HIV-1 viral blips are associated with repeated and increasingly high levels of cell-associated HIV-1 RNA transcriptional activity.

OBJECTIVE: Some HIV+ patients, virally suppressed on ART, show occasional 'blips' of detectable HIV-1 plasma RNA. We used a new highly sensitive assay of cell-associated HIV-1 RNA to measure transcriptional activity in PBMCs and production of infectious virus from the viral reservoir, in patients with and without 'blips'. DESIGN/METHODS: RNA and DNA extracted from cells in 6 ml of peripheral blood, from suppressed patients with one to two 'blip' episodes over the past 2 years of ART (n = 55), or no 'blips' (n = 52), were assayed for HIV-1 RNA transcripts and proviral DNA targeting the highly conserved 'R' region of the LTR. Follow-up samples were also collected. Purified CD4+ T cells were cultured with anti-CD3/CD28/CD2 T-cell activator to amplify transcription and measure replication competent virus. RESULTS: HIV-1 RNA transcripts ranged from 1.3 to 5415 copies/106 white blood cells. 'Blip' patients had significantly higher levels vs. without blips (median 192 vs. 49; P = 0.0007), which correlated with: higher levels of inducible transcripts after activation in vitro, sustained higher HIV-1 transcription levels in follow-up samples along with increasing HIV-1 DNA in some, and production of replication-competent HIV-1. CONCLUSION: Viral 'blips' are significant reflecting higher transcriptional activity from the reservoir and contribute to the reservoir over time. This sensitive assay can be used in monitoring the size and activity of the HIV-1 reservoir and will be useful in HIV-1 cure strategies.

Lack of correlation between three commercial platforms for the evaluation of human immunodeficiency virus type 1 (HIV-1) viral load at the clinically critical lower limit of quantification.

BACKGROUND: Concordance in plasma HIV-1 viral load quantification at the lower limit of quantification (LLOQ) is crucial for current commercial assays. OBJECTIVE: To compare the performance of three commercial viral load assays and carry out a correlation study with the Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 test, the Roche Cobas Amplicor HIV-1 MONITOR test, and the Abbott RealTime HIV-1 assay. STUDY DESIGN: Assay agreement was analyzed using linear regression and Bland-Altman plots. Concordance near the clinically critical LLOQ was measured by Cohen's kappa statistics. Intra-assay precision was assessed, and assay reproducibility was measured at 50copies/mL across all three platforms. RESULTS: While good overall correlation was observed between the assays (r≥0.93), quantitative differences exceeded 0.5log(10)copies/mL among paired results in 3.7 to 8.3% of specimens. The degree of concordance between the assays near the LLOQ was unsatisfactory, with Cohen's kappa ranging from 0.14 to 0.38. The intra-assay precision of the Abbott RealTime HIV-1 assay ranged from 0.04 to 0.15 (SD log(10)) and 1.34% to 8.37% (CV). Reproducibility at 50copies/mL for RealTime HIV-1, TaqMan, and Amplicor was 10.05, 11.04 and 5.07 (% CV), respectively. CONCLUSION: Although good correlation was observed between the assays across their linear range, their concordance at the clinically critical LLOQ was poor. The accurate quantification of low-level viremia remains elusive, and the lack of correlation of these assays presents a challenge to the interpretation of such results and in the clinical management of HIV-infected patients.