Enzyme-Linked Immunosorbent Assay for Serological Diagnosis of ...

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antibodies to Trypanosoma cruzi in patients with treated or untreated Cha- gas' disease. ... Peralta, J. M., M. G. Teixeira, W. G. Shreffler, J. B. Pereira, J. M. Burns,.
JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 2001, p. 4390–4395 0095-1137/01/$04.00⫹0 DOI: 10.1128/JCM.39.12.4390–4395.2001 Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Vol. 39, No. 12

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-Me´rieux 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

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 TcFELISA 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. 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 diagnosis of the disease (1, 4). Among the existing serologic procedures, 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 commercially 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 leading frequently to false-positive results (2). Furthermore, parasite 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 evaluation of a series of antigens that may be useful in diagnosis and vaccine development (10, 14). Recent studies employing mixtures of synthetic peptides and/or recombinant antigens demonstrated an increase in assay sensitivity (11, 14, 16).

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 Diseases of the World Health Organization, Chagas’ disease is still considered an important world public health problem. Migration 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-related, 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 findings permits a highly accurate definition of the chagasic patient. Direct detection of the parasite in the blood by micros* Corresponding author. Mailing address: Tropical Medicine Institute, Av. Dr. Eneas de Carvalho Aguiar, 470, CEP 05403-140, Sa˜o Paulo, Brazil. Phone: 55 11 30850416. Fax: 55 11 30623622. E-mail: [email protected]. 4390

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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 different T. cruzi-specific peptides. Sensitivity, specificity, and inter- 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

TABLE 1. Calculation of the Youden coefficient using results obtained for group I and group III sera with different suggested cutoff values Suggested cutoffa

0.113 (2 0.143 (3 0.173 (4 0.203 (5 0.233 (6 0.263 (7 a

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 hemagglutination (IHA), and conventional ELISA. The patients were clinically and epidemiologically 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 immunologically 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 Ceara´). The remaining five samples were obtained from Peruvian patients. All patients were living in areas where leishmaniasis 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 nonchagasic 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 doublestranded 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; Invitrogen, 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 antihuman immunoglobulin G conjugate (biolab-Me´rieux 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

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SD) SD) SD) SD) SD) SD)

No. of sera with TcF-ELISA result Reactive

Not reactive

110 103 102 101 101 101

141 148 149 150 150 150

J index

1.028 1.015 1.008 1.000 1.000 1.000

Average OD of the group III sera ⫹ SD.

ready-for-use solution of tetramethylbenzidine-H2O2 (Intergen Co.) was pipetted 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 sensitivity (17). Reproducibility of TcF antigen and TcF-ELISA. To evaluate the reproducibility 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 nonchagasic patients tested in triplicate during 5 consecutive days. Other serologic assays. Conventional serology was performed by employing commercial tests for indirect immunofluorescence (IMUNOCRUZI; biolabMe´rieux S.A.), indirect hemagglutination (HEMACRUZIⱕ; biolab-Me´rieux S.A.), and an ELISA with total T. cruzi extract as antigen (BIOELISACRUZI; biolab-Me´rieux S.A.). All assays were performed according to respective instructions 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 evaluated. For the samples from patients with leishmaniasis, the TcFELISA presented significantly fewer false-positive results than the other tests, thus suggesting a higher specificity. The positivity 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

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TABLE 2. Sensitivity and specificity of the TcF-ELISA and the conventional assays after testing sera from chagasic patients, healthy individuals, and leishmaniasis patients No. of sera with result by indicated assaya Sample set

TcF-ELISA

Chagasic patients (n ⫽ 101) Leishmaniasis patients (n ⫽ 39) Healthy individuals (n ⫽ 150)

b

IHAc

IIFd

ELISA (total antigen)e

Reactive

Not reactive

Reactive

Not reactive

Reactive

Not reactive

Reactive

Not reactive

101 2 0

0 37 150

101 25 0

0 14 150

101 28 0

0 11 150

101 10 0

0 29 150

a

For each assay, the reactivities of 101 of 101 sera were correctly identified (sensitivity, 100%). 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). b

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 obtained 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 (TcFELISA) 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 BIOELISACRUZI, 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 reproducibility when the ELISA-TcF was performed for 5 consecutive days was 1.78%. DISCUSSION The serologic diagnosis of Chagas’ disease remains problematic, 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 diagnosis 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

use of two assays based on different principles. This strategy improved the precision of the results and minimized the problems 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 development 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 evaluated (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) antigens, 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 represents 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, alternatively, 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 development of an ELISA that employs TcF, one such artificially

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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 compared 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

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-

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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 BIOELISACRUZIⱕ. 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 reactivity 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 recommended 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 intertest CV of 1.78%, lower than the 2% accepted limit for ELISA. Both values are considered excellent for an immunoenzymatic assay. Our results demonstrate the applicability of the TcF antigen for the serological diagnosis of Chagas’ disease. Further evaluations that employ serum samples obtained

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. REFERENCES 1. Camargo, M. E. 1992. An appraisal of Chagas’ disease serodiagnosis, p. 165–178. In S. Wendell, Z. Brener, M. E. Camargo, and A. Rassi (ed.), Chagas’ disease (American trypanosomiasis): its impact on transfusion and clinical medicine. ISBT Brazil 92. Sociedade Brasileira de Hematologia e Hemoterapia, Sa˜o Paulo, Brazil. 2. Chiller, T. M., M. A. Samudio, and G. Zoulek. 1990. IgG antibody reactivity with Trypanosoma cruzi and Leishmania antigens in sera of patients with Chagas’ disease and leishmaniasis. Am. J. Trop. Med. Hyg. 43:650–656. 3. Ferreira, A. W., Z. R. Belem, M. E. G. Moura, and M. E. Camargo. 1991. Aspectos da padronizac¸˜ao de testes sorolo ´gicos para doenc¸a de Chagas: um teste imunoenzima´tico para a triagem de doadores de sangue. Rev. Inst. Med. Trop. S. Paulo 33:(2)123–128. 4. Ferreira, A. W., and S. L. M. Avila. (ed.). 2001. Diagno ´stico laboratorial das principais doenc¸as infecciosas e auto-imunes, 2nd ed., p. 241–249. Guanabara Koogan S. A., Rio de Janeiro, Brazil. 5. Houghton, R. L., D. R. Benson, L. D. Reynolds, P. D. McNeill, P. R. Sleath, M. J. Lodes, Y. A. Skeiky, D. A. Leiby, R. Badaro, and S. G. Reed. 1999. A multi-epitope synthetic peptide and recombinant protein for the detection of antibodies to Trypanosoma cruzi in radioimmunoprecipitation-confirmed and consensus-positive sera. J. Infect. Dis. 179:1226–1234. 6. Houghton, R. L., D. R. Benson, L. D. Reynolds, P. D. McNeill, P. R. Sleath, M. J. Lodes, Y. A. Skeiky, R. Badaro, A. U. Krettli, and S. G. Reed. 2000. Multiepitope synthetic peptide and recombinant protein for the detection of antibodies to Trypanosoma cruzi in patients with treated or untreated Chagas’ disease. J. Infect. Dis. 181:325–330. 7. Krieger, M. A., E. Almeida, W. Oeleman, J. J. Lafaille, J. Borges-Pereira, H. Krieger, M. R. Carvalho, and S. Goldemberg. 1992. Use of recombinant antigens for the accurate immunodiagnosis of Chagas’ disease. Am. J. Trop. Med. Hyg. 46:427–434. 8. Mocayo, A. 1992. Chagas’ disease. Epidemiology and prospects for interruption of transmission in the Americas. World Health Stat. Q. 45:276–279. 9. Oelemann, W. M. R., M. G. M. Teixeira, G. C. Verissı´mo Da Costa, et al.

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