Cerebrospinal fluid PCR: A new approach for the diagnosis of CNS sporotrichosis

Oliveira, Manoel Marques Evangelista; Muniz, Mauro de Medeiros; Almeida‐Paes, Rodrigo; Zancopé‐Oliveira, Rosely Maria; Freitas, Andréa d’Avila; Lima, Marco Antônio; Gutierrez-Galhardo, Maria Clara; Freitas, Dayvison Francis Saraiva

doi:10.1371/journal.pntd.0008196

Cited by 11 publications

(25 citation statements)

References 9 publications

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“…Nested-PCR has been successfully used in clinical samples, where Ophiostomataceae members besides Sporothrix spp. are unlikely to occur [ 25 , 26 ]. The present study demonstrates that this methodology is not appropriate for environmental studies, due to the frequent amplification of DNA from saprobe fungi.…”

Section: Discussionmentioning

confidence: 99%

“…Nested-PCR was performed in order to detect low amounts of Sporothrix DNA [ 25 , 26 ]. The reaction mixture consisted of a total volume of 25 µL, with final concentrations of 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 1.5 mM MgCl 2 , 0.4 µM concentrations of primers ( Table S1 ), 1.5 U of Taq DNA polymerase (Invitrogen, Waltham, MA, USA), and 200 µM concentration of each dNTP (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

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Beyond Domestic Cats: Environmental Detection of Sporothrix brasiliensis DNA in a Hyperendemic Area of Sporotrichosis in Rio de Janeiro State, Brazil

Almeida-Silva

Rabello

Scramignon-Costa

et al. 2022

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In Brazil, sporotrichosis has transitioned from a rural to urban disease, driven by a shift in the initiation of infection from the accidental inoculation of organic matter to the traumatic implantation of the fungus by cats. Since the emergence of zoonotic sporotrichosis caused by Sporothrix brasiliensis, investigations have largely ignored the environmental habitat of the pathogen due to its association with domestic cats. Therefore, we investigated 18 environmental samples collected from rural areas of two cities where zoonotic sporotrichosis is endemic, but where domestic cats are scarce. We utilized traditional culture methods, and samples were also examined with two molecular methods used for the clinical diagnosis of sporotrichosis: a nested-PCR targeting the ITS region and a species-specific PCR targeting the calmodulin gene. No Sporothrix colonies were identified by traditional culture methods. However, the nested-PCR and the species-specific PCR for S. brasiliensis were positive for 18 and 5 samples, respectively. Sequencing revealed that positive results with the nested-PCR were due to non-specific amplification of other Ophiostomatales DNA, rather than Sporothrix spp. Three of the five amplicons from the species-specific PCR were suitable for sequencing and confirmed the presence of S. brasiliensis DNA. Hence, we confirmed that S. brasiliensis, as with other Sporothrix species, has an environmental habitat. Our findings underscore the challenges of nested-PCR for Sporothrix environmental studies and highlight that sequencing must follow PCR protocols to definitively identify Sporothrix spp. in environmental samples.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Beyond Domestic Cats: Environmental Detection of Sporothrix brasiliensis DNA in a Hyperendemic Area of Sporotrichosis in Rio de Janeiro State, Brazil

Almeida-Silva

Rabello

Scramignon-Costa

et al. 2022

JoF

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“…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. brasiliensis and 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.…”

Section: Discussionmentioning

confidence: 99%

“…For the genus-level PCR identification of Sporothrix sp. in FFPE specimens, a nested PCR was performed according to a previously described method based on the amplification of the 18S ribosomal RNA (ITS) ( 40 ) with slight modifications ( 41 ).…”

Section: Methodsmentioning

confidence: 99%

“…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.…”

Section: Introductionmentioning

confidence: 99%

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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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The Historical Burden of Sporotrichosis in Brazil: a Systematic Review of Cases Reported from 1907 to 2020

et al. 2021

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Cerebrospinal fluid PCR: A new approach for the diagnosis of CNS sporotrichosis

Cited by 11 publications

References 9 publications

Beyond Domestic Cats: Environmental Detection of Sporothrix brasiliensis DNA in a Hyperendemic Area of Sporotrichosis in Rio de Janeiro State, Brazil

Beyond Domestic Cats: Environmental Detection of Sporothrix brasiliensis DNA in a Hyperendemic Area of Sporotrichosis in Rio de Janeiro State, Brazil

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

The Historical Burden of Sporotrichosis in Brazil: a Systematic Review of Cases Reported from 1907 to 2020

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