title: Use of mycological, nested PCR, and real-time PCR methods on BAL fluids for detection of Aspergillus fumigatus and A. flavus in solid organ transplant recipients.
abstract: Invasive aspergillosis continues to be a significant cause of morbidity and mortality in solid organ transplant (SOT) recipients. A reliable and early diagnostic method is needed to improve survival. In this study, four methods direct microscopy, culture, nested PCR on internal transcribed spacer region, and TaqMan real-time PCR targeted β-tubulin gene were examined for the detection of Aspergillus fumigatus and A. flavus in sixty-four bronchoalveolar lavage (BAL) fluids that were obtained from SOT recipients. Direct examination with 20 % KOH (potassium hydroxide) and culture on mycological media were also performed. Of the 64 samples, seven (10.9 %) were positive in direct examination (five with septate hyphae and two with aseptate hyphae), and 15 (23 %) had positive culture including five A. flavus, four A. niger, two Penicillium spp., two Rhizopus spp., one Fusarium spp. and one mixed A. flavus/A. niger. Twenty five (39 %) samples had positive nested PCR with A. flavus and 6 (9.4 %) with A. fumigatus-specific primers. Only eight (12.5 %) had positive real-time PCR for A. flavus and nine (14 %) for A. fumigatus. The incidence of aspergillosis in these patients included proven invasive pulmonary aspergillosis (IPA) in two (3 %), probable IPA in 14 (22 %), possible IPA in 38 (59 %), and not IPA in 10 (16 %). A. flavus was the most common cause of pulmonary aspergillosis (PA) in the study. The results suggest that because nested PCR is too sensitive it may increase the number of false-positive results and is not recommended for BAL samples for diagnosis of PA. Although further studies with significant number of proved positive/negative standard BAL samples are necessary for better evaluation, the novel multiplex real-time PCR developed in the study could be promising as a valid diagnostic method for IPA.
authors.affiliation: Department of Medical Parasitology and Mycology, School of Medicine, Ghaem Hospital, Mashhad University of Medical Sciences, Mashhad, Iran.
authors.forename: Hossein
authors.initials: H
authors.lastname: Zarrinfar
authors.forename: Hossein
authors.initials: H
authors.lastname: Mirhendi
authors.forename: Koichi
authors.initials: K
authors.lastname: Makimura
authors.forename: Kazuo
authors.initials: K
authors.lastname: Satoh
authors.forename: Hossein
authors.initials: H
authors.lastname: Khodadadi
authors.forename: Omolbanin
authors.initials: O
authors.lastname: Paknejad
mesh_terms: D000328:Adult; D001228:Aspergillosis / Q000175:diagnosis* / Q000382:microbiology; D001231:Aspergillus flavus / Q000235:genetics / Q000302:isolation & purification*; D001232:Aspergillus fumigatus / Q000235:genetics / Q000302:isolation & purification*; D001992:Bronchoalveolar Lavage Fluid / Q000382:microbiology*; D003430:Cross-Sectional Studies; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D025202:Molecular Diagnostic Techniques / Q000379:methods; D009172:Mycology / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014180:Transplantation; D019737:Transplants; D055815:Young Adult
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