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The aspHS gene as a new target for detecting Aspergillus fumigatus during infections by quantitative real-time PCR
Ana Abad-Diaz-De-Cerio1, Jimena V Fernandez-Molina, Andoni Ramirez-Garcia
1Department of Immunology, Microbiology and Parasitology, Faculty of Science and Technology, University of the Basque Country (UPV/EHU), Leioa, Spain.
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.
Insights
A new quantitative real-time PCR (qPCR) method targets the aspHS gene for specific detection of Aspergillus fumigatus, the primary cause of invasive aspergillosis (IA). This rapid diagnostic tool aids in early IA diagnosis by identifying the pathogen in clinical samples.
Area of Science:
- Medical Microbiology
- Molecular Diagnostics
- Infectious Diseases
Background:
- Invasive aspergillosis (IA) is a severe fungal infection with high mortality, often due to diagnostic challenges.
- Aspergillus fumigatus is the predominant species responsible for IA.
- Early and accurate diagnosis is crucial for effective patient management.
Purpose of the Study:
- To develop a novel, specific, and rapid diagnostic method for Aspergillus fumigatus detection.
- To validate the aspHS gene as a target for quantitative real-time PCR (qPCR) in diagnosing IA.
- To assess the assay's performance in detecting A. fumigatus in various biological samples.
Main Methods:
- Design and implementation of a specific qPCR assay targeting the aspHS virulence gene.
- Development of specific primers and Taqman hydrolysis probes for high-throughput detection.
- Inclusion of a chimeric internal amplification control (IC) to monitor for PCR inhibition.
- Testing the assay with diverse microbial DNA, infected mouse tissues, and human bronchoalveolar lavage (BAL) samples.
Main Results:
- The developed qPCR assay demonstrated high specificity for detecting Aspergillus fumigatus.
- The assay successfully differentiated between germinated and non-germinated A. fumigatus conidia.
- The method showed potential for early diagnosis, distinguishing viable infectious agents.
- Effectiveness was validated across various sample types, including simulated clinical specimens.
Conclusions:
- The aspHS gene is a promising novel target for the specific and rapid detection of Aspergillus fumigatus.
- The developed qPCR assay offers a sensitive tool for early diagnosis of invasive aspergillosis.
- Further optimization is needed to address PCR inhibitors present in clinical samples like BAL fluid for robust clinical application.
