Direct PCR of lesions suggestive of sporotrichosis in felines

Gonsales, Fernanda Fidelis; Fernandes, Natália Coelho Couto de Azevedo; Mansho, Wilson; Montenegro, Hildebrando; Benites, Nilson Roberti

doi:10.1590/1678-4162-11743

Cited by 8 publications

(18 citation statements)

References 18 publications

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“…The control group of 15 non‐ Sporothrix ‐infected animals (healthy animals) were negative for Sporothrix in our assay. Our triplex‐probe qPCR assay was in partial agreement with the positivity achieved with culture ( Kappa = 0.53±0.09; 95% CI = 0.354−0.723; fair agreement), and the species‐specific PCR ( Kappa = 0.62±0.09; 95% CI = 0.443−0.811; moderate agreement) carried out previously by Gonsales et al (2020), reinforcing the advantage of our qPCR assay to detect and discriminate the main agents of sporotrichosis (Supplementary Table S7).…”

Section: Resultssupporting

confidence: 87%

“…There were 9 (12.9%) dry lesions and 61 (87.1%) wet lesions regarding the type of injury. Sixteen (22.9%) cats had already started treatment with itraconazole at the time of sampling, one of the cats had 1 week of treatment, and the others had months of treatment (ranging from 2 to 9 months) (Gonsales et al., 2019; Gonsales et al, 2020). Lesion samples for culture and PCR were collected with sterile swabs.…”

Section: Methodsmentioning

confidence: 99%

“…Lesion samples for culture and PCR were collected with sterile swabs. Specimens for culture were stored in Stuart's medium, and samples for PCR were stored in dry tubes without the medium (Gonsales et al, 2020).…”

Section: Methodsmentioning

confidence: 99%

“…Afterwards, a slide stained with lactophenol was prepared for microscopic confirmation of the genus; the visualization of hyaline septate hyphae and single‐celled conidia on clusters of denticles (daisy bouquet) confirmed the genus (Rodrigues et al, 2020). DNA from the swab samples was extracted and purified using a DNeasy Blood and Tissue kit (Qiagen, Crawley, UK), following the manufacturer's instructions, resulting in DNA eluted in 200 μl of buffer at the end of the process (Gonsales et al., 2020). DNAs from 15 cats with no signs of sporotrichosis or other mycotic diseases (control group) were obtained from São Paulo as described elsewhere (Rodrigues et al., 2015b).…”

Section: Methodsmentioning

confidence: 99%

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Development and evaluation of a multiplex qPCR assay for rapid diagnostics of emerging sporotrichosis

Terra

Gonsales

Carvalho

et al. 2021

Transbounding Emerging Dis

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Sporothrix schenckii and related species are the agents of human and animal sporotrichosis. Routine diagnoses using classical mycological approaches are unspecific due to overlapping phenotypes. As the frequency and prevalence of sporotrichosis increases worldwide, developing specific, sensitive and cost‐effective diagnostic tools is essential to understand the distribution patterns, map‐affected areas and promote specific public health strategies to mitigate future outbreaks. Polymorphisms among the β‐tubulin gene were exploited to speciate S. brasiliensis, S. schenckii and S. globosa in a one‐tube multiplex probe‐based qPCR assay. A panel of 84 Sporothrix revealed 100% specificity (AUC = 1.000, 95% CI = 0.971–1.000, p < .0001) without cross‐reacting with other medically relevant fungi, human, feline or murine DNA. Speciation via multiplex qPCR matched phylogenetic identification (Kappa = 1.0; 95% CI = 1.0–1.0; very good agreement), supporting its use as a reliable alternative to DNA sequencing. Remarkably, the lower limit of detection was 3 copies of the target for all species. As a proof of concept, we used swabs of wound exudate of 70 cats suspected of sporotrichosis to reveal an overwhelming occurrence of S. brasiliensis in 69 specimens (sensitivity = 98.57%; 95%CI: 92.3–100.0 and specificity = 100%; 95% CI = 78.2–100). In comparison to culture, qPCR showed a larger area under the curve (AUC = 0.993±0.007; 95% CI = 0.944–1.000; p < .0001; Youden's index = 0.9857), supporting that qPCR is an essential tool for accurately detect Sporothrix DNA directly from clinical samples, thus accelerating the diagnosis of sporotrichosis. Moreover, our multiplex qPCR system has the potential to increase diagnostic capacity in Sporothrix‐affected areas, helping the local animal health agent or veterinarian to quickly identify and isolate new cases, which will likely benefit thousands of patients infected every year worldwide.

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Section: Resultssupporting

confidence: 87%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Development and evaluation of a multiplex qPCR assay for rapid diagnostics of emerging sporotrichosis

Terra

Gonsales

Carvalho

et al. 2021

Transbounding Emerging Dis

Self Cite

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“…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.…”

Section: Introductionmentioning

confidence: 99%

Nested PCR for the Diagnosis of Feline Sporotrichosis From Formalin-Fixed and Paraffin-Embedded Samples Using Different DNA Extraction Protocols

et al. 2022

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Sporotrichosis is a chronic, cosmopolitan granulomatous mycosis that affects humans and animals. The infection is caused by the dimorphic fungi Sporothrix sp. The aims of the present study were to evaluate, standardize and validate a nested PCR technique using two DNA purification kits for the extraction of DNA from formalin fixed and paraffin-embedded tissues (FFPE) for Sporothrix sp. detection. FFPE mycological culture pellet samples of different Sporothrix species (S. chilensis, S. mexicana, S. pallida, S. globosa, S. brasiliensis and S. schenckii) were used as positive controls and clinical FFPE tissue samples of animals positive for Cryptococcus sp., Leishmania infantum and Histoplasma sp. were used as negative controls. Ten clinical FFPE skin samples from cats with sporotrichosis were used to validate the nested PCR. These samples were cut into two distinct paraffin sectioning protocols (5 and 16 μm thick). The paraffin sections were subjected to two different DNA extraction kits (chemical and thermal extractions). A nested PCR was performed on the extracted DNA to identify the genus Sporothrix. The chemical extraction protocol with the 5 μm thick paraffin section was more effective in extracting DNA from Sporothrix sp. from FFPE samples and the nested PCR technique showed the highest sensitivities (100% in the positive controls and of 50% in the skin samples of cats) and specificity (100%). Therefore, the nested PCR using this protocol has great potential to be applied in Sporothrix sp. diagnosis in FFPE samples of cats.

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