ViroSeq HIV-1 Genotyping System Research Articles

Performance of the Applied Biosystems HIV-1 Genotyping Kit with Integrase.

HIV genotyping is used to assess HIV susceptibility to antiretroviral drugs. The Applied Biosystems HIV-1 Genotyping Kit with Integrase (AB kit, Thermo Fisher Scientific) detects resistance-associated mutations (RAMs) in HIV protease (PR), reverse transcriptase (RT), and integrase (IN). We compared results from the AB kit with results obtained previously with the ViroSeq HIV-1 Genotyping System. DNA amplicons from the AB kit were also analyzed using next-generation sequencing (NGS). HIV RNA was extracted using the MagNA Pure 24 instrument (Roche Diagnostics; 96 plasma samples, HIV subtype B, viral load range: 530-737,741 copies/mL). FASTA files were generated from AB kit data using Exatype (Hyrax Biosciences). DNA amplicons from the AB kit were also analyzed by NGS using the Nextera XT kit (Illumina). Drug resistance was predicted using the Stanford HIV Drug Resistance Database. The mean genetic distance for sequences from ViroSeq and the AB kit was 0.02% for PR/RT and 0.04% for IN; 103 major RAMs were detected by both methods. Four additional major RAMs were detected by the AB kit only. These four major RAMs were also detected by NGS (detected in 18.1%-38.2% of NGS reads). NGS detected 27 major RAMs that were not detected with either of the Sanger sequencing-based kits. All major RAMs detected with ViroSeq were detected with the AB kit; additional RAMs were detected with the AB kit only. DNA amplicons from the AB kit can be used for NGS for more sensitive detection of RAMs.

Prevalence of Transmitted Drug Resistance among HIV-1 Patients in the Aegean Region: Results from the Western part of Turkey.

This study aimed to analyze the antiretroviral drug resistance in antiretroviral treatment-naïve HIV-positive patients in the Aegean Region of Turkey from 2012 to 2019. The study included 814 plasma samples from treatment-naïve HIV-positive patients. Drug resistance analysis was performed by Sanger sequencing (SS) between 2012-2017 and by next-generation sequencing sequencing (NGS) between 2018-2019. SS was used to analyze resistance mutations in the protease (PR) and reverse transcriptase (RT) gene regions using a ViroSeq HIV-1 Genotyping System. PCR products were analyzed with an ABI3500 GeneticAnalyzer (Applied Biosystems). The sequencing of the HIV genome in the PR, RT, and integrase gene regions was carried out using MiSeq NGS technology. Drug resistance mutations and subtypes were interpreted using the Stanford University HIV-1 drug resistance database. Transmitted drug resistance (TDR) mutation was detected in 34/814 ( 4.1 %) samples. Non-nucleoside reverse transcriptase inhibitor (NNRTI), nucleoside reverse transcriptase inhibitor (NRTI), and protease inhibitor (PI) mutations were identified in 1.4 % (n =12), 2.4 % (n =20), and 0.3 % (n = 3) of samples, respectively. The most common subtypes were B (53.1 % ), A (10.9%), CRF29_BF (10.6%), and B + CRF02_AG (8,2%). The most common TDR mutations were E138A (3.4%), T215 revertants (1.7%), M41L (1.5%), and K103N (1.1%). Transmitted drug resistance rate in the Aegean Region is compatible with national and regional data. Routine surveillance of resistance mutations may guide the safe and correct selection of initial drug combinations for antiretroviral therapy. The identification of HIV-1 subtypes and recombinant forms in Turkey may contribute to international molecular epidemiological data.

Diversity of HIV-1 Subtypes and Transmitted Drug-resistance Mutations Among Minority HIV-1 Variants in a Turkish Cohort.

The World Health Organization (WHO) recommends the surveillance of transmitted drug resistance mutations (TDRMs) to ensure the effectiveness and sustainability of HIV treatment programs. Our aim was to determine the TDRMs and evaluate the distribution of HIV-1 subtypes using and compared next-generation sequencing (NGS) and Sanger-based sequencing (SBS) in a cohort of 44 antiretroviral treatment-naïve patients. All samples that were referred to the microbiology laboratory for HIV drug resistance analysis between December 2016 and February 2018 were included in the study. After exclusions, 44 treatment-naive adult patients with a viral load of >1000 copies/mL were analyzed. DNA sequencing for reverse transcriptase and protease regions was performed using both DeepChek ABL single round kit and Sanger-based ViroSeq HIV-1 Genotyping System. The mutations and HIV-1 subtypes were analyzed using the Stanford HIVdb version 8.6.1 Genotypic Resistance software, and TDRMs were assessed using the WHO surveillance drug-resistance mutation database. HIV-1 subtypes were confirmed by constructing a maximum-likelihood phylogenetic tree using Los Alamos IQ-Tree software. NGS identified nucleos(t)ide reverse transcriptase inhibitor (NRTI)-TDRMs in 9.1 % of the patients, non-nucleos(t)ide reverse transcriptase inhibitor (NNRTI)-TDRMs in 6.8 % of the patients, and protease inhibitor (PI)-TDRMs in 18.2 % of the patients at a detection threshold of ≥ 1 %. Using SBS, 2.3 % and 6.8 % of the patients were found to have NRTI- and NNRTI-TDRMs, respectively, but no major PI mutations were detected. M41L, L74I, K65R, M184V, and M184I related to NRTI, K103N to NNRTI, and N83D, M46I, I84V, V82A, L24I, L90M, I54V to the PI sites were identified using NGS. Most mutations were found in low-abundance (frequency range: 1.0 % - 4.7 %) HIV-1 variants, except M41L and K103N. The subtypes of the isolates were found as follows; 61.4 % subtype B, 18.2 % subtype B/CRF02_AG recombinant, 13.6 % subtype A, 4.5 % CRF43_ 02G, and 2.3 % CRF02_AG. All TDRMs, except K65R, were detected in HIV-1 subtype B isolates. The high diversity of protease site TDRMs in the minority HIV-1 variants and prevalence of CRFs were remarkable in this study. All minority HIV-1 variants were missed by conventional sequencing. TDRM prevalence among minority variants appears to be decreasing over time at our center.

Evaluation of a Novel In-house HIV-1 Genotype Drug Resistance Assay using Clinical Samples in China.

BackgroundHIV drug resistance poses a major challenge for anti-retroviral treatment (ART) and the prevention and control of HIV epidemic.ObjectiveThe study aims to establish a novel in-house assay with high efficiency, named AP in- house method, that would be suitable for HIV-1 drug resistance detection in China.MethodsAn in-house HIV-1 genotyping method was used to sequence the partial pol gene from 60 clinical plasma samples; the results of our test were compared with a commercial ViroSeq HIV-1 genotyping system.ResultsAmong sixty samples, 58(96.7%) were successfully amplified by AP in-house method, five of them harbored viral load below 1,000 copies/ml. The genotype distribution was 43.1% CRF07_BC (25/58), 39.7% CRF01_AE (23/58), 6.9% CRF55_01B (4/58), 5.2% subtype B (3/58) and 5.2% CRF08_BC (3/58). Compared with that of the ViroSeq system, the consistent rate of these nucleotides and amino acids obtained by AP in-house method was up to 99.5 ± 0.4% and 99.5 ± 0.4%, respectively. A total of 290 HIV-1 drug resistance mutations were identified by two methods, including 126 nucleoside reverse transcriptase inhibitors (NRTIs), 145 non-nucleoside reverse transcriptase inhibitors (NNRTIs) and 19 protease inhibitors (PIs) resistance mutations. Out of them, 94.1% (273/290) were completely concordant between the AP in-house method and the ViroSeq system. ConclusionOverall, the evaluation of AP in-house method provided comparable results to those of the ViroSeq system on diversified HIV-1 subtypes in China.

Prevalent subtypes and one-year outcomes of an HIV-cohort from an urban Philippine center.

Circulating HIV subtypes in the Philippines have increasingly diversified, potentially affecting treatment. We monitored outcomes of a treatment-naïve cohort and their virus subtype prevalence.Retrospective/prospective study cohort.HIV-I-REACT clinic patients co-enrolled in the Virology Quality Assurance Program (RUSH-VQA) from 7/2017-6/2019 were included. Relevant demographic and laboratory information were collected. The ViroSeq HIV-1 Genotyping System v.3 and HIV-1 Integrase Genotyping Kit identified protease-reverse transcriptase and integrase drug resistance mutations (DRM). Sequence subtyping followed using the Stanford University Drug Resistance Database and the REGA HIV-1 Subtyping Tool v.3. The jpHMM HIV-1 Tool and REGA HIV-1 Subtyping Tool provided additional subtype analysis of this cohort's 5’LTR-VIF regions after Sanger sequencing. One-year outcomes included virologic suppression, mortality, and follow-up.86/88 patients were males. Median age was 30 (range 19–65) years; 61/88 were MSM. 15/85 carried baseline DRM. ViroSeq-generated sequences included subtypes CRF01_AE (66/85), B (14/85), and newer recombinants (4/85). Extensive sequencing (n = 71) of the 5’-LTR-GAG-Pol genes showed CRF01_AE (n = 50), subtype B (n = 7), and other recombinants (n = 13). Bootstrap analysis identified 7 pairs of highly related strains. Discordant DRM appeared in 2/7 pairs, where 1/2 strains displayed DRM. After 1 year, 87 individuals were alive, with 19 lost to care. Viral load (VL) was repeated for only 31/77 (40.2%). Follow-up CD4 testing for 39/77 (50.6%) showed an increase to a median of 327 cells/mm3.Our cohort currently carries subtype CRF01_AE (∼68%–70%), followed by subtype B and CRF01_AE/B recombinants. Outcomes were favorable, regardless of subtype after 1 year on cART.

Antiretroviral drug use and HIV drug resistance in female sex workers in Tanzania and the Dominican Republic.

ObjectiveFemale sex workers (FSW) have increased risk of HIV infection. Antiretroviral treatment (ART) can improve HIV outcomes and prevent HIV transmission. We analyzed antiretroviral (ARV) drug use and HIV drug resistance among HIV-positive FSW in the Dominican Republic and Tanzania.MethodsPlasma samples collected at study entry with viral loads >1,000 copies/mL were tested for ARV drugs and HIV drug resistance. ARV drug testing was performed using a qualitative assay that detects 22 ARV drugs in five classes. HIV genotyping was performed using the ViroSeq HIV-1 Genotyping System. Phylogenetic analyses were performed to determine HIV subtype and assess transmission clusters.ResultsAmong 410 FSW, 144 (35.1%) had viral loads >1,000 copies/mL (DR: n = 50; Tanzania: n = 94). ARV drugs were detected in 36 (25.0%) of 144 samples. HIV genotyping results were obtained for 138 (95.8%) cases. No transmission clusters were observed in either country. HIV drug resistance was detected in 54 (39.1%) of 138 samples (31/35 [88.6%] with drugs detected; 23/103 [22.3%] without drugs detected); 29/138 (21.0%) had multi-class resistance (MCR). None with MCR had integrase strand transfer inhibitor resistance. In eight cases, one or more ARV drug was detected without corresponding resistance mutations; those women were at risk of acquiring additional drug resistance. Using multivariate logistic regression, resistance was associated with ARV drug detection (p<0.001), self-reported ART (full adherence [p = 0.034]; partial adherence [p<0.001]), and duration of HIV infection (p = 0.013).ConclusionsIn this cohort, many women were on ART, but were not virally suppressed. High levels of HIV drug resistance, including MCR, were observed. Resistance was associated with detection of ARV drugs, self-report of ART with full or partial adherence, and duration of HIV infection. These findings highlight the need for better HIV care among FSW to improve their health, reduce HIV drug resistance, and decrease risk of transmission to others.

Performance of a high-throughput next-generation sequencing method for analysis of HIV drug resistance and viral load.

To evaluate the performance of a high-throughput research assay for HIV drug resistance testing based on whole genome next-generation sequencing (NGS) that also quantifies HIV viral load. Plasma samples (n = 145) were obtained from HIV-positive MSM (HPTN 078). Samples were analysed using clinical assays (the ViroSeq HIV-1 Genotyping System and the Abbott RealTime HIV-1 Viral Load assay) and a research assay based on whole-genome NGS (veSEQ-HIV). HIV protease and reverse transcriptase sequences (n = 142) and integrase sequences (n = 138) were obtained using ViroSeq. Sequences from all three regions were obtained for 100 (70.4%) of the 142 samples using veSEQ-HIV; results were obtained more frequently for samples with higher viral loads (93.5% for 93 samples with >5000 copies/mL; 50.0% for 26 samples with 1000-5000 copies/mL; 0% for 23 samples with <1000 copies/mL). For samples with results from both methods, drug resistance mutations (DRMs) were detected in 33 samples using ViroSeq and 42 samples using veSEQ-HIV (detection threshold: 5.0%). Overall, 146 major DRMs were detected; 107 were detected by both methods, 37 were detected by veSEQ-HIV only (frequency range: 5.0%-30.6%) and two were detected by ViroSeq only. HIV viral loads estimated by veSEQ-HIV strongly correlated with results from the Abbott RealTime Viral Load assay (R2 = 0.85; n = 142). The NGS-based veSEQ-HIV method provided results for most samples with higher viral loads, was accurate for detecting major DRMs, and detected mutations at lower levels compared with a method based on population sequencing. The veSEQ-HIV method also provided HIV viral load data.

Antiretroviral Drug Use and HIV Drug Resistance Among Young Women in Rural South Africa: HPTN 068.

Antiretroviral (ARV) drugs are used for HIV treatment and prevention. We analyzed ARV drug use and HIV drug resistance in a cohort of young women in rural South Africa enrolled in the HIV Prevention Trials Network (HPTN) 068 study, which evaluated the use of a cash transfer conditional on school attendance to reduce HIV incidence. ARV drug testing was performed using plasma samples from 2526 young women. This included 2526 enrollment samples (80 HIV-infected and 2446 HIV-uninfected) and 162 seroconversion samples (first HIV-positive study visit). Testing was performed using a qualitative assay that detects 20 ARV drugs from 5 drug classes. HIV drug resistance testing was performed with the ViroSeq HIV-1 Genotyping System for samples that had HIV viral loads ≥400 copies per milliliter. At enrollment, ARV drugs were detected in 10 (12.5%) of 80 HIV-infected young women. None of 2446 HIV-uninfected young women had ARV drugs detected at enrollment. ARV drugs were also detected in 16 (9.9%) of 162 seroconverters. At enrollment, 9 (13.4%) of 67 young women with genotyping results had HIV drug resistance; resistance was also detected in 9 (6.9%) of 131 seroconverters with genotyping results. Most of the HIV-infected young women in this cohort from rural South Africa were not taking ARV drugs, suggesting they were unaware of their HIV status or were not in care. HIV drug resistance was detected in young women with both prevalent and new HIV infection.

Human immunodeficiency virus type 1 drug resistance in a subset of mothers and their infants receiving antiretroviral treatment in Ouagadougou, Burkina Faso.

The emergence of HIV-1 drug resistance (HIVDR) is a public health problem that affects women and children. Local data of HIVDR is critical to improving their care and treatment. So, we investigated HIVDR in mothers and infants receiving antiretroviral therapy (ART) at Saint Camille Hospital of Ouagadougou, Burkina Faso. This study included 50 mothers and 50 infants on ART. CD4 and HIV-1 viral load were determined using FACSCount and Abbott m2000rt respectively. HIVDR was determined in patients with virologic failure using ViroSeq HIV-1 Genotyping System kit on the 3130 Genetic Analyzer. The median age was 37.28 years in mothers and 1.58 year in infants. Sequencing of samples showed subtypes CRF02_AG (55.56%), CRF06_cpx (33.33%) and G (11.11%). M184V was the most frequent and was associated with highlevel resistance to 3TC, FTC, and ABC. Other mutations such as T215F/Y, D67N/E, K70R, and K219Q were associated with intermediate resistance to TDF, AZT, and 3TC. No mutation to LPV/r was detected among mothers and infants. The findings of HIVDR in some mothers and infants suggested the change of treatment for these persons.

Phylogenetic Analysis of HIV from People Who Inject Drugs in Indonesia, Ukraine, and Vietnam: HPTN 074

Background: HPTN 074 evaluated an HIV prevention intervention for persons who inject drugs (PWID) that included support for HIV and substance use treatment. The study enrolled HIV-infected index participants and HIV-uninfected injection partners (up to five per index) in Indonesia, Ukraine, and Vietnam. Seven partners acquired HIV infection during the study. We analyzed the relationship between HIV strains in this cohort. Methods: HIV from index participants and their partners was sequenced using the ViroSeq HIV-1 Genotyping System. Phylogenetic trees were constructed using RAxML v8.2.10. Pol sequence clusters were identified that had a genetic distance ≤1.5% with ≥90% branch support. Next generation sequencing (NGS, env region) was also performed for the seven seroconverters and the corresponding index participants (index-partner pairs) and from other index participants in pol sequence clusters. Participants were classified as having linked infections if their env sequences formed a distinct monophyletic cluster with ≥90% bootstrap support Findings: Phylogenetic analysis included pol sequences from 445 of 502 index participants and all seven HIV-infected partners. The median pairwise genetic distance at the three study sites ranged from 2.9% to 3.3% with no evidence of large discrete subclades. Eighteen pol clusters were identified, including two of the seven index-partner pairs, one pair of partners enrolled with different indexes, and 15 index-only groups including 36 participants (2-7 index participants per cluster). NGS-env analysis confirmed linkage for both index-partner clusters, the partner-partner cluster, and 11 index-index clusters. Interpretation: Only two of the seven index-partner pairs in this study had genetically-linked HIV infections. Clusters were also identified that included two partners enrolled with different indexes, and groups of 2-7 index participants. These findings suggest that individuals in these PWID communities have exposure to multiple sources of HIV infection. This should be considered when designing strategies for HIV prevention in these high-risk populations. Trial Registration Number: In this report, we analyzed HIV from PWID enrolled in a randomized clinical trial conducted in Indonesia, Ukraine, and Vietnam (HIV Prevention Trials Network [HPTN] 074) Funding Statement: National Institute of Allergy and Infectious Diseases (NIAID), National Institute on Drug Abuse (NIDA), and Office of AIDS Research, of the National Institutes of Health (NIH). Declaration of Interests: None of the authors has a conflict of interest or potential conflict of interest, with the following exceptions: Susan Eshleman has collaborated on research studies with investigators from Abbott Laboratories (distributor of the ViroSeq HIV-1 Genotyping System); Abbott Laboratories has provided reagents for collaborative research studies. Ethics Approval Statement: Written informed consent was signed by each participant in the HPTN 074 trial. The study was approved by institutional review boards and ethics committees of the participating institutions in the United States and at each study site.

HIV Antiretroviral Resistance and Transmission in Mother–Infant Pairs Enrolled in a Large Perinatal Study

BackgroundDetection of antiretroviral (ARV) resistance in HIV-infected individuals is not uncommon and may be particularly problematic in HIV-infected pregnant women as it can lead to infant infection with resistant strains. To better evaluate the effect of drug resistance mutations (DRMs) on HIV mother-to-child transmission (MTCT), we determined the prevalence of DRMs in a subset of mother–infant pairs enrolled in a multi-center trial of infant prophylaxis among women not receiving ARVs during the current pregnancy.MethodsA case–control design of 1:4 (1 transmitter to 4 nontransmitters) was utilized to evaluate ARV resistance as a predictor of HIV MTCT in specimens obtained from mother–infant pairs. Secondary objectives included identification of potential risk factors associated with the presence of DRMs. Viroseq HIV-1 Genotyping System was performed on mother–infant specimens to assess for mutations that might result in a substantial reduction in drug susceptibility and clinical outcome, as determined by the Stanford HIV Drug Resistance Database.ResultsOne hundred and forty infants were infected. Of these, 123 HIV infected mother–infant pairs and 483 of 560 women who did not transmit HIV had amplifiable HIV nucleic acid enabling ARV resistance testing. A wide variety of DRMs were detected (Figure 1). Sixty (10%) of 606 women had clinically relevant DRMs; 12 (2%) had DRMs against more than 1 ARV class. Among 123 HIV− infected infants, 13 (11%) had clinically relevant DRMs with 3 (2%) harboring DRMs against more than 1 ARV class. Of 13 infants with DRMs, 10 (77%) were infected in utero. In univariate and multivariate analyses, DRMs in mothers were not associated with increased risk of HIV MTCT (AOR 0.79, 95% CI 0.38–1.5). Log HIV viral load was the only predictor of MTCT (OR 1.4, 95% CI 1.2−1.6). The presence of DRMs in mothers who transmitted was strongly associated with the presence of DRMs in infants (P < 0.001).ConclusionIn infected pregnant women without ARV exposure during their current gestation, the presence of pre-existing DRMs with a wide diversity was noted. DRMs do not increase the risk of HIV MTCT. However, if women with DRMs are not virologically suppressed they are likely to transmit resistant mutations even without selective ARV pressure, thus complicating treatment options.Disclosures All authors: No reported disclosures.

Prevalence of HIV-1 Subtypes and Antiretroviral Drug Resistance Mutations in Nepal.

There have been very few reports of HIV-1 subtypes and drug resistance mutations (DRMs) from Nepal which is geographically located between two high-prevalence HIV-1 infection countries, China and India. The aim of this study was to determine prevalence of acquired and transmitted DRMs and HIV-1 subtypes in Nepal. Thirty-five HIV-1 seropositive samples from central region of Nepal were collected in 2011. The subjects were divided into two groups, antiretroviral (ARV) drug naïve group (n=15) and antiretroviral treatment (ART) group (n=20), 90% (18/20) of them received zidovudine, lamivudine and nevirapine (AZT/3TC/NVP) regimen. HIV pol (protease and reverse transcriptase regions) nucleotide sequences were analyzed by Viroseq HIV-1 Genotyping System. Nearly full-length genomic (NFLG) sequences of 10 samples were performed. NFLG genotyping revealed that 80% of samples were infected with subtype C and 20% with recombinants (C/D/H and C/A). Phylogenetic analysis of 35 pol sequences from Nepal were subtype C. The prevalence of acquired DRMs to NNRTIs and NRTIs was 15% (3/20). DRMs to NVP, K103N and V179D, and to NRTIs were observed at 11.1% (2/18) and 5% (1/20), respectively. The prevalence of DRMs to rilpivirine for E138A/G was 5.7%. The minor protease inhibitors (PI) associated mutations (A71T/V and T74S) were observed in 5/35 (14.3%) subjects. This is the first report of NFLG HIV-1 genomic sequences and DRMs from Nepal. National surveillance of HIV DRMs to ARVs and molecular epidemiology study should be done annually for better prevention and treatment of HIV infection in Nepal.

Опыт использования филогенетического анализа в эпидемиологическом расследовании случая заражения ВИЧ-инфекцией

Objective of the study was to investigate a criminal case of infection with HIV, applying molecular-genetic analysis of blood plasma samples from an estimated source of an infection and the recipient for evaluation of probability of epidemiological connection between them. Materials and methods. The study involved genotyping and phylogenetic analysis of nucleotide sequences of HIV-1 variants, isolated from patients in the investigated group and the control one (19 nucleotide sequences of the HIV-1 from the patients living in the Saratov region, and 15 nucleotide sequences from GenBank). Genotyping was performed using the commercial ViroSeq HIV-1 Genotyping System. The sub-typing of HIV-1 strains was carried out on-line, through the COMET HIV-1/2 and HCV and REGA HIV-1 Sybtyping Tool programs. Phylogenetic analysis of nucleotide sequences was carried out by Mega software, version 5.2. Phylogenetic trees were constructed; nucleotide distances were calculated by Kimura method (bootstrap level 1000). Results and conclusions. Virus variants, isolated from the studied samples, were defined as HIV-1 A subtype. Performed phylogenetic analysis showed that nucleotide sequences of the studied samples authentically grouped on the phylogenetic tree, forming a common cluster, which mismatched that of control group. Calculation of the genetic distance testifies that the genetic relation between the samples within the investigated group is higher, than between the same samples and those of the control group. Thus, by means of phylogenetic analysis it is shown that the strains received from an estimated source of infection and the recipient are genetically closer to each other, than to the strains from the group of comparison. In this regard, it is possible to claim with a big share of confidence that probability of epidemiological connection between them exists.

Workflow Optimization of the ViroSeqTM Human Immunodeficiency Virus-1 (HIV-1) Genotyping System

Background Current therapy guidelines recommend resistance testing to guide therapy in antiretroviral ARV treatment naive patients as well as patients with suboptimal virologic responses or virologic failure Objective This study evaluated workflow optimization and automation improvements to the ViroSeq HIV Genotyping System through the use of the semi automated extraction platforms Abbott Molecular Inc m sp and bioM eacute rieux Inc NucliSens easyMAG for sample extraction NanoDrop Spectrophotometer Thermo Scientific CA and Agilent Bioanalyzer Agilent Technologies Germany for amplicon quantitation and Applied Biosystems Instruments ABI Life Technologies Ca for increased sequencing throughput Table Methods Clinical plasma samples were processed in parallel with Viroseq HIV Genotyping System per manufacturer instructions along with above described automated options Resistance associated mutations from the different approaches were evaluated and assessed for clinical impact Results All samples tested in this study gave valid results regardless of the procedure used Samples extracted with m sp demonstrated resistance associated mutations in samples while complete drug resistance concordance was observed in samples Ninety one percent of the samples extracted with easyMAG had concordance at the nucleotide level and concordance for drug resistance mutations No apparent difference was observed in sequence quality between manual and semi automated extractions and comparable drug resistance profiles were obtained regardless of the extraction method Conclusion The ViroSeq HIV Genotyping System is compatible with the semi automated extraction systems alternative amplicon quantitation devices as well as with high throughput sequencing platform such as the ABI sequencer This study also indicated that the assay can accurately sequence HIV virus below copies mL Significant workflow and turnaround time advantages were observed in this study with the automation of the Viroseq HIV Genotyping System

Performance of an in-house human immunodeficiency virus type 1 genotyping system for assessment of drug resistance in Cuba.

As commercial human immunodeficiency virus type 1 drug resistance assays are expensive, they are not commonly used in resource-limited settings. Hence, a more affordable in-house procedure was set up taking into account the specific epidemiological and economic circumstances of Cuba. The performance characteristics of the in-house assay were evaluated using clinical samples with various subtypes and resistance patterns. The lower limit of amplification was determined on dilutions series of 20 clinical isolates and ranged from 84 to 529 RNA copies/mL. For the assessment of trueness, 14 clinical samples were analyzed and the ViroSeq HIV-1 Genotyping System v2.0 was used as the reference standard. The mean nucleotide sequence identity between the two assays was 98.7% ± 1.0. Additionally, 99.0% of the amino acids at drug resistance positions were identical. The sensitivity and specificity in detecting drug resistance mutations was respectively 94.1% and 99.5%. Only few discordances in drug resistance interpretation patterns were observed. The repeatability and reproducibility were evaluated using 10 clinical samples with 3 replicates per sample. The in-house test was very precise as nucleotide sequence identity among paired nucleotide sequences ranged from 98.7% to 99.9%. The acceptance criteria were met by the in-house test for all performance characteristics, demonstrating a high degree of accuracy. Subsequently, the applicability in routine clinical practice was evaluated on 380 plasma samples. The amplification success rate was 91% and good quality consensus sequences encoding the entire protease and the first 335 codons in reverse transcriptase could be obtained for 99% of the successful amplicons. The reagent cost per sample using the in-house procedure was around € 80 per genotyping attempt. Overall, the in-house assay provided good results, was feasible with equipment and reagents available in Cuba and was half as expensive as commercial assays.

Pitfalls of antiretroviral drug resistance genotyping of HIV-1 Group M and Group N from Cameroon by sequenced-based assays.

Background:HIV-1 genotyping for antiretroviral drug resistance mutations (DRMs) were developed based basically on subtype B HIV-1 Group M, which represents only 10% of HIV strains worldwide. In sub-Saharan Africa, non-B subtypes HIV-1 largely predominate and HIV-1 genetic diversity could affect the performance of drug resistance genotyping assays. We compared prospectively the performance of the ViroSeq® and Trugene® genotyping assays to detect DRM in HIV-1-infected adult patients living in Douala, Cameroun.Materials and Methods:DRM in protease (P) and reverse transcriptase (RT) genes were assessed in parallel using both ViroSeq® and Trugene® assays in plasma samples from 45 first-line antiretroviral treatment-experienced patients in Douala, Cameroon.Results:Trugene HIV-1 Genotyping Assay® (Siemens Health Care Diagnostics, NY, USA) and ViroSeq HIV-1 Genotyping System®(Celera Diagnostics, CA, USA) assessed equivalently antiretroviral DRMs in P and RT genes from non-B HIV-1 Group M in 44 Cameroonian adults in virological failure; Trugene® was slightly more sensitive than ViroSeq® (100% vs. 91%). One patient infected by HIV-1 Group N was successfully amplified only by the Trugene HIV-1 Genotyping assay®, while ViroSeq HIV-1 Genotyping System v2.0® assay could not.Conclusion:Results showed the higher performance of the Trugene® system to detected and amplify P and RT genes targeting DRM to the principal antiretroviral drugs used in sub-Saharan Africa. Discrepancies between the results of HIV viral load assays and molecular tests should alert clinicians and virologists to the possibility of infection by an atypical variant virus, especially in Central Africa where very broad HIV-1 genetic diversity exists.

Early clinical response and presence of viral resistant minority variants: a proof of concept study.

IntroductionTraditional genotyping assays detect viral variants present in at least 15–25% of the entire virus population. We tested the Next generation GS Junior System (NGS) setted with a detection limit of 0.05% and evaluated the clinical relevance of low prevalent mutations.MethodsNGS was performed on the plasma of 26 infected individuals who started a TDF/FTC/RPV (15 subjects) or TDF/FTC/EFV (11 subjects) cART after a routine HIV-1 drug-resistance negative test by Viroseq HIV-1 Genotyping System. Amplicon Sequencing of HIV-1 RT and PR Plate (Roche) was performed following the manufacturer's instructions. HIV-1 variants were analyzed by a specific HIV-1 tool by AVA software v. 2.7. The updated IAS resistance mutations list (March 2013) was considered for the analysis of resistance positions. Patients were followed testing viral load and immunologic parameters.ResultsTwenty four males and two females with a mean age of 43 years were included. Twenty-one were nave for cART. At baseline, median HIV-RNA was 4.57 log copies/mL (range 2.15–6.57) and CD4 count 315 cells/mcL (range 16–648). In 18 patients, NGS did not detect any additional variant relevant for the selected cART compared to population genotyping. In the remaining eight patients resistance conferring mutations to part of the ongoing regimen were detected. Single mutations E138K (two cases) and M184V in three distinct patients and V90I+G190E; M184V+A98S; Y215F+V118I+T215I; L210S+T215I+F227L; and A62V+D67G+K70N+188H in the remaining five subjects. In all cases, the mutation prevalence was inferior to 5%. The mean daily reduction of VL was −3759 copies/mL in patients without NGS detected mutations and −1045 copies/mL in those with mutations. The median KM estimates for reaching an HIV-RNA blood level <50 copies/mL were 127 days and 161 days, respectively. One patient without baseline resistance selected for M184I+E138K+T215I (NGS) after four months of TDF/FTC/RPV therapy.ConclusionsNGS detected low-frequency HIV-1 variants harbouring RT drug resistance mutations that could have affected the therapy outcome. However, viral decay in an early cART phase was not affected by the presence of resistant minority variants. The low prevalence of the detected mutation, the limited effect on the combination regimen and the potency of cART components could be possible explanations of our findings. Longer follow-up and larger casuistries are needed to determine the clinical relevance of NGS in routine clinical practice and eventually define a clinically relevant mutations' prevalence.

An international multicenter study on HIV-1 drug resistance testing by 454 ultra-deep pyrosequencing

The detection of mutant spectra within the viral quasispecies is critical for therapeutic management of HIV-1 infections. Routine clinical application of ultrasensitive genotyping requires reproducibility and concordance within and between laboratories. The goal of the study was to evaluate a new protocol on HIV-1 drug resistance testing by 454 ultra-deep pyrosequencing (454-UDS) in an international multicenter study. Sixteen blinded HIV-1 subtype B samples were provided for 454-UDS as both RNA and cDNA with viral titers of 88,600–573,000 HIV-1 RNA copies/ml. Eight overlapping amplicons spanning protease (PR) codons 10–99 and reverse transcriptase (RT) codons 1–251 were generated using molecular barcoded primers. 454-UDS was performed using the 454 Life Sciences/Roche GS FLX platform. PR and RT sequences were analyzed using 454 Life Sciences Amplicon Variant Analyzer (AVA) software. Quantified variation data were analyzed for intra-laboratory reproducibility and inter-laboratory concordance. Routine population sequencing was performed using the ViroSeq HIV-1 genotyping system. Eleven laboratories and the reference laboratory 454 Life Sciences sequenced the HIV-1 sample set. Data presented are derived from seven laboratories and the reference laboratory since severe study protocol execution errors occurred in four laboratories leading to exclusion. The median sequencing depth across all sites was 1364 reads per position (IQR=809–2065). 100% of the ViroSeq-reported mutations were also detected by 454-UDS. Minority HIV-1 drug resistance mutations, defined as HIV-1 drug resistance mutations identified at frequencies of 1–25%, were only detected by 454-UDS. Analysis of 10 preselected majority and minority mutations were consistently found across sites. The analysis of drug-resistance mutations detected between 1 and 10% demonstrated high intra- and inter-laboratory consistency in frequency estimates for both RNA and prepared cDNA samples, indicating robustness of the method. HIV-1 drug resistance testing using 454 ultra-deep pyrosequencing results in an accurate and highly reproducible, albeit complex, approach to the analysis of HIV-1 mutant spectra, even at frequencies well below those detected by routine population sequencing.

Performance of the ViroSeq HIV-1 genotyping system v2.0 on HIV-1 strains circulating in Senegal

The objective of this study was to investigate the performance of the ViroSeq HIV-1 Genotyping System v2.0 on HIV-1 non-B strains identified in Senegalese patients. The study involved 150 patients, and genotyping was performed using the ViroSeq HIV-1 Genotyping System v2.0 or an in-house method developed by the French National Agency on AIDS Research AC11 when the ViroSeq HIV-1 Genotyping System v2.0 failed. The sequences were edited to assess the performance of sequencing primers at their presumed binding regions. The Polymorphism was studied in the regions between the sequences of Senegalese patients and the subtype B strains used as references. The phylogenetic analysis showed a predominance of CRF02_AG (88/150; 58.7%) and the circulation of 11 subtypes/CRFs, 16 unique recombinant forms (URFs) and one unclassified sample. The amplification and sequencing rates were 98% (147/150) and 96.6% (142/147), respectively. This study showed that only primer B exhibited 100% success, while the failure rate ranged from 1.4% to 71.4% for the other primers (D: 71.4%, A and H: 12.2%, F: 7.5%, G: 5.5% and C: 1.4%). These findings suggest the need for an alternative method or alternative primers for non-B strains that were not sequenced successfully using the ViroSeq HIV-1 Genotyping System v2.0.

Characterization of HIV-1 antiretroviral drug resistance after second-line treatment failure in Mali, a limited-resources setting.

We describe the outcomes of second-line drug resistance profiles and predict the efficacy of drugs for third-line therapy in patients monitored without the benefit of plasma HIV-1 RNA viral load (VL) or resistance testing. We recruited 106 HIV-1-infected patients after second-line treatment failure in Mali. VL was determined by the Abbott RealTime system and the resistance by the ViroSeq HIV-1 genotyping system. The resistance testing was interpreted using the latest version of the Stanford algorithm. Among the 106 patients, 93 had isolates successfully sequenced. The median age, VL and CD4 cells were respectively 35 years, 72 000 copies/mL and 146 cells/mm(3). Patients were exposed to a median of 4 years of treatment and to six antiretrovirals. We found 20% of wild-type viruses. Resistance to etravirine was noted in 38%, to lopinavir in 25% and to darunavir in 12%. The duration of prior nucleos(t)ide reverse transcriptase inhibitor exposure was associated with resistance to abacavir (P < 0.0001) and tenofovir (P = 0.0001), and duration of prior protease inhibitor treatment with resistance to lopinavir (P < 0.0001) and darunavir (P = 0.06). Long duration of therapy prior to failure was associated with high levels of resistance and is directly related to limited access to VL monitoring and delayed switches to second-line treatment, precluding efficacy of drugs for third-line therapy. This study underlines the need for governments and public health organizations to recommend the use of VL monitoring and also the availability of darunavir and raltegravir for third-line therapies in the context of limited-resource settings.