Voluntary non-remunerated repeat blood donors are perceived to be safer than the first time blood donors. This study was planned for follow-up of previous hepatitis C virus (HCV) test results of anti-HCV enzyme-linked immunosorbent assay (ELISA) reactive repeat blood donors. The aim was to suggest a protocol for re-entry of the blood donors who are confirmed HCV negative by nucleic acid test (NAT) and recombinant immunoblot assay (RIBA). A group of repeat voluntary donors were followed retrospectively who became reactive on a cross sectional study and showed HCV reactivity while donating blood regularly.
Material and Methods:
A total of 51,023 voluntary non remunerated blood donors were screened for anti-HCV ELISA routinely. If anybody showed positivity, they were tested by two ELISA kits (screening and confirmatory) and then confirmed infection status by NAT and or RIBA. The previous HCV test results of repeat donors reactive by anti-HCV ELISA were looked back from the records. Data of donors who were repeat reactive with single ELISA kit (in the present study) were analyzed separately from those reactive with two ELISA kits (in the present study).
In this study, 140 (0.27%) donors who were reactive by anti HCV ELISA were included. Out of them, 35 were repeat voluntary donors and 16 (11.43%) were reactive with single ELISA kit. All 16 donors were reactive by single ELISA kit occasionally in previous donations. Their present ELISA positive donations were negative for HCV NAT and RIBA. A total of 19 (13.57%) donors were reactive with two ELISA kits. In their previous donations, the donors who were reactive even once with two ELISA kits were consistently reactive by the same two ELISA kits in their next donations also.
Donor sample reactive by only single ELISA kit may not be considered as infectious for disposal as they were negative by NAT and or RIBA. One time ELISA positivity was found probably due to ELISA kit specificity and sensitivity. Donors reactive with two ELISA kit should be discarded as there is a high positivity with NAT/ RIBA. However, donors reactive by two ELISA kits and negative by NAT and RIBA should be followed up and may not be deferred permanently.
Anti-HCV ELISA; repeat voluntary blood donor; occult infections; donor follow-up; nucleic acid test; recombinant immunoblot assay
The specificities of four assays for hepatitis C virus (HCV) were compared by using units from volunteer blood donors. Upon Food and Drug Administration licensure of the first immunoassay for anti-HCV, EIA-1, units previously deemed acceptable for transfusion and all subsequent blood donations were screened. EIA-1 repeat-reactive (RR) units were tested for HCV by a second-generation enzyme-linked immunoassay (EIA-2) and by a four-antigen recombinant immunoblot assay (RIBA II) and for HCV RNA by reverse transcriptase polymerase chain reaction. All HCV RNA-positive samples were reactive by both RIBA II and EIA-2. All RIBA II-reactive sera were EIA-2 RR. In EIA-1, 0.45% of the prescreened units and 0.59% of the prospectively screened donors were RR. Of these, 52.5 and 54%, respectively, were EIA-2 RR, 71.4 and 69% of the EIA-2 RR units were reactive on RIBA II, and 93 and 88% of the RIBA II-reactive samples were HCV RNA positive. When the sample/cutoff ratio for EIA-2 was greater than 5, 91% of the samples were RIBA II reactive and 82% of the samples were HCV RNA positive. None of EIA-2 RR units with a sample/cutoff ratio of < 5 was RIBA II reactive or HCV RNA positive. In conclusion, RIBA II and RT PCR results are highly concordant. A sample/cutoff ratio of > 5 in EIA-2 is a good discriminator for the likelihood of a true HCV infection on the basis of RT PCR and RIBA II assays.
The interpretation of “indeterminate” results of the recombinant immunoblot assay (RIBA) is a particularly sensitive issue for Transfusion Services, and donors with such a serological condition require long-term follow-up.
Materials and methods
In the Immunohaematology and Transfusion Medicine Division of Umberto I University Hospital (Rome, Italy), 102,979 donor blood units were screened for hepatitis C virus (HCV) antibodies by enzyme-linked immunosorbent assay (ELISA) over a 5-year period (01.01.2000 – 31.12.2004). Since 24.10.2001, HCV -RNA testing was added. All samples repeatedly reactive by ELISA were then submitted to a HCV confirmatory assay (RIBA).
Among the 102,979 donors we found 271 positive to HCV ELISA testing. The results of the RIBA assay for these donors were negative in 178 (65.7%) cases, positive in 28 (10.3%) and indeterminate in 65 (24.0 %).
Of the 65 subjects with an indeterminate pattern, 24 completed a sufficient follow-up (median 25 months; range, 6 – 52), during which some (n=8; 33%) converted to a negative status, some (n=16; 67%) maintained their reactivity pattern, but none became seropositive for HCV.
The HCV-RIBA indeterminate status may indicate either a non-specific reaction (false positive) or a real pre-existing or initial infection and does not, therefore, enable a prediction of outcome. The use of HCV genomic assays (nucleic acid amplification testing), which are more specific than antibody-based assays (ELISA, RIBA), therefore improves HCV blood donor testing by allowing an accurate interpretation of such primary assays.
hepatitis C virus (HCV); HCV-RIBA indeterminate; blood donors
A solid phase recombinant-immunoblot-assay(RIBA) is often used to determine the specificity of antibody to hepatitis-C-virus(anti-HCV). The RIBA result is recorded as positive, negative or indeterminate. The interpretation of RIBA indeterminate reactivity and its significance to patients and blood donors are unclear. We attempted to address the clinical relevance of RIBA-indeterminate reactions in the context of the natural history of HCV infection in a prospectively followed cohort of anti-HCV positive blood donors.
STUDY-DESIGN AND METHODS
Donor demographics, HCV exposure history, humoral and cell-mediated immunity(CMI) to HCV were compared in 15 RIBA-indeterminates, 9 chronic-HCV-carriers and 8 spontaneously-recovered subjects. Serum samples were tested for the presence of anti-HCV by a liquid phase Luciferase-Immunoprecipitation-System(LIPS) assay. CMI was assessed by IFN-γ-ELISpot assay.
In the quantitative LIPS assay, the sum of antibody responses to 6 HCV-antigens showed significant (p<0.001) step-wise diminution progressing downward from chronic-carriers to spontaneously-recovered to RIBA-indeterminates. CMI responses in RIBA-indeterminates were similar to spontaneously-recovered subjects, and greater than chronic-carriers and negative controls (p<0.008). A parenteral risk factor was identified in 13% of RIBA-indeterminates as compared with 89% of chronic-carriers and 87% of spontaneously-recovered subjects. On average, donors in the RIBA-indeterminate group were older than the other groups.
The combined CMI and LIPS results suggest that persistent RIBA-indeterminate reactions generally represent waning anti-HCV responses in persons who have recovered from a remote HCV infection. In such cases, detectable antibody may ultimately disappear leaving no residual serologic evidence of prior HCV infection, as previously reported in a minority of long-term HCV-recovered subjects.
HCV; RIBA indeterminate; HCV infection spontaneously recovered; Chronic HCV infection; RIBA 3.0; Cell-mediated immunity; IFNγ; Luciferase immunoprecipitation system (LIPS) assay
A retrospective study was carried out to assess the performance of hepatitis C diagnostic assays in our laboratory, and to determine the prevalence of hepatitis C among blood donors at the Sultan Qaboos University Hospital.
From 1991 to 2001, approximately 55,000 serum samples collected from blood donors and patients were submitted to our laboratory for testing. All sera were screened for antibodies to hepatitis C virus (HCV) by three successive generations of the enzyme-linked immunosorbent assay (ELISA). Anti-HCV positive sera were further tested by recombinant immunoblot assay (RIBA). Reverse-transcriptase polymerase chain reaction (RT-PCR) for HCV RNA was carried out on a limited number (241) of ELISA positive samples.
Out of 30012 samples from blood donors that were screened for anti-HCV, 272 (0.91%) were positive. Of these, 46.5% were confirmed positive by RIBA. The proportion of patient sera that were confirmed positive varied from 95% among intravenous drug users to 81% in patients with hepatitis to 70% in those with haemoglobinopathies. HCV RNA was detected in 67%, 6%, and 0% of the RIBA positive, indeterminate and negative samples respectively.
Based on RIBA, the prevalence of anti-HCV among blood donors in Oman is close to 0.5%. In our experience, RIBA-positivity is predictive of HCV infection in two thirds of subjects, and HCV infection is highly unlikely in those who are RIBA-negative. The experience at SQUH with three types of HCV assays has enabled the laboratory to develop a test algorithm, starting with screening anti-HCV ELISA.
hepatitis C virus; ELISA; RIBA; polymerase chain reaction
The significance of seroconversion as detected by an ELISA screening test for hepatitis C virus (HCV) antibody with a negative supplemental/confirmatory recombinant immunoblot assay (RIBA) result was investigated. Of 118,220 established West Midlands blood donors with at least one negative HCV antibody screen, 43 had seroconverted in 1994 according to the ELISA but had negative RIBA-3 results. The paired archive serum samples of the pre- and postseroconversion donations of 29 seroconverting donors were tested by nested polymerase chain reaction (PCR) for the detection of HCV RNA. All 58 samples were negative by PCR. The absence of detectable viraemia in all tested seroconverting donors suggests that HCV infection was not responsible for seroconversion by ELISA.
In a study designed to assess the prevalence of antibodies to hepatitis C virus (HCV) in pregnant women, anti-HCV positivity in French pregnant women was twice as high as that found in French blood donors. Positive ELISA 2 results were confirmed by positive RIBA 2 in most subjects, and seven of nine RIBA 2 positive patients also tested positive for HCV-RNA by PCR. High rates of anti-HCV positivity were seen among immigrant pregnant women, partly because of false positive results with ELISA 2. RIBA 2 results suggested that the prevalence of HCV infection was not any higher in immigrant compared with French pregnant women.
Samples reactive by first-generation recombinant immunoblot assay (RIBA) to detect antibody to hepatitis C virus (anti-HCV) (RIBA-1 [Chiron, Calif.]) remained reactive by a second-generation test (RIBA-2) for HCV antibodies. A total of 75% of specimens indeterminate by RIBA-1 became reactive, 12.5% were nonreactive, and 12.5% remained indeterminate by RIBA-2. Among RIBA-1-nonreactive specimens, 12.0% became positive and 5.1% became indeterminate by RIBA-2. The antigens c33c and c22-3 have increased the sensitivity of RIBA-2.
Hepatitis C virus (HCV) antibodies were detected in 85.9% of the samples by commercial enzyme immunoassay (EIA) kits. Most EIA-positive samples were reactive (80%) by the RIBA HCV test (Ortho Diagnostics). Samples with optical density values greater than or equal to 2.0 were mostly reactive (87%) by RIBA HCV test, in contrast to those with values less than or equal to 1.0 (6.6%). Samples which were indeterminate by the RIBA HCV test were positive (88.4%) by HCV neutralization EIA (Abbott Laboratories), along with 29.4% of samples which were nonreactive by the RIBA HCV test.
The performance of a new automated analyzer for the processing and interpretation of the RIBA Strip Immunoblot Assay (SIA), used in the diagnosis of hepatitis C virus (HCV) infection, was evaluated. Laboratory performance of the RIBA SIA was compared with that of two manually processed supplementary anti-HCV tests (RIBA HCV 3.0 SIA and INNO-LIA HCV Antibody III). Specificity of the automated processing of SIA was 100% for 90 selected anti-HCV-negative samples. On the other hand, 119 of 120 (99.2%) previously confirmed anti-HCV-positive samples were also positive when assayed on the automated processor. Results for all specimens except one (51 of 52) were concordant for manual and automated RIBA, while 15 of 68 sera tested with automated RIBA and the INNO-LIA assay showed different patterns of reactivity. Three HCV sensitivity panels and one seroconversion panel were also compared. The results show a high sensitivity for SIA NS3- and NS5-encoded antigens. Moreover, data obtained for the anti-HCV seroconversion panel and for samples with borderline or discordant anti-HCV enzyme-linked immunosorbent assay results suggest that bands with a relative intensity of >0.5 on the automated analyzer (theoretically negative) should be evaluated with care. Coefficients of variability ranged from 9 to 14.8% in an interassay reproducibility study. Overall, the performance of the automated analysis of SIA is comparable to that of the manual RIBA assay. The new automated processor for SIA bands proved to be sensitive and specific. Its use makes the optical scoring of bands unnecessary by indicating relative intensity values, which could be particularly useful in the follow-up care of anti-HCV-positive patients receiving antiviral therapy.
Surveillance for hepatitis C virus (HCV) is limited by the challenge of differentiating between acute and chronic infections. In this study, we evaluate a cross-sectional testing strategy that identifies individuals with acute HCV infection and we estimate HCV incidence. Anti-HCV-negative persons from four populations with various risks, i.e., blood donors, Veterans Administration (VA) patients, young injection drug users (IDU), and older IDU, were screened for HCV RNA by minipool or individual sample nucleic acid testing (NAT). The number of detected viremic seronegative infections was combined with the duration of the preseroconversion NAT-positive window period (derived from analysis of frequent serial samples from plasma donors followed from NAT detection to seroconversion) to estimate annual HCV incidence rates. Projected incidence rates were compared to observed incidence rates. Projected HCV incidence rates per 100 person-years were 0.0042 (95% confidence interval [95% CI], 0.0025 to 0.007) for blood donors, 0.86 (95% CI, 0.02 to 0.71) for VA patients, 39.8 (95% CI, 25.9 to 53.7) for young IDU, and 53.7 (95% CI, 23.4 to 108.8) for older IDU. Projected rates were most similar to observed incidence rates for young IDU (33.4; 95% CI, 28.0 to 39.9). This study demonstrates the value of applying a cross-sectional screening strategy to detect acute HCV infections and to estimate HCV incidence.
A comparison between the CHIRON RIBA hepatitis C virus (HCV) processor and manual systems was performed by using 88 specimens repeatedly reactive by the second-generation HCV enzyme-linked immunosorbent assay (ELISA) (HCV 2.0 ELISA) and 111 random specimens from volunteer donors. For the second-generation RIBA HCV strip immunoblot assay (SIA) (RIBA HCV 2.0 SIA), test results correlated strongly between the manual and the automated runs (kappa value, 0.937). For the RIBA HCV 3.0 SIA, the correlation of the test results was also high (kappa value, 0.899). Among the specimens with positive results by RIBA HCV 2.0 and 3.0 SIAs, there was a very strong concordance of the test results between the manual and the automated runs with regard to the reactive bands. Nine samples had discordant results between the manual and the automated runs; this was probably attributable to increased variability in antigen scores close to the cutoff values for both tests. Run-to-run and within-run testing by the CHIRON RIBA HCV Processor System showed a very low rate of conflicting values. In conclusion, the CHIRON RIBA HCV Processor System is capable of performing RIBA HCV 2.0 and 3.0 SIAs accurately with minimal operator involvement. In addition, the CHIRON RIBA HCV Processor System shows excellent reproducibility, with the potential for operator-to-operator and site-to-site variability being greatly reduced. Our data indicate that this novel methodology may be very useful for supplemental anti-HCV testing of specimens repeatedly reactive by ELISA in routine clinical assessments and epidemiologic evaluations.
Until lately, anti-HBc antibodies were considered an effective marker for occult Hepatitis B virus (HBV) infection and have served their role in improving blood safety. But, with the development of advanced tests for HBV DNA detection, the role of anti-HBc in this regard stands uncertain.
Materials and Methods:
Anti-HBc and HBsAg ELISA and ID-NAT tests were run in parallel on donor blood samples between April 1, 2006 and December 31, 2010 at the Department of Transfusion Medicine, Indraprastha Apollo Hospitals, New Delhi. A positive ID-NAT was followed by Discriminatory NAT assay.
A total of 94 247 samples were tested with a total core positivity rate of 10.22%. We identified nearly 9.17% of donors who were reactive for anti-HBc and negative for HBsAg and HBV DNA. These are the donors who are potentially non-infectious and may be returned to the donor pool.
Although anti HBc testing has a definite role in improving blood safety, centers that have incorporated NAT testing may not derive any additional benefit by performing anti-HBc testing, especially in resource-limited countries like ours.
Anti-HBc; HBsAg; ID-NAT
BACKGROUND: A viral cause of idiopathic pulmonary fibrosis (IPF) was recently suggested by a Japanese study in which a high prevalence of anti-hepatitis C virus (HCV) antibodies was detected. A subsequent British study failed to confirm these results. METHODS: Antibodies to HCV were evaluated in 60 patients with IPF, 130 patients with non-interstitial lung disease, and in 4614 blood donors. HCV-RNA and HCV genotypes were evaluated in the anti-HCV positive patients with IPF. Anti-HCV antibodies were evaluated by ELISA and confirmed by recombinant immunoblotting assay (RIBA). HCV-RNA and genotypes were detected by reverse transcriptase polymerase chain reaction (PCR). RESULTS: Eight patients with IPF had anti-HCV antibodies detected by ELISA (13.3%). In the blood donor control group the prevalence of HCV antibodies was lower (0.3%). In patients with non-interstitial lung disease HCV antibody prevalence was 6.1%. In all eight patients with IPF found to be anti-HCV positive by ELISA, HCV antibodies were also detected by RIBA. Furthermore, all were HCV-RNA positive by PCR assay. HCV genotypes were identified in four of these eight patients. In all four genotype II was present and in two it was associated with genotype III and/or genotype IV. In the remaining four cases the genotype was not identified. CONCLUSION: Italian patients with IPF show an increased prevalence (approximately 13%) of HCV infection and viral replication, but the prevalence of anti-HCV antibodies does not differ from other lung diseases.
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.
In a study using the current first generation Ortho ELISA, a second generation Ortho ELISA, and the RIBA HCV strip immunoblot assay, all patients who were strongly positive for antihepatitis C virus (HCV) on ELISAs (OD490 > 3) were reactive to RIBA for multiple bands. While all ELISA false positive samples had low or intermediate OD490 values, RIBA confirmed HCV reactivity in 50% of the patients reactive to ELISA with a low suspicion of HCV infection, thus suggesting that RIBA HCV strip immunoblot assay will be most useful for patients who react weakly positive or intermediate to ELISA.
The prevalence of antibodies against hepatitis C virus (HCV) was studied in hemophiliacs, hemodialysis patients, intravenous drug abusers, female prisoners, homosexuals, individuals with no markers of recent hepatitis A or B virus infections and normal individuals (federal public servants), by an enzyme immunoassay (Ortho Diagnostic Systems Inc). Repeat positive samples were further tested by recombinant immunoblot assay (riba) HCV (Chiron Corp, California). The number of samples positive for antibodies to HCV (anti-HCV) was higher with enzyme immunoassay than by riba HCV in most cases. A high prevalence of anti-HCV was detected in hemophiliacs by both enzyme immunoassay (68.8%) and riba HCV (53.7%). Among intravenous drug abusers and female prisoners the prevalence rates for anti-HCV were 42.8% and 29.8%, respectively, by riba HCV; the results with enzyme immunoassay were only slightly higher. The prevalence rate was also high by both tests (54.2%) in hemodialysis patients’ sera taken during 1980–82, when many cases of non-A,non-B hepatitis were suspected in this group. In contrast, only 14.1% of sera taken during 1990 were positive by riba HCV. In individuals with no markers of recent hepatitis A or B infections, 13.4% were positive by enzyme immunoassay, whereas only 4.5% were reactive by riba HCV. The lowest prevalence was seen in homosexuals (2.3%) and normal individuals (1.2%) by riba HCV. These results indicate a high prevalence of anti-HCV in high risk groups tested in Canada.
Enzyme immunoassay; Hepatitis C virus; Non-A,non-B hepatitis
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.
The diagnostic performances of three commercially available recombinant immunoblot assays (RIBAs) for anti-hepatitis C virus antibody were evaluated on 50 ORTHO-HCV RIBA-2 (RIBA-2)-indeterminate serum samples. Concordant interpretations were obtained with the three tests in 60% of the samples, with 56% positive, 2% indeterminate, and 2% negative results. Considering test performance in regard to the number of remaining indeterminate results, analyzing sera by RIBA-3, INNO-LIA HCV Ab III, and DECISCAN HCV reduced the number of samples reacting indeterminately to 40, 6, and 8%, respectively. The three serum samples classified as indeterminate in the INNO-LIA HCV Ab III as well as three of four serum samples interpreted as indeterminate in the DECISCAN HCV and 16 of 20 samples classified as indeterminate in the RIBA-3 were hepatitis C virus RNA positive by PCR. This study clearly shows the good performance of the three tests as confirmatory assays compared with that of the RIBA-2. However, according to the manufacturers' criteria of positivity, the INNO-LIA HCV Ab III and DECISCAN HCV appeared to be more suitable than the RIBA-3 for interpreting serum samples found indeterminate in the RIBA-2.
The clinical significance of specimens with low sample-to-cutoff (S/Co) ratios in the Ortho VITROS chemiluminescence assay (CIA) for detection of antibodies to hepatitis C virus (HCV) was evaluated. In one study of 482 CIA-reactive samples, none of the 83 samples with S/Co ratios of <5 was HCV RNA positive. In a subsequent study, 332 samples with S/Co ratios of between 1 and 20 were tested with the recombinant immunoblot assay (RIBA). None of the 163 samples with S/Co ratios of <5 was RIBA positive, 83% were RIBA negative, and 28 samples (18%) were RIBA indeterminate. HCV RNA and/or clinical evidence of hepatitis was not found in the 27 indeterminate cases examined. These results show that over 99% of samples with very low S/Co ratios (≤5) have no evidence of HCV infection. Therefore, we suggest that the HCV antibody testing algorithm for the VITROS assay might be modified to eliminate supplemental testing of samples with very low S/Co ratios.
Plasma samples from 1,182 patients undergoing primary liver transplantation were tested for anti-hepatitis C (HCV) virus by two methods: Ortho HCV ELISA Test System (EIA) and Chiron RIBA HCV Test System (RIBA II). The EIA results, 0 or +, were recorded first, followed by RIBA results, N = negative, P = positive, or I = indeterminate. Concordant results—0N, + P, + I—were found in 1,076 (91%), and discordant results were found in 106 (9%). The EIA optical density did not relate to concordant or discordant results. Band patterns were described by stating the band position (1, 2, 3, or 4) and insetting a dash (−) if no band was visualized. Most + P samples fell into two patterns: 47% showed all four bands, pattern 1234, and 15% showed the two-band pattern, 34. When the EIA was negative, 0P, the opposite was seen: 8% showed the 1234 pattern and 81% showed the 34 pattern. There were 226 samples that formed bands (+ P, 149; 0P, 31; + I, 15; 0I, 31). The frequency of bands was as follows: 4, 32%; 3, 31%; 2, 19%; and 1, 18%. Band 2 and the EIA test detected antibodies to the same c100-3 fragment and showed 74% concordance. No explanation is apparent for the lower concordance rate here than that between the EIA test and bands 3 = 96% or 4 = 88%. The EIA and RIBA II tests, together with positive liver function tests and abnormal tissue pathologic findings, provide a basis for the diagnosis of HCV.
Hepatitis C; Non-A,non-B hepatitis; Anti-HCV; RIBA II band patterns
Background.A total of 738 volunteer blood donors who were positive for anti–hepatitis C virus (HCV) were assessed for risk factors and outcomes for up to 15 years within the study and up to 54 years from the estimated onset of infection.
Methods.A third-generation recombinant immunoblot assay (RIBA) was performed to distinguish true from false anti-HCV reactivity. Findings of HCV polymerase chain reaction classified subjects as having chronic HCV infection or as having recovered. Liver biopsy specimens were staged by Ishak fibrosis score and graded by histologic activity index.
Results.Of 738 anti-HCV–positive subjects, 469 (64%) had positive RIBA results, 217 (29%) had negative results, and 52 (7%) had indeterminate results. Primary independent risk factors were injection drug use (odds ratio [OR], 35.0; P < .0001), blood transfusion (OR, 9.9; P < .0001), and intranasal cocaine use, including 79 “snorters” who repeatedly denied injection drug use or blood transfusion (OR, 8.5; P < .0001). Classification and regression tree and random forest analyses confirmed these risk factors. A total of 384 RIBA-positive donors (82%) were HCV RNA positive; of these, liver biopsy specimens from 185 (48%) showed no fibrosis in 33%, mild fibrosis in 52%, bridging fibrosis in 12%, and cirrhosis in 2% a mean duration of 25 years after infection. Analysis of 63 repeat biopsy specimens showed that 8% progressed ≥2 Ishak stages over 5 years (mean progression, 0.06 Ishak stages/year).
Conclusions.Injection drug use and blood transfusion before 1990 are dominant risk factors for HCV acquisition; intranasal cocaine use may be a surreptitious route of parenteral spread. After a mean of 25 years of HCV infection, histologic outcomes were relatively mild: 85% had no or mild fibrosis, and only 2% had cirrhosis. Nearly one-fifth spontaneously recovered.
One hundred and four clinical specimens from provincial public health laboratories were tested for antibody to hepatitis C virus (HCV) envelope protein (anti-E2). To evaluate the effect of hypervariability of E2 region on anti-E2 assay, 49 recombinant immunoblot assay (RIBA) 3.0 positive samples were genotyped. All 49 genotyped samples were positive for anti-E2. Eight of 12 (67%) indeterminate, HCV RNA positive samples were anti-E2 reactive. Nine of 30 (30%) indeterminate, HCV RNA negative samples were also positive for anti-E2. Anti-E2 was detected in two of 13 (15%) RIBA-negative and enzyme immunoassays-positive samples. Although small number of samples were tested, the results showed that it may be possible to resolve indeterminate samples with the anti-E2 assay.
Core antigen (C22p); Envelope antigen (E2); Hepatitis virus C; Recombinant immunoblot assay (RIBA)
The specificity of first-generation enzyme-linked immunosorbent assays (ELIAs) for antibody detection in individuals with hepatitis C virus (HCV) infection has been questioned in some pathological situations. We observed a surprisingly high prevalence of anti-HCV antibodies in alcoholic patients, and thus, false-positive reactions in anti-HCV tests were strongly suspected. The introduction of new epitopes, particularly a core protein, C22 (second-generation tests), seems to increase the sensitivity of anti-HCV detection. In order to study the specificity of the second-generation tests, 60 serum samples from alcoholic patients found to be positive by the first-generation anti-HCV ELISA (Ortho) were reexamined by a second-generation anti-HCV enzyme immunoassay (Abbott) and a recombinant immunoblot assay (RIBA II; Chiron). Fifteen serum samples gave contradictory results when they were tested by the two assays. We performed nested polymerase chain reactions (PCRs) to confirm that the discrepancies that we observed could be due to the presence of low levels of anti-HCV antibodies, which were detected by a more sensitive test, or to unspecific positive reactions. Nested PCR revealed the presence of HCV RNA sequences in all anti-HCV-positive sera or sera that were weakly positive by ELISA. Anti-HCV positive by RIBA II was always correlated with the presence of viral RNA in serum, but HCV RNA was detected in RIBA II-negative sera. These results indicate that the specificity of the second-generation tests is an important improvement but that an HCV infection can still persist without detectable antibodies. PCR remains the reference assay to clear up controversial serology results and to detect HCV infection in patients with no anti-HCV-detectable immune response.
To evaluate whether a new enzyme immunoassay developed for the simultaneous detection of hepatitis C virus (HCV) core antigen (Ag) and anti-HCV antibodies (anti-HCV Ab) (Monolisa HCV Ag/Ab ULTRA; Bio-Rad) could improve the early detection of HCV infection, we compared its sensitivity to that of anti-HCV, HCV core Ag, and HCV RNA assays. The populations studied included 12 blood donor samples positive for HCV RNA and HCV core Ag but negative for anti-HCV antibodies and 23 hemodialysis patients who developed anti-HCV Ab (seroconversion) during the follow-up. From these 23 individuals, 83 samples sequentially collected prior to seroconversion and 108 samples collected after seroconversion were tested. Six of 12 blood donations were positive by the HCV Ag/Ab assay. In the hemodialysis cohort, the 24 HCV RNA-negative samples were negative by the HCV Ag/Ab assay and 23 of the 59 HCV RNA-positive samples (39%) were positive. The HCV Ag/Ab assay detected HCV infection on average 21.6 days before the most sensitive antibody assay. The HCV Ag/Ab assay did not detect HCV infection as early as the HCV RNA assay (mean delay, 30.3 days) or HCV Ag assay (mean delays, 27.9, and 16.3 days by the HCV core Ag quantification assay and the HCV Ag blood screening assay, respectively). This new assay provides a notable improvement for the early detection of HCV infection during the so-called window period compared with anti-HCV Ab assays and could be a useful alternative to HCV RNA detection or HCV core Ag assays for diagnosis or blood screening when nucleic acid technologies or HCV core Ag detection are not implemented.