gms | German Medical Science

Dermatophytes are a group of fungi able to invade keratin-comprised tissues like stratum corneum, hair and nails. They are the most common cause of contagious diseases and the leading cause for onychomycoses and dermatomycoses. While conventional diagnosis relies on selective culture and KOH-microscopy, genetic analyses can increase diagnostic sensitivity and specificity while reducing the necessary time to diagnosis.

For this project, samples with the presumptive clinical diagnosis of dermato-/onychomycosis from three tertiary care hospitals in Vienna were evaluated. The samples were sent to the Department of Laboratory Medicine, Division of Clinical Microbiology at the General Hospital of Vienna (Medical University of Vienna) mostly as epithelial, nail or hair samples. Specimens were analyzed using selective fungal culture (Sabouraud dextrose agar from Mast Diagnostica, DE), 15% potassium hydroxide [KOH] microscopical testing, a commercial multiplex real time-PCR (“FTD Dermatophytes” by Fast-Track Diagnostics) and a panfungal PCR with subsequent sequencing of the ITS-region. The combination of selective culture and KOH-microscopy was considered as diagnostic standard. Additionally, in a second step a combined gold standard was calculated (defined as positive in any of the above mentioned tests) and compared to the above mentioned assays. Accuracy, sensitivity, marginal homogeneity (using McNemar’s test) and inter-rater agreement (using Cohen’s kappa) were calculated.

Of 195 unique samples, the number of samples testing positive for dermatophytes was n=47 (26%), n=60 (34%) and n=15 (8%) for selective culture or KOH-microscopy [culture/KOH], the multiplex real time-PCR [rt-PCR] and PCR of the ITS-region with subsequent sequencing [sequencing], respectively. Compared to culture/KOH, the rt-PCR showed a sensitivity of 73% and an accuracy of 78% (95%-CI: 71–83%). McNemar’s test showed no significant difference in homogeneity after correction for multiple testing (using the Bonferroni-method). When comparing sequencing to culture/KOH, sensitivity was only 22%, whereas accuracy was 78% (95%-CI: 71–83%). McNemar’s test showed highly significant differences between culture/KOH and sequencing. When comparing each test to the combined gold standard, sensitivity was 63%, 81% and 19%, whereas accuracy was 85% (95%-CI: 78–89%), 92% (95%-CI: 87-95%) and 66% (95%-CI: 59–73%) for culture/KOH, rt-PCR and sequencing, respectively. The negative predictive value was 79%, 88% and 63% and the Cohen’s kappa was 0.66, 0.83 and 0.22 for culture/KOH, rt-PCR and sequencing, respectively.

Our results show that the use of rt-PCR is a valuable addition to classical microbiological testing for dermatophytes while drastically decreasing the time necessary until diagnosis. The sequencing of the ITS-region can be used to identify individual species in a sample, although sensitivity and inter-rater agreement are significantly lower compared to other methods.