Sensitive, Subtype Independent HIV-1 PCR Assays for Assessment of Residual Viremia and Total HIV-1 DNA

Abstract

Background: Correct measurements of residual viremia and reservoir size are crucial in HIV-1 eradication trials and there is a need for sensitive and automated assays. The increasing worldwide diversity of HIV-1 subtypes stresses the importance of subtype independent assays. Methods:We added an ultra-centrifugation step to the COBAS AmpliPrep/COBAS TaqMan HIV-1 test, version 2.0 and evaluated assay sensitivity. A previously published method for quantification of total HIV-1 DNA was also evaluated. Nineteen clinical samples from virologically suppressed cART treated patients infected with various HIV-1 subtypes were analysed. The relationship between plasma HIV-1 RNA and HIV-1 DNA in Peripheral Blood Mononuclear Cells (PBMC) was studied. Results:The HIV-1 RNA PCR assay reached a limit of quantification of 3 copies/ml of plasma. The total HIV-1 DNA assay allowed quantification to 3 copies/million cells with an intra-and inter-assay coefficient of variation of 13.2% and 23.2% respectively. All subtypes studied were quantified by the HIV-1 RNA PCR assay and the HIV-1 DNA assay quantified all subtypes except CRF_AE. No significant correlation was observed between plasma HIV-1 RNA and total HIV-1 DNA in PBMC.Conclusions: By modification of a commercial assay, we achieved a sensitive quantification of plasma HIV-1 RNA that could be used to assess residual viremia. Sensitive quantification of total HIV-1 DNA in PBMC was also demonstrated. The assays were subtype independent to a large extent, making them useful for research and clinical use in settings where a variety of HIV-1 subtypes is present.