caa a22 4500 606156038 CHVBK 20210128100606.0 cr unu---uuuuu 210128e20150701xx s 000 0 eng 10.1007/s10096-015-2363-4 doi (NATIONALLICENCE)springer-10.1007/s10096-015-2363-4 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) [Elektronische Daten] [M. Mentasti, D. Kese, F. Echahidi, S. Uldum, B. Afshar, S. David, J. Mrazek, R. De Mendonça, T. Harrison, V. Chalker] Prompt detection of Legionella pneumophila is essential for rapid investigation of legionellosis. Furthermore, as the majority of L. pneumophila infections are caused by serogroup 1 (sg1) strains, rapid identification of such strains can be critical in both routine and outbreak scenarios. The ESCMID Study Group for Legionella Infections (ESGLI) was established in 2012 and immediately identified as a priority the validation of a reliable, easy to perform and interpret, cost-effective qPCR assay to standardise the detection of L. pneumophila DNA amongst members. A novel L. pneumophila assay targeting the mip gene was designed and combined with previously published methodologies amplifying the sg1 marker (wzm) and the green fluorescent protein gene (gfp) internal process control. The resulting triplex assay was validated internationally on the three qPCR platforms used by the majority of European Legionella reference laboratories: ABI 7500 (Life Technologies), LightCycler 480 Instrument II (Roche) and Rotor-Gene Q (Qiagen). Clinical and EQA specimens were tested together with a large panel of strains (251 in total) to validate the assay. The assay proved to be 100% specific for L. pneumophila and sg1 DNA both in silico and in vitro. Efficiency values for mip and wzm assays ranged between 91.97 and 97.69%. Limit of detection values estimated with 95% confidence were adopted for mip and wzm assays on all three qPCR platforms. Inhibition was not observed. This study describes a robust assay that could be widely implemented to standardise the molecular detection of L. pneumophila among ESGLI laboratories and beyond. Crown Copyright, 2015 Mentasti M. Public Health England, 61 Colindale Avenue, NW9 5EQ, London, UK aut Kese D. University of Ljubljana, Ljubljana, Slovenia aut Echahidi F. Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel (VUB), Brussels, Belgium aut Uldum S. Statens Serum Institut, Copenhagen, Denmark aut Afshar B. Public Health England, 61 Colindale Avenue, NW9 5EQ, London, UK aut David S. Public Health England, 61 Colindale Avenue, NW9 5EQ, London, UK aut Mrazek J. Institute of Public Health, Ostrava, Czech Republic aut De Mendonça R. University Hospital Erasme, Brussels, Belgium aut Harrison T. Public Health England, 61 Colindale Avenue, NW9 5EQ, London, UK aut Chalker V. Public Health England, 61 Colindale Avenue, NW9 5EQ, London, UK aut European Journal of Clinical Microbiology & Infectious Diseases Springer Berlin Heidelberg 34/7(2015-07-01), 1387-1393 0934-9723 34:7<1387 2015 34 10096 https://doi.org/10.1007/s10096-015-2363-4 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s10096-015-2363-4 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Mentasti M. Public Health England, 61 Colindale Avenue, NW9 5EQ, London, UK aut NATIONALLICENCE 700 1- Kese D. University of Ljubljana, Ljubljana, Slovenia aut NATIONALLICENCE 700 1- Echahidi F. Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel (VUB), Brussels, Belgium aut NATIONALLICENCE 700 1- Uldum S. Statens Serum Institut, Copenhagen, Denmark aut NATIONALLICENCE 700 1- Afshar B. Public Health England, 61 Colindale Avenue, NW9 5EQ, London, UK aut NATIONALLICENCE 700 1- David S. Public Health England, 61 Colindale Avenue, NW9 5EQ, London, UK aut NATIONALLICENCE 700 1- Mrazek J. Institute of Public Health, Ostrava, Czech Republic aut NATIONALLICENCE 700 1- De Mendonça R. University Hospital Erasme, Brussels, Belgium aut NATIONALLICENCE 700 1- Harrison T. Public Health England, 61 Colindale Avenue, NW9 5EQ, London, UK aut NATIONALLICENCE 700 1- Chalker V. Public Health England, 61 Colindale Avenue, NW9 5EQ, London, UK aut NATIONALLICENCE 773 0- European Journal of Clinical Microbiology & Infectious Diseases Springer Berlin Heidelberg 34/7(2015-07-01), 1387-1393 0934-9723 34:7<1387 2015 34 10096