title: Establishment and application of real-time quantitative PCR for diagnosing invasive aspergillosis via the blood in hematological patients: targeting a specific sequence of Aspergillus 28S-ITS2.
abstract: BACKGROUND
  Invasive aspergillosis (IA) is an important cause of morbidity and mortality in immunocompromised individuals. This study was conducted to identify a desirable target DNA sequence for the diagnosis of aspergillosis using real-time quantitative polymerase chain reaction (qPCR).
  
  
  METHODS
  Genomic DNA was extracted from Aspergillus, Candida, and bacteria species, and qPCR was applied to validate a partial ribosomal DNA 28S-ITS2 sequence. Ethylenediaminetetraacetic acid-anticoagulated blood samples were collected from 72 febrile hematological patients, while total DNA was isolated from plasma and whole blood for the Aspergillus qPCR. The results were analyzed using a receiver operating characteristic curve. All cases were evaluated using the revised European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) diagnostic criteria.
  
  
  RESULTS
  Use of qPCR yielded positive results for 15 Aspergillus species but negative results for Candida species, bacterial strains, and human DNA. The limit of detection was one copy per microliter of DNA. Analytical sensitivity and specificity were six copies of DNA and 100%, respectively. The standard curve showed that qPCR was reliable for Aspergillus detection and that significantly more DNA copies were obtained from whole blood than from plasma (P < 0.001). At a cut-off value ≥ 25 copies/μL, the diagnostic sensitivity and specificity for IA using 28S-ITS2 qPCR were 90.9% and 73.4%, respectively.
  
  
  CONCLUSIONS
  The use of qPCR with whole blood to detect and verify the 28S-ITS2 sequence is a specific and useful way to diagnose IA.
authors.affiliation: Department of Hematology and BMT center, Chinese PLA General Hospital, 28 Fuxing Road, Beijing 100853, China. chunhuiliyu@yahoo.com.
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.forename: Li
authors.initials: L
authors.lastname: Gao
authors.forename: Yi
authors.initials: Y
authors.lastname: Ding
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Xu
authors.forename: Minhang
authors.initials: M
authors.lastname: Zhou
authors.forename: Wenrong
authors.initials: W
authors.lastname: Huang
authors.forename: Yu
authors.initials: Y
authors.lastname: Jing
authors.forename: Honghua
authors.initials: H
authors.lastname: Li
authors.forename: Lili
authors.initials: L
authors.lastname: Wang
authors.forename: Li
authors.initials: L
authors.lastname: Yu
mesh_terms: D000328:Adult; D000368:Aged; D001230:Aspergillus / Q000235:genetics* / Q000302:isolation & purification; D002648:Child; D002675:Child, Preschool; D004271:DNA, Fungal / Q000032:analysis / Q000235:genetics; D005260:Female; D019337:Hematologic Neoplasms / Q000097:blood / Q000382:microbiology*; D006801:Humans; D055744:Invasive Pulmonary Aspergillosis / Q000175:diagnosis* / Q000382:microbiology / Q000473:pathology; D057230:Limit of Detection; D008297:Male; D008875:Middle Aged; D012372:ROC Curve; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D055815:Young Adult
chemical_list: D004271:DNA, Fungal