Indeterminate results of the second-generation hepatitis C virus (HCV) recombinant immunoblot assay: significance of high-level c22-3 reactivity and influence of HCV genotypes.

A second-generation recombinant immunoblot assay (RIBA 2.0) is used in the United States to confirm infection with hepatitis C virus (HCV) in samples that are anti-HCV (enzyme immunoassay) positive. In some cases, indeterminate results of RIBA 2.0, which are defined as reactivity to a single antigen species or reactivity limited to two proteins derived from the same coding region of the HCV genome, are encountered. This study was performed to establish the significance of indeterminate RIBA 2.0 results in relation to HCV RNA detection, high positivity for the c22-3 band, and the HCV genotype as determined by direct DNA sequencing. Ninety-six samples with indeterminate RIBA 2.0 results were studied. HCV RNA was detected in 21 of 34 (62%) samples with high reactivity to c22-3 and in 8 of 62 (13%) samples with low reactivity to c22-3. The HCV genotype distribution in samples that were RIBA 2.0 indeterminate and HCV RNA positive was significantly different from that in samples of a control group with positive results for both the RIBA 2.0 and HCV PCR. These results suggest that highly positive c22-3 samples are likely to be associated with HCV viremia and that infection with less common HCV genotypes is more commonly associated with indeterminate RIBA 2.0 results.     

Clinical significance of serum hepatitis C virus (HCV) RNA as marker of HCV infection.

We have evaluated the clinical significance of hepatitis C virus (HCV) RNA determination by analyzing a group of 221 hospitalized patients with abnormal liver function tests. Serum HCV RNA was detected by "nested" PCR amplification followed by nonisotopic hybridization. Of the 200 (90.5%) patients with anti-HCV-positive enzyme-linked immunosorbent assay results, 152 (76%) were RIBA reactive, 47 (23.5%) had indeterminate results, and 1 (0.5%) was nonreactive. Of the 180 (90%) patients positive for anti-HCV and HCV RNA, 138 (76.7%) were RIBA reactive and 42 (23.3%) were RIBA indeterminate. The pattern of RIBA reactivity did not correlate with the presence of HCV RNA. Elevated alanine aminotransferase levels were associated neither with the presence of viremia nor with the RIBA pattern. Histological findings consistent with non-A non-B hepatitis correlated with the presence of HCV RNA but not with the RIBA pattern. HCV RNA was detected in 11 of 21 (52.4%) anti-HCV-negative patients. These 11 patients were either immunosuppressed or in the prodromic phase of acute hepatitis C. Circulating HCV RNA can therefore be described as being predictive of virus-induced liver damage in anti-HCV-positive patients and may be useful in the diagnosis of HCV infection in anti-HCV-negative immunosuppressed patients or in those with early acute infection.     

Interpretation of Ortho HCV Ab ELISA test results by chiron HCV recombinant immunoblot assay]

HCV infections are diagnosed by determining the circulating antibodies to the C 100 recombinant viral antigen using the ELISA method. Cut-off analysis from normal subjects and well documented NANBH patients suggests that screening of a low risk group such as blood donors might yield a relatively high ratio of false positives. An immunoblot assay (Chiron RIBA) using 3 recombinant antigens, C 100, 5-1-1 and SOD has been developed for evaluating the ELISA reactives as an additional, more specific assay. In the RIBA testing 51.5% were reactive and 28.5% were indeterminate in ELISA positive donor specimens, and 79.5% were reactive and 8.0% were indeterminate in ELISA positive non-A, non-B hepatitis patients specimens. These findings coincide with the ratio of theoretically calculated true positive. In a study done by Ortho U.S.A. viral RNA were detected in 70% of RIBA reactive, 33% of indeterminate and 3.6% non-reactive specimens by polymerase chain reaction (PCR). Furthermore, an advanced system using another immunogenic region of viral polyprotein including c33c encoded in NS3 has been on trial to evaluate the possibility of confirming HCV infection and detecting seroconversion at an earlier stage.     

Study on reliability of commercially available hepatitis C virus antibody tests.

The serodiagnosis of hepatitis C virus (HCV) infection was analyzed by a recombinant immunoblot assay (RIBA) with recombinant proteins encoded by the viral RNA isolated from our patients in Hamburg, Germany. The HCV RNA was amplified by PCR, and proteins encoded by the viral core and the NS3, NS4, and NS5 regions were expressed subsequently in Escherichia coli. The results obtained with our UKE RIBA were compared with the results of the Abbott HCV second-generation enzyme immunoassay (EIA). Serum samples from 270 patients, which were sent to us on the suspicion of HCV hepatitis and which were negative for hepatitis A virus and hepatitis B virus antibodies, were examined. In 227 cases (84.1%), there were identical positive (204 cases, 75.6%) or negative (23 cases, 8.5%) results in both tests. In 32 cases (11.9%), the reactive Abbott second-generation HCV EIA results could not be confirmed by the UKE RIBA and the HCV PCR. In follow-up studies conducted over 1 year, these results did not change. In three cases (1.1%), the UKE RIBA presented a positive result while the Abbott second-generation HCV EIA was negative. All three cases were positive in the HCV PCR and showed seroconversion in an HCV EIA 4 to 6 weeks later. In addition, 33 patient serum samples were examined by UKE RIBA in parallel with the Ortho RIBA 2.0. In three cases (9.1%), a positive Ortho RIBA 2.0 result could not be confirmed by the UKE RIBA and the HCV PCR. All three patients were free of complaints. The UKE RIBA showed also a smaller number of indeterminate results (3.0%) than the Ortho RIBA 2.0 (24.2%). This comparison study demonstrates that the commercially available HCV antibody tests should be further improved.     

Use of aminotransferase, hepatitis C antibody, and hepatitis C polymerase chain reaction RNA assays to establish the diagnosis of hepatitis C virus infection in a diagnostic virology laboratory.

Clinical and therapeutic decisions for hepatitis C virus (HCV) infection depend on factors that include documentation of past infection as well as identification of those who might benefit from antiviral chemotherapy with systemic interferon. To evaluate the ability of a diagnostic laboratory to accurately identify such patients, we compared results obtained with serum transaminase assays, two HCV antibody assays (enzyme immunoassay [EIA] and immunoblot), and a polymerase chain reaction (PCR)-based assay for HCV RNA using a group of consecutively submitted samples within our university-based diagnostic virology laboratory and sera from a population of random blood donors. One hundred percent of specimens with R values of greater than 3.0 in the HCV EIA were positive in the confirmatory immunoblot. However, 25% of specimens with EIA R values of between 1.0 and 3.0 were not confirmed by either recombinant immunoblot assay (RIBA) or RNA PCR assay (false-positive specimens). A significant correlation (P less than 0.01) between increasing reactivity in the RIBA and positivity in the RNA PCR assay was found. The incidence of HCV viremia, as determined by the RNA PCR assay, was 73% for confirmed seropositive specimens, 33% for seropositive specimens with indeterminate RIBA results, 12% for seronegative specimens obtained from infected patients, and 2.0% for seronegative specimens obtained from uninfected blood donors. In contrast, serum transaminase testing did not correlate with the RNA PCR assay for HCV. Use of the EIA and immunoblot assay followed by RNA PCR testing will identify most patients who are viremic with HCV.     

Significance of indeterminate third-generation hepatitis C virus recombinant immunoblot assay.

Indeterminate hepatitis C virus (HCV) third-generation recombinant immunoblot assay (RIBA3.0; Ortho Diagnostic Systems) patterns were arbitrarily defined by the manufacturer as the detection of only one antibody out of the four that were sought, namely, c100 (NS4 encoded), c22 (core encoded), c33c (NS3 encoded), and NS5 (NS5 encoded). The aims of the present study were (i) to determine the prevalence of indeterminate RIBA3.0 patterns in patients consecutively tested for anti-HCV antibodies in a university hospital; (ii) to evaluate the significance of these patterns in terms of viral replication, liver disease, and risk factors for HCV; and (iii) to get an insight into the mechanism underlying this peculiar immune response. Among 3,074 serum samples consecutively tested for anti-HCV antibodies, 588 were found to be positive by screening assays. Fifty-nine of them (10%) were RIBA3.0 indeterminate and were compared with 59 RIBA3.0-positive ones. Thirty-one RIBA3.0-indeterminate and 53 RIBA3.0-positive serum samples were HCV RNA positive by PCR (53 versus 90%; P < 10(-6). RIBA3.0-indeterminate and RIBA-3.0-positive patients with positive PCR results were not significantly different for the prevalence of risk factors for HCV infection and elevated serum alanine aminotransferase activities. Immunosuppression, attributable to coexisting human immunodeficiency virus infection, organ transplantation, or the administration of immunosuppressive drugs, was significantly more frequent in PCR-positive, RIBA3.0-indeterminate patients than in PCR-negative, RIBA3.0 indeterminate patients (P < 0.001) and PCR-positive patients with a positive RIBA3.0 result (P < 0.01). The distribution of HCV genotypes did not differ significantly between HCV RNA-positive patients with indeterminate or positive RIBA3.0 results. In conclusion, the prevalence of indeterminate RIBA3.0 patterns in virology laboratories is about 10%; in about half of these patients HCV replication is detected by PCR; the main factor responsible for indeterminate RIBA3.0 patterns could be immunosuppression, whereas HCV genotypes do not seem to play major role.     

Poor performance of hepatitis C antibody tests in hospital patients in Uganda

Most hepatitis C testing in Uganda is performed using commercial rapid strip assays (RSA) to detect antibodies to hepatitis C virus (anti‐HCV), rather than enzyme immunoassays (EIA). The prevalence of hepatitis C antibodies in a Ugandan hospital population was determined using both methods to test their accuracy using nucleic acid testing (NAT) as a reference. Sera from 380 consecutive hospitalized Ugandan patients were tested for anti‐HCV using an RSA in Uganda, with subsequent automated third‐generation EIA testing in the United States, followed by NAT. Recombinant immunoblot assays (RIBA) were used as a supplementary test to detect anti‐HCV epitopes. Overall, anti‐HCV was detected in 48/380 (13%) by one or both antibody tests. Anti‐HCV was detected in 19 (5.0%) patients by RSA and in 33 (8.7%) patients by EIA; only four patients were anti‐HCV positive by both methods. Fourteen of the 48 anti‐HCV positive patients had detectable serum HCV RNA, 7 each by bDNA assay or by PCR. RSA detected only 7 of 14 HCV RNA positive sera. Of 29 RNA negative but anti‐HCV positive patients tested by RIBA, only two were anti‐HCV positive; 27 were anti‐HCV negative or indeterminate. Anti‐HCV testing by RSA and/or EIA was neither sensitive nor specific for detection of ongoing HCV infection in hospitalized Ugandan patients. Our findings underscore the importance of confirmatory nucleic acid testing, which, despite its increased cost, appears essential to manage African patients with HCV. J. Med. Virol. 82:1371–1378, 2010. © 2010 Wiley‐Liss, Inc.     

Prevalence of hepatitis C virus infections among heterosexuals with multiple partners

A study among heterosexual men and women with multiple sexual partners was carried out to assess the seroprevalence of antibody against hepatitis C virus (HCV). The 468 participants were recruited among visitors to the Clinic for Sexually Transmitted Diseases in Amsterdam. Sera were tested by an enzyme-linked immunosorbent assay (ELISA; Ortho), a recombinant-based immunoblot assay (RIBA; Chiron), and the polymerase chain reaction (PCR). A total of 468 persons were tested, and seven (1.5%) were found ELISA positive. Another 25 (5%) were ELISA indeterminate. Six of the seven ELISA-positive cases were RIBA positive. Further serum samples from five HCV ELISA-positive persons were tested by PCR, and four were found to be positive. The HCV ELISA-positive/RIBA-indeterminate reaction was PCR negative. None of the 17 RIBA-tested sera of the ELISA-indeterminate group yielded a positive result. There was a good correlation between an ELISA optical density/cut-off ratio greater than 2 and a positive RIBA result. The risk factor for HCV appeared to be the type of sexual partner, i.e., belonging to a "high-risk" group for human immunodeficiency virus infection and origin from hepatitis B-endemic countries. It is concluded that HCV may be transmitted through heterosexual contact but probably with low efficiency.     

Antibody to hepatitis C virus second envelope (HCV-E2) glycoprotein: A new marker of HCV infection closely associated with viremia

The second envelope protein (E2) of the hepatitis C virus (HCV) was cloned and expressed in Chinese hamster ovary (CHO) cells. This E2 glycoprotein was purified using ion exchange and lectin chromatography and used to construct an enzyme immunoassay for HCV E2 antibodies. The assay was shown to have good specificity, and detection of E2 antibodies was positively correlated (97.3%) to the presence of HCV RNA in serum and plasma. A high concordance between HCV 2.0 and E2 EIA reactivities was also observed. E2 antibody was the first serological marker to appear in 3/5 HCV seroconversion panels. This work demonstrated that 42.4% of core and 15.4% of NS3 indeterminate specimens also contained antibodies to E2, suggesting that HCV infection had occurred in these individuals. The E2 antibody assay was used to evaluate HCV 2.0 EIA-positive, HCV 3.0 EIA-negative plasma donors with indeterminate reactivity on RIBA HCV 2.0 or MATRIX HCV 1.0. Several HCV 3.0-negative specimens were shown to contain E2 antibodies in addition to an original indeterminate serological marker, primarily core. It is concluded that anti-E2 is a useful marker for determining HCV infection, and that the presence of antibodies to two nonoverlapping viral gene products suggests true HCV exposure. New HCV 3.0 blood screening tests should detect HCV 2.0-positive donors who present with an indeterminate pattern by RIBA or MATRIX and who also carry E2 antibodies. © 1995 Wiley-Liss, Inc.     

A comparison between one first generation and three second generation anti-HCV ELISAs: an investigation in high- and low-risk subjects in correlation with recombinant immunoblot assay and polymerase chain reaction

One first generation assay (manufactured by Ortho, test I) and 3 second generation anti-HCV ELISAs (manufactured by Ortho, Abbott, and UBI, tests II-IV) were compared. Sera from 4 different sources were used: (1) intravenous drug-users (IVDUs, n = 50), (2) blood donors (n = 1055), (3) all clinical samples from one day of routine anti-HCV testing (n = 89), (4) hemodialysis patients previously found negative by test I but clinically suspected to have a HCV infection (n = 11). Confirmatory anti-HCV tests were carried out with a second generation recombinant immunoblot assay (RIBA II). In sera positive exclusively by test IV, one antibody consumption test (UBI HCV Neutralization EIA) and one further immunoblot assay (INNO-LIA HCV Ab) were used. PCR for HCV RNA was carried out on all hemodialysis patient sera and in the RIBA II positive blood donor sera. The second generation ELISAs discriminated 11 more positive samples than the first generation test (2 IVDUs, 5 blood donors, 4 clinical samples). The 9 sera from blood donors and clinical samples were all RIBA II positive or indeterminate. The second generation tests thus showed increased sensitivity. The second generation tests also showed increased specificity in that 4 samples that were positive by test I but negative by the second generation tests, were also negative by RIBA II. With few exceptions, all RIBA II-positive and most of the indeterminate samples were positive by the second generation ELISAs. With few exceptions, all the RIBA II-negative samples were negative by the second generation ELISAs. Eleven blood donor sera were positive by test IV exclusively where RIBA II and other supplementary assays were negative. The recently introduced second generation anti-HCV ELISAs were found to have a higher sensitivity than the first generation test. The tests also showed a good concordance with the exception of test IV in the group of blood donor sera.     

Prevalence of false-positive hepatitis C antibody results,National Health and Nutrition Examination Study (NHANES) 2007–2012

BackgroundScreening large numbers of persons in a population with low prevalence of a disease leads to many false-positives. However, populations with low HCV prevalence may sometimes be recommended for HCV screening, for instance patients or healthcare workers after a possible healthcare-related exposure.ObjectivesWe determined the percentage of true vs false-positive HCV antibody (anti-HCV) test results among 2007–2012 participants in the National Health and Nutrition Examination Study (NHANES), a nationally representative study with approximately 1% HCV infection prevalence, much lower than in groups typically recommended for HCV screening.Study designAnti-HCV test confirmation was performed using a recombinant immunoblot assay (RIBA) test and follow-up HCV RNA testing.ResultsOverall, of 22,359 NHANES participants tested, 479 (2%) were anti-HCV screening reactive and 477 were tested for RIBA; of these 323 (68%) confirmed as true positive and 105 (22%) were false-positives. Many others (49, 10%) were RIBA indeterminate and likely false-positive. Because of these false positive tests, the overall prevalence of chronic infection among those testing anti-HCV screening reactive was much lower (218, 51%) than would be expected due to disease clearance alone (approximately 80%).ConclusionsAll screening anti-HCV positive tests should be followed by an HCV RNA test, in order to confirm whether the patient has current infection so that infected persons can be referred to care and treatment to avoid the significant morbidity and mortality associated with chronic HCV infection.     

Clinical evaluation of the signal-to-cutoff ratios of hepatitis C virus antibody screening tests used in China

The clinical diagnosis of hepatitis C virus (HCV) infection is important to direct an accurate course of therapy. Previous studies have reported a correlation between the signal-to-cutoff (S/CO) ratios of the anti-HCV screening test and confirm HCV infections for American anti-HCV screening kits as well as for those in China. It is currently unknown whether clinical laboratories use the same threshold S/CO ratios under routine conditions and if these values are acceptable for the analysis of Chinese samples. A total of 336 anti-HCV screening-test-positive serum samples were tested in duplicate using different lots of three most commonly used enzyme immunoassay (EIA) kits available in China. Samples were also tested using the Architect Anti-HCV chemiluminescent microparticle immunoassay (CMIA) kit and measured for HCV RNA. Recombinant immunoblot assays (RIBA) were additionally performed on samples with HCV RNA-negative results with RIBA HCV 3.0. The relationship between S/CO ratios and confirmed HCV infection rates were analyzed. The threshold S/CO ratio for each screening kit correlated with the ≥ 95% positive predictive value was InTec 12.0, KHB 4.0, Wantai 5.0, and Abbott Architect 5.0. Therefore, the same threshold S/CO ratios for manufactured domestically EIA kits was difficult to attain. A multi-center study with a large sample size is required to identify a uniform threshold S/CO ratio for use in different diagnostic laboratories. Alternatively, individual laboratories may be required to establish threshold S/CO ratios in their own laboratories to obtain consistent diagnostic results.     

Confirmation of second generation anti-hepatitis C virus enzyme immunoassays by antigenic cross-validation.

AIM: To determine if a scheme for validating enzyme immunoassay (EIA) results could be devised that did not require costly and methodically elaborate supplemental assays. METHODS: Samples (n = 525) from patients with haemophilia A, leukaemia, and chronic liver disease and at increased risk of hepatitis C virus infection were tested by EIA-1 (Ortho Diagnostics), an assay which uses recombinant HCV fusion proteins as antigens, and by EIA-2 (United Biomedical), an assay based on synthetic HCV oligopeptide antigens. RESULTS: Samples (n = 193) were repeatedly reactive in both EIAs. Of these, 190 (98%) yielded reactivities in both of two supplemental assays used, one an immunoblot assay (RIBA) using recombinant HCV polypeptides similar to EIA-1 antigens, and the other a neutralisation EIA (EIA-2N) based on antigenic competition with HCV peptides similar to EIA-2 antigens. The three samples not reactive in supplemental tests exhibited low EIA optical density (OD) values (signal/cutoff ratios of less than 3). Hence, all specimens reactive and yielding high OD values in both EIAs were also reactive in supplemental assays. Twenty four samples were reactive in EIA-1 only and nine (38%) of these were reactive in RIBA. Fourteen of the 15 (93%) specimens reactive in EIA-1 but not RIBA were derived from patients with chronic liver dysfunction. Two samples were reactive in EIA-2 only, of which one was reactive in EIA-2N and none in RIBA. CONCLUSIONS: Compared with EIA-2, EIA-1 yielded more validated reactive samples and resulted in more non-validated reactivities. It is therefore suggested that for clinical diagnosis: (i) EIA-1 be used for anti-HCV testing and EIA-2 for validation of EIA-1 reactivities; (ii) samples concordantly reactive in EIA-1 and EIA-2 and displaying high OD readings be considered HCV antibody positive without supplemental testing; (iii) supplemental testing by RIBA be limited to samples reactive in EIA-1 but equivocal or unreactive in EIA-2 and those concordantly reactive but exhibiting low absorbance readings.     

Role of signal-to-cutoff ratios in hepatitis C virus antibody detection

We reviewed results from 12,800 samples tested for hepatitis C virus (HCV) antibody detection in our laboratory by screening (Ortho chemiluminescence immunoassay [CIA]) and supplemental tests (Chiron recombinant immunoblot assay [RIBA]). We found that a signal-to-cutoff (S/Co) ratio of 10.3 was, in our setting, the most efficient cutoff point to improve the diagnostic algorithm of HCV infection.     

Coxsackie B virus infections in women with miscarriage

Serum samples from 1,765 consecutive Sardinian blood donors, negative for hepatitis B surface antigen (HBsAg) and for antibodies to human immunodeficiency virus (HIV) (anti-HIV), were evaluated for the presence of antibodies to hepatitis C virus (anti-HCV) by second-generation ELISA. Anti-HCV was detected in 25 (1.45%) of the 1,765 donors examined. Anti-HCV was found in 15 of the 1,690 (0.9%) donors with normal alanine aminotransferase (ALT) and in 10 of the 75 (13%) donors with elevated ALT (P < 0.0001). Of the 15 anti-HCV-positive donors with normal ALT, only five (33%) were confirmed to be positive by second-generation RIBA, six (40%) were indeterminate, while four (27%) were RIBA negative. HCV RNA, as detected by polymerase chain reaction (PCR) using a set of primers from the 5′-noncoding region, was found in six of the 15 (40%) donors with normal ALT, including five RIBA, positive and one indeterminant. Of the 10 anti-HCV-positive donors with elevated ALT, all were RIBA positive and eight (80%) had detectable HCV RNA. Thus, among ELISA-reactive donors, those with elevated ALT had a significantly higher probability of being positive for secondgeneration RIBA and HCV RNA compared to those with normal ALT levels (P = 0.028). None of the 65 donors with elevated ALT but negative for anti-HCV by ELISA had detectable serum HCV RNA, as compared to eight of 10 anti-HCV ELISApositive donors (P < 0.0001). However, although negative for HBsAg, 12 of the 65 (18%) had serum HBV DNA by PCR. This study demonstrates that the combined use of second-generation ELISA and RIBA anti-HCV assays is highly effective in identifying HCV infection, whereas the specificity of ELISA alone for the screening of blood donors with normal ALT values appears to be limited. In contrast, in donors with elevated ALT levels, there is a positive correlation between second-generation assays (ELISA and RIBA) and HCV viremia. The high proportion of inapparent HBV infection in blood donors with elevated ALT levels underlines the importance of this test for the prevention of transfusion-associated viral hepatitis.     

Improved diagnosis of chronic hepatitis C virus infection by detection of antibody to multiple epitopes: confirmation by antibody to synthetic oligopeptides.

Serum samples from 226 patients covering a wide spectrum of liver disease were tested for antibodies to hepatitis C virus (HCV) using both first and second generation enzyme linked immunosorbent assays. Selected sera were also tested by peptide immunoassays, by the four-antigen recombinant immunoblot assay (RIBA II), and for viral genome by the polymerase chain reaction. Antibody to c100-3 was detected in 61% of patients with chronic non-A, non-B (NANB) hepatitis and/or 46.5% with presumed NANB-related cirrhosis by the first generation test. These figures increased to 77% and 58% when antibodies to recombinant structural and non-structural HCV antigens were sought by the second generation assay. Supplemental testing against peptide Sp75 and Sp65/sp67 confirmed that reactivity of sera by second generation assays was due to antibodies to the additional structural and non-structural antigens. Samples negative by the first generation assay were not confirmed by the supplemental assay using peptides Sp75 and Sp65/Sp67. HCV RNA was detected in 60% of the anti-HCV positive sera tested, most of which were also RIBA II positive. Our findings confirm that the introduction of the structural and non-structural antigens, especially the putative nucleocapsid protein, improves sensitivity of detection of antibodies to HCV, and facilitates diagnosis in patients with "cryptogenic" chronic hepatitis.