Verification and application of qPCR and viability-qPCR for Legionella monitoring in evaporative cooling systems...

To date, in many countries the only legally valid method for evaporative cooling system (ECS) monitoring is the culture method. However, a duration of up to 14 days and a risk of underestimation of Legionella concentrations are seen as limitations of cultivation methods. Rapid cultivation-independent methods are an important step towards a more practicable monitoring of ECS to quickly control interventions if elevated concentrations of Legionella are found. Two commercial kits for quantitative polymerase chain reaction (qPCR) and viability-qPCR (v-qPCR) were studied, comprising sample filtration and DNA extraction. Cryopreserved Legionella pneumophila were established as calibration standard with intact (ILC) and total Legionella count (TLC) determined by flow cytometry before conducting spiking experiments in commercial mineral water and artificial process water. Final assessment was carried out using real ECS samples. Recovery and robustness ranged from 86 to 108 % for qPCR with a drop to 40-60 % for v-qPCR when compared to direct extraction, possibly attributable to cell damage during sample concentration. All methods including culture did perform well regarding linearity with R² ≥ 0.95 for most trials. Detected concentrations in comparison to spiked Legionella counts differed with culture averaging 25 ± 7 % of spiked ILC and v-qPCR being closest to spiked concentrations with 65-144 %. In comparison, qPCR was several fold above spiked TLC concentrations. For real ECS samples Legionella spp. were detected in concentrations above 10³ GU/100 mL by v-qPCR in 70-92 % of samples, depending on the kit used. Most of these samples were either culture-negative or not evaluable on agar plates. This study showed that a cryopreserved bacterial standard based examination is applicable and can be used for future v-qPCR verification. For assessment of differences in results between culture and v-qPCR/qPCR in ECS samples expert knowledge about the operating mode and used analytical methods is required. Guidelines addressing this issue could be a solution.