Application of Legionella pneumophila-specific quantitative real-time PCR combined with direct amplification and sequence-based typing in the diagnosis and epidemiological investigation of Legionnaires’ disease
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The detection of Legionella pneumophila DNA in clinical specimens using quantitative real-time polymerase chain reaction (qPCR) combined with direct sequence-based typing (SBT) offers rapid confirmation and timely intervention in the investigation of cases of Legionnaires’ disease (LD). We assessed the utility of a specific L. pneumophila qPCR assay targeting the macrophage infectivity potentiator (mip) gene and internal process control with three clinical specimen types from confirmed LD cases. The assay was completely specific for L. pneumophila, as demonstrated by positive results for 39/39 strains from all subspecies and 16 serogroups. No cross-reaction was observed with any of the 54 Legionella non-pneumophila (0/69 strains) or 21 non-Legionella (0/58 strains). All L. pneumophila culture-positive respiratory samples (81/81) were qPCR-positive. Of 80 culture-negative samples tested, 47 (58.8%) were qPCR-positive and none were inhibitory. PCR was significantly more sensitive than culture for samples taken ≤2 days of hospitalisation (94.7% vs. 79.6%), with the difference being even more marked for samples taken between 3 and 14 days (79.3% vs. 47.8%). Overall, the sensitivity of the qPCR was ∼30% greater than that of culture and direct typing on culture-negative PCR-positive samples resulted in full 7-allele profiles from 23/46, 5 to 6 alleles from 8/46 and ≥1 allele from 43/46 strains.
KeywordsqPCR Assay Respiratory Sample Macrophage Infectivity Potentiator Internal Process Control American Type Culture Collection 33152T
We thank all the diagnostic laboratories for the submission of the clinical specimens. We also gratefully acknowledge Rod Ratcliff, Institute of Medical and Veterinary Science, Adelaide, Australia, for the provision of unpublished Legionella mip sequence data, Clare Ling, Health Protection Agency (HPA) Microbiology Services for the purified genomic DNA from M. tuberculosis and Stefano Conti, Statistics Unit, HPA—Health Protection Services for the assistance with the Probit analyses.
This work was supported, in part, by funding from the European Centre for Disease Prevention and Control, “Laboratory support for surveillance of Legionnaires’ disease at European level”. Preliminary results from this study were presented at Legionella 2009, Institut Pasteur, Paris, France, 13–17 October 2009, abstract P38 and the Society for General Microbiology Spring Meeting, Harrogate International Centre, Harrogate, UK, 30 March–2 April 2009, abstract HAR29/06.
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