Sporotrichosis is a chronic cosmopolitan granulomatous mycosis, caused by the thermodimorphic fungus of the genus Sporothrix, which affects humans and a wide variety of animals, especially cats and dogs (1–4). Since 1998, there has been a large number of cases of sporotrichosis in Rio de Janeiro. Approximately 5,000 cases of sporotrichosis in humans (1998–2015), 5113 cases in cats (1998–2018), and 244 in dogs (1998–2014) were registered at the Evandro Chagas National Institute of Infectious Diseases (INI) of the Oswaldo Cruz Foundation (Fiocruz), one of the main reference center for the treatment of the disease in Brazil (3, 5, 6). The classical transmission of sporotrichosis occurs via traumatic implantation of vegetal or organic matter of soil contaminated with Sporothrix sp. conidia into the skin (7). In addition, zoonotic transmission through infected animals can occur (8). In most cats and dogs, the infection is acquired after fights with other infected cats and a contaminated environment (3, 6). Sporotrichosis is caused by pathogenic species of Sporothrix, including S. brasiliensis, S. schenckii sensu stricto (s. str.), S. globosa, S. luriei, S. pallida, S. mexicana, and S. chilensis (9–15). S. schenckii and S. globosa are the species generally associated with the classical (non-zoonotic) transmission route, while the species S. brasiliensis is associated with zoonotic sporotrichosis transmitted by cats (11, 15–18) in Brazil, mainly in Rio de Janeiro (19, 20), and in Argentina (21) and Paraguay (22). Cats are the animal species most affected by sporotrichosis caused by S. brasiliensis. This disease can lead to death of cats and its main clinical signs in these animals are multiple skin nodules and ulcers, mucosal lesions, enlarged lymph nodes and respiratory signs (20). The transmission of S. brasiliensis from an infected cat to humans, cats and dogs occurs through the bite, scratch, or contact with the exudate of cutaneous lesions (22).

The reference standard method for diagnosing animal sporotrichosis is mycological culture from clinical samples, such as biopsies or exudate from animal skin lesions or nasal swabs, with a presumptive diagnosis using microscopic examinations, such as cytopathology and histopathology (23). However, fungal growth in culture may not be observed and there may be microbiological contamination. It is a laborious and often time-consuming methodology, in addition to requiring considerable knowledge for the correct morphological identification of fungal species (24, 25). Formalin-fixed and paraffin-embedded (FFPE) tissue specimens represent an extremely valuable sample source for prospective and retrospective studies (26). If mycological culture is not possible for logistical or technical reasons, and if there are no alternative samples available other than FFPE material, polymerase chain reaction (PCR)-based molecular diagnostic approaches may be used (27).

PCR methodologies have recently been developed with the aim of improving the sensitivity and specificity of diagnostic tests for sporotrichosis. In addition, PCR allows the rapid identification of pathogenic Sporothrix sp., which is critical for the early treatment of sick cats, increasing the chances of clinical cure and reducing the risk of zoonotic transmission of Sporothrix sp. by these animals (28, 29). For the molecular identification of Sporothrix sp., PCR-based methodologies such as RFLP (restriction fragment length polymorphism) (30, 31), RAPD (random amplified polymorphic DNA) (32), PCR M13 fingerprinting and PCR T3B fingerprinting (33–35), partial DNA sequencing of the transcription region internal ribosomal RNA (ITS) (36), partial DNA sequencing of the calmodulin (CAL) (11, 37, 38), β-tubulin (βtub) (37) and chitin synthase (CHS) region (11, 39), nested PCR (40, 41) and PCR targeting the topoisomerase II gene (42, 43) have been developed. In these methodologies, DNA sequences in genomic loci encoding proteins, such as CAL (11, 37, 38), βtub (37) and CHS (11), in addition to another biomarker such as the ITS region (36) are used as molecular markers to differentiate between Sporothrix species. In cats, some of these PCR-based techniques were successfully applied to detect Sporothrix sp. in fungal isolates obtained by culture of clinical fresh samples (16, 20, 44) or directly from fresh clinical samples (45, 46). Direct detection of Sporothrix in FFPE skin samples from cats was only described by Bernhardt et al. (47), who used a fungal broad-range PCR assay targeting the internal transcribed spacer 2 (ITS 2) followed by amplicon sequencing. However, the sensitivity of this test was not evaluated (47).

Obtaining high-quality and pure PCR products from DNA extracted from FFPE tissue is a difficult task, because, in general, DNA in this material is scarce, degraded and contains substances that inhibit the amplification reaction, such as formalin, or inhibit proteinase K used in the extraction procedure, such as xylene (48). Prior to this study, the detection of Sporothrix sp. DNA by nested PCR in FFPE tissue samples was investigated only once, but using human samples (49, 50). There is still no standardized and reproducible nested PCR methodology for the definitive diagnosis of animal sporotrichosis using FFPE tissues. The aims of the present study were: to analyze and standardize a nested PCR technique for the detection of Sporothrix sp. in FFPE Sporothrix spp. culture pellets, using two kits for DNA extraction (chemical and thermal extractions) and two different types of paraffin sections; to validate the results using clinical FFPE samples from cats; and to identify at the genus-level all pathogenic species of Sporothrix and clinical samples by nested PCR.

The FFPE pellet samples were sectioned using both paraffin sectioning protocols (eight 5 μm thick sections and one 16 μm thick section). No statistical difference between the weights of the paraffin sectioning protocols was observed (p > 0.05). The median weight of the FFPE samples in the eight 5 μm thick sectioning protocol and in the single 16 μm thick section was 15.1 mg and 8.5 mg, respectively.

The comparison of the DNA extraction protocols in the FFPE mycological culture pellet samples based on the concentration and purity of the DNA using both paraffin sectioning protocols (5 and 16 μm) is shown in the Table 1A. There was no statistical difference in the DNA concentration between the extraction kits (p = 0.8203 in 5 μm and p = 1.000 in 16 μm). There was also no statistical difference in the DNA concentration between the tested sectioning protocols (Table 1A). The degree of purity of the extracted DNA was higher in the thermal DNA extraction protocol compared to the chemical extraction protocol using the 16 μm thick section (p = 0.009) (Table 1B).

Using the chemical DNA extraction protocol, nested PCR technique was able to detect the 152bp fragment of all six Sporothrix species tested in FFPE pellet samples using both paraffin sectioning protocols (100% sensitivity) (Figure 1). The sensitivity of the thermal extraction in the 5 and 16 μm sectioning protocols was 83 and 50%, respectively (Table 1C). Both FFPE DNA extraction protocols showed 100% specificity for Sporothrix sp. and no cross-reactions were observed, demonstrated by the absence of amplified product in nested PCR in FFPE samples of Cryptococcus sp., Leishmania infantum and Histoplasma sp. (Figure 1). In clinical FFPE samples from cats, the comparison of the DNA extraction protocols and the sensitivity of nested PCR are shown in Tables 2A–C. There was no statistical difference in the DNA concentration between the extraction kits in clinical samples (p = 0.0483 in 5 μm and p = 0.4316 in 16 μm). The nested PCR technique showed 50% sensitivity in chemical extraction and 30% sensitivity in thermal DNA extraction at the 5 μm thick sectioning protocol (Table 2C). Two clinical FFPE samples from 2009 and three from 2015 tested negative by nested PCR in all paraffin sectioning and DNA extraction protocols. Four samples from 2017 and one sample from 2015 amplified in at least one of the paraffin sectioning or DNA extraction protocols.

FFPE tissues have several advantages for diagnostic purposes, as they are easy to handle and transport, their processing is low-cost and they can be stored for a long time at room temperature (26). However, obtaining DNA from this type of sample in sufficient quantity and quality using extraction techniques is a challenge (56). Several preanalytical factors can influence the analysis of nucleic acids in FFPE samples, such as biospecimen fixation, specimen size, block storage conditions, section thickness, section storage, and others (57). The 5 μm thick sections are commonly used in the analysis of different types of FFPE tissues (47, 58–63). The 16 μm thick sections were tested in this work as an alternative to produce samples with a smaller amount of paraffin, which contains PCR inhibitors (64), in an attempt to increase the sensitivity of the methods. In the present study, the chemical DNA extraction protocol demonstrated the best performance compared to the thermal protocol, mainly in 5 μm thick sectioning protocol.

Although the DNA concentration was similar in both extraction and paraffin sectioning protocols and the purest DNA was observed in thermal DNA extraction (16 μm section), the nested PCR assay demonstrated the superiority of the chemical DNA extraction protocol by the positivity observed in all FFPE pellet samples tested. The chemical extraction kit used in our study was also used by Sarnecka et al. (63) with FFPE tumor tissues. These authors obtained an amount of DNA similar to the present study (median of 13.20 ng/μL) and a DNA purity with the same median (A₂₆₀/A₂₈₀ = 1.90) in the 5 μm thick sectioning protocol. However, in the study of Sarnecka et al. (63), molecular analyzes were not performed and the performance of this kit was inferior to the other tested (automated extraction) (63). Higher DNA concentrations ranging from 12.3 to 618.9 ng were observed in another study with FFPE fungal samples using the same chemical DNA extraction kit with one 25 μm thick paraffin section and modifications such as longer incubation time with proteinase K and another cell lysis protocol (27).

In this work, the nested PCR methodology performed was similar to that described by Hu et al. (40) based on amplification of the 18S region of ribosomal RNA (ITS), using specific primers for Sporothrix sp. A semi-nested PCR methodology was developed for the diagnosis of human sporotrichosis in tissues embedded in paraffin with high sensitivity and specificity, but few cases were analyzed and there was a lack of details that make its reproducibility difficult (49). Also, the authors used the same chemical DNA extraction kit but it was not reported how the paraffin blocks were sectioned (49). Hu et al. (40) described a nested PCR assay for the detection of S. schenckii for diagnosing cutaneous sporotrichosis in frozen skin tissues (40). Based on them, Hayashi et al. (49) designed new primers that increased the sensitivity and specificity (100% and 98.7%, respectively) of their semi-nested PCR assay. In the present study, we used the same Sporothrix sp. specific primer sets, described by Hu et al. (40), and we were able to detect this fungus in FFPE samples.

Another important outcome in this work was the standardization of the methodology using a master mix solution to perform the PCR mixture, which facilitated the performance of the assays and the diagnosis of sporotrichosis in the clinical laboratory. The DNA yield from FFPE mycological culture pellet samples was considered ideal, as a standardization of the DNA concentration was performed in our study, but the highest degree of DNA purity was only 44.4%, within the purity criteria, observed in the thermal extraction protocol using the 16 μm sectioning protocol. This result may have been influenced by the low amount of material in the paraffin blocks. However, the nested PCR assay showed that even with low DNA purity in FFPE samples, it was possible to obtain high sensitivity and specificity. This indicates that in FFPE samples, the degree of DNA purity can be more variable than in fresh or frozen samples, with a range of purity larger than the one considered (A₂₆₀/A₂₈₀ = 1.8 – 2.0). Furthermore, the nested PCR was capable to identify at the genus-level all the pathogenic species of Sporothrix tested (S. chilensis, S. mexicana, S. pallida, S. globosa, S. brasiliensisand S. schenckii). Oliveira et al. (41) also used the same sets of primers to detect the Sporothrix sensu lato in cerebrospinal fluid, therefore, the nested amplification of the 18S rRNA gene fragment can detect all the Sporothrix species of the Sporothrix complex. In order to ensure no cross-reaction with other pathogens frequently found in feline and canine skin cases, Cryptococcus sp., Leishmania infantum and Histoplasma sp. FFPE tissue samples was included. As Sporothrix species have a large geographic distribution (65), this protocol can be applied in different regions of the world regardless of the species associated with the cases.

As Rio de Janeiro, Brazil is a hyperendemic region of sporotrichosis associated with feline zoonotic transmission, clinical FFPE samples from cats with sporotrichosis were used to validate our findings with the FFPE pellet samples. Some studies describe a high concentration of DNA extracted from paraffin blocks, but they differ in relation to the type of clinical sample tested, size of tissue embedded in paraffin, DNA extraction protocol and histological sections (66–69). Nechifor-Boilă et al. (67) considered the concentration of 100 ng/μL as an ideal cut-off point for a good DNA concentration in FFPE carcinoma samples. Our study with FFPE skin biopsy samples from cats with sporotrichosis showed a large variability in DNA concentration (0.10–73.70 ng/μL). However, our data corroborate with the previous study by Nechifor-Boilă et al. (67), since we validated our PCR method using a DNA concentration of 100–120 ng/μL, as the ideal one for the amplification of our target. In our validation study with clinical samples, the highest degree of purity was observed in FFPE tissues from cats using the chemical DNA extraction kit at the 16 μm paraffin sectioning protocol (50%) and the thermal DNA extraction kit at the 5 μm section (50%). Considering the clinical FFPE samples, negative results in the nested PCR were only observed in the oldest samples (from 2009 to 2015). Therefore, these negative results may have been influenced by the time of storage of these FFPE samples, because DNA degradation in FFPE samples increases considerably after 4–6 years of storage (70).

Despite the low DNA purity observed in the clinical samples, the nested PCR assay showed higher sensitivity in clinical FFPE samples from cats when using 5 μm paraffin sectioning protocol and the chemical extraction protocol (50% sensitivity). Ricci et al. (71) demonstrated PCR positivity of 74.2% in FFPE skin samples from patients with paracoccidioidomycosis, showing that in FFPE samples from skin lesions the sensitivity of the PCR technique is reduced. Our results were similar to Lysen et al. (70), who demonstrated PCR sensitivity of 54% for fungal identification from FFPE human tissues. Bernhardt et al. (47) detected DNA from Sporothrix in 2 of 52 (3.8%) FFPE skin samples from cats with histologically confirmed cutaneous and subcutaneous mycoses. Lau et al. (72) demonstrated higher sensitivity of a panfungal PCR assay in fresh frozen tissues compared to FFPE tissues for identifying different fungi in human and animal samples (fresh tissue: 96.8%; FFPE: 87.5% of culture-proven cases / fresh tissue: 100%; FFPE: 54.5% of histologically proven cases). The results of these authors (72) can be explained by the decreased quality, quantity, and size of DNA extracted from FFPE tissues compared to frozen tissues (73). However, FFPE samples can be preserved without refrigeration, making them easier to transport and store in areas with limited infrastructure and enable retrospective analysis (73, 74). In addition, due to maintenance, space, and labor requirements, storing FFPE tissues at room temperature for lengthy periods of time is more cost-effective than preserving frozen tissues at ultra-low temperatures (73, 74).

Despite the satisfactory results observed in our study by the nested PCR assay, it is necessary to search for alternatives to obtain FFPE samples with higher concentration and purity of DNA, such as the collection of a larger fragment from skin lesions of cats, in order to obtain a greater fungal load in these samples. Adjustments in DNA extraction protocols to reduce inhibitors and contaminants could also be pursued. Although we used specific primers for the amplification of the Sporothrix genus in combination with mycological culture, the standard reference method for the diagnosis of sporotrichosis, sequencing the amplicons would have improved our molecular analysis. Furthermore, the statistical tests may have been impaired due to the limited sample size.

In conclusion, the effective chemical DNA extraction of Sporothrix sp. DNA from FFPE samples using 5 μm thick paraffin sectioning protocol and the good sensitivity of the nested PCR assay indicates that the protocol herein presented have a great potential to be applied in Sporothrix sp. diagnosis in FFPE samples.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.

The animal study was reviewed and the protocols in this study were approved by the Animal Use Ethics Committee of the Oswaldo Cruz Foundation (CEUA-FIOCRUZ permit numbers L-041/06; LW-25/11; LW-32/12; LW-14/16; LW-12/17 and LW-17/17). This commission was established by FIOCRUZ Deliberative Council Resolution No. 004/2010 of January 21, 2010, and CIAEP No.463/2015 in compliance with National Council of Animal Experimentation Control (CONCEA).

RL, RM, and MO designed the study. RL carried out the experiments. RL, SP, and RM organized the database. RM and MO designed the methodology. RO performed the statistical analysis. SP, RO, and RM supervised the study. RM and MO raised funds. RL wrote the original draft. RL, RM, SP, RO, and MO reviewed and edited the manuscript. All authors contributed to the article and approved the submitted version.

This study was supported by the State Funding Agency Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ - Grants: JCNE E-26/201.433/2021; JCNE E-26/203.301/2017; E-26/201.737/2019), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq - Grant Proc. 409227/2016-1; Proc. 309682/2018-5; Proc. 312238/2020-7) and in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.