Rapid and accurate diagnosis of dermatophytosis is essential for appropriate treatment and antifungal stewardship. However, fungal culture lacks sensitivity and requires prolonged incubation, potentially delaying targeted therapy. We evaluated the diagnostic performance of the EurobioPlex DermaScreen multiplex PCR assay in routine laboratory practice compared with direct microscopic examination (DE) and fungal culture.

This single-center pragmatic study included 1383 specimens for epidemiological analysis and 134 consecutive clinical samples for diagnostic evaluation. PCR results were compared with fungal culture as the primary reference standard and with a composite reference (culture and/or positive DE) in secondary analysis. Sensitivity, specificity, predictive values, and Cohen’s kappa (κ) were calculated. Paired proportions were compared using McNemar’s test.

The overall culture positivity rate was 22.9%, with Trichophyton rubrum representing 68.5% of isolates. Using culture as reference, PCR sensitivity was 96.2% (95% CI 80.4–99.9) in DE-positive and 88.2% (95% CI 63.6–98.5) in DE-negative specimens. In DE-negative samples, negative predictive value reached 96.4% (95% CI 87.7–99.6). Agreement between PCR and culture was fair (κ = 0.21–0.39), with significant discordance by McNemar testing ( p ≤ 0.006), mainly due to PCR-positive/culture-negative results. Using the composite reference ( n = 57), PCR sensitivity was 89.5% (95% CI 78.5–96.0).

In routine practice, multiplex PCR demonstrated high sensitivity and strong rule-out capacity, particularly in DE-negative specimens. Integration into specimen-adapted diagnostic algorithms may facilitate earlier diagnostic decision-making. These findings support the complementary use of multiplex PCR alongside conventional mycological methods in selected clinical contexts.