Assessment of fluorescent in situ hybridization and PCR-based methods for rapid identification of Burkholderia cepacia complex organisms directly from sputum samples.
Several species within the Burkholderia cepacia complex (BCC) have emerged as significant opportunistic pathogens of patients with cystic fibrosis (CF). BCC infection is typically associated with a poor clinical prognosis and decreased survival. These factors, combined with the existence of highly transmissible epidemic strains, have resulted in strict segregation of BCC- and non-BCC-infected patients to minimize cross infection. Accurate and rapid diagnosis of infections is essential to enable appropriate patient management. However, the rapidly evolving taxonomy of BCC poses a considerable challenge to diagnostics. In the present study, we assessed a commercially available fluorescent in situ hybridization (FISH) assay (seaFAST Cystic Fibrosis I kit) and a novel rRNA gene-based PCR assay for the rapid identification of BCC-positive sputa, irrespective of the BCC species. We report that, while the FISH assay fails to identify all BCC species, it does identify the majority of species, including the two most clinically relevant species, B. multivorans and B. cenocepacia. The sensitivity of the assay applied to sputum was limited by nonspecific background fluorescence. While sputum processing was optimized to minimize background, the resulting sensitivity for BCC detection was 8 x 10(5) CFU/ml. In contrast, the novel PCR assay reported herein exhibits 100% sensitivity and specificity for all BCC species and can detect 10(4) CFU/ml when applied to sputum. This novel rRNA gene-based assay is currently the most sensitive BCC-specific PCR assay for the detection of BCC direct from clinical samples and as such is a valuable addition to the field of BCC diagnostics.
Brown
A R
AR
Cystic Fibrosis Group, Centre for Infectious Diseases, University of Edinburgh, The Chancellor's Building, Edinburgh EH16 4SB, United Kingdom. alan.brown@ed.ac.uk
Govan
J R W
JR
D015373:Bacterial Typing Techniques; D019121:Burkholderia Infections / Q000382:microbiology; D042602:Burkholderia cepacia complex / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D003550:Cystic Fibrosis / Q000382:microbiology*; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D011933:Reagent Kits, Diagnostic; D013183:Sputum / Q000382:microbiology*; D013997:Time Factors
D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S; D011933:Reagent Kits, Diagnostic