PCR as a Diagnostic Tool for the Rapid Detection of Burkholderia cepacia in the Sputum of Patients with Cystic Fibrosis ♦ 1973

Abstract

Culture detection of Burkholderia cepacia in sputum from patients with cystic fibrosis (CF) is problematic. The difficulties may be partially due to the recent demonstration that isolates currently identified as B. cepacia consist of several species, the “B. cepacia complex.” In addition, growth of B. cepacia may require 72h or more, and species from other genuses grow on selective media, requiring further testing to establish an accurate identification. Improvements in the detection of B. cepacia would not rely on culture, would detect the organism in low quantities, and would be specific to B. cepacia. The objectives of this study were twofold. The first goal was to determine if B. cepacia could be detected directly from sputum samples using PCR and the second goal was to compare the PCR detection with the standard culture detection. We examined sputum samples from two CF centers serving children and adults. Following liquefaction of the sputa using N-acetyl-L-cysteine, DNA was isolated and analyzed in PCRs with three different primer pairs directed toward bacterial ribosomal RNA loci. Two primer pairs were putatively specific for B. cepacia. The other pair, which universally amplifies a band from all bacteria, served as a control. Sputum samples were obtained from 219 patients and analyzed independently by culture and by PCR to detect B. cepacia. The analyses were performed blinded with respect to each other. Results of the PCR with sputa demonstrated that the primers directed to the 16S rRNA loci demonstrated approximately 95% concordance with culture results and were more specific than those amplifying the 16S-23S spacer region. In addition, the 16S primer pair putatively identified B. cepacia in seven patients whose sputa were culture negative at this time. Of these culture negative patients, five had sputum that was culture positive for B. cepacia either prior or subsequent to this study. The results of this study indicate the utility of PCR as a diagnostic method for the rapid identification of B. cepacia in the sputum of both child and adult CF patients. We anticipate that improvements in our taxonomic understanding may allow the design of more specific primers for detection of each species of the B. cepacia complex in sputum.