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

The European Directive 2020/2184 concerning the quality of water for human consumption now includes <i>Legionella</i> 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 <i>Legionella pneumophila</i>. 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 <i>Legionella pneumophila</i> 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 <i>Legionella</i> (<i>P</i> < 0.0001).IMPORTANCE<i>Legionella</i> 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 <i>Legionella</i> prevention has made an important contribution to <i>Legionella</i> surveillance. According to the European Directive 2020/2184, techniques already known can be used for <i>Legionella</i> 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.

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Journal
Applied and Environmental Microbiology
Published
2025-05-29
DOI
https://doi.org/10.1128/aem.00236-25
Primary Topic
Legionella and Acanthamoeba research
Type
article
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article

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

Osvalda De Giglio, Silvia Livi, Alessio Facciolà, Sandra Cristino et al.
Applied and Environmental Microbiology
Legionella and Acanthamoeba research
article

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

Osvalda De Giglio, Silvia Livi, Alessio Facciolà, Sandra Cristino, M Talini, Eduardo A. Ceccarelli, Donella Gestri, Marinella Franchi, C. Della Croce, Laura De Lellis, Anna Maria Spagnolo, A. Moschin, Pasqualina Laganà, Simona Spiteri, P. Miana, Antonella Agodi, Claudio Ottaviano, Fabio Ferrari, Anna Giammanco, Chiara Romano, Andrea Caprini, E. Ballarini, Maria Giovanna Guiso, Sabina Palmieri, Antonino Bella, Maria Luisa Ricci, Melania Dragone, Mariagabriella Viggiani, A. Rossi, Antonella Mansi, Giusy Diella, Ivan Corradi, Teresa Fasciana, Maria Scaturro, Maria Anna Coniglio, M. Cavallaro, G N R Vespa, Annalisa Bargellini, Isabella Marchesi, Anna Maria Marcelloni, Stefano Della Sala, Maria Teresa Montagna, Elisabetta Graziano, Daria Barberis, Antonella Felice, Fabrizia Helfer, Florida Damasco, Valentina Coroneo, Anna Maria Marella, Giovanna La Vecchia, Annalisa Grucci, Massimiliano Ingrassia, Maria Luisa Cristina, Antonietta Girolamo, Maria Cristina Rota, Adriana Sanna, Marina Foti, Mariangela Pagano, M. Fortin, Francesca Borney, Maria Teresa Buratto, Gaetano Caricato, Filomena Casaburi, Giovanna Tozzi, Daniela De Mirto, Marianna Mari, Giorgia Lugarini, Rossana Amari
article en

Abstract

The European Directive 2020/2184 concerning the quality of water for human consumption now includes <i>Legionella</i> 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 <i>Legionella pneumophila</i>. 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 <i>Legionella pneumophila</i> 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 <i>Legionella</i> (<i>P</i> < 0.0001).IMPORTANCE<i>Legionella</i> 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 <i>Legionella</i> prevention has made an important contribution to <i>Legionella</i> surveillance. According to the European Directive 2020/2184, techniques already known can be used for <i>Legionella</i> 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.

Applied and Environmental MicrobiologyVol. 91(6)
University of Messina (IT), Agenzia Regionale per la Protezione Ambientale del Piemonte (IT), University of Udine (IT), University of Cagliari (IT), Istituto Superiore di Sanità (IT), University of Catania (IT), Veritas (Italy) (IT), Istituto per lo Studio e la Prevenzione Oncologica (IT), DataMED (Italy) (IT), Azienda Sanitaria Unità Locale di Reggio Emilia (IT), RINA Consulting - Centro Sviluppo Materiali S.p.A. (Italy) (IT), Centro Ricerche Produzioni Animali (IT), Ospedali Riuniti Marche Nord (IT), Agenzia Regionale Prevenzione e Ambiente della Regione Emilia-Romagna (IT), Istituto Nazionale per l'Assicurazione Contro gli Infortuni sul Lavoro (IT), StemMed (United States) (US), Agenzia Regionale per la Protezione dell'ambiente ligure (IT), Accademia di Belle Arti di Catanzaro (IT), European Society of Clinical Microbiology and Infectious Diseases (CH), Tecnologie Avanzate (Italy) (IT), Osservatorio Astronomico della Regione Autonoma Valle d'Aosta (IT), Novamont (Italy) (IT), Agenzia Sanitaria Regionale Abruzzo (IT), University of Bari Aldo Moro (IT), University of Genoa (IT), Advanced Research Projects Agency - Energy (US), European Automobile Manufacturers Association (BE), University of Bologna (IT)
Bentham-Moxon Trust
Clean water and sanitation
Openalex Percentile: Top 90%
Legionella and Acanthamoeba research
DOIOpen Access PDFOpenAlex
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