Dataset Information

Evaluation of a cost effective in-house method for HIV-1 drug resistance genotyping using plasma samples.

ABSTRACT:

Objectives

Validation of a cost effective in-house method for HIV-1 drug resistance genotyping using plasma samples.

Design

The validation includes the establishment of analytical performance characteristics such as accuracy, reproducibility, precision and sensitivity.

Methods

The accuracy was assessed by comparing 26 paired Virological Quality Assessment (VQA) proficiency testing panel sequences generated by in-house and ViroSeq Genotyping System 2.0 (Celera Diagnostics, US) as a gold standard. The reproducibility and precision were carried out on five samples with five replicates representing multiple HIV-1 subtypes (A, B, C) and resistance patterns. The amplification sensitivity was evaluated on HIV-1 positive plasma samples (n?=?88) with known viral loads ranges from 1000-1.8 million RNA copies/ml.

Results

Comparison of the nucleotide sequences generated by ViroSeq and in-house method showed 99.41±0.46 and 99.68±0.35% mean nucleotide and amino acid identity respectively. Out of 135 Stanford HIVdb listed HIV-1 drug resistance mutations, partial discordance was observed at 15 positions and complete discordance was absent. The reproducibility and precision study showed high nucleotide sequence identities i.e. 99.88±0.10 and 99.82±0.20 respectively. The in-house method showed 100% analytical sensitivity on the samples with HIV-1 viral load >1000 RNA copies/ml. The cost of running the in-house method is only 50% of that for ViroSeq method (112$ vs 300$), thus making it cost effective.

Conclusions

The validated cost effective in-house method may be used to collect surveillance data on the emergence and transmission of HIV-1 drug resistance in resource limited countries. Moreover, the wide applications of a cost effective and validated in-house method for HIV-1 drug resistance testing will facilitate the decision making for the appropriate management of HIV infected patients.

SUBMITTER: Chaturbhuj DN

PROVIDER: S-EPMC3922725 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Publications

Evaluation of a cost effective in-house method for HIV-1 drug resistance genotyping using plasma samples.

Chaturbhuj Devidas N DN Nirmalkar Amit P AP Paranjape Ramesh S RS Tripathy Srikanth P SP

PloS one 20140212 2

<h4>Objectives</h4>Validation of a cost effective in-house method for HIV-1 drug resistance genotyping using plasma samples.<h4>Design</h4>The validation includes the establishment of analytical performance characteristics such as accuracy, reproducibility, precision and sensitivity.<h4>Methods</h4>The accuracy was assessed by comparing 26 paired Virological Quality Assessment (VQA) proficiency testing panel sequences generated by in-house and ViroSeq Genotyping System 2.0 (Celera Diagnostics, U ...[more]

PMID: 24533056

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Project description:BackgroundHIV genotyping has had a significant impact on the care and treatment of HIV/AIDS. At a clinical level, the test guides physicians on the choice of treatment regimens. At the surveillance level, it informs policy on consolidated treatment guidelines and microbial resistance control strategies. Until recently, the conventional test has utilized the Sanger sequencing (SS) method. Unlike Next Generation Sequencing (NGS), SS is limited by low data throughput and the inability of detecting low abundant drug-resistant variants. NGS can improve sensitivity and quantitatively identify low-abundance variants; in addition, it has the potential to improve efficiency as well as lowering costs when samples are batched. Despite the NGS benefits, its utilization in clinical drug resistance profiling is faced with mixed reactions. These are largely based on a lack of a consensus regarding the quality control strategy. Nonetheless, transitional views suggest validating the method against the gold-standard SS. Therefore, we present a validation report of an NGS-based in-house HIV genotyping method against the SS method in Uganda.ResultsSince there were no established proficiency test panels for NGS-based HIV genotyping, 15 clinical plasma samples for routine care were utilized. The use of clinical samples allowed for accuracy and precision studies. The workflow involved four main steps; viral RNA extraction, targeted amplicon generation, amplicon sequencing and data analysis. Accuracy of 98% with an average percentage error of 3% was reported for the NGS based assay against the SS platform demonstrating similar performance. The coefficient of variation (CV) findings for both the inter-run and inter-personnel precision showed no variability (CV ≤ 0%) at the relative abundance of ≥ 20%. For both inter-run and inter-personnel, a variation that affected the precision was observed at 1% frequency. Overall, for all the frequencies, CV registered a small range of (0-2%).ConclusionThe NGS-based in-house HIV genotyping method fulfilled the minimum requirements that support its utilization for drug resistance profiling in a clinical setting of a low-income country. For more inclusive quality control studies, well-characterized wet panels need to be established.

Dataset Information

Evaluation of a cost effective in-house method for HIV-1 drug resistance genotyping using plasma samples.

Objectives

Design

Methods

Results

Conclusions

Publications

Evaluation of a cost effective in-house method for HIV-1 drug resistance genotyping using plasma samples.

Similar Datasets

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