Barbital

SUMMARY

Chromoblastomycosis (CBM) is a chronic subcutaneous infection caused by several dematiaceous fungi. The most commonly etiological agent found in Brazil is Fonsecaea pedrosoi, which appears as thick walled, brownish colored cells with transverse and longitudinal division in the lesions, called "muriform cells". This disease is found worldwide but countries like Madagascar and Brazil have highest incidence. Diagnosis is made by clinical, direct and histopathologic examination and culture of specimens. Scrological tests have been used to identify specific antibodies against Fonsecaea pedrosoi antigens, as well as immunotechniques have been used for CBM serological identification and diagnosis. In the present study double immunodiffusion (DID), countcrimmunoelcctrophoresis (CIE) and immunoenzymatic test (ELISA) have been used to evaluate humoral immune response in patients with CBM caused by F. pedrosoi. Metabolic antigen was used for immunoprecipitation tests (DID and CIE) while somatic antigen for ELISA. Our results demonstrated 53% sensitivity and 96% specificity for DID, while CIE presented 68% sensitivity and 90.5% specificity. ELISA demonstrated 78% sensibility and 83% specificity. Serological tests can be a useful tool to study different aspects of CBM, such as helping differential diagnosis, when culture of the pathogenic agent is impossible.

KEYWORDS: Chromoblastomycosis; Fonsecaea pedrosoi; Serologie tests and antigen.

INTRODUCTION

Chromoblastomycosis (CBM) is a chronic infection of skin and subcutaneous tissue caused by dematiaceous fungi. The causal agents arc normally found as saphrophytes in soil and vegetation and infection follows traumatic implantation17.

The most important agent of CBM in Brazil is Fonsecaea pedrosoi. It can be considered endemic in certain regions1,3,4,13,22,24,27,28,29,30. Diagnosis is accomplished by clinical, direct and histopathologic examination and culture of specimens. The fungus appears in lesions as thick walled, brownish colored cells with transverse and longitudinal division, also known as "muriform cells" or "sclerotic cells"17.

Although serological tests are of great help in establishing diagnosis and helping monitorization of therapy of deep mycoses, they have not been routinely used for CBM. The first demonstration of specific circulating antibodies in K pedmsoi-caused CBM was performed using conidia precipitation5. DID and CIE were used to analyze sera of CBM caused by Cladophialophora carrionii. It was demonstrated that metabolic antigens presented higher specificity and results depended on the use of fresh sera, since storage for long period (two years) causes looses or inactivation of the antibodies. These authors recommended the implantation of this methodology for routine immunodiagnosis of chromoblastomycosis based on the results and on the ease to perform the tests32,33.

The exoantigen test was used for serological identification of several dematiaceous fungi and it is considered a useful tool to distinguish CBM agents8,13,21,23.

The ELISA has been extensively used to detect either antigens or antibodies in infectious diseases. Investigators from Madagascar2,9 reported the successful use of ELISA to study sera from human CBM cases, most of which caused by K pedrosoi. They reported that ELISA test was reproducible, gave satisfactory results and allowed biologic diagnosis of CBM, even when culture of the pathogenic agent was not possible. ELISA, using somatic antigen from F. pedrosoi and C. carrionii reference strains, presented 87% sensitivity and 92.3% specificity. The same test was used to detect circulating antibodies in patients with C. carrionii-caused CBM. This test proved to be a useful tool for the differential scrodiagnosis of this infection.

In the present study, the authors evaluated sensitivity and specificity of DID, CIE and ELISA in detecting anti- F. pedrosoi antibodies in sera of patients with CBM caused by this fungus.

MATERIAL AND METHODS

Sera: Sixty sera samples obtained from patients with CBM caused by F. pedrosoi and followed at the "Clinica Dermatologica do Hospital das Clinicas da Universidade de SSo Paulo, Brasil" were analyzed. Diagnosis was confirmed by the presence of sclerotic cells in lesions (direct exam) and growth of F. pedrosoi in culture. Hyperimmune serum was produced by inoculation of two New Zealand males, 3 kg in weight rabbits with F. pedrosoi metabolic antigen, according to GARCIA et al.n. Scrum was precipitated by caprilic acid according to the methodology described by McKINNEY & PARKINSON20. Sera from 36 sporothricosis (SPT) patients, 34 cutaneous leishmaniasis (CL) patients and 48 from healthy blood donors (HBD) were used as controls.

Metabolic Antigen (Met-Ag): Culture filtrate of F. pedrosoi sample # 884 from Institute de Mcdicina Tropical de Sao Paulo Collection was used to produce Met-Ag. This sample was cultured on Sabouraud-agar at 25 °C for 10 days. The inoculum was prepared in 0.85% saline, to reach McFarland's scale 5. Five ml of the suspension was incubated in 250 ml of Sabouraud-broth at 25 °C for 30 days, under constant shaking. Culture was killed by addition of thimerosal at 1:5000 and filtrated through Whatman paper #1, according to VIDAL et al.31. The filtrate was concentrated (antigen) and maintained at 4 °C until use.

Somatic antigen (Som-Ag): Mycclial mats were washed in phosphate buffer solution (PBS) three times and mechanically disrupted using ice and glass bits. The suspension was left at 4 °C for two days followed by centrifuging at 3000 rpm for 30 minutes, according to GARCIA". The supernatant (antigen) was frozen at -20 °C until use.

Antigen protein content was determined according to LOWRY et al.19.

Double immunocliffusion (DID): Glass slides (25 X 75 mm) were covered with 3 ml of sodium citrate agar 1% and left at 4 °C for three hours. A seven-well template was stamped twice on the surface of each slide. Met-Ag was placed in the central well and sera samples in the surrounding six wells. The slides were incubated for 48 hours at room temperature and washed for further 48 hours using saline solution. A stove was used to dry the slides by evaporation. Slides were stained using 0.4% Coomassic Brilliant Blue (Sigma) in acetic acid 10% solution. Precipitation lines indicated positivity.

Counterimmunoelectrophoresis (CIE): The same slides used for DID were covered with barbital-buffered agarose (pH 8.2), to produce wells with 3 mm in depth. Thirteen ?? of Met-Ag and sera were placed in the wells. The slides were submitted to electrophoresis with barbital buffer (pH 8.2), 4 volts/cm, for 90 minutes through filter paper. Gels were processed as described for DID.

ELISA: Indirect ELISA was performed according to DEL NEGRO et al.1. For standardization purposes different aspects were evaluated: a) Antigen concentration: Somatic antigen optimal protein concentration was found to be 2.5 µg/ml, as demonstrated by dilution in carbonate/ bicarbonate buffer (pH 9.6); b) both positive and negative sera were used at 1:40 dilution (previously titrated) and each sample was analyzed in triplicate; c) Conjugate: a goat anti-human (reactive against IgG) peroxidase conjugate (Sigma) was diluted 1:700 (optimal concentration). Each experiment was performed including a step with 1% Bovine Serum Albumine (BSA). Optical density (OD) was determined using a 490 nm filter. Cut off value was determined as the average OD of control sera added two standard deviations, d) Sensitivity and specificity were calculated on homologous (60 CBM) and heterologous (36 SPT, 34 CL and 48 HBD) sera samples, according to LINNET18.

RESULTS

Hyperimmune scrum reacted both with Met-Ag by DID and CIE up to a 1:32 dilution. Met-Ag protein content was 4.90 mg/ml while for Som-Ag it was 0.70 mg/ml. Hyperimmunc serum showed 0.186 mg/ml protein content.

DID: 32 (53%) out of 60 CBM sera samples tested positive. Only 3 (8.3%) of 36 SPT sera and 3 (8.8%) out 34 CL sera tested positive. None of the 48 HBD sera tested positive. DID presented 53% sensitivity and 96% specificity.

CIE: 41 (68%) out of 60 CBM sera samples tested positive. Six (16.7%) out of 36 SPT sera, 6 (17.6%) out of 34 CL sera tested positive exclusively when undiluted sera samples were used. None of 48 HBD sera tested positive. CIE presented 68% sensitivity and 90.5% specificity. Table 1 describes all values of sensitivity and specificity of the different tests, against the all sera sample groups studied.

ELISA: Figure 1 demonstrates results about ELISA. Cut-off value was determined as 0.122 OD (black line). ELISA tested positive in 45 CBM sera, 10 SPT sera, 8 CL sera and 3 HBD sera. This test showed 78% of sensitivity and 83% of specificity.

DISCUSSION

Although serological tests arc not routinely used for CBM diagnosis, numerous papers on this matter have appeared in the literature. A wide array of different dematiaceous fungi antigens is currently available for use in serologie tests 23,5,6,9,10,14,16,24,28,29. The large experience using MetAg in gel precipitation serologie tests for diagnosis of deep mycosis led us to test this type of antigen using DID and CIE. BUCKEY & MURRAY5, testing CBM Met-Ag by DID, demonstrated that almost all patients developed precipitating antibodies at some phase of the disease.

VILLALBA & YEGRES33 emphasized that precipitation reactions tend to present low sensitivity because of the low amount of circulating antibodies found in CBM. These authors also reported cross-reactivity with health controls' sera and SPT patients' sera, which accounted for low specificity, a finding also observed by ISHIZAKI et al.15, who studied hyperimmune anti-Spomthix schenckii serum against different fungal antigens, including F. pedmsoi. VILLALBA32 testing Met-Ag by CIE demonstrated that this antigen presented higher specificity for F. pedmsoi than Som-Ag. ROMERO et al.26 evaluated sera samples of patients with CBM caused by Cladophialophora carrionii using DID and CIE. According to these authors DID presented higher specificity and sensitivity than CIE. In contrast, the present data showed that CIE presented superior sensitivity, but lower specificity.

In the present study ELISA was able to identify specific antibodies against F. pedmsoi Som-Ag in 45 (75%) of 60 CBM sera. It was 78% sensitive and 83% specific. In 1993, ANDRIANTSIMAHAVANDY et al.2 used ELISA to test sera of CBM patients from Madagascar. The authors used Som-Ag of both, F. pedmsoi and C. carrionii, the most prevalent species in the island. Based on 86% reproducibility and 65% specificity they concluded that ELISA, using F. pedmsoi and C. carrionii Som-Ag, was a satisfactory tool for biologic diagnosis of CBM. ESTERRE et al.9 used ELISA to analyse 136 sera samples obtained from 43 CBM patients enrolled in a one-year therapeutic trial of terbinafine. Sensitivity reached 87% and specificity 92.3%. ROMERO et al.21 evaluated ELISA test for CBM caused by C. carrionii in Venezuela using Som-Ag. Results were also encouraging (82.5% sensitivity and 81.8% specificity).

The data presented demonstrate that serological tests can be a useful tool to study CBM caused by F. pedmsoi specially when the culture of the pathogenic agent is not possible and on the basis of the results, the serological tests can be a useful tool to help a serological identification, immunological evaluate and monitoring the efficiency of treatment on CBM, after future studies. The identification of specific antigenic fractions is currently under investigation and results are promising.

RESUMO

Tccnicas de imunoprecipitacao c ELISA na dctcccao de anticorpos anti-Fonsecaea pedrosoi na cromoblastomicosc

Cromoblastomicosc (CBM) e infeccao subcutanea causada por varios fungos demaceos. O agente mais importante no Brasil e Fonsecaea pedrosoi, que se aprcsenta nas lesoes como celulas de coloracao acastanhada, com divisao transversal e longitudinal, originando as denominadas "celulas muriformes". Esta infeccao apresenta carater universal, mas paises como Madagascar e Brasil apresentam alla incidencia. O diagnostico e realizado atraves dos exames clinico, direto e histopatologico, acompanhado de cultura e identificacao do agente etiologico. Os testes sorologicos foram aplicados para identificar anticorpos especificos frente a antigenes de Fonsecaea pedrosoi e varias metodologias tem sido emprcgadas para identificacao sorologica e o diagnostico da CBM. Neste estudo, avaliamos reacocs de imunodifusao dupla (DID), contraimunoeletroforese (CIE) e teste imunoenzimatico (ELISA) para avaliar a resposta imune humoral na CBM causada por F. pedrosoi. Utilizamos antigeno metabolico para DID e CIE e antfgeno somatico para ELISA. Nossos dados rcvclaram 53% e 68% de sensibilidade e 96% e 90,5% de espccificidade, respectivamente. O teste de ELISA demonstrou 78% de sensibilidade e 83% de especificidade. Estes resultados indicam que as reacoes sorologicas podem ser uma ferramenta util no auxflio diagnostico desta infeccao, quando a cultura do agente nao for possivcl.

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Received: 4 june 2003

Accepted: 23 October 2003

Monica Scarpclli Martinelli VIDAL(I), Luis Guillierme Martins de CASTRO(2), Sonia Cristina CAVALCANTE(I) & Carlos da Suva LACAZ(I)[dagger]

1) Laboratorio de Micologia Medica do Instituto de Medicina Tropical de Sao Paulo/ LIM 53/HC FMUSP, Sao Paulo, SP, Brasil.

2) Clfnica Dermatotologica do Hospital das Clinicas da FMUSP, Sao Paulo, SP, Brasil.

[dagger] ln memoriam

Correspondence to: Monica S.M. Vidai, MD. Laboratorio de Micologia Medica do Instituto de Mcdicina Tropical de Sao Paulo/Univcrsidade de Sao Paulo. Av. Dr. Eneas Carvalho de Aguiar 500, terreo, 05403-000 Sao Paulo, SP, Brasil. Email: movida@usp.br

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