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Highly specific and sensitive, immunoblot-detected 54 kDa

antigen from Fonsecaea pedrosoi

M. S. M. VIDAL*, L. G. M. CASTRO%, S. C. CAVALCANTE* & C. S. LACAZ*$

*Laboratorio de Micologia Medica, Instituto de Medicina Tropical de Sao Paulo and%Divisao de Dermatologia, Hospital das

Clnicas, Faculdade de Medicina, Universidade de Sao Paulo, Brazil

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 Sao 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 approximately54 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 antigenic fraction, chromoblastomycosis, Fonsecaea pedrosoi,

serology

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 pedro-

soi (now F. pedrosoi) conidia against CBM patients

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 immunodiffu-

sion (DID) and immunoelectrophoresis (IE) to test

Cladophialophora carrionii (formerly Cladosporium

carrionii), Phialophora verrucosa and Fonsecaea pedro-

soi antigens against rabbit-produced specific hyperim-

mune sera. They demonstrated antigens of the three

species cross-reacted. Numerous 7.6/78.5-kDa frac-tions 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 immuno-

blotting (IB).$ Deceased April 2002.

Correspondence: Monica S. M. Vidal, Instituto de Medicina Tropical

de Sao Paulo, Laboratorio de Micologia Medica, Av. Dr Eneas de

Carvalho Aguiar, 500 Sao Paulo, SP, Brazil. Tel.: '/55 11 3066 7046;

Fax: '/55 11 3062 3622; E-mail: [email protected]

Received 24 June 2003; Accepted 18 November 2003

2004 ISHAM DOI: 10.1080/13693780310001654337

Medical Mycology December 2004, 42, 511/515

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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

Clnicas, University of Sao Paulo Medical School.

Diagnosis of CBM was confirmed by a positive culture

in all cases and by the presence of muriform cells in10% 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.

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 Sao Paulo Collection). This sample was

cultured on Sabouraud agar at 258C for 10 days. The

inoculum was prepared in 0.85% saline, according toscale 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 What-

man paper no. 1 and kept at 48C until use [12].

F. pedrosoi eluate antigen (El-Ag)

Immunoelectrophoresis (IE) reaction disclosed an ano-

dic 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

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 andsubmitted 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].

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)

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 nitrocellu-

lose 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 deter-

mined 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.

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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SDS/PAGE

Both metabolic and eluate F. pedrosoi antigens sub-

mitted to electrphoresis presented two fractions, weigh-

ing 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.

Discussion

Serological reactions are not routinely used for diag-

nosis of CBM because direct exam and histology have

proved efficacious. A better understanding of hostimmune response and identification of specific antigens

of CBM-causing fungi may prove helpful.

Antigen preparation methodology and standardiza-

tion 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 antigenswas 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, Albornoz 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. ped-

rosoi/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

Table 1 Sensitivity and specificity 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 Totalspecificity

Specificity

(SPT and CL)$

Specificity

(HBD)%

IE MET-Ag 57.0% 100.0% 100.0% 100.0%

(34/60) (0/70) (0/48) (0/118)

IB EL-Ag 54 kDa 96.7% 100.0% 100.0% 100.0%

(58/60) (0/70) (0/48) (0/118)

IB EL-Ag 66 kDa 71.7% 74.3% 100.0% 84.7%

(43/60) (18/70) (0/48) (18/118)

*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.

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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of 54 and 66 kDa were identified (Fig. 2). Both

fractions were also present in crude F. pedrosoi/Met-Ag.

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 thisvalue 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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