title: Quantitative multiplexed detection of common pulmonary fungal pathogens by labeled primer polymerase chain reaction.
abstract: BACKGROUND
  Invasive fungal infections are an important cause of morbidity and mortality among immunocompromised patients.
  
  
  OBJECTIVE
  To design and evaluate a multiplexed assay aimed at quantitative detection and differentiation of the 5 molds that are most commonly responsible for pulmonary infections.
  
  
  METHODS
  Using labeled primer polymerase chain reaction chemistry, an assay was designed to target the 5.8S and 28S ribosomal RNA genes of Aspergillus spp, Fusarium spp, Scedosporium spp, and members of the order Mucorales ( Rhizopus oryzae , Rhizopus microsporus, Cunninghamella bertholletiae, Mucor circinelloides, Lichtheimia corymbifera, and Rhizomucor pusillus). This assay was split into 2 multiplexed reactions and was evaluated using both samples seeded with purified nucleic acid from 42 well-characterized clinical fungal isolates and 105 archived samples (47 blood [45%], 42 bronchoalveolar lavage fluid [40%], and 16 tissue [15%]) collected from rabbit models of invasive pulmonary fungal infections.
  
  
  RESULTS
  Assay detection sensitivity was less than 25 copies of the target sequence per reaction for Aspergillus spp, 5 copies for Fusarium spp and Scedosporium spp, and 10 copies for the Mucorales. The assay showed quantitative linearity from 5 × 10(1) to 5 × 10(5) copies of target sequence per reaction. Sensitivities and specificities for bronchoalveolar lavage fluid, tissue, and blood samples were 0.86 and 0.99, 0.60 and 1.00, and 0.46 and 1.00, respectively.
  
  
  CONCLUSIONS
  Labeled primer polymerase chain reaction permits rapid, quantitative detection and differentiation of common agents of invasive fungal infection. The assay described herein shows promise for clinical implementation that may have a significant effect on the rapid diagnosis and treatment of patients' severe infections caused by these pulmonary fungal pathogens.
authors.affiliation: From the Department of Pathology, St. Jude Children's Research Hospital, Memphis, Tennessee (Drs Gu, Buelow, and Hayden); and the Transplantation-Oncology Infectious Diseases Program, Weill Cornell Medical Center, New York, New York (Drs Petraitiene, Petraitis, and Walsh).
authors.forename: Zhengming
authors.initials: Z
authors.lastname: Gu
authors.forename: Daelynn R
authors.initials: DR
authors.lastname: Buelow
authors.forename: Ruta
authors.initials: R
authors.lastname: Petraitiene
authors.forename: Vidmantas
authors.initials: V
authors.lastname: Petraitis
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Walsh
authors.forename: Randall T
authors.initials: RT
authors.lastname: Hayden
mesh_terms: D000818:Animals; D001230:Aspergillus / Q000235:genetics; D004195:Disease Models, Animal; D005260:Female; D005670:Fusarium / Q000235:genetics; D005800:Genes, Fungal; D006801:Humans; D008172:Lung Diseases, Fungal / Q000175:diagnosis* / Q000382:microbiology*; D025202:Molecular Diagnostic Techniques / Q000379:methods / Q000706:statistics & numerical data; D009090:Mucorales / Q000235:genetics; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D016533:Mycological Typing Techniques / Q000379:methods / Q000706:statistics & numerical data; D012331:RNA, Fungal / Q000032:analysis / Q000235:genetics; D012339:RNA, Ribosomal, 28S / Q000032:analysis / Q000235:genetics; D012340:RNA, Ribosomal, 5.8S / Q000032:analysis / Q000235:genetics; D011817:Rabbits; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D021681:Scedosporium / Q000235:genetics; D012680:Sensitivity and Specificity
chemical_list: D012331:RNA, Fungal; D012339:RNA, Ribosomal, 28S; D012340:RNA, Ribosomal, 5.8S