# Q4ddPCR: a flexible, 4-target assay for high-resolution HIV reservoir profiling

## TL;DR

The paper introduces Q4ddPCR, a new assay that improves the accuracy of measuring HIV reservoirs by targeting four regions of the virus's genome.

## Contribution

The novel contribution is the development of a multi-target droplet digital PCR assay that enhances HIV reservoir quantification and specificity.

## Key findings

- Q4ddPCR reduces quantification gaps and assay dropout to 5% in measuring HIV reservoirs.
- The assay closely correlates with viral outgrowth and tracks reservoir decay in longitudinal studies.
- Multi-target readouts reveal clonal reservoir dynamics undetectable by existing methods.

## Abstract

Precise and scalable quantification of the intact HIV reservoir is critical for advancing curative strategies. Current reservoir assays, such as the intact proviral DNA assay (IPDA), are limited by quantification failures or misclassification of defective proviruses due to HIV sequence heterogeneity. Q4ddPCR is a modular, droplet digital PCR simultaneously targeting four conserved regions in the HIV genome to improve specificity, reduce quantification gaps, and provide multi-layered readouts. It comprises two configurations: one fully based on Q4PCR primer/probes and one combining IPDA with gag and pol primer/probes from Q4PCR. We benchmark Q4ddPCR against 3650 near full-length proviral sequences from 13 virally suppressed people with HIV (PWH) generated by Q4PCR. Q4ddPCR closely matches sequence-confirmed reservoir measurements, and multi-probe readouts reveal clonal reservoir dynamics not detectable by IPDA. Q4ddPCR enables intact reservoir quantification in 95% of samples across four independent cohorts and in 16 PWH, strongly correlates with viral outgrowth. In longitudinal samples from 42 participants over the first 4.5 years on antiretroviral therapy (ART), Q4ddPCR reports lower proviral frequencies and a steeper decline in intact proviral DNA compared to IPDA. Collectively, our findings confirm key predictions from mathematical modeling, demonstrating that multi-target assays improve specificity and more accurately capture intact reservoir dynamics.

Here, the authors develop Q4ddPCR, a high-throughput assay to quantify genetically intact HIV reservoirs by targeting four regions, and demonstrate that it reduces assay dropout to 5%, tracks reservoir decay, and closely correlates with viral outgrowth.