Evaluation of a Novel Serotyping System for Hepatitis C Virus: Strong Correlation with Standard Genotyping Methodologies

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1995, p Vol. 33, No /95/$ Copyright 1995, American Society for Microbiology Evaluation of a Novel Serotyping System for Hepatitis C Virus: Strong Correlation with Standard Genotyping Methodologies V. DIXIT, 1 * S. QUAN, 2 P. MARTIN, 1 D. LARSON, 2 M. BREZINA, 1 R. DINELLO, 2 K. SRA, 2 J. Y. N. LAU, 3 D. CHIEN, 2 J. KOLBERG, 2 A. TAGGER, 4 G. DAVIS, 3 A. POLITO, 2 AND G. GITNICK 1 Division of Digestive Diseases, UCLA School of Medicine, Los Angeles, California ; Chiron Corporation, Emeryville, California ; Section of Hepatobiliary Diseases, University of Florida College of Medicine, Gainesville, Florida ; and Istituto di Virologia, University of Milan, Milan, Italy 4 Received 3 April 1995/Returned for modification 30 May 1995/Accepted 22 August 1995 Direct sequencing and analysis of viral genomes are definitive methods for identifying various hepatitis C virus (HCV) genotypes. However, HCV genome sequencing methods are cumbersome and unsuitable for analyzing large numbers of clinical samples. We have developed a convenient, reliable, and reproducible RIBA strip immunoblot assay system for determining HCV serotype. Briefly, the assay consists of an immunoblot strip on which there are five lanes of immobilized serotype-specific HCV peptides from the nonstructural (NS-4) and core regions of the genomes of HCV types 1, 2, and 3. HCV serotype is deduced by determining the greatest intensity of reactivity to the NS-4 serotype-specific HCV peptide band in relation to the intensity of the human immunoglobulin G internal control bands on each strip. HCV core peptide reactivity is used only in the absence of NS-4 reactivity. We used this assay to successfully serotype a high percentage of sera from well-documented HCV-infected patients. Our serotyping results correlated 99% with the findings from the standard restriction fragment length polymorphism genotyping methods. Less than 5% of the serum samples were untypeable. For a selected group of alpha interferon-treated patients we observed that the nonresponders (76.2%) and a majority of the responders who relapsed (72.2%) had type 1 HCV infection. A small population (n 8) of complete responders was split 3:4:1 as type 1, type 2, and type 3, respectively. Our data indicate that this new serotyping assay has the potential to be a highly specific and reliable method for typing of HCV infection in patients. Choo et al. s identification and mapping of the hepatitis C virus (HCV) genome (1) was the first step in the development of reliable and accurate diagnostic assays for HCV infection. The earliest HCV assays had a serious drawback, i.e., a high rate of occurrence of false-negative and -positive results (12). Supplemental assays such as the RIBA (Chiron Corporation) HCV 2.0 or 3.0 strip immunoblot assay (SIA) are usually necessary to discriminate between true and false positives for repeatedly reactive in anti-hcv enzyme-linked immunosorbent assays (ELISAs) (14). Recent studies have shown that HCV exhibits considerable structural heterogeneity (9, 11). Nucleic acid sequence analyses of HCV isolates from different parts of the world have revealed an overall nucleic acid sequence diversity of 30% (9, 11). Such analyses have identified at least six well-characterized HCV genotypes worldwide, numbered 1 to 6 (8, 10). Within each genotype, closely related strains or subtypes are identified by subscripts a, b, etc. There are some important geographical variations in genotype prevalence. Genotypes 1a, 1b, 2a, 2b, and 3a are the most common genotypes found in the United States and Western Europe, whereas 1b, 2a, and 2b are most frequently found in Japan and Taiwan. Type 3 is most prevalent in India, Bangladesh, and other parts of Asia. Type 4 is found most commonly in the Middle East, type 5 is found most commonly in South Africa, and type 6 is found most commonly in Hong Kong and Macau (2, 6, 10). We have performed serial monitoring of HCV patients with the RIBA HCV 2.0 SIA and have observed some interesting * Corresponding author. Mailing address: UCLA School of Medicine, Department of Medicine, 675 Circle Dr. South, MRL Room 1240, Los Angeles, CA Phone: (310) Fax: (310) patterns in the intensity of reactivity to the recombinant antigens on the RIBA strips. Samples from some patients showed high-level reactivity to all four antigens, while those from other patients reacted weakly or not at all to some antigens and strongly to others. Some have postulated that this variability in RIBA HCV antigen reactivity may be due to HCV heterogeneity or to the viral load (3, 15). Furthermore, some HCV patients showed diminished antibody reactivity to certain antigens as a consequence of their response to the alpha interferon (2, 6). It has been suggested that variations in RIBA HCV antigen reactivity among alpha interferon responders or nonresponders may correspond to heterogeneity of HCV strains or to differences in viral load (6, 10, 15). Preliminary HCV genotyping analyses by different groups worldwide suggest that HCV type 1 is the least responsive to alpha interferon therapy (5), while HCV types 2 and 3 appear to respond best to alpha interferon therapy (6, 10, 13). HCV types 4 to 6 are less well studied and are probably found in only a very small percentage of U.S. patients (4). Current methodologies for determining HCV genotypes are complex and impractical for routine clinical use. They require time-consuming comparison of HCV nucleic acid sequences (6, 10). Their reliability may be further compromised if viral RNA is lost in the serum or plasma through storage or improper laboratory handling or if it is absent in the blood circulation during sample collection. A convenient, reliable, and reproducible serological test for determining HCV serotype would be of great clinical benefit. It might allow us to predict disease severity, prognosis, and likelihood of response to antiviral agents such as alpha interferon. We have developed a serotyping strip based on RIBA methodology that distinguishes HCV serotypes 1, 2, and 3. Briefly, the RIBA HCV serotyping strip is different from the RIBA HCV 2.0 or 3.0 SIA 2978

2 VOL. 33, 1995 HCV SEROTYPING BY RIBA SIA METHODOLOGY 2979 FIG. 1. Chiron RIBA HCV serotyping SIA strip diagram. strip in that instead of incorporating recombinant HCV antigens from the NS-4 and core regions of the virus, the serotyping strip contains five different serotype-specific peptide sequences taken from the NS-4 regions and two peptide sequences taken from serotype-specific peptide segments of the core regions of the HCV genomes for types 1, 2, and 3. A detailed description of the serotyping strip is provided below in Materials and Methods. When used in combination with RIBA HCV 2.0 and 3.0 SIAs, the new serotyping strip offers a powerful tool for reliably differentiating the type of HCV infection. We report our preliminary results obtained by using this newly developed serotyping strip to distinguish HCV serotypes 1, 2, and 3. MATERIALS AND METHODS Chiron RIBA SIA HCV serotyping assay. The new Chiron RIBA SIA system, as shown in Fig. 1, consists of a nitrocellulose solid support on which are immobilized five lanes of serotype-specific HCV peptides (lanes 2 to 6) and two lanes of high- and low-level immunoglobulin G (IgG) controls (lanes 1 and 7, respectively). The IgG controls are used to assess the level of RIBA SIA reactivity. The remaining lanes are coated with HCV peptides as follows: Lane 2 contains two synthetic peptides from the NS-4 regions of HCV subtypes 1a and 1b. Lane 3 contains two synthetic peptides from the NS-4 regions of subtypes 2a and 2b. Lane 4 contains a peptide with the consensus sequences from the NS-4 region of subtypes 3a and 3b. Lane 5 contains a peptide synthesized from consensus sequences of the core regions of the HCV subtypes 1a and 1b. Lane 6 contains one peptide from the consensus sequence of the core regions of HCV subtypes 2a and 2b. The core peptides 1a and 1b are identical, as are 2a and 2b. The procedure for performing the serotyping assay is similar to previous RIBA HCV assays with the following modifications. Briefly, 20 l of each specimen to be serotyped is added to a tube containing a serotyping strip and 1 ml of RIBA HCV 3.0 SIA specimen diluent that contains a peptide (20 g/ml) from the nonspecific sequences of the NS-4 region common to both HCV types 1 and 3. The remainder of the assay procedure is exactly as for the RIBA HCV 3.0 assay: a 4-h incubation with the specimen at room temperature, followed by a 30-min incubation in specimen diluent, two washes in wash buffer, a 10-min incubation in conjugate followed by three washes in wash buffer, and a 15-min incubation in substrate followed by two washes with deionized water. Algorithm for HCV serotyping. The band reactivity pattern on the serotyping strip is interpreted by comparing the intensity of each serotype-specific HCV peptide band with the intensities of the human IgG (level I and level II) internal control bands on each strip (Fig. 1). The intensity of the HCV peptide bands is scored in relation to the intensities of the internal IgG controls. An HCV peptide band intensity equal to the intensity of the level I IgG control band is scored as 1. Band intensity greater than that of level I IgG but less than that of level II IgG is scored as 2. Band intensity equal to that of level II IgG is scored as 3. Band intensity greater than that of level II IgG is scored as 4. Band intensity less than that of the level I IgG control band is scored as, and the absence of any band reactivity is scored as. HCV serotype is determined primarily on the basis of 1 or greater reactivity to the NS-4 serotype-specific HCV peptide bands (Fig. 2). HCV core peptide reactivity of 1 or greater is used only in the absence of NS-4 reactivity. For example, if the greatest NS-4 band reactivity is to the NS-4 (1a plus 1b) band, the interpretation is serotype 1; if the greatest NS-4 reactivity is to the NS-4 (2a plus 2b) band, the interpretation is serotype 2; and if the greatest NS-4 band reactivity is to the NS-4 (3) band, the interpretation is serotype 3 (regardless of the HCV core peptide reactivity). In the absence of NS-4 reactivity, the core reactivity is used to determine serotype as follows. (i) If the greatest core reactivity is to the core 1 peptide band, the serotype is either 1 or 3 (i.e., there is cross-reactivity between type 1 and type 3 HCV antibody to the core peptide, making it impossible to distinguish between the two types in the absence of NS-4 reactivity). (b) If the greatest core reactivity is to the core 2 antigen band, the serotype is 2. In the presence of equal reactivities to two or more NS-4 bands, or to the two core antigen bands, dilution of the specimen, followed by reinterpretation of the results, is required. Approximately 6% of the tested (n 975) required dilution for the serotype assay. A 1:10 dilution is usually used for showing identical reactivities towards the NS-4 peptides, and a 1:2 dilution is usually used for showing identical reactivities towards the core peptides. It is conceivable that coinfection by more than one genotype is possible. However, this serotyping assay provides information on the predominant serotype. The rare specimen that shows no antibody reactivity to the serotyping peptides is untypeable. The algorithm for HCV serotyping is presented in Fig. 2. Study. Specimens previously assayed with the RIBA HCV 2.0 SIA as positive or indeterminate were obtained from the Chiron reference laboratory and used to evaluate the new HCV serotyping assay. These were obtained from a population of blood donors (n 5,543). Additional serum from alpha interferon-treated patients were obtained from the University of California, Los Angeles, and the University of Florida, Gainesville, with Human Subject Protection Committee approved consent. All tested were well documented and were collected and stored according to an established protocol to prevent contamination and degradation. Genotype verification of serotyped samples. Genotype verification of the results generated by the Chiron RIBA HCV serotyping assay was performed by restriction fragment length polymorphism (RFLP) or line probe assay (LiPA) analysis. RFLP techniques have been described elsewhere in detail (7). Briefly, HCV typing was carried out after amplification of extracted HCV RNA by a nested PCR assay. The primers were deduced from well-conserved areas of the 5 untranslated region of the HCV genome. For RFLP analysis, PCR amplicons were first generated from the 5 untranslated region. The amplicons were digested with restriction enzymes HaeIII-RsaI and MvaI-HinfI to categorize HCV into various genotypes. Subtypes 1a and 1b were further distinguished by restriction digestion with BstUI. Restriction digestion with ScrFI was used to differentiate between 2a and 2b and between 3a and 3b. An LiPA was performed according to the manufacturer s instructions (Innogenetics, Zwijnaarde, Belgium) as described elsewhere (5). Briefly, first-round PCR products were amplified with PCR primers 5 end labeled with biotin and homologous to conserved regions in the 5 untranslated region of the HCV genome. Two microliters of the first-round product used for RFLP-based genotyping was used as a template for further amplification for LiPA typing. Hybridized PCR products were then detected by means of streptavidin-alkaline phosphatase immunochemical study using nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate as substrates. The LiPA strips were then analyzed, and the genotype was assigned according to the pattern of PCR product hybridization. Statistical analysis. Percentages were compared by means of Fisher s exact test. Differences were considered significant if the P value was 0.05 or less. FIG. 2. Algorithm for HCV serotyping.

3 2980 DIXIT ET AL. J. CLIN. MICROBIOL. TABLE 1. Correlation between RIBA HCV serotyping results and genotyping results for previously genotyped samples a Genotype samples of RIBA HCV serotype b : or3 RESULTS samples untypeable c 1 52 (55.9) 3 (3.2) 2 (2.2) 2 1 (1.1) 15 (16.1) 2 (2.2) 3 16 (17.2) 2 (2.2) a n 93. b A total of 84 (90.3%) of 93 samples were serotyped, and 99% agreement with genotype results was achieved. Five (5.4%) of the 93 samples were serotype 1 or 3. c A total of 4 (4.3%) of 93 samples were untypeable. Correlation between RIBA HCV serotyping and genotyping results. The RIBA HCV serotype results for 93 were compared with RFLP-derived and/or LiPA genotype findings. The results are presented in Table 1. We were able to reliably serotype 84 of 93 (90.3%). For these 84 we obtained a 99% agreement with the genotype data. Of the 84 HCV serotyped, 55.9% were type 1, 16.1% were type 2, and 17.2% were type 3. Only 4.3% of the (4 of 93) were untypeable by the serotyping method (Table 1). RIBA HCV 2.0 SIA band patterns for anti-hcv-positive. The distribution of band pattern reactivity for 5,543 RIBA HCV 2.0 SIA positive or indeterminate is shown in Table 2. In order to resolve the approximately 32% RIBA HCV 2.0 SIA indeterminates in this large cohort, these were further analyzed with a more sensitive and specific RIBA HCV 3.0 SIA. This assay eliminated many of the samples that were found to be indeterminate by RIBA HCV 2.0 SIA, yielding a cohort of 3,462 that were either positive or indeterminate by RIBA 3.0 SIA. The distribution of RIBA HCV 2.0 SIA band patterns for these HCV is presented in Table 3. Forty-eight percent of the 3,462 RIBA HCV 3.0 SIA-positive or -indeterminate were found to be highly positive (four-band reactivity) for HCV antibodies. The proportions of RIBA HCV 3.0 SIA-positive or -indeterminate that were three-band, two-band, and oneband (c22) positive were 9, 33, and 10%, respectively. Specimens from each of the four-, three-, two-, and one-band (RIBA HCV 2.0 c22-indeterminate and RIBA HCV 3.0 c22- indeterminate or -positive) groups were selected for serotype analysis using the new RIBA HCV serotyping strip. The results are presented below. Serotyping of four-band-positive RIBA HCV 2.0 SIA. Forty-eight four-band-positive serum samples were randomly selected from our specimen inventory and serotyped by RIBA HCV 2.0 SIA band pattern TABLE 2. Distribution of RIBA HCV 2.0 SIA band patterns in blood donor Four band...1,644 (30) Three band (5) Two band...1,158 (21) One band (c22)...1,786 (32) Other (12) Total...5,543 TABLE 3. Distribution of RIBA HCV 2.0 SIA band patterns in found to be positive or c22 indeterminate by RIBA HCV 3.0 SIA RIBA HCV 2.0 SIA band pattern Four band...1,644 (48) Three band (9) Two band...1,158 (33) One band (c22) (10) Total...3,462 the assay that we developed. This cohort of RIBA HCV 2.0 SIA four-band-positive sera represented approximately 48% of the total RIBA HCV 3.0 SIA-positive or -indeterminate patient population in this study (Table 3). Using the serotyping strip, we successfully typed 96% of these (Table 4). Only 4% of the were untypeable. These 4% untypeable four-band-positive represent approximately 4% of the 48% four-band-positive HCV or approximately 1.92% of the total cohort of RIBA HCV 3.0 SIApositive or -indeterminate in this study (Table 3). Of the serotyped, the majority, 83.3%, were serotype 1. Serotypes 2 and 3 comprised 2.1 and 10.4%, respectively (Table 4). Serotyping of three-band-positive RIBA HCV 2.0 SIA. Thirty-four three-band-positive serum samples from our specimen inventory were randomly selected for serotyping. This cohort of RIBA HCV 2.0 SIA three-band-positive sera represented approximately 9% of the total RIBA HCV 3.0 SIA-positive or -indeterminate patient population in this study (Table 3). Using the serotyping strip, we successfully typed 94% of these (Table 4). The 6% untypeable threeband-positive represent approximately 6% of the 9% three-band-positive HCV or approximately 0.54% of the total cohort of RIBA HCV 3.0 SIA-positive or -indeterminate in this study (Table 3). Of the serotyped, the majority, 70.6%, were serotype 1. Serotypes 2 and 3 comprised 5.9 and 17.7%, respectively (Table 4). Serotyping of two-band-positive RIBA HCV 2.0 SIA. We serotyped sixty two-band-positive RIBA HCV 2.0 SIA from our specimen inventory. This cohort of RIBA HCV 2.0 SIA two-band-positive sera represented approximately 33% of the total RIBA HCV 3.0 SIA-positive or -indeterminate in our study (Table 3). We were successful in serotyping 57% of the (Table 4). Seventeen percent of the samples were serotyped as either type 1 or type 3. Twenty-seven percent of the total serum TABLE 4. HCV serotype distribution for having different RIBA HCV 2.0 SIA band patterns RIBA HCV 2.0 band pattern of serotype: or 3 untypeable Four-band positive a 40 (83.3) 1 (2.1) 5 (10.4) 0 (0) 2 (4.2) Three-band positive b 24 (70.6) 2 (5.9) 6 (17.7) 0 (0) 2 (5.9) Two-band positive c 13 (21.7) 14 (23.3) 7 (11.7) 10 (16.7) 16 (26.7) One-band positive 6 (14.3) 1 (2.4) 9 (21.4) 3 (7.1) 23 (54.8) (c22 indeterminate) d a n 48. b n 34. c n 60. d n 42.

4 VOL. 33, 1995 HCV SEROTYPING BY RIBA SIA METHODOLOGY 2981 Patient group TABLE 5. Interferon treatment Genotype of RIBA HCV serotype: or 3 Sustained responders a 1 3 (37.5) 2 4 (50.0) 3 1 (12.5) untypeable Nonsustained responders b 1 13 (72.2) 1 (5.6) 2 1 (5.6) 1 (5.6) 3 2 (11.1) Nonresponders c 1 16 (76.2) 1 (4.8) 1 (4.8) 2 2 (9.5) 3 1 (4.8) a n 8. Genotype concordance, 8 of 8 (100%); number of serotyped, 8 of 8 (100%). b n 18. Genotype concordance, 16 of 17 (94%); number of serotyped, 17 of 18 (94%). c n 21. Genotype concordance, 19 of 19 (100%); number of serotyped, 19 of 21 (90%). in this group were untypeable. However, these 27% untypeable (16 of 60 patients) represent approximately 27% of the 33% of the HCV patients with two-band-positive sera or 8.9% of the total cohort of HCV patients in this study (Table 3). Most of the 57% of the that were successfully serotyped were type 1 (21.7%) and type 2 (23.3%), and only 11.7% were type 3 (Table 4). Serotyping of c22-indeterminate RIBA HCV 2.0 SIA. We tested 42 RIBA HCV 2.0 SIA c22-indeterminate with the new serotyping strip. This cohort of RIBA HCV 2.0 SIA c22-indeterminate and RIBA HCV 3.0 SIA c22-indeterminate or -positive represented approximately 10% of the RIBA HCV 3.0 SIA-positive or -indeterminate (Table 3) in this study. Using the serotyping strip, we successfully typed 38% of these c22-indeterminate. Nearly 55% of the in this group were untypeable. However, it should be noted that these 55% represent approximately 55% of the 10% RIBA HCV 2.0 c22- indeterminate and RIBA HCV 3.0 c22-positive or -indeterminate HCV or 5.5% of the total cohort of HCV in this study (Table 3). Of those serotyped, 14.3% were type 1, 2.4% were type 2, and 21.4% were type 3. Seven percent of the were serotyped as either HCV type 1 or HCV type 3 (Table 4). HCV serotyping of from alpha interferon-treated patients. We serotyped from eight patients who were sustained responders to alpha interferon treatment. We found that 37.5% of this cohort were HCV serotype 1, 50% were HCV type 2, and 12.5% were HCV type 3 (Table 5). When this serotype analysis was verified by standard RFLP genotyping methods, 100% concordance was obtained (Table 5). A cohort of 18 patients who responded to alpha interferon treatment but relapsed after termination of treatment was also studied. We were able to reliably serotype the from 17 (94%) of these 18 patients. The majority were HCV type 1 (72.2%). HCV types 2 and 3 comprised 5.6 and 11.1% of this population, respectively (Table 5). When HCV serotyping results were verified by RFLP genotyping analysis, we found 94% concordance with our results. We also studied 21 individuals who did not respond to alpha interferon treatment. The serotyping results are presented in Table 5. We were able to successfully serotype the from 19 (90%) of these 21 patients with a 100% concordance with RFLP-determined genotypes. The from the majority (76.2%) of these nonresponders to alpha interferon treatment were serotyped as HCV type 1. Small percentages (9.5 and 4.8%) of the were serotyped as HCV types 2 and 3, respectively. The specimen from only 1 (5%) of 21 patients was typed as being either HCV type 1 or HCV type 3, and the specimen from only 1 patient (5%) was untypeable. DISCUSSION Direct sequencing of viral RNA is currently considered the definitive method for HCV genotyping analysis (10). However, application of this technique is not practical in a clinical setting (6). RFLP and LiPA analyses are less cumbersome but still require selective PCR amplification of the HCV genome. Despite their wide use, PCR methodologies are not standardized, and the results may be affected by poor specimen collection and storage (16). Thus, the complexity of existing HCV genotyping assays limits their applicability outside a research setting. We here describe a novel method that uses RIBA HCV SIA methodology for serological assessment of HCV genotypes, which is referred to as serotyping in this paper. Essentially, RIBA HCV SIA methodology detects antibodies to individual HCV proteins and/or peptides. In the present RIBA HCV serotyping assay, highly serotype-specific peptides from the NS-4 and core regions of HCV types 1, 2, and 3 are immobilized on a nitrocellulose strip (Fig. 1), where they may react with antibodies in the patient s serum. Antibodies to these specific HCV peptides bind to the RIBA strip to form a dark band at the site of the IgG antibody-antigen complex. The RIBA strip is then interpreted for HCV serotype according to the algorithm illustrated in Fig. 2. The RIBA HCV SIA methodology is highly reproducible and reliable. The RIBA HCV 2.0 SIA has been established as the supplemental assay for confirming the presence of HCV antibodies in found to be repeatedly reactive in HCV screening ELISAs. This convenient methodology easily lends itself to automation and can be used to test large numbers of patient samples. To evaluate the accuracy of our RIBA HCV serotyping assay, we compared our results with genotyping results, as obtained by the RFLP and LiPA methodologies, using from patients who were clinically and/or serologically confirmed as having HCV infection. We tested a total of 93 HCV serum samples and were able to reliably serotype 84 (90.3%). Five were serotyped as either 1 or 3, and only four were untypeable. We observed a 99% concordance between our serotyping data (Table 1) and the RFLP and/or LiPA genotyping data obtained from the same set of. Thus, our serotyping results were as reliable as those of the more cumbersome genotyping analysis. Our serotyping was also far easier to perform, requiring no special preparation of the sera. During the course of our ongoing studies of HCV, we noticed that sera from normal blood donor populations, which tested positive by anti-hcv ELISA, showed variable antigen reactivity in the RIBA HCV 2.0 SIA. We observed RIBA HCV 2.0 SIA patterns that ranged from highly positive (four-band) reactivity to indeterminate (one-band [c22]) reactivity. Therefore, a large cohort of blood donor serum that were positive or indeterminate in the RIBA HCV 2.0 SIA were studied. Since this second-generation RIBA confirmatory assay can result in false-positive values, especially for indeterminate with weak antibody reactivities, we further subjected our cohort to third-generation RIBA HCV 3.0 SIA

5 2982 DIXIT ET AL. J. CLIN. MICROBIOL. analysis. The third-generation RIBA HCV 3.0 SIA is highly sensitive and specific, and we used it to exclude samples that could be falsely positive for HCV. Of this final cohort of RIBA HCV 3.0-positive or -indeterminate nearly half (48%) were highly positive (four-band reactivity), 9% were three-band positive, 33% were two-band positive, and 10% were indeterminate (one-band [c22] positive) by the RIBA HCV 2.0 SIA. This interesting distribution of RIBA HCV 2.0 SIA band patterns (ranging from highly positive to indeterminate) for HCV-infected sera led us to hypothesize that such variation in RIBA HCV 2.0 SIA reactivity might be due to the variability in the HCV genotypes, as others have reported (7). Accordingly, we examined the correlation between RIBA HCV 2.0 SIA reactivity and HCV serotype. As seen in Table 4, a large proportion of RIBA HCV 2.0 SIA four-band-positive samples were serotype 1, compared with lower proportions of samples showing three-, two-, or one-band reactivity. If we consider serotype 1 as a group, a large proportion of these HCV-positive blood donor showed four-band reactivity versus serotypes 2 and 3 over the entire study (P 0.01). We also examined the correlation between RIBA HCV 2.0 reactivity and serotype for RIBA HCV 2.0 SIA one- and twoband-positive samples. Here, in contrast to the situation with four-band-positive samples, a much larger proportion were serotype 2 or serotype 3. Since the HCV 2.0 SIA was developed on the basis of HCV genotype 1a antigens, it is conceivable that genetic heterogeneity of HCV has an impact on the utility of such diagnostic tests. As the majority of patients in the United States are infected with genotype 1 (4) and the core antigen used in the assay is conserved across the different genotypes, the magnitude of this problem should not be great and the current algorithm interpretation should not be changed. However, the problem does pose challenges to the development of better serological assays to cover the heterogeneity of the HCV genome. Even though large proportions of the two- and one-band samples in the RIBA HCV SIA were untypeable, it is still valid to conclude that for two-band reactives, the serotype distribution is approximately 2:2:1 for serotypes 1, 2, and 3, respectively, for the that can be serotyped. As for the one-band-reactive, the distribution of the serotypes is mostly between serotypes 1 and 3 for the that can be serotyped. The clinical implication of this observation is that one should suspect a certain percentage of serotype 2 or 3 HCV infection if the patient is RIBA HCV 2.0 SIA two- or one-band reactive, since serotypes 2 and 3 have been suggested to respond to alpha interferon therapy better than serotype 1. In summary, the total percentage of untypeable in our cohort of 150 blood donor that were either RIBA HCV 3.0 SIA positive or c22 indeterminate was approximately 17% (i.e., 2% four-band-positive plus 0.5% three-band-positive plus 9% two-band-positive plus 5.5% one-band-positive ). For this group we were able to serotype 83.14% of the total number of in our study. The large number of untypeable blood donor, especially among the two-band-positive and one-band-indeterminate, may be due to the absence of antibodies towards the serotype peptides in this population of asymptomatic. In contrast, when we examined a cohort of 93 from well-documented HCV-positive patients who normally have higher levels of circulating HCV antibodies, we found that we were able to serotype approximately 90% of the. In this group the proportion of untypeable was approximately 4%. Furthermore, these serotyping results were found to be 99% concordant compared with results from definitive genotyping methodology (Table 1). Thus, this assay is a reliable tool for accurately serotyping HCV in both blood donors and patients with chronic HCV infection (3, 6). In our examination of the serotype and genotype distribution for HCV patients undergoing alpha interferon therapy, we found that the majority (50%) of sustained responders were serotyped as having HCV type 2 infection. HCV serotype 1 was found in 37.5% of the patients, and a small percentage of patients (12.5%) was determined to have HCV serotype 3. For this group of sustained responders, we successfully serotyped from all eight patients (100%) and RFLP genotype evaluation showed a 100% concordance with our serotyping data (Table 5). In a cohort of nonsustained responders, from the majority of patients (72.2%) were serotyped as HCV type 1. The specimen from one patient (5.5%) was serotyped as HCV type 2, and those from 2 of 18 patients (11.1%) were serotyped as type 3. For this group we successfully serotyped from 17 of 18 patients (94%), and the genotype evaluation was 94% in concordance with the serotyping results (Table 5). For 21 nonresponders to alpha interferon therapy, the majority (76.2%) of the were determined to be HCV serotype 1. Two (9.5%) were classified as serotype 2, and one (4.8%) was classified as serotype 3 (Table 5). Only one specimen (4.8%) in this group was classified as either serotype 1 or serotype 3, and one (4.8%) was untypeable. Thus, we successfully serotyped from 19 of 21 patients (90%), with a concordance of 100% with genotyping. Overall, the serotype and genotype results showed a high degree of concordance for these patients. Our results also agree with previous assessments of the distribution of various HCV genotypes among responders and nonresponders to alpha interferon therapy (2, 6, 15). In addition to viral load, several investigators have reported the clinical implications of specific viral genotype in determining disease severity and responsiveness to alpha interferon therapy (2, 6, 10). For example, HCV type 1b, a prevalent HCV genotype in the Western world, is associated with cirrhosis and hepatocellular carcinoma (6). HCV type 1b infection is also reported to be associated with a longer disease duration and with nonresponsiveness to alpha interferon therapy (6). Our data for alpha interferon responders support this observation. With concurrent HCV genotype 1 and serotype 1 (Table 5) we found that only 3 (9.4%) of 32 patients were sustained responders to alpha interferon treatment. These studies suggest that the RIBA HCV serotyping SIA may be of value in predicting a patient s responsiveness to alpha interferon. In conclusion, we have developed a simple and accurate assay for HCV serotyping. The serotyping results obtained were in close concordance with results of genotype evaluation by established RFLP and/or LiPA methodologies. Further evaluation of this novel serotyping assay in other laboratories is now necessary for its validation as a useful tool for assessing the effectiveness of therapy in HCV patients. This test should prove useful in providing the epidemiological data to monitor the spread and natural history of HCV infection. REFERENCES 1. Choo, Q.-L., G. Kuo, A. J. Weiner, L. R. Oberby, D. W. Bradley, and M. Houghton Isolation of cdna derived from a blood-borne non-a, non-b hepatitis genome. 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