OIE Procedure for Validation and Certification of Diagnostic Assays

sheet Name of the diagnostic kit: TeSeETM WESTERN BLOT Manufacturer: Bio-Rad OIE Approval number: 20090105 Date of Registration: May 2009 Disease: Prion associated diseases (Scrapie, Bovine Spongiform Encephalopathy, Chronical Waste Disease) Pathogen Agent: Abnormal prion protein PrP Type of Assay: The TeSeETM WESTERN BLOT kit is based on the Western blotting technique and allows detection of abnormal prion protein PrP after protease digestion. Purpose of Assay: Certified by the OIE in May 2009 as fit for the post mortem detection of Transmissible Spongiform Encephalopathies (TSEs) in cattle (Bovine Spongiform Encephalopathy, BSE), in ovine and caprine (BSE and scrapie), and in cervids (Chronic Wasting Disease, CWD) and as validated fit for the following purposes: 1. To confirm TSE suspected positive samples detected at the screening laboratories in countries with active/passive surveillance programmes. Any sample with a negative result according to the TeSeETM WESTERN BLOT assay interpretation criteria, following a positive rapid test result, should be tested with one of the other OIE certified confirmatory methods, Immunohistochemistry (IHC) or Scrapie-associated fibrils (SAF)Immunoblot; 2. To confirm the prevalence of infection with one of the TSE associated diseases (BSE, scrapie, CWD) in the context of an epidemiological survey in a low prevalence country; 3. To estimate prevalence of infection to facilitate risk analysis (e.g. surveys, implementation of disease control measures) and to assist the demonstration of the efficiency of eradication policies. Species and Specimen: Bovine (obex), Cervids (obex and lymph nodes) and ovine (obex) 1. Information on the kit Information is available on Bio-Rad Website, at www.bio-rad.com 2. Summary of validation studies STAGE 1 Validation – Analytical characteristics Calibration Since no reference standard is available, the TeSeETM WESTERN BLOT assay was calibrated against the Bio-Rad TeSeE rapid test (TeSeE immunoassay: sandwich ELISA format), on a panel of Immunohistochemistry (IHC) confirmed positive samples, including Abstract sheet – Bio-Rad TeSeETM WESTERN BLOTsheet – Bio-Rad TeSeETM WESTERN BLOT weak positive and atypical scrapie samples. The TeSeETM WESTERN BLOT could detect all positive samples. Repeatability: Both within run and between run repeatability were evaluated. Within run: The results were perfectly reproducible when testing 32 times within the same day, a panel of 1 negative and 3 positive samples. Within run repeatability was confirmed by the Reference Laboratory for European Union (CRL), VLA, Weybridge, UK, within assay study. Between run: The results were perfectly reproducible when testing a panel of 1 negative and 3 positive samples, in duplicate, for 20 days. Between serial repeatability is verified for every reagent batch produced through the final quality control procedure performed in the manufacturing site with a specifically designed Quality Control sample panel. Analytical specificity: As specimens collected on animals infected both prion and other pathogen are difficult to obtain, analytical specificity was not studied. Analytical sensitivity: The TeSeETM WESTERN BLOT assay was demonstrating the same sensitivity than the Bio-Rad TeSeE S/G Rapid test (ELISA) and than the OIE scrapie-associated fibrils (SAF) blot method. STAGE 2 Validation – Diagnostic characteristics Field samples from passive or active surveillance programs were used for the evaluation studies of the TeSeETM WESTERN BLOT assay. The status (positive or negative) of these samples had been determined with one of the European Commission approved screening methods and/or IHC reference method. The ability of the TeSeETM WESTERN BLOT assay to confirm their status was evaluated. The same evaluation approach was used in all evaluations performed. Study I Internal evaluation at Bio-Rad, France Analytical sensitivity on ovine and bovine characterized samples from veterinary reference laboratories Diagnostic specificity on ovine and bovine samples from French slaughterhouses (animal without any clinical sign) classified as negative with a rapid screening tests (TeSeETM ELISA) Diagnostic sensitivity on ovine samples from French flocks with natural cases of TSE tested with the TeSeETM ELISA and IHC; Study II External evaluation at CRL, VLA, Weybridge, UK Diagnostic specificity and sensitivity on bovine samples from UK passive surveillance Diagnostic specificity and sensitivity retrospective study on bovine samples from UK Diagnostic specificity and sensitivity on ovine samples from UK active and passive surveillance; Bio-Rad-TeSeETMWESTERN BLOT Abstract Sheet Version 1 – August 2009 Page 2 of 8 Abstract sheet – Bio-Rad TeSeETM WESTERN BLOTsheet – Bio-Rad TeSeETM WESTERN BLOT Study III External study at CODA-CERVA, Belgium: prospective study on bovine samples from routine screening and retrospective study on previously BSE positive samples from the laboratory routine; Study IV External study at IZW Berlin, Germany: retrospective study on 104 cervid samples (deer and elk) from Germany, USA and Canada, characterized by IHC including 64 CWD positive samples; Study V External study at AFSSA, France: retrospective study on scrapie positive ovine samples choosen randomly among the scrapie diagnosed cases between 2002 and 2004 in France (positive with the French reference AFSSA Western blot method and with the TeSeETM Sheep/Goat ELISA); Study VI External retrospective study at AFSSA, France: Arsac et al, Acta Neuropathol., July 2007: retrospective study on BSE positive obex collected in France during active and passive surveillance between 2001 and 2003 (confirmed positive with the AFSSA Western blot and analysed with IHC in case of discrepancy between rapid test and Western blot); Study VII External retrospective study at AFSSA, France: Arsac et al, EID January 2007: retrospective study on 54 atypical scrapie positive samples detected by the rapid test TeSeETM ELISA, collected in France during the scrapie surveillance program between 2002 and 2004; Study VIII External retrospective study on BSE positive samples (including atypical BSE cases) at CRL, VLA, Weybridge: Terry et Al., Veterinary Record, June 2007: retrospective study on 5 BSE positive confirmed samples (including one French H type from AFSSA and one H type from UK) and 1 scrapie positive confirmed sample; Study IX CFIA Ottawa (external study): retrospective study on 50 positive elk brain samples, 40 positive White Tailed Deer brain, 65 positive White Tailed Deer retropharyngeal lymph node samples; Study X USDA Ames Iowa (external study): retrospective study on 53 positive deer retropharyngeal lymph node samples. Threshold determination The threshold was determined according to the following criteria: Negative samples: They do not show any signal, since PrP was totally digested by proteinase K. Positive samples: • Classical positive samples: They show a typical 3 band pattern, demonstrating digestion of PrP and transformation of the disease specific prion protein into a proteinase resistant core fragment with reduced molecular mass following removal of N-terminus part of the protein. The two higher bands correspond to mono and di-glycosylated forms (27-30 kDa) while the lower band corresponds to the un-glycosylated form of the protein. • Nor 98 positive samples: They show an atypical glycoprofile. A lower band is visible at approximately 12kDa, while other upper bands are not located at the same positions compare to classical scrapie cases. • Other samples that should be repeated: The samples for which only the di-glycosylated band is detected and the samples for which only the di-glycosylated band and the monoglycosylated band are detected. Bio-Rad-TeSeETMWESTERN BLOT Abstract Sheet Version 1 – August 2009 Page 3 of 8 Abstract sheet – Bio-Rad TeSeETM WESTERN BLOTsheet – Bio-Rad TeSeETM WESTERN BLOT Diagnostic sensitivity (DSn) and specificity (DSp) estimates with 95% confidence limits (Cl) TeSeETM WESTERN BLOT Target Species : Bovine Diagnostic sensitivity N DSn CI 315 99.0 % 97.2 – 99.8 Diagnostic specificity N DSp CI 282 99.3 % 97.5 – 99.9 TeSeETM WESTERN BLOT Target Species : Ovine Diagnostic sensitivity N DSn CI 306 98.0 % 95.7 – 99.3 Diagnostic specificity N DSp CI 141 100 % 97.4 – 100 TeSeETM WESTERN BLOT Target Species : Cervid Diagnostic sensitivity N DSn CI 272 100 % 98.65 – 100 Diagnostic specificity N DSp CI 40 100 % 91.2 – 100 Agreement between tests The TeSeETM WESTERN BLOT assay was evaluated on different animal species (bovine, ovine, goat and cervids), in several National Reference laboratories, and in comparison with the confirmation methods currently in used in those laboratories. Here are the performances of the TeSeETM WESTERN BLOT assay: Bio-Rad-TeSeETMWESTERN BLOT Abstract Sheet Version 1 – August 2009 Page 4 of 8 Abstract sheet – Bio-Rad TeSeETM WESTERN BLOTsheet – Bio-Rad TeSeETM WESTERN BLOT TeSeETM WESTERN BLOT versus VLA Hybrid WB Bovine Diagnostic sensitivity N DSn CI 223 100 % 98,4 % 100 % Diagnostic specificity N DSp CI 50 100 % 92,9 % 100 % TeSeETM WESTERN BLOT versus AFSSA WB Bovine Diagnostic sensitivity N DSn CI 52 100 % 93,1 % 100 % Diagnostic specificity N DSp CI 25 84 % 63,9 % 95,5 % TeSeETM WESTERN BLOT versus IHC Ovine Diagnostic sensitivity N DSn CI 211 99 % 96,4 % 99,9 % Diagnostic specificity N DSp CI 71 100 % 94,9 % 100 % TeSeETM WESTERN BLOT versus VLA Hybrid WB Ovine Diagnostic sensitivity N DSn CI 73 100 % 95,1 % 100 % Diagnostic specificity N DSp CI 61 100 % 94,1 % 100 % Bio-Rad-TeSeETMWESTERN BLOT Abstract Sheet Version 1 – August 2009 Page 5 of 8 Abstract sheet – Bio-Rad TeSeETM WESTERN BLOTsheet – Bio-Rad TeSeETM WESTERN BLOT TeSeETM WESTERN BLOT versus SAF Immunoblot OIE Ovine Diagnostic sensitivity N DSn CI 0 NC NC Diagnostic specificity N DSp CI 54 (*) 0 % NC (*): the 54 discrepant samples were all confirmed positive by IHC (atypical scrapie) TeSeETM WESTERN BLOT versus AFSSA WB Ovine Diagnostic sensitivity N DSn CI 40 100 % 91,2 % 100 % Diagnostic specificity N DSp CI 0 NC NC TeSeETM WESTERN BLOT versus IHC Cervids Diagnostic sensitivity N DSn CI 272 100 % 98.65 % 100 % Diagnostic specificity N DSp CI 40 100 % 91,2 % 100 % STAGE 3 Validation Reproductibility The CRL regularly issues proficiency testing panels for a range of TSE tests. The samples consist of homogenised brain material presented as 50% tissue/water homogenates. The summarised results for confirmatory blotting rounds issued in 2007 are given below. CRL Scrapie confirmatory blotting Proficiency test round A panel of 5 samples (1 strong positive, 2 weak positives [all classical scrapie] and 2 negatives) was issued to 23 European laboratories in November 2007. 10 sets of results were returned by laboratories using the BioRad TeSeETM WESTERN BLOT. All of these 10 laboratories identified all samples correctly. Bio-Rad-TeSeETMWESTERN BLOT Abstract Sheet Version 1 – August 2009 Page 6 of 8 Abstract sheet – Bio-Rad TeSeETM WESTERN BLOTsheet – Bio-Rad TeSeETM WESTERN BLOT CRL BSE confirmatory blotting Proficiency test round A panel of 5 samples (1 strong positive, 1 medium positive, 1 weak positive [all classical BSE] and 2 negatives) was issued to 21 European laboratories in October 2007. Eight sets of results were returned by laboratories using the BioRad TeSeETM WESTERN BLOT. All of these 8 labs identified all samples correctly. In addition, reproducibility of the TeSeETM WESTERN BLOT was evaluated with the Bio-Rad control panel including 2 negative ovine samples, 1 negative bovine sample, 1 CWD positive sample, 3 scrapie positive samples and 1 BSE positive sample. The panel was provided to 3 TSE Reference laboratories: VLA in UK, CFIA in Canada and NVI in Norway. The results from the 3 sites were compared to the results obtained in the Bio-Rad facilities. All of these 3 laboratories identified all samples correctly (the BSE positive sample was not evaluated by the CFIA-ACIA laboratory in Ottawa since this laboratory is not a Reference laboratory for BSE in Canada). STAGE 4 Validation Applications The TeSeE WESTERN BLOT assay is only for use in National Reference Laboratories (NRLs) and Community Reference laboratories (CRLs). The TeSeE WESTERN BLOT assay can be used for different types of applications: as a confirmatory assay for the confirmation of TSE suspected cases, in cattle (BSE), small ruminants e.g. sheep/goat (BSE/Scrapie) and cervids (CWD). The diagnostic regimen includes several steps at both screening laboratories and Reference laboratories. • at the screening laboratory: initial screening with one of the approved rapid test. If the sample turns positive (= initial reactive), the test needs to be repeated in duplicate with the same assay and with the same sample (sample homogenate). If one of the two duplicates or the two turn positive, the sample is then considered as “suspect”. All remaining material (sample homogenate + remaining tissues) are sent to the Reference laboratory for confirmation. • at the reference laboratory: A negative result with the TeSeETM WESTERN BLOT means that the test sample does not contain detectable PrPres by TeSeETM WESTERN BLOT assay. However, as very low levels of PrPres may not be detected, such a result does not exclude the possibility of infection. Any sample with a negative result according to the TeSeETM WESTERN BLOT assay interpretation criteria, following a positive rapid test result, should be tested with one of the other OIE certified confirmatory methods, Immunohistochemistry (IHC) or SAF-Immunoblot. Any sample with a reproducible positive result according to the test interpretation critera must be verified in accordance with current legal regulation. as a rapid method for the determination of the prevalence of infection with one of the TSE associated diseases (BSE, scrapie, CWD) in the context of an epidemiological survey in a low prevalence country.