title: Detection, identification, and distribution of fungi in bronchoalveolar lavage specimens by use of multilocus PCR coupled with electrospray ionization/mass spectrometry.
abstract: As pulmonary fungal infections continue to increase due to an increasing number of immunocompromised patients, rapid detection and accurate identification of these fungal pathogens are critical. A broad fungal assay was developed by incorporating broad-range multilocus PCR amplification and electrospray ionization/mass spectrometry (PCR/ESI-MS) to detect and identify fungal organisms directly from clinical specimens. The aims of this study were to evaluate the performance of PCR/ESI-MS for detection, identification, and determination of the distribution of fungal organisms in bronchoalveolar lavage (BAL) fluid specimens. The BAL fluid specimens submitted for fungal culture at Vanderbilt University Medical Center between May 2005 and October 2011 were included. Cultures and identification were done using standard procedures. In addition, DNA was extracted from BAL fluid specimens, and fungal DNA amplification/identification were performed by PCR/ESI-MS. The results were compared with those of the standard cultures. A total of 691 nonduplicated BAL fluid specimens with sufficient leftover volume for molecular testing were evaluated using PCR/ESI-MS. Among them, 134 specimens (19.4%) were positive for fungi by both culture and PCR/ESI-MS testing. Of the dual-positive specimens, 125 (93.3%) were positive for Candida and Aspergillus species, with concordances between culture and PCR/ESI-MS results being 84 (67.2%) at the species level and 109 (87.2%) at the genus level. In addition, 243 (35.2%) and 30 (4.3%) specimens were positive only by PCR/ESI-MS or by culture, respectively (odds ratio [OR] = 11.95, 95% confidence interval [CI] = 7.90 to 18.17, P = 0.0000). Codetection of fungal organisms was noted in 23 (3.3%) specimens by PCR/ESI-MS, which was significantly higher than the 4 (0.6%) in which they were noted by culture (OR = 5.91, 95% CI = 1.93 to 20.27, P = 0.0002). Among 53 specimens in which cultures failed because of bacterial overgrowth, at least one fungus was identified in 26 specimens (47.3%) by PCR/ESI-MS. PCR/ESI-MS provides an advanced tool for rapid and sensitive detection, identification, and determination of the distribution of fungal organisms directly from BAL fluid specimens. Moreover, it detected fungal organisms in specimens in which cultures failed because of bacterial overgrowth. The clinical relevance of the significantly higher detection rate of fungal organisms by PCR/ESI-MS merits further investigation.
authors.affiliation: Department of Laboratory Medicine, Inje University College of Medicine, Busan, South Korea.
authors.forename: Jeong Hwan
authors.initials: JH
authors.lastname: Shin
authors.forename: Raymond
authors.initials: R
authors.lastname: Ranken
authors.forename: Susan E
authors.initials: SE
authors.lastname: Sefers
authors.forename: Robert
authors.initials: R
authors.lastname: Lovari
authors.forename: Criziel D
authors.initials: CD
authors.lastname: Quinn
authors.forename: Shufang
authors.initials: S
authors.lastname: Meng
authors.forename: Heather E
authors.initials: HE
authors.lastname: Carolan
authors.forename: Donna
authors.initials: D
authors.lastname: Toleno
authors.forename: Haijing
authors.initials: H
authors.lastname: Li
authors.forename: Jeong Nyeo
authors.initials: JN
authors.lastname: Lee
authors.forename: Charles W
authors.initials: CW
authors.lastname: Stratton
authors.forename: Christian
authors.initials: C
authors.lastname: Massire
authors.forename: Yi-Wei
authors.initials: YW
authors.lastname: Tang
mesh_terms: D001992:Bronchoalveolar Lavage Fluid / Q000382:microbiology*; D005658:Fungi / Q000737:chemistry / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008172:Lung Diseases, Fungal / Q000175:diagnosis*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009172:Mycology / Q000379:methods*; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*