anti-HCV Immunoblot Research Articles

Anti-HCV immunoblot indeterminate results in blood donors: non-specific reactivity or past exposure to HCV?

The significance of anti-HCV immunoblot (IB) indeterminate results can be difficult to determine. We analysed results for blood donors tested on the MP Diagnostics HCV Blot 3.0 IB assay to determine whether indeterminate results representing past exposure to HCV could be distinguished from those due to non-specific reactivity. Results for all donors tested by IB during the study period (July 2010 to December 2013) were included in this study. Of 131 donors tested by IB, 34 (26.0%) were negative, 38 (29.0%) were indeterminate, and 59 (45.0%) were positive. There was no significant difference in IB band reactivity strength between indeterminate and positive donors. The PRISM HCV chemiluminescent immunoassay (ChLIA) sample to cut-off (s/co) ratio distribution for the indeterminate donors was significantly higher than for those with biological false reactivity (P=0·037), but significantly lower than for donors who were IB positive/HCV RNA negative (P<0·001) or IB not tested/HCV RNA positive (P<0·001). Of donors available for follow-up, 53.1% of the indeterminate group disclosed a putative risk factor for HCV infection compared to 39.4% (P<0·001) for the IB-negative group, 76.6% (P=0·065) for the IB-positive group and 83.4% (P<0·001) for the HCV RNA-positive group. The results of this study indicate that PRISM ChLIA s/co ratios >2·00 with IB indeterminate results predict exposure to HCV, particularly in the presence of putative risk factors for HCV infection. These findings may be applied to optimizing counselling of donors with indeterminate HCV results.

Detection limit of architect hepatitis C core antigen assay in correlation with HCV RNA, and renewed confirmation algorithm for reactive anti-HCV samples

Backgroundhepatitis C infections are detected by anti-HCV screening tests. Reactive anti-HCV results give no information about the presence or absence of hepatitis C viruses, or of unspecific reactivity. To obtain information about the viral load, HCV RNA measurements, following a reactive anti-HCV result, are performed in well equipped and specialised laboratories. Anti-HCV immunoblots are the only means to exclude non specific reactivity. The measurement of HCV core antigen (HCV-Ag), as an alternative to HCV RNA, is discussed, as it can be analysed on the same instrument as anti-HCV. ObjectivesThe detection limit of HCV-Ag is crucial to use it in lieu of HCV RNA, in regard of the different genotypes. A renewed algorithm is proposed to exclude unspecific reactivity of anti-HCV. Study designsSamples were tested on Architect i2000SR (Abbott) for anti-HCV and HCV-Ag. HCV RNA measurements were obtained by Cobas Ampliprep/Taqman (Roche) or m2000rt® (Abbott). Results and ConclusionsComparison between HCV-Ag and HCV RNA from 126 samples of 101 patients with chronic hepatitis C gave linear regression R2 0.89, slope 0.885 and intercept −2.258, which were independent of the genotypes. The detection limit of HCV-Ag was between 2.4 and 4.5Log10IU/mL. A renewed algorithm for confirmation of reactive anti-HCV results is proposed: active or resolved hepatitis C infections or false reactivity can be differentiated by sequenced reflex testing due to HCV-Ag, anti-HCV immunoblot and HCV RNA.

Evidence for Novel Hepaciviruses in Rodents

Hepatitis C virus (HCV) is among the most relevant causes of liver cirrhosis and hepatocellular carcinoma. Research is complicated by a lack of accessible small animal models. The systematic investigation of viruses of small mammals could guide efforts to establish such models, while providing insight into viral evolutionary biology. We have assembled the so-far largest collection of small-mammal samples from around the world, qualified to be screened for bloodborne viruses, including sera and organs from 4,770 rodents (41 species); and sera from 2,939 bats (51 species). Three highly divergent rodent hepacivirus clades were detected in 27 (1.8%) of 1,465 European bank voles (Myodes glareolus) and 10 (1.9%) of 518 South African four-striped mice (Rhabdomys pumilio). Bats showed anti-HCV immunoblot reactivities but no virus detection, although the genetic relatedness suggested by the serologic results should have enabled RNA detection using the broadly reactive PCR assays developed for this study. 210 horses and 858 cats and dogs were tested, yielding further horse-associated hepaciviruses but none in dogs or cats. The rodent viruses were equidistant to HCV, exceeding by far the diversity of HCV and the canine/equine hepaciviruses taken together. Five full genomes were sequenced, representing all viral lineages. Salient genome features and distance criteria supported classification of all viruses as hepaciviruses. Quantitative RT-PCR, RNA in-situ hybridisation, and histopathology suggested hepatic tropism with liver inflammation resembling hepatitis C. Recombinant serology for two distinct hepacivirus lineages in 97 bank voles identified seroprevalence rates of 8.3 and 12.4%, respectively. Antibodies in bank vole sera neither cross-reacted with HCV, nor the heterologous bank vole hepacivirus. Co-occurrence of RNA and antibodies was found in 3 of 57 PCR-positive bank vole sera (5.3%). Our data enable new hypotheses regarding HCV evolution and encourage efforts to develop rodent surrogate models for HCV.

753 INSULIN RESISTANCE IS INDEPENDENTLY ASSOCIATED WITH ADVANCED LIVER FIBROSIS AND HIGH BODY MASS INDEX IN HIV/HCV-COINFECTED PATIENTS

Results: Among 145 patients with serologically silent occult HCV infection 45 (31%) tested anti-HCVcore-positive at the time of the diagnostic biopsy. Anti-HCVcore was detected in 143/145 (98.6%) chronic hepatitis C patients but in none of the 140 patients with HCV-unrelated liver disease (P< 0.001). A serological follow-up was conducted in occult HCV-infected patients (every 3−6 months for at least 12 months). Among 23 anti-HCVcorepositive patients at baseline, 18 remained antibody-reactive on all occasions tested; anti-HCVcore fluctuated in 4 (from positive to negative and viceversa) and was lost in one patient. Similarly, among 31 anti-HCVcorenegative at baseline, 17 remained antibody non-reactive; however, 4 patients seroconverted to anti-HCVcore and remained so whereas antiHCVcore became reactive in 10 patients on at least one occasion. Thus, a total of 59/145 (40.7%) patients with occult HCV infection reacted in the anti-HCVcore assay at any time-point analyzed, including 14 initially non-reactive patients. By supplemental anti-HCV immunoblot assay 16/83 (19.2%) sera reacted weakly with a single core-peptide band (indeterminate test result) of which 10/16 (62.5%) reacted in the anti-HCVcore ELISA. According to anti-HCVcore status, occult HCV-infected patients who tested anti-HCVcore-positive showed significantly greater scores of necroinflammation (P = 0.046) and percentages of HCVRNA-positive hepatocytes (P = 0.004) compared with the anti-HCVcore-negative counterparts. Conclusion: Anti-HCVcore is detectable in 40% patients with serologically silent occult HCV infection. Anti-HCVcore testing improves serodiagnosis of HCV infection in patients lacking serum anti-HCV and HCVRNA using commercial tests. This anti-HCVcore assay may be useful in tracking subclinical HCV infections.

The significance of third-generation HCV RIBA-indeterminate, RNA-negative results in voluntary blood donors screened with sequential third-generation immunoassays.

One of the problems associated with the use of anti-HCV immunoblot assays is the interpretation of indeterminate results without detectable HCV RNA. The purpose of this study was to examine the significance of third-generation RIBA (RIBA-3)-indeterminate, RNA-negative results in voluntary blood donors. Since June 2000 all Australian Red Cross Blood Service testing sites have used an anti-HCV sequential immunoassay testing strategy whereby donors who are reactive on the primary screening immunoassay are tested on a secondary immunoassay and if reactive on both assays, further tested by immunoblot. From the four testing sites that use RIBA-3, the result profiles of donors who were RIBA-3-indeterminate, HCV RNA-negative were analyzed. From 2,661,786 donations screened for anti-HCV during the study period, 102 RIBA-3-indeterminate, RNA-negative donors were identified, most of whom were reactive to either c33p (69.6%) or c22p (27.5%). The RIBA-3-indeterminate, RNA-negative donors showed a significantly higher screening immunoassay signal strength to assay cutoff (S/CO) distribution than those with biologic false-reactive (BFR) results (1.853 vs. 1.524, p < 0.05) but a significantly lower distribution than RIBA-3-positive, RNA-negative (1.853 vs. 4.546, p < 0.05) or RNA-positive (1.853 vs. 6.467, p < 0.05) donors. The RIBA-3-indeterminate, RNA-negative donors showed a similar distribution of c33c and c22p band intensities compared with RIBA-3-positive, RNA-negative donors but significantly lower distribution of band strengths compared to the RIBA-3-positive, RNA-positive group. Compared to the indeterminate donors with previous anti-HCV-negative or BFR results, the indeterminate donors not previously screened for anti-HCV showed higher immunoassay S/CO ratio distributions, a higher proportion with c22p reactivity (16.2% vs. 36.7%), and higher frequency of risk factors (46.4% vs. 75.0%). Our analysis suggests that a combination of indicators can be used to help clarify RIBA-3-indeterminate, RNA-negative results. Specifically, donors with high S/CO ratios on a screening immunoassay, RIBA-3 reactivity to c22p or c33c with band intensity of 2+ or greater, without a previous history of negative or BFR donations and with an identifiable risk factor, have a high probability of representing true anti-HCV rather than nonspecific reactivity.

Immunoblot as a supplemental test to detect antibodies to hepatitis C virus in blood donors

Supplemental tests using Immunoblot are recommended to improve specificity of anti-HCV by ELISA. In Brazil immunoblot is not officially recommended. Aiming to identify EIA false-positive rate 70 positive EIA anti-HCV blood donors were submitted to 3rd generation immunoblot at Hemocentro of Mato Grosso State where polymerase chain reaction tests are not performed. There were 44 (62.9%) immunoblot-positive, 22 (31.4%) negative and 4 (5.7%) indeterminate. Anti-HCV immunoblot can distinguish blood donors with false-positive ELISA from those who need medical assessment. Our data suggest that immunoblot could be useful in Brazilian blood banks where molecular biology tests are not available.

Efficacy of different second- and third-generation assays for detection of hepatitis C virus antibodies in plasma and sera also tested by polymerase chain reaction

Sera or plasma from 52 blood donors, initially reactive by Ortho HCV EIA 2.0 were tested by other immunoassays (Abbott HCV EIA, UBI HCV EIA and Innotest HCV EIA). Positivity was confirmed by confirmatory assays (RIBA 2.0 or RIBA 3.0 and Inno-LIA or Inno-LIA III); hepatitis C virus (HCV) RNA was detected by reverse transcription nested polymerase chain reaction (RT-nested PCR). Forty-four (84.6%) were repeatedly positive by Ortho, 41 (78.8%) by Abbot, 37 (71.2%) by Innotest and 36 (69.2%) by UBI. When tested for RIBA 2.0, 35 (67.3%) were positive. Only 26 sera (50%) were positive for HCV RNA. None of the Innotest or UBI negative sera were positive for HCV RNA nor for confirmatory tests. UBI and Innotest seem more reliable for detection of antibodies against HCV, and combined application of anti-HCV immunoblot assay and HCV RNA detection by PCR is required for confirmation of HCV infection.