%0 Journal Article
%T Proposal of a quantitative PCR-based protocol for an optimal Pseudomonas aeruginosa detection in patients with cystic fibrosis
%+ Laboratoire de Parasitologie et Mycologiede [CHRU Brest]
%+ Laboratoire Universitaire de Biodiversité et Ecologie Microbienne (LUBEM)
%+ Département de Médecine Interne et Pneumologie
%+ Centre d'Investigation Clinique (CIC - Brest)
%+ Centre de Ressources et de Compétences de la Mucoviscidose (CRCM)
%A Le Gall, Florence
%A Le Berre, Rozenn
%A Rosec, Sylvain
%A Hardy, Jeanne
%A Gouriou, Stéphanie
%A Boisramé-Gastrin, Sylvie
%A Vallet, Sophie
%A Rault, Gilles
%A Payan, Christopher
%A Héry-Arnaud, Geneviève
%Z This study was supported by a grant from the French Cystic Fibrosis Association "Vaincre la Mucoviscidose" (contract No. RCO 1773).
%< avec comité de lecture
%@ 1471-2180
%J BMC Microbiology
%I BioMed Central
%V 13
%N 1
%P 143
%8 2013
%D 2013
%K Pseudomonas aeruginosa
%K Cystic fibrosis
%K qPCR
%K Early detection
%Z Life Sciences [q-bio]/Microbiology and ParasitologyJournal articles
%X Background
The lung of patients with cystic fibrosis (CF) is particularly sensitive to Pseudomonas aeruginosa. This bacterium plays an important role in the poor outcome of CF patients. During the disease progress, first acquisition of P. aeruginosa is the key-step in the management of CF patients. Quantitative PCR (qPCR) offers an opportunity to detect earlier the first acquisition of P. aeruginosa by CF patients. Given the lack of a validated protocol, our goal was to find an optimal molecular protocol for detection of P. aeruginosa in CF patients.
Methods
We compared two formerly described qPCR formats in early detection of P. aeruginosa in CF sputum samples: a qPCR targeting oprL gene, and a multiplex PCR targeting gyrB and ecfX genes.
Results
Tested in vitro on a large panel of P. aeruginosa isolates and others gram-negative bacilli, oprL qPCR exhibited a better sensitivity (threshold of 10 CFU/mL versus 730 CFU/mL), whereas the gyrB/ecfX qPCR exhibited a better specificity (90% versus 73%). These results were validated ex vivo on 46 CF sputum samples positive for P. aeruginosa in culture. Ex vivo assays revealed that qPCR detected 100 times more bacterial cells than culture-based method did.
Conclusion
Based on these results, we proposed a reference molecular protocol combining the two qPCRs, which offers a sensitivity of 100% with a threshold of 10 CFU/mL and a specificity of 100%. This combined qPCR-based protocol can be adapted and used for other future prospective studies.
%G English
%2 https://inserm.hal.science/inserm-00837789/document
%2 https://inserm.hal.science/inserm-00837789/file/1471-2180-13-143.pdf
%L inserm-00837789
%U https://inserm.hal.science/inserm-00837789
%~ INSERM
%~ UNIV-BREST
%~ LUBEM
%~ IBSAM