Hepatitis C virus RNA in blood donor sera detected by the polymerase chain reaction: Comparison with supplementary hepatitis C antibody assays

Abstract

The low specificity of screening ELISAs for antibodies to hepatitis C virus in blood donors has called for confirmatory tests. Two types of supplementary antibody assays are available, recombinant immunoblot assays (RIBA-1 and RIBA-2) and an antibody consumption test referred to as a neutralization assay. Amplification of viral nucleic acid by the polymerase chain reaction (PCR) provides an antibody independent mode of detecting viral infection. We applied reverse transcription-double PCR to detect an HCV 5'-noncoding viral RNA sequence in serum specimens and compared PCR findings with confirmatory antibody tests. This study includes sera from 37 blood donors found positive by the Ortho anti-HCV (C100-3) ELISA out of 14,591 donations. Of the 37 positive sera, 8 were positive by RIBA-1 and 1 further by RIBA-2. Seven of the RIBA positive sera contained HCV RNA by PCR. Among the 8 indeterminate and the 21 negative donor sera by RIBA-1, no PCR positive serum was found. The 37 anti-HCV positive donor sera identified by Ortho ELISA were also tested by Abbott anti-HCV (C100-3) ELISA whereby 22 were positive. Of these 22 sera plus 1 further with ELISA OD just below cutoff, 8 were positive by the "neutralization assay," (Abbott Laboratories, North Chicago, IL, USA) and 6 of these, including the borderline serum, were PCR positive. One of the two neutralizable but PCR negative sera was RIBA positive and the other was indeterminate. However, one ELISA (Abbott Laboratories) positive (OD 1.99) serum was not neutralizable but nevertheless contained HCV RNA by PCR.(ABSTRACT TRUNCATED AT 250 WORDS)

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

“…In addition to the three recombinant antigens mentioned above, this test also utilizes the recombinant protein 5-1-1 and superoxide dismutase (SOD) 27 . Recently, a more sensitive third-generation of RIBA (RIBA-3) has been introduced and uses the same antigens as the EIA-3.…”

Seroconversion to Hepatitis C Virus in Dialysis Patients: A Retrospective and Prospective Study

“…Polymerase chain reaction (PCR) was used for demonstration of HCV RNA in serum from patients with indeterminate RIBA II reac tions using the method described by Widell et al [14], Retrospective testing of stored serum samples was performed in patients found to be anti-HCV positive for determination of the time of seroconversion to HCV in relation to treatment on dialysis and trans fusions. All patients had been tested regularly for hepatitis B markers during treatment on dialysis, and sera were kept at -20°C during at least 5 years in the laboratory.…”

Hepatitis C Virus Genotypes among Blood Donors and Their Recipients in Iceland Determined by the Polymerase Chain Reaction

“…The second thermocycling of the typing PCR consisted of 25 cycles in a Perkin-Elmer thermocycler under stringent conditions as follows: melting at 94°C for 1 min, annealing at 63 °C for 1 min and elongation at 72 °C for 1.5 min. Lastly, the final amplification product was mixed with bromphenol blue, electrophoresed in an agarose gel, stained with ethidium bromide, visualized and photographed as previously de scribed [12].…”

“…The viral RNA was extracted from 100 pi of serum using the acid guanidinium thiocyanate-phenol-chlo roform method of Chomczynski and Sacci [11] with some improve ments [12], Water controls were included between each sample and weak-positive controls after every six samples.…”