Assessment of early virological response to antiviral therapy. by comparing four assays for HCV RNA quantitation using

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1 Journal of Medical Virology 2006;78: Halfon Page Assessment of early virological response to antiviral therapy by comparing four assays for HCV RNA quantitation using the international unit standard: implications for clinical management of patients with chronic hepatitis C virus infection Dr Philippe Halfon 1*, Guillaume Pénaranda 2, Dr Marc Bourlière 3, Dr Hacène Khiri 1, Dr Marie-France Masseyeff 4, Dr Denis Ouzan 4 1 Alphabio Laboratory, Marseille, France 2 Department of Biostatistics and Epidemiology, CDL Pharma, Marseille, France 3 Saint Joseph Hospital, Marseille, France 4 Arnault Tzanck Institute, Saint Laurent du Var, France Abbreviations: HCV: Hepatitis C Virus. HIV: Human Immunodeficiency Virus. Hepatitis B virus (HBV). SVR: Sustained Virological response. * 23 rue friedland, Marseille, France. philippe.halfon@alphabio.fr Telephone: (33) Fax: (33) Keywords: HCV treatment, HCV RNA quantitation, International Unit Standard, Real Time Polymerase Chain Reaction

2 Journal of Medical Virology 2006;78: Halfon Page Abstract. WHO International Standards for nucleic acid tests are widely used to compare the different assays used in HCV RNA quantitation.the aim of the study was to assess the impact of the international unit standard for measuring HCV RNA in the management of patients with chronic hepatitis C virus (HCV) infection. Twenty-seven naïve patients infected chronically by HCV were treated with ribavirin plus PEG-interferon-alfa-2b for 48 weeks. SVR was obtained for 16 patients (the other were non-responders). For HCV RNA quantitation, four assays were performed: Versant HCV RNA 3.0 (Bayer), Real time PCR (TaqMan, Roche), LCx HCV RNA (Abbott), and Cobas Amplicor-Monitor v2 (Roche). Considering a 2-log decline at week 12 after the beginning of therapy, discordant results were found with the four HCV RNA methods in predicting SVR or non-response. At weeks 4 and week 12, significant differences were observed between Versant HCV RNA 3.0 vs PCR HCV Taqman, Versant HCV RNA 3.0 vs LCx HCV RNA, Cobas Monitor Amplicor HCV 2.0 vs LCx HCV RNA, and Cobas Monitor Amplicor HCV 2.0 vs PCR HCV Taqman (p<0.001). The HCV RNA cutoff, given a 100% negative predictive value at week 4 and week 12, differed with the assays used to quantify HCV RNA, despite the use of the IU/ml units. Eighty-nine percent of serum values for HCV RNA were concordant by the IU standard. All assays, however, failed to detect HCV RNA in some cases. Despite the use of IU, HCV-infected patients might be monitored with only one assay.

3 Journal of Medical Virology 2006;78: Halfon Page Introduction. Several studies have investigated the value of monitoring serum HCV RNA during treatment as a predictor of response or non-response to antiviral therapy [Berg et al., 2003; Halfon et al., 1998; Herrmann et al., 2003; Jessner et al., 2001; Jessner et al., 2002; Lam et al., 1997; Layden et al., 2003; Neumann et al., 2000; Nguyen et al., 1996; Zeuzem et al., 2001]. European and American consensus conferences recommended the use of the HCV RNA decline to manage the patients during treatment [1999; 2002]. It was suggested that treatment can be discontinued if HCV RNA level does not decrease by 2 log at week 12 [Fried et al., 2002; Hadziyannis and H, 2002]. The data for these recommendations were generated in pivotal trials using centralized laboratories. General application may be difficult due to the lack of standardization of previous HCV RNA quantitation methods [Pawlotsky, 2002]. The most common methods available for detecting and quantifying HCV RNA are based on polymerase chain reaction (PCR) and on the signal amplification technique (branched DNA). A semi-automated PCR assay, the complete bioanalytical system (COBAS) Amplicor Monitor, is commonly used by many laboratories. Recently two other assays were developed, the LCx assay and real time PCR (Taqman); both have a high sensitivity (ie the lower limit for detecting HCV RNA) and a great dynamic range. The principle of realtime PCR techniques is to detect amplicon synthesis and to deduce the amount of viral genomes in the starting clinical sample during the PCR reaction rather than at the end [Pawlotsky, 2002]. Real-time PCR will probably become the standard for HCV RNA detection and quantitation in the future. The main advantage of these tests is that each has been standardized, which makes results by different laboratories comparable. Many other in-house PCR assays have been developed by reference and hospital laboratories, but they lack such standardization. To make the quantitation of HCV RNA comparable among the different assays, the National Institute for Biological Standards and Controls and the World Health Organization developed and certified a uniform standard for measuring HCV RNA in International Units [Pawlotsky et al., 2000]. Despite the introduction of International Units, discrepancies may occur when patients are monitored using different types of

4 Journal of Medical Virology 2006;78: Halfon Page assays [Shiffman et al., 2003]. In that study, authors observed false positive and false negative results as well as variations of HCV RNA level of more than 1 to 2 log international units. These results may have an impact on the management of patients receiving interferon therapy, for example, when base treatment decisions are made using a single HCV RNA determination. The first aim of this study was to assess the impact of using the international unit standard for measuring HCV RNA in the management of patients with chronic HCV infection by determining HCV RNA viral kinetics during the first twelve weeks of treatment using four commercial HCV RNA quantitation assays. The second objective was to investigate the prognostic relevance of HCV RNA viral load at week 2, week 4, and week 12 for virological response to antiviral therapy. 83

5 Journal of Medical Virology 2006;78: Halfon Page Materials and Methods. Patients A total of 27 consecutive patients were enrolled just before starting interferon and ribavirin therapy for chronic HCV infection at the hepatology and gastroenterology department of the Arnault Tzanck Institute (St Laurent du Var, France). The mean age of the population was 44 years. Of the patients, 20 were male and 20 were genotype 1. All tested positive for HCV RNA and negative for HIV. Other causes of chronic hepatitis were excluded by appropriate serological testing and liver histology. HCV genotype was determined by sequence analysis using the HCV genotyping Trugene assay (Bayer, Puteaux, France) [Halfon et al., 2001]. All patients were treated with ribavirin 400 mg twice a day, orally, plus PEG-interferonalfa-2b (1.5 µg/kg subcutaneous injection once a week) for 48 weeks. Written informed consent was obtained from each patient, and the study was approved by the local Ethics committee in accordance with the 1975 declaration of Helsinki. Virological response was assessed by a qualitative HCV RNA assay with a lower sensitivity of 50 IU/ml (HCV Amplicor 2.0 Roche Diagnostics, Meylan, France). According to the qualitative HCV RNA results, patients were defined as virologic sustained responders (HCV RNA negative 6 months after the end of therapy) and the remaining patients were classified as non-responders. Serum for HCV RNA analysis A total of 486 serum samples were obtained from the 27 patients. Each of the specimens was divided into four aliquots and frozen to 80 C within 2 hours of collection [Halfon et al., 1996]. Each aliquot was used for HCV RNA quantitation by the following four assays: Versant HCV RNA 3.0 (Bayer, Puteaux, France) with a detection limit of 615 IU/ml[Trimoulet et al., 2002]; Real time PCR HCV Taqman (Roche Diagnostics, Meylan, France) with a detection limit of 10 IU/ml[Germer et al., 2005]; LCx HCV RNA quantitative assay (Abbott Laboratories, Rungis, France) with a detection limit of 23 IU/ml [Leckie et al., 2004]; and the Cobas Monitor Amplicor HCV 2.0 (Roche

6 Journal of Medical Virology 2006;78: Halfon Page Diagnostics, Meylan, France) with a detection limit of 603 IU/ml [Lee et al., 2000]. HCV RNA was measured at five time points: baseline, week 2, week 4day 30, week 12day 90 after the beginning of therapy, and 6 months after discontinuation of therapy. Values of HCV RNA are reported in IU/ml. All the assays were done in Alphabio laboratory. Statistical analysis Values are expressed as median [range] or as mean [95% confidence interval]. Correlations between assays were evaluated by linear regression. Data comparison was assessed with Wilcoxon-test for quantitative variables. A p value of <0.05 was considered significant. 124

7 Journal of Medical Virology 2006;78: Halfon Page Results. Virological response Among the 27 patients, 20 were HCV genotype 1 and 7 were genotype 2-3, 16 had a complete sustained virological response (SVR), and 11 were non-responders. Figure 1 shows the median values for serum HCV RNA at baseline, week 2, week 4, and week 12 for each of the four assays. Significant differences were observed in median values at baseline between Versant HCV RNA 3.0 vs LCx HCV RNA, Versant HCV RNA 3.0 vs Cobas Monitor Amplicor HCV 2.0, LCx HCV RNA PCR vs HCV Taqman, LCx HCV RNA vs Cobas Monitor Amplicor HCV 2.0, and PCR HCV Taqman vs Cobas Monitor Amplicor HCV 2.0 (Wilcoxon p<0.05). The median Versant HCV RNA 3.0 assay results at week 4 and week 12 were 2.93logIU/ml [1.54;4.78] and 2.82logIU/ml [1.54;5.80] respectively. The median PCR HCV Taqman results were 2.64logIU/ml [1.00;6.35] and 1.00logIU/ml [1.00;5.05]. The median LCx HCV RNA results were 1.65logIU/ml [1.35;5.66] and 1.35logIU/ml [1.35;4.16], and the median Cobas Monitor Amplicor HCV 2.0 results were 2.78logIU/ml [2.47;5.23] and 2.78logIU/ml [2.78;4.24]. At week 4 and week 12, significant differences were observed between Versant HCV RNA 3.0 vs PCR HCV Taqman, Versant HCV RNA 3.0 vs LCx HCV RNA, and Cobas Monitor Amplicor HCV 2.0 vs PCR HCV Taqman (Wilcoxon p<0.05). Relations between assays The relations between assays are shown in Figure 2. A method comparison demonstrated a correlation coefficient of 0.81 between Versant HCV RNA 3.0 and PCR HCV Taqman (Figure 2A), 0.87 between the LCx HCV RNA assay and PCR HCV Taqman (Figure 2B), 0.81 between the LCx HCV RNA assay and Versant HCV RNA 3.0 (Figure 2C), 0.83 between Cobas Monitor Amplicor HCV 2.0 and Versant HCV RNA 3.0 (Figure 2D), 0.73 between Cobas Monitor Amplicor HCV 2.0 and PCR HCV Taqman, and 0.80 between Cobas Monitor Amplicor HCV 2.0 and the LCx HCV RNA assay. The discordance between assays for detection of HCV RNA is summarized in

8 Journal of Medical Virology 2006;78: Halfon Page Table 1: 0.4% to 6% of the 486 samples tested were discordant for detection of HCV RNA. The mean Versant HCV RNA 3.0 assay result was 0.74log IU/ml [0.59;0.89] higher than the mean PCR HCV Taqman result, 1.04log IU/ml [1.02;1.06] higher than the mean LCx HCV RNA result, and 0.05log IU/ml [-0.01;0.11] higher than mean Cobas Monitor Amplicor HCV 2.0 assay. The mean PCR HCV Taqman assay result was 0.30log IU/ml [0.17;0.43] higher than the mean LCx HCV RNA assay, but 0.69log IU/ml [0.48;0.90] lower than the Cobas Monitor Amplicor HCV 2.0 assay. The mean Cobas Monitor Amplicor HCV 2.0 assay was 0.99log IU/ml [0.91:1.07] higher than mean LCx HCV RNA assay. Effect of variable HCV RNA results on the management of Chronic HCV Of the 486 samples analyzed, 61% (296/486) of values were within 1 log unit, 1% (6/486) of values were more than 2 log units, and 38% (184/486) of values were between 1 and 2 log units of difference between assays (Table 2). This variability did not depend on HCV genotype. Of the serum values for HCV RNA, 89% were concordant (<2Log >2Log) by the International Unit standard regardless of which virological assay was used. In some cases, virus was undetectable by one of the assays but it was detectable by another assay (Table 3). Considering a 2-log drop of difference in viral load, 6 patients presented discordant value according to the antiviral response at week 12 (Figure 3). Overall positive predictive value and negative predictive value for Versant HCV RNA 3.0, PCR HCV Taqman, LCx HCV RNA, and Cobas Monitor Amplicor HCV 2.0 were respectively: 100% and 64%, 100% and 62%, 50% and 60%, and 43% and 60%. For Versant HCV RNA 3.0, a negative predictive value (NPV) of 100% was reached by using cutoff levels of 4.72, 3.28, and 3.28 for HCV RNA viral load at week 2, week 4, and week 12 respectively (Table 4). By applying these cutoff levels at week 2, 4, and 12, treatment could be discontinued early in 4 (36%), 5 (46%), and 6 (55%) virological non-responders respectively. For PCR HCV Taqman, an optimal NPV of 100% was reached by using cutoff levels of 4.79, 3.02, and 1.01 for HCV RNA viral load at week 2, week 4, and week 12 respectively. By applying these cutoff levels at week 2, 4, and 12,

9 Journal of Medical Virology 2006;78: Halfon Page treatment could be discontinued early in 4 (36%), 1 (9%), and 6 (55%) virological nonresponders respectively. For LCx HCV RNA assay, cutoff levels of 4.14, 2.41, and 1.35 for HCV RNA viral load at week 2, week 4, and week 12 were found. By applying these cutoff levels at week 2, 4, and 12, treatment could be discontinued early in 6 (55%), 1 (9%), and 7 (64%) virological non-responders respectively. For Cobas Monitor Amplicor HCV 2.0 assay, cutoff levels of 4.39, 2.78 and 2.78 for HCV RNA viral load at week 2, week 4, and week 12 were found. By applying these cutoff levels at week 2, 4, and 12, treatment could be discontinued early in 3 (27%), 3 (27%), and 7 (64%) virological nonresponders respectively.

10 Journal of Medical Virology 2006;78: Halfon Page Discussion. This study shows that HCV RNA results vary according to the method used and reports on the consequences for management of chronic HCV during antiviral therapy. The study evaluated for the first time four widely available assays frequently used to measure HCV RNA in clinical practice. Despite the use of international units (IU) recently established to standardize the HCV RNA viral load measured with the different assays [Saldanha, 1999], only 58% of the 486 samples tested had values were within 1 log unit; 2% of values were over 2 log units and 40% of values were between 1 and 2 log units of difference between assays. This variability was not dependent on HCV genotype. The main limitation of this study is the low number of patients studied. However, 486 samples were tested for analysis using the four HCV RNA assays (ie 4 points per patients). The adoption of the IU standard has reduced interassay variability by providing a single reference point to which these assays are now calibrated. Shiffman and coll first reported false positive and false negative results as well as variations of HCV RNA level of more than 1 to 2 log IU, which can occur with any assay and suggested these results may have an impact on the management of the patients receiving interferon therapy [Shiffman et al., 2003]. Based on pivotal trials in large multicenter studies, positive and negative predictions of SVR using viral load kinetics have been established and are now used for recommendations on antiviral therapy management by the American and European international consensus conference[1999; 2002; Fried et al., 2002; Hadziyannis and H, 2002]. The ability to predict either a positive or negative therapeutic response is of obvious benefit to clinicians and patients [Jessner et al., 2003; Rosen et al., 2002]. Positive predictive evidence early in the course of treatment could be used to reinforce the importance of compliance in ensuring a successful outcome. Conversely, negative predictive capability would allow clinicians to discontinue therapy early during the course of treatment, which would save health care resources and, more important, could prevent drug-related adverse events. The data for the proposal algorithm in the management of antiviral therapy in patients with chronic hepatitis C were mainly based

11 Journal of Medical Virology 2006;78: Halfon Page on measurement of viral load by only one assay for HCV RNA quantitation (Cobas Monitor Amplicor HCV 2.0) [1999; Hadziyannis and H, 2002]. A dynamic prediction model based on the virological response at weeks 4 and 12 showed that the outcome of combination therapy with peginterferon alfa-2a and ribavirin is dependent on the rapidity of viral response[1999; Fried et al., 2002; Hadziyannis and H, 2002; Pawlotsky, 2002; Pawlotsky et al., 2000]. In the present study, considering a 2- log decline at week 12 after the beginning of therapy, discordant results were found with the four HCV RNA methods in predicting SVR or non-response. At week 4 and week 12, significant differences were observed between Versant HCV RNA 3.0 vs PCR HCV Taqman, Versant HCV RNA 3.0 vs LCx HCV RNA, Cobas Monitor Amplicor HCV 2.0 vs LCx HCV RNA, and Cobas Monitor Amplicor HCV 2.0 vs PCR HCV Taqman (p<0.001). From 0.4% to 6% of the samples tested were discordant for detection of HCV RNA. The HCV RNA cutoff, given a 100% of negative predictive value at week 4 and week 12, differed with the assays used to quantify HCV RNA, despite the use of the IU/ml units. These differences have many explanations. One is related to the variable nature of these assays with reference to the IU standard. Thus, before the introduction of the IU standard, 1 IU turned out to be equivalent to 2.4 copies/ml with the Amplicor assay, 3.4 copies with NGI assay, and 5.4 meq/ml by Versant HCV RNA 3.0 [Saldanha, 1999]. Another difference among these assays is the lower and upper limit for quantitation of HCV RNA. The IU standard does not address this issue. Regarding the comparison of the mean values of the assay, the HCV RNA viral load assessed by the Versant HCV RNA 3.0 assay was higher that those found with the other assays in a interval range comprise in 0.05 to 1.04 log IU/ml. This finding differs from that of another study comparing the LCx HCV RNA assay to the Bayer Versant HCV RNA 3.0 assay [Bertuzis et al., 2005]. Significant differences in viral load were observed at week 4 and week 12, between Versant HCV RNA 3.0 vs PCR HCV Taqman, Versant HCV RNA 3.0 vs LCx HCV RNA, Cobas Monitor Amplicor HCV 2.0 vs LCx HCV RNA, and Cobas Monitor Amplicor HCV 2.0 vs PCR HCV Taqman (p<0.001). The correlation coefficients between the different assays in the present study were

12 Journal of Medical Virology 2006;78: Halfon Page between 0.73 and This finding agrees with others in this field [Bertuzis et al., 2005; Lunel et al., 1999; Veillon et al., 2003]. The present study was not designed to determine which of these assays were superior for use in clinical practice. All four assays failed to detect HCV RNA in 0.4% to 6% of the 486 samples, mainly due to the different sensitivities of the assays. The assays were performed in the same laboratory with a rigorous, closely monitored process. Duplicate analysis of serum HCV RNA was performed when results of virological testing were not consistent with clinical findings and when results were discordant between the assays. These variations observed between the different assays cannot be imputed to intra or interassay reproducibility. The decision to abandon therapy on the basis of HCV RNA levels calls for assays that are accurate and reliable. Examples of how variation in HCV RNA could lead to erroneous decisions in patient management are illustrated in Figure 3. Considering a 2- log drop of difference in viral load, 6 of 27 patients presented discordant value according to the antiviral response at week 12. Four non-responder patients were not correctly classified at week 12 depending on their HCV RNA viral load decline. Two complete responders were classified as non-responders or SVR depending on their HCV RNA viral load decline. Berg and coll. suggested that an absolute value of more than IU/ ml at week 12 should be used instead of a 2 log decline to consider a patient a non-responder and to discontinue the treatment [Berg et al., 2003]. In the present study, the absolute value, the NPV, at week 4 and week 12 differed according to the assay used to assess the HCV RNA viral load and it was higher than the under limit of each assay. Of the 486 samples analyzed, 58% of values were within 1 log unit, 2% of values were more than 2 log units, and 40% of values were between 1 and 2 log units of difference between assays (Table 2). This variability did not depend on HCV genotype. Of the serum values for HCV RNA, 89% were concordant (<2Log >2Log) by the IU standard regardless of which virological assay was used. These discordances agree with those reported by [Shiffman et al., 2003]. Relative to their study, however, the number of

13 Journal of Medical Virology 2006;78: Halfon Page patients classified within 1 log unit was higher (90% vs 58%); mainly due to the comparison of four assays instead of three. In summary, the present study has shown that despite the use of IU standard, false positive and false negative results as well as significant variations in HCV RNA can occur and that these results can affect treatment. Regardless the knowledge by clinicians and virologist that absolute values obtained by different assays are different; this study highlights the fact that HCV RNA viral load decline, assessed by different assays during antiviral therapy, can give different results with an important clinical therapeutic decision This study validates the study from [Shiffman et al., 2003] which confirms that it is unwise in clinical practice to base important treatment decisions on a single HCV RNA determination. HCV-infected patients might be monitored with only one assay method. This recommendation must be validated in larger prospective trials with different HCV quantitation assays. 297

14 Journal of Medical Virology 2006;78: Halfon Page Conflicts of interest Authors declare that they do not have conflict of interest with other parts. Financial support Authors declare that there was no source of financial support for this study.

15 Journal of Medical Virology 2006;78: Halfon Page References EASL International Consensus Conference on Hepatitis C. Paris, 26-28, February 1999, Consensus Statement. European Association for the Study of the Liver. J Hepatol 30(5): National Institutes of Health Consensus Development Conference Statement: Management of hepatitis C: June 10-12, Hepatology 36(5 Suppl 1):S3-20. Berg T, Sarrazin C, Herrmann E, Hinrichsen H, Gerlach T, Zachoval R, Wiedenmann B, Hopf U, Zeuzem S Prediction of treatment outcome in patients with chronic hepatitis C: significance of baseline parameters and viral dynamics during therapy. Hepatology 37(3): Bertuzis R, Hardie A, Hottentraeger B, Izopet J, Jilg W, Kaesdorf B, Leckie G, Leete J, Perrin L, Qiu C, Ran I, Schneider G, Simmonds P, Robinson J Clinical performance of the LCx HCV RNA quantitative assay. J Virol Methods 123(2): Fried MW, Shiffman ML, Reddy KR, Smith C, Marinos G, Goncales FL, Jr., Haussinger D, Diago M, Carosi G, Dhumeaux D, Craxi A, Lin A, Hoffman J, Yu J Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. N Engl J Med 347(13): Germer JJ, Harmsen WS, Mandrekar JN, Mitchell PS, Yao JD Evaluation of the COBAS TaqMan HCV test with automated sample processing using the MagNA pure LC instrument. J Clin Microbiol 43(1): Hadziyannis S, H C Peginterferon alfa-2a (40KD) (PEGASYS) in combination with ribavirin (RBV): Efficacy and safety results from a phase III, randomized, double-blind, multicentre study examining effect of duration of treatment and RBV dose. 37th Annual Meeting of the European Association for the Study of the Liver (EASL). Madrid, Spain. Halfon P, Bourliere M, Halimi G, Khiri H, Bertezene P, Portal I, Botta-Fridlund D, Gauthier AP, Jullien M, Feryn JM, Gerolami V, Cartouzou G Assessment of spontaneous fluctuations of viral load in untreated patients with chronic hepatitis C by two standardized quantitation methods: branched DNA and Amplicor Monitor. J Clin Microbiol 36(7): Halfon P, Khiri H, Gerolami V, Bourliere M, Feryn JM, Reynier P, Gauthier A, Cartouzou G Impact of various handling and storage conditions on quantitative detection of hepatitis C virus RNA. J Hepatol 25(3): Halfon P, Trimoulet P, Bourliere M, Khiri H, de Ledinghen V, Couzigou P, Feryn JM, Alcaraz P, Renou C, Fleury HJ, Ouzan D Hepatitis C virus genotyping based on 5' noncoding sequence analysis (Trugene). J Clin Microbiol 39(5): Herrmann E, Lee JH, Marinos G, Modi M, Zeuzem S Effect of ribavirin on hepatitis C viral kinetics in patients treated with pegylated interferon. Hepatology 37(6): Jessner W, Gschwantler M, Steindl-Munda P, Hofer H, Watkins-Riedel T, Wrba F, Mueller C, Gangl A, Ferenci P Primary interferon resistance and treatment response in chronic hepatitis C infection: a pilot study. Lancet 358(9289): Jessner W, Stauber R, Hackl F, Datz C, Watkins-Riedel T, Hofer H, Gangl A, Kessler H, Ferenci P Early viral kinetics on treatment with pegylated interferon-alpha-2a in chronic hepatitis C virus genotype 1 infection. J Viral Hepat 10(1): Jessner W, Watkins-Riedel T, Formann E, Steindl-Munda P, Ferenci P Hepatitis C viral dynamics: basic concept and clinical significance. J Clin Virol 25 Suppl 3:S31-39.

16 Journal of Medical Virology 2006;78: Halfon Page Lam NP, Neumann AU, Gretch DR, Wiley TE, Perelson AS, Layden TJ Dose-dependent acute clearance of hepatitis C genotype 1 virus with interferon alfa. Hepatology 26(1): Layden TJ, Layden JE, Ribeiro RM, Perelson AS Mathematical modeling of viral kinetics: a tool to understand and optimize therapy. Clin Liver Dis 7(1): Leckie G, Schneider G, Abravaya K, Hoenle R, Johanson J, Lampinen J, Ofsaiof R, Rundle L, Shah S, Frank A, Toolsie D, Vijesurier R, Wang H, Robinson J Performance attributes of the LCx HCV RNA quantitative assay. J Virol Methods 115(2): Lee SC, Antony A, Lee N, Leibow J, Yang JQ, Soviero S, Gutekunst K, Rosenstraus M Improved version 2.0 qualitative and quantitative AMPLICOR reverse transcription-pcr tests for hepatitis C virus RNA: calibration to international units, enhanced genotype reactivity, and performance characteristics. J Clin Microbiol 38(11): Lunel F, Cresta P, Vitour D, Payan C, Dumont B, Frangeul L, Reboul D, Brault C, Piette JC, Huraux JM Comparative evaluation of hepatitis C virus RNA quantitation by branched DNA, NASBA, and monitor assays. Hepatology 29(2): Neumann AU, Lam NP, Dahari H, Davidian M, Wiley TE, Mika BP, Perelson AS, Layden TJ Differences in viral dynamics between genotypes 1 and 2 of hepatitis C virus. J Infect Dis 182(1): Nguyen TT, Sedghi-Vaziri A, Wilkes LB, Mondala T, Pockros PJ, Lindsay KL, McHutchison JG Fluctuations in viral load (HCV RNA) are relatively insignificant in untreated patients with chronic HCV infection. J Viral Hepat 3(2): Pawlotsky JM Molecular diagnosis of viral hepatitis. Gastroenterology 122(6): Pawlotsky JM, Bouvier-Alias M, Hezode C, Darthuy F, Remire J, Dhumeaux D Standardization of hepatitis C virus RNA quantification. Hepatology 32(3): Rosen HR, Ribeiro RR, Weinberger L, Wolf S, Chung M, Gretch DR, Perelson AS Early hepatitis C viral kinetics correlate with long-term outcome in patients receiving high dose induction followed by combination interferon and ribavirin therapy. J Hepatol 37(1): Saldanha J Saldanha J, Lelie N, Heath A. Establishment of the first international standard for nucleic acid amplification technology (NAT) assays for HCV RNA. WHO Collaborative Study. Group Vox Sang. 76 (3): Shiffman ML, Ferreira-Gonzalez A, Reddy KR, Sterling RK, Luketic VA, Stravitz RT, Sanyal AJ, Garrett CT, De Medina M, Schiff ER Comparison of three commercially available assays for HCV RNA using the international unit standard: implications for management of patients with chronic hepatitis C virus infection in clinical practice. Am J Gastroenterol 98(5): Trimoulet P, Halfon P, Pohier E, Khiri H, Chene G, Fleury H Evaluation of the VERSANT HCV RNA 3.0 assay for quantitation of hepatitis C virus RNA in serum. J Clin Microbiol 40(6): Veillon P, Payan C, Picchio G, Maniez-Montreuil M, Guntz P, Lunel F Comparative evaluation of the total hepatitis C virus core antigen, branched-dna, and amplicor monitor assays in determining viremia for patients with chronic hepatitis C during interferon plus ribavirin combination therapy. J Clin Microbiol 41(7): Zeuzem S, Herrmann E, Lee JH, Fricke J, Neumann AU, Modi M, Colucci G, Roth WK Viral kinetics in patients with chronic hepatitis C treated with standard or peginterferon alpha2a. Gastroenterology 120(6):

17 Journal of Medical Virology 2006;78: Halfon Page Figure Legends. Figure 1: Histogram of the median values and 95 th percentile bars for serum HCV RNA at baseline, week 2, week 4, and week 12 for the four assays: Versant HCV RNA 3.0, PCR HCV Taqman, LCx HCV RNA, and Cobas Monitor Amplicor HCV 2.0. Figure 2: Relations between the different assays for genotype 1 patients. 2A represents values measured by Versant HCV RNA 3.0 and PCR HCV Taqman assay. 2B shows values measured by LCx HCV RNA and PCR HCV Taqman assay. 2C shows values measured by Versant HCV RNA 3.0 and LCx HCV RNA. 2D shows values measured by Versant HCV RNA 3.0 and Cobas Monitor Amplicor HCV 2.0 assay. 2E represents values measured by Cobas Monitor Amplicor HCV 2.0 and PCR HCV Taqman, and 2F represents values measured by Cobas Monitor Amplicor HCV 2.0 and LCx HCV RNA. Lines represents the r=1 correlation with ± 0.5log unit interval. Figure 3: Patients 1 (3A) and 2 (3B) are sustained virological responders and the differences in VL with Versant HCV RNA 3.0 (diamonds), PCR HCV Taqman (squares) and LCx HCV RNA (triangles) assays were over 2 log; however, the difference in VL with the Cobas Monitor Amplicor HCV 2.0 (circles) is under 2 log. Patient 3 (3C) is a non-responder and the differences in VL with LCx HCV RNA and Cobas Monitor Amplicor HCV 2.0 are under 2 log; however, differences in VL with Versant HCV RNA 3.0 and PCR HCV Taqman are over 2 log. Patient 4 (3D) is a non-responder and the differences in VL with Versant HCV RNA 3.0, PCR HCV Taqman, and Cobas Monitor Amplicor HCV 2.0 are under 2 log; however, the difference in VL with LCx HCV RNA is over 2 log. Patient 5 (3E) is a non-responder and the difference in VL with Versant HCV RNA 3.0 is under 2 log; however differences in VL with PCR HCV Taqman, LCx HCV RNA and Cobas Monitor Amplicor HCV 2.0 are over 2 log. Patient 6 (3F) is a nonresponder and the difference in VL with Cobas Monitor Amplicor HCV 2.0 is under 2 log; however, differences in VL with Versant HCV RNA 3.0, PCR HCV Taqman and LCx HCV RNA are over 2 log. According to the prediction of SVR or non-response, discordant results were found with the four HCV RNA methods.

18 Journal of Medical Virology 2006;78: Halfon Page Tables Table 1: Discordant cases between assays for detection of HCV RNA HCV RNA undetected by HCV RNA detected by Versant 3.0 Taqman LCx Cobas Monitor 2.0 Versant HCV RNA 3.0 (n, %) - 15 (6%) 11 (4.5%) 4 (1.7%) Mean log ± SE (IU/ml) ± ± ± 0.18 Range (IU/ml) PCR HCV Taqman (n, %) 1 (0.4%) Mean log ± SE (IU/ml) Range (IU/ml) LCx HCV RNA (n, %) 0 4 (1.7%) - 0 Mean log ± SE (IU/ml) ± Range (IU/ml) Cobas Monitor Amplicor HCV 2.0 (n, %) 3 (1.6%) 12 (4.9%) 9 (3.7%) - Mean log ± SE (IU/ml) 2.78 ± ± ± Range (IU/ml)

19 Journal of Medical Virology 2006;78: Halfon Page Table 2: Number of samples in which HCV RNA varied by at least 1 log unit by the various assays (samples N=486) Versant 3.0 vs Taqman Versant 3.0 vs LCx Taqman vs LCx Versant 3.0 vs Cobas Monitor 2.0 Taqman vs Cobas Monitor 2.0 LCx Vs Cobas Monitor 2.0 < 1Log unit difference between assays Genotype Genotype Non Log units difference between assays Genotype Genotype Non > 2Log units difference between assays Genotype Genotype Non

20 Journal of Medical Virology 2006;78: Halfon Page Table 3: Viral load difference between baseline and week12 for the different assays. Considering a 2log difference in viral load, the * indicate the discordant value according to the antiviral response. Week 12 Patient Response To therapy Genotype Versant 3.0 TaqMan LCx Cobas Monitor Responder * Viral load 2 Responder * difference between 3 Non- Responder * 2.42* baseline and week 12 4 Non- Responder * 1.63 (Log IU/ml) 5 Non- Responder * 3.46* 2.51* 6 Non- Responder * 5.01* 3.64*

21 Journal of Medical Virology 2006;78: Halfon Page Table 4:Values of viral load for which NPV of non-response is 100% Versant HCV RNA 3.0 PCR HCV Taqman LCx HCV RNA Cobas Monitor Amplicor HCV 2.0 VL (Log IU) VL (IU) VL (Log IU) VL (IU) VL (Log IU) VL (IU) VL (Log IU) VL (IU) Week Week Week

22 Journal of Medical Virology 2006;78: Halfon Page Figure

23 Journal of Medical Virology 2006;78: Halfon Page Figure

24 Journal of Medical Virology 2006;78: Halfon Page Figure 3 471