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.
Acknowledgments
Technical and human support provided by SGIker (UPV/EHU, MICINN, GV/EJ, ERDF and ESF) is gratefully acknowledged. We are grateful to Ideas Need Communicating Language Services for improving the use of English in the manuscript.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper.
This work was partly funded by the UPV/EHU (grants GIU0820 and UFI11/25), and the Government of the Basque Country (grants IT343-10, S-PC09UN04, and S-PC11UN007). Jimena V. Fernandez Molina has been supported by Pre-doctoral Research Grants of the University of Basque Country (UPV/EHU).
This paper was first published online on Early Online on 21 January 2013.