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Open Access Highly Accessed Methodology article

Proposal of a quantitative PCR-based protocol for an optimal Pseudomonas aeruginosa detection in patients with cystic fibrosis

Florence Le Gall12, Rozenn Le Berre13, Sylvain Rosec4, Jeanne Hardy1, Stéphanie Gouriou1, Sylvie Boisramé-Gastrin1, Sophie Vallet12, Gilles Rault5, Christopher Payan12 and Geneviève Héry-Arnaud12*

Author Affiliations

1 EA 3882-Laboratoire de Biodiversité et d’Ecologie Microbienne (LUBEM), SFR 148 ScInBioS, Faculté de Médecine, Université de Brest, Brest F-29200, France

2 Département de Bactériologie-Virologie, Hygiène Hospitalière et Parasitologie-Mycologie, CHRU Brest, Brest F-29200, France

3 Département de Médecine Interne et Pneumologie, CHRU Brest, Brest F-29200, France

4 INSERM-CIC 0502, CHRU Brest, Brest F-29200, France

5 Centre de Perharidy, CRCM, Roscoff F-29680, France

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BMC Microbiology 2013, 13:143  doi:10.1186/1471-2180-13-143

Published: 21 June 2013

Abstract

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.

Keywords:
Pseudomonas aeruginosa; Cystic fibrosis; qPCR; Early detection