AI Summary & Key Takeaways
AI InsightFurther validation with clinically positive CSF samples is required before clinical implementation.
📌 3 Key Takeaways
Abstract
Aims: Most commercial multiplex polymerase chain reaction (PCR) panels primarily detect bacterial and viral pathogens and usually include only Cryptococcus among fungal targets. However, other fungi such as Candida and Aspergillus are increasingly recognized as important causes of central nervous system (CNS) infections, especially in immunocompromised patients. This study aimed to preliminarily develop a multiplex real-time PCR approach for the detection of selected fungal targets in cerebrospinal fluid (CSF)-based samples. The assay was initially designed as a SYBR Green-based screening method; however, because overlapping melting temperatures limited reliable target discrimination, a three-target probe-based confirmation step was subsequently incorporated for Candida spp., Aspergillus spp., and Fusarium spp. Methods: Target-specific primers were used for Candida spp., Aspergillus spp., Fusarium spp., Mucor spp., Rhizopus spp., and Trichosporon spp. in the SYBR Green-based screening step. In addition, probe-based primer/probe sets were evaluated for Candida spp., Aspergillus spp., and Fusarium spp. to improve target-specific discrimination. The assay was evaluated using CSF-based simulated samples and tested against selected bacterial backgrounds. Results: The SYBR Green-based screening step produced amplification signals for the selected fungal targets, but melting curve analysis alone was insufficient for reliable discrimination because of overlapping Tm values. In the probe-based format, Candida spp., Aspergillus spp., and Fusarium spp. generated target-specific signals in their assigned fluorescence channels, with no off-target amplification in the tested bacterial background. These findings support the added value of probe-based confirmation while highlighting the preliminary nature of the assay. Conclusion: This preliminary study suggests that an in-house multiplex real-time PCR approach may provide a feasible framework for detecting selected fungal targets in CSF-based simulated samples. While SYBR Green-based amplification enabled initial screening, overlapping melting temperatures limited reliable target discrimination. Probe-based testing in a limited subset suggested a potential role in reducing interpretive ambiguity. Further validation with clinically positive CSF samples is required before clinical implementation.