Single ELISA is Adequate, Even When Results are Weak

A single enzyme linked immunosorbent assay (ELISA) determination is all that is needed for the diagnosis of hepatitis C virus infection, according to a report from France.

Researcher Jean-Michel Pawlotsky and colleagues suggest that, due to advances in the assay, confirmation of positive or weakly positive ELISAs with immunoblot-based confirmatory assays is not needed.

The first-generation group of anti-hepatitis C virus (HCV) ELISAs lacked sensitivity as well as specificity, and for this reason confirmatory assays based on immunoblot testing were developed and systematically used to confirm positive samples.

Since the early 1990s, however, ELISA assays have considerably improved, and the second- or third-generation tests available today are both highly sensitive and specific. Second- or third-generation immunoblot tests are still used for confirmation in most laboratories, however.

The aim of this study was to determine whether a double ELISA determination and confirmation of positive ELISA results with immunoblot assays are still useful in clinical laboratories performing routine diagnosis of HCV infection (“What Strategy Should Be Used for Diagnosis of Hepatitis C Virus Infection in Clinical Laboratories?” Hepatology, June 1998;27(6):1700-1702).

Anti-HCV antibodies were sought in 3,014 consecutive unselected samples with two different ELISAs. An immunoblot-based confirmatory assay (RIBA3.0) was performed in the samples with at least one ELISA positive or weakly positive. HCV RNA was evaluated using HCV polymerase chain reaction (PCR) in the samples with a weakly positive ELISA, discrepant results of the two ELISAS, or an indeterminate RIBA3.0 pattern.

The two ELISAs gave concordant results in 2,957 (98.1 percent) of the 3,014 samples (negative in 87.9 percent, positive in 11.8 percent, and weakly positive in 0.3 percent), and discrepant results in 57 (1.9 percent).

RIBA3.0 was positive in 338 of the 350 ELISA-positive samples (96.6 percent) and indeterminate in 12. Six of them were PCR-positive.

Among the eight weakly positive samples, one was RIBA3.0-positive, six were RIBA3.0-indeterminate, and one was RIBA3.0-negative; all were PCR-negative.

Among the 57 samples with discrepant ELISA results, four were RIBA3.0-positive (none were PCR-positive), 22 were RIBA3.0-indeterminate (one was PCR-positive), and 31 were RIBA3.0-negative (six were PCR-positive). In these cases, the clinical context and PCR detection of HCV RNA allowed for definitive classification.

Pawlotsky et al. proposed the adoption of a cost-saving diagnostic procedure for patient suspected of having HCV infection or for those with a risk for parenterally acquired viral infections. They suggested that:

† Screening should be based on one single second- or third-generation ELISA.

“Confirmation of positive ELISAs by ELISA on a second different sample might be useful to avoid false-positive results owing to sampling or processing errors,” they wrote.

†No immunoblot-based confirmatory assay is needed.

† HCV RNA detection by PCR should be performed in the samples with an OD ratio in ELISA between 1 and 2, or when knowing the replicating status of HCV is needed for clinical decisions. This includes the classical indications of qualitative HCV RNA PCR, i.e., seronegative acute or chronic hepatitis of unknown cause, chronic liver disease with several possible causes including the presence of HCV antibodies, chronic hepatitis C with repeatedly normal alanine aminotransferase activity, diagnosis of HCV infection in babies born to HCV infected mothers, and antiviral therapy monitoring.

“This diagnostic procedure has been recently recommended by the French Consensus Conference on HCV,” Pawlotsky et al. wrote. “In our laboratory, where about 8,000 HCV serological tests are performed every year, this would allow a savings of U.S. $65,000 in reagent costs per year, not including materials and lab work. Similar evaluations of diagnostic strategies in the setting of blood donation screening are now needed.”

The corresponding author for this study is Jean-Michel Pawlotsky, Service de Bacteriologi-Virologie, Hopital Henri Mondor, 51 avenue du marechal de Lattre de Tassigny, 94010 Creteil, France. – by Salynn Boyles, Senior Editor