Clinical Evaluation of a Low Cost, In-House Developed Real-Time RT-PCR Human Immunodeficiency Virus Type 1 (HIV-1) Quantitation Assay for HIV-1 Infected Patients

Palvinder Kaur,Evelyn Koay,Sue Yuen Wee,Kuan Kiat Chew,Mei Ting Tan,Eng Lee Tan,Masafumi Inoue,Oon Tek Ng,Yee Sin Leo,Kah Ying Ng,Juergen Pipper,Joe Kwan Yap,Wei Xin Khong,Anil Kumar

doi:10.1371/journal.pone.0089826

Abstract

ObjectivesHIV-1 viral quantitation is essential for treatment monitoring. An in-house assay would decrease financial barriers to access.Materials and MethodsA real-time competitive RT-PCR in house assay (Sing-IH) was developed in Singapore. Using HXB2 as reference, the assay's primers and probes were designed to generate a 183-bp product that overlaps a portion of the LTR region and gag region. A competitive internal control (IC) was included in each assay to monitor false negative results due to inhibition or human error. Clinical evaluation was performed on 249 HIV-1 positive patient samples in comparison with the commercially available Generic HIV Viral Load assay. Correlation and agreement of results were assessed for plasma HIV-1 quantification with both assays.ResultsThe assay has a lower limit of detection equivalent to 126 copies/mL of HIV-1 RNA and a linear range of detection from 100–1000000 copies/mL. Comparative analysis with reference to the Generic assay demonstrated good agreement between both assays with a mean difference of 0.22 log10 copies/mL and 98.8% of values within 1 log10 copies/mL range. Furthermore, the Sing-IH assay can quantify HIV-1 group M subtypes A–H and group N isolates adequately, making it highly suitable for our region, where subtype B and CRF01_AE predominate.ConclusionsWith a significantly lower running cost compared to commercially available assays, the broadly sensitive Sing-IH assay could help to overcome the cost barriers and serve as a useful addition to the currently limited HIV viral load assay options for resource-limited settings.

Highlights

With an estimated 34 million people living with HIV worldwide and 1.7 million deaths in 2011 [1], Human Immunodeficiency Virus Type 1 (HIV-1) is a global pandemic that remains one of the world’s most serious health challenges
The assay has a lower limit of detection equivalent to 126 copies/mL of HIV-1 RNA and a linear range of detection from 100–1000000 copies/mL
We developed a viral load assay with internal control, targeting a 189-bp region starting from 39 end of the LTR region to part of 59 of the gag region of HIV-1 virus (PCT Patent Application No PCT/SG2010/000257)

Summary

Introduction

With an estimated 34 million people living with HIV worldwide and 1.7 million deaths in 2011 [1], HIV-1 is a global pandemic that remains one of the world’s most serious health challenges. More than 10 million individuals receiving HIV-1 antiretroviral therapy, many of which living in low- and middleincome countries [2]. HIV-1 viral quantitation remains limited in many affected regions, especially in resource-limited settings [7]. The running cost of commercially available viral load assays is prohibitively expensive: ranging from US$20–$160 per viral load test. The prohibitive high running cost of commercially available viral load assays has impeded their routine application in most resource-limited settings [7,9]

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