Comparison of Quantitative HCV RNA Assays in Chronic Hepatitis C

Transcription

1 MICROBIOLOGY AND INFECTIOUS DISEASE Original Article Comparison of Quantitative HCV RNA Assays in Chronic Hepatitis C SHERAJ JACOB, MD, DEBORAH BAUDY, ELIZABETH JONES, LIZHE XU, PhD, ANDREW MASON, MBBS, FREDRIC REGENSTEIN, MD, AND ROBERT P. PERRILLO, MD We compared the relative sensitivities of first-and-second generation branched nucleotide assays (Quantiplex HCV RNA 1.0 and 2.0, respectively, Chiron, Emeryville, Calif) for the detection of hepatitis C virus (HCV) RNA to that of a commercially available quantitative reverse transcriptase polymerase chain reaction (RT-PCR) method (Monitor, Roche Molecular Systems, Nutley, NJ) in 53 patients with chronic hepatitis C. The sensitivities of the second-generation branched DNA (bdna) and RT-PCR assays were similar (91% and 92%, respectively), and both were significantly more sensitive (P<.001) than the first-generation method. Moreover, both assays detected HCV RNA in all 11 patients with type 2a, 2b, or 3a geno- types vs 45% with the HCV RNA 1.0 bdna assay. We examined 174 serum samples by the bdna 2.0 and RT-PCR assays. Major quantification differences were noted on a given specimen with the RT- PCR method reporting values an average 41-fold lower (range, fold) than those obtained with the bdna assay. We conclude that both methods can be used to detect HCV RNA in patients who are infected with the genotypes that are most commonly encountered in the United States. The HCV RNA 2.0 bdna assay may offer advantages when attempting to quantify high-level viremia. (Key words: Hepatitis C virus; RNA; quantitative RNA; hepatitis C virus genotypes) Am J Clin Pathol 1997,107: Hepatitis C virus (HCV), a small single-stranded RNA virus, is the major causative agent of transfusion-associated hepatitis. 1,2 The nucleotide sequence data of various HCV isolates have led to the designation of several genotypes. 3-6 Genetic variants of HCV have been classified into six major genotypes, some with several subtypes, on the basis of nucleotide sequence homology and phylogenetic analysis. 7 In chronically infected individuals, HCV genotype and serum HCV RNA levels are clinically relevant because certain genotypes, such as types 2 and 3, and low level viremia are more frequently associated with a sustained response to therapy with interferon-alfa Moreover, evidence indicates that genotype lb may be associated with a more aggressive course histologically 13 Commercially available methods for quantification of HCV RNA are now available. Methods to quantitate HCV RNA are based on an adaptation of the reverse-transcriptase polymerase chain reaction (RT-PCR) or a signal amplification strategy that From the Section of Gastroenterology and Hepatology, Ochsner Clinic, Nezv Orleans, Louisiana. Manuscript received July 31,1996; revision accepted September 25,1996. Address reprint requests to Dr Perrillo: 1514 Jefferson Hwy, New Orleans, LA involves the use of enzymatically labeled branched nucleotide (bdna) Investigators have found that quantitation with RT-PCR is not highly reproducible, because the efficiency of the reverse transcription and amplification steps may vary, and a number of falsenegative and false-positive test results may be found. 14 ' Moreover, the efficiency of the hybridization and transcription steps could also result in nonlinearity of results when high levels of virus are present. Whereas a lack of specificity is not a problem in the bdna assay, it does not have the inherent sensitivity of RT-PCR. 16,17 An assay that reliably detects HCV RNA in serum or plasma and that is linear over a broad range of values would be useful in predicting and monitoring the response of patients during and after therapy. The first-generation bdna assay (Quantiplex HCV RNA, 1.0, Chiron, Emeryville, Calif) has been refined with a new set of oligonucleotide probes that are based on sequence variations among different HCV isolates. This modification has been shown to enhance equal hybridization efficiencies to all known genotypes. 20 This second-generation bdna assay (Quantiplex HCV RNA 2.0) and a quantitative RT- PCR method for the detection of HCV RNA (Monitor RT-PCR, Roche Molecular Systems, Nutley, NJ) have recently become available for clinical use, but limited 362

2 JACOB ET AL 363 Hepatii CRNA cross comparison of these assays has been made and virtually no data are available in serial samples collected from interferon-treated patients. The primary objective of the current study, therefore, was to compare the HCV RNA 1.0, HCV RNA 2.0, and the quantitative RT-PCR assays in baseline serum samples of 53 patients with chronic hepatitis C, 24 of whom were serially studied before, during, and after interferon therapy. A second objective of the study was to compare the efficiency of detection of HCV RNA by the three methods in regard to the hepatitis genotype. Clinical MATERIALS AND METHODS Specimens The study included 174 serum samples derived from 53 patients with chronic hepatitis C. All serum specimens were removed from the clot within 4 hours, frozen and stored at -20 to -70 C. Each of these patients had persistent abnormal serum alanine transaminase values and had positive results for antibody to HCV by enzyme-linked immunoassay (Abbott Laboratories, North Chicago, 111). In most instances, a definite risk for exposure to HCV and hepatic biopsy-proved evidence for chronic hepatitis C was available. Serial serum samples were available in 24 patients who were treated with interferon-alfa. The serial samples were derived before, during (at 4- week intervals), and after therapy with interferon-alfa 2b (Intron A, Schering-Plough, Kenilworth, NJ). Each of the baseline serum samples from these 53 patients was evaluated for genotype by line probe assay (Inno- LIPA, Immunogenetics, Ghent, Belgium) or restriction fragment length polymorphism to determine the effect genotype had on the ability to detect HCV RNA by the various assays. HCV RNA Quantification by bdna Technology Serum HCV RNA was quantitatively determined by a signal amplification method based on bdna, according to the manufacturer's instructions. 17,20 Briefly, duplicate 50-uL serum samples are added to wells of a 96-well plate. Lysis and inhibition of ribonucleases are accomplished by the addition of a buffer containing proteinase K and detergent. The buffer also contains two sets of oligonucleotide probes; one set mediates capture of the target nucleic acid probes on the microwell and another set binds the amplifier molecule (bdna) to the target. After overnight incubation at 53 C and washing, multiple synthetic bdna molecules are added. Conjugated alkaline phosphatase probes are then annealed to the immobilized complex, resulting in signal amplification when the chemiluminescent substrate dioxetane is added. The visible light output is measured in a luminometer. The results from a luminometer are reported in relative luminescence units, which are a measurement of the amount of light emitted from each HCV capture well. To obtain HCV RNA quantitative results, the mean relative luminescence units of each specimen is compared with the standard curve. The final result is reported in HCV RNA megaequivalents (hereafter referred to as MEq/mL). One megaequivalent is the amount of HCV RNA that generates a level of light emission equivalent to that generated by 10 6 copies of an RNA standard as previously described. 20 The quantification limit for the Quantiplex HCV RNA 1.0 assay is 0.35 HCV RNA genomic MEq/mL; for the Quantiplex HCV RNA 2.0 assay, the limit is 0.2 HCV RNA MEq/mL. HCV RNA Quantification by RT-PCR Technology The HCV RNA Monitor test includes a quantitation standard of known copy number that is coamplified with the target and is used to assign the copy level to the specimen. Briefly, the method is as follows: 100 pl of serum or plasma processed within 3 hours of collection or stored at -20 C is extracted, together with a quantitation standard with guanidine thiocyanate, and the RNA is recovered by isopropanol precipitation. The RNA is resuspended in a buffer containing manganese necessary for the PCR. The quantitation standard is a synthetic RNA molecule with primer binding sites identical to the HCV target RNA and a unique probe sequence specific for the quantitation standard molecule. For the amplification step, the 5' noncoding region of the HCV genome, a 244 base pair sequence defined by the primers KY80 and KY78 is reverse transcribed and amplified by PCR in a single reaction. The reaction uses the thermostable recombinant enzyme rlth DNA polymerase, which has reverse transcriptase and DNA polymerase activity in the presence of manganese. The KY78 primer is biotinylated at the 5' end and together with KY80 yields amplification products. Selective amplification of target RNA from clinical samples is achieved by the use of AmpErase, which digests contaminating HCV sequences that contain deoxyuridine triphosphate as a result of previous amplification procedures. After amplification, the amplification products are denatured and transferred to a microwell detection plate containing separate Vol.1 No. 3

3 364 MICROBIOLOGY AND INFECTIOUS DISEASE Original Article wells coated with HCV-specific and quantitation-standard specific oligonucleotide probes. Detection probes for the HCV target and quantitation standards coat the microwell plate. To measure the HCV and quantification standards over a broad dynamic range, fivefold serial dilutions of the amplification product are made in the HCV-specific and quantitation-standard specific wells of the microwell plate. The bound, biotinylated amplification products are quantitated with an avidinhorseradish peroxidase conjugate and a colorimetric reaction for horseradish peroxidase. The HCV RNA copy number is then calculated from the known input copy number of the quantitation standard RNA and the ratio of total optical density (450 nm) generated by the HCV and the quantitation standard. The sensitivity of the Monitor RT-PCR assay is about 100 to 500 HCV RNA genomes per milliliter of serum. HCV Genotyping All 53 patients were genotyped by two commonly used genotyping systems. These methods use RT-PCR with generic primers based on the HCV 5' UT region after which the PCR product is annealed to strips with genotype-specific probes. The HCV genotype nomenclature used in this report is that proposed by an international panel and is the most accepted of all nomenclature systems. 7 The line probe assay was used to assess HCV genotyping as previously described. 21 Briefly, the 5' UT region was amplified using "nested" PCR with biotinylated primers. The labeled amplicon was allowed to hybridize with probes derived from various HCV genotypes mounted on a strip. After stringent washing, streptavidin labeled with alkaline phosphatase was used to trace the hybridized products, and nitroblue tetrazolium and 5-bromo-4-chloro- 3-indoyl-phosphate were used as substrates. The restriction fragment length polymorphism analysis was performed as described previously 6 HCV genotypes were determined from nested PCR products using primers derived from the HCV 5' untranslated region. Briefly, the 5' UT was reverse transcribed and amplified by nested PCR using outer primers (antisense, 5'-TCATGGTGCACGGTCTACGAGACCT-3'; sense, 5'-CTGTGAGGAACTACTGTCTT-3') and inner primers (anti-sense, 5'-CACTCGCAAGCAC- TATCAGGCAGT-3'; sense, 5'-TCACGCA- GAAAGCGTCTAG-3') as described previously. 22 The amplicons were then digested by two sets of restriction enzymes HAeIII/Rsa I and Mva I/Hinf I to differentiate HCV into various major genotypes (types 1-6). Subtypes la/c and lb were further differentiated by restriction with the enzyme BstU I. Subtypes 2a/c and 2b and subtypes 3a and 3b were further differentiated by digestion with ScrFI. RESULTS Table 1 shows the overall results with the three quantitative HCV RNA assays. Of the 53 baseline samples, 31 (58%) were positive by the bdna 1.0 assay, 48 (90%) by the bdna 2.0 assay, and 49 (92%) by the RT- PCR assay. The bdna 2.0 assay and the RT-PCR assays were significantly more sensitive than the bdna 1.0 assay (P<.001). When all 174 serum samples derived from these patients were compared, the percentage of positive results by the bdna 1.0, bdna 2.0, and RT- PCR assays were 38%, 72%, and 69%, respectively, with the lower frequencies reflecting the effects of interferon-alfa therapy. In general, the correlation was good between the results of all three methods in the patients who were analyzed before, during, and after therapy (Fig 1), but higher levels of virus were consistently detectable by the bdna method (Fig 2). An attempt was made to genotype all 53 patients, and 49 patients had similar results obtained by line TABLE 1. POSITIVE TEST RESULTS OBTAINED BY THE THREE QUANTITATIVE HCV RNA ASSAYS Baseline Positive, No. (%) Total Positive, No. (%) bdna 1.0* (58) (38) bdna 2.0* (91) (72) HCV = hepatitis C virus; bdna = branched nucleotide assay; RT-PCR = reverse transcriptase polymerase chain reaction. Quantiplex HCV RNA 1.0 and 2.0 assays from Chiron, Emeryville, Calif. + Monitor RT-PCR from Roche Molecular Systems, Nutley, NJ. RT-PCR (92) (69) TABLE 2. COMPARISON OF THE THREE QUANTITATIVE HCV RNA ASSAYS IN RELATION TO GENOTYPE* Genotype n bdnal.0* (%) bdna2.0 f (%) RT-PCR* (%) 15 (65) 11 (73) 0(0) 4(57) 1(50) 31 (63) 23 (100) 14 (93) 7 (100) 48 (98) 23 (100) 14 (93) 7 (100) 48 (98) HCV = hepatitis C virus; bdna = branched nucleotide assay; RT-PCR = reverse transcriptase polymerase chain reaction. *Exclusive of 4 patients in whom genotype could not be clearly delineated. Data are reported as No. (%). + Quantiplex HCV RNA 1.0 and 2.0 assays from Chiron, Emeryville, Calif. ^Monitor RT-PCR from Roche Molecular Systems, Nutley, NJ. AJCP- 1997

4 JACOB ET AL 365 Hepatitis C RNA HCV RNA Eq/mL HCV RNA Copies/mL HCV RNA Eq/mL 1X10*. HCV RNA Copies/mL r 1X10* r 1X10* 1x10' 1x10' 1x10' 1X10' ixio" Not Detectable - I I I I 0 -T- r Weeks -e- HCV HCV 2.0 -B- Monitor FIG 1. Hepatitis C virus (HCV) RNA levels in a patient who was treated with interferon-alfa (IFN) as measured by branched nucleotide (bdna; Quantiplex HCV RNA 1.0 and 2.0, Chiron, Emeryville, Calif) 1.0 and 2.0) and reverse transcriptase polymerase chain reaction (Monitor, Roche Molecular Systems, Nutley, NJ). Note the similarities in the curves during and after interferon treatment and the differences in the values reported by the two methods. Eq/mL = genome equivalents per nil One megaequivalent (MEq) equals 1 genomic equivalent X 10. Weeks -O-HCV HCV 2.0 -B- Monitor FIG 2. Hepatitis C virus (HCV) RNA levels in a patient who was treated with interferon-alfa. Note the consistently higher levels that were detected with either of the two branched DNA assays (Quantiplex HCV RNA 1.0 and 2.0; Chiron, Emeryville, Calif Monitor reverse transcriptase polymerase chain reaction from Roche Molecular Systems, Nutley, NJ). Eq/mL refers to genome equivalents per ml. One megaequivalent (MEq) equals 1 genomic equivalent X probe assay and restriction fragment length polymorphism. The characteristics of the three quantitative HCV RNA assays among the patients in whom there was agreement on genotypic classification are shown in Table 2. The bdna 2.0 and RT-PCR assays detected all 11 patients with genotypes 2a, 2b, or 3a compared with only 5 of 11 (45%) by the first generation signal amplification assay (P=.0124, Fisher's exact test). Of the 174 serum samples, 110 were negative by the first generation bdna assay, of which 61 (55%) of 110 were positive with the bdna 2.0 assay and 55 (51%) of 108 were positive by the RT-PCR assay (Table 3). Discrepancies between the two more sensitive methods were observed in 32 of the 174 samples, including 18 (10%) in which the bdna 2.0 method was positive and 14 (8%) in which the opposite applied (Table 4). The HCV RNA values obtained by the bdna 2.0 and RT-PCR assays were plotted on a graph (Fig 3). Overall, the results reveal a persistent bias of the numbers generated by the RT-PCR method compared with those from bdna, with the RT-PCR values an average of 41-fold lower than the bdna values. This bias seemed to become larger as the RNA concentration increased (from about fivefold at bdna levels below 1 MEq/mL to about 50-fold at bdna levels around 100 MEq/mL), indicating a possible saturation effect in RT-PCR. Monitor HCV (copies per ml) 1X10% 1x10 s, 1x10% ixi os 1x1o 2 - o a Quantiplex* quantification limit ^ ~-^~ o o - f»?l o g o c? oo 0 o <> o i OS > 000 O 00 i i i 1111 i i i i i 11 < i i i i i i 1x10" 1X10' 1x10* Quantiplex HCV RNA 2.0 Assay (bdna) (genome equivalents per ml) O Monitor quantification limit FIG 3. Results of hepatitis C virus (HCV) RNA quantitation in 174 samples by branched nucleotide fl)dna) 2.0 and reverse transcriptase polymerase chain reaction (RT-PCR; Monitor, Roche Molecular systems, Nutley, NJ) assays. The solid line represents the identity line, where all determinations should fall if a perfect correlation between the two methods was achieved. RT-PCR numbers are an average 41-fold lower (range, 0 to 703-fold) than those reported by bdna. (Quantiplex, Chiron, Emeryville, Calif). DISCUSSION Access to a sensitive and reliable assay for HCV RNA is useful to the clinician for judging the likelihood of response to therapy with interferon-alfa and in determining whether a virologic response has Vol. 107 No. 3

5 366 MICROBIOLOGY AND INFECTIOUS DISEASE Article occurred. 8,11,23 Moreover, measurement of HCV RNA permits better understanding of the relationship between viral load and the natural history of chronic HCV infection. Differences in the detection rates among the available quantitative methods could be based on differences in the efficiency of hybridization of HCV RNA to complementary nucleotide sequences in these assays. 18 Therefore, we believe that comparison of two frequently used commercially available means of HCV RNA quantitation was important. From this study, it is apparent that the second generation branched DNA assay has much greater sensitivity compared with the first generation assay, which to date has been the source of much of the published data. Although multiple probes in the bdna 1.0 assay were designed to include the sequence variations present in the diverse HCV genotypes, hybridization efficiencies vary among HCV genotypes, causing this assay to have a small but consistent bias toward detection of the more prevalent genotypes la and lb. This is a potentially important finding because genotypes other than la and lb are found in nearly a third of patients with chronic hepatitis C in the United States and western Europe. 24 ' 25 For this reason, a modified set of oligonucleotide probes was used to enhance the efficiency of binding among the various genotypes in the bdna 2.0 assay. The data from the current study suggest that the bdna 2.0 assay provides a more equivalent quantification of HCV viremia with the different HCV genotypes, as indicated by the increased detection rate of this assay compared with the 1.0 version. These data agree with the findings of Detmer et al. 20 Our study suggests that genotypes other than la and lb are also better detected by RT-PCR, qualitatively, when compared with the bdna 1.0 assay. Despite the different quantification limits defined for the bdna (0.2 MEq/mL) and the RT-PCR (500 copies /ml) assays, the clinical sensitivity observed in this study was quite similar (91% and 92%, respectively). However, we observed that the values reported by these two assays on a given specimen were quite different, with the RT-PCR method reporting values an average 41-fold lower (range, 0 to 703- fold) than those obtained with the bdna 2.0 assay. These apparent differences could be explained by a difference in the unit definition of the two assays. Although the "gold standard" used in the assignment of the bdna standards has been well documented, 26 no report exists on the unit assignment of RT-PCR. It is also possible that the lower quantification values TABLE 3. bdna 2.0 AND RT-PCR ASSAY RESULTS ON SAMPLES THAT WERE NEGATIVE BY THE bdna 1.0 ASSAY* bdna 2.0 RT-PCR No. of samples analyzed Positive samples, No. (%) 61(55) 55(51) bdna = branched nucleotide assay; RT-PCR = reverse transcriptase polymerase chain reaction. Quantiplex HCV RNA 1.0 and 2.0 assays from Chiron, Emeryville, Calif; Monitor RT- PCR from Roche Molecular Systems, Nutley, NJ. TABLE 4. COMPARISON OF THE bdna 2.0 AND RT-PCR ASSAYS IN 174 SAMPLES* HCV RNA 2.0 RT-PCR Positive Negative Positive Negative bdna = branched nucleotide assay; RT-PCR = reverse transcriptase polymerase chain reaction; HCV = hepatitis C virus. *Quantiplex HCV RNA 1.0 and 2.0 assays from Chiron, Emeryville, Calif; Monitor RT- PCR from Roche Molecular Systems, Nutley, NJ. reported by RT-PCR were due to RNA loss during the sample extraction process required for RT-PCR, whereas serum samples are tested directly in the bdna procedure. Additional studies are needed to establish the quantification accuracy of RT-PCR. Previous studies have indicated that the Monitor RT-PCR assay is incapable of detecting more than 5 x 10 5 molecules of HCV RNA without a previous dilution step. 27 Taken together, this suggests that the bdna 2.0 assay may have practical advantages in a busy clinical laboratory when a quantitative rather than qualitative test is needed in a serum sample with high viral copy numbers as in immunosuppressed patients. While both methods can be used in other circumstances to monitor infection, select patients for therapy, and evaluate efficacy of treatment regimens, our study confirms that the two assays are not interchangeable and restriction to one method is necessary when interpreting serial results in individual patients. REFERENCES 1. Choo QL, Kiro G, Weiner AJ, et al. Isolation of a cdna clone derived from a blood borne non-a, non-b viral hepatitis genome. Science. 1989;244: Choo QL, Weiner AJ, Overby LR, et al. Hepatitis C virus: the major causative agent of viral non-a, non-b hepatitis. Br Med Bull. 1990;46:423^41. AJCP* 1997

6 JACOB ET AL 367 Hepatitis C RNA 3. Kubo Y, Takeuchi K, Boonmar S, et al. A cdna fragment of hepatitis C virus isolated from an implicated donor of post transfusion non-a, non-b hepatitis in Japan. Nucleic Acids Res. 1989;17: Choo QL, Richman KH, Han JH, et al. Genetic organization and diversity of the hepatitis C virus. Proc Natl Acad Sci USA 1991;88: Takamizava A, Mori C, Fuke I, et al. Structure and organisation of the hepatitis C virus genome isolated from human carriers./ Virol. 1991;65: Nakao T, Enomoto N, Takada N, Takada A, Dati T. Typing of hepatitis C virus genomes by restriction fragment length polymorphism. / Gen Virol. 1991;72: Simmonds P, Alberti A, Alter HJ, et al. A proposed system for the nomenclature of hepatitis C viral genotypes. Hepatology. 1994;19: Lau JYN, Davis GL, Kniffen J, et al. Significance of serum hepatitis C virus RNA levels in chronic hepatitis C. Lancet. 1993;341: Hayashi J, Ohmiya M, Kishihara N, et al. A statistical analysis of predictive factors of response to human lymphoblastoid interferon in patients with chronic hepatitis C. Am ] Gastroenterol. 1994;89: Matsumoto A, Tanaka E, Suzuki T, Ogata H, Kiyosawa K. Viral and host factors that contribute to efficacy of interferonalpha 2a therapy in patients with hepatitis C. Dig Dis Sci. 1994;39: Mita E, Hayashi N, Hagiwara H,, et al. Predicting interferon therapy efficacy from hepatitis C virus genotype and RNA titer. Dig Dis Sci. 1994;39: Magrin S, Craxi A, Fabiano C, et al. Serum hepatitis C virus (HCV)-RNA and response to alpha interferon in anti HCV positive chronic hepatitis. / Med Virol. 1992;38: Pozzato G, Kaneko S, Morretti M, et al. Different genotypes of hepatitis C virus are associated with different severity of liver disease. / Med Virol. 1994;43: Brechot C. Polymerase chain reaction: a new tool for the study of viral infections in hepatology. / Hepatol. 1990;11: Lin HJ, Hollinger FB, Mizokami M, Shi N. A polymerase chain reaction assay for hepatitis C virus RNA using a single tube for reverse transcription and serial rounds of amplification with nested primer pair, j Med Virol. 1992;3: Sanchez-Pescador R, Sheridan PJ, Detmer JJ, et al. Quantitative detection of HCV RNA sera using a chemiluminescent signal amplification oligonucleotide probe assay. Hepatology. 1992;16:588. Abstract. 17. Urdea MS, Horn T, Fultz TJ, et al. Branched DNA amplification multimers for the sensitive, direct detection of human hepatitis viruses. Nucleic Acids Res. 1991;24: Zaaijier HL, Cuypers HT, Reesnik WH, et al. Reliability of polymerase chain reaction for detection of hepatitis C virus. Lancet. 1993;341: Simmonds P, Zhang LQ, Watson HG, et al. Hepatitis C quantification and sequencing in blood products, haemophiliacs and drug users. Lancet. 1990;336: Detmer J, Lagier R, Flynn J, et al. Accurate quantification of hepatitis C virus (HCV) RNA from all HCV genotypes by using branched-dna technology. / Clin Microbiol. 1996;34: Stuyver L, Rossau R, Wyseur A, et al. Typing of hepatitis C virus isolates and characterization of new subtypes using a line probe assay. / Gen Virol. 1993;74: McOmish F, Yap PL, Dow BC, et al. Geographical distribution of hepatitis C virus genotypes in blood donors: an international collaborative study. / Clin Microbiol. 1994;32: Davis GL. Predictors of response to interferon treatment of chronic hepatitis C. I Hepatol. 1994;21: Lau JYN, Mizokami M, Kolberg JA, et al. Application of six hepatitis C virus genotyping systems to sera from chronic hepatitis C patients in the United States. / Infect Dis. 1995;171: Dusheiko G. Genetic diversity of hepatitis C virus: implications for pathogenesis, treatment, and prevention. Lancet. 1995;345: Collins ML, Zayati C, Detmer JJ, et al. Preparation and characterization of RNA standards for use in quantitative branched DNA hybridization assays. Anal Biochem. 1995;226: Roth KW, Lee JH, Ruster B, Zeuzem S. Comparison of two quantitative hepatitis C virus reverse transcriptase PCR assays. / Clin Microbiol. 1996;34: Vol. 107 No. 3