title: Identification of Pseudomonas aeruginosa, Burkholderia cepacia complex, and Stenotrophomonas maltophilia in respiratory samples from cystic fibrosis patients using multiplex PCR.
abstract: A multiplex PCR method was developed to identify P. aeruginosa, B. cepacia complex, and S. maltophilia directly in sputum and oropharyngeal samples from CF patients. One hundred and six patients (53 male, and 53 female) attending our pulmonology clinic were studied from September 2000-April 2001. Two hundred and fifty-seven samples were cultured in selective media and submitted to multiplex PCR reactions, using three primer pairs targeting specific genomic sequences of each species, with an additional primer pair targeting a stretch of ribosomal 16S DNA, universal for bacteria, to act as a control. P. aeruginosa was isolated by culture in 56% of samples, B. cepacia complex in 4.3%, and S. maltophilia in 2.7%, while multiplex PCR identified P. aeruginosa in 78.7%, B. cepacia complex in 3.9%, and S. maltophilia in 3.1% of samples. Multiplex PCR results were verified by PCR reactions using different species-specific primers described in the literature and DNA sequencing of amplicons from a few samples. Comparing to culture results, the sensitivity and specificity values of multiplex PCR for bacterial identification were, respectively, 97.2% and 45.5% for P. aeruginosa, 45.5% and 97.9% for B. cepacia complex, and 40% and 97.6% for S. maltophilia. All 10 multiplex PCR-positive results for B. cepacia complex were confirmed using other species-specific primers described in the literature, while this approach confirmed results for S. maltophilia identification in 7/8 samples (87.5%). Sequencing of amplicons from samples culture-negative but multiplex PCR-positive for P. aeruginosa and B. cepacia complex confirmed their identity, while minor nucleotide differences among amplicons ruled out the hypothesis of PCR contamination.
authors.affiliation: Instituto da Criança Prof. Pedro de Alcântara, Hospital das Clínicas, University of São Paulo Medical School, São Paulo, Brazil. vicres@usp.br
authors.forename: Luiz V F
authors.initials: LV
authors.lastname: da Silva Filho
authors.forename: Adriana F
authors.initials: AF
authors.lastname: Tateno
authors.forename: Luciana de F
authors.initials: Lde F
authors.lastname: Velloso
authors.forename: José E
authors.initials: JE
authors.lastname: Levi
authors.forename: Silvana
authors.initials: S
authors.lastname: Fernandes
authors.forename: Christina N O
authors.initials: CN
authors.lastname: Bento
authors.forename: Joaquim C
authors.initials: JC
authors.lastname: Rodrigues
authors.forename: Sonia R T S
authors.initials: SR
authors.lastname: Ramos
mesh_terms: D000293:Adolescent; D000328:Adult; D016956:Burkholderia cepacia / Q000235:genetics* / Q000302:isolation & purification*; D002648:Child; D002675:Child, Preschool; D003550:Cystic Fibrosis / Q000382:microbiology*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis; D005260:Female; D006801:Humans; D008297:Male; D009960:Oropharynx / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D011550:Pseudomonas aeruginosa / Q000235:genetics* / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D013183:Sputum / Q000382:microbiology; D020615:Stenotrophomonas maltophilia / Q000235:genetics* / Q000302:isolation & purification*
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial