S10.1d Molecular diagnosis and antifungal susceptibility of Colombian clinical isolates of the Sporothrix spp complex
Authors: Cifuentes Erika Sánchez , Álzate Martha Úran , Ochoa Juan Guillermo McEwen , Velasquez Margarita , Molina Diana , Álzate Maria del Pilar Jiménez 
Published: September 2022

Abstract

S10.1 Antifungal dosing in children and adolescents, September 24, 2022, 10:30 AM - 12:00 PM

Objective
To detect by chitinase PCR and Species-Specific PCR (SS PCR) the DNA of Sporothrix spp in fresh tissue and Formalin Fixed Paraffin Embedded (FFPE) tissue from patients with sporotrichosis and to evaluate the susceptibility to antifungal of Sporothrix spp isolates obtained from patients diagnosed with sporotrichosis in the Medical Mycology group of the Faculty of Medicine at the University of Antioquia.

Materials & Methods
At the Medical Mycology group service, 26 patients with suspected sporotrichosis were evaluated between 2018 and 2022. Samples of the skin lesions (fresh tissue) were taken for molecular tests and microbiological culture.
Regarding the FFPE tissue, 87 samples stored by different histopathological diagnosis centers from 1976 to 2022 were chosen: 45 samples had a histopathological diagnosis of sporotrichosis, and 42 samples of other diseases with involvement in subcutaneous tissue, were used as controls.
DNAs previously extracted from fresh tissue and FFPE tissue were used to perform chitinase nested PCR and SS nested PCR. The chitinase nested PCR amplifies a 209 bp fragment for the genus Sporothrix. For species identification, species-specific primers were used (SS PCR), which amplify a 331 bp sequence for S schenckii s. str. and 243 bp for S. globosa of the calmodulin gen. For the SS PCR in tissues, a nested PCR was implemented using the primers CAL1 and CAL2 for the external sequence and the species-specific primers for the internal sequences.
The antifungal susceptibility tests were performed according to the Clinical and Laboratory Standards Institute (CLSI) M27-A3 protocol for yeasts. Six antifungals were used: itraconazole, terbinafine, voriconazole, posaconazole, amphotericin B, and fluconazole.

Results
The culture was positive for Sporothrix spp in 15 (58%) patients and the chitinase nested PCR was positive in 14 of these, with a sensitivity of 93% and a specificity of 91%. In 11 patients, both the culture and the chitinase nested PCR were negative.
SS nested PCR was applied to the 14 DNAs with positive chitinase PCR, of which seven were positive for S. schenckii s. str. and one for S. globosa. For the other six samples, the results of this PCR were negative. The results of these PCR were confirmed by the identification of species from the isolates recovered in culture: 13 were identified as S. schenckii s. str and 1 as S. globosa.
Of the 45 FFPE tissue samples with histopathological diagnosis of sporotrichosis, the chitinase PCR was positive for 25 (55%) of these and all FFPE tissue samples were negative for SS nested PCR.

Conclusions
Chitinase nested PCR had a sensitivity of 93% and a specificity of 91% with respect to culture, in samples from fresh tissue. This PCR also has a good performance when it is applied to a good-quality DNA obtained from FFPE tissue, hence, PCR positivity decreased in samples stored for ˃15 years. The results of the nested SS PCR are encouraging, however, it is necessary to improve this test, which we believe is affected by the type of sample used for DNA extraction, so further studies are required.