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. 2024 Feb 18;10(2):159.
doi: 10.3390/jof10020159.

Identification of Chromoblastomycosis and Phaeohyphomycosis Agents through ITS-RFLP

Gabriel S M Sousa  1 , Rodrigo S De Oliveira  2 , Alex B De Souza  2 , Ruan C Monteiro  3 , Elaine P T E Santo  2 , Luciano C Franco Filho  2 , Silvia H M Da Silva  2
Affiliations

Identification of Chromoblastomycosis and Phaeohyphomycosis Agents through ITS-RFLP

Gabriel S M Sousa et al. J Fungi (Basel). .

Abstract

Chromoblastomycosis (CBM) and phaeohyphomycosis (FEO) are infections caused by melanized filamentous fungal agents, primarily found in tropical and subtropical regions. Both infections pose significant challenges for the correct identification of the causative agent due to their morphological similarity, making conventional methods of morphological analysis highly subjective. Therefore, molecular techniques are necessary for the precise determination of these species. In this regard, this study aimed to contribute to a new methodology based on PCR-RFLP for the identification of agents causing CBM and FEO. Sequences from the Internal Transcribed Spacer (ITS) region were used to identify potential restriction enzyme sites in silico, followed by in vitro validation using the selected restriction enzymes. The obtained results were compared with species identification through morphological analyses and sequencing. The results demonstrated that the PCR-RFLP applied in this study accurately identified two major agents of chromoblastomycosis, Fonsecaea pedrosoi and Fonsecaea monophora, as well as Cladophialophora bantiana and Exophiala dermatitidis, both causative agents of phaeohyphomycosis. In this context, the proposed assay can complement current methods for identifying these species, aiding in diagnosis, and contributing to the proper management of these infections.

Keywords: Herpotrichiellaceae; PCR-RFLP; chormoblastomycosis; molecular; phaeohyphomycosis; phenotypic; sequencing.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Workflow employed in this study.
Figure 2
Figure 2
In silico analysis of the cleavage site positions for the Hhal and HaeIII enzymes, identifying the locations of the sites and the organization of fragmentation in single and double digestion.
Figure 3
Figure 3
Comparison of fragmentation patterns obtained with enzymatic restriction using HhaI in silico and in vitro. A: Fonsecaea pedrosoi; B: Fonsecaea monophora; C: Cladophialophora bantiana; D: Exophiala dermatitidis.
Figure 4
Figure 4
Comparison of fragmentation patterns obtained with enzymatic restriction using HhaI and HaeIII. A: Fonsecaea monophora; B: Fonsecaea pedrosoi; C: Cladophialophora bantiana; D: Exophiala dermatitidis.
See this image and copyright information in PMC

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