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

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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.