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Next Article 
Clinical and Diagnostic Laboratory Immunology, November 2001, p. 1039-1043, Vol. 8, No. 6
1071-412X/01/$04.00+0 DOI: 10.1128/CDLI.8.6.1039-1043.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Antibodies to an Epitope from the Cha Human
Autoantigen Are Markers of Chagas' Disease
Núria
Gironès,1
Clara I.
Rodríguez,1
Beatriz
Basso,2
José M.
Bellon,3
Salvador
Resino,3
M. Angeles
Muñoz-Fernández,3
Susana
Gea,4
Edgardo
Moretti,2 and
Manuel
Fresno1,*
Centro de Biología Molecular,
CSIC-UAM, Universidad Autónoma de Madrid,1
and Servicio de Inmunología, Hospital Gregorio
Marañón,3 Madrid, Spain, and
Servicio Nacional de Chagas2 and
Facultad de Ciencias Químicas, Universidad Nacional de
Córdoba,4 Córdoba, Argentina
Received 12 March 2001/Returned for modification 7 June
2001/Accepted 17 July 2001
 |
ABSTRACT |
Chagas' disease is a prevalent disease in South America that is
thought to have an autoimmune etiology. We previously identified human
Cha as a new autoantigen recognized by chagasic sera. Those sera
recognized an epitope spanning amino acids 120 to 129 of Cha, named R3.
In the present study we have used the synthetic R3 peptide for the
detection of serum immunoglobulin G antibodies from patients at
different stages of Chagas' disease, including a therapeutically
treated group. The immunoreactivity with R3 by enzyme-linked
immunosorbent assay (ELISA) showed 92.4% sensitivity and 100%
specificity for Chagas' disease sera. This sensitivity and specificity
were higher than for any other autoantigen described to date. No
anti-R3 antibodies were detected in sera from
Leishmania-infected or idiopathic dilated cardiomyopathy
patients or healthy controls from the same areas. Moreover, anti-R3
antibody reactivity detected by ELISA correlated with conventional
serological tests as indirect immunofluorescence and ELISA assays with
Trypanosoma cruzi extracts and other diagnostic tests as
indirect hemagglutination. The levels of anti-R3 antibodies increased
with progression and symptomatology of Chagas' disease. More
interestingly, a statistically significant fall in anti-R3 antibody
titer was observed in patients treated with antiparasitic drugs. Those
results suggest that the presence of anti-R3 antibodies is a highly
specific marker of Chagas' disease and that R3 ELISA could be helpful
in the diagnosis and monitoring of this disease.
| |
INTRODUCTION |
Chagas' disease, which is caused by
the protozoan parasite Trypanosoma cruzi, affects several
million people in Central and South America (3).
Approximately 30% of infected persons develop symptoms of the disease
in their lifetime, which include cardiomyopathy, neuropathies, and
dilatation of colon or esophagus (27). The finding of a
T-cell-rich inflammatory mononuclear cell infiltrate and the scarcity
of parasites in heart lesions questioned the direct participation of
T. cruzi in chronic Chagas' cardiomyopathy (CCC) and
suggested the possible involvement of autoimmunity (24), although this remains a hotly debated issue (13).
Natural infections occur via the triatomid insect vector and have been
almost abolished through vector control programs. Congenital transmission and transfusion of blood from infected donors have become
the major routes of transmission of Chagas' disease, and blood bank
testing is now necessary in many countries. Diagnosis of T. cruzi infection often requires a combination of some of the
commercially available tests (15). Traditional methods of parasite detection such as xenodiagnosis and hemocultures have low
sensitivity and require long periods of time to carry out. Recently,
PCR amplification of nuclear or kinetoplast DNA has been shown to be
very sensitive (2, 4, 25, 31). However, PCR is not yet
feasible for blood bank testing in many of the areas where Chagas'
disease is endemic. At present, the best way of diagnosing an
indeterminate or chronic T. cruzi infection is the serologic
detection of antibodies directed against the parasite. Usually, two
tests based on different methodologies are required, indirect
immunofluorescence (IIF) and indirect hemagglutination (IHA), with the
results confirmed by a third test, an enzyme-linked immunosorbent assay
(ELISA) (15). Thus, specificity of Chagas' disease
diagnosis is still a problem.
Previously, we isolated a human antigen recognized by chagasic sera,
named Cha. The epitope of Cha recognized by chagasic sera was mapped to
amimo acids 120 to 129 (the R3 peptide) (11). We studied
whether the R3 peptide of the Cha autoantigen could be used as a marker
of the disease. For this, we studied the reactivity of chagasic sera,
including sera from patients at different clinical stages, against the
R3 peptide in ELISA. In addition, we compared the R3 ELISA with other
available tests.
| |
MATERIALS AND METHODS |
Synthetic peptides.
Peptides R3 (MRQLDTNVERRALGEIQNV) from
human Cha and S1 (STPSTPADSSAHSTPSTPV) from T. cruzi shed
acute-phase antigen were synthesized on an Applied Biosystems
synthesizer model 431A. Peptides were purified by high-pressure liquid
chromatography and checked for accuracy by mass spectrometry.
Human sera.
A total of 79 sera from patients with chronic
Chagas' disease from Venezuela and Argentina were tested. Of those, 50 were from patients at different clinical stages, including chronic patients treated with antiparasite drugs (Radanil or Lampit). Sera from
Argentina were obtained from the Servicio Nacional de Chagas Argentina,
and sera from Venezuela were a gift from J. Sequí (Centro de
Investigación Clínica, Instituto Carlos III, Madrid).
Nonchagasic patients included 10 healthy individuals from an area were
Chagas' disease is endemic (EHS samples) (Servicio Nacional de Chagas,
Argentina), 10 individuals infected with the Leishmania
parasite whose antigens cross-react with T. cruzi (kindly supplied by C. Alonso, Centro de Biología Molecular, Madrid), and 6 patients with nonchagasic cardiomyopathy with diagnosis of
idiopathic dilated cardiomyopathy (IDC), a disease with similar cardiac
symptoms as CCC (kindly supplied by Barbieri, Chagas Center and
Regional Pathology, Santiago del Estero, Argentina).
ELISA.
ELISA with total T. cruzi antigens was
performed in microtiter plates covered with soluble antigens following
the directions of the manufacturer (T. cruzi, Biozima-Ch,
Polychaco, Argentina). The sera were diluted 1:100. The second antibody
was monoclonal anti-human immunoglobulin G (IgG) labeled with
horseradish peroxidase. Hydrogen peroxide-tetramethylbenzidine was used
for color development, and the reaction was stopped with 2 N
H2SO4. The developed color was measured in a microplate reader at 495 nm.
An ELISA was developed against peptides R3 and S1. The binding of the
peptides to the ELISA microtiter plates (Maxisorp; Nunc) was carried
out using 2 to 20 µg/ml of peptide in carbonate buffer (pH 9.6) in a
final volume of 50 µl and incubating the plates overnight at 4°C.
Blocking was carried out in phosphate-buffered saline (PBS) containing
3% low-fat dry milk and 0,2% Tween-20 for 1 h at room
temperature. Sera from chagasic patients were added at 1:100 dilutions
in PBS-1% low fat dry milk-0,05% Tween-20 to the plates and then
incubated overnight at 4°C. Wells were washed three times for 10 min
with the same buffer and subsequently incubated with goat anti-human
IgG (heavy and light chains) horseradish peroxidase-conjugated
antibodies (Pierce) for 1 h at room temperature and washed five
times. Color development was accomplished by incubation with
o-phenylenediamine (Sigma) for 30 min and measured at 450 nm in a
microplate reader.
IHA.
The IHA was performed essentially as described
(18) using commercial kits (Polychaco), and following the
instructions of the manufacturer.
IIF.
T. cruzi epimastigotes were fixed with
glutharaldehyde, washed, and incubated with human sera and anti-human
IgG conjugated with fluorescein isothiocyanate following the method
described (10).
Statistical analysis.
We evaluated the accuracy of the ELISA
technique against the R3 peptide in function of specificity and
sensitivity. For the ELISA against the R3 peptide, a receiver operating
characteristics (ROC) curve was used to choose the best cutoff.
The cutoff level with the best specificity and sensitivity was an
optical density at 450 nm (OD450) of 0.122. Spearman correlation analysis, a variant of the Pearson correlation
coefficient, was also used. The sign of the R coefficient
indicates the sense of the association, being direct when there is a
positive sign and inverse when negative. A value of 0 indicates no relationship.
| |
RESULTS |
R3 ELISA of chronic chagasic sera.
A total of 105 serum
samples were analyzed. Those included sera from 79 persons in the
chronic stage of Chagas' disease. Figure 1 shows that the majority of chagasic
sera, 73 of 79 (92.4%), had IgG against R3 above the cutoff level
(OD450 of 0.122), with 58 of 79 sera (73%)
having ELISA OD values above the cutoff (greater than 1). The
specificity of the R3 peptide was analyzed with serum samples from
nonchagasic patients, including EHS, and patients with other parasitic
diseases such as leishmaniasis or unrelated cardiomyopathy such as IDC.
All nonchagasic sera showed OD values below the cutoff level (Fig. 1).
The statistical analysis showed that the assay was 92.4% sensitive and
100% specific for chagasic sera, while it showed no sensitivity for
the nonchagasic controls (leishmaniasis, IDC, and EHS sera) (Table
1).
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FIG. 1.
Reactivity of sera from chronic chagasic patients
against the R3 peptide. The reactivity of 79 sera with positive
serology for Chagas' was tested in ELISA with the R3 peptide. Ten sera
from individuals infected with Leishmania infantum, 6 sera from patients suffering (IDC, and samples from 10 healthy
individuals living in an area were Chagas' disease is endemic (EHS)
were used as controls. Reactivity is expressed as OD at 450 nm. The
horizontal dashed line represents the cutoff value.
|
|
Correlation of anti-R3 IgG antibody levels with Chagas' disease
progression.
Next, we analyzed the titers of anti-R3 antibodies in
sera from patients in different stages of clinical disease. The ELISA reactivity against R3 peptide was compared in serum samples from patients in different stages of Chagas' disease (11 patients with and
33 patients without symptoms) and 10 serum samples of EHS. Although it
was not statistically significant, we observed an increase in the mean
anti-R3 titer (from 0.8 to 1.1) with progression of disease, since
antibody titer was higher in symptomatic than in asymptomatic chagasic
patients (Fig. 2A). We included in the analysis an ELISA based on the S1 peptide, which corresponds to the
C-terminal repeats of the shed acute-phase antigen of T. cruzi, a highly immunogenic T. cruzi antigen. The
reasons to include it were twofold: shed acute-phase antigen has been
considered one of the most immunogenic antigens and the best one for
the acute phase, and to compare the R3 peptide ELISA with another peptide-based ELISA (not based on parasite extracts) against which IgG
prevalence in samples from chronic patients has been reported (23). Noteworthy, the titers of anti-S1 antibodies were
much lower than anti-R3 titers in all patients.
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FIG. 2.
Correlation of anti-R3 IgG antibodies with
symptomatology and treatment. (A) Correlation of IgG antibody levels
against R3 and S1 peptides with Chagas' disease progression. Sera were
grouped as control (EHS), asymptomatic, and symptomatic and plotted
against the mean OD490 ± standard deviation (SD)
values obtained with the R3 (solid circles) and S1 (solid triangles)
peptide ELISA. The number of individual sera tested in each group is
indicated. Sera were assayed at 1:100 dilution. (B) Correlation of IgG
antibody levels against R3 and S1 peptides with the treatment of
asymptomatic chagasic patients. The results obtained with sera from
untreated and treated chagasic patients were plotted against the mean
OD490 ± SD values obtained with the R3 (solid
circles) and S1 (solid triangles) peptide ELISA. The number of
individuals in each group is indicated.
|
|
Relationship of anti-R3 IgG antibody levels to treatment of
chagasic patients.
The titers of antibodies against the R3 peptide
and S1 peptide were also tested in a group of 19 asymptomatic chagasic
patients treated with nifurtimox (Lampit) or benznidazol (Radanil) and compared to 30 sera from untreated patients of the same asymptomatic group. A statistically significant decrease in the mean reactivity against R3 was observed in patients treated with either Lampit or
benznidazol (P < 0.01). The analysis of the S1
reactivity, although lower in magnitude, also showed a statistically
significant decrease in the OD values after treatment
(P < 0.01), (Fig. 2B).
Comparison of R3 peptide ELISA with other serological tests.
The results obtained with ELISA of the R3 or S1 peptides were compared
with the results of other commercially available diagnostic tests,
including IFA, IHA, and ELISA with crude parasite extract, and
statistical analyses were performed using the Spearman test. Correlation between the R3 ELISA and IIF (Fig.
3A) and IHA (Fig. 3B) titers as well as
with conventional ELISA (Fig. 3C) showed indexes of 0.61, 0.71, and
0.64, respectively (P < 0,001). Although the S1 ELISA
also showed a direct correlation with IIF, IHA, and ELISA, the indexes
were smaller than with the R3 ELISA (0.39, 0.5, and 0.35, respectively,
with P < 0.01 to 0.001).
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FIG. 3.
Correlation of IgG antibodies against R3 and S1 peptides
by ELISA with other serological tests (IIF, IHA, and parasite ELISA).
Individual sera were grouped based on the results (titer inversa) of
each serological test and plotted against the mean of
OD490 ± SD values obtained with the R3 (solid
circles) and S1 (solid triangles) peptide ELISA. The number of serum
samples in each group is indicated. (A) Correlation with IIF. A value
of 0 is negative and indicates titers <1:32. (B) Correlation with IHA.
A value of 0 is negative (titer < 1:16). (C) Correlation with a
commercial ELISA using crude T. cruzi extract at 1:100
serum dilution.
|
|
| |
DISCUSSION |
Despite programs of insect control, diagnosis of Chagas'
diseases in blood bank screening is necessary nowadays in order to avoid spreading the infection. Currently, the detection of positive samples for T. cruzi infection involves a combination of
different tests. A recurrent problem is the presence of false-positives due to cross-reactions with sera from patients with other infectious diseases, such as leishmaniasis.
Here, we have tested the potential of the R3 peptide of Cha, a recently
described autoantigen (11), as a marker of Chagas' disease. The presence of specific autoantibodies is used as marker of
the disease and of disease progression in several pathologies of
autoimmune etiology (7, 8, 29). Here, we report the first
description of the usefulness of such a test for the diagnosis of
Chagas' disease. We performed a statistical analysis of the reactivity
against the R3 peptide of Cha and found that it is a good marker of the
disease. The R3 peptide ELISA showed 92.4% sensitivity and 100%
specificity for chronic chagasic sera in comparison with sera from
individuals infected with Leishmania and patients suffering
IDC and EHS, sera which were below the cutoff value. Although many
autoantigens have been described during T. cruzi infection,
the percentage of recognition among chagasic sera was lower (6,
9) than for the R3 peptide. The R3 peptide was not recognized by
sera from IDC patients, a disease clinically similar to CCC. Very
interestingly, this result is contrary to results obtained with other
autoantigens which are recognized by both chagasic and IDC sera
(5) and indicates the high specificity of the R3 peptide
as a marker of Chagas' disease.
Currently employed serological tests for Chagas' disease include some
commercially available enzyme immunoassays, based on T. cruzi extracts, as well as IIF and IHA assays. These assays are
still widely used but often lead to false-positives or -negatives due
to subjective interpretation. However, those tests gave between 7.5%
and 17.5% positive results for sera from
Leishmania-infected patients as well as false-positives and
-negatives (15). Many enzyme immunoassays using either
crude antigen or recombinant T. cruzi proteins have been
recently described with the aim to circumvent this problem (12,
14, 16, 17, 19, 20, 22, 28). Those tests have specificities
ranging from 93 to 100% and sensitivities of 97 to 100%, although few
of these are really employed in the field. The test described here has
similar sensitivity and specificity. Moreover, statistically
significant correlations of recognition of the R3 by chagasic sera with
commercially available tests (IIF, IHA, and ELISA based on the
parasite) were found. Our results indicated that all sera positive for
T. cruzi infection, usually diagnosed by IHA and IIF, are
recognized by the anti-R3 test. Anti-R3 antibody titer tended to
increase with disease progression (mean OD increased from 0.8 to 1.1)
although the differences were not statistically significant because of
the spreading of the OD values (from approximately 0.2 to 1.5). We
observed an inverse correlation between nifurtimox/Lampit treatment and
anti-R3 antibody titers in asymptomatic patients. This inverse
correlation was observed as well with IIF, IHA, and ELISA based on the
parasite antigen. Thus, the IgG titers against R3 of positive sera
showed a statistically significant decrease in treated patients.
The R3 peptide ELISA showed a stronger titer and correlation with other
tests than an ELISA against the S1 peptide, which contains the highly
immunogenic C-terminal repeats of shed acute-phase antigen of T. cruzi and is considered a major antigen (23). This
result indicated that a stronger reactivity against R3 of Cha than
against S1 of shed acute-phase antigen develops during infection. On
the other hand, T. cruzi is a polymorphic parasite, and
different strains may circulate in different areas. Our results gave
similar results when the sera were divided according to their origin
(Venezuela and Argentina) (not shown). Another problem that some
serologic tests based on T. cruzi antigens present is derived from the cross-reactivity of anti-T. cruzi
antibodies with other endemic parasites such as Leishmania
(1) and Trypanosoma rangeli (12, 21, 26,
30). R3 is not recognized by sera from
Leishmania-infected patients. Although we have not
specifically tested reactivity of R3 with sera from T. rangeli-infected individuals, we think this is unlikely by the
nature of the test, since these patients have no autoantibodies.
In summary, our results indicate that the detection of antibodies
against R3 peptide could be used as a confirmation marker of Chagas'
disease. Due to the direct correlation with symptoms and inverse
correlation with treatment, it can be used to help monitor the clinical
status of patients.
| |
ACKNOWLEDGMENTS |
This work was supported by grants from Instituto de
Cooperación Iberoamericana, Ministerio de Educación y
Cultura, Fondo de Investigaciones Sanitarias, Comunidad Autónoma
de Madrid, and Fundación Ramón Areces.
We thank María Chorro and Lucía Horrillo for their
excellent technical assistance.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Centro de
Biología Molecular, CSIC-UAM, Universidad Autónoma de
Madrid, Cantoblanco, E-28049 Madrid, Spain. Phone: 34-91-3978413. Fax:
34-91-3974799. E-mail: Mfresno{at}cbm.uam.es.
| |
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Clinical and Diagnostic Laboratory Immunology, November 2001, p. 1039-1043, Vol. 8, No. 6
1071-412X/01/$04.00+0 DOI: 10.1128/CDLI.8.6.1039-1043.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
