Molecular Identification of Etiological Agents of Chromoblastomycosis in Costa Rica"
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ACTA SCIENTIFIC MICROBIOLOGY (ISSN: 2581-3226) Volume 3 Issue 5 May 2020 Research Article Daniela Jaikel-VíquezMolecular1,2 Identification*, Stefany Lozada-Alvarado of Etiological1,3 and Agents Lorena of Chromoblastomycosis in Costa Rica Uribe-Lorío4 Received: 1 Published: Section of Medical Mycology, Department of Clinical Microbiology and Immunology, March 10, 2020 Daniela School of Microbiology, University of Costa Rica, Ciudad Universitaria Rodrigo Facio April 14, 2020 Jaikel-Víquez., et al. Brenes, San Pedro, Costa Rica © All rights are reserved by 2Center for Research in Tropical Diseases (CIET), Faculty of Microbiology, University of Costa Rica, Ciudad Universitaria Rodrigo Facio Brenes, San Pedro, Costa Rica 3Clinical Laboratory and Blood Bank, University of Costa Rica (LCBSUCR), University of Costa Rica, San José, Costa Rica 4Center for Research in Cellular and Molecular Biology (CIBCM), School of Agronomy, University of Costa Rica, Ciudad Universitaria Rodrigo Facio Brenes, San Pedro, Costa Rica *Corresponding Author: Daniela Jaikel-Víquez, Section of Medical Mycology, Department of Clinical Microbiology and Immunology, School of Microbiology, University of Costa Rica, Ciudad Universitaria Rodrigo Facio Brenes, San Pedro, Costa Rica. Abstract Fonsecaea pedrosoi and Cladophialophora Chromoblastomycosis is the second most frequently reported subcutaneous mycosis in Costa Rica. It is caused by dematiaceous carrionii Fonsecaea monophora fungi belonging to the family Herpotrichiellaceae (Order Chaetothyriales), especially by - . However, it is important to note that is able to disseminate and cause cerebral phaeohyphomycosis. - Thus, five clinical isolates deposited in the Fungal Collection of the School of Microbiology of the University of Costa Rica were ana lyzed. The isolates were characterized macroscopically and microscopically after grown in potato dextrose agar. Genetic identifica F. pedrosoi F. monophora Rhinocladiella aquaspersa R. aquaspersa tion was performed via amplification and sequencing of the ITS (internal transcription spacer) region. The isolates were identified as F. pedrosoi and F. monophora (n = 3), (n = 1) and (n = 1). Hence, we report for the first time that - is an etiological agent of chromoblastomycosis in Costa Rica and confirm the presence of both in the country. Therefore, we recommend the usage of molecular techniques to identify these pathogens since there is a risk of fungal dis Keywords: Fonsecaea monophora; Fonsecaea pedrosoi; Rhinocladiella aquaspersa semination in our patients. Chromoblastomycosis; Chromomycosis; ; Subcutaneous Infections Abbreviation dematiaceous fungi. It usually affects lower limbs and is slowly IntroductionITS: internal transcription spacer evolving as the infection grows approximately one centimeter per year. A papule or plaque appears at the site of inoculation, which then acquires a verrucous form. Older lesions can ulcerate, become Chromoblastomycosis is a worldwide disease, however, most - infected secondarily, and eventually culminate in squamous cell cases are reported in tropical and subtropical areas, especially in - carcinoma. Also, lesions in the form of atrophic plaque have been Latin America [1-5]. It is the second most frequently reported sub described. In direct examinations (potassium hydroxide or histo cutaneous mycosis in Costa Rica, surpassed only by sporotrichosis logical stains) Medlar bodies are observed [1-5]. [6,7]. Thus, in 1953, Costa Rica ranked third in incidence (n = 53 Fonsecaea cases), below Brazil and Cuba [2]. The etiologic agents are from the Herpotrichiellaceae family Cladophialophora carrionii, Phialophora verrucosa, Exophiala (order Chaetothyriales) among which are the species It is a cutaneous and subcutaneous infection that is obtained jeanselmei and Rhinocladiella aquaspersa spp., by traumatic inoculation of organic material contaminated with [8-15]. In regard to the Citation: Daniela Jaikel-Víquez., et al. Acta Scientific Microbiology “Molecular Identification of Etiological Agents of Chromoblastomycosis in Costa Rica". 3.5 (2020): 45-49. Molecular Identification of Etiological Agents of Chromoblastomycosis in Costa Rica 46 Fonsecaea - - genus , the usage of molecular biology has discerned heated in the flame, without boiling. Finally, the preparation was F. pedrosoi, F. monophora and F. nubica that it is composed of several species, being those of medical im observed under a microscope (400x) so that the typical morphol portance: [16]. This is of ogy of each fungus could be observed [20]. F. pedrosoi DNA extraction and amplification great importance because at the microscopic level these fungi are ® Tissue kit and F. nubica - very similar, however, the pathology they cause differs. F. monophora are exclusive causative agents of chromoblastomy DNA extraction was perform using The Nucleo Spin - cosis, while can cause other clinical conditions such (Macherey-Nagel, Germany), following manufacturer procedure. - et al as cerebral phaeohyphomycosis [17-19] and/or spread to lymph The internal transcription spacer (ITS) was amplificated accord F. monophora nodes, lungs, kidneys and liver [17]. Hence, the diversity and com ing to the protocol described by White., . (1990) [21], using the following pair of primers: ITS1 (5’-TCC GTA GGT GAA CCT GCG plexity of the clinical manifestations caused by is the - reason why various groups of researchers, worldwide, have been G-3 ‘) [forward] and ITS4 (5’ -TCC TCC GCT TAT TGA TAT GC-3 ‘) F. pedrosoi given the task of characterizing at the molecular level all those [reverse]. The PCR products were sent to Macrogen Inc. (South Ko et al. , analyzed 20 isolates previously identified as , by light microscopy rea), where the sequencing process was carried out. The sequences F. monophora techniques. For example, Yaguchi (2007) [17] obtained were reviewed and edited using the BioEdit v 7.0.5.2 [22] and aligned with reference sequences from the NCBI database in Japanese strains of which 100% were identified as - F. pedrosoi n F. monophora - and 21 Latin American strains, of which 47.62% (n = 10) were MEGA [23]. Multiple sequence alignments were performed with et al identified as and 33.33% ( = 7) as . Simi ClustalX [24]. Phylogenetic trees were constructed using Bayes et al lar results were published by de Hoog., . (2004) [10] and by ian Inference through the MrBayes 3.2.6 program; 1000 replicates were analyzed; the resampling percentage was reflected in each Najafzadeh., . (2009) [11]. - Resultsbranch of and the tree. Discussion To date, no work has been carried out in Costa Rica that con et al - Clinical isolates and phenotypic characterization tinue with these international initiatives. This is essential since the F. mo- study of Yaguchi . (2007) [17] analyzed three Costa Rican clini - nophora cal isolates out of which one was molecularly identified as Chromoblastomycosis is the second most frequently reported , thus proving the existence of this fungus in our country. subcutaneous mycosis in Costa Rica, preceded only by sporotricho sis. As for its epidemiology, most cases occur in adult male patients In turn, it is very important to know which are the causative agents - of this pathology and to evaluate whether these fungi can also be and the lesions are located in the lower limbs [14]. The isolates potential agents of invasive phaeohyphomycosis. Therefore, the analyzed in this study mostly come from male patients, but it is im present study aims to identify by molecular biology techniques the portant to highlight that the majority of the patients had lesions isolated chromoblastomycosis causing fungi from the collection of in their upper limbs; situation which, although not common, has also been reported in the literature [12,25,26]. Demographic data the Medical Mycology Laboratory of the School of Microbiology, - of the patients is found in table 1. Only the isolate FOPE 26 had MaterialsUniversity of and Costa Methods Rica. information of the year in which it was deposited in the fungal col Clinical isolates lection: 2002. Macroscopic morphology of the fungal cultures was - composed of black and velvety texture colonies. At the microscopic Five Costa Rican clinical isolates, from patients suffering chro level, the isolates FOPE 02, 17, 22 and 26 presented dematiaceous, - moblastomycosis were analyzed; these isolates were deposited septate mycelium with the presence of branched conidiophores, in the Fungal Collection of the Section of Medical Mycology of the simple acroteca-type conidiophores and/or phyalid-type conid - School of Microbiology, University of Costa Rica. The fungi were iophores; meanwhile, FOPE 04 presented dematiaceous, septate Fonsecaea pedrosoi kept in potato dextrose agar (PDA), with a layer of mineral oil and mycelium with the presence of only simple acroteca-type conidio and F. monophora at room temperature (20 - 30°C). CBS 659.76 phores (Figure 1). Molecular identification CBS 269.37 were used as control strains. Phenotypic characterization - - Prior to this work, Costa Rican studies focused only on the mor mycosis A drop of clear lactophenol reagent was placed on a slide and phological identification of the etiological agents of chromoblasto then a small portion of the fungal colony was placed on top of it, [8,13,14], where those fungi that presented dematiaceous, then, the preparation was covered with a coverslip and slightly Citation: Daniela Jaikel-Víquez., et al. Acta Scientific Microbiology “Molecular Identification of Etiological Agents of Chromoblastomycosis