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Introduction: The source and significance of residual plasma HIV-1 RNA detection during suppressive ART remain controversial. It has been proposed that nevirapine (NVP)-based regimens achieve a greater HIV-1 RNA suppression than regimens containing a protease inhibitor (PI). The aim of this study was to compare the effect of receiving NVP- vs PI-based ART on the virological markers of HIV persistence in peripheral blood., Material and Methods: The study population comprised 161 HIV-1 infected patients receiving either NVP-based (n=81) or PI-based (n=80) ART and showing a HIV-1 RNA load stably suppressed <40 copies/mL for median of 5.2 years (IQR 2.2-8.0). Residual viraemia was detected by real-time PCR with 50% and 95% detection thresholds of 1 and 3 HIV-1 RNA copies/mL, respectively. Cell-associated (CA) unspliced HIV-1 RNA, total HIV-1 DNA and 2 LTR circles were quantified in peripheral blood mononuclear cells (PBMCs) using droplet digital PCR. Groups were compared by standard non-parametric tests; factors associated with HIV-1 detection were analyzed by univariate regression analysis and generalized linear models (SPSS® V22 and Rstudio)., Results: Plasma HIV-1 RNA was detected in 37/81 (45.7%) and 47/80 (58.8%) subjects on NVP- and PI-based ART, with median (IQR) levels of 5 (3-6) and 5 (3-8) copies/mL, respectively. HIV-1 RNA detection was associated with shorter duration of suppressive ART regardless of treatment arm (p=0.007), and lower CD4 nadir (p=0.015). HIV-1 DNA levels were median 282 (120-484) and 213 (87-494) copies/106 PBMCs in the two groups respectively, and were lowest (<100 copies/106 PBMCs) in subjects with lower plasma HIV-1 RNA (p=0.049), CA unspliced HIV-1 RNA (p=0.0001), 2 LTR circles (p=0.005) and pre-ART HIV-1 RNA load (p=0.0001)., Conclusions: In this comprehensive characterization of patients on long-term suppressive ART, we did not observe evidence for a greater suppressive activity of NVP-based over PI-based therapy on plasma and intracellular markers of virus persistence. Overall excellent correlation was observed between the markers, allowing the identification of a subset of treated patients with low HIV-1 expression as an important cohort for future HIV cure studies.

Introduction: In HIV-infected patients on combination antiretroviral therapy (cART), the detection of episomal HIV 2-LTR circles is a potential marker for ongoing viral replication. Quantification of 2-LTR circles is based on quantitative PCR or more recently on digital PCR assessment, but is hampered due to its low abundance. Sample pre-PCR processing is a critical step for 2-LTR circles quantification, which has not yet been sufficiently evaluated in patient derived samples., Materials and Methods: We compared two sample processing procedures to more accurately quantify 2-LTR circles using droplet digital PCR (ddPCR). Episomal HIV 2-LTR circles were either isolated by genomic DNA isolation or by a modified plasmid DNA isolation, to separate the small episomal circular DNA from chromosomal DNA. This was performed in a dilution series of HIV-infected cells and HIV-1 infected patient derived samples (n=59). Samples for the plasmid DNA isolation method were spiked with an internal control plasmid., Results: Genomic DNA isolation enables robust 2-LTR circles quantification. However, in the lower ranges of detection, PCR inhibition caused by high genomic DNA load substantially limits the amount of sample input and this impacts sensitivity and accuracy. Moreover, total genomic DNA isolation resulted in a lower recovery of 2-LTR templates per isolate, further reducing its sensitivity. The modified plasmid DNA isolation with a spiked reference for normalization was more accurate in these low ranges compared to genomic DNA isolation. A linear correlation of both methods was observed in the dilution series (R2=0.974) and in the patient derived samples with 2-LTR numbers above 10 copies per million peripheral blood mononuclear cells (PBMCs), (R2=0.671). Furthermore, Bland-Altman analysis revealed an average agreement between the methods within the 27 samples in which 2-LTR circles were detectable with both methods (bias: 0.3875±1.2657 log10)., Conclusions: 2-LTR circles quantification in HIV-infected patients proved to be more accurate with a modified plasmid DNA isolation procedure compared to total genomic DNA isolation. This method enables the processing of more blood cells, thus enhancing quantification accuracy and sensitivity. An improved quantification of 2-LTR circles will contribute to the better understanding of ongoing replication in the HIV reservoir of patients on cART.