conferring resistance to HIV drugs, with non-nucleoside analogue reverse transcriptase inhibitor (NNRTI)-resistance associated mutations as the majority (92%). Frequent NNRTI-resistance associated mutations were K103N (n = 11), V106M (n = 5) and G190A (n = 5). Prevalent nucleoside analogue reverse transcriptase inhibitors-resistance associated mutations were M184V (n = 17), K70R (n = 7) and D67N (n = 6). Dual-class resistance was observed in 16 (64%) samples. Thirteen samples (52%) had at least one thymidine analogue-resistance associated mutation (TAM). Three samples (12%) had T69D mutation with at least 1 TAM. Two samples (8%) had at least one mutation associated with protease inhibitor resistance. Age [aOR = 0.94, 95% CI (0.90–0.97), p < 0.001] and occupation [aOR = 0.35, 95% CI (0.12–1.04), p = 0.059] associated with VF. In conclusion, HIV drug resistance is common among people failing antiretroviral therapy. Resistance testing will help to guide switching of HIV drugs.

Use of a mutation-specific genotyping method to assess for HIV-1 drug resistance in antiretroviral-naïve HIV-1 Subtype C-infected patients in Botswana

Background:HIV-1 drug resistance poses a major threat to the success of antiretroviral therapy. The high costs of available HIV drug resistance assays prohibit their routine usage in resource-limited settings. Pan-degenerate amplification and adaptation (PANDAA), a focused genotyping approach based on quantitative PCR (qPCR), promises a fast and cost-effective way to detect HIV drug resistance mutations (HIVDRMs).  Given the high cost of current genotyping methods, we sought to use PANDAA for screening key HIVDRMs in antiretroviral-naïve individuals at codons 103, 106 and 184 of the HIV-1 reverse transcriptase gene. Mutations selected at these positions have been shown to be the most common driver mutations in treatment failure. Methods:A total of 103 samples from antiretroviral-naïve individuals previously genotyped by Sanger population sequencing were used to assess and verify the performance of PANDAA. PANDAA samples were run on the ABI 7500 Sequence Detection System to genotype the K103N, V106M and M184V HIVDRMs. In addition, the cost per sample and reaction times were compared.Results:Sanger population sequencing and PANDAA detected K103N mutation in three (2.9%) out of 103 participants.  There was no evidence of baseline V106M and M184V mutations observed in our study. To genotype the six HIVDRMs it costs approximately 40 USD using PANDAA, while the reagents cost per test for Sanger population sequencing is approximately 100 USD per sample. PANDAA was performed quicker compared to Sanger sequencing, 2 hours for PANDAA versus 15 hours for Sanger sequencing.Conclusion:The performance of PANDAA and Sanger population sequencing demonstrated complete concordance. PANDAA could improve patient management by providing quick and relatively cheap access to drug-resistance information.

External Quality Assessment Scheme for HIV-1 Drug-Resistance Genotyping in Thailand.

The efficacy of antiretroviral (ARV) therapy can be compromised by the emergence and transmission of HIV-1 drug-resistant strains. HIV-1 drug-resistance (DR) genotypic testing thus plays an important role in the selection of optimal treatment regimens for HIV-infected individuals. Given the complexities of the testing procedures and the variety of approaches used, there is considerable potential for results to vary between laboratories. In Thailand, the national External Quality Assessment (EQA) scheme assesses the DR genotype testing performance of laboratories. Here, we evaluated the performance of laboratories in nucleotide sequencing and compared drug-resistance-associated mutations (DRMs) in the HIV-1 protease (PR) and reverse transcriptase (RT) genes during 2010-2015. The EQA samples in the 12 panels showed predominance for the CRF01_AE (85%) and subtype B (15%). Fourteen laboratory datasets were generated: eight using TruGene (TG), two using ViroSeq (VS), and four using in-house (IH) assays. All IH and VS laboratories had penalty scores <7, whereas five of the eight TG laboratories had fluctuating penalty scores. Moreover, seven and six TG laboratories could not amplify the two identical samples, 10B and 10E samples, or the CRF01_AE. Our findings demonstrate the requirement for laboratory participation in the ongoing EQA program and the optimization of kit assays using CRF01_AE samples. Our results also indicate that one advantage of participation is that the laboratories can monitor and investigate the source of laboratory errors.

Lower genetic variability of HIV-1 and antiretroviral drug resistance in pregnant women from the state of Par\xe1, Brazil

BackgroundThe present study aimed to describe the genetic diversity of HIV-1, as well as the resistance profile of the viruses identified in HIV-1 infected pregnant women under antiretroviral therapy in the state of Pará, Northern Brazil.MethodsBlood samples were collected from 45 HIV-1 infected pregnant to determine the virus subtypes according to the HIV-1 protease (PR) gene and part of the HIV-1 reverse transcriptase (RT) gene by sequencing the nucleotides of these regions. Drug resistance mutations and susceptibility to antiretroviral drugs were analyzed by the Stanford HIV Drug Resistance Database.ResultsOut of 45 samples, only 34 could be amplified for PR and 30 for RT. Regarding the PR gene, subtypes B (97.1%) and C (2.9%) were identified; for the RT gene, subtypes B (90.0%), F (6.7%), and C (3.3%) were detected. Resistance to protease inhibitors (PI) was identified in 5.8% of the pregnant, and mutations conferring resistance to nucleoside reverse transcriptase inhibitors were found in 3.3%, while mutations conferring resistance to non-nucleoside reverse transcriptase inhibitors were found in 3.3%.ConclusionsThese results showed a low frequency of strains resistant to antiretroviral drugs, the prevalence of subtypes B and F, and the persistent low transmission of subtype C in pregnant of the state of Pará, Brazil.

Prevalence of HIV-1 drug resistance in treated patients with viral load >50copies/mL: a 2014 French nationwide study.

Surveillance of HIV-1 resistance in treated patients with a detectable viral load (VL) is important to monitor, in order to assess the risk of spread of resistant viruses and to determine the proportion of patients who need new antiretroviral drugs with minimal cross-resistance. The HIV-1 protease and reverse transcriptase (RT) and integrase genes were sequenced in plasma samples from 782 consecutive patients on failing antiretroviral regimens, seen in 37 specialized centres in 2014. The genotyping results were interpreted using the ANRS v24 algorithm. Prevalence rates were compared with those obtained during a similar survey conducted in 2009. The protease and RT sequences were obtained in 566 patients, and the integrase sequence in 382 patients. Sequencing was successful in 60%, 78%, 78% and 87% of patients with VLs of 51-200, 201-500, 501-1000 and >1000 copies/mL, respectively. Resistance to at least one antiretroviral drug was detected in 56.3% of samples. Respectively, 3.9%, 8.7%, 1.5% and 3.4% of patients harboured viruses that were resistant to any NRTI, NNRTI, PI and integrase inhibitor (INI). Resistance rates were lower in 2014 than in 2009. Resistance was detected in 48.5% of samples from patients with a VL between 51 and 200 copies/mL. In France in 2014, 90.0% of patients in AIDS care centres were receiving antiretroviral drugs and 12.0% of them had VLs >50copies/mL. Therefore, this study suggests that 6.7% of treated patients in France might transmit resistant strains. Resistance testing may be warranted in all treated patients with VL > 50 copies/mL.

Deep Sequencing of HIV-1 RNA and DNA in Newly Diagnosed Patients with Baseline Drug Resistance Showed No Indications for Hidden Resistance and Is Biased by Strong Interference of Hypermutation.

Deep sequencing of plasma RNA or proviral DNA may be an interesting alternative to population sequencing for the detection of baseline transmitted HIV-1 drug resistance. Using a Roche 454 GS Junior HIV-1 prototype kit, we performed deep sequencing of the HIV-1 protease and reverse transcriptase genes on paired plasma and buffy coat samples from newly diagnosed HIV-1-positive individuals. Selection was based on the outcome of population sequencing and included 12 patients with either a revertant amino acid at codon 215 of the reverse transcriptase or a singleton resistance mutation, 4 patients with multiple resistance mutations, and 4 patients with wild-type virus. Deep sequencing of RNA and DNA detected 6 and 43 mutations, respectively, that were not identified by population sequencing. A subsequently performed hypermutation analysis, however, revealed hypermutation in 61.19% of 3,188 DNA reads with a resistance mutation. The removal of hypermutated reads dropped the number of additional mutations in DNA from 43 to 17. No hypermutation evidence was found in the RNA reads. Five of the 6 additional RNA mutations and all additional DNA mutations, after full exclusion of hypermutation bias, were observed in the 3 individuals with multiple resistance mutations detected by population sequencing. Despite focused selection of patients with T215 revertants or singleton mutations, deep sequencing failed to identify the resistant T215Y/F or M184V or any other resistance mutation, indicating that in most of these cases there is no hidden resistance and that the virus detected at diagnosis by population sequencing is the original infecting variant.

Evaluation of Automatic Analysis of Ultradeep Pyrosequencing Raw Data to Determine Percentages of HIV Resistance Mutations in Patients Followed-Up in Hospital.

A major obstacle to using next generation sequencing (NGS) technology in clinical routine practice is reliable data analysis. Thousands of sequences need to be aligned and validated, to exclude sequencing artifacts and generate accurate results. We compared two analysis pipelines for Roche 454 ultradeep pyrosequencing (UDPS) raw data generated from HIV-1 clinical samples: a commercial and fully automated Web-based software NGS HIV-1 Module (SmartGene, Zug, Switzerland) vs. the Amplicon Variant Analyzer software (AVA, 454 Life Sciences; Roche). Results were also compared to those obtained with Sanger sequencing. HIV-1 reverse transcriptase and protease genes from 34 plasma samples were submitted to Sanger sequencing and GS Junior UDPS. Raw UDPS data (sff files) from all samples were analyzed with AVA 2.7 software plus manual review of the alignments and the fully automated SmartGene NGS HIV-1 Module prototype (SMG). Results obtained with both analysis pipelines showed good correlation (85.0%). Divergent results were mainly observed at homopolymer positions, such as K101, where the frame-aware alignment and error corrections of the automated approach were more efficient and more accurate, both in terms of detecting and quantifying drug resistance mutations. Our study shows that NGS data can easily be analyzed via a fully automated analysis pipeline, here the SmartGene NGS HIV-1 Module, thus minimizing the need for manual review of alignments by the user, otherwise essential to ensure accurate results. Such automated analysis pipelines may facilitate the adoption of NGS platforms in the routine clinical laboratory.

The prevalence and determinants of drug-resistance-associated mutations in the HIV-1-infected MSM population of Henan Province in China.

To estimate the prevalence of human immunodeficiency virus (HIV) drug resistance (DR) in a population of men who have sex with men (MSM) from Henan Province of China and to identify the DR-associated HIV-1 mutations in these MSM. The HIV-positive status of the MSM subjects in this study was confirmed using ELISA and Western blotting. The MSM subjects were classified into non-treatment group (n = 106) and treatment group (n = 313). CD4(+) T-lymphocyte counts were obtained by flow cytometry, and viral load was measured by branched DNA (bDNA) signal amplification assay. HIV-1 genotypic resistance tests were performed by sequence analysis of the HIV-1 protease and reverse transcriptase genes. In the non-treatment group, 15 patients (14.2 %) displayed DR to non-nucleoside reverse transcriptase inhibitor (NNRTI). In the treatment group, the failure rate of viral suppression was 38.33 % and the DR rate was 33.2 %, which was higher than the rate observed in the non-treatment group (P < 0.05). The incidence of mutations corresponding to NNRTI resistance was significantly higher than the incidence of mutations corresponding to nucleoside reverse transcriptase inhibitor (NRTI) resistance (32.9 % vs. 26.5 %) in the cohort. After antiretroviral therapy (ART), the frequencies of K103N, G190A, Y181C, and V106A mutations were highly elevated. Logistic regression analysis results showed that duration of treatment, poor treatment compliance, drug abuse and homosexual orientation are the major risk factors for DR in this MSM population (all P < 0.05). Our results showed that DR-associated mutations in the HIV-1-infected MSM population increased significantly after ART. Furthermore, duration of treatment, poor treatment compliance, drug abuse and homosexual orientation were identified as the risk factors for DR in the MSM population from Henan Province in China.

Genotypic Variability of HIV-1 Reverse Transcriptase Gene from Long-Term Antiretroviral-Experienced Patients in Kenya.

There is continuous need to track genetic profiles of HIV strains circulating in different geographic settings to hasten vaccine discovery and inform public health and intervention policies. We partially sequenced the reverse transcriptase region of the HIV-1 pol gene from a total of 54 Kenyan patients aged 18-56 years who continued highly active antiretroviral treatment (HAART) for between 8 and 102 months. Subtyping was done using both the JPHMM tool and phylogenetic method. HIV-1 subtype A1 was the predominant strain in circulation, representing 57.4% and 70.4% of all isolates as determined by JPHMM and phylogenetic methods, respectively. Subtypes D (14.8%, 7.4%), C (5.6%, 9.3%), and A2 (0%, 5.6%) were determined at respective prevalence by both methods. JPHMM identified 22.2% of the isolates as recombinants. This surveillance focused on the RT gene and reaffirms the predominance of subtype A and an increasing proportion of recombinant strains in the Kenyan epidemic.

Appearance of drug resistance-associated mutations in human immunodeficiency virus type 1 protease and reverse transcriptase derived from drug-treated Indonesian patients.

Although HIV-1 drug resistance is a major obstacle in Indonesia, information on drug resistance is limited. In this study, the viral subtype and appearance of drug resistance mutations in the HIV-1 protease (PR) and reverse transcriptase (RT) genes were determined among drug-treated, HIV-1-infected patients in Surabaya. HIV-1 patients who received antiretroviral therapy (ART) more than 2 years were randomly recruited regardless of the viral load or ART failure. Fifty-eight HIV-1 PR genes and 53 RT genes were sequenced. CRF01_AE viruses were identified as the predominant strain. Major drug resistance mutations were not detected in the PR genes. In contrast, 37.7% (20/53) of the participants had one or more major drug resistance mutations in the RT genes, predominantly M184V (28.3%), K103N (11.3%), and thymidine analogue mutations (TAMs) (20.8%). The high prevalence of drug resistance mutations in RT genes indicated the necessity of monitoring the effectiveness of ART in Indonesia.

Comparison of HIV-1 drug resistance profiles generated from novel software applications for routine patient care.

IntroductionClinical laboratories performing routine HIV-1 genotyping antiviral drug resistance (DR) testing need reliable and up-to-date information systems to provide accurate and timely test results to optimize antiretroviral treatment in HIV-1-infected patients.Materials and MethodsThree software applications were used to compare DR profiles generated from the analysis of HIV-1 protease (PR) and reverse transcriptase (RT) gene sequences obtained by Sanger sequencing assay in 100 selected clinical plasma samples from March 2013 through May 2014. Interpretative results obtained from the Trugene HIV-1 Genotyping assay (TG; Guidelines v17.0) were compared with a newly FDA-registered data processing module (DPM v1.0) and the research-use-only ViroScore-HIV (VS) software, both of which use the latest versions of Stanford HIVdb (SD v7.0) and geno2pheno (G2P v3.3) interpretive algorithms (IA). Differences among the DR interpretive algorithms were compared according to drug class (NRTI, NNRTI, PI) and each drug. HIV-1 tropism and integrase inhibitor resistance were not evaluated (not available in TG).ResultsOverall, only 17 of the 100 TG sequences obtained yielded equivalent DR profiles among all 3 software applications for every IA and for all drug classes. DPM and VS generated equivalent results with >99.9% agreement. Excluding AZT, DDI, D4T and rilpivirine (not available in G2P), ranges of agreement in DR profiles among the three IA (using the DPM) are shown in Table 1. ConclusionsSubstantial discrepancies (<75% agreement) exist among the three interpretive algorithms for ETR, while G2P differed from TG and SD for resistance to TDF and TPV/r. Use of more than one DR interpretive algorithm using well-validated software applications, such as DPM v1.0 and VS, would enable clinical laboratories to provide clinically useful and accurate DR results for patient care needs.