P.N. Lelie

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.

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.

New hepatitis B virus mutant form in a blood donor that is undetectable in several hepatitis B surface antigen screening assays

BACKGROUND: Envelope mutant forms of hepatitis B virus (HBV), impairing HBV antibody recognition, have been reported with mutations in single or multiple sites of the hepatitis B surface antigen (HBsAg) group‐ specific “a” determinant. Blood donors infected with such an HBsAg mutant form of HBV may escape detection by HBsAg screening assays and therefore may affect the safety of the blood supply. CASE REPORT: A repeat blood donor became HBsAg‐reactive in an enzyme immunoassay. Confirmatory testing yielded negative results for HBsAg in a radioimmunoassay and in four enzyme immunoassays used in blood donor screening. The specificity of the HBsAg reactivity in the first enzyme immunoassay was confirmed by HBsAg neutralization with antibody to HBsAg. Additional HBV confirmatory test results were positive for antibody to hepatitis B core antigen and antibody to hepatitis B e antigen; negative for antibody to HBsAg and for hepatitis B e antigen; and positive for HBV DNA. DNA sequence analysis of the “a” determinant region of HBsAg revealed amino acid substitutions from Q (Gln) to R (Arg) at codon 129 and from M (Met) to T (Thr) at codon 133. CONCLUSION: This case illustrates the presence of HBsAg mutant forms of HBV in a West European blood donor population that were undetected by several HBsAg screening assays. Adaptation of HBsAg screening is indicated to overcome deficiencies in sensitivity in detecting HBsAg mutant forms of HBV. Screening for antibody to hepatitis B core antigen or HBV DNA may also detect blood donors infected with HBsAg mutant forms of HBV

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.