Stella Quan

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.

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.

Improved Detection of Antibodies to Hepatitis C Virus Using a Second Generation Elisa

A screening assay for the detection of antibodies to hepatitis C virus (HCV); ORTHO HCV ELISA Test System, Second Generation, was compared with the currently licensed c100-3 based test (ORTHO HCV ELISA Test System). The second generation ELISA differs from the c100-3 based assay in that it detects circulating antibodies to both structural (nucleocapsid) and non-structural (NS3/NS4) HCV proteins. Specimens tested consisted of a cohort of 35 patients diagnosed with non-A, non-B hepatitis (NANBH) and 3971 presumably healthy volunteer blood donors. Second generation ELISA demonstrated significantly greater clinical sensitivity in patients with acute phase NANBH (80% vs. 60%) as well as chronic disease (88% vs. 72%). Additional specimens reactive only in second generation ELISA, demonstrated reactivity to HCV antigens c33c and/or c22-3 in supplemental testing by the Chiron HCV RIBA Assay System. The second generation ELISA also detected additional RIBA reactive volunteer blood donors (0.18% of the population tested) that were nonreactive in first generation ELISA. This data indicated that second generation ELISA would detect approximately 2 additional anti-HCV reactive donors per 1,000 screened. Specificities obtained with this low risk population were 99.6% for first generation and 99.7% for second generation ELISA.

High prevalence of cagA-positive strains in Helicobacter pylori-infected, healthy, young Chinese adults

Background: Cytotoxin‐associated gene A (cagA) has been implicated as a potential pathogenic marker for Helicobacter pylori‐induced severe gastroduodenal diseases. Although the prevalence of cagA‐positive strains has been reported in patient populations from developed countries, only limited information from developing countries is available.

Typing of hepatitis C virus antibody with specific peptides in seropositive blood donors and comparison with genotyping of viral RNA

Background and objectives: Serotyping of antibody to hepatitis C virus (anti‐HCV) with specific peptides has been developed as an alternate method for typing HCV infections. The method does not require a prior amplification of viral RNA sequences from the sample. Identification of the viral genotype may be relevant for prognosis and clinical management. Materials and methods: We used a previously described HCV serotyping assay (RIBA™ HCV Serotype SIA kit, Chiron Corp.) to investigate the serotype in 191 samples from blood donors selected for anti‐HCV patterns (positive and indeterminate), ALT levels, and the presence or absence of viral RNA. The serotypes were compared with the genotypes obtained from typing the 5′‐noncoding region of the viral RNA in 82 viremic samples. Results: We were able to obtain the viral serotype in 85% (114/134) of samples positive for anti‐HCV but in only 3.5% (2/57) of the indeterminates. Lack of anti‐NS4 in the sample was significantly associated with both untypable results and the presence of HCV serotypes other than serotype 1. The overall correlation with genotyping was 78% (64/82), rising to 95.5% (64/67) if only samples that could be both genotyped and serotyped were considered. The assay was easy to perform, gave reactivity patterns easy to interpret, and performed with high proficiency on anti‐HCV‐positive samples lacking detectable levels of viral RNA. Conclusions: This is a practical and useful method for typing HCV infections in the clinical setting. The poor ability of the Core peptides to give the serotype in samples lacking anti‐NS4 and the lack of specific peptides to recognize HCV types other than 1, 2, and 3 are, however, some aspects of the method that need improvement in the future.

New Strip Immunoblot for the Confirmation of HTLV‐I/II Infection

In The Netherlands, anti-human T-cell lymphotropic virus type I (HTLV-I) blood donor screening became mandatory in January 1993. Donations reactive in the enzymelinked immunosorbent assay (ELISA) screening test are confirmed with Western blot analysis (WB). Only WB-positive or indeterminate blood donors are notified and retested by polymerase chain reaction (PCR) [1]. In accumulated data of the Dutch Blood Banks, only 2% of donors repeatedly positive in the anti-HTLV-I/II ELISA were WB-positive and all of these were also PCR-positive. However, 75% of the ELISA-positive blood donors have indeterminate WB results. In these cases the ELISA reactivities are nonspecific since all WB-indeterminate donors are negative in PCR [2]. Current WB confirmation thus leads to the notification and often to the deferral of many WB-indeterminate blood donors as well as to high costs for PCR testing, illustrating the need for a more specific serological confirmatory assay. The aim of our study was to evaluate a newly developed anti-HTLV-I/II Recombinant Immunoblot Assay (RIBA) to confirm samples reactive to screening tests with a special emphasis on the ability of this test to resolve WB-indeterminate results in blood donors, without compromising the sensitivity.

Humoral immune response to hepatitis C after liver transplantation: assessment of a new recombinant immunoblot assay

OBJECTIVE:The immune control of infection with hepatitis C virus (HCV) is poorly understood; vigorous antibody responses to viral proteins seem to coexist with the virus and thus whether they are neutralizing remains controversial. HCV-related liver failure is the leading indication for orthotopic liver transplantation (OLT) worldwide. Attenuated antibody responses in immunosuppressed patients and decreased reliability compared to assessment of HCV RNA has hampered the use of antibody testing post-OLT. The goals of this current analysis were twofold: to determine the sensitivity of a prototype strip immunoblot assay (RIBA 3.0, Chiron Diagnostics) for the diagnosis of HCV post-OLT; to determine if there was a correlation between antibody response and severity of histological recurrence.METHODS:The study was comprised of 76 HCV-positive individuals divided into three patient groups: liver allograft recipients with evidence of mild or no histological recurrence (n = 52), liver allograft recipients with evidence of severe HCV recurrence and allograft cirrhosis (n = 12), and nontransplant patients being enrolled in an induction interferon trial (n = 12). All transplant patients had histological follow-up of at least 1 yr.RESULTS:Sixty of the 64 (94%) HCV-positive OLT recipients had 1+ reactivity to two or more recombinant antigens; three of the patients who lacked a detectable response had minimal histological recurrence and one had severe recurrence. All nontransplant patients demonstrated 4+ reactivity to at least two antigens, and 55/64 (86%) OLT recipients demonstrated this same level of reactivity. Seven of the nine patients lacking this high level of reactivity had evidence of minimal recurrence. Furthermore, the mean (± SEM) level of antibody reactivity for c100 (p = 0.04) and NS5 (p = 0.01) were significantly lower for patients with mild recurrence after OLT, compared to the other groups. The level of antibody reactivity was unrelated to HCV genotype or viral load.CONCLUSIONS:The recently developed RIBA 3.0 assay for detection of antibodies to HCV appears to be highly sensitive for the diagnosis of HCV post-OLT. In general, the level of antibody reactivity was comparable in the transplant patients and in nonimmunosuppressed controls. The pathogenic implications of the relatively diminished humoral response in patients with mild recurrence post-OLT are discussed.

Concordance between hepatitis C virus serotyping assays

Summary. Hepatitis C virus testing has evolved from a simple enzyme‐linked immunosorbent assay (ELISA) to complex molecular tests including qualitative and quantitative polymerase chain reaction (PCR) as well as multiple methods to determine geno and serotypes. Serotyping assays have been described and are being further refined to aid describing the epidemiology of hepatitis C virus (HCV) infection and may have a role in predicting response treatment. This study describes the concordance between two serotyping assay systems, a recombinant immunoblot assay Chiron RIBATM Strip Immunoblot Assay (SIA) and a more competitive ELISA peptide assay, using PCR as the standard. Serotype was successfully determined in 144/202 (71%) patients by the Murex ELISA assay and 179/202 (89%) by the Chiron strip immunoblot assay (SIA) assay (P < 0.001). Concordance between restriction fragment length polymorphism (RFLP) and the Murex assay was 139/144 (97%) between RFLP and the Chiron SIA was 171/179 (96%) and between the Murex and Chiron SIA was 136/144 (94%). These assays provided a reliable, simple, and rapid method of determining HCV serotype.

Automated RIBATM HCV Strip Immunoblot Assay: A Novel Tool for the Diagnosis of Hepatitis C Virus Infection in Hemodialysis Patients

Hemodialysis (HD) patients remain a high-risk group for hepatitis C virus (HCV) infection. Serological assays (enzyme-linked immunosorbent assays, ELISAs) are the only tests currently approved by the Food and Drug Administration in the United States for the diagnosis of HCV. The RIBATM HCV Strip Immunoblot Assay (SIA) is an established method for supplemental testing of repeat reactive hepatitis C ELISA patients on HD. However, the current manual procedure is labor intensive, requiring subjective band scoring and result interpretation. Recently, the automated CHIRON® RIBATM HCV Processor System has been designed to perform RIBA supplemental testing. The CHIRON RIBA HCV Processor System consists of a bench-top instrument that provides objective evaluation of the RIBA immunoblot strips, by measuring the light differentially reflected from the developed bands and white background, creating a density of reflectance. The CHIRON RIBA HCV Processor System assesses the intensity of each of the reactive bands in relation to the intensity of the internal control bands on each RIBA HCV strip. Comparison between processor and manual protocols was performed using a large (n = 200) cohort of ELISA 3.0 HCV negative and positive patients on maintenance HD. The test characteristics of RIBA HCV 3.0 SIA were identical with manual and automated runs. The relative intensity values of antigenic bands by the CHIRON RIBA HCV 3.0 Processor System between anti-HCV positive and negative patients were significantly different; only 15 of 784 (1.9%) antigenic bands had borderline reactivities. The correlation of test results between manual and automated runs was very high (kappa value 0.989). Among positive results by RIBA HCV 3.0 SIA, there was a strong concordance between manual and automated runs with regard to the pattern of reactivity (kappa value 0.943). The discordant results between manual and automated protocols were attributable to increased variability of antigen scores close to the cutoff value for both tests. In conclusion, the CHIRON RIBA HCV 3.0 Processor System is capable of performing RIBA HCV 3.0 SIA in the HD population accurately with minimal operator involvement. The test characteristics of RIBA HCV 3.0 SIA were identical by manual and automated runs. There was a strong correlation between the results of the manual and automated runs; the few discordant results between the two procedures were mostly due to increased variability of antigen scores close to the cutoff value for both tests. The Centers for Disease Control and Prevention in the USA have recently included chronic HD patients among those persons for whom routine HCV testing is recommended; HCV-infected patients on HD often have a high rate of indeterminate results by manual RIBA technology which is operator dependent for band scoring and result interpretation. The CHIRON RIBA HCV 3.0 Processor System may be very useful for supplemental anti-HCV testing of ELISA repeat reactive specimens in clinical practice within dialysis units.

THE IMPACT OF HCV GENOTYPES ON SEROREACTIVITY AGAINST HCV ANTIGENS IMMOBILIZED ON THE RIBA 3.0 SIA

To the Editor: Hepatitis C virus (HCV) is known for its genetic heterogeneity and has been classified into six major genotypes based on phylogenetic analysis (1). The diverse genetic heterogeneity also translates into variations of the encoded viral proteins, which may impact on virologic behavior, host-virus interaction, and the viral epitopes recognized by the host B and T cells. The current serologic assay for diagnosing HCV infection is based on the detection of host antibody against relatively conserved HCV viral polypeptides. Recent studies have shown differences in seroreactivity between HCV genotypes based on the commercial assays, and suggested that a large proportion of epitopes of the type la or lb recombinant proteins used in the current assays are genotype specific (2). The antigens used for Ortho HCV version 3.0 ELISA and RIBA TM HCV 3.0 Strip Immunobtot assay (SIA) were not modified to improve the detection of various genotypes. However, the sensitivity of these assays for the detection of seroreactivities in patients infected with different HCV genotypes has not been extensively investigated. To determine the impact of HCV genotypes on seroreactivity in patients infected with different HCV genotypes, 108 patients infected with different HCV genotypes were studied. The HCV genotypes in these patients were determined by both restriction fragment length polymorphism and a line probe assay (InnoLiPA-HCV, Innogenetics, Ghent, Belgium) (3). Seroreactivities to HCV antigens from structural and non-structural regions were evaluated with RIBA TM HCV 3.0 SIA according to manufacturers instructions. RIBA TM HCV 3.0 SIA strip contains four HCV antigen lanes coated with 5 different capture HCV-encoded recombinant antigens/peptides: lane 1 with cl00(p) and 5-1-1(p); lane 2 with c33c; lane 3 c22(p); and lane 4 with NS5; in addition to a fifth lane with a recombinant human superoxide dismutase (hSOD) as a control for anti-SOD reactivities. Seroreactivity seen on developed strip was graded from - (minus) to 4+ according to product insert. Table 1 shows the results. Most patients had either strong reactivity or no reactivity to a particular antigen, and hence the results are given as 0 (no reactivity), 1 to 3+, and 4+. Two patients infected with HCV type 4 were non-reactive to all four antigens, and they both had severe renal insufficiency (serum creatinine 6.2 and 8.4 mg/dl), suggesting that host factors may play a role in the non-reactivity in these two patients. Sequencing of the HCV peptide for HCV core(p) region (a.a. 10-53) by reverse transcription-polymerase chain reaction and dideoxy chain termination sequencing showed no specific amino acid substitutions at this region to account for the loss of seroreactivity, further supporting the hypothesis that the non-reactivity was related to the host factors in these two patients. In general, antibody reactivity to c33c and c22(p) was strong and found to be present in 104/108 (96.3%) of the chronic HCV patients tested, demonstrating that the sequence selected for c33c and c22(p) in the RIBA TM HCV 3.0 SIA was conserved for its B-cell reactivity amongst the various genotypes, and offering the ability to detect the presence of these antibodies in the specimens. Antibody reactivities towards cl00(p)/5-1-1(p) occurred in >90% of genotypes 1, 2, 3, 5, and 6 specimens, but in only 12/20 (60.0%) of the type 4 specimens. The prevalence of antibody reactivity towards NS5 ranged from 75% to 95% in the various genotype specimens. With the current set of data, non-reactivity to c-100(p) and 5-1l(p) in patients with established HCV infection suggested genotypes 2, 3 or 4 infection. As all but two of the 108 patients genotyped as type 1 to type 6 were detected by RIBA TM HCV 3.0 SIA, the current RIBA HCV 3.0 SIA is satisfactory for confirming the diagnosis of HCV infection in most HCV genotypes.