Diagnosis of hepatitis C virus in Brazilian blood donors using a reverse transcriptase nested polymerase chain reaction: comparison with enzyme immunoassay and recombinant protein immunoblot assay

Abstract

SUMMARYScreening blood donations for anti-HCV antibodies and alanine aminotransferase (ALT) serum levels generally prevents the transmission of hepatitis C virus (HCV) by transfusion. The aim of the present study was to evaluate the efficiency of the enzyme immunoassay (EIA) screening policy in identifying potentially infectious blood donors capable to transmit hepatitis C through blood transfusion. We have used a reverse transcriptase (RT)-nested polymerase chain reaction (PCR) to investigate the presence of HCV-RNA in blood donors. The prevalence of HCV-RNA positive individuals was compared with the recombinant immunoblot assay (RIBA-2) results in order to assess the usefulness of both tests as confirmatory assays. Both tests results were also compared with the EIA-2 OD/C ratio (optical densities of the samples divided by the cut off value). ALT results were expressed as the ALT quotient (qALT), calculated dividing the ALT value of the samples by the maximum normal value (53UI/l) for the method. Donors (n=178) were divided into five groups according to their EIA anti-HCV status and qALT: group A (EIA³3, ALT<1), group B (EIA³3, ALT>1), group C (1£EIA<3, ALT<1), group D (1£EIA<3, ALT>1) and group E (EIA£0.7). HCV sequences were detected by RT-nested PCR, using primers for the most conserved region of viral genome. RIBA-2 was applied to the same samples. In group A (n=6), all samples were positive by RT-nested PCR and RIBA-2. Among 124 samples in group B, 120 (96.8%) were RIBA-2 positive and 4 (3.2%) were RIBA-2 indeterminate but were seropositive for antigen c22.3. In group B, 109 (87.9%) of the RIBA-2 positive samples were also RTnested PCR positive, as well as were all RIBA-2 indeterminate samples. In group C, all samples (n=9) were RT-nested PCR negative: 4 (44.4%) were also RIBA-2 negative, 4 (44.4%) were RIBA-2 positive and 1 (11.1%) was RIBA-2 indeterminate. HCV-RNA was detected by RT-nested PCR in 3 (37.5%) out of 8 samples in group D. Only one of them was also RIBA-2 positive, all the others were RIBA-2 indeterminate. All of the group E samples (controls) were RT-nested PCR and RIBA-2 negative. Our study suggests a strong relation between anti-HCV EIA-2 ratio ³ 3 and detectable HCV-RNA by RT-nested PCR. We have also noted that blood donors with RIBA-2 indeterminate presented a high degree of detectable HCV-RNA using RT-nested PCR (75%), especially when the c22.3 band was detected.

Cost-effective analysis of different algorithms for the diagnosis of hepatitis C virus infection

“…Reflex supplemental testing could be limited to screening test-positive samples with ratios below the cutoff value. In studies conducted in October 2000 using the second-generation HCV ELISA assay, the authors obtained s/co ratio >3.0 as cut-off and high concordance (96%) with IB-positive tests (10). In our samples, algorithm A was able to diagnose 46% (238/517) of the samples as true positive only by ELISA (s/ co ratio ≥6).…”

Low-Positive Anti-Hepatitis C Virus Enzyme Immunoassay Results: An Important Predictor of Low Likelihood of Hepatitis C Infection

“…Because no further testing was done in ϳ40% of patients, it is possible that some of the patients with negative RIBA results were, in fact, HCV RNA-positive. However, several studies have shown that a negative result in the third-generation RIBA is virtually never associated with positive HCV RNA results except in the setting of acute hepatitis C infection (17,36,37 ).…”

“…Although EIA tests provide a quantitative absorbance result [often reported as the signal to cutoff (S/C) ratio], they are usually reported simply as positive or negative. Several studies using first-and second-generation anti-HCV EIA tests have shown that samples with absorbance values just slightly above the cutoff value have a significantly greater likelihood of representing false-positive results compared with those with higher values (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17).…”

“…The two most helpful markers were alanine aminotransferase (ALT) activity and antibody to the hepatitis B virus core antigen (antiHBc) (21)(22)(23)(24). Several studies have shown that there are differences in the frequency of increased ALT activity and positive anti-HBc between blood donors with low and high absorbance values for anti-HCV (4,9,14,17 ).…”

Chemiluminescence Assay Improves Specificity of Hepatitis C Antibody Detection

“…Although usually reported as positive or negative, the results are actually measured as an absorbance signal that is compared with that of a cutoff value; results above the cutoff are reported as positive, whereas those below the cutoff are called negative. Several studies in the blood bank literature have shown that positive samples with a low signal-to-cutoff (S/C) ratio are usually found to be negative for anti-HCV antibodies by recombinant immunoblot assay (RIBA) (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16). To minimize the likelihood of false-positive anti-HCV results, the CDC has recommended confirmation of all anti-HCV results by either RIBA or HCV RNA assays (17 ).…”