Journal of Medical Virology 65:505±509 (2001)

Hepatitis C Viral Genotype In¯uences the ClinicalOutcome of Patients With Acute PosttransfusionHepatitis C

Shinn-Jang Hwang,1* Shou-Dong Lee,2 Rei-Hwa Lu,2 Chen-Wei Chu,2 Jaw-Ching Wu,2

Shiau-Ting Lai,3 and Full-Young Chang2

1Department of Family Medicine, Veterans General Hospital-Taipei, National Yang-Ming University School of Medicine,Taipei, Taiwan, Republic of China2Division of Gastroenterology, Department of Medicine, Veterans General Hospital-Taipei, National Yang-MingUniversity School of Medicine, Taipei, Taiwan, Republic of China3Department of Surgery, Veterans General Hospital-Taipei, National Yang-Ming University School of Medicine, Taipei,Taiwan, Republic of China

Most patients with an acute infection of hepatitisC virus (HCV) will develop chronic hepatitis, andonly about 15±20% of the cases will resolvespontaneously. The mechanism for the differentoutcomes in patients with acute HCV infectionremains unclear. HCV genotype has been recog-nized as an important factor affecting the clinicalcourse and outcome of chronic hepatitis Cpatients. In order to evaluate the role of HCVgenotype in the clinical course and outcome ofacute posttransfusion hepatitis C, 67 patientswith acute posttransfusion hepatitis C from aprospective study of posttransfusion non-A, non-B hepatitis were enrolled. Thirty-nine patients(58.2%) were HCV genotype 1b. Among the 67patients with acute posttransfusion hepatitis C,53 (79.1%) progressed to chronic hepatitis. Sig-ni®cantly more patients with genotype 1b thannon-1b genotypes developed chronic hepatitis(89.7% vs. 64.3%; P� 0.019). There was nosigni®cant difference in gender, mean age,amount of transfused blood, hepatitis symp-toms, jaundice, incubation period, peak serumalanine transaminase, or serum HCV RNA titerbetween patients with HCV genotype 1b and non-1b infections. Patients who developed chronichepatitis had a signi®cantly greater incidence ofgenotype 1b infection (66.0% vs. 28.6%;P�0.013) and a longer incubation period (7.3weeks vs. 5.4 weeks; P�0.052) than patientswhose infection was resolved. Patients with agenotype 1b infection that resolved itself sponta-neously all had an incubation period of less than 6weeks. Multivariate logistic regression analysisrevealed that genotype 1b and an incubationperiod �6 weeks were signi®cant predictivefactors for the development of chronic hepatitis.

Therefore, the HCV genotype can in¯uence theoutcome of patients with acute HCV infection.J. Med. Virol. 65:505±509, 2001.ß 2001 Wiley-Liss, Inc.

KEY WORDS: clinical outcome; genotype,viral; incubation period; viralhepatitis C, acute

INTRODUCTION

Most patients infected with hepatitis C virus (HCV)will develop chronic hepatitis, and only 15±20 % of thecases will resolve spontaneously [Lee et al., 1991a;Alter et al., 1997; Seeff, 1997]. The mechanism for thedifferent outcomes after HCV infection remainsunclear. However, it has been postulated that hostfactors, such as the acute T-cell immune response toHCV infection, and viral factors, such as quasispecies ofthe HCV genome, may play a role [Farci et al., 1992,2000; Weiner et al., 1992; Sakamoto et al., 1994; Tsaiet al., 1998; Cerny et al., 1999]. The HCV genotype hasbeen implicated in determining the clinical outcome ofpatients with chronic hepatitis C [Dusheiko et al., 1994;Pozzato et al., 1994; Qu et al., 1994; Lau et al., 1995;Nousbaum et al., 1995; Simmonds, 1995]. Patients withHCV genotype 1b infection have been noted to have amore aggressive clinical course of the disease and apoorer response to interferon and/or ribavirin treat-ment than patients infected with other genotypes[Camps et al., 1993; Hino et al., 1994; Chemello et al.,

*Correspondence to: Dr. Shinn-Jang Hwang, Department ofFamily Medicine, Veterans General Hospital-Taipei, 201 Shih-PaiRoad, Section 2, Taipei, 11217, Taiwan.

Accepted 12 January 2001

ß 2001 WILEY-LISS, INC.

1994; Serfaty et al., 1994]. It has also been shown thatHCV genotype 1b is more prevalent than othergenotypes in patients with severe liver conditions, suchas cirrhosis and hepatocellular carcinoma [De Mitriet al., 1995]. Whether the HCV genotype plays animportant role in the clinical outcome of acute HCVinfection has not been reported previously and thusremains of clinical interest.

It is dif®cult to evaluate the factors in¯uencingthe clinical outcome of acute HCV infection becausepatients with acute hepatitis C are dif®cult to identify.There is no seromarker for acute hepatitis C. Inaddition, patients are usually asymptomatic. Afterscreening for antibody to HCV (anti-HCV) in blooddonors, acute posttransfusion hepatitis C is foundrarely. Patients with acute sporadic hepatitis C arealso dif®cult to identify because the time of exposure isusually uncertain, and there is a lack of pre-infectedserum samples for con®rmation. Thus, patients withacute hepatitis C can only be diagnosed correctly in aprospective study of posttransfusion hepatitis con-ducted before screening blood donors for anti-HCV.The clinical outcome of acute hepatitis C infection andthe factors affecting this outcome can be clearlyevaluated in these patients. A prospective posttransfu-sion non-A, non-B hepatitis study was conducted fromMarch 1981 to June 1992 before screening for anti-HCVin blood donors. The aim of the present study was toevaluate the role of HCV genotype in the clinicaloutcome of patients with acute hepatitis C who wereenrolled from our previous prospective study.

PATIENTS AND METHODS

Patients With Posttransfusion Hepatitis

Patients with acute posttransfusion hepatitis C wererecruited from a prospective posttransfusion study inTaiwan [Lee et al., 1991a,b]. Brie¯y, 497 consecutivepatients who had cardiovascular surgery and bloodtransfusions at Taipei Veterans General Hospital,between March 1981 and June 1992, were followedprospectively. Patients who had elevated serum ala-nine transaminase (ALT) levels and who tested positivefor serum hepatitis B surface antigen (HBsAg) wereexcluded. During the study, serum samples were ob-tained before transfusion. Further serum samples werecollected weekly during the ®rst month after transfu-sion, then biweekly for the next 2 months, and followedby monthly up to 6 months and bimonthly up to 1 year.The serum samples collected were stored in aliquots atÿ708C until analyzed. Acute posttransfusion hepatitiswas diagnosed when, from 2 weeks to 6 months aftertransfusion, two consecutive serum ALT levels exceed-ed two times the upper normal value (> 90 U/l) insamples drawn at an interval >2 weeks. Acutehepatitis C was diagnosed based on seroconversion ofanti-HCV or HCV RNA after transfusion. Cases inwhich there was serologic evidence of acute hepatitis Aor B, cytomegalovirus, or Epstein-Barr virus infectionwere excluded. Patients exhibiting other likely causes

of hepatitis (drug-related or heart failure) were alsoexcluded. In total, 67 (13.5%) of 497 patients whoreceived blood transfusions developed acute hepatitisC and were enrolled in the present study. Chronichepatitis C was de®ned as elevated serum ALT levelsand positive serum HCV RNA one year after transfu-sion.

During the study period, patients returned for bloodtests as scheduled. Symptoms and signs of hepatitis,such as malaise, poor appetite, nausea, and tea coloredurine, were recorded at each visit. The incubationperiod was de®ned as the time from transfusion to the®rst elevation of serum ALT. Patients were recorded asjaundiced when they had a serum bilirubin level>2 mg/ml. Peak serum ALT was de®ned as the highest serumALT during the acute phase of hepatitis. Quantitativeserum HCV RNA and HCV genotype were determinedat the time of peak serum ALT.

Laboratory Tests

Liver biochemical tests, including ALT and totalbilirubin, were measured by an autoanalyzer (HitachiModel 736 Automatic Analyzer, Tokyo, Japan). Serumanti-HCV was measured by a second-generation en-zyme immunoassay that contained both HCV structureand non-structure antigens (Abbott Laboratories,Chicago, IL). HBsAg was measured by using a radio-immunoassay (Abbott Laboratories). Quantitativemeasurements of serum HCV RNA were performed bythe branched DNA signal ampli®cation (bDNA) assay(QuantiplexTM 2.0, Chiron Corp., Emeryville, CA),following the manufacturer's instructions [Chan et al.,1995]. The assay produced results in terms of numbersof HCV genome equivalents per ml (Eq/ml). Thedetection limit of this assay was 200,000 Eq/ml.Samples below the detection limit of the bDNA assaywere further tested by home-brew reverse transcrip-tion-nested polymerase chain reaction (RT-nestedPCR), as described previously [Hwang et al., 1993].HCV genotyping was performed using RT-nested PCRwith type-speci®c primers [Okamoto et al., 1992, 1993],and results were reported as types 1a, 1b, 2a, 2b,and 3a, based on Simmonds' classi®cation [Simmondset al., 1994].

Data in the text and tables are expressed as mean-�standard deviation (SD). Results were comparedbetween groups depending on the type of data analyzedusing the chi-square test, Fisher's exact test, Student'st-test, or the Mann-Whitney test. Multivariate stepwiselogistic regression (SPSS, Inc. Chicago, IL) was carriedout to evaluate the predictive values of the patients'clinical, biochemical, and virological features asso-ciated with the development of chronic hepatitis. Forall tests, results with P values less than 0.05 (two-tailtest) were considered signi®cant statistically.

RESULTS

Sixty-seven patients (47 men, 20 women) whoful®lled the criteria of acute posttransfusion hepatitis

506 Hwang et al.

C were enrolled in the present study. The mean age ofthese 67 patients was 42�16 years (range: 13 to 74years). The mean amount of transfused blood that thepatients received was 27�23 units (range: 1 to 137units). The mean incubation period was 7.8�3.2 weeks(range: 2 to 16 weeks) and the mean peak serum ALTlevel was 660�411 U/l (range: 91 to 1,704 U/l).Symptoms and signs of hepatitis were noted in 36(53.7%) patients, and jaundice was noted in 18 (26.9%)cases. None of these patients developed fulminatinghepatitis. HCV genotyping showed that one patient(1.5%) was type 1a, 39 (58.2%) patients were type 1b, 10(14.9%) patients were type 2a, 11 (16.4%) patients weretype 2b, three (4.5%) patients were of a mixed type (twowith types 1a and 1b, one with types 2a and 2b), andthree (4.5%) patients were unable to be classi®ed by thecurrent method. None of these 67 patients receivedinterferon or antiviral treatment during the acutephase of hepatitis C.

Among the 67 acute hepatitis C patients, 53 (79.1%)developed chronic hepatitis. Of these, signi®cantlymore were infected with genotype 1b than non-1bgenotypes (89.7% vs. 64.3%, P�0.019). The chronicityrate of patients with genotype 1b infection (89.7%) washigher than that for genotype 2a (70.0%, P� 0.25) orgenotype 2b (63.6%, P�0.052) infection, but thedifference did not reach statistical signi®cance. Therewas no signi®cant difference in gender, mean age,

amount of transfused blood, incubation period, thepresence of hepatitis symptoms or jaundice, peakserum ALT, or serum HCV RNA titer between patientswith HCV genotype 1b and non-1b infection (Table I).When patients who developed chronic hepatitis werecompared with those whose infection resolved sponta-neously, a signi®cant difference was found in theincidence of genotype 1b infection (66.0% vs. 28.6%,P� 0.013) and the length of incubation period (7.3weeks vs. 5.4 weeks, P�0.052). All four patients with agenotype 1b infection that resolved spontaneously hadan incubation period of less than 6 weeks. There was nosigni®cant difference in gender, mean age, amount oftransfused blood, the presence of hepatitis symptoms orjaundice, peak serum ALT, or serum HCV RNA titerbetween patients who developed chronic hepatitis andcases that were resolved spontaneously (Table II).

Gender, mean age, amount of transfused blood,incubation period, the presence of hepatitis symptomsor jaundice, HCV genotype (genotype 1b/non-1b), andserum HCV RNA titer were all selected as independentvariables in a logistic regression analysis, with thedevelopment of chronic hepatitis as the dependentvariable. The continuous variables were transformed tocategorical variables with the cut-points determined bythe receiver operating characteristic curve. Multivari-ate logistic regression analysis revealed genotype 1b(odds ratio: 4.8, 95% con®dence interval: 1.1 to 21.9,

TABLE I. Comparison of Demographic and Clinical Data Between Patients With HCVGenotype 1b and Non-1b Genotype in 67 Patients With Acute Posttransfusion Hepatitis C*

Genotype 1b(n� 39)

Genotype non-1b(n� 28) P value

Sex (male/female) 28/11 19/9 0.728Mean age (years) 50.4�16.3 54.5�15.3 0.298Amount of transfused blood (unitsa) 29.2�26.4 24.6�17.6 0.418Chronicity 35(89.7%) 18(64.3%) 0.019Hepatitis symptoms (yes/no) 20/19 16/12 0.635Jaundice (yes/no) 13/26 5/23 0.159Incubation period (weeks) 7.1�3.8 6.5�3.1 0.503Peak serum ALT (IU/l) 725�429 569�373 0.118Serum HCV RNA (log Eq/ml) 5.84�1.10 5.60�0.93 0.376

*Data were expressed as mean�SD. ALT, alanine transaminase; HCV, hepatitis C virus.aone unit�250 cc.

TABLE II. Comparison of Demographic and Clinical Data Between Patients With ResolvedHepatitis and Developed Chronicity in 67 Patients With Acute Posttransfusion Hepatitis C*

Resolvedhepatitis (n� 14)

Developedchronicity (n� 53) P value

Sex (male/female) 7:7 40/13 0.099Mean age (years) 54.1�14.5 51.6�16.3 0.588Amount of transfused blood (unitsa) 29.2�26.4 24.6�17.6 0.418Genotype 1b/ non-1b 4/10 35/18 0.013Hepatitis symptoms (yes/no) 6/8 30/23 0.359Jaundice (yes/no) 3/11 15/38 0.743Incubation period (weeks) 5.4�3.0 7.3�3.6 0.052Peak serum ALT (IU/l) 601�452 676�403 0.577Serum HCV RNA (log Eq/ml) 5.67�0.86 5.76�1.08 0.749

*Data were expressed as mean�SD. ALT, alanine transaminase; HCV, hepatitis C virus.aone unit�250 cc.

HCV Genotype in Acute Hepatitis C 507

P�0.040) and an incubation period � 6 weeks (oddsratio: 5.5, 95% con®dence interval: 1.0 to 29.3,P�0.046) were signi®cant independent predictivefactors for developing chronic hepatitis.

DISCUSSION

HCV, an RNA virus related to the Flaviviridae, has ahigh spontaneous mutation rate. As a result, differentgenotypes and subtypes have been characterized. Todate, at least six major HCV genotypes and more than50 subtypes have been identi®ed throughout the world[Purcell, 1997]. Further studies have revealed thatHCV isolates consist of heterogeneous mixtures ofgenetically different but closely related variants (qua-sispecies), even in patients with a single HCV subtypeinfection [Martell et al., 1992; Simmonds, 1995].

HCV infection accounts for most cases of transfusion-associated non-A, non-B hepatitis seen before thescreening of blood donors with anti-HCV. Patientsinfected with HCV usually develop chronic hepatitis.According to this prospective follow-up study, 79.1% ofthose patients who contracted an HCV infectionthrough blood transfusion developed chronic hepatitis.This high rate of chronicity was consistent withprevious prospective posttransfusion studies fromWestern countries [Alter et al., 1997]. The persistentviral infection has been attributed to the existence ofHCV quasispecies, which escape the host immuneclearance [Farci et al., 1992; Weiner et al., 1992;Sakamoto et al., 1994; Tsai et al., 1998; Farci et al.,2000]. In addition, viral persistence may be due to aweak antiviral immune response, leading to an inabil-ity to eradicate HCV from infected cells [Cerny et al.,1999].

The HCV genotype is known to in¯uence the clinicalcourse and outcome of patients with chronic hepatitisC. In patients with severe liver diseases, such ascirrhosis or hepatocellular carcinoma, HCV genotype1b is more prevalent than other genotypes [De Mitriet al., 1995]. In addition, patients with HCV genotype1b infection are relatively more resistant to interferon/ribavirin treatment. However, to date, the role of HCVgenotype in the clinical outcome of acute HCV infectionhas not been determined. Previous studies did notreveal the HCV genotype to be an important factor indetermining the clinical outcome of acute HCV infec-tion, mostly due to the small number of cases analyzed.It is essential to study large enough groups of patientsto be able to perform multivariate analysis for sepa-rately analyzing the contribution of in¯uences on theoutcome. Our study has revealed that acute posttrans-fusion hepatitis C patients with HCV genotype 1binfection have a signi®cantly higher chronicity ratethan patients with non-1b genotypes. Multivariatelogistic regression analysis also shows thatgenotype 1b is a signi®cant independent predictor forthe development of chronic hepatitis. Patients withHCV genotype 1b infection have the highest rate ofchronicity, probably due to an increased number of

HCV quasispecies in these patients, which may escapeimmune surveillance by the host [Tsai et al., 1998;Farci et al., 2000].

Our study also revealed that the length of theincubation period was another signi®cant independentfactor in predicting chronicity in patients with acutehepatitis C. Patients with HCV genotype 1b infectionwhose acute hepatitis resolved itself, all had a shortincubation period (<6 weeks). These ®ndings mayindicate that early host immune responses facilitate theclearing of HCV. Recent animal studies have revealedthat an early and durable multispeci®c cytotoxic Tlymphocyte response during acute hepatitis C clearedthe HCV infection, and may thus support our hypoth-esis [Missale et al., 1996; Cooper et al., 1999]. Inconclusion, HCV genotype can in¯uence the outcomefor patients with acute HCV infection. Patients withHCV genotype 1b infection and an incubation periodgreater than 6 weeks will develop chronic hepatitis.

ACKNOWLEDGMENTS

This study was support by a grant (NSC87-2314-B-075-078) from the National Science Council, Taiwan.The authors thank nurse Ming-Huei Ho for collectingserum specimens, and Pearl Chen for her technicalassistance.

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