C.L. van der Poel

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.

Infectivity of blood seropositive for hepatitis C virus antibodies

Stored serum samples from 5150 blood product transfusions and 383 recipients were tested for antibodies to hepatitis C virus (anti-HCV) by a recombinant enzyme-linked immunosorbent assay (ELISA) as part of a prospective study on post-transfusion non-A, non-B hepatitis (NANBH). Donor cofactors associated with HCV infectivity of anti-HCV-positive blood products were raised alanine aminotransferase concentrations (6 of 9 infective vs 1 of 26 not infective); a mean ELISA optical density/cut-off ratio greater than or equal to 2 (7 of 9 vs 9 of 26); both preceding factors (together in 6 blood products, all of which transmitted infection); and persistent donor anti-HCV seropositivity. Use of anti-HCV screening to prevent post-transfusion NANBH was compared with measurement of alanine aminotransferase concentrations: a corrected efficacy of 63% and 65%, a specificity of 93% and 64%, and a positive predictive value of 16.2% and 3.6% were found, respectively; 0.7% or 3.8% of blood donations, respectively, would be discarded. Blood donor screening for anti-HCV is recommended to reduce the incidence of post-transfusion NANBH.

ANTI-HEPATITIS C ANTIBODIES AND NON-A, NON-B POST-TRANSFUSION HEPATITIS IN THE NETHERLANDS

In a prospective study carried out in the Netherlands (1984-86) to establish the incidence of post-transfusion hepatitis non-A, non-B (PTH-NANB) in patients undergoing open heart surgery, 393 patients received 5315 blood product transfusions. PTH-NANB developed in 9 patients (index cases); stored serum samples from these patients and from 9 control patients, matched for age, sex, and number of blood product transfusions, as well as serum samples of all implicated blood products, were selected retrospectively. Sera were tested under code with a radioimmunoassay for the detection of antibodies to hepatitis C virus (anti-HCV). PTH-NANB patients received 151 blood product transfusions and control patients 140. 4 of 9 PTH-NANB patients (3/5 chronic, 1/4 acute resolved hepatitis) and 0/9 controls seroconverted. 7 of the transfusions given to PTH-NANB patients but none of those given to control patients were anti-HCV positive. In 7 of 9 serum sets from PTH-NANB index cases plus implicated donors, either a donor or the recipient was anti-HCV positive. Among the donors implicated in transmission of PTH-NANB there was a strong correlation between raised alanine aminotransferase levels and the presence of anti-HCV antibodies.

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.

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.

Evaluation of automated nucleic acid extraction devices for application in HCV NAT.

BACKGROUND: To further improve the safety of the blood supply, various national blood transfusion organizations presently use or are in the process of implementing routine HCV NAT in minipools. According to the Committee for Proprietary Medicinal Products (CPMP) of the European Union, the HCV NAT detection limit of the assay should be 100 IU per mL (270 geq/mL) for testing initial plasma pools. Paul Ehrlich Institute (PEI) regulations stipulate that 5000 IU per mL (13,500 geq/mL) must be detected to calculate the amount contributed by individual donations composing the minipool. The sensitivity for HCV RNA extraction achieved by three commercially available laboratory kits was compared.