Recombinant Immunoblot Assay Results Research Articles

The diagnostic significance of hepatitis C virus antibody levels for chronic hepatitis C virus infection.

Although anti-hepatitis C virus (HCV) assay is widely used to screen for HCV infection, it has a high false-positive (FP) rate in low-risk populations. We investigated the accuracy of anti-HCV signal-to-cutoff (S/CO) ratio to distinguish true-positive (TP) from FP HCV infection. We retrospectively analyzed 77,571 patients with anti-HCV results. A receiver operating characteristic (ROC) curve analysis was performed to evaluate the diagnostic accuracy of anti-HCV S/CO ratio in anti-HCV positive patients. Overall, 1,126 patients tested anti-HCV positive; 34.7% of patients were FP based on HCV RNA and/or recombinant immunoblot assay (RIBA) results. The age and sex-adjusted anti-HCV prevalence was 1.22%. We identified significant differences in serum aspartate transaminase and alanine transaminase levels, anti-HCV S/CO ratio, and RIBA results between groups (viremia vs. non-viremia, TP vs. FP). Using ROC curves, the optimal cutoff values of anti-HCV S/CO ratio for HCV viremia and TP were 8 and 5, respectively. The area under the ROC curve, sensitivity, specificity, positive and negative predictive values were 0.970 (95% CI, 0.959-0.982, p < 0.001), 99.7%, 87.5%, 87.4%, and 99.7%, respectively, for predicting HCV viremia at an anti-HCV S/CO ratio of 8 and 0.987 (95% CI, 0.980-0.994, p < 0.001), 95.3%, 94.7%, 97.1%, and 91.4%, respectively, for TP HCV infection at an anti-HCV S/CO ratio of 5. No patients with HCV viremia had an anti-HCV S/CO ratio below 5. The anti-HCV S/CO ratio is highly accurate for discriminating TP from FP HCV infection and should be considered when diagnosing HCV infections.

HCV genotype 6 prevalence, spontaneous clearance and diversity among elderly members of the Li ethnic minority in Baisha County, China.

The epidemiology of hepatitis C virus varies widely across geographical regions and ethnic groups. Our previous study showed that 6 strains isolated from Baisha County, Hainan Island, China, were all new genotype 6 (gt6) subtypes which differed significantly from subtypes of other regions. In the current study, we conducted a comprehensive epidemiological survey of HCV in the Li ethnic group, native to Baisha County. Anti-HCV antibodies were detected by 2 independent ELISAs in all participants, and positive results confirmed by the recombinant immunoblot assay (RIBA) and HCV RNA viral loads were measured. Univariate chi-square test and multivariable logistic regression analyses were used to determine the risk factors for HCV infection and spontaneous clearance rates. Indeterminate RIBA results were excluded or included in analyses; consequently, findings were expressed as a range. Direct sequencing of partial regions within NS5B and E1 was employed for genotyping. Among 1682 participants, 117 to 153 were anti-HCV positive (7.0%-9.1%), with 42.7%-52.6% confirmed to have cleared infection. Anti-HCV positivity was associated with older age (≥60years) (OR=0.02, 95% CI 0.01-0.05, P<0.01) and surgery (OR=2.75, 95% CI 1.36-5.57, P<0.01), with no significant difference found between the HCV infection group and the HCV spontaneous clearance group. The gt6 subtype distribution characteristics of Baisha County were unique, complex and diverse. The sequences did not cluster with known gt6 subtypes but formed 4 Baisha community-specific groups. HCV infection in members of the Li minority ethnic group is characterized by high prevalence rates in the elderly, high spontaneous clearance rates and broad gt6 diversity.

Characterization of differential antibody production against hepatitis C virus in different HCV infection status.

BackgroundThe Centers for Disease Control and Prevention (CDC) issued an update on hepatitis C virus (HCV) testing approach, in which it omitted the use of recombinant immunoblot assay (RIBA) in the diagnostic algorithm and recommended that future studies are needed to evaluate the performance of HCV testing without RIBA. As Egypt has the highest prevalence of HCV worldwide, we aimed to evaluate the value of RIBA in HCV testing in a high prevalence population. Our objective was to clarify whether enzyme linked immunosorbent assay (ELISA) anti-HCV signal-to-cutoff (S/CO) ratios were able to discriminate true positive from false positive anti-HCV antibody status and to evaluate the role of RIBA in solving this problem which may lead to a redefined strategy for diagnosis of HCV infection. Our second objective was to elucidate the effects of different HCV peptides of both structural and non-structural proteins on the humoral immune response to HCV infection. MethodsThe current study drew results from 167 individuals divided into three groups: Group I: included 77 HCV antibody positive (ELISA) high risk health care workers (HCW), Group II: included 56 presumably uninfected individuals who showed normal liver enzymes, negative HCV RNA and were asymptomatic. Their ELISA HCV antibody S/C ratio ranged from 0.9 to <5. Group III: included 34 patients enrolled from outpatient clinics of Ain Shams Hospital with persistent viral replication, elevated liver enzymes, and chronic HCV related liver disease. All study participants were assessed for the presence of anti-HCV antibodies by 3rd generation ELISA which was confirmed by RIBA.ResultsInterpreting the results of both ELISA and RIBA together, false positive results were highly significantly increased in HCW when compared with the other two groups. Indeterminate and false negative results were only found in the presumably uninfected group. For differentiated antibody responses by RIBA, chronic HCV cases had the highest frequency of positive antibody response to core peptides while the presumably uninfected group had the lowest. Antibody response to E2 was found less frequently in chronic cases than Core 1, Core 2 and NS3. The specific antibody response to the different HCV peptides showed the same distribution of frequencies in both chronic HCV cases and the presumably uninfected individuals with the chronic cases having the highest frequencies. This distribution was different from the HCW. The most evident difference was the reaction towards NS3 which was the highest antibody producing peptide in chronic HCV and presumably uninfected individuals whereas in HCW Core1 was the highest.ConclusionThe HCV antibody immunoblot assay (RIBA) is still necessary for the detection of false positive cases which can occur quite frequently in countries of high prevalence as Egypt. Indeterminate RIBA results indicate a waning antibody response in elderly individuals who recovered from previous or distant HCV infection.

Correlation Analysis Between Protein Phosphatase-1 Regulatory Inhibitor Subunit-11 Polymorphism and Hepatitis C Virus Spontaneous Clearance

Objective To explore the association of protein phosphatase-1 regulatory inhibitor subunit-11 (PPP1R11) polymorphism and hepatitis C virus (HCV) spontaneous clearance.Methods From January 2009 to May 2013,a total of 328 blood donors whose blood samples were anti-HCV positive with enzyme-linked immunosorbent assay (ELISA) were included into this study.According to follow-up results,they were divided into spontaneous clearance group whose HCV RNA were negative but anti-HCV recombinant immunoblot assay results were positive (n =134),and chronic infection group whose HCV RNA were positive (n=198).Polymerase chain reaction with sequence specific primer(PCR-SSP) was performed to determine the genotype of rs3807031 of PPP1R11.Statistical software SPSS was used to analyze the association between genotype of rs3807031 and HCV spontaneous clearance.Results The average age of donors in spontaneous clearance group was (28.8± 10.2) years old,which was significantly lower than that of chronic infection group whose average age was (31.0±9.0) years old (t=2.135,P =0.034).Higher percentage of female was observed in spontaneous clearance group compared to chronic infection group (35.8％ vs 21.6％ ; x2 =7.994,P=0.005).Importantly,rs3807031 A allele composed 30.6％ and 22.4％,while C allele composed 69.4％ and 77.6％ in spontaneous clearance and chronic infection group,respectively.The frequencies of rs3807031 alleles were statistically different between these two groups (x2 =5.538,P =0.023).Multivariate unconditional logistic regression analysis showed that gender (OR=1.957,95％CI:1.118-3.224,P=0.008) and genotype of rs3807031 (OR=0.650,95％CI:0.450-0.973,P =0.021) are independent factors associated with HCV spontaneous clearance.Conclusions PPP1R11 rs3807031 is associated with HCV spontaneous clearance,which has the potential to predict the spontaneous clearance of HCV infection.Our results provide information to the research on the mechanism and molecular marker of HCV spontaneous clearance. Key words: Hepatitis C, chronic; Hepacivirus; Genotype; Polymorphism, single nucleotide

Indeterminate RIBA results were associated with the absence of hepatitis C virus RNA (HCV-RNA) in blood donors

Hepatitis C virus (HCV) infection is diagnosed by the presence of antibodies and is supplemented by confirmatory testing methods, such as recombinant immunoblot assay (RIBA) and HCV-RNA detection. This study aimed to evaluate the efficacy of RIBA testing to diagnose HCV infection in blood donors positive for anti-HCV antibodies. A total of 102 subjects positive for anti-HCV determined by enzyme-linked immunosorbent assay (ELISA) at the Hematology and Hemotherapy Foundation of Bahia (HEMOBA) were later assessed with new samples using the Abbott Architect anti-HCV test (Abbott Diagnostics, Wiesbaden, Germany), the RIBA III test (Chiron RIBA HCV 3.0 SIA, Chiron Corp., Emeryville, CA, USA), the polymerase chain reaction (PCR; COBAS® AMPLICOR HCV Roche Diagnostics Corp., Indianapolis, IN, USA) and line probe assay (LiPA - Siemens, Tarrytown, NY, USA) genotyping for HCV diagnosis. Of these new samples, 38.2% (39/102) were positive, 57.8% (59/102) were negative and 3.9% (4/102) were indeterminate for anti-HCV; HCV-RNA was detected in 22.5% (23/102) of the samples. RIBA results were positive in 58.1% (25/43), negative in 9.3% (4/43) and indeterminate in 32.6% (14/43) of the samples. The prevailing genotypes were 1 (78.3%, 18/23), 3 (17.4%, 4/23) and 2 (4.3%, 1/23). All 14 samples with indeterminate RIBA results had undetectable viral loads (detection limit ≤50 IU/mL). Of these samples, 71.4% (10/14) were reevaluated six months later. Eighty percent (8/10) of these samples remained indeterminate by RIBA, and 20% (2/10) were negative. In this study, individuals with indeterminate RIBA results had no detectable HCV-RNA.

Evaluation of a new rapid test kit to detect hepatitis C virus infection

This study investigated the performance characteristics and diagnostic usefulness of a new rapid device test (RDT) for HCV (SD Bioline, Korea). A total of 200 specimens were used in this study and to assess cross-reactivity, five hepatitis B surface antigen (HBsAg) positive, five anti-hepatitis B surface antibody (anti-HBs) positive, five rheumatoid factor (RF) positive, and six samples from multiple myeloma patients were tested. The early detection capability of the test was assessed using seroconversion panels. Sensitivity and specificity were calculated compared with a recombinant immunoblot assay (RIBA). Sixty-six cases were positive by the RIBA, while 52 cases were positive using the new RDT. The sensitivity and specificity of the new RDT were 78.8% (95% CI: 71.2–86.8%) and 100%, respectively. The kappa value for the agreement between the new RDT and RIBA results was 0.831 (95% CI: 0.746–0.916). The early detection capability of the new RDT and a HCV EIA were similar, with the same window period. The new RDT did not cross-react with HBsAg, anti-HBs, RF and immunoglobulins. In conclusion, the SD Bioline HCV RDT has superior sensitivity and specificity than the GENEDIA® HCV Rapid LF that is used in Korea. This assay can be used for HCV screening, especially in small hospitals, without the financial burden of expensive equipment.

A 25-Year Study of the Clinical and Histologic Outcomes of Hepatitis C Virus Infection and Its Modes of Transmission in a Cohort of Initially Asymptomatic Blood Donors

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. 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. 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). 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.

Improved Reflexive Testing Algorithm for Hepatitis C Infection Using Signal-to-Cutoff Ratios of a Hepatitis C Virus Antibody Assay

Chemiluminescence immunoassay (CIA) is used to detect hepatitis C virus (HCV) antibody status on the basis of signal-to-cutoff (S/Co) ratios. Positive results of antibody to HCV (anti-HCV) are followed by either recombinant immunoblot assay (RIBA) to confirm anti-HCV positivity or reverse transcription (RT)-PCR to detect viremia. We hypothesized that by analyzing S/Co ratios, we could determine a strategy to reduce unnecessary supplementary testing in our population. CIA was performed to screen for anti-HCV, and positive results were followed up with RT-PCR testing. Negative RT-PCR results were followed up with RIBA, whereas positive RT-PCR results were assumed to be RIBA positive. ROC curves were analyzed to determine the optimal S/Co ratios to predict HCV infection. We determined the S/Co ratios on 34 243 veteran patient samples. We found that with the CIA method 9.0% of patients had positive test results for anti-HCV. An S/Co ratio <3.0 ruled out active HCV infection and exposure with 100% negative predictive value. When the S/Co ratio was ≥20.0, positive predictive values were 98.5% compared with RIBA results, and 81.0% compared with RT-PCR results. RIBA is not necessary to confirm negative or positive CIA anti-HCV if the S/Co ratio is <3.0 or ≥20.0, respectively. To confirm HCV exposure, samples with an S/Co ratio between 3.0 and 19.9 should be followed up with RIBA unless PCR testing has been performed and the result is positive. Samples with an S/Co ratio ≥20.0 or positive RIBA results should be further tested by RT-PCR to determine HCV viremia status.

Evaluation of the persistence and characteristics of indeterminate reactivity against hepatitis C virus in blood donors

A substantial number of individuals are excluded from blood donation due to indeterminate results in screening tests for hepatitis C virus antibody (anti-HCV). Disclosure of the characteristics of the indeterminate serologic pattern could optimize the testing and the management and care of blood donors. Ninety-two former blood donors deferred from blood donation due to consistent reactivity in anti-HCV enzyme immunoassay and indeterminate HCV recombinant immunoblot assay (RIBA) results were retested for anti-HCV to examine the extent of disappearance of reactivity. In addition, they were screened for selected viral, immunologic, and inflammatory variables to detect possible causes of the test reactivity pattern. After a median observation time of 75 months, 66 of 92 individuals had persistent indeterminate HCV RIBA results. Reactivity against the nonstructural NS5 antigen was the most frequent finding. A significant association was detected both between indeterminate reactivity in HCV RIBA and against the NS5 antigen independently and detectable antibody against adenovirus. Novel indeterminate reactivity showed increased prevalence during autumn and winter months. Indeterminate HCV RIBA reactivity in blood donors persists over years. Increased prevalence during autumn and winter and association to detection of adenovirus antibody indicate that viral infection and cross-reactivity are etiologic factors in indeterminate HCV RIBA reactivity. Further prospective studies are needed to confirm the results.

Hepatitis C infection and risk of malignant lymphoma

The association between hepatitis C virus (HCV) infection and risk of malignant lymphoma remains controversial, perhaps due to small-sized studies and low prevalence of HCV in the general population. On the basis of a large Danish-Swedish population-based case-control study, 2,819 lymphoma patients and 1,856 controls of second-generation Danish-Swedish origin were screened for HCV infection using an enzyme-linked immunosorbent assay and a confirming recombinant immunoblot assay (RIBA) test. Positive samples were tested with real-time PCR for the presence of HCV RNA. The association between HCV infection and risk of malignant lymphoma was assessed by logistic regression. When intermediate RIBA test results were interpreted as positive, anti-HCV antibody positivity was associated with a nonsignificant increased risk of non-Hodgkin lymphoma (NHL) overall (odds ratio (OR) = 2.2; 95% confidence interval (CI) 0.9-5.3; n = 20 cases), of B-cell lymphomas combined (OR = 2.4 [1.0-5.8]; n = 20) and of lymphoplasmacytic lymphoma (OR = 5.2 [1.0-26.4]; n = 2). No patients with T-cell or Hodgkin lymphoma were HCV-positive. A more conservative definition of HCV positivity (disregarding intermediate RIBA results) resulted in an OR = 1.6 (0.3-8.5; n = 5) for NHL overall. When the definition was further restricted to require HCV RNA positivity, OR was 1.7 (0.2-16.2; n = 3) for NHL overall. Our findings from a population with a low prevalence of HCV suggest a positive association between HCV and risk of NHL, in particular of B-cell origin.

Low Prevalence of Hepatitis C Virus Antibody in Men Who Have Sex with Men Who Do Not Inject Drugs

It is well documented that injection drug users (IDUs) have a high prevalence of antibodies to hepatitis C virus (HCV). Sexual transmission of HCV can occur, but studies have shown that men who have sex with men (MSM) without a history of injection drug use are not at increased risk for infection. Still, some health-care providers believe that all MSM should be routinely tested for HCV infection. To better understand the potential role of MSM in risk for HCV infection, we compared the prevalence of antibody to HCV (anti-HCV) in non-IDU MSM with that among other non-IDU men at sexually transmitted disease (STD) clinics and human immunodeficiency virus (HIV) counseling and testing sites in three cities. During 1999-2003, public health STD clinics or HIV testing programs in Seattle, San Diego, and New York City offered counseling and testing for anti-HCV for varying periods to all clients. Sera were tested using enzyme immunoassays, and final results reported using either the signal-to-cutoff ratio or recombinant immunoblot assay results. Age, sex, and risk information were collected. Prevalence ratios and 95% confidence intervals were calculated. Anti-HCV prevalence among IDUs (men and women) was between 47% and 57% at each site, with an overall prevalence of 51% (451/887). Of 1,699 non-IDU MSM, 26 (1.5%) tested anti-HCV positive, compared with 126 (3.6%) of 3,455 other non-IDU men (prevalence ratio 0.42, 95% confidence interval 0.28, 0.64). The low prevalence of anti-HCV among non-IDU MSM in urban public health clinics does not support routine HCV testing of all MSM.

Integration of nucleic acid amplification test results into hepatitis C virus supplemental serologic testing algorithms: implications for donor counseling and revision of existing algorithms

The routine use of hepatitis C virus (HCV) nucleic acid amplification testing (NAT) donor screening assays has provided an opportunity for revision of the current HCV supplemental testing algorithm, which requires that recombinant immunoblot assay (RIBA) be performed on every HCV enzyme immunoassay (EIA)-repeat-reactive donation. The FDA has approved variance requests to use a new algorithm that eliminates the need to perform RIBA when HCV NAT results are reactive. Data are provided in support of this new algorithm. HCV EIA (including signal-to-cutoff optical density ratio), RIBA, and NAT data were compiled from 33.2 million donations screened over an approximately 4-year period by the American Red Cross and Blood Systems Laboratories. Further, donations having specific combinations of HCV EIA, RIBA, and minipool (MP) NAT results were evaluated, with more sensitive individual-donation (ID) NAT, to construct improved counseling messages for donors. Of 47,041 EIA-repeat-reactive donations, 49.3 percent were RIBA-positive, 17.1 percent RIBA-indeterminate, and 33.5 percent RIBA-negative. NAT-reactive rates were 79.2, 2.5, and 0.18 percent for RIBA-positive, -indeterminate, and -negative donations, respectively. The new algorithm classified an additional 1 percent of donations as HCV-infected while at the same time detecting all infections classified as HCV-infected under the current algorithm. An additional 2.4 percent of RIBA-positive, MP NAT-nonreactive donations were reactive when a frozen-thawed aliquot was retested by ID NAT. Integrating HCV NAT results with RIBA results for purposes of donor notification allows more appropriate counseling messages to be given to EIA-repeat-reactive donors. The new HCV supplemental algorithm is an acceptable alternative to the current algorithm because it provides equivalent or superior accuracy in formulating donor counseling messages and may also result in reduced costs and more timely notification of infected donors.

Unaltered lymphocyte subsets in hepatitis C virus-seropositive blood donors.

Lymphocyte subsets were evaluated by dual-color flow cytometry in whole blood specimens from 35 blood donors who were seropositive on enzyme-linked immunosorbent assay (ELISA) for hepatitis C virus (HCV) and whose sera reacted in a four-antigen recombinant immunoblot assay (RIBA) (referred to as the HCV+R group), 15 donors who were seropositive on ELISA for HCV with indeterminate or negative RIBA results (the HCV+I/N group), and 25 HCV-seronegative controls (HCV-group). The cell subsets assessed included natural killer cells, B cells, T cells, CD4 and CD8 subsets of T cells, and T-cell subsets defined by the coexpression of markers that appear (HLA-DR, CD25, CD38) or disappear (CD45RA) after activation. A one-way analysis of variance revealed no significant differences among the three study groups. These findings show that, unlike cytomegalovirus- and human immunodeficiency virus-positive individuals, HCV-positive individuals do not exhibit lymphocyte alterations indicative of the immune activation caused by chronic viral infection.

Recombinant immunoblot assay for hepatitis C antibody in patients with posttransfusion non-A, non-B hepatitis.

In a prospective study of 287 patients who received blood transfusion, 26 who were found positive for hepatitis C antibody (anti-HCV) by an enzyme-linked immunosorbent assay (ELISA) were studied by a recombinant immunoblot assay (RIBA). Nineteen of the 26 patients had posttransfusion non-A, non-B (NANB) hepatitis. Sixteen (84.2%) of the 19 patients with hepatitis had positive results by RIBA, 2 had indeterminate results, and 1 was negative. By contrast, five of the 7 recipients without hepatitis were negative, 1 indeterminate, and 1 positive by RIBA. Those with negative RIBA results had significantly lower optical density (OD) readings by ELISA than those with positive RIBA tests. Therefore, patients without hepatitis or lower OD value have a higher false-positive rate in anti-HCV ELISA than did those with a high OD value or with evidence of hepatitis.