Cell-associated (CA) HIV-1 RNA is considered a potential marker for assessment of viral reservoir dynamics and antiretroviral therapy (ART) response in HIV-infected patients. Recent studies employed sensitive seminested real-time quantitative (q)PCR to quantify CA HIV-1 RNA. Digital PCR has been recently described as an alternative PCR-based technique for absolute quantification with higher accuracy compared to qPCR. Here, a comparison was made between the droplet digital PCR (ddPCR) and the seminested qPCR for quantification of unspliced (us) and multiply spliced (ms) CA HIV-1 RNA. Synthetic RNA standards and CA HIV-1 RNA from infected patients on and off ART (N = 34) were quantified with both methods. Correlations were observed between the methods both for serially diluted synthetic standards (usRNA: R2 = 0.97, msRNA: R2 = 0.92) and patient-derived samples (usRNA: R2 = 0.51, msRNA: R2 = 0.87). Seminested qPCR showed better quantitative linearity, accuracy and sensitivity in the quantification of synthetic standards than ddPCR, especially in the lower quantification ranges. Both methods demonstrated equally high detection rate of usRNA in patient samples on and off ART (91%), whereas ddPCR detected msRNA in larger proportion of samples from ART-treated patients (p = 0.13). We observed an average agreement between the methods for usRNA quantification in patient samples, albeit with a large standard deviation (bias = 0.05±0.75 log10). However, a bias of 0.94±0.36 log10 was observed for msRNA. No-template controls were consistently negative in the seminested qPCR, but yielded a positive ddPCR signal for some wells. Therefore, the false positive signals may have affected the detection power of ddPCR in this study. Digital PCR is promising for HIV nucleic acid quantification, but the false positive signals need further attention. Quantitative assays for CA HIV RNA have the potential to improve monitoring of patients on ART and to be used in clinical studies aimed at HIV eradication, but should be cross-validated by multiple laboratories prior to wider use.

Digital PCR is rapidly gaining interest in the field of molecular biology for absolute quantification of nucleic acids. However, the first generation of platforms still needs careful validation and requires a specific methodology for data analysis to distinguish negative from positive signals by defining a threshold value. The currently described methods to assess droplet digital PCR (ddPCR) are based on an underlying assumption that the fluorescent signal of droplets is normally distributed. We show that this normality assumption does not likely hold true for most ddPCR runs, resulting in an erroneous threshold. We suggest a methodology that does not make any assumptions about the distribution of the fluorescence readouts. A threshold is estimated by modelling the extreme values in the negative droplet population using extreme value theory. Furthermore, the method takes shifts in baseline fluorescence between samples into account. An R implementation of our method is available, allowing automated threshold determination for absolute ddPCR quantification using a single fluorescent reporter.

The ability to persist long term in latently infected CD4 T cells represents a characteristic feature of HIV-1 infection and the predominant barrier to efforts aiming at viral eradication and cure. Yet, increasing evidence suggests that only small subsets of CD4 T cells with specific developmental and maturational profiles are able to effectively support HIV-1 long-term persistence. Here, we analyzed how the functional polarization of CD4 T cells shapes and structures the reservoirs of HIV-1-infected cells. We found that CD4 T cells enriched for a Th1/17 polarization had elevated susceptibilities to HIV-1 infection in ex vivo assays, harbored high levels of HIV-1 DNA in persons treated with antiretroviral therapy, and made a disproportionately increased contribution to the viral reservoir relative to their contribution to the CD4 T memory cell pool. Moreover, HIV-1 DNA levels in Th1/17 cells remained stable over many years of antiretroviral therapy, resulting in a progressively increasing contribution of these cells to the viral reservoir, and phylogenetic studies suggested preferential long-term persistence of identical viral sequences during prolonged antiretroviral treatment in this cell compartment. Together, these data suggest that Th1/17 CD4 T cells represent a preferred site for HIV-1 DNA long-term persistence in patients receiving antiretroviral therapy. A lthough antiretroviral therapy (ART) is highly effective in suppressing HIV-1 replication and clinical HIV-1 disease manifestations, small reservoirs of HIV-1-infected cells persist despite treatment and can lead to rapid viral rebound once treatment is stopped (1, 2). Identifying the functional and phenotypic characteristics of cell subsets that harbor replication-competent virus during suppressive antiretroviral therapy is a critical step for developing interventional strategies to target residual viral reservoirs. Most available evidence supports the notion that latently infected CD4 T cells containing chromosomally integrated but transcriptionally silent HIV-1 DNA represent the most dominant cell population responsible for HIV persistence despite treatment (3-5). However, it is increasingly recognized that such cells represent a phenotypically diverse cell population that consists of a variety of different cell subsets with distinct developmental profiles and functional properties (6-10), divergent levels of permissiveness to HIV-1 infection and latency, and, most likely, different susceptibilities to clinical strategies or pharmaceutical agents aiming at reversing viral latency and inducing HIV-1 eradication.From a developmental perspective, memory CD4 T cell evolution can be described as a hierarchical process during which immature, long-lived cells undergo progressive commitment to more differentiated cell types (11). Recent data suggest that the initial, most immature population of memory CD4 T cells consists of a small number of extremely long-lasting cells that phenotypically express a mix of naive and memory cell markers and display ...

The persistence of a reservoir of latently infected CD4 T cells remains one of the major obstacles to cure HIV. Numerous strategies are being explored to eliminate this reservoir. To translate these efforts into clinical trials, there is a strong need for validated biomarkers that can monitor the reservoir over time in vivo. A comprehensive study was designed to evaluate and compare potential HIV-1 reservoir biomarkers. A cohort of 25 patients, treated with suppressive antiretroviral therapy was sampled at three time points, with median of 2.5 years (IQR: 2.4–2.6) between time point 1 and 2; and median of 31 days (IQR: 28–36) between time point 2 and 3. Patients were median of 6 years (IQR: 3–12) on ART, and plasma viral load (<50 copies/ml) was suppressed for median of 4 years (IQR: 2–8). Total HIV-1 DNA, unspliced (us) and multiply spliced HIV-1 RNA, and 2LTR circles were quantified by digital PCR in peripheral blood, at 3 time points. At the second time point, a viral outgrowth assay (VOA) was performed, and integrated HIV-1 DNA and relative mRNA expression levels of HIV-1 restriction factors were quantified. No significant change was found for long- and short-term dynamics of all HIV-1 markers tested in peripheral blood. Integrated HIV-1 DNA was associated with total HIV-1 DNA (p<0.001, R² = 0.85), us HIV-1 RNA (p = 0.029, R² = 0.40), and VOA (p = 0.041, R2 = 0.44). Replication-competent virus was detected in 80% of patients by the VOA and it correlated with total HIV-1 DNA (p = 0.039, R² = 0.54). The mean quantification difference between Alu-PCR and VOA was 2.88 log10, and 2.23 log10 between total HIV-1 DNA and VOA. The levels of usHIV-1 RNA were inversely correlated with mRNA levels of several HIV-1 restriction factors (TRIM5α, SAMHD1, MX2, SLFN11, pSIP1). Our study reveals important correlations between the viral outgrowth and total and integrated HIV-1 DNA measures, suggesting that the total pool of HIV-1 DNA may predict the size of the replication-competent virus in ART suppressed patients.