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62 Molecular diagnosis of Pseudomonas aeruginosa infection in culture-negative samples from cystic fibrosis patients

62 Molecular diagnosis of Pseudomonas aeruginosa infection in culture-negative samples from cystic fibrosis patients

Authors :
Mehdi Amiri
Sonia Pasquaroli
Francesca Biavasco
Gianmarco Mangiaterra
Carla Vignaroli
Natalia Cirilli
Esther Manso
Source :
Journal of Cystic Fibrosis. 14:S73
Publication Year :
2015
Publisher :
Elsevier BV, 2015.

Abstract

Pseudomonas aeruginosa infection is the main cause of morbidity and mortality in cystic fibrosis (CF) patients. Cultural methods, the gold standard for bacterial detection, are hampered by alginate overproduction and the possible presence of dormant cells. This work aimed at developing a sensitive and specific diagnostic tool capable of detecting P. aeruginosa in culture-negative sputum from CF patients. DNA was extracted with the QIAamp DNA kit after cell spin-down to remove free DNA. Real-time PCR assays targeting 3 species-specific genes, ecfX, gyrB and oprL, were developed using previously designed and new primers. The LOD was determined by spiking different amounts of P. aeruginosa ATCC9027 in a negative sputum sample. The PCR protocol targeting oprL was found to be unreliable, whereas those targeting ecfX and gyrB proved to be effective (LOD 60 cells/ml and 102 cells/ml, respectively). Examination of 33 randomly selected culture-negative samples yielded 6 positive samples from 3 patients: all 6 were ecfX-positive and 2 were positive for ecfX and gyrB. After 1 and 5 months additional samples from patients 1 and 2 were culture-positive; patient 3 received a 4-month antibiotic cycle and 2 months later he resulted still culture-negative and PCR-positive. These findings highlight the value of the Real-time PCRs developed, especially the one targeting ecfX, in diagnosing P. aeruginosa infection in CF samples. Protocol effectiveness was confirmed by the eventual cultural recovery of P. aeruginosa in the two PCR-positive patients, who were found to have chronic CF. Findings also seem to provide further support for a role of dormant forms in recurrent CF infection.

Details

ISSN :
15691993
Volume :
14
Database :
OpenAIRE
Journal :
Journal of Cystic Fibrosis
Accession number :
edsair.doi...........f20108cb15ec09a685171cb10a5f12cc
Full Text :
https://doi.org/10.1016/s1569-1993(15)30239-3