title: Rapid presumptive identification of Burkholderia pseudomallei with real-time PCR assays using fluorescent hybridization probes.
abstract: Burkholderia pseudomallei (the etiologic agent of melioidosis) can cause pyogenic or granulomatous lesions in almost any organ. Septicemia has a case fatality rate of >40%. Early diagnosis and appropriate antibiotic therapy are crucial for survival, but cultivation, biochemical identification, and conventional PCR of B. pseudomallei are time consuming. We established real-time PCR assays using fluorescent hybridization probes targeting the 16S rDNA, the flagellin C (fliC) and the ribosomal protein subunit S21 (rpsU) genes. The test sensitivity and specificity were assessed with a representative panel of 39 B. pseudomallei, 9 B. mallei, 126 other Burkholderia strains of 29 species, and 45 clinically relevant non-Burkholderia organisms. The detection limit for the 16S rDNA, fliC, and rpsU assay was 40, 40, and 400 genome equivalents per reaction, however, in spiked blood samples it was 300, 300, and 3000, respectively. Specificity, positive and negative predictive value of the assays was 100%. In conclusion, we recommend the use of the 16S rDNA and/or fliC real-time PCR assays for the rapid identification of B. mallei and B. pseudomallei in positive blood cultures or from suspicious bacterial colonies.
authors.affiliation: Federal Armed Forces Institute of Microbiology, Neuherbergstrasse 11, Munich 80937, Germany. herbert.tomaso@web.de
authors.forename: Herbert
authors.initials: H
authors.lastname: Tomaso
authors.forename: Tyrone L
authors.initials: TL
authors.lastname: Pitt
authors.forename: Olfert
authors.initials: O
authors.lastname: Landt
authors.forename: Sascha
authors.initials: S
authors.lastname: Al Dahouk
authors.forename: Holger C
authors.initials: HC
authors.lastname: Scholz
authors.forename: Emil C
authors.initials: EC
authors.lastname: Reisinger
authors.forename: Lisa D
authors.initials: LD
authors.lastname: Sprague
authors.forename: Ilka
authors.initials: I
authors.lastname: Rathmann
authors.forename: Heinrich
authors.initials: H
authors.lastname: Neubauer
mesh_terms: D016957:Burkholderia pseudomallei / Q000235:genetics / Q000302:isolation & purification*; D015342:DNA Probes; D004275:DNA, Ribosomal / Q000097:blood / Q000235:genetics; D005408:Flagellin / Q000097:blood / Q000235:genetics; D005456:Fluorescent Dyes; D006801:Humans; D008554:Melioidosis / Q000175:diagnosis*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012269:Ribosomal Proteins / Q000097:blood / Q000235:genetics; D012680:Sensitivity and Specificity
chemical_list: D015342:DNA Probes; D004275:DNA, Ribosomal; D005456:Fluorescent Dyes; D012269:Ribosomal Proteins; C032643:ribosomal protein S21; D005408:Flagellin