CROI 2019 Abstract eBook

Abstract eBook

Poster Abstracts

558 REPLICATE APTIMA ASSAY FOR QUANTIFYING RESIDUAL PLASMA VIREMIA IN INDIVIDUALS ON ART Sonia Bakkour 1 , Xutao Deng 1 , Peter Bacchetti 2 , Mars Stone 1 , AndrewWorlock 3 , Scott Hauenstein 3 , Steven G. Deeks 2 , Michael P. Busch 1 , for the RAVEN Program 1 Blood Systems Research Institute, San Francisco, CA, USA, 2 University of California San Francisco, San Francisco, CA, USA, 3 Hologic Corporation, Bedford, MA, USA Background: Quantification of residual low-level viremia in research participants on anti-retroviral therapy (ART) and during curative interventions requires ultrasensitive plasma HIV RNA assays. Current single copy assays based on ultracentrifugation are limited in throughput. Methods: The Aptima HIV-1 Quant Assay is performed on a fully automated platform using 0.5 ml sample with limits of detection (LOD) of 12 cp/ml and quantitation (LOQ) of 30 cp/ml. To detect lower-level viremia, the instrument can generate 9 replicates (reps) per 5 ml plasma input, with the option of loading multiple 5 ml aliquots to further enhance sensitivity. To validate this approach, samples [4 plasma samples from blood donors with acute infection (2 each subtype B and C) as well as the WHO 3rd international standard] with quantified working stock low viral loads (VL), ranging from 16 to 291 cp/ml, were serially diluted in defibrinated plasma to ~0.2 cp/ml, and tested in 38-90 reps per dilution. A Poisson model-based hybrid algorithmwas developed to estimate the viral RNA copy number. The replicate testing strategy (45 reps) was then applied to 102 apheresis-derived plasma samples from 50 well-suppressed RAVEN study participants on ART. Results: For each of the 5 serially diluted samples, estimated concentrations were calculated using standard limiting dilution analysis (LDA) software and they ranged from no underestimation to underestimation of the expected VL by up to 2-fold, reflecting imperfect sensitivity for detection of a single copy. The ratio between expected and estimated VLs was 1.6 with 95% CI 1.04-2.45. Using the replicate testing approach with 45 reps, requiring 25 ml plasma, the median VL in the well-suppressed RAVEN cohort (N=50 participants) was 0.54 cp/ml (range 0.07-13 cp/ml). All 50 participants had detectable low-level viremia in at least one longitudinal visit (range 1-6 visits spanning up to 18 months). At 0.54 cp/ml, the false negative rate was estimated to be 21.7% and 4.7%with 9 and 18 reps, respectively. The figure shows the impact of rep number on precision of low VL estimates, with higher confidence interval widths at 9 relative to 18 reps. Conclusion: Quantification of low-level viremia can be achieved based on reactive/non-reactive digital readouts on multiple replicates of the Aptima assay via Poisson analysis, with a correction factor that accounts for imperfect sensitivity. Viremia can be detected in all or most individuals on long-term ART, although most have VL <1 cp/ml.

1 University of Pittsburgh, Pittsburgh, PA, USA, 2 Harvard University, Boston, MA, USA, 3 University of North Carolina at Chapel Hill, Chapel Hill, NC, USA, 4 Massachusetts General Hospital, Boston, MA, USA Background: The correlation between single copy plasma HIV-1 RNA assays (research tests) and less sensitive but automated, FDA-cleared plasma HIV-1 RNA assays is not well-defined. We examined this association by testing plasma with both a single copy qRT-PCR assay and the Abbott M2000 automated, commercial platform. Methods: The single copy qRT-PCR assay targeting integrase (iSCA) was performed as published (Cillo, J Clin Micro 2016) with a limit of detection (LoD) 0.4 cp/mL for a 5 mL sample tested. iSCA results were classified as HIV-1 RNA “detected” or “not detected”. The FDA-cleared Abbott M2000 RealTime HIV-1 Viral Load assay has a LoD of 40 cp/mL for a 1.0 mL sample. Results below 40 cp/mL were reported as either <40 cp/mL detected but not quantifiable (<40 target detected) or target not detected (TND). Plasma samples obtained at entry into the ACTG A5321 cohort study were tested with both assays. Participants were on suppressive ART with HIV RNA <40 cp/mL by the Abbott assay. Results: Participants are mostly men (82%), median age of 49, and median of 7 years on ART. Paired samples from 309 participants were tested with both assays. 52% of iSCA results had undetectable HIV-1 RNA; the undetectable iSCA results were primarily (94%) <0.4 cp/mL; nine were <0.5 to <1.1 cp/mL because of lower sample volume. By Abbott M2000, 17% of samples were <40 target detected and 83%were TND. Of the samples TND by Abbott, 43% had HIV-1 RNA detected by iSCA. Of the samples <40 target detected by Abbott, 73% had detectable HIV-1 RNA by iSCA (Figure; p<0.001). Results were similar excluding nine with lower iSCA plasma volume, categorizing iSCA as <0.4 vs. ≥0.4 cp/ mL: 44%≥0.4 cp/mL if TND by Abbott and 73%≥0.4 cp/mL if target detected (p<0.001). Conclusion: 73% of plasma samples with an Abbott HIV-1 RNA result of <40 cp/mL target detected also had HIV-1 RNA detected by iSCA, whereas 43% of samples that were TND by Abbott had HIV-1 RNA detected by iSCA. The difference between <40 cp/mL target detected and TND by Abbott has meaningful information, and can be used to estimate the likelihood of HIV-1 RNA detectability by iSCA. The strong association between the results of both assays indicates that a high-throughput automated assay such as Abbott M2000 could be used in epidemiologic investigations of low-level viremia and to screen for changes in low-level viremia following therapeutic interventions, thereby reducing the need for more labor-intensive research single copy assays.

Poster Abstracts

559 THE USE OF EXTERNAL QUALITY-ASSURANCE DATA TO COMPARE HIV-1 RNA ASSAY PERFORMANCE Cheryl Jennings 1 , Daniel J. Zaccaro 2 , Amy Couzens 2 , Salvatore R. Scianna 1 , Donald J. Brambilla 3 , James W. Bremer 1 1 Rush University Medical Center, Chicago, IL, USA, 2 RTI International, Research Triangle Park, NC, USA, 3 RTI International, Rockville, MD, USA Background: The NIAID Virology Quality Assurance (VQA) program provides well-characterized quality control materials (QCMs) for HIV-1 RNA proficiency testing and assay validations to participating labs as part of an external quality assurance (EQA) program. Seventy-eight labs from 22 countries currently participate in this program using a variety of assays. Data generated for purposes of proficiency or assay validation were used to evaluate HIV-1 RNA cross-platform performance. Methods: Data generated on Roche TaqMan (RT), Abbott RealTime (AR), Roche cobas (RC), and Cepheid GeneXpert (GX) HIV-1 RNA assays were included in this

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