Droplet Digital PCR and PCR-free BIOSensors for the diagnosis of resistance-associated SNPs in Pneumocystis jirovecii
( DDBIOS )

Research Project: 2024-04-01 - 2027-03-31
Total sum awarded: €698 532

Pneumocystis jirovecii is a respiratory fungus, whose infection can lead to the development of Pneumocystis pneumonia (PcP), especially in immunosuppressed patients. There are no available systems for its in vitro culture – currently used diagnostic methods are based mainly on molecular techniques which enable detection and analysis of single nucleotide polymorphisms (SNPs) within genes encoding targets for agents widely used in PcP prevention and treatment. SNPs development may result from the previous use of such agents and be associated with antimicrobial resistance (AMR). However, PCR-based methods still have several limitations and new tools which enable rapid and sensitive pathogen identification and SNPs assessment are required. The aim of the proposed research is the development and evaluation of two methods – droplet digital PCR and PCR-free biosensors – in the diagnosis of P. jirovecii, as well as in the quantitative and qualitative analysis of SNPs within four pathogen genes potentially associated with AMR (DHPS, DHFR, IMPDH, CYB), in biological samples collected from patients at high risk of infection. The final objective of the project is to create the database based on the results obtained – the distribution of identified SNPs and their ratio to the wild type, in relation to the examined patients’ data, will allow to determine the potential consequences of the use of specific drugs on AMR emergence, as well as the possible impact of such mutations on the effectiveness of treatment in the context of given infection risk factors.

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  • Marta Kicia, Wroclaw Medical University, Poland (Coordinator)
  • Enrique Calderón, University of Sevilla, Spain (Partner)
  • Solène Le Gal, University of Brest, France (Partner)

Pneumocystis jirovecii is a unicellular, opportunistic fungus occurring in human lungs, whose infection can lead to the development of Pneumocystis pneumonia in susceptible individuals, especially those with immunosuppression. The widespread use of prophylaxis may lead to the emergence of mutations in pathogen’s genome, which can be potentially associated with the development of its resistance, and thus patient’s susceptibility to infection despite the use of preventive measures. Due to the lack of available in vitro research methods on this issue, access to tools enabling not only rapid, sensitive and specific P. jirovecii diagnosis, but also identification of mutations potentially associated with its resistance is required. The aim of this project is therefore to develop solutions based on droplet digital PCR and PCR-free biosensors - techniques meeting these criteria, which would allow the detection of P. jirovecii DNA in respiratory specimens from patients at risk, at the same time enabling qualitative and quantitative characterization of mutations in genes encoding targets for most commonly used anti-Pneumocystis agents. The planned result of our research is to create the database of detected mutations potentially associated with pathogen’s resistance, in correlation with the data of the examined patients. This would allow to determine the potential consequences of the use of specific therapeutics in patients at risk, as well as the possible influence of these mutations on the effectiveness of anti-Pneumocystis prophylaxis / treatment in an infected individual.

Call

2023 - Development of innovative strategies, tools, technologies, and methods for diagnostics and surveillance of antimicrobial resistance