Abstract Book of EAVLD2012 - eavld congress 2012

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S4 - O - 03 EVALUATION OF THE DIAGNOSTIC PERFORMANCE OF PEPTIDE COCKTAILS IN THE INTERFERON GAMMA ASSAY FOR DIAGNOSIS OF TUBERCULOSIS IN CATTLE Björn Schröder 1 , Roland Hardegger 1 , Marcus G. Doherr 2 , Jean-Louis Moyen 3 , Eamonn Gormley 4 and Alex J. Raeber 1 1 Prionics AG, Schlieren, Switzerland, 2 Vetsuisse Faculty, University of Bern, Switzerland, 3 Laboratoire Départemental d’Analyse et de Recherche, Coulounieix-Chamiers, France, 4 University College Dublin, Ireland, Tuberculosis, Bovigam, defined antigens, peptide cocktail Introduction Cattle infected with bovine tuberculosis (bTB) still represent a serious regulatory and health concern in a many countries. For more than two decades, eradication programs for bTB have relied on early detection and removal of infected cattle using the intradermal skin test as primary test and the in vitro interferon gamma (IFN-) assay as an ancillary assay. The interferon gamma assay is generally considered to be more sensitive than the skin test while the specificity is slightly lower. To increase the specificity of the IFN- assay, defined mycobacterial antigens such as ESAT-6 and CFP-10 have been evaluated as alternative stimulation antigens in blood cultures and have shown promising results although sensitivities were slightly lower than with tuberculin stimulation. Combining additional specific mycobacterial antigens with ESAT-6 and CFP-10 has been proposed to bring the sensitivity of antigen cocktails to the levels obtained with tuberculin and at the same time achieving specificities comparable to the skin test. We have developed peptide cocktails based on ESAT-6 and CFP-10 (1, 2, 3) and compared their performance to tuberculins in the IFN- assay for diagnosis of bTB in field studies in Ireland and France. Materials & methods Lelystad tuberculin PPD antigens (avian and bovine) (Prionics, The Netherlands) or a synthetic peptide cocktail were used for stimulation of whole blood cultures. Following stimulation, plasma was harvested and IFN-γ was measured with the BOVIGAM ® 2G (Prionics, Switzerland) sandwich enzyme immunoassay. The peptide cocktail Prionics ® PC-HP (Prionics, Switzerland) contained overlapping peptides derived from ESAT-6 and CFP- 10 and in addition peptides derived from Rv3615c and 3 other mycobacterial proteins. Diagnostic sensitivity was assessed in naturally bTB-exposed animals in Ireland. Diagnostic specificity was evaluated in France in animals from 12 herds free of bTB according to OIE criteria. Estimates for the test characteristics in the absence of a true gold standard were derived by a Bayesian model as described by Branscum et al. 2005 (4). Results Estimates of sensitivity and specificity (median and 95% confidence intervals) are shown in Table 1. In France 390 animals have been included into the specificity trial. Specificity estimates for the BOVIGAM ® 2G with tuberculin PPD from Lelystad was calculated with 84% (95%CI: 81% - 87%) whereas using the peptide cocktail PC-HP for whole blood stimulation a specificity estimate of 94% (95%CI: 92% - 96%) was computed. The difference in the specificity estimates of PPD and PC-HP was highly significant (p < 0.05; two-tailed exact Fisher test). Sensitivity was evaluated from the sample of 201 animals from Ireland, with an apparent prevalence (depending on test) between 16% and 31%. Sensitivity in this trial was calculated for Lelystad PPD with 89% (95%CI: 75% - 97%) whereas with PC- HP a comparable sensitivity of 85% (95%CI: 63% - 96%) was obtained. Table 1: Comparison of diagnostic performance estimates of BOVIGAM ® 2G using PPD or antigen peptide cocktail PC-HP for stimulation of blood. Sensitivity Estimates 95% CI Specificity Estimates 95% CI PPD 89% 75 - 97% 84% 81 - 87% PC-HP 85% 64 - 96% 94% 92 - 96% Discussion & conclusions Since 1991 when the BOVIGAM ® IFN- test was approved as an official test for bTB in Australia and later on in the European Union and the US, numerous field trials were undertaken to determine its diagnostic performance. In a review published by de la Rua-Domenech et al. (5) the sensitivity of the IFN- test varied between 73.0% and 100%, with a median value of 87.6% while its median specificity was calculated with 96.6%, with a range of 85.0–99.6%. Using a Bayesian modelling approach, we have compared the diagnostic performance of the peptide cocktail Prionics ® PC-HP with tuberculin PPD for the stimulation of blood cultures in the BOVIGAM ® 2G IFN- assay. Our results suggest that the sensitivity of the peptide cocktail PC-HP is not significantly different from the sensitivity with PPD whereas peptide cocktail PC-HP resulted in a specificity that was significantly higher than stimulation with PPD. Overall, we conclude from these studies that peptide cocktails provide superior performance compared to tuberculin PPD in the BOVIGAM ® 2G assay which opens up new possibilities for highly specific diagnostic tools in the eradication of bTB. References 1. Buddle, BM, Ryan, TJ, Pollock, JM, Andersen, JM, and de Lisle, GW (2001). Use of ESAT-6 in the interferon- test for diagnosis of bovine tuberculosis following skin testing. Vet Microbiol, 80, 37-46 2. Sidders, B, Pirson, C, Hogarth, PJ, Hewinson, RG, Stoker, NG, and Vordermeier, HM (2008). Screening of highly expressed mycobacterial genes identifies Rv3615c as a useful differential diagnostic antigen for the Mycobacterium tuberculosis complex. Infect Immun, 76, 3932-3939 3. Schiller, I, Vordermeier, HM, Waters, WR, Palmer, M, Thacker, T, Whelan, A, Hardegger, R, Marg-Haufe, B, Raeber, A and Oesch, B (2009). Assessment of Mycobacterium tuberculosis OmpATb as a novel antigen for the diagnosis of bovine tuberculosis. Clin Vaccine Immunol, 16,1314-21 4. Branscum, AJ, Gardner, IA and Johnson WO (2005). Estimation of diagnostic-test sensitivity and specificity through Bayesian modeling. Prev Vet Med 68, 145-163 5. De la Rua-Domenech, R, Goodchild, A.T., Vordermeier, H.M., Hewinson, R.G., Christiansen, K.H., Clifton-Hadley, R.S. (2006) Ante mortem diagnosis of tuberculosis in cattle: a review of the tuberculin tests, gamma-interferon assay and other ancillary diagnostic techniques." Res Vet Sci 81: 190-210
S4 - O - 04 MATRIX-ASSISTED LASER DESORPTION IONIZATION-TIME OF FLIGHT MASS SPECTROMETRY (MALDI-TOF MS) IN A VETERINARY DIAGNOSTIC LABORATORY Annet E. Heuvelink, Refke Peerboom, Erik van Engelen, Abdul A. Hassan GD Animal Health Service, Deventer, The Netherlands MALDI-TOF, bacterial identification, veterinary laboratory Introduction Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) has emerged as a new tool for fast and reliable identification of microorganisms (). We evaluated the performance of MALDI-TOF MS for the routine identification of bacteria in our veterinary diagnostic laboratory. Materials & methods Mass spectra were acquired using a Microflex LT mass spectrometer and analysed by MALDI Biotyper 3.0 software (Bruker Daltonik GmbH, Bremen, Germany). First, we determined the accuracy of MALDI-TOF MS identification by testing 66 bacterial strains of culture collections, in duplicate spots on the MALDI target plate. Second, we performed a prospective validation study, including 262 isolates collected in the routine laboratory: 156 isolates from post-mortem examinations of mammals and 106 mastitis pathogens from cow’s milk. The isolates were identified by conventional biochemical methods in parallel with MALDI-TOF MS, measured in duplicate spots. A selection of Gram-positive cocci, including coagulase-negative staphylococci (CNS) and Streptococcus uberis isolates, were tested by both direct colony testing and the extended direct transfer method described by the manufacturer, comprising a rapid partial extraction with 70% aqueous formic acid. Finally, the repeatability and the robustness of identification of bacteria by MALDI-TOF MS were evaluated. The repeatability was determined with five reference strains, by testing 10 cfu from one plate, in one run, by one technician. The robustness was evaluated by testing the effect of (a) refrigerated storage of fresh cultures grown on agar plates (for 24 or 48 h), (b) the use of selective solid media, and (c) prolonged incubation (for 48 or 72 h) either or not followed by a refrigeration step. For each of the species included in the robustness test, one reference strain and five field isolates were tested. some isolates, especially those of S. uberis, more not reliable identifications were obtained when performed with cultures stored for 48 h in the refrigerator or incubated for 48 h, and especially for 72 h prior to identification. Discussion & conclusions The present results show that MALDI-TOF MS is a reliable and rapid method for identification of most bacterial species encountered in our routine veterinary laboratory. No misidentifications occurred, only “no reliable identifications”. For the majority of the isolates direct colony testing can be performed without extraction. For identification of presumptive CNS isolates, it is recommended to use the simple partial extraction procedure with 70% aqueous formic acid following the instructions of the manufacturer. One of the additional benefits of MALDI-TOF MS analysis is the identification of CNS species. Species level identification of CNS will help to elucidate sources, transmission mechanisms and the impact of different CNS species on cow health, productivity and milk quality, and this knowledge may lead to species-specific infection control measures. References 1. Bizzini A, Greub G. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, a revolution in clinical microbial identification. Clin. Microbiol. Infect. 2010, 16(11), 1614-1619. 2. Wieser A, Schneider L, Jung J, Schubert S. MALDI-TOF MS in microbiological diagnostics-identification of microorganisms and beyond (mini review). Appl. Microbiol. Biotechnol. 2012, 93(3), 965-974. Results Of the 66 reference strains tested, 79% were correctly identified to species level. The remaining strains were identified to genus level (14%) or could not be identified (7%). The latter strains belonged to species not included in the BioTyper 3.0 database. For 90% of the 156 isolates from post-mortem examinations of mammals matching identifications at species level were obtained between MALDI-TOF MS and biochemical methods, 8% were correctly identified to genus level, and for 2% no reliable identifications were achieved. Eighty-nine% of the MALDI-TOF MS identifications of 106 mastitis pathogens were concordant with standard biochemical identifications. Ten% of the mastitis pathogens were identified to species level by MALDI-TOF MS (mainly CNS), while standard biochemical identifications of these isolates were at genus level only. For one isolate the MALDI-TOF MS result diverged from the biochemical identification; MALDI- TOF MS identified the isolate as Klebsiella pneumoniae, a qualitatively good result at species level, while the API 20E (bioMérieux, Marcy l’Etoile, France) result was Klebsiella oxytoca, with a doubtful profile. Most bacteria tested in this study could be successfully identified at species level using the direct colony testing method. However, for 70% of the CNS isolates the partial extraction procedure showed to be superior. For S. uberis the superiority of the extended direct transfer method appeared to be less pronounced, being observed for only 32% of the isolates. The repeatability of MALDI-TOF MS identification of the reference strains of Escherichia coli, Clostridium perfringens, Staphylococcus aureus, and Salmonella Typhimurium was 100%. For the Streptococcus suis reference strain it was 90%, with 10% being not reliably identified. In general, MALDI-TOF MS identification results were not altered when performed with cultures stored for 24 or 48 h at refrigeration temperatures or with cultures tested after 48 or 72 h of incubation, nor when selective agars were used. However, for
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2 nd CONGRESSOFTHEEUROPEAN ASSOCIAT
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Welcome Dear colleagues, Welcome to
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S3-P-06 KELLER SELINA S3-P-07 KÖNI
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