New multiplex PCR based methodology for the diagnosis of Aspergillus species directly from clinical samples

J Carvalho-Pereira1, J Springer2, MJ Buítrago3, J Löffler2, C Pais1, P Sampaio1

Author address: 

1Centre of Molecular and Environmental Biology (CBMA), University of Minho, Braga, Portugal 2Medizinische Klinik II, IIUniversitätklinikum Würzburg, Germany 3Mycology Department, Instituto de Salud Carlos III, Madrid, Spain

Abstract: 

Purpose: Aspergillus are opportunistic pathogenic fungus that cause invasive infections in immunocompromised patients, known as invasive aspergillosis (IA). Due to the delay in clinical diagnosis IA presents high morbidity and mortality rates. The rapid and correct identification of the species from the Aspergillus complex is essential since the susceptibility to antifungal drugs is different. In clinical microbiology laboratories several biochemical and serological methodologies are available, however all techniques present low specificity and sensibility. We developed a new methodology based in a multiplex PCR analysis, for the specific identification of four of the most clinically important Aspergillus species, namely Aspergillus fumigatus, A. flavus, A. niger and A. terreus.

Methods: This methodology is based on the design of a multiplex PCR reaction panel for species identification. This panel combines fluorescence with molecular weight of specific PCR fragments for the identification of A. fumigatus, A. flavus, A. niger and A. terreus. The PCR fragments obtained are then analysed by capillary electrophoresis and GeneScan fragment analysis. This methodology was optimized using DNA extracted from strains previously identified and, in order to optimize the methodology to identify Aspergillus sp. DNA from clinical samples we also used serum from healthy donors spiked with different concentrations of DNA from A. fumigatus and A. terreus and DNA extracted from blood of IA patients. DNA was extracted by using the QIAmp Ultrasens Virus Kit (QIAGEN).

Results: The optimization of the method by using DNA from known strains belonging to the target species, and strains form other species, showing 100% of specificity. The calculated yield from DNA extracted from serum spiked with DNA from A. fumigatus and A. terreus was of around 80%. This DNA was then used to determine the sensitivity of the technique and results showed that we were able to obtain amplification products with 20pg of the total DNA extracted. Results obtained with DNA extracted from blood of IA patients, showed that although very few samples tested so far, we were able to detect the specific DNA amplification in 75% (3 out of 4 samples). However, several optimizations are being performed, expand even further the limit of detection, such as the increase of the number of PCR cycles to 45.

Conclusion: This new methodology is a promising method that combines the molecular weigh of a specific marker with a fluorescent dye, allowing the identification of the four most important Aspergillus species involved in IA. It is fast, accurate and reproducible and due to the design of the identification panel it is able to identify the species involved in mixed infections.

 

2018

Full conference title: 

The 8th Advances Against Aspergillus, Lisbon Conference Center, Lisbon, Portugal
    • AAA 8th (2018)