Within-Subtype HIV-1 Polymorphisms and Their Impacts on Intact Proviral DNA Assay (IPDA) for Viral Reservoir Quantification

The Intact Proviral DNA Assay (IPDA) is widely used to quantify genome-intact HIV proviruses in people living with HIV, but viral sequence diversity has been observed to cause assay failures due to primer/probe mismatches. Adapted for subtype C, IPDA-BC is a modified version of the IPDA validated on South African HIV-1 subtype C. India is also impacted by subtype C, but IPDA performance within-subtype across geographical regions is not well studied. We analyzed Indian (IN) and South African (ZA) subtype C sequences in silico, hypothesizing that IPDA-BC may underperform with IN viruses. Primer/probe binding was predicted using three increasingly stringent nucleotide mismatch criteria, whose sensitivity and specificity were evaluated against experimental IPDA outcomes. Phylogenetic analyses confirmed that IN and ZA subtype C sequences form distinct clusters with significant compartmentalization (<i>p</i> < 0.003). Across criteria, up to 6-10% decreases in primer/probe binding were observed in IN versus ZA, with the <i>env</i> forward primer being the most affected. These criteria showed low sensitivity (18-53%) and variable specificity (67-100%) in predicting experimental outcomes. In conclusion, even within subtype, HIV-1 variation across geographical regions may impact IPDA performance, underscoring the need for improved predictive models to guide assay design for global HIV cure research.