Detection of hepatitis C virus and parvovirus B19 in human plasma pools by nucleic-acid amplification techniques - Trends in results of EDQM proficiency testing studies from 2004 to 2018.

The European Directorate for the Quality of Medicines & HealthCare (EDQM) has run proficiency testing schemes on the detection of viral contaminants in human plasma pools by nucleic-acid amplification techniques since 1999 for hepatitis C virus and since 2004 for parvovirus B19. A retrospective analysis was performed to assess their impact and identify trends and progress in the results obtained by participating laboratories over a 15-year span, from 2004 to 2018. The results demonstrate that overall performance improved over that time, especially among the regular participants. Participation in these proficiency testing schemes is therefore recommended for all interested control laboratories. This analysis also shows that hepatitis C virus detection now seems well established compared to that of parvovirus B19, which still appears more challenging.

Detection of West Nile virus RNA (lineages 1 and 2) in an external quality assessment programme for laboratories screening blood and blood components for West Nile virus by nucleic acid amplification testing.

BACKGROUND: A second Italian external quality assessment programme was run in 2011 to assess the performance of blood transfusion centres in detecting West Nile virus RNA in plasma. MATERIALS AND METHODS: Each participant received two panels containing negative samples and samples positive for West Nile virus lineages 1 and 2, some of which with a viral concentration close to or below the 95% limit of detection of the respective commercial nucleic acid amplification test assay: the PROCLEIX WNV assay or the Cobas TaqScreen West Nile virus test. RESULTS: Eleven laboratories took part in the external quality assessment programme. All of them correctly identified the positive samples with a viral concentration above the 95% limit of detection. No false positive results or pre-/post-analytical errors were observed. DISCUSSION: The External quality assessment programme run in 2011 allowed participants to assess the performance of the nucleic acid amplification test methods applied in their seasonal routine screening of blood donations. The results confirm the 95% limit of detection reported by the test kits' manufacturers for both West Nile virus lineages.

Interlaboratory study to evaluate the performance of laboratories involved in West Nile virus RNA screening of blood and blood components by nucleic acid amplification testing in Italy.

BACKGROUND: An Italian interlaboratory study was run in 2010 to assess the performance of Blood Transfusion Services in detecting the genome of West Nile virus (WNV) in plasma. MATERIALS AND METHODS: Each laboratory received a panel of samples containing four samples negative for WNV and six positive samples with a nominal viral concentration close to or below the 95% detection limit of two commercially available nucleic acid amplification tests (NAT) for WNV, the PROCLEIX® WNV kit and the Cobas® TaqScreen West Nile Virus kit. RESULTS: Ten laboratories took part in the study. All correctly identified the positive samples with a viral concentration above the 95% detection limit. No pre- or post-analytical errors were observed. CONCLUSIONS: The interlaboratory study run in 2010 allowed participants to assess the performance of the NAT methods applied in their seasonal routine screening of blood donations.

Quantification of hepatitis C virus (HCV) RNA in a multicenter study: implications for management of HCV genotype 1-infected patients.

Assessment of the viral load in hepatitis C virus (HCV) genotype 1-infected patients is critical before, during, and after antiviral therapy. In patients achieving a rapid virological response at week 4 of treatment, the viral load at the baseline is considered a predictive criterion of a sustained virological response 24 weeks after the discontinuation of treatment. A >or=2-log(10) drop in the viral load at week 12 of treatment (early virological response) triggers the continuation of therapy. We organized a multicenter study (MS) for diagnostic laboratories involved in the quantification of HCV RNA. Commercial assays, including two based on real-time reverse transcription-PCR (TaqMan system), and in-house methods, were used by the 61 participants. The overall reproducibility of the commercial quantitative nucleic acid amplification techniques (qNAT) was acceptable. As the intermethod variability among commercial qNAT for HCV RNA was still present, the manufacturers of these test kits should join efforts to harmonize the means of quantification of HCV RNA. This study also shows that caution should be exercised when the baseline viral load is evaluated and when the 2-log(10) reduction after 12 weeks of therapy is interpreted. Finally, this MS confirms the higher sensitivity of the commercial qNAT based on the TaqMan system, making them the elective assays for the monitoring of therapy.

Collaborative study for the calibration of HCV RNA, HBV DNA and HIV RNA reference preparations against the relative international standards.

We organised a collaborative study to calibrate three new ISS reference preparations (ISS: Istituto Superiore di Sanità), one for HCV RNA, one for HIV RNA and one for HBV DNA, to be used for nucleic acid amplification techniques (NAT) in blood testing. Serial dilution of the ISS reference preparations and the respective international standards were tested in different days by each participating laboratory using two commercial NAT assays. Data were collected by the ISS for statistical analysis. Based on the mean potency of the HCV RNA and HIV RNA preparations, calculated from the results provided by the 12 participating laboratories, a definitive concentrations of 5700 IU/mL and 4000 IU/mL, respectively, were assigned to the reference materials. On the contrary, it was not possible to obtain a consensus titre for the HBV DNA reference material. These new Italian reference preparations (HCV RNA ISS/1005 and HIV RNA ISS/1005) calibrated against the respective international standards are available free of charge to any laboratory upon request.

Collaborative study for the calibration of a new Italian HCV RNA reference preparation against the international standard.

We organised a collaborative study to calibrate a new Italian reference preparation for HCV RNA, the ISS 0102 reagent, to be used for plasma pools testing. This preparation will replace the previous one, the ISS 0498 reagent, as we are running short of it. The ISS 0102 reagent was obtained by appropriately diluting an HCV RNA-positive donation. Every participant in the collaborative study received four coded panels, each consisting of 5 semi-logarithmic dilutions of the international standard, 5 semi-logarithmic dilutions of the ISS 0102, and 2 samples of a negative plasma pool. Based on the results provided by the 22 participating laboratories, an HCV RNA concentration of 4500 IU/ml was assigned to the reference material. This preparation is available free of charge to any laboratory upon request.

Overestimation of the hepatitis C virus RNA content of reference preparations by the AMPLICOR HCV monitor test, version 2.0.

An evaluation of the AMPLICOR hepatitis C virus (HCV) monitor test, version 2.0 (Roche Diagnostics), was carried out to investigate whether this test overestimates the HCV RNA content of reference preparations. Satisfactory accuracy was observed when the World Health Organization HCV international standard was included in the assay and a modified formula was used to calculate the viral content.

[Optimisation and establishing of serological methods for the potency testing of immunglobulins against Clostridium tetani-toxin]

The quality control of human tetanus immunglobulin requires animal experiments according to European Pharmacopoeia monograph 398. The potency estimation has to be done in a toxin neutralisation test in mice (MNT) or guinea pigs. Immunoassays could also be used if they show a suitable sensitivity and specificity. The first results of our study verify that an indirect enzyme linked immunosorbent assay (ELISA), a rocket immunelectrophoresis (RIE) and a toxin binding inhibition test (ToBI) could be used as serological alternativ methods to the MNT. Studies on the reproducibility of the in vitro methods resulted inter-assay coefficients of variation between 2 and 27%. The ELISA is more sensitive (limit of detectability: 0,005 IE/ml) than the ToBI (0,04 IE/ml) and the RIE (5 IE/ml). The transferability of the ELISA to other labs is proofed. The transferability of the RIE and the ToBI will be tested in the near future.