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

THE AMERICAN JOURNAL OF GASTROENTEROLOGY © 2003 by Am. Coll. of Gastroenterology Published by Elsevier Inc.

Vol. 98, No. 5, 2003 ISSN 0002-9270/03/$30.00 doi:10.1016/S0002-9270(03)00184-9

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 Mitchell L. Shiffman, M.D., Andrea Ferreira-Gonzalez, Ph.D., K. Rajender Reddy, M.D., Richard K. Sterling, M.D., Velimir A. Luketic, M.D., R. Todd Stravitz, M.D., Arun J. Sanyal, M.D., Carleton T. Garrett, M.D., Ph.D., Maria De Medina, M.S.P.H., and Eugene R. Schiff, M.D. Hepatology Section, USA and Division of Molecular Diagnostics, Virginia Commonwealth University Health System–Medical College of Virginia, Richmond, Virginia; Division of Gastroenterology and Hepatology, University of Pennsylvania, Philadelphia, Pennsylvania; and Center for Liver Diseases, University of Miami, Miami, Florida

OBJECTIVES: The present study was performed to evaluate the impact of the international unit standard for measuring HCV RNA in the management of patients with chronic hepatitis C virus (HCV) infection. METHODS: The three assays used were Amplicor Monitor PCR, the National Genetics Institute PCR assay, and branched chain DNA. HCV RNA was measured at four time points (baseline, 3 months after the start of therapy, at the end of treatment, and 6 months after discontinuation of therapy) in 106 consecutive patients who received interferon and ribavirin for chronic HCV. RESULTS: The mean age of the patients was 44 yr. Of the patients, 62% were male, 24% were African American, 38% had bridging fibrosis or cirrhosis, and 75% were HCV genotype 1. Of the 424 samples analyzed, 82– 89% of values were within 1 log unit and 85–92% were within 2 log units by the various assays. This variability was not dependent upon HCV genotype. HCV RNA was undetectable in 1.4 – 6.8% of samples when virus was detected by another assay. The mean HCV RNA in these discordant samples was 1.47– 6.33 log IU/ml (30 –2,100,000 IU/ml). CONCLUSIONS: These data demonstrate that approximately 90% of serum values for HCV RNA were within 1 log unit by the international unit standard regardless of which virological assay was used. However, false positive and false negative results as well as variations in the HCV RNA level of more than 1 to 2 log units can occur with any of the assays, and these results may have an impact upon the management of patients receiving interferon therapy. It is Presented in part at the Annual Meeting of the American Association for the Study of Liver Diseases, November 11–14, 2001, Dallas, TX.

therefore unwise in clinical practice to base important treatment decisions upon a single HCV RNA determination. (Am J Gastroenterol 2003;98:1159 –1166. © 2003 by Am. Coll. of Gastroenterology)

INTRODUCTION Measuring HCV RNA has become essential in the management of patients with chronic hepatitis C virus (HCV) infection. Virological testing is used to document infection in patients who test positive for HCV antibodies (anti-HCV) and to confirm that HCV has become undetectable during and after therapy (1– 4). Measuring HCV RNA before initiating treatment has been used to assess the likelihood of achieving a response and in determining the duration of interferon-based therapy (1–3, 5– 8). Monitoring changes in HCV RNA within the first 4 –24 wk after initiating interferon and ribavirin has recently been shown to identify patients who are unlikely to become HCV RNA negative and in whom treatment can be discontinued (2, 3, 9 –15). The most common methods available for detecting and quantifying HCV RNA are based upon polymerase chain reaction (PCR) and the branched chain DNA (b-DNA) technique (1–3). A semiautomated PCR assay, the complete bioanalytical system (COBAS) Amplicor Monitor, is used by many laboratories (2, 3). Many other reference and hospital laboratories have developed their own “in house” PCR assay for measuring HCV. The most well known of these is the HCV Superquant developed and marketed by the National Genetics Institute (16, 17). One of the greatest limitations in managing patients with chronic HCV infection has been the lack of standardization for these virological assays. Assays differ widely in their

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sensitivity (i.e., the lower limit for detection of HCV RNA) and their dynamic range (2, 3, 8, 18, 19). Such variability has not only been observed with different assays but when different laboratories perform the same assay. To address these limitations, the National Institute for Biological Standards and Controls and the World Health Organization have recently developed and certified a uniform standard for measuring HCV RNA in international units (20). The present study assessed serum HCV RNA, in international units, by three commonly used commercial assays in more than 100 consecutive patients receiving interferon and ribavirin for treatment of chronic HCV. The efficacy and limitations of virological testing in the management of patients with chronic HCV is discussed.

MATERIALS AND METHODS Patient Population A total of 106 consecutive patients were enrolled into this study just before starting interferon and ribavirin therapy for chronic HCV infection at the Virginia Commonwealth University Health System–Medical College of Virginia (VCUHS/MCV). The mean age of the study population was 42.2 yr (range 27– 69 yr). Of the patients, 62% were male and 24% were African American. All tested positive for HCV RNA and negative for HIV. All other causes of chronic hepatitis were excluded by appropriate serological testing and liver histology. HCV genotype was determined by the InnoLipa assay (Immunogenetics, Zwijnaarde, Belgium) (21). Non–type 1 HCV genotypes were present in 25% of patients. This included one patient each with HCV genotypes 4 and 6. HCV genotype was mixed or could not be determined in five patients. These samples were included in the non–type1 genotype group. All patients were treated with interferon (3 mU three times weekly) and ribavirin (1000 –1200 mg/day) for 6 –12 months. The duration of therapy was based upon HCV genotype (5). Serum samples were obtained for determination of HCV RNA just before the initiation of treatment (baseline), 3 months after the initiation of treatment, at the end of treatment, and 6 months after the discontinuation of therapy (EOF). Serum ALAT (ALT) was measured at baseline and at monthly intervals during treatment. The study protocol was approved by the Committee on the Conduct of Human Investigations at the VCUHS/MCV. Handling and Evaluation of Serum for HCV RNA Analysis A total of 424 serum samples were obtained from the 106 patients. Each of the specimens was divided into three aliquots and frozen at ⫺70oC within 120 min of collection. One aliquot was sent to National Genetics Institute (NGI, Foster City, CA) for determination of HCV RNA by their Superquant PCR assay. All procedures for collection and shipping of these samples, as recommended by NGI, were followed. Values for HCV RNA were reported by NGI (in

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copies/ml). These values were converted to international units (IU) using a fixed conversion factor of 3.4 copies/IU as recommended by this reference laboratory. A second aliquot was shipped frozen to the Hepatology Clinical Laboratory at the University of Miami, where HCV RNA was determined by the b-DNA assay version 3 (Bayer Diagnostics, Emeryville, CA). Values for HCV RNA by this assay were reported in IU/ml. A third aliquot was analyzed for HCV RNA by the COBAS Amplicor Monitor PCR assay version 2 (Roche Molecular Systems, Branchberg, NJ) at the Molecular Diagnostics Laboratory of the VCUHS/MCV. Values for HCV RNA by this assay were also reported in IU/ml. The handling, processing, analysis, and reporting of results was in accordance with the standard practices used by these three clinical laboratories. Samples were not diluted or retested to confirm discordant results recognized after the data were analyzed. The interassay variability for each of these tests has either been previously published or is available from the manufacturer (1–3, 8, 16 –19, 22, 23). Statistical Analysis Values were reported as mean ⫾ SE. The significance between mean values was assessed by Student’s t test, the Mann-Whitney rank sum test, or the Kruskal-Wallis oneway analysis of variance on ranks as appropriate. The ␹2 test was used to determine whether the frequency of observations between groups was significant. A p value of ⬍0.05 was considered to be significant.

RESULTS Virological Response Figure 1 illustrates the mean values for serum HCV RNA at baseline, 3 months, end of treatment (either 6 or 12 months), and end of follow-up (6 months after discontinuation of treatment) for each of the three assays. The mean value for serum HCV RNA at these times was 5.94, 2.53, 2.63, and 3.93 log IU/ml, respectively. These values were not significantly different when measured by the three assays. This was also the case when patients were grouped by genotype (1 vs non-1). Depending upon the assay used, HCV RNA was undetectable at 3 months in 47–51% of patients, at the end of treatment (either 6 months or 12 months) in 45–55%, and at the end of follow-up in 33–36%. Relationship Among HCV RNA Assays The relationship among the three HCV RNA assays is illustrated in Figure 2. Figures 2A and 2B compare the Amplicor and b-DNA assays. Figures 2C and 2D compare the Amplicor and NGI assays. Similar results were observed when the b-DNA and NGI assays were compared (data not shown). The three parallel lines in this series of figures represent the unit line and 0.5 log unit above and below the unit line. Although some scatter existed, 82– 89% of values for HCV RNA were within 1 log unit and 85–92% were within 2 log units by the various assays (Table 1). The

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Figure 1. Mean values for serum HCV RNA at baseline, 3 months, at the end of treatment (EOT), and 6 months after the end of therapy (EOF) for each of the three assays. All patients were treated with interferon and ribavirin for 6 –12 months as recommended based on HCV genotype. Circles represent values obtained by Amplicor PCR; squares represent values obtained by b-DNA assay; and diamonds represent values obtained by NGI PCR assay.

degree of variation observed in HCV RNA among the three assays was similar for HCV genotype 1 and non-1. As is evident from Figure 2, HCV RNA was undetectable in several samples by one of the assays when virus was detected in the same sample by another assay. This discordance between assays for detection of HCV RNA is summarized in Table 2. Overall, 1.4 – 6.8% of the 464 samples tested were discordant for detection of HCV RNA. Six and eight samples that were undetectable by Amplicor PCR had HCV RNA detected by the b-DNA and NGI PCR assays, which varied between 2.90 – 6.33 and 1.47– 6.17 log IU/ml (790 –2,100,000 and 30 –1,500,000 IU/ml), respectively. Similar findings were observed for those samples that were undetectable by b-DNA and NGI PCR. Affect of Variable HCV RNA Results on the Management of Chronic HCV Specific examples of how discrepancies in detecting HCV RNA could adversely affect the treatment administered to patients with chronic HCV are presented in Figures 3 and 4. In Figure 3A, serum HCV RNA was undetectable 3 months after the initiation of interferon ribavirin therapy by both the Amplicor and b-DNA assays. However, at 6 months, HCV RNA was detectable by the b-DNA assay at a value of 4.99 log IU/ml (97,000 IU/ml). This value was most likely a false positive result, as all subsequent values by both assays were undetectable for HCV RNA. The treatment was therefore classified as a sustained virological response. However, if the patient had been monitored only by b-DNA, treatment would have been discontinued at month 6 according to current treatment recommendations (5, 12, 14, 15), and the ability to achieve a sustained virological response would not have been realized. In Figure 3B, values for HCV RNA were similar at baseline and after 3 months of interferon and ribavirin

therapy by the Amplicor and NGI PCR assays. However, at 6 months, HCV RNA was undetectable by NGI but was positive by Amplicor at a value of 3.05 log IU/ml (1122 IU/ml). Further follow-up confirmed that the patient was positive for HCV RNA at the end of treatment, and therefore this patient was classified as a nonresponder. The undetectable HCV RNA reported by NGI PCR at month 6 was therefore most likely a false negative. Thus, if this patient were monitored by Amplicor PCR, treatment would have been discontinued for nonresponse at month 6. In contrast, if monitored by NGI PCR, treatment would have continued for 12 months in accordance with current treatment recommendations (5, 12, 14, 15). Figure 4 illustrates how variations in HCV RNA among the various assays could adversely affect treatment. At baseline, HCV RNA titer was very similar by both the Amplicor and NGI PCR assays. At 3 months, HCV RNA had declined by both assays and was undetectable at both 6 months and the end of treatment, only to relapse by the end of follow-up (Fig. 4A). A closer look at the values for HCV RNA at baseline and 3 months in this patient is illustrated in Figure 4B. According to the Amplicor PCR assay, HCV RNA declined by 2.2 log units during the first 12 wk of therapy. However, the decline in HCV RNA during this time was only 1.2 log units by the NGI PCR assay. According to recent recommendations (12, 14, 15), treatment would have been discontinued at 3 months if HCV RNA had been monitored by the NGI assay (a decline in HCV RNA titer by ⬍2 log units at 12 wk) but would have been continued if the patient had been monitored by Amplicor PCR.

DISCUSSION Clinicians rely heavily upon virological testing to guide therapy for patients with chronic HCV infection. In the past

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Figure 2. Relationship among the three qualitative assays to measure HCV RNA. Values measured by Amplicor PCR and the b-DNA assay for patients with HCV genotype 1 (A) and non-1 (B) infection. Values measured by the Amplicor PCR and NGI PCR assay for patients with HCV genotype 1 (C) and non-1 (D) infection. Lines in each part of figure represent the 1:1 correlation ⫾ 0.5 log unit.

this was problematic, as the reporting of HCV RNA varied widely among different assays and laboratories (1–3, 8, 18, 19). In contrast, standardization of virological assays ac-

cording to the international unit should yield similar values for HCV RNA regardless of the methodology and laboratory used (20). The present study was therefore performed to

Table 1. Samples in Which HCV RNA Titer Varied by More Than 1 to 2 Log Units By the Various Assays

⬎1 Log unit difference between assays Genotype 1 Genotype non-1 1–2 Log units difference between assays Genotype 1 Genotype non-1 ⬎2 Log units difference between assays Genotype 1 Genotype non-1

Amplicor PCR vs b-DNA

Amplicor PCR vs NGI PCR

b-DNA vs NGI PCR

39 (12.2%) 11 (10.6%)

58 (18.1%) 18 (17.3%)

47 (14.7%) 12 (11.5%)

14 (4.4%) 2 (1.9%)

17 (5.3%) 2 (1.9%)

19 (5.9%) 2 (1.9%)

25 (7.8%) 9 (8.7%)

41 (12.8%) 16 (15.4%)

28 (8.8%) 10 (9.6%)

Total number of samples analyzed ⫽ 424; genotype 1 ⫽ 320; genotype non-1 ⫽ 104.

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Table 2. Samples in Which Assays Were Discordant for Detection of HCV RNA Assay in Which HCV RNA Was Detectable Amplicor PCR Amplicor undetectable (n, %) Mean log titer ⫾ SE (IU/ml) Range (IU/ml) b-DNA undetectable (n, %) Mean log titer ⫾ SE (IU/ml) Range (IU/ml)

20 (4.7%) 4.51 ⫾ 0.34 2.33–6.69

NGI undetectable (n, %) Mean log titer ⫾ SE (IU/ml) Range (IU/ml)

29 (6.8%) 4.95 ⫾ 0.25 2.33–6.69

b-DNA

NGI PCR

6 (1.4%) 4.64 ⫾ 0.61 2.90–6.33

8 (1.9%) 3.10 ⫾ 0.68 1.47–6.17 8 (1.9%) 2.57 ⫾ 0.53 1.47–6.11

16 (3.8%) 4.84 ⫾ 0.34 2.77–6.70

n ⫽ number of all samples in which the assay failed to detect HCV RNA when this was detected by at least one of the other assays. Total number of samples tested ⫽ 424.

evaluate the impact of the international unit (IU) standard for measuring HCV RNA on the management of patients with chronic HCV. Our results suggest that adoption of the IU standard provides excellent reliability when measuring HCV RNA regardless of the assay used. Between 82% and 89% of ⬎420 samples tested had values for HCV RNA that were within 1 log unit by the three assays (Table 1). A recently published study seems to confirm our observations, at least for the Amplicor and NGI assays (24). In the prior study, these two assays were also shown to be linear and to yield similar results up to HCV RNA concentrations of 600,000 IU/ml by Amplicor, the equivalent of approximately 1,000,000 copies/ml with the NGI assay. This study evaluated three assays that are widely available and frequently used to measure HCV RNA in clinical practice. COBAS Amplicor PCR, a semiautomated standardized device, was developed by Roche Molecular Systems and has recently been approved by the United States Food and Drug Administration to confirm HCV infection (22). A version of this device, Amplicor Monitor, is used to measure HCV RNA by several reference and hospital laboratories (2, 3, 22). The National Genetics Institute, an

independent reference laboratory, has developed its own PCR assay to measure HCV RNA. This laboratory has been used by pharmaceutical companies and independent investigators to assess HCV RNA in several large clinical trials (16, 17, 25, 26). The branched chain DNA assay is a standardized device manufactured by Bayer Diagnostics. This assay detects and quantifies HCV RNA using a novel branched DNA and detection probe system in which the detection probe, rather than the virus substrate, is amplified (1–3, 27, 28). FDA approval for this device was recently granted. The sensitivity and dynamic ranges of these assays to detect HCV RNA have been reported by individual laboratories and by the manufacturers of these products (1–3, 8, 16 –19, 23, 27, 28). PCR has a sensitivity for detection of HCV RNA to a value of 100 –1,000 IU/ml and a dynamic range of 600,000 to several million IU/ml depending upon the specific methodology used, the manner by which the assay was calibrated by the individual laboratory, and whether the sample was diluted prior to analysis. The bDNA technique was thought to have a broader dynamic range but reduced sensitivity when compared with PCR.

Figure 3. Change in serum HCV RNA measured by two different assays from representative patients during and after interferon ribavirin therapy. Circles represent values determined by Amplicor PCR; squares represent values measured by the b-DNA assay; diamonds represent values measured by the NGI PCR assay.

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Figure 4. Change in serum HCV RNA measured by two different assays from a representative patient during interferon ribavirin therapy. Circles represent values determined by Amplicor PCR; diamonds represent values measured by the NGI assay.

Before the development of the IU standard the amount of HCV RNA reported within each sample varied significantly depending upon the assay used (2, 3, 8, 18, 19). This, in part, was secondary to the variable nature by which these assays compared to the IU standard. For example, 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 b-DNA (20). As a result, the amount of virus reported for any given sample was always 1.5–2 fold greater when measured by b-DNA than by NGI, which in turn was always greater than with Amplicor. The adoption of the IU standard has reduced interassay variability by providing a single reference point to which these assays are now calibrated. Another difference among the three assays is the upper limit for quantification of HCV RNA (2, 3, 8, 18, 19). This was greatest with the b-DNA assay but was limited to only 600,000 –1,000,000 IU/ml for many PCR assays. Although the IU standard does not address this issue, it is important to realize that values for HCV RNA as high as 5– 8 million IU/ml are still within 1 log unit and therefore not significantly different from the maximal value reported by some PCR assays. These two explanations account for the close linear relationship among the Amplicor, NGI, and b-DNA assays observed in this study (Fig. 2 and Table 1). The present study was not designed to determine which of these assays was superior for use in clinical practice. Such a study would require the identification of a single assay as the “gold standard” that could detect and quantify HCV RNA with 100% accuracy. Unfortunately, none of these three assays fulfilled this criterion. All three failed to detect HCV RNA in 1.4 – 6.8% of samples when virus was detectable by one or both of the other assays (Table 2). Furthermore, the inability to detect HCV RNA was not related to varying degrees of sensitivity among the assays or confined to a single genotype. The mean serum level for HCV RNA ranged from 1.47 to 6.70 log IU/ml (30 –5,000,000 IU/ml) when virus was undetectable by at least one of the other assays (Table 2). Which of these results was correct, and which was either false positive or false negative, cannot be

determined with absolute certainty. Measurement of HCV RNA by each of these assays relies upon numerous operator dependent steps that include sample processing, shipping, and preparation before performing amplification and detection of virus. It is well known that ribonucleases in plasma affect the stability of RNA viruses, and that alterations in sample preparation and viral extraction may either yield false negative results or affect the amount of HCV RNA detected by any assay. Furthermore, cross-contamination of samples in the laboratory may lead to false positive results (1–3, 29 –31). It is therefore likely that most cases of discordance in detection of HCV RNA, observed in up to 6.8% of samples, and in the variation in HCV RNA level by ⬎1 to 2 log units, observed in up to 15% of samples, were the result of random errors in sample processing and preparation rather than in the sensitivity of or actual technique used to amplify and detect virus by these assays. The routine use of virological testing in the management of patients with chronic HCV has emerged from large clinical trials conducted by the pharmaceutical industry to determine the efficacy of various interferons with or without ribavirin to eradicate virus (14, 16, 17, 24, 25, 32–35). Virological testing in these studies was a rigorous, closely monitored process. Multiple samples were collected at frequent intervals, additional aliquots of serum were prepared and stored, and samples were reanalyzed when the results of virological testing were not consistent with clinical findings. In contrast, virological testing in clinical practice has none of these safeguards. Samples are prepared and transported to reference or hospital-based laboratories where they are analyzed, and the results are reported without the benefit of clinical oversight. Duplicate samples are not available to verify results that seem inconsistent with the clinical course of patients, as illustrated in Figure 3. The present study was conducted with this in mind. Samples from more than 100 consecutive patients receiving treatment for chronic HCV were collected, processed, and analyzed by three separate laboratories in accordance with routine recommended procedures. Samples were not reanalyzed when virological

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results were inconsistent with the clinical course of the patient (Fig. 3) or after discordant results were detected during data analysis (Tables 1 and 2). The present study therefore illustrates the impact— both positive and negative—that virological testing may have on the clinical management of patients with chronic HCV. Recent studies have demonstrated that a decline in the serum level of HCV RNA by ⬎2 log units from baseline within 12 wk of initiating peginterferon and ribavirin can accurately identify patients with a high likelihood for sustained virological response (12, 14, 15). If these patients do not require dose reduction and remain compliant with treatment, a sustained virological response of 75% can be achieved. In contrast, the failure to achieve a 2-log reduction in HCV RNA from baseline within 12 wk of initiating therapy was highly predictive of continued nonresponse, and it has been proposed that such patients discontinue therapy if this pattern of nonresponse is observed (14, 15). However, the decision to abandon therapy based on the assessment of serum HCV RNA levels demands that the assay relied upon to make this clinical decision is accurate and reliable. It is therefore important to recognize that measurements of HCV RNA varied by ⬎1 log unit in 11–18% of samples, by ⬎2 log units in 8 –15% of samples, and was discordant for detection of HCV RNA in 1.4 – 6.8% of samples tested (Tables 1 and 2). Examples of how such variation in HCV RNA could lead to erroneous decisions in patient management are illustrated in Figures 3 and 4. These data do not suggest that one assay is superior to any other for measuring HCV RNA. These variations and random false negative results occurred in all three assays evaluated and, given the complexity of measuring HCV RNA, it is unlikely that a perfect assay with 100% reproducibility will ever exist. In summary, the present study has demonstrated that the IU standard has successfully reduced the variability that was previously observed when HCV RNA was measured by different assays and laboratories. Approximately 90% of samples tested by three different assays in three different laboratories were within 1 log unit for HCV RNA. However, it is important to recognize that false positive and false negative results as well as significant variations in HCV RNA can occur, and these results may affect patient treatment. As a result, it is probably unwise for clinical decisions regarding early discontinuation of therapy to be based on a single HCV RNA determination as recently recommended (14, 15). Rather, these data strongly suggests that more frequent—not less frequent—HCV RNA testing should be performed during the first several months (i.e., months 2– 4) after initiation of therapy for chronic HCV infection to ensure that correct clinical decisions regarding the continuation or discontinuation of therapy are rendered.

ACKNOWLEDGMENTS This work was supported in part by National Institutes of Health grant ROM-RO1-R00065 to the Clinical Research

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Center at the Virginia Commonwealth University Health System, National Institutes of Health contract N01-DK-92322 to M.L.S., and a grant-in-aid from Roche Molecular Systems. Reprint requests and correspondence: Mitchell L. Shiffman, M.D., Hepatology Section, Virginia Commonwealth University Health System, Medical College of Virginia, Box 980341, Richmond, VA 23298. Received Apr. 12, 2002; accepted Oct. 16, 2002.

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