A SuperSelective primer-based real-time PCR platform for hypersensitive detection of azole resistance in Cryptococcus...

Cryptococcus neoformans is the leading cause of fungal meningitis with limited treatment options, making early and accurate diagnosis critical for improved patient outcomes. 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 resistance. 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 of 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 into our SSP-PCR platform further enhanced amplification specificity, enabling selective detection of the ERG11 Y145F (A434T) mutation, which is known to confer high azole resistance. Using genomic DNA from in vitro cultures and mouse lung tissues infected with either WT strain H99 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, this is the first study that utilizes the SSP-PCR platform to provide a robust and ultrasensitive molecular approach for the detection of azole resistance in C. neoformans, which provides a proof-of-concept methodology for its potential high-throughput clinical screening applications.

Invasive fungal infections are a growing public health threat causing over 1.5 million deaths annually, with cryptococcal meningitis accounting for over 15% of HIV/AIDS-related mortality. The problem is aggravated by limited treatment options and emerging drug resistance. Long-term use of fungistatic azoles like fluconazole promotes the emergence of azole resistance and 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 the detection of multiple SNP mutations in a single reaction. Our SSP-PCR platform offers a powerful molecular approach for identifying azole resistance with the potential to improve diagnostic precision and guide antifungal therapy in clinical settings.