title: The aspHS gene as a new target for detecting Aspergillus fumigatus during infections by quantitative real-time PCR.
abstract: Invasive aspergillosis (IA) is a serious nosocomial infection caused by Aspergillus spp. which has a high mortality rate due to the fact, among other factors, that it is difficult to diagnose. Within the Aspergillus genus, A. fumigatus is the main species causing IA. We propose a virulence factor, the aspHS gene, as a novel target for the specific detection of A. fumigatus by quantitative real-time PCR (qPCR). This target gene encodes a haemolysin, which is overexpressed in vivo during infection. We have designed specific primers and hydrolysis (Taqman) probes for the detection of this target and a chimeric internal amplification control (IC), designed to detect false negative results due to PCR inhibition. This qPCR assay was tested with DNA extracted from a wide collection of microorganisms, tissues from infected mice, and human bronchoalveolar lavage (BAL) samples. Results showed that it, together with the DNA extraction method, could detect A. fumigatus with high specificity. Furthermore, it can distinguish between germinated (first step to the development of infection) and non-germinated conidia (not detected). Our data indicate that these techniques could be sufficiently sensitive and rapid to help clinicians establish an earlier diagnosis, but the presence of PCR inhibitors in clinical samples such as BAL fluids needs to be addressed.
authors: Department of Immunology, Microbiology and Parasitology, Faculty of Science and Technology, University of the Basque Country (UPV/EHU), Leioa, Spain.
authors: Ana
authors: A
authors: Abad-Diaz-De-Cerio
authors: Jimena V
authors: JV
authors: Fernandez-Molina
authors: Andoni
authors: A
authors: Ramirez-Garcia
authors: Javier
authors: J
authors: Sendino
authors: Fernando L
authors: FL
authors: Hernando
authors: Javier
authors: J
authors: Pemán
authors: Javier
authors: J
authors: Garaizar
authors: Aitor
authors: A
authors: Rementeria
mesh_terms: D000818:Animals; D001228:Aspergillosis / Q000175:diagnosis*; D001232:Aspergillus fumigatus / Q000235:genetics / Q000302:isolation & purification*; D017931:DNA Primers / Q000235:genetics; D005260:Female; D005656:Fungal Proteins / Q000235:genetics*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D025202:Molecular Diagnostic Techniques / Q000379:methods* / Q000592:standards; D009172:Mycology / Q000379:methods* / Q000592:standards; D015345:Oligonucleotide Probes / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity; D037521:Virulence Factors / Q000235:genetics*
chemical_list: D017931:DNA Primers; D005656:Fungal Proteins; D015345:Oligonucleotide Probes; D037521:Virulence Factors