A Robust PCR Protocol for HIV Drug Resistance Testing on Low-Level Viremia Samples

Gupta, Shivani; Taylor, Tracy; Patterson, Aileen P.; Liang, Binhua; Bullard, Jared; Sandstrom, Paul; Domselaar, Gary Van; Ji, Hezhao

doi:10.1155/2017/4979252

Cited by 13 publications

(11 citation statements)

References 12 publications

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“…In contrast, our assay is effective to 500 cp/ml for all examined major group M subtypes with no modifications required. Although not presented here, this platform is amendable to include IN genotyping which only requires combining PR/RT and IN amplicons prior to library prep 30 HyDRA web already possesses the capacity to analyze IN sequence data if provided in the input Fastq files. In addition, our library prep followed the streamlined Nextera™ library prep protocol using a bead-based normalization completely satisfactory for generating coverage of at least 20,000× across the PR/RT amplicon region.…”

Section: Discussionmentioning

confidence: 99%

“…RT-PCR conditions were performed as follows: 50 °C for 30 minutes, 94 °C for 2 minutes, 40 cycles of 94 °C for 20 seconds, 50 °C for 30 seconds and 68 °C for 90 seconds, and a final extension at 68 °C for 5 minutes. The same RT-PCR conditions were also used to prepare amplicons for Sanger sequencing 30 .…”

Section: Methodsmentioning

confidence: 99%

“…The primers used were as follows: PR_F2 5′-CTTTARCTTCCCTCARATCACTCT-3′ (HXB2 loci 2243–2266) and RT_R2 5′-CTTCTGTATGTCATTGACAGTCC-3′ (HXB2 loci 3304–3326). Nested-PCR conditions were performed as follows: 98 °C for 30 seconds, 35 cycles at 98 °C for 10 seconds, 62 °C for 20 seconds and 72 °C for 40 seconds, followed by a final extension at 72 °C for 10 minutes 30 .…”

Section: Methodsmentioning

confidence: 99%

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A MiSeq-HyDRA platform for enhanced HIV drug resistance genotyping and surveillance

Taylor

Lee

Nykoluk

et al. 2019

Sci Rep

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Conventional HIV drug resistance (HIVDR) genotyping utilizes Sanger sequencing (SS) methods, which are limited by low data throughput and the inability of detecting low abundant drug resistant variants (LADRVs). Here we present a next generation sequencing (NGS)-based HIVDR typing platform that leverages the advantages of Illumina MiSeq and HyDRA Web. The platform consists of a fully validated sample processing protocol and HyDRA web, an open web portal that allows automated customizable NGS-based HIVDR data processing. This platform was characterized and validated using a panel of HIV-spiked plasma representing all major HIV-1 subtypes, pedigreed plasmids, HIVDR proficiency specimens and clinical specimens. All examined major HIV-1 subtypes were consistently amplified at viral loads of ≥1,000 copies/ml. The gross error rate of this platform was determined at 0.21%, and minor variations were reliably detected down to 0.50% in plasmid mixtures. All HIVDR mutations identifiable by SS were detected by the MiSeq-HyDRA protocol, while LADRVs at frequencies of 1~15% were detected by MiSeq-HyDRA only. As compared to SS approaches, the MiSeq-HyDRA platform has several notable advantages including reduced cost and labour, and increased sensitivity for LADRVs, making it suitable for routine HIVDR monitoring for both patient care and surveillance purposes.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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A MiSeq-HyDRA platform for enhanced HIV drug resistance genotyping and surveillance

Taylor

Lee

Nykoluk

et al. 2019

Sci Rep

Self Cite

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“…Of the blood samples obtained from the 401 cases enrolled in the case-control study, 344 were successfully amplified and sequenced. The remaining 57 samples failed to amplify possibly due to low viral load secondary to receiving antiretroviral treatment or due to genomic diversity attributed to quasispecies in an individual (Debyser et al, 1998;Gupta et al, 2017). Out of 344 cases, socio-demographic information was available for 321 sequences, while information for 23 cases was missing.…”

Section: Study Populationmentioning

confidence: 99%

Phylogenetic and Drug-Resistance Analysis of HIV-1 Sequences From an Extensive Paediatric HIV-1 Outbreak in Larkana, Pakistan

et al. 2021

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IntroductionIn April 2019, an HIV-1 outbreak among children occurred in Larkana, Pakistan, affecting more than a thousand children. It was assumed that the outbreak originated from a single source, namely a doctor at a private health facility. In this study, we performed subtype distribution, phylogenetic and drug-resistance analysis of HIV-1 sequences from 2019 outbreak in Larkana, Pakistan.MethodsA total of 401 blood samples were collected between April–June 2019, from children infected with HIV-1 aged 0–15 years recruited into a case-control study to investigate the risk factors for HIV-1 transmission. Partial HIV-1 pol sequences were generated from 344 blood plasma samples to determine HIV-1 subtype and drug resistance mutations (DRM). Maximum-likelihood phylogenetics based on outbreak and reference sequences was used to identify transmission clusters and assess the relationship between outbreak and key population sequences between and within the determined clusters. Bayesian analysis was employed to identify the time to the most recent common recent ancestor (tMRCA) of the main Pakistani clusters.ResultsThe HIV-1 circulating recombinant form (CRF) 02_AG and subtype A1 were most common among the outbreak sequences. Of the treatment-naïve participants, the two most common mutations were RT: E138A (8%) and RT: K219Q (8%). Four supported clusters within the outbreak were identified, and the median tMRCAs of the Larkana outbreak sequences were estimated to 2016 for both the CRF02_AG and the subtype A1 clusters. Furthermore, outbreak sequences exhibited no phylogenetic mixing with sequences from other high-risk groups of Pakistan.ConclusionThe presence of multiple clusters indicated a multi-source outbreak, rather than a single source outbreak from a single health practitioner as previously suggested. The multiple introductions were likely a consequence of ongoing transmission within the high-risk groups of Larkana, and it is possible that the so-called Larkana strain was introduced into the general population through poor infection prevention control practices in healthcare settings. The study highlights the need to scale up HIV-1 prevention programmes among key population groups and improving infection prevention control in Pakistan.

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“…Research for well-optimized PCR protocols which has a broad HIV-1 subtype coverage, a wide range of viral load span, high sensitivity and reproducibility is a promising research area. [177]…”

Section: Expert Opinionmentioning

confidence: 99%

The dawn of precision medicine in HIV: state of the art of pharmacotherapy

Kodidela

Wang

et al. 2018

Expert Opinion on Pharmacotherapy

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Introduction: Combination antiretroviral therapy reduces viral load to under the limit of detection, successfully decreasing HIV-related morbidity and mortality. Due to viral mutations, complex drug combinations and different patient response, there is an increasing demand for individualized treatment options for patients. Areas covered: This review first summarizes the pharmacokinetic and pharmacodynamic profile of clinical first-line drugs, which serves as guidance for antiretroviral precision medicine. Factors which have influential effects on the drug efficacy and thus precision medicine are discussed: patients’ pharmacogenetic information, virus mutations, comorbidities and immune recovery. Furthermore, strategies to improve the application of precision medicine are discussed. Expert opinion: Precision medicine for antiretroviral therapy requires comprehensive information on the drug, virus and clinical data from the patients. The clinically available genetic tests are a good starting point. To better apply precision medicine, deeper knowledge of drug concentrations, HIV reservoirs, and efficacy associated genes, such as polymorphisms of drug transporters and metabolizing enzymes, are required. With advanced computer-based prediction systems which integrate more comprehensive information on pharmacokinetics, pharmacodynamics, pharmacogenomics and the clinically relevant information of the patients, precision medicine will lead to better treatment choices and improved disease outcomes.

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A Robust PCR Protocol for HIV Drug Resistance Testing on Low-Level Viremia Samples

Cited by 13 publications

References 12 publications

A MiSeq-HyDRA platform for enhanced HIV drug resistance genotyping and surveillance

A MiSeq-HyDRA platform for enhanced HIV drug resistance genotyping and surveillance

Phylogenetic and Drug-Resistance Analysis of HIV-1 Sequences From an Extensive Paediatric HIV-1 Outbreak in Larkana, Pakistan

The dawn of precision medicine in HIV: state of the art of pharmacotherapy

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