Background
Intact proviral DNA assays (IPDA) are high-throughput methods to quantify intact and defective HIV proviruses during ART, with the former providing estimates of rebound-competent HIV reservoir. However, most IPDAs have been designed for subtype B and have ~28% false negative rate due to polymorphisms in the primer/probe sites1. The cross-subtype IPDA (CS-IPDA) was designed to tolerate diversity across subtypes A, B, C, D and CRF01_AE,2 but has not yet been validated on large cohorts.
Methods
CS-IPDA is a multiplex droplet-digital PCR assay that targets 3 genomic regions (gag, pol and env) of HIV. The number of triple-positive droplets is used to estimate intact proviruses, while single- and dual-positive droplets are indicative of defective proviruses. We used the CS-IPDA on PBMCs collected during ART from 191 children living with HIV from 2 cohorts in Kenya, where subtypes A, C and D are prevalent. Each sample was tested in 3 or more replicates and included here if >1e5 cell equivalents (median 4.2e5, range 1e5 – 4.3e6) were tested and if the shearing index was <40% (median 3%, range 2% – 31%).
Results
Across all 696 samples collected longitudinally from 191 ART-treated participants, there was a median 1.4e3 defective (range: 0 – 6.8e4) and 145 intact (range: 0 – 5.2e4) HIV DNA copies per million T cells. In 91 (13.1%) samples there were no intact proviruses detected, which could indicate intact levels truly below detection, or false negatives due to primer/probe mismatches. In 75 (10.7%) samples intact HIV DNA was undetectable, however all 3 genomic targets were detected in the defective proviral population (in single- or dual-positive droplets), suggesting the negative intact result was not due to polymorphisms causing failure of the PCR to detect a region of the provirus. In only 16 (2.3%) of the 696 samples tested, the CS-IPDA failed to detect any signal for one of the targets, likely due to polymorphisms in the primers and/or probe binding sites.
Conclusions
The CS-IPDA has a very low (<3%) false-negative rate due to PCR failure in samples from Kenya, where the most prevalent global subtypes circulate, suggesting it effectively tolerates diversity across multiple subtypes.
References:
1. Kinloch, NN, et al. (2021) HIV-1 diversity considerations in the application of the Intact Proviral DNA Assay (IPDA). Nat Commun 12, 165.
2. Cassidy, NAJ, et al. (2022) HIV reservoir quantification using cross-subtype multiplex ddPCR. iScience 25, 103615.
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