MULTICENTER STUDY USING STANDARDIZED PROTOCOLS AND REAGENTS FOR EVALUATION OF REPRODUCIBILITY OF PCR-BASED FINGERPRINTING OF ACINETOBACTER SPP

Seven laboratories in six European countries examined 40 isolates belo nging to the Acinetobacter calcoaceticus-Acinetobacter baumannii compl ex to investigate whether standardized protocols and quality-controlle d reagents could produce reliable, discriminatory, and reproducible PC R-based fingerprinting results, Four PCR protocols with different prim ers (primers DAF4, ERIC-2, M13, and REP1 + REP2) were used. The epidem iological conclusions reached by the participating laboratories were s ubstantially correct, with 96.4% of the total isolate grouping allocat ions agreeing with the consensus view, All laboratories identified the main epidemiological clusters, and each laboratory also identified tw o non-outbreak-related isolates. There were no significant differences between the isolate grouping results obtained by the different protoc ols and with the different primers, Visual comparison indicated that t he standardized protocols and reagents yielded reproducible fingerprin t patterns, but with some variations in particular band intensities. M inor variations in fingerprint profiles were detected, but computer-as sisted analysis of PCR fingerprints obtained on agarose gels demonstra ted that 88.3 to 91.6% (depending on the source of DNA) of the pattern s clustered correctly, while 96.4 to 98.9% of the patterns clustered c orrectly following automated high-resolution laser fluorescence analys is. Correlation of the patterns for isogenic isolates ranged from 83.3 to 86.6% but was slightly better (mean correlation, 87.1%) for centra lly prepared DNA extracts than for DNA extracts prepared by individual laboratories (mean correlation, 84.7%). It was concluded that indepen dently produced PCR fingerprint patterns can be obtained reproducibly for Acinetobacter spp. at the practical level if (i) quality-controlle d reagents, (ii) standardized extraction of DNA, and (iii) standardize d amplification conditions are used.