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.

Abstract

OBJECTIVES: HIV-1 viral quantitation is essential for treatment monitoring. An in-house assay would decrease financial barriers to access. MATERIALS AND METHODS: A 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. RESULTS: 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. 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. CONCLUSIONS: With 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.