Comparison of digital PCR platforms and semi-nested qPCR as a tool to determine the size of the HIV reservoir

Abstract

HIV persists in latently infected cells of patients on antiretroviral therapy (ART). This persistent proviral DNA reservoir is an important predictor of viral rebound upon therapy failure or interruption and forms a major obstacle towards cure. Accurate quantification of the low levels of persisting HIV DNA may aid patient monitoring and cure research. Digital PCR is a promising tool that enables direct absolute quantification with high sensitivity. With recent technological advances, several platforms are available to implement digital PCR in a clinical setting. Here, we compared two digital PCR platforms, the Quantstudio 3D (Life Technologies) and the QX100 (Bio-Rad) with a semi-nested qPCR on serial HIV DNA dilutions and DNA isolated from PBMCs of ART-suppressed patients. All three methods were able to detect target to the lowest levels of 2.5 HIV DNA copies. The QX100 excelled in having the least bias and highest precision, efficiency and quantitative linearity. Patient sample quantifications by the QX100 and semi-nested qPCR were highly agreeable by Bland-Altman analysis (0.01 ± 0.32 log10). Due to the observation of false-positive signals with current digital PCR platforms however, semi-nested qPCR may still be preferred in a setup of low quantity detection to discriminate between presence or absence of HIV DNA.

The Future of Digital Polymerase Chain Reaction in Virology

“…If reaction volumes for qPCR and dPCR are equal, sensitivity of both platforms have been shown to be comparable [47]. Notably, false-positive partitions have been encountered on several dPCR platforms and can cause a decrease in sensitivity [15,16,45,46]. These false positives seem assaydependent and may also differ between the dPCR platform used [15,46].…”

“…For HIV, Strain et al [16] showed a 4-to 20-fold decrease in coefficient of variation (CV) for HIV pol and 2-LTR quantification. When ddPCR was compared with a seminested qPCR for HIV quantification, ddPCR was more precise for measuring HIV DNA [46], but showed an equal precision for the quantification of HIV RNA forms [15]. For CMV, Hayden et al [12] and Sedlak et al [47] found a higher precision at high CMV concentrations, although equal precision was found for lower levels of CMV.…”

“…Notably, false-positive partitions have been encountered on several dPCR platforms and can cause a decrease in sensitivity [15,16,45,46]. These false positives seem assaydependent and may also differ between the dPCR platform used [15,46]. For diagnostic purposes, it will be necessary for each new application to compare the sensitivity of the dPCR with that of the current gold-standard assay, and to assess the clinical significance of these results.…”

“…It is widely agreed that dPCR has a good quantitative linearity over a wide dynamic range [12,14,46,59,60]; however, it should be noted that the dynamic range of any dPCR device depends on the amount of partitions per reaction. Quantification by dPCR can only be performed when a number of partitions remain negative.…”

Total HIV-1 DNA, a Marker of Viral Reservoir Dynamics with Clinical Implications

“…More recently, an assay that dispenses with the extraction step has been proposed (70). Recent technical progress has permitted the development of HIV DNA quantification by digital droplet PCR (ddPCR), which does not require an external quantification standard (71)(72)(73)(74)(75)(76)(77). However, some false-positive signals have been observed with ddPCR, which can be a problem when this method is used for diagnosis (76).…”