title: Incidence of pulmonary aspergillosis and correlation of conventional diagnostic methods with nested PCR and real-time PCR assay using BAL fluid in intensive care unit patients.
abstract: BACKGROUND
  Although the incidence of invasive aspergillosis in the intensive care unit (ICU) is scarce, it has emerged as major problems in critically ill patients. In this study, the incidence of pulmonary aspergillosis (PA) in ICU patients has evaluated and direct microscopy and culture has compared with nested polymerase chain reaction (PCR) and real-time PCR for detection of Aspergillus fumigatus and A. flavus in bronchoalveolar lavage (BAL) samples of the patients.
  
  
  METHODS
  Thirty BAL samples obtained from ICU patients during a 16-month period were subjected to direct examinations on 20% potassium hydroxide (KOH) and culture on two culture media. Nested PCR targeting internal transcribed spacer ribosomal DNA and TaqMan real-time PCR assay targeting β-tubulin gene were used for the detection of A. fumigatus and A. flavus.
  
  
  RESULTS
  Of 30 patients, 60% were men and 40% were women. The diagnosis of invasive PA was probable in 1 (3%), possible in 11 (37%), and not IPA in 18 (60%). Nine samples were positive in nested PCR including seven samples by A. flavus and two by A. fumigatus specific primers. The lowest amount of DNA that TaqMan real-time PCR could detect was ≥40 copy numbers. Only one of the samples had a positive result of A. flavus real-time PCR with Ct value of 37.5.
  
  
  CONCLUSIONS
  Although a significant number of specimens were positive in nested PCR, results of this study showed that establishment of a correlation between the conventional methods with nested PCR and real-time PCR needs more data confirmed by a prospective study with a larger sample group.
authors.affiliation: Department of Medical Parasitology and Mycology, Ghaem Hospital, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
authors.forename: Hossein
authors.initials: H
authors.lastname: Zarrinfar
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: Hossein
authors.initials: H
authors.lastname: Mirhendi
mesh_terms: D000328:Adult; 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; D004271:DNA, Fungal / Q000032:analysis; D005260:Female; D006801:Humans; D015994:Incidence; D007362:Intensive Care Units; D008297:Male; D008875:Middle Aged; D009172:Mycology / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods*; D055732:Pulmonary Aspergillosis / Q000175:diagnosis / Q000453:epidemiology
chemical_list: D004271:DNA, Fungal