Chromoblastomycosis

EtiologyWhen CBM is defined as an implantation mycosis leading to the hyperproliferation of host tissue, combined with the presence of a fungal pathogenic phase in the form of muriform cells, most of the agents of this disease are members of a single order in the fungal kingdom, the Chaetothyriales. Within this order is a single family, the Herpotrichiellaceae. The restricted distribution of CBM, with only the single exception of Chaetomium (32), indicates that this host-fungus interaction is highly specific because CBM is nearly exclusively found in patients with fully functional immunity. The less specific counterpart disease caused by black fungi, PHM, usually involves a course with tissue necrosis rather than proliferation, has a much wider spectrum of causative agents throughout the fungal kingdom, and is associated mostly with immune disorders.

The Chaetothyriales are particularly known by the genus Exophiala, comprising so-called “black yeasts,” which are able to reproduce by budding. The majority of their relatives, including all the CBM agents, are strictly filamentous. Melanin is consistently present in reproductive and vegetative cells, and therefore, colonies of Chaetothyriales are typically olivaceous, dark gray, or black shades due to the presence of dihydroxynaphthalene (DHN)-derived melanin, a hydrophobic, negatively charged compound with a high molecular weight produced by phenolic and/or indolic oxidative polymerization (33). Growth of the Chaetothyriales is invariably slow. Generic distinction is made by the morphology of their clonal mode of reproduction. In Rhinocladiella, conidia are produced sympodially on elongate cellular extensions; in Fonsecaea, they are clustered on denticles and arranged in short chains; in the Cladophialophora genus, they are arranged in long, dry chains; in Phialophora and in Cyphellophora, they are produced in slimy heads through collarettes, with Cyphellophora being differentiated by curved, mostly septate conidia; in Veronaea, long conidiophores produce sympodial, two-celled conidia; and in Exophiala, the cells producing conidia are annellidic, while the delivered cells show intensive budding. Different morphotypes may occur next to each other in a single strain, and the genera outlined above lack phylogenetic significance. Species of the same genus are often morphologically indistinguishable from each other; for reliable distinction of species, sequencing of diagnostic genes is necessary (34). On the other hand, although the number species related to the etiology of CBM has increased after molecular taxonomy, no clinical or therapeutic association has been attributed to the new genotyping of species.

Molecular PhylogenyToday, the guiding principle to display taxonomic relationships among fungi is molecular phylogeny, since phenotypic characteristics are poorly informative. Partial ribosomal DNA (rDNA) large-subunit (LSU) sequences are sufficiently conserved to show relationships at the ordinal or family level. Pathogenic species are polyphyletic within the Chaetothyriales, being dispersed all over the tree. The main agents of CBM are limited to three clusters. A Fonsecaea cluster, nested in the “bantiana clade” (34, 35), contains prevalent agents of CBM, Fonsecaea pedrosoi and F. monophora (36, 37) (Fig. 1). Uncommon, recently described agents in this clade are F. nubica and F. pugnacius (38, 39). Other fungi in the bantiana clade are Cladophialophora species related to C. bantiana, the main agent of primary brain infection, and several species isolated from disseminated infections but also some saprobes that are not known to be involved in human disease (40).

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FIG 1

Phylogeny of a representative selection of species of the Chaetothyriales, based on the confidently aligned LSU rDNA D1-D2 domains of LSU sequences constructed by the maximum likelihood method implemented in MEGA 5.10. Bootstrap values of >80% from 500 resampled data sets are shown with branches. Morphologies of the species concerned are shown at the right. Exophiala spinifera was used as the outgroup.

Cladophialophora carrionii (41, 42) is located in a separate cluster (“carrionii clade”) along with the recently described species C. samoensis, causing chromoblastomycosis in Samoa (33). This clade also includes Phialophora verrucosa, which has already been isolated from CBM lesions (40, 43, 44).

Rhinocladiella aquaspersa, another consistent agent of CBM (45–48), is located at a significant distance from both clades and is currently not assigned to any phylogenetic group within the Herpotrichiellaceae. Occasional infections by Exophiala species have been reported, which mostly cause other types of infections, i.e., Exophiala jeanselmei, E. dermatitidis, and E. spinifera (8, 49–54), each located in separate clades (Fig. 1). All agents are flanked by species that cause other types of disease and by environmental species (55, 56).

Molecular identification of individual species is done with the rDNA internal transcribed spacer (ITS) region (35). For distinction of closely related Fonsecaea or Phialophora species, an additional gene such as translation elongation factor 1α (TEF1) or a partial β-tubulin gene (BT2) may be recommended (35, 57) (Fig. 2). The cytochrome P450 cluster involved in melanin synthesis and hydrocarbon degradation might play an important role in the virulence of the Chaetothyriales, which probably differs from the other virulence factors of fungi reported previously, such as Lac and HmgA (58), and other virulence factors (59–65). Likewise, the ACT1, BT2, and Cdc42 genes are effectively involved in cell cycle stages and the formation of the actin cytoskeleton (35), which has been related to morphogenetic switching to muriform cells, which are considered the invasive phase of agents of CBM (66) and which are also used for species distinction (35, 57).

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FIG 2

(a to c) Exophiala dermatitidis CBS 748.88. (a) Colony on malt extract agar (MEA) after 3 and 4 weeks of incubation at 30°C; (b) conidial head; (c) conidiophore and conidia. (d to f) Exophiala spinifera CBS 899.68. (d) Colony on MEA after 3 weeks of incubation; (e) conidiophore and conidia clustered at the apex of the conidiophore; (f) conidiophore and liberated conidia. (g to i) Cladophialophora carrionii CBS 166.54. (g) Colony on MEA after 3 weeks of incubation; (h) branching conidial system and conidial chains; (i) conidial chains. (j to l) Fonsecaea pedrosoi CBS 273.66. (j) Colony on MEA after 3 weeks of incubation; (k) conidiophores and conidia; (l) phialides and conidia. (m to o) Phialophora verrucosa BMU 07506. (m) Colony on MEA; (n) phialides and conidia; (o) flask-shaped phialides and conidia. (p to r) Rhinocladiella aquaspersa CBS 122635. (p) Colony on MEA after 3 weeks of incubation; (q) conidiophore and conidia; (r) young conidia and conidiophore. All cultures were incubated at 30°C.

BiodiversityThe genus Fonsecaea comprises four species that cause CBM: F. pedrosoi, F. monophora, F. nubica, and F. pugnacius. Fonsecaea monophora and F. pugnacius show significant neurotropism, eventually leading to dissemination to the brain and other organs (38, 39, 41) or causing primary brain infection without skin lesions, which are clinical forms of PHM, because no muriform cells are seen in tissues (40, 67). All species of Fonsecaea have felt-like, gray-olivaceous colonies. Hyphae are regular, melanized, and branched in the apical part. Terminal cells show 1 to 4 denticles, each bearing a single-celled, broadly clavate conidium, which in turn produces 1 to 2 smaller conidia on denticles (34). Additionally, particularly in media poor in nutrients, phialides with slimy heads of conidia emerging from large collarettes may be produced (Fig. 2). The taxonomy of Fonsecaea was revised previously by de Hoog et al. (41) and Najafzadeh et al. (57). New species such as F. nubica and F. pugnacius were identified with sequences of the ITS and cdc42, BT2, and ACT1 genes, eventually supplemented with amplified fragment length polymorphism (AFLP) profiles (38, 39). Pathogenic species of Fonsecaea present optimum development at 33°C, with a thermotolerance of growth at 37°C. These cardinal temperatures are slightly higher than those of strictly environmental species (56).

The two Cladophialophora species causing CBM, i.e., C. carrionii and C. samoensis, form grayish-green, dry colonies that profusely sporulate, with conidia being arranged in long, densely branched chains composing a shrub-like conidial system (Fig. 2). Also, in these species, phialophora-like conidia may be produced on nutritionally poor media. Species are phenotypically identical; distinction is made by ITS sequencing (33). Phialophora verrucosa is monomorphic for flask-shaped phialides with large, dark, funnel-shaped collarettes at the top, from which slimy heads of ellipsoidal, one-celled conidia are produced. Colonies are olivaceous-black and grow moderately rapidly (24, 25, 34, 68). Disseminated forms of the disease have also been reported but without unambiguous muriform cells in tissue (44), and thus, they may be considered PHM. Some similar cases in China concerned patients with CARD9 mutations (69).

Rhinocladiella aquaspersa forms erect, dark brown, well-differentiated conidiophores, which produce abundant conidia alongside the terminal parts of conidiophores (45, 70). Conidia are subhyaline, ellipsoidal to clavate, and produced from darkened scars. As such, the species is recognizable by microscopic morphology. Colonies are restricted, velvety, and olivaceous-black. Most cases have been reported from South America (46–48).

Exophiala is the only genus of the Chaetothyriales that produces a yeast-like phase. Liberated cells inflate and reproduce by budding or become subspherical and gradually change over to hyphae via a string of more or less inflated cells known as the “torulose mycelium,” which is highly diagnostic for Exophiala. Colonies are black, slow growing, and initially mucous and often become dry and hyphal with age, giving the impression that the colony has become contaminated. Exophiala spinifera has long, erect conidiophores with long annellated zones, and E. dermatitidis has several very short annellated zones next to each other on a single cell (34). Almost all other Exophiala species have to be distinguished by sequencing (55, 71, 72).

The currently recognized increased diversity of species related to CBM has limited clinical or epidemiological consequences, since risk factors and treatment regimens are largely the same for all species.

Potential Environmental Sources of InfectionMelanized fungi and their relatives are also denominated “dematiaceous,” “phaeoid,” or simply “black” fungi (30, 67, 73). This denomination refers to fungi containing melanin in their cell walls microscopically visible by a gross brown, olivaceous, or black pigmentation period. Although many authors claimed to have obtained melanized fungi from natural sources, in most of those reports, no molecular identification was performed. Agents of CBM in the Chaetothyriales constitute only a small fraction of this group. Members of this order are enriched where toxic monoaromates are prevalent. Particularly, human-made niches are occupied, such as wood treated by phenolic preservatives, toxic mine waste, or oil-polluted soils. In nature, monoaromates are found in trace amounts in plant debris, thorns, and wood cortex, which provide microhabitats for these fungi (74). There is a strong correlation between the traumatic implantation of potential natural sources of infection and CBM lesions (74–80). This route of infection may be supported by clinical reports of patients exhibiting the presence of fragments of plant material at the site where they experienced a previous trauma. On rare occasions, wood fragments containing muriform cell-like structures were histopathologically observed in patients with CBM (77, 81–84).

CBM is strongly associated with agricultural activities, which further underscores the occupational nature of this disease (16). During labor activities, individuals living in areas of endemicity are probably infected through diverse traumas related to environmental materials (80, 84–88) (Table 2). Remarkably, however, etiological agents of CBM are difficult to recover from their environment due to their saprobiotic lifestyle: direct isolation by the use of selective methods usually yields saprobic counterparts of the pathogens, while the pathogens are almost exclusively restricted to warm-blooded hosts. Ruben et al. and Fernández-Zeppenfeldt et al. reported the identification of Cladophialophora in cactus plants (83, 89), but de Hoog et al. showed that this was a molecular sibling, C. yegresii (33). Vicente et al. reported the identification of mainly saprobic Fonsecaea species such as F. minima and F. erecta in plant thorns close to the habitat of patients with CBM due to F. pedrosoi, while pathogenic strains were recovered only exceptionally (56, 74, 76). In 1937, Conant thought that Cadophora americana recovered from wood pulp was identical to Phialophora verrucosa, which potentially caused CBM (90), but recently, Feng et al. proved that the P. verrucosa complex contains clinical species next to environmental species (91). Phenotypically identified isolates of F. pedrosoi and C. carrionii have been isolated from plant debris, grass, tree cortex, and also abandoned wasp nests (75–78, 92–97). After the isolation of a strain identified as F. pedrosoi from a spiny plant (Mimosa pudica), Salgado et al. suggested that it might be a probable natural source of CBM infection in the north of Brazil (74), but molecular proof was not provided. Several species of Palmacea (palm trees) have also been recognized as natural habitats of melanized fungi (72–75). A probable infection source was described in an area of endemicity in the State of Maranhão, located on the border of the Brazilian Amazon rainforest, where several agricultural communities were working on harvesting babassu (Orbignya phalerata), a wild palmacea specimen (78–80). Local dwellers collect the babassu nuts, which are rich in oil and are a well-known component used by industrial companies for the manufacture of international beauty products. Members of the Chaetothyriales are indeed enriched on babassu shell fragments, which are hypothetically considered a risk factor for the development of cutaneous lesions after labor trauma (78, 80, 84). The invasive potential of agents differs significantly between species and is as yet only fragmentarily understood. CBM is a human disease, and the scarce reports of this infection in other animals, such as amphibians and mammals, are considered PHM, because typical muriform cells were lacking (98–101).

Given the above-described ambiguous results, we hypothesize that saprobic and opportunistic counterparts may occur in a single environmental sample but occupy different microhabitats. Due to this nutritional deviation, saprobic species can be isolated by standard methods (56, 74), while opportunists and pathogens are selected when samples are enriched via a mammalian vector (75). We also hypothesize that both invasive and noninvasive fungi may be present in the same environmental sample, where they each have a different microhabitat. Methods are available to isolate saprobic fungi from the environment, but selective methods for pathogens are needed. The use of mammalian vectors, as done previously by Gezuele, Mok, and others, should be repeated with the support of state-of-the-art identification methods (74–78).

Geographic DistributionThe species most frequently associated with CBM belong to the genera Fonsecaea and Cladophialophora. Infections due to Rhinocladiella are less frequent, while a few cases were associated with members of the Phialophora and/or Exophiala genus. F. pedrosoi and C. carrionii infections are normally observed in tropical and subtropical areas of endemicity around the world. There are several reports addressing the reservoir of the most common CBM agents: C. carrionii occurs in semiarid areas, whereas Fonsecaea pedrosoi is associated with humid climates (3, 9, 11–13, 85, 86) (Fig. 3).

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FIG 3

Global distribution of chromoblastomycosis based on reported case series.

Similarly to most of the endemic mycoses, CBM is not a notifiable disease. As a consequence, there is no precise assessment of either the incidence or prevalence of this mycosis. Instead, data gathered from surveys and case series suggest that the incidence of CBM ranges from 1:6,800 (Madagascar) to 1:8,625,000 (United States) (3, 16). The disease is prevalent in tropical and subtropical regions of the planet, mainly between latitudes of 30°N and 30°S. The majority of cases are reported from Latin America, the Caribbean, Africa, and Asia. Brazil, Mexico, Venezuela, India, Australia, and southern China report the majority of the series of patients.

Chromoblastomycosis in the AmericasAlthough Lane and Medlar described one of the first cases of CBM in the United States in 1915 (24, 25), the United States is not an area where this mycosis is endemic. The second case of CBM in the United States was not described until 1933 in Texas; this patient presented with ulcerated and nodular lesions on the right foot (96). Over subsequent decades, scattered cases have been described, particularly from Texas and Louisiana (84, 100–107).

In comparison, implantation mycoses in Mexico are a frequent health problem. Sporotrichosis and CBM (108) are the most common forms of implantation mycosis in this country. A review of the cases (109) reported until 2013 identified 603 mycologically and/or histologically proven cases, supporting the premise that Mexico should be considered a region where the disease is highly endemic (110, 111). Fonsecaea pedrosoi is the most common etiological agent of CBM (95.8%), although disease caused by C. carrionii, P. verrucosa, R. aquaspersa, and E. spinifera has also been reported (12, 111). Most strains were identified by morphology; data from only a few of these reports have been confirmed by molecular methods.

The disease has also been described throughout Central America, from Guatemala to Panama (111, 112). Large numbers of cases have been described in Costa Rica (113, 114), Panama (115), Honduras (111, 116), El Salvador (112), Nicaragua, and Guatemala (12), in decreasing order. Most cases arise in the tropical rainforest. The epidemiological data are similar to those for Mexico, with the prevailing fungus isolated being F. pedrosoi, although other species have also been reported. Among these reports was the exceptional report of CBM caused by Chaetomium funicola in Chiriquí, western Panama (117).

In the Caribbean, unlike Central America, disease is the main cause of implantation mycosis; around 600 cases have been reported (113). Of these cases, 450 patients from the Dominican Republic were described by Isa-Isa (114). Most of these patients were from the humid southern forest region, which is considered to be a focus of endemicity, similar to the Brazilian Amazonian region or Madagascar (118, 119). Approximately 100 cases have been reported in Cuba (37, 120), and fewer cases have been reported in Puerto Rico (121), Jamaica, and Haiti (114, 122). While F. pedrosoi is the most common etiological agent of CBM in the Caribbean, its molecular sibling F. monophora may also occur, as was uncovered in Cuba (37) only after sequencing. Cladophialophora carrionii was encountered in Puerto Rico (121).

In South America, most of the cases of CBM have been described in Brazil. With the exception of Chile, CBM has been reported in all South American countries. After Brazil, Venezuela and Colombia account for most cases of CBM. Within Brazil, CBM is endemic in many geographical areas, especially in the northern regions, where 872 cases were retrospectively reported during the last decades (3, 79, 123, 124). Although 332 cases were reported from other provinces, a significant decrease in the number of new CBM cases has been observed, especially in the southern regions of this country (125, 126). The mean annual incidences of cases of CBM reported in Brazil were 6.4/year (71 cases/11 years) for the state of Paraná (southern region), 5.9/year (325 cases/55 years) for Pará 45 (northern region), 4.3/year (13 cases/3 years) for Maranhão (northeastern region), and 2.6/year (73 cases/28 years) for Rio Grande do Sul (southern region) (8, 13, 123–126). The principal etiological agent of CBM in Brazil is F. pedrosoi (which may include its molecular siblings), followed by sporadic reports of P. verrucosa and E. spinifera.

Following a pattern similar to that observed for paracoccidioidomycosis, the number of new cases of CBM is decreasing in some Brazilian regions. This is thought to be a consequence of several modifications of agricultural methods, including the massive use of agricultural azole fungicides and progressive agriculture mechanization, resulting in a diminution of risk factors due to occupational exposure (127–129).

Chromoblastomycosis in Asia and OceaniaJapan has the highest incidence of CBM among populations in Asia (1/416,000). The first Japanese case was described in 1930 by Kano, who reported a female patient with an unusual CBM-like infection in the facial region, caused by Hormiscium dermatitidis (currently Exophiala dermatitidis) (130). Since the first description, several Japanese authors reported ∼700 cases of CBM infections (130–133). Several hundred cases have been reported from Mainland China since the first description by Yew in 1951 (134, 135). The cases were distributed all over the country, covering more than 21 provinces. The highest prevalence rates were found for Guangdong Province (84/196) and Shandong Province (38/196) based on literature published from 1990 to 2015 (136). There may be areas of hyperendemicity; for example, in a study of CBM conducted by Dai et al. in Zhangqiu City, Shandong Province, up to 300 cases of CBM were found in 1998 (135).

India has long been known for autochthonous CBM infections, with more than 100 cases being reported since the first description in 1950 (137, 138). In other counties, such as Sri Lanka, Pakistan, Thailand, and Malaysia, CBM cases have regularly been reported, demonstrating its endemic character in South and East Asia (139–144).

In Oceania, CBM was first described in Australia, and to date, approximately 200 cases have been reported. CBM in Australia is caused mainly by C. carrionii, due to the prevailing arid climatic conditions. There have been a few reported cases from other countries in Oceania, such as New Zealand and Solomon Islands (145–148).

Chromoblastomycosis in AfricaMadagascar represents the most important focus of CBM described to date in the world. Retrospective data collected by Brygoo and Segretain and Esterre et al. at the Institut Pasteur of Madagascar during a 40-year period revealed one of the world's largest case repositories of CBM, consisting of 1,323 confirmed cases observed between 1955 and 1995 (19, 20, 85, 86). Those authors described two distinct ecosystems in the island, one in the north, with a tropical rainforest climate, where F. pedrosoi predominates, and other in the south, with an arid and dry geographic region, where C. carrionii causes 41% of chromoblastomycotic infections. The isolation of the latter species from the Malagasy spiny desertic region suggests that continuous deforestation, in order to produce charcoal and for house construction, constitutes an environmental risk factor associated with this disease (85, 86). Unfortunately, updated epidemiological studies from the African continent are scarce, and the actual burden of CBM in Africa may be underestimated.

Chromoblastomycosis in EuropeCBM is a disease observed mainly in tropical and subtropical regions (Latin America, Asia, and Africa), but there are many imported cases in Europe. A recent review of CBM in Europe revealed a total of 31 probable cases (149). The authors of that study suggested that the disease was considered to be autochthonous in some cases. One of these cases was an infection acquired in a mine from locally harvested mine wood and caused by F. monophora (149). As CBM is uncommon in Europe, cases may have been misdiagnosed as cutaneous tuberculosis, squamous cell carcinoma, psoriasis, PHM, and sporotrichosis or other infectious and noninfectious conditions that may mimic CBM.

Demographics and Risk FactorsJudging from most of the reported case series, CBM involves mainly adult males. The sex distribution in a case series reported in the southern region of Brazil showed that disease was prevalent in males (4:1) (126). In two other studies involving 390 cases in the same region, the sex ratio distribution was much higher for males (17:1) (123, 124). The difference related to sex distribution in patients with CBM may be related to hormonal protection, as observed for paracoccidioidomycosis patients (150). In these systemic mycoses, females are protected from clinical manifestations by β-estradiol, whereas in CBM, hormonal protection may be related to progesterone (150, 151). Cytosol receptors have been identified in P. verrucosa, and its in vitro growth was found to be influenced by progesterone and testosterone hormones but not by estradiol (152). Previous explanations of skewed male/female ratios, such as different occupational risks of agricultural labor, are largely erroneous.

In the Amazon, the age distribution ranged from 25 to 85 years, with the most affected group being between 41 and 70 years old (86%) (123, 124). In Mexico, in a series of 603 cases, the disease predominated in adult males (66%) >38 years of age. Although the age range extended from 9 years to 90 years, children were rarely affected (1.2%) (109). In China, the majority of CBM patients are males (6.7:1), with a mean age of 54.75 years (range, 10 to 81 years) (136). Although CBM in children is infrequent, from 1992 to 2004, 22 cases (aged 2 to 19 years) of C. carrionii infections were reported in the semiarid state of Falcón, Venezuela, (88).

Genetic susceptibility may also participate in adaptations of the etiological agents to the host tissue environment. In a Brazilian study, 32 nonconsanguineous white CBM patients and 77 healthy controls who were matched according to gender, age, ethnic background, profession, and geographical region were studied for the distribution of HLA-A, -B, -C, -DR, and -DQ. The frequency of only HLA-A29 was significantly increased. This antigen was present in 28% of patients, as opposed to 4% of the controls (P = 0.03) (153). These findings suggest a possible genetic susceptibility to CBM. The relative risk to develop disease for patients carrying HLA-A29 was estimated to be 10. In another study conducted in Venezuela, Yegres-Rodriguez also indicated a genetic susceptibility to CBM in the population of the endemic area of Falcon State (154).

Chromoblastomycosis is considered an occupational disease around the world, affecting farm laborers, gardeners, lumberjacks, vendors of farm products, and other workers exposed to contaminated soil and plant materials (3, 12, 13, 16). Similarly to eumycotic mycetoma infections, the lack of protective shoes, gloves, or garments in association with poor hygienic habits and deficient nutrition may favor the development of clinical forms of CBM after infection by implantation (13, 16, 125). As reported in Madagascar, other groups of laborers besides charcoal producers deal with environmental occupational hazards (78–80, 85, 86). For instance, there is some evidence in India that CBM may be acquired during manual work in black tea cultivation at the Gardens of Assam, northeast region, and also in rubber plantations in the central districts of Kerala and nearby Western Ghats (155). Less frequently, CBM has been observed in immunosuppressed hosts, usually in solid-organ transplant recipients and in association with neoplastic diseases (156). Similarly to implantation mucormycosis after natural disasters, several cases of CBM were reported in the United States after Hurricane Ike (157, 158).

Cell Morphology and ArchitectureMelanized fungi are polymorphic organisms. Due to their plasticity and adaptability to several organic and inorganic environments, melanized fungi may show a great diversity in their morphology. When causing PHM, in clinical specimens, the etiological agents may present a series of morphological shapes, isolated or in combination: septated (toruloid) hyphae, pseudohyphae, and yeast-like and vesicular components. When causing black grain mycetoma, melanized fungi usually present as dark black grains composed of short distorted hyphae associated with vesicular elements (3, 67, 73). It is believed that hyphae and conidia are found abundantly in nature and are easily replicated in simple media such as Sabouraud agar (56, 77). In contrast, resistance forms are usually found only under extreme environmental stress conditions, such as very high or very low temperatures, extreme pHs, and nutrient-deficient soils. These resistance forms may also survive in rocks and in plants (164, 165).

After transcutaneous implantation, propagules of CBM agents present a unique cellular and morphological plasticity. During infection, cell differentiation becomes meristematic, with isodiametric swelling and cross-septation. The resulting muriform cells (166) have also been denominated “Medlar bodies” or, alternatively, “copper pennies,” “chromo or fumagoid bodies,” and “sclerotic or meristematic cells” (118). “Meristematic” is a botanical term for the expansion of cells, and for black fungi, it is used mostly for the description of clumpy thalli of rock-inhabiting extremophiles (167). The word meristem comes from the Greek word merizein, meaning to divide. The term “sclerotic” is related to “sclerotia,” which are made of compacted masses of latent hyphae (168). The term “muriform cell” is restricted to cells of the Chaetothyriales with meristematic growth that serve as invasive forms in living tissue, either human or plant (169). Muriform cells may be single or clustered. They have a round-to-polyhedral form in a darkly pigmented thick wall with transverse and longitudinal cross-walls. The muriform cell is considered to be a mechanism for evolutionary adaptation to enable survival inside the microenvironment of the host (118). It is directly associated with an intense granulomatous response as well as with the evasion of immune mechanisms signaling the onset of disease chronicity. The time of conversion from conidia to muriform cells in in vitro studies was estimated to be 6 days (58, 170).

The muriform cell arrangement in tissue depicts an optimal surface/volume ratio favoring significant melanin deposition. Fonsecaea pedrosoi requires a low concentration (0.1 mM; pH 2.5) of Ca²⁺ to differentiate from mycelia to muriform cells, indicating that the ion concentration may be important in the process of transitioning during CBM (58, 171, 172). Muriform cells collected from lesional tissues can easily differentiate into hyphae and conidia in vitro or can be maintained as muriform cells under harsh conditions of low pH and nonoptimal nutritional sources in liquid medium (170). It has been shown that the survival of resistance forms and consequently the emergence of clinical disease caused by F. pedrosoi may be strongly associated with the presence of muriform cells and an invasive form inside host tissue (173). Among other factors, melanin is also strongly associated with the process of transition. Furthermore, muriform cells remain a significant differential diagnostic tool to distinguish between CBM and the semantically closely related PHM, in which typically muriform elements are not detected (174). Muriform cells are highly resistant to immune system attack, and therefore, better knowledge of this differentiation process may permit the proposal of different and more efficient therapeutic approaches against CBM (175, 176).

Microbial adherence and hydrophobicity are two of the most important determinants of fungal pathogenesis (176, 177). For CBM, infectious forms may stick to epithelial tissue inside the host, leading to the differentiation of muriform cells that resist killing by the host and permit the evolution of chronic granulomatous inflammation. The presence of extracellular hydrophilic molecules of polysaccharides may enhance this phenomenon because of hydrophobicity. The production of adhesive conidia by phialides of F. pedrosoi, Exophiala dermatitidis, E. spinifera, and Cladophialophora carrionii suggests binding to arthropod or other invertebrate vectors. Thus, a detailed review of the fungal cell wall structure and membrane organization is important to improve knowledge about pathogen-host interactions. Cell wall glycoproteins and glycolipids appear to function as epitopes, indicating their use in immunodiagnosis and potential therapy focused on the stimulation of the humoral response.

Ceramide monohexosides (CMHs) have been identified in membranes and cellular walls of pathogenic and nonpathogenic fungi, presenting a unique ceramide moiety compared to those of mammalian CMHs (177, 178). CMHs seem to have a pivotal function in the host immune response, and they may be associated with fungal differentiation. Cerebroside expression in CBM cells has been strongly associated with muriform cell transition and melanin deposition. In particular, structural but not immunogenic CMH diversity has been observed among different forms of F. pedrosoi. Identical CMH structures have been demonstrated for the conidial and mycelial forms, while the CMH moiety in muriform cells possesses an additional −OH group bound to the backbone of the lipid structure. Furthermore, muriform cells maintain their resistance to CMH antibodies, while their recognition occurs only at melanin-depleted cell wall regions. Three possibilities have been raised based on the above-described results: first, CMHs are absent from muriform cells; second, CMH may have a different structure on muriform cells that would interfere with the recognition of monoclonal antibodies (MAbs); and third, the melanin structure on muriform cells impairs the linkage of anti-CMH antibodies to the membrane target (178). Indeed, chemical removal of melanin by alkali augmented the reaction of anti-CMH antibodies with muriform cells.

The glycoprotein structure of conidial cells of F. pedrosoi consists of two linear polymers, i.e., residual α-(1→6)-bound mannose and β-(1→6)-bound galactofuranose, with replacements of both polymers by an α-(1→2) linkage with residues of glucose. The structure of the glycoprotein additionally contains large amounts of O-linked oligosaccharides, particularly a hexanose, in a comb-like structure (179). O-mannosylation defines the form of the cell wall and cellular specialization and participates in the virulence of the fungus (180, 181). Therefore, the specific above-mentioned structure might play a crucial role in clinics, in strategies for the development of new drugs, and for the detection of F. pedrosoi in the future (179).

Protein phosphorylation and dephosphorylation are important for immunomodulation, influencing the host reaction to invading fungal pathogens (176, 182). The contact and invasion of F. pedrosoi in epithelial cells and macrophages may be associated with the activity of fungal protein kinases (183). In particular, inhibitors of protein kinase, such as genistein and stauporine, when used for pretreating either macrophages or F. pedrosoi conidia before infection, may inhibit cellular invasion. Notably, pretreatment of conidia had an effect simply on interactions with epithelial cells, with no influence on macrophages (183). Specific peptidase inhibitors (PIs) against human immunodeficiency virus significantly affect peptidase secretion and growth of F. pedrosoi, interfering with fungus-host cellular contacts (184). Peptidase activity was impaired in a dose-dependent way, with nelfinavir producing the greatest inhibitory result. The growth of Fonsecaea pedrosoi was importantly affected after exposure to PIs, while the conidial structure presented significant morphological changes comprising cytoplasm invagination, cell wall detachment, widening of fungal vacuoles, and anomalous cell division (184).

Virulence FactorsThermotolerance is one of the important virulence factors among members of the Chaetothyriales. Pathogenic Fonsecaea species have an optimum growth temperature of 33°C and a maximum growth temperature of 37°C (185). These cardinal temperatures are slightly higher than those of strictly environmental species. In comparison, Cladophialophora bantiana, which infects the central nervous system and the respiratory system of humans, can grow at 40°C (186, 187). Similar differences in optimum and maximum growth temperatures seem to guide the predilection for either cold- or warm-blooded hosts in Exophiala species (188).

The rigid cellular wall of the fungus contrasts with the dynamic flux of structural molecules necessary for differentiation, growth, and adaptation to the host. Different membrane and cell wall components may contribute to CBM fungal virulence, including melanin, chitin synthases (CHSs), a set of hydrolytic enzymes (phosphatases, phospholipases, lipases, esterases, ecto-ATPases, peptidases, DNases, ureases, and gelatinases), lipids, galactomannans, and cerebrosides. The differentiation of hyphae and conidia into muriform cells may contribute to virulence, considering the increase of the thickness of the wall and the formation of muriform cells that resist host responses.

Lipids or lipid-free cell wall fractions extracted from different CBM agents induce significant granulomatous reactions in mice (189, 190). Indeed, live muriform cells have a high capacity for the induction of Langerhans giant-cell formation in vitro (185). Acid-labile galactofuranosyl residues are responsible for the immunogenic property of galactomannans isolated from three Fonsecaea species (191). Cerebrosides, the major neutral glycosphingolipids of fungal cells, composed of a monosaccharide linked to an N-acyl sphingoid ceramide, elicit antibody production that inhibits F. pedrosoi growth and enhances the mouse macrophage-killing function (192).

Chitin is formed by a beta-1,3-linked glucan and polymers of N-acetylglucosamine occurring in different conformations, suggesting a role in fungal structure and differentiation (193). Chitin synthesis depends on the enzymatic activity of chitin synthases I, II, and III, which participate in cytokinesis, septum synthesis, and bud scar formation (194), respectively, with each one having its catalytic activity encoded by different genes, CHS1, CHS2, and CHS3, respectively (195), that have conserved sequences in F. pedrosoi (196). The black yeast Exophiala dermatitidis, occasionally involved in CBM (166), possesses a structural gene for a class V CHS, WdCHS5 (194). WdCHS5 is essential for fungal growth at 37°C and the expansion of muriform cells, indicating a critical importance of the class V CHS for virulence (197). Muriform cells of F. pedrosoi were demonstrated to have a chitin-like component that mediates Th17 development by inhibiting dectin-1, impairing the immune response, and contributing to CBM chronicity (32) (Fig. 4).

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FIG 4

CBM immunology. Epidermal Langerhans cells (LCs) are at the front line of defense and may respond differently to conidia or meristematic cells. (A) After recognition of conidia, engulfment and destruction occur, followed by inhibition of costimulatory molecules. Conidial antigens may be presented to T cells, which activates B cells to produce antibodies against CBM pathogens. (B) If muriform cells are present, although there is no phagocytosis by LCs, naive T cells may be activated and proliferate, amplifying the immune response. (C) Recognition of conidia by dendritic cells (DCs) can be made by the C-type lectin dectin-2 or Mincle. However, the dectin-2 pathway leads to T-cell activation with proliferation of Th1 and Th17 cells, while Mincle signaling inhibits the same process, in a clear demonstration of PRR antagonism of the immune response following recognition of CBM fungi by DCs.

MelaninMelanin is a complex, hydrophobic, negatively charged macromolecule that includes indolic or phenolic polymers (32, 73). In CBM agents, this polymer can be derived from either l-3,4-dihydroxyphenylalanine (l-DOPA) or DHN (32, 60). Melanin is broadly found in nature. It is considered to be an immunologically active compound functioning as an important virulence factor in different pathogenic fungi (32, 198). Three possible mechanisms have been proposed to be associated with its contribution to the enhanced resistance of fungi against immune host cells: (i) protection against proteolytic enzymes, (ii) protection against oxygen or nitrogen derivatives, and (iii) reduction of phagocytosis. Melanin derived from DHN is synthesized by the polyketide path, a route starting with acetyl coenzyme A (acetyl-CoA) (199). Interestingly, the inhibition of this specific biochemical process with tricyclazole may be used to induce morphological changes in the cellular wall of F. pedrosoi leading to diminished resistance of the fungus to mechanical lysis and macrophage killing (200, 201). Melanin is mainly stored, as revealed by transmission electron microscopy, in concentric layers in intracellular vesicles, known as melanosomes, similarly to mammalian cells (201). Further studies have demonstrated that F. pedrosoi produces not only cell wall-associated but also extracellular melanin, pyomelanin, which may accumulate, e.g., in mutants of the tyrosine degradation pathway, as observed for spontaneous mutants of F. monophora (202). In particular, melanin may be detected inside phagocytic vacuoles together with engulfed fungi.

During infection, melanin deposition from F. pedrosoi interferes with nitric oxide (NO) production and inhibits phagocytosis (202), which is not reverted with gamma interferon (IFN-γ) and lipopolysaccharide (LPS). This phagocytosis inhibition may also be observed after in vitro incubation with whole melanized cell walls. The immunomodulating role of melanin has also been shown for CMH recognition by antibodies on specific regions of the fungal cell wall where melanin was poorly expressed, implying that inhibition of melanin biosynthesis by drugs might render muriform cells more vulnerable to antibodies against CMH (190). As demonstrated by flow cytometry and immunofluorescence analyses, antibodies to melanin were reactive with mycelia, conidia, and muriform cells along with ghost particles (203). Muriform cells derived from clinical specimens were immunogenic, leading to interactions with antibodies. Opsonization of Fonsecaea pedrosoi conidia gradually led to increased polymorphonuclear leukocyte (PMN) attack and phagocytosis (178).

Extracellular Enzymes and MetabolitesProteolytic enzymes, such as peptidases, have multiple tasks in consecutive stages of the pathogen-host interaction enabling the bypass of the host defense, leading to either digestion or host surface destruction. Proteolytic enzymes can be detrimental to various components of the host defense mechanisms, leading to immunological escape or antimicrobial resistance (204, 205). Kneipp et al. demonstrated that muriform cells, in comparison to conidia and mycelia, have high phosphatase activity associated with pathogenicity (206, 207). Phosphorylated substrates were hydrolyzed by surface ectophosphatases. The activity of the enzymes was increased on low-acidic-pH compounds and was blocked by specific compounds such as sodium fluoride and sodium molybdate (207).

A series of hydrolytic enzymes is produced and secreted by CBM agents. The walls of F. pedrosoi muriform cells present higher phosphatase activity than do conidia or hyphae (207), and phosphatase activity enhances host cell adhesion in both F. pedrosoi (206) and Rhinocladiella aquaspersa (208). The cytolytic effect of ATP suggests that ecto-ATPases found at the surface of F. pedrosoi may favor fungus survival in hostile environments such as the human body (209, 210). Peptidases secreted by F. pedrosoi are able to cleave human plasma proteins, such as immunoglobulins and albumin, and components of the matrix, such as fibronectin, while metalloproteinase inhibitors impair conidial growth and differentiation (211) These findings were recently confirmed in Phialophora verrucosa (212). HIV aspartyl peptidase inhibitors strongly abrogate aspartyl proteolytic activity (204), greatly affect F. pedrosoi ultrastructure, diminish adhesion to epithelial cells, and increase susceptibility to killing by macrophage cells, indicating possible therapeutic use in CBM patients (184).

Innate Immune ResponseAmong the cells of innate immunity, macrophages seem to have an important function in regulating fungal growth. CBM fungal cells can be detected in intracytoplasmic vacuoles of skin macrophages (213). Sotto et al. investigated the cellular immune response, especially the distribution of antigens and antigen-presenting cells (APCs), in lesional biopsy specimens of patients with CBM (214). Notably, most antigens were observed as homogeneous or granulated material in the cytoplasm of macrophages, indicating that phagocytes are involved in innate immunity against CBM agents (214). Similar antigens accumulated in hypertrophic FXIIIa⁺ dendritic cells (DCs) (214). However, chronic granulomatous infectious diseases are usually characterized by numerous macrophages on lesional tissue. On the other hand, activated macrophages seem to have a fungistatic rather than fungicidal role in CBM, enabling the survival and proliferation of F. pedrosoi inside macrophages. In particular, Rozental et al. demonstrated a fungistatic role of activated macrophages in delaying germ tube and hypha formation (215). Macrophage fungicidal activity is dependent on the etiological agent of CBM (216). Higher digestive activity was observed in cases of F. pedrosoi, C. carrionii, and R. aquaspersa infections than in infections by other microorganisms (216). Phagocytosis mediated by complement was more significant in R. aquaspersa and P. verrucosa than in F. pedrosoi infection and was suppressed by mannan; killing was significant only in R. aquaspersa. These findings indicate fungicidal activity of resident macrophages against R. aquaspersa but little or no activity against C. carrionii, P. verrucosa, and F. pedrosoi.

Bocca et al. showed impaired macrophage function upon F. pedrosoi infection (217), with inhibition of NO production, even after culture with IFN-γ and LPS. In addition, decreases in the levels of CD80 (B7-1) and major histocompatibility complex class II (MHC-II) have been shown. da Silva et al. reported that phagocytosis of CBM fungi was cell type dependent (171). Langerhans cells (LCs) isolated from the skin of BALB/c mice were phagocytic only against conidia and not against muriform cells of F. pedrosoi. In addition, maturation of LCs, evaluated by the amounts of CD40 and CD86, was blocked only by conidia, demonstrating a significant function of the innate immunity of LCs in infection by F. pedrosoi. Judging from these results, muriform cells induce disease exacerbation with a Th1 response, and conidia divert this to a Th2 antibody response (Fig. 4).

The cytokine profile depends on the severity of CBM. Mazo Fávero Gimenes et al. demonstrated that patients with more severe clinical forms produced predominantly interleukin-10 (IL-10) with inhibition of IFN-γ, resulting in low-level induction of T-cell proliferation (218), while patients with mild CBM presented increased IFN-γ and decreased IL-10 production with effective T-cell proliferation (199). Mild forms of CBM favor a Th1 profile due to a good immunological response that may inhibit disease development, while, in contrast, moderate forms of CBM trigger an intermediate response between Th1 and Th2. Furthermore, CBM patients secrete large amounts of tumor necrosis factor alpha (TNF-α). Furthermore, F. pedrosoi and R. aquaspersa induce macrophage IL-1 secretion, whereas C. carrionii triggers IL-6 production, suggesting that IL production is fungal species specific (218).

Sousa et al. showed that in severe CBM, patients presented more IL-10 and less HLA-DR and costimulatory molecules than did patients with mild CBM (219). Immune therapy with anti-IL-10 or with recombinant IL-12 upregulated HLA-DR and costimulatory molecules, thus reestablishing an antigen-specific Th1 cellular response. These results also demonstrate different profiles of monocytes from CBM patients with distinct clinical forms (219). The impact of chemotherapy for CBM on the cellular immune response was analyzed by Gimenes et al., who evaluated the production of IL-10, TNF-α, and IFN-γ as well as the proliferation of peripheral blood mononuclear cells (PBMCs) from patients with CBM at different time points of therapy (220). In this study, after treatment for 6 months, cells from CBM patients proliferated after contact with fungal antigens, producing high IFN-γ levels. After 1 year of treatment, T-cell proliferation and IFN-γ secretion were diminished, followed by IL-10 augmentation. da Silva et al. showed that CBM patients presented large serum amounts of transforming growth factor β (TGF-β), which were decreased after therapy with itraconazole (ITZ) for 3 months, correlating these findings with fast clinical enhancement (221). On the other hand, after therapy for 6 to 12 months, the amounts of TGF-β increased to the levels found before therapy, which was clinically correlated with slow enhancement in clinics and the persistence of fungal cells and fibrotic lesions (221).

The absence of fungal identification mediated by Toll-like receptors (TLRs) led to flawed cytokine production against F. pedrosoi, which was restored after the addition of TLR ligands in vitro, followed by in vivo protection from infection in an animal model (222). Chronicity developed after failure of costimulation of pattern recognition receptors (PRRs) (206). In cases of Fonsecaea pedrosoi, initial fungal recognition is mediated only by C-type lectin receptors (CLRs), leading to the flawed stimulation of proinflammatory compounds. TLR costimulation restored inflammation in response to F. pedrosoi, also requiring signaling by CLRs through the Syk/CARD9 pathway. Administration of exogenous TLR ligands facilitated the clearance of fungal infection in vivo (222).

PMNs are the main regulatory cells of the characteristic CBM granuloma (223). Abscesses rich in PMNs show abrogated engulfment of muriform fungi, whose in situ existence is thought to be a crucial factor in the chronicity of the inflammatory reaction (118). PMNs appear to competently eliminate F. pedrosoi extracellularly by using antibody-independent mechanisms, including reactive oxygen species production and the liberation of substrates (224, 225). Host receptors, such as β-glucans, may also participate in the fungicidal role of PMNs by ligation to dectin-1 (226) (Fig. 4).

DCs are significant immune regulators in several fungal infections. Mature DCs present a unique ability to prime and polarize naive lymphocytes toward a Th1 response, while immature DCs enhance a Th2 response, inducing immune tolerance. DCs are responsible for surveillance and primary protection upon invasion of melanized fungi on the skin (227). Patients with severe CBM may have T-helper-cell activation and an upregulation of HLA-DR and costimulatory molecules enhanced by DCs. The authors of that study concluded that an altered T-cell response in F. pedrosoi infection induces fungal disease exacerbation (227) (Table 3).

Adaptive Immune ResponseCell-mediated immunity (CMI) shows increased antigenic specificity and memory but develops more slowly than innate immunity (58, 176). Stimulation of CD4⁺ cells by macrophages responsible for the engulfment of F. pedrosoi is essential to prevent CBM development (228). Impairment of cell-mediated immunity in CBM patients led to an inefficient reaction to fungally derived antigens, resulting in the maintenance of CBM-related fungi in lesional skin (229). A delayed-type hypersensitivity (DTH) reaction in F. pedrosoi CBM indicates that inflammation may be mediated by T cells (227). The immunophenotypic profile associated with CMI in the chronic granulomatous process leading to CBM cutaneous lesions showed predominantly macrophages but also T-helper and cytotoxic T cells, besides B cells (85, 230).

A study in nude mice inoculated with F. pedrosoi revealed more diffuse granulomas during infection, with a haphazard fungal distribution, corroborating the crucial function of T cells in CBM control (231). CD4⁺ lymphocytes have a role in controlling CBM by secreting IFN-γ in order to increase cellular immunity responses against F. pedrosoi (218, 220, 232). Upon mouse inoculation with live conidia, increased T-helper-cell entrance into lymph nodes was observed (232). After inoculation of F. pedrosoi into the peritoneum of mice depleted of T helper or cytotoxic T cells, a high fungal burden was observed in the spleen and liver of CD4⁺-depleted animals, in contrast to CD8⁺-depleted mice, which were not affected. Furthermore, CD4⁺-depleted mice had decreased DTH and produced smaller amounts of IFN-γ than wild-type animals (232).

The Th17 function in CBM is not well defined. Interleukin-17 and IL-22 seem to play a critical role in mucocutaneous host defense (233). CARD9 mutations in four patients with PHM presented significantly decreased cytokine production and fewer Th17 cells, with a diminished immune response to P. verrucosa (69, 234) (Fig. 4).

The humoral immune response is less frequently investigated for CBM infection, since previous studies have shown that this arm of the host defense may not be effective against melanized fungus infections (58). Esterre et al. demonstrated a decrease in antibody titers after antifungal therapy in CBM patients (235). For the severe form of CBM, increased production of IgG has been observed. A specific humoral immune response may develop in individuals who live in regions of endemicity and who were previously exposed to the fungus, but this does not clearly correlate with the severity of CBM (86). This corresponds to data from a previous study in which levels of antibodies against neutrophils correlated with CBM presenting chronic and widespread lesions (236). More than 1 year after finishing CBM treatment, patients may present positive serology.

Some antibodies may be protective against CBM. Antibodies against F. pedrosoi melanin enhance the efficacy of phagocytes in inhibiting fungal growth (237). Some patients locally become achromic during the evolution of the disease or upon treatment, indicating that antimelanin antibodies have cross-reacted with human melanocytes, a phenomenon known as vitiligo (Fig. 5).

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FIG 5

Vitiligo and chromoblastomycosis. (A) A 62-year-old Brazilian man presented with a 10-year history of a slow-growing plaque lesion, which started as a small nodule near his navel and spread centrifugally until it reached 30 cm in diameter. Concomitantly, he noted the presence of interwoven achromic patches, which extended to other regions of his skin after 5 years of having the disease restricted to the abdominal area. (B) A skin scraping from the plaque lesion revealed the presence of muriform cells. (C and D) After 3 weeks of culture in Mycosel, a grayish colony grew (C), and micromorphology depicted Fonsecaea pedrosoi structures (D). (E and F) An association of CBM and vitiligo has not been reported, but the presence of antimelanin antibodies in CBM patients is known. Most of the cases that were treated with itraconazole were healed, leaving an achromic patch (F) in place of the previous verrucous/plaque lesions (E). This patient developed vitiligo after CBM, which could be related to the presence of antimelanin antibodies.

Coupling of an antiglucosylceramide (anti-GlcCer) MAb on F. pedrosoi conidia diminished the fungal burden, enhancing the engulfment and destruction of F. pedrosoi by murine cells. Among other factors, immunoglobulin levels may interfere with the host response against melanized fungus (58) (Table 4).

Initial Cutaneous LesionsAfter several kinds of micro- or macrotraumatic wounds, infection initiates after infectious propagules of the etiological agents gain entrance through the cutaneous barrier, usually in exposed and nonprotected areas of the body (3, 5, 12, 13). Feet, knees, lower legs, and hands are the most common sites, but infections of other regions, such as the trunk, nose, ears, eyelids, shoulders, and buttocks, have also been reported (8, 11–13, 85, 86, 125, 238). The period between inoculation and the onset of the initial lesion is uncertain and may range from weeks to months; some patients do not recall any inoculation.

The initial lesion may begin as an erythematous macular skin lesion and progresses to a pink and smooth papular lesion. With time, it may manifest as a papulosquamous lesion and evolve with polymorphic aspects, which may be confused with several infectious and noninfectious diseases (Fig. 6A). The initial lesion may spread locally and produce satellite lesions. At this point, pruritus is the main clinical manifestation. At this early stage, the patient mostly does not seek medical help; this stage is rarely seen by the clinician, and if not diagnosed, the initial lesions may progress while assuming several types of clinical forms with different grades of severity (9, 239, 240) (Fig. 6 and 7).

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FIG 6

Clinical types of lesions observed in patients with chromoblastomycosis. (A) Initial lesion with a 3-month duration in the lower leg. (B) Confluent nodular lesions on the knee. (C) Tumoral (cauliflower-like) lesion on the posterior part of the foot. (D) Cicatricial lesion with verruca showing serpiginous and verrucous contours. (E) Hyperkeratotic verrucous lesion on the sole of the foot. (F) Soft violaceous plaque lesion in the root of the thigh.

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FIG 7

Lesions of chromoblastomycosis with different severity grades. (A) Mild forms; (B) moderate forms; (C) severe forms.

Clinical Classification and SeveritySimilarly to leprosy, leishmaniasis, and other parasitic diseases, CBM and PHM represent two poles of a wide spectrum of diseases caused by distinct species of melanized fungi (118). Clinically, the boundaries of the spectrum are imprecise. Both diseases may depict plaque and nodular lesions types, and some etiological agents may cause both types of infections. Both infections can be found in either immunocompetent or immunosuppressed hosts, but CBM is prevalent in immunocompetent patients, while PHM is mostly not (Fig. 8). Earlier literature reports referred to an involvement of deep organic sites such as brain and lungs as CBM, but such cases should be considered PHM (241–243). CBM affects mainly the skin and the subcutaneous tissue; dissemination to deeper organs is extremely rare (39, 244).

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FIG 8

Spectrum of fungal diseases caused by melanized fungi. PHM, phaeohyphomycosis.

Several species of the Herpotrichiellaceae are related to the etiology of CBM, but no link with a specific type of lesion or mild to severe grades of this disease has been associated with any etiological agent.

Dissemination is hypothesized to occur slowly by continuous spread, leading to new satellite lesions, while noncontiguous or remote-site lesions may result from autoinoculation due to itching. Lymphatic spread to somewhat remote sites has been described in a few cases (245, 246). After months or even years, if not surgically removed, the initial CBM lesion evolves, assuming polymorphic clinical aspects.

To describe the wide clinical spectrum of CBM lesions, several classifications were proposed (247–249). Among these, the classification introduced by Arturo Carrión in 1950 is still valid and very helpful for clinicians facing this mycosis because it is based upon dermatological definitions of cutaneous lesions (249). Over time, this classification has been used by several authors for different types of lesions: nodular, tumoral (cauliflower-like), verrucous, scarring, and plaque (3, 239, 249) (Table 5). Recently, Lu et al. (87) referred to pseudovacuolar and eczematous types in patients with a short time of evolution and showing mild to moderate severities of disease and a favorable response to therapy. In more severe and advanced cases, patients may present a combination of lesion types, one of which may predominate (Table 5 and Fig. 6). In view of patient management and a favorable disease prognosis, CBM lesions must be classified according to the predominant clinical type and severity grade (3) (Table 5 and Fig. 7). The different grades of severity are related mainly to the time of evolution, the site involved, the patient's hygienic habits, the patient's compliance with antifungal therapy, and impaired innate host defense mechanisms, including primary immunodeficiencies such as CARD9 mutations (69, 234).

Complications and SequelaeUnlike other implantation mycoses such as sporotrichosis and mycetoma, CBM is limited to subcutaneous tissues, and it does not affect fascia, tendons, muscles, and osteoarticular sites (3, 108). However, CBM progresses slowly and by contiguity produces fibrotic changes and lymphatic stasis, leading to lymphedema, which in some cases resembles elephantiasis. Secondary recurrent bacterial infection is another frequently observed complication of CBM. This process exacerbates the commitment of lymphatic vessels (12–15, 246).

Initial lesions and mild forms are oligosymptomatic, and usually, they do not lead to medical consultation and do not interfere with the patient's daily activities. With time, pruritus is the predominant complaint, which may be intense and accompanied by local pain in patients depicting moderate clinical forms. If not treated, with time, the severity increases, and CBM lesions are associated with complications such as edema and secondary bacterial infections, leading to a limitation of or an inability to work (13). During therapy, CBM lesions may present an intense fibrotic reaction resulting in scarring. Facial lesions may produce eyelid retraction, leading to several grades of ectropion, xerophthalmia, and keratitis. Associations of CBM with several infectious diseases have been reported, including osteomyelitis, paracoccidioidomycosis, leishmaniasis, and leprosy (126, 250–254). These coinfections may increase the progression of both diseases, resulting in prolonged antifungal therapy and increased toxicity related to the respective therapies.

In advanced cases, chronic lymphedema, ankylosis, and malignant transformation are observed (13, 15, 246, 255–259). The latter is the most aggressive and disabling CBM-associated complication, leading mostly to squamous cell carcinoma (255–259). Recently, Azevedo et al. reported seven cases from Brazil and reviewed another 10 reported cases (259). The mean duration of CBM was 23 years, ranging from 5 to 36 years. Neoplastic transformation occurred independently of the applied antifungal therapy, and most patients underwent curative amputation. According to those authors, it is difficult to establish if the association of CBM, chronic inflammation, and bacterial infection may play a role as a carcinogenic or cocarcinogenic factor (259). Figures 9 and 10 depict the clinical aspects of the most frequent complications and sequelae related to CBM.

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FIG 9

Complications and sequelae related to severe forms of chromoblastomycosis. (A) Chronic lymphedema hyperkeratotic lesions in the upper limb. (B) Ectropium, secondary bacterial infection, and facial lymphedema. (C) Ankylosis of the knee. (D) Neoplastic transformation of a foot lesion. Shown is an ulcerative lesion with chronic bacterial infection. (E) Skin biopsy specimen taken 80 months later showing a well-differentiated epidermoid carcinoma with typical nuclear atypias and “corn pearls” in the middle of neoplastic cell blocks. Shown is an HE-stained section at a ×400 magnification. (F) Vegetant and papillomatous lesions resulting from the association of chromoblastomycosis with neoplastic lesions.

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FIG 10

Differential diagnosis of chromoblastomycosis. (A) Coccidioidomycosis; (B) paracoccidioidomycosis; (C) phaeohyphomycosis; (D) sporotrichosis; (E) deep dermatophytosis associated with CARD9 homozygous mutation; (F) verrucous tuberculosis; (G) Bowen disease; (H) mossy foot; (I) mycosis fungoid.

Differential DiagnosisCBM lesions are chronic, indolent, and clinically polymorphic. They can mimic a wide spectrum of diseases with infectious and noninfectious causes. It is important to differentiate CBM from other endemic diseases occurring in several geographic areas, especially in patients presenting with prolonged cutaneous and or subcutaneous lesions. Diagnosis must be confirmed by histopathology and mycological examination (Table 6 and Fig. 11).

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FIG 11

Laboratory diagnosis of chromoblastomycosis. (A) Skin scrapes and biopsy specimens should be taken from the “black dot” area (arrows). (B and C) Direct examination shows muriform cells (B), which may germinate and form filaments near the cutaneous surface (C). (D to F) In HE-stained sections, tissue reaction with hyperkeratosis, pseudoepitheliomatous hyperplasia (D), may be observed, associated with neutrophilic microabscesses containing muriform cells (E), which may also be found in Langerhans cells (F).

MycologyThe diagnosis of CBM requires laboratory confirmation by direct mycological examination and/or histopathology. The visualization of muriform cells in clinical specimens is compulsory for confirmation of the diagnosis of this disease. Pigmented fungal elements may easily be found superficially at the lesion, and they look like small black dots (cayenne pepper appearance). These structures observed by the naked eye represent small hematic crusts, cellular debris, and fungal structures resulting from transepithelial elimination (Fig. 11A). Skin scrapings containing crusts, cellular debris, and tissue fragments are clarified by using a 10 to 40% potassium hydroxide (KOH) solution. Single or clustered muriform cells are depicted as round to polyhedral (chestnut-like) cells with a diameter of 5 to 12 μm. They are typically dark pigmented, thick walled, and crossed by both transverse and longitudinal septa resembling a brown brick wall (Fig. 11B). The calcofluor staining method may be helpful for diagnosis if fungal cells are scarce.

Muriform cells near the surface may germinate with filaments (Fig. 11C). The sensitivity of direct examination ranges from 90 to 100%. This method is fast, easy, and inexpensive. Therapy may be started upon the demonstration of muriform cells; however, culture identification is important because Fonsecaea species may be less sensitive to antifungals than C. carrionii (260, 261). In addition, identification may contribute to data on the epidemiology and biodiversity of the etiological agents worldwide (262).

When grown in routine culture media, most of the causative agents of CBM tend to form slow-growing, dark-pigmented colonies. Exceptions are sporadic cases caused by Exophiala spp., which may show an initial black-yeast aspect. CBM agents are not inhibited by cycloheximide or chloramphenicol, enabling the use of selective media to avoid rapidly growing contaminants. The incubation time is up to 6 weeks. Initial colonies are deep green, becoming velvety and darkening with time. In contrast to black yeasts, CBM agents lack an initial yeast phase. Microscopic examination allows identification to the genus level (Fig. 2). Further identification to the species level requires molecular sequencing, for which the rDNA ITS barcoding gene is recommended. In addition, taxonomic studies may apply specific genes, such as those encoding β-tubulin, translation elongation factor 1α (35, 37, 54), and others.

The tissular response is not specific in CBM specimens, and it may be similar to the tissue reactions observed for most implantation mycoses. Histopathological examination of tissue shows muriform cells that may be inside giant cells or multinucleate giant cells of the Langerhans giant-cell type, identified by routine hematoxylin-eosin (HE) staining. Gomori-Grocott and Fontana-Masson stains are sensitive for the detection of fungal cells where fungal elements are scarce. Hyperkeratosis, pseudoepitheliomatous hyperplasia of the epidermis, pyogranulomatous reactions, and irregular acanthosis alternating with areas of atrophy are the most important histological characteristics of CBM (143, 223, 263). The dermis typically contains a dense granulomatous inflammatory infiltrate, with different grades of fibrosis, associated with mononuclear cells (histiocytes, lymphocytes, and plasma cells), epithelioid cells, giant cells, and polymorphonuclear cells (Fig. 11D to F). Two different kinds of inflammatory responses were suggested by d'Avila et al.: amorphous suppurative granulomas and true tuberculoid granulomas (230). Suppurative granulomas demonstrated pseudoepitheliomatous hyperplasia, microabscesses with large numbers of fungi, higher numbers of dermal capillary vessels, and fibrosis. True tuberculoid granulomas demonstrated atrophy of the epidermis or light acanthosis, well-formed granulomas with Langerhans giant cells, epithelioid cell lymphocytes, abscesses, and microabscesses. Fragmented fungal elements may be observed in the cytoplasm of giant multinucleated cells.

ImmunodiagnosisSimilarly to other implantation mycoses, serological and intradermal tests have not been standardized for CBM and are not used in the routine laboratory. However, according to data from in-house serological studies, such tests may be helpful for seroepidemiological and diagnostic purposes. Esterre et al. developed an enzyme-linked immunosorbent assay (ELISA) technique (235). Those authors obtained positive reactions in 6.2% of samples in western Madagascar, showing the existence of asymptomatic individuals. Vidal et al. studied 60 serum samples from Brazilian patients with CBM by immunodiffusion and ELISA for IgG anti-Fonsecaea antibodies (237). They observed variable positivity with both techniques but a specificity of 90%. Intradermal reactions prepared with culture filtrates (chromomycin) were also employed for epidemiological surveys, suggesting the presence of delayed hypersensitivity to CBM infection in healthy individuals living in areas of endemicity (264, 265).

Treatment with Physical MethodsIn most cases, the various modalities of physical methods that are available are used as adjuvant therapy in combination with antifungal agents and include surgery, thermotherapy, laser therapy, and photodynamic therapy (PDT).

Conventional SurgeryWithout a doubt, surgery is the best physical method for the treatment of CBM. Excisional surgery is strongly recommended for all initial small and well-delimitated cutaneous lesions. Surgery may also be used in conjunction with ITZ or terbinafine (TBF) treatment. Scattered reports of surgical removal of larger lesions in association with skin grafting are available, but there is a risk that this might lead to dissemination of the infection. Other physical methods, such as Moh's surgery (268) and iontophoresis, are no longer used (12, 13, 240).

CryotherapyCryotherapy or cryosurgery uses liquid nitrogen, the coldest cryogenic agent (−196°C) with the greatest freezing capability, to stimulate inflammatory reactions and necrosis of the affected tissue. Cryotherapy is recommended mostly for small lesions. With larger lesions (>15 cm²), cryotherapy should be performed in sections and at different time intervals. Larger skin folds should be avoided in order to prevent secondary fibrosis and reduce the risk of sequelae such as retractile scars. This method can be applied by using a cotton-tipped applicator or spray devices (269, 270). The freezing time for CBM cryotherapy with liquid nitrogen ranges from 30 s to 4 min according to the extent of the lesion. This therapy is also recommended for patients who have previously been treated with antifungal agents that led to a reduction of the size of the lesions. The most frequent adverse events related to cryotherapy include local pain, edema, blisters, postinflammatory hypopigmentation, hypertrophic scars, and secondary bacterial infection. In order to avoid dissemination of lesions to adjacent areas after cryotherapy, antifungal drugs, e.g., ITZ or TBF, should be administered in combination with physical methods (271, 272). In general, cryotherapy is convenient, cost-effective, and efficient but requires perseverance from both the patient and the physician.

Heat TherapyThe maximum growth temperature of causative pathogens of CBM is 42 to 46°C, and therefore, the application of heat therapy, either in combination therapy or as monotherapy, yields favorable results. Heat therapy has particularly been applied in Japan (273, 274). A 1-month application of a disposable chemical pocket warmer occluded with a bandage over the lesions 24 h per day resulted in an improvement of lesions and negative microscopic examination and culture results (275, 276). Flattening of lesions can be observed within 4 days, while complete resolution is achieved within ∼2 months (275). A follow-up biopsy after 3 months showed cicatrized lesions (277). However, the use of other heat sources, such as electric bed warmers, as monotherapy is insufficiently effective and should be used in combination with other physical therapeutic modalities (278). Local heat therapy for 2 h per day combined with the administration of posaconazole (PCZ) at 400 mg twice per day for an 8-month period led to a significant reduction of lesions, while the combination of heat therapy for 12 h/day and terbinafine (125 to 250 mg daily) led to negative direct examination and culture results within 2 weeks (278, 279). Additional surgical therapy was necessary in recalcitrant cases. Heat therapy is a potential therapeutic option that deserves further clinical investigation.

Laser TherapyA CO₂ laser emits a 10,600-nm wavelength, promoting photocoagulation. Currently, it is the only laser with very high continuous-wave power (280, 281). With its high precision, minimal tissue damage, and hemostatic capacities, the carbon dioxide laser is an ideal and very useful nonselective ablative laser. Lasers have been applied both as monotherapy and in combination with other treatment modalities (280–282).

Photodynamic TherapyPDT is a recent therapeutic modality with an effect on CBM similar to that on actinic keratosis or other types of skin cancer. PDT combines visible-light photons of an appropriate wavelength to stimulate intracellular molecules of a photosensitizer (283, 284). The activation of the photosensitizer produces several reactive molecules, including oxygen species, which leads to target cell damage. Antifungal PDT has been successfully used to treat fungal infections such as those caused by dermatophytes, Candida species, and Aspergillus niger. Lyon et al. reported PDT with a red light-emitting diode (LED) light with a low-cost methylene blue photosensitizer against CBM (283). After 6 PDT sessions, this well-tolerated procedure resulted in substantial improvement and complete remission of the lesions (285). Also, combination therapy with antifungals and PDT has been reported. Hu et al. combined oral terbinafine treatment with weekly 5-aminolevulinic acid (ALA)-PDT in a case of CBM; apparent clinical improvement was achieved within less than a year, and no recurrence was observed (286). PDT is minimally invasive, with few side effects, and may shorten the time of treatment. Adverse effects are mild, such as burning sensations, stinging, or pain. For patients experiencing intense pain and discomfort, the application of a fan, cooling sprays, or analgesics or adjustment of the irradiation time may be considered. PDT is a promising treatment option for a better quality of life for patients. The effectiveness of PDT against the most common species causing CBM has been demonstrated in an in vitro study (284). However, the antimicrobial effects of PDT appeared to serve no useful purpose when the light was turned off, so sequential systemic antifungal agents are necessary (284).

In Vitro Antifungal SusceptibilityThe clinical outcome for infected patients is determined by the intrinsic antifungal activity of the drug used but also its pharmacological profile and the clinical presentation and severity of the infection. In this scenario, there is a clear need for standardization of in vitro susceptibility testing to assist the clinician in achieving optimal treatment (73). There are concerns regarding the reproducibility and clinical correlation of MIC results generated by different methods used on a worldwide scale (287–289). In general, the determination of in vitro susceptibility to antifungal drugs is useful to evaluate intrinsic microbiological resistance, but usually, it is not useful for the prediction of the patient's clinical response. A further problem is the use of hyphae and conidia rather than muriform cells as the inoculum. Muriform cells are believed to be highly resistant and may respond differently to exposure to antifungal drugs. Antifungal sensibility test results should always be interpreted with care.

Despite the above-described limitations, numerous studies suggest that Fonsecaea species are highly susceptible in vitro to several triazole compounds, including ITZ, voriconazole (VCZ), PCZ, and isavuconazole (ISA), but not to fluconazole (FCZ), 5-flucytosine (5-FC), and amphotericin B (AMB) (290–292). There are a few studies addressing putative differences in antifungal susceptibility between species of the same genus. Najafzadeh et al. tested Fonsecaea pedrosoi, F. monophora, and F. nubica, and they all exhibited low MIC values for ITZ, VCZ, PCZ, and ISA. Higher MIC values were documented for all species against d-AMB, anidulafungin (ANI), caspofungin (CAS), and FCZ, while MICs values for VCZ and ISA showed 1- to 2-fold-higher dilutions for F. pedrosoi than in F. nubica and F. monophora. PCZ exhibited significant in vitro activity against all species tested, indicating that this expanded-spectrum azole has a potential role in the treatment of CBM (289).

TBF also shows in vitro activity against Fonsecaea and other etiological agents of CBM (287). This allylamine derivative was employed to treat CBM patients in Japan and Madagascar, with good clinical response (293), but more data are needed, including comparative trials, to more precisely determine the role of this antifungal in the therapy of CBM.

Cladophialophora carrionii and prevalently environmental Cladophialophora species (C. yegresii, C. saturnica, and C. immunda) are susceptible in vitro to triazoles. Deng et al. revealed high susceptibility values of PCZ, ISA, VCZ, ITZ, and TBF, with slightly higher triazole MIC values for isolates from Latin America than for those from other continents (292). Antifungal combination therapy is sometimes used for severe and invasive infections. Some authors have documented a good response of the combination of ITZ plus 5-FC for refractory CBM cases. No synergism or antagonism against melanized fungi was observed when d-AMB was combined with ITZ or TBF (291). A synergistic interaction was noted for only a single C. carrionii isolate with the combination of TBF and ITZ.

In summary, ITZ, PCZ, VCZ, and ISA currently exhibit the best in vitro activity against agents of CBM, whereas d-AMB, FCZ, and the echinocandins usually have limited activity. Secondary antifungal resistance is apparently uncommon but should be suspected when patients are not responding to or are relapsing under correct regimens of antifungal therapy. Ideally, serum triazole levels should be monitored during the course of therapy.

First-Line TherapyAccording to several open and noncomparative clinical trials, ITZ is the standard therapy for CBM, and it is also the most commonly used antifungal drug. ITZ cure rates range from 15 to 80% (12, 13, 15, 266, 294). The compound is an antimold triazole with a favorable safety profile (295, 296). As for other triazoles, ITZ inhibits the biosynthesis of cell membrane ergosterol via 14-α-sterol demethylase, a cytochrome P450 oxidase coenzyme (295). The loss of ergosterol generates defective cell membranes that lose fluidity and permeability. In contrast to ketoconazole (KTZ), ITZ does not affect human steroidogenesis, including the adrenal response to testosterone and corticotrophin during prolonged periods of continuous therapy (296). The capsule formulation of ITZ shows clinically significant activity against most CBM agents, although it is more effective against C. carrionii than against F. pedrosoi (289, 290). Doses for adults and adolescents of 200 to 400 mg/day are usually recommended. The duration of treatment varies; however, most cases show improvement within 8 to 10 months (266). Although complete clinical and mycological cure is achieved, this drug is administered mostly until criteria of cure have been observed (13, 15) or lesion reduction has occurred, and cryosurgery can subsequently be used to remove the remaining active cutaneous lesions. Clinical results with ITZ have been variable, limited mainly by gut absorption deficiencies and, consequently, low plasma levels and tissue concentrations, for which reason serum therapeutic drug monitoring is recommended. Success with ITZ was also reported for 6 to 12 months of pulse therapy consisting of sequential periods of 1 week per month of daily administration of 400 mg per day (297, 298). Unfortunately, comparative clinical trials to support this approach are lacking. Drug-drug interactions due to competitive inhibition of the cytochrome P450 3A4 enzyme system by ITZ also need to be addressed in patients receiving other medicines that are metabolized through this pathway.

Terbinafine is the second most frequently used antifungal agent for the treatment of CBM. Terbinafine has cure rates that are similar to those with ITZ (278, 293, 299). It is an orally administered allylamine derivative with fungistatic and fungicidal effects through the inhibition of squalene-epoxidase, which interferes with ergosterol biosynthesis and fungal membrane function. Unlike triazole derivatives, which are metabolized through the cytochrome P450 3A4 pathway, TBF is metabolized through the cytochrome P450 2D6 pathway. Thus, drug-drug interactions are minimal for this allylamine compound. In general, TBF shows good in vitro activity against most etiological agents of CBM. The recommended doses are 250 to 500 mg/day; the duration of treatment varies, until mycological cure or resolution of skin lesions is achieved. TBF may be advantageous over ITZ in that TBF shows fewer drug-drug interactions and may exert a relevant in vitro antifibrotic action (118, 293, 300).

Combined Systemic Antifungal TreatmentCombination therapy with systemic antifungal drugs has been used in the salvage therapy scenario for patients with invasive refractory mycoses. A combination of antifungal agents and/or physical methods usually is the last therapeutic option for refractory or advanced clinical presentations of CBM. On the other hand, there is no strong evidence to support the superiority of the combination of two systemic antifungal agents for the treatment of CBM.

The combination of ITZ and TBF is often used in patients presenting refractory disease. In some cases of CBM where ITZ and TBF were used for a prolonged time and failed, Gupta et al. used both drugs in an alternate modality with alternate weeks of ITZ and TBF. This can rescue some cases (301). In vitro studies have not demonstrated a synergism or antagonism of this combination (291). This combined therapy is utilized for very special cases that have been unresponsive to previous treatments. A few patients depicting moderate to severe clinical forms were treated with the combination of ITZ and 5-FC, with excellent results. The main issue is that in most of the countries where CBM is present, 5-FC is not available. Moreover, patients need to take a large number of pills per day, resulting in more noncompliance (302–304).

Role of Other TriazolesThe development of new antifungal drugs for invasive fungal infections favored their use in the therapy of most endemic mycoses, including CBM. Among the recent expanded-spectrum triazoles, PCZ is the best potential option for treatment of all clinical presentations of CBM, including severe or refractory clinical forms (305, 306). The broad in vitro antifungal spectrum of PCZ includes most of the melanized fungi causing CBM and PHM (289, 290). In addition, the PCZ oral solution is characterized by better pharmacodynamic and pharmacokinetic profiles than those of the ITZ capsule formulation. The recommended dose of the oral suspension of PCZ in patients with CBM is 800 mg/day divided into two doses for long periods of therapy (304). It is expected that in the future, recently licensed PCZ formulations, that is, oral tablets and intravenous solutions, may also be evaluated in PHM and CBM patients. Special emphasis should be focused on delayed-release PCZ tablets. In neoplastic patients, this formulation seems to be minimally affected by factors such as food ingestion, increased gastric pH, impaired motility, or mucositis. Recent data revealed that delayed-release PCZ tablets may also achieve higher average plasmatic concentrations than those achieved with the oral solution and are well tolerated in healthy individuals (307, 308). The combination of PCZ and 5-FC or TBF may be a potential therapeutic armamentarium for refractory cases.

Oral VCZ was also tested in a few cases to treat refractory forms of this disease. Although good clinical results have been achieved with this drug, adverse effects such as visual disturbances and photosensitive cutaneous reactions are not uncommon (309). Isavuconazole, a newly licensed broad-spectrum triazole for the treatment of invasive aspergillosis and mucormycosis, was recently tested for efficacy and tolerability in a small number of patients with PHM and CBM who participated in an international multicentric clinical trial. Similarly to other triazoles, ISA is very effective in vitro against melanized fungi, and it may be another therapeutic option for patients with CBM in the future (ClinicalTrials.gov registration no. NCT00634049).

Abandoned Antifungal AgentsSeveral antifungal regimens have been used in the therapy of patients with CBM in the past with varying results. This group of agents has been used when conventional treatment has failed. The most important agent in this group has been cholecalciferol or vitamin D₂ at doses of 600,000 IU per week. Potassium iodide alone has minimal effects, and the use of oral thiabendazole seems to be ineffective. Treatment with intravenous d-AMB alone or combined with 5-FC has not been used since the introduction of ITZ during the 1980s. In the past, several authors had also recommended d-AMB by intravenous, intra-arterial, or intralesional injection, with limited success; however, due to prolonged treatment, it easily produces adverse events such as arteritis, necrosis, and local pain. In addition, when the drug was discontinued, infection almost always reactivated. Other drugs used with variable results and severe side effects are ketoconazole, topical 5-fluorouracil, and topical ajoene (15).

Adjuvant TherapyIn recent years, there have been several reports of small case series evaluating the combination of antifungal drugs such as ITZ or TBF with immunoadjuvant compounds such as glucan (310) and topical imiquimod (311). Adjuvant therapy was used mostly in more severe and refractory cases, so the results are widely variable, but in general, in some cases, cure can be obtained, and in others, an important reduction of lesions has been achieved. Glucan is an injectable formulation of (1→3)-β-polyglucoside obtained from Saccharomyces cerevisiae in order to activate the Th2 immunophenotype, resulting in higher levels of IL-10 and lower levels of IFN-γ (312). This kind of therapy has been used with success in some cases of leishmaniasis and disseminated paracoccidioidomycosis (313). A few patients have been treated in Brazil with triweekly subcutaneous injections of glucan combined with 200 to 400 mg of ITZ, with some degree of clinical response (310).

Imiquimod is a synthetic amine similar to guanosine, an imidazoquinoline, with an immunomodulatory effect with antitumoral action, enhanced innate and acquired immunity, and powerful antiviral activity. This compound is known by its TLR7-activating property (314). Although there is only a single report of its use for the therapy of CBM, 5% imiquimod applied 4 to 5 times weekly appeared to be a potential adjunctive agent when combined with itraconazole. Side effects are rare, including itching and burning sensations. After prolonged use, a lichenoid infiltrate may develop as a reflection of increased local immunity. Siqueira et al. (315) developed an experimental DNA-hsp65 vaccine to stimulate adaptive immunity, with promising results not only for therapy but also for CBM prophylaxis in animal models. This drug has been combined with itraconazole, with an important improvement of experimental cutaneous lesions (315).