title: Accurate identification of members of the Burkholderia cepacia complex in cystic fibrosis sputum.
abstract: The Burkholderia cepacia complex (BCC) is a group of closely related species which includes opportunistic pathogens causing chronic respiratory infections in immunocompromised patients, or individuals affected by cystic fibrosis (CF). Other Burkholderia species causing infection in the CF population are Burkholderia gladioli and Burkholderia pseudomallei. Traditional phenotypic analyses have been demonstrated to be inadequate for reliable identifications of isolates of BCC and B. gladioli. A pan-genomic analysis approach was used to design species-specific probes for Burkholderia cenocepacia, B. cepacia, Burkholderia multivorans, Burkholderia vietnamiensis, Burkholderia ambifaria, Burkholderia dolosa, Burkholderia pyrrocinia and B. gladioli. Multiplex real-time PCR assay was developed and tested using sputum specimens collected from CF patients spiked with Burkholderia species. The assay exhibited 100% sensitivity for all eight target species and detected 10(2) to 10(3)  CFU ml(-1) when applied to spiked sputum. Our PCR assay resulted highly specific for each of the Burkholderia species tested, allowing discrimination among Burkholderia and non-Burkholderia pathogens. Analysis carried out on 200 sputa positive for the presence of Burkholderia revealed that PCR assay and recA sequencing were fully comparable for identification of Burkholderia at the level of species.
  
  
  CONCLUSIONS
  Burkholderia cepacia complex (BCC) has a complex taxonomic organization and its identification is a challenge for microbiology laboratories. Nonidentification or misidentification of BCC isolates represent a problem in epidemiology and treatment of cystic fibrosis patients. The high specificity and sensitivity of the multiplex Real-time PCR assay developed in this study indicates its potential to be a rapid and reliable method for the detection of Burkholderia at the level of species from sputum samples of cystic fibrosis patients.
authors.affiliation: Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università degli Studi di Napoli Federico II, Napoli, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Martinucci
authors.affiliation: Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università degli Studi di Napoli Federico II, Napoli, Italy.
authors.forename: E
authors.initials: E
authors.lastname: Roscetto
authors.affiliation: Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università degli Studi di Napoli Federico II, Napoli, Italy.
authors.forename: V D
authors.initials: VD
authors.lastname: Iula
authors.affiliation: Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università degli Studi di Napoli Federico II, Napoli, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Votsi
authors.affiliation: Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università degli Studi di Napoli Federico II, Napoli, Italy.
authors.forename: M R
authors.initials: MR
authors.lastname: Catania
authors.affiliation: Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università degli Studi di Napoli Federico II, Napoli, Italy.
authors.forename: E
authors.initials: E
authors.lastname: De Gregorio
mesh_terms: D001483:Base Sequence; D019121:Burkholderia Infections / Q000382:microbiology; D042602:Burkholderia cepacia complex / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D003550:Cystic Fibrosis / Q000382:microbiology*; D006801:Humans; D016867:Immunocompromised Host; D008297:Male; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D011938:Rec A Recombinases / Q000235:genetics*; D012141:Respiratory Tract Infections / Q000382:microbiology; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D013183:Sputum / Q000382:microbiology*
chemical_list: D011938:Rec A Recombinases
keywords: Burkholderia cepacia complex; Burkholderia spp; molecular diagnostics; multiplex TaqMan real-time PCR; respiratory infections