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.