Advances and challenges in tissue-based HIV reservoir quantification

PURPOSE OF REVIEW: Peripheral blood provides an accessible but incomplete representation of the HIV reservoir. This review summarizes recent advances in PCR-based quantification of HIV persistence in human tissues, with particular emphasis on the intact proviral DNA assay (IPDA), the methodological challenges introduced by tissue sampling, and complementary approaches that extend the biological information obtained from IPDA. RECENT FINDINGS: IPDA and related droplet digital PCR assays are increasingly being applied to human tissues, including lymph nodes, brain, gut, and adipose tissue, often in combination with orthogonal assays to address specific biological questions. Recent studies highlight important considerations in interpreting these measurements, particularly the identity and number of cells used as the denominator, DNA shearing, and spatial representativeness of the sampled tissue. These issues complicate comparisons across tissues and with peripheral blood, particularly when different cell populations or sampling depths are analyzed. Newer multitarget droplet digital PCR assays increase the genomic information used to infer proviral intactness, while complementary approaches provide information on viral genotype, cellular identity, clonality, and functional state. SUMMARY: Recent applications of IPDA to human tissues highlight important considerations in cellular denominator, sampling depth, DNA shearing, and spatial sampling that influence the interpretation of reservoir measurements across anatomical compartments. At the same time, combining IPDA with complementary assays is extending tissue studies beyond quantification toward characterization of the virus, infected cell, clonality, and functional state. Future approaches that preserve the relationship between the virus, its host cell, and its tissue environment will further advance the study of HIV persistence in tissues.