H. T. M. Cuypers

Confirmation of hepatitis C virus infection by new four-antigen recombinant immunoblot assay

A new four-antigen recombinant immunoblot assay (4-RIBA) for confirmation of hepatitis C virus (HCV) C-100 enzyme-linked immunosorbent assay (ELISA) reactivity was tested in stored serum samples (1984-86) of blood donors and recipients and compared with results from polymerase chain reaction (PCR) analysis of fresh (1990) plasma samples in donors and recipients from the original study. Of 37 HCV C-100 ELISA-positive blood products, 8 were 4-RIBA positive, of which 7 were implicated in post-transfusion non-A, non-B hepatitis (PT-NANBH) and/or PCR confirmed recipient HCV infection. Of 9 recipients with PT-NANBH, 8 were reactive in 4-RIBA (6 positive and 2 indeterminate). With fresh plasma samples, 3 donors and 6 recipients who were 4-RIBA positive were also PCR positive. 4 4-RIBA indeterminate and 78 4-RIBA negative samples of donors and recipients were PCR negative. Of 6 4-RIBA positive recipients, 5 were PCR positive four to six years later. 1.6% of the 383 recipients became chronically infected with HCV. The new 4-RIBA represents a candidate confirmation test to discriminate between infective and non-infective HCV C-100 ELISA-positive blood donors.

Reliability of polymerase chain reaction for detection of hepatitis C virus

The polymerase chain reaction (PCR) is used to detect hepatitis C virus (HCV) RNA, and the results of this assay may have a bearing on management of patients. We tested 31 laboratories for performance of HCV PCR with a coded panel that comprised 4 HCV-positive plasma samples, 6 HCV-negative samples, and two dilution series of HCV-positive plasma. 15 (48%) laboratories had faultless results with both dilution series, and 16 (52%) laboratories reported erroneous results with one or both series. 10 (32%) laboratories had faultless results when testing undiluted plasma samples, 11 (35%) produced a false-negative result with a weak-positive sample, and 10 (32%) produced false negative and/or false positive results. Only 5 (16%) laboratories performed faultlessly with the entire panel of samples. Reports of presence of HCV should be interpreted with care until reliable HCV-RNA detection becomes widely available.

Sexual transmission of hepatitis C virus

We tested 50 heterosexual partners of hepatitis C viraemic (HCV) individuals, using second generation HCV antibody assays and a validated polymerase chain reaction assay. In none of them were HCV antibodies or HCV-RNA detected. The median duration of the sexual relationship was 13 years. This study, with the most sensitive techniques for detection of HCV, indicates that the risk of sexual transmission of HCV is absent or very low.

Look-back study of infectivity of anti-HCV ELISA-positive blood components

The infectivity of blood components from donors who were later found to be anti-HCV ELISA-positive was investigated in recipients who were enrolled in a look-back programme. Recipients received ELISA-positive blood components from donors who were PCR-positive and/or RIBA-2-positive (n = 22, group A) or PCR-negative and indeterminate or negative on RIBA-2 (n = 105, group B). 26 of 32 (81%) recipients of group A donors and none of 140 of group B were HCV-infected. All stored serum samples of previous donations (n = 172) of group A donors were anti-HCV-positive in RIBA-3, indicating a chronic carrier state of HCV in these donors.

Recombinant immunoblot assay reaction patterns and hepatitis C virus RNA in blood donors and non-A, non-B hepatitis patients

To establish the value of the second‐generation recombinant immunoblot assay (RIBA‐2) and cDNA polymerase chain reaction (cDNA PCR) for confirmation of hepatitis C virus (HCV) infection, anti‐HCV reaction patterns and the presence of HCV RNA were examined in 610 blood donors and 255 non‐A, non‐B hepatitis patients who were positive or indeterminate in RIBA‐2. Of RIBA‐2‐positive donors (n = 191) and patients (n = 224), 75.4 and 89.7 percent, respectively, were HCV RNA positive. The most frequently observed anti‐HCV recognition patterns in HCV RNA‐positive donors and patients were c22, c33c, and c100 and/or 5‐1‐1 (67.3%, 57.7%) and c22, c33c (24.8%, 29.3%). Among subjects with a RIBA‐2‐indeterminate result, HCV RNA was detected more often in patients (n = 31) than in donors (n = 419): 67.7 and 2.1 percent, respectively. In viremic persons with single‐band reactivity in RIBA‐2, this reactivity was always directed against either c22 or c33c. HCV RNA was detected by cDNA PCR in none of 162 persons with only anti‐c100 and/or anti‐5‐1‐1 reactivity. Therefore, RIBA‐2 reactivity against c100 in combination with 5‐1‐1 should not be considered positive but indeterminate. In RIBA‐2‐indeterminate persons, HCV RNA detection is necessary for reliable confirmation of HCV infection.

Early Antihepatitis C Virus Response with Second—Generation C200/C22 ELISA

Detection of early antibody to hepatitis C virus (HCV) by a new second‐generation C200/C22 anti‐HCV enzyme‐linked immunosorbent assay (ELISA) and a four‐antigen recombinant immunoblot assay (4‐RIBA) was compared with the first‐generation anti‐HCV C100 ELISA using sequential serum samples of 9 recipients who were infected with HCV, as detected by polymerase chain reaction after transfusion of blood products. Within 26 weeks after transfusion, 9/9 (100%) recipients seroconverted with C200/22 ELISA, and 6/9 (67%) seroconverted with C100 ELISA. Compared with C100 ELISA, C200/C22 ELISA seroconversion occurred simultaneously in 3 cases, 5–6 weeks earlier in 3 other cases, and 20 weeks earlier in 1 case. Seven of 9 (78%) recipients became positive, and 2/9 (22%) became indeterminate with 4‐RIBA. In 8 cases with clinical posttransfusion hepatitis non‐A, non‐B (PTH‐NANB), anti‐HCV C200/C22 ELISA seroconversion occurred 2–17 (mean 6) weeks after the onset of hepatitis. In 6 cases of PTH‐NANB, anti‐HCV C100 ELISA seroconversion occurred 2–26 (mean 9) weeks after the onset of hepatitis. It is concluded that the second‐generation C200/C22 ELISA is more sensitive than the C100 ELISA for the detection of antibody during early HCV infection. Indeterminate 4‐RIBA results are found in the early phase of HCV infection.

Enhanced Sensitivity of a Second Generation ELISA for Antibody to Hepatitis C Virus

A second generation ELISA for combined detection of antibodies to three hepatitis C virus (HCV) recombinant proteins, i.e. C100, C33c and core, was compared with a first generation anti‐HCV ELISA in which only antibodies to C100 are detected. The results of the ELISAs were evaluated in 225 haemophilia patients (panel A) and 44 patients with non‐A, non‐B (NANB) hepatitis (panel B). HCV infection was established by cDNA‐polymerase chain reaction (PCR) (in panel B only) and by studying the anti‐HCV reaction patterns in 4 separate ELISAs for detection of antibodies to the recombinant proteins C100, C33c, core and a combination of two synthetic peptides sp67/65 derived from the C100 region. The sensitivity for the detection of HCV infection had increased from 0.92 [95% confidence interval (CI): 0.87–0.95] to 1.00 (95% CI: 0.98–1.00) in haemophiliacs and from 0.84 (95% CI: 0.66–0.95) to 1.00 (95% CI: 0.89–1.00) in NANB hepatitis patients when the second generation ELISA was used instead of the first generation ELISA. Concurrently the chance of a false negative result was reduced in panel A and B from 0.37 to 0 and from 0.28 to 0, respectively. Analysis of anti‐HCV reaction patterns revealed that 172 of 206 (83.5%) anti‐HCV ELISA‐reactive haemophilia patients had antibodies to all 4 antigens tested. In the NANB hepatitis patients 18 of 31 (58.1%) anti‐HCV ELISA‐reactive subjects reacted with 4 antigens. In the PCR tested panel of NANB hepatitis patients 2 subjects who showed antibody reactivity to only one antigen and 5 patients with reactivity to 2 antigens were PCR‐positive. On the other hand, 1 NANB hepatitis patient showed antibody reactivities to all 4 antigens tested, but was PCR‐negative. Antibodies to core were the most prevalent in the two populations studied. We conclude that the introduction of the second generation anti‐HCV ELISA will significantly reduce the likelihood of unrecognized HCV infection.

Detection of hepatitis C viral RNA sequences in fresh and paraffin-embedded liver biopsy specimens of non-A, non-B hepatitis patients

In this study methods of HCV-RNA detection in fresh frozen and formalin-fixed, paraffin-embedded liver biopsies are described. Of 22 untreated chronic non-A, non-B hepatitis patients and 6 control patients, a plasma sample and part of a liver biopsy were freshly frozen for hepatitis C virus (HCV) cDNA-PCR. From 16 of the same non-A, non-B hepatitis patients and from 5 of the same control patients formalin-fixed, paraffin-embedded liver tissue from the same biopsy was available also for HCV cDNA-PCR. In 13 of 22 non-A, non-B hepatitis patients HCV-RNA could be detected in plasma as well as in liver tissue. In the other 9 non-A, non-B hepatitis patients and in 6 control patients, no HCV-RNA was detectable in either plasma or liver tissue. The comparison between HCV cDNA-PCR results in fresh frozen versus formalin-fixed, paraffin-embedded liver biopsies showed that although detection of HCV-RNA in both correlated 100% the quantity of HCV-RNA was lower in the formalin-fixed, paraffin-embedded liver biopsies of 5 of 8 patients for whom end-point dilution titration of liver RNA was performed. We conclude that using the procedures described HCV-RNA can be reliably detected in both fresh-frozen and formalin-fixed, paraffin-embedded liver biopsies and that HCV cDNA-PCR in liver tissue may become an important assay, especially for monitoring anti-viral therapy.

Sensitivity and Specificity of Three Third‐Generation Anti‐Hepatitis C Virus ELISAs

Three commercially available 3rd‐generation anti‐HCV ELISAs (Abbott, Murex and Ortho) were evaluated in various serum panels: (A) blood donor samples (n = 403) with 1st‐ or 2nd‐generation anti‐HCV ELISA (various manufacturers) positive test results; (B) non‐A, non‐B hepatitis patients (n = 212); (C) multitransfused patients (n = 253); (D) serial dilutions of HCV confirmed (RIBA and PCR) positive blood donors (n = 24), and (E) first‐time blood donors (n = 1,055). All samples of panels A, B and C were tested in PCR and RIBA‐2. In panels A, B and C, 398 samples were HCV PCR positive: all were detected by Abbott and Ortho, and 397 (99.7%) by Murex. The sample missed by the Murex ELISA showed an isolated anti‐C33c reactivity in RIBA‐2. In panels A–C, 442 samples were RIBA‐2 positive and all were detected by the 3 tests. With Probit analysis on results of panel D, no significant difference in sensitivity was observed between the 3 evaluated ELISAs. Specificities of Abbott, Murex and Ortho in 1,055 blood donors were 99.7, 99.3 and 99.9%, respectively (NS, χ2). We conclude that the sensitivity and specificity of the 3 ELISAs are comparable although the C33c antigen in the Murex VK47 test should be improved.

Analysis of genomic variability of hepatitis C virus

The anti-C100-enzyme-linked immunosorbent assay, the new four-antigen antibody recombinant immunoblot assay, and detection of viral RNA sequences by copy DNA-polymerase chain reaction were used to establish the course of hepatitis C virus (HCV) infection in recipients of HCV-infected blood products. Different patterns of infection were observed: (1) persistent HCV infection with and without chronic hepatitis, and with acute resolved hepatitis; and (2) acute resolved hepatitis with clearance of HCV. In order to determine whether different infection- and anti-HCV recognition patterns are correlated to differences in viral nucleotide sequences, we compared sequences in the NS3 region between isolates from recipient(s) and their infective donors. Based on these comparisons we conclude that in The Netherlands two types of molecular variants circulate; one resembling the Japanese prototype isolate JH1, and the other the HCV-1 isolate from the U.S.A. The difference in sequence homology between the two types is approximately 24%. Comparison of sequences of donors and involved recipients determined in isolates prepared from blood samples four years after transfusion revealed that viral RNA sequences are strongly conserved (greater than 96.8%) in the NS3 region. These data indicate that the observed differences in anti-HCV immune response patterns between recipients are more a reflection of their immune reactivity than of divergence of viral strains.