Duplex digital PCR assay on microfluidic chamber arrays for total HIV DNA reservoir quantification in persons with HIV.

HIV reservoir latently persists in people with HIV (PWH) despite long-term antiretroviral therapy (ART). Total HIV DNA quantification is commonly employed as a surrogate marker to assess the size of the viral reservoir. We developed a duplex digital PCR assay to easily quantify the total HIV DNA in PWH on the microfluidic automated Absolute Q™ digital PCR platform. We assessed the linearity, specificity, sensitivity, and precision targeting the HIV LTR and the human RPP30 gene. We evaluated the assay in clinical samples from 50 PWH on ART and six ART-naïve PWH. The assay showed good linearity (R² = 0.977, p < 0.0001) and a 95% lower limit of detection of 79.7 HIV DNA copies/10⁶ cells. Repeatability and reproducibility were acceptable for 1,250 copies/10⁶ cells (CV = 8.7% and 10.9%) but higher variability for 150 copies/10⁶ cells (CV = 26.9% and 19.9%). Total HIV reservoir was detected in all PWH samples, with statistically significant differences between ART-treated and ART-naïve PWH (p < 0.0001). Positive strong correlation (rho = 0.868, p < 0.0001) was observed between the HIV reservoir in CD4 + T cells and PBMCs from 15 ART-treated PWH. Our assay expands the range of digital PCR platforms available for accurate HIV reservoir quantification.