Development, Validation and Clinical Evaluation of a Low Cost In-House HIV-1 Drug Resistance Genotyping Assay for Indian Patients

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.

Long-Range HIV Genotyping Using Viral RNA and Proviral DNA for Analysis of HIV Drug Resistance and HIV Clustering

“…The CDC supplies WHO-designated and CDC-supported President's Emergency Plan for AIDS Relief (PEPFAR) Genotyping Laboratories with the ATCC HIV-1 Drug Resistance Genotyping kit (46) for drug resistance testing. Many experienced genotyping laboratories have developed their own in-house amplification and sequencing protocols (11,(47)(48)(49)(50)(51)(52)(53)(54)(55)(56), including identification of minor viral variants that are normally missed by commercial genotyping kits (57-61). All of these approaches generally include smaller and more restricted regions for testing HIV-1 drug resistance.…”

Cost analysis of implementing HIV drug resistance testing in Kenya: a case study of a service delivery site at a tertiary level hospital in Kenya

“…The cost estimate represents all the inputs required for performing HIVDR testing including; capital, personnel, reagents, consumables, quality assurance program and service contracts for the laboratory equipment for performing 1000 tests per year. Previous studies did not include all cost categories, making it difficult to compare costs for offering drug resistant tests across many laboratories ( Acharya et al , 2014 ; Alemán et al , 2015 ; Inzaule et al , 2013 ; Novitsky et al , 2015 ). In this study, we offer a framework for performing laboratory cost analyses that makes it easy to compare cost categories between different laboratories.…”

“…Some of the studies performed in other parts of the world found considerably lower reagents and consumables costs than those estimated by this study. For instance, the costs reported in India and Cuba were $85.00 and $87.80, respectively ( Acharya et al , 2014 ; Alemán et al , 2015 ). Conversely, one study reported higher reagent costs than those found in the present study; the estimated cost was $139.75 per test ( Novitsky et al , 2015 ).…”

“…This is in keeping with other studies evaluating the cost of HIVDR testing. Other studies report $59.88 (52.92%) and $50 (58.82%) as the cost for the sequencing step ( Acharya et al , 2014 ; Inzaule et al , 2013 ). The one-way sensitivity analysis performed illustrates a cost saving opportunity, for example through negotiating lower reagent prices and maximizing utilization of the sequencing platform, therefore ensuring sustainable use of health financing resources.…”

“…Some of the essential costs when performing a costing analysis include equipment costs, personnel costs, and utilities costs. Previous studies have included reagents cost and consumables cost in their estimations, excluding major cost categories ( Acharya et al , 2014 ; Inzaule et al , 2013 ; Novitsky et al , 2015 ; Zhou et al , 2011 ). A detailed cost analysis provides a deeper understanding of the costs of HIVDR to the health systems.…”