Evaluation of genotyping methods for tracking the spread of Sporothrix during epidemics and outbreaks

Sporotrichosis, a neglected mycosis caused by Sporothrix species, has shown notable increases in prevalence and geographic spread, driven largely by the rise of cat-transmitted cases. Robust molecular tools are crucial for tracking this emergence. This study compared the performance of four genotyping methods-calmodulin (CAL) sequencing, amplified fragment length polymorphism (AFLP), simple sequence repeat (SSR), and T3B-random amplified polymorphic DNA (T3B-RAPD)-in differentiating Sporothrix, determining intraspecific diversity, and recognizing genotypes, using a collection of 53 isolates. CAL sequencing, with 108 variable sites and 17 haplotypes, was highly effective for species identification (bootstrap values: 92-100) but offered limited insight into intraspecific diversity (H = 0.351-0.897). AFLP analysis, particularly with primer combination #5 (Rp = 40.6415; H = 0.3306), showed greater resolving power and identified intraspecific subgroups. A panel of 15 SSRs demonstrated superior strain differentiation (MI = 0.9153; PIC = 0.9094; D = 0.7424). T3B-RAPD showed moderate diversity (H = 0.3837; PIC = 0.3101), aligning with CAL for species differentiation but exhibiting limitations in detailed genetic analysis (Rp = 7.1320). The AFLP markers showed high congruence with each other (up to r = 88.4 %) and with the SSRs (up to r = 79.41 %), whereas they were poorly concordant with CAL and T3B (r = 47.05-69.87 %). A strategy using AFLP or SSR is recommended to dissect both deep- and fine-scale genetic structures, whereas CAL and T3B are suitable for species identification, particularly in resource-limited settings. This approach enhances molecular epidemiology and surveillance efforts, supporting the effective tracking of Sporothrix outbreaks and guiding public health interventions.