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Journal of Clinical Microbiology, April 2009, p. 981-987, Vol. 47, No. 4
0095-1137/09/$08.00+0     doi:10.1128/JCM.02071-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Evaluation of a Nested-PCR-Derived Sequence-Based Typing Method Applied Directly to Respiratory Samples from Patients with Legionnaires' Disease {triangledown}

Christophe Ginevra,1,2,3,4 Marie Lopez,3,4 Françoise Forey,3,4 Monique Reyrolle,3,4 Helene Meugnier,3,4 François Vandenesch,1,2,3,4 Jerome Etienne,1,2,3,4 Sophie Jarraud,1,2,3,4 and Maëlle Molmeret1,2,3,4*

Université de Lyon, Lyon, France,1 INSERM, U851, 21 Avenue Tony Garnier, Lyon, F-69007, France,2 Université Lyon 1, IFR128, Lyon, France,3 Hospices Civils de Lyon, Faculté de Médecine Laënnec, Lyon, France4

Received 27 October 2008/ Returned for modification 22 December 2008/ Accepted 11 February 2009

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.

* Corresponding author. Mailing address: Université de Lyon, INSERM U851, Faculté de Médecine Laennec, 7 rue Guillaume Paradin, 69372 Lyon cedex 08, France. Phone: 33 (0) 478 77 86 57. Fax: 33 (0) 478 77 86 58. E-mail: maelle.molmeret{at}univ-lyon1.fr

{triangledown} Published ahead of print on 18 February 2009.

Journal of Clinical Microbiology, April 2009, p. 981-987, Vol. 47, No. 4
0095-1137/09/$08.00+0     doi:10.1128/JCM.02071-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Coscolla, M., Gonzalez-Candelas, F. (2009). Direct Sequencing of Legionella pneumophila from Respiratory Samples for Sequence-Based Typing Analysis. J. Clin. Microbiol. 47: 2901-2905 [Abstract] [Full Text]  


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