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Am. J. Trop. Med. Hyg., 66(1), 2002, pp. 91102

Copyright  2002 by The American Society of Tropical Medicine and Hygiene

EVALUATION OF ANTIGENS FROM VARIOUS LEISHMANIA SPECIES IN A WESTERN

BLOT FOR DIAGNOSIS OF AMERICAN TEGUMENTARY LEISHMANIASIS
CELY CRISTINA MARTINS GONC
ALVES, EDNA MARIA VISSOCI REICHE, BENICIO ALVES DE ABREU FILHO,
NIA CRISTINA FELIZARDO, KAROLINE ROCHA MAIA, RAFAEL
THAIS GOMES VERZIGNASSI SILVEIRA, TA
PADOVESI, BENEDITO PRADO DIAS FILHO, SHIDUCA ITOW JANKEVICIUS, AND
COSTACURTA, EVANDRO JOSE
VITOR JANKEVICIUS
JOSE
Laborato rio de Tripanosomatdeos, Centro de Ciencias Biologicas, Universidade Estadual de Londrina, Londrina, Parana, Brazil;
Hospital Universitario Regional Norte do Parana, Centro de Ciencias da Saude, Universidade Estadual de Londrina, Londrina,
Parana, Brazil; Departamento de Analises Clnicas da Universidade Estadual de Maringa, Maringa, Parana, Brazil; Ambulatorio
do Hospital da Sociedade Filantropica Humanitas, Sao Jeronimo da Serra, Parana, Brazil

Abstract. A Western blot method that uses antigens from culture promastigote forms of Leishmania (Viannia)
braziliensis, L. (Leishmania) amazonensis, L. (Leishmania) tropica, and a trypanosomatid (strain 268T) isolated from
naturally infected tomatoes was evaluated for laboratory diagnosis of American tegumentary leishmaniasis (ATL).
Serum samples were obtained from 108 patients with ATL (group I), 23 chagasic patients (group II), 32 patients with
other diseases (group III), and 78 healthy individuals (group IV). The overall analysis showed a sensitivity of 76.90%,
90.40%, 78.50%, and 87.90%, a specificity of 100%, 93.80%, 87.80%, and 77.10%, a positive predictive value of
100%, 94.00%, 89.50%, and 72.50%, a negative predictive value of 75.70%, 90.00%, 75.40%, and 90.20%, and a
concordance coefficient kappa of 0.7358, 0.8400, 0.6491, and 0.6287 for L. (V.) braziliensis, L. (L.) amazonensis, L.
(L.) tropica, and strain 268T antigens, respectively. The antigenic profile recognized by serum samples from patients
with ATL and with Chagas disease permits serologic distinction between these infections.
in the clinical laboratory.2022 The parasites Trypanosoma
cruzi and Leishmania species share antigenic determinants
that cause significant cross-reactions in serologic tests, thus
impairing accurate laboratory diagnosis of the specific infections.23,24 Other trypanosomatid-derived antigens, which
also share antigenic determinants with Leishmania, are easily produced and contain epitopes recognized by human antibodies. They could be an alternative source of antigens for
studying the humoral immune response in leishmaniasis and,
perhaps, an alternative laboratory diagnostic method with
minimal expense and no risk of laboratory infection.25 We
report the results of Western blot studies with antigens derived from Leishmania (Viannia) braziliensis, L. (Leishmania) amazonensis, L. (Leishmania) tropica, and a trypanosomatid (strain 268T) isolated from naturally infected tomatoes
and serum samples from humans with ATL, those with other
diseases, and healthy individuals (controls).

INTRODUCTION
The flagellated protozoa of the Trypanosomatidae family
comprises important human parasites, mainly Trypanosoma
cruzi, the pathogen that causes Chagas disease and various
species of Leishmania, which are the etiologic agents of a
spectrum of diseases known as cutaneous, tegumentary, and
visceral leishmaniasis.1 They are all endemic in many countries in South America and their geographic distribution
overlaps in many areas. American tegumentary leishmaniasis (ATL) is transmitted by the sand fly vector (Phlebotominae of the genus Lutzomyia) to a suitable host in which
Leishmania invades host macrophages, starting a localized
chronic granulomatous lesion at the sand fly bite site.2 The
diagnosis of ATL is based mainly on the clinical picture and
the identification of the parasite from skin or bucco-pharyngeal lesions.3 The parasitologic methods have always been
considered first-choice procedures for the diagnosis of leishmaniasis due to their specificity of 100%, although with variable sensitivities.46 However, in some instances it is very
difficult to demonstrate the presence of parasites7,8 and immunodiagnosis then becomes an important alternative for
demonstrating the presence of the parasite.9,10 The main line
of defense against infection in ATL is the cell immune response. The delayed intradermal Montenegro test is the best
method for evaluation,11,12 but present infections are not distinguished from past infections.1,13 The humoral immune response occurs only during the active phase of infection, with
the appearance of low titers of antibodies, representing a
temporary response.14,15
Several techniques have been developed for the serologic
diagnosis of leishmaniasis. These include an immunofluorescence assay (IFA), an enzyme-linked immunosorbent assay (ELISA), and the Western blot.1618 The Western blot
technique is highly sensitive and specific, and provides more
information about the parasite antigenic profile.19 Crude extracts of cultured promastigote forms of Leishmania species
are the current source of antigen for seroimmunologic assays

MATERIALS AND METHODS

This study was reviewed and approved by the Internal
Scientific Commission and the Bioethics in Research Committee of the Universidade Estadual de Londrina (UEL),
Londrina, Parana, Brazil.
Human sera. The serum samples used were divided into
four groups.
Group I. One hundred eight serum samples were obtained
from individuals with a clinical diagnosis of ATL defined by
an active lesion and coming from endemic areas. Twentynine serum samples were from the Universidade Estadual de
Maringa (UEM) (Maringa, Parana, Brazil) from patients reactive by an IFA, and positive by the Montenegro skin test
and microscopy. The other 79 serum samples were obtained
from patients reactive in at least one conventional laboratory
test for ATL, such as the IFA, the Montenegro skin test, or
microscopy: 74 from patients seen at the Outpatient Department (Ambulatorio) of the Hospital das Clnicas, Universidade Estadual de Londrina (UEL) (Londrina, Parana, Brazil)

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and five from the Outpatient Department (Ambulatorio) of

the Hospital da Sociedade Filantropica Humanitas (HSFH)
(Sao Jeronimo da Serra, Parana, Brazil).
Group II. Twenty-three serum samples were obtained
from patients seen at the Hospital Universitario Regional
Norte do Parana (HURNP) with a serologic diagnosis of
Chagas disease (by IFA and ELISA) and without lesions
suggestive of leishmaniasis.
Group III. Thirty-two serum samples were obtained from
patients seen at HURNP with a laboratory diagnosis of other
infectious or autoimmune diseases: toxoplasmosis (3), syphilis (5), antinuclear antibodies (8), microsomal antibodies
(5), streptococcus infection (5), anti-dsDNA antibodies (2),
and leprosy (4). None of these patients had any lesions suggestive of leishmaniasis.
Group IV. Seventy-eight serum samples were obtained
from donors at the hemotherapy unit of the HURNP. They
were seronegative for Chagas disease, antibodies to hepatitis C, hepatitis B surface antigen, hepatitis B virus core
antigen, antibodies to human immunodeficiency virus, and
antibodies to human virus T cell lymphotrophic virus types
1/2, and had negative Venereal Disease Research Laboratory
test results and normal levels of alanine aminotransferase.
They were used as negative controls.
Serologic tests for leishmaniasis. Indirect immunofluorescence assay. This test was performed according the standards used by each institution (UEL, UEM, or HSFH). In
the serum samples from HURNP and HSFH, the titers of
antibodies against Leishmania were measured using fixed L.
braziliensis or L. donovani promastigotes on commercially
available slides (Lio Serum; Industria e Comercio de Equipamentos e Produtos para Laboratorio Ltda., Ribeirao Preto,
Brazil and Cecon, Sao Paulo, Brazil) and fluorescein-conjugated goat anti-human IgG (Laborclin; Produtos para Laboratorio Ltda, Pinhais, Parana, Brazil and The Binding Site
Ltd., Birmingham, United Kingdom) as a second antibody
at an initial serum dilution of 1:20. The serum samples that
came from UEM were assayed using fixed L. (V.) braziliensis (M11272) originally isolated from a patient with cutaneous leishmaniasis and fluorescein-conjugated goat anti-human IgG (Biolab Merieux, Rio de Janeiro, Brazil) as a second antibody at an initial serum dilution of 1:20.
The Montenegro skin test. In the patients seen at HURNP
and HSFH, the Montenegro skin test was performed using
commercially available antigens of L. braziliensis or L. donovani promastigotes (Reagentes Biologicos Pimenta Abreu
Ltda., Sao Paulo, Brazil). In the patients seen at UEM, antigens from L. (V.) braziliensis (M11272) were used. The
reactions were performed by injecting intradermally 0.1 ml
of antigen into the forearm. The test was read 4872 hr later
and induration 5 mm in diameter was considered positive.
Parasites. Leishmania (V.) braziliensis strain M11272
(serodeme 1) was originally isolated at UEM from a patient
with cutaneous leishmaniasis and identified as L. (V.) braziliensis by monoclonal antibodies and isoenzyme analysis
by Dr. J. J. Shaw (Instituto Evandro Chagas, Belem, Para,
Brazil). This strain was maintained by continuous passages
in hamsters by UEM. Leishmania (L.) amazonensis was
maintained by successive passages into biphasic medium at
the Laboratorio de Tripanossomatdeos of UEL. Leishmania
(L.) tropica (strain ATCC 30012) was maintained by suc-

cessively passages into biphasic medium at the Laboratorio

de Tripanossomatdeos of UEL. A trypanosomatid (strain
268T) was isolated from tomatoes (Lycopersicon esculentum) naturally infected in the Londrina region (Parana, Brazil) by the Laboratorio de Tripanossomatdeos of UEL in
1989. This parasite was maintained by successive passages
into glucose yeast peptone meat infusion (GYPMI) medium.26
Preparation of Leishmania and trypanosomatid isolated from tomato (strain 268T) antigens. The leishmanial
and strain 268T antigens were derived from culture promastigotes forms. L. (L.) tropica and L. (L.) amazonensis were
grown in liver infusion tryptose medium (LIT)27 containing
10% fetal calf serum (Sigma Chemical Co., St. Louis, MO)
at 25C without shaking. Leishmania (V.) braziliensis was
grown in the BAB (blood agar base; Oxoid USA Inc, Columbia, MD) biphasic medium with 15% rabbit defibrinated
blood) in a tissue culture flask28 with an LIT overlay.29 The
cells were cultured at 25C without shaking. Strain 268T was
cultured in GYPMI medium at 28C without shaking. The
cells were harvested in the late log phase.30,31 Antigens for
the Western blot test were prepared by previously published
methods.3234 The cells were pelleted by centrifugation at
3,200 g for 10 min at 4C, and washed four times with
150 mM phosphate-buffered saline (PBS), pH 7.2, by centrifugation at 3,200 g for 10 min at 4C. The sediment
was resuspended in sample buffer (0.125 M Tris-HCl, 4%
sodium dodecyl sulfate [SDS], 20% glycerol, 5% -mercaptoethanol, 0.042% bromophenol blue, pH 6.8) and boiled for
5 min. The suspension was centrifuged at 3,200 g for 30
min at 4C, and the supernatant was divided into aliquots
and stored at 20C up to the time of use. Protein concentration in the antigenic suspension was determined by the
Bradford method35 using bovine serum albumin (1,000 g/
ml) as a standard.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The discontinuous SDS buffer system36
was used with 0.8 mm-thick slab gels with a 7.515.0%
polyacrylamide gradient in the running gel. The stacking gel
was 5% acrylamide and had one narrow (1 cm wide) lane
and one wide (8.6 cm wide) lane. The narrow lane was used
for the molecular weight markers and the wide lane was used
for 200300 l of freshly solubilized parasite antigens containing 1 mg of protein equivalent per gel. Carbonic anhydrase (29 kD), egg albumin (45 kD), and bovine serum albumin (66 kD) (SDS-200 Kit; Sigma Chemical Co.) were
used as molecular weight markers. The molecular weight
markers were diluted in the same sample buffer as the parasite antigens, boiled for 5 min, and applied to the gel. Running buffer (0.025 M Tris-base, 0.92 M glycine, 0.1% SDS,
pH 8.8) was added and the samples were subjected to electrophoresis at 110 V (25 mA) until the stain front reached
the bottom of the gel. The gel was then stained with Coomassie brilliant blue R 250 for proteins at room temperature
overnight.37 The gel was dried between two wet cellophane
sheets and 10% gelatin (w/v) for 72 hr at room temperature.
The relative molecular weight (MWr) of each Leishmania
and strain 268T electrophoresed protein fraction was determined.38 The gel was also mounted in a protein transfer apparatus and subjected to electroimmunotransfer blotting.
Enzyme-linked immunoelectrotransfer blot (Western

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WESTERN BLOT FOR TEGUMENTARY LEISHMANIASIS

FIGURE 1. Western blot analysis of sodium dodecyl sulfatepolyacrylamide gel electrophoresis of promastigote forms of the Leishmania
(Viannia) braziliensis strain recognized by A, serum samples from patients with American tegumentary leishmaniasis (ATL) (group I, lanes
110); B, serum samples from patients with Chagasdisease (group II, lanes 1013), patients with other diseases (group III, lanes 69), and
healthy individuals (group IV, lanes 15). MW molecular weight markers; P positive control serum; N negative controls serum; kDa
kilodaltons.

blot). The polypeptides in the gel were transferred to nitrocellulose sheets (pore size 0.45 m, Probind 45 membrane
roll, Pharmacia Biotech, San Francisco, CA) at 400 mA (48
V) at 4C for 3 hr in a Trans Blot apparatus (Electrophoresis
Power Supply EPS-600; Pharmacia Biotech). This method
was performed according to the procedure of Towbin and
others39 with some modifications.40 The nitrocellulose sheet
was cut into vertical 5-mm strips. The strips were blocked
with a solution of 5% defatted (skim) milk (Molico, Nestle,
Sao Paulo, Brazil), 0.1% Tween 20 in PBS, pH 7.2, for 2 hr
at room temperature with constant shaking, rinsed with PBS,
and treated for 1 hr at room temperature with constant shaking with serum samples diluted 1:50 in PBS containing 5%
defatted milk and 0.1% Tween 20. The strips were then
washed three times (10 min/wash) with PBS containing
0.1% Tween 20, and treated for 1 hr at room temperature
with a peroxidase-labeled, affinity-purified rabbit anti-human
IgG conjugate (Sigma Chemical Co.) diluted 1:2,000 in PBS,
pH 7.2, containing 5% defatted milk and 0.1% Tween 20.
After three additional washes (10 min/wash), the substrate
(30% H2O2, 0.3% 4-chloro-1-naphthol in methanol dissolved
in PBS) was added. The color was allowed to developed for
30 min at room temperature in the dark. The strips were
then rinsed with distilled water, dried, and photographed.
The results were compared visually with the positive and
negative controls run in parallel. Human serum was omitted
in some nitrocellulose strips as a control for nonspecific
binding of the conjugate to antigens.
The reactivity criterion used for the interpretation of the
Western Blot results was based on the antigenic bands that
were recognized most frequently by the serum samples assayed.41,42 A serum sample was considered positive when it
recognized at least one antigenic band from a group of three
with the highest frequency of recognition. The result was

considered negative when the serum sample showed no reactivity with these diagnostic antigenic bands.
Statistical analysis. Evaluation of Western blot results
was based on sensitivity, specificity, positive and negative
predictive values, and the kappa index concordance.43 Sensitivity was defined as the number of samples with a Western
blot-positive result/the number of samples with a positive
diagnosis by conventional methodology (group I) 100.
Specificity was defined as the number of samples with a
Western blot-negative result/the number of samples with a
negative diagnosis for leishmaniasis (groups II, III, and IV)
100. The EpiI-Info program (version 6.04 b) (Centers for
Disease Control and Prevention, Atlanta, GA) was used for
statistical analysis.
RESULTS
Gel electrophoresis. The soluble protein profiles of the
parasites obtained by SDS-PAGE showed at least 41 major
protein bands to L. (V.) braziliensis with relative MWr values
ranging from 13 kD to 150 kD, 39 bands to L. (L.) amazonensis with MWr values from 13 kD to 140 kD 39 bands to
L. (L.) tropica with MWr values ranging from 13 kD to 120
kD, and 44 bands to strain 268T with MWr values from 16
kD to 170 kD.
Immunologic detection of antigens from various Leishmania species and strain 268T by Western blot. Several
antigenic bands of the various Leishmania species and strain
268T were recognized by the serum samples from groups I,
II, III, and IV at different frequencies and intensities of reaction and exhibit a complex pattern of reactivity (Figures
14).
The average number of bands recognized by the serum
samples from groups I, II, III, and IV of the Leishmania (V.)

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FIGURE 2. Western blot analysis of sodium dodecyl sulfatepolyacrylamide gel electrophoresis of promastigote forms of the Leishmania
(Leishmania) amazonensis strain recognized by A, serum samples from patients with American tegumentary leishmaniasis (ATL) (group I,
lanes 114); B, serum samples from patients with Chagas disease (group II, lanes 48), patients with other diseases, (group III, lanes 13),
and healthy individuals (group IV, lanes 913). P positive control serum; N negative control serum; MW molecular weight markers;
kDa kilodaltons.

braziliensis, L. (L.) amazonensis, L. (L.) tropica, and strain

268T antigens in Western blots are shown in Table 1.
Leishmania (V.) braziliensis. All 41 L. (V.) braziliensis
polypeptides stained with Coomassie brilliant blue in SDSPAGE showed reactivity with at least one serum sample
from groups I, II, III, and/or IV in Western blots.
The serum samples from patients with ATL (group I) recognized 34 antigenic bands of L. (V.) braziliensis and the
most frequently recognized bands (diagnostic for ATL) are

shown in Table 2. In this group, 5.60% of the serum samples

recognized no band, 62.80% recognized between one and
seven bands, and the remainder recognized more than seven
bands. Serum samples from patients with Chagas disease
(group II) showed a strong intensity of reaction to several
antigen bands of L. (V.) braziliensis, with 73.90% recognizing between eight and 14 bands and only 4.35% showing no
reactivity. In this group, Western blotting detected antibodies
that recognized bands with MWr values ranging from 13 kD

FIGURE 3. Western blot analysis of sodium dodecyl sulfatepolyacrylamide gel electrophoresis of promastigote forms of the Leishmania
tropica strain recognized by A, serum samples from patients with American tegumentary leishmaniasis (ATL) (group I, lanes 113); B, serum
samples from patients with Chagas disease (group II, lanes 48), patients with other diseases (group III, lanes 13), and healthy individuals
(group IV, lanes 915). P positive control serum; N negative control serum; MW molecular weight markers; kDa kilodaltons.

95

WESTERN BLOT FOR TEGUMENTARY LEISHMANIASIS

FIGURE 4. Western blot analysis of sodium dodecyl sulfatepolyacrylamide gel electrophoresis of promastigotes forms of the 268T strain
recognized by A, serum samples from patients with American tegumentary leishmaniasis (ATL) (group I, lanes 112); lane 13 whole cell
proteins stained with Ponceau, and patients with other diseases (group III, lanes 1416); B, serum samples from patients with Chagas disease
(group II, lanes 16), and healthy individuals (group IV, lanes 711). P positive control serum; N negative control serum; MW
molecular weight markers; kDa kilodaltons.

to 120 kD. The most reactive bands (diagnostic for Chagas

disease) are shown in Table 2. In serum samples from patients with other diseases (group III), no band was recognized by 31.30%, while 53.10% recognized between one and
seven bands by Western blotting. The serum samples from
patients with a positive diagnosis for leprosy recognized a
smaller number of components, with MWr values between
58 kD and 120 kD. Serum samples from healthy persons
(group IV) reacted faintly with bands with MWr values between 13 kD and 150 kD. Thirty-two percent of the samples
showed no reactivity, 56.40% recognized between one and
three bands, and the remaining 11.60% recognized up to six
bands.
The recognition of at least one of the ATL diagnostic antigenic bands was considered a positive result. In the 108
serum samples from patients with ATL, 79.63% had positive
results for ATL and 20.37% had negative results in the L.
(V.) braziliensis Western blot (Table 3). Cross-reactivity was
found in 30.43% of the serum samples from patients with
Chagas disease (group II) in which the ATL diagnostic
bands (63 kD and 58 kD) were recognized. Among the se-

rum samples from patients with other diseases (group III),

15.63% had positive results for ATL and 84.37% had negative results. Serologic cross-reactions seen in 10.00% of the
serum samples from patients with autoimmune diseases occurred with the ATL diagnostic band (63 kD). Serum samples from patients with a positive diagnosis for leprosy
showed faint reactivity with the ATL bands (63 kD and 58
kD) (Table 4). None of the serum samples from healthy individuals showed positive results for ATL by Western blotting.
Leishmania (L.) amazonensis. Twenty-nine of the 39 L.
(L.) amazonensis polypeptides stained with Coomassie brilliant blue showed reactivity with serum samples from groups
I, II, III, and/or IV. Fourteen of these showed reactivity with
group I sera; 7.60% of the serum samples showed no reactivity with any band, 71.60% recognized between one and

TABLE 2
Leishmanial antigenic bands most frequently reactive with human
sera from groups I and II defined as diagnostic antigenic bands
Molecular weights of bands (% recognition)

TABLE 1
Mean number of antigenic bands recognized by human sera from
groups I, II, III, and IV in a Western blot with antigens of various
Leishmania species and the 268T strain

Antigen extract

L. (V.) braziliensis
L. (L.) amazonensis

Mean number of bands recognized by sera of

Antigen extract

Group I*

L. (V.) braziliensis
L. (L.) amazonensis
L. (L.) tropica
268T

6.00
3.80
5.60
7.10

* Samples
Samples
Samples
Samples

from
from
from
from

Group II

9.74
9.20
6.60
13.20

Group III

Group IV

3.15
0.53
2.50
4.00

1.53
0.55
0.25
1.00

patients with a diagnosis of American tegumentary leishmaniasis (ATL).

patients with Chagas disease.
patients with other diseases.
healthy individuals.

L. (L.) tropica
268T

Group I*

42
58
63
40
50
74
44
50
58
32
38
50

kD
kD
kD
kD
kD
kD
kD
kD
kD
kD
kD
kD

(53.70%)
(39.80%)
(63.90%)
(75.50%)
(72.00%)
(75.50%)
(53.70%)
(49.30%)
(47.80%)
(44.00%)
(53.00%)
(79.40%)

Group II

30
38
72
28
35
66
28
38
40
28
32
40

kD
kD
kD
kD
kD
kD
kD
kD
kD
kD
kD
kD

(78.30%)
(73.90%)
(73.90%)
(90.00%)
(90.00%)
(90.00%)
(90.00%)
(80.00%)
(80.00%)
(80.00%)
(60.00%)
(70.00%)

* Samples from patients with a diagnosis of American tegumentary leishmaniasis.

Samples from patients with Chagas disease.

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TABLE 3
Results obtained in the Western blot with Leishmania (Viannia) braziliensis for the diagnosis of American tegumentary leishmaniasis (ATL)
with serum samples from groups I, II, III, and IV
Western blot*
P
Serum
samples

No. of serum
samples

Laboratory diagnosis

Group I
Group II
Group III

Group IV

ATL
Chagas disease
Antinuclear antibodies
Microsomal antibodies
Anti-streptolysin O
Antinuclear anti-dsDNA
Toxoplasmosis
Syphilis
Leprosy
Healthy individuals

No.

108
23
8
5
5
2
3
5
4
78

86
7
0
1
0
1
0
0
3
0

79.63
30.43
0.00
20.00
0.00
50.00
0.00
0.00
75.00
0.00

No.

22
16
8
4
5
1
3
5
1
78

20.37
69.57
100.00
80.00
100.00
50.00
100.00
100.00
25.00
100.00

* P positive when the sample recognized at least one antigenic band from a group of three with the highest frequency; N negative when the sample showed no reactivity.
Samples from patients with a diagnosis of American tegumentary leishmaniasis (ATL).
Samples from patients with Chagas disease.
Samples from patients with other diseases.
Samples from healthy individuals.

five bands, and 20.80% recognized between six and eight

bands. The major bands recognized most frequently (diagnostic for ATL) are shown in Table 2. In the serum samples
from group II, Western blotting detected antibodies that
showed intense reactivity with bands with MWr values ranging from 13 kD to 170 kD. Among these serum samples,
80.00% recognized between six and 10 bands and 20.00%
recognized between 13 and 15 bands. The most frequently
recognized bands (diagnostic for Chagas disease) are shown
in Table 2. From serum samples of group III, 76.50% did
not recognize any band, 17.60% recognized one band, and

5.90% recognized six bands. The serum samples with a positive reactivity for syphilis showed no reactivity. Of the serum samples from group IV, 55.00% did not recognize any
band and the remaining 45.00% recognized a few antigens
(a maximum of two bands). Cross-reactivity was found in
30.00% of the serum samples from patients with Chagas
disease, which recognized an ATL diagnostic band (40 kD)
(Table 4). Based on the positivity criterion established,
88.70% of group I sera had positive results and 11.30% had
negative results (Table 5). Although serum samples from patients in groups III and IV recognized some bands, these

TABLE 4
Results obtained in the Western blot with the diagnostic antigenic bands and serum samples from groups I, II, III, and IV
Frequency of recognition of human sera from
Antigen extract

Leishmania (Viannia) braziliensis

Leishmania (Leishmania) amazonensis

Leishmania (Leishmania) tropica

268T

Protein fraction

30
38
42
58
63
72
28
30
35
40
50
74
28
30
38
44
50
58
28
32
38
40
50
66

kD
kD
kD
kD
kD
kD
kD
kD
kD
kD
kD
kD
kD
kD
kD
kD
kD
kD
kD
kD
kD
kD
kD
kD

Group I*

Group II

Group III

Group IV

35.20%
28.70%
53.70%
39.80%
63.90%
34.30%
0.00%
0.00%
0.00%
75.50%
72.00%
75.50%
0.00%
41.80%
7.50%
53.70%
49.30%
47.80%
11.80%
44.00%
53.00%
14.70%
79.40%
00.00%

78.30%
73.90%
0.00%
4.30%
26.00%
73.90%
90.00%
80.00%
90.00%
30.00%
0.00%
0.00%
90.00%
70.00%
80.00%
0.00%
30.00%
0.00%
80.00%
60.00%
40.00%
70.00%
50.00%
50.00%

9.40%
6.30%
0.00%
6.30%
15.60%
3.10%
0.00%
0.00%
0.00%
0.00%
0.00%
0.00%
11.80%
29.40%
29.40%
5.90%
5.90%
0.00%
17.60%
0.00%
5.90%
5.90%
0.00%
17.60%

0.00%
0.00%
0.00%
0.00%
0.00%
0.00%
0.00%
0.00%
0.00%
0.00%
0.00%
0.00%
0.00%
0.00%
0.00%
0.00%
0.00%
0.00%
15.00%
0.00%
0.00%
10.00%
5.00%
0.00%

* Samples from patients with a diagnosis of American tegumentary leishmaniasis (ATL).

Samples from patients with Chagas disease.
Samples from patients with other diseases.
Samples from healthy individuals.
The percentages in bold represent the fractions with the highest recognition for the sera ATL (group I) and Chagas disease (group II).

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TABLE 5
Results obtained in the Western blot with Leishmania (Leishmania) amazonensis for the diagnosis of American tegumentary leishmaniasis
(ATL) with serum samples from groups I, II, III, and IV
Western blot*
P
Serum
samples

Group I
Group II
Group III

Group IV

Laboratory diagnosis

ATL
Chagas disease
Antinuclear antibodies
Microsomal antibodies
Antinuclear anti-dsDNA
Syphilis
Leprosy
Healthy individuals

No. of serum
samples

53
10
5
2
1
5
4
20

No.

N
%

47
3
0
0
0
0
0
0

No.

88.70
30.00
0.00
0.00
0.00
0.00
0.00
0.00

6
7
5
2
1
5
4
20

11.30
70.00
100.00
100.00
100.00
100.00
100.00
100.00

* P positive when the sample recognized at least one antigenic bands from a group of three with the highest frequency; N negative when the sample showed no reactivity.
Samples from patients with a diagnosis of American tegumentary leishmaniasis (ATL).
Samples from patients with Chagas disease.
Samples from patients with other diseases.
Samples from healthy individuals.

samples showed no reactivity with the ATL diagnostic antigenic bands of L. (L.) amazonensis and the test was considered 100.00% negative for both of these groups.
Leishmania (L.) tropica. Thirty-six of the 39 Leishmania
(L.) tropica polypeptides stained with Coomassie brilliant
blue showed reactivity by Western blotting with at least one
of the serum samples from groups I, II, III, and/or IV; 34 of
these were recognized by serum samples from patients in
group I (ATL). A total of 13.40% of the serum samples in
group I recognized no band, while 59.60% recognized between one and seven bands. The most frequently recognized
bands (diagnostic for ATL) are shown in Table 2. In group
II, several antigenic bands of L. (L.) tropica between 15 kD
and 74 kD were strongly recognized. Thirty percent of these
serum samples recognized between three and five bands and
70.00% recognized between seven and nine bands. The most
frequently recognized major bands (diagnostic for Chagas
disease) are shown in Table 2. In the serum samples from
patients in group III, Western blotting detected antibodies
that recognized bands with MWr values ranging from 16 kD
to 150 kD; 29.40% of the serum samples showed no reactivity, but 58.80% recognized between one and four bands.
Eighty percent of the serum samples from group IV showed
no reactivity and 20.00% recognized up to two antigenic

bands of L. (L.) tropica and had weak immune reactivity.

Cross-reactivity was found in 30.00% of the serum samples
from patients with Chagas disease (group II), which recognized an ATL band with an MWr value of 50 kD (Table
4). From patients with other diseases (group III), 5.90% had
positive results and 94.10% had negative results for ATL.
Serologic cross-reactions occurred with the same band (50
kD) in 20.00% of the serum samples from patients with a
positive diagnosis for syphilis. No serum samples from
healthy individuals showed positive results by Western blotting for ATL. Based on the positivity criterion, 77.61% of
the serum samples from patients with ATL had positive results and 22.39% had negative results by Western blotting
(Table 6).
Strain 268T. Forty-one of the 44 polypeptides detected
in strain 268T were recognized by at least one serum from
groups I, II, III, and/or IV. The samples from group I recognized 31 antigenic bands, with 2.90% showing no reactivity, 56.00% recognizing between one and seven bands,
38.20% recognizing between eight and 13 bands, and 2.90%
recognizing 18 bands. The most reactive bands (diagnostic
for ATL) are shown in Table 2. In group II, Western blotting
detected bands with MWr values ranging from 13 kD to 150
kD that showed high intensity of reactivity; 10.00% of the

TABLE 6
Results obtained in the Western blot with Leishmania (Leishmania) tropica for the diagnosis of American tegumentary leishmaniasis (ATL)
with serum samples from groups I, II, III, and IV
Western blot*
P
Serum
samples

Group I
Group II
Group III

Group IV

Laboratory diagnosis

ATL
Chagas disease
Antinuclear antibodies
Microsomal antibodies
Antinuclear anti dsDNA
Syphilis
Leprosy
Healthy individuals

No. of serum
samples

67
10
5
2
1
5
4
20

No.

52
3
0
0
0
1
0
0

N
%

77.60
30.00
0.00
0.00
0.00
20.00
0.00
0.00

No.

15
7
5
2
1
4
4
20

22.40
70.00
100.00
100.00
100.00
80.00
100.00
100.00

* P positive when the sample recognized at least one antigenic bands from a group of three with the highest frequency; N negative when the sample showed no reactivity with the
antigenic bands.
Samples form patients with a diagnosis of American tegumentary leishmaniasis (ATL).
Samples from patients with Chagas disease.
Samples from patients with other diseases.
Samples from healthy individuals.

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TABLE 7
Results obtained in the Western blot with the 268T strain for the diagnosis of American tegumentary leishmaniasis (ATL) with serum samples
from groups I, II, III, and IV
Western blot*
P
Serum
samples

Group I
Group II
Group III

Group IV

Number of
serum samples

Laboratory diagnosis

ATL
Chagas disease
Antinuclear antibodies
Microsomal antibodies
Antinuclear anti dsDNA
Syphilis
Leprosy
Healthy individuals

34
10
5
2
1
5
4
20

No.

30
8
0
1
0
0
0
1

N
%

No.

88.20
80.00
0.00
50.00
0.00
0.00
0.00
5.00

4
2
5
1
1
5
4
19

11.80
20.00
100.00
50.00
100.00
100.00
100.00
95.00

* P positive when the sample recognized at least one antigenic bands from a group of three with the highest frequency; N negative when the sample showed no reactivity.
Samples from patients with a diagnosis of American tegumentary leishmaniasis (ATL).
Samples from patients with Chagas disease.
Samples from patients with other diseases.
Samples from healthy individuals.

serum samples recognized five bands, 50.00% recognized

between eight and 13 bands, and the remaining 40.00% recognized between 16 and 18 bands. The most frequently recognized bands (diagnostic for Chagas disease) are shown in
Table 2. In group III, 47.00% of the serum samples did not
recognize any bands, 35.60% recognized between one and
five bands, 11.80% recognized between 10 and 11 bands,
and 5.90% recognized 32 bands. Samples from patients with
a positive diagnosis for syphilis showed no reactivity. Sera
from healthy persons (group IV) reacted with polypeptides
with MWr values ranging from 13 kD to 105 kD; 45.00%
of the samples showed no reactivity, 20.00% recognized between one and three bands, and 35.00% recognized between
four and six bands. Extensive cross-reactivity was found in
the serum samples from patients with Chagas disease, in
which 80.0% had a positive result (recognizing all the ATL
diagnostic bands of 50 kD, 38 kD, and 32 kD). In the group
III, a total of 5.90% of sera from patients with autoimmune
diseases showed cross-reactivity with an ATL band (38 kD)
and the sera from patients with a positive diagnosis for syphilis or leprosy showed no reactivity. Five percent of sera
from healthy individuals (group IV) recognized an ATL band
(50 kD) (Table 4). In accordance with the positivity criterion,
88.20% of the serum samples from patients with ATL had
positive results and 11.80% had negative results (Table 7).
Table 8 shows the sensitivity (95% confidence interval),
specificity, positive and negative predictive values, and the
kappa concordance coefficient of the antigen extracts from

L. (V.) braziliensis, Leishmania (L.) amazonensis, Leishmania (L.) tropica, and strain 268T. The results showed that no
band recognized by 100% of the sera from patients with ATL
was found using the four antigens analyzed by Western blotting.
DISCUSSION
Western blotting can provide detailed information about a
parasites antigenic structure, which is very useful in the
standardization of more sensitive and specific diagnostic procedures. This method has used soluble extracts,15,18,20,21,44 purified membrane proteins,45 or recombinant proteins as antigens.4648
The immunoblot results obtained with soluble protein extracts of Leishmania species and strain 268T showed a complex reactivity pattern (Figures 14), in which most (93%)
of the polypeptide fractions of parasites were recognized by
at least one serum sample from the individuals tests. Similarly complex profiles were found15,49 when the human antibody specificity to antigens of L. (V.) braziliensis and L.
infantum was analyzed by Western blotting.
The protein fractions recognized by serum samples varied
with the different parasite strains and different individuals
recognized different protein fractions of the same parasite.
Variability has been observed50 in T cell stimulation in sera
from patients with ATL. This could be explained in part by
the complexity of the antigenic constitution of each parasite

TABLE 8
Evaluation of the comparative results of the Western blot with serum from patients with American tegumentary leishmaniasis, Chagas disease,
other diseases, and healthy controls

Antigen

L. (V.) braziliensis
L. (L.) amazonensis
L. (L.) tropica
268T

Sensitivity

Specificity

Positive predictive
value

Negative
predictive value

76.90%
(67.6084.20%)
90.40%
(78.2096.40%)
78.50%
(66.2087.30%)
87.90%
(70.9096.00%)

100.0%
(94.20100.0%)
93.80%
(81.8098.40%)
87.80%
(74.5094.90%)
77.10%
(62.3087.50%)

100.0%
(94.50100.0%)
94.00%
(82.5098.40%)
89.50%
(77.8095.60%)
72.50%
(55.9084.90%)

75.70%
(66.10%83.40%)
90.00%
(77.4096.30%)
75.40%
(62.0085.50%)
90.20%
(75.9096.80%)

The values in parentheses represent 95% confidence interval.

Concordance
kappa
coefficient

Statistical Z
(P 0.0000)

0.7358

10.40

0.8400

8.41

0.6491

7.00

0.6287

5.75

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and by the genetic background of each individual, which

together influence the immune response.22,51
Western blotting showed that serum samples from patients
with ATL recognized 31 bands (13150 kD) from L. (V.)
braziliensis, 34 bands (13150 kD) from L. tropica, and 31
bands (17170 kD) from the 268T strain. Similar results
were observed52 with serum samples from patients with
American visceral leishmaniasis, which recognized 36 antigenic components (14.5123 kD) and with serum samples
from patients with cutaneous leishmaniasis, which recognized 23 soluble proteins (18.5115 kD) from a L. donovani
chagasi extract.
Only 14 of 39 polypeptide fractions from L. (L.) amazonensis that stained with Coomassie brilliant blue were recognized by serum samples from patients with ATL. The reactivity (immune staining) of the serum samples with L. (L.)
amazonensis seemed less intense but more homogeneous,
with the majority (75.50%) of the serum samples from group
I recognizing the same fractions (Figure 2). The low intensity of immunostaining of fractions from L. (L.) amazonensis
could be explained by the long period (more than four years)
of maintenance in artificial culture medium, leading to lower
virulence as demonstrated in animal models,53 less L. donovani chagasi antigen, and a concomitant decrease in the gp
63 content of the promastigote form. However, this was not
observed in L. tropica that was also maintained for many
years in vitro, in which serum samples from patients with
ATL recognized various fractions of the protein extract of
this strain (Figure 3).
The 63-kD protein fraction of L. (L.) amazonensis was not
recognized by sera from group I (ATL). Similarly, only
19.4% of these sera recognized the 63-kD antigen of L. tropica. The majority (63.89%) of ATL-positive sera recognized the 63-kD protein of L. (V.) braziliensis. This protein
was identified as a glycoprotein by staining with periodic
acid-Schiff, indicating that it could be the main promastigote
surface molecule (MWr 6065 kD) common to all Leishmania species. This surface antigen may be involved in the
development of the infective stage31 in promastigote forms
of L. (V.) braziliensis, in which there is a progressive increase in the expression of a 65-kD surface antigen (gp 63)
in the stationary growth phase.31
A comparison of the MWr values of protein fractions is a
complex process, and many factors such as composition of
the culture media, parasite growth phase, concentration of
SDS and 2-mercaptoethanol in the sample buffer, separation
of soluble fractions from pellets, SDS-PAGE gel size, polyacrylamide concentration, and the molecular weight markers
used can influence the fraction migration and determination
of the MWr values.34 Thus, direct comparisons of molecular
weights of antigens cited in the literature are always very
difficult and must be done with caution.
In spite of the complex recognition pattern, the protein
fractions most reactive with ATL-positive sera (Table 4)
have relatively low molecular weights (2874 kD). This is
similar to results found for L. infantum (1490 kD)49 and L.
chagasi (26116 kD).20 Relatively high molecular weights
were found for the most frequently reacting protein fractions
of L. braziliensis panamensis (78120 kD)18 and L. (V.) braziliensis (69250 kD).20
When we compared individual reactive protein fractions,

99

a 60-kD protein from L. (V.) braziliensis had been previously

identified by human sera from patients with ATL,15 whereas
we detected a 63-kD antigen (Table 4). In the L. (L.) amazonensis extract, a 74-kD fraction was recognized by 75.47%
of the ATL-positive sera, whereas in L. tropica and strain
268T, the recognition was much lower (Table 4). Similar
results were obtained with a fraction of 7073 kD from L.
donovani chagasi extracts that was recognized by 89.47%
of sera from patients with visceral leishmaniasis.20
Fractions of 40 kD, 42 kD, 44 kD, and 38 kD were detected in extracts of L. (L.) amazonensis, L. (V.) braziliensis,
L. tropica, and strain 268T, respectively (Table 4). All were
recognized by more than 50.00% of the ATL-positive sera.
A 42-kD protein was purified from the membrane of L. amazonensis, and it was shown that this protein (La gp 42) was
recognized by sera from patients with different forms of
leishmaniasis caused by L. major, L. donovani, and L. chagasi, and was also recognized by sera from patients with
Chagas disease.45 Analysis of the N-terminal sequence of
La gp 42 showed homology with gp 46/M-2.54 This protein
belongs to the promastigote surface antigen complex-2,
which has been found in all Leishmania species studied to
date, except for L. braziliensis.55 A 42-kD antigen was identified by sera from patients with cutaneous and visceral leishmaniasis in an L. major extract.21 The immune response induced by a recombinant protein from L (L.) amazonensis (33
kD [Larp33]) was also evaluated. Western blotting showed
that Larp33 was a 40-kD protein expressed in L. (L.) amazonensis and L. (V.) brazilliensis.47
A protein with an MWr of 50 kD, which was recognized
by more than 50.00% of the ATL-positive sera, was detected
in extracts from L. (L.) amazonensis, L. tropica, and strain
268T (Table 4). However, only 23.00% of the ATL-positive
sera recognized the 50-kD fraction in the L. (V.) braziliensis
extract. In cutaneous leishmaniasis caused by L. major, more
than 90.00% of positive sera recognized a fraction of 50
kD.56 The investigators suggest that this fraction could be a
promastigote surface protein (gp 63) with an MWr of 50,000
(determined under non-reducing conditions). It is well
known that some of the surface antigens are active proteases
(gp 63). The proteolytic activity of a wide range of trypanosomatids was determined and many trypanosomatids (7
genera and 11 species) have various proteolytic activities,
including some cross-reactive promastigote surface protein
(gp 63).57
An antigenic profile that enables one to discriminate positive from negative serologic results with regard to ATL,
Chagas disease, other diseases, and healthy controls is urgently needed. A diagnostic antigen recognized by 100% of
positive sera is not available. The principal antigen used depends on the expression of this diagnostic antigen because
even a highly immunogenic antigen, if poorly expressed,
will react as a weak band.58 The complex molecular constitution of the parasite, associated with limitations in the analysis of the results obtained by different techniques, lead to
a lack of consensus in the definition of the principal antigens
in Leishmania. It was possible to correlate the presence of
the parasite in dogs with the recognition of different antigenic fractions detected; statistical analysis was used to select the bands with a stronger correlation and define a criterion of positivity based on the recognition of these bands.34

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Based on these criteria, five bands were selected and the test
result was considered positive when at least one band was
recognized. A sensitivity of 95.80% and a specificity of
100.00% were obtained with this system. However, it is not
always possible to correlate parasitologic and serologic results. Another approach is to define a specific antigenic component (e.g., a 94-kD L. infantum antigen) whose detection
with sera from a dog with visceral leishmaniasis gave a sensitivity of 95.45% and a specificity of 100%.33 In the same
L. infantum extract, 14-kD and 16-kD fractions were detected by sera from a patient with visceral leishmaniasis sera
showed recognition frequencies of 92.00% and 95.00%, respectively. When combined, they gave a recognition frequency of 100.00%, a sensitivity of 100.00%, and a specificity of 98.00%.49 However, such a level of a recognition of
antigenic fractions is not always achieved.
The average number of recognized antigenic fractions
from the different strains was always higher for serum samples from group II (Chagas disease) than from group I
(ATL) (Table 1). Analysis of cross-reactivity in human infections caused by L. (V.) braziliensis, T. cruzi, and L. chagasi showed that sera from chagasic patients recognized 24
bands with homologous antigen, 13 bands with L. chagasi
antigen, and 17 with Leishmania (V.) braziliensis antigen.24
The sera from patients with visceral leishmaniasis recognized 29 bands with homologous antigen, 14 with T. cruzi
antigen, and 10 bands with Leishmania (V.) braziliensis antigen. Sera from individuals with ATL recognized 17 bands
with homologous antigen, 10 bands with T. cruzi antigen,
and four bands with L. chagasi antigen. A serum sample
was considered positive for ATL by Western blotting when
it recognized at least one band among the three with the
highest recognition frequency in each strain analyzed. Western blotting showed a sensitivity of 84.90% and a specificity
of 91.10% with L. (V.) braziliensis, a sensitivity of 90.40%
and a specificity of 93.80% with L. (L.) amazonensis, and a
sensitivity of 78.50% and a specificity of 87.80% with L.
(L.) tropica. A specificity of 77.10% was obtained by Western blotting with a distant trypanosomatid parasite of tomato
(strain 268T). Our results showed that the L. (L.) amazonensis soluble antigen is more adequate for the diagnosis of ATL
by Western blotting test than the antigen from L. (V.) braziliensis (Table 8).
Cross-reactivity is still one of the biggest problems in clinical serology. With the L. (V.) braziliensis strain, the sera
from chagasic patients showed cross-reactivity with the 63kD fraction. This findings differs from that of another study,
which reported that the 60-kD fraction is specific for Leishmania.15 Other sera (autoimmune diseases and leprosy) also
recognized the gp 63 antigen. Another 22 L. donovani chagasi antigens (18.5112 kD) were recognized by sera from
Chagas disease patients and a small number of antigens
(32110 kD) were reactive with sera from patients with leprosy and tuberculosis.52
We observed cross-reactivity with the 40-kD L. (L.) amazonensis fractions with sera from chagasic patients, as previously described.45 Studies of human IgG reactivity with
antigens of T. cruzi and Leishmania revealed cross-reactivity
of serum samples from chagasic patients with a 38-kD antigen of L. b. panamensis, but the profiles of the sera detected with the T. cruzi and Leishmania antigens permit clear

serologic differentiation of these parasitoses.23 Cross-reactivity of sera from patients with ATL and Chagas disease in
the diagnosis of visceral leishmaniasis, mainly with the 70kD antigen, and the serologic differentiation of these infections, has also been reported.20 Conversely, an L. braziliensis
braziliensis 72-kD antigen was described that had no crossreactivity with sera from patients with Chagas disease.44 Extensive cross-reactivity was observed among the strain 268T
antigens and serum samples from patients with Chagas disease, and at a lower frequency among patients with autoimmune diseases and sera from healthy individuals. A comparison of indirect immunofluorescence and the intradermal
reaction in the diagnosis of leishmaniasis give a statistically
significant agreement value (Z 9.72, P 0.01) but a poor
kappa index (0.32).59 An epidemiologic study of visceral
leishmaniasis examined 50 unselected subjects living in a
high risk area for visceral leishmaniasis simultaneously by
means of a skin test and Western blotting.60 The criterion of
positivity in the Western blot used was based on the presence
of antibodies directed against the L. infantum 14-kD and/or
16-kD antigens. The agreement of the two techniques (agreement percentage 82.00%) does not consider the influence
of agreement purely by chance.43 We analyzed the agreement
of Western blotting with a positive diagnosis for ATL by
means of clinical signs and classical laboratory tests (parasitologic, skin test, and IFA) using the kappa statistic, which
corrects for the agreement expected by chance.43 The overall
agreement between the Western blotting and these other
techniques results in a kappa index of 0.8400 for L. (L.)
amazonensis, 0.7358 for L. (V.) braziliensis, 0.6491 for L.
(L.) tropica, and 0.6287 for 268T (Table 8).
Our results showed that all strains studied showed crossreactivity with serum samples from patients with Chagas
disease, which is consistent with the findings of previously
published reports23,24,52,61 and indicates the presence of common epitopes between Leishmania and T. cruzi. The overall
cross-reactivity observed in serum samples from patients
with positive diagnosis for ATL and in non-related microorganisms and other conditions indicates antigenic similarity
between Leishmania and bacteria such as Mycobacterium
species, as demonstrated by significant cross-reactivity in serologic tests.51,52,59,60,62
We did not find any common antigenic band with 100%
recognition that was diagnostic for all sera from patients
with ATL. The more specific fractions for Leishmania antigens were 42 kD for L. (V.) braziliensis, 50 kD for L. (V.)
amazonensis, and 58 kD for L. (L.) tropica with a frequency
of recognition of 53.70%, 72%, and 47.80%, respectively.
For strain 268T, none of the most reactive fractions were
specific and there was cross-reactivity in at least one of the
serum samples from groups II, III, or IV, albeit at a lower
frequency (Table 4).
Western blotting is a sensitive test for detecting antibodies
to Leishmania.18,62 It can also confirm false-positive results
obtained by an ELISA.63 It allows specific serodiagnosis of
visceral and mucocutaneous leishmaniasis in patients living
in nonendemic areas, and appears to be superior to the classic IFA.21 The use of this test has been assayed in following
chemotherapy and cure.13 Our results showed that some proteins were specific for the serum samples from patients with
Chagas disease (Table 2). The major bands with apparent

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molecular weight of 30 kD for L. (V.) braziliensis, 35 kD

for L. (L.) amazonensis, 28 kD for (L.) tropica, and 28 kD
for strain 268T were recognized by these sera at a high frequency of 78.30%, 90.00%, 90.00% and 80.00%, respectively (Table 4). These fractions permit clear differentiation
of leishmaniasis from Chagas disease, mainly with L. (L.)
amazonensis extracts. It has also been reported that sera
from chagasic patients recognized Leishmania antigens with
MWr values of 124, 107, 92, 59, and 32 kD.64
Our results show that it is possible to obtain recognition
profiles that enable one to diagnose ATL and differentiate
leishmaniasis from Chagas disease. Thus, a mixed human
infection of T. cruzi and Leishmania species can be detected
by Western blotting. This method should benefit from the
use of recombinant antigens.
Acknowledgments: We thank J. C. Dalmas and T. Matsuo for statistical analysis.
Financial support: This investigation received financial support from
the Conselho Nacional de Desenvolvimento Cientfico e Tecnologico
(CNPq) e Coordenadoria de Pos Graduacao da Universidade Estadual de Londrina.
Authors addresses: Cely Cristina Martins Goncalves, Rua Garibaldi
Deliberador 483/33, Londrina CEP 86050-170, Parana, Brazil. Edna
Maria Vissoci Reiche, Bencio Alves De Abreu Filho, Thais Gomes
Verzignassi Silveira, Tania Cristina Felizardo, Karoline Rocha Maia,
Rafael Costacurta, Evandro Jose Padovesi, Benedito Prado Dias Filho, Shiduca Itow Jankevicius, and Jose Vitor Jankevicius, Centro de
Ciencias Biologicas, Universidade Estadual de Londrina, Campus
Universitario, Caixa Postal 6001, CEP 86051, Londrina, Parana,
Brazil.

REFERENCES
, Wirth DF, 1993. Leishmaniasis. Warren
1. Sacks DL, Louis JA
KS, ed. Immunology and Molecular Biology of Parasitic infections. Boston: Blackwell Scientific Publications 237268.
2. Chang KP, 1990. Cell biology of Leishmania. Wyler DJ, ed.
Modern Parasite Biology. Cellular, Immunological and Molecular Aspects. New York: W. H. Freeman and Company 79
80.
3. Palma G, Guitierrez Y, 1991. Laboratory diagnosis of Leishmania. Clin Lab Med 11: 909922.
4. Weigle AK, Davalos M, Heredia P, Molineros R, Saravia NG,
DAlessandro A, 1987. Diagnosis of ATL leishmaniasis in
Colombia: a comparison of seven methods. Am J Trop Med
Hyg 36: 489496.
5. Navin TR, Arana FE, de Merida AM, Arana BA, Catillo AL,
Silvers DN, 1990. Cutaneous leishmaniasis in Guatemala:
comparison of diagnostic methods. Am J Trop Med Hyg 42:
3642.
6. Chico ME, Guderian RH, Cooper PJ, Armijos R, Grogl M,
1995. Evaluation of a direct immunofluorescent antibody
(DIFMA) test using Leishmania genus-specific monoclonal
antibody in the routine diagnosis of cutaneous leishmaniasis.
Rev Soc Bras Med Trop 28: 99103.
7. Cuba-Cuba CA, Llanos-Cuentas EA, Marsden PD, 1986. Failure
to detect circulating Leishmania in mucocutaneous leishmaniasis due to Leishmania braziliensis braziliensis. Trans R Soc
Trop Med Hyg 80: 346.
8. Saravia, NG, Valderrama L, Labrada M, Holgun CN, Navas C,
Palma G, Weigle KA, 1989. The relationship of Leishmania
braziliensis subspecies and immune response to disease expression in New World leishmaniasis. J Infect Dis 159: 725
735.
9. Price MS, Silvers DN, 1977. New world leishmaniasis: serologic aids to diagnosis. Arch Dermatol 113: 14151416.
10. Kar K, 1995. Serodiagnosis of Leishmaniasis. Crit Rev Microbiol 21: 123152.

101

11. Furtado T, 1980. Criterios para o diagnostico da leishmaniose

tegumentar americana. An Bras Dermatol 55: 8186.
12. Sampaio RNR, Rocha RAA, Marsden PD, Cuba CC, Barreto
AC, 1980. Leishmaniose tegumentar americana-casustica do
Hospital Escola da Universidade de Braslia. An Bras Dermatol 55: 6976.
13. Delgado O, Guevara P, Silva S, Belfort E, Ramirez JL, 1996.
Follow-up of a human accidental infection by Leishmania
(Viannia) braziliensis using conventional immunologic techniques and polymerase chain reaction. Am J Trop Med Hyg
55: 267272.
14. Behin R, Jacques L, 1984. Immune response to Leishmania. Crit
Rev Trop Med 2: 141188.
15. Masuda A, Nascimento SF, Guerra CS, Paranhos GS, Ferreira
AW, 1989. Analysis of the specificity of human antibodies to
antigens of Leishmania braziliensis braziliensis. Rev Inst Med
Trop Sao Paulo 31: 228234.
16. Walton BC, Brooks WH, Arjona I, 1972. Serodiagnosis of
American leishmaniasis by indirect fluorescent antibody test.
Am J Trop Med Hyg 21: 296299.
17. Garcia-Miss MR, Andrade-Narvaes FJ, Esquivel-Vinas RE,
Simmons-Diaz EB, Canto-Lara SB, Cruz-Ruiz AL, 1990. Localized cutaneous leishmaniasis (chicleros ulcer) in Mexico:
sensitivity and specificity of ELISA for IgG antibodies to
Leishmania mexicana mexicana. Trans R Soc Trop Med Hyg
84: 356358.
18. Izasa DM, Restrepo M, Mosca W, 1997. Immunoblot analysis
of Leishmania panamensis antigens in sera of patients with
American cutaneous leishmaniasis. J Clin Microbiol 35:
30433047.
19. Hermann KL, Erdman DD, 1995. Lennette EH, Lennette DA,
Lennette ET, eds. Diagnostic Procedures for Viral, Rickettsial
and Clamydial Infections: Diagnosis by Serologic Assays.
Washington, DC: American Health Association, 6, 129134.
20. Evans TG, Krug EC, Wilson ME, Vasconcelos AW, Alencar JE,
Pearson RD, 1989. Evaluation of antibody responses in American visceral leishmaniasis by ELISA and Immunoblot. Mem
Inst Oswaldo Cruz 84: 157166.
21. Bogdan C, Stosiek N, Fuchs H, Rollinghoff M, Solbach W,
1990. Detection of potentially diagnostic leishmanial antigens
by Western blot analysis of sera from patients with kala-azar
or multilesional cutaneous leishmaniasis. J Infect Dis 162:
14171418.
22. Jaffe CL, Shor R, Trau H, Passwell JH, 1990. Parasite antigens
recognized by patients with cutaneous leishmaniasis. Clin Exp
Immunol 80: 7782.
23. Chiller TM, Samudio MA, Zoulek G, 1990. IgG antibody reactivity with Trypanosoma cruzi and Leishmania antigens in
sera of patients with Chagas disease and leishmaniasis. Am
J Trop Med Hyg 43: 650656.
24. Vexenat AC, Santana JM, Teixeira ARL, 1996. Cross-reactivity
of antibodies in human infections by the kinetoplastid protozoa Trypanosoma cruzi, Leishmania chagasi and Leishmania
(Viannia) braziliensis. Rev Inst Med Trop Sao Paulo 38: 177
185.
25. Monteon VM, Gusman-Rojas L, Negrete-Garcia C, Rosales-Encina JL, Lopez PAR, 1997. Serodiagnosis of American trypanosomosis by using nonpathogenic trypanosomatid antigen.
J Clin Microbiol 35: 33163319.
26. Itow Jankevicius S, Almeida ML, Jankevicius JV, Cavazzana M
Jr, Attias M, de Souza W, 1993. Axenic cultivation of trypanosomatids found in corn (Zea mays) and in phytophagous
hemipterans (Leptoglossus zonatus, coreidae) and their experimental transmission. J Eukaryot Microbiol 40: 576581.
27. Camargo ME, 1964. Growth and differentiation in Trypanosoma
cruzi. I. Origin of metacyclic trypanosomes in liquid media.
Rev Inst Med Trop Sao Paulo 6: 93100.
28. Romero GG, Arana M, Lopez M, Montoya I, Bohl R, Campos
M, Arevalo J, Llanos A, 1987. Characterization of Leishmania
species from Peru. Trans R Soc Trop Med Hyg 81: 1424.
29. Guimaraes MCS, Celeste BJ, Castilho EA, Mineo JR, Diniz,
JMP, 1981. Immunoenzymatic assay (ELISA) in mucocutaneous leishmaniasis, kala-azar, and Chagas disease: an epimastigote Trypanosoma cruzi antigen able to distinguish be-

tmed 65_527 Mp_102

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Friday Feb 01 2002 02:58 PM

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102

30.
31.

32.

33.

34.

35.
36.
37.

38.
39.

40.

41.

42.

43.
44.

45.

46.

47.

GONC
ALVES AND OTHERS

tween anti-Trypanosoma and anti-Leishmania antibodies. Am

J Trop Med Hyg 30: 942947.
Sacks DL, Perkins PV, 1984. Identification of an infective stage
of Leishmania promastigotes. Science 223: 14171419.
Kweider M, Lemesre JL, Darcy F, Kusnierz JP, Capron A, Santoro F, 1987. Infectivity of Leishmania braziliensis promastigotes is dependent on the increasing expression of a 65,000dalton surface antigen. J Immunol 138: 299305.
Maizels RM, Blaxter ML, Robertson BD, Selkirk ME, 1991.
Parasite Antigens, Parasite Genes: A Laboratory Manual For
Molecular Parasitology. Cambridge, United Kingdom: Cambridge University Press, 2526.
Neogy AB, Vouldouks I, Silva OA, Tselentis Y, Lascombe JC,
Segalen T, Rzepka D, Monjour L, 1992. Serodiagnosis and
screening of canine visceral leishmaniasis in an endemic area
of Corsica: applicability of a direct aglutination test and immunoblot analysis. Am J Trop Med Hyg 47: 772777.
Aisa MJ, Castillejo S, Gallego M, Fisa R, Riera MC, Colmenares M, Torras S, Roura, X, Sentis J, Portus M, 1998. Diagnostic potential of Western blot analysis of sera from dogs
with leishmaniasis in endemic areas and significance of the
pattern. Am J Trop Med Hyg 58: 154159.
Bradford MM, 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248254.
Laemmli UK, 1970. Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 227: 680
685.
Goding W, Handman E, 1983. Electrophoretic analysis of protein antigens. Genes and Antigens of Parasites: A Laboratory
Manual: Morel CM, ed. Rio de Janeiro: Fundacao Oswaldo
Cruz, 338383.
Jankevicius JV, 1983. Glicoprotenas de Superfcie em Tripanosomatideos: Identificacao Bioqumica e Antigenicidade.
Sao Paulo: Tese (Mestrado) Escola Paulista de Medicina.
Towbin H, Staehelin T, Gordon J, 1979. Electrophoretic transfer
of proteins from polyacrylamide gels to nitrocellulose sheets:
procedure and some applications. Proc Natl Acad Sci USA
76: 43504354.
Morales G, Carrillo G, Requena JM, Guzman F, Gomez LC,
Patarroyo ME, Alonso C, 1997. Mapping of the antigenic
determinants of the Leishmania infantum gp 63 protein recognized by antibodies elicited during canine visceral leishmaniasis. Parasitology 114: 507516.
Teixeira MGM, Borges Pereira J, Netizert E, Souza MLNX,
Peralta JM, 1994. Development and evaluation of an enzyme
linked immunotransfer blot technique for serodiagnosis of
Chagas disease. Trop Med Parasitol 45: 308312.
Reiche EMV, Cavazzana M Jr, Okamura H, Tagata EC, ItowJankevicius S, Jankevicius JV, 1998. Evaluation of the Western blot in the confirmatory serologic diagnosis of Chagasdisease. Am J Trop Med Hyg 59: 750756.
Kramer MS, Feinstein AR, 1981. Clinical biostatistics. LIV. The
biostatistics of concordance. Clin Pharmacol Ther 29: 111
123.
Kutner S, Pellerin P, Breniere F, Desjeux P, Dedet JP, 1991.
Antigenic specificity of the 72-kilodalton major surface glycoprotein of Leishmania braziliensis braziliensis. J Clin Microbiol 29: 595599.
Burns JM, Scott JM, Carvalho EM, Russo DM, March CJ, van
Ness KP, Reed SG, 1991. Characterization of a membrane
antigen of Leishmania amazonensis that stimulates human immune responses. J Immunol 146: 742748.
Shreffler WG, Burns JM, Badaro R, Ghalib HW, Button LL,
McMaster WR, Reed S, 1993. Antibody responses of visceral
leishmaniasis patients to gp 63, a major surface glycoprotein
of Leishmania species. J Infect Dis 167: 426430.
Fernandes AP, Herrera EC, Mayrink W, Gazinelli RT, Liu WY,
Costa CA, Tavares CAP, Melo MN, Michalick MSM, Gentz
R, Nascimento E, 1997. Immune responses induced by a
Leishmania (Leishmania) amazonensis recombinant antigen
in mice and lymphocytes from vaccinated subjects. Rev Inst
Med Trop Sao Paulo 39: 7178.

48. Ozenzoy S, Ozbel Y, Turgay N, Alkan ZA, Gul K, GilmanSachs A, Chang KP, Reed SG, Ozcel MA, 1998. Serodiagnosis and epidemiology of visceral leishmaniasis in Turkey.
Am J Trop Med Hyg 59: 362369.
49. Mary C, Lamouroux D, Dunan S, Quilici M, 1992. Western blot
analysis of antibodies to Leishmania infantum antigens: potential of the 14-kD and 16-kD antigens for diagnosis and
epidemiologic purposes. Am J Trop Med Hyg 47: 764771.
50. Zalis MG, Schubach A, Oliveira Neto M, Costa DA, Jaffe C,
Lopes VG, Barcinski MA, 1988. The cell immune response
of patients with cutaneous leishmaniasis to leishmanial antigen with different molecular weights. Mem Inst Oswaldo Cruz
83(suppl I): 117.
51. Pappas MG, McGreevy PB, Hajkowski R, Hendriks LD, Oster
CN, Hockmeyer WT, 1983. Evaluation of promastigote and
amastigote antigens in the indirect fluorescent antibody test
for American cutaneous leishmaniasis. Am J Trop Med Hyg
32: 12601267.
52. Santos JI, Morgado MG, Galvao-Castro B, 1987. Human visceral leishmaniasis: analysis of the specificity of humoral immune response to polypeptides of Leishmania donovani chagasi. Am J Trop Med Hyg 37: 263270.
53. Wilson ME, Hardin KK, Donelson JE, 1989. Expression of the
major surface glycoprotein of Leishmania donovani chagasi
in virulent and attenuated promastigotes. J Immunol 143:
678684.
54. Kahl LP, McMahon-Pratt D, 1987. Structural and antigenic characterization of a species- and promastigote- specific Leishmania mexicana amazonensis membrane protein. J Immunol
138: 15871595.
55. Handman E, Osborn AH, Symons F, Driel R, Cappai R, 1995.
The Leishmania promastigote surface antigen 2 complex is
differentially expressed during the parasite life cycle. Mol
Biochem Parasitol 74: 189200.
56. Jaffe CL, Rachamim N, Sarfstein R, 1990. Characterization of
two proteins from Leishmania donovani and their use for vaccination against visceral leishmaniasis. J Immunol 144: 699
706.
57. Branquinha MH, Vermelho AB, Goldenberg, S, Bonaldo MC,
1996. Ubiquity of cysteine- and metalloproteinase activities
in a wide range of trypanosomatids. J Eukaryot Microbiol 43:
131135.
58. Stolf AMS, 1992. Trypanosoma cruzi antigens in serodiagnosis.Wendel S, Brener Z, Camargo ME, Rassi A, eds. Chagas
Disease (American Trypanosomiasis): Its Impact on Transfusion and Clinical Medicine. Sao Paulo: ISTB 92, 195205.
59. Barbosa Santos EG, Marzochi MCA, Conceicao NF, Brito CM,
Pacheco RS, 1998. Epidemiological survey on canine population with the use of immunoleish skin test in endemic areas
of human American cutaneous leishmaniasis in the state of
Rio de Janeiro, Brazil. Rev Inst Med Trop Sao Paulo 40: 41
47.
60. Marty P, Lelievre A, Quaranta JF, Rahal A, Gari-Toussaint M,
Fichoux Y, 1994. Use of the leishmanin skin test and Western
blot analysis for epidemiological studies in visceral leishmaniasis areas: experience in a highly endemic focus in AlpesMaritimes (France). Trans R Soc Trop Med Hyg 88: 658
659.
61. Araujo FG, 1986. Analysis of Trypanosoma cruzi antigens
bound by specific antibodies and by antibodies to related Trypanosomatids. Infect Immun 53: 179185.
62. Fargeas C, Hommel M, Maingon R, Dourado C, Monsigny M,
Mayer R, 1996. Synthetic peptide-based enzyme-linked immunosorbent assay for serodiagnosis of visceral leishmaniasis. J Clin Microbiol 34: 241248.
63. Correia da Costa JM, Neogy AB, Vouldouks I, Sampaio Silva
ML, Gentilini M, Monjour L, 1996. Antigenic components of
partially purified antigens of Leishmania donovani infantum
recognized by sera from dogs with asymptomatic or active
visceral leishmaniasis. Am J Trop Med Hyg 55: 511515.
64. Chiaramonte MG, Zwirner NW, Caropresi SL, Taranto NJ, Malchiodi EL, 1996. Trypanosoma cruzi and Leishmania spp. human mixed infection. Am J Trop Med Hyg 54: 271273.