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Background  The current gold standard for diagnosing onychomycosis is direct microscopic examination and culturing. Fungal culture is a time‐consuming procedure, while direct microscopy of potassium hydroxide ( KOH ) mounts suffers from low sensitivity. More rapid and sensitive methods for the diagnosis of onychomycosis are in high demand.    Objective  To establish an effective method for the diagnosis of onychomycosis by assessing the efficacies of fungal fluorescent staining and internal transcribed spacer ( ITS ) ribosomal  DNA  ( rDNA ) polymerase chain reaction ( PCR )‐based sequencing.    Methods  A total of 204 clinical specimens from patients with suspected onychomycosis were analysed. The gold standard for a true positive sample was positive by  KOH , culturing or both methods. All specimens were also tested by fungal fluorescent staining and  ITS rDNA PCR ‐based sequencing. We compared the detection, sensitivity and specificity for these two methods with conventional methods.    Results  In total, 126 (62%) and 102 (50%) were detected by fluorescent staining and  PCR ‐based sequencing, respectively. According to the conventional diagnostic standard, the sensitivity of fluorescent staining and  PCR ‐based sequencing was 97% and 78%, respectively, and specificities of 89% and 90%, respectively. Use of fluorescence enhanced the sensitivity of direct examination by 12% compared with  KOH .  PCR ‐based sequencing increased the sensitivity by 6% compared with culturing.    Conclusions  Fluorescence microscopy has a higher sensitivity for the detection of fungi in nail specimens compared with  KOH  and can be used as a rapid screening tool.  PCR ‐based sequencing was faster and more sensitive compared with culture and when used in conjunction with fluorescence microscopy resulted in higher efficiency.

Comparison of fungal fluorescent staining and ITS rDNA PCR ‐based sequencing with conventional methods for the diagnosis of onychomycosis