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Onychomycosis (OM) is a widespread infection requiring prolonged treatment with potential side effects. Diagnostic certainty is therefore essential before initiating antifungal therapy. Molecular biology has already shown benefits in reducing the time to diagnosis, providing technical ease, and increasing sensitivity for the respective species that molecular tests can detect. Nevertheless, causative agents are numerous, and culture remains essential, particularly for detecting non-dermatophytes mold infections. This study compared the performance of three different diagnostic strategies: conventional culture technique, the multiplex DermaGenius® 2.0 PCR (DG), and a mixed PCR/culture algorithm guided by the result of direct examination with calcofluor (DEC). The mixed algorithm (MA) prioritizes DG PCR and DEC as the primary diagnostic tools, supplemented by selective sample inoculation when mycelial elements are visualized in DEC and when DG PCR fails to detect any fungus or identifies a fungus with morphology differing from that observed in DEC (filamentous fungi versus yeasts). With only 13% of samples requiring inoculation, MA emerged as the most effective strategy, demonstrating significantly higher sensitivity (98.18%; p < 0.001) compared to single-method approaches (78.18% for DG PCR alone and 74.55% for culture alone) while maintaining a specificity comparable to DG PCR (100%). This new approach saves time in result delivery, requires fewer human resources, and increases diagnostic accuracy to better meet the needs of clinicians.

From early 2020, a high demand for SARS-CoV-2 tests was driven by several testing indications, including asymptomatic cases, resulting in the massive roll-out of PCR assays to combat the pandemic. Considering the dynamic of viral shedding during the course of infection, the demand to report cycle threshold (Ct) values rapidly emerged. As Ct values can be affected by a number of factors, we considered that harmonization of semi-quantitative PCR results across laboratories would avoid potential divergent interpretations, particularly in the absence of clinical or serological information. A proposal to harmonize reporting of test results was drafted by the National Reference Centre (NRC) UZ/KU Leuven, distinguishing four categories of positivity based on RNA copies/mL. Pre-quantified control material was shipped to 124 laboratories with instructions to setup a standard curve to define thresholds per assay. For each assay, the mean Ct value and corresponding standard deviation was calculated per target gene, for the three concentrations (107, 105 and 103 copies/mL) that determine the classification. The results of 17 assays are summarized. This harmonization effort allowed to ensure that all Belgian laboratories would report positive PCR results in the same semi-quantitative manner to clinicians and to the national database which feeds contact tracing interventions.