title: Rapid identification of fungal pathogens in BacT/ALERT, BACTEC, and BBL MGIT media using polymerase chain reaction and DNA sequencing of the internal transcribed spacer regions.
abstract: We report a direct polymerase chain reaction/sequence (d-PCRS)-based method for the rapid identification of clinically significant fungi from 5 different types of commercial broth enrichment media inoculated with clinical specimens. Media including BacT/ALERT FA (BioMérieux, Marcy l'Etoile, France) (n = 87), BACTEC Plus Aerobic/F (Becton Dickinson, Microbiology Systems, Sparks, MD) (n = 16), BACTEC Peds Plus/F (Becton Dickinson) (n = 15), BACTEC Lytic/10 Anaerobic/F (Becton Dickinson) (n = 11) bottles, and BBL MGIT (Becton Dickinson) (n = 11) were inoculated with specimens from 138 patients. A universal DNA extraction method was used combining a novel pretreatment step to remove PCR inhibitors with a column-based DNA extraction kit. Target sequences in the noncoding internal transcribed spacer regions of the rRNA gene were amplified by PCR and sequenced using a rapid (24 h) automated capillary electrophoresis system. Using sequence alignment software, fungi were identified by sequence similarity with sequences derived from isolates identified by upper-level reference laboratories or isolates defined as ex-type strains. We identified Candida albicans (n = 14), Candida parapsilosis (n = 8), Candida glabrata (n = 7), Candida krusei (n = 2), Scedosporium prolificans (n = 4), and 1 each of Candida orthopsilosis, Candida dubliniensis, Candida kefyr, Candida tropicalis, Candida guilliermondii, Saccharomyces cerevisiae, Cryptococcus neoformans, Aspergillus fumigatus, Histoplasma capsulatum, and Malassezia pachydermatis by d-PCRS analysis. All d-PCRS identifications from positive broths were in agreement with the final species identification of the isolates grown from subculture. Earlier identification of fungi using d-PCRS may facilitate prompt and more appropriate antifungal therapy.
authors.affiliation: PathWest Laboratory Medicine WA, Department of Microbiology and Infectious Diseases, Royal Perth Hospital, Perth 6847, Western Australia, Australia. todd.pryce@health.wa.gov.au
authors.forename: Todd M
authors.initials: TM
authors.lastname: Pryce
authors.forename: Silvano
authors.initials: S
authors.lastname: Palladino
authors.forename: Diane M
authors.initials: DM
authors.lastname: Price
authors.forename: Dianne J
authors.initials: DJ
authors.lastname: Gardam
authors.forename: Peter B
authors.initials: PB
authors.lastname: Campbell
authors.forename: Keryn J
authors.initials: KJ
authors.lastname: Christiansen
authors.forename: Ronan J
authors.initials: RJ
authors.lastname: Murray
mesh_terms: D003365:Costs and Cost Analysis; D003470:Culture Media; D004271:DNA, Fungal / Q000032:analysis*; D021901:DNA, Intergenic / Q000235:genetics; D006801:Humans; D003904:Mitosporic Fungi / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D009181:Mycoses / Q000175:diagnosis*; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic
chemical_list: D003470:Culture Media; D004271:DNA, Fungal; D021901:DNA, Intergenic