The global burden of chromoblastomycosis

Background Chromoblastomycosis (CBM), represents one of the primary implantation mycoses caused by melanized fungi widely found in nature. It is characterized as a Neglected Tropical Disease (NTD) and mainly affects populations living in poverty with significant morbidity, including stigma and discrimination. Methods and findings In order to estimate the global burden of CBM, we retrospectively reviewed the published literature from 1914 to 2020. Over the 106-year period, a total of 7,740 patients with CBM were identified on all continents except Antarctica. Most of the cases were reported from South America (2,619 cases), followed by Africa (1,875 cases), Central America and Mexico (1,628 cases), Asia (1,390 cases), Oceania (168 cases), Europe (35 cases), and USA and Canada (25 cases). We described 4,022 (81.7%) male and 896 (18.3%) female patients, with the median age of 52.5 years. The average time between the onset of the first lesion and CBM diagnosis was 9.2 years (range between 1 month to 50 years). The main sites involved were the lower limbs (56.7%), followed by the upper limbs (19.9%), head and neck (2.9%), and trunk (2.4%). Itching and pain were reported by 21.5% and 11%, respectively. Malignant transformation was described in 22 cases. A total of 3,817 fungal isolates were cultured, being 3,089 (80.9%) Fonsecaea spp., 552 (14.5%) Cladophialophora spp., and 56 Phialophora spp. (1.5%). Conclusions and significance This review represents our current knowledge on the burden of CBM world-wide. The global incidence remains unclear and local epidemiological studies are required to improve these data, especially in Africa, Asia, and Latin America. The recognition of CBM as NTD emphasizes the need for public health efforts to promote support for all local governments interested in developing specific policies and actions for preventing, diagnosing and assisting patients.

Introduction Chromoblastomycosis (CBM), together with mycetoma, represents one of the primary implantation mycoses caused by melanized or black fungi widely found in nature that may infect agricultural workers after transcutaneous inoculation during their daily activities [1][2][3][4]. Chromoblastomycosis is primarily an occupational disease associated with a considerable social stigma and severe personal and family socioeconomic consequences [1,3,5,6]. It is mainly caused by Fonsecaea spp., followed by Cladophialophora, Phialophora, and Rhinocladiella. The genera Fonsecaea includes three closely related siblings represented by F. pedrosoi, F. monophora, and F. nubica. The genus Cladophilophora spp. contains two related siblings: C. carrionii that may be found in clinical samples and nature, whereas C. yegresii is exclusively found in the environment [1,[7][8][9][10][11][12]. These agents present some peculiarities in terms of geographic distribution and ecological niches. The clinical manifestations and therapeutic response of the patients differs by infecting fungus.
CBM is nowadays characterized as a Neglected Tropical Disease (NTD) because (a) it mainly affects populations living in poverty causing significant morbidity and mortalityincluding stigma and discrimination; (b) it is mostly found in tropical and sub-tropical areas; (c) it may be controlled or eradicated by applying one or more of the five public health strategies adopted by the Department for Control of NTDs; (d) it has been neglected by research when it comes to developing new diagnostics, medicines, and other control tools [1,[3][4][5]. The process of recognizing CBM as NTD began at the meeting held in São Luís, state of Maranhão, Brazil, in 2011, when the disease's centenary was celebrated. After an application by the Global Action Fund for Fungal Infections with support from the governments of Brazil and Madagascar, World Health Organization (WHO) incorporated CBM into the NTD portfolio in category B in 2017, together with mycetoma and other deep mycoses [1,7].
In most endemic areas, health services do not have professionals trained in the early diagnosis and clinical management of CBM. Skills in skin biopsy, direct microscopy, histopathology with fingal stains fungal culture is often lacking. Effective antifungal treatment rarely included in universal health coverage and government insurance. Long term itraconazole at 400mg daily or variable terbinafine dose are not be available in many countries and is expensive and requires monitoring [1,3,[6][7][8][9]. Patients are usually diagnosed after several years of clinical manifestations, and medication is unavailable or unaffordable, two factors that may increase the risk of sequelae and further social stigma [1,3]. In addition, CBM in some patients is complicated by continuous bacterial co-infection and later neoplastic transformation of the CBM lesions into epidermoid carcinoma may occur [1,3] The global incidence of CBM remains unclear. Only a few population epidemiology studies have been done. This mycosis is not a mandatory notifiable disease and most of the literature consists of case reports or small series incompletely characterized. Although several authors suggest that the CBM global burden may be comparable to mycetoma, its geographic distribution and incidence rates in different endemic areas have never been widely characterized in the medical literature.
We conducted a comprehensive systematic review of all medical literature published between 1914 and 2020 to characterize better the prevalence rates and geographic distribution of CBM in all continents. Data generated in this paper corroborate the WHO recognition of CBM as a NTD and provides helpful support for all local governments interested in developing specific policies and actions for preventing, diagnosing, and assisting patients with CBM [1,[5][6][7]13,14].

Methods
Our plan for literature review included the selection of all articles addressing the epidemiology of chromoblastomycosis in the world that were published in four different languages (English, Spanish, French and Portuguese) between 1914 and 2020 and listed in the PubMed (https:// www.ncbi.nlm.nih.gov/pubmed/) and Bireme (http://portal.revistas.bvs.br/) with access to "LILACS", "IBECS", "MEDLINE", "Cochrane Library" and "SciELO" databases. The terms used to select papers included "chromoblastomycosis", "chromomycosis", "neglected mycoses", "subcutaneous mycoses" or "implantation fungal infections". Letters to the editor and abstracts available published in congress or conferences were also searched and identified. The literature review was complemented by reviewing the reference lists of all studies selected to be sure that we did not miss any relevant references. Due to the large number of single case reports published in some highly endemic countries, papers from Mexico, Brazil, Venezuela, Colombia, Madagascar, India, China, Japan, and Australia were only included if they reported at least 5 patients. Review papers were selected only to find references to original papers to avoid case duplication [2,15]. All papers in this comprehensive review were able to meet the main diagnostic criteria of CBM: presence of dark pigmented and thick-walled muriform cells in a biological sample. Whenever there was doubt about this finding, the paper was excluded from the analysis.
Epidemiological and clinical data of all cases of CBM were collected using a standard clinical form. The variables that were systematically assessed along the literature review included year and country of publication, the number of cases, the period of cases collection, age, gender, history of cutaneous trauma and previous agricultural work, time from onset of symptoms to diagnosis (years), symptoms, clinical pattern of the lesions and severity of the disease, malignant transformation and clinical management (physical methods such as surgery, thermotherapy, laser therapy, and photodynamic therapy; antifungal drugs with itraconazole, terbinafine, iodide, flucytosine) [1,3].
To determine the prevalence rate of CBM in each country, we used the method described by Van de Sande [2]. The number of reported cases along each year in all countries was divided by the total population of each country in the selected period. Population data for each country in each collection period was extracted from the website www.indexmundi.com/facts/ indicators/SP.POP.TOTL/compare#country=ma) [2]. As an example 71 cases of CBM were reported between 1978 and 1993 in Sri Lanka, with a mean of 4.73 cases/year. The average population of this country in this period was 16,283,921 inhabitants. In this case, the prevalence of CBM in Sri Lanka was defined as 0.29 cases per 1 million inhabitants.

Results
Our review identified a total of 208 articles that were published in English (119 articles), Spanish (42 articles), French (39 articles), and Portuguese (8 articles), accounting for 7,740 cases of CBM on all continents except Antarctica. The main characteristics of CBM are illustrated by countries and continents, as summarized in Tables 1 and S1. The worldwide distribution and prevalence of CBM cases are shown in

Chromoblastomycosis in South America
A total of 51 articles described 2,619 patients that were reported in Venezuela (1,167  caused by C. carrionii in Venezuela is underestimated in the indexed literature, specially in Falcón state, and it should be greater than 500. The highest prevalence rates of the disease per 1 million inhabitants were observed in Venezuela, French Guyana, Colombia, and Paraguay. The primary epidemiologic and clinical data of all CBM cases reported in the region were summarized in Tables 1 and S1. The highest prevalence rates of CBM per 1 million inhabitants were observed in Mayotte Island, Madagascar, Gabon, and Reunion Island. The primary epidemiologic and clinical data of all CBM cases reported in the region were summarized in Tables 1 and S1.  [180][181][182][183], Indonesia (13 cases) [184][185][186][187], South Korea (9 cases) [188][189][190][191][192][193][194][195][196], Pakistan (2 cases) [197,198], and Philippines, Bangladesh, Laos, Vietnam and Iraq each one with 1 case [199][200][201][202]  Some authors describe the latter cases are doubtful because these genera do not belong to known agents of CBM and may concern misidentifications [1,3]. Some strains of Fonsecaea spp. (27 strains) were subjected to molecular identification, showing F. monophora in 21, F. pedrosoi in 5, and F. nubica in 1 case [165,175,201]. The highest prevalence rates of CBM per 1 million inhabitants were observed in Sri Lanka, Laos, Taiwan, Japan, Malaysia, and Nepal. The primary epidemiologic and clinical data of all CBM cases reported in the region were summarized in Tables 1 and S1.
The authors described 133 male and 18 female patients, exhibiting one of the highest male: female rates in all studies analyzed. They mentioned the previous rural activities or a history of trauma in 40.8% (20 out 49 cases) and 42.9% (18 out 42 cases), respectively. Histological findings were described for 23 (13.7%) patients. Cultures from the lesions yielded 43 fungal agents represented by Fonsecaea spp. (29; 67.5%) and Cladophialophora spp. (14; 32.5%). Cladophialophora spp. was found only in Australia. Fonsecaea spp. was widely distributed throughout Oceania, especially in the southeast coastal area of Queensland, Australia [203,205,[208][209][210][211]. The highest prevalences of CBM per 1 million inhabitants were observed in New Caledonia and the Solomon Islands. The primary epidemiologic and clinical data of all CBM cases reported in the region were summarized in Tables 1 and S1.

Chromoblastomycosis in Europe
Excluding two publications from Russia and Finland that were written in their native languages, which precludes our analysis of data, we were able to evaluate only 35 cases of CBM published on the European continent.

Consolidated worldwide CBM data
A total of 7,740 cases of CBM was described in five continents. The authors described 4,022 (81.7%) male and 896 (18.3%) female patients. The median age was 52.5 years (range between 2-93 years), and the average time between the onset of the first lesion and CBM diagnosis was 9.2 years (range between 1 month to 50 years). The authors mentioned exposure to rural activities and history of trauma for 56.8% (1,895 out 3,334 cases) and 33.1% (568 out 1,717 cases), respectively. Histological findings were described for 2,125 (27.8%) patients.
The presence of immunosuppressive diseases at the time of diagnosis of CBM was reported in only 16 (0.2%) cases, with solid organ transplantation the most common condition (kidney, heart transplantation), followed by HIV infection, rheumatoid arthritis, systemic lupus erythematosus, bladder neoplasia, celiac disease, pernicious anemia, and non-Hodgkin lymphoma [

Discussion
The true burden of CBM is not known. A lack of national surveillance systems checking for CBM in sentinel centers does not exist [1,2,232,233]. This paper represents the most comprehensive review of CBM cases published between 1914 and 2020, providing data to partially characterize the relative burden of this neglected implantation mycosis in different countries and the main clinical and mycological characteristics of the affected patients.
Our review showed that CBM has been widely described on all continents over the last eight decades and thrives in areas where access to adequate sanitation, clean water, and healthcare is limited. Regardless of the country considered, CBM is diagnosed in people who live in remote and rural areas and affects some of the world's poorest and most marginalized communities, predominantly in Africa, Asia, and America [1,2,232]. Rural areas in developing countries highly endemic for CBM generally present high informal employment arrangements, low human development index, and lack of appropriate social protection systems for agriculture workers. In most countries, surveillance practices for personal protective equipment (PPE) in agriculture are unknown, and their use in rural areas is woefully inadequate and requires more attention. The lack of protective shoes, gloves, or garments associated with poor hygienic habits and insufficient nutrition may favor development of CBM after infection by implantation [1][2][3][4][5][6][7]14,[234][235][236]. It is not known if there are other factors affecting the development of CBM in particular individuals or disease expression and severity. For example, an inability of toll like receptor 7 (TLR7) to recognize and respond appropriately to the causative fungi could underly the progressive nature of CBM in some patients [237].
Although the route of acquisition of CBM agents is by traumatic inoculation, most of the series did not track the source of infection once clinical manifestations as several years usually elapse after trauma when the patients and the lesion(s) grows very slowly. We were not capable to analyze data related to trauma or characterization of rural jobs as this information were not available in most papers [1,3,6,18,19,27,35,56,70,116,165,238,239].
Although the diagnosis of CBM does not rely on expensive and sophisticated laboratory tools, the disease remains neglected by all health systems, making the time of diagnosis too long (mean of 9 years). This aspect certainly impacts in morbidity, including disease progression, risk of superinfection and malignant transformation [8,56,68,99,167,175,240,241,242].
Considering the insidious progression of the fungal disease, patients continue their labor and social activities for many years before having the diagnosis made [1,3,5,48,176].
Notably, the disease is mostly observed in males probably due to different environmental exposition and possible protection of women by endogenous steroids [1,5]. This hypothesis needs further investigation and validation. However, in some countries the prevalence in women is high probably due to their involvement in agricultural activities [16,43,44,116,122,[166][167][168]199]. Lower limbs are the most common site affected, although some countries frequently reported lesions in upper limbs due to local practice of carrying wood or other agricultural materials in arms or shoulders [16,19,20,165,180,182,204,205,228,229].
In the present review, we adopted the CBM Carrión classification for skin lesions considered the most consistent and comprehensive description of dermatological lesions with updated nomenclature [1,3,65,68]. As expected, warty lesions (29%), tumors (27%), infiltrative plaques (23%), and nodules (12%) were the most common pattern of CBM, but polymorphic lesions may also be found, especially in patients with a long history and chronic evolution of the process [52,56,65,67,68,99,165,169,175]. The main symptoms were itching (21%) and pain (11%), with local edema rarely reported by most authors [6,7,9,21,28,30]. Of note, the pattern of skin lesions is not linked to the etiological agent of CBM.
Some series have shown that secondary bacterial infections and lymphedema are concerns. Uncommonly, malignant transformation may occur, especially in patients with a long history of CBM diagnosis [1,3,54,67,68,76,99,103,116,119,167,[240][241][242]. CBM progresses slowly, produces fibrotic changes and lymphatic stasis. Secondary recurrent bacterial infection exacerbates the involvement of lymphatic vessels, resembling elephantiasis. Severe forms of CBM disable and disfigure patients much more frequently than they kill, and are multifactorial [243,244]. Affected people live decades with disability, stigma and social withdrawal. Disability Adjusted Life-Years (DALY) lost due to CBM has not been comprehensively evaluated in endemic areas [1,3-5, [189][190][191]. Molecular studies showed that Fonsecaea pedrosoi is the main species within this genera, and it is found practically in all countries where CBM has been reported. This species causes almost exclusively subcutaneous disease, with rare visceral involvement [11,12,16,68]. Disseminated forms of the disease have also been reported but without unambiguous muriform cells in tissue (44), and thus, they may be considered phaeohyphomycosis. Fonsecaea monophora is widely distributed, with high prevalence, especially in Asia and subtropical or temperate countries. F. monophora, together with F. pugnacius, can cause disseminated CBM or phaeohyphomycosis with visceral impairment [165, 245,246].
Finally, Fonsecaea nubica is also widely distributed in Asia, but current studies showed that Madagascar might be the country with the highest number of CBM caused by this species [239].
The second main etiological agent of CBM is C. carrionii, with Venezuela, Madagascar, Australia, India, and China the countries most affected [44,45,48,49,99,165,170,[203][204][205][206][207][208]. This agent is typically found in arid and semi-arid climates, with average yearly temperatures of 24˚C, scarce rainfall (up to 600 annual mL) and is located at moderate altitude (up to 500 m) [ [1,247,248]. Interestingly, some therapies have been abandoned, such as cholecalciferol, thiabendazole, intravenous amphotericin, ketoconazole, and topical 5-fluorouracil. Due to the low cost, potassium iodide has been used in some countries, especially in Cuba and India [19,23,170]. Adjuvant therapy for improve the cellular immune response with topical imiquimod or intramuscular glucan was used mostly in more severe and refractory cases [249][250][251].

Conclusions
Despite all limitations, our study provides a comprehensive review of clinical and therapeutic aspects of CBM and an estimate of the prevalence of the disease in each country. Our maps have shown CBM to be widespread in five different continents, specially in Latin America, Africa and Asia. Countries such as Madagascar, Gabon, Indian Ocean Islands (Comoro and Reunion), Costa Rica, Dominican Republic, Venezuela, French Guiane, and Island of Oceania (New Caledonia) are the countries with the highest incidence densities in the world. CBM in world is probably more common than expected. The disease especially affects men (81.7%), with an average delay of 9.2 years between onset and diagnosis. The mean age was 57.1 years (range 2-93 years), being the lower and upper limbs the most compromised sites. Verrucous, tumoral and plaque represent the main dermatological patterns. Fonsecaea spp. is the main etiological agent, being widely distributed on all continentes and responsible for more than 80% of cases. This review allows the understanding of a gap in epidemiological, diagnostic and therapeutic data. There is an urgent need to create and implement social protection policies for vulnerable populations and national programs for the diagnosis and treatment of the disease.
Supporting information S1