Comparison of Three Different FDA-Approved Plasma HIV-1 RNA Assay Platforms

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1 JCM Accepted Manuscript Posted Online 10 June 2015 J. Clin. Microbiol. doi: /jcm Copyright 2015, American Society for Microbiology. All Rights Reserved Comparison of Three Different FDA-Approved Plasma HIV-1 RNA Assay Platforms Confirm the Virologic Failure Endpoint of 200 copies/ml Despite Improved Assay Sensitivity Christina M. Lalama 1 #, Cheryl Jennings 2, Victoria A. Johnson 3, Robert W. Coombs 4, John E. McKinnon 5, James W. Bremer 2, Bryan R. Cobb 6, Gavin A. Cloherty 7, John W. Mellors 8, Heather J. Ribaudo 1, for the AIDS Clinical Trials Group 1 Harvard T.H. Chan School of Public Health, Boston, MA, USA; 2 Rush University Medical Center, Chicago, IL, USA; 3 Birmingham Veterans Affairs Medical Center and University of Alabama at Birmingham School of Medicine, Birmingham, AL, USA; 4 University of Washington, Seattle, WA, USA; 5 Henry Ford Hospital, Detroit, MI, USA; 6 Roche Molecular Systems, Pleasanton, CA, USA; 7 Abbott Molecular, Des Plaines, IL, USA; 8 University of Pittsburgh, Pittsburgh, PA, USA Running Title: Comparing FDA-Approved Plasma HIV-1 Assay Platforms #Corresponding Author: Christina M. Lalama, lalama@sdac.harvard.edu 1

2 Abstract Discrepancies between HIV-1 RNA results assayed by different FDA-approved platforms have been reported. Plasma samples collected from 332 randomly selected clinical trial participants during second year of antiretroviral treatment were assayed with 3 FDA-approved platforms: UltraSensitive Roche Amplicor Monitor, v1.5 [Monitor], Abbott RealTime HIV-1 Test/m2000 [Abbott] and Roche TaqMan HIV-1 Test, v2.0 [TaqMan]. Samples from 61 additional participants with confirmed HIV-1 RNA >50 c/ml during trial follow-up were also included. Endpoints were HIV-1 RNA quantification vs. >50 c/ml at an individual sample level (primary) and determination of confirmed virologic failure (VF) from longitudinal samples. 389 participants had results obtained from all assays on at least one sample (median=6). Proportions of results >50 c/ml were 19% [Monitor], 22% [TaqMan] and 25% [Abbott]. Despite indication of strong agreement (Cohen s kappa: ), Abbott was more likely to detect HIV-1 RNA >50 c/ml than Monitor (matched pair odds ratio [mor]=4.2; modified Obuchowski p<0.001) and TaqMan (mor=2.1; p<0.001); TaqMan was more likely than Monitor (mor=2.6; p<0.001). Although strong agreement in classifying VF across assay comparisons (kappa: ), at a 50 c/ml threshold, differences in the probability of VF classification (in the same direction as primary) were apparent (all McNemar s p<0.007). At a 200 c/ml VF threshold, no differences between assays were apparent (all p>0.13). Despite strong agreement among assays, significant differences were observed with respect to detecting HIV-1 RNA >50 c/ml and identifying VF at the 50 c/ml threshold. This has important implications for the definition of VF in clinical trials and clinical practice. 2

3 Introduction Recent advances in real-time PCR technology have improved the sensitivity and precision of HIV-1 RNA assays. However, initial implementation of these newer assay technologies into the clinical setting raised concerns about more frequent detection of HIV-1 RNA above a threshold near the lower limits of quantification in participants with prior viral suppression (1). These observations have created uncertainty about how best to manage patients with newly detected HIV-1 RNA above the quantification limit. Based on the assay quantification limits of previous generation assays, a plasma HIV-1 RNA threshold of 50 copies/ml is commonly used in clinical practice as a determination of virologic failure, although data reflected in recent US treatment guidelines suggest that HIV-1 RNA levels up to 200 copies/ml may not be indicative of treatment failure (2). The optimal management of patients with HIV-1 RNA between 50 and 200 copies/ml remains unclear (3). Although further enhancements to assay technology have been made to address some of these initial concerns (4), comparative evaluations are needed between the older UltraSensitive Roche COBAS Amplicor HIV-1 Monitor Test, version (v) 1.5 assay (lower limit of quantification (LLOQ) = 50 c/ml) and the newer Abbott RealTime HIV-1 Test on the m2000 system (LLOQ = 40 c/ml) and the Roche COBAS AmpliPrep / COBAS TaqMan HIV-1 Test, v2.0 (LLOQ = 20 c/ml). The relative performance of these platforms close to each platform s quantification limit and the commonly used 50 copies/ml threshold for defining virologic failure (with and without confirmation) is uncertain and is important to ascertain, as is each platform s impact on the choice of a 3

4 threshold for virologic failure endpoints. Such comparative evaluations were performed as part of this retrospective study. We also wanted to evaluate the utility of detectable not quantifiable results in predicting subsequent virologic failure Materials and Methods Study Design Stored plasma from participants randomized to 2 or 3 nucleoside reverse-transcriptase inhibitor (NRTI) plus efavirenz (EFV) or lopinavir/ritonavir (LPV/r) as part of AIDS Clinical Trial Group (ACTG) studies A5095 and A5142 (5, 6) was utilized. To provide a study sample that reflected stable virologic suppression with HIV-1 RNA breakthrough indicative of virologic failure, stored longitudinal plasma samples from study participants collected during year 2 of trial follow-up were targeted. Specifically, all available plasma samples from trial weeks for 332 randomly selected participants who remained on their initial regimen through 48 weeks of trial follow-up were included. To enhance power to evaluate the secondary outcome related to classification of confirmed virologic failure, year 2 longitudinal samples were included from 61 additional participants who experienced confirmed (2 consecutive) HIV-1 RNA > 50 copies/ml during weeks of trial follow-up (based on real-time testing using UltraSensitive Roche Amplicor Monitor RT PCR, v1.0/1.5). All samples were assayed with 3 FDA-approved platforms: UltraSensitive Roche COBAS Amplicor HIV-1 Monitor Test, v1.5 [Roche Monitor] assay, the Abbott RealTime HIV-1 Test on the m2000 system [Abbott RT/m2000] assay, and the Roche COBAS AmpliPrep / COBAS TaqMan HIV-1 Test, v2.0 [Roche TM] assay. To eliminate inter-lab variation, all assays were performed by one laboratory (DAIDS 4

5 90 91 Virology Quality Assurance (VQA) Laboratory, Rush University Medical Center, Chicago, IL) Study Outcomes The primary outcome was the quantification of HIV-1 RNA versus > 50 copies/ml at an individual sample level. The secondary outcome included two virologic failure endpoints defined as confirmed (2 consecutive) HIV-1 RNA levels > 50 copies/ml and > 200 copies/ml. Statistical Analysis For all three pairwise assay comparisons, assay agreement was assessed with Cohen s kappa using resampling to account for repeated measures as needed (7). Assay differences were evaluated with tests for marginal homogeneity accounting for repeated measures as appropriate (modified Obuchowski (8) and McNemar s) and quantified with a matched pair odds ratio (mor). Only time points with complete data for a pair of assays being compared were included. Separately for each assay, the association of a recent HIV-1 RNA result above the assay quantification limit and subsequent confirmed virologic failure (based on all available longitudinal HIV-1 RNA results for the respective assay) was examined as a time-updated covariate in a Cox proportional hazards model. The observed failure time was measured from trial randomization to the date of the first of the two consecutive HIV-1 RNA measurements above the confirmed virologic failure threshold; participants 5

6 not experiencing confirmed virologic failure during the sampled follow-up period were censored at the date of their last available HIV-1 RNA result. At each failure time, the recent HIV-1 RNA level for each participant in the risk set at that time was used. Participants with only one available HIV-1 RNA result and those whose virologic failure occurred at the first time point with an available HIV-1 RNA result were excluded. The discriminatory properties (sensitivity, specificity and positive predictive value) of a prior HIV-1 RNA result above the assay quantification limit prior to virologic failure or censoring for predicting subsequent confirmed virologic failure were estimated separately for each assay. All p-values and confidence intervals (CI) presented were nominal, unadjusted for multiple comparisons. SAS version 9.2 (SAS Institute Inc, Cary, NC) was used for all analyses. Results Data Availability Of the 393 participants selected for the study sample, 3 had de-consented use of stored samples and 1 had no adequate samples available, leaving 389 participants with a total 2207 stored samples. After exclusion of 12 samples due to inadequate condition or volume, a total of 2195 stored longitudinal plasma samples were assayed with the 3 FDA-approved platforms (Figure 1). Among the 389 participants, there were 1 to 10 HIV-1 RNA results per participant (median of 6). The assays yielded 2137 unique time points with results from all 3 assays and 58 unique time points missing results from 1 or 6

7 of the 3 assays. The majority of the missing data for Roche Monitor was associated with the fact that the upper limit of detection for the Roche Monitor test was 100,000 copies/ml compared to 10,000,000 copies/ml for Abbott RT/m2000 and Roche TM, and there was insufficient volume of plasma to permit repeat testing on a diluted sample. Other factors that contributed to missing data across the three platforms included failed assays, suspected plasma pooling error (multiple samples received from the repository had to be pooled prior to testing to evaluate volume and ensure a homogeneous specimen), and suspected sample switch or inadequate sample volume. Participant Characteristics The demographic and health characteristics of the evaluable study population (n=389) prior to initiating ART (pre-art) were as follows: 76% were male (n=295), 41% were non-hispanic white (n=160), 38% were non-hispanic black (n=146) and 19% were Hispanic (n=74); median pre-art age was 37 years. Median HIV-1 RNA level and CD4 T-cell count were 4.79 log 10 copies/ml and 185 cells/mm 3, respectively (Table 1). Quantification of HIV-1 RNA versus > 50 copies/ml At an individual sample level, the proportion of HIV-1 RNA results >50 copies/ml were 19%, 22% and 24-25% for Roche Monitor, Roche TM and Abbott RT/m2000, respectively (Figure 2). Despite strong agreement in all pairwise assay comparisons (Cohen s kappa: ), evidence of systematic differences in the probability of an HIV-1 RNA result >50 copies/ml was apparent (all modified Obuchowski test p <0.001). Specifically, Abbott RT/m2000 was four times more likely to quantify an HIV-1 RNA 7

8 result >50 copies/ml than Roche Monitor (mor = 4.2), and was more than twice as likely to quantify an HIV-1 RNA result >50 copies/ml compared to Roche TM (mor = 2.1); Roche TM was more than twice as likely compared to Roche Monitor (mor = 2.6) Confirmed Virologic Failure At a 50 copies/ml threshold, 31%, 35% and 38% of participants were classified as experiencing confirmed virologic failure from longitudinal samples with the Roche Monitor, Roche TM and Abbott RT/m2000, respectively. There was high agreement in classifying confirmed virologic failure across all assay comparisons (Cohen s kappa: ; Figure 3A). However, a significant difference in the probability of confirmed virologic failure classification was apparent (all McNemar s test p<0.007), where discordance was due to either none or only one HIV-1 RNA result being >50 copies/ml by the other assay. Specifically, the relative odds of identifying a confirmed virologic failure was at least four times higher with Abbott RT/m2000 and Roche TM, respectively, compared to Roche Monitor (mor = 5.3, 95% CI = [2.4, 11.9] and mor = 4.0, 95% CI = [1.5, 10.7], respectively), and more than twice as high with Abbott RT/m2000 than with Roche TM (mor = 2.9, 95% CI = [1.3, 6.3]). By contrast, with a threshold of 200 copies/ml, between 23-24% of subjects were classified as experiencing confirmed virologic failure across the three assays, and no differences between assays were apparent (all McNemar s test p>0.13; Figure 3B). HIV-1 RNA Result above the Assay Quantification Limit 8

9 With the 50 copies/ml virologic failure threshold, while a higher risk of confirmed virologic failure was observed with a recent HIV-1 RNA result above the assay quantification limit for the Roche TM (>20 copies/ml) and Abbott RT/m2000 (>40 copies/ml), the association was statistically significant only for the Roche TM (HR [95% CI] =1.80 [1.07, 3.01], p=0.027, time-updated Cox proportional hazards model; Table 2A). Since the Roche Monitor has a quantification limit of 50 copies/ml, a hazard ratio could not be calculated for the 50 copies/ml virologic failure threshold. For all three assays, a recent HIV-1 RNA result above the respective assay quantification limit was associated with a higher risk of confirmed virologic failure at the 200 copies/ml threshold (p 0.001; Table 2B). Despite these associations, a prior HIV-1 RNA result above the respective assay limit did not have good precision for predicting a subsequent confirmed virologic failure. Specifically, across the three assays, the positive predictive value (PPV) was no higher than 38% (95% CI=[28%, 49%]) with a 50 copies/ml threshold (for Abbott RT/m2000, PPV [95% CI] =30 [18, 44]% for Roche Monitor and 29 [22, 38]% for Roche TM; Figure 4A) and no higher than 30% (95% CI=[20%, 41%]) with a 200 copies/ml threshold (for Roche Monitor, PPV [95% CI] =22 [15, 30]% for Abbott RT/m2000 and 20 [14, 27]% for Roche TM; Figure 4B). Examination of the utility of a prior HIV-1 RNA result further categorized as detectable and quantifiable, detectable but not quantifiable, or undetectable (reference) for predicting confirmed virologic failure yielded similar results. Specifically, at the 200 copies/ml virologic failure threshold, a recent detectable and quantifiable result was associated with a higher risk of confirmed virologic failure compared to a recent 9

10 undetectable result (HR [95% CI]: Roche Monitor = 3.24 [1.60, 6.54]; Abbott RT/m2000 = 2.45 [1.40, 4.31]; Roche TM = 4.21 [2.15, 8.26]); however, a prior detectable and quantifiable result did not have good precision for predicting a subsequent confirmed virologic failure (PPV [95% CI]: Roche Monitor = 30 [20, 41]%; Abbott RT/m2000 = 21 [15, 29]%; Roche TM = 21 [15, 27]%). At the 50 copies/ml virologic failure threshold, a significant association was only seen for Roche TM, which identified the highest number of recent detectable (quantifiable and not quantifiable) HIV-1 RNA results (HR [95% CI], PPV [95% CI]: Roche TM = 2.66 [1.49, 4.75], 30 [23, 38]%; Abbott RT/m2000 = 1.86 [0.99, 3.53], 37 [27, 48]%). For the comparison of detectable but not quantifiable versus undetectable, the only notable finding was for Roche TM with a 50 copies/ml virologic failure threshold; a detectable but not quantifiable result was associated with a higher risk of failure (HR [95% CI]: Roche TM = 1.82 [1.10, 3.01]; Abbott RT/m2000 = 1.25 [0.79, 1.97]), but did not have good precision for predicting a subsequent confirmed virologic failure (PPV [95% CI] =21 [16, 27]% for Roche TM and 24 [19, 31]% for Abbott RT/m2000). Discussion In this study, more than 2000 stored longitudinal plasma samples collected during year two of ART in ACTG clinical trials were assayed using Roche Monitor, Abbott RT/m2000 and Roche TM platforms and showed strong agreement in all pairwise comparisons at the individual sample level. Despite this strong agreement, significant differences in quantification of HIV-1 results >50 copies/ml between assays were apparent with a higher odds of results >50 copies/ml for the Abbott/m2000 compared to 10

11 Roche Monitor and Roche TM and, in turn, Roche TM with a higher odds over Roche Monitor. Similar findings were apparent across all assay comparisons with respect to identification of confirmed virologic failure based on consecutive results >50 copies/ml. Differences across the assays were not apparent, however, when virologic failure was defined using a 200 copies/ml threshold on consecutive results. As well, recent studies assessed the correlation between paired assay results at low viral loads (including the assays examined here), and found that the correlation between assays was lower at lower HIV-1 RNA results and that discordance decreased when a 200 copies/ml threshold was used versus a 50 copies/ml threshold (9, 10). These findings were not completely unexpected based on previous studies that have evaluated sources of variability in HIV-1 RNA assays (11-13), including the variability at the low end of the reportable range (14). Limit of detection for an assay refers strictly to the ability of an assay to detect the HIV-1 RNA in a sample with a >95% detection rate. A limit of quantitation, on the other hand, refers to the HIV-1 RNA concentration where the precision of replicate sample testing is deemed acceptable. For instance, the VQA HIV- 1 RNA proficiency testing program currently uses a total assay standard deviation target of 0.15 log10 based on the expectation that the assay should be able to detect a 5-fold difference in HIV-1 RNA concentration. For real-time PCR assays, including Abbott /m2000 and Roche TM, the minimal nominal value that can be included in precision analyses is 100 copies/ml; for historical endpoint PCR assays such as Roche Monitor, that nominal value was 200 copies/ml. Data show that samples with nominal values below this cutoff have higher standard deviations (VQA unpublished data); data published by Ruelle et al. show similar observations for Roche TM and Abbott/m

12 (14). These findings add further support to the recommendation for using a threshold of >200 copies/ml as evidence of virologic failure (2) Although a recent HIV-1 RNA result above the assay threshold result was associated with a higher risk of confirmed virologic failure at a 200 copies/ml threshold across all assays, this association was only apparent at the 50 copies/ml threshold with the Roche TM platform. An association with a detectable but not quantifiable result with Roche TM and virologic failure at the 50 copies/ml threshold was also noted, but no associations with detectable but not quantifiable results were apparent with the other platforms. Despite these significant population-level associations (when apparent), at an individual level, an HIV-1 RNA result above the respective assay limit or detectable but not quantifiable had low predictive value for subsequent confirmed virologic failure across all platforms at both the 50 copies/ml and 200 copies/ml thresholds (PPVs 38%). Whereas Havlir et al. has shown that transient increases in HIV-1 RNA (blips) are not associated with virologic failure among patients who have achieved initial viral suppression below 50 copies/ml (15), other studies have reported associations between low level viremia and subsequent risk of virologic failure (16, 17). The predictive value of these findings at an individual level have not been reported. Our findings indicate that a single detectable (regardless of quantifiability) HIV-1 RNA result has insufficient positive predictive value for subsequent confirmed virologic failure to warrant a change in treatment regimen, although standard recommendations for review of medication adherence and appropriate counseling should be followed. 12

13 Strengths of this study are the large number of samples tested and the standardized procedures under which they were collected and stored. These samples were collected during year 2 of ART from participants in two treatment-naïve randomized ACTG studies of initial ART; therefore, all participants were on ART the same length of time and the collection of samples was done within the same clinical trial network using the same procedures throughout with sample storage maintained at a central specimen repository. Although these samples were more than 6 years old when tested, no significant loss of HIV RNA across years of storage was seen when the VQA compared the data generated by the two platforms of the Roche Monitor assay: microwell plate done in real time during trial follow-up versus COBAS done for this study (data not shown). In addition, the testing done for these analyses was performed in a single laboratory, thus minimizing inter-lab variation. Further, the testing laboratory was blinded to any prior HIV-1 RNA quantification for the samples. A key limitation of this study is that the true level of HIV-1 RNA in any given sample to which each assay can be compared is unknown. So while we can evaluate differences, it is unclear if one assay is to be preferred over another with respect to accuracy of measurement. Further, when examining the association between detectable and quantifiable recent HIV-1 RNA results and subsequent confirmed virologic failure, subjects are excluded from the risk set at the time of the first of the two HIV-1 RNA results above the virologic failure threshold. The exclusion of these inherently detectable and quantifiable results that were subsequently confirmed as virologic failures is necessary for identifiability for statistical analysis. However, it will naturally 13

14 underestimate the association that is of interest clinically; faced with a single detectable and quantifiable result, what is the likelihood that a patient s regimen is failing? A sensitivity analysis was performed to examine the robustness of our conclusions in light of this limitation. Specifically, this analysis included subjects in the risk set up to and including the initial virologic failure sample; subsequent failure was then determined based on whether virologic failure was defined by the either the current or subsequent result. Since all subjects with virologic failure then contribute two failure results to the analysis, robust variance estimation was used. While this analysis naturally strengthened the association between detectable HIV-1 RNA results and subsequent virologic failure (findings across all assays and both thresholds were statistically significant), the discriminative properties of single detectable and quantifiable result remained relatively poor (all PPVs 68% for 50 copies/ml threshold and all PPVs 46% for 200 copies/ml). In conclusion, this study revealed a high degree of agreement between three FDAapproved HIV-1 RNA platforms assays, but despite this strong agreement, significant difference were observed between assays with respect to quantifying HIV-1 RNA >50 copies/ml that translated into differences in the determination of confirmed virologic failure >50 copies/ml; however, these differences were not apparent at the 200 copies/ml threshold. These results have important implications for the definition of virologic failure in clinical trials and clinical practice a virologic failure threshold of >200 copies/ml that is confirmed by a follow-up viral load evaluation is supported by our findings. 14

15 Acknowledgements This project described was supported by Award Numbers UM1 AI and AI38858 from the National Institute of Allergy and Infectious Diseases (NIAID). This work was also supported by the Statistical and Data Management Center of the AIDS Clinical Trials Group (UM1 AI068634; AI38855) and the Virology Quality Assurance Program (HHSN C, HHSN C) from NIAID. The content is solely the responsibility of the authors and does not necessarily represent the official views of NIAID or the National Institutes of Health. Roche Molecular Systems and Abbott Molecular supplied dedicated viral load assay kit lots. We gratefully thank the A5095 and A5142 teams, the AIDS Clinical Trial Unit personnel and the study volunteers for their participation. VAJ has had Roche laboratory equipment with training support. RWC has served on Roche and Abbott Molecular expert panels. JEM has received grant funding from Abbott Molecular. BRC is an employee of Roche Molecular Systems. GAC is an employee of Abbott Molecular. JWM is a consultant to Gilead Sciences and holds share options in RFS Pharmaceuticals. HJR has served on a Roche expert panel. No other conflicts are reported. 15

16 336 References Lima V, Harrigan R, Montaner JSG Increased reporting of detectable plasma HIV-1 RNA levels at the critical threshold of 50 copies per milliliter with Taqman assay in comparison to the Amplicor assay. J. Acquir. Immune Defic. Syndr. 51: HHS Panel on Antiretroviral Guidelines for Adult and Adolescents. 06 October Guidelines for the use of antiretroviral agents in adults and adolescents. United States Department of Health and Human Sciences, Washington, DC Thompson MA, Aberg JA, Hoy JF, Telenti A, Benson C, Cahn P, Eron JJ, Günthard HF, Hammer SM, Reiss P, Richman DD, Rizzardini G, Thomas DL, Jacobsen DM, Volberding PA Antiretroviral treatment of adult HIV infection: 2012 recommendations of the International AIDS Society USA panel. JAMA 308: Pas S, Rossen JW, Schoener D, Thamke D, Pettersson A, Babiel R, Schutten M Performance evaluation of the new Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 test version 2.0 for quantification of human immunodeficiency virus type 1 RNA. J. Clin. Microbiol. 48: Gulick RM, Ribaudo HJ, Shikuma CM, Lalama C, Schackman BR, Meyer WA, Acosta EP, Schouten J, Squires KE, Pilcher CD, Murphy RL, Koletar SL, Carlson M, Reichman RC, Bastow B, Klingman KL, Kuritzkes DR; AIDS Clinical 16

17 Trials Group A5095 Study Team Three- vs four-drug antiretroviral regimens for the initial treatment of HIV-1 infection: a randomized controlled trial. JAMA 296: Riddler SA, Haubrich R, DiRienzo AG, Peeples L, Powderly WG, Klingman KL, Garren KW, George T, Rooney JF, Brizz B, Lalloo UG, Murphy RL, Swindells S, Havlir D, Mellors JW; AIDS Clinical Trials Group Study A5142 Team Class-sparing regimens for initial treatment of HIV-1 infection. N. Engl. J. Med. 358: Follmann D, Proschan M, Leifer E Multiple outputation: inference for complex clustered data by averaging analyses from independent data. Biometrics 45: Yang Z, Sun X, Hardin JW A note on the tests for clustered matched-pair binary data. Biom. J. 52: Swenson LC, Cobb B, Geretti AM, Harrigan PR, Poljak M, Seguin-Devaux C, Verhofstede C, Wirden M, Amendola A, Boni J, Bourlet T, Huder JB, Karasi JC, Zidovec Lepej S, Lunar MM, Mukabayire O, Schuurman R, Tomazic J, Van Laethem K, Vandekerckhove L, Wensing AM; International Viral Load Assay Collaboration Comparative performances of HIV-1 RNA load assays at low viral load levels: results of an international collaboration. J. Clin. Microbiol. 52:

18 Amendola A, Marsella P, Bloisi M, Forbici F, Angeletti C, Capobianchi MR Ability of two commercially available assays (Abbott RealTime HIV-1 and Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 Version 2.0) to quantify low HIV-1 RNA Levels (<1,000 copies/milliliter): comparison with clinical samples and NIBSC working reagent for nucleic acid testing assays. J. Clin. Microbiol. 52: Brambilla D, Reichelderfer PS, Bremer JW, Shapiro DE, Hershow RC, Katzenstein DA, Hammer SM, Jackson B, Collier AC, Sperling RS, Fowler MG, Coombs RW The contribution of assay variation and biological variation to the total variability of plasma HIV-1 RNA measurements. AIDS 13: Yen-Lieberman B, Brambilla D, Jackson B, Bremer J, Coombs R, Cronin M, Herman S, Katzenstein D, Leung S, Lin HJ, Palumbo P, Rasheed S, Todd J,Vahey M, Reichelderfer P Evaluation of a quality assurance program for quantitation of human immunodeficiency virus type 1 RNA in plasma by the AIDS clinical trials group virology laboratories. J. Clin. Microbiol. 34: Jennings C, Harty B, Granger S, Wager C, Crump JA, Fiscus SA, Bremer JW Cross-Platform Analysis of HIV-1 RNA data generated by a multicenter assay validation study with wide geographic representation. J. Clin. Microbiol. 50: Ruelle J, Debaisieux L, Vancutsem E, De Bel A, Delforge ML, Piérard D, Goubau P HIV-1 low level viraemia assessed with 3 commercial realtime PCR assays show high variability. BMC Infect. Dis. 12:

19 Havlir DV, Bassett R, Levitan D, Gilbert P, Tebas P, Collier AC, Hirsch MS, Ignacio C, Condra J, Günthard HF, Richman, DD, Wong JK Prevalence and predictive value of intermittent viremia with combination HIV therapy. JAMA 286: Doyle T, Smith C, Vitiello P, Cambiano V, Johnson M, Owen A, Phillips AN, Geretti AM Plasma HIV-1 RNA detection below 50 copies/ml and risk of virologic rebound in patients receiving highly active antiretroviral therapy. Clin. Infect. Dis. 54: Henrich TJ, Wood BR, Kuritzkes DR Increased risk of virologic rebound in patients on antiviral therapy with detectable HIV load <49 copies/ml. PLoS One 7: e doi: /journal.pone Downloaded from on July 5, 2018 by guest 19

20 TABLE 1: Pre-ART participant characteristics Characteristic Total (N=389) Pre-ART age (years) Mean (s.d.) 38 (9) Median (Q1 - Q3) 37 (32-43) Min - Max <30 69 (18%) (39%) (35%) (8%) Race/ethnicity Non-Hispanic white 160 (41%) Non-Hispanic black 146 (38%) Hispanic (Regardless of Race) 74 (19%) Other 9 (2%) Sex Male 295 (76%) Female 94 (24%) Pre-ART HIV-1 RNA (log 10 copies/ml) Mean (s.d.) 4.9 (0.7) Median (Q1 - Q3) 4.8 ( ) Min - Max (copies/ml) <10, (11%) 10,000-29, (19%) 30,000-99, (31%) 100, , (19%) 300, (21%) Pre-ART CD4+ T-cell count (cells/mm 3 ) Mean (s.d.) 215 (182) Median (Q1 - Q3) 185 (63-302) Min - Max 1-1, (22%) (32%) (26%) (20%) 20

21 TABLE 2: Time to confirmed virologic failure with the effect of a recent HIV-1 RNA result above the assay quantification limit Assay Quantification limit (copies/ml) (A) 50 copies/ml virologic failure threshold N Total no. samples prior to virologic failure/censoring Above limit At or below limit No. of events Hazard ratio [95% CI] P-value Roche Monitor [0.00,.] 0.99 Abbott RT/m [0.90, 3.21] 0.10 Roche TM [1.07, 3.01] Assay Quantification limit (copies/ml) (B) 200 copies/ml virologic failure threshold N Total no. samples prior to virologic failure/censoring Above limit At or below limit No. of events Hazard ratio [95% CI] P-value Roche Monitor [1.55, 6.07] Abbott RT/m [1.67, 4.84] <0.001 Roche TM [1.90, 5.58] <0.001 A subject may be counted more than once. A hazard ratio could not be calculated since assay quantification limit is 50 copies/ml. 21

22 427 Figure Legends 428 Figure 1. Disposition of samples and participants Figure 2. Assay comparisons of the quantification of HIV-1 RNA level ( versus > 50 copies/ml) at the individual sample level. P-values were calculated with the modified Obuchowski test. mor=matched pair odds ratio; kappa=cohen s kappa based on a resampling technique. Figure 3. Assay comparisons of the determination of confirmed virologic failure from longitudinal samples. Panel A: Assay comparisons with a confirmed virologic failure threshold of 50 copies/ml. Panel B: Assay comparisons with a confirmed virologic failure threshold of 200 copies/ml. P-values were calculated with the McNemar s test. VF=confirmed virologic failure; mor=matched pair odds ratio; CI=confidence interval; kappa=cohen s kappa. Figure 4. Positive predictive value (PPV) of an HIV-1 RNA result above the assay quantification limit for predicting confirmed virologic failure from longitudinal samples. The quantification limits are 50 c/ml, 40 c/ml and 20 c/ml for Roche Monitor, Abbott RT/m2000 and Roche TM, respectively. Panel A: PPV of each assay for confirmed virologic failure threshold of 50 copies/ml. Panel B: PPV of each assay for confirmed virologic failure threshold of 200 copies/ml. CI=exact confidence interval. 22

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