L E Prescott

Distribution of Hepatitis C Virus Genotypes Determined by Line Probe Assay in Patients with Chronic Hepatitis C Seen at Tertiary Referral Centers in the United States

Hepatitis C virus (HCV) has a high spontaneous mutation rate with an estimated frequency of 1.4 to 1.9 103 mutations per nucleotide per year [1-3]. As a result, HCV exists as a heterogeneous group of viruses sharing approximately 70% homology. On the basis of nucleotide sequence homology, HCV has been classified into six major genotypes and a series of subtypes. A proposed consensus system for nomenclature, based on sequence homology in at least two regions with confirmation by phylogenetic tree analysis, has been adopted by most HCV investigators [4]. The relation between this nomenclature and other commonly used nomenclature systems is shown in Table 1. Table 1. Commonly Used Hepatitis C Virus Genotype Nomenclature Systems* Various methods have been used for HCV genotyping, including genomic amplification and sequencing [10, 14-16], polymerase chain reaction (PCR) with genotype-specific primers [5, 8], restriction fragment length polymorphism of the PCR amplicons [13, 17], differential hybridization [18], and serologic genotyping [11, 19]. Genomic amplification and sequencing, followed by sequence comparison and phylogenetic tree construction for confirmation, is currently considered the gold standard; the genomic regions commonly used for this approach include the HCV core region, envelope 1, and nonstructural region 5 (NS5). This method, however, is expensive and labor intensive, making it difficult to study HCV genotypes in a large number of patients. Several alternative methods have been proposed. Okamoto and colleagues [5] and Chayama and coworkers [8] have described PCR done with genotype-specific primers derived from the HCV core region and from NS5, respectively. These methods rely on the use of genotype-specific primers that anneal to sequences unique to specific HCV genotypes. Genotype-specific primers derived from the HCV core region allowed the differentiation of HCV types 1a, 1b, 2a, and 2b [5]; genotype-specific primers derived from NS5 allowed the differentiation of HCV types 1a, 1b, 2a, 2b, and 3b. Restriction fragment length polymorphism of the PCR amplicons relies on the presence of unique and genotype-specific nucleotide substitutions that are recognized and digested by restriction enzymes into fragments that can be separated be gel electrophoresis [13, 17]. The 5 untranslated region (5 UTR) is commonly used for this approach. Hybridization of PCR amplicon mounted on a solid phase using genotype-specific probes has also been used for HCV genotyping [18]. Finally, serologic approaches have also been used to determine HCV genotypes. These approaches rely on the different amino acid sequences encoded by different nucleotide sequences from different HCV genotypes. Patients infected with different HCV genotypes may therefore have antibodies directed to genotype-specific amino acid sequences. Polypeptides and synthetic peptides derived from nonstructural region 4 (NS4), as well as synthetic peptides derived from the HCV core region, have been used for serologic genotyping [11, 19]. These peptides are used to coat enzyme immunoassay plates, and specific binding by a serum specimen is determined by peptide competition. The HCV genotypes assigned by this method are commonly referred to as serotypes. Conventionally, the term HCV serotype refers to different viral types determined by a panel of cross-neutralization antibodies. Because recognition of genotype-specific peptides by a patients antibody response reflects the difference in amino acid sequences and thus nucleotide sequences, the results of this approach are more appropriately referred to as serologically defined genotypes or serologic genotypes. The concordance among these different HCV genotyping methods has been questioned. In a previous study [20], we compared six different genotyping methods and found that most of them had high concordance with each other. The only method that we identified as unsuitable for U.S. patients was PCR with genotype-specific primers derived from the HCV core region, proposed by Okamoto and colleagues [11]. This method gave false-positive signals for HCV type 1b in patients who were actually infected with types 1a and 3a [20]. Because all molecular biological genotyping methods rely on PCR as a first step, those systems based on the 5 UTR, the most conserved genomic region, should be the most sensitive. Recently, a new genotyping system based on reverse hybridization of the labeled PCR amplicon derived from the 5 UTR (line probe assay, INNO-LiPA HCV, Innogenetics, Ghent, Belgium) was developed and has been used widely by investigators in Europe [21, 22]. Whether this line probe assay is also useful in patients with chronic HCV infection in the United States is unknown. In this study, we sought to 1) assess the concordance of this line probe assay with other assays on a panel of well-characterized serum specimens to verify the line probe assays validity; 2) determine the distribution of various HCV genotypes in a large sample of patients with chronic hepatitis C seen in tertiary referral centers in the United States; and 3) evaluate the clinical characteristics of patients infected with different HCV genotypes. Methods Patients We studied 438 patients from three different groups (Table 2). All patients were from the United States, had chronic hepatitis C, and were seen in tertiary referral centers. Most had been referred by their physicians or by a gastroenterologist or hepatologist for inclusion in experimental antiviral therapy programs. The first group consisted of 137 patients with chronic HCV infection whose serum specimens had previously been characterized with six different genotyping methods [20]. The specimens of these patients were studied to verify the validity of the line probe assay. The second group consisted of 248 patients from nine centers in the United States who had participated in a randomized, controlled study of interferon- therapy. One of these 248 patients was negative for the antibody to HCV (anti-HCV), and another had no serum specimen available, which left a total of 246 patients for this study. A careful review of the serum bank showed that 40 patients were included in both group 1 and group 2. To avoid duplication, we used only the specimen obtained from each patient before interferon- therapy was started. For patients for whom serum specimens obtained before treatment on the day of study entry were not available, specimens collected 1 or 2 months before treatment were used for HCV RNA quantitation and genotyping. A previous study [23] has shown that the viremia levels of patients with chronic HCV infection usually remain about the same over time. The details of the role of HCV genotype as a predictor of subsequent response to interferon- therapy will be discussed in another report (Lindsay KL and colleagues. In preparation). The third group consisted of 95 patients seen at the University of Florida in a prospective study of immune-mediated mechanisms of hepatocellular damage. Table 2. Clinical, Biochemical, and Histologic Characteristics of Study Patients All patients studied were seropositive for anti-HCV and had had abnormal serum aminotransferase levels for at least 6 months. None had biochemical or serologic evidence of other causes of liver disease; in particular, all were seronegative for hepatitis B surface antigen. Duration of HCV infection was established by detailed history taking and was estimated by the clinical investigator (available in 258 patients). Antibody to Hepatitis C Virus and Detection and Quantitation of Hepatitis C Virus RNA We detected anti-HCV by using second-generation enzyme immunoassay (Ortho Diagnostics, Raritan, New Jersey or Abbott Diagnostics, North Chicago, Illinois). Serum specimens were tested for HCV RNA by reverse transcription nested PCR with primers derived from the highly conserved 5 UTR and quantitated by branched DNA (bDNA) signal amplification assay (Quantiplex HCV RNA, version 1.0, Chiron Corp., Emeryville, California) as described previously [24, 25]. All reverse transcription PCR assays were done in a single laboratory; the performance of the PCR assays has been previously verified to have specificity and sensitivity of 100% against a coded serum panel prepared by the Chiron Corporation. Serum HCV RNA levels were measured by bDNA signal amplification assay (bDNA, Quantiplex HCV, version 1.0, Chiron Corp.). Because the bDNA assay underestimates HCV RNA levels in patients infected with HCV types 2 and 3, the appropriate correction factors (times 3 for type 2 and 2 for type 3) were applied to obtain accurate levels of viremia [26-28]. The bDNA assay accurately measures HCV RNA levels for HCV types 1, 4, 5, and 6; thus, no conversion was necessary for these types. Hepatitis C Virus Genotyping The details of the six HCV genotyping methods used to characterize the serum specimens of group 1 have been described previously [20]. These methods are PCR with genotype-specific primers based on the HCV core region and the genomic region of NS5, restriction fragment length polymorphism based on the 5 UTR, direct sequencing of the NS5B region, and serologic genotyping based on NS4 recombinant and synthetic peptides. The line probe assay was used to assess HCV genotyping as previously described [21]. Briefly, the 5 UTR 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. To ensure that this assay was consistently concordant with other tests, 342 specimens were tested by restriction fragment length polymorphism of the PCR amplicon generated from the 5 UTR, and 339 specimens were tested by a serologic genotyping assay based on the

Early Acquisition of TT Virus (TTV) in an Area Endemic for TTV Infection

TT virus (TTV) is widely distributed, with high frequencies of viremia in South America, Central Africa, and Papua New Guinea. The incidence and timing of infection in children born in a rural area of the Democratic Republic of Congo was investigated. TTV viremia was detected in 61 (58%) of 105 women attending an antenatal clinic and in 36 (54%) of 68 infants. Most infants acquired the infection at >/=3 months postpartum. Surprisingly, TTV infection was detected in a large proportion of children with TTV-negative mothers (13 [43%] of 30). Nucleotide sequences of TTV-infected children were frequently epidemiologically unlinked to variants detected in the mother. These three aspects contrast with the maternal transmission of hepatitis G virus/GB virus C in this cohort and suggest an environmental source of TTV infection comparable to hepatitis A virus and other enterically transmitted infections.

Absence of hepatitis C virus transmission but frequent transmission of HIV-1 from sexual contact with doubly-infected individuals

Hepatitis C virus (HCV) is transmitted through infected blood and blood products, but evidence of other routes of transmission is less clearly understood. In a study designed to examine human immunodeficiency virus (HIV) transmission, the prevalence of HCV has also been measured. Sixty-one couples were analysed, 30 in which partners were at risk through sexual contact alone, of whom 12 (40%) became infected with HIV and none with HCV. Thirty-one partners were exposed sexually and additionally through intravenous drug use. Of these, 16 (52%) became infected with HIV and 25 (80%) contracted HCV infection. These findings support the evidence of others that HCV is only rarely transmitted by sexual intercourse in heterosexual relationships and that HIV is not a co-factor for HCV transmission.

Hepatitis C virus (HCV) genotypes and chronic liver disease in Pakistan

Hepatitis C virus (HCV) is classified into different types depending on nucleotide sequence variability. Detailed information on the distribution of various HCV genotypes in some geographical areas is available but little is known about Pakistan. In this study, a 5’ non‐coding region (NCR)‐based restriction fragment length polymorphism (RFLP) genotyping assay was used to investigate the genotype distribution in a large series of HCV‐infected patients in Karachi, Pakistan. Serum samples from 74 hepatitis B surface antigen (HBsAg)‐negative patients with a clinical diagnosis of chronic liver disease (60 patients) and hepatocellular carcinoma (HCC) (14 patients) were assayed for anti‐HCV antibody by second generation enzyme immunoassay and 48 were confirmed anti‐HCV‐positive (33 males, 15 females). Other causes of chronic liver disease (e.g. haemochromatosis, Wilsons disease and immunemediated injury) were ruled out. Liver biopsy was done in 27/48 anti‐HCV‐positive patients and in all HCC patients. Genotypes were determined for 45/48 anti‐HCV‐positive study patients; 39/45 (87%) were type 3; four (9%) were type 1; one was type 2; and one was type 5. Past blood transfusion was the main identifiable risk factor found in 10 patients, all type 3. Seven of the 14 HCC patients were anti‐HCV positive, (six were type 3). Most patients with hepatitis C presented with established cirrhosis and complications of portal hypertension and liver failure. In conclusion: (i) genotype 3 is the most common isolate in HCV‐associated chronic liver disease in Pakistan; (ii) a significant proportion of HBsAg‐negative cirrhotics are non‐B, non‐C in aetiology; and (iii) half of the patients with HCC have serological evidence of HCV infection.

Genotype, Viral Load and Age as Independent Predictors of Treatment Outcome of Interferon-α2a Treatment in Patients with Chronic Hepatitis C

Patients with chronic hepatitis C respond differently when treated with interferon. We randomized 116 patients with chronic hepatitis C in order to compare two dosage regimens of recombinant interferon alpha 2a:3 MIU x 3 per week for 6 months (arm A) or 6 MIU x 3 per week for 3 months and then 3 MIU x 3 per week for 3 months (arm B). There were no significant differences concerning outcome between the two dose regimens: sustained clearance of HCV viremia 6 months after the end of treatment was obtained in 12/59 (20%) in group A compared with 18/57 (32%) in group B (p = 0.24). In patients with genotype 1a, 4/31 (13%), in genotype 1b, none of 9 (0%), 9/15 (60%) in genotype 2, and 17/58 (29%) in genotype 3, showed sustained clearance of HCV viremia 6 months after the end of treatment (p = 0.002). In a stepwise logistic regression analysis, only pretreatment viral load (p = 0.0001), genotype (p = 0.001) and age (p = 0.04) were identified as independent predictors of sustained clearance of HCV viremia. Liver histology as assessed by Knodell index was significantly improved in patients with sustained HCV RNA response 6 months after the end of treatment (5.2 +/- 2.2 vs 2.6 +/- 2.2, p < 0.001), but not in responders with relapse or in non-responders. In conclusion, stepwise logistic regression analysis showed that viral load, HCV genotype and age were the only independent predictors for sustained HCV RNA response.

Relevance of RIBA-3 supplementary test to HCV PCR positivity and genotypes for HCV confirmation of blood donors

HCV antibody screening of 624,910 blood donations resulted in 3,832 samples being referred for confirmation. All were tested by RIBA‐3 with 2,710 negative, 945 indeterminate and 177 positive results. HCV RNA was detected by PCR in an average of 69.5% of RIBA‐3 positives (4 bands 84.1%; 3 bands 74.1%; 2 bands 34.1%) and only 0.53% of RIBA‐3 indeterminates. Eighty‐four percent of samples with a total RIBA‐3 band intensity score (maximum 16) of ≥8 were PCR positive compared with only 22% of those with a score of <8. Total mean band intensities for HCV genotype 1 samples (n = 65) were 13.2, genotype 2 (n = 17) 11.4 and genotype 3 (n = 65) 11.2 with type 1 samples showing greater reactivity with c100 and c33 antibodies. No PCR positive type 1 samples were found with RIBA‐3 total band scores less than 8, no PCR positive type 2 samples less than 6, whilst PCR positive type 3 samples were found with scores as low as 2. NS5 indeterminates were the most common (40.2%) single band pattern but yielded no PCR positive samples, followed by c33 (23.3%) with one PCR positive and c100 (20.2%) with one PCR positive whilst c22 indeterminates were least common (16.3%) but included three PCR positive donors. All five RIBA‐3 indeterminate PCR positive donors were type 3.

Influence of viraemia and genotype upon serological reactivity in screening assays for antibody to hepatitis C virus.

Detection of antibody to recombinant proteins derived from hepatitis C virus (HCV) genotype 1 represents the principal method for diagnosis of HCV infection. A method was developed for quantifying antibody reactivity in two third‐generation enzyme immunoassays (Ortho EIA 3.0 and Murex VK48), and the influence of viraemia, HCV genotype, and host factors such as age, gender, and risk group upon antibody levels were investigated in a consecutive series of 117 anti‐HCV‐positive volunteer blood donors. Viraemic donors (as assessed by the polymerase chain reaction; PCR) showed significantly higher levels of anti‐HCV by the Ortho EIA than those who were nonviraemic (adjusted mean difference of 10.1 fold after multiple regression analysis). The only other factor to influence significantly antibody level was genotype, where it was found that donors infected with type 1 showed 4 to 4.5 times greater serological reactivity by the Ortho assay than those infected with type 2 or 3. Antibody levels by the Ortho assay correlated closely to those detected by the Murex VK48 assay, and similar differences between PCR‐positive and negative donors and between those infected with different genotypes were found. Differences in serological reactivity between genotypes indicate that a large proportion of epitopes of the type 1a or 1b recombinant proteins used in current assays are genotype specific. Variation in sensitivity of screening assays for different genotypes is of potential concern when used in countries where non‐type 1 genotypes predominate in the blood donor or patient population.

Sequence diversity of TT virus in geographically dispersed human populations

TT virus (TTV) is a newly discovered DNA virus originally classified as a member of the Parvoviridae. TTV is transmitted by blood transfusion where it has been reported to be associated with mild post-transfusion hepatitis. TTV can cause persistent infection, and is widely distributed geographically; we recently reported extremely high prevalences of viraemia in individuals living in tropical countries (e.g. 74% in Papua New Guinea, 83% in Gambia; Prescott & Simmonds, New England Journal of Medicine 339, 776, 1998). In the current study we have compared nucleotide sequences from the N22 region of TTV (222 bases) detected in eight widely dispersed human populations. Some variants of TTV, previously classified as genotypes 1a, 1b and 2, were widely distributed throughout the world, while others, such as a novel subtype of type 1 in Papua New Guinea, were confined to a single geographical area. Five of the 122 sequences obtained in this study (from Gambia, Nigeria, Papua New Guinea, Brazil and Ecuador) could not be classified as types 1, 2 or 3, with the variant from Brazil displaying only 46-50% nucleotide (32-35% amino acid) sequence similarity to other variants. This study provides an indication of the extreme sequence diversity of TTV, a characteristic which is untypical of parvoviruses.

Detection and clinical features of hepatitis C virus type 6 infections in blood donors from Hong Kong

The genotype distribution of hepatitis C virus (HCV) was investigated in 212 viraemic blood donors from Hong Kong. A subset of the samples was investigated using three different genotyping assays to establish the accuracy of each in this population. These assays were restriction fragment length polymorphism (RFLP) of amplified 5′ noncoding region (5′NCR) sequences, RFLP of the core region, and a serotyping assay using peptides from two antigenic regions of NS4.

Sequence analysis of hepatitis C virus variants producing discrepant results with two different genotyping assays

Methods for identifying the genotype of hepatitis C virus (HCV) in clinical specimens are frequently based upon the direct characterisation of viral RNA sequences by polymerase chain reaction (PCR) amplification, or by serologically based methods, in which the infecting genotype is inferred from the pattern of antibody reactivity to type‐specific peptides or recombinant proteins used as antigens in an Enzyme Linked Immunosorbent Assay (ELISA). Although genotyping by direct, PCR‐based methods show generally highly concordant results with the genotype inferred from serological typing assays (>95% agreement), there exist a small number of samples that produce discrepant results. To investigate the underlying reasons for the discrepancies, we obtained eleven samples from haemophiliacs and four samples from patients with chronic hepatitis C that produced discordant results between a PCR based assay (InnoLipa I and II) and a serotyping assay (Murex HC02). Nucleotide sequences in the 5′noncoding region (5′NCR), core, and NS4 region were used to identify the genotype of the circulating virus and to identify amino acid changes in NS4 that might alter antigenicity. In 14 samples, sequence analysis of all three regions was concordant with the results of the InnoLipa assay. There were few if any amino acid substitutions in NS4 that might have accounted for the discrepant serotyping results, which were found predominantly in samples from individuals with a history of multiple exposure to HCV. It remains unclear whether the detection of antibody in such discrepant samples corresponds to previous expression of a different genotype than detected by PCR, or whether the virus population in plasma is more restricted in genotype diversity than the population in the liver or at other sites of viral replication. J. Med. Virol. 53:237–244, 1997.