Medical Mycology December 2004, 42, 511 /515
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*$
*Laboratório de Micologia Médica, Instituto de Medicina Tropical de São Paulo and %Divisão de Dermatologia, Hospital das
Clı́nicas, Faculdade de Medicina, Universidade de São 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 São 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,
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: Mônica S. M. Vidal, Instituto de Medicina Tropical
de São Paulo, Laboratório de Micologia Médica, Av. Dr Enéas de
Carvalho Aguiar, 500 São Paulo, SP, Brazil. Tel.: /55 11 3066 7046;
Fax: /55 11 3062 3622; E-mail: [email protected]
$
Deceased April 2002.
– 2004 ISHAM
chromoblastomycosis,
Fonsecaea
pedrosoi,
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
512
Vidal et al.
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
Clı́nicas, University of São 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.
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 São 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].
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].
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
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
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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Immunoblot-detected antigen from F. pedrosoi
513
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)*
Total
specificity§
Control sera
Specificity
(SPT and CL)$
Specificity
(HBD)%
IE
MET-Ag
57.0%
(34/60)
100.0%
(0/70)
100.0%
(0/48)
100.0%
(0/118)
IB
EL-Ag 54 kDa
96.7%
(58/60)
100.0%
(0/70)
100.0%
(0/48)
100.0%
(0/118)
IB
EL-Ag 66 kDa
71.7%
(43/60)
74.3%
(18/70)
100.0%
(0/48)
84.7%
(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.
SDS /PAGE
Discussion
Both metabolic and eluate F. pedrosoi antigens submitted to electrphoresis presented two fractions, weighing approximately 54 and 66 kDa (Fig. 2).
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, Albornóz 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
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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514
Vidal et al.
Fig. 3 54 and 66 kDa fractions recognized by
CBM sera through immunoblotting.
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-Granet’s 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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