PCR-based detection and identification of Burkholderia cepacia complex pathogens in sputum from cystic fibrosis patients

Citation
A. Mcdowell et al., PCR-based detection and identification of Burkholderia cepacia complex pathogens in sputum from cystic fibrosis patients, J CLIN MICR, 39(12), 2001, pp. 4247-4255
Citations number
34
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Clinical Immunolgy & Infectious Disease",Microbiology
Journal title
JOURNAL OF CLINICAL MICROBIOLOGY
ISSN journal
0095-1137 → ACNP
Volume
39
Issue
12
Year of publication
2001
Pages
4247 - 4255
Database
ISI
SICI code
0095-1137(200112)39:12<4247:PDAIOB>2.0.ZU;2-N
Abstract
PCR amplification of the recA gene followed by restriction fragment length polymorphism (RFLP) analysis was investigated for the rapid detection and i dentification of Burkholderia cepacia complex genomovars directly from sput um. Successful amplification of the B. cepacia complex recA gene from cysti c fibrosis (CF) patient sputum samples containing B. cepacia genomovar I, B urkholderia multivorans, B. cepacia genomovar III, Burkholderia stabilis, a nd Burkholderia vietnamiensis was demonstrated. In addition, the genomovar identifications determined directly from sputum were the same as those obta ined after selective culturing. Sensitivity experiments revealed that recA- based PCR could reliably detect B. cepacia complex organisms to concentrati ons of 10(6) CFU g of sputum(-1). To fully assess the diagnostic value of t he method, sputum samples from 100 CIT patients were screened for B. cepaci a complex infection by selective culturing and recA-based PCR. Selective cu lturing identified 19 samples with presumptive B. cepacia complex infection , which was corroborated by phenotypic analyses. Of the culture-positive sp utum samples, 17 were also detected directly by recA-based PCR, while 2 sam ples were negative. The isolates cultured from both recA-negative sputum sa mples were subsequently identified as Burkholderia gladioli. RFLP analysis of the recA amplicons revealed 2 patients (12%) infected Kith B. multivoran s, 11 patients (65%) infected with B. cepacia genomovar III-A, and 4 patien ts (23%) infected with B. cepacia genomovar III-B. These results demonstrat e the potential of recA-based PCR-RFLP analysis for the rapid detection and identification of B. cepacia complex genomovars directly from sputum. Wher e the sensitivity of the assay proves a limitation, sputum samples can be a nalyzed by selective culturing followed by recA-based analysis of the isola te.