title: Use of real-time PCR with multiple targets to identify Pseudomonas aeruginosa and other nonfermenting gram-negative bacilli from patients with cystic fibrosis.
abstract: Pseudomonas aeruginosa and other gram-negative isolates from patients with cystic fibrosis (CF) may be difficult to identify because of their marked phenotypic diversity. We examined 200 gram-negative clinical isolates from CF respiratory tract specimens and compared identification by biochemical testing and real-time PCR with multiple different target sequences using a standardized combination of biochemical testing and molecular identification, including 16S rRNA partial sequencing and gyrB PCR and sequencing as a "gold standard." Of 50 isolates easily identified phenotypically as P. aeruginosa, all were positive with PCR primers for gyrB or oprI, 98% were positive with exotoxin A primers, and 90% were positive with algD primers. Of 50 P. aeruginosa isolates that could be identified by basic biochemical testing, 100% were positive by real-time PCR with gyrB or oprI primers, 96% were positive with exotoxin A primers, and 92% were positive with algD primers. For isolates requiring more-extensive biochemical evaluation, 13 isolates were identified as P. aeruginosa; all 13 were positive with gyrB primers, 12 of 13 were positive with oprI primers, 11 of 13 were positive with exotoxin A primers, and 10 of 13 were positive with algD primers. A single false-positive P. aeruginosa result was seen with oprI primers. The best-performing commercial biochemical testing was in exact agreement with molecular identification only 60% of the time for this most difficult group. Real-time PCR had costs similar to those of commercial biochemical testing but a much shorter turnaround time. Given the diversity of these CF isolates, real-time PCR with a combination of two target sequences appears to be the optimum choice for identification of atypical P. aeruginosa and for non-P. aeruginosa gram-negative isolates.
authors.affiliation: Department of Laboratory Medicine, Therapeutics Development Network Resource Center for Microbiology, Children's Hospital and Regional Medical Center, University of Washington, Seattle, Washington, USA.
authors.forename: Xuan
authors.initials: X
authors.lastname: Qin
authors.forename: Julia
authors.initials: J
authors.lastname: Emerson
authors.forename: Jenny
authors.initials: J
authors.lastname: Stapp
authors.forename: Lynn
authors.initials: L
authors.lastname: Stapp
authors.forename: Patrick
authors.initials: P
authors.lastname: Abe
authors.forename: Jane L
authors.initials: JL
authors.lastname: Burns
mesh_terms: D003365:Costs and Cost Analysis; D003550:Cystic Fibrosis / Q000382:microbiology*; D006090:Gram-Negative Bacteria / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000302:isolation & purification*; D013997:Time Factors