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 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 in Costa Rica. It is caused by dematiaceous carrionii monophora fungi belonging to the family (Order ), 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; ; 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 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, , 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 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 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 in Costa Rica". 3.5 (2020): 45-49. Molecular Identification of Etiological Agents of Chromoblastomycosis in Costa Rica

47 Patient GenBank Demographics Preliminary Isolate access identification* Anatomical code Gender site of the lesion Fonsecaea Rhinocladiella NI FOPE 02 sp. MH374865 Female Arm Male FOPE 04 Fonsecaea sp. MH374866 Arm (elbow) Fonsecaea Male FOPE 17 sp. MH374872 Little finger Fonsecaea Male FOPE 22 sp. MH374867 Right ankle FOPETable 26 1: sp. MH374868 Leg

Preliminary identification of the isolates analyzed and demographic data of patients with chromoblastomycosis from which they were obtained. Figure 1: *: Microscopic identification of the saprophytic phase. Phylogenetic tree constructed with the ITS sequence obtained by Bayesian Inference. 1000 replicates were analyzed, NI: Not Indicated. - the bootstrap percentage is shown on each branch of the tree. The septated mycelium with the presence of two or three of the follow red triangles show the Costa Rican isolates. The forms of asexual - ing types of asexual sporulation: branched conidiophores, simple sporulation of the aetiological agents of chromoblastomycosis, Fonsecaea - acroteca-type conidiophores and simple phyalid-type conidio found in this study are: (A) branched conidiophore, (B, D) simple et al studied Fonsecaea phores were classified as members of the genus . How acroteca-type conidiophore and (C) phyalid-type conidiophore. ever, in 2004, de Hoog., .[10] isolates based - on the analysis of their ITS sequences and random amplification F. pedrosoi and F. of polymorphic DNA (RAPD), finding a high diversity and propos theses). The percentage of replicate trees in which the associated monophora ing the separation of the genus in two species: taxa clustered together in the bootstrap test (1000 replicates) are F. nubica and Fonsecaea multimorphosa . Over the years, new studies have defined two more shownF. pedrosoi next to the branches. - species: . so it is not is the most frequently isolated species from chro F. pedrosoi and F. nubica - moblastomycosis lesions worldwide [10,11,17,31,32], Species-level identification is related to the pathology they F. monophora and F. multimorphosa et al , re- cause, with been exclusive agents of chro surprising that 60% of the isolates from this study were identified F. pedrosoi to F. mo- moblastomycosis, and causing as this species. On the other hand, Yaguchi., . (2007) [17] - nophora cerebral phaeohyphomycosis in immunocompetent patients and classified a clinical isolate from Costa Rica from Cryptococ- felines, respectively [17,19,27]. On the other hand, fungal infec (GenBank access code AB117978 by ITS and AB253501 by cus and are mostly as- tions at the brain level caused by hyaline fungi such as cytochrome b sequencing), evidencing its presence in the country; spp., spp., data which was corroborated in this study. In turn, being Costa Rica a tropical country that has a high microbial diversity, it is expected sociated with immunosuppressive patients [28-30]. - that the other genera that cause this disease are also present. For C. carrionii The sequences obtained were deposited in GenBank. The ac example, Mora and collaborators reported in 2010 the first Costa R. cess codes are shown in table 1. The phylogenetic analysis was Rican clinical case of chromoblastomycosis caused by aquaspersa performed using type strain sequences in order to estimate with [26] and we are currently describing the first case caused by greater certainty the of the clinical isolates. The analysis . F. pedrosoi F. monophora and n grouped the isolates sequences under study into three groups: n as R. aquaspersa = 3 were identified as , n = 1 as = 1 Regarding the phylogenetic analysis, the tree obtained showed (Figure 1). The evolutionary history was inferred a separation of genera and species in clades with good statistical - using Bayesian Inference. The consistency index is (0.953271), the support (100% bootstrap). The retention index shows that the - retention index is (0.985549), and the composite index is 0.959884 differences in the tree are in the internal nodes and that the iso (0.939495) for all sites and parsimony-informative sites (in paren lates under study were generated from the same common ancestor.

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

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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

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Citation: Daniela Jaikel-Víquez., et al. Acta Scientific Microbiology

“Molecular Identification of Etiological Agents of Chromoblastomycosis in Costa Rica". 3.5 (2020): 45-49.