scholarly journals Are We Ready for NGS HIV Drug Resistance Testing? The Second “Winnipeg Consensus” Symposium

Viruses ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 586 ◽  
Author(s):  
Hezhao Ji ◽  
Paul Sandstrom ◽  
Roger Paredes ◽  
P. Richard Harrigan ◽  
Chanson J. Brumme ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Clinical Care ◽  
Resistance Testing ◽  
Quantitative Detection ◽  
Resistance Mutations ◽  
Hiv Drug Resistance ◽  
Global Challenge ◽  
Symposium Series ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

HIV drug resistance is a major global challenge to successful and sustainable antiretroviral therapy. Next-generation sequencing (NGS)-based HIV drug resistance (HIVDR) assays enable more sensitive and quantitative detection of drug-resistance-associated mutations (DRMs) and outperform Sanger sequencing approaches in detecting lower abundance resistance mutations. While NGS is likely to become the new standard for routine HIVDR testing, many technical and knowledge gaps remain to be resolved before its generalized adoption in regular clinical care, public health, and research. Recognizing this, we conceived and launched an international symposium series on NGS HIVDR, to bring together leading experts in the field to address these issues through in-depth discussions and brainstorming. Following the first symposium in 2018 (Winnipeg, MB Canada, 21–22 February, 2018), a second “Winnipeg Consensus” symposium was held in September 2019 in Winnipeg, Canada, and was focused on external quality assurance strategies for NGS HIVDR assays. In this paper, we summarize this second symposium’s goals and highlights.

scholarly journals Limited marginal utility of deep sequencing for HIV drug resistance testing in the age of integrase inhibitors

2018 ◽  
Author(s):  
Ronit Dalmat ◽  
Negar Makhsous ◽  
Gregory Pepper ◽  
Amalia Magaret ◽  
Keith R. Jerome ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Virological Failure ◽  
Sanger Sequencing ◽  
Low Frequency ◽  
Resistance Testing ◽  
Antiviral Resistance ◽  
Resistance Mutations ◽  
Hiv Drug Resistance ◽  
Next Generation Sequencing Ngs

AbstractHIV drug resistance genotyping is a critical tool in the clinical management of HIV infections. Although resistance genotyping has traditionally been conducted using Sanger sequencing, next-generation sequencing (NGS) is emerging as a powerful tool due to its ability to detect lower frequency alleles. However, the value added from NGS approaches to antiviral resistance testing remains to be demonstrated. We compared the variant detection capacity of NGS versus Sanger sequencing methods for resistance genotyping of 144 drug resistance tests (105 protease-reverse transcriptase tests and 39 integrase tests) submitted to our clinical virology laboratory over a four-month period in 2016 for Sanger-based HIV drug resistance testing. NGS detected all true high frequency drug resistance mutations (>20% frequency) found by Sanger sequencing, with greater accuracy in one instance of a Sanger-detected false positive. Freely available online NGS variant callers Hydra and PASeq were superior to Sanger methods for interpretations of allele linkage and automated variant calling. NGS additionally detected low frequency mutations (1-20% frequency) associated with higher levels of drug resistance in 30/105 (29%) of protease-reverse transcriptase tests and 4/39 (10%) of integrase tests. Clinical follow-up of 69 individuals for a median of 674 days found no difference in rates of virological failure between individuals with and without low frequency mutations, although rates of virological failure were higher for individuals with drug-relevant low frequency mutations. However, all 27 individuals who experienced virological failure reported poor adherence to their drug regimen during preceding follow-up time, and all 19 who subsequently improved their adherence achieved viral suppression at later time points consistent with a lack of clinical resistance. In conclusion, in a population with low antiviral resistance emergence, NGS methods detected numerous instances of minor alleles that did not result in subsequent bona fide virological failure due to antiviral resistance.ImportanceGenotypic antiviral resistance testing for HIV is an essential component of the clinical microbiology and virology laboratory. Next-generation sequencing (NGS) has emerged as a powerful tool for the detection of low frequency sequence variants (allele frequencies <20%). Whether detecting these low frequency mutations in HIV contributes to improved patient health, however, remains unclear. We compared NGS to conventional Sanger sequencing for detecting resistance mutations for 144 HIV drug resistance tests submitted to our clinical virology laboratory and detected low frequency mutations in 24% of tests. Over approximately two years of follow-up for 69 patients for which we had access to electronic health records, no patients had virological failure due to antiviral resistance. Instead, virological failure was entirely explained by medication non-adherence. While larger studies are required, we suggest that detection of low frequency variants by NGS presents limited marginal clinical utility when compared to standard of care.

scholarly journals External Quality Assessment Program for Next-Generation Sequencing-Based HIV Drug Resistance Testing: Logistical Considerations

Viruses ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 556 ◽  
Author(s):  
Hezhao Ji ◽  
Neil Parkin ◽  
Feng Gao ◽  
Thomas Denny ◽  
Cheryl Jennings ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Next Generation Sequencing ◽  
Quality Assessment ◽  
External Quality Assessment ◽  
Resistance Testing ◽  
Next Generation ◽  
External Quality ◽  
Hiv Drug Resistance ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Next-generation sequencing (NGS) is likely to become the new standard method for HIV drug resistance (HIVDR) genotyping. Despite the significant advances in the development of wet-lab protocols and bioinformatic data processing pipelines, one often-missing critical component of an NGS HIVDR assay for clinical use is external quality assessment (EQA). EQA is essential for ensuring assay consistency and laboratory competency in performing routine biomedical assays, and the rollout of NGS HIVDR tests in clinical practice will require an EQA. In September 2019, the 2nd International Symposium on NGS HIVDR was held in Winnipeg, Canada. It convened a multidisciplinary panel of experts, including research scientists, clinicians, bioinformaticians, laboratory biologists, biostatisticians, and EQA experts. A themed discussion was conducted on EQA strategies towards such assays during the symposium. This article describes the logistical challenges identified and summarizes the opinions and recommendations derived from these discussions, which may inform the development of an inaugural EQA program for NGS HIVDR in the near future.

scholarly journals Next-Generation Sequencing for HIV Drug Resistance Testing: Laboratory, Clinical, and Implementation Considerations

Viruses ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 617 ◽  
Author(s):  
Santiago Ávila-Ríos ◽  
Neil Parkin ◽  
Ronald Swanstrom ◽  
Roger Paredes ◽  
Robert Shafer ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Next Generation Sequencing ◽  
Clinical Care ◽  
Pcr Amplification ◽  
Resistance Testing ◽  
Sample Type ◽  
Next Generation ◽  
Hiv Drug Resistance ◽  
Drug Resistance Testing ◽  
Generation Sequencing

Higher accessibility and decreasing costs of next generation sequencing (NGS), availability of commercial kits, and development of dedicated analysis pipelines, have allowed an increasing number of laboratories to adopt this technology for HIV drug resistance (HIVDR) genotyping. Conventional HIVDR genotyping is traditionally carried out using population-based Sanger sequencing, which has a limited capacity for reliable detection of variants present at intra-host frequencies below a threshold of approximately 20%. NGS has the potential to improve sensitivity and quantitatively identify low-abundance variants, improving efficiency and lowering costs. However, some challenges exist for the standardization and quality assurance of NGS-based HIVDR genotyping. In this paper, we highlight considerations of these challenges as related to laboratory, clinical, and implementation of NGS for HIV drug resistance testing. Several sources of variation and bias occur in each step of the general NGS workflow, i.e., starting material, sample type, PCR amplification, library preparation method, instrument and sequencing chemistry-inherent errors, and data analysis options and limitations. Additionally, adoption of NGS-based HIVDR genotyping, especially for clinical care, poses pressing challenges, especially for resource-poor settings, including infrastructure and equipment requirements and cost, logistic and supply chains, instrument service availability, personnel training, validated laboratory protocols, and standardized analysis outputs. The establishment of external quality assessment programs may help to address some of these challenges and is needed to proceed with NGS-based HIVDR genotyping adoption.

scholarly journals HIV Drug Resistance Mutations Detection by Next-Generation Sequencing during Antiretroviral Therapy Interruption in China

Pathogens ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 264
Author(s):  
Miaomiao Li ◽  
Shujia Liang ◽  
Chao Zhou ◽  
Min Chen ◽  
Shu Liang ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Next Generation Sequencing ◽  
Antiretroviral Therapy ◽  
Detection Threshold ◽  
Next Generation ◽  
Resistance Mutations ◽  
Hiv Drug Resistance ◽  
Drug Resistance Mutations ◽  
Therapy Interruption ◽  
Generation Sequencing

Patients with antiretroviral therapy interruption have a high risk of virological failure when re-initiating antiretroviral therapy (ART), especially those with HIV drug resistance. Next-generation sequencing may provide close scrutiny on their minority drug resistance variant. A cross-sectional study was conducted in patients with ART interruption in five regions in China in 2016. Through Sanger and next-generation sequencing in parallel, HIV drug resistance was genotyped on their plasma samples. Rates of HIV drug resistance were compared by the McNemar tests. In total, 174 patients were included in this study, with a median 12 (interquartile range (IQR), 6–24) months of ART interruption. Most (86.2%) of them had received efavirenz (EFV)/nevirapine (NVP)-based first-line therapy for a median 16 (IQR, 7–26) months before ART interruption. Sixty-one (35.1%) patients had CRF07_BC HIV-1 strains, 58 (33.3%) CRF08_BC and 35 (20.1%) CRF01_AE. Thirty-four (19.5%) of the 174 patients were detected to harbor HIV drug-resistant variants on Sanger sequencing. Thirty-six (20.7%), 37 (21.3%), 42 (24.1%), 79 (45.4%) and 139 (79.9) patients were identified to have HIV drug resistance by next-generation sequencing at 20% (v.s. Sanger, p = 0.317), 10% (v.s. Sanger, p = 0.180), 5% (v.s. Sanger, p = 0.011), 2% (v.s. Sanger, p < 0.001) and 1% (v.s. Sanger, p < 0.001) of detection thresholds, respectively. K65R was the most common minority mutation, of 95.1% (58/61) and 93.1% (54/58) in CRF07_BC and CRF08_BC, respectively, when compared with 5.7% (2/35) in CRF01_AE (p < 0.001). In 49 patients that followed-up a median 10 months later, HIV drug resistance mutations at >20% frequency such as K103N, M184VI and P225H still existed, but with decreased frequencies. The prevalence of HIV drug resistance in ART interruption was higher than 15% in the survey. Next-generation sequencing was able to detect more minority drug resistance variants than Sanger. There was a sharp increase in minority drug resistance variants when the detection threshold was below 5%.

scholarly journals Next Generation Sequencing for HIV-1 Drug Resistance Testing—A Special Issue Walkthrough

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 340
Author(s):  
Rami Kantor
Keyword(s):  
Drug Resistance ◽  
Next Generation Sequencing ◽  
Resistance Testing ◽  
Next Generation ◽  
Special Issue ◽  
Global Challenge ◽  
Drug Resistance Testing ◽  
Generation Sequencing ◽  
Hiv 1

Drug resistance remains a global challenge in the fight against the HIV pandemic [...]

scholarly journals HIV Drug Resistance Testing by High-Multiplex “Wide” Sequencing on the MiSeq Instrument

2015 ◽  
Vol 59 (11) ◽  
pp. 6824-6833 ◽  
Author(s):  
H. R. Lapointe ◽  
W. Dong ◽  
G. Q. Lee ◽  
D. R. Bangsberg ◽  
J. N. Martin ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Sanger Sequencing ◽  
Illumina Miseq ◽  
Quality Data ◽  
Resistance Testing ◽  
Study Cohort ◽  
Miseq Sequencing ◽  
Resistance Mutations ◽  
Hiv Drug Resistance ◽  
Drug Resistance Testing

ABSTRACTLimited access to HIV drug resistance testing in low- and middle-income countries impedes clinical decision-making at the individual patient level. An efficient protocol to address this issue must be established to minimize negative therapeutic outcomes for HIV-1-infected individuals in such settings. This is an observational study to ascertain the potential of newer genomic sequencing platforms, such as the Illumina MiSeq instrument, to provide accurate HIV drug resistance genotypes for hundreds of samples simultaneously. Plasma samples were collected from Canadian patients during routine drug resistance testing (n= 759) and from a Ugandan study cohort (n= 349). Amplicons spanning HIV reverse transcriptase codons 90 to 234 were sequenced with both MiSeq sequencing and conventional Sanger sequencing methods. Sequences were evaluated for nucleotide concordance between methods, using coverage and mixture parameters for quality control. Consensus sequences were also analyzed for disparities in the identification of drug resistance mutations. Sanger and MiSeq sequencing was successful for 881 samples (80%) and 892 samples (81%), respectively, with 832 samples having results from both methods. Most failures were for samples with viral loads of <3.0 log10HIV RNA copies/ml. Overall, 99.3% nucleotide concordance between methods was observed. MiSeq sequencing achieved 97.4% sensitivity and 99.3% specificity in detecting resistance mutations identified by Sanger sequencing. Findings suggest that the Illumina MiSeq platform can yield high-quality data with a high-multiplex “wide” sequencing approach. This strategy can be used for multiple HIV subtypes, demonstrating the potential for widespread individual testing and annual population surveillance in resource-limited settings.

scholarly journals Dry Panels Supporting External Quality Assessment Programs for Next Generation Sequencing-Based HIV Drug Resistance Testing

Viruses ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 666 ◽  
Author(s):  
Marc Noguera-Julian ◽  
Emma R. Lee ◽  
Robert W. Shafer ◽  
Rami Kantor ◽  
Hezhao Ji
Keyword(s):  
Drug Resistance ◽  
Next Generation Sequencing ◽  
Quality Assessment ◽  
External Quality Assessment ◽  
Resistance Testing ◽  
External Quality ◽  
Hiv Drug Resistance ◽  
Drug Resistance Testing ◽  
Ngs Data ◽  
Generation Sequencing

External quality assessment (EQA) is a keystone element in the validation and implementation of next generation sequencing (NGS)-based HIV drug resistance testing (DRT). Software validation and evaluation is a critical element in NGS EQA programs. While the development, sharing, and adoption of wet lab protocols is coupled with the increasing access to NGS technology worldwide, rendering it easy to produce NGS data for HIV-DRT, bioinformatic data analysis remains a bottleneck for most of the diagnostic laboratories. Several computational tools have been made available, via free or commercial sources, to automate the conversion of raw NGS data into an actionable clinical report. Although different software platforms yield equivalent results when identical raw NGS datasets are analyzed for variations at higher abundance, discrepancies arise when variations at lower frequencies are considered. This implies that validation and performance assessment of the bioinformatics tools applied in NGS HIV-DRT is critical, and the origins of the observed discrepancies should be determined. Well-characterized reference NGS datasets with ground truth on the genotype composition at all examined loci and the exact frequencies of HIV variations they may harbor, so-called dry panels, would be essential in such cases. The strategic design and construction of such panels are challenging but imperative tasks in support of EQA programs for NGS-based HIV-DRT and the validation of relevant bioinformatics tools. Here, we present criteria that can guide the design of such dry panels, which were discussed in the Second International Winnipeg Symposium themed for EQA strategies for NGS HIVDR assays.

scholarly journals External Quality Assessment for Next-Generation Sequencing-Based HIV Drug Resistance Testing: Unique Requirements and Challenges

Viruses ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 550 ◽  
Author(s):  
Emma R. Lee ◽  
Feng Gao ◽  
Paul Sandstrom ◽  
Hezhao Ji
Keyword(s):  
Drug Resistance ◽  
Next Generation Sequencing ◽  
Quality Assessment ◽  
Sanger Sequencing ◽  
External Quality Assessment ◽  
Resistance Testing ◽  
Next Generation ◽  
External Quality ◽  
Hiv Drug Resistance ◽  
Generation Sequencing

Over the past decade, there has been an increase in the adoption of next generation sequencing (NGS) technologies for HIV drug resistance (HIVDR) testing. NGS far outweighs conventional Sanger sequencing as it has much higher throughput, lower cost when samples are batched and, most importantly, significantly higher sensitivities for variants present at low frequencies, which may have significant clinical implications. Despite the advantages of NGS, Sanger sequencing remains the gold standard for HIVDR testing, largely due to the lack of standardization of NGS-based HIVDR testing. One important aspect of standardization includes external quality assessment (EQA) strategies and programs. Current EQA for Sanger-based HIVDR testing includes proficiency testing where samples are sent to labs and the performance of the lab conducting such assays is evaluated. The current methods for Sanger-based EQA may not apply to NGS-based tests because of the fundamental differences in their technologies and outputs. Sanger-based genotyping reports drug resistance mutations (DRMs) data as dichotomous, whereas NGS-based HIVDR genotyping also reports DRMs as numerical data (percent abundance). Here we present an overview of the need to develop EQA for NGS-based HIVDR testing and some unique challenges that may be encountered.

scholarly journals Pretreatment HIV drug resistance spread within transmission clusters in Mexico City

2019 ◽  
Vol 75 (3) ◽  
pp. 656-667
Author(s):  
Margarita Matías-Florentino ◽  
Antoine Chaillon ◽  
Santiago Ávila-Ríos ◽  
Sanjay R Mehta ◽  
Héctor E Paz-Juárez ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Mexico City ◽  
Continuous Monitoring ◽  
Low Frequency ◽  
Genetic Network ◽  
Genetic Distances ◽  
Resistance Mutations ◽  
Hiv Drug Resistance ◽  
Genetic Transmission ◽  
Generation Sequencing

Abstract Background Pretreatment HIV drug resistance (HIVDR) to NNRTIs has consistently increased in Mexico City during the last decade. Objectives To infer the HIV genetic transmission network in Mexico City to describe the dynamics of the local HIV epidemic and spread of HIVDR. Patients and methods HIV pol sequences were obtained by next-generation sequencing from 2447 individuals before initiation of ART at the largest HIV clinic in Mexico City (April 2016 to June 2018). Pretreatment HIVDR was estimated using the Stanford algorithm at a Sanger-like threshold (≥20%). Genetic networks were inferred with HIV-TRACE, establishing putative transmission links with genetic distances &lt;1.5%. We examined demographic associations among linked individuals with shared drug resistance mutations (DRMs) using a ≥ 2% threshold to include low-frequency variants. Results Pretreatment HIVDR reached 14.8% (95% CI 13.4%–16.2%) in the cohort overall and 9.6% (8.5%–10.8%) to NNRTIs. Putative links with at least one other sequence were found for 963/2447 (39%) sequences, forming 326 clusters (2–20 individuals). The inferred network was assortative by age and municipality (P &lt; 0.001). Clustering individuals were younger [adjusted OR (aOR) per year = 0.96, 95% CI 0.95–0.97, P &lt; 0.001] and less likely to include women (aOR = 0.46, 95% CI 0.28–0.75, P = 0.002). Among clustering individuals, 175/963 (18%) shared DRMs (involving 66 clusters), of which 66/175 (38%) shared K103N/S (24 clusters). Eight municipalities (out of 75) harboured 65% of persons sharing DRMs. Among all persons sharing DRMs, those sharing K103N were younger (aOR = 0.93, 95% CI 0.88–0.98, P = 0.003). Conclusions Our analyses suggest age- and geographically associated transmission of DRMs within the HIV genetic network in Mexico City, warranting continuous monitoring and focused interventions.

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