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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
- 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.
- Subjects :
- Pulmonary and Respiratory Medicine
Pseudomonas aeruginosa
medicine.drug_class
business.industry
Cell
Antibiotics
Gold standard (test)
medicine.disease
medicine.disease_cause
Cystic fibrosis
Microbiology
chemistry.chemical_compound
medicine.anatomical_structure
chemistry
Pediatrics, Perinatology and Child Health
medicine
Sputum
medicine.symptom
business
Gene
DNA
Subjects
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