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Assessing suitability of next-generation viral outgrowth assays as proxies for classic QVOA to measure HIV-1 latent reservoir size.

  • Author(s): Stone, Mars
  • Rosenbloom, Daniel
  • Bacchetti, Peter
  • Deng, Xutao
  • Dimapasoc, Melanie
  • Keating, Sheila
  • Bakkour, Sonia
  • Richman, Douglas
  • Mellors, John
  • Deeks, Steven
  • Lai, Jun
  • Beg, Subul
  • Siliciano, Janet
  • Pagliuzza, Amélie
  • Chomont, Nicolas
  • Lackman-Smith, Carol
  • Ptak, Roger G
  • Busch, Michael P
  • Reservoir Assay Validation and Evaluation Network (RAVEN) Study Group
  • et al.

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BACKGROUND:Evaluations of HIV curative interventions require reliable and efficient quantification of replication-competent latent reservoirs (LR). The "classic" quantitative viral outgrowth assay (QVOA) has been regarded as "gold standard," although prohibitively resource- and labor-intensive. We compared six "next-gen" VOA employing PCR or ultrasensitive p24 to assess their suitability as scalable proxies for QVOA. METHODS:Next-gen VOA were compared to classic QVOA using single leukapheresis-derived samples from five ART-suppressed HIV+ participants and one HIV- control; each lab tested blinded batches of three frozen and one fresh sample. Markov chain Monte Carlo methods estimated extra-Poisson variation at aliquot, batch, and lab levels. Models also estimated the effect of testing frozen versus fresh samples. RESULTS:Next-gen VOA had similar estimates of variation to QVOA. Assays with ultrasensitive readout reported higher IUPM than classic QVOA. Within-batch testing had 2.5-fold extra-Poisson variation (95%CI 2.1,3.5) for next-gen assays. Between-lab variation increased extra-Poisson variation to 3.4-fold (95% CI 2.6,5.4). Frozen storage did not substantially alter IUPM (-18%(-52%,+39%)). CONCLUSIONS:The data offer cautious support for use of next-gen VOA as proxies for more laborious QVOA, while providing greater sensitivities and dynamic ranges. Measurement of LR in eradication strategies would benefit from high throughput and scalable assays.

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