Endemic Chromoblastomycosis Caused Predominantly By
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Endemic Chromoblastomycosis Caused Predominantly by Fonsecaea nubica, Madagascar1 Tahinamandranto Rasamoelina, Danièle Maubon, Malalaniaina Andrianarison, Irina Ranaivo, Fandresena Sendrasoa, Njary Rakotozandrindrainy, Fetra A. Rakotomalala, Sébastien Bailly, Benja Rakotonirina, Abel Andriantsimahavandy, Fahafahantsoa R. Rabenja, Mala Rakoto Andrianarivelo, Muriel Cornet, Lala S. Ramarozatovo Chromoblastomycosis is an implantation fungal infec- plant material from thorns or wood splinters or by soil tion. Twenty years ago, Madagascar was recognized as contamination of an existing wound (1,2). The causative the leading focus of this disease. We recruited patients agents are mainly Fonsecaea spp., Cladophialophora spp., in Madagascar who had chronic subcutaneous lesions and Rhinocladiella spp. However, rare cases caused by suggestive of dermatomycosis during March 2013– other genera, such as Phialophora spp. or Exophiala spp., June 2017. Chromoblastomycosis was diagnosed in 50 have been reported (1,3). As is the case for other implan- (33.8%) of 148 patients. The highest prevalence was in tation mycoses, chromoblastomycosis lesions are locat- northeastern (1.47 cases/100,000 persons) and southern ed mainly on the lower limbs, particularly on the dorsal (0.8 cases/100,000 persons) Madagascar. Patients with face of the feet, ankles, and legs (1,4–6). chromoblastomycosis were older (47.9 years) than those Infection is caused by a lack of protective clothing without (37.5 years) (p = 0.0005). Chromoblastomyco- or shoes for persons working in rural areas in which sis was 3 times more likely to consist of leg lesions (p = 0.003). Molecular analysis identifiedFonsecaea nubica spiny plants are common. Chromoblastomycosis is in 23 cases and Cladophialophora carrionii in 7 cases. linked to poverty and is highly prevalent in low-income Of 27 patients who underwent follow-up testing, none resource countries. It was the first fungal infection to be were completely cured. We highlight the persistence of a recognized as a neglected tropical disease (along with high level of chromoblastomycosis endemicity, which was mycetoma, which is not exclusively of fungal origin) (7). even greater at some locations than 20 years ago. We The clinical manifestation of chromoblastomy- used molecular tools to identify the Fonsecaea sp. strains cosis is polymorphous but is dominated by verru- isolated from patients as F. nubica. cous and tumoral lesions resembling cauliflower. No clinical particularity associated with the fungal hromoblastomycosis is a chronic, implantation, species or genera has been described (1,4,5,7). In- Cfungal disease caused by melanized fungi from a fection begins with development of muriform cells variety of genera of the order Chaetothyriales. This dis- in the skin, provoking a granulomatous immune ease is included in a group of melanized infections and response. Muriform cells are specific to chromo- easily identifiable by verrucous lesions that eventually blastomycosis and described as large brown, thick- lead to cauliflower-like eruptions on the skin. Infection walled, compartmented cells. They are also found is acquired traumatically by implantation of infected in the infected plants assumed to be the source of Author affiliations: Université d’Antananarivo, Antananarivo, human contamination. Madagascar (T. Rasamoelina, N. Rakotozandrindrainy, Albeit belonging to the same order, the species F.A. Rakotomalala, B. Rakotonirina, A. Andriantsimahavandy, found in these plants and the soil are different from the M. Rakoto Andrianarivelo); Université Grenoble Alpes, Grenoble, pathogenic ones (4,5,7,8). Infection involves >1 nod- France (D. Maubon, S. Bailly, M. Cornet); Hôpital Universitaire ules that develop into verrucous, hyperkeratotic, or Joseph Raseta Befelatanana, Antananarivo (M. Andrianarison, papillomatous lesions or plaques. The lesions progress I. Ranaivo, F. Sendrasoa, F.R. Rabenja, L.S. Ramarozatovo); slowly, over a period of 2–20 years and become highly Centre Hospitalier Universitaire de Befelatanana, Antananarivo 1 (L.S. Ramarozatovo) Preliminary results from this study were presented at the 20th International Society for Human and Animal Mycology DOI: https://doi.org/10.3201/eid2606.191498 Conference; June 29–July 5, 2018; Amsterdam, the Netherlands. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 26, No. 6, June 2020 1201 RESEARCH disabling because of development of elephantiasis- in this study (Table 1). Cases were identified after a type edema or superinfections. Itching and scratching monthly consultation between the clinicians of the favor dissemination (1). The usual diagnosis relies on Department of Dermatology-Rheumatology of Jo- detection of the muriform cells in superficial samples, seph Ravoahangy Befelatanana University Hospital which is sufficient to confirm chromoblastomycosis. Center in Antananarivo and the teams of mycologists However, culture, yielding black fungi, is required to from the Charles Mérieux Infectiology Center and identify the causative agent at the species level by mor- Université Grenoble Alpes. phologic and molecular analyses (1,5). Chromoblastomycosis predominates in tropi- Clinical Samples cal and subtropical regions, and most reported cases We obtained consent from patients and collected are from Latin America (Brazil, Mexico, and Venezu- specimens consisting of biopsy material or flakes of ela), the Caribbean (Dominican Republic and Cuba), skin. We then sent samples to the laboratory of the Africa, Asia (India, Japan, and southern China) and Charles Mérieux Infectiology Center of Antanana- Australia (1). In Madagascar, studies conducted by ivo, where they were processed immediately or after Institut Pasteur during 1955–1994 provided an inven- 24–48 hours of storage at 2°C–8°C. tory of the number of cases of chromoblastomycosis and identified this country as the leading focus of Mycological Analysis chromoblastomycosis worldwide. The mean annual incidence was estimated to be ≈1 cases/200,000 per- Cultures sons during this period. The most commonly isolated We performed direct microscopic examination of agents were Fonsecaea pedrosoi in the humid tropical clinical specimens with and without Chlorazol Black areas and Cladophialophora carrionii in the semiarid staining to detect muriform cells, which are typical zones of the southern Madagascar (9–11). of chromoblastomycosis (2). We then used samples Since 2013, we have established a cross-sectional to inoculate Sabouraud medium supplemented with study to document the current epidemiology of im- chloramphenicol, on which samples were incubat- plantation mycoses in Madagascar, including chro- ed at 30°C for 2–3 weeks. For positive cultures, we moblastomycosis (12). Clinical diagnosis and fungal morphologically identified fungal isolates, extracted identification were confirmed by using molecular bi- DNA, and froze the culture at -80°C. ology methods. We describe the current prevalence and clinical manifestation of chromoblastomycosis in Molecular Analysis Madagascar and patient outcomes. We also report the We used the QIAamp DNA Blood Mini Kit species-level identification, genetic relatedness, and (QIAGEN, https://www.qiagen.com) for DNA puri- antifungal susceptibility of clinical isolates. fication from clinical samples and fungal colonies. We performed PCR amplification in 2 steps. In the first Materials and Methods step, we used 2 panfungal PCRs targeting internal transcribed spacer (ITS) regions with primers ITS1/ Study Design and Patient Recruitment ITS4 and D1D2 regions with primers NL-1/NL-4 and We conducted a cross-sectional study as described NL-3/NL-4 (13–15). In the second step, we used a C. (12). We recruited patients with clinically suspected carrionii–specific PCR, primers Ccar-F 5′-ATCGCT- chromoblastomycosis or another chronic dermato- GCGAAGCGTCTCG-3′ and Ccar-R 5′-ACCGTC- mycosis during March 2013–June 2017 at the Derma- CAACACCAAGCACAGG-3′, and specific Fonsecaea tology Department of the Joseph Raseta Befelatanana sp. and PCR primers that have been described (16). University Hospital in Antananarivo or during ad- We sequenced panfungal PCR products by LGC Ge- vanced consultation campaigns in districts (Figure nomics GmbH, https://www.nucleics.com) by using 1, panel A). A clinical and demographic information the same primers as for amplification. form was completed for each participant. This study We aligned the sequences obtained for panfungal was approved by the Ethics Committee for Biomedi- PCR with reference sequences in the International So- cal Research of the Ministry of Public Health of Mad- ciety of Human and Animal Mycology Barcoding Da- agascar (authorization no. 66-MSANP/CE). tabase (http://its.mycologylab.org) for the ITS region and the National Center for Biotechnology Information Case Definition (NCBI; https://www.ncbi.nlm.nih.gov) database for We provide clinical, mycological, histological, sever- the D1D2 and ITS regions (17). We constructed a phylo- ity and prognostic criteria used for classifying cases genetic tree by using MEGA7 software (https://www. 1202 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 26, No. 6, June 2020 Endemic Chromoblastomycosis, Madagascar Figure 1. Recruitment of patients for study of chromoblastomycosis and prevalence by region, Madagascar, March 2013–June 2017. A) Recruitment sites (green triangles). Region of Sava: 1) Centre Hospitalier de Référence Régionale, Sambava District; 2) Centre Hospitalier de District and Hôpital Adventiste, Andapa District,