Special Issue "Next Generation Sequencing for HIV Drug Resistance Testing"

A special issue of Viruses (ISSN 1999-4915). This special issue belongs to the section "Viral Immunology, Vaccines, and Antivirals".

Deadline for manuscript submissions: closed (31 May 2020).

Special Issue Editor

Dr. Rami Kantor
E-Mail Website
Guest Editor
Brown University; Providence, RI, USA
Interests: HIV drug resistance; treatment monitoring and transmission

Special Issue Information

Dear Colleagues,

Drug resistance remains a global challenge in the fight against the HIV pandemic, as recognized by the World Health Organization and the United Nations. Where feasible, guidelines recommend testing for drug resistance before and during antiretroviral therapy to guide regimen selection. Where less available, such testing is used only in specific populations, and where even less accessible, it is used for public health surveillance. Sanger sequencing, available since the 1970s, has conventionally been used for HIV drug resistance testing in clinical care and research. More recently developed next-generation sequencing (NGS) methods are increasingly used in diverse circumstances, including for HIV drug resistance testing. Whether these technologies can and should replace Sanger sequencing for HIV drug resistance testing is unclear, mostly due to associated financial, infrastructure, and logistical challenges. This topic was recently discussed at the International Symposium on External Quality Assurance Strategies for NGS-based HIV drug resistance testing, convened in Winnipeg, Canada in September 2019. In this Special Issue, I invite Symposium participants to highlight related opportunities and challenges that were discussed in the Symposium. I also invite other experts and interested individuals to contribute to this discussion. Deliberations will emphasize logistical and implementation needs and considerations in incorporation of NGS for HIV drug resistance testing, towards clarifying this existing gap and helping clinical, public health and programmatic resolutions.

Dr. Rami Kantor
Guest Editor

Manuscript Submission Information

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Keywords

  • HIV drug resistance
  • Next-generation sequencing

Published Papers (11 papers)

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Editorial

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Editorial
Next Generation Sequencing for HIV-1 Drug Resistance Testing—A Special Issue Walkthrough
Viruses 2021, 13(2), 340; https://doi.org/10.3390/v13020340 - 22 Feb 2021
Viewed by 405
Abstract
Drug resistance remains a global challenge in the fight against the HIV pandemic [...] Full article
(This article belongs to the Special Issue Next Generation Sequencing for HIV Drug Resistance Testing)

Research

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Article
Multi-Laboratory Comparison of Next-Generation to Sanger-Based Sequencing for HIV-1 Drug Resistance Genotyping
Viruses 2020, 12(7), 694; https://doi.org/10.3390/v12070694 - 27 Jun 2020
Cited by 8 | Viewed by 1011
Abstract
Next-generation sequencing (NGS) is increasingly used for HIV-1 drug resistance genotyping. NGS methods have the potential for a more sensitive detection of low-abundance variants (LAV) compared to standard Sanger sequencing (SS) methods. A standardized threshold for reporting LAV that generates data comparable to [...] Read more.
Next-generation sequencing (NGS) is increasingly used for HIV-1 drug resistance genotyping. NGS methods have the potential for a more sensitive detection of low-abundance variants (LAV) compared to standard Sanger sequencing (SS) methods. A standardized threshold for reporting LAV that generates data comparable to those derived from SS is needed to allow for the comparability of data from laboratories using NGS and SS. Ten HIV-1 specimens were tested in ten laboratories using Illumina MiSeq-based methods. The consensus sequences for each specimen using LAV thresholds of 5%, 10%, 15%, and 20% were compared to each other and to the consensus of the SS sequences (protease 4–99; reverse transcriptase 38–247). The concordance among laboratories’ sequences at different thresholds was evaluated by pairwise sequence comparisons. NGS sequences generated using the 20% threshold were the most similar to the SS consensus (average 99.6% identity, range 96.1–100%), compared to 15% (99.4%, 88.5–100%), 10% (99.2%, 87.4–100%), or 5% (98.5%, 86.4–100%). The average sequence identity between laboratories using thresholds of 20%, 15%, 10%, and 5% was 99.1%, 98.7%, 98.3%, and 97.3%, respectively. Using the 20% threshold, we observed an excellent agreement between NGS and SS, but significant differences at lower thresholds. Understanding how variation in NGS methods influences sequence quality is essential for NGS-based HIV-1 drug resistance genotyping. Full article
(This article belongs to the Special Issue Next Generation Sequencing for HIV Drug Resistance Testing)
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Article
Development and Application of Performance Assessment Criteria for Next-Generation Sequencing-Based HIV Drug Resistance Assays
Viruses 2020, 12(6), 627; https://doi.org/10.3390/v12060627 - 10 Jun 2020
Cited by 3 | Viewed by 960
Abstract
Next-generation sequencing (NGS)-based HIV drug resistance (HIVDR) assays outperform conventional Sanger sequencing in scalability, sensitivity, and quantitative detection of minority resistance variants. Thus far, HIVDR assays have been applied primarily in research but rarely in clinical settings. One main obstacle is the lack [...] Read more.
Next-generation sequencing (NGS)-based HIV drug resistance (HIVDR) assays outperform conventional Sanger sequencing in scalability, sensitivity, and quantitative detection of minority resistance variants. Thus far, HIVDR assays have been applied primarily in research but rarely in clinical settings. One main obstacle is the lack of standardized validation and performance evaluation systems that allow regulatory agencies to benchmark and accredit new assays for clinical use. By revisiting the existing principles for molecular assay validation, here we propose a new validation and performance evaluation system that helps to both qualitatively and quantitatively assess the performance of an NGS-based HIVDR assay. To accomplish this, we constructed a 70-specimen proficiency test panel that includes plasmid mixtures at known ratios, viral RNA from infectious clones, and anonymized clinical specimens. We developed assessment criteria and benchmarks for NGS-based HIVDR assays and used these to assess data from five separate MiSeq runs performed in two experienced HIVDR laboratories. This proposed platform may help to pave the way for the standardization of NGS HIVDR assay validation and performance evaluation strategies for accreditation and quality assurance purposes in both research and clinical settings. Full article
(This article belongs to the Special Issue Next Generation Sequencing for HIV Drug Resistance Testing)
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Review

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Review
Application of a Sanger-Based External Quality Assurance Strategy for the Transition of HIV-1 Drug Resistance Assays to Next Generation Sequencing
Viruses 2020, 12(12), 1456; https://doi.org/10.3390/v12121456 - 17 Dec 2020
Cited by 1 | Viewed by 482
Abstract
The National Institute of Allergy and Infectious Diseases (NIAID) Virology Quality Assurance (VQA) established a robust proficiency testing program for Sanger sequencing (SS)-based HIV-1 drug resistance (HIVDR) testing in 2001. While many of the lessons learned during the development of such programs may [...] Read more.
The National Institute of Allergy and Infectious Diseases (NIAID) Virology Quality Assurance (VQA) established a robust proficiency testing program for Sanger sequencing (SS)-based HIV-1 drug resistance (HIVDR) testing in 2001. While many of the lessons learned during the development of such programs may also apply to next generation sequencing (NGS)-based HIVDR assays, challenges remain for the ongoing evaluation of NGS-based testing. These challenges include a proper assessment of assay accuracy and the reproducibility of low abundance variant detection, intra- and inter-assay performance comparisons among laboratories using lab-defined tests, and different data analysis pipelines designed for NGS. In collaboration with the World Health Organization (WHO) Global HIVDR Laboratory Network and the Public Health Agency of Canada, the Rush VQA program distributed archived proficiency testing panels to ten laboratories to evaluate internally developed NGS assays. Consensus FASTA files were submitted using 5%, 10%, and 20% variant detection thresholds, and scored based on the same criteria used for SS. This small study showed that the SS External Quality Assurance (EQA) approach can be used as a transitional strategy for using NGS to generate SS-like data and for ongoing performance while using NGS data from the same quality control materials to further evaluate NGS assay performance. Full article
(This article belongs to the Special Issue Next Generation Sequencing for HIV Drug Resistance Testing)
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Review
Quality Control of Next-Generation Sequencing-Based HIV-1 Drug Resistance Data in Clinical Laboratory Information Systems Framework
Viruses 2020, 12(6), 645; https://doi.org/10.3390/v12060645 - 14 Jun 2020
Cited by 2 | Viewed by 1203
Abstract
Next-generation sequencing (NGS) in HIV drug resistance (HIVDR) testing has the potential to improve both clinical and public health settings, however it challenges the normal operations of quality management systems to be more flexible due to its complexity, massive data generation, and rapidly [...] Read more.
Next-generation sequencing (NGS) in HIV drug resistance (HIVDR) testing has the potential to improve both clinical and public health settings, however it challenges the normal operations of quality management systems to be more flexible due to its complexity, massive data generation, and rapidly evolving protocols. While guidelines for quality management in NGS data have previously been outlined, little guidance has been implemented for NGS-based HIVDR testing. This document summarizes quality control procedures for NGS-based HIVDR testing laboratories using a laboratory information systems (LIS) framework. Here, we focus in particular on the quality control measures applied on the final sequencing product aligned with the recommendations from the World Health Organization HIV Drug Resistance Laboratory Network. Full article
(This article belongs to the Special Issue Next Generation Sequencing for HIV Drug Resistance Testing)
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Review
Next-Generation Sequencing for HIV Drug Resistance Testing: Laboratory, Clinical, and Implementation Considerations
Viruses 2020, 12(6), 617; https://doi.org/10.3390/v12060617 - 05 Jun 2020
Cited by 4 | Viewed by 1078
Abstract
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 [...] Read more.
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. Full article
(This article belongs to the Special Issue Next Generation Sequencing for HIV Drug Resistance Testing)
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Other

Perspective
Fact and Fiction about 1%: Next Generation Sequencing and the Detection of Minor Drug Resistant Variants in HIV-1 Populations with and without Unique Molecular Identifiers
Viruses 2020, 12(8), 850; https://doi.org/10.3390/v12080850 - 04 Aug 2020
Cited by 2 | Viewed by 946
Abstract
Next generation sequencing (NGS) platforms have the ability to generate almost limitless numbers of sequence reads starting with a PCR product. This gives the illusion that it is possible to analyze minor variants in a viral population. However, including a PCR step obscures [...] Read more.
Next generation sequencing (NGS) platforms have the ability to generate almost limitless numbers of sequence reads starting with a PCR product. This gives the illusion that it is possible to analyze minor variants in a viral population. However, including a PCR step obscures the sampling depth of the viral population, the key parameter needed to understand the utility of the data set for finding minor variants. Also, these high throughput sequencing platforms are error prone at the level where minor variants are of interest, confounding the interpretation of detected minor variants. A simple strategy has been applied in multiple applications of NGS to solve these problems. Prior to PCR, individual molecules are “tagged” with a unique molecular identifier (UMI) that can be used to establish the actual sample size of viral genomes sequenced after PCR and sequencing. In addition, since PCR generates many copies of each sequence tagged to a specific UMI, a template consensus sequence (TCS) can be created from the many reads of each template, removing virtually all of the method error. From this perspective we examine our own use of a UMI, called Primer ID, in the detection of minor drug resistant variants in HIV-1 populations. Full article
(This article belongs to the Special Issue Next Generation Sequencing for HIV Drug Resistance Testing)
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Commentary
Dry Panels Supporting External Quality Assessment Programs for Next Generation Sequencing-Based HIV Drug Resistance Testing
Viruses 2020, 12(6), 666; https://doi.org/10.3390/v12060666 - 20 Jun 2020
Cited by 3 | Viewed by 837
Abstract
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 [...] Read more.
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. Full article
(This article belongs to the Special Issue Next Generation Sequencing for HIV Drug Resistance Testing)
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Commentary
Are We Ready for NGS HIV Drug Resistance Testing? The Second “Winnipeg Consensus” Symposium
Viruses 2020, 12(6), 586; https://doi.org/10.3390/v12060586 - 27 May 2020
Cited by 2 | Viewed by 1083
Abstract
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. [...] Read more.
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. Full article
(This article belongs to the Special Issue Next Generation Sequencing for HIV Drug Resistance Testing)
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Opinion
External Quality Assessment Program for Next-Generation Sequencing-Based HIV Drug Resistance Testing: Logistical Considerations
Viruses 2020, 12(5), 556; https://doi.org/10.3390/v12050556 - 18 May 2020
Cited by 5 | Viewed by 1080
Abstract
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 [...] Read more.
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. Full article
(This article belongs to the Special Issue Next Generation Sequencing for HIV Drug Resistance Testing)
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Commentary
External Quality Assessment for Next-Generation Sequencing-Based HIV Drug Resistance Testing: Unique Requirements and Challenges
Viruses 2020, 12(5), 550; https://doi.org/10.3390/v12050550 - 16 May 2020
Cited by 7 | Viewed by 1164
Abstract
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, [...] Read more.
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. Full article
(This article belongs to the Special Issue Next Generation Sequencing for HIV Drug Resistance Testing)
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