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The HIV proviral reservoir is the major barrier to cure. The predominantly replication-defective proviral landscape makes the measurement of virus that is likely to cause rebound upon ART-cessation challenging. To address this issue, novel assays to measure intact HIV proviruses have been developed. The Intact Proviral DNA Assay (IPDA) is a high-throughput assay that uses two probes to exclude the majority of defective proviruses and determine the frequency of intact proviruses, albeit without sequence confirmation. Quadruplex PCR with four probes (Q4PCR), is a lower-throughput assay that uses limiting dilution long distance PCR amplification followed by qPCR and near-full length genome sequencing (nFGS) to estimate the frequency of sequence-confirmed intact proviruses and provide insight into their clonal composition. To explore the advantages and limitations of these assays, we compared IPDA and Q4PCR measurements from 39 ART-suppressed people living with HIV. We found that IPDA and Q4PCR measurements correlated with one another but frequencies of intact proviral DNA differed by approximately 19-fold. This difference may be in part due to inefficiencies in long distance PCR amplification of proviruses in Q4PCR, leading to underestimates of intact proviral frequencies.  In addition, nFGS analysis within Q4PCR explained that some of this difference is explained by proviruses that are classified as intact by IPDA but carry defects elsewhere in the genome. Taken together, this head-to-head comparison of novel intact proviral DNA assays provides important context for their interpretation in studies to deplete the HIV reservoir and shows that together the assays bracket true reservoir size.

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