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

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.