A SuperSelective primer-based real-time PCR Platform for hypersensitive detection of azole heteroresistance in Cryptococcus neoformans

Cryptococcus neoformans is the leading cause of fungal meningitis with limited treatment options, making early and accurate diagnosis critical for improved patient outcome. Current diagnostic methods for cryptococcosis rely largely on capsule antigen detection and fungal culture, which are time-consuming and unable to identify mutation-driven antifungal heteroresistance. In this study, we have developed a SuperSelective primer-based PCR (SSP-PCR) platform for the rapid and specific detection of azole resistance-associated single-nucleotide polymorphisms (SNPs). We demonstrate that SSP-PCR reliably detected a single copy mutant allele in the presence of excess wild-type (WT) DNA, with sensitivity reaching a 1:104 mutant-to-WT ratio. Incorporating molecular beacon (MB) probes with our SSP-PCR platform further enhanced amplification specificity, enabling selective detection of the ERG11 Y145F (A434T) mutation that is known to cause high azole resistance. Using genomic DNA from in vitro cultures and mouse lung tissues infected with either WT strain H99 strain or a fluconazole-hyper-resistant mrl1 clinical isolate that carries the ERG11(A434T) mutation, or both strains, we successfully detected the A434T mutant allele in both settings. Moreover, our SSP-PCR simultaneously identified ERG11(A434T) and the multi-azole resistance-associated ERG11(G1885A) mutant alleles in a single-tube duplex reaction. Collectively, the SSP-PCR platform provides a robust and ultrasensitive molecular approach for the detection of azole resistance and heteroresistance in C. neoformans, with strong potential for high-throughput clinical screening applications. ImportanceInvasive fungal infections are a growing public health threat, causing over 1.5 million deaths annually, with cryptococcal meningitis accounting for over 15% HIV/AIDS related mortality. The problem is aggravated by limited treatment options and emerging of drug resistance. Long-term use of fungistatic azoles like fluconazole promotes the emergence of azole heteroresistance, contributing to clinical treatment failure. Current diagnostic assays often fail to detect resistance-associated mutations within heterogeneous fungal populations, limiting their clinical utility. In this study, we developed a SuperSelective primer-based PCR (SSP-PCR) platform for the rapid and ultrasensitive detection of azole resistance associated single-nucleotide polymorphisms (SNPs) in Cryptococcus neoformans. By integrating molecular beacon probes, this assay achieves high specificity and enables simultaneously detection of multiple SNP mutations in a single reaction. Our SSP-PCR platform offers a powerful molecular approach for identifying azole resistance and heteroresistance, with strong potential to improve diagnostic precision and guide antifungal therapy in clinical settings.