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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  opr L gene, and a multiplex PCR targeting  gyr B and  ecf X genes.    Results  Tested  in vitro  on a large panel of  P. aeruginosa  isolates and others gram-negative bacilli,  opr L qPCR exhibited a better sensitivity (threshold of 10 CFU/mL  versus  730 CFU/mL), whereas the  gyr B /ecf X 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.

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