Interlaboratory comparison of culture- and PCR-based methods for Legionella pneumophila detection in drinking water samples

Abstract

The European Directive 2020/2184 concerning the quality of water for human consumption now includes Legionella among the pathogens for assessment in domestic water systems. It states that "for risk-based verification and to complement spread-plate culture methods, rapid culture methods, non-culture-based methods, and molecular-based methods may be used." In this study, 33 laboratories across Italy analyzed a number of unique water samples ranging from 10 to 30 for the presence of Legionella pneumophila. All laboratories used the standard spread-plate culture method, 31 laboratories also performed the Legiolert rapid liquid culture method (IDEXX Laboratories), and 27 out of 33 performed the DI-Check Legionella pneumophila real-time PCR method (Diatheva). In all, 23 laboratories executed all three methods. Data generated from 817 samples were collected and statistically analyzed. The Legiolert method allowed analysis with a smaller sample volume (100 mL and 10 mL), compared to the standard culture method with which it was shown to be comparable with K agreement values of 0.785 and 0.840 in the two mentioned volumes, respectively. The standard real-time PCR method was more sensitive (93%) than the spread-plate culture method. Sensitivity values of 95.2% and 98.8% were also obtained by comparing two new real-time PCR procedures with the spread-plate culture method, tested to shorten the analysis, and the standard culture method. Finally, data obtained from the analysis of drinking water samples with the spread-plate culture method using both Buffered charcoal yeast extract and non-selective glycine vancomycin polymyxin cycloheximide media showed the greater capacity of the latter in the recovery of Legionella (P < 0.0001).IMPORTANCELegionella is a waterborne fastidious pathogen that occasionally infects humans and can cause a severe form of pneumonia, called Legionnaires' disease (LD), which, if not identified in a timely manner, can progress to multiorgan failure. The trend for LD cases is steadily rising, and prevention and control of water system contamination is the only way to stop or prevent the spread of further cases. In Italy, since 2005, a network of regional reference laboratories for Legionella prevention has made an important contribution to Legionella surveillance. According to the European Directive 2020/2184, techniques already known can be used for Legionella detection in addition to the standard culture method. To ensure the reliability of the results and guarantee the correct risk evaluation, a comparison between the standard culture method and real-time PCR and the Legiolert rapid liquid culture method was required.

Keywords: BCYE; EU Directive 2020/2184; GVPC; ISO 11731; Legiolert; Legionella; Legionella pneumophila; Legionnaires disease; drinking water testing; real-time PCR.

Fig 1

(A) Graphical representation of ISO17994 analysis of data generated during the trial. The mean relative difference for all comparisons is above “0.” In three out of four comparisons, the lower confidence limit was above the upper threshold (10%) recommended for drinking water, indicating that Legiolert is more sensitive. (B) Summary of ISO 17994 mean relative difference analysis comparing the Legiolert 100 mL (717 samples) and 10 mL (272 samples) tests versus the spread-plate method on BCYE and GVPC media (817 samples).