HarveyJ. Alter

Transmissible agent in non-A, non-B hepatitis.

Plasma or serum from 4 patients with acute or chronic non-A, non-B post-transfusion hepatitis (P.T.H.) and from a blood-donor implicated in two cases of P.T.H. was inoculated into 5 chimpanzees. Biochemical and histological evidence of hepatitis developed in these 5 chimpanzees but not in a control animal. The mean incubation period in the chimpanzees was 13.4 weeks, compared with 7.7 weeks in the 4 patients with P.T.H. The peak alanine aminotransferase (A.L.T.) levels in the 5 chimpanzees were 265, 212, 219, 70, and 62 I.U./l. Histological changes ranged from mild to conspicuous hepatitis and generally correlated with the degree of A.L.T. elevation. There was no evidence of clinical disease and all animals went on to biochemical and histological recovery. There was no serological evidence of type A or type B hepatitis. Hepatitis was transmitted by serum derived from patients with chronic as well as acute hepatitis, strongly suggesting a chronic carrier state for the agent responsible for non-A, non-B hepatitis. Non-A, non-B hepatitis thus seems to be due to a transmissible agent which can persist and remain infectious for long periods.

EFFECT OF HEPATITIS G VIRUS INFECTION ON CHRONIC HEPATITIS C

Hepatitis C virus (HCV) is a major cause of acute and chronic hepatitis throughout the world [1-3]. A new virus, tentatively called hepatitis G virus (HGV), was recently cloned and sequenced [4]. This virus is closely related to HCV in genomic structure; like HCV, HGV is transmitted through transfusion and may be associated with acute and chronic hepatitis [4-6]. We analyzed the role of HGV infection in patients with chronic hepatitis C, including patients treated with interferon-. Methods Patients We enrolled 189 patients with chronic hepatitis C who were seen at Shinshu University Hospital, Matsumoto, Japan (122 men and 67 women; mean age, 51.1 11.0 years). All patients were positive for antibody to HCV according to a second-generation assay and were negative for hepatitis B virus (HBV) surface antigen and antibody to human immunodeficiency virus. No patients had hepatocellular carcinoma or a history of alcohol intake exceeding 80 g/d. Of the 189 patients, 101 (66 men and 35 women; mean age, 50.0 11.2 years) had been retrospectively selected so that our sample would include all patients who had received a single course of interferon- therapy between October 1991 and December 1993. The remaining 88 (47%) patients (56 men and 32 women; mean age, 52.5 10.7 years) were consecutively selected from patients with chronic hepatitis C who had been followed for more than 1 year and had had liver biopsy within the same period but had not received antiviral therapy. Interferon- 2a had been administered at a dosage of 9 million U daily for 2 weeks, followed by 9 million U three times a week for 22 weeks (total dose, 720 million U). Treated patients had histologic examination within the 6 months before interferon- therapy was initiated and were followed for at least 6 months after therapy was completed. For all patients, serum samples were obtained at the time of liver biopsy and were stored at 70C until testing. For patients receiving interferon- therapy, serum samples were also collected just before therapy began, just after therapy was completed, and 6 months after therapy was completed. Serum alanine aminotransferase levels (normal range, 5 to 45 IU/L) were measured at liver biopsy and, in patients receiving interferon- therapy, at least once every 4 weeks during therapy and follow-up. The grade (extent of hepatic inflammation and hepatocellular destruction) and stage (degree of fibrosis) of liver histologic findings [7] were judged by three authors; the final diagnosis was established by consensus. Investigators involved in separate portions of the study were blinded to the results of other portions. Serologic Markers of and Molecular Assays for Hepatitis C Virus RNA Levels of HCV antibody, HBV surface antigen, and antibody to human immunodeficiency virus were measured by using commercially available second-generation enzyme-linked immunosorbent assays (Abbott Laboratories, North Chicago, Illinois). Serum levels of HCV RNA were measured by using nested reverse-transcription polymerase chain reaction (PCR) with primers in the 5 noncoding region [8] and were quantified by using the branched-DNA signal amplification assay [9, 10]. The detection limit of the branched-DNA assay was set at 105.7 equivalents/mL. Hepatitis C virus genotypes were tested by nested PCR using genotype-specific primers of core region [11] and were categorized according to the classification system of Simmonds and colleagues [12]. Serum Levels of Hepatitis G Virus RNA Levels of HGV RNA in serum were measured by using reverse-transcription PCR as described elsewhere [5]. Total nucleic acids were extracted from 50 mL of serum. After reverse transcription, 45 PCR cycles (for detection) or 35 PCR cycles (for quantitation) were done with primers in the putative NS5 region of the HGV RNA genome. The PCR products were analyzed by dot-blot hybridization with a 32P-labeled oligonucleotide probe. The sensitivity of this assay system was 20 RNA copies/mL of starting serum. Quantitative measurement of HGV RNA levels was done using standards of known HGV RNA levels. Statistical Analysis Statistical analyses were done using the Student t-test, the Mann-Whitney U test, the Wilcoxon rank-sum test, the chi-square test, the Fisher exact test, and the Somers D test. A P value of 0.05 or less indicated statistical significance. Results Clinical Features and Hepatitis C Virus Markers Twenty-one (11.1%) of the 189 enrolled patients were positive for HGV RNA. The rate of detection of HGV RNA was similar in the subgroup of 88 untreated patients (12.5%; 11 of 88) and the 101 patients who received interferon- therapy (9.9%; 10 of 101). Mean age and the number of men and women were also similar in the two subgroups. Thus, the clinical features of patients who had chronic hepatitis C could be compared with those of patients who had HCV and HGV infection without respect to interferon- therapy (Table 1). Patients with HGV infection were significantly younger than those without HGV infection. Other clinical features did not differ between HGV RNA-positive and HGV RNA-negative patients (Table 1). Table 1. Clinical Features in HGV RNA-Positive and HGV RNA-Negative Patients with Chronic Hepatitis C* Hepatitis C virus genotype and serum HCV RNA level were compared between the 21 patients with HGV RNA and the 52 patients without HGV RNA who were randomly selected from the 168 HGV-negative persons. The HCV genotypes and HCV RNA levels were distributed similarly in the two groups (Table 2). Table 2. Markers of HCV and Response of HCV to Interferon- in HGV RNA-Positive and HGV RNA-Negative Patients with Chronic Hepatitis C* Effect of Hepatitis G Virus Infection on Response of Hepatitis C Virus to Interferon- Therapy Of the 101 patients receiving interferon-, 36 had a sustained loss of HCV RNA and normalization of alanine aminotransferase levels; they were therefore considered to have responded to interferon-. The remaining 65 patients were positive for HCV RNA 6 months after completing therapy and thus were classified as nonresponders. The rate of HCV response to interferon- did not differ between patients with and those without HGV infection (Table 2). Response of Hepatitis G Virus to Interferon- Response of HGV to interferon- was analyzed in 9 of 10 patients with HGV infection. The serum HGV RNA level decreased during interferon- therapy in all 9 patients. The geometric mean titer of HGV RNA just after interferon- therapy (mean, 6.3 RNA copies/mL; range, 0.0 to 5000 RNA copies/mL) was significantly (P = 0.008; Wilcoxon rank-sum test) lower than the titer just before therapy (mean, 3200 RNA copies/mL; range, 20 to 1 000 000 RNA copies/mL). Two patients had a sustained loss of HGV RNA 6 months after discontinuation of therapy. In the remaining 7 patients, HGV RNA level increased after cessation of therapy to levels similar to those just before therapy. Thus, the sustained response rate of HGV (22%; 2 of 9 patients) was lower than but not significantly different from (P > 0.2; Fisher exact test) the sustained response rate of HCV (36%; 36 of 101 patients). However, a dichotomy was seen in the response to interferon-; two patients who had a sustained loss of HGV RNA did not clear HCV RNA, and three patients who had a sustained loss of HCV RNA did not clear HGV RNA. In the latter three HCV responders, alanine aminotransferase levels remained normal despite the reappearance of HGV viremia. Discussion Approximately 10% of patients with chronic hepatitis C are also infected with HGV [5, 6]. Although the precise routes of HGV transmission have not been established, this agent is parenterally transmitted through blood transfusion and exposure to shared needles during injection drug use [4]. In our study, the frequency of previous blood transfusion was similar in patients with HGV and HCV co-infection and patients with hepatitis C alone. The apparent link between HGV and HCV infections probably reflects common exposures and transmission patterns rather than a specific interdependence of the two agents. Patients with chronic hepatitis often harbor more than one hepatitis agent [4, 13-15], and important interactions between HBV and HCV have been documented [15]. The relation between HCV and HBV replication is reciprocal: Increasing replication of one agent can diminish the replication of the other. The key question underlying our study is whether coexistent HGV infection alters the level of viremia, clinical course, or treatment response of HCV infection. Although it is unclear whether our findings are applicable to non-Japanese persons, we found no evidence of such an effect on any of these variables. The following findings support this claim. First, the HCV RNA level in patients with HGV and HCV infection was the same as in patients with HCV infection alone. In addition, Nakatsuji and colleagues [5] reported that the serum level of HGV RNA did not differ between these two groups of patients. These two studies showed no evidence of unidirectional or bidirectional viral interference between HGV and HCV. Second, no evidence suggested that HGV infection increased the severity of hepatitis C. When patients with chronic hepatitis C were compared with those who had HCV and HGV infection, no differences were seen in the mean alanine aminotransferase level or liver histologic findings. Further, in five co-infected patients in whom HGV and HCV responses to interferon- therapy were dissociated, hepatic inflammation after discontinuation of therapy seemed to depend on HCV replication, not on HGV replication. These data suggest that HGV has limited pathogenicity compared with HCV, and they are consistent with previous observations that HGV-positive blood donors were no more likely to have elevated alanine aminotransferase than were HGV-negative donors [4]. Several viral and host factors have been reported to influence the response of HCV to interferon- [8, 16-18]. We found no effect of HGV on the HCV respo

IMPORTANCE OF WESTERN BLOT ANALYSIS IN PREDICTING INFECTIVITY OF ANTI-HTLV-III/LAV POSITIVE BLOOD

Stored donor and recipient sera from prospective studies of post-transfusion hepatitis were analysed for the presence of human T-cell lymphotropic virus type-III/lymphadenopathy associated virus (HTLV-III/LAV) antibodies as determined by enzyme-linked immunosorbent assays (ELISA). Of 3961 donor samples given to 461 patients, only 2 (0.05%) contained specific HTLV-III/LAV antibodies as determined by an avidin-biotin-enhanced western blot tech nique. Anti-HTLV-III/LAV was measured before and 3 and 6 months after transfusion in 295 recipients of anti-HTLV-III-negative blood, 7 recipients of ELISA-positive blood which was western blot negative, and 2 recipients of ELISA-positive blood confirmed as specific by western blot. Only the last 2 recipients became infected with HTLV-III/LAV, as assessed by antibody seroconversion (p less than 0.0001). Serocon version occurred early (6 and 8 weeks after transfusion) and was characterised first by antibody to p24 and later by antibody to p41. AIDS has not developed in either patient, but one has a T4/T8 ratio of 0.4 and impaired mitogen responses; the second patient has no evidence of immune dysfunction 4 years after exposure. This study confirms that HTLV-III/LAV infection can be transmitted by blood transfusion and supports the advisability of anti-HTLV-III/LAV testing of all blood donors. It also confirms the validity of western blot testing for HTLV-III/LAV specificity and suggests that ELISA-positive, western-blot-negative blood may not be infectious.

WHAT DO WESTERN BLOT INDETERMINATE PATTERNS FOR HUMAN IMMUNODEFICIENCY VIRUS MEAN IN EIA-NEGATIVE BLOOD DONORS?

To investigate the specificity of western blot indeterminate (WBi) patterns for antibodies to the human immunodeficiency virus type 1 (HIV-1), 100 enzyme immunoassay (EIA) negative donors from whom prospectively obtained recipient sera were available were tested by WB. 20 were WBi, with p24 being the predominant (70%) and generally the only band. Among recipients of WBi blood, 36% were WBi in their 6 month post-transfusion sample, but so were 42% of a control population that had received only WB-negative blood. When serial samples from recipients with a WBi pattern were tested on two occasions, only 35% of results were reproducible. No reciepient of WBi blood became EIA positive, true positive for WB, positive for HIV-1 antigen, or positive for EIA reactivity against recombinant p24 or gp41. The polymerase chain reaction was negative for gag and env HIV-1 sequences in all donors and recipients. Thus WBi patterns are exceedingly common in randomly selected donors and recipients and such patterns do not correlate with the presence of HIV-1 or the transmission of HIV-1 from donor to recipient.

RELATION OF HEPATITIS-B-ANTIGEN SUBTYPES IN SYMPTOM-FREE CARRIERS TO GEOGRAPHICAL ORIGIN AND LIVER ABNORMALITIES

Abstract Hepatitis-B-antigen (HBAg) subtypes and possible liver abnormalities were investigated in 63 symptom-free HBAg-positive blood-donors in Toronto. 38 (60%) had the determinant d, while 25 (40%) had y. Most donors who were subtype adw were born in Canada, China, Germany, and the West Indies, whereas most donors who were ayw originated from Mediterranean countries. All 8 donors who were adr were from China. Thus the subtype of chronic HBAg carriers appeared to be related to their country of origin rather than their country of residence. Evidence of liver dysfunction, mainly raised serum-glutamic-pyruvictransaminase and liver-biopsy abnormalities were almost equally common in the adw, adr, and the ayw groups (30%, 25%, and 26%, respectively). 14 HBAg-positive, first-degree relatives of 9 blood-donors were also subtyped; all had the same subtype as the index case to whom they were related.

RISK OF TRANSFUSING BLOOD CONTAINING ANTIBODY TO HEPATITIS-B SURFACE ANTIGEN

Abstract 362 blood-transfusion recipients, whose sera were initially negative for hepatitis-B antigen (HB s Ag), were prospectively followed for clinical and serological evidence of exposure to hepatitis-B virus (H.B.V.) and for the development of hepatitis unrelated to H.B.V. None of the donor units received by these patients contained detectable HB s Ag. Of the 362 transfusion recipients, 23 (6%) developed 25 episodes of hepatitis; only 4 of these 25 episodes were serologically related to H.B.V. Based on the absence of antibody to HB s Ag anti-HB s ) prior to transfusion, 278 of the patients were considered susceptible to H.B.V. infection. Of these susceptible patients, 133 received at least one unit of blood containing anti-HB s ; when compared with the 145 who did not receive anti-HB s , there was no significant difference in biochemical or overt hepatitis B (3/133 vs. 1/145), in serological response to H.B.V. 5/133 vs. 5/145), or in hepatitis unrelated to H.B.V. 11/133 vs. 6/145). It is concluded that blood containing detectable anti-HB s carries no increased risk of transmitting hepatitis B compared with blood which lacks this antibody.

GAMMA-GLOBULIN FOR HEPATITIS-VIRUS B PREVENTION OR EXTENSION ?

Abstract Inferential evidence is presented that attenuation of hepatitis-B-antigen (H.B. Ag)-positive hepatitis may favour the progression of acute to chronic liver disease and the development of a persistent H.B. Ag carrier state. These considerations may be relevant to the use of antibody to the hepatitisB antigen (anti-H.B. Ag) in the prophylaxis of hepatitis B. Based on preliminary encouraging results, controlled trials to test the efficacy of anti-H.B. Ag are being planned or are in progress. It is urged that these trials go beyond questions relevant to acute, overt hepatitis and specifically examine the possible induction of chronic liver disease and a persistent viral carrier state.