Methods of low-level detection of HIV-1: Validation of a real time PCR for total DNA quantification and a sensitive subtype independent plasma RNA PCR
Tomas Mellberg1, Jon Krabbe2, Maria Buzon3, Ulrika Noborg2, Magnus Lind2, Magnus Gisslén1, Bo Svennerholm2
Affiliates: 1Institute of Biomedicine, Department of Infectious Diseases, University of Gothenburg, Sweden, 2Institute of Biomedicine, Department of Clinical Virology, University of Gothenburg, Sweden, 3Irsicaixa AIDS Institute, Badalona, Spain
Correct measurement of low-grade viremia and estimation of reservoir size are crucial in eradication trials and there is a need for sensitive, automated and subtype-independent assays.
We added an ultracentrifugation step prior to the routine plasma HIV-1 RNA RT-PCR to improve sensitivity. Comparison of assay performance and specificity was performed using a dilution series. Validation of a previously published method for quantification of total HIV-1 DNA was performed and correlation between plasma HIV-1 RNA and DNA in peripheral blood mononuclear cells (PBMC) was studied in patients on effective antiretroviral treatment (cART).
The HIV-1 RNA assay reached a sensitivity of 2-3 copies/ml (lowest copy number 2.3 copies/ml). The total HIV-1 DNA assay had a sensitivity of 3 copies/million cells (lowest copy number 1.4 copies/million cells) with a variation coefficient of 12% (intra-assay) and 21% (inter-assay). All subtypes represented were quantifiable by the HIV-1 RNA assay and the DNA assay managed to quantify all subtypes except CRF_AE. No correlation was found between residual plasma HIV-1 RNA and total DNA in PBMC.
We present sensitive assays for quantification of plasma HIV-1 RNA and total DNA in PBMC. The lack of correlation between plasma HIV-1 RNA and total total DNA indicate that this combination of assays is not sufficient to estimate the latent viral burden in patients on cART. The assays were sub-type independent to a large extent and are important for their possible utility in research and clinical settings with a variety of sub-types present.