Accuracy of real-time PCR and digital PCR for the monitoring of total HIV DNA under prolonged antiretroviral therapy

Abstract

Total HIV DNA is a standard marker to monitor the HIV reservoir in people living with HIV. We investigated HIV DNA quantification accuracy by a real-time PCR kit (qPCR) and digital PCR (dPCR) method within the same set of primers and probes. Among 48 aviremic patients followed for up to 7 years with qPCR, the mean coefficient of variation of total HIV DNA between two successive measurements was 77% (± 0.42log10 HIVDNA copies/106 PBMC). The total HIV DNA quantified by the two PCR methods has a high correlation (0.99 and 0.83, for 8E5 and PLHIV samples, respectively), but we observed better repeatability and reproducibility of the dPCR compared to the qPCR (CV of 11.9% vs. 24.7% for qPCR, p-value = 0.024). Furthermore, we highlighted a decay of the number of HIV copies in the 8E5 cell line qPCR standard over time (from 0.73 to 0.43 copies per cell), contributing to variations of HIV DNA results in patients whose HIV reservoir should be theoretically stabilized. Our study highlighted that absolute quantification of total HIV DNA by dPCR allows more accurate monitoring of the HIV reservoir than qPCR in patients under prolonged antiretroviral therapy.

Development of a droplet digital polymerase chain reaction assay for the sensitive detection of total and integrated HIV-1 DNA

“…Considering the distance to the nearest Alu element in each infected cell is different, [27] and the actual HIV copies in ACH-2 cells may be less than one copy per cell just as in other cell lines such as 8E5. [28] Actually, the quanti cation of integrated HIV DNA is challenging due to the natural heterogeneity of HIV-1 integration, which leads to poor PCR ef ciency. [29] Measurement of total and integrated HIV DNA can provide insights into how reservoirs are formed and maintained.…”

A new multiplexed magnetic capture—Droplet digital PCR tool for monitoring wildlife population health and pathogen surveillance

“…In general, experiments comparing qPCR and ddPCR assays for the detection of low-concentration targets show that ddPCR outperforms qPCR with a lower LOD and LOQ and less variation overall among replicates (Nyaruaba et al, 2021;Renault et al, 2022;Yang et al, 2020).…”

A new multiplexed magnetic-capture - droplet digital PCR (ddPCR) tool for monitoring wildlife population health and pathogen surveillance

“…In general, experiments comparing qPCR and ddPCR assays for the detection of lowconcentration targets show that ddPCR outperforms qPCR with a lower LOD and LOQ and less variation overall among replicates (Nyaruaba et al, 2021;Renault et al, 2022;Yang et al, 2020).…”

“…In general, experiments comparing qPCR and ddPCR assays for the detection of low-concentration targets show that ddPCR outperforms qPCR with a lower LOD and LOQ and less variation overall among replicates (Nyaruaba et al, 2021; Renault et al, 2022; Yang et al, 2020). Our assays are consonant with this observation, as the variation (%CV) in our ddPCR assays is consistently lower than the variation indicated in the publications from which the primers were derived (when applicable; Cuttell et al, 2012; Forde, Orsel, et al, 2016; Lorente-Leal et al, 2019), which mostly used qPCR.…”