- Email: [email protected]
Late seroconversion and high chronicity rate of hepatitis C virus infection in patients with hematologic disorders
Annals of Oncology 4: 229-234, 1993. © 1993 Kluwer Academic Publishers. Printed in the Netherlands.
Original article Late seroconversion and high chronicity rate of hepatitis C virus infection in patients with hematologic disorders A. Gruber,1 H. Norder,2 L. Magnius,2 M. Rotzen,3 C. Rubio,4 L. Grillner3 & M. Bjorkholm1 'Division of Medicine, Section of Hematology and Immunology; 2National Bacteriological LMboratory; 'Department of Clinical Microbiology, Section of Virology; 'Department of Pathology, Karolinska Hospital, Stockholm, Sweden
months. Seventeen patients with resistant or relapsed acute leukemia were treated with combination chemotherapy durBackground: Patients with hematologic disorders requiring ing the acute or chronic phase of hepatitis. Suppression of the repeated blood and platelet transfusions are at high risk for inflammatory activity as reflected by a decrease of serum aminotransferase levels was recorded during the subsequent development of post-transfusion non-A, non-B hepatitis. Patients and methods: Fifty-five patients with hematologic pancytopenic period. diseases and post-transfusion non-A, non-B hepatitis were Conclusions: Hepatitis C has a high chronicity rate in pastudied. Sera were assayed for hepatitis C virus (HCV) anti- tients with hematologic disorders which parallels the situabodies with a second-generation enzyme-linked immuno- tion of hepatitis B in the immunocompromised host. Furtherassay. Sera from 40 patients were examined for the presence more, like the situation in hepatitis B, the hosts' immune of HCV RNA with a nested PCR method. response to infection seems to be involved in the pathogeneResults: The clinical picture of acute non-A, non-B hepa- sis of liver injury. Time to seroconversion may be prolonged titis did not differ from that described in other patient and detection of HCV RNA is therefore important for diaggroups: however, progression to chronic hepatitis was very nosis. common (95%). Thirty-eight (95%) of 40 patients, whose sera were analysed both serologically and for the presence of HCV RNA had verified HCV infections. In some patients Key words: immunosuppression, hematologic diseases, hepatime to seroconversion was prolonged, up to more than 14 titis C, chronic active hepatitis, non-A and non-B hepatitis Summary
Introduction
be detected within 1-3 weeks after exposure in patients subsequently developing hepatitis C [14]. Patients with hematologic disorders often require multiple red cell and platelet transfusions during periods of primary, or chemotherapy-induced bone marrow failure. We found post-transfusion hepatitis to be a common complication in this patient group and followed its clinical course in 55 patients. Sera from all patients were assayed with the second-generation antiHCV test and in 35 patients a first-generation test was also used. In addition sera from 40 patients were examined for the presence of HCV RNA with a nested PCR method.
Following the cloning of a part of the hepatitis C virus (HCV) genome and the subsequent development of an antibody test (anti-HCV) it has been shown that approximately 90% of post-transfusion non-A, non-B hepatitis is caused by HCV [1-4]. About 90% of posttransfusion non-A, non-B hepatitis occurs within 5-12 weeks after transfusion but incubation periods of up to 6 months have been reported [5]. The clinical course of acute non-A, non-B hepatitis is generally mild. A majority of the patients are asymptomatic and only about 20% will develop jaundice [6]. However, 30%-70% of patients with post-transfusion non-A, non-B hepatitis will show elevated aminotransferases after 6 months (chronic hepatitis) and at least 20% of these patients Patients and methods will eventually develop cirrhosis of the liver [6-10]. Seroconversion determined by the first-generation Patients anti-HCV enzyme-linked immunoassays (EIA) occurs within 3 months in about 60% of the patients and 90% The study included 55 patients with various hematologic disorders seroconvert within 12 months [11]. With the second- and post-transfusion non-A, non-B hepatitis (Table 1). All patients generation anti-HCV assay which is both more specific had normal aminotransferase levels prior to transfusion. The diagnosis of non-A, non-B hepatitis was based on a rise in serum alanine and sensitive the time interval to seroconversion was aminotransferase (ALT) for more than one month to at least 2.0 found to be shorter [12, 13]. By the use of the poly- Ukat/L (normal <0.7 (ikat/L) in the absence of only other possible merase chain reaction (PCR) method HCV RNA may cause for the elevation (such as hepatitis A, B, CMV or EBV infec-
230 Table 1. Clinical data of patients and results of R1BA II and HCV RNA determinations. Pt no
Age at onset of hepatitis/
Diagnosis
Onset hepatitis month/ year
Followup time, years
RJBA II
NHL AML ITP ITP ITP ITP NHL NHL ALL AA AA CML NHL AML AA AML HD ALL AML AML MDS AML AML AML ALL ALL AML AUL ITP AA AML CML
05/-73 04/-78 06/-81 11/-81 O5/-83 07/-83 09/-85 06/-85 01/-86 04/-86 06/-86 1 1/-87 03/-88 04/-88 06/-88 06/-88 01/-89 02/-89 03/-89 03/-89 04/-89 05/-89 06/-89 09/-89 10/-89 10/-89 11/-89 11/-89 11/-89 11/-89 12/-89 01/-90 03/-90 05/-90 05/-90 07/-90 08/-90 08/-90 08/-90 08/-90 09/-90 10/-90 10/-90 10/-90 01/-91 01/-91 02/-91 03/-91 05/-9I 05/-91 07/-91 08/-91 09/-91 10/-91 10/-91
18.4 13.7
+
sex
36/M 32/F 27/M 38/F 43/F 47/F 51/M 51/M 28/F 25/M 1 1 54/M 12 18/F 13 55/F 14 54/F 15 53/M 52/F 16 17 47/F 18 44/M 19 43/M 20 81/F 21 84/F 22 20/F 41/F 23 24 47/F 25 59/F 26 45/M 27 69/M 25/M 28 56/M 29 30 46/F 31 74/M 32 39/F 75/F 33 34 44/F 35 16/F 36 66/M 37 24/F 38 27/M 74/M 39 40 25/M 41 33/F 42 36/F 43 19/M 44 75/M 45 53/M 46 49/F 47 51/F 68/F 48 38/M 49 63/F 50 51 57/F 52 28/M 53 64/F 54 78/F 55 43/F 1 2 3 4 5 6 7 8 9 10
AML CML ALL AML AA ALL AUL CML ALL ALL HD MDS AML AML MDS MDS AML ALL AML AA AML AML MDS
9.9
10.2 8.7 8.5
4.8' 6.6 5.1 5.7 5.5
0.6° 1.5° 3.5° 3.5
0.6° 2.51.5° 1.2* 0.6"
HCV RNA
+ + + + (+) + +
++ + ++ ++ ND +
+
+
+ +
++ ++ ND
+ + +
ND ++ ++
+ +
+ ND
ND
ND ND
2.8
+
-
1.4* 0.3"
+ + + +
ND ND + ND
2.3
1.50.3" 0.8" 0.9" 2.2 2.2
0.3° 2.0 1.8 1.7 1.7
1.4" 1.4 .4 .0" .4 .0° ().2" .3 .3 .0
0.6' 0.9 0.7 0.5
0.5" 0.2 0.4 0.3
0.1" 0.3
ND
+ + + + + + + +
ND ND ND ++ + ++
•+•
-
+ (+) (+) +
++ ++ + + -HI-
ND
+ + + + -
++ ++ + ++ ++ ++ ++ ++ ++ ++ -H-
AML: acute myelocytic leukemia, ALL: acute lymphocytic leukemia, AUL: acute undifferentiated leukemia, MDS: myelodysplastic syndrome, CML: chronic myelocytic leukemia, NHL: non Hodgkin lymphoma, HD: Hodgkin's disease, AA: aplastic anemia, ITP: immune mediated thrombocytopenia, ND: not done, R1BA II (+) indeterminate result (positive in one band), HCV RNA ++ - positive after first, + — positive after second PCR round. * Deceased patient.
tion, or ongoing treatment with hepatotoxic drugs). The onset of hepatitis was recorded between 1985 and 1991 in 49 of the patients and between 1973 and 1983 in the remaining 6. Fifty-two patients had received platelet and/or red cell transfusions. The median number of units transfused 2-26 weeks prior to the onset of hepatitis was 28 (range 2-189). Three patients (nos. 3, 4, 6) had been treated with intravenous gammaglobulin for immune-mediated thrombocytopenia. The median age of patients at the onset of hepatitis was 44 years (range 16-84): 32 patients were female and 23 male. The median follow-up time after detection of the hepatitis was 1.2 years (range 0.1-18.6). Chronic hepatitis was defined as an elevation of ALT for more than 6 months with at least one value exceeding five times the upper limit of normal [7, 8). The clinical course in the 11 patients whose hepatitis became clinically manifest prior to 1987 was evaluated retrospectively. The remaining patients were followed prospectively. Anti-HCV assay Before June 1991 the patients' sera were analysed by a first-generation test, anti-HCV EIA (Abbott Laboratories, Chicago, IL, USA) which is based on the non-structural (NS) c 100-3 antigen only. Later sera were tested by a second-generation anti-HCV EIA (Abbott Laboratories), which includes antigens from the core (c22) and NS3 and NS4 regions of the genome, and earlier samples were reanalysed using this test. At least one serum sample from each antiHCV-positive patient was analysed by a complementary recombinant immunobinding assay (RJBA II; Ortho Diagnostic Systems, Raritan, NJ, USA). This assay qualitatively detects antibodies against structural (c22) and NS3 and NS4 (c33, cl00-3 and, 5-1-1) antigens. The tests were performed according to the manufacturer's instructions. Reactivity against two or more of the antigens in RIBA II was required to confirm a positive anti-HCV test, while reactivity against only one antigen was considered indeterminate. Patients without detectable HCV antibodies at initial analysis were repeatedly tested thereafter. When anti-HCV positivity was established at the initial test, frozen stored sera, if available, were retrospectively analysed. Detection of HCV RNA by PCR Forty patients were still alive in 1991 and their sera were analysed for the presence of HCV RNA sequences by a nested PCR method [15, 16]. Sera from five patients were analysed on consecutive occasions, before, during and after treatment with combination chemotherapy. By amplifying the HCV RNA sequence during two PCR rounds the sensitivity of the assay is increased approximately 1,000 times [17]. Serum samples were fresh-frozen and kept at -70*C until tested. RNA was prepared from 100 u.1 serum by precipitation with polyethylene/sodium dodecyl sulfate followed by phenol extraction 115]. The RNA was dissolved in 10 u.1 distilled water which had been treated with diethylpyrocarbonate. Five u,l dissolved RNA was used for cDNA synthesis using cloned Moloney murine leukemia virus reverse transcriptase (Bethesda Research Laboratories, Md, USA), and as primer the antisense NCR2 oligonucleotide was used [16]. In the first round of PCR primers NCR1 and NCR2 were used, and 5 u,l reaction mixture from the first round were used in a second PCR using NCR3 and NCR4 as the inner primers [16|. In addition to the standard precautions, a negative plasma was tested after each tested sample during RNA extraction, reverse transcription and amplification to exclude false positive results in the PCR due to cross contamination.
Liver biopsies Fourteen patients underwent liver biopsy using the Menghini technique during the chronic hepatitis phase. The specimens were fixed
231 in 10% formalin and embedded in paraffin. Sections (4 u.m) were stained with haematoxylin and eosin, periodic acid-Schiff after previous treatment with diastase and with Sirus stain. The histological findings were classified according to the degree of portal inflammation andfibrosis[18].
10
8
8 |
6
Results Anti-HCV Fifty-one of the 55 patients had been followed serologically for at least 3 months after the onset of hepatitis. Thirty-eight (75%) of the 51 had detectable antibodies to HCV with the second-generation antibody test, 35 of those were positive and 3 had an indeterminate (all 3 anti-c22 positive) reaction in RIBA II. Thirty of the thirty-eight anti-HCV-positive patients were negative in at least one preceding test. The remaining eight patients were positive in the first analysis performed and no sera for retrospective analysis were available. The exact time to seroconversion could not be determined in most patients. However, 11 patients who had verified HCV infections (positive HCV RNA and/or later seroconversion and RIBA II positivity) were anti-HCVnegative for more than 3 months after increased ALT levels were observed (4 pts 4, 2 pts 5, 2 pts 6, 1 pt 7, 1 pt 9 and 1 pt 14 months). Sera from 35 patients, which initially had been assayed with the first-generation anti-HCV test, were reanalysed with the second-generation assay. Twelve samples were positive and 10 negative by both assays. Thirteen sera with HCV antibodies detectable by the second-generation assay (10 RIBA II positive, 3 indeterminate) were negative by the first-generation test.
420
o o •••
91
•at
HCV RNA Fig. 1. Relationship between ALT levels and HCV RNA. Positive PCR after first (++) and after second (+) rounds. Anti-HCV positive (•), anti-HCV negative (o) samples.
or two amplifications of HCV RNA sequences, the ALT levels ranged from normal to high (Fig.l). Clinical course
During the acute infection phase most patients were asymptomatic. Ten patients (18%) were jaundiced. None of the patients suffered a fulminant hepatitis. Thirteen patients (23%) had an elevation of ALT to more than 20 ukat/L. The median of the maximal ALT elevation was 11.3 jikat/L (range 2.7->48 ukat/L). After the acute phase, which in most patients lasted for about one month, the aminotransferase levels fluctuated. Forty-four patients were followed for more than 6 months (median 20 months) and of those, 42 (95%) progressed to chronic hepatitis (95% confidence interHCV RNA determination val 85%-99%). In two of 42 patients (nos. 30 and 35) with elevation of ALT for more than 6 months, hepatiSera from 40 patients were analysed for the presence of tis resolved spontaneously after 18 months (normal HCV RNA. The duration of hepatitis at the time of ALT levels for three months, no detectable HCV RNA sampling ranged from one week to 18.5 years (median and in pat. no. 35 disappearance of detectable anti1 year). Thirty-one samples were HCV RNA-positive bodies). (22 after the first and 9 after the second amplification) and 9 were negative. Twenty-one of the 31 HCV RNA- Liver histopathology positive patients were anti-HCV-positive (19 RIBA II positive and 2 indeterminate) and 10 were anti-HCV- In most patients liver biopsies were not performed due negative. The duration of hepatitis at the time of blood to severe bleeding abnormalities. Six of the 14 patients sampling in the 10 HCV RNA-positive/anti-HCV- who underwent biopsy had cirrhosis of the liver (Table negative patients was less than 3 months in 7 patients 2). Five of 7 patients who had a duration of hepatitis and 6, 7 and 14 months in the remaining 3. Four of 9 for more than three years at the time of biopsy had proHCV RNA-negative patients had recovered from their gressed to cirrhosis. One patient (no. 14) with AML progressed from acute hepatitis without fibrosis to cirhepatitis. Thirty-eight (95%) of 40 patients with post-transfu- rhosis within one year after contracting hepatitis. sion non-A, non-B hepatitis, whose sera were analysed both for the presence of antibodies to components of Hepatitis and anti-leukemic treatment HCV and HCV RNA had verified HCV infections. ALT was normal or only slightly elevated in sera from Seventeen patients with post-transfusion hepatitis (6 patients who were HCV RNA-negative. In contrast, during the acute infection) and acute lymphocytic or among patients who were HCV RNA-positive after on myelocytic leukemia were given anti-leukemic treat-
232 Table 2. Liver biopsy findings in 14 patients studied during the chronic phase of hepatitis C. Pt no 1 3 4 6 8 7 9 32 33 38 43 40 14 17
Diagnosis
NHL ITP ITP ITP NHL NHL ALL CML AML ALL HD CML AML HD
Duration of hepatitis at time of biopsy, years
Portal inflammation
Fibrosis
18.6 11.0 7.4 6.8 4.0 3.6 3.6 1.7 1.6 1.6 1.3 1.2 0.9 0.9
10-1 CO
r50
Q
-40
6-
-30
un
c CD
I
1 C\J
c
4
h20
-10 -0'
-10
0
10 Time (Days)
20
••§ CO
a. "o <
30
Fig. 2. ALT levels in two patients with hepatitis C before and after combination chemotherapy treatment for ALL in relapse (pt no 25, °) and AML in relapse (pt no 23, •). Chemotherapy was started on Portal inflammation: 0 — None, 1 - Mild (sprinkling of inflammatory dayO. cells in < l / 3 of portal tracts), 2 - Moderate (increased inflammatory cells in 1/3-2/3 of portal tracts), 3 — Marked (dense packing of inflammatory cells in > 2/3 of portal tracts). In one study 82% of blood donors who were antiFibrosis: 0 — None, 1 — Fibrous portal expansion, 2 — BridgingHCV-positive by a first-generation test were implicated fibrosis, 3 ~ Cirrhosis. Patient number corresponds to number in in HCV transmission [22] and the screening of blood Table 1. For explanation of abbreviations see Table 1.
ment due to relapsing or resistant disease. Conventional doses of cytostatic drugs used for the treatment of leukemia were used (amsacrine, etoposide, mitoxantrone, araC, cyclophosphamide). The myelosuppressive effect of treatment was not augmented compared to that seen in patients without hepatitis. Neither did the hepatitis deteriorate during treatment as revealed by aminotransferase levels. In contrast, most patients experienced diminished ALT levels during chemotherapy. This is illustrated by the serum ALT levels of 2 patients, measured before, during and after chemotherapy (Fig. 2). The reduction of ALT levels was not accompanied by a decrease of viral replication as measured by nested PCR. Samples from 4 of 5 patients analysed for HCV RNA before, during and after chemotherapy remained strongly positive (positive after one amplification) and in the serum from one patient there was an increase of viral sequences following cytostatic treatment (two amplifications needed prior to, but only one after chemotherapy to detect HCV RNA).
Discussion
The results of this study show that almost all of our cases of post-transfusion non-A, non-B hepatitis were caused by HCV, i.e. 95% if measured as anti-HCVpositivity and/or detectable HCR RNA. Although the carrier rate of HCV is relatively low in the blood-donor population, 0.4%-1.2% in the Western world as determined by the use of a first-generation anti-HCV assay [19-21], patients who require multiple transfusions run a substantial risk of becoming infected.
products for HCV antibodies has reduced the incidence of post-transfusion hepatitis [23, 24]. Such screening of blood products, using the first-generation anti-HCV assay, was introduced at our hospital in April 1991 and two months later with the second-generation assay. However, two of the patients with HCV RNA-positive hepatitis in this series received only red cell and platelet units which were tested with the second-generation anti-HCV assay. Furthermore, 10 additional patients have developed transfusion-induced hepatitis C since this study was concluded. Thus, also after the introduction of screening of blood units with the second-generation anti-HCV assay, transmission of HCV seems to remain an important problem. Similar findings were reported in another study where only 13 of 19 HCV RNA-positive sera from blood donors were indentified as positive with current serological assays [25]. In the present series, samples from 13 patients which were negative with the first-generation assay were positive when reanalysed with the second-generation test, confirming the higher sensitivity and decreased time to seroconversion with the second-generation anti-HCV assay [4,13]. The clinical picture of the acute HCV infection in this group of patients suffering from hematologic disorders did not differ from that described in other series consisting mainly of patients who underwent surgery [6, 7, 9]. However, almost all patients (95%) progressed to chronic hepatitis, as compared to 30%-70% of patients with post-transfusion non-A, non-B hepatitis following surgery [7-10). A high chronicity rate (26 of 28 patients, 93%), of post-transfusion non-A, non-B hepatitis has also been reported in renal transplant patients [26]. The high incidence of chronicity is presumably
233
related to immunosuppressive treatment after renal transplantation and in our patients treatment with cytostatic drugs or antithymocyte-globulin and cyclosporin A known to impair preferably cell-mediated immune functions, allowing high replication of virus. In parallel, a high frequency of chronic infection in immunocompromised hosts has also been reported in patients with chronic hepatitis B infection [27-29]. Treatment with cytostatic drugs during both the acute and the chronic stages of infection was well tolerated, suggesting that the capacity of the liver to metabolise drugs was not substantially impaired [30]. We also observed a suppression of the inflammatory activity, measured as a lowering of serum ALT levels, unaccompanied by a decrease in viremia, during chemotherapy and the subsequent period of pancytopenia. This finding indicates that the inflammatory response to HCV infection is one mechanism behind hepatocyte damage. Reactivation of, and also fulminant hepatitis C after withdrawal of immunosuppressive therapy has been reported [31, 32] and is well documented in hepatitis B [29, 33]. The mechanism is an increase in viral replication during the period of immunosuppression resulting in a severe liver damage when the immune system is reconstituted and reacts to the infected hepatocytes [28]. Consequently, it is important to screen patients who are to be treated with immunosuppressive therapy for ongoing HCV infection. One may speculate that treatment with alpha-interferon or ribavirin, which have been shown to be effective in the treatment of chronic HCV infection in some patients [34, 35], may inhibit viral replication during periods of immunosuppression. An impaired immune response might also explain the prolonged period (up to 14 months) without detectable antibodies which is seen in some of the HCV RNA-positive patients. HCV RNA has been shown to be present in sera even before the clinical onset of hepatitis [14]. Sera from three of our patients were analysed within one week after onset of hepatitis and all were positive for HCV RNA. It is apparent that the detection of HCV RNA is important for diagnosis in this patient group often with a prolonged time to development of measurable antibody levels. The high proportion of patients who progressed to manifest cirrhosis of the liver may also be explained by a high level of viral replication but the results have to be confirmed in larger patient series. Comparison of serum ALT levels and results of HCV RNA by PCR indicated that HCV RNA-negative patients generally have normal or only slightly increased ALT levels. However, normal ALT levels were also seen both in patients with low and high levels of HCV RNA, which could explain the limited value of ALT testing in blood donors to prevent transmission of non-A, non-B hepatitis [36]. We conclude that in patients with hematologic diseases, hepatitis C often progresses to chronic disease and that the time to seroconversion may be prolonged,
both probably due to an impaired immune response, and that the detection of HCV RNA by PCR is important for diagnosis in this patient group. Finally, paralleling the situation in hepatitis B, the inflammatory component of the response to infection in hepatitis C is likely to be one of the pathogenetic mechanisms behind hepatocyte damage. References 1. Choo QL, Kuo G, Weiner AJ et al. Isolation of a cDNA clone derived from a blod-borne non-A, non-B viral hepatitis genome. Science 1989; 244: 359-61. 2. Kuo G, Choo QL, Alter HJ et al. An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis. Science 1989; 244: 362-4. 3. Esteban JI, Gonzalez A, Hernandez JM et al. Evaluation of antibodies to hepatitis C virus in a study of transfusion-associated hepatitis. N Engl J Med 1990; 323: 1107-12. 4. Aach RD, Stevens CE, Hollinger FB et al. Hepatitis C virus infection in post-transfusion hepatitis. An analysis with first- and second-generation assays. N Engl J Med 1991; 325: 1325-9. 5. Dienstag JL. Non-A, non-B hepatitis. I. Recognition, epidemiology, and clinical features. Gastroenterology 1983; 85: 43962. 6. Dienstag JL, Alter HJ. Non-A, non-B hepatitis: Evolving epidemiologic and clinical perspective. Semin Liver Dis 1986; 6: 67-81. 7. Koretz RL, Stone O, Gitnick GL. The long-term course of non-A, non-B post-transfusion hepatitis. Gastroenterology 1980; 79: 893-8. 8. Mattsson L, Weiland O, Glaumann H. Long-term follow-up of chronic post-transfusion non-A, non-B hepatitis: Clinical and histological outcome. Liver 1988; 8: 184-8. 9. Feinman SV, Berris B, Bojarski S. Posttransfusion hepatitis in Toronto, Canada. Gastroenterology 1988; 95:464-9. 10. Lee SD, Hwang SJ, Lu RH et al. Antibodies to hepatitis C virus in prospectively followed patients with posttransfusion hepatitis. J Infect Dis 1991; 163: 1354-7. 11. Tremolada F, Casarin C, Tagger A et al. Antibody to hepatitis C virus in post-transfusion hepatitis. Ann Intern Med 1991; 114:277-81. 12. Van Der Poel CL, Cuypers HTM, Reesink HW et al. Confirmation of hepatitis C virus infection by new four-antigen recombinant immunoblot assay. Lancet 1991; 337: 317-19. 13. Mattsson L, Grillner L, Weiland O. Seroconversion to hepatitis C virus antibodies in patients with acute post transfusion nonA, non-B hepatitis in Sweden with a second-generation test. Scand J Infect Dis 1992; 24: 15-20. 14. Farci P, Alter HJ, Wong D et al. A long-term study of hepatitis C virus replication in non-A, non-B hepatitis. N Engl J Med 1991; 325: 98-104. 15. Garson JA, Tuke PW, Makris M et al. Demonstration of multiple viraemia patterns in haemophiliacs treated with hepatitis C virus contaminated factor VIII concentrates. Lancet 1990; 336: 1022-5. 16. Garson JA, Ring C, Tuke P et al. Enhanced detection of PCR of hepatitis C virus RNA. Lancet 1990; 336: 878-9. 17. Garson J A, Tedder RS, Briggs M et al. Detection of hepatitis C viral sequences in blood donations by 'nested' polymerase chain reaction and prediction of infectivity. Lancet 1990; 335: 1419-22. 18. Knodell RG, Ishak KG, Black WC et al. Formulation and application of a numerical scoring system for assessing histological activity in asymptomatic chronic active hepatitis. Hepatology 1981; 1:431-5. 19. Ferroni P, Tagger A, Ribero ML et al. Antibodies to hepatitis C virus in blood donors. Eur J Epidemiol 1990; 6: 326-8.
234 20. Dawson GJ, Lesniewski RR, Stewart JL et al. Detection of antibodies to hepatitis C virus in U.S. blood donors. J Clin Microbiol 1991; 29: 551-6. 21. Richards C, Holland P, Kuramoto K et al. Prevalence of antibody to hepatitis C virus in a blood donor population. Transfusion 1990; 31:109-13. 22. Esteban JI, Lopez-Talavera JC, Genesca J et al. High rate of infectivity and liver disease in blood donors with antibodies to hepatitis C virus. Ann Intern Med 1991; 115:443-9. 23. Japanese red cross non-A, non-B hepatitis research group. Effect of screening for hepatitis C virus antibody and hepatitis B virus core antibody on incidence of post-transfusion hepatitis. Lancet 1991; 338: 1040-1. 24. Nelson K, Donahue J, Munoz A et al. Risk of hepatitis C virus (HCV) infection in cardiac surgery patients and effectiveness of screening. Third International Symposium on HCV 1991; Abstract: B 66. 25. Sugitani M, Inchauspe G, Shindo M et al. Sensitivity of serologjcal assays to identify blood donors with hepatitis C viraemia. Lancet 1992; 339: 1018-19. 26. LaQuaglia MP, Tolkoff-Rubin NE, Dienstag JL. Impact of hepatitis on renal transplantation. Transplantation 1981; 32: 504-7. 27. Scullard GH, Smith CI, Merigan TC et al. Effects of immunosuppressive therapy on viral markers in chronic active hepatitis B. Gastroenterology 1981; 81: 987-1. 28. Dienstag JL. Viral hepatitis in the compromised host. In Rubin RH Young (ed): Clinical approach to infection in the compromised host, 2nd ed, New York, Plenum. 1986; 325-45. 29. Pinto PC, Hu E, Bernstein-Singer M et al. Acute hepatic injury after the withdrawal of immunosuppressive chemotherapy in patients with hepatitis B. Cancer 1990; 65: 878-84.
30. Powis G. Effect of human renal and hepatic disease on the pharmacokinetics of anticancer drugs. Cancer Treat Rev 1982; 9:85-124. 31. Fan FS, Tzeng CH, Hsiao KI et al. Withdrawal of immunosuppressive therapy in allogeneic bone marrow transplantation reactivates chronic viral hepatitis C. Bone Marrow Transplant 1991; 8:417-20. 32. Kanamori H, Fukawa H, Maruta A et al. Case report: Fulminant hepatitis C viral infection after allogeneic bone marrow transplantation. Am J Med Sci 1992; 303: 109-11. 33. Pariente EA, Goudeau A, Dubois F et al. Fulminant hepatitis due to reactivation of chronic hepatitis B virus infection after allogeneic bone marrow transplantation. Dig Dis Sci 1988; 33: 1185-91. 34. Davis G, Recombinant alpha-interferon treatment of non-A, non-B (type C) hepatitis: Review of studies and recommendations for treatment. J Hepatol (suppl) 1990; 11: 72-7. 35. Anderson J, Reichard O. A possible alternative for the treatment of chronic non-A, non-B hepatitis. Scand J Infect Dis 1990; 22: 509. 36. Alter HJ, Purcell RH, Holland PV et al. Donor transaminase and recipient hepatitis. JAMA 1981; 246:630-4. Received 24 August 1992; accepted 17 November 1992. Correspondence to: Astrid Gruber, M.D. Division of Medicine Section of Hematology and Immunology Karolinska Hospital 10401 Stockholm, Sweden
Original article Late seroconversion and high chronicity rate of hepatitis C virus infection in patients with hematologic disorders A. Gruber,1 H. Norder,2 L. Magnius,2 M. Rotzen,3 C. Rubio,4 L. Grillner3 & M. Bjorkholm1 'Division of Medicine, Section of Hematology and Immunology; 2National Bacteriological LMboratory; 'Department of Clinical Microbiology, Section of Virology; 'Department of Pathology, Karolinska Hospital, Stockholm, Sweden
months. Seventeen patients with resistant or relapsed acute leukemia were treated with combination chemotherapy durBackground: Patients with hematologic disorders requiring ing the acute or chronic phase of hepatitis. Suppression of the repeated blood and platelet transfusions are at high risk for inflammatory activity as reflected by a decrease of serum aminotransferase levels was recorded during the subsequent development of post-transfusion non-A, non-B hepatitis. Patients and methods: Fifty-five patients with hematologic pancytopenic period. diseases and post-transfusion non-A, non-B hepatitis were Conclusions: Hepatitis C has a high chronicity rate in pastudied. Sera were assayed for hepatitis C virus (HCV) anti- tients with hematologic disorders which parallels the situabodies with a second-generation enzyme-linked immuno- tion of hepatitis B in the immunocompromised host. Furtherassay. Sera from 40 patients were examined for the presence more, like the situation in hepatitis B, the hosts' immune of HCV RNA with a nested PCR method. response to infection seems to be involved in the pathogeneResults: The clinical picture of acute non-A, non-B hepa- sis of liver injury. Time to seroconversion may be prolonged titis did not differ from that described in other patient and detection of HCV RNA is therefore important for diaggroups: however, progression to chronic hepatitis was very nosis. common (95%). Thirty-eight (95%) of 40 patients, whose sera were analysed both serologically and for the presence of HCV RNA had verified HCV infections. In some patients Key words: immunosuppression, hematologic diseases, hepatime to seroconversion was prolonged, up to more than 14 titis C, chronic active hepatitis, non-A and non-B hepatitis Summary
Introduction
be detected within 1-3 weeks after exposure in patients subsequently developing hepatitis C [14]. Patients with hematologic disorders often require multiple red cell and platelet transfusions during periods of primary, or chemotherapy-induced bone marrow failure. We found post-transfusion hepatitis to be a common complication in this patient group and followed its clinical course in 55 patients. Sera from all patients were assayed with the second-generation antiHCV test and in 35 patients a first-generation test was also used. In addition sera from 40 patients were examined for the presence of HCV RNA with a nested PCR method.
Following the cloning of a part of the hepatitis C virus (HCV) genome and the subsequent development of an antibody test (anti-HCV) it has been shown that approximately 90% of post-transfusion non-A, non-B hepatitis is caused by HCV [1-4]. About 90% of posttransfusion non-A, non-B hepatitis occurs within 5-12 weeks after transfusion but incubation periods of up to 6 months have been reported [5]. The clinical course of acute non-A, non-B hepatitis is generally mild. A majority of the patients are asymptomatic and only about 20% will develop jaundice [6]. However, 30%-70% of patients with post-transfusion non-A, non-B hepatitis will show elevated aminotransferases after 6 months (chronic hepatitis) and at least 20% of these patients Patients and methods will eventually develop cirrhosis of the liver [6-10]. Seroconversion determined by the first-generation Patients anti-HCV enzyme-linked immunoassays (EIA) occurs within 3 months in about 60% of the patients and 90% The study included 55 patients with various hematologic disorders seroconvert within 12 months [11]. With the second- and post-transfusion non-A, non-B hepatitis (Table 1). All patients generation anti-HCV assay which is both more specific had normal aminotransferase levels prior to transfusion. The diagnosis of non-A, non-B hepatitis was based on a rise in serum alanine and sensitive the time interval to seroconversion was aminotransferase (ALT) for more than one month to at least 2.0 found to be shorter [12, 13]. By the use of the poly- Ukat/L (normal <0.7 (ikat/L) in the absence of only other possible merase chain reaction (PCR) method HCV RNA may cause for the elevation (such as hepatitis A, B, CMV or EBV infec-
230 Table 1. Clinical data of patients and results of R1BA II and HCV RNA determinations. Pt no
Age at onset of hepatitis/
Diagnosis
Onset hepatitis month/ year
Followup time, years
RJBA II
NHL AML ITP ITP ITP ITP NHL NHL ALL AA AA CML NHL AML AA AML HD ALL AML AML MDS AML AML AML ALL ALL AML AUL ITP AA AML CML
05/-73 04/-78 06/-81 11/-81 O5/-83 07/-83 09/-85 06/-85 01/-86 04/-86 06/-86 1 1/-87 03/-88 04/-88 06/-88 06/-88 01/-89 02/-89 03/-89 03/-89 04/-89 05/-89 06/-89 09/-89 10/-89 10/-89 11/-89 11/-89 11/-89 11/-89 12/-89 01/-90 03/-90 05/-90 05/-90 07/-90 08/-90 08/-90 08/-90 08/-90 09/-90 10/-90 10/-90 10/-90 01/-91 01/-91 02/-91 03/-91 05/-9I 05/-91 07/-91 08/-91 09/-91 10/-91 10/-91
18.4 13.7
+
sex
36/M 32/F 27/M 38/F 43/F 47/F 51/M 51/M 28/F 25/M 1 1 54/M 12 18/F 13 55/F 14 54/F 15 53/M 52/F 16 17 47/F 18 44/M 19 43/M 20 81/F 21 84/F 22 20/F 41/F 23 24 47/F 25 59/F 26 45/M 27 69/M 25/M 28 56/M 29 30 46/F 31 74/M 32 39/F 75/F 33 34 44/F 35 16/F 36 66/M 37 24/F 38 27/M 74/M 39 40 25/M 41 33/F 42 36/F 43 19/M 44 75/M 45 53/M 46 49/F 47 51/F 68/F 48 38/M 49 63/F 50 51 57/F 52 28/M 53 64/F 54 78/F 55 43/F 1 2 3 4 5 6 7 8 9 10
AML CML ALL AML AA ALL AUL CML ALL ALL HD MDS AML AML MDS MDS AML ALL AML AA AML AML MDS
9.9
10.2 8.7 8.5
4.8' 6.6 5.1 5.7 5.5
0.6° 1.5° 3.5° 3.5
0.6° 2.51.5° 1.2* 0.6"
HCV RNA
+ + + + (+) + +
++ + ++ ++ ND +
+
+
+ +
++ ++ ND
+ + +
ND ++ ++
+ +
+ ND
ND
ND ND
2.8
+
-
1.4* 0.3"
+ + + +
ND ND + ND
2.3
1.50.3" 0.8" 0.9" 2.2 2.2
0.3° 2.0 1.8 1.7 1.7
1.4" 1.4 .4 .0" .4 .0° ().2" .3 .3 .0
0.6' 0.9 0.7 0.5
0.5" 0.2 0.4 0.3
0.1" 0.3
ND
+ + + + + + + +
ND ND ND ++ + ++
•+•
-
+ (+) (+) +
++ ++ + + -HI-
ND
+ + + + -
++ ++ + ++ ++ ++ ++ ++ ++ ++ -H-
AML: acute myelocytic leukemia, ALL: acute lymphocytic leukemia, AUL: acute undifferentiated leukemia, MDS: myelodysplastic syndrome, CML: chronic myelocytic leukemia, NHL: non Hodgkin lymphoma, HD: Hodgkin's disease, AA: aplastic anemia, ITP: immune mediated thrombocytopenia, ND: not done, R1BA II (+) indeterminate result (positive in one band), HCV RNA ++ - positive after first, + — positive after second PCR round. * Deceased patient.
tion, or ongoing treatment with hepatotoxic drugs). The onset of hepatitis was recorded between 1985 and 1991 in 49 of the patients and between 1973 and 1983 in the remaining 6. Fifty-two patients had received platelet and/or red cell transfusions. The median number of units transfused 2-26 weeks prior to the onset of hepatitis was 28 (range 2-189). Three patients (nos. 3, 4, 6) had been treated with intravenous gammaglobulin for immune-mediated thrombocytopenia. The median age of patients at the onset of hepatitis was 44 years (range 16-84): 32 patients were female and 23 male. The median follow-up time after detection of the hepatitis was 1.2 years (range 0.1-18.6). Chronic hepatitis was defined as an elevation of ALT for more than 6 months with at least one value exceeding five times the upper limit of normal [7, 8). The clinical course in the 11 patients whose hepatitis became clinically manifest prior to 1987 was evaluated retrospectively. The remaining patients were followed prospectively. Anti-HCV assay Before June 1991 the patients' sera were analysed by a first-generation test, anti-HCV EIA (Abbott Laboratories, Chicago, IL, USA) which is based on the non-structural (NS) c 100-3 antigen only. Later sera were tested by a second-generation anti-HCV EIA (Abbott Laboratories), which includes antigens from the core (c22) and NS3 and NS4 regions of the genome, and earlier samples were reanalysed using this test. At least one serum sample from each antiHCV-positive patient was analysed by a complementary recombinant immunobinding assay (RJBA II; Ortho Diagnostic Systems, Raritan, NJ, USA). This assay qualitatively detects antibodies against structural (c22) and NS3 and NS4 (c33, cl00-3 and, 5-1-1) antigens. The tests were performed according to the manufacturer's instructions. Reactivity against two or more of the antigens in RIBA II was required to confirm a positive anti-HCV test, while reactivity against only one antigen was considered indeterminate. Patients without detectable HCV antibodies at initial analysis were repeatedly tested thereafter. When anti-HCV positivity was established at the initial test, frozen stored sera, if available, were retrospectively analysed. Detection of HCV RNA by PCR Forty patients were still alive in 1991 and their sera were analysed for the presence of HCV RNA sequences by a nested PCR method [15, 16]. Sera from five patients were analysed on consecutive occasions, before, during and after treatment with combination chemotherapy. By amplifying the HCV RNA sequence during two PCR rounds the sensitivity of the assay is increased approximately 1,000 times [17]. Serum samples were fresh-frozen and kept at -70*C until tested. RNA was prepared from 100 u.1 serum by precipitation with polyethylene/sodium dodecyl sulfate followed by phenol extraction 115]. The RNA was dissolved in 10 u.1 distilled water which had been treated with diethylpyrocarbonate. Five u,l dissolved RNA was used for cDNA synthesis using cloned Moloney murine leukemia virus reverse transcriptase (Bethesda Research Laboratories, Md, USA), and as primer the antisense NCR2 oligonucleotide was used [16]. In the first round of PCR primers NCR1 and NCR2 were used, and 5 u,l reaction mixture from the first round were used in a second PCR using NCR3 and NCR4 as the inner primers [16|. In addition to the standard precautions, a negative plasma was tested after each tested sample during RNA extraction, reverse transcription and amplification to exclude false positive results in the PCR due to cross contamination.
Liver biopsies Fourteen patients underwent liver biopsy using the Menghini technique during the chronic hepatitis phase. The specimens were fixed
231 in 10% formalin and embedded in paraffin. Sections (4 u.m) were stained with haematoxylin and eosin, periodic acid-Schiff after previous treatment with diastase and with Sirus stain. The histological findings were classified according to the degree of portal inflammation andfibrosis[18].
10
8
8 |
6
Results Anti-HCV Fifty-one of the 55 patients had been followed serologically for at least 3 months after the onset of hepatitis. Thirty-eight (75%) of the 51 had detectable antibodies to HCV with the second-generation antibody test, 35 of those were positive and 3 had an indeterminate (all 3 anti-c22 positive) reaction in RIBA II. Thirty of the thirty-eight anti-HCV-positive patients were negative in at least one preceding test. The remaining eight patients were positive in the first analysis performed and no sera for retrospective analysis were available. The exact time to seroconversion could not be determined in most patients. However, 11 patients who had verified HCV infections (positive HCV RNA and/or later seroconversion and RIBA II positivity) were anti-HCVnegative for more than 3 months after increased ALT levels were observed (4 pts 4, 2 pts 5, 2 pts 6, 1 pt 7, 1 pt 9 and 1 pt 14 months). Sera from 35 patients, which initially had been assayed with the first-generation anti-HCV test, were reanalysed with the second-generation assay. Twelve samples were positive and 10 negative by both assays. Thirteen sera with HCV antibodies detectable by the second-generation assay (10 RIBA II positive, 3 indeterminate) were negative by the first-generation test.
420
o o •••
91
•at
HCV RNA Fig. 1. Relationship between ALT levels and HCV RNA. Positive PCR after first (++) and after second (+) rounds. Anti-HCV positive (•), anti-HCV negative (o) samples.
or two amplifications of HCV RNA sequences, the ALT levels ranged from normal to high (Fig.l). Clinical course
During the acute infection phase most patients were asymptomatic. Ten patients (18%) were jaundiced. None of the patients suffered a fulminant hepatitis. Thirteen patients (23%) had an elevation of ALT to more than 20 ukat/L. The median of the maximal ALT elevation was 11.3 jikat/L (range 2.7->48 ukat/L). After the acute phase, which in most patients lasted for about one month, the aminotransferase levels fluctuated. Forty-four patients were followed for more than 6 months (median 20 months) and of those, 42 (95%) progressed to chronic hepatitis (95% confidence interHCV RNA determination val 85%-99%). In two of 42 patients (nos. 30 and 35) with elevation of ALT for more than 6 months, hepatiSera from 40 patients were analysed for the presence of tis resolved spontaneously after 18 months (normal HCV RNA. The duration of hepatitis at the time of ALT levels for three months, no detectable HCV RNA sampling ranged from one week to 18.5 years (median and in pat. no. 35 disappearance of detectable anti1 year). Thirty-one samples were HCV RNA-positive bodies). (22 after the first and 9 after the second amplification) and 9 were negative. Twenty-one of the 31 HCV RNA- Liver histopathology positive patients were anti-HCV-positive (19 RIBA II positive and 2 indeterminate) and 10 were anti-HCV- In most patients liver biopsies were not performed due negative. The duration of hepatitis at the time of blood to severe bleeding abnormalities. Six of the 14 patients sampling in the 10 HCV RNA-positive/anti-HCV- who underwent biopsy had cirrhosis of the liver (Table negative patients was less than 3 months in 7 patients 2). Five of 7 patients who had a duration of hepatitis and 6, 7 and 14 months in the remaining 3. Four of 9 for more than three years at the time of biopsy had proHCV RNA-negative patients had recovered from their gressed to cirrhosis. One patient (no. 14) with AML progressed from acute hepatitis without fibrosis to cirhepatitis. Thirty-eight (95%) of 40 patients with post-transfu- rhosis within one year after contracting hepatitis. sion non-A, non-B hepatitis, whose sera were analysed both for the presence of antibodies to components of Hepatitis and anti-leukemic treatment HCV and HCV RNA had verified HCV infections. ALT was normal or only slightly elevated in sera from Seventeen patients with post-transfusion hepatitis (6 patients who were HCV RNA-negative. In contrast, during the acute infection) and acute lymphocytic or among patients who were HCV RNA-positive after on myelocytic leukemia were given anti-leukemic treat-
232 Table 2. Liver biopsy findings in 14 patients studied during the chronic phase of hepatitis C. Pt no 1 3 4 6 8 7 9 32 33 38 43 40 14 17
Diagnosis
NHL ITP ITP ITP NHL NHL ALL CML AML ALL HD CML AML HD
Duration of hepatitis at time of biopsy, years
Portal inflammation
Fibrosis
18.6 11.0 7.4 6.8 4.0 3.6 3.6 1.7 1.6 1.6 1.3 1.2 0.9 0.9
10-1 CO
r50
Q
-40
6-
-30
un
c CD
I
1 C\J
c
4
h20
-10 -0'
-10
0
10 Time (Days)
20
••§ CO
a. "o <
30
Fig. 2. ALT levels in two patients with hepatitis C before and after combination chemotherapy treatment for ALL in relapse (pt no 25, °) and AML in relapse (pt no 23, •). Chemotherapy was started on Portal inflammation: 0 — None, 1 - Mild (sprinkling of inflammatory dayO. cells in < l / 3 of portal tracts), 2 - Moderate (increased inflammatory cells in 1/3-2/3 of portal tracts), 3 — Marked (dense packing of inflammatory cells in > 2/3 of portal tracts). In one study 82% of blood donors who were antiFibrosis: 0 — None, 1 — Fibrous portal expansion, 2 — BridgingHCV-positive by a first-generation test were implicated fibrosis, 3 ~ Cirrhosis. Patient number corresponds to number in in HCV transmission [22] and the screening of blood Table 1. For explanation of abbreviations see Table 1.
ment due to relapsing or resistant disease. Conventional doses of cytostatic drugs used for the treatment of leukemia were used (amsacrine, etoposide, mitoxantrone, araC, cyclophosphamide). The myelosuppressive effect of treatment was not augmented compared to that seen in patients without hepatitis. Neither did the hepatitis deteriorate during treatment as revealed by aminotransferase levels. In contrast, most patients experienced diminished ALT levels during chemotherapy. This is illustrated by the serum ALT levels of 2 patients, measured before, during and after chemotherapy (Fig. 2). The reduction of ALT levels was not accompanied by a decrease of viral replication as measured by nested PCR. Samples from 4 of 5 patients analysed for HCV RNA before, during and after chemotherapy remained strongly positive (positive after one amplification) and in the serum from one patient there was an increase of viral sequences following cytostatic treatment (two amplifications needed prior to, but only one after chemotherapy to detect HCV RNA).
Discussion
The results of this study show that almost all of our cases of post-transfusion non-A, non-B hepatitis were caused by HCV, i.e. 95% if measured as anti-HCVpositivity and/or detectable HCR RNA. Although the carrier rate of HCV is relatively low in the blood-donor population, 0.4%-1.2% in the Western world as determined by the use of a first-generation anti-HCV assay [19-21], patients who require multiple transfusions run a substantial risk of becoming infected.
products for HCV antibodies has reduced the incidence of post-transfusion hepatitis [23, 24]. Such screening of blood products, using the first-generation anti-HCV assay, was introduced at our hospital in April 1991 and two months later with the second-generation assay. However, two of the patients with HCV RNA-positive hepatitis in this series received only red cell and platelet units which were tested with the second-generation anti-HCV assay. Furthermore, 10 additional patients have developed transfusion-induced hepatitis C since this study was concluded. Thus, also after the introduction of screening of blood units with the second-generation anti-HCV assay, transmission of HCV seems to remain an important problem. Similar findings were reported in another study where only 13 of 19 HCV RNA-positive sera from blood donors were indentified as positive with current serological assays [25]. In the present series, samples from 13 patients which were negative with the first-generation assay were positive when reanalysed with the second-generation test, confirming the higher sensitivity and decreased time to seroconversion with the second-generation anti-HCV assay [4,13]. The clinical picture of the acute HCV infection in this group of patients suffering from hematologic disorders did not differ from that described in other series consisting mainly of patients who underwent surgery [6, 7, 9]. However, almost all patients (95%) progressed to chronic hepatitis, as compared to 30%-70% of patients with post-transfusion non-A, non-B hepatitis following surgery [7-10). A high chronicity rate (26 of 28 patients, 93%), of post-transfusion non-A, non-B hepatitis has also been reported in renal transplant patients [26]. The high incidence of chronicity is presumably
233
related to immunosuppressive treatment after renal transplantation and in our patients treatment with cytostatic drugs or antithymocyte-globulin and cyclosporin A known to impair preferably cell-mediated immune functions, allowing high replication of virus. In parallel, a high frequency of chronic infection in immunocompromised hosts has also been reported in patients with chronic hepatitis B infection [27-29]. Treatment with cytostatic drugs during both the acute and the chronic stages of infection was well tolerated, suggesting that the capacity of the liver to metabolise drugs was not substantially impaired [30]. We also observed a suppression of the inflammatory activity, measured as a lowering of serum ALT levels, unaccompanied by a decrease in viremia, during chemotherapy and the subsequent period of pancytopenia. This finding indicates that the inflammatory response to HCV infection is one mechanism behind hepatocyte damage. Reactivation of, and also fulminant hepatitis C after withdrawal of immunosuppressive therapy has been reported [31, 32] and is well documented in hepatitis B [29, 33]. The mechanism is an increase in viral replication during the period of immunosuppression resulting in a severe liver damage when the immune system is reconstituted and reacts to the infected hepatocytes [28]. Consequently, it is important to screen patients who are to be treated with immunosuppressive therapy for ongoing HCV infection. One may speculate that treatment with alpha-interferon or ribavirin, which have been shown to be effective in the treatment of chronic HCV infection in some patients [34, 35], may inhibit viral replication during periods of immunosuppression. An impaired immune response might also explain the prolonged period (up to 14 months) without detectable antibodies which is seen in some of the HCV RNA-positive patients. HCV RNA has been shown to be present in sera even before the clinical onset of hepatitis [14]. Sera from three of our patients were analysed within one week after onset of hepatitis and all were positive for HCV RNA. It is apparent that the detection of HCV RNA is important for diagnosis in this patient group often with a prolonged time to development of measurable antibody levels. The high proportion of patients who progressed to manifest cirrhosis of the liver may also be explained by a high level of viral replication but the results have to be confirmed in larger patient series. Comparison of serum ALT levels and results of HCV RNA by PCR indicated that HCV RNA-negative patients generally have normal or only slightly increased ALT levels. However, normal ALT levels were also seen both in patients with low and high levels of HCV RNA, which could explain the limited value of ALT testing in blood donors to prevent transmission of non-A, non-B hepatitis [36]. We conclude that in patients with hematologic diseases, hepatitis C often progresses to chronic disease and that the time to seroconversion may be prolonged,
both probably due to an impaired immune response, and that the detection of HCV RNA by PCR is important for diagnosis in this patient group. Finally, paralleling the situation in hepatitis B, the inflammatory component of the response to infection in hepatitis C is likely to be one of the pathogenetic mechanisms behind hepatocyte damage. References 1. Choo QL, Kuo G, Weiner AJ et al. Isolation of a cDNA clone derived from a blod-borne non-A, non-B viral hepatitis genome. Science 1989; 244: 359-61. 2. Kuo G, Choo QL, Alter HJ et al. An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis. Science 1989; 244: 362-4. 3. Esteban JI, Gonzalez A, Hernandez JM et al. Evaluation of antibodies to hepatitis C virus in a study of transfusion-associated hepatitis. N Engl J Med 1990; 323: 1107-12. 4. Aach RD, Stevens CE, Hollinger FB et al. Hepatitis C virus infection in post-transfusion hepatitis. An analysis with first- and second-generation assays. N Engl J Med 1991; 325: 1325-9. 5. Dienstag JL. Non-A, non-B hepatitis. I. Recognition, epidemiology, and clinical features. Gastroenterology 1983; 85: 43962. 6. Dienstag JL, Alter HJ. Non-A, non-B hepatitis: Evolving epidemiologic and clinical perspective. Semin Liver Dis 1986; 6: 67-81. 7. Koretz RL, Stone O, Gitnick GL. The long-term course of non-A, non-B post-transfusion hepatitis. Gastroenterology 1980; 79: 893-8. 8. Mattsson L, Weiland O, Glaumann H. Long-term follow-up of chronic post-transfusion non-A, non-B hepatitis: Clinical and histological outcome. Liver 1988; 8: 184-8. 9. Feinman SV, Berris B, Bojarski S. Posttransfusion hepatitis in Toronto, Canada. Gastroenterology 1988; 95:464-9. 10. Lee SD, Hwang SJ, Lu RH et al. Antibodies to hepatitis C virus in prospectively followed patients with posttransfusion hepatitis. J Infect Dis 1991; 163: 1354-7. 11. Tremolada F, Casarin C, Tagger A et al. Antibody to hepatitis C virus in post-transfusion hepatitis. Ann Intern Med 1991; 114:277-81. 12. Van Der Poel CL, Cuypers HTM, Reesink HW et al. Confirmation of hepatitis C virus infection by new four-antigen recombinant immunoblot assay. Lancet 1991; 337: 317-19. 13. Mattsson L, Grillner L, Weiland O. Seroconversion to hepatitis C virus antibodies in patients with acute post transfusion nonA, non-B hepatitis in Sweden with a second-generation test. Scand J Infect Dis 1992; 24: 15-20. 14. Farci P, Alter HJ, Wong D et al. A long-term study of hepatitis C virus replication in non-A, non-B hepatitis. N Engl J Med 1991; 325: 98-104. 15. Garson JA, Tuke PW, Makris M et al. Demonstration of multiple viraemia patterns in haemophiliacs treated with hepatitis C virus contaminated factor VIII concentrates. Lancet 1990; 336: 1022-5. 16. Garson JA, Ring C, Tuke P et al. Enhanced detection of PCR of hepatitis C virus RNA. Lancet 1990; 336: 878-9. 17. Garson J A, Tedder RS, Briggs M et al. Detection of hepatitis C viral sequences in blood donations by 'nested' polymerase chain reaction and prediction of infectivity. Lancet 1990; 335: 1419-22. 18. Knodell RG, Ishak KG, Black WC et al. Formulation and application of a numerical scoring system for assessing histological activity in asymptomatic chronic active hepatitis. Hepatology 1981; 1:431-5. 19. Ferroni P, Tagger A, Ribero ML et al. Antibodies to hepatitis C virus in blood donors. Eur J Epidemiol 1990; 6: 326-8.
234 20. Dawson GJ, Lesniewski RR, Stewart JL et al. Detection of antibodies to hepatitis C virus in U.S. blood donors. J Clin Microbiol 1991; 29: 551-6. 21. Richards C, Holland P, Kuramoto K et al. Prevalence of antibody to hepatitis C virus in a blood donor population. Transfusion 1990; 31:109-13. 22. Esteban JI, Lopez-Talavera JC, Genesca J et al. High rate of infectivity and liver disease in blood donors with antibodies to hepatitis C virus. Ann Intern Med 1991; 115:443-9. 23. Japanese red cross non-A, non-B hepatitis research group. Effect of screening for hepatitis C virus antibody and hepatitis B virus core antibody on incidence of post-transfusion hepatitis. Lancet 1991; 338: 1040-1. 24. Nelson K, Donahue J, Munoz A et al. Risk of hepatitis C virus (HCV) infection in cardiac surgery patients and effectiveness of screening. Third International Symposium on HCV 1991; Abstract: B 66. 25. Sugitani M, Inchauspe G, Shindo M et al. Sensitivity of serologjcal assays to identify blood donors with hepatitis C viraemia. Lancet 1992; 339: 1018-19. 26. LaQuaglia MP, Tolkoff-Rubin NE, Dienstag JL. Impact of hepatitis on renal transplantation. Transplantation 1981; 32: 504-7. 27. Scullard GH, Smith CI, Merigan TC et al. Effects of immunosuppressive therapy on viral markers in chronic active hepatitis B. Gastroenterology 1981; 81: 987-1. 28. Dienstag JL. Viral hepatitis in the compromised host. In Rubin RH Young (ed): Clinical approach to infection in the compromised host, 2nd ed, New York, Plenum. 1986; 325-45. 29. Pinto PC, Hu E, Bernstein-Singer M et al. Acute hepatic injury after the withdrawal of immunosuppressive chemotherapy in patients with hepatitis B. Cancer 1990; 65: 878-84.
30. Powis G. Effect of human renal and hepatic disease on the pharmacokinetics of anticancer drugs. Cancer Treat Rev 1982; 9:85-124. 31. Fan FS, Tzeng CH, Hsiao KI et al. Withdrawal of immunosuppressive therapy in allogeneic bone marrow transplantation reactivates chronic viral hepatitis C. Bone Marrow Transplant 1991; 8:417-20. 32. Kanamori H, Fukawa H, Maruta A et al. Case report: Fulminant hepatitis C viral infection after allogeneic bone marrow transplantation. Am J Med Sci 1992; 303: 109-11. 33. Pariente EA, Goudeau A, Dubois F et al. Fulminant hepatitis due to reactivation of chronic hepatitis B virus infection after allogeneic bone marrow transplantation. Dig Dis Sci 1988; 33: 1185-91. 34. Davis G, Recombinant alpha-interferon treatment of non-A, non-B (type C) hepatitis: Review of studies and recommendations for treatment. J Hepatol (suppl) 1990; 11: 72-7. 35. Anderson J, Reichard O. A possible alternative for the treatment of chronic non-A, non-B hepatitis. Scand J Infect Dis 1990; 22: 509. 36. Alter HJ, Purcell RH, Holland PV et al. Donor transaminase and recipient hepatitis. JAMA 1981; 246:630-4. Received 24 August 1992; accepted 17 November 1992. Correspondence to: Astrid Gruber, M.D. Division of Medicine Section of Hematology and Immunology Karolinska Hospital 10401 Stockholm, Sweden