Development, validation and clinical evaluation of a low cost in-house HIV-1 drug resistance genotyping assay for Indian patients.

Arpan Acharya,Minal Wani,Rabindra Nath Misra,Parth Shah,Pratap N Mukhopadhyaya,Salil Vaniawala,Julian W Tang

doi:10.1371/journal.pone.0105790

Abstract

Human Immunodeficiency Virus-1 (HIV-1) drug resistance genotyping assay is a part of clinical management of HIV-1 positive individuals under treatment with highly active antiretroviral therapy (HAART). Routine monitoring of drug resistance mutations in resource limited settings like India is not possible due to high cost of commercial drug resistance assays. In this study we developed an in-house, cost effective HIV-1 drug resistance genotyping assay for Indian patients and validated it against the US-FDA-approved ViroSeq HIV-1 drug resistance testing system. A reference panel of 20 clinical samples was used to develop and validate the assay against ViroSeq HIV-1 drug resistance testing system which was subsequently used to genotype a clinical panel of 225 samples. The Stanford HIV database was used to identify drug resistant mutations. The analytical sensitivity of the assay was 1000 HIV-1 RNA copies/ml of plasma sample while precision and reproducibility was 99.68±0.16% and 99.76±0.18% respectively. One hundred and one drug resistant mutations were detected by the in-house assay compared to 104 by ViroSeq system in the reference panel. The assay had 91.55% success rate in genotyping the clinical panel samples and was able to detect drug resistant mutations related to nucleoside reverse transcriptase inhibitor (NRTI), non-nucleoside reverse-transcriptase inhibitor (NNRTI) as well as protease inhibitor (PI) classes of antiretroviral drugs. It was found to be around 71.9% more cost effective compared to ViroSeq genotyping system. This evaluation of the assay on the clinical panel demonstrates its potential for monitoring clinical HIV-1 drug resistance mutations and population-based surveillance in resource limited settings like India.

Highlights

Introduction of antiretroviral therapy (ART) all over the world has dramatically improved the health condition of Human Immunodeficiency Virus-1 (HIV-1) infected individuals [1]
In this study we report a cost effective HIV-1 drug resistance genotyping assay for Indian population that is validated against US-FDA approved ViroSeq Genotyping System 2.0 (Celera Diagnostics, USA) and demonstrate its efficiency in identifying all drug resistant mutations related to nucleoside reverse transcriptase inhibitor (NRTI), nucleoside reverse-transcriptase inhibitor (NNRTI) and protease inhibitor (PI) classes of antiretroviral drugs
The primers used in the study were designed using a database of HIV-1 pol gene sequences reported from India and archived at NCBI GenBank, USA

Summary

Introduction

Introduction of antiretroviral therapy (ART) all over the world has dramatically improved the health condition of HIV-1 infected individuals [1]. This is reflected by way of sharp fall in mortality and morbidity associated with HIV-1/AIDS [2]. As HIV-1 drug resistance is one of the formidable causes for treatment failure and choice for drugs are limited, there is need for a cost effective HIV-1 drug resistance monitoring system in resource limited settings like India [5]. Information regarding emergence and prevalence of HIV-1 drug resistance mutations is important in guiding the national strategy for implementing ART program in resource limited settings with limited treatment options [11]

Methods

Results

Conclusion