title: Molecular detection and species-specific identification of medically important Aspergillus species by real-time PCR in experimental invasive pulmonary aspergillosis.
abstract: Diagnosis of invasive pulmonary aspergillosis (IPA) remains a major challenge to clinical microbiology laboratories. We developed rapid and sensitive quantitative PCR (qPCR) assays for genus- and species-specific identification of Aspergillus infections by use of TaqMan technology. In order to validate these assays and understand their potential diagnostic utility, we then performed a blinded study of bronchoalveolar lavage (BAL) fluid specimens from well-characterized models of IPA with the four medically important species. A set of real-time qPCR primers and probes was developed by utilizing unique ITS1 regions for genus- and species-specific detection of the four most common medically important Aspergillus species (Aspergillus fumigatus, A. flavus, A. niger, and A. terreus). Pan-Aspergillus and species-specific qPCRs with BAL fluid were more sensitive than culture for detection of IPA caused by A. fumigatus in untreated (P < 0.0007) and treated (P ≤ 0.008) animals, respectively. For infections caused by A. terreus and A. niger, culture and PCR amplification from BAL fluid yielded similar sensitivities for untreated and treated animals. Pan-Aspergillus PCR was more sensitive than culture for detection of A. flavus in treated animals (P = 0.002). BAL fluid pan-Aspergillus and species-specific PCRs were comparable in sensitivity to BAL fluid galactomannan (GM) assay. The copy numbers from the qPCR assays correlated with quantitative cultures to determine the pulmonary residual fungal burdens in lung tissue. Pan-Aspergillus and species-specific qPCR assays may improve the rapid and accurate identification of IPA in immunocompromised patients.
authors: Transplantation-Oncology Infectious Diseases Program, Weill Medical College of Cornell University, 1300 York Ave., Rm. A-421, New York, NY 10065, USA. thw2003@med.cornell.edu
authors: Thomas J
authors: TJ
authors: Walsh
authors: Mark C
authors: MC
authors: Wissel
authors: Kevin J
authors: KJ
authors: Grantham
authors: Ruta
authors: R
authors: Petraitiene
authors: Vidmantas
authors: V
authors: Petraitis
authors: Miki
authors: M
authors: Kasai
authors: Andrea
authors: A
authors: Francesconi
authors: Margaret P
authors: MP
authors: Cotton
authors: Johanna E
authors: JE
authors: Hughes
authors: Lora
authors: L
authors: Greene
authors: John D
authors: JD
authors: Bacher
authors: Pradip
authors: P
authors: Manna
authors: Martin
authors: M
authors: Salomoni
authors: Steven B
authors: SB
authors: Kleiboeker
authors: Sushruth K
authors: SK
authors: Reddy
mesh_terms: D000818:Animals; D001230:Aspergillus / Q000235:genetics / Q000302:isolation & purification*; D058491:Bacterial Load; D001992:Bronchoalveolar Lavage Fluid / Q000382:microbiology; D017931:DNA Primers / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000235:genetics; D004195:Disease Models, Animal; D005260:Female; D055744:Invasive Pulmonary Aspergillosis / Q000175:diagnosis* / Q000382:microbiology*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009172:Mycology / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics; D011817:Rabbits; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors
chemical_list: D017931:DNA Primers; D021903:DNA, Ribosomal Spacer; D015345:Oligonucleotide Probes