Since mutations or gene deletions occur on PCR target sequences, they could decrease the sensitivity of the method [29]. Moreover, horizontal genetic transfer with other bacterial species present in the CF lung niche can impact upon the specificity
of the PCR [14]. In a prospective TSA HDAC GNS-1480 manufacturer multicenter study, we aimed to assess the role of PCR for the early detection of P. aeruginosa in CF patients; we evaluated two qPCRs in detection of P. aeruginosa: a simplex qPCR targeting oprL gene [30], and a multiplex qPCR, targeting gyrB and ecfX genes [14]. The sensitivity and the specificity of both qPCRs were initially evaluated testing a large panel of P. aeruginosa isolates and closely related non-P. aeruginosa gram-negative bacilli isolates from PKC412 mouse CF patients. Then, the two different
qPCRs ability in detection of P. aeruginosa were tested ex vivo, i.e in CF sputum samples. Finally, we were able to propose a promising reference protocol combining these two qPCRs for an optimal detection of P. aeruginosa in clinical setting. Methods Bacterial collection Thirty-six P. aeruginosa isolates, including mucoid and non mucoid forms, were obtained from 31 sputum samples of CF patients and from 5 samples of non CF patients (blood, n = 1; stool, n = 1; urine, n = 1; sputum, n = 1; peritoneal fluid, n = 1), attending three French University Hospitals, the CHRU of Brest (n = 3), the CHU of Nantes (n = 26), and the GHSR Avelestat (AZD9668) of Saint Pierre, La Réunion (n = 2). The reference strain P. aeruginosa CIP 76.110 was also included in the study. Forty-one closely related non-P. aeruginosa gram-negative bacillus isolates were collected, including 26 obtained from sputum samples of CF patients, and 15 from clinical samples of non CF patients (n = 13) or environmental samples (n = 2). Sixteen species were represented: Achromobacter xylosoxidans (n = 9), P. putida (n = 5), Stenotrophomonas maltophilia (n = 5), Burkholderia cepacia (n = 4), B. multivorans (n = 3), B. gladioli (n = 2), Chryseobacterium indologenes (n = 2), Elizabethkingia meningoseptica (n = 2), P. stutzeri (n = 2), B. cenocepacia (n = 1), Flavimonas oryzihabitans
(n = 1), Pandoraea pnomenusa (n = 1), P. fluorescens (n = 1), Ralstonia picketti (n = 1), Roseomonas spp. (n = 1), and Shewanella putrefaciens (n = 1). Identification of bacterial isolates was previously conducted based on phenotypical and morphological criteria (colony morphology, pigmentation, lactose fermentation, oxidase activity checked with 1% tetramethyl p-phenylenediamine dihydrochloride, sensitivity to antibiotics). Atypical P. aeruginosa isolates, for which difficulties of identification were encountered, were further analyzed with biochemical tests [API 20NE system (bioMérieux, Marcy l’Etoile, France), ID 32GN (bioMérieux)], or with the gram-negative bacillus identification card on VITEK 2 Compact (bioMéreux). All non- P.