Medical Mycology December 2004, 42, 511 /515

Highly specic and sensitive, immunoblot-detected 54 kDa antigen from Fonsecaea pedrosoi
M. S. M. VIDAL*, L. G. M. CASTRO%, S. C. CAVALCANTE* & C. S. LACAZ*$ *Laborato rio de Micologia Me dica, Instituto de Medicina Tropical de Sa o Paulo and %Divisa o de Dermatologia, Hospital das Cl nicas, Faculdade de Medicina, Universidade de Sa o Paulo, Brazil

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Chromoblastomycosis (CBM) is a chronic subcutaneous mycosis caused by a group of different dematiaceous fungi, first described by Rudolph in 1914. In Brazil there is a clear predominance of Fonsecaea pedrosoi . Sixty sera samples obtained from patients with F. pedrosoi- caused CBM were analysed. Sera obtained from 36 sporothricosis (SPT) patients, 34 cutaneous leishmaniasis (CL) patients and from 48 blood donors (HBD) were used as control. F. pedrosoi metabolic antigen was obtained from F. pedrosoi sample no. 884 (Instituto de Medicina Tropical de Sa o Paulo Collection). IE reaction disclosed an anodic migrating arch, which was eluted and used as antigen. Both metabolic and eluate F. pedrosoi antigens were submitted to SDS PAGE and two fractions, weighing approximately 54 and 66 kDa were identified. The 66-kDa fraction reacted against 43 of 60 CBM (71.7%) sera samples and was recognized by 10 SPT and eight CL sera (15.3%). No reactivity was observed against HBD sera. The 54-kDa fraction reacted against 58 of 60 CBM sera (96.7% sensitivity) and was not recognized by HBD, SPT nor CL sera (100% specificity). Such high sensitivity and specificity levels suggest this antigenic fraction is immunodominant and might prove a useful tool for further studies on F. pedrosoi- caused CBM.
/

Keywords serology

antigenic

fraction,

chromoblastomycosis,

Fonsecaea

pedrosoi ,

Introduction
Chromoblastomycosis (CBM) is a chronic fungal infection of the skin and subcutaneous tissue caused by a group of different dematiaceous fungi, first described by Rudolph in 1914 [1,2]. In Brazil the most common agent is Fonsecaea pedrosoi [2 /4]. The first published studies on serologic aspects of CBM appeared in 1927, when Montpellier and Catanei [5] evaluated agglutination reaction of Phialophora pedrosoi (now F. pedrosoi ) conidia against CBM patients

Received 24 June 2003; Accepted 18 November 2003 Correspondence: Mo nica S. M. Vidal, Instituto de Medicina Tropical de Sa rio de Micologia Me dica, Av. Dr Ene as de o Paulo, Laborato Carvalho Aguiar, 500 Sa o Paulo, SP, Brazil. Tel.: '/55 11 3066 7046; Fax: '/55 11 3062 3622; E-mail: movida@usp.br $ Deceased April 2002.

sera. A few years later, Martin et al. [6] demonstrated that complement fixation reaction-detected, specific antibodies, decreased during treatment. In 1970, Cooper and Schneidau [7] used double immunodiffusion (DID) and immunoelectrophoresis (IE) to test Cladophialophora carrionii (formerly Cladosporium carrionii ), Phialophora verrucosa and Fonsecaea pedrosoi antigens against rabbit-produced specific hyperimmune sera. They demonstrated antigens of the three species cross-reacted. Numerous 7.6 /78.5-kDa fractions of F. pedrosoi antigens were detected through electrophoresis with polyacrilamide gel (SDS /PAGE) by Ibraim-Granet et al . [8,9]. Esterre et al . [10,11] studied 136 sera obtained from CBM patients from Madagascar using immunoenzymatic test, enzyme linked immunoadsorbent assay (ELISA) and immunoblotting (IB).
DOI: 10.1080/13693780310001654337

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Materials and methods

Sixty sera samples obtained from patients with F. pedrosoi- caused CBM were analysed. Patients were followed at the Dermatology Clinic, Hospital das Cl nicas, University of Sa o Paulo Medical School. Diagnosis of CBM was confirmed by a positive culture in all cases and by the presence of muriform cells in 10% KOH cleared specimens or in H&E histological sections. Sera from 36 sporothricosis patients (SPT), 34 cutaneous leishmaniasis patients (CL) and 48 healthy blood donors (HBD) were used as control.

supernatant was used as antigen and kept at /208C until use.

Immunoelectrophoresis (IE)
The glass slides were covered by 6 ml barbital-buffered agarose (pH 8.2) and were left at 48C for 3 h and 15 ml Met-Ag, [conc. )/20], were placed in the well and submitted to electrophoresis with barbital buffer (pH 8.2) at 4 V/cm for 1.5 h. All sera samples (CBM, SPT, CL and HBD) were placed in the second well and incubated at room temperature for 48 h. The slides were washed with saline solution for 48 h, dried by evaporation in a stove and stained by 0.4% Coomassie Brilliant Blue (Sigma) in 10% acetic acid solution. All CBM sera samples were tested against S. schenckii metabolic antigen. Presence of a precipitation arch indicated positivity [13].

F. pedrosoi metabolic antigen (Met-Ag) Culture filtrate was obtained from F. pedrosoi sample no. 884 (isolated from a patient Instituto de Medicina Tropical de Sa o Paulo Collection). This sample was cultured on Sabouraud agar at 258C for 10 days. The inoculum was prepared in 0.85% saline, according to scale 5 of McFarland and 5 ml of the suspension was inoculated into 250 ml Sabouraud broth at 258C for 30 days, under constant shaking. The culture was killed by addition of thimerosal at 1:5000 [final]. The filtrate was concentrated by evaporation, filtrated through Whatman paper no. 1 and kept at 48C until use [12].

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SDS /PAGE
The optimal conditions for El-Ag were as follows: duodecil sulfate polyacrylamide gel electrophoresis with 12% acrylamide gel carried out for 2 h at 30 mA, on Mini-Protean II (BioRad) [14].

Immunoblotting (IB)
F. pedrosoi eluate antigen (El-Ag) Immunoelectrophoresis (IE) reaction disclosed an anodic migrating arch (Fig. 1), which was dissected from the agarose gel using a scalpel. The gel fragment containing the arch was incubated in saline solution, at 48C in PBS for 7 days. After centrifuging, the IB was used to test the presence of specific antibodies against the El-Ag. Protein transfer was performed with 40 V overnight at 48C in glycine buffer (pH 8.6), on Trans-Blot System (BioRad). Incubation of nitrocellulose paper was performed with sera (1:20 dilution), before the addition of a goat anti-human (IgG) peroxidase conjugate (Sigma) diluted 1:2000. Reaction evaluation was performed by addition of 3,3?diaminobenzidine to this buffer (pH 7.5) with hydrogen peroxidase [14].

Sensitivity and specificity

Sensitivity and specificity of the reactions were determined according to Linnet [15].

Results
IE
Thirty-four out of 60 CBM sera samples recognized an anodic migrating arch (Fig. 1). None of the control sera samples by IE recognized the arch. IE demonstrated 57.0% sensitivity and 100.0% specificity (Table 1). CBM sera sample did not recognize S. schenckii antigen.
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Fig. 1 Immunoelectrophoresis of CBM serum demonstraing an anodic migrating arch, which was eluted for obtention of El-Ag. 1) sporothricosis patient serum (control) 2) chromoblastomycosis patient serum 3) F. pedrosoi Met-Ag

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Table 1 Sensitivity and specicity values obtained for IE and IB with Fonsecaea pedrosoi Met-Ag and El-Ag against CBM and control sera Test Antigen Sensitivity (CMB)* Control sera Specificity (SPT and CL)$ IE IB IB MET-Ag EL-Ag 54 kDa EL-Ag 66 kDa 57.0% (34/60) 96.7% (58/60) 71.7% (43/60) 100.0% (0/70) 100.0% (0/70) 74.3% (18/70) Specificity (HBD)% 100.0% (0/48) 100.0% (0/48) 100.0% (0/48) 100.0% (0/118) 100.0% (0/118) 84.7% (18/118) Total specificity

*Reactive CBM sera/total CBM sera; $reactive SPT and CL sera/total SPT and CL sera; %reactive HBD sera/total HBD sera; reactive control sera/total (SPT'/CL'/HBD) control sera; IE, immunoelectrophoresis; IB, immunoblotting; MET-Ag, F. pedrosoi metabolic antigen; El-Ag, F. pedrosoi eluate antigen; CBM, chromoblastomycosis; SPT, sporothricosis; CL, cutaneous leishmaniasis; HBD, health blood donors.

SDS /PAGE
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Discussion
Serological reactions are not routinely used for diagnosis of CBM because direct exam and histology have proved efficacious. A better understanding of host immune response and identification of specific antigens of CBM-causing fungi may prove helpful. Antigen preparation methodology and standardization of the different reactions have varied widely, making it difficult to compare results. Some authors used metabolic antigens (precipitated or not), while others analysed cell extracts obtained by different techniques such as DID and CIE [16 /19,21], IE [7], ELISA and IB [8,10,11,20,22]. Cooper and Scheneidau [6] studied several antigens of CBM-causing dematiaceous fungi (F. pedrosoi, C. carrionii and P. verrucosa ) using ID and IE. Some degree of cross-reactivity between the different antigens was noted. Cross-reactivity was a common finding among the antigens obtained from these three species, but F. pedrosoi fractions were clearly more specific. IE of a F. pedrosoi /Met-Ag disclosed an isolated anodic migrating arch (Fig. 1). In 1984, Alborno z et al . [23] described an anodic migrating arch in S. schenckii metabolic antigen (S arch). It was recognized by 100% of SPT sera analysed by IE. In order to study a possible cross-reactivity between sporothicosis S arch and the anodic migration arch present in F. pedrosoi /Met-Ag, we tested the 60 CBM sera against S. schenckii metabolic antigen and 36 SPT sera against F. pedrosoi /Met-Ag. Results showed a total absence of reactivity, indicating a high degree of specificity of the F. pedrosoi arch. In order to obtain a F. pedrosoi -specific antigenic fraction, characterization of the IE detected arch was carried out. The arch present in the agarose gel (El-Ag) was eluted for electrophoretic analysis (SDS /PAGE) and two fractions, with approximate molecular weight

Both metabolic and eluate F. pedrosoi antigens submitted to electrphoresis presented two fractions, weighing approximately 54 and 66 kDa (Fig. 2).

IB
The 66-kDa fraction reacted against 43 of 60 CBM (71.7%) sera samples and was recognized by 10 SPT and eight CL sera (25.7%). No reactivity was observed against HBD sera. The 54-kDa fraction reacted against 58 of 60 CBM (96.7%) sera (Fig. 3) and was not recognized by HBD, SPT nor CL sera. IB sensitivity and specificity against CBM, SPT, CL and HBD sera appear in Table 1.

Fig. 2 SDS-PAGE, gel 12%. Demonstrating 66 and 54 kDa fractions in F. pedrosoi Met-Ag and El-Ag. a) molecular weight standard b) F. pedrosoi Met-Ag c) F. pedrosoi El-Ag

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Fig. 3 54 and 66 kDa fractions recognized by CBM sera through immunoblotting.

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of 54 and 66 kDa were identified (Fig. 2). Both fractions were also present in crude F. pedrosoi /MetAg. In 2000, Esterre et al . [11] demonstrated by IB the presence of four fractions in F. pedrosoi /Met-Ag. Three fractions (26, 36 and 40 kDa) were present in C. carrionii Met-Ag, while a 18.5 kDa was F. pedrosoi specific. These fractions were not detected in the present study. This finding may be explained by differences in methodology of obtaining the Met-Ag, such as incubation period (10 /15 days vs. 30 days) and F. pedrosoi isolate (sample IPM-A8 versus IMTSP 884). Ibrahim-Granet et al . [8] used electrophoresis to study the protein profile of antigens obtained from several isolates of F. pedrosoi. The protein fractions ranged from 7.6 to 78.5 kDa. We believe that both 66- and 54-kDa fractions identified in the present study correspond to the 67- and 55-kDa fractions identified by Ibrahim-Granets group three years later [9]. In these studies the 18.5-kDa fraction was not mentioned. Both 54- and 66-kDa fractions were tested by IB against 60 CBM, 36 SPT, 34 CL and 48 HBD sera. The 66-kDa fraction demonstrated 71% sensitivity (43/60) and 84.7% specificity (Table 1). Specificity against HBD sera was 100% while against SPT/CL sera this value decreased to 74.3%. The 54-kDa fraction presented more impressive results. Sensitivity reached 96.7% and specificity 100%, both for SPT/CL and HBD sera. Such high levels of sensitivity and specificity suggest this antigenic fraction is immunodominant and might prove a useful tool for further studies on CBM.

Authors note
This paper is the result of one of the last studies in which Professor Carlos da Silva Lacaz took active part; the authors therefore dedicate its publication to his memory.

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