title: Evaluation of a nested-PCR-derived sequence-based typing method applied directly to respiratory samples from patients with Legionnaires' disease.
abstract: Sequence-based typing (SBT) is a powerful method based on the sequencing of seven genes of Legionella pneumophila isolates. SBT performed directly on clinical samples has been used only in a limited number of cases. In our study, its efficiency was tested with 63 legionellosis respiratory samples. Sixty-three clinical samples, which included 23 samples from sporadic cases and 40 collected during four French outbreaks, confirmed by culture or urinary antigen testing and all positive by L. pneumophila quantitative PCR were subtyped by SBT according to the European Working Group for Legionella Infections standard scheme. Only 28.6% of the samples provided nucleotide sequences by SBT. Nested-PCR-based SBT (NPSBT) applied to the same respiratory samples was thus evaluated with new PCR primers surrounding the first set of primers used for the SBT. Sequencing results were obtained with 90.5% of the samples. Complete allelic profiles (seven genes sequenced) were obtained for 3.2% versus 53.9% of the samples by SBT and NPSBT, respectively. More importantly, of the 28 culture-negative samples, only 4 did not give any sequencing results. Taken together, NPSBT applied directly to clinical specimens significantly improved epidemiological typing compared to the initial SBT, in particular when no isolates are available.
authors.affiliation: Université de Lyon, Lyon, France.
authors.forename: Christophe
authors.initials: C
authors.lastname: Ginevra
authors.forename: Marie
authors.initials: M
authors.lastname: Lopez
authors.forename: Françoise
authors.initials: F
authors.lastname: Forey
authors.forename: Monique
authors.initials: M
authors.lastname: Reyrolle
authors.forename: Helene
authors.initials: H
authors.lastname: Meugnier
authors.forename: François
authors.initials: F
authors.lastname: Vandenesch
authors.forename: Jerome
authors.initials: J
authors.lastname: Etienne
authors.forename: Sophie
authors.initials: S
authors.lastname: Jarraud
authors.forename: Maëlle
authors.initials: M
authors.lastname: Molmeret
mesh_terms: D015373:Bacterial Typing Techniques / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D005602:France; D005838:Genotype; D006801:Humans; D016952:Legionella pneumophila / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D007877:Legionnaires' Disease / Q000175:diagnosis* / Q000382:microbiology*; D017720:Molecular Epidemiology / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D017931:DNA Primers