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CHROMOBLASTOMYCOSIS

SOURCE: http://emedicine.medscape.com/article/1092695-print AUTHORS:Robert A Schwartz, MD, MPH, Professor and Head, Dermatology, Professor of Pathology, Pediatrics, Medicine, and Preventive Medicine and Community Health, UMDNJNew Jersey Medical School; Eugeniusz Baran, MD, PhD, Professor, Department of Dermatology, Venereology and Allergology, Head, Clinic of Dermatology and Venereology, Wroclaw Medical University, Poland LAST UPDATED: July 8, 2010

INTRODUCTION Background Chromoblastomycosis is a chronic fungal infection of the skin and the subcutaneous tissue caused by traumatic inoculation of a specific group of dematiaceous fungi (usually Fonsecaea pedrosoi, Phialophora verrucosa, Cladosporium carrionii, or Fonsecaea compacta) through the skin.1 Several cases of infection by Exophiala species have appeared in the literature.2 Chromoblastomycosis is classified among the subcutaneous mycoses and is ubiquitous; however, the prevalence is higher in rural populations in countries with a tropical or subtropical climate, such as Madagascar in Africa and Brazil in South America. Nomenclature Since its identification in the early 1910s, the name of the disease has been frequently misused to encompass other infections caused by dematiaceous fungi. More recently, the advent of immunosuppressive therapies and diseases brought more confusion because of the identification of new agents and clinical settings. With the introduction of the concept of phaeohyphomycosis by McGinnis in 1983,3 differentiation among these diseases became more obvious. The features of chromoblastomycosis are distinctive enough to consider chromoblastomycosis an independent clinical entity. The infection should not be confused with mycoses, such as mycetoma or phaeohyphomycosis, caused by other dematiaceous fungi. Nowadays, the term chromoblastomycosis is restricted to the cases in which sclerotic cells are present in tissue. Sclerotic cells, also known as Medlar bodies, are globe-shaped, cigar-colored, thick-walled structures that are 4-12 m in diameter (see the image below).

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Sclerotic cells on a potassium hydroxide preparation.

Medlar first described them in 1915.4 These structures multiply by septation, and they induce a purulent and granulomatous inflammatory reaction in tissue (see Histologic Findings). In 1992,5 the International Society for Human and Animal Mycology (ISHAM) recommended that the best name to define the disease was chromoblastomycosis, which Terra et al coined in 1922.6 History Contrary to what appears in some textbooks, chromoblastomycosis was first described by Max Rudolph in 1914 and not by Lane 7 or Medlar4 in 1915. In 1987, Castro and Castro8 reported that Max Rudolph, a German physician living in Brazil, published a preliminary communication where the first 6 cases of the disease were described. Rudolph was also able to isolate a dark-colored fungus from 4 of 6 patients; this fungus grew in culture as a dark grey-to-blackcolored furlike colony. Rudolph believed this fungus to be a type of blastomycete, and he successfully inoculated the disease in 4 white rats and 2 monkeys. Surprisingly, he did not describe the histologic aspects of the disease or the pathognomonic sclerotic cells, which both Lane and Medlar described 1 year later. In 1928, Hoffman9 mentioned that in Cuba in 1908 Guiteras had observed 10 cases of a disease known as chapa in which the clinical aspects resembled those of chromoblastomycosis. Unfortunately, those cases were not published. In 1920, 2 Brazilian physicians,

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Pedroso and Gomes,10 published 4 cases that had been under observation for many years, the first one since 1911. According to them, all 4 cases were caused by P verrucosa. Two years later, in 1922, Brumpt11 concluded that the agents isolated by Pedroso and Gomes could not be classified as Phialophora species, and he coined the denomination Hormodendrum pedrosoi, later renamed F pedrosoi by Negroni12 in 1936. By 1930, new cases had been described outside the American continent in France, Sumatra, and Poland.13 Four different genera are now widely accepted to cause chromoblastomycosis: F pedrosoi, P verrucosa, C carrionii, and F compacta.14 Rare cases of chromoblastomycosis caused by Rhinocladiella aquaspersa and Exophiala species have also been reported, allowing the inclusion of these species among those that cause the disease.15,16,17,18,19 Pathophysiology The infection usually results from a traumatic cutaneous injury that is often not remembered or realized by the patient. The agents often gain entry into the human body by contact with wood splinters or thorns. The fungi most commonly reported as causing chromoblastomycosis are F pedrosoi, C carrionii, and P verrucosa. A small number of cases due to F compacta, R aquaspersa, and different species of Exophiala have also been reported. In 2007, Chaetomium funicola was identified as a cause of chromoblastomycosis in Panama.20 Several authors have demonstrated that a number of different dematiaceous fungi can be isolated from nature. In 1937, Conant21 demonstrated that a fungus called Cadophora americana, which was isolated from timber, was actually the same organism as P verrucosa. Later isolations were obtained in several countries from materials, such as plants, palm trees, grass, and soil.18 C carrionii has been isolated from cactaceae, both from the thorns and from the medullae, in Venezuela.22 C carrionii is the most common agent of chromoblastomycosis in that country, and trauma due to plants is believed to inoculate the skin with the fungus. The fungal cells in human tissue present with the same features as those observed in the inner portions of the plants. The lesions develop slowly at the site of implantation, producing a warty nodule, which tends to be limited to the skin and the subcutaneous tissue. Over the years, the nodule grows centripetally. In many instances, the central parts of the lesion heal, leaving ivorycolored scars. The disease tends to spread to neighboring healthy skin,

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forming plaques, which, at times, can involve a whole limb. When nodular lesions predominate over the plaques, the disease assumes a typical cauliflower aspect. Both lymphatic dissemination and cutaneous dissemination have been described. A new species of Fonsecaea, Fonsecaea nubica, morphologically similar to F pedrosoi and F monophora, has been described in association with chromoblastomycosis.23 Rhinocladiella aquaspersa may also be a causative agent.24 Frequency International The disease has been described worldwide, but the incidence is greater in tropical and subtropical regions located between 30 N and 30 S.25 An estimated 20% of cases are found in temperate climates, such as in Japan,26 Canada,27 Finland,28,29 and Romania and the Czech Republic.13 Chromoblastomycosis most commonly affects male agricultural workers in rural regions of North America, Central America, and South America, but chromoblastomycosis does occur worldwide, including Europe, England, India, South Africa, and Australia. The highest incidence of chromoblastomycosis in the world is in the African country of Madagascar, and a large number of cases have been reported in Japan. A survey in the Brazilian Amazon disclosed an extremely high incidence of chromoblastomycosis (1.6 cases per 10,000 population) in a village called Monte Negro, in the state of Rondonia. Countries with the highest number of cases are Madagascar and Brazil. Several different African and Latin American countries, such as Gabon, Colombia, Venezuela, Cuba, the Dominican Republic, and Mexico, also have high prevalence rates.

F pedrosoi infection is most commonly observed in humid climates, whereas C carrionii infection is normally found in dry areas.
Farming is the most common occupation in patients with chromoblastomycosis.30

Fonsecaea monophora is the predominant etiologic agent of chromoblastomycosis in southern China.31 Mortality/Morbidity Mortality due to chromoblastomycosis is a rare event.

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Morbidity relates directly to the severity of the disease. Initially, in the papule or nodule phase, the disease is asymptomatic. When the nodules coalesce, forming large plaques and sometimes involving the whole limb, complications may appear. Common complications include ulceration, lymphedema, and secondary infection. Race No racial predilection is reported. Sex Most series reports indicate a clear male predominance, although a small number of reports describe similar male-to-female ratios. Approximately 70% of cases are seen in men. The explanation for this male predominance is not clear, but men are assumed to be more commonly involved in agricultural work and are more prone to inflict injuries on themselves, thereby causing self-inoculation, rather than women who are supposedly more dedicated to house jobs. The possible inhibitory effect of female hormones on the growth of fungi may partially explain the relatively low number of cases in females. Age Patients with the disease are most commonly aged 30-50 years. The period between inoculation and disease development is believed to last years, explaining the scarcity of children with chromoblastomycosis. The disease is rarely found in children exposed to the same environmental conditions as adults. CLINICAL History Patients usually do not report discomfort, and they tend to not seek medical care until secondary infection or elephantiasis ensues. Physical The disease appears at the site of a previous often unnoticed or unremembered trauma to the skin. After several years, a small, raised, erythematous, asymptomatic papule develops. As the chromoblastomycosis lesion develops over years, it assumes a scaly

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and infiltrated aspect, generally leading to either of the 2 most common clinical variants: nodular or plaque. In both types, the surface is verrucous, and lesions spread laterally to contiguous healthy tissue. Note the images below.
Chromoblastomycosis, tumoral form. Chronic disease led to elephantiasis and involvement of the entire lower limb.

Plaque lesion on the foot. The verrucous aspect of the lesion differentiates it from other infectious dermatoses that may present as a verrucous lesion, namely, cutaneous leishmaniasis, sporotrichosis, cutaneous tuberculosis, and cutaneous mycobacteriosis.

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In general, the disease remains localized to the area of the initial infection or neighboring skin, but lymphatic and hematogenous dissemination may occur, producing metastatic lesions away from the primary site. The nodular type of lesion normally develops into verrucous, pedunculated, cauliflowerlike florets, while the plaque type spreads peripherally, with an active, raised border, leaving a central healed area with atrophic and yellowish scar tissue. On the surface of both types of clinical variants, numerous black dots may be observed where the causative organisms are preferentially found. Hemopurulent material covering small ulcerations is commonly observed. Cutaneous localized annular chromoblastomycosis has been described as well-circumscribed, slow-growing, annular, papulosquamous or papulosquamous-verrucous patches or plaques with no regression despite the use of topical antifungals.32 These plaques may be atrophic.33 Secondary infection with bacteria is common, giving the lesion a characteristic ill odor. Secondary infection is believed to be important in the genesis of lymph stasis and consequently of elephantiasis. In old cases, lesions in different stages of development can be found. Extracutaneous spread may occur rarely, due to hematogenic and lymphatic dissemination. Contiguous spread to the underlying bone may produce an osteolytic lesion.34 The sites most commonly affected are the lower extremities, especially the feet. The hands, the arms, and the buttocks are also frequently involved, and sporadic reports mention lesions on the ears, the face, and the breasts. Morphological variants occur. Rarely, chromoblastomycosis may resemble sporotrichosis in having verrucous nodules and a lymphatic distribution on the forearm expanding into verrucous plaques.24 Chromoblastomycosis may also be evident as a large cauliflowerlike mass.35 Causes The isolation of the causative fungi from nature on several different occasions has added to the demonstration that fungi gain entry into the host's body through traumatic inoculation1 ; this finding also confirmed that populations at risk are rural workers who walk barefoot in endemic regions where the causative agents are found. A Brazilian study has suggested a link between the occurrence of lesions on the buttocks and the habit of sitting on babau (Orbignya phalerata) shells. The authors have failed to isolate the causative

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fungus from the shells.36 In 2004, Salgado et al37 were able to isolate the same F pedrosoi from a thorny bush (Mimosa pudica) and from a chromoblastomycosis patient. The lesion had developed after an accident at the site where those bushes grew. On the other hand, in 1994, Zeppenfeldt et al22 demonstrated that certain cactaceae from the Falcon state in Venezuela had fungi not only on their surfaces and thorns but also in the medulla. Not coincidentally, the fungus was identified as C carrionii, which is the most commonly found organism in chromoblastomycosis cases in that country. The histologic characteristics of the fungus in plant tissue resembled that found in humans. Experimental inoculation of C carrionii in goats, performed n Venezuela, demonstrated that although chromoblastomycosis affecting that genus of mammals has never been reported, C carrionii caused cellular reactions in goats identical to those seen in humans at the first stages of infection.38 In 1989, Tsuneto et al39 demonstrated a higher frequency of HLA-A29 in patients with chromoblastomycosis; this finding suggests that genetics might have an influence in the acquisition of the disease. DIFFERENTIAL DIAGNOSES Leishmaniasis Sporotrichosis Squamous Cell Carcinoma

Other Problems to Be Considered When clinically presenting as a verrucous plaque, chromoblastomycosis may mimic several dermatoses that also manifest as a verrucous plaque; these conditions include cutaneous leishmaniasis, sporotrichosis, cutaneous tuberculosis or mycobacteriosis, and squamous cell carcinoma. Chromoblastomycosis-induced lymphedema may mimic elephantiasis. Cutaneous infection with capnodialean melanized fungi, which often thrive in extreme environments like rock surfaces and hypersaline microhabitats, may mimic chromoblastomycosis; however, capnodialean melanized fungi demonstrate hyphae and 40 chlamydosporelike conidia in tissue.

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WORKUP Laboratory Studies Among the possible laboratory tests to be obtained, direct examination of 10% potassium hydroxide cleared lesion scrapings is by far the most useful. Typical, thick-walled, cigar-colored, sclerotic cells, also known as Medlar bodies, are readily seen. Although these cells are pathognomonic of chromoblastomycosis, they do not give specific information on the agent. Eventually, dematiaceous hyphae can also be observed. Identifying the agent is easier when the specimens collected include the black dots present in the lesions. In 2005, Miranda et al41 suggested the use of vinyl adhesive tape for collecting and identifying some types of deep-seated mycoses in which the infectious agents can be observed in the horny layer of the epidermis in transepidermal elimination events. Note the image below.

Sclerotic cells on a potassium hydroxide preparation.

In culture, each causative agent produces a similar pattern, that of a slow-growing, dark, velvety colony with a black obverse. Identification of individual species is handled in the usual manner by observing various characteristics, including conidia production. Note the image below.

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Micromorphology of Cladosporium carrionii (left) and Fonsecaea pedrosoi (right), the 2 most commonly isolated agents in chromoblastomycosis.

Serologic tests are exclusively used for research matters, and they are not routinely used or available. A recently isolated, highly specific and sensitive immunoblotting method depicted a 54-kd antigen from F pedrosoi42 that may prove to be helpful in the study of the disease. Note the image below.

Culture of Fonsecaea pedrosoi on Sabouraud agar. The black velvety colony has the same macroscopic appearance as the colonies of other chromoblastomycosiscausing agents (eg,Cladosporium carrionii, Fonsecaea compacta, Phialophora verrucosa, Rhinocladiella aquaspersa, Exophiala species).

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An enzyme-linked immunosorbent assay (ELISA) using a somatic antigen of C carrionii was tested to serologically follow cases of chromoblastomycosis due to C carrionii before, during, and after therapy. ELISA proved to be a valuable tool for the diagnosis and follow-up of patients with chromomycosis (due to C carrionii). According to the authors43 in 2005, the use of an ELISA might be useful to establish remission criteria in chromoblastomycosis caused by C carrionii. Duplex polymerase chain reaction is a rapid and specific assay for identification of Fonsecaea isolates, mainly for the strains that are difficult to identify using morphologic methods.44 Imaging Studies Bone involvement is not a typical finding in chromoblastomycosis. Long-lasting lesions may lead indirectly to bone changes, making it necessary to evaluate possible bone compromise with regular radiography, especially when lesions are old, located at sites with scarce subcutaneous tissue, or associated with lymphedema. Lymphoscintigraphy has been used to evaluate lymphedema, but it is not routinely used.45 Histologic Findings In cutaneous lesions, the cigar-colored fungi are easily seen in hematoxylin and eosinstained sections. No special stains are needed. The typical finding is a pseudoepitheliomatous hyperplasia of the epidermis with a diffuse, lymphomononuclear inflammatory infiltrate in the dermis. The tissue response to the fungus is typically mixed. True and pure abscesses or microabscesses, granulomas, granulomatous reactions, and abscesses surrounded by a granulomatous reaction with giant cells may be seen in the same section.46 Inside the giant cells, brown-colored, thick-walled fungal cells may be seen. These muriform fungal cells may be single, 2-celled, or multiple-celled; this feature is a result of multiplication by septation rather than budding. Note the image below.

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Hematoxylin and eosinstained section shows typical sclerotic cells inside an abscess. Sclerotic cells present as round, thick-walled, cigar-colored structures.

Transepidermal elimination of the fungal cells is the histologic counterpart of the black dots clinically evident in chromoblastomycosis lesions. An unusual dermal response has been described by Jawitz et al47 in 2007, consisting of dermal effacement by a spindle cell proliferation arranged in sweeping fascicles. Staging Staging of the disease is especially useful for academic matters. Because treatment is still difficult, several papers in the literature describe the results obtained with different therapeutic methods. Unfortunately, most papers are single case reports, and follow-up tends to be short. The lack of uniformity in staging contributes to the low reproducibility of the proposed regimens. In 1999, Castro devised an elegant, clinically based, physician friendly index to stage chromoblastomycosis. The severity index is based on the extension of the diseased area, the number of lesions, the presence of complications (eg, lymphedema, ulceration, secondary infection), and the unresponsiveness to previous treatments. According to this scoring system, patients are classified as having mild (up to 3 points), moderate (4-6 points), or severe (7-10 points) disease. A scoring system for staging chromoblastomycosis is as follows: Area of lesions: Small lesions up to 25 cm2 are 1 point. Medium lesions larger than 25 cm2 and smaller than 100 cm2 are 2 points. Lesions larger than 100 cm2 are 3 points.

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Number of lesions: A single lesion is 1 point. One to 5 lesions is 2 points. More than 5 lesions or metastatic lesions is 3 points. Complications (1 point for each complication present): Lymphedema is 1 point. Ulceration is 1 point. Secondary infection is 1 point. Resistance to previous treatment or previous unsuccessful treatment is 1 point. TREATMENT

Medical Care One of the most characteristic features of chromoblastomycosis is its refractoriness to treatment. Treatment options include oral itraconazole (as monotherapy or with oral flucytosine [5-FC]), locally applied heat therapy, cryosurgery, and combination therapy.48 Therapeutic success is related to the causative agent, as well as the clinical form and severity of the chromoblastomycosis. Several authors indicate itraconazole as the best choice of therapy.18,49,50 Daily doses range from 200-400 mg, and results vary greatly. Adverse effects are not common, but efficacy is not as high as one would desire. Severe cases should be treated for several years. The authors' experience in treating more than 25 patients with varying degrees of severity for up to 5 years shows that itraconazole produces dramatic improvement after a few months of therapy; however, a complete cure is rarely reached, especially in severe cases. These results might be because of the predominantly fungistatic mechanism of action of the drug. In numerous cases, drug withdrawal led to relapse. Although few studies have been published, the association of itraconazole and 5-FC is promising.51 As with 5-FC and amphotericin B, itraconazole and 5-FC produce a synergistic effect. Multidrug therapy for chromoblastomycosis seems to be an interesting approach and may also be used with cryosurgery. In 1996, Esterre et al52 presented interesting results when using terbinafine to treat more than 100 patients in Madagascar. Similar to that of itraconazole, the drug presented below optimal results, it is exceedingly expensive, and treatment lasts several months. To date, no reports on the association of terbinafine and itraconazole or terbinafine and 5-FC have appeared in the literature. Posaconazole (Noxafil), a new triazole derivative, has been experimentally used to treat chromoblastomycosis. Posaconazole has recently received approval from the US Food and Drug Administration for prophylaxis against invasive Aspergillus and Candida infections in

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patients at high risk because of severe immunosuppression. The results of isolated cases suggest that outcomes may be slightly superior to those obtained by itraconazole or terbinafine (Unpublished data on file, Dr. Shikanai-Yasuda, Department of Infectious Diseases, Univ. So Paulo). According to Keating53 in 2005, posaconazole at 800 mg/d was associated with an overall success rate of 82% in persons with refractory chromoblastomycosis. Heat therapy is another treatment. Especially in Japan, the use of pocket warmers has proven successful in the treatment of a limited number of cases. Apparently, an increase in skin temperature somehow impairs fungal development.54 Surgical Care Cryosurgery with liquid nitrogen can be used to treat chromoblastomycosis, especially localized lesions. Several reports have appeared in the literature showing that the method has been used on almost every continent with good results. Freezing time in one study varied from 30 seconds to 4 minutes, and the number of cycles varied from 1 to more than 40. All localized lesions responded extremely well to treatment, with no relapse for as long as 15 years, a follow-up period unparalleled in the literature. Three patients with generalized lesions attained clinical and mycologic remission for as long as 26 months, and 3 had significant improvement without cure.55 Multiple treatment modalities are often combined, such as long courses of antifungals, surgical excision, and destructive physical therapies, because chromoblastomycosis is one of the most difficult deep mycotic infections to eradicate.56 MEDICATION Overall results of the various treatments in one Mexican study were as follows: 31% of the conditions were cured, 57% improved, and 12% did not respond to treatment. Optimal results were obtained with cryosurgery for small lesions; itraconazole for large ones; and, in some cases, the combination of both.57 Combination therapy with itraconazole and terbinafine during the early stages of treatment of chromoblastomycosis caused by F monophora has been suggested.58 Chromoblastomycosis may also be cured by prolonged topical application of tolerable heat from pocket warmers. It may take as long as 6 months or more.59

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Antifungals Mechanism of action usually involves inhibiting pathways (eg, enzymes, substrates, transport) necessary for sterol/cell membrane synthesis or altering the permeability of the cell membrane (polyenes) of the fungal cell.

Itraconazole (Sporanox) Fungistatic activity. Synthetic triazole antifungal agent that slows fungal cell growth by inhibiting cytochrome P450dependent synthesis of ergosterol, a vital component of fungal cell membranes. Results may take several weeks to be noted, and treatment may last several months or years.

Dosing
Adult 100 mg PO bid/tid or 200 mg PO bid, depending on response and severity Pediatric Not established for chromoblastomycosis

Interactions
With cisapride, can cause cardiac rhythm abnormalities and death; may increase digoxin levels; affects metabolism of drugs processed by cytochrome P450-3A enzyme system; antacids may reduce absorption; edema may occur with coadministration of calcium channel blockers (eg, amlodipine, nifedipine); hypoglycemia may occur with sulfonylureas; may increase tacrolimus and cyclosporine plasma concentrations with high doses; rhabdomyolysis may occur with coadministration of HMG-CoA reductase inhibitors (eg, lovastatin, simvastatin); coadministration may increase plasma levels of midazolam or triazolam; phenytoin and rifampin may reduce levels (phenytoin metabolism may be altered)

Contraindications
Documented hypersensitivity; altered liver function

Precautions
Pregnancy C - Fetal risk revealed in studies in animals but not established or not studied in humans; may use if benefits outweigh risk to fetus Precautions Caution in hepatic insufficiencies or in patients with high alcohol intake

Terbinafine (Lamisil)

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Member of allylamine family and believed to be a fungicidal agent. Inhibits ergosterol synthesis via squalene epoxidase, resulting in decreased ergosterol levels and increased concentrations of squalene.

Dosing
Adult 500-1000 mg/d PO for several weeks Pediatric Not established

Interactions
Cimetidine, rifampin, and cyclosporine may interact (rarely); toxicity may increase with coadministration of rifampin and cimetidine; coadministration with cyclosporine may decrease cyclosporine levels

Contraindications
Documented hypersensitivity

Precautions
Pregnancy B - Fetal risk not confirmed in studies in humans but has been shown in some studies in animals Precautions Generally well tolerated; adverse effects are primarily GI, including hepatobiliary dysfunction; transient loss of sense of taste is rare but specific adverse effect; blood dyscrasias, Stevens-Johnson syndrome, and ocular disturbances are described; minimally inhibits cytochrome P450 enzyme pathway Flucytosine (Ancobon) 5-FC has proven useful when used in association with oral itraconazole. Should not be used as monotherapy.

Dosing
Adult 50-100 mg/kg/d PO divided q6h Pediatric Not indicated

Interactions
Amphotericin B may increase toxicity of 5-FC; cytosine may inactivate 5-FC

Contraindications
Documented hypersensitivity

Precautions
Pregnancy C - Fetal risk revealed in studies in animals but not established or not studied in humans; may use if benefits outweigh risk to fetus Precautions

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Caution in bone marrow suppression; adjust dose in renal impairment

FOLLOW-UP Further Inpatient Care Lymphedema and secondary infection or ulcerated lesions are common complications of chromoblastomycosis on the lower limbs. Physiotherapy and lymphatic drainage are useful in preventing lymphedema. Ulcers and secondary infection are dealt with by appropriate nursing care. Oral antibiotics may be used. Complications The most common complications of chromoblastomycosis are ulceration; secondary infection; and lymphedema, leading to elephantiasis. Rare cases of malignant transformation (squamous cell carcinoma) of chromoblastomycosis have been documented in the literature. Prognosis The prognosis for chromoblastomycosis tends to be good, especially for small and localized lesions. When the affected area is large, as in severe cases, cure is difficult, although control is easily achieved. Cicatricial and unaesthetic scars are the rule after the disease is eliminated. MISCELLANEOUS Medicolegal Pitfalls No medical/legal pitfalls are of interest for chromoblastomycosis.
REFERENCES 1. Rubin HA, Bruce S, Rosen T, McBride ME. Evidence for percutaneous inoculation as the mode of transmission for chromoblastomycosis. J Am Acad Dermatol. Nov 1991;25(5 Pt 2):9514. [Medline]. 2. Castro LG, Pimentel ER, Lacaz CS. Treatment of chromomycosis by cryosurgery with liquid nitrogen: 15 years' experience. Int J Dermatol. May 2003;42(5):408-12. [Medline]. 3. McGinnis MR. Chromoblastomycosis and phaeohyphomycosis: new concepts, diagnosis, and mycology. J Am Acad Dermatol. Jan 1983;8(1):1-16. [Medline]. 4. Medlar EM. A cutaneous infection caused by a new fungus Phialophora verrucosa with a study of the fungus. J Med Res. 1915;32:507-22. 5. Odds FC, Arai T, Disalvo AF, et al. Nomenclature of fungal diseases: a report and recommendations from a Sub-Committee of the International Society for Human and Animal Mycology (ISHAM). J Med Vet Mycol. 1992;30(1):1-10. [Medline]. 6. Terra F, Torres M, Fonseca Filho O. Novo tipo de dermatite verrucosa; micose por Acrotheca com associado de leishmaniose. Brasil Medico. 1922;36:363-8. 7. Lane CG. A cutaneous disease caused by a new fungus Phialophora verrucosa. J Cutan Dis. 1915;33:840-6. 8. Castro RM, Castro LG. On the priority of description of chromomycosis. Mykosen. Sep 1987;30(9):397-403. [Medline]. 9. Hoffman WH. Die Chromoblastomykose in Kuba. Arch Schiffs u Tropen Hyg. 1928;32:485-7. 10. Pedroso A, Gomes JM. 4 casos de dermatite verrucosa produzida pela Phialophora verrucosa. Ann Paulistas de Medicina e Cirurgia. 1920;11:53-61. 11. Brumpt E. Prs de Parasitologie. 3rd ed. Paris, France: Masson; 1922:1105. 12. Negroni R. Estudio del primer caso argentino de cromomicosis, Fonsecaea (Negroni) pedrosoi (Brumpt) 1921. Rev Inst Bacteriol. 1936;7:419-26.

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Rippon JW. Chromoblastomycosis and related dermal infections caused by dematiaceous fungi. In: Medical Mycology. The Pathogenic Fungi and the Pathogenic Actinomycetes. 2nd ed. Philadelphia, Pa: WB Saunders; 1982:249-76. Lacaz CS, Porto E, Martins JEC. Fungos, actinomicetos e algas de interesse medico. In: Micologia Medica. 8th ed. Sao Paulo, Brazil: Sarvier; 1991:373-86. South DA, Brass C, Stevens DA. Chromohyphomycosis. Treatment wit ketoconazole. Arch Dermatol. May 1981;117(5):311-2. [Medline]. Naka W, Harada T, Nishikawa T, Fukushiro R. A case of chromoblastomycosis: with special reference to the mycology of the isolated Exophiala jeanselmei. Mykosen. Oct 1986;29(10):44552. [Medline]. Barba-Gomez JF, Mayorga J, McGinnis MR, Gonzalez-Mendoza A. Chromoblastomycosis caused by Exophiala spinifera. J Am Acad Dermatol. Feb 1992;26(2 Pt 2):367-70. [Medline]. Queiroz-Telles F, Purim KS, Fillus JN, et al. Itraconazole in the treatment of chromoblastomycosis due to Fonsecaea pedrosoi. Int J Dermatol. Nov 1992;31(11):805-12. [Medline]. Padhye AA, Hampton AA, Hampton MT, Hutton NW, Prevost-Smith E, Davis MS. Chromoblastomycosis caused by Exophiala spinifera. Clin Infect Dis. Feb 1996;22(2):3315. [Medline]. Piepenbring M, Caceres Mendez OA, Espino Espinoza AA, Kirschner R, Schofer H. Chromoblastomycosis caused by Chaetomium funicola: a case report from Western Panama. Br J Dermatol. Nov 2007;157(5):1025-9. [Medline]. Conant NF. The occurrence of a human pathogenic fungus as a saprophyte in nature. Mycologia. 1937;29:597-8. Zeppenfeldt G, Richard-Yegres N, Yegres F. Cladosporium carrionii: hongo dimorfico en cactaceas de la zona endemica para la cromomicosis en Venezuela. Rev Iberoam Micol. 1994;11:61-3. Najafzadeh MJ, Sun J, Vicente V, Xi L, van den Ende AH, de Hoog GS. Fonsecaea nubica sp. nov, a new agent of human chromoblastomycosis revealed using molecular data. Med Mycol. Mar 22 2010; [Medline]. Badali H, Bonifaz A, Barrn-Tapia T, Vzquez-Gonzlez D, Estrada-Aguilar L, Cavalcante Oliveira NM, et al. Rhinocladiella aquaspersa, proven agent of verrucous skin infection and a novel type of chromoblastomycosis. Med Mycol. Jan 29 2010;[Medline]. Brygoo ER, Destombes P. Epidemiologie de la chromoblastomycose humaine. Bull Inst Pasteur. 1975;74:219-43. Nishimoto K. Chromomycosis in Japan. Ann Soc Belg Med Trop. Sep 1981;61(3):405-12. [Medline]. Berger L, Langeron M. Sur un type noveau de chromomycose observ au Canada (Torula bergeri n. sp). Ann Parasit Hum Comp. 1949;24:574-99. Putkonen T. [Chromomycosis in Finland. The possible role of the Finnish sauna in its spreading]. Hautarzt. Nov 1966;17(11):507-9. [Medline]. Sonck CE. Chromomycosis in Finland. Dermatology. 1975;19:189-93. Pradhan SV, Talwar OP, Ghosh A, Swami RM, Shiva Raj KC, Gupta S. Chromoblastomycosis in Nepal: a study of 13 cases. Indian J Dermatol Venereol Leprol. May-Jun 2007;73(3):1768. [Medline]. Xi L, Sun J, Lu C, et al. Molecular diversity of Fonsecaea (Chaetothyriales) causing chromoblastomycosis in southern China. Med Mycol. Feb 2009;47(1):27-33. [Medline]. Salgado CG, da Silva MB, Yamano SS, Salgado UI, Diniz JA, da Silva JP. Cutaneous localized annular chromoblastomycosis. J Cutan Pathol. Feb 2009;36(2):257-61. [Medline]. El Euch D, Mokni M, Haouet S, Trojjet S, Zitouna M, Ben Osman A. [Erythemato-squamous papular and atrophic plaque on abdomen: chromoblastomycosis due to Fonsecaea pedrosoi]. Med Trop (Mars). Feb 2010;70(1):81-3. [Medline]. Sharma NL, Sharma VC, Mahajan V, Shanker V, Sarin S. Chromoblastomycosis with underlying osteolytic lesion. Mycoses. Nov 2007;50(6):517-9. [Medline]. Sharma A, Hazarika NK, Gupta D. Chromoblastomycosis in Sub-Tropical Regions of India. Mycopathologia. Jan 22 2010; [Medline]. Silva CM, da Rocha RM, Moreno JS, et al. [The coconut babau (Orbignya phalerata martins) as a probable risk of human infection by the agent of chromoblastomycosis in the State of Maranho, Brazil]. Rev Soc Bras Med Trop. Jan-Mar 1995;28(1):49-52. [Medline]. Salgado CG, da Silva JP, Diniz JA, et al. Isolation of Fonsecaea pedrosoi from thorns of Mimosa pudica, a probable natural source of chromoblastomycosis. Rev Inst Med Trop Sao Paulo. JanFeb 2004;46(1):33-6. [Medline]. Martinez EC, Rey Valeiron C, Yegres F, Reyes R. [The goat: approach to an animal model in human chromomycosis]. Invest Clin. Jun 2005;46(2):131-8. [Medline]. Tsuneto LT, Arce-Gomez B, Petzl-Erler ML, Queiroz-Telles F. HLA-A29 and genetic susceptibility to chromoblastomycosis. J Med Vet Mycol. 1989;27(3):181-5. [Medline]. Campolina SS, Caligiorne RB, Rezende-Silva S, Hahn RC, De Hoog GS. A skin infection mimicking chromoblastomycosis by a Capnodialean fungus. Med Mycol. Feb 2009;47(1):81-5. [Medline]. Miranda MF, Silva AJ. Vinyl adhesive tape also effective for direct microscopy diagnosis of chromomycosis, lobomycosis, and paracoccidioidomycosis. Diagn Microbiol Infect Dis. May 2005;52(1):39-43. [Medline].

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Vidal MS. Estudo imunoquimco de um antigeno de Fonsecaea pedrosoi e padronizao de teiicas sorologicas para cromoblastomicose causada por este fungo [dissertation/master's thesis]. Sao Paulo, Brazil: University of Sao Paulo; 2002. Oberto-Perdigon L, Romero H, Perez-Blanco M, Apitz-Castro R. [An ELISA test for the study of the therapeutic evolution of chromoblastomycosis by Cladophialophora carrionii in the endemic area of Falcon State, Venezuela]. Rev Iberoam Micol. Mar 2005;22(1):39-43. [Medline]. de Andrade TS, Cury AE, de Castro LG, Hirata MH, Hirata RD. Rapid identification of Fonsecaea by duplex polymerase chain reaction in isolates from patients with chromoblastomycosis. Diagn Microbiol Infect Dis. Mar 2007;57(3):267-72. [Medline]. Ogawa MM. Cromoblastomicose: Teraplicas e alternativos linfocintilograpicas. Sao Paulo, Brazil: Tese de Mestrado, Escola Paulista de Medicina; 2001. Salfelder K, de Liscano TR, Sauerteig E. Atlas of Fungal Pathology. Kluwer/Dordrecht; 1990:14550. Jawitz RS, Calder KB, Turner LM, Schlauder S, Morgan MB. Cryptic "chromo-fibroma". Am J Dermatopathol. Dec 2007;29(6):573-5. [Medline]. Queiroz-Telles F, Esterre P, Perez-Blanco M, Vitale RG, Salgado CG, Bonifaz A. Chromoblastomycosis: an overview of clinical manifestations, diagnosis and treatment. Med Mycol. Feb 2009;47(1):3-15. [Medline]. Restrepo A, Gonzalez A, Gomez I, Arango M, de Bedout C. Treatment of chromoblastomycosis with itraconazole. Ann N Y Acad Sci. 1988;544:504-16. [Medline]. Graybill JR. Future directions of antifungal chemotherapy. Clin Infect Dis. Mar 1992;14 Suppl 1:S170-81. [Medline]. Pradinaud R, Bolzinger T. Treatment of chromoblastomycosis. J Am Acad Dermatol. Nov 1991;25(5 Pt 1):869-70. [Medline]. Esterre P, Inzan CK, Ramarcel ER, et al. Treatment of chromomycosis with terbinafine: preliminary results of an open pilot study. Br J Dermatol. Jun 1996;134 Suppl 46:33-6; discussion 40. [Medline]. Keating GM. Posaconazole. Drugs. 2005;65(11):1553-67; discussion 1568-9. [Medline]. Kinbara T, Fukushiro R, Eryu Y. Chromomycosis--report of two cases successfully treated with local heat therapy. Mykosen. Dec 1982;25(12):689-94. [Medline]. Pimentel ER, Castro LG, Cuce LC, Sampaio SA. Treatment of chromomycosis by cryosurgery with liquid nitrogen: a report on eleven cases. J Dermatol Surg Oncol. Jan 1989;15(1):72-7. [Medline]. Ameen M. Managing chromoblastomycosis. Trop Doct. Apr 2010;40(2):65-7. [Medline]. Bonifaz A, Carrasco-Gerard E, Saul A. Chromoblastomycosis: clinical and mycologic experience of 51 cases. Mycoses. 2001;44(1-2):1-7. [Medline]. Zhang J, Xi L, Lu C, et al. Successful treatment for chromoblastomycosis caused by Fonsecaea monophora: a report of three cases in Guangdong, China. Mycoses. Mar 2009;52(2):17681. [Medline]. Tagami H, Ginoza M, Imaizumi S, Urano-Suehisa S. Successful treatment of chromoblastomycosis with topical heat therapy. J Am Acad Dermatol. Apr 1984;10(4):615-9. [Medline].

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CHROMOBLASTOMYCOSIS
SOURCE:http://www.mycology.adelaide.edu.au/Mycoses/Subcutaneous/Chromoblastomycos is/ AUTHOR: David Ellis LAST PUBLISHED: March 03, 2011

Description: A mycotic infection of the cutaneous and subcutaneous tissues characterised by the development in tissue of dematiaceous (brownpigmented), planate-dividing, rounded sclerotic bodies. Infections are caused by the traumatic implantation of fungal elements into the skin and are chronic, slowly progressive and localised. Tissue proliferation usually occurs around the area of inoculation producing crusted, verrucose, wart-like lesions. World-wide distribution but more common in bare footed populations living in tropical regions. Aetiological agents include various dematiaceous hyphomycetes associated with decaying vegetation or soil, especially Phialophora verrucosa, Fonsecaea pedrosoi, F. compacta and Cladophialophora carrionii. Clinical manifestations: Lesions of chromoblastomycosis are most often found on exposed parts of the body and usually start a small scaly papules or nodules which are painless but may be itchy. Satellite lesions may gradually arise and as the disease develops rash-like areas enlarge and become raised irregular plaques that are often scaly or verrucose. In long standing infections, lesions may become tumorous and even cauliflower-like in appearance. Other prominent features include epithelial hyperplasia, fibrosis and microabscess formation in the epidermis. Chromoblastomycosis must be distinguished from other cutaneous fungal infections such as blastomycosis, lobomycosis, paracoccidioidomycosis and sporotrichosis. It may also mimic protothecosis, leishmaniasis, verrucose tuberculosis, certain leprous lesions and syphilis. Mycological and histopathological investigations are essential to confirm the diagnosis.

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Chronic verrucous chromoblastomycosis of the hand due to Cladophialophora Note: tissue hyperplasia forming a white verrucoid cutaneous lesion. In Australia, chromoblastomycosis due to C. carrionii occurs mostly on the hands and arms of timber and cattle workers in humid tropical forests.

carrionii.

Laboratory diagnosis: 1. Clinical Material: Skin scrapings and/or biopsy. 2. Direct Microscopy: (a) Skin scrapings should be examined using 10% KOH and Parker ink or calcofluor white mounts; (b) Tissue sections should be stained using H&E, PAS digest, and Grocott's methenamine silver (GMS). Interpretation: The presence in tissue of brown pigmented, planate-dividing, rounded sclerotic bodies from a patient with supporting clinical symptoms should be considered significant. Remember direct microscopy or histopathology does not offer a specific identification of the causative agent. Note: direct microscopy of tissue is necessary to differentiate between

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chromoblastomycosis and phaeohyphomycosis where the tissue morphology of the causative organism is mycelial.

Skin scrapings from a patient with chromoblastomycosis mounted in 10% KOH and Parker ink solution showing characteristic brown pigmented, planate-dividing, rounded sclerotic bodies.

H&E stained section showing characteristic dark brown sclerotic cells which divide by binary fission and not by budding. Note all agents of chromoblastomycosis form these sclerotic bodies in tissue.

3. Culture: Clinical specimens should be inoculated onto primary isolation media, like Sabouraud's dextrose agar.

Cultures of the aetiologic agents of chromoblastomycosis are typically olivaceous-black with a suede-like surface.

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Interpretation: The dematiaceous hyphomycetes involved are well recognised as environmental fungi, therefore a positive culture from a non-sterile specimen, such as sputum or skin, needs to be supported by clinical history and direct microscopic evidence in order to be considered significant. Culture identification is the only reliable means of distinguishing these fungi. 4. Serology: There are currently no commercially available serological procedures for the diagnosis of chromoblastomycosis. 5. Identification: Culture characteristics and microscopic morphology are important, especially conidial morphology, the arrangement of conidia on the conidiogenous cell and the morphology of the conidiogenous cell. Cellotape flag and/or slide culture preparations are recommended. Causative agents:

Cladophialophora carrionii, Fonsecaea pedrosoi, Phialophora verrucosa


Management: The treatment of chromoblastomycosis has been exceedingly difficult. Successful surgical excision requires the removal of a margin of uninfected tissue to prevent local dissemination. Flucytosine with or without thiabendazole has been extensively used in the past. However both itraconazole [400 mg/day] and terbinafine [500 mg/ day] for 6 to 12 months have been used successfully for the treatment of chromoblastomycosis.
REFERENCES Ajello L and R.J. Hay. 1997. Medical Mycology Vol 4 Topley & Wilson's Microbiology and Infectious Infections. 9th Edition, Arnold London. Esterre P, Inzan CK, Ramarcel ER, Andriantsimahavandy A, Ratsioharana M, Pecarrere JL, Roig R, Treatment of chromomycosis with terbinafine: preliminary results of an open pilot study. Br J Dermatol (1996) 134 (Suppl. 46):33-36. Kwon-Chung KJ and JE Bennett 1992. Medical Mycology Lea & Febiger. Richardson MD and DW Warnock. 1993. Fungal Infection: Diagnosis and Management. Blackwell Scientific Publications, London. Rippon JW. 1988. Medical Mycology WB Saunders Co.

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BIOLOGY AND PATHOGENESIS OF FONSECAEA PEDROSOI, THE MAJOR ETIOLOGIC AGENT OF CHROMOBLASTOMYCOSIS
SOURCE: http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6976.2007.00077x/full AUTHORS: Andr L.S. Santos1, Vanila F. Palmeira1,2, Sonia Rozental3, Lucimar F. Kneipp4, Leonardo Nimrichter1, Daniela S. Alviano2, Marcio L. Rodrigues1, Celuta S. Alviano2 DATE PUBLISHED: July 23, 2007
Abstract

Fonsecaea pedrosoi is the principal etiologic agent of chromoblastomycosis, a fungal

disease whose pathogenic events are poorly understood. Treatment of the disease presents poor effectiveness and serious side effects. The disease is epidemiologically important in several regions, which has stimulated studies focused on the biology and pathogenic potential of its major causative agent. In this review, we summarize the current knowledge on the biological aspects of F. pedrosoi, including cell differentiation and pathogenic mechanisms during the interaction of fungi with different hosts' elements.

Introduction Chromoblastomycosis (or chromomycosis) is a chronic fungal disease usually limited to the skin and subcutaneous tissues. Chromoblastomycosis is caused by a list of dematiaceous fungi sharing as a common feature the constitutive synthesis and deposition of melanin at fungal cell wall (Rippon, 1988; De Hoog et al., 2000; Brandt & Warnock, 2003). The mycosis begins with a transcutaneous trauma allowing hyphal fragments and conidial forms to penetrate. It occurs preferentially in humans, although some cases of chromoblastomycosis have been reported in other mammals (McGinnis, 1983; Rippon, 1988; De Hoog et al., 2000; Fondati et al., 2001). The etiologic agents of chromoblastomycosis are Fonsecaea pedrosoi, Fonsecaea compacta (a morphological variety of F. pedrosoi), Phialophora verrucosa, Cladophialophora (Cladosporium) carrionii and Rhinocladiella aquaspersa (Caligiorne et al., 1999). Recently, Exophiala jeanselmei and Exophiala spinifera have also been reported as agents of chromoblastomycosis (Silva et al., 2005; Develoux et al., 2006; Tomson et al., 2006). Geographic distribution and occurrence of chromoblastomycosis Although the disease has been diagnosed on all continents, it occurs more frequently in humid tropical and subtropical regions of America, Asia and Africa (Rippon, 1988; Kwon-Chung & Bennett, 1992; Brandt & Warnock, 2003). Major sites of chromoblastomycosis occurrence include

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Brazil (Silva et al., 1999; Minotto et al., 2001), Mexico (Bonifaz et al., 2001), Venezuela (Perez-Blanco et al., 2006), Japan (Fukushiro, 1983) and Madagascar (Esterre et al., 1996). In Brazil, the Amazon region has been considered as the main chromoblastomycosis endemic area (Silva et al., 1999; Marques et al., 2006). The infection generally affects individuals who are engaged in farming and males are more commonly affected (c. 70% of cases). The explanation for this predominance is not clear, but men are assumed to be more commonly involved in agricultural work and are more prone to inflict injuries on themselves, thereby causing self-inoculation. Moreover, the possible inhibitory effect of female hormones on the fungal growth may partially explain the relatively low number of cases in women (Hernandez-Hernandez et al., 1995; Silva et al., 1999). No racial predilection is reported. Patients with the disease are most commonly aged 3050 years. The period between inoculation and disease development is believed to last years (Kwon-Chung & Bennett, 1992; Silva et al., 1999; Brandt & Warnock, 2003). A correlation with HLA-A29 antigen suggests that genetic factors may play a role as well (Tsuneto et al., 1989). The sites most commonly affected are the lower extremities, especially the feet, which are more likely to be in contact with infected materials, soil and plants or rotting wood (Rippon, 1988). Hands, arms and the buttocks are also frequently involved, and sporadic reports mention lesions on the ears, cornea, neck, face, breasts, chest and abdomen (Bittencourt et al., 1994; Minotto et al., 2001; Hfling-Lima et al., 2005; Salgado et al., 2005). Pathophysiology and diagnosis of chromoblastomycosis Initial lesions of chromoblastomycosis are usually erythematous papules, which gradually enlarge to display varying morphologies such as verrucous nodules, cauliflower-like tumors and psoriasis-like plaques (Richard-Blum et al., 1998). In the later stages of infection, dissemination may occur by extension of the lesions as satellites along the lymph vessels or by autoinoculation through scratching (Kondo et al., 2005). The main complication is secondary infection, which may be frequent and lead to gross lymphoedema and elephantiasis (Silva & Ekizlerian, 1985; Silva et al., 1999; Kondo et al., 2005). When observed under the microscope, histological sections are shown to be composed of an external layer of fibrous tissue and an internal layer of thick granulomatous inflammatory tissue containing polymorphonuclear leukocytes, histiocytes and multinucleated giant cells (Silva & Ekizlerian, 1985; Brandt & Warnock, 2003). In some cases, especially the long standing ones, the presence of chronic inflammatory reaction and fibrous cicatricial tissue offer a favourable condition for the development of malignant skin neoplasm (Caplan, 1968; Foster & Harris, 1987; Dos

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Santos Gon & Minelli, 2006). Systemic invasion following chromoblastomycosis is very rare. However, the occurrence of F. pedrosoi in brain abscesses was reported in immunosuppressed and immunocompetent individuals (McGinnis, 1983; Al-Hedaithy et al., 1988; Santosh et al., 1995; Nobrega et al., 2003; Saberi et al., 2003). Laboratory diagnosis of chromoblastomycosis is easily accomplished because the sclerotic bodies can be identified by direct examination and histological study of the crusts and exudates of the lesions (Kwon-Chung & Bennett, 1992). Sclerotic bodies are easily observed in 10% potassium hydroxide preparations or in hematoxylin-eosin stained tissues. These fungal forms are often seen in the deeper portions of the lesion, whereas hyphae and budding cells may be present at the surface of the lesions (Bonifaz et al., 2001). Sclerotic bodies may be found individualized, in clusters or within giant cells. For cultures, parts of the biopsy sample could be used for growth in Sabouraud-glucose agar supplemented with antibiotics. Small black colonies usually appear within 2 weeks. Species identification is based on conidiogenesis during fungal growth in different media, including potato glucose agar (Dixon & Polak-Wyss, 1991). No serological procedures are available for the diagnosis of chromoblastomycosis. Recently, molecular approaches using the gene coding for ribosomal subunits were described as promising tools for dematiaceous fungi taxonomy (Caligiorne et al., 1999, 2005; Abliz et al., 2003, 2004; De Hoog et al., 2004; Tanabe et al., 2004). If not diagnosed in the early stages, the course of chromoblastomycosis can evolve to a chronic state involving difficulties in managing therapy due to the recrudescent nature of the disease, potential association with the growth of epidermoid carcinoma in affected areas, poor quality of life, and work incapacity for the infected individuals (Brandt & Warnock, 2003). Immune response against chromoblastomycosis Neutrophils and macrophages are key cells in the immune response against chromoblastomycosis. The typical granulomatous reaction observed in patients with the disease is regulated by polymorphonuclear neutrophils (PMNs) (Uribe et al., 1989). Macrophage-derived cytokine production and killing of F. pedrosoi has been described (Alviano et al., 2004a, b; Nimrichter et al., 2004; Hayakawa et al., 2006), although there is evidence demonstrating that fungal structures can down-modulate the effectiveness of macrophages in vitro (Farbiarz et al., 1992; Bocca et al., 2006). The mechanisms of host defense in chromoblastomycosis have not been satisfactorily investigated. It remains unknown whether humoral immunity plays a significant role in fungal disease control, although antibodies with direct antimicrobial action have been detected in sera of infected individuals (Alviano et al., 2004a; Nimrichter et al., 2005).

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Individuals from endemic areas with previous exposure to the fungus develop a specific humoral response, although it has not been clearly correlated with the course of the disease (Esterre et al., 1997). The period required for serological conversion is variable, and some patients may present positive serology for more than 1 year after the end of antifungal treatment (Esterre et al., 2000). The role of cellular immunity in chromoblastomycosis has been more deeply investigated in the last years. It has been suggested that cellmediated immunity in patients with long-standing chromoblastomycosis is somehow impaired and, as a result, these individuals became unable to develop an efficient immune reaction to fungal antigens, contributing to the persistence of the fungus in tissue (Fuchs & Pecher, 1992). Delayedtype hypersensitivity reactions can be used to indicate T-cell-mediated inflammatory responses to either exogenous antigens or autoantigens (Brown, 1993), and are important determinants of previous exposure to fungal elements. This parameter is often used to evaluate the host cellular immune response to a wide range of pathogens, including F. pedrosoi (Corbellini et al., 2006). There are few studies evaluating the immunophenotype of the cellular elements related to cell-mediated immunity involved in the inflammatory response to chromoblastomycosis in skin lesions. Common cellular subtypes include T (CD4+ and CD8+) and B lymphocytes and numerous macrophages (Esterre et al., 1991; D'Avila et al., 2003). Sotto et al. (2004) demonstrated that skin macrophages accumulate antigens from F. pedrosoi in their cytoplasm as homogeneous or granular material. Similar phenomena were detected in factor XIIIa+ dendrocytes and Langerhans cells, suggesting a role of these cells as antigen-presenting cells in chromoblastomycosis. Studies in athymic mice infected with F. pedrosoi showed that, during the course of infection, granulomas became diffuse and confluent with random fungal distribution, supporting a role for T-cell-mediated immune response (Ahrens et al., 1989). In fact, two recent studies showed that CD4+ lymphocytes are potentially key cells for the control of chromoblasto-mycosis (Gimenes et al., 2005; Teixeira de Sousaet al., 2006). Immunization of mice with living F. pedrosoi conidia produced a high influx of CD4+ cells into draining lymph node (Teixeira de Sousa et al., 2006). These cells were purified from mice and, in vitro, proliferated after restimulation with specific antigen (designated as chromoAg, which corresponds to the cell-free culture supernatant obtained after F. pedrosoi growth in Sabouraud medium) with an associated production of IFN-. The use of mice lacking CD4+ and CD8+ cells in a chromoblastomycosis model revealed that fungal burden in the liver and spleen of intraperitoneally infected mice is higher in the absence of CD4+ cells, but not influenced by CD8+lymphocytes. Moreover, mice lacking CD4+ cells, but not CD8+ cells, presented diminished delayed-type hypersensitivity. These animals also produced lower amounts of IFN-

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when compared with wild-type mice, whereas the levels of this cytokine in mice lacking CD8+ cells were not altered (Teixeira de Sousa et al., 2006). In humans with severe forms of the disease, a high IL-10 production and low levels of IFN- are usually observed (Gimenes et al., 2005). T cells from these individuals fail to proliferate in vitro after induction with chromoAg. In contrast, patients with mild forms of disease predominantly produce IFN- and low levels of IL-10; an efficient ability to proliferate is observed in T-cell populations from these patients. Collectively, these results may suggest that CD4+ T cells secreting IFN- are needed to induce protective immunity against F. pedrosoi (Gimenes et al., 2005; Teixeira de Sousa et al., 2006). The host receptors involved in the immune response to chromoblastomycosis still remain to be characterized. In vitro studies demonstrated that, in the presence of chromoblastomycosis agents, the expression of MHC-II and CD8 molecules in macrophages is down modulated (Hayakawa et al., 2006). In addition, phagocytosis of F. pedrosoi conidia by Langerhans cells results in an inhibited expression of CD40 and B7-2 (Silva et al., 2007). Direct binding of fungal antigens to host receptors, however, remains to be demonstrated. Melanin, a major F. pedrosoi antigen, is potentially involved in the activation of Toll-like receptor (TLR) 4 with consequent production of the pro-inflammatory chemokine IL-8 (El-Obeid et al., 2006). The knowledge on the structure of cell wall components of F. pedrosoi and other chromoblastomycosis agents is still limited, but the stimulation of TLR responses by classic fungal molecules such as -glucans (Levitz, 2004) indicates that such immune mechanisms may be involved in the innate response against chromoblastomycosis. -Glucans are also ligands involved in the activation of the fungicidal activity of neutrophils through binding to dectin-1 (Kennedy et al., 2007), which has been recently associated with the control of fungal infections (Dennehy & Brown, 2007). Finally, mannose-bearing structures, which were previously described as surface structures of F. pedrosoi (Alviano et al., 2003), could also have the potential to activate mannose receptors and help the host to control chromoblastomycosis. Treatment of chromoblastomycosis Chromoblastomycosis treatment is difficult and most therapeutic attempts provide only a modest success rate (Bonifaz et al., 1997, 2004; Esterre & Queiroz-Telles, 2006). Moreover, the response to oral antimycotic drugs is limited (Minotto et al., 2001). Several treatment regimes have been used to treat chromoblastomycosis but long-term efficacy is still too low to allow specialists to elect a drug of choice (McGinnis, 1983). Itraconazole (200400 mg day1 for a long period, usually between 6 and 12 months) is considered as an alternative; however, many patients do not show clinical improvement (Andrade et al., 2004). For some cases, the best therapeutic strategy in cases of chromoblastomycosis seems to

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be a combination of two drugs chosen according to the results of prior antifungal susceptibility testing (Poirriez et al., 2000), such as the combination of amphotericin B and 5-fluorocytosine, itraconazole and 5fluorocytosine (Bonifaz et al., 1997), or itraconazole and terbinafin (Gupta et al., 2002). The widespread use of antifungal associated with long-term treatment might lead to fungal resistance producing relapses during therapy (Espinel-Ingroff, 1997; Gimenes et al., 2005; Esterre & Queiroz-Telles, 2006). Tests in vitro revealed that caspofungin is active against F. pedrosoi (Del Poeta et al., 1997). On the other hand, fluconazole and ketoconazole have no practical activity (De Bedout et al., 1997; Andrade et al., 2004). Other alternative therapies include physical methods such as conventional surgery, laser surgery, thermotherapy and cryotherapy (Bonifazet al., 1997; Poirriez et al., 2000; Castro et al., 2003). Among them, surgical treatment appears to be the most effective choice to manage chromoblastomycosis. Surgery, electrodessication and cryosurgery are more effective in early stages (Lupi et al., 2005). Local heat can reduce substantially the extension of the lesions and the need for extensive surgeries. A successful therapy of F. pedrosoi infection was observed through maintenance of the surface temperature at 46C for at least 5 h daily for 2 months and the use of a carbon dioxide laser (Hira et al., 2002). Disabled or deformed limbs may require amputation (Elgart, 1996). Fonsecaea pedrosoi

Fonsecaea pedrosoi is one of the most frequent causative agents of

chromoblastomycosis (Rippon, 1988; Kwon-Chung & Bennett, 1992; Brandt & Warnock, 2003). Although considered a worldwide-distributed fungus, F. pedrosoi is mostly found in tropical regions. Its natural niche is the same described for other dematiaceous species, which comprise soil, rotten wood and decomposing plant material (Rippon, 1988; Kwon-Chung & Bennett, 1992; Brandt & Warnock, 2003; Salgado et al., 2004; Marques et al., 2006). In culture, F. pedrosoi grows slowly and produces restricted, flat to raised and folded, velvety to cottony colony texture at 25C. The colonies mature usually in 14 days and are olivaceous to brown-black in color (Fig. 1). The filamentous appearance is maintained upon cultivation at 25C, 30C or 37C. Fonsecaea produces septate, dark brown hyphae and erect conidiophores that highly branch at apices (Fig. 2). The conidia (1.53.0 2.56.0 m) are brown and barrel-shaped (Fig. 2). Usual Fonsecaea conidiogenesis includes patterns of conidial formation observed in three other genera, namely Cladosporium-, Phialophora- and Rhinocladiellatype (Kwon-Chung & Bennett, 1992; Rippon, 1998). Inside the host the fungus differentiates to form major spherical cells, with a thick and deeply pigmented cell wall; these cells are known as sclerotic, muriform,

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fumagoid or Medlar cells/bodies (Fig. 2) (Kwon-Chung & Bennett, 1992; Brandt & Warnock, 2003; Gimenes et al., 2005; Franzen et al., 2006).

Figure 1. Cultures of Fonsecaea pedrosoi on Sabouraud dextrose agar. A velvety, dark fungal colony of F. pedrosoi grown in a Petri dish is shown in (a). Heaped and granular colonies with a black surface are shown in (b). An aqueous solution of melanin, the dark pigment of F. pedrosoi, is shown in (c).

Figure 2. Morphologies of Fonsecaea pedrosoi observed by light (a, b) and scanning electron microscopy (c). Mycelial cells, including conidial formation (arrows), are shown in (a). Inset in (a) shows isolated conidia. Sclerotic cells, the parasitic forms of F. pedrosoi, are shown in (b). Inset in (b) shows disarticulated sclerotic cells. Scanning electron microscopic images of each fungal form and the potential forms of differentiation are shown in (c). Scale bars: 10 m (a and b) and 1 m (c).

Cellular differentiation

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Fonsecaea pedrosoi is a valuable model in cell biology. As discussed

above, its life cycle comprises different morphological states that include reproduction structures (conidia) and fungal forms usually found in the saprophytic (mycelia) and parasitic stage (sclerotic bodies) (Fig. 2). In principle, any of these fungal forms can generate all of the others, except for the transition of sclerotic cells into conidia (Lopez Martinez & MendezTovar, 2007) (Fig. 2). The mechanisms involved in the morphological transitions of F. pedrosoi are still unknown, although the knowledge on the experimental control of these processes has improved in the last years. Many factors are involved in fungal morphogenesis, including nutritional elements, temperature and aerobic conditions (Szaniszlo et al., 1983; Osherov & May, 2001; van Burik & Magee, 2001). In F. pedrosoi, cultivation under static conditions preferentially induces growth as hyphal forms, whereas agitation for short periods (25 days) stimulates conidia formation. Attempts to induce the formation of sclerotic bodies in vitro using different approaches have been reported periodically (Reiss & Nikerson, 1974; Szaniszlo et al., 1976; McGinnis, 1983; Cooper, 1985; Ibrahim-Granet et al., 1987; Matsumoto & Ajello, 1991; Alviano et al., 1992). However, the difficulty in inducing sclerotic cells in vitro has resulted in a lack of physiological studies with the tissue forms of F. pedrosoi when compared with the other morphological stages of the fungus (Kwon-Chung & Bennett, 1992; Mendoza et al., 1993). Acidic ranges of pH are considered to be key inducers of the formation of sclerotic cells (Cooper, 1985; Ibrahim-Granet et al., 1987). Additionally, as described by Alviano et al. (1992), a major population of disarticulated sclerotic bodies (90%) is formed after growth for 14 days at 37C in acidic medium supplemented with propranolol (800 M). This study was further validated by Silva et al. (2002) who demonstrated that sclerotic cells formed under these conditions are morphologically and antigenically similar to those obtained in vivo. It has also been described that F. pedrosoi requires low concentrations of Ca2+ (0.1 mM) at pH 2.5 to switch almost 100% from mycelium to sclerotic cells, suggesting that ion concentration in human tissues could participate in the process of dimorphism during chromoblastomycosis (Mendoza et al., 1993). In fact, Ca2+ and propranolol induce differentiation in many other eukaryotic cells through the activation of different signaling pathways (Wallukat, 2002; Breitwieser, 2006). Although the conditions that induce morphological transitions and consequent sclerotic cell formation during infection are unknown, a potential physiological inducer of the F. pedrosoi dimorphism was suggested in a recent study (Alviano et al., 2003). Platelet activator factor (PAF), a naturally occurring phospholipid that has been shown to induce differentiation of animal (Chao & Olson, 1993) and microbial (Rodrigues et al., 1996; Lopes et al., 1997) cells, was reported to stimulate the conversion of mycelial to sclerotic forms of F. pedrosoi

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(Alviano et al., 2003). Electron microscopy techniques confirmed that PAF induced the formation of typical sclerotic cells, as previously described with propranolol (Alviano et al., 1992). In addition, several biochemical parameters were evaluated in PAF and propranolol-induced cells. They both presented similar profiles of fatty acid constituents, polysaccharide composition and surface carbohydrate expression (Alviano et al., 2003). The addition of PAF or propranolol to mycelial cultures similarly stimulated ectophosphatase activity, supporting a correlation between morphogenesis and surface expression of phosphatases (Kneipp et al., 2003), as described later in this review. Sclerotic cells are extremely resistant to destruction by immunological action (Esterre et al., 1993; Rosen & Overholt, 1996; Hamza et al., 2003), which contributes to the persistence of F. pedrosoi inside the human host and development of a chronic disease. In this context, inhibition of the differentiation of F. pedrosoi mycelia into the parasitic sclerotic forms could be an interesting alternative for the design of antifungal drugs to be used in chromoblastomycosis. Therefore, the understanding of this process and, consequently, how this morphological transition can be inhibited may allow the design of alternative and efficient therapies against chromoblastomycosis. Surface and secreted structures ofF. pedrosoi Adhesins and interaction of F. pedrosoi with host cells A common feature in systemic mycoses is the formation of a pathogeninduced granulomatous reaction, comprising polymorphonuclear leukocytes (PMNs), monocytes and macrophages (Hirsch & Johnson, 1984; Esterre et al., 1993). Therefore, besides the host cells regularly present in infected tissues, effector cells that are recruited to the sites of infection are key elements in the immune response to subcutaneous mycoses (Hayakawa et al., 2006). In this section, the most relevant aspects of the interaction of F. pedrosoi with phagocytes or epithelial cells will be discussed. Farbiarz et al. (1990) demonstrated that mouse peritoneal resident macrophages have little or no cytotoxic activity against F. pedrosoiconidia. During the phagocytic process, pigmented particles, supposedly melanin, are released by the fungus. Interestingly, these particles were detected by electron microscopy in phagocytic vacuoles containing ingested fungi as well as in other small cytoplasmic vacuoles. As described later in this review, secreted pigments activate neutrophils and induce the production of antimicrobial antibodies (Alviano et al., 2004a), which may have an impact on chromoblastomycosis pathogenesis. The protective ability of cell wall-associated melanin against phagocytes will be also discussed. Rozental et al. (1994) showed that activated macrophages also failed to kill F. pedrosoi conidia, although they caused a significant delay in germ

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tube and hyphae formation. In that study, the oxidative activity of activated macrophages was studied by light and transmission electron microcopy. Oxygen intermediate derivatives could be observed either in the plasma membrane or in association with fungi-containing vacuoles (Rozental et al., 1994). Another study revealed that, in contrast to macrophages, PMN killed F. pedrosoi even in the absence of serum and specific antibodies (Fig. 3). PMN-derived fungal killing occurred extracellularly after short periods of interaction (Rozental et al., 1996). Cytochemical observations allowed the establishment of a close correlation between the oxidative burst and the extracellular cytotoxicity of PMNs, as previously reported by Nathan et al. (1979). Importantly, peroxidase-containing granules in neutrophils were found to be fused with the parasitophorous vacuole, indicating that the toxic products of H2O2 cleavage are involved in F. pedrosoi killing.
Figure 3. Thin sections showing different steps of the interaction of Fonsecaea pedrosoi with PMNs. (A) A conidial cell (c) in the initial processes of phagocytosis by a PMN, including pseudopod emission. A previously ingested conidium (dc) was destroyed by the same PMN. (B) Two PMNs closely associated surrounding a conidial cell (c). Fragments of a conidial cell wall, probably originated from PMN-mediated fungal lysis, were detected in a PMN vacuole (C; asterisk). Scale bars: 1 m.

Microbial adherence is one of the most important determinants of pathogenesis. This step is normally followed by microbial internalization, which can be used by the pathogen to replicate inside the host or evade the immune response (Verstrepen & Klis, 2006). It has been demonstrated that F. pedrosoi can be detected in the cytoplasm of cytochalasin-treated macrophages and epithelial cells, suggesting that the fungus presents an invasive ability (Farbiarz et al., 1990; Limongi et al., 1997). Apparently, invasion of host cells by F. pedrosoi is preceded by an adhesive step that is mediated by a lectin-like surface adhesin, as described below. Limongi et al. (1997) used Chinese hamster ovary (CHO) cells presenting different mutations in their glycosylation patterns (Stanley, 1984, 1985) to study the process of adhesion of F. pedrosoi to host molecules. This

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study suggested that conidial cells express lectin-like adhesins with affinity for mannose and N-acetylglucosamine (GlcNAc). The mannose residues were apparently involved in the initial adhesion step of F. pedrosoi to host cells, while GlcNAc units were supposedly required for fungal internalization (Limongi et al., 1997). The surface distribution of F. pedrosoi lectins was confirmed using fluorescein isothiocyanate (FITC)labeled neoglycoproteins [bovine serum albumin (BSA)mannose and BSAGlcNAc] (Limongi et al., 2001). Interestingly, the expression of surface sites for mannose and GlcNAc binding was higher when the fungus was cultivated at 37C than 28C, suggesting that the putative adhesins can be over expressed during infection. In addition, the binding of mannose/GlcNAc-containing neoglycoproteins to F. pedrosoi conidia was inhibited by previous incubation of fungal cells with chloroquine and/or trypsin (Limongi et al., 2001). This observation confirms the protein nature of the carbohydrate-binding molecule and indicates that the adhesins are not recycled to be expressed on conidial surface, as previously described by Tietze et al. (1980) for macrophages. Cell wall extracts from F. pedrosoi conidia, obtained by mechanical cell disruption, were purified by affinity chromatography on mannose- or GlcNAc-agarose columns. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed the occurrence of a single band with a molecular mass corresponding to 50 kDa in eluates from both resins, suggesting that the same adhesin recognizes both carbohydrates (Limongi et al., 2001). A similar ligand-binding specificity to the mannose/GluNAG receptor was earlier described by Stahl et al. (1978). Protein phosphorylation and dephosphorylation plays a key role in the response of host cells to invading pathogens (Mendes-Giannini et al., 2005). In F. pedrosoi, the attachment to and invasion of epithelial cells and macrophages has been suggested to be linked to the action of protein kinases, including those from fungal cells (Limongi et al., 2003). Pretreatment of macrophages, epithelial cells or conidia with the protein kinase inhibitors staurosporine, genistein and calphostin before infection significantly decreased cell invasion by F. pedrosoi. Interestingly, the pretreatment of conidial cells with the inhibitory drugs arrested only the invasion process of epithelial cells, having no effect on the interaction with macrophages (Limongi et al., 2003). These results suggest that the protein kinase-associated machinery of the fungus is not necessary for its entry in phagocytic cells. The protein kinase apparatus, however, is required for invasion of nonphagocytic cells, suggesting an active process of penetration. Immunofluorescence assays using monoclonal antiphosphotyrosine antibodies revealed an accumulation of phosphorylated tyrosine residues at the site of attachment of F. pedrosoi to the host cell, although no labeling was observed after fungal internalization. Simultaneous labeling of the host cell with phalloidin showed that actin filaments also participate in this process. The staining of phosphothreonine was weaker and only observed in macrophage phagocytic vacuoles containing the fungus (Limongi et al., 2003,

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Supplementary figure http://jmm.sgmjournal.org). It may be inferred that tyrosine phosphorylation during this process is an early, local and short-life event, whereas threonine phosphorylation is a later and transient phenomenon, difficult to detect by the methods used in the study of Limongi et al. (2003). These results show that signal transduction networks involving protein kinases and protein phosphatase activities could modulate crucial events during F. pedrosoi infections. Melanin Fungal melanins are negatively charged, hydrophobic and of high molecular weight, arising by the oxidative polymerization of phenolic and/or indolic precursors (Nosanchuk & Casadevall, 2003). Melanin has been detected in several pathogens, including Candida albicans(MorrisJones et al., 2005), Cryptococcus neoformans (Chaskes & Tyndall, 1978), Paracoccidioides brasiliensis (Gomez et al., 2001),Sporothrix schenckii (Romero-Martinez et al., 2000; Morris-Jones et al., 2003), Histoplasma capsulatum (Nosanchuk et al., 2002), Blastomyces dermatitidis (Nosanchuk et al., 2004) and Aspergillus fumigatus (Tsai et al., 2001). The ability of pathogenic fungi to produce melanin is associated with virulence. In C. neoformans, melanin protects fungal cells against oxidative agents and also by inhibiting cell-mediated responses (Mednick et al., 2005). Melanin can interfere with complement activation and reduce the susceptibility to antifungal agents (reviewed by Nosanchuk & Casadevall, 2003). In A. fumigatus, B. dermatitidis, H. capsulatum, P. brasiliensis and S. schenckii, melanin was detected in vitro and in vivo, but its role in fungal infections is still unclear. The ability of F. pedrosoi to produce secreted (Fig. 1) or cell-wallassociated melanin-like components has been widely reported (Farbiarzet al., 1990; Alviano et al., 1991, 2004a; Franzen et al., 1999, 2006; Cunha et al., 2005). The constituents of the melanin complex from mycelial forms of F. pedrosoi were partially characterized in an early report (Alviano et al., 1991). Melanin-associated components mainly comprised fatty acids, polysaccharides and proteins. Mannose, rhamnose, galactose and glucose were the sugar constituents, whereas lipid components included even-numbered, saturated and unsaturated fatty acids ranging from C16 to C18. Aspartic and glutamic acids, leucine, glycine and alanine were the major amino acids (Alviano et al., 1991). The pigment was mainly accumulated on large alkali-extractable, electron-dense cytoplasmic bodies and, apparently, on the outer layer of the cell wall. In fact, a close association between the increase of cytoplasmic electron-dense granules, acidic organelles and pigmentation of the fungal cultures was further reported (Franzen et al., 1999). In addition, transmission electron microscopy revealed that melanin synthesis in F. pedrosoi involves the formation of melanosome-like compartments, as described for mammalian cells (Seiji et al., 1963; Quevedo et al., 1987; Marks & Seabra, 2001; Raposo & Marks, 2002).

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The biosynthesis of melanin in many fungal pathogens, including F. pedrosoi, leads to the accumulation of the pigment as a highly resistant structure underlying the cell wall (Rosas et al., 2000; Gomez & Nosanchuk, 2003; Nosanchuk & Casadevall, 2003). Because of this combination between cellular distribution and high resistance against different chemicals, the digestion of melanized fungi with proteases, glycanases, denaturant and hot concentrated acid yields melanin particles that retain the shape of fungal cells, which were therefore called melanin ghosts (Rosas et al., 2000). The digestion of pigmented F. pedrosoi also yields particles similar in size and shape to intact cells (Alviano et al., 2004a) (Fig. 4). These results associated with immunofluorescence analysis provided evidence that melanin synthesis in F. pedrosoi follows the previously described pattern of wall-associated pigment accumulation in fungal cells (Fig. 4). Interestingly, secreted particles of melanin can also be isolated from F. pedrosoi cultures (Fig. 1). These extracellular molecules were recognized by sera from individuals with chromoblastomycosis (Alviano et al., 2004a), which agreed with previous studies demonstrating that fungal melanins are immunogenically active compounds (Nosanchuk et al., 1998; Rosas et al., 2000).

Figure 4. Cell wall expression of melanin in Fonsecaea pedrosoi. A comparison between intact mycelial cells (A) and melanin-rich residues (ghosts) obtained after several steps of chemical and enzymatic hydrolyses followed by treatment with denaturing agents (B) suggests that melanin probably plays a key role in maintaining cell shape. Insets in (A and B) show the macroscopic aspects of mycelial cells before (a) and after (b) treatment with NaOH, showing that the pigment is alkali-extractable (b). Polyclonal antibodies to melanin bound to the surface of conidia (C), confirming that these fungal forms also express the pigment at their surface. Scale bars: 1 m.

The biosynthesis of fungal melanins derives from different precursors. Most commonly, fungal pigments derive from 1,8-dihydroxynaphthalene, generating the so-called dihydroxynaphthalene-melanins (reviewed

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by Gomez & Nosanchuk, 2003). This is the biological pathway used by Aspergillus nidulans, Aspergillus niger, A. fumigatus, Alternaria alternata, Cladosporium carrionii, Exophiala (Wangiella) dermatitidis, Exophiala jeanselmei, F. compacta, Hendersonulla toruloidii, Phaeoannellomyces werneckii, Phialophora richardsiae, Phialophora verrucosa, Xylohypha bantiana and S. schenckii (reviewed by Gomez & Nosanchuk, 2003). In contrast, C. neoformans produces eumelanin via a polyphenoloxidase (a laccase), which catalyzes a one-step oxidation of dihyroxyphenols to quinone intermediates that subsequently autooxidize to form melanin (Rhodes et al., 1982). Supplementation of F. pedrosoi cultures with pesticide tricyclazole results in defective pigmentation (Cunha et al., 2005). As tricyclazole inhibits melanization in fungal cells by blocking the dihydroxynaphthalenepathway of melanin synthesis, it has been concluded that F. pedrosoi produces the pigment using this pathway (Cunha et al., 2005). Although treatment with tricyclazole did not influence fungal viability (Franzen et al., 2006), it impaired the well-known ability of F. pedrosoi to destroy the host macrophages (Farbiarz et al., 1990). In fact, tricyclazoletreated conidia are more susceptible to the antimicrobial effects of macrophages (Cunha et al., 2005). This effect may be related to the noticeable morphological differences induced by tricyclazole, as recently described by Franzen et al. (2006). Melanin has been shown to be produced and released during the infection by F. pedrosoi (Farbiarz et al., 1990). As stated above, sera from patients with chromoblastomycosis reacted with the soluble form of the F. pedrosoi melanin (Alviano et al., 2004a). Antibodies to melanin were purified based on their similar affinity by insoluble pigment residues and used in immunological and cell growth assays. As demonstrated by immunofluorescence and flow cytometry analyses, melanin-binding antibodies were shown to be reactive with pigmented conidia, mycelia and sclerotic cells, as well as the ghost particles (Alviano et al., 2004a). Sclerotic bodies obtained from patients' lesions were similarly recognized by the antibodies. In phagocytosis assays, F. pedrosoi conidia were opsonized in the presence of antibodies to melanin, resulting in increased ingestion and killing by human and animal neutrophils (Alviano et al., 2004a). Even in the absence of host effector cells, antibodies to melanin inhibited in vitro growth of conidial and sclerotic cells. After antibody treatment, only 2.8% of conidia remained viable, indicating a highly effective antibody-mediated toxicity. Sclerotic cells were also inhibited, but to a lesser extent. This observation may be a consequence of the fact that sclerotic bodies typically form large aggregates (Alviano et al., 1992), which could impair the access of external ligands to inner cells. The above results agree with previous data demonstrating that, in C. neoformans, administration to lethally infected mice of monoclonal antibodies (mAbs) to melanin significantly improved survival (Rosas et al.,

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2001). In the Cryptococcus model, melanin-binding mAbs completely abrogated cell growth, whereas there was no effect on the replication of nonmelanized cells. Taken together, these results demonstrate that melanin is a key cell wall polymer that could be targeted by antifungal agents. The phagocytosis levels of conidia or sclerotic cells by neutrophils are enhanced in the presence of the fungal melanin, suggesting that melanin particles could also activate phagocytic cells (Alviano et al., 2004a). In fact, oxidative burst of neutrophils in the presence of F. pedrosoi cells, phagocytosis of nonrelated fungi (C. albicans) by neutrophils and ingestion and killing of F. pedrosoi conidia by different phagocytic cells were enhanced in the presence of soluble melanin (Alviano et al., 2004a). These observations agreed with previous reports demonstrating that melanins or their precursors influence the activation of macrophages (D'Acquisto et al., 1995) and the proliferation and differentiation of human keratinocytes and fibroblasts (Blinova et al., 2003). The ability of F. pedrosoi melanin to activate phagocytes, however, was contradicted in recent studies (Bocca et al., 2006). In a model including IFN-- and lipopolysaccharide-stimulated macrophages, the pigment inhibited nitric oxide production (Bocca et al., 2006). According to these authors, this effect could be related to the inability of the host to clear F. pedrosoi, leading to a chronic disease. Sialic acid metabolism and F. pedrosoi pathogenesis Sialic acids are a family of monosaccharides comprising several derivatives of neuraminic acid (Schauer, 1982). Their presence in many glycoconjugates as terminal monosaccharides confers biologically diverse activities to cell surfaces (Schauer & Kamerling, 1997). In fungal cells, sialic acids seem to contribute to microbial pathogenesis by protecting fungi against phagocytosis (reviewed by Alviano et al., 1999) or promoting adhesion to extracellular matrix proteins (Wasylnka et al., 2001). Sialic acids were identified as cell surface components of conidia and mycelia of F. pedrosoi (Souza et al., 1986; Alviano et al., 2004b). Nacetylneuraminic acid (Neu5Ac) was the only derivative found in the mycelium phase, whereas conidia contained both N-glycolylneuraminic acid (Neu5Gc) and Neu5Ac (Souza et al., 1986). In an early report, sialic acids were characterized as key determinants of morphogenesis and cellular integrity in F. pedrosoi, as inferred from the marked alterations in the hyphal morphology observed after enzymatic removal of these sugars (Souza et al., 1986). The expression of sialoglycoconjugates in F. pedrosoi conidia, mycelia and sclerotic cells was analyzed using sialic acid-binding viruses (influenza A and C virus strains), sialidase treatment and lectin-binding assays (Alviano et al., 2004b) (Fig. 5). Treatment of conidia and mycelia

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with FITC-labeled lectins revealed the presence of surface sialoglycoconjugates containing 2,3- and 2,6-sialylgalactosyl sequences. Conidial cells also expressed cell wall sialylated structures containing 9-Oacetyl-N-acetylneuraminic acid (Neu5,9Ac2). The sialylated structures in F. pedrosoi were apparently glycoproteins, as demonstrated by Western blotting analysis (Alviano et al., 2004b). In cellular extracts from conidia and mycelia, sialylated proteins with molecular masses corresponding to 40 and 56 kDa were detected by lectin binding. An additional band of 77 kDa was detected in conidial protein extracts, suggesting an association between sialic acid expression and morphogenesis (Alviano et al., 2004b). Sialic acids and sialylated glycoproteins were not detected in F. pedrosoi sclerotic cells obtained from in vitro or in vivo sources (Alviano et al., 2004b), suggesting that these cells lack sialic acid metabolism. The possibility that the conventional techniques for sialic acid detection are not useful with these cells cannot be ruled out either. It is still unclear how sialic acids influence fungal pathogenesis. There is, however, some evidence supporting the hypothesis that sialic acids could help fungal cells to escape host defenses by protecting them against phagocytosis (Oda et al., 1983; Rodrigues et al., 1997; Alviano et al., 2004b). In fact, enzymatic removal of sialic acids from the surface of F. pedrosoi conidia resulted in increased phagocytosis by human neutrophils from healthy individuals (Alviano et al., 2004b). In this context, the role of surface sialoglycoconjugates in the infection by F. pedrosoi may be similar to that proposed for other infections (Rodrigues et al., 1997), in which sialic acid, expressed by the infecting propagules, could protect fungi against destruction by host cells until differentiation into the parasitic form. In sclerotic cells, the enhanced expression of melanin increases the resistance against the action of effector immune cells (Esterre et al., 1993; Rosen & Overholt, 1996; Hamza et al., 2003) and the sialic acid expression is not necessary. Sialidases are enzymes that remove terminal sialic acids from sialylated glycoconjugates (Schauer & Kamerling, 1997). These enzymes have a potential role in many microbial infections (Jost et al., 2001), but their role in the metabolism of fungal cells is controversial (Uchidaet al., 1974; Royal et al., 1984; Roggentin et al., 1999). Recently, Alviano et al. (2004b) demonstrated that mycelia and conidia, but not sclerotic cells of F. pedrosoi, produce surface and secreted sialidase activity. The role of sialidases in F. pedrosoi infection is unknown, but removal of sialic acids from cell-surface glycoproteins and glycolipids alters the profiles of glycosylation in fungal and host cells, which could result in the exposition of additional sites for interaction with conidia and mycelial forms.

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Figure 5. Surface expression of sialic acids in Fonsecaea pedrosoi. Sialic acid expression was probed with the sialic acid-specific FITC-labeled lectin LFA. Detection of surface sialic acids is more evident in conidial (A) and mycelial (B) cells than in sclerotic bodies (C). Sialidase-treated cells are shown in insets. Panels A, B and C show fungal cells observed under differential interferential contrast, while panels a, b and c show the same cells observed under the fluorescence mode. Scale bars: 10 m.

Fonsecaea pedrosoi cell wall lipids: glycosylceramides


Glycosylceramides [cerebrosides, monohexosylceramides (CMH) or GlcCer/GalCer] are neutral glycosphingolipids (GSL) synthesized by a variety of organisms, including animal, plant and fungi (Lynch et al., 1992; Barreto-Bergter et al., 2004; Nimrichter et al., 2004). They are composed of a monosaccharide, normally glucose or galactose, in 1ortho-betaglycosidic linkage with the primary alcohol of an N-acyl sphingoid (ceramide). Significant structural differences have been observed between fungal CMH and their counterparts in animal cells, indicating a dissimilar biosynthetic pathway. CMH have been implicated in several cellular functions, such as cell growth, intracellular signaling, microbial adhesion, apoptosis and protein sorting (Hakomori, 2003; Nimrichter et al., 2005). In fungal organisms, CMH has been related with sorting of molecules to cell surface, cell differentiation, growth and pathogenicity (Rodrigues et al., 2000, 2007; Toledo et al., 2001;

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Levery et al., 2002; Pinto et al., 2002; Da Silva et al., 2004; Nimrichter et al., 2005; Rittershaus et al., 2006). Chromatographic methods and MS demonstrated that CMH is synthesized by F. pedrosoi. Major CMH from mycelial and conidial forms ofF. pedrosoi were purified and characterized as N-2-hydroxyhexadecanoyl-1-D-glucopyranosyl-9-methyl-4,8-sphingadienine (Nimrichteret al., 2004, 2005) (Fig. 6), the conserved structure usually found in fungal organisms (Barreto-Bergter et al., 2004). Immunofluorescence analysis using a mAb against F. pedrosoi CMH, demonstrated a very interesting profile of surface reactivity of these cells, in which only growing conidia or mycelia were stained. Other fungal pathogens presenting a similar distribution of CMH at their surface had their differentiation process inhibited by the anti-CMH antibody, confirming an association between CMH expression and fungal growth (Rodrigues et al., 2000; Pinto et al., 2002). To confirm this hypothesis, F. pedrosoi conidia were incubated with the anti-CMH mAb and the growth of treated cells, as well as their ingestion by macrophages, was further analyzed (Nimrichter et al., 2004). The treatment with anti-CMH mAb killed at least 60% of the conidial population. In addition, pretreatment of conidia with this antibody also rendered fungal cells more susceptible to killing by murine macrophages, revealing an opsonic activity of the mAb (Nimrichter et al., 2004). Collectively, these results demonstrated for the first time that, besides their immediate antifungal action, CMH-binding antibodies can help host cells to eliminate internalized fungi increasing both phagocytosis and macrophage microbicidal activity (Nimrichter et al., 2004). These data indicate that antibodies to CMH could have a potential to be used in passive immunization therapies. In addition, if fungal CMH have the ability to effectively elicit protective antibody immune responses, they could be tested as vaccine components.

Figure 6. Fonsecaea pedrosoi cerebrosides. Analysis of CMH purified from each fungal form by electrospray ionization-MS (ESI-MS) revealed a previously unknown structural diversity, since a tri-hydroxylated ceramide moiety was detected in conidia and sclerotic bodies. ESI-MS spectra of CMH from mycelial (a), conidial (b) and sclerotic (c) forms are shown, as well as the structures of the previously described molecule (d) and the newly discovered cerebroside (e).

The activity of anti-CMH antibodies against the parasitic form of F. pedrosoi was also tested to support their potential ability to control

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chronic chromoblastomycosis. However, mAb-treated sclerotic cells remained 100% viable. This result was in agreement with the immunofluorescence data, showing that these cells are not recognized by the anti-CMH antibodies (Nimrichter et al., 2004). Furthermore, the adhesion index of mAb-treated sclerotic cells was similar to that observed when the antibodies were not added. Three possibilities were then raised: (1) CMH could be absent on sclerotic cells, (2) CMH could be produced by these cells, although presenting a distinct structure that would impair its recognition by the mAb, or (3) the high melanization of sclerotic cells would form a protective shield, blocking the access of the antibody to the CMH. All the possibilities were investigated and are discussed below (Nimrichter et al., 2005). CMH from sclerotic cells were purified and finely characterized. In contrast to several fungal pathogens in which CMH have been studied, molecules from sclerotic cells of F. pedrosoi had an interesting structural diversity. Although the conserved structure, N-2-hydroxyhexadecanoyl-1-D-glucopyranosyl-9-methyl-4,8-sphingadienine, was found in these cells, the major CMH species contained an additional hydroxyl group, bound to its long-chain base (Fig. 6). Therefore, the expression of CMH on the parasitic forms of F. pedrosoi was confirmed. Interestingly, this structurally different CMH was also recognized in enzyme-linked immunosorbent assays by the mAb produced against the conserved structure. However, a substantial decrease in this recognition was detected, which could explain the lack of reactivity experimentally shown by immunofluorescence assays (Nimrichter et al., 2005). The last question raised was the potential blockage of antibody recognition due to melanin expression. In fact, sclerotic cells are heavily pigmented (Silva et al., 2002). In addition, it is well known that this polymer is able to block the access of several molecules through the cell wall of fungal cells (Nosanchuk & Casadevall, 2006). A detailed microscopic analysis revealed that antibody binding to CMH was only detectable in cell wall regions where melanization was not evident. Accordingly, removal of melanin from the surface resulted in a strong reaction with the anti-CMH antibody, confirming that other cell wall components affect the exposure of these lipids to external ligands. These results led to the conclusion that melanization could affect binding and, consequently, the susceptibility of fungal cells to antimicrobial compounds (Nimrichter et al., 2005). The above data confirmed previous studies revealing that fungal CMH and its biosynthetic pathway are potential targets for the development of new antifungal drugs (Thevissen et al., 2005). The recent data discussed here suggest that anti-CMH is an effective antifungal agent in vitro. However, the melanin shield formed at the cell surface, especially in this pathogen, is clearly a limiting factor. The alternative use of small antimicrobial molecules, supposedly able to penetrate the melanin shield and bind CMH, could be a promising option to control fungal growth. In this regard, it has been recently demonstrated that plant defensins specifically recognize fungal CMH and cause microbial killing

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(Thevissen et al., 2004). Alternatively, drugs that inhibit melanin synthesis, already discussed in this review, could be administered together with the anti-CMH mAb, increasing the efficacy of these antibodies. Ectoenzymes, pathogenesis and morphogenesis Ecto- or extracytoplasmic enzymes are molecules whose catalytic site faces the extracellular medium (Hunter, 1995; Meyer-Fernandes, 2002; Rast et al., 2003). In F. pedrosoi, ecto-phosphatases and ecto-ATPases (E-type) were characterized as cell wall enzymes (Kneipp et al., 2003, 2004; Collopy-Junior et al., 2006). Below we describe their biochemical and potential biological roles. The ability of F. pedrosoi to produce surface ecto-phosphatase activities was first suggested by the ability of intact mycelia to hydrolyze phosphorylated substrates, such as p-nitrophenylphosphate and phosphorylated amino acids (Kneipp et al., 2003, 2004). Enzyme activity was higher at acidic pHs and was strongly reduced by classical inhibitors of acid phosphatases such as sodium orthovanadate, sodium molybdate and sodium fluoride. The cell wall distribution of the mycelial as well as conidial enzyme was confirmed by transmission electron microscopy, which showed electron-dense cerium phosphate deposits on this cellular compartment (Kneipp et al., 2003, 2004) (Fig. 7).

Figure 7. Surface expression of acid phosphatases in Fonsecaea pedrosoi. Conidial cells were incubated in the absence (a) or presence (b) of -glycerophosphate, a phosphatase substrate. Phosphate precipitates are clearly visible on the cell wall in (b) (arrows). Scale bars: 1 m.

Ecto-phosphatase activity in F. pedrosoi seems to be associated with parasitism, as sclerotic cells showed much higher activities than conidia and mycelia (Kneipp et al., 2003). In addition, a recent isolate from a human case of chromoblastomycosis also showed increased enzyme activity (Kneipp et al., 2003), suggesting that the expression of surface phosphatases may be related to pathogenesis. In further studies, a model of differential ecto-phosphatase expression induced by exogenous

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phosphate (Pi) confirmed this hypothesis (Kneipp et al., 2004). Following the description that the conidial ecto-phosphatase was strongly inhibited by Pi, Kneipp et al. (2004)showed that cultivation of conidial cells in the absence of exogenous Pi resulted in a 130-fold increase in the surface phosphatase activity. These conidia presented adhesion levels to host up to sevenfold higher than fungal cells cultivated in regular conditions. Pretreatment of fungal cells with sodium orthovanadate, an irreversible inhibitor of conidia acid ecto-phosphatase, reduced the adhesive capacity of conidia (Kneipp et al., 2004). Taken together, these results indicate that ecto-phosphatase, besides its possible functions in the biology of fungal cells, may contribute to the adhesion of F. pedrosoi conidia to host cells, a first and crucial step in the establishment of chromoblastomycosis. The ability of F. pedrosoi to hydrolyze phosphorylated amino acids apparently varies according with the cell differentiation. A correlation between morphogenesis and surface expression of phosphatases in fact seems to occur in F. pedrosoi. Besides the observation that sclerotic cells, conidia and mycelia express different levels of ecto-phosphatase activity (Kneipp et al., 2003), it was demonstrated that propranolol and PAF, two distinct inducers of cellular differentiation of F. pedrosoi (Alviano et al., 1992, 2003), significantly stimulated the activity of surface phosphatases (Alviano et al., 2003). The signaling pathways involving the activity of ecto-phosphatases and cell differentiation in F. pedrosoi remain to be characterized. Ecto-ATPases are also present at the cell surface of F. pedrosoi (CollopyJunior et al., 2006). Using ATP as a substrate, an Mg2+-stimulated ectoATPase activity in mycelial forms was characterized. This enzyme reaches its optimum activity at alkaline pHs and was not sensitive to inhibitors of phosphatases and intracellular ATPases (P-, V- and F-type ATPases). The surface localization of the Mg2+-stimulated ATPase in F. pedrosoi was determined using of 4,4-diisothiocyanostylbene-2,2-disulfonic acid (DIDS), a membrane impermeant inhibitor (Meyer-Fernandes et al., 1997; Collopy-Junior et al., 2005), and suramin, an inhibitor of ectoATPases (Ziganshin et al., 1995). Based on the fact that mycelial cells presented levels of enzyme activity much higher than those observed in conidia and sclerotic cells, a correlation between ecto-ATPase activity and differentiation in F. pedrosoi was also suggested (Collopy-Junior et al., 2006). However, knowledge of the relevance of these enzymes in fungal biology or pathogenesis is still very preliminary. Extracellular proteolytic enzymes The secretion of molecules, especially hydrolytic enzymes, to the extracellular environment may represent an important adaptive mechanism during the life cycle of a variety of microorganisms, including fungi (van Burik & Magee, 2001; Monod et al., 2002; Naglik et al., 2003).

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Although knowledge about surface structures has been increasing very rapidly, little is known about molecules secreted by F. pedrosoi. Many human pathogenic fungal species produce secreted peptidases that are directly involved in different stages of microorganismhost interactions (Hube, 2000; Monod et al., 2002; Naglik et al., 2003; Rodrigues et al., 2003; Santos & Soares, 2005; Santos et al., 2006; Silvaet al., 2006a, b). The peptidases include all enzymes that catalyze the cleavage of peptide bonds in proteins, digesting these macromolecules into peptides or free amino acids (Rao et al., 1998). In F. pedrosoi, the detection of proteolytic activity in the culture supernatant was the first step in the understanding of the possible roles of this class of enzymes in metabolic and infection-related processes. The production of extracellular peptidases by F. pedrosoi, a largely unexplored research field, was initially studied using conidial cells grown on chemically defined and complex culture media (Palmeira et al., 2006a). Two distinct classes of extracellular peptidases capable of degrading soluble BSA were identified. In the supernatant derived from conidia cultivated in chemically defined medium, an aspartyl peptidase activity was detected. This enzyme was only active at acidic pH ranges, reaching its optimal activity at pH 4.0 and totally sensitive to pepstatin A, a wellknown specific aspartyl peptidase inhibitor. Conversely, conidia cultivated in Kauffman complex medium secreted a distinct peptidase whose activity was restrained by some metallopeptidase inhibitors, such as ethylene glycol tetraacetic acid and 1,10-phenanthroline (Palmeira et al., 2006a). These results suggested that the medium composition could modulate the synthesis and secretion of distinct proteolytic enzymes by F. pedrosoi conidia as previously observed for other pathogenic fungi (Monod et al., 2002; Naglik et al., 2003; Santos et al., 2006; Silva et al., 2006a, b). The existence of two biochemically distinct secreted peptidases could be advantageous for the adaptation of F. pedrosoi to different environments, including host tissues. As discussed previously, disseminated chromoblastomycosis was already reported, but there is no information available in the current literature about the mechanism by which fungal forms invade other tissues. In this context, these clinical reports are consistent with the secretion of tissuedegrading enzymes, such as extracellular peptidases. Peptidases are believed to contribute to microbial virulence by destroying host tissues and digesting immunologically important proteins, such as antibodies and complement factors (Hube & Naglik, 2001; Monod et al., 2002; Naglik et al., 2003). Using F. pedrosoi conidial cells, it was verified that the secreted aspartyl peptidase was capable of hydrolyzing important human serum proteins including albumin, nonimmune IgG, fibrinogen and hemoglobin. Key extracellular matrix components such as laminin, fibronectin and type IV collagen were also cleaved by the conidial aspartyl peptidase (Palmeira, 2006). Consequently, we speculate that the

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extracellular proteolytic enzyme produced by F. pedrosoi conidial cells could have a role in the dissemination of this fungus inside the host. Proteolytic enzymes have been consistently associated with fungal morphogenesis and virulence of several human pathogens including C. albicans (reviewed by Naglik et al., 2003), A. fumigatus (Monod et al., 1999) and P. brasiliensis (Singh, 2000). In F. pedrosoi, the morphological transition of conidia to mycelia is considered to be an essential step during the fungal life cycle. For this reason, the extracellular proteolytic profile of mycelial forms of F. pedrosoi was also analyzed. Supernatant collected from mycelia cultivated in chemically defined conditions also presented an aspartyl proteolytic activity; however, presenting optimal hydrolytic activity at pH 2.0 (Palmeira et al., 2006b). These results could suggest a differential expression of extracellular aspartyl peptidases that is directly dependent on the F. pedrosoi morphological stage, which could have an impact on the pathogenesis of chromoblastomycosis. Such refined control of enzyme activity has been described in the secreted aspartyl peptidase (Sap) family produced by C. albicans. For example, Sap2 and 3 acts mainly at pH values around 4.0 and 2.0, respectively, whereas Saps4-6 are more efficient at physiological pHs (reviewed by Naglik et al., 2003). This provides C. albicans with a wide range of proteolytic activities, a property that may be essential for the specific adaptation of individual Saps to different host environments (reviewed by Hube, 2000; Naglik et al., 2003). Interestingly, a recent human F. pedrosoi isolate (Mag strain) produced levels of secreted aspartyl proteolytic activity around fourfold higher than that observed in a laboratory-adapted strain (5VPL), suggesting that the expression of secreted aspartyl-type peptidases may be stimulated by interaction with the host (Palmeira et al., 2006b). Aspartyl peptidases are widely distributed in nature and participate in the control of several biological processes (Dash et al., 2003), including infectious processes (Coombs et al., 2001; Casolari et al., 2004; Pozio & Morales, 2005). In this context, several inhibitors of various aspartyl peptidases, as well as the biochemical and biophysical properties of the enzymes, have been investigated in the last decade (reviewed by Dash et al., 2003). The most prominent examples in this regard are the HIV proteolytic inhibitors used during highly active antiretroviral therapy (HAART), which mimic endogenous peptides and thereby block the activity of the HIV aspartyl peptidase (Sepkowitz, 1998). Interestingly, HIV aspartyl peptidase inhibitors have a direct effect over C. albicans Saps, whose involvement in pathogenicity has been partially elucidated (Palella et al., 1998; Cassone et al., 1999; Naglik et al., 2003). The effect of saquinavir, ritonavir, indinavir and nelfinavir, four distinct HIV aspartyl peptidase inhibitors commonly used in the HAART, on the secreted proteolytic activity of F. pedrosoi cells was recently evaluated (Palmeira, 2006; Palmeira et al., 2006b). These compounds inhibited the extracellular aspartyl proteolytic activity produced by both conidial and

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mycelial forms in a dose-dependent manner. In general, the inhibitory activities of these four HIV peptidase inhibitors were comparable to those exerted by pepstatin A, a prototypal inhibitor of aspartyl peptidases. All inhibitors acted in the micromolar range, in contrast to the nanomolar range that is necessary to inhibit the HIV enzymes. This observation probably reflects a lower affinity of this drug for the fungal aspartyl peptidases, in comparison with their high affinity for HIV peptidases (Kohl et al., 1988). Peptidases are apparently key regulators of the in vitro growth in F. pedrosoi (Palmeira, 2006; Palmeira et al., 2006b). Pepstatin A demonstrated a fungicidal activity against conidia in a dose-dependent fashion. Accordingly, the HIV aspartyl peptidase inhibitors mentioned above also abrogated the growth of conidial forms as well as its transformation to mycelial cells (Fig. 8). Drug-treated fungi presented irreversible ultrastructural alterations, including invagination of the plasmatic membrane and cell wall damage. Furthermore, the aspartyl peptidase inhibitors drastically reduced the indices of adhesion and endocytosis during the interaction of conidia with different animal cell lineages, including epithelial cells, fibroblasts and macrophages, suggesting the relevance of aspartyl-type peptidases in the fungihost cells interaction (Palmeira, 2006).

Figure 8. Pepstatin A, a n inhibitor of aspartyl peptidases, controls the conidiummycelium transition in Fonsecaea pedrosoi. Conidial cells were grown for 7 days in the absence () or in the presence of 10 M pepstatin A (+) and analyzed by light microscopy. A clear inhibition of fungal growth and differentiation is observed. Scale bars: 10 m.

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Conclusions and remarks In the last two decades our understanding about chromoblastomycosis and its major etiological agent, F. pedrosoi, has advanced considerably. Nonetheless, a standard procedure to treat chromoblastomycosis is still not available, although several studies indicate that the disease is more prevalent than initially thought. The capacity to differentiate into sclerotic cells makes F. pedrosoi an exceptional model in cell biology studies. As discussed here, synthetic drugs and environmental conditions can induce the formation of sclerotic cells, but physiological molecules such as Ca+2 and PAF are also able to induce this differentiation process. Dimorphism apparently includes the expression of other biochemical markers such as cell wall phosphatases, ATPases and glycosphingolipids. We believe that the knowledge of the dimorphic mechanisms of F. pedrosoi can contribute with the understanding of how fungal cells undergo through morphological transitions, but can also reveal new therapeutic targets of chromoblastomycosis. The pathogenesis of F. pedrosoi is apparently multifactorial and involves surface/secreted molecules of different chemical natures. The expression of F. pedrosoi cell wall adhesins are up regulated when the temperatures change from 28C (natural environment) to 37C (mammalian parasitism), increasing the invasive ability of the fungus. Secreted peptidases are also putatively involved not only with fungal metabolism but also with its dissemination inside the host. The expression and/or activity of these enzymes are modulated by environmental conditions, including substrate availability. Physiological substrates for F. pedrosoi peptidases include serum and extracellular matrix proteins. Melanin, which is constitutively expressed by F. pedrosoi, apparently has ambiguous functions. Although it helps fungal cells to survive inside macrophage, it induces the production of opsonic, antifungal antibodies and activates different phagocytes. Other structures with potential roles in F. pedrosoi pathogenesis include glycosphingolipids, sialic acids and the enzyme sialidase. The knowledge of F. pedrosoi cell biology and biochemistry seems to be of great potential for the future treatment of chromoblastomycosis. As summarized above, peptidases, glycosphingolipids and melanin are promising targets for the action of new antifungal drugs. Synthetic drugs or natural products affecting the expression and biological functions of these molecules could, therefore, represent new alternatives to control chromoblastomycosis in human patients.
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CHROMOBLASTOMYCOSIS: CLINICAL PRESENTATION AND MANAGEMENT


SOURCE: http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2230.2009.03415.x/full AUTHOR: M. Ameen DATE PUBLISHED: December 2009 in Clinical and Experimental Dermatology Volume 34, Issue 8, pages 849854

Summary Chromoblastomycosis is an important subcutaneous mycotic infection that is endemic worldwide but more common in the tropics. It is caused by the traumatic inoculation of the skin with pigmented saprophytic moulds, and the principal infective species are Fonsecaea pedrosoi and Cladophialophora carrionii. Although chromoblastomycosis is not known to be fatal, it is characteristically chronic, and can be complicated by lymphatic damage and neoplastic transformation. It is one of the more difficult-to-treat mycoses, and a variety of antifungal regimens often combined with various physical treatments can be used.

Introduction Chromoblastomycosis, also known as chromomycosis, is a chronic fungal infection of cutaneous and subcutaneous tissues. It was first described in 1920 in Brazil by Pedroso and Gomes1 as a verrucous dermatitis of infectious origin. It is caused by pigmented (dematiaceous) fungi that are prevalent worldwide. However, the disease is most common in tropical and subtropical areas of Latin America, the Caribbean, Africa and Asia, with particular foci in the Amazon region of Brazil, Madagascar, Mexico, the Dominican Republic, Venezuela and India. It is also regularly reported in Japan and Australia.2,3 Aetiology and clinical presentation The most common aetiological agents are of the genera Fonsecaea, Phialophora and Cladophialophora, which are found as saprophytes in soil and plants. Fonsecaea pedrosoi is the commonest agent found in tropical forests characterized by high rainfall, such as the Amazon, as well as temperate regions of Latin America (Table 1).4Cladophialophora carrionii is the most important agent in dry countries and desert regions such as Australia, South Africa and Cuba.2

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Table 1. Principal aetiological species of chromoblastomycosis.

Fonsecaea pedrosoi

Most common agent worldwide (7090%). Responsible for majority of cases in Brazil, Mexico, Northern Madagascar and Japan Predominant agent in dry endemic regions. Main species in Australia, Southern Madagascar, South Africa and Cuba Also an important phaeohyphomycosis cause of

Cladophialophora carrionii

Phialophora verrucosa Fonsecaea compacta, Exophiala jeanselmei, Exophiala spinifera, Rhinocladiella aquaspersa

Rare agents of chromoblastomycosis

Infection arises from the traumatic implantation of these fungi into the skin. Consequently, the disease is more frequently seen in agriculturalists, labourers and those who are barefoot. It has a male predominance, and can affect any age but is rarely seen before adolescence. It most commonly affects the limbs, particularly the lower legs and feet. Unusual sites of infection such as the genitalia and nose have also been reported.5 Chromoblastomycosis is characteristically slow to develop. Early lesions may resemble a dermatophyte infection or begin as a papule. These then progress into plaques (Fig. 1a), nodules, or verrucous and exophytic lesions. After many years, lesions may develop into tumoral, cauliflower-like masses (Fig. 1b) or may extend centripetally, leaving central areas of scarring. Lesions may also heal leaving sclerotic plaques or keloids (Fig. 1c). Disease is usually localized but satellite lesions can develop from autoinoculation through scratching and from lymphatic dissemination. Skin lesions may be mildly pruritic or asymptomatic unless there are complications, which include ulceration, secondary bacterial infection, and lymphoedema (Fig. 1d). Rarely, chronic lesions may be complicated by malignant transformation into squamous cell carcinoma. Chromoblastomycosis remains confined to the subcutaneous fat and does not invade underlying muscle or bone except in cases of immunosuppression.2,6 Extracutaneous haematogenous dissemination to regional lymph nodes, lungs and brain has rarely been reported,5 although some authors believe that these cases may actually represent phaeohyphomycosis.7 The principal differential diagnosis of verrucous-like lesions of chromoblastomycosis includes verrucous variants of leishmaniasis and tuberculosis, sporotrichosis and verrucous carcinoma.2

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(a)

(c)

(b)

(d)

Figure 1. (a) Plaque-like lesion overlying elbow demonstrating the characteristic black dots; (b) tumoral and satellite lesions on the upper arm; (c) healing of lesions with antifungal therapy leaving sclerotic scarring; (d) fibrosis and episodes of bacterial infection cause lymphatic damage and lymphoedema, apparent in this case after resolution of lesions with prolonged drug treatment.

Diagnosis Direct microscopy in 10% potassium hydroxide of scrapings taken from the lesion reveals round, brown, thick-walled, multiseptate sclerotic cells that are pathognomonic of chromoblastomycosis, irrespective of the causative species (Fig. 2a). These are known as muriform cells, Medlar bodies or copper pennies, and they divide by binary fission. Specimens are more likely to yield a positive result if they include the black dots visible on the surface of the lesion, which represent transepidermal elimination of the fungal agents (Fig. 1a). Culture enables species identification (Fig. 2b) but given that the causative agents are characteristically slow-growing fungi, it may be

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inconclusive due to poor morphological differentiation. PCR assays have been developed for the identification of Fonsecaea species andC. carrionii.8,9 Histopathological examination shows a granulomatous process and marked epithelial hyperplasia with transepithelial elimination and the presence of muriform cells (Fig. 2c). Serological tests such as ELISA can be useful in evaluating response to treatment, but like PCR, are not widely available in most endemic settings.10 (a) (b)

(c)

Figure 2. (a) Direct examination of skin scrapings in a potassium hydroxide mount, showing characteristic brown, multiseptate Medlar bodies; (b) culture of Fonsecaea pedrosoi; (c) characteristic Medlar bodies or copper pennies within a mixed granulomatous and lymphohistiocytic infiltrate with neutrophils (haematoxylin and eosin, original magnification 400).

Treatment Chromoblastomycosis is associated with low cure rates and high relapse rates, particularly in chronic and extensive disease. Treatment choice and outcome depend on the aetiological agent, the size and extent of the lesions, the clinical topography, and the presence of complications (dermal fibrosis and oedema may reduce antifungal levels in tissue).7 Despite being the most common aetiological agent,F. pedrosoi appears to be less sensitive to antifungal therapy

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than either C. carrionii or Phialophora verrucosa.3,11 In patients who present early with small lesions, the goal of treatment should be cure. However, in the case of extensive disease, even years of continuous drug treatment may fail to clear the lesions, and a more realistic goal in such cases may be disease reduction and control in order to prevent complications. Clinical cure may be defined as complete resolution of lesions, usually leaving sclerotic scarring. Mycological cure is defined as negative results on microscopy and culture. Management consists of long courses of antifungal chemotherapy often combined with physical treatments such as surgery, cryotherapy and thermotherapy (Table 2). Studies report highly variable rates of clinical and mycological cure, ranging from 15% to 80%.11 Small and localized lesions may be surgically excised with wide margins. Antifungals are often given before surgery to decrease the size of the lesion, and continued afterwards to prevent any risk of relapse. Curettage and electrodessication is not recommended because it may promote lymphatic spread.11 Cryotherapy with liquid nitrogen, and thermotherapy (applying local heat to produce controlled temperatures of 4245 C, which inhibits fungal growth) using a variety of methods including benzene pocket warmers and pocket handkerchief-type warmers, carry minimal risks of adverse effects and are relatively inexpensive treatment options, but they are only suitable for solitary and limited lesions. Approximately 67 treatments with cryotherapy are usually required.12,13 Thermotherapy is less commonly used, and the few published reports of its use originate from Japan. It requires daily application of heat directly onto the lesions for several hours for 26 months.14,15 The antifungals that have shown greatest efficacy are itraconazole (200400 mg daily) and terbinafine (5001000 mg daily) given for at least 612 months, preferably at the higher doses if tolerated.6,16 19 Both drugs have shown high in vitro activity against the causative agents of chromoblastomycosis.20,21 Pulse itraconazole (400 mg daily for 1 week every month) has been shown to be as effective as the conventional daily regimen, reducing the total drug dosage required and possibly increasing compliance.22,23 There are fewer studies evaluating treatment with terbinafine, and the studies that do exist had small patient numbers. However, they showed high tolerability and efficacy of terbinafine, especially against F. pedrosoi, even in imidazole-refractory cases.16 Dual therapy with itraconazole and terbinafine is recommended as the drugs appear to act synergistically and are well-tolerated in combination. However, as yet there are few studies describing the use of this regimen.24,25
Table 2. Therapeutic regimens for chromoblastomycosis.

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Treatment regimen 1.

Species (n = number of cases)

Therapy response

Reference

C., Cladophialophora; F., Fonsecaea; P., Phialophora.

F. pedrosoi(n = 46)
Itraconazole 200 400 mg once daily

31% cured improved

and

57%

Bonifaz et al.6

F. pedrosoi(n = 30)

Clinical and mycological cure in: 90% with mild/moderateQueirozdisease, 44% with severeTelleset al.17 disease Cure after 12 months (n = 4),Ungpakorn & > 50% improvement (n = 2) Reangchainam23 Marked clinical improvement 83% Esterre et al.16 after 12 months

Itraconazole pulse therapy 400 mg once F. pedrosoi(n = 6) daily for 1 week monthly

F. pedrosoi(n = 37) C.after 24 months; carrionii (n = 9) mycological cure


Terbinafine once daily 500 mg

F. pedrosoi(n = 3) P.Clinical and mycological cure Bonifaz et al.18 verrucosa (n = 1) after mean of 7 months F. pedrosoi(n = 2) C.Clinical cure after 48 months Xibao et al.19 carrionii (n = 2) (37.560 g cumulative dose)

Itraconazole (200 400 mg once daily) and terbinafine (250F. pedrosoi(n = 4) 1000 mg once daily) combination therapy Posaconazole once daily 800 mg

Clinical and mycological cure in 3 cases after mean ofGupta et al.24 6 months Cure in 4 cases after mean of Negroni et al.28 269 days Cure after mean cryotherapy sessions of 3
Bonifaz et al.12

F. pedrosoi(n = 6) F. pedrosoi(n = 4)

Cryotherapy

F. pedrosoi(n = 22)

Clinical and mycological cure in 41%, and clinical cure in Castro et al.13 further 36% (mean of 7 sessions, range 122)

Of the other antifungals, ketoconazole is not recommended as prolonged treatment at high doses is associated with toxicity. Fluconazole is also not recommended as in vitro studies have shown that it has little activity against black fungi.21 Flucytosine (converted into 5-fluorouracil in fungal cells) was an early treatment that showed some degree of efficacy, but it is associated with a high risk of developing resistance, and is hepatotoxic and myelotoxic.26 With the emergence of newer antifungals it is rarely used nowadays except for resistant cases. Amphotericin B monotherapy is ineffective, and even in combination with other antifungals, results are generally poor, but a

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combination of amphotericin B and flucytosine has shown efficacy, with in vitro studies indicating synergistic activity between the two drugs.27 The new second-generation triazoles such as posaconazole and voriconazole are promising drugs in the management of deep cutaneous mycoses, but experience is limited by their prohibitive high costs in endemic settings. To date, there is only a single report of the use of posaconazole, in which five of six cases refractory to standard antifungal therapies achieved cure.28 Conclusion Treating chromoblastomycosis remains a challenge. Response to treatment depends on the infective agent, and the extent and chronicity of disease. Although long-term courses of antifungals remain the cornerstone of treatment, published studies to date have consisted only of single-centre case series with limited numbers. There is a need for comparative trials and further evaluation of combination antifungal and pulse itraconazole therapies in particular. In vitro studies have demonstrated synergy between itraconazole and terbinafine,24,29 and pulse regimens may circumvent any risks of toxicity associated with long-term treatment. Chemotherapy may be successfully combined with thermotherapy, either cryotherapy or heat therapy. Cryotherapy is advocated especially for small lesions. Surgical excision of small lesions must always be combined with chemotherapy both before and after surgery. In the management of extensive disease, a more realistic goal may be to control disease activity and contain further spread, thereby minimizing the risks of complications such as secondary bacterial infection, lymphatic damage and neoplastic transformation. Learning points Chromoblastomycosis is a chronic, deep cutaneous mycosis endemic mainly in the tropics and subtropics. It is caused by traumatic inoculation of saprophytic fungi, principally of the genera Fonsecaea, Phialophora and

Cladophialophora.

Anybody site may be affected but it most commonly affects the distal limbs. Clinically, chromoblastomycosis presents as plaques or verrucous nodules that are characteristically very slow-growing. Further lesions develop secondary to traumatic autoinoculation or lymphatic spread.

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Diagnosis is usually made easily by direct microscopy, which reveals the characteristic muriform cells. Culture enables species identification. Long courses of oral antifungals are the cornerstone of treatment. High-dose itraconazole (200400 mg daily) and/or terbinafine (5001000 mg daily) are recommended. There are preliminary data for the efficacy of posaconazole, one of the novel triazoles. Chemotherapy is usually combined with surgical excision of small lesions and/or cryosurgery or thermotherapy. Complications include secondary bacterial infection, ulceration, lymphoedema, and the development of squamous cell carcinoma in chronic lesions.

REFERENCES 1. Pedroso A, Gomes JM. Sobre quatro casos de dermatite verrucosa produzida por Phialophora verrucosa [in Portuguese]. An Paul Med Cir 1920; 11: 531. 2. Lupi O, Tyring SK, McGinnis MR. Tropical dermatology: fungal tropical diseases. J Am Acad Dermatol 2005; 53: 93151. 3. Lpez Martnez R, Mndez Tovar LJ. Chromoblastomycosis. Clin Dermatol 2007; 25: 18894. 4. Sanche Se Sutton DA, Rinaldi MG. Dematiaceous fungi. In: Clinical Mycology (AnaissieEJ, McGinnisMR, PfallerMA, eds). New York: Churchill-Livingstone, 2003: 32551. 5. Sharma NL, Sharma RC, Grover PS et al. Chromoblastomycosis in India. Int J Dermatol 2002; 41: 1620. 6. Bonifaz A, Carrasco-Gerard E, Sal A. Chromoblastomycosis; clinical and mycologic experience of 51 cases. Mycoses 2001; 44:17. 7. Esterre P, Queiroz-Telles F. Management of chromoblastomycosis: novel perspectives. Curr Opin Infect Dis 2006; 19: 14852. 8. Abliz P, Fukushima K, Takizawa K, Nishimura K. Specific oligonucleotide primers for identification of Cladophialophora carrionii, a causative agent of chromoblastomycosis. J Clin Microbiol 2004; 42: 4047. 9. De Andrade TS, Cury AE, De Castro LG et al. Rapid identification of Fonsecaea by duplex polymerase chain reaction in isolates from patients with chromoblastomycosis. Diagn Microbiol Infect Dis 2007; 57: 26772. 10. Oberto-Perdign L, Romero H, Prez-Blanco M, Apitz-Castro R. An ELISA test for the study of the therapeutic evolution of chromoblastomycosis by Cladophialophora carrionii in the endemic area of Falcon State, Venezuela [in Spanish]. Rev Iberoam Micol 2005; 22: 3943. 11. Bonifaz A, Paredes-Solis V, Saul A. Treating chromoblastomycosis with systemic antifungals. Expert Opin Pharmacother 2004; 5:24754. 12. Bonifaz A, Martinez-Soto E, Carrasco-Gerard E, Peniche J. Treatment of chromoblastomycosis with itraconazole, cryosurgery, and a combination of both. Int J Dermatol 1997; 36: 5427. 13. Castro LG, Pimentel ER, Lacaz CS. Treatment of chromomycosis by cryosurgery with liquid nitrogen: 15 years experience. Int J Dermatol 2003; 42: 40812. 14. Tagami H, Ginoza M, Imaizumi S, Urano-Suehisa S. Successful treatment of chromoblastomycosis with topical heat therapy. J Am Acad Dermatol 1984; 10: 61519. 15. Hiruma M, Kawada A, Yoshida M, Kouya M. Hyperthermic treatment of chromomycosis with disposable chemical pocket warmers. Report of a successfully treated case, with a review of the literature. Mycopathologia 1993; 122: 10714. 16. Esterre P, Inzan CK, Ramarcel ER et al. Treatment of chromomycosis with terbinafine: preliminary results of an open pilot study.Br J Dermatol 1996; 134 (Suppl. 46): 336. 17. Queiroz-Telles F, McGinnis MR, Salkin I, Graybill JR. Subcutaneous mycoses. Infect Dis Clin North Am 2003; 17: 5985. 18. Bonifaz A, Sal A, Paredes-Solis V et al. Treatment of chromoblastomycosis with terbinafine: experience with four cases. J Dermatolog Treat 2005; 16: 4751. 19. Xibao Z, Changxing L, Quan L, Yuqing H. Treatment of chromoblastomycosis with terbinafine: a report of four cases. J Dermatolog Treat 2005; 16: 1214. 20. McGinnis MR, Pasarell L. In vitro evaluation of terbinafine and itraconazole against dematiaceous fungi. Med Mycol 1998; 36:2436.

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21. Caligiorne RB, Resende MA, Melillo PH et al. In vitro susceptibility of chromoblastomycosis and 22. Kumarasinghe SP, Kumarasinghe MP. Itraconazole pulse therapy in chromoblastomycosis. Eur J 23. Ungpakorn R, Reangchainam S. Pulse itraconazole 400 mg daily in the treatment of
chromoblastomycosis. Clin Exp Dermatol2006; 31: 2457. phaeohyphomycosis agents to antifungal drugs. Med Mycol 1999; 37: 4059.

Dermatol 2000; 10: 2202.

24. Gupta AK, Taborda PR, Sanzovo AD. Alternate week and combination itraconazole and terbinafine 25. 26. 27. 28. 29.

therapy for chromoblastomycosis caused by Fonsecaea pedrosoi in Brazil. Med Mycol 2002; 40: 529 34. Zhang J, Xi L, Lu C et al. Successful treatment for chromoblastomycosis caused by Fonsecaea monophora: a report of three cases in Guangdong, China. Mycoses 2009; 52: 17681. Lopez CF, Alvarenga RJ, Cisalpino EO et al. Six years experience in treatment of chromomycosis with 5-fluorocytosine. Int J Dermatol 1978; 17: 41418. Park SG, Oh SH, Suh SB et al. A case of chromoblastomycosis with an unusual clinical manifestation caused by Phialophora verrucosa on an unexposed area: treatment with a combination of amphotericin B and 5-flucytosine. Br J Dermatol 2005; 152:5604. Negroni R, Tobon A, Bustamante B et al. Posaconazole treatment of refractory eumycetoma and chromoblastomycosis. Rev Inst Med Trop Sao Paulo 2005; 47: 33946. Yu J, Li R, Zhang M et al. In vitro interaction of terbinafine with itraconazole and amphotericin B against fungi causing chromoblastomycosis in China. Med Mycol 2008; 46: 7457.

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Case Report

LYMPHANGITIC CHROMOBLASTOMYCOSIS
SOURCE: http://www.ijdvl.com/text.asp?2006/72/6/443/29342 AUTHORS: K. Muhammed1, G. Nandakumar1, K.K. Asokan2, P. Vimi1 - 1Department of Dermatology and Venereology, Medical College, Kozhikode, Kerala, India 2Department of Microbiology, Medical College, Kozhikode, Kerala, India DATE PUBLISHED: Year 2006 in Indian J Dermatol Venereol Leprol 2006;72:443-5 Abstract Chromoblastomycosis (CM), a chronic subcutaneous mycosis, is caused by several dematiaceous fungi, the most common being Fonsecaea pedrosoi . It usually occurs in the lower extremities following traumatic implantation of the organisms. We are reporting a case of chromoblastomycosis on the right lower limb in a sporotrichoid pattern caused by F. pedrosoi . The pattern was probably due to lymphatic spread that seems to be one of the rare presentations. The histopathology showed typical muriform or medlar bodies both intracellularly and extracellularly within the granuloma. Culture revealed sporulating anisms ( Cladosporium and Rhinocladiella type) by a combination method, characteristic of F. pedrosoi . Our case responded well to itraconazole.

Introduction Chromoblastomycosis (CM) is a chronic granulomatous mycotic infection of the skin and subcutaneous tissue caused by pigmented fungi, the most common being F. pedrosoi.1 It typically occurs on the exposed surfaces of the lower leg following traumatic implantation of the organisms.2,3,4 The lesions can involve other sites either by direct spread, autoinoculation5 or by hematogenous spread.6 The lymphatics may also play a role in disseminating the infection.1,7,8 We are reporting a case of CM caused by F. pedrosoi with an lymphatic spread. Case report A 40-year-old male agricultural worker of low socioeconomic status weighing 43 kg presented with a nonhealing ulcer on the right big toe and multiple nodules over the anterior aspect of the right leg and foot [Figure-1], of one-year duration arranged in a linear fashion. He initially developed a nontender nodule on the dorsum of the right big toe which later got ulcerated. Subsequently, after three months, about five nodules started to appear in a linear fashion on the right foot and leg of size ranging from 1 to 2.5 cm in diameter with verrucous surface. He gave history of trauma at the site of initial lesion on the big toe. There was no regional lymphadenopathy.

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His baseline blood and urine examination, renal and liver function tests were normal. Scrapings from the lesion and tissue smear did not demonstrate any organisms. Histopathology of the lesion showed pseudo-carcinomatous hyperplasia with neutrophilic micro abscesses in the epidermis. The subepidermal region showed granuloma composed of pithelioid cells and Langhan's giant cells. Muriform bodies were present intracellularly [Figure-2] as well as extracellularly. Fungal culture showed velvety dark gray to brown colonies with black pigment on the reverse [Figure-3]. Microscopic examination of the cultured organism showed sporulation by a combination method, with acrogenous conidia with short branching chains (Cladosporium type) and oval conidia at irregular positions on tips and sides of conidiospores ( Rhinocladiella type), characteristic of F. pedrosoi [Figure-4]. This confirmed the diagnosis of chromoblastomycosis. We treated the patient with oral itraconazole 100 mg daily. Skin lesions had regressed by 80% after six months of therapy. However, the patient died due to ischemic heart disease.

Figure 1. Nonhealing ulcer on the right big toe and multiple nodules over the anterior aspect of the right foot and leg

Figure 2. Extracellular and intracellular muriform bodies (x400)

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Figure 3. Fungal culture showing velvety dark gray to brown colonies with black reverse

Figure 4. Microscopic examination (x100) of cultured organisms showing acrogenous conidia with short branching chains (cladosporium type) and oval conidia on tips and sides of conidiospores (rhinocladiella type)

Discussion CM is caused by several species of dematiaceous fungi and is usually confined to one of the lower extremities and affects only skin and subcutaneous tissue, though the draining lymph glands may participate in the pathological process.8 The CM lesion may be verrucous with central scarring (tuberculoid), severe scarring with a serpiginous border (syphiloid), scaly (psoriasifiorm) or indurated with fistula formation (mycetomatoid).9 Carrion described five morphologic types consisting of nodular, tumorous, verrucous, plaque and cicatricial lesions.9 The diagnosis of CM should be confirmed either by direct microscopy of the scrapings from the lesion in 20% KOH when thick-walled dark brown tissue forms of the fungus (fumagoid bodies/ muriform bodies/ copper penny bodies) are seen; by histological examination of a biopsy specimen with granulomatous reaction and spores; or by culture of scrapings or biopsy material.9 Usually, histopathology reveals acanthosis and may demonstrate pseudo-carcinomatous hyperplasia. Neutrophils and giant cells may be seen infiltrating the epidermis with occasional formation of microabscesses. The dermis reveals a granulomatous tissue reaction with a mixed focal or diffuse inflammatory infiltrate consisting of lymphocytes, neutrophils, monocytes, plasma cells, eosinophils and giant cells of the foreign body and Langhan's type. The muriform bodies may be seen both intracellularly and extracellularly.

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In our case both histopathological and mycological studies confirmed the diagnosis of CM caused by F. pedrosoi . However, the clinical presentation was atypical. The patient developed the lesion in a sporotrichoid pattern. Though CM can have a lymphatic mode of spread with satellite lesions, presentation in a sporotrichoid fashion is very rare. Carrion and Koppisch reported a case with well-documented involvement of the subcutaneous, lymphatic and muscular tissue.3 The involvement of lymphatic vessels (sporotrichosis-like) was observed by Fraga, Almaida, Aleixo and Palominos.6 After this, no recent reports of sporotrichoid type of CM are available,3,4 except the description by Bopp et al .10 in which a vegetating plaque in the foot associated with inflammatory nodules on the leg was shown. Our patient's response to itraconazole (ITZ) was good. Hyle reported two cases treated successfully with 100 mg of daily ITZ therapy.11 Yu treated CM with 100 mg ITZ for 15 months with complete mycological and clinical recovery.12 Even though the current regimen of therapy is with 200-400 mg of ITZ alone or in combination with cryosurgery, considering the low body weight and socioeconomic status, we started the patient on 100 mg of ITZ with good response. The present case is a good reminder that CM should be kept at the back of the mind in the differential diagnosis of sporotrichoid lesions.
REFERENCES 1. 2. Milam CP, Fenske NA. Chromoblastomycosis. Dermatol Clin 1989;7:219-25. Wortman PD. Concurrent chromoblastomycosis caused by Fonsecaea pedrosoi and actinomycetoma caused by Nocardia braseliensis. J Am Acad Dermatol 1995;32:390-2. 3. Lupi O, Tyring SK, McGinnis MR. Tropical dermatology: Fungal tropical diseases. J Am Acad Dermatol 2005;53:931-51. 4. Minnotto R, Bernardi CD, Mallmann LF, Edelweiss MI, Scrofernker ML. Chromoblastomycosis: A review of 100 cases in the state of Rio Grande do Sul, Brazil. J Am Acad Dermatol 2001;44:585-92. 5. Rippon JW. The subcutaneous mycosis. In : Rippon JW, editor. Medical Mycology. Philadelphia: WB Saunders; 1974. p. 229-47. 6. Azulay RD, Serraya J. Hematogenous dissemination in chromoblastomycosis: Report of a generalized case. Arch Dematol 1967;95:57-60. 7. Derbes VJ, Friedman I. Chromoblastomycosis. Dermatol Tropica 1964;3:201. 8. Carrion AL, Koppisch E. Observation of dermatomycosis in Puerto Rico. J Pub Health Trop Med 1933;9:169-93. 9. Vollum DI. Chromomycosis: A review. Br J Dermatol 1977;96: 454-8. 10. Bopp C, Vetoratto, Boreli D. Chromoblastomycosis caused by new species: Taeniolella boppi . Med Cutan Ibero Lat Am 1983;11:221-6. 11. Hyle T. Treatment of chromoblastomycosis with itraconazole. Br J Dermatol 1985;112:728-9. 12. Yu R. Successful treatment of chromoblastomycosis with itraconazole. Mycoses 1995;38:79-83.

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Case Report

SOURCE: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2917698/?tool=pubmed AUTHORS: Young-Min Son, M.D., Hong-Kyu Kang, M.D., So-Young Na, M.D., HyeYoung Lee, M.D., Jin-Ok Baek, M.D., Jong-Rok Lee, M.D., Joo-Young Roh, M.D.,* and Yiel-Hea Seo, M.D.# - Department of Dermatology, Gachon University of Medicine and Science, Gil Medical Center, Incheon, Korea, #Department of Laboratory Medicine, Gachon University of Medicine and Science, Gil Medical Center, Incheon, Korea, *Corresponding author. DATE PUBLISHED: December 9, 2009. Abstract Chromoblastomycosis is a chronic fungal disease of the skin and subcutaneous tissues caused by a group of dematiaceous (black) fungi. The most common etiologic agents are Fonsecaea pedrosoi and Cladophialophora carrionii, both of which can be isolated from plant debris. The infection usually follows traumatic inoculation by a penetrating thorn or splinter wound. Several months after the injury, painless papules or nodules appear on the affected area; these papules then progress to scaly and verrucose plaques. We report a case of chromoblastomycosis caused by Phialophora richardsiae, which has been rarely associated with chromoblastomycosis. The case involved a 43-year-old male, who for the past 2 months had noted an erythematous, pustulous plaque that was somewhat dark brown in color on his right shin; the plaque also had intermittent purulent discharge and crust formation. On histopathological examination, chronic granulomatous inflammation and sclerotic cells were seen. The tissue fungus culture grew out the typical black fungi of P. richardsiae, which was confirmed by polymerase chain reaction. The patient has been treated with a combination of terbinafine and itraconazole for 3 months with a good clinical response.

CHROMOBLASTOMYCOSIS CAUSED BY PHIALOPHORA RICHARDSIAE

INTRODUCTION Chromoblastomycosis, also called chromomycosis, is a subcutaneous chronic mycosis caused by a group of dematiaceous (black) fungi1,2. It occurs worldwide, but is most commonly seen in tropical and subtropical regions2-4. The skin lesions are polymorphic and must be differentiated from those associated with other clinical conditions5-7. Chromoblastomycosis has been noted and thus well documented in patients who are immunosuppressed after renal transplantation or who are otherwise on corticosteroid therapy5,7. An increased correlation of chromoblastomycosis with malignant diseases has also been noted6. Five causative fungi have been identified:Fonsecaea pedrosoi is the most prevalent, and Phialophora verrucosa, Cladosporium carrionii, Fonsecaea compacta, and Rhinocladiella aquaspersa also occur in descending order of frequency8-10. Less frequently, chromoblastomycosis is caused by Cladophialophora

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arxii, Exophiala spinifera, Exophiala dermatitidis, Exophiala jeanselmei and Wangiella dermatitidis6,7. Phialophora richardsiae is one of the many dematiaceous fungi that

can be isolated from plant material and soil9,11,12. It is a common contaminant of wood and is an industrial nuisance, causing bluing of wood pulp and paper13,14. P. richardsiae and other members of the genus Phialophora can cause disease in humans, most often manifested as an infectious subcutaneous granuloma or abscess15,16. Infection usually occurs on exposed body parts through traumatic skin inoculation with contaminated vegetable matter13-16. Chromoblastomycosis is notoriously difficult to treat, with no one form of treatment being uniformly successful. Small lesions can be removed with wide and deep excision. Cryotherapy, topical heat therapy, systemic medications, and a combination of the above have been reported to be effective. We report a 43-year-old man with chromoblastomycosis caused by P. richardsiae who was successfully treated with a combination of terbinafine and itraconazole. CASE REPORT A 43-year-old male construction worker presented with a 2-month history of a slowly enlarging lesion on his right shin. The lesion had the appearance of an erythematosquamous plaque, with a slightly verrucous appearance and irregular shape. The patient's symptoms included mild itching. The lesion first appeared 2 months ago in the form of a papule that gradually extended to its current shape and had purulent drainage. Based on the clinical findings, the presumptive diagnoses of sporotrichosis and an atypical mycobacterial infection were made. On examination, the right shin showed a dark brownish, hyperkeratotic, erythematous, fluctuating plaque with focal purulent discharge, crust formation, and several conglomerated firm nodules (Fig. 1A). The patient's general condition was otherwise good. A skin biopsy was obtained from the verrucous plaque, and tissue fungus and open pus cultures were established. The histopathologic findings revealed hyperkeratosis, pseudoepitheliomatous hyperplasia, and a mixed granulomatous inflammatory cell infiltrate consisting of lymphocytes, histiocytes and multinucleated giant cells with sclerotic bodies in the dermis (Fig. 2). There was a focal micro-abscess in the deep dermis and periodic acid-Schiff (PAS) staining revealed a fungal colony in the micro-abscess (Fig. 2A). Tissue culture on Sabouraud's dextrose agar at room temperature showed growth of heaped and fuzzy colonies with a red-to-dark brown surface (Fig. 3). Staining with lactophenol-cotton blue after slide culture on corn meal agar showed a cluster of conidia accumulating at the apex of the phialides with flared collarettes, giving the appearance of a vase of flowers (Fig. 3). The

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significant characteristics were the distinct septate hyphae and many flask-shaped, polymorphic phialides with large, flaring collarettes producing 2 types of conidia (regularly-sized globose or irregularlysized cylindrical phialoconidia). The 2 types of conidia were either separate or present in the same ball. Based on these morphologic features, the fungus was identified as P. richardsiae, which was confirmed by polymerase chain reaction (PCR). Ribosomal DNA internal transcribed spacer (ITS) domains were amplified using the following primers: ITS-1 (TCC GTA GGT GAA CCT GCG G) and ITS-4 (TCC TCC GCT TAT TGA TAT GC). PCR for M. tuberculosis and other atypical mycobacteria using DNA from tissue specimens were negative.

Fig. 1. (A) Hyperkeratotic, erythematous black pustular plaque with crust formation on the right shin. (B) The skin lesion gradually improved after 3 months of combination treatment with itraconazole and terbinafine.

Fig. 2 (A) Skin biopsy specimen showing hyperkeratosis, pseudoepitheliomatous hyperplasia, and a mixed dermal inflammatory cell infiltrate consisting of lymphocytes, and histiocytes (H&E, 40). Inset: Periodic acid-Schiff stain reveals a fungal colony in a micro-abscess (100). (B) Mixed inflammatory cell infiltrate with large, dark-brown, sclerotic bodies (arrow) in the dermis (H&E, 400). Inset: Sclerotic bodies are found within giant cells (H&E, 400).

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Fig. 3 Slide culture and lactophenol-cotton blue stain showed a cluster of conidia accumulating at the apex of the phialides with flared collarettes, giving the appearance of a vase of flowers. Inset: Culture on Sabouraud's dextrose agar at room temperature showed growth of heaped and fuzzy dark brown colored colonies.

Under the diagnosis of chromoblastomycosis caused by P. richardsiae, oral itraconazole (200 mg/day) was administered. Three months after monotherapy, the lesion showed a partial response but continued to have purulent discharge, so terbinafine (500 mg/day) was added. Three months after initiating combination treatment, the patient had a good clinical response (Fig. 1B). DISCUSSION Chromoblastomycosis is a dermal mycosis caused by a group of dematiaceous (black) fungi and characterized by the presence of verrucous-nodular lesions that are usually located on one of the limbs1,6. The infection usually occurs through traumatic skin inoculation, with the majority of lesions occurring on the feet and legs of outdoor workers7,10. The lack of a history of trauma or inoculation injury in most patients may be due to a long latency period from the time of injury to the development of the lesion9,12. Under these circumstances, many patients may not recall a previous injury, especially if the injury was minor6-9. Our patient denied a history of trauma to the area. The only pertinent history was that he had worked on a construction site for several years. The initial lesion of chromoblastomycosis is usually solitary and unilateral, presenting as a small, pink, smooth-surfaced, papular skin lesion4,6. The papules gradually increase over a few weeks and may have a scaly surface4,5. With time, the initial lesion may evolve into several types of skin lesions, leading to a polymorphic clinical

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appearance (nodular, tumorous, plaque, cicatricial and verrucous types)7,8. Histologically, the tissue response in chromoblastomycosis is not specific and may be similar to other deep mycoses6,8. Hyperkeratosis and pseudoepitheliomatous hyperplasia are the main features within the stratum corneum and epidermis3,8. Several micro-abscesses, with or without pigmented fungal elements, may be present in the epidermis3,5. The tissue response to the fungi is typically mixed, consisting of micro-abscesses and a granulomatous response with giant cells11,12. The hallmark of the disease is the presence of muriform cells embedded in the granulomas, as well as a suppurative tissue reaction3,6. Muriform cells have been referred to as sclerotic cells, or as fumagoid, chromo, or Medlar bodies3,6,8. They are likened to "copper pennies" that may lie either singly or in chains or clusters 3,4. The histopathologic specimen of our patient showed a mixed granulomatous inflammatory cell infiltrate and sclerotic cells with multinucleated giant cells. On the mycologic examination, 3 morphologic patterns of sporulation or conidiation exist, which are referred to as Phialophoralike, Cladosporium-like, andRhinocladiella-like6,9,11. The Phialophora type of sporulation gives rise to a flask-shaped phialide, which occurs either at the end or on the side of the hyphae10,11. The conidia are formed and accumulate at the flask's distal end and resemble 'flowers in a vase'9-11. Phialophora richardsiae in our case showed only Phialophora-like sporulation. Reports on P. richardsiae occurring in humans are rather rare14,15. There are about 25 species in the genus Phialophora but only 5 species, i.e., P. verrucosa, P. richardsiae, P. repens, P. parasitica and P. cyanescens, that cause human infection11,12. These are classified according to the growth rate and color of the colonies, the shape of the phialide and collarette, and the color and shape of the conidia10-12. P. richardsiae most closely resembles P. verrucosa, but these species can be differentiated by careful microscopic morphologic examination10,14. The phialides of P. verrucosa are generally short and distinctly urn-shaped, with a broad base and a cup-like collarette10,12,16. In contrast, P. richardsiae has longer, more slender and tapering phialides that, when mature, terminate in a flat, saucer-shaped collarette14,16. The differential diagnosis of chromoblastomycosis is broad and includes many infectious and non-infectious possibilities. There are 3 kinds of diseases caused by black fungi (chromoblastomycosis, phaeohypho-mycosis, and mycetoma)6,7,10. Chromoblastomycosis and phaeohypho-mycosis represent 2 poles of a spectrum of diseases caused by melanized fungi10,12. Clinically, the boundaries of this spectrum are unclear, and both infections may be caused by the same

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etiologic agents9-11. Although either immunocompetent and immunosuppressed hosts may present with both types of infections, chromoblastomycosis is usually observed in immunocompetent hosts, while phaeohyphomycosis is not9,10,12. The host immune status may play an important role in the clinical evolution of these diseases. The present case of a P. richardsiae infection was identified as a phaeo-hyphomycosis rather than a chromoblastomycosis, because the infection remained localized and subepidermal14,16. Phaeohyphomycosis does not have sclerotic bodies, which represent a vegetative form of the fungus10,11 Furthermore, hyphal elements, rather than muriform and yeast cells, were found in the affected tissues14-16. Thus, our case is unique because the patient's chromoblastomycosis lesion was caused by a rarely isolated dematiaceous fungus, P. richardsiae. Invasive disease is unusual and occurs only in patients who are severely immunocompromised13,15. Conditions associated with deficient cell-mediated immunity appear to increase the risk of acquiring infection with P. richardsiae, but a normal host may also become infected as in our patient's case13-16. Due to the marked chronicity of the lesions and the well-known resistance of many cases to simple therapies, it is difficult to treat chromoblastomycosis17,18. The preferred treatment is usually surgical excision of small and localized lesions with wide surgical margins to prevent local recurrence. For deep or extensive cutaneous involvement however, surgical treatment is often not feasible 6,18. Prolonged treatment with systemic antifungal agents alone or in combination for deep or extensive lesions provides the best chance of a cure, but antifungal therapy forPhialophora infections has been generally disappointing8,9,12. It is important to know the clinical type of chromoblastomycosis in order to offer a more accurate prognosis for the patient presenting with this difficult-to-treat disease9,19. Despite many reports showing different clinical types with diverse cure rates, there is no study showing a correlation between a specific clinical type and the cure of chromoblastomycosis17-19. Traditional therapy includes continuous dosing with oral itraconazole or terbinafine, and there have been reports that itraconazole is a favorable drug for treatment of chromoblastomycosis19,20. However, in some cases, monotherapy has been ineffective or is associated with a less than acceptable response; there have also been reports on the development of resistance to itraconazole17,18. To achieve a good response rapidly and prevent microbiologic resistance, combination therapy with itraconazole and terbinafine may be effective during the early stages of treatment because of the synergistic effect6,17,18. In our case, itraconazole monotherapy did not result in a clinical response; however, after administration of terbinafine in combination with itraconazole, the skin lesion was markedly improved.

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The lesions of chromoblastomycosis usually respond to surgical excision, and surgery is currently considered the treatment of choice17,20. Our patient had a low income and made his living by working on a construction site, so he declined surgical excision. The clinical presentation, risk factors, and response to therapy of infections due to P. richardsiae are similar to those of other agents against chromoblastomycosis9,16,18. Further observations, especially in the area of antifungal susceptibility testing, are needed to determine whether a specific etiologic diagnosis is important in the management of these infections.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. Hay RJ. Deep fungal infections. In: Wolff K, Goldsmith LA, Katz SI, Gilchrest BA, Paller AS, Leffell DJ, editors. Fitzpatrick's dermatology in general medicine. 7th ed. New York: McGraw-Hill; 2008. pp. 18331834. James WD, Berger TG, Elston DM. Andrews' diseases of the skin: clinical dermatology. 10th ed. Philadelphia: Saunders Elsevier; 2006. pp. 323324. Elder DE, Elenitsas R, Johnson BL, Jr, Murphy GF, Xu X. Lever's histopathology of the skin. 10th ed. Philadelphia: Lippincott-Raven; 2009. pp. 607608. Kim HU, Son GY, Ihm CW. A case of chromoblastomycosis showing a good response to itraconazole. Ann Dermatol. 1997;9:5154. Suh MK, Sung YO, Yoon KS, Ha GY, Kim JR. A case of chromoblastomycosis caused by Fonsecaea pedrosoi. Korean J Dermatol. 1996;34:832836. Lpez Martnez R, Mndez Tovar LJ. Chromoblastomycosis. Clin Dermatol. 2007;25:188194. [PubMed] Minotto R, Bernardi CD, Mallmann LF, Edelweiss MI, Scroferneker ML. Chromoblastomycosis: a review of 100 cases in the state of Rio Grande do Sul, Brazil. J Am Acad Dermatol. 2001;44:585592. [PubMed] Salgado CG, da Silva MB, Yamano SS, Salgado UI, Diniz JA, da Silva JP. Cutaneous localized annular chromoblastomycosis. J Cutan Pathol. 2009;36:257261. [PubMed] Brandt ME, Warnock DW. Epidemiology, clinical manifestations, and therapy of infections caused by dematiaceous fungi. J Chemother. 2003;15(Suppl 2):3647. [PubMed] De Hoog GS, Queiroz-Telles F, Haase G, Fernandez-Zeppenfeldt G, Attili Angelis D, Gerrits Van Den Ende AH, et al. Black fungi: clinical and pathogenic approaches. Med Mycol. 2000;38(Suppl 1):243 250. [PubMed] Vicente VA, Attili-Angelis D, Pie MR, Queiroz-Telles F, Cruz LM, Najafzadeh MJ, et al. Environmental isolation of black yeast-like fungi involved in human infection. Stud Mycol.2008;61:137144. [PubMed] Kimura M, Goto A, Furuta T, Satou T, Hashimoto S, Nishimura K. Multifocal subcutaneous phaeohyphomycosis caused by Phialophora verrucosa. Arch Pathol Lab Med. 2003;127:91 93.[PubMed] Pitrak DL, Koneman EW, Estupinan RC, Jackson J. Phialophora richardsiae infection in humans.Rev Infect Dis. 1988;10:11951203. [PubMed] Ikai K, Tomono H, Watanabe S. Phaeohyphomycosis caused by Phialophora richardsiae. J Am Acad Dermatol. 1988;19:478481. [PubMed] Lieb DF, Smiddy WE, Miller D, Cooperman EW. Case report: Fungal endophthalmitis caused by Phialophora richardsiae. Retina. 2003;23:406407. [PubMed] Guho E, Bonnefoy A, Luboinski J, Petit JC, de Hoog GS. Subcutaneous granuloma caused by Phialophora richardsiae: case report and review of the literature. Mycoses. 1989;32:219223. [PubMed] Ranawaka RR, Amarasinghe N, Hewage D. Chromoblastomycosis: combined treatment with pulsed itraconazole therapy and liquid nitrogen cryotherapy. Int J Dermatol. 2009;48:397400. [PubMed] Bonifaz A, Sal A, Paredes-Solis V, Araiza J, Fierro-Arias L. Treatment of chromoblastomycosis with terbinafine: experience with four cases. J Dermatolog Treat. 2005;16:4751. [PubMed] Xibao Z, Changxing L, Quan L, Yuqing H. Treatment of chromoblastomycosis with terbinafine: a report of four cases. J Dermatolog Treat. 2005;16:121124. [PubMed] Kumarasinghe SP, Kumarasinghe MP. Itraconazole pulse therapy in chromoblastomycosis. Eur J Dermatol. 2000;10:220222. [PubMed]

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Case Report

CHROMOBLASTOMYCOSIS AFTER A LEECH BITE COMPLICATED BY MYIASIS


SOURCE: http://www.biomedcentral.com/1471-2334/11/14 AUTHORS: Gnther Slesak1,2 , Saythong Inthalad1,3 , Michel Strobel4, Matthias Marschal5 , Martin JR Hall6 and Paul N Newton7,8 - 1 SFE Medical Project, Luang Namtha, Lao PDR,2 Tropenklinik Paul-Lechler-Krankenhaus, 72076 Tbingen, Germany, 3 Luang Namtha Provincial Hospital, Luang Namtha, Lao PDR, 4 Institut de la Francophonie pour la Mdecine Tropicale, Vientiane, Lao PDR,5 Interfakultres Institut fr Mikrobiologie und Infektionsmedizin, Tbingen, Germany, 6 Department of Entomology, Natural History Museum, Cromwell Road, London, UK, 7 Wellcome Trust-Mahosot Hospital-Oxford Tropical Medicine Research Collaboration, Microbiology Laboratory, Mahosot Hospital, Vientiane, Lao PDR, 8 Centre for Clinical Vaccinology and Tropical Medicine, Churchill Hospital, University of Oxford, Oxford, England, UK DATE PUBLISHED: 12 January 2011 in BMC Infectious Diseases 2011 Abstract Background Chromoblastomycosis is a chronic mycotic infection, most common in the tropics and subtropics, following traumatic fungal implantation. Case presentation A 72 year-old farmer was admitted to Luang Namtha Provincial Hospital, northern Laos, with a growth on the left lower leg which began 1 week after a forefoot leech bite 10 years previously. He presented with a cauliflower-like mass and plaque-like lesions on his lower leg/foot and cellulitis with a purulent tender swelling of his left heel. Twenty-two Chrysomya bezziana larvae were extracted from his heel. PCR of a biopsy of a left lower leg nodule demonstrated Fonsecaea pedrosoi, monophora, or F. nubica. He was successfully treated with long term terbinafin plus itraconazole pulsetherapy and local debridement. Conclusions Chromoblastomycosis is reported for the first time from Laos. It carries the danger of bacterial and myiasis superinfection. Leech bites may facilitate infection.

Background Chromoblastomycosis is a worldwide chronic infection of the skin and subcutaneous tissue, most commonly found in tropical and subtropical areas. It is mainly caused by the fungal genera Fonsecaea, Phialophora and Cladophialophora that are saprophytes in soil and plants1-3. Fonsecaea pedrosoi is the commonest agent found in

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tropical rain forests1. Infection occurs by traumatic cutaneous implantation of fungi1, for example by skin abrasion from wood or thorns and rarely by an insect or leech bite4,5. The lower limbs are most commonly infected and the nodular and/or verrucous plaques can develop centripetal satellite lesions. The most frequent complication is bacterial secondary infection, but malignancies have also been recorded1,2,6. Diagnosis can be made by direct microscopic demonstration of pathognomonic brown sclerotic cells (also called fumagoid or muriform cells) in skin scrapings1-3. Case Presentation An otherwise healthy 72-year-old Khmu farmer was admitted in August 2009 at Luang Namtha Provincial Hospital, northern Lao PDR (Laos), with a painful massive growth on his lower left leg, preventing walking. A red nodule developed one week after a leech bite on the dorsum of the left foot and over ten years, this painless, non-itchy growth spread up to his knee. Three days before admission he developed a painful left ankle with discharge from his heel. On examination he was oriented, afebrile (axillary 37.5C) with normal vital signs but cauliflower-like masses and several centripetally verrucous oval plaque-like lesions on his left lower leg and foot, with erythema and warmth and a purulent ulcerative very tender swelling of his left heel (Figure 1 and 2). He was thought initially to have leprosy or skin cancer, but skin scrapings from the left lower leg lesions revealed typical brownish, round, thick-walled, multiseptate sclerotic cells in a wet film, confirmed with the 10% potassium hydroxide technique1-3 (Figure 3 and 4). Left lower leg and foot radiographs showed no evidence of bone involvement. He was treated with oral cloxacillin and metronidazole for 1 week, followed by co-trimoxazole, and local iodine-based antiseptics. Bacterial culture of wound discharge grew Escherichia coli susceptible to co-trimoxazole by disc diffusion testing (according to CLSI guidelines7). During wound dressing on day 3, 22 maggots (fly larvae) were discovered in the heel wound (Figure 5,) and identified as third instar larvae of the Old World screwworm fly, Chrysomya bezziana (Diptera: Calliphoridae)8. Due to the pathognomonic microscopic findings of sclerotic cells he was diagnosed with chromoblastomycosis and started on itraconazole 400 mg/d monthly pulse therapy5 on day 18 and a surgical debridement of all skin lesions was performed on day 21.

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Figure 1. Lower legs of the patient at presentation with typical lesions on his left foot that spread centripetally up to his knee.

Figure 2. Patient's heel with groups of deeply burrowed larvae and their black caudal ends.

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Figure 3. Characteristic brownish sclerotic cells in skin scrapings (100 objective with oil, wet film).

Figure 4. Sclerotic cells with hyphae (10% potassium hydroxide technique).

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5. Third instar larvae of Old World screwworm fly, Chrysomya bezziana recovered from the patient's heel with the characteristic bands of black Figure cuticular spines which help to resist extraction.

In order to confirm the diagnosis, and as fungal cultures were not available in Laos, identification by PCR was attempted at Tbingen from heated and ethanol-treated tissue. DNA extraction was performed from each of three tissue samples of about 3 mm in diameter from the patient's lower leg. Fungal DNA was amplified with two different PCR-protocols, using primers of the internal transcribed spacer (ITS) 1 and 4 region9,10 and the conserved 18S subunit of the rRNA gene11 with 2.5 l of DNA-extract applied in each PCR reaction. PCR-products were subsequently sequenced as described11 and compared with Basic Local Alignment Search Tool (BLAST). PCR using ITS primers remained negative in all 3 samples, whereas 18S rRNAPCR was positive in one of the 3 samples. Subsequent sequencing revealed 100% similarity with Fonsecaea pedrosoi, monophora, and F. nubica. After 4 months, oral terbinafin (500 mg/day, later 750 mg/day) for 9 months was added. Local terbinafin ointment was also applied for 6 months. The patient's left lower leg and foot healed without lesions but with some residual swelling (Figure 6).

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Figure 6. Healed skin lesions 8 months after initiation of antifungal treatment and surgical debridement.

Discussion Chromoblastomycosis has been reported from neighbouring Thailand and China5,12, and it is likely to be endemic in Laos. Its slow growth and variable appearance may result in confusion with skin cancer, eczema, psoriasis, or leprosy, as in our patient. Direct microscopic identification of muriform/sclerotic cells is diagnostic1-3 but doctors have to be aware of this differential diagnosis. Treatment remains challenging, especially in financially-poor countries. Antifungals needed to be given for at least 6-12 months, often combined with physical treatments such as surgery, cryotherapy and thermotherapy. Cure rates range from 15% to 80%1. Despite being the most common aetiological agent, F. pedrosoi appears to be less sensitive to antifungal therapy than either C. carrionii or P. verrucosa1,2. However, terbinafin has shown high tolerability and efficacy especially against F. pedrosoi, even in imidazole-refractory cases13 and dual therapy with itraconazole and terbinafin is recommended1. Which species of Fonsecaea was responsible remains unclear in our patient. The gene sequence analysis using 18S primers, in contrast to ITS primers, which have been used for Fonsecaeaspecies identification in pure cultures14-16, cannot distinguish between Fonsecaea pedrosoi, F. monophora and F. nubica17. The finding that only one sample was positive with 18S primers might be due to a very low fungal load or an alteration during the sample processing and transport from remote northern Laos. PCR with 18S primers may have a higher sensitivity for the detection of Fonsecaea spp. from biopsy material compared to ITS

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primers, however, PCR diagnosis of Fonsecaea spp. is not yet standardized. It is possible that the patient acquired the infection via the leech bite or subsequently through the wound. The injection of platelet inhibitors during leech bites18 might impair the local skin immunity increasing the risk for fungal infections. The coagulation system overlaps with the immune system and many acute-phase proteins of inflammation are also involved in the coagulation process. Platelet microbiocidal proteins and konocidins have shown to exert strong efficacy against bacteria and fungi19. To our knowledge this is the second report of chromoblastomycosis associated with a leech bite5. Since leeches are typically found in humid tropical areas, as is chromoblastomycosis, there might be an important but overlooked association. Leech bites have been associated with Aeromonas hydrophilia infections, complicating 2.4% to 20% of medical leech therapies20, but might also be involved in the pathogenesis of other tropical soil-related infections, such as Chromobacterium violaceum septicaemia21. Ingested blood in leeches' digestive tract can contain various pathogens, including HIV and hepatitis B, that, although unproven, could be re-injected into another host by regurgitation during the manipulation of leech removal22. Bacterial superinfection is a relatively common complication of chromoblastomycosis1,2 but myiasis does not appear to have been reported. Cochliomyia hominivorax, Chrysomya bezziana, and Wohlfahrtia magnifica are the most common flies worldwide causing human wound myiasis. Chrysomya bezziana is the most common cause in India and Southeast Asia23. Although scientific evidence from Laos is scarce, old clinical descriptions indicate that myiasis has been a common poverty-related complication of neglected wounds in remote areas of Laos24. Massive tissue destruction, bone erosion, and death can occur from infestation by Old World screwworm larvae23. Our patient had only three of the reported predisposing factors for wound myiasis including advanced age, poor social condition and hygiene, but did not have diabetes, vascular occlusive disease, alcoholism, mental/psychiatric illness, or physical disability23. However, the skin lesions of chromoblastomycosis could facilitate fly eggs deposition and might represent another risk factor for human wound myiasis. Although a potentially serious complication, the larval infestation and associated pain had the benefit here of prompting our patient to seek medical attention, which led to the root cause of his ten-year condition being successfully diagnosed and treated.

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Conclusions Chromoblastomycosis is reported for the first time from Laos and should be considered in the differential diagnosis of chronic skin diseases. Leech bites might facilitate infection and bacterial superinfection and myiasis may occur.
REFERENCES 1. Ameen M: Chromoblastomycosis: clinical presentation and management. Clin Exp Dermatol 2009, 34:849-854. 2. Bonifaz A, Carrasco-Gerard E, Sal A: Chromoblastomycosis: clinical and mycologic experience of 51 cases. Mycoses 2001, 44:1-7. 3. Queiroz-Telles F, Esterre P, Perez-Blanco M, Vitale RG, Salgado CG, Bonifaz A: Chromoblastomycosis: an overview of clinical manifestations, diagnosis and treatment. Med Mycol 2009, 47:3-15. 4. Sauerteig E, Hernndez R, Salfelder K, Bastidas C: Acute chromoblastomycosis provoked by an insect bite in an immunosuppressed patient. Mycoses 1998, 41:191-194. 5. Ungpakorn R, Reangchainam S: Pulse itraconazole 400 mg daily in the treatment of chromoblastomycosis. Clin Exp Dermatol 2006, 31:245-247. 6. Minotto R, Bernardi CDV, Mallmann LF, Edelweiss MIA, Scroferneker ML: Chromoblastomycosis: a review of 100 cases in the state of Rio Grande do Sul, Brazil. J Am Acad Dermatol 2001, 44:585-592. Clinical and Laboratory Standards Institute: Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard. In . Tenth edition. Wayne, PA: Clinical and Laboratory Standards Institute; 2009. Approved Standard M02-A10. 7. Hall MJR: New World screwworm (Cochliomyia hominivorax) and Old World screwworm (Chrysomya bezziana). Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (mammals, birds and bees) Sixth edition. OiE, World Organisation for Animal Health. Paris, France; 2008, 1:265-275. Chapter 2.1.10 8. Loeffler J, Hebart H, Bialek R, Hagmeyer L, Schmidt D, Serey FP, Hartmann M, Eucker J, Einsele H: Contaminations occurring in fungal PCR assays. J Clin Microbiol 1999, 37:1200-1202. 9. Leinberger DM, Schumacher U, Autenrieth IB, Bachmann TT: Development of a DNA microarray for detection and identification of fungal pathogens involved in invasive mycoses. J Clin Microbiol 2005, 43:4943-53. 10. Einsele H, Hebart H, Roller G, Lffler J, Rothenhfer I, Mller CA, Bowden RA, Van Burik JA, Engelhard D, Kanz L, Schumacher U:Detection and identification of fungal pathogens in blood by using molecular probes. J Clin Microbiol 1997, 35:1353-60. 11. Zhang J, Xi L, Lu C, Li X, Xie T, Zhang H, Xie Z, Sybren DH: Successful treatment for chromoblastomycosis caused byFonsecaea monophora: a report of three cases in Guangdong, China.Mycoses 2008, 52:176-181. 12. Esterre P, Inzan CK, Ramarcel ER, Andriantsimahavandy A, Ratsioharana M, Pecarrere JL, Roig P: Treatment of chromomycosis with terbinafine: preliminary results of an open pilot study. Br J Dermatol 1996, 134(Suppl 46):33-36. 13. Xi L, Sun J, Lu C, Liu H, Xie Z, Fukushima K, Takizawa K, Najafzadeh MJ, De Hoog GS: Molecular diversity of Fonsecaea (Chaetothyriales) causing chromoblastomycosis in southern China. Med Mycol 2009, 47:27-33. 14. De Andrade TS, Cury AE, de Castro LG, Hirata MH, Hirata RD: Rapid identification of Fonsecaea by duplex polymerase chain reaction in isolates from patients with chromoblastomycosis. Diagn Microbiol Infect Dis 2007, 57:267-272. 15. Sun J, Najafzadeh MJ, Vicente V, Xi L, de Hoog GS: Rapid detection of pathogenic fungi using loopmediated isothermal amplification, exemplified by Fonsecaea agents of chromoblastomycosis. J Microbiol Methods 2010, 80:19-24. 16. Najafzadeh MJ, Sun J, Vicente V, Xi L, van den Ende AH, de Hoog GS: Fonsecaea nubica sp. nov a new agent of human chromoblastomycosis revealed using molecular data. Med Mycol 2010, 48:8006. 17. White TC, Berny MA, Robinson DK, Yin H, DeGrado WF, Hanson SR, McCarty OJT: The leech product saratin is a potent inhibitor of platelet integrin 21 and von Willebrand factor binding to collagen. FEBS J 2007, 274:1481-1491. 18. Yeaman MR: Platelets in defense against bacterial pathogens. Cell Mol Life Sci 2010, 67:525-544. 19. Ardehali B, Hand K, Nduka C, Holmes A, Wood S: Delayed leech-borne infection with Aeromonas hydrophilia in escharotic flap wound. J Plast Reconstr Aesthet Surg 2006, 59:94-95. 20. Slesak G, Douangdala P, Inthalad S, Silisouk J, Vongsouvath M, Sengduangphachanh A, Moore CE, Mayxay M, Matsuoka H, Newton PN: Fatal Chromobacterium violaceum septicaemia in northern Laos, a modified oxidase test and post-mortem forensic family G6PD analysis. Ann Clin Microbiol Antimicrob 2009, 8:24.

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21. 22. 23.

Nehili M, Ilk C, Mehlhorn H, Ruhnau K, Dick W, Njayou M: Experiments on the possible role of leeches as vectors of animal and human pathogens: a light and electron microscopy study. Parasitol Res 1994, 80:277-290. McGraw TA, Turiansky GW: Cutaneous myiasis. J Am Acad Dermatol 2008, 58:907-926. Dooley TA: The edge of tomorrow. In . New York: Farrar, Straus & Cudahy; 1958.

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CHROMOBLASTOMYCOSIS ASSOCIATED WITH FONSECAEA PEDROSOI IN A CARPENTER HANDLING EXOTIC WOODS


SOURCE:http://anagen.ucdavis.edu/142/case_reports/chromoblastomycosis/menezes.html AUTHORS: Nuno Menezes1, Paulo Varela1, Antnia Furtado2, Antonio Couceiro2, Ismlia Calheiros3, Laura Rosado4, Gioconda Mota1, Armando Baptista1 - 1Servio de Dermatologia do Centro Hospitalar V.N. de Gaia (CHVNG), Portugal, 2Servio de Anatomia Patolgica do CHVNG, Portugal, 3Servio de Patologia Clnica do CHVNG, Portugal 4Instituto de Sade Dr. Ricardo Jorge, Portugal DATE PUBLISHED: Year 2008 in Dermatology Online Journal 14 (2): 9 Abstract Chromoblastomycosis is a rare, hard to diagnose disease that arises mostly in the tropics, especially in humid areas, affecting mainly males and rural workers. It is characterized by verrucous plaques or nodules that are slow growing and attributed to infection by different pigmented (dematiaceous) fungi. Usually the infection develops after injury, being primarily located on the lower extremities. The authors present the case of a healthy, 60-year-old man observed with a one year history of an erythematous violaceous 5cm tumor located on the right thigh. A biopsy specimen for histopathology revealed single or clustered brown pigmented cells, with a single or double septum and thick cell walls. Cultural smears showed growth of Fonsecaea Pedrosoi. The patient was treated with oral itraconazole (200mg per day), with a good response and clinical cure in 6 months that left only an atrophic scar.

The term chromoblastomycosis was first used by Terra et al. in cases of polymorphic fungal disease of the lower limbs presenting with nodules or verrucous plaques with hyperkeratosis and acanthosis of the affected epithelial tissues, which could develop into complications such as ulceration, lymphedema and squamous cell carcinoma1,2. Chromoblastomycosis is a subcutaneous mycosis caused by a variety of black or dematiaceous fungi3 that assume the shape of sclerotic, muriform structures known as Medlar bodies4. ethiologic agents belong to the Fonsecaea, Phialophora, Rhinocladiella and Cladosporium genera4,5. The causal agents are The

normally found as saprophytes in the soil, wood and vegetation3. They are more frequent in the tropics but have a worldwide distribution7. The vast majority of cases are attributed to Fonsecaea pedroso4,6. In Portugal there have been cases both of native7 and imported8 origin. Nowadays the number of cases in north Europe is growing because of

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the increasing number of steam bath facilities, which are normally made of tropical wood9. The inoculation usually occurs after trauma, usually in exposed areas, particularly in the lower limbs, therefore being more frequent in rural areas1,4,10,11. The disease is more common among men 30-50 years old4,6. If not diagnosed early the infection has a chronic evolution, being difficult to treat and with frequent recurrences. Sometimes complications such as bacterial super-infection can lead to lymphedema and elephantiasis and occasionally to squamous cell carcinoma at affected sites. Lymphatic and hematogenic dissemination and brain metastases have been observed rarely1,5,12. Clinical synopsis A previously healthy 60-year-old male carpenter who had never traveled outside Portugal was evaluated for a 5cm erythematoviolaceous tumoral lesion involving the external surface of the right thigh, with a one year evolution (Fig. 1).

Figure 1

A skin biopsy was obtained, which showed intense pseudoepitheliomatous hyperplasia and a mixed dermal granulomatous response, with small neutrophil abscesses and organized granulomae of the giant-cell type, inside which groups of fungal cells were seen. The fungal cells were characteristically divided in several planes by thick septa (muriform or sclerotic cells) (Fig. 2).

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Figure 2

A skin biopsy fragment was then cultured in a Sabouraud medium with chloramphenicol and incubated at 25C, showing growth of black colonies, whose microscopic exam showed numerous elliptical to oval spores with thick septa, long and oval conidia confined to the upper part of the cell, and multiple phialides with terminal collarette (Fig. 3). The diagnosis of chromoblastomycosis attributed to Fonsecaea pedrosoi was also confirmed by rRNA sequence analysis.

Figure 3

We started treatment with itraconazole PO 100mg bid reducing to 100mg per day after 3 months due to marked clinical improvement. After 5 months of treatment it was possible to see only a violaceous cicatricial area and treatment was suspended (Fig. 4). After a followup of 18 months the patient is still without any clinical recurrence.

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Figure 4

Discussion Our patient denied any travel to foreign countries or contact with people arriving from tropical countries. We think it is likely that the source of infection was the professional contact with tropical woods, probably from a minor trauma. This represents one more case of this rare disease in developed countries (less than 10 cases reported in Portugal)7,10,11 and to our knowledge the first to occur in a carpenter. Diagnosis is accomplished by Medlar bodies isolation either in pathologic or microbiological exams. They are defined as rounded, brown structures, with 5-10mm in diameter, thick wall and internal septation5,11,13. Clinically, the diagnosis is suggested by warty plaques, nodules, or tumors, located in the lower limbs of a patient living or arriving from a tropical country. Differential diagnosis must be done with other fungal infections (such as sporotrichosis or lobomycosis), cutaneous tuberculosis, Hansen disease, late syphilis, cutaneous leishmania, squamous cell carcinoma, amelanotic melanoma, and other dermatologic malignancies4,10.

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The clinical suspicion should then be confirmed by pathologic, cultural and microscopic examinations10. Recent technology was developed that allows species confirmation with rRNA sequence analysis6,14.

Fonsecaea pedroso has the ability to produce melanin, which will be

an immunogenic structure that together with the fungal antigens will induce a humoral and cellular response, which will be responsible by the epidermis pseudoepitheliomatous hyperplasia with transepidermal fungal elimination. This phenomenon is clinically seen by the observation of black dots at the surfaces of the lesion7,11,13. Treatment is difficult and not well established. Small lesions in hidden places can be treated by simple surgical excision with large margins followed by systemic treatment with an oral anti-fungal drug to avoid dissemination4,7. More often, medical treatment is preferred with systemic anti-fungal agents. Fonsecaea pedrosoi, the most common pathogenic agent, is also more resistant to treatment, especially in protracted cases15. Usually, itraconazole (200-400mg/day PO for 6 months) is the first choice of therapy, with an 80-90 percent rate of success. This rate may increase if an association of 5-flucytosine is used16,17,18. Terbinafine, fluconazole, ketoconazole, and thiabendazole have been used17,18,19,20,21,22,23. Intravenous amphotericin B (up to 1mg/kg daily) is recommended in extensive cases16. Voriconazole efficacy is being tested, although its use is limited by serious side-effects23. Topical heat, cryotherapy, and CO2-laser ablation also have been described as treatment options24,25,26,27. The more resistent situations can be treated with combination of agents like CO2 laser with topical heat28; combination of cryotherapy, shaving, oral 5-fluorocytosine, and oral amphotericin B16 or itraconazole plus cryotherapy29. In our patient we had excellent results with slightly lower doses than those described in the literature (itraconazole 200mg/day for 3 months plus 100mg/day for more 2 months).
REFERENCES 1. 2. 3. 4. 5. 6. Terra F, Torres M, Fonseca Filho O, Ara Leo AE. Novo tipo de dermatite verrucosa: micose por Acrotheca com associao de leishmaniose. Brasil Med 1922;36: 363-8 Minotto R, Bernardi CD, Mallmann LF, Edelweiss MI, Scroferneker ML. Chromoblastomycosis: A review of 100 cases in the state of Rio Grande do Sul, Brazil. J Am Acad Dermatol 2001;44: 585-92. Vidal MS, de Castro LG, Cavalecate SC, Lacaz Cda S. Immunoprecipitation techniques and elisa in the detection of anti-Fonsecaea Pedrosoi antibodies in chromoblastomycosis. Rev. Inst. Med. Trop. S. Paulo 2003;45(6): 315-318. Vieira MR, Martins ML. Micoses subcutneas. Trab Soc Port Dermatol Venerol 2004;62 (2): 137-67. Pang KR, Wu JJ, Huang DB, Tyring SK. Subcutaneous fungal infections. Dermatologic Therapy 2004;17(6): 523-31. Tanabe H, Kawasaki M, Mochizuki T, Ishizaki H. Species identification and strain typing of Fonsecaea Pedrosoi using ribosomal RNA gene internal transcribed spacer regions. Nippon Ishinkin Gakkai Zasshi 2004;45(2):105-12.

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7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.

Clerins I, Mariano A, Tellechea O, Reis JP, Velho R, Figueiredo A. romomicose por Exophiala Jeanselmei em doente transplantado renal. Trab Soc Port Dermatol Venerol 2000;58(2): 241-47. Matos J, Figueiredo A, Eiriz E, Velho R, Baptista AP. Cromomicose (2 casos autctones). Trab Soc Port Dermatol Venerol 1990;48: 43-47. Verde SF, Nobre G, Oliveira A. Cromomicose, primeiro caso descrito em Portugal (oriundo de Angola). Trab Soc Port Dermatol Venerol 1976;34: 135-46. Guerras Rodrigo F, Sequeira H. Micoses subcutneas e sistmicas. Trab Soc Port Dermatol Venerol 2000;58 (4): 459-91. McGinnis MR. Cromoblastomycoses and phaeohyphomycoses: New concepts, diagnosis and mycology. J Am Acad Dermatol 1983;8(1): 1-16. Nobrega JP, Rosemberg S, Adami AM, Heins-Vaccari EM, Lacaz Cda S, de Brito T. Fonsecaea pedrosoi cerebral phaeohyphomycosis ("chromoblastomycosis"). First human culture-proven case reported in Brazil. Rev Inst Med Trop S. Paulo 2003;45(4): 217-220. Brandt ME, Warnock DW. Epidemiology, clinical manifestations, and therapy of infections caused by dematiaceous fungi. J Chemother 2003;15 Suppl 2: 36-47. Vidal MS, de Castro LG, Cavalecate SC, Lacaz Cda S. Immunoprecipitation techniques and ELISA in the detection of anti-Fonsecaea pedrosoi antibodies in chromoblastomycosis. Rev Inst Med Trop S. Paulo 2003;45(6): 315-18. Poirriez J, Breuillard F, Francois N, Fruit J, Sendid B, Gross S, Dei-Cas E. A case of chromoblastomicosis treated by a combination of cryotherapy, shaving, oral 5-fluorocytosine and oral amphotericin B. Am J Trop Med Hyg 2000;63(1, 2): 61-63. Hay R.J. Deep Fungal Infections. In: Freedberg IM, Eisen AZ, Wolff K, Austen KF, Goldsmith LA, Katz SI, editors. Dermatology in General Medicine, 6th ed. Mc Graw-Hill, New York, 2003, 2018-34. Esterre P, Inzan CK, Ramarcel ER, Andriantsimahavandy A, Ratsioharana M, Pecarrere JL, Roig P. Treatment of chromomycosis with terbinafine preliminary results of an open pilot study. Br J Dermatol 1996, 134 (Suppl. 46): 33-36. Sobera JO, Elewski BE; Fungal Diseases. In: Bolognia JL, Jorizzo JL, Rapini RP, editors. Dermatology, 1st ed, Londres. Mosby, 2003, 1171-98. Vivas JRC, Torres-Rodrguez JM. Sensibilidad de hongos miceliares dematiceos a diez antifngicos empleando un mtodo de difusin en agar. Rev Iberoam Micol 2001; 18: 113-17. Mc Burney EI. Chromoblastomycosis treatment with ketoconazole. Cutis 1982;30(6): 746-8 Guerriero C, De Simone C, Tulli A. A case of chromoblastomycosis due to Phialophora verrucosa responding to treatment with fluconazole. Eur J Dermatol 1998;8(3): 167-8. Olle-Goig JE, Domingo J. Case of chromomycosis treated with thiabendazole. Trans R Soc Trop Med Hyg 1983;77(6): 773-4. Radford SA, Johnson EM, Warnock DW. In Vitro Studies of Activity of Voriconazole (UK-109,496), a New Triazole Antifungal Agent, against Emerging and Less-Common Mold Pathogens. Antimicrobial Agents and Chemotherapy 1997; 41: 841-843. Tagami H, Ohi M, Aoshima T, Moriguchi M, Suzuki N, Yamada M. Topical heat therapy for cutaneous chromomycosis. Arch Dermatol 1979;115(6): 740-1. Tagami H, Ginoza M, Imaizumi S, Urano-Suehisa S. Successful treatment of chromoblastomycosis with topical heat therapy. J Am Acad Dermatol 1984;10(4): 615-9. Bonifaz A, Martinez-Soto E, Carrasco-Gerard E, Peniche J. Treatment of chromoblastomycosis with itraconazole, cryosurgery and a combination of both. Int J Dermatol 1997;36: 542-547. Kuttner BJ, Siegle RJ. Treatment of chromomycosis with a CO2 laser. J Dermatol Surg Oncol 1986;12(9): 965-8. Hira K, Yamada H, Takahashi Y, Ogawa H. Successful treatment of chromomycosis using carbon dioxide laser associated with topical heat applications. J Eur Acad Dermatol Venereol. 2002 May;16(3): 273-5. Kullavanijaya P, Rojanavanich V. Successful treatment of chromoblastomycosis due to Fonsecaea pedrosoi by the combination of itraconazole and cryotherapy. Int J Dermatol 1995;34(11): 804-7.

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