Development and evaluation of a real-time RT-PCR assay for quantification of cell-free human immunodeficiency virus type 2 using a Brome Mosaic Virus internal control

Abstract

Quantification of cell–free virus in plasma is important for monitoring disease progression and for assessing the response to antiretroviral therapy in both human immunodeficiency type 1 and type 2 (HIV-1, HIV-2) infections. Although commercial assays suitable for HIV-1 quantification have been used for more than a decade, no commercial assays are yet available for the measurement of cell-free HIV-2. We have therefore developed a novel real-time RT-PCR assay which, unlike previously described ‘in house’ assays, incorporates a Brome Mosaic Virus (BMV) internal control to minimise the risk of generating false-negative or falsely low results due to unrecognised problems with viral RNA purification, cDNA synthesis or PCR amplification. The assay has a dynamic range of >5 log 10, detects the clinically important HIV-2 subtypes A and B with high sensitivity and shows no cross reactivity with HIV-1. The 95% detection limit is ∼100 HIV-2 RNA copies/ml and both the inter-assay and intra-assay variability are low (CV% at 1.8 × 10 5 copies/ml, 13.3% and 5.7%, respectively). Overall, plasma HIV-2 RNA was detected in 38% of 167 unselected HIV-2 antibody-positive samples analysed over a 2 year period. The assay described provides an ideal system for studying viral replication in HIV-2 infected patients and for monitoring antiretroviral therapy.