Rapid PCR-based diagnosis of superficial mycoses with ROC-optimized cutoffs

Area of Science:

• Mycology
• Infectious Diseases
• Molecular Diagnostics

Background:

• Superficial fungal infections are common, affecting 25% of the global population.
• Current diagnostic methods, fluorescence microscopy and fungal culture, have limitations in sensitivity and turnaround time, respectively.
• There is an urgent need for rapid and accurate diagnostic tools to guide antifungal therapy and combat resistance.

Purpose of the Study:

• To determine the optimal cycle threshold (Ct) cutoff for a novel multiplex PCR assay (iFIND).
• To evaluate the diagnostic performance of a combined strategy using microscopy and iFIND for superficial mycoses.
• To compare the diagnostic accuracy and turnaround time of the combined strategy with conventional methods.

Main Methods:

• Prospective study of 113 patients with suspected superficial mycoses.
• Specimens were analyzed using fluorescence microscopy, fungal culture, and the automated iFIND multiplex PCR assay.
• Receiver operating characteristic (ROC) analysis was used to determine the optimal Ct cutoff, with internal bootstrap validation.
• Positive percent agreement (PPA), negative percent agreement, and overall percent agreement were calculated against a composite reference standard.

Main Results:

• The optimal Ct cutoff for iFIND was determined to be 32.66 (AUC = 0.746), with robust validation.
• The combined microscopy + iFIND strategy achieved a PPA of 65.6%, superior to microscopy alone (50.0%) and comparable to culture (75.0%).
• The iFIND assay demonstrated excellent discrimination for *Epidermophyton* (AUC = 0.824) and *Trichophyton* (AUC = 0.834), with a turnaround time of <3 hours.

Conclusions:

• The iFIND assay is a rapid and accurate tool for diagnosing superficial fungal infections.
• The microscopy + iFIND strategy offers a practical, culture-comparable alternative with significantly reduced turnaround time.
• This combined approach facilitates same-day treatment decisions, potentially reducing unnecessary antibiotic use and addressing antifungal resistance.