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

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.