Focus on the ability to quantify low HIV-1 RNA levels (<1000 copies/ml) by two commercially

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1 JCM Accepts, published online ahead of print on 26 March 2014 J. Clin. Microbiol. doi: /jcm Copyright 2014, American Society for Microbiology. All Rights Reserved Focus on the ability to quantify low HIV-1 RNA levels (<1000 copies/ml) by two commercially available assays (Abbott Real-time HIV-1 and the Roche COBAS Ampliprep/COBAS TaqMan HIV-1 v2.0): comparison with clinical samples and NIBSC Working Reagent for NAT assays Alessandra Amendola #, Patrizia Marsella, Maria Bloisi, Federica Forbici, Claudio Angeletti and Maria R Capobianchi. All Authors Affiliation: National Institute for Infectious Diseases Lazzaro Spallanzani Rome, Italy Running Head: Comparison between assays at low HIV-1 RNA levels. # Address correspondence to: Alessandra Amendola, PhD, Laboratory of Virology, National Institute for Infectious Diseases Lazzaro Spallanzani, Via Portuense 292, Rome, Italy. Tel: ; fax: ; alessandra.amendola@inmi.it Text word count: 3369 Abstract word count: 247 Number of tables: 2 Number of figures: 3 Key words: HIV-1 RNA, low viremia levels, assay comparison. Abbreviations: VL (HIV-1 RNA viral load); LVL (low HIV-1 viremia levels); Abbott assay (Abbott Real-time HIV-1 assay); Roche assay (Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 v2.0 assay); Guideline (GL); Antiretroviral therapy (ART). 1

2 26 ABSTRACT Concordance between molecular assays may be sub-optimal at low HIV-1 viremia levels (<1000 copies/ml), therefore it may be difficult to define and compare virologic endpoints for successful therapy and treatment failure. We compared two commercial assays (Abbott Real-time HIV-1 and Roche COBAS Ampliprep/TaqMan HIV-1 v2.0) for their ability to detect and quantify low viremia levels. Comparison was performed using 167 residual clinical samples with values ranging from notdetected to 1000 copies/ml as measured by the Abbott assay, and NIBSC HIV-1 RNA Working Reagent 1 for NAT assays, serially diluted to span a range between 1 and 1000 copies/ml. Quantitative results were compared using Lin s concordance correlation coefficient and Bland- Altman procedure. Concordance on qualitative results was measured by Cohen s kappa statistic. With clinical samples, the degree of inter-assay concordance of qualitative results at 40 copies/ml HIV-1 RNA threshold was substantial (k=0.762); the correlation among quantified samples was suboptimal (concordance correlation coefficient=0.728; p<0.0001); the mean difference of values between Roche and Abbott assays was log 10 copies/ml. With HIV-1 RNA Working Reagent 1 for NAT assays, results provided by Roche assay were, on average, 3 times higher than expected, while Abbott assay showed high accuracy. The Roche assay was highly sensitive, being able to detect down to 3.5 copies/ml HIV-1 RNA with 95% of probability. Performance characteristics of each molecular assay should be taken into account when HIV-1 RNA threshold values for virologic suppression, virologic failure, persistent low viremia levels, etc., are defined and indicated to support clinical decisions. 47 2

3 48 Background Assessment of HIV-1 RNA in HIV-infected patients is a powerful parameter in monitoring viral suppression and drug efficacy during antiretroviral therapy (ART) Current international guidelines (GL) and clinical trials define virologic suppression the achievement and the maintenance of HIV-1 RNA below 50 copies/ml or to levels below assay limits as the virological endpoint of successful ART: in fact, below this threshold, patients have the lowest morbidity and mortality probabilities (1-5). By contrast, HIV RNA threshold for defining virological failure (VF) differs across GL. In fact, World Health Organization GL define VF as a viral load (VL) value persistently exceeding 1000 copies/ml (two consecutive VL measurements within a three-month interval) after at least six months of using ART (1). Instead, British HIV Association (BHIVA), International Antiviral Society (IAS), European AIDS Clinical Society (EACS) and Department of Health and Human Services (DHHS) GL consider as VF confirmed VL >400, or >200, >50 or >48 copies/ml after suppression, respectively (2-5). Low HIV-1 viremia levels (LVL) ( copies/ml) represent clinically relevant values in virologic monitoring of HIV-infected patients under ART, because they may dictate the need of antiretroviral regimen changes, due to early appreciation of therapy failure (1-5). It is accepted that an increase of HIV-1 RNA VL from suppressed condition (below 50 copies/ml) to levels between 50 and 1000 copies/ml may predict virological failure, with a gradation of rebound risk that directly correlates with amplitude of viremia. In particular, in patients under suppressive ART single measurements of HIV-1 RNA between 500 and 1000 copies/ml seems to be associated with higher rebound risk, with respect to lower values (i.e. below 500 copies/ml). To rule out blip or laboratory artifacts, VL increases below 500 copies/ml would benefit from confirmation by repeated testing (6-7). 3

4 Furthermore, even the detection of HIV-RNA values below 50 copies/ml (residual viremia, RV) is receiving increased attention, because it may announce beforehand a viral rebound risk, in a manner that is directly proportional to the extent of RV (8-12) In this evolving context, it is important to consider performance features of diagnostic assays used for VL measurements and monitoring. In fact, although most studies have displayed that the currently available real-time PCR tests are fairly similar, showing high sensitivity, specificity, reproducibility and good correlation in the relatively high range of quantification (13-15), poor inter-assay concordance has been well described exactly at LVL (16-21). This last issue is an important matter of debate in relation to the need of establish the univocal and shared definition of VF and other virologic definitions (such as virologic suppression, virologic rebound and persistent LVL ), because if molecular tests are not in perfect agreement, it becomes difficult to define univocal virologic thresholds. Therefore, the performance characteristics of diagnostic systems used to measure the VL need to be taken into account in deciding when to intervene in the management of HIV-1 infection. Given the great interest about VF definition and LVL in ART-treated patients for the implications it may have in the clinical setting and decisions, we analyzed performance characteristics of two widely used assays for HIV-1 RNA measurement, in order to clarify the differences between them at LVL. We compared Abbott Real-Time HIV-1 (referred to as Abbott assay in this paper) and Roche COBAS Ampliprep/COBAS TaqMan HIV-1 v2.0 (referred to as Roche assay in this paper) assays on the ability to detect and quantify clinical specimens with LVL (from not-detected to 1000 copies/ml, as established with Abbott assay). In addition, the HIV-1 RNA Working Reagent 1 for NAT assays (HIV-WR1), diluted from 1053 to 1 copies/ml, was evaluated with both assays. For VL concentrations below 40 copies/ml, the comparison was conducted in two ways: 1) considering HIV-1 RNA values from 20 to 40 copies/ml with Roche as 4

5 detected <40copies/ml, in order to align them with the Abbott assay results; 2) using the modified protocol of Abbott assay (limit of detection, LOD: 19 copies/ml) (22), in order to compare data <40 copies/ml with the Roche assay Materials and Methods Clinical samples: Clinical samples: 167 clinical plasma samples were selected among those (n=11583) submitted to the Laboratory of Virology at the National Institute for Infectious Diseases L. Spallanzani, Rome, for routine monitoring of HIV-1 viremia, during the period between April and October Routine measurements with Abbott assay was performed on fresh plasma samples, after one night storage at 4 C, according to manufacturer s instructions. Among all samples tested with Abbott assay, those having HIV-1 RNA levels ranging from not detected to 1000 copies/ml and sufficient residual volume were selected and directly tested with Roche assay, without freezing, in the same working day. More in detail, to avoid VL decay between the two measurements, all plasma samples subjected to routine monitoring of HIV-1 viremia were removed from the Abbott m2000sp automatic extractor immediately after sampling (about 1 hour from the start of the extraction protocol) and were maintained at 4 C until the end of amplification procedure and reading of Abbott assay results; overall, the samples remained at 4 C for about 6 hours before starting of Roche assay. The elaboration of the results for the study purpose was performed after complete anonymization of the samples HIV-1 RNA assays: Viral load assays were used according to manufacturer instructions. The Abbott assay (Abbott Real-time HIV-1, Abbott Molecular Inc., Des Plaines, IL, USA) was 5

6 performed on the m2000 TM sp/rt instruments. The stated linear quantification range of the standard version is 40-10,000,000 copies/ml (0.6ml protocol) and the reported LOD is 40 copies/ml. For samples with HIV-1 RNA values below 40 copies/ml, a modified protocol of Abbott assay, showing a LOD of 19 copies/ml, was used (22). The modified protocol of Abbott assay was obtained by introducing some changes to the standard protocol, namely: using a new calibration curve to cover low HIV-1 RNA concentrations ( copies/ml); reducing the volume of internal control; adopting "open-mode" software for quantification. Performance characteristics of the modified version were previously described, showing high accuracy and precision (22). The Roche assay (Roche COBAS Ampliprep/COBAS TaqMan HIV-1 v2.0, Roche Molecular Systems Inc., Pleasanton, CA, USA) was performed on the docked configuration of the Cobas/TaqMan 96 instrument. The reported quantification range of the current 2.0 version is 20-10,000,000 copies/ml and the declared LOD is 20 copies/ml. HIV-1 subtype: HIV-1 subtype assignment was retrieved from the HIV-pol sequences, in the context of drug resistance mutation pattern assessment, and was available for 80 of the 167 (47.9%) patients: 73 were B subtype; 2 A1; 1 AG,B; 2 C, 1 CRF02/AG; 1 F1. For subtype establishment, HIV-1 pol sequences were aligned in Bio-Edit and compared to reference sequences for major HIV-1 subtypes and circular recombinant forms (CRFs), available at Los Alamos database ( Subtype classification was confirmed also by the REGA ( and the COMET subtyping tools ( Quality control material: Multiple replicates of a dilution panel were prepared from the HIV-WR1 (NIBSC code: 99/ , This is a working preparation whose use is intended for monitoring the performance of diagnostic assays over a period of time. It 6

7 consists of HIV-1 subtype B virus, diluted in pooled human plasma, with an assigned value of 3.56 log 10 IU/ml, calculated against the 1 st International Standard for HIV-1 RNA. For performance analyses here described, HIV-WR1 was diluted with HIV-1 negative human plasma (Basematrix; Boston Biomedica Inc., W. Bridgwater, MA) to obtain the concentration indicated in copies/ml, after considering the common conversion factor of 1 IU as 0.6 copies, according to Abbott and Roche manufacturers declarations. For the comparison between the two assays, five twofold dilutions (from 1053 down to 66 copies/ml) of HIV-WR1 were prepared and three replicates for each dilution were tested. For the comparison at VL values lower than 40 copies/ml, eight twofold dilutions (from 128 down to 1 copies/ml) of HIV-WR1 were prepared and 7 replicates for each dilution were analyzed. Data analysis: All VL data were analysed as log 10 -transformed values. In the correlation analysis, the VL data were lined up to 1.59 log 10 copies/ml (39 copies/ml) if detected, below 40 copies/ml, or to 0 log 10 if not-detected. Concordance on qualitative results was measured by Stuart-Maxwell test and Cohen s kappa statistic. The correlation between quantitative results was computed as concordance correlation coefficient (ccc) of measurements according to Lin (23). The agreement between assays was assessed with Bland-Altman s procedure (24). A probit model was used to describe the response probability of the assays as function of concentration of HIV-1 RNA (25). Applying the inverse of the estimated function, we computed the value of the independent variable, with the relative 95% confidence interval, corresponding to a desired response probability (26). In this way was also computed the analytical sensitivity (i.e. the minimal concentration of HIV-1 RNA detected with response probability of 95%, LOD) of both assays (including the modified Abbott protocol) using the HIV-WR1 dilutions

8 167 Results Assay comparison on clinical samples. A comparative evaluation between Abbott and Roche assays on the ability to quantify LVL of HIV-1 RNA was performed using 167 residual clinical samples, with HIV-1 RNA levels ranging from not-detected to 1000 copies/ml with Abbott assay. The degree of inter-assay concordance of qualitative results at 40 copies/ml HIV-1 RNA threshold was substantial (Cohen s k statistic=0.762). The proportion of samples with HIV-1 RNA resulted not-detected was similar with the two assays (34.7% with Abbott vs 36.5% with Roche); the percentage of samples detected above and below 40 copies/ml was similar as well (65.3% with Abbott vs 63.5% with Roche). However, among samples with detected HIV-1 RNA, Roche assay provided a significantly higher percentage of samples quantified 40 copies/ml, as compared to Abbott assay (42.5% vs 35.9%, p=0.0116, Stuart-Maxwell test). Discordant samples between detected above 40 copies/ml and detected below 40 copies/ml with the two assays were 19 (11.4%) and resulted as follows: all the four samples quantified by Abbott resulted detected <40copies/ml with Roche; among the 15 samples quantified by Roche, 10 resulted detected <40 copies/ml (6.0%) and five not-detected (3.0%) with Abbott. HIV-1 subtype was known for 11 out of 19 (57.8%) discordant samples, and all of them were subtype B (Table 1). Considering the 56 samples with VL precisely quantified ( 40 copies/ml) with both assays, the distribution of values is shown in Figure 1A. Mean values obtained with Roche ( log 10 copies/ml) were log 10 copies/ml (95% limits of agreement: , 0.816) higher than mean values obtained with Abbott ( log 10 copies/ml), with a Lin s ccc inter-assay concordance of 0.728, p< (Figure 1B). A difference exceeding >0.3 log 10 copies/ml was observed in 21 (12.6%) samples (19 with Roche and 2 with Abbott higher results); a difference exceeding >0.5 log 10 copies/ml was observed in 9 (5.4%) samples (8 with Roche and 8

9 with Abbott higher results) (Figure 1C). Overall, in more than half of samples (30, 18.0%), higher levels of quantification were observed with Roche (Figure 1C). To see if HIV-1 non-b subtype could affect the comparison between assays, the analysis was restricted to 73 samples with known HIV-1 B subtype. Similar and significant differences in HIV-1 RNA measurements between Abbott and Roche were observed. In fact, the concordance of qualitative results at 40 copies/ml HIV-1 RNA threshold concerned 84.92% of samples (Cohen s k statistic=0.684), with 53.42% samples below and 31.50% above the cut-off with both assays. In addition, considering the 34 subtype B specimens with HIV-1 RNA detected (from detected <40 copies/ml to quantified) with both assays, the mean VL value obtained with Roche ( log 10 copies/ml) was log 10 copies/ml (95% limits of agreement: , 0.471) higher than that obtained with Abbott ( log 10 copies/ml) with a Lin s ccc inter-assay concordance of 0.590, p< In six (8.2%) samples, a difference exceeding >0.3 log 10 copies/ml was observed, with Roche results higher; a difference exceeding >0.5 log 10 copies/ml was also observed in other six (8.2%) samples (five with Roche results higher and one with Abbott results higher). Concordance between Abbott and Roche assays was also examined for HIV-1 RNA cut-offs higher than 40 copies/ml. The interassay concordance for HIV-1 RNA levels at 50 copies/ml was 89.81% (with 58.68% of samples below and 31.13% above threshold with both assays). Increasing the HIV-1 RNA cut-off to 200 and 400 copies/ml, the percentages of concordant samples with both assays tended to increase up to 92.20% and 95.20%, respectively. Assay comparison with the NAT assays reference standard HIV-WR1. To compare the two assays in the ability to quantify LVL, serial dilutions of the HIV-WR1, covering the range of 66 to 1053 copies/ml, have been analyzed. The results show an overestimation of the HIV-WR1 by 9

10 Roche at all concentrations, while Abbott results are in line with the expected values (Table 2). Despite high Pearson correlation coefficient between assays (r=0.961, p<0.0001), concordance between two assays was very low (Lin s ccc=0.498; p<0.0001), due to a considerable distance of HIV-WR1 measured values from those on the line of the perfect concordance, as shown in Figure 2A. Bland-Altman plot among HIV-WR1 expected and obtained values, shows that data produced by Roche assay were systematically higher (0.522 log 10 copies/ml; 95% limits of agreement: to 0.697) than those expected (Figure 2B), while Abbott assay gave results almost perfectly aligned ( log 10 copies/ml; 95% limits of agreement: to 0.128) with the expected values (Figure 2C). Next, we focused our attention to a lower range of HIV-1 RNA concentration. In this case, the comparison was performed using the modified protocol of the Abbott assay (reported LOD: 19 copies/ml) (22), in order to use assays with presumed comparable sensitivity. We first reassessed the actual analytical sensitivity of the two assays (i.e. the minimal concentration of HIV-1 RNA detected with response probability of 95%, LOD), using 7 replicates of each two-fold dilution (from 128 to 1 copies/ml) of the HIV-WR1. By probit analysis, the minimal concentration of HIV-1 RNA detected with probability response of 95% resulted 3.5 copies/ml with the Roche assay, while it was confirmed to be 19 copies/ml with the modified Abbott assay (22). Considering the range of copies/ml, the correlation between the two assays (Roche and modified Abbott) was even lower (Lin s ccc = 0.404, p<0.0001), despite high Pearson correlation coefficient (r=0.816, p<0.0001) (Figure 3A). Bland-Altman plot of expected vs obtained values (Figure 3B and 3C) confirmed again the VL overestimation by the Roche assay (0.501 log 10 copies/ml; 95% limits of agreement: to 0.897), compared to modified Abbott ( log 10 copies/ml; 95% limits of agreement: to 0.296). 10

11 Comparison at higher HIV-1 RNA thresholds. In an attempt to better clarify differences between the VL values given by the two assays at the clinically important VL level of 50 copies/ml, we directly compared the results obtained with the two assays using a subgroup of clinical samples and of HIV-WR1 dilutions whose Abbott VL concentration was around 50 copies/ml (clinical samples: range copies/ml, n=9; HIV-WR1: range copies/ml, n=6), and compared the mean values obtained with both assays. The mean HIV RNA values with Abbott were 54 and 50 copies/ml for clinical samples and HIV-WR-1 dilutions, as compared to 134 and 168 copies/ml with Roche assay, with a difference of 2.48 and 3.36 fold, respectively (p=0.022 and <0.001). When restricting the clinical sample comparison to subtype B (n=5), a similar difference was observed (mean Abbott vs Roche: 53 vs 114 copies/ml, p=0.037). Downloaded from on May 4, 2018 by guest 11

12 252 Discussion In this study, we compared the characteristics of two diagnostic assays widely diffused for the quantification of HIV-1 RNA, in a range of values important for therapeutic decisions, i.e. below 1000 copies/ml. The study was based on both clinical samples and on serial dilutions of the HIV- WR1. The results indicate tha,t in the range of copies/ml HIV-1 RNA, the correlation between the two assays is lower than that described at higher concentrations (Lin s ccc=0.728, p<0.0001), and poor concordance at the clinically relevant value of 50 copies/ml is confirmed, both with clinical samples and the HIV-WR1. As both assays have been optimized for HIV-1 B subtype, the HIV-WR1 represents a single subtype B sample and half of the patients were infected by HIV-1 B subtype, including most of those with discordant results, the HIV-1 B subtype does not seem to be crucial (or the sole factor) in influencing the different performances of these molecular systems (in fact, 11 of 19 patients with discordant results resulted infected with HIV-1 B subtype and 7 patients with HIV-1 non-b subtypes were similarly measured with both assays). In addition, using the HIV-WR1 preparation, we observed that, within the range of HIV-1 RNA concentrations under analysis, Roche assay produced HIV-1 RNA values about 3-fold higher than those obtained with the Abbott assay. On the other hand, Abbott assay provided HIV-WR1 RNA results that were perfectly aligned with the expected ones, with both the standard protocol (in the range down to 40 copies/ml) and the modified protocol (down to 19 copies/ml) (22). Therefore, it is reasonable that the higher values obtained with Roche assay may be due to overestimation. On the other hand, according to the probit analysis, Roche assay showed higher analytical sensitivity, being able to detect the presence of HIV-1 RNA at values well lower than the claimed LOD (20 copies/ml), precisely until 3.5 copies/ml, in agreement with recent data (27). Overall, our results on performance characteristics of these two assays at LVL are aligned with similar data previously reported with clinical samples (20, 28-30) and with 2 nd International HIV-1 12

13 RNA WHO Standard (31). In particular, although there is a good agreement between the commonly used diagnostic assays for detecting both HIV-1 B and non-b subtypes along the common range of detection (18, 32-36), the inter-assay correlation is lower at the lower limit of quantification (16-18, 29-30, 37-38). Also the concordance between samples with detected or "not-detected" VL is rather low (32-34), with both clinical samples (11), 2 nd International HIV-1 RNA WHO Standard and commercial test panels for HIV-1 (16, 19-20). In line with a recent multicenter comparison study (30), we also observed that inter-assay concordance for a threshold at 200 copies/ml was much higher (92.20%) than when setting the threshold at the clinically relevant value of 50 copies/ml (89.81%). In the light of the reported differences in HIV-1 RNA quantification and of the different sensitivity among the existing commercial HIV-1 VL assays, most GL recommend the use of a single method for therapeutic monitoring of individual patients (1-5). Furthermore, several current GL advice not to change ART in HIV-1 infected patients when VL remains below copies/ml (1-5), taking into account the possibility that it may represent a blip (6-7) and considering also the suboptimal performances and the poor inter-assay concordance at LVL. On the other hand, under ART, careful evaluation of persistent HIV-1 RNA in the range of copies/ml is recommended, since viral evolution and drug-resistance mutations may accompany low level viral replication, subsequently leading to virologic failure (39-42). However, also performance characteristics of each individual diagnostic assay should be carefully taken into account when VL values are considered and clinical intervention is speculated. For example, when incomplete virologic response is suspected (such as in the presence of repeated low levels of HIV-1 RNA), it is important to consider that Roche assay produces HIV-1 RNA quantification levels log 10 higher than other real-time-based assays (20, 43-45). For instance, by extrapolating the data from our comparison of clinical samples and HIV-WR1 with HIV-RNA values close to 50 copies/ml, we may foresee that a 13

14 viremia value measured with Roche assay that would be considered VF might correspond to a suppressed viremia value with the Abbott assay (i.e. 120 vs 40 copies/ml). It is evident that, in such a case, the clinical decision would be different depending on the assay, and it may benefit from repeated measurement. LVL represents a pivotal focus in the clinical management, as it would led to consequences such as different interpretations of therapy efficacy, possibly leading to relevant changes in therapeutic schemes, and the lack of correlation among assays may represent a challenge to the interpretation of the results. However, the clinical implication of the present findings are not fully appreciated, and clinical follow-up would be necessary to establish whether the differences between the assays are significant enough to make a large difference in treatment decisions. Large clinical study would be necessary to establish whether GL should take into consideration performance characteristics of each assay when making statements about HIV- 1 RNA cut-offs for clinical decisions. 14

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18 TaqMan assays for quantification of human immunodeficiency virus type 1 group M non-b subtypes. J. Clin. Microbiol. 47(5): Naeth G, Ehret R, Wiesmann F, Braun P, Knechten H, Berger A Comparison of HIV-1 viral load assay performance in immunological stable patients with low or undetectable viremia. Med Microbiol Immunol; 202: 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, Lepej SZ, Lunar MM, Mukabayire O, Schuurman R, Tomazic J, Van Laethem K, Vandekerckhove L, Wensing AM Comparative performance of HIV-1 RNA load assays at low viral load levels: Results of an international collaboration. J Clin Microbiol. Published online ahead of print doi: /jcm Glaubitz J, Sizmann D, Simon CO, Hoffmann KS, Drogan D, Hesse M, Lang G, Kroeh M, Simmler P, Dewald M, Haberhausen G, Lindauer A, Beyser K, Reber A, Baumeister A, Wolf E, Jaeger H, Babiel R Accuracy to 2nd International HIV-1 RNA WHO Standard: assessment of three generations of quantitative HIV-1 RNA nucleic acid amplification tests. J. Clin. Virol. 50: Taylor N, Schmid I, Egle A, Greil R, Patsch W, Oberkofler H Initial evaluation of the Roche COBAS TaqMan HIV-1 v2.0 assay for determining viral load in HIV-infected individuals. Antivir Ther. 14(8): Sloma CR, Germer JJ, Gerads TM, Mandrekar JN, Mitchell PS, Yao JD Comparison of the Abbott realtime human immunodeficiency virus type 1 (HIV-1) assay to the Cobas AmpliPrep/Cobas TaqMan HIV-1 test: workflow, reliability, and direct costs. J. Clin. Microbiol. 47(4): Sire JM, Vray M, Merzouk M, Plantier JC, Pavie J, Maylin S, Timsit J, Lascoux-Combe C, Molina JM, Simon F, Delaugerre C Comparative RNA quantification of HIV-1 group M and non-m with the Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 v2.0 and Abbott Real-Time HIV-1 PCR assays. J. Acquir. Immune Defic. Syndr.; 56: Wall GR, Perinpanathan D, Clark DA Comparison of the QIAGEN artus HIV-1 QS-RGQ test with the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 test v2.0. J Clin Virol. 55(1): Garcia-Diaz A, Labbett W, Clewley GS, Guerrero-Ramos A, Geretti AM Comparative evaluation of the Artus HIV-1 QS-RGQ assay and the Abbott RealTime HIV-1 assay for the quantification of HIV-1 RNA in plasma. J. Clin. Virol. 57(1):

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20 480 Acknowledgments 481 This work was partially supported by grants from Italian Ministry of Health (Ricerca Corrente) Funding: Grants from Italian Ministry of Health (Ricerca Corrente) Competing interests: None. Ethical approval: Not required. Downloaded from on May 4, 2018 by guest 20

21 489 Figures and Tables Figure 1: Log 10 -transformed VL results of 56 clinical samples given as >1.60 log 10 copies/ml (>40 copies/ml) both with Abbott and Roche assays. (A) Dashed line indicates the 1.60 log 10 copies/ml (40 copies/ml) cut-off value; continuous line shows mean VL. (B) Concordance graph of the observed data, with the linear regression line (continuous) and the line of perfect concordance (dashed). (C) Bland-Altman plot of VL values obtained with the 2 assays, with the linear regression line (continuous) and the line of perfect agreement (dashed). Table 1: HIV-1 subtype of 11 out of 19 clinical samples with discordant VL results according to Abbott and Roche assays. Table 2: Results obtained with Abbott and Roche assays on HIV-WR1 (diluted from 66 to 1053 copies/ml). Figure 2: Log 10 -transformed HIV-RNA results of HIV-WR1, diluted to obtain 1053 to 66 copies/ml and measured with Abbott and Roche assays (3 replicates/dilution). (A) Concordance graph of the observed data, with the linear regression line (continuous) and the line of perfect concordance (dashed). (B) Bland-Altman plot of obtained and expected HIV-RNA values of HIV-WR1 produced by Roche, with the linear regression line (continuous) and the line of perfect agreement (dashed). (C) Bland-Altman plot of obtained and expected HIV-RNA values 21

22 of HIV-WR1 produced by Abbott, with the linear regression line (continuous) and the line of perfect agreement (dashed) Figure 3: Log 10 -transformed VL results of HIV-WR1, diluted to obtain 128 to 16 copies/ml and measured with modified Abbott and Roche assays (7 replicates/dilution). (A) Concordance graph of HIV-RNA results, with the linear regression line (continuous) and the line of perfect concordance (dashed). (B) Bland-Altman plot between obtained and expected HIV RNA values obtained with Roche, with the linear regression line (continuous) and the line of perfect agreement (dashed). (C) Bland-Altman plot of HIV RNA values obtained with modified Abbott, with the linear regression line (continuous) and the line of perfect agreement (dashed)

23 532 Figure 1 A HIV RNA (log 10 copies/ml) 533 Abbott (log 10 copies/ml) B Abbott Roche Downloaded from difference Roche minus Abbott C Roche (log 10 copies/ml) on May 4, 2018 by guest 535 mean (log 10 copies/ml) 23

24 536 Table Patient ID HIV Subtype Abbott Roche (HIV-1 RNA copies/ml) ID 1 B <40 Copies/ml 198 ID 20 B <40 Copies/ml 40 ID 22 B ID 31 B ID35 B <40 Copies/ml 57 ID 38 B <40 Copies/ml 68 ID 43 B <40 Copies/ml 90 ID 44 B <40 Copies/ml 240 ID 48 B <40 Copies/ml 116 ID 69 B 127 <20 Copies/ml ID 71 B

25 553 Table HIV-WR1 Abbott Roche Nominal values (log10cp/ml) (cp/ml) Number of values Median (log 10 cp/ml) (Minimum- Maximum) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) Mean (log 10 cp/ml) Std deviation Std Error Lower 95% CI of mean , Upper 95% CI of mean Coefficient of variation 9.06% 3.06% 0.86% 1.92% 3.45% 1.67% 1.32% 0.54% 1.80% 1.16% Mean difference vs nominal values

26 566 Figure 2 A Abbott (log 10 copies/ml) 567 difference (Roche minus expected values) B Roche (log 10 copies/ml) copies/ml Downloaded from difference (Abbott minus expected values) C mean (log 10 copies/ml) on May 4, 2018 by guest 569 mean (log 10 copies/ml) 26

27 570 Figure 3 A 571 modified Abbott (log 10 copies/ml) difference (Roche minus expected values) B Roche (log 10 copies/ml) copies/ml Downloaded from difference ( modified Abbott minus expected values) C mean (log 10 copies/ml) on May 4, 2018 by guest 573 mean (log 10 copies/ml) 27