Quality Assessment Of Hepatitis C Virus Nucleic Acid Amplification Methods - An International Proficiency Study

The European Medicine Evaluation Agency (EMEA) and the European Pharmacopoeia (EP) intend to prescribe HCV RNA testing of plasma pools, with a required assay detection limit of 100 International Units per milliliter (IU/ml). The feasibility of this requirement was examined in the international Viral Qualify Control (VQC) programme. In this collaborative study a coded HCV RNA proficiency panel composed of 4 negative, 2 positive samples, and 2 dilution series was distributed to 88 laboratories, of which 81 submitted 141 result sets. After exclusion of 33% of data sets with false-positive or false-negative results, the sensitivity of the applied nucleic acid amplification techniques (NAT) was examined on half-log dilution series of the EUROHEP genotype 1 and VQC genotype 1 HCV RNA standards. The overall 95% and 50% detection end points of proficient laboratories on the EUROHEP standard were for in-house PCR assays (n = 36) 2,700 geq/ml and 90 geq/ml; for Roche AMPLICOR(R) tests (n = 27) 3,000 geq/ml and 370 geq/ml, and for AMPLICOR(R) assay runs with modified sample preparation (n = 19) 2,200 geq/ml and 70 geq/ml, respectively. The respective percentages of PCR reactive results at the EMEA detection limit of 100 IU/ml (equivalent to 4.5-log dilution of VQC standard) were for in-house PCR assays 66%; for AMPLICOR(R) tests 44%, and for modified AMPLICOR(R) assays 89%. The proposed EMEA detection limit was not achieved in 35/95 (37%) test runs of proficient laboratories. In a second study at CLB the VQC proficiency panel was tested in 10 assay runs for validation of the HCV RNA detection method for testing of plasma pools. For this purpose the NucliSens silica particle RNA extraction method of Organon Teknika is combined with the Roche AMPLICOR(R) HCV RNA test. Testing 2 mi volumes of the EUROHEP genotype 1 standard dilutions we found a 95% and 50% hit rate cut-off point of 50 geq/ml and 4 geq/ml, respectively. These results demonstrate that the proposed EMEA/EP requirement for sensitivity of HCV RNA NAT assays in plasma pools can be fulfilled when improved nucleic acid extraction technology is used.