Validation and in-field testing of a new on-site qPCR system for quantification of Legionella pneumophila according to ISO/TS 12869:2012 in HVAC cooling towers.

Legionella pneumophila, found in engineered water systems such as HVAC cooling towers, poses a significant public health risk. Culture, though routinely used to quantify L. pneumophila, has several disadvantages including long turnaround time, low sensitivity, and inter-laboratory variability. In this study, we validated the performance of an on-site quantitative polymerase chain reaction (qPCR) detection system for L. pneumophila in accordance with International Standards Organization Technical Specification 12869:2012. We evaluated specificity, limit of detection and quantification, and calibration curve linearity. Additionally, we evaluated whole system recovery and robustness using samples taken from taps and evaporative cooling towers. We then compared the system's performance against laboratory culture and laboratory qPCR across 53 cooling towers in a 12-week in-field study. We found that concordance between on-site qPCR and culture was both laboratory- and site/sample-dependent. Comparison of laboratory qPCR with on-site qPCR revealed that laboratory results were highly variable and showed little concordance. Some discordance may be explained by time delay between sample collection and testing ('shipping effect') which may lead to inaccurate reporting. Overall, our study highlights the value of on-site qPCR detection of L. pneumophila, demonstrates that laboratories are prone to misreporting results due to shipping effects, and reveals significant discordance between laboratory qPCR and culture.

Oxacillin susceptibility testing of coagulase-negative staphylococci using the disk diffusion method and the Vitek GPS-105 card.

One hundred and ninety-three isolates of coagulase-negative staphylococci (CoNS) were tested against oxacillin by agar dilution, disk diffusion, and Vitek (GPS-105 card), and the presence of the mecA gene determined by multiplex PCR. The results obtained by all testing methods were in agreement for 190 isolates. Two mecA-negative isolates (S. lugdunensis and S. haemolyticus) had MICs of < or = 0.25 microg/ml by agar dilution and Vitek but were resistant by disk diffusion. One mecA-positive isolate was resistant by Vitek and disk diffusion but had an agar dilution MIC of < or = 0.25 microg/ml. For the species of CoNS tested, oxacillin susceptibility results obtained with the Vitek GPS-105 card and disk diffusion correlated well with results obtained by National Committee for Clinical Laboratory Standards agar dilution and with the presence of the mecA gene.