JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 2001, p. 4390–4395 Vol. 39, No.

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0095-1137/01/$04.00⫹0 DOI: 10.1128/JCM.39.12.4390–4395.2001
Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Enzyme-Linked Immunosorbent Assay for Serological Diagnosis

of Chagas’ Disease Employing a Trypanosoma cruzi
Recombinant Antigen That Consists of Four Different Peptides
A. W. FERREIRA,1,2* Z. R. BELEM,1 E. A. LEMOS,1 S. G. REED,3 AND A. CAMPOS-NETO3
Biolab-Mérieux S/A-Sao Paulo1 and Tropical Medicine Institute Sao Paulo,2 Sao Paulo, Brazil, and
Corixa Corporation and Infectious Disease Research Institute, Seattle, Washington3
Received 7 May 2001/Returned for modification 12 July 2001/Accepted 17 September 2001

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Serological tests to detect Trypanosoma cruzi antibodies have been used for screening blood donors, for
epidemic studies, and for diagnosis of probably infected persons. Among different tests, the enzyme-linked
immunosorbent assay (ELISA) with total, semipurified, or synthetic antigens has been widely used, mainly due
to its easy automation. Aiming to improve serological studies concerning Chagas’ disease, we have developed
and evaluated a new test, the TcF-ELISA, using an artificially engineered recombinant antigen, which contains
tandem sequences of different T. cruzi-specific peptides. The sensibility of the TcF-ELISA was determined with
101 serum samples from chagasic patients well-defined by clinical and epidemiological criteria. The specificity
was determined with 39 serum samples from leishmaniasis or kala-azar patients and 150 serum samples from
nonchagasic blood donors from Sao Paulo, Brazil. The TcF-ELISA showed 100% sensitivity and 98.94% of
specificity. Compared with conventional ELISA (with semipurified T. cruzi epimastigote antigens), the TcF-
ELISA showed advantages; for example, it distinguishes better between reagent and nonreagent serum and
provides better precision and a lower occurrence of leishmaniasis cross-reactions. Our studies demonstrate
high reproducibility between two different lots of the TcF ELISA and its applicability for the serological
diagnosis of Chagas’ disease.

Chagas’ disease is caused by the flagellated protozoa
Trypanosoma cruzi and is an endemic infection in Central and
South America that affects 16 to 18 million individuals (8).
Over the last years, intensive eradication campaigns directed
against the triatomine vectors responsible for Chagas’ disease
transmission have been carried out in most of the cities of the
Southern Cone (i.e., Argentina, Brazil, Chile, Paraguay, and
Uruguay). As a consequence, vector transmission of T. cruzi
diminished drastically, especially in rural areas, and does not
exist today in many regions where the infection used to be
endemic. However, the transfusion of parasite-containing
blood continues to be an important way of transmission (8, 12,
13).
According to the Division for the Control of Tropical Dis-
eases of the World Health Organization, Chagas’ disease is still
considered an important world public health problem. Migra-
tion and immigration of people led to a spread of the disease
beyond the geographical borders of Latin America, and it has
been detected in Europe, Asia, and the United States. Because
Chagas’ disease has become predominantly transfusion-re-
lated, a systematic screening of blood donors is useful not only
in Latin America but also in developed countries that receive
immigrants from areas of endemicity (8, 13).
Several strategies exist for the diagnosis of Chagas’ disease.
The association of clinical, epidemiological, and serologic find-
ings permits a highly accurate definition of the chagasic pa-
tient. Direct detection of the parasite in the blood by micros-
* Corresponding author. Mailing address: Tropical Medicine Insti-
tute, Av. Dr. Eneas de Carvalho Aguiar, 470, CEP 05403-140, São
Paulo, Brazil. Phone: 55 11 30850416. Fax: 55 11 30623622. E-mail:
clawsmbf@usp.br.
copy, hemoculture, xenodiagnosis, or PCR is highly specific
and confirms the existence of an infection (1, 4). However,
these procedures are technically and operationally demanding.
In addition, as a consequence of the pathology of the disease,
direct detection is not very sensitive during the indeterminate
and chronic phases of Chagas’ disease (1, 4).
On the other hand, serologic tests that detect antibodies
specific for antigens expressed by the different developmental
stages of the parasite are well-suited for a fast and easy diag-
nosis of the disease (1, 4). Among the existing serologic pro-
cedures, the enzyme-linked immunosorbent assays (ELISAs)
are the tests of choice due to their capacity to be automated
and to permit the testing of a great number of serum samples
in a short time. Additionally, the results obtained are precise
since readings are objective, and test validation criteria and
interpretation of results are rigorous. The majority of the com-
mercially available ELISAs employ antigens obtained by lysis
of epimastogote or trypomastigote forms of T. cruzi (3, 9).
These tests are sensitive but often fail to distinguish between T.
cruzi-specific and Leishmania sp.-specific antibodies, thus lead-
ing frequently to false-positive results (2). Furthermore, para-
site culture conditions and antigen purification protocols are
difficult to standardize and therefore lead often to variations
between different lots. In an attempt to improve the serological
diagnosis of Chagas’ disease and to avoid the problems of assay
specificity and antigen obtainment, molecular techniques, such
as gene cloning and expression, and the in vitro synthesis of the
corresponding peptides allowed the identification and evalua-
tion of a series of antigens that may be useful in diagnosis and
vaccine development (10, 14). Recent studies employing mix-
tures of synthetic peptides and/or recombinant antigens dem-
onstrated an increase in assay sensitivity (11, 14, 16).

4390
VOL. 39, 2001
The present study reports the development and evaluation
of the TcF-ELISA. This assay uses an artificially engineered
recombinant antigen which contains tandem sequences of dif-
ferent T. cruzi-specific peptides. Sensitivity, specificity, and in-
ter- and intra-assay variability of the TcF-ELISA were assessed
by using serum samples obtained from either blood donors or
patients with a thorough clinical and epidemiological diagnosis
of either Chagas’ disease or leishmaniasis. In a second step, the
diagnostic performance of the test was compared to that of
other assays normally employed in blood bank screening.
MATERIALS AND METHODS
Serum samples. A total of 290 serum samples were employed in this study.
The sera were subdivided in three distinct groups (groups I to G III).
(i) Group I (n ⴝ 101): samples from chagasic patients that were positive for
T. cruzi antibodies by indirect immunofluorescence (IIF), indirect hemaggluti-
nation (IHA), and conventional ELISA. The patients were clinically and epide-
miologically well-defined and living in areas in Brazil and Argentina where
Chagas’ disease was until recently endemic. Of the 101 sera, 27 were obtained
from Brazilian patients with chagasic cardiopathy; 37 were from patients of the
Institute Mario Fatala Chaben, Buenos Aires, Argentina; and 37 were obtained
from patients residing in the state of Minas Gerais, Brazil.
(ii) Group II (n ⴝ 39): samples from patients with clinically and immunolog-
ically defined leishmaniasis or kala-azar. Of the 39 samples from patients with
clinically and immunologically defined leishmaniasis or kala-azar, a total of 34
samples originated from Brazilian patients (10 from the state of Sao Paulo, 11
from the state of Minas Gerais, 4 from the state of Maranhao, 5 from the state
of Pernambuco, and 4 from the state of Ceará). The remaining five samples were
obtained from Peruvian patients. All patients were living in areas where leish-
maniasis is endemic.
(iii) Group III (n ⴝ 150): serum samples obtained from nonchagasic blood
donors from the city of Sao Paulo, Brazil. The samples obtained from noncha-
gasic blood donors from Sao Paulo are representative of the Brazilian population
since the larger part of the donors migrated to Sao Paulo from various different
Brazilian states.
Recombinant antigen. The antigen TcF (T. cruzi fusion protein) was obtained
from Corixa Corp. (Seattle, Wash.). This antigen is a recombinant protein that
comprises a linear assembly of four previously described serologically active T.
cruzi peptides, namely, PEP-2 (11), TcD (15), TcE (16), and TcLo1.2 (5). The
recombinant protein was created as described (5) using a synthetic double-
stranded DNA that corresponded to the peptide sequences organized in tandem
as PEP-2–TcD–TcE–TcLo1.2 and with a hexahistidine tag at the amino terminus
(GDKPSPFGQAAAGDKPSPFGQAKTAAPPAKTAAPPAKTAAPPA-KAAI
APAKAAAAPAKAATAPAGTSEEGSRGGSSMPSGTSEEGSRGGSSMPA).
The synthetic DNA was bound into the pT7 vector following subcloning into the
pET expression vector. The expression of the recombinant protein was achieved
after transformation of BLR(DE3) pLys Escherichia coli. Recombinant protein
was purified by affinity chromatography using a nickel column (Pro-bond; In-
vitrogen, Carlsbad, Calif.). Purified protein was contained with undetectable
levels of endotoxin (less than 10 endotoxin units/mg of protein) as detected by
the Limulus amebocyte assay.
TcF-ELISA. The TcF-ELISA was developed after standardization of the solid
phase, sample dilution, reaction times, and the dilution of the conjugate and the
chromogenic substrate. The wells of polystyrene plates (Polysorb flat-bottom
plates; Nalge-Nunc, Roskilde, Denmark) were sensitized with 75 ng of the
recombinant TcF-antigen dissolved in 100 ␮l of sodium carbonate-bicarbonate
buffer (0.05 M, pH 9.6). After an incubation overnight at room temperature, the
plates were blocked with 1% bovine serum albumin (fraction V; Sigma, St. Louis,
Mo.) in phosphate-buffered saline (PBS) (pH 7.2) and subsequently washed once
with PBS–0.05% Tween 20 (PBS-T), followed by two washes with distilled water.
The sensitized plates were stabilized with a proprietary antioxidant solution,
dried overnight at 40°C in an oven with circulating air, and wrapped in aluminum
foil together with a desiccant. For the assay, 200 ␮l of sample dilution buffer
(PBS, bovine serum albumin [0.1%], Tween 20 [0.1%]) was placed into the wells
of the sensitized plate and 10 ␮l of serum was added, resulting in a final dilution
of approximately 1/20. After an incubation at 37°C for 30 min, the plates were
washed four times with PBS-T, and 100 ␮l of peroxidase-labeled sheep anti-
human immunoglobulin G conjugate (biolab-Mérieux S.A.) was added to each
well. The conjugate dilution was previously established. After another incubation
at 37°C for 30 min, plates were washed four times with PBS-T and 100 ␮l of
CHAGAS’ DISEASE SEROLOGY WITH TcF ANTIGEN 4391
TABLE 1. Calculation of the Youden coefficient using results
obtained for group I and group III sera with different suggested
cutoff values
No. of sera with TcF-ELISA
Suggested cutoffa result J index
Reactive Not reactive
0.113 (2 SD) 110 141 1.028
0.143 (3 SD) 103 148 1.015
0.173 (4 SD) 102 149 1.008
0.203 (5 SD) 101 150 1.000
0.233 (6 SD) 101 150 1.000
0.263 (7 SD) 101 150 1.000
a
Average OD of the group III sera ⫹ SD.
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ready-for-use solution of tetramethylbenzidine-H2O2 (Intergen Co.) was pipet-
ted into each well. The reaction was stopped after 30 min at 37°C by adding 100
␮l of 2 M H2SO4. The optical densities (OD) were read at 450 nm with a
reference filter of 620 nm. Each assay plate contained five negative and two
positive samples as controls. The results were considered valid when the negative
controls had OD values of less then 0.200 and the positive controls had OD
values above 0.800. The cutoff value was established after calculation of the
Youden coefficient (J index), in order to obtain maximum specificity and sensi-
tivity (17).
Reproducibility of TcF antigen and TcF-ELISA. To evaluate the reproducibil-
ity of the TcF antigen provide by Corixa Corp., two different lots were tested by
ELISA, using a well-defined serum panel from chagasic patients (nine samples)
and nonchagasic patients (seven samples). Plastic plates were sensitized with 75
ng of TcF antigen per well, as previous defined.
The reproducibility of the ELISA TcF intratest and intertest were performed
with six serum samples from chagasic patients and 6 serum samples from non-
chagasic patients tested in triplicate during 5 consecutive days.
Other serologic assays. Conventional serology was performed by employing
commercial tests for indirect immunofluorescence (IMUNOCRUZI; biolab-
Mérieux S.A.), indirect hemagglutination (HEMACRUZIⱕ; biolab-Mérieux
S.A.), and an ELISA with total T. cruzi extract as antigen (BIOELISACRUZI;
biolab-Mérieux S.A.). All assays were performed according to respective instruc-
tions provided by the manufacturer. Serum samples were diluted 1/20 for all
commercial tests.
RESULTS
The cutoff value for the TcF-ELISA was established by using
the Youden coefficient. Between 2 and 7 standard deviations
(SD) were added to the average OD obtained for the group III
sera (n ⫽ 150), and the Youden coefficient was calculated to
obtain maximum sensitivity and specificity for the group I sera
(n ⫽ 101). The average OD obtained for group III sera was
0.053 with a SD of 0.030. These values were used to calculate
the J index (Table 1).
According to the result shown in Table 1, the cutoff for the
TcF-ELISA was defined as 0.200, corresponding to the average
OD of the group III sera plus 4.5 SD (J index ⫽ 1.004). Table
2 shows the results obtained for the sera from all three groups
by using the preestablished cutoff value. The sensitivity and
specificity of TcF-ELISA and conventional tests were evalu-
ated.
For the samples from patients with leishmaniasis, the TcF-
ELISA presented significantly fewer false-positive results than
the other tests, thus suggesting a higher specificity. The posi-
tivity was 5.1% (2 of 39) for the TcF-ELISA, 25.6% (10 of 39)
for the BIOELISACRUZI, 71.8% (28 of 39) for IIF, and
64.1% (25 of 39) for IHA. All assays identified all chagasic sera
samples, thus presenting a sensitivity of 100%.
In Fig. 1, the OD of the TcF-ELISA are compared to those
4392 FERREIRA ET AL.
TABLE 2. Sensitivity and specificity of the TcF-ELISA and the conventional assays after testing sera from chagasic patients, healthy
individuals, and leishmaniasis patients
b
Sample set TcF-ELISA
Reactive Not reactive
Chagasic patients (n ⫽ 101) 101 0 101
Leishmaniasis patients (n ⫽ 39) 2 37
Healthy individuals (n ⫽ 150) 0 150
a
For each assay, the reactivities of 101 of 101 sera were correctly identified (sensitivity, 100%).
b
Relative specificity, 98.94% (187 of 189 sera).
c
Relative specificity, 86.77% (167 of 189 sera).
d
Relative specificity, 85.18% (161 of 189 sera).
e
Relative specificity, 94.71% (179 of 189 sera).
obtained with the conventional ELISA, BIOELISACRUZIⱕ.
Group I samples gave an average OD of 1.655 ␳ 0.643, whereas
the BIOELISACRUZIⱕ that employs total T. cruzi antigen
showed an average of 0.597 ␳ 0.238. For the group II samples,
obtained from Brazilian and Peruvian patients with cutaneous
or tegument leishmaniasis or kala-azar, the average OD ob-
tained in the TcF-ELISA was 0.122 ⫾ 0.118, where 2 out of 39
samples presented OD values above the predetermined cutoff
of 0.200. For the same group, the BIOELISACRUZI was
positive for 10 out of 39 sera when the interpretation criteria
were applied that consider positive all samples with an OD
higher than the cutoff and consider doubtful all samples with
an OD of ⫾ 20% of the cutoff (“gray zone”). Sera belonging to
group III had average OD values of 0.053 ␳ 0.030 (TcF-
ELISA) and 0.052 ⫾ 0.030 (BIOELISACRUZI) and therefore
were classified by both tests as true negatives. Taken together,
with the TcF-ELISA the average OD for positive sera was
about three times higher than that obtained with the BIOEL-
ISACRUZI, thus demonstrating a better discrimination of
positive and negative sera than the latter assay.
The immunological reproducibility of two different lots of
TcF antigen preparation in ELISA is presented in Fig. 2. No
significant differences in the OD were observed when sera
from chagasic and nonchagasic patients were tested. Intratest
and intertest reproducibility for ELISA-TcF were evaluated
and the coefficient of variation (CV) was determined. The
maximum CV obtained for intratest reproducibility when the
serum samples in ELISA-TcF was performed in triplicate was
1.37%. The maximum CV obtained for inter-test reproducibil-
ity when the ELISA-TcF was performed for 5 consecutive days
was 1.78%.
DISCUSSION
The serologic diagnosis of Chagas’ disease remains problem-
atic, although various kits available show a high degree of
precision (1, 4, 10). Differences in the developmental stage of
the parasite and in the procedures used for extraction and
purification of antigenic components, as well as in the assay
protocol itself, present a permanent source variation for the
final product, leading to difficulties in the industrial production
of reproducible lots. In order to improve the serological diag-
nosis of Chagas’ disease, the Special Programme for Research
and Training in Tropical Diseases of the UNDP/World Health
Organization recommended in 1982 for each serum sample the
J. CLIN. MICROBIOL.
No. of sera with result by indicated assaya
IHAc IIFd ELISA (total antigen)e
Reactive Not reactive Reactive Not reactive Reactive Not reactive
0 101 0 101 0
25 14 28 11 10 29
0 150 0 150 0 150
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use of two assays based on different principles. This strategy
improved the precision of the results and minimized the prob-
lems of sensitivity and specificity associated with the available
tests (8, 12). Usually, when a single test is used, sensitivity and
specificity vary around 98.91 and 98.52%, respectively. When
two or three tests are employed, sensitivity increases to 100%.
However, specificity values normally remain or diminish, thus
leading to a significant percentage of inconclusive results that
are frequently observed in clinical laboratories and blood
banks (1, 4, 12). Therefore, various research groups around the
world tried to identify more defined immunodominant and
antigenic fractions of T. cruzi that are highly sensitive and
specific in immunological tests. With the development of the
techniques of molecular biology, various proteins were isolated
and sequenced, and their corresponding genes were cloned
and subsequently used to produce recombinant proteins. Their
applicability in diagnosis as well as in our understanding of the
host-parasite interactions leading to new ways of vaccine de-
velopment has been widely reported (7, 9, 10, 11, 14, 16).
Among the synthetic peptides that have been evaluated, TcD
and PEP2 are outstanding. Both peptides are derived from
repetitive sequence parts of T. cruzi antigens and were shown
to be sensitive and specific in the diagnosis of acute and
chronic Chagas’ disease in studies carried out in different Latin
American countries (6, 11). However, although specific, the
peptides demonstrated limited sensitivity in immunoenzymatic
and immunoradiometric assays when employed separately, just
like other recombinant fractions that were previously evalu-
ated (11, 14, 16). In recent years, several groups reported an
improvement in sensitivity when sera were simultaneously
tested with sets of synthetic (11) and recombinant (14) anti-
gens, reaching 99.7% and 100%, respectively, without affecting
their excellent specificity that was previously reported. The use
of mixtures of peptides and recombinant proteins requires a
rigorous standardization and close monitoring of the solid
phase during the antigen adsorption step in order to warrant
reproducible performance of the different lots. The fusion of
different antigenic peptides into a single stable molecule rep-
resents an alternative that permits a uniform and reproducible
adsorption without loss of the individual diagnostic properties
observed. Either such a molecule can be synthesized or, alter-
natively, its synthetic corresponding DNA can be cloned, thus
permitting the large-scale production and purification of the
protein at low costs. In the present work we report the devel-
opment of an ELISA that employs TcF, one such artificially
VOL. 39, 2001
FIG. 1. Distribution of OD obtained with the TcF-ELISA and the BIOELISACRUZI for the serum samples from group I (chagasic patients),
group II (leishmaniasis patients), and group III (blood donors).
engineered recombinant T. cruzi molecule produced by Corixa
Corp. Our results demonstrate that this approach is a viable
strategy. The TcF-ELISA was shown to be 100% sensitive for
sera obtained from chagasic patients and showed an increased
specificity for samples from patients with leishmaniasis com-
pared to conventional serologic tests that use total T. cruzi
antigen. A clear difference can be observed when the OD
obtained with the TcF-ELISA and BIOELISACRUZI are
compared for group II samples. The BIOELISACRUZI
showed OD values close to the cutoff for the majority of the
samples and gave 10 false-positive results. On the other hand,
the TcF-ELISA gave only two false-positive results, with OD
values slightly above the cutoff, and the other sera from that
group gave OD values comparable to that obtained for the
CHAGAS’ DISEASE SEROLOGY WITH TcF ANTIGEN 4393
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blood donor samples. The donor samples used in this study
represent a population with a low prevalence of infection, and
the OD values obtained for all sera are well below the cutoff.
These findings make the TcF-ELISA highly suitable for the
diagnosis of chagasic patients in areas in Latin America were
leishmaniasis and Chagas’ disease coexist and mixed infections
are frequent.
The TcF-ELISA showed an absolute reactivity three times
higher than the BIOELISACRUZI, thus permitting a better
discrimination of positive and negative results and avoiding the
occurrence of indeterminate ones. For the BIOELISACRUZI
the definition of a gray zone (20% of the cutoff) in which
samples are classified as indeterminate is extremely important
for guaranteeing sensitivity, since the average OD value ob-
4394 FERREIRA ET AL.
FIG. 2. Comparison of immunological reactivity of two different lots of TcF antigen by ELISA with sera from chagasic and nonchagasic
patients. A reference filter of 620 nm was used.
served with this test for group I sera was only 2.7 times higher
than the cutoff. As a consequence, the number of sera with OD
values close to the cutoff is much higher for the BIOELISA-
CRUZIⱕ. The gray zone permits all sera with an OD of
⬎0.175 to be considered initially reactive. With the exception
of one serum, the average OD obtained for chagasic samples
with the TcF-ELISA was 8.2 times higher than the cutoff.
In the present study we evaluated two different lots of the
TcF antigen, both of which presented the same level of reac-
tivity and adsorption to the solid phase. These findings indicate
that the production of the fusion molecule is reproducible. In
order to assess the stability of the TcF-ELISA, the kit was
stored for 7 and 15 days at 37°C and subsequently evaluated
with a panel of chagasic sera. When the results were compared
to those obtained with a kit that was stored under the recom-
mended condition of 8 to 10°C, the OD values decreased 5 and
10%, respectively. These findings indicate that the fusion of the
peptides into a single molecule and its production by cloning
result in a stable antigen that can be homogeneously adsorbed
to a solid phase, thus avoiding the problems related to the use
of a mixture of single peptides. In order to assess the inter and
intratest reproducibility, reactive sera were tested in triplicate
on 5 consecutive days. Analysis of the CV obtained from the
OD indicated a maximum intratest CV of 1.37% and an in-
tertest CV of 1.78%, lower than the 2% accepted limit for
ELISA. Both values are considered excellent for an immu-
noenzymatic assay. Our results demonstrate the applicability of
the TcF antigen for the serological diagnosis of Chagas’ dis-
ease. Further evaluations that employ serum samples obtained
J. CLIN. MICROBIOL.
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in different areas in Latin America and a large number of
blood donor samples and compare the performance of the
antigen with those of conventional assays should be able to
confirm and validate the diagnostic value of the test.
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