title: 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.
abstract: 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.
authors.affiliation: Respiratory and Systemic Infection Laboratory, Microbiological Services Division, Health Protection Agency, 61 Colindale Avenue, London, NW9 5EQ, UK.
authors.forename: M
authors.initials: M
authors.lastname: Mentasti
authors.forename: N K
authors.initials: NK
authors.lastname: Fry
authors.forename: B
authors.initials: B
authors.lastname: Afshar
authors.forename: C
authors.initials: C
authors.lastname: Palepou-Foxley
authors.forename: F C
authors.initials: FC
authors.lastname: Naik
authors.forename: T G
authors.initials: TG
authors.lastname: Harrison
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D006801:Humans; D016952:Legionella pneumophila / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D007877:Legionnaires' Disease / Q000175:diagnosis* / Q000453:epidemiology / Q000382:microbiology*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D017720:Molecular Epidemiology / Q000379:methods; D058889:Molecular Typing / Q000379:methods*; D019696:Peptidylprolyl Isomerase / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D001426:Bacterial Proteins; C512922:Mip protein, Legionella pneumophila; D019696:Peptidylprolyl Isomerase