{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T19:01:16Z","timestamp":1790103676333,"version":"4.0.1"},"reference-count":34,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2026,9,17]],"date-time":"2026-09-17T00:00:00Z","timestamp":1789603200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pathogens"],"abstract":"<jats:p>The laboratory diagnosis of Legionnaires\u2019 disease (LD) remains a significant challenge due to the limitations of current methods. Most LD cases are diagnosed through the detection of Legionella urine antigen using commercial assays that are usually specific just for Legionella pneumophila (Lp) serogroup 1. Isolating Lp through culture is rarely done and difficult to achieve, especially due to two important issues: the low sensitivity of the method and the fact that it is difficult to obtain respiratory secretions from a significant proportion of patients. Real-time PCR (qPCR) has significantly improved LD diagnosis, indicating that molecular methods based on Legionella DNA detection can be highly effective. This study aimed to adapt a qPCR assay to a digital PCR (dPCR) assay, taking advantage of the more sensitive and more sophisticated dPCR technology. In particular, the study focused on 42 respiratory specimens, including bronchoalveolar lavage, tracheal aspirates, and sputum samples, with a particular focus on culture-negative specimens presumed to contain low concentrations of Legionella DNA, which were considered particularly suitable for assessing the potential of dPCR to improve the diagnosis of Legionnaires\u2019 disease. The results of culture, qPCR, and dPCR of respiratory samples were compared with each other and with data from urinary antigen tests (UATs) available for the corresponding patients. Overall, dPCR detected Legionella DNA in additional respiratory specimens compared with qPCR, corresponding to a 19% increase in the number of qPCR-negative specimens yielding a positive dPCR result. dPCR also detected Legionella DNA in specimens from patients with negative urinary antigen results. These findings suggest that dPCR may have potential as a complementary molecular approach for improving Legionella DNA detection in respiratory specimens, particularly in culture-negative samples. However, the clinical significance of dPCR-positive\/qPCR-negative results could not be independently confirmed in this study and requires further investigation.<\/jats:p>","DOI":"10.3390\/pathogens15090989","type":"journal-article","created":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T17:56:41Z","timestamp":1790099801000},"page":"989","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Can Digital PCR Enhance the Detection of Legionella pneumophila in Legionnaires\u2019 Disease Cases?"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1377-178X","authenticated-orcid":false,"given":"Fabiola","family":"Mancini","sequence":"first","affiliation":[{"name":"Department of Infectious Diseases, Istituto Superiore di Sanit\u00e0, 00161 Rome, Italy"},{"name":"Public Health Wales NHS Trust, Cardiff CF10 4BZ, UK"},{"name":"ESCMID Study Group for Legionella Infections (ESGLI), 4051 Basel, Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Enza Bruna","family":"Bonazza","sequence":"additional","affiliation":[{"name":"Department of Infectious Diseases, Istituto Superiore di Sanit\u00e0, 00161 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6615-4227","authenticated-orcid":false,"given":"Antonino","family":"Bella","sequence":"additional","affiliation":[{"name":"Department of Infectious Diseases, Istituto Superiore di Sanit\u00e0, 00161 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Antonietta","family":"Girolamo","sequence":"additional","affiliation":[{"name":"Department of Infectious Diseases, Istituto Superiore di Sanit\u00e0, 00161 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Massimo","family":"Mentasti","sequence":"additional","affiliation":[{"name":"Public Health Wales NHS Trust, Cardiff CF10 4BZ, UK"},{"name":"ESCMID Study Group for Legionella Infections (ESGLI), 4051 Basel, Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maria Luisa","family":"Ricci","sequence":"additional","affiliation":[{"name":"Department of Infectious Diseases, Istituto Superiore di Sanit\u00e0, 00161 Rome, Italy"},{"name":"Public Health Wales NHS Trust, Cardiff CF10 4BZ, UK"},{"name":"ESCMID Study Group for Legionella Infections (ESGLI), 4051 Basel, Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maria","family":"Scaturro","sequence":"additional","affiliation":[{"name":"Department of Infectious Diseases, Istituto Superiore di Sanit\u00e0, 00161 Rome, Italy"},{"name":"Public Health Wales NHS Trust, Cardiff CF10 4BZ, UK"},{"name":"ESCMID Study Group for Legionella Infections (ESGLI), 4051 Basel, Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,9,17]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Sancha, D.L., Cotos, S.A., S\u00e1nchez, L.M., and Mu\u00f1oz Bellido, J.L. (2024). Present and Future Applications of Digital PCR in Infectious Diseases Diagnosis. Diagnostics, 14.","DOI":"10.3390\/diagnostics14090931"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Quan, P.L., Sauzade, M., and Brouzes, E. (2018). dPCR: A technology review. Sensors, 18.","DOI":"10.3390\/s18041271"},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Oon, Y.L., Oon, Y.S., Ayaz, M., Deng, M., Li, L., and Song, K. (2023). Waterborne pathogens detection technologies: Advances, challenges, and future perspectives. Front. Microbiol., 14.","DOI":"10.3389\/fmicb.2023.1286923"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"107294","DOI":"10.1016\/j.mimet.2025.107294","article-title":"Diagnostic tools for detection of pathogens and diseases in plantation crops","volume":"239","author":"Sarmah","year":"2025","journal-title":"J. Microbiol. Methods"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"117566","DOI":"10.1016\/j.cca.2023.117566","article-title":"Clinical value of droplet digital PCR in the diagnosis and dynamic monitoring of suspected bacterial bloodstream infections","volume":"550","author":"Li","year":"2023","journal-title":"Clin. Chim. Acta"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"143","DOI":"10.1186\/s13071-023-05756-7","article-title":"Digital PCR: Modern solution to parasite diagnostics and population trait genetics","volume":"16","year":"2023","journal-title":"Parasit. Vectors"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"63","DOI":"10.1186\/s13054-022-03948-8","article-title":"Digital PCR applications for the diagnosis and management of infection in critical care medicine","volume":"26","author":"Merino","year":"2022","journal-title":"Crit. Care"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"e24152","DOI":"10.1002\/jcla.24152","article-title":"Coronavirus Disease 2019 (COVID-19): Emerging detection technologies and auxiliary analysis","volume":"36","author":"Chen","year":"2022","journal-title":"J. Clin. Lab. Anal."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"37","DOI":"10.1016\/j.ymeth.2026.03.013","article-title":"Digital PCR as a potential reference measurement procedure to support monkeypox virus\/Orthopoxvirus external quality assessment schemes","volume":"251","author":"Falak","year":"2026","journal-title":"Methods"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"345210","DOI":"10.1016\/j.aca.2026.345210","article-title":"Standardized digital PCR assay validation using PCR-ValiPal, demonstrated in cross-platform quantification of bovine papilloma virus","volume":"1394","author":"Gleerup","year":"2026","journal-title":"Anal. Chim. Acta"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"124269","DOI":"10.1016\/j.envres.2026.124269","article-title":"Real-time outlier detection in digital PCR data for wastewater-based pathogen surveillance","volume":"298","author":"McLeod","year":"2026","journal-title":"Environ. Res."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"129381","DOI":"10.1016\/j.talanta.2026.129381","article-title":"Piezoelectric printing-based droplet digital PCR for noninvasive and sensitive detection of Helicobacter pylori in stool samples","volume":"302","author":"Li","year":"2026","journal-title":"Talanta"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"121074","DOI":"10.1016\/j.cca.2026.121074","article-title":"Multi-omics precision diagnosis of brucellosis: Advances in biomarker discovery and clinical application","volume":"590","author":"Cai","year":"2026","journal-title":"Clin. Chim. Acta"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"51","DOI":"10.1186\/s13613-024-01252-y","article-title":"Severe Legionnaires\u2019 disease","volume":"14","author":"Rello","year":"2024","journal-title":"Ann. Intensive Care"},{"key":"ref_15","unstructured":"European Centre for Disease Prevention and Control (2023). Legionnaires\u2019 disease. ECDC. Annual Epidemiological Report for 2021, ECDC."},{"key":"ref_16","first-page":"29","article-title":"I risultati del sistema di sorveglianza nazionale della legionellosi coordinato dall\u2019Istituto Superiore di Sanit\u00e0, anno 2023","volume":"5","author":"Iera","year":"2024","journal-title":"Boll. Epidemiol. Naz."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1016\/j.mimet.2015.06.014","article-title":"Evaluation of a new lateral flow test for detection of Streptococcus pneumoniae and Legionella pneumophila urinary antigen","volume":"116","author":"Uldum","year":"2015","journal-title":"J. Microbiol. Methods"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"e01429-19","DOI":"10.1128\/JCM.01429-19","article-title":"Method Comparison of the ImmuView L. pneumophila and L. longbeachae Urinary Antigen Test with the BinaxNOW Legionella Urinary Antigen Card for Detection of Legionella pneumophila Serogroup 1 Antigen in Urine","volume":"58","author":"Badoux","year":"2020","journal-title":"J. Clin. Microbiol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"70","DOI":"10.1016\/j.ymeth.2026.02.014","article-title":"Clinical assessment of the diagnostic performance of the LIAISON Legionella urinary antigen assay","volume":"249","author":"Cusato","year":"2026","journal-title":"Methods"},{"key":"ref_20","first-page":"1800032","article-title":"Improvement of Legionnaires\u2019 disease diagnosis using real-time PCR assay: A retrospective analysis, Italy, 2010 to 2015","volume":"50","author":"Ricci","year":"2018","journal-title":"Euro Surveill."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"401","DOI":"10.1128\/JCM.02675-15","article-title":"Diagnostic Accuracy of PCR Alone and Compared to Urinary Antigen Testing for Detection of Legionella spp.: A Systematic Review","volume":"54","author":"Avni","year":"2016","journal-title":"J. Clin. Microbiol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1275","DOI":"10.1093\/cid\/cit504","article-title":"Impact of routine systematic polymerase chain reaction testing on case finding for Legionnaires\u2019 disease: A pre-post comparison study","volume":"57","author":"Murdoch","year":"2013","journal-title":"Clin. Infect. Dis."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1387","DOI":"10.1007\/s10096-015-2363-4","article-title":"Design and validation of a qPCR assay for accurate detection and initial serogrouping of Legionella pneumophila in clinical specimens by the ESCMID Study Group for Legionella Infections (ESGLI)","volume":"34","author":"Mentasti","year":"2015","journal-title":"Eur. J. Clin. Microbiol. Infect. Dis."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"276","DOI":"10.11613\/BM.2012.031","article-title":"Interrater reliability: The kappa statistic","volume":"22","author":"McHugh","year":"2012","journal-title":"Biochem. Med."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"51","DOI":"10.1111\/j.1348-0421.2002.tb02676.x","article-title":"Cross-reactivity and sensitivity of two Legionella urinary antigen kits, Biotest EIA and Binax NOW, to extracted antigens from various serogroups of L. pneumophila and other Legionella species","volume":"46","author":"Okada","year":"2002","journal-title":"Microbiol. Immunol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"2763","DOI":"10.1128\/JCM.38.7.2763-2765.2000","article-title":"Evaluation of the Binax and Biotest urinary antigen kits for detection of Legionnaires\u2019 disease due to multiple serogroups and species of Legionella","volume":"38","author":"Benson","year":"2000","journal-title":"J. Clin. Microbiol."},{"key":"ref_27","unstructured":"CDC (2026, May 26). Laboratory Testing for Legionella, Available online: https:\/\/www.cdc.gov\/legionella\/php\/laboratories."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"95","DOI":"10.1128\/CMR.00029-14","article-title":"Current and emerging Legionella diagnostics for laboratory and outbreak investigations","volume":"28","author":"Mercante","year":"2015","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"379","DOI":"10.1111\/j.1469-0691.2009.02812.x","article-title":"Quantitative real-time PCR tests for diagnostic and prognostic purposes in cases of legionellosis","volume":"16","author":"Maurin","year":"2010","journal-title":"Clin. Microbiol. Infect."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1299","DOI":"10.1007\/s15010-021-01720-8","article-title":"Members of the CAPNETZ study group. Evaluation of a multiplex PCR screening approach to identify community-acquired bacterial co-infections in COVID-19: A multicenter prospective cohort study of the German competence network of community-acquired pneumonia (CAPNETZ)","volume":"49","author":"Rothe","year":"2021","journal-title":"Infection"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"7141","DOI":"10.1007\/s11033-021-06704-3","article-title":"Occurrence of the Legionella species in the respiratory samples of patients with pneumonia symptoms from Ahvaz, Iran; first detection of Legionella cherrii","volume":"48","author":"Ghorbani","year":"2021","journal-title":"Mol. Biol. Rep."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1670","DOI":"10.1373\/clinchem.2013.211045","article-title":"Tolerance of droplet-digital PCR vs real-time quantitative PCR to inhibitory substances","volume":"59","author":"Dingle","year":"2013","journal-title":"Clin. Chem."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"115702","DOI":"10.1016\/j.bios.2023.115702","article-title":"Fluorescence-coded logarithmic-dilution digital droplet PCR for ultrawide-dynamic-range nucleic acid quantification","volume":"241","author":"Shi","year":"2023","journal-title":"Biosens. Bioelectron."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"8604","DOI":"10.1021\/ac202028g","article-title":"High-throughput droplet digital PCR system for absolute quantitation of DNA copy number","volume":"83","author":"Hindson","year":"2011","journal-title":"Anal. Chem."}],"container-title":["Pathogens"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-0817\/15\/9\/989\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T18:05:17Z","timestamp":1790100317000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-0817\/15\/9\/989"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,17]]},"references-count":34,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2026,9]]}},"alternative-id":["pathogens15090989"],"URL":"https:\/\/doi.org\/10.3390\/pathogens15090989","relation":{},"ISSN":["2076-0817"],"issn-type":[{"value":"2076-0817","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,17]]}}}