Archives of Medical Research 44 (2013) 302e306

PRELIMINARY REPORT

Revisiting the Clinical and Histopathological Aspects of Patients withChromoblastomycosis from the Brazilian Amazon Region

Carla Avelar-Pires,a Juarez Antonio Simoes-Quaresma,a,b Geraldo Mariano Moraes-de Macedo,b

Marilia Brasil-Xavier,a,b and Arival Cardoso-de Britob

aCentro de Ciencias Biologicas e da Saude, Universidade do Estado do Para, Belem, PA, BrazilbNucleo de Medicina Tropical, Universidade Federal do Para, Belem, PA, Brazil

Received for publication April 1, 2013; accepted April 15, 2013 (ARCMED-D-13-00183).

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de Medicina Tropical

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Background and Aims. Chromoblastomycosis is a chronic fungal infection caused byspecies of the family Dematiaceae. Fonsecaea pedrosoi is the most common etiologicalagent. The objective of this study was to describe the epidemiological and mycologicalprofile of patients with chromoblastomycosis from the Amazon region of Brazil and tocorrelate the clinical forms with the histopathological findings and severity criteria.

Methods. Sixty-five patients were submitted to mycological (direct, culture, and micro-culture) and histopathological (hematoxylin-eosin staining) examination. Severity of thedisease was classified according to the criteria proposed by Carri�on in 1950.

Results. Most patients were males (93.8%) and laborers (89.2%). There was a predomi-nance of verrucous lesions (55.4%), which were mainly found on the lower limbs(81.5%). Two major types of tissue reaction were observed: a granulomatous reactioncharacterized by the formation of suppurative granulomas rich in fungal cells, which werealmost always seen in verrucous lesions, and a reaction characterized by the formation oftuberculoid granulomas with few parasites, which were mainly found in well-delimitederythematous plaque-like and cicatricial lesions ( p 5 0.0001). A peculiar type of orga-nized mycotic granuloma was observed in 20 subjects. Suppurative granulomas weremore frequently detected in severe lesions ( p 5 0.0189) and in lesions with a durationof O10 years ( p 5 0.0408).

Conclusions. These results suggest that verrucous lesions present a less competentinflammatory tissue response than patients who develop a well-formed tuberculoidreaction. The latter is associated with a more effective immune response as observedin the limited clinical forms of chromoblastomycosis. � 2013 IMSS. Published byElsevier Inc.

Key Words: Chromoblastomycosis, Inflammation, Granuloma, Histopathology.

Introduction

Chromoblastomycosis (CBM) is a chronic fungal diseasethat affects the skin and subcutaneous tissue. Classifiedas a deep mycosis, infection occurs by traumatic implanta-tion into the skin of pigmented fungi of different species of

equests to: Dr. Arival Cardoso-de Brito, Nucleo

, Universidade Federal do Para, Av. Generalissimo

l, Belem-PA-Brazil, 66085-31; E-mail: acdebrito@

nt matter. Copyright � 2013 IMSS. Published by Elsevier.1016/j.arcmed.2013.04.008

the family Dematiaceae, which are found in soil and plants(1e5).

The Amazon region concentrates the largest number ofcases of CBM in Brazil (2). This fact can be explainedby social and environmental factors that contribute to main-tain the fungus viable in nature, as well as by work activi-ties during which individuals have frequent contact withplants and fruits and thus remain exposed to the pathogen.Despite its chronic evolution, in the last 5 years few studieshave characterized the clinical, histopathological andinflammatory features of CBM, and these investigations

Inc.

303Chromoblastomycosis from the Brazilian Amazon Region

only involved small samples. In this respect, studies areneeded to better understand the range of CBM and the clin-ical, mycological, inflammatory and histopathologicalfeatures of the disease in the Amazon region where thenumber of cases is significant.

Therefore, the objective of the present study was todescribe the clinical and histopathological profile of patientswith CBM from the State of Para, Amazon region, and tocorrelate the clinical forms with the inflammatory and histo-pathological findings and severity criteria.

Materials and Methods

Patients

A descriptive study was conducted on 65 patients withCBM seen at the outpatient clinic of the Dermatology Ser-vice, N�ucleo de Medicina Tropical, Universidade Federaldo Par�a (UFPA) between January 2000 and July 2007.

The criteria proposed by Carri�on in 1950 were used forthe clinical diagnosis, which divide CBM lesions into fivedifferent types: verrucous, nodular, plaque-like, cicatricialand tumorous (6).

We excluded patients with immunosuppressive diseases,negative mycological examination and histopathology neg-ative for chromoblastomycosis.

The study was approved by the Ethics Committee ofNucleo de Medicina Tropical, UFPA.

Mycological Examination

For direct examination, material was scraped from thesurface of the lesions, especially in the dark areas knownas black dots, with scalpel blades. The material wasmounted on a slide and cleared in 10% KOH.

For primary isolation of the fungus, the material wascultured on Sabouraud-dextrose agar for 4 weeks. Aftergrowth of the dematiaceous fungus, the colonies were trans-ferred to potato agar for filamentation.

For fungal microculture on slides, small segments of thecolonies in a 4-mm thick potato agar block were placed ona slide and covered with a sterile coverslip.

Figure 1. Clinical appearance of lesions in chromoblastomycosis. (A) Injury ver

online version of this article.)

Histopathology

Skin biopsies were obtained from each patient with a 4-mmpunch or scalpel. Next, 4-mm histological sections werecut from the paraffin-embedded material and stained withhematoxylin-eosin for morphological analysis.

Results

In the present study, most patients were male (93.8%) andlaborers (89.2%) from the countryside of Para State. Patientages ranged from 45 to 65 years (49.2%). The lesions mostfrequently affected the lower limbs (81.5%).

According to the classification of Carri�on (1950), therewas a predominance of verrucous lesions found in morethan half the cases (55.4%), followed by plaque-like(26.2%), nodular (7.7%), cicatricial (6.2%), and tumorouslesions (4.6%) (Figures 1A and 1B).

Direct mycological examination was performed in 86.2%of patients and was positive in most cases (83.1%). Growthof Fonsecaea pedrosoi was detected in 77.8% of the cultures.

Elimination of the fungus was observed in 12 cases(18.5%). Fungal structures were detected in the dermis ofall cases, with these structures being present in the papillarydermis of 30.8% of these patients.

Odds ratio analysis showed that the presence of thefungus in the papillary dermis increased the chance oftransepidermal elimination by five times ( p 5 0.0187).

Irregular hyperplasia was observed in most cases (60%)and pseudoepitheliomatous hyperplasia was seen in 40%.

Multiple logistic regression analysis showed that lesionswith a duration ofO10 years have a 3.2 times higher chanceof being severe and this chance increases to 4.9 times inthe case of verrucous lesions. Moreover, older lesions(O10 years) have a 5.5 times higher chance of containinga suppurative granuloma and this chance increases to19.32 times if these lesions are severe.

Two major types of inflammatory response wereobserved in the case series studied: one characterized bya tendency to form suppurative granulomas (46.5%) andthe other by a predominance of tuberculoid granulomas(33.3%). In addition to these two classical types, a peculiar

rucosa. (B) Lesions in atrophic plaque. (A color figure can be found in the

Figure 2. Histopathology of suppurative granuloma with frequent neutrophils (A) and tuberculoid type of granuloma showing the presence of histiocytes and

mononuclear cells (B). (A color figure can be found in the online version of this article.)

304 Avelar-Pires et al./ Archives of Medical Research 44 (2013) 302e306

type, called organized mixed mycotic granuloma, wasobserved in 20.2% of cases (Figures 2A and 2B).

In patients with the verrucous form of CBM, histo-pathological analysis revealed an intense granulomatousinflammatory infiltrate in the papillary and reticular dermis,which consisted of numerous neutrophils, lymphocytes,eosinophils, and histiocytes. In addition, there was a ten-dency to form suppurative granulomas that contained fewerepithelioid cells than tuberculoid granulomas, the periph-eral mantle of lymphocytes was absent, and there werelarge numbers of viable fungal ( p 5 0.0025). Significantformation of tuberculoid cells containing numerous epithe-lioid and multinucleated cells and a mantle of lymphocyteswas observed in patients with clinically well-delimitedlesions, especially those with erythematous plaque-likelesions and even those with nodular and cicatricial lesions( p !0.0001) (Figure 2).

Clusters of neutrophils indicating microabscess forma-tion were observed in a large number of cases (83.1%)and the chance of occurrence of these clusters was 11 timeshigher in patients with verrucous lesions ( p 5 0.0176).

Discussion

Analysis of aspects related to the prevalence of CBMshowed a preference of the disease for male workers fromrural areas. This finding might be related to the naturalhabitat of the pathogen, which is found in soil and vegeta-tions, and to the activities of the population that mainlyworks in the agricultural sector. The lesions most frequentlyaffected the lower limbs. These areas are classically moreexposed to traumatic inoculation of the fungus into the skin,especially when unprotected, and give rise to the develop-ment of CBM lesions (1,4,6e8).

The polymorphism of CBM lesions has raised theinterest of various investigators who, at distinct times,proposed different clinical classifications. However, theclassification of Carri�on is the most accepted and mostwidely used (9e13). Using this classification, we observeda predominance of verrucous lesions, in agreement with the

studies of Matte et al. and Minotto et al. As described forother mycoses, this polymorphism may be attributed in partto differences in the immune response within the tissuemicroenvironment where fungus-immune system interac-tions occur. However, further studies are needed to clarifythese aspects (14).

The epidermis plays an active role in the elimination offungi in some mycoses. In CBM, the epidermis has been re-ported to be highly active and pseudoepitheliomatoushyperplasia is the most common histological finding(15e19). In contrast, in the present series the most markedepidermal alteration was irregular hyperplasia. Thesedifferences between studies support the fact that this histo-pathological feature should not be used as a diagnosticcriterion because it is not always found in all lesions andits presence depends on the type of lesion from which thefragment is removed.

Histopathologically, CBM is characterized by chronic,frequently granulomatous, inflammation that affects thedermis to variable extents and consists of suppurative gran-ulomas, microabscesses and, rarely, tuberculoid granulomas.However, this presentation is not exclusive of the disease butis also found in other infectious skin diseases. Therefore, thehistopathological diagnosis of CBM can only be confirmedby detection of fungal structures (6,16,18,20).

Two major types of inflammatory response wereobserved in the present case series. One reaction was char-acterized by a tendency to form suppurative granulomasand the other by the predominance of tuberculoid granu-lomas. In addition to these two classical types of granu-lomas, there was a type called organized mixed mycoticgranuloma (20). This type of granuloma mainly occurs inCBM and in paracoccidioidomycosis. Its higher frequencyin these two diseases may be a consequence of the markedpresence of polymorphonuclear cells that modify the tissueresponse in these mycoses. In addition, ineffective attemptsof phagocytosis by unproductive giant cells that start todisintegrate lead to cytoplasmic degeneration and marginal-ization of cell nuclei to the periphery where they formcomplete arches until total destruction of these giant cells

305Chromoblastomycosis from the Brazilian Amazon Region

and the release of fungal structures. The latter attract morepolymorphonuclear cells and a new microabscess isformed, restarting the cycle (17). In paracoccidioidomyco-sis, some studies have demonstrated that the interaction ofthe fungus with the immune system leads to the inhibitionof dendritic cells. This impairs the activation of macro-phages due to the lack of effective activation of T helperlymphocytes which, in turn, would secrete IFN-ʏ that acti-vates macrophages and giant cells. This subversion of theimmune response in terms of the adequate activation ofdendritic cells has also been described in Jorge Lobo’sdisease, with the observation of the lack of activation ofLangerhans cells in the lesions (19). However, the role ofdendritic cells in CBM needs further investigation.

D’Avila et al. observed a correlation between the clinicalforms of CBM and its different histopathological expres-sions (18). According to the author, the verrucous form(more frequent) is characterized by the formation of suppu-rative granulomas and the presence of numerous fungalstructures in the lesions. In contrast, cicatricial lesions arecharacterized by the presence of well-formed tuberculoidgranulomas with epithelioid cells, Langhans cells, theabsence of microabscesses, and few fungal structures inthe lesion. In the present study, the histopathological find-ings obtained for patients with the verrucous form weresimilar to those reported by D’Avila and were characterizedby the presence of an intense granulomatous inflammatoryinfiltrate in the papillary and reticular dermis, which con-sisted of numerous neutrophils, lymphocytes, eosinophils,and histiocytes (18). In addition, there was a tendency toform suppurative granulomas that contained fewer epithe-lioid cells than tuberculoid granulomas, the peripheralmantle of lymphocytes was absent, and large numbers ofviable fungal structures were seen (10,11,15,18). It isbelieved that this persistence and multiplication capacityof fungal cells in the dermis, as well as the exuberantinflammatory response seen in the verrucous form, are asso-ciated with the inability to develop a well-formed and morecompetent tuberculoid granulomatous reaction which, inturn, is intimately related to the immune response and cyto-kine cascade in the lesions. The fact that verrucous lesionsmainly present suppurative inflammation, which is sugges-tive of less competent cellular immunity, may be the reasonwhy these lesions become more severe, grow larger, andpresent a longer duration due to the difficulty of treatment.

In the present study, significant formation of tubercu-loid granulomas containing numerous epithelioid andmultinucleated cells and a mantle of lymphocytes wasobserved in patients with clinically well-delimited lesions,mainly erythematous plaque-like lesions but also nodularand cicatricial lesions. These lesions also containeda smaller number of fungi and the fungal structures weregenerally found inside giant cells. This finding seems tobe related to a more competent immune response sinceexacerbation of cellular immunity, activation of Th1

populations and production of proinflammatory cytokinesmediated by the induction of IL-12 are observed in tuber-culoid granulomas (19).

Most fungi simultaneously induce a response of the poly-morphonuclear system, which gives rise to suppurativeinflammation characterized by infiltration of neutrophils,and components of the mononuclear system (9,12,20).Mechanisms resembling those acting in other deep mycosesmay trigger the development of a granulomatous response inCBM (20). Once the dematiaceous fungus penetrates thedermis, it first induces a nonspecific immune reaction andlater triggers a specific response. Macrophages, which arethe dominant cell population in this distinct pattern ofchronic inflammatory reaction (granulomatous reaction),can survive for months or years in tissues. Once activated,these cells secrete biologically active products that causetissue damage and fibrosis. Specifically in CBM, some inves-tigators such as D’�Avila et al. and Sotto et al. have focused onthe interaction between the fungus and host, and the findingspoint to a predominantly cellular immune response that isalways accompanied by the activation of macrophages thatare normally involved in fungal phagocytosis (13,18).However, phagocytosis seems to be compromised in thesepatients and the death of fungal cells rarely occurs. In situpersistence of the fungus is believed to be the main factorresponsible for the morbidity and exuberant lesions seen inCBM (13,14,17). Studies suggest that the formation ofa granulomatous tissue reaction is related to the pattern ofthe cellular immune response developed by the host (20).

It should be noted that CBM without complications isa mycosis that does not severely compromise the patient’ssystemic condition and these patients should thereforebe able to satisfactorily respond to the invading agent.However, this is not what is observed. Furthermore, severalstudies on infectious diseases have shown that the prog-ression of damage in a certain organ or target tissue isintimately related to the in situ interaction between themicrobial agent and immune system and not only tothe capacity of the immune system to respond to the pres-ence of the infectious agent. The reasons for the lack ofdestruction of the fungus in CBM are still unknown andthe heterogeneity of the lesions depending on the microbici-dal capacity of the host immune system justifies the compar-ison of CBM with other infectious diseases characterized bya broad spectrum of clinical and pathological severity (20).

Although numerous reports have investigated thephysiopathology of fungal infections over the last years,further studies are needed to better characterize this groupof diseases, particularly parasite-host interactions and theimmune response, and to correlate the findings with theclinical evolution.

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