- Email: [email protected]
Outcome of an outbreak of acute hepatitis C among healthy volunteers participating in pharmacokinetics studies
Outcome of an Outbreak of Acute Hepatitis C Among Healthy Volunteers Participating in Pharmacokinetics Studies Alberto Larghi,1 Massimo Zuin,1 Andrea Crosignani,1 Maria Lisa Ribero,3 Cristina Pipia,1 Pier Maria Battezzati,1 Giorgio Binelli,4 Francesco Donato,5 Alessandro Remo Zanetti,2 Mauro Podda,1 and Alessandro Tagger2 We identified 15 patients with acute hepatitis C (AHC) among 29 healthy volunteers participating in 2 consecutive pharmacokinetics studies. Molecular techniques were used to determine the relatedness of viral strains, whereas clinical and virologic follow-up was started to establish the course and outcome of the acute infection. After presentation, serum liver enzymes and HCV RNA were monitored weekly for 4 months, then monthly for at least 12 months. Liver biopsy was performed 6 to 12 months after AHC diagnosis. Phylogenetic analysis of coding regions for the envelope glycoproteins E1 and E2 was performed. At presentation, all 15 patients tested HCV RNA–positive and had HCV genotype 2c. Phylogenetic analysis indicated a common source of infection. Fourteen patients agreed to be followed prospectively. Infection resolved spontaneously in 8 patients, HCV RNA becoming undetectable by 4 to 5 months after the presumed time of infection in 5 of them and by 8, 13, and 24 months in the remaining 3. Six patients developed chronic infection. Liver biopsies performed in 9 subjects who were HCV RNA–positive 6 months after AHC diagnosis revealed that the prevalent histologic finding was lobular inflammation. In conclusion, our homogeneous cohort showed a wide spectrum of clinical, virologic and histologic features, and, more importantly, short-term outcome differed noticeably despite the common source of infection. (HEPATOLOGY 2002;36:993-1000.)
See Editorial on Page 1020
H
epatitis C virus (HCV) infection has a worldwide distribution1,2 and has been recognized as a major cause of chronic hepatitis, cirrhosis, and end-stage liver disease.3 Although the understand-
Abbreviations: HCV, hepatitis C virus; AHC, acute hepatitis C; ALT, alanine aminotransferase; HBV, hepatitis B virus; HIV, human immunodeficiency virus; HGV, hepatitis G virus; HAV, hepatitis A virus; ELISA, enzyme-linked immunosorbent assay; UPGMA, unweighted pair group method with arithmetic mean; UTR, untranslated region; NS3, nonstructural protein 3; NS5b, nonstructural protein 5b. From the 1Division of Internal Medicine, Department of Medicine, Surgery and Dentistry, Ospedale San Paolo, San Paolo School of Medicine; the 2Institutes of Virology and 3Hygiene, University of Milan; the 4Dipartimento di Biologia Strutturale e Funzionale, University of Insubria, Varese; and the 5Institute of Hygiene, University of Brescia, Brescia, Italy. Received November 19, 2001; accepted July 17, 2002. Supported in part by grants from MURST (No. 12-1-21, 1996 and No.12-124, 1997), Italy. Alberto Larghi is currently a first-year Fellow of the Department of Digestive and Liver Diseases, College of Physicians and Surgeons at Columbia University, 630 West 168th Street, P & S 10-508, New York, NY 10032. Address reprint requests to: Mauro Podda, M.D., Division of Internal Medicine, Department of Medicine, Surgery and Dentistry, San Paolo School of Medicine, University of Milan, Ospedale San Paolo, Via di Rudinı` 8, 20142 Milan, Italy. E-mail: [email protected]; fax: (39) 02-89123960. Copyright © 2002 by the American Association for the Study of Liver Diseases. 0270-9139/02/3604-0027$35.00/0 doi:10.1053/jhep.2002.36129
ing of many aspects of HCV infection has improved significantly,4 its natural history has not yet been fully defined.5 Recent data from retrospective and prospective studies indicate that HCV acquired from parenteral exposure to contaminated anti-D immune globulin is followed by low rates of progression to chronic infection and to histologic cirrhosis.6,7 However, because prospective studies have been performed almost exclusively in the transfusion setting, few data are available on the clinical course and outcome of acute hepatitis C resulting from other sources of parenteral exposure.8-16 Indeed, with the significant decrease in cases of posttransfusion hepatitis,17-19 other modes of transmission have gained importance, but the disease often remains undiagnosed during the acute phase, which is usually asymptomatic.3-5 Therefore, the prospective observation of cohorts of subjects in whom acute hepatitis C (AHC) has been recognized at an early stage would supply valuable information on the shortterm outcome of the infection. We report an outbreak of HCV infection shown by phylogenetic analysis of the coding regions for the envelope glycoproteins E1 and E2 of HCV among subjects infected in the process of participating in pharmacokinetics studies, and we report their clinical, virologic, and histologic outcome. 993
994
LARGHI ET AL.
Patients and Methods Patients and Clinical Setting. In July, 1995, 4 health care workers from a nursing home were referred to our center after the diagnosis of acute hepatitis. Alanine aminotransferase (ALT) levels in these patients were elevated 28 to 51 days before presentation to our center. The subjects were part of a group of 29 health care workers who had taken part as healthy volunteers in 2 consecutive studies conducted between February and July, 1995, that investigated the pharmacokinetics of an oral antihypertensive drug and of an oral nonsteroidal anti-inflammatory drug. Four weeks before enrollment in either study, subjects had undergone biochemical and hematologic screening tests, including serum liver enzyme activity. Blood tests for serological markers of HCV infection, hepatitis B virus (HBV) infection, and human immunodeficiency virus (HIV) infection were not included in the study protocols, nor had serum samples been stored in the laboratory. All 29 subjects were invited to attend our center for blood testing and clinical evaluation. All but 2 subjects agreed to undergo clinical, biochemical, virologic, and, when indicated, histologic evaluation. They presented to our center for the first time between July 2 and August 1, 1995. Case Definition and Procedures. Diagnosis of AHC was based on the finding of ALT level of at least 10 times the upper normal limit in the presence of HCV RNA. The incubation period tentatively was defined as the time between apparent exposure and the first ALT value twice the upper limit of the normal range. Other potential causes of liver disease were excluded. In particular, HBV DNA and hepatitis G virus (HGV) RNA as well as serologic markers of acute HBV and hepatitis A virus (HAV) infection were investigated in all patients. Subjects who were resulted HCV RNA–positive after the initial visit underwent weekly blood samplings for ALT and HCV RNA determination during the first 4 months. Subsequently, patients were tested monthly for at least 12 months. Antibodies against HCV (anti-HCV) were sought every week until their detection. Subjects who had tested HCV RNA–negative were followed on a monthly basis for at least 12 months and underwent the same biochemical and virologic determinations. Liver biopsy was scheduled to be performed 6 to 12 months after AHC diagnosis if serum HCV RNA was still detectable. Histologic diagnosis was made with both hematoxylin and eosin- and reticulin-stained liver sections according to a recent international scoring system for chronic hepatitis.20 Samples were read by a pathologist who was blind to the clinical data.
HEPATOLOGY, October 2002
After the first year, follow-up visits were scheduled every 4 months for all patients who tested positive for HCV RNA. Interferon treatment was offered to all patients with persistent HCV infection and increased serum ALT activity. The protocol was approved by the bioethics committee of the San Paolo Hospital in Milan and by the Local Health Authority. Laboratory Methods. For laboratory determinations other than routine measurements, serum samples were stored at ⫺80°C. Anti– hepatitis B core antigen antibodies, hepatitis B surface antigen, and anti– hepatitis A immunoglobulin M antibodies were measured in serum samples (Axym, Abbott, North Chicago, Ill.). Antibodies to HCV were detected by a third-generation enzymelinked immunosorbent assay (ELISA) (HCV 3.0 ELISA; Ortho Diagnostic Systems, Raritan, NJ) and recombinant immunoblot assay (Chiron RECOMBINANT IMMUNOBLOT ASSAY 3; Ortho Diagnostic Systems). HCV RNA was detected by in-house reverse transcriptase polymerase chain reaction, with nested primers of the 5⬘noncoding region of the HCV genome. The sensitivity of this assay was 100 HCV RNA copies per mL of serum, measured by testing serially diluted samples in parallel with branched DNA version 2.0 assay (Quantiplex HCV RNA, Bayer Diagnostics, Emerville, CA). The HCV RNA load was determined longitudinally with the bDNA version 2.0 (linearity range 0.2-120 ⫻ 106 genome equivalents/mL) according to the manufacturer’s instructions. Mean HCV RNA genome equivalents per mL were calculated by dividing the total count of viral loads greater than 0.2 ⫻ 106 genome equivalents/mL by the total number of determinations after seroconversion. Samples negative by bDNA, but positive by reverse transcriptase polymerase chain reaction, were arbitrarily assigned a value of 0.1 ⫻ 106 HCV RNA genome equivalents/mL. After reverse transcription with primer E1818 (antisense: 5⬘ GTC GAG GKG SGT AGT GCC AG 3⬘, nt 1818 to 1799), the first round of polymerase chain reaction used primers E1278 (sense: 5⬘ RTM WCG GGH CAC CGC ATG G 3⬘, nt 1278 to 1296) and E1818, and the second-round primers E1300 (sense: 5⬘ GG GAC ATG ATG ATG AAC TGG 3⬘, nt 1300 to 1319) and E1604 (antisense: 5⬘ GTG CCA RCT GCC RTT GGT G 3⬘, nt 1604 to 1586). We performed direct-sequence analysis of nested polymerase chain reaction products of 258 bp (nt 1329 to 1586), encompassing the 3⬘ end of HCV envelope 1 (E1: nt 1329 to 1490) and the 5⬘ end of envelope 2 (E2: nt 1491 to 1586), which includes the hypervariable region 1. The polymerase chain reaction products were gel purified and sequenced on an Applied Biosystems ABI 373 automated sequencer with the
HEPATOLOGY, Vol. 36, No. 4, 2002
LARGHI ET AL.
995
Table 1. Characteristics on Presentation of the 16 Subjects Who Tested Positive for HCV RNA Subject
Sex
Age
ALT*
Anti-HCV
Date† of Presentation
Date† of Diagnosis of Acute Hepatitis
Presumed Date† of Infection‡
1 2 3 4 7 8 9 12 15 16 17 20 21 27 28 29
F M F F M M F F F F M F F F F F
45 33 21 23 34 41 35 30 22 25 31 30 31 35 29 35
15 92 656 34 167 29 219 52 16 427 42 10 1128 1142 10 188
Pos Pos Pos Pos Pos Pos Neg Neg Pos Pos Pos Neg Pos Neg Neg Neg
July 17 July 2 July 11 July 11 July 2 July 27 July 19 July 17 July 13 July 2 July 17 July 19 Aug 1 July 19 July 19 July 19
ND June 2 July 11 May 24 July 11 Sept 11 July 25 Aug 23 June 26 July 2 May 26 Sept 6 Aug 1 July 19 Aug 8 Aug 8
Feb 22 Feb 22 Feb 22 Feb 22 Mar 1 Mar 1 May 21 May 21 Apr 5 Apr 5 Apr 5 May 21 May 21 June 4 June 4 June 4
Abbreviation: ND, not documented. *Normal values ⬍42 IU/L. †All the dates refer to 1995. ‡Inferred from the date of participation in the pharmacokinetics studies.
PRISM dye terminator cycle sequencing kit (PE Applied Biosystems, Foster City, CA). Sequences were analyzed with the Sequencher 3.0 analysis program (Gene Codes Corporation, Ann Arbor, MI). Attribution of the HCV genotype was performed on the basis of the sequence obtained. For phylogenetic analysis, 16 sequences, each 258 nt long, obtained from the 16 HCV RNA–positive patients involved in the outbreak were compared with 20 sequences (19 of genotype 2c and 1 of genotype 2a) derived from a previous study21 taken as unrelated controls, and with 5 prototype sequences sampled from GenBank (D00944 and D10075 for genotype 2a, D10077 and D10988 for genotype 2b, D50409 for genotype 2c). The whole set of 41 sequences was resampled by bootstrapping 1,000 times and then analyzed with the PHYLIP package, v.3.5c.22 Bootstrapping of the original data set was performed to obtain a measure of the confidence we can have in a particular phylogenetic feature, such as a given branch of the tree. In effect, the random variation obtained by analyzing bootstrapped data sets is typical of the variation one would get from collecting new data sets.23 The degree of divergence between the sequences was estimated by the Kimura 2-parameter method, with a transition-transversion ratio of 2:0 (DNADIST of the PHYLIP package).24 The set of distance matrices was then analyzed by UPGMA (Unweighted Pair Group Method with Arithmetic Mean) as implemented by the NEIGHBOR program of the PHYLIP package. The final unrooted consensus tree was drawn by the CONSENSE program of the same package.
HGV RNA Detection. Reverse transcriptase polymerase chain reaction amplification was used, with nested primers from 5⬘ untranslated region (UTR), nonstructural protein (NS)3 helicase, and NS5b regions to detect HGV RNA. The methods are described in detail elsewhere.25 HBV DNA Detection. Nucleic acid was extracted from 100 L of serum with the proteinase K-sodium dodecyl sulphate-phenol-chloroform method. HBV DNA was amplified with nested primers as described previously.26
Results Sixteen of the 27 subjects who agreed to be evaluated tested positive for serum HCV RNA on presentation to our center. Nucleotide sequence analysis of E1 revealed they were all infected with HCV genotype 2c. Ten were anti-HCV positive and 9 out of the 16 subjects had elevated serum ALT levels. The 6 HCV RNA–positive subjects who were anti-HCV–negative at presentation to our center seroconverted within 1 to 6 weeks. Subjects’ characteristics are shown in Table 1. All serum samples tested negative for HBV DNA and HGV RNA, as well as for serologic markers of acute HBV and HAV infection. Of the 16 HCV RNA–positive subjects, 8 showed serum ALT levels higher than 10 times the upper normal limit at the time of presentation or within the previous 2 months, 7 showed a progressive elevation of serum liver enzymes during the first 2 months of follow-up, and 1 subject (patient 1) had persistently normal ALT levels in
996
LARGHI ET AL.
association with intermittently positive serum HCV RNA throughout the observation period. Therefore, AHC was documented in 15 out of the 16 subjects who tested HCV RNA–positive at presentation. AHC was clinically asymptomatic in all subjects but 1 (patient 12), who experienced mild and transient malaise, nausea, and vomiting parallel to the elevation of ALT. The aligned sequences of 258 nt, encompassing the E1 and E2 regions, obtained from the 16 HCV RNA–positive patients, were bootstrapped and a phylogenetic tree based on the UPGMA method was obtained, as shown in Fig. 1. The most evident feature of the tree is the clustering of all patients involved in the outbreak (denoted by PT#). The bootstrap value of 1,000/1,000 of the node leading to the PT cluster lends statistical robustness to the interpretation that all of the patients involved in the outbreak were infected by a common source. Prototype sequences of genotypes 2a and 2b also form separate clusters. The BsHCC197 unrelated control, attributed to genotype 2a on the basis of its sequence, correctly clusters with the 2a prototypes. The other unrelated control, BsHCC226, although attributed to genotype 2c on the basis of its sequence features, exhibited a degree of polymorphism sufficient to cluster apart. All 11 subjects who were HCV RNA–negative on presentation had normal serum ALT values, and none developed acute hepatitis during follow-up. Anti-HCV antibodies were detected in one subject at presentation. Biochemical and Virologic Course During the First Year of Follow-Up. One of the 15 subjects with AHC refused to be followed further after the first visit (patient 17). The biochemical and virologic course of HCV infection during the first year of follow-up in the remaining 14 subjects with AHC is summarized in Table 2. Infection resolved spontaneously in seven subjects. In 5 of these (Nos. 2, 3, 4, 21, and 27), HCV RNA became undetectable 4 to 5 months after the presumed time of infection and preceded ALT normalization by 2 to 12 weeks. A representative case is shown in Fig. 2A. In two other subjects, the disappearance of serum HCV RNA occurred eight (patient 12) and thirteen months (patient 8) after the presumed time of infection and was associated with ALT normalization. All seven subjects were followed for 6 to 8 months after the last positive polymerase chain reaction and were HCV RNA–negative in 3 serum samples collected 2 months apart. Low HCV RNA loads (range ⬍ 0.2 ⫻ 106 – 0.39 ⫻ 106 genome equivalents/mL) were detected during the viremic phase in all of them. Seven other subjects did not clear the virus during the first year of follow-up. In four cases, HCV RNA remained persistently detectable (Nos. 7, 9, 16, and 20). Serum ALT activity was persistently abnormal in three of them (a
HEPATOLOGY, October 2002
Fig. 1. Phylogenetic tree analysis comparing coding sequences in the HCV regions for the envelope glycoproteins E1 and E2 from 41 isolates. For phylogenetic analysis, 16 sequences, 258 nt long, obtained from the 16 HCV RNA–positive patients involved in the outbreak were compared with 20 sequences (19 of genotype 2c and 1 of genotype 2a) originating from a previous study21 taken as unrelated controls, and 5 prototype sequences sampled from GenBank (D00944 and D10075 for genotype 2a, D10077 and D10988 for genotype 2b, D50409 for genotype 2c). Bootstrap values greater than 800/1,000 are reported at the nodes. Accession numbers are as follows: for the 5 sequences taken from GenBank D00944 (HPCPOLP or HC-J6), D10075 (HPCHCJ5 or HC-J5), D10077 (HPCHCJ7 or HC-J7), D10988 (HPCJ8G or HC-J8), D50409 (BEBE-1); for the sequences originated from the patients (PT) involved in the outbreak, AF237634 to AF237649; for the sequences originated from the controls (BSHCCX and BSCOX), AF142385 to AF142404.
representative case in Fig. 2B), while in one case (patient 9), a complete and sustained normalization of serum transaminase levels occurred after 10 months of followup. In the remaining three subjects (Nos. 15, 28, and 29), HCV RNA was detected intermittently over the initial 6-month period of follow-up with ALT levels paralleling HCV RNA fluctuations (a representative case in Fig. 2C).
HEPATOLOGY, Vol. 36, No. 4, 2002
LARGHI ET AL.
Table 2. Biochemical and Virologic Course of HCV Infection in 14 of the 15 Subjects With Acute Hepatitis C During the First Year of Follow-up ALT
HCV RNA Clearance (n ⫽ 7) Persistently positive (n ⫽ 4) Intermittently positive (n ⫽ 3) All subjects (n ⫽ 14)
Normalized
Fluctuating
Persistently Abnormal
7 1 0 8
0 0 3 3
0 3 0 3
997
positive six months after the diagnosis of acute hepatitis. Overall, the predominant histologic finding was lobular inflammation. A mild necroinflammatory activity was also detected in three subjects (Nos. 8, 12 and 15) in
NOTE. Patient 17 was lost at follow-up after the first visit.
At the end of 12 months of follow-up, HCV RNA became persistently positive in two subjects, while the fluctuating pattern persisted in the third subject (patient 15). Titers ranging from 0.2 to 2.0 ⫻ 106 genome equivalents/mL (mean 1.1 ⫻ 106 genome equivalents/mL) were detected during the first year of follow-up, both in the four cases (Nos. 7, 9, 16, and 20) who were persistently HCV RNA–positive and in the three cases (Nos. 15, 28, and 29) who were intermittently HCV RNA–positive. Additional Follow-Up. Fourteen patients with AHC were further observed with a median follow-up period of 45 months (range 33-53 months) from the time of diagnosis. All seven patients in whom infection resolved spontaneously within 13 months of the presumed time of infection remained persistently HCV RNA–negative with normal ALT levels throughout the follow-up period. Among the seven patients with persistent (Nos. 7, 9, 16, and 20) or intermittent (Nos. 15, 28, and 29) viremia during the first year of follow-up, all but one (patient 15) were HCV RNA positive for at least 12 months thereafter. In patient 15 infection resolved spontaneously 24 months after the presumed time of infection, and HCV RNA was persistently undetectable and ALT levels were normal in three subsequent serum samples collected at four-monthly intervals. Among the six patients with chronic infection, one (patient 9) had persistently normal ALT levels whereas five patients had persistently elevated ALT throughout the second year of follow-up. Of these five patients, three (Nos. 20, 28, and 29) started a 12-month course of interferon alfa at the dose of 3 MU thrice weekly 15 to 18 months after presentation to our center. All showed sustained virologic response, and liver biopsies performed six months after the end of therapy in two subjects (Nos. 20 and 29) showed improvement in the histologic activity score (from 5 to 3 and from 9 to 2, respectively). Histologic Examination. The results of the histologic examination are shown in Table 3. Liver biopsy was performed in all nine subjects who were still HCV RNA
Fig. 2. Representative cases of different biochemical and virologic courses in an outbreak of AHC. ALT values are expressed as multiples of the upper limits of the normal range. The light vertical bars indicate negative assays for HCV RNA, and the dark bars positive assays. (A) A case of spontaneous clearance of HCV infection is reported. (B) A case with persistently abnormal ALT levels and persistently positive HCV RNA is described. (C) A case of intermittently positive HCV RNA associated with fluctuations of serum transaminase levels is shown. The effects of interferon alfa administration (3 MU thrice weekly for 12 months) are also described.
998
LARGHI ET AL.
HEPATOLOGY, October 2002
Table 3. Histologic Outcome of Acute Hepatitis C in 9 Patients Who Tested Still HCV RNA Positive 6 Months After the Diagnosis of Acute Hepatitis Liver Histology
Subject
HCV RNA Course
ALT Course
Periportal Necrosis
Lobular Necrosis
Portal Inflammation
Fibrosis
Knodell HAI Score
7 9 16 20 15 28 29 8 12
PP PP PP PP IP IP IP C C
PA N PA PA F F F N N
1 1 3 0 1 0 4 0 0
4 3 3 4 3 3 3 2 3
1 0 3 1 1 1 1 0 1
0 1 1 0 1 0 1 0 0
6 5 10 5 6 4 9 2 4
Abbreviations: PP, persistently positive; IP, intermittently positive; C, clearance; PA, persistently abnormal; N, normalized; F, fluctuating. NOTE. Patient 15 with intermittent viremia during the first year of follow-up cleared HCV RNA 24 months after the presumed time of infection.
whom infection resolved spontaneously 2-12 months after liver biopsy.
Discussion Primary HCV infection is poorly characterized because most patients are asymptomatic and, therefore, it is rarely diagnosed.3-5 The clinical picture of AHC has mainly been described in transfused patients whereas for other modes of infection such as needle stick or accidental exposure it is not well known. The present study describes the occurrence and outcome of an outbreak of HCV infection in a group of subjects who took part in two pharmacokinetics studies as healthy volunteers. This outbreak allowed us to perform a detailed study of the clinical course of acute HCV infection in a cohort of subjects infected by a parenteral route other than blood transfusion. The temporal clustering of the cases linked participation in the pharmacokinetics studies to the development of AHC. The demonstration of a high degree of homology among the sequences of the viral region encompassing E1 and E2 in all of the subjects studied strengthens the probability of a common source of infection. The value of the double-pronged approach of molecular and phylogenetic techniques in providing evidence of the occurrence of HCV outbreaks27,28 and documenting the transmission of HCV infection29-31 has been reported previously. The course of transmission remains unclear, but we may speculate that the outbreak started from patient 1, who was already HCV RNA–positive at presentation but without documented AHC. She may have had a pre-existing infection characterized by viremia with normal serum ALT levels (i.e., an HCV carrier32). Despite the fact that the patients included in this study were exposed to a common source of infection, the incu-
bation period and the outcome of the acute phase of the disease were highly variable, probably reflecting the biologic variability of HCV infection. The average incubation period was 10 weeks, but the range was 6 to 28 weeks. The clinical and virologic course also varied, both in patients in whom infection resolved spontaneously and in those developing chronic hepatitis. This suggests that host factors play a major role in conditioning the outcome of acute HCV infection.33,34 Patients with viral clearance and those with persistent infection showed 2 distinct patterns of viremia. Patients whose HCV RNA levels became undetectable exhibited lower viral loads during the course of infection compared with those who remained persistently viremic, but a specific threshold value that predicts the outcome of infection could not be detected. HCV RNA load was not related with the time of viral clearance; indeed, 2 patients with low viral load cleared the virus more than 6 months after infection (8 and 13 months); in another case, relatively high levels of viremia were found before clearance at 24 months. This finding suggests that longer periods of time than generally considered may be required to establish whether HCV infection has progressed to chronicity.35 Hepatitis C is a disease with a high rate of progression to chronicity. Estimates indicate that nearly 85% of patients develop chronic HCV infection after acute infection.36 The finding that in our setting only 43% (95% confidence interval: 18% to 71%) of the patients became chronically infected may indicate that the tendency to chronicity of HCV infection is lower than expected. However, the validity of this difference is questionable given the small number of patients examined and the wide, unstable confidence interval. Similar rates of chronic infection (55%) were found in young women who had received HCV-contaminated
HEPATOLOGY, Vol. 36, No. 4, 2002
anti-D immunoglobulins6,7 and in children who acquired posttransfusion hepatitis C after cardiac surgery.37 These rates are lower than those reported in community-based investigations of acute HCV infection38 or in analyses of patients who were infected through transfusion as adults.15 The reason for the relatively high rate of spontaneous clearance in our cohort as well as in women infected by anti-D immunoglobulins and children with posttransfusion hepatitis is uncertain. Differences in host immune responses have been implicated, but data on age-related immune responses to HCV infection are not available. The presence of genotype 2c as the source of outbreak also may have contributed to the better short-term prognosis in the present study; however, the impact of HCV genotypes on disease progression is still a matter of debate, and this is particularly true for the acute setting, in which there is a limited quantity of data available.35,39,40 In conclusion, the sequence analysis of the HCV hypervariable region 1 supplied strong evidence for transmission of HCV from a common source of infection, leading to an outbreak of hepatitis C among participants in pharmacokinetics studies. Acute HCV infection showed highly variable clinical, virologic and histologic features. The clinical course of infection was characterized by lower rates of chronicity than generally reported and by late clearance in a few cases. Our results point to the need for future research aimed at better defining the role of viral and host factors in determining the chronicity of HCV infection and the rate of disease progression. Acknowledgment: The authors thank Nora V. Bergasa, MD, for careful help in revising the manuscript and Marco Maggioni, MD, for the histologic evaluation.
References 1. Alter MJ. Epidemiology of hepatitis C in the West. Semin Liver Dis 1995; 15:5-14. 2. Mansell CJ, Locarnini SA. Epidemiology of hepatitis C in the East. Semin Liver Dis 1995;15:15-32. 3. Hoofnagle JH. Hepatitis C: the clinical spectrum of disease. HEPATOLOGY 1997;26(Suppl):15S-20S. 4. Di Bisceglie AM. Hepatitis C. Lancet 1998;351:351-355. 5. Seeff LB. Natural history of hepatitis C. HEPATOLOGY 1997;26(Suppl): 21S-28S. 6. Kenny-Walsh E. Clinical outcomes after hepatitis C infection from contaminated anti-D immune globulin. N Engl J Med 1999;340:1228-1233. 7. Wiese M, Berr F, Lafrenz M, Porst H, Oesen U. Low frequency of cirrhosis in a hepatitis C (genotype 1b) single-source outbreak in Germany: a 20year multicenter study. HEPATOLOGY 2000;32:91-96. 8. Garson JA, Tuke PW, Makris M, Briggs M, Machin SJ, Preston FE, Tedder RS. Demonstration of viraemia patterns in haemophiliacs treated with hepatitis-C-virus contaminated factor VIII concentrates. Lancet 1990;336:1022-1025. 9. Alter MJ, Margolis HS, Krawczynski K, Judson FN, Mares A, Alexander WJ, Hu PY, et al. The natural history of community-acquired hepatitis C in the United States. N Engl J Med 1992;327:1899-1905.
LARGHI ET AL.
999
10. Farci P, Alter HJ, Wong D, Miller RH, Shih JW, Jett B, Purcell RH. A long-term study of hepatitis C virus replication in non-A, non-B hepatitis. N Engl J Med 1991;325:98-104. 11. Di Bisceglie AM, Goodman ZD, Ishak KG, Hoofnagle JH, Melpolder JJ, Alter HJ. Long-term clinical and histopathological follow-up of chronic posttransfusion hepatitis. HEPATOLOGY 1991;14:969-974. 12. Tremolada F, Casarin C, Alberti A, Drago C, Tagger A, Ribero ML, Realdi G. Long-term follow-up study of non-A, non-B (type C) post-transfusion hepatitis. J Hepatol 1992;16:273-281. 13. Koretz RL, Abbey H, Coleman E, Gitnick G. Non-A, non-B post-transfusion hepatitis: looking back in the second decade. Ann Intern Med 1993;119:110-115. 14. Mattsson L, Sonnerborg A, Weiland O. Outcome of acute symptomatic non-A, non-B hepatitis: a 13-year follow-up study of hepatitis C virus markers. Liver 1993;13:274-278. 15. Barrera JM, Bruguera M, Ercilla MG, Gil C, Celis R, Gil MP, del Valle Onorato M, et al. Persistent hepatitis C viremia after acute self-limiting post-transfusion hepatitis C. HEPATOLOGY 1995;21:639-644. 16. Seeff LB, Hollinger FB, Alter HJ, Wright EC, Cain CM, Buskell ZJ, Ishak KG, et al. Long-term mortality and morbidity of transfusion-associated non-A, non-B, and type C hepatitis: A National Heart, Lung, and Blood Institute collaborative study. HEPATOLOGY 2001; 33: 455-463. 17. Donahue JG, Munoz A, Ness PM, Brown DE, Yawn DH, McAllister HA, Reitz BA, et al. The declining risk of post-transfusion hepatitis C virus infection. N Engl J Med 1992;327:369-373. 18. Schreiber GB, Busch MP, Kleinman SH, Korelitz JJ. The risk of transfusion-transmitted viral infections. N Engl J Med 1996;334:1685-1690. 19. Alter MJ. The epidemiology of hepatitis C. HEPATOLOGY 1997;26(Suppl): 62S-65S. 20. Desmet VJ, Gerber M, Hoofnagle JH, Manns M, Scheuer PJ. Classification of chronic hepatitis: diagnosis, grading and staging. HEPATOLOGY 1994;19:1513-1520. 21. Tagger A, Donato F, Ribero ML, Chiesa R, Portera G, Gelatti U, Albertini A, et al. Case-control study on hepatitis C virus (HCV) as a risk factor for hepatocellular carcinoma: the role of HCV genotypes and the synergism of hepatitis B virus and alcohol. Brescia HCC Study. Int J Cancer 1999;81: 695-699. 22. Felsenstein J. PHYLIP (Phylogeny Inference Package) version 3.5c. Department of Genetics, University of Washington, Seattle, 1993. 23. Felsenstein J. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 1985;39:783-791. 24. Brown AJL. Methods in evolutionary analysis of viral sequences. In: Morse SS, eds. The evolutionary biology of viruses. New York, NY: Raven Press; 1994, pp 75-84. 25. Tagger A, Donato F, Ribero ML, Chiesa R, Tomasoni V, Portera G, Gelatti U, et al. A case-control study on GB virus-C/hepatitis G virus infection and hepatocellular carcinoma. Brescia HCC Study. HEPATOLOGY 1997;26:1653-1657. 26. Tagger A, Ribero ML, Larghi A, Donato F, Zuin M, Chiesa R, Benetti G, et al. Prevalence of GB virus-C/hepatitis G virus infection in patients with cryptogenic chronic liver disease and in patients with primary biliary cirrhosis or Wilson’s disease. Am J Gastroenterol 1999;94:484-488. 27. Power JP, Lawlor E, Davidson F, Holmes EC, Yap PL, Simmonds P. Molecular epidemiology of an outbreak of infection with hepatitis C virus in recipients of anti-D immunoglobulin. Lancet 1995;345:1211-1213. 28. Widell A, Christensson B, Wiebe T, Schalen C, Hansson HB, Allander T, Persson MA. Epidemiologic and molecular investigation of outbreaks of hepatitis C virus infection on a pediatric oncology service. Ann Intern Med 1999;130:130-134. 29. Ohto H, Terazawa S, Sasaki N, Sasaki N, Hino K, Ishiwata C, Kako M, et al. Transmission of hepatitis C virus from mothers to infants. N Engl J Med 1994;330:744-750. 30. Esteban JI, Gomez J, Martell M, Cabot B, Quer J, Camps J, Gonzalez A, et al. Transmission of hepatitis C virus by a cardiac surgeon. N Engl J Med 1996;334:555-560. 31. Suzuki K, Mizokami M, Lau JY, Mizoguchi N, Kato K, Mizuno Y, Sodeyama T, et al. Confirmation of hepatitis C virus transmission through
1000
32.
33.
34.
35.
LARGHI ET AL.
needlestick accidents by molecular evolutionary analysis. J Infect Dis 1994; 170:1575-1578. Alberti A, Morsica G, Chemello L, Cavalletto D, Noventa F, Pontisso P, Ruol A. Hepatitis C viraemia and liver disease in symptom-free individuals with anti-HCV. Lancet 1992;340:697-698. Missale G, Bertoni R, Lamonaca V, Valli A, Massari M, Mori C, Rumi MG, et al. Different clinical behaviors of acute hepatitis C virus infection are associated with different vigor of the anti-viral-cell-mediated immune response. J Clin Invest 1996;98:706-714. Thursz M, Yallop R, Goldin R, Trepo C, Thomas HC. Influence of MHC class II genotype on outcome of infection with hepatitis C virus. Lancet 1999;354:2119-2124. Villano SA, Vlahov D, Nelson KE, Cohn S, Thomas DL. Persistence of viremia and the importance of long-term follow-up after acute hepatitis C infection. HEPATOLOGY 1999;29:908-914.
HEPATOLOGY, October 2002
36. Marcellin P. Hepatitis C: the clinical spectrum of the disease. J Hepatol 1999;31(Suppl 1):9-16. 37. Vogt M, Lang T, Fro¨ sner G, Klinger C, Sendl AF, Zeller A, Wiebecke B et al. Prevalence and clinical outcome of hepatitis C infection in children who underwent cardiac surgery before the implementation of blood-donor screening. N Engl J Med 1999;341:866-870. 38. Alter MJ, Kruszon-Moran D, Nainan OV, McQuillan GM, Gao F, Moyer LA, Kaslow RA, et al. The prevalence of hepatitis C virus infection in the United States, 1988 through 1994. N Engl J Med 1999; 341:556-562. 39. Amoroso P, Rapicetta M, Tosti ME, Mele A, Spada E, Buonocore S, Lettieri G, et al. Correlation between virus genotype and chronicity rate in acute hepatitis C. J Hepatol. 1998;28:939-944. 40. Mondelli MU, Silini E. Clinical significance of hepatitis C virus genotypes. J Hepatol 1999;31(Suppl):65-70.
See Editorial on Page 1020
H
epatitis C virus (HCV) infection has a worldwide distribution1,2 and has been recognized as a major cause of chronic hepatitis, cirrhosis, and end-stage liver disease.3 Although the understand-
Abbreviations: HCV, hepatitis C virus; AHC, acute hepatitis C; ALT, alanine aminotransferase; HBV, hepatitis B virus; HIV, human immunodeficiency virus; HGV, hepatitis G virus; HAV, hepatitis A virus; ELISA, enzyme-linked immunosorbent assay; UPGMA, unweighted pair group method with arithmetic mean; UTR, untranslated region; NS3, nonstructural protein 3; NS5b, nonstructural protein 5b. From the 1Division of Internal Medicine, Department of Medicine, Surgery and Dentistry, Ospedale San Paolo, San Paolo School of Medicine; the 2Institutes of Virology and 3Hygiene, University of Milan; the 4Dipartimento di Biologia Strutturale e Funzionale, University of Insubria, Varese; and the 5Institute of Hygiene, University of Brescia, Brescia, Italy. Received November 19, 2001; accepted July 17, 2002. Supported in part by grants from MURST (No. 12-1-21, 1996 and No.12-124, 1997), Italy. Alberto Larghi is currently a first-year Fellow of the Department of Digestive and Liver Diseases, College of Physicians and Surgeons at Columbia University, 630 West 168th Street, P & S 10-508, New York, NY 10032. Address reprint requests to: Mauro Podda, M.D., Division of Internal Medicine, Department of Medicine, Surgery and Dentistry, San Paolo School of Medicine, University of Milan, Ospedale San Paolo, Via di Rudinı` 8, 20142 Milan, Italy. E-mail: [email protected]; fax: (39) 02-89123960. Copyright © 2002 by the American Association for the Study of Liver Diseases. 0270-9139/02/3604-0027$35.00/0 doi:10.1053/jhep.2002.36129
ing of many aspects of HCV infection has improved significantly,4 its natural history has not yet been fully defined.5 Recent data from retrospective and prospective studies indicate that HCV acquired from parenteral exposure to contaminated anti-D immune globulin is followed by low rates of progression to chronic infection and to histologic cirrhosis.6,7 However, because prospective studies have been performed almost exclusively in the transfusion setting, few data are available on the clinical course and outcome of acute hepatitis C resulting from other sources of parenteral exposure.8-16 Indeed, with the significant decrease in cases of posttransfusion hepatitis,17-19 other modes of transmission have gained importance, but the disease often remains undiagnosed during the acute phase, which is usually asymptomatic.3-5 Therefore, the prospective observation of cohorts of subjects in whom acute hepatitis C (AHC) has been recognized at an early stage would supply valuable information on the shortterm outcome of the infection. We report an outbreak of HCV infection shown by phylogenetic analysis of the coding regions for the envelope glycoproteins E1 and E2 of HCV among subjects infected in the process of participating in pharmacokinetics studies, and we report their clinical, virologic, and histologic outcome. 993
994
LARGHI ET AL.
Patients and Methods Patients and Clinical Setting. In July, 1995, 4 health care workers from a nursing home were referred to our center after the diagnosis of acute hepatitis. Alanine aminotransferase (ALT) levels in these patients were elevated 28 to 51 days before presentation to our center. The subjects were part of a group of 29 health care workers who had taken part as healthy volunteers in 2 consecutive studies conducted between February and July, 1995, that investigated the pharmacokinetics of an oral antihypertensive drug and of an oral nonsteroidal anti-inflammatory drug. Four weeks before enrollment in either study, subjects had undergone biochemical and hematologic screening tests, including serum liver enzyme activity. Blood tests for serological markers of HCV infection, hepatitis B virus (HBV) infection, and human immunodeficiency virus (HIV) infection were not included in the study protocols, nor had serum samples been stored in the laboratory. All 29 subjects were invited to attend our center for blood testing and clinical evaluation. All but 2 subjects agreed to undergo clinical, biochemical, virologic, and, when indicated, histologic evaluation. They presented to our center for the first time between July 2 and August 1, 1995. Case Definition and Procedures. Diagnosis of AHC was based on the finding of ALT level of at least 10 times the upper normal limit in the presence of HCV RNA. The incubation period tentatively was defined as the time between apparent exposure and the first ALT value twice the upper limit of the normal range. Other potential causes of liver disease were excluded. In particular, HBV DNA and hepatitis G virus (HGV) RNA as well as serologic markers of acute HBV and hepatitis A virus (HAV) infection were investigated in all patients. Subjects who were resulted HCV RNA–positive after the initial visit underwent weekly blood samplings for ALT and HCV RNA determination during the first 4 months. Subsequently, patients were tested monthly for at least 12 months. Antibodies against HCV (anti-HCV) were sought every week until their detection. Subjects who had tested HCV RNA–negative were followed on a monthly basis for at least 12 months and underwent the same biochemical and virologic determinations. Liver biopsy was scheduled to be performed 6 to 12 months after AHC diagnosis if serum HCV RNA was still detectable. Histologic diagnosis was made with both hematoxylin and eosin- and reticulin-stained liver sections according to a recent international scoring system for chronic hepatitis.20 Samples were read by a pathologist who was blind to the clinical data.
HEPATOLOGY, October 2002
After the first year, follow-up visits were scheduled every 4 months for all patients who tested positive for HCV RNA. Interferon treatment was offered to all patients with persistent HCV infection and increased serum ALT activity. The protocol was approved by the bioethics committee of the San Paolo Hospital in Milan and by the Local Health Authority. Laboratory Methods. For laboratory determinations other than routine measurements, serum samples were stored at ⫺80°C. Anti– hepatitis B core antigen antibodies, hepatitis B surface antigen, and anti– hepatitis A immunoglobulin M antibodies were measured in serum samples (Axym, Abbott, North Chicago, Ill.). Antibodies to HCV were detected by a third-generation enzymelinked immunosorbent assay (ELISA) (HCV 3.0 ELISA; Ortho Diagnostic Systems, Raritan, NJ) and recombinant immunoblot assay (Chiron RECOMBINANT IMMUNOBLOT ASSAY 3; Ortho Diagnostic Systems). HCV RNA was detected by in-house reverse transcriptase polymerase chain reaction, with nested primers of the 5⬘noncoding region of the HCV genome. The sensitivity of this assay was 100 HCV RNA copies per mL of serum, measured by testing serially diluted samples in parallel with branched DNA version 2.0 assay (Quantiplex HCV RNA, Bayer Diagnostics, Emerville, CA). The HCV RNA load was determined longitudinally with the bDNA version 2.0 (linearity range 0.2-120 ⫻ 106 genome equivalents/mL) according to the manufacturer’s instructions. Mean HCV RNA genome equivalents per mL were calculated by dividing the total count of viral loads greater than 0.2 ⫻ 106 genome equivalents/mL by the total number of determinations after seroconversion. Samples negative by bDNA, but positive by reverse transcriptase polymerase chain reaction, were arbitrarily assigned a value of 0.1 ⫻ 106 HCV RNA genome equivalents/mL. After reverse transcription with primer E1818 (antisense: 5⬘ GTC GAG GKG SGT AGT GCC AG 3⬘, nt 1818 to 1799), the first round of polymerase chain reaction used primers E1278 (sense: 5⬘ RTM WCG GGH CAC CGC ATG G 3⬘, nt 1278 to 1296) and E1818, and the second-round primers E1300 (sense: 5⬘ GG GAC ATG ATG ATG AAC TGG 3⬘, nt 1300 to 1319) and E1604 (antisense: 5⬘ GTG CCA RCT GCC RTT GGT G 3⬘, nt 1604 to 1586). We performed direct-sequence analysis of nested polymerase chain reaction products of 258 bp (nt 1329 to 1586), encompassing the 3⬘ end of HCV envelope 1 (E1: nt 1329 to 1490) and the 5⬘ end of envelope 2 (E2: nt 1491 to 1586), which includes the hypervariable region 1. The polymerase chain reaction products were gel purified and sequenced on an Applied Biosystems ABI 373 automated sequencer with the
HEPATOLOGY, Vol. 36, No. 4, 2002
LARGHI ET AL.
995
Table 1. Characteristics on Presentation of the 16 Subjects Who Tested Positive for HCV RNA Subject
Sex
Age
ALT*
Anti-HCV
Date† of Presentation
Date† of Diagnosis of Acute Hepatitis
Presumed Date† of Infection‡
1 2 3 4 7 8 9 12 15 16 17 20 21 27 28 29
F M F F M M F F F F M F F F F F
45 33 21 23 34 41 35 30 22 25 31 30 31 35 29 35
15 92 656 34 167 29 219 52 16 427 42 10 1128 1142 10 188
Pos Pos Pos Pos Pos Pos Neg Neg Pos Pos Pos Neg Pos Neg Neg Neg
July 17 July 2 July 11 July 11 July 2 July 27 July 19 July 17 July 13 July 2 July 17 July 19 Aug 1 July 19 July 19 July 19
ND June 2 July 11 May 24 July 11 Sept 11 July 25 Aug 23 June 26 July 2 May 26 Sept 6 Aug 1 July 19 Aug 8 Aug 8
Feb 22 Feb 22 Feb 22 Feb 22 Mar 1 Mar 1 May 21 May 21 Apr 5 Apr 5 Apr 5 May 21 May 21 June 4 June 4 June 4
Abbreviation: ND, not documented. *Normal values ⬍42 IU/L. †All the dates refer to 1995. ‡Inferred from the date of participation in the pharmacokinetics studies.
PRISM dye terminator cycle sequencing kit (PE Applied Biosystems, Foster City, CA). Sequences were analyzed with the Sequencher 3.0 analysis program (Gene Codes Corporation, Ann Arbor, MI). Attribution of the HCV genotype was performed on the basis of the sequence obtained. For phylogenetic analysis, 16 sequences, each 258 nt long, obtained from the 16 HCV RNA–positive patients involved in the outbreak were compared with 20 sequences (19 of genotype 2c and 1 of genotype 2a) derived from a previous study21 taken as unrelated controls, and with 5 prototype sequences sampled from GenBank (D00944 and D10075 for genotype 2a, D10077 and D10988 for genotype 2b, D50409 for genotype 2c). The whole set of 41 sequences was resampled by bootstrapping 1,000 times and then analyzed with the PHYLIP package, v.3.5c.22 Bootstrapping of the original data set was performed to obtain a measure of the confidence we can have in a particular phylogenetic feature, such as a given branch of the tree. In effect, the random variation obtained by analyzing bootstrapped data sets is typical of the variation one would get from collecting new data sets.23 The degree of divergence between the sequences was estimated by the Kimura 2-parameter method, with a transition-transversion ratio of 2:0 (DNADIST of the PHYLIP package).24 The set of distance matrices was then analyzed by UPGMA (Unweighted Pair Group Method with Arithmetic Mean) as implemented by the NEIGHBOR program of the PHYLIP package. The final unrooted consensus tree was drawn by the CONSENSE program of the same package.
HGV RNA Detection. Reverse transcriptase polymerase chain reaction amplification was used, with nested primers from 5⬘ untranslated region (UTR), nonstructural protein (NS)3 helicase, and NS5b regions to detect HGV RNA. The methods are described in detail elsewhere.25 HBV DNA Detection. Nucleic acid was extracted from 100 L of serum with the proteinase K-sodium dodecyl sulphate-phenol-chloroform method. HBV DNA was amplified with nested primers as described previously.26
Results Sixteen of the 27 subjects who agreed to be evaluated tested positive for serum HCV RNA on presentation to our center. Nucleotide sequence analysis of E1 revealed they were all infected with HCV genotype 2c. Ten were anti-HCV positive and 9 out of the 16 subjects had elevated serum ALT levels. The 6 HCV RNA–positive subjects who were anti-HCV–negative at presentation to our center seroconverted within 1 to 6 weeks. Subjects’ characteristics are shown in Table 1. All serum samples tested negative for HBV DNA and HGV RNA, as well as for serologic markers of acute HBV and HAV infection. Of the 16 HCV RNA–positive subjects, 8 showed serum ALT levels higher than 10 times the upper normal limit at the time of presentation or within the previous 2 months, 7 showed a progressive elevation of serum liver enzymes during the first 2 months of follow-up, and 1 subject (patient 1) had persistently normal ALT levels in
996
LARGHI ET AL.
association with intermittently positive serum HCV RNA throughout the observation period. Therefore, AHC was documented in 15 out of the 16 subjects who tested HCV RNA–positive at presentation. AHC was clinically asymptomatic in all subjects but 1 (patient 12), who experienced mild and transient malaise, nausea, and vomiting parallel to the elevation of ALT. The aligned sequences of 258 nt, encompassing the E1 and E2 regions, obtained from the 16 HCV RNA–positive patients, were bootstrapped and a phylogenetic tree based on the UPGMA method was obtained, as shown in Fig. 1. The most evident feature of the tree is the clustering of all patients involved in the outbreak (denoted by PT#). The bootstrap value of 1,000/1,000 of the node leading to the PT cluster lends statistical robustness to the interpretation that all of the patients involved in the outbreak were infected by a common source. Prototype sequences of genotypes 2a and 2b also form separate clusters. The BsHCC197 unrelated control, attributed to genotype 2a on the basis of its sequence, correctly clusters with the 2a prototypes. The other unrelated control, BsHCC226, although attributed to genotype 2c on the basis of its sequence features, exhibited a degree of polymorphism sufficient to cluster apart. All 11 subjects who were HCV RNA–negative on presentation had normal serum ALT values, and none developed acute hepatitis during follow-up. Anti-HCV antibodies were detected in one subject at presentation. Biochemical and Virologic Course During the First Year of Follow-Up. One of the 15 subjects with AHC refused to be followed further after the first visit (patient 17). The biochemical and virologic course of HCV infection during the first year of follow-up in the remaining 14 subjects with AHC is summarized in Table 2. Infection resolved spontaneously in seven subjects. In 5 of these (Nos. 2, 3, 4, 21, and 27), HCV RNA became undetectable 4 to 5 months after the presumed time of infection and preceded ALT normalization by 2 to 12 weeks. A representative case is shown in Fig. 2A. In two other subjects, the disappearance of serum HCV RNA occurred eight (patient 12) and thirteen months (patient 8) after the presumed time of infection and was associated with ALT normalization. All seven subjects were followed for 6 to 8 months after the last positive polymerase chain reaction and were HCV RNA–negative in 3 serum samples collected 2 months apart. Low HCV RNA loads (range ⬍ 0.2 ⫻ 106 – 0.39 ⫻ 106 genome equivalents/mL) were detected during the viremic phase in all of them. Seven other subjects did not clear the virus during the first year of follow-up. In four cases, HCV RNA remained persistently detectable (Nos. 7, 9, 16, and 20). Serum ALT activity was persistently abnormal in three of them (a
HEPATOLOGY, October 2002
Fig. 1. Phylogenetic tree analysis comparing coding sequences in the HCV regions for the envelope glycoproteins E1 and E2 from 41 isolates. For phylogenetic analysis, 16 sequences, 258 nt long, obtained from the 16 HCV RNA–positive patients involved in the outbreak were compared with 20 sequences (19 of genotype 2c and 1 of genotype 2a) originating from a previous study21 taken as unrelated controls, and 5 prototype sequences sampled from GenBank (D00944 and D10075 for genotype 2a, D10077 and D10988 for genotype 2b, D50409 for genotype 2c). Bootstrap values greater than 800/1,000 are reported at the nodes. Accession numbers are as follows: for the 5 sequences taken from GenBank D00944 (HPCPOLP or HC-J6), D10075 (HPCHCJ5 or HC-J5), D10077 (HPCHCJ7 or HC-J7), D10988 (HPCJ8G or HC-J8), D50409 (BEBE-1); for the sequences originated from the patients (PT) involved in the outbreak, AF237634 to AF237649; for the sequences originated from the controls (BSHCCX and BSCOX), AF142385 to AF142404.
representative case in Fig. 2B), while in one case (patient 9), a complete and sustained normalization of serum transaminase levels occurred after 10 months of followup. In the remaining three subjects (Nos. 15, 28, and 29), HCV RNA was detected intermittently over the initial 6-month period of follow-up with ALT levels paralleling HCV RNA fluctuations (a representative case in Fig. 2C).
HEPATOLOGY, Vol. 36, No. 4, 2002
LARGHI ET AL.
Table 2. Biochemical and Virologic Course of HCV Infection in 14 of the 15 Subjects With Acute Hepatitis C During the First Year of Follow-up ALT
HCV RNA Clearance (n ⫽ 7) Persistently positive (n ⫽ 4) Intermittently positive (n ⫽ 3) All subjects (n ⫽ 14)
Normalized
Fluctuating
Persistently Abnormal
7 1 0 8
0 0 3 3
0 3 0 3
997
positive six months after the diagnosis of acute hepatitis. Overall, the predominant histologic finding was lobular inflammation. A mild necroinflammatory activity was also detected in three subjects (Nos. 8, 12 and 15) in
NOTE. Patient 17 was lost at follow-up after the first visit.
At the end of 12 months of follow-up, HCV RNA became persistently positive in two subjects, while the fluctuating pattern persisted in the third subject (patient 15). Titers ranging from 0.2 to 2.0 ⫻ 106 genome equivalents/mL (mean 1.1 ⫻ 106 genome equivalents/mL) were detected during the first year of follow-up, both in the four cases (Nos. 7, 9, 16, and 20) who were persistently HCV RNA–positive and in the three cases (Nos. 15, 28, and 29) who were intermittently HCV RNA–positive. Additional Follow-Up. Fourteen patients with AHC were further observed with a median follow-up period of 45 months (range 33-53 months) from the time of diagnosis. All seven patients in whom infection resolved spontaneously within 13 months of the presumed time of infection remained persistently HCV RNA–negative with normal ALT levels throughout the follow-up period. Among the seven patients with persistent (Nos. 7, 9, 16, and 20) or intermittent (Nos. 15, 28, and 29) viremia during the first year of follow-up, all but one (patient 15) were HCV RNA positive for at least 12 months thereafter. In patient 15 infection resolved spontaneously 24 months after the presumed time of infection, and HCV RNA was persistently undetectable and ALT levels were normal in three subsequent serum samples collected at four-monthly intervals. Among the six patients with chronic infection, one (patient 9) had persistently normal ALT levels whereas five patients had persistently elevated ALT throughout the second year of follow-up. Of these five patients, three (Nos. 20, 28, and 29) started a 12-month course of interferon alfa at the dose of 3 MU thrice weekly 15 to 18 months after presentation to our center. All showed sustained virologic response, and liver biopsies performed six months after the end of therapy in two subjects (Nos. 20 and 29) showed improvement in the histologic activity score (from 5 to 3 and from 9 to 2, respectively). Histologic Examination. The results of the histologic examination are shown in Table 3. Liver biopsy was performed in all nine subjects who were still HCV RNA
Fig. 2. Representative cases of different biochemical and virologic courses in an outbreak of AHC. ALT values are expressed as multiples of the upper limits of the normal range. The light vertical bars indicate negative assays for HCV RNA, and the dark bars positive assays. (A) A case of spontaneous clearance of HCV infection is reported. (B) A case with persistently abnormal ALT levels and persistently positive HCV RNA is described. (C) A case of intermittently positive HCV RNA associated with fluctuations of serum transaminase levels is shown. The effects of interferon alfa administration (3 MU thrice weekly for 12 months) are also described.
998
LARGHI ET AL.
HEPATOLOGY, October 2002
Table 3. Histologic Outcome of Acute Hepatitis C in 9 Patients Who Tested Still HCV RNA Positive 6 Months After the Diagnosis of Acute Hepatitis Liver Histology
Subject
HCV RNA Course
ALT Course
Periportal Necrosis
Lobular Necrosis
Portal Inflammation
Fibrosis
Knodell HAI Score
7 9 16 20 15 28 29 8 12
PP PP PP PP IP IP IP C C
PA N PA PA F F F N N
1 1 3 0 1 0 4 0 0
4 3 3 4 3 3 3 2 3
1 0 3 1 1 1 1 0 1
0 1 1 0 1 0 1 0 0
6 5 10 5 6 4 9 2 4
Abbreviations: PP, persistently positive; IP, intermittently positive; C, clearance; PA, persistently abnormal; N, normalized; F, fluctuating. NOTE. Patient 15 with intermittent viremia during the first year of follow-up cleared HCV RNA 24 months after the presumed time of infection.
whom infection resolved spontaneously 2-12 months after liver biopsy.
Discussion Primary HCV infection is poorly characterized because most patients are asymptomatic and, therefore, it is rarely diagnosed.3-5 The clinical picture of AHC has mainly been described in transfused patients whereas for other modes of infection such as needle stick or accidental exposure it is not well known. The present study describes the occurrence and outcome of an outbreak of HCV infection in a group of subjects who took part in two pharmacokinetics studies as healthy volunteers. This outbreak allowed us to perform a detailed study of the clinical course of acute HCV infection in a cohort of subjects infected by a parenteral route other than blood transfusion. The temporal clustering of the cases linked participation in the pharmacokinetics studies to the development of AHC. The demonstration of a high degree of homology among the sequences of the viral region encompassing E1 and E2 in all of the subjects studied strengthens the probability of a common source of infection. The value of the double-pronged approach of molecular and phylogenetic techniques in providing evidence of the occurrence of HCV outbreaks27,28 and documenting the transmission of HCV infection29-31 has been reported previously. The course of transmission remains unclear, but we may speculate that the outbreak started from patient 1, who was already HCV RNA–positive at presentation but without documented AHC. She may have had a pre-existing infection characterized by viremia with normal serum ALT levels (i.e., an HCV carrier32). Despite the fact that the patients included in this study were exposed to a common source of infection, the incu-
bation period and the outcome of the acute phase of the disease were highly variable, probably reflecting the biologic variability of HCV infection. The average incubation period was 10 weeks, but the range was 6 to 28 weeks. The clinical and virologic course also varied, both in patients in whom infection resolved spontaneously and in those developing chronic hepatitis. This suggests that host factors play a major role in conditioning the outcome of acute HCV infection.33,34 Patients with viral clearance and those with persistent infection showed 2 distinct patterns of viremia. Patients whose HCV RNA levels became undetectable exhibited lower viral loads during the course of infection compared with those who remained persistently viremic, but a specific threshold value that predicts the outcome of infection could not be detected. HCV RNA load was not related with the time of viral clearance; indeed, 2 patients with low viral load cleared the virus more than 6 months after infection (8 and 13 months); in another case, relatively high levels of viremia were found before clearance at 24 months. This finding suggests that longer periods of time than generally considered may be required to establish whether HCV infection has progressed to chronicity.35 Hepatitis C is a disease with a high rate of progression to chronicity. Estimates indicate that nearly 85% of patients develop chronic HCV infection after acute infection.36 The finding that in our setting only 43% (95% confidence interval: 18% to 71%) of the patients became chronically infected may indicate that the tendency to chronicity of HCV infection is lower than expected. However, the validity of this difference is questionable given the small number of patients examined and the wide, unstable confidence interval. Similar rates of chronic infection (55%) were found in young women who had received HCV-contaminated
HEPATOLOGY, Vol. 36, No. 4, 2002
anti-D immunoglobulins6,7 and in children who acquired posttransfusion hepatitis C after cardiac surgery.37 These rates are lower than those reported in community-based investigations of acute HCV infection38 or in analyses of patients who were infected through transfusion as adults.15 The reason for the relatively high rate of spontaneous clearance in our cohort as well as in women infected by anti-D immunoglobulins and children with posttransfusion hepatitis is uncertain. Differences in host immune responses have been implicated, but data on age-related immune responses to HCV infection are not available. The presence of genotype 2c as the source of outbreak also may have contributed to the better short-term prognosis in the present study; however, the impact of HCV genotypes on disease progression is still a matter of debate, and this is particularly true for the acute setting, in which there is a limited quantity of data available.35,39,40 In conclusion, the sequence analysis of the HCV hypervariable region 1 supplied strong evidence for transmission of HCV from a common source of infection, leading to an outbreak of hepatitis C among participants in pharmacokinetics studies. Acute HCV infection showed highly variable clinical, virologic and histologic features. The clinical course of infection was characterized by lower rates of chronicity than generally reported and by late clearance in a few cases. Our results point to the need for future research aimed at better defining the role of viral and host factors in determining the chronicity of HCV infection and the rate of disease progression. Acknowledgment: The authors thank Nora V. Bergasa, MD, for careful help in revising the manuscript and Marco Maggioni, MD, for the histologic evaluation.
References 1. Alter MJ. Epidemiology of hepatitis C in the West. Semin Liver Dis 1995; 15:5-14. 2. Mansell CJ, Locarnini SA. Epidemiology of hepatitis C in the East. Semin Liver Dis 1995;15:15-32. 3. Hoofnagle JH. Hepatitis C: the clinical spectrum of disease. HEPATOLOGY 1997;26(Suppl):15S-20S. 4. Di Bisceglie AM. Hepatitis C. Lancet 1998;351:351-355. 5. Seeff LB. Natural history of hepatitis C. HEPATOLOGY 1997;26(Suppl): 21S-28S. 6. Kenny-Walsh E. Clinical outcomes after hepatitis C infection from contaminated anti-D immune globulin. N Engl J Med 1999;340:1228-1233. 7. Wiese M, Berr F, Lafrenz M, Porst H, Oesen U. Low frequency of cirrhosis in a hepatitis C (genotype 1b) single-source outbreak in Germany: a 20year multicenter study. HEPATOLOGY 2000;32:91-96. 8. Garson JA, Tuke PW, Makris M, Briggs M, Machin SJ, Preston FE, Tedder RS. Demonstration of viraemia patterns in haemophiliacs treated with hepatitis-C-virus contaminated factor VIII concentrates. Lancet 1990;336:1022-1025. 9. Alter MJ, Margolis HS, Krawczynski K, Judson FN, Mares A, Alexander WJ, Hu PY, et al. The natural history of community-acquired hepatitis C in the United States. N Engl J Med 1992;327:1899-1905.
LARGHI ET AL.
999
10. Farci P, Alter HJ, Wong D, Miller RH, Shih JW, Jett B, Purcell RH. A long-term study of hepatitis C virus replication in non-A, non-B hepatitis. N Engl J Med 1991;325:98-104. 11. Di Bisceglie AM, Goodman ZD, Ishak KG, Hoofnagle JH, Melpolder JJ, Alter HJ. Long-term clinical and histopathological follow-up of chronic posttransfusion hepatitis. HEPATOLOGY 1991;14:969-974. 12. Tremolada F, Casarin C, Alberti A, Drago C, Tagger A, Ribero ML, Realdi G. Long-term follow-up study of non-A, non-B (type C) post-transfusion hepatitis. J Hepatol 1992;16:273-281. 13. Koretz RL, Abbey H, Coleman E, Gitnick G. Non-A, non-B post-transfusion hepatitis: looking back in the second decade. Ann Intern Med 1993;119:110-115. 14. Mattsson L, Sonnerborg A, Weiland O. Outcome of acute symptomatic non-A, non-B hepatitis: a 13-year follow-up study of hepatitis C virus markers. Liver 1993;13:274-278. 15. Barrera JM, Bruguera M, Ercilla MG, Gil C, Celis R, Gil MP, del Valle Onorato M, et al. Persistent hepatitis C viremia after acute self-limiting post-transfusion hepatitis C. HEPATOLOGY 1995;21:639-644. 16. Seeff LB, Hollinger FB, Alter HJ, Wright EC, Cain CM, Buskell ZJ, Ishak KG, et al. Long-term mortality and morbidity of transfusion-associated non-A, non-B, and type C hepatitis: A National Heart, Lung, and Blood Institute collaborative study. HEPATOLOGY 2001; 33: 455-463. 17. Donahue JG, Munoz A, Ness PM, Brown DE, Yawn DH, McAllister HA, Reitz BA, et al. The declining risk of post-transfusion hepatitis C virus infection. N Engl J Med 1992;327:369-373. 18. Schreiber GB, Busch MP, Kleinman SH, Korelitz JJ. The risk of transfusion-transmitted viral infections. N Engl J Med 1996;334:1685-1690. 19. Alter MJ. The epidemiology of hepatitis C. HEPATOLOGY 1997;26(Suppl): 62S-65S. 20. Desmet VJ, Gerber M, Hoofnagle JH, Manns M, Scheuer PJ. Classification of chronic hepatitis: diagnosis, grading and staging. HEPATOLOGY 1994;19:1513-1520. 21. Tagger A, Donato F, Ribero ML, Chiesa R, Portera G, Gelatti U, Albertini A, et al. Case-control study on hepatitis C virus (HCV) as a risk factor for hepatocellular carcinoma: the role of HCV genotypes and the synergism of hepatitis B virus and alcohol. Brescia HCC Study. Int J Cancer 1999;81: 695-699. 22. Felsenstein J. PHYLIP (Phylogeny Inference Package) version 3.5c. Department of Genetics, University of Washington, Seattle, 1993. 23. Felsenstein J. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 1985;39:783-791. 24. Brown AJL. Methods in evolutionary analysis of viral sequences. In: Morse SS, eds. The evolutionary biology of viruses. New York, NY: Raven Press; 1994, pp 75-84. 25. Tagger A, Donato F, Ribero ML, Chiesa R, Tomasoni V, Portera G, Gelatti U, et al. A case-control study on GB virus-C/hepatitis G virus infection and hepatocellular carcinoma. Brescia HCC Study. HEPATOLOGY 1997;26:1653-1657. 26. Tagger A, Ribero ML, Larghi A, Donato F, Zuin M, Chiesa R, Benetti G, et al. Prevalence of GB virus-C/hepatitis G virus infection in patients with cryptogenic chronic liver disease and in patients with primary biliary cirrhosis or Wilson’s disease. Am J Gastroenterol 1999;94:484-488. 27. Power JP, Lawlor E, Davidson F, Holmes EC, Yap PL, Simmonds P. Molecular epidemiology of an outbreak of infection with hepatitis C virus in recipients of anti-D immunoglobulin. Lancet 1995;345:1211-1213. 28. Widell A, Christensson B, Wiebe T, Schalen C, Hansson HB, Allander T, Persson MA. Epidemiologic and molecular investigation of outbreaks of hepatitis C virus infection on a pediatric oncology service. Ann Intern Med 1999;130:130-134. 29. Ohto H, Terazawa S, Sasaki N, Sasaki N, Hino K, Ishiwata C, Kako M, et al. Transmission of hepatitis C virus from mothers to infants. N Engl J Med 1994;330:744-750. 30. Esteban JI, Gomez J, Martell M, Cabot B, Quer J, Camps J, Gonzalez A, et al. Transmission of hepatitis C virus by a cardiac surgeon. N Engl J Med 1996;334:555-560. 31. Suzuki K, Mizokami M, Lau JY, Mizoguchi N, Kato K, Mizuno Y, Sodeyama T, et al. Confirmation of hepatitis C virus transmission through
1000
32.
33.
34.
35.
LARGHI ET AL.
needlestick accidents by molecular evolutionary analysis. J Infect Dis 1994; 170:1575-1578. Alberti A, Morsica G, Chemello L, Cavalletto D, Noventa F, Pontisso P, Ruol A. Hepatitis C viraemia and liver disease in symptom-free individuals with anti-HCV. Lancet 1992;340:697-698. Missale G, Bertoni R, Lamonaca V, Valli A, Massari M, Mori C, Rumi MG, et al. Different clinical behaviors of acute hepatitis C virus infection are associated with different vigor of the anti-viral-cell-mediated immune response. J Clin Invest 1996;98:706-714. Thursz M, Yallop R, Goldin R, Trepo C, Thomas HC. Influence of MHC class II genotype on outcome of infection with hepatitis C virus. Lancet 1999;354:2119-2124. Villano SA, Vlahov D, Nelson KE, Cohn S, Thomas DL. Persistence of viremia and the importance of long-term follow-up after acute hepatitis C infection. HEPATOLOGY 1999;29:908-914.
HEPATOLOGY, October 2002
36. Marcellin P. Hepatitis C: the clinical spectrum of the disease. J Hepatol 1999;31(Suppl 1):9-16. 37. Vogt M, Lang T, Fro¨ sner G, Klinger C, Sendl AF, Zeller A, Wiebecke B et al. Prevalence and clinical outcome of hepatitis C infection in children who underwent cardiac surgery before the implementation of blood-donor screening. N Engl J Med 1999;341:866-870. 38. Alter MJ, Kruszon-Moran D, Nainan OV, McQuillan GM, Gao F, Moyer LA, Kaslow RA, et al. The prevalence of hepatitis C virus infection in the United States, 1988 through 1994. N Engl J Med 1999; 341:556-562. 39. Amoroso P, Rapicetta M, Tosti ME, Mele A, Spada E, Buonocore S, Lettieri G, et al. Correlation between virus genotype and chronicity rate in acute hepatitis C. J Hepatol. 1998;28:939-944. 40. Mondelli MU, Silini E. Clinical significance of hepatitis C virus genotypes. J Hepatol 1999;31(Suppl):65-70.