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Journal of Clinical Microbiology, September 1998, p. 2423-2427, Vol. 36, No. 9
Institute of Microbiology,
Received 23 February 1998/Returned for modification 6 April
1998/Accepted 2 June 1998
Chagas' disease is a common cause of morbidity in Latin American
countries. In Brazil, naturally occurring transmission of its etiologic
agent, Trypanosoma cruzi, has been almost completely abolished through effective control programs aimed at the triatomid insect vector. Thus, transfusion of blood from infected donors has
become the major route for contracting Chagas' disease due to the
socioeconomically motivated migration of residents from areas where the
disease is endemic to the larger urban centers. Therefore, the
employment of screening tests is mandatory for all blood banks
throughout the country. We compared the diagnostic performances of
three commercially available screening assays used in routine testing
in Brazilian blood banks: the Abbott Chagas antibody enzyme immunoassay
(Abbott Laboratórios do Brasil, São Paulo), the
BIOELISACRUZI kit (Biolab-Mérieux, Rio de Janeiro, Brazil), and
the BIOZIMA Chagas kit (Polychaco S.A.I.C., Buenos Aires, Argentina).
The evaluation was performed with sera obtained from chagasic patients
and healthy residents of four different areas in Brazil where Chagas'
disease is either endemic or emergent and where clinical manifestations
of the disease and circulating parasite strains vary. The results
obtained with each kit were compared to matched in-house enzyme-linked
immunosorbent assay and immunofluorescence assay data obtained for each
sample. Depending on the area under investigation, the three commercial
kits produced specificity values between 93.3 and 100.0%, sensitivity
values between 97.7 and 100%, and accuracies ranging from 93.6 to
100.0%.
The protozoan parasite
Trypanosoma cruzi is the etiologic agent of Chagas'
disease, which is endemic throughout Latin America and which is a major
cause of morbidity and death in the affected countries. According to
World Health Organization estimates (31), 16 to 18 million
people are infected by the parasite and about 50,000 chagasic patients
die each year from the disease. In Brazil, the area in which the
disease is endemic extends over 17 states in the northeastern,
southeastern, southern, and central western regions (21),
but successful vector control programs have abolished almost completely
the natural transmission of T. cruzi by its reduviid
insect vector. Recent studies reported few chagasic patients younger
than 12 years in the state of Minas Gerais (8, 20). Apart
from vectorial transmission, Chagas' disease can be contracted either
orally (39), congenitally (23), or by transfusion
of blood from an infected donor (38). Due to socioeconomic
factors, the migration of infected people from the areas in which the
disease is endemic to the urban centers is very frequent, and blood
transfusion has become the principal way of infection, accounting for
an estimated 20,000 new cases per year in Brazil, a country with five
to six million blood transfusions per year (21). Therefore,
efficient donor screening is very important in order to identify and
discard contaminated blood without negatively affecting the country's blood supply.
T. cruzi infection is lifelong, and after a short and
mostly asymptomatic acute phase, during which the parasites can be
detected in blood smears, patients enter the indeterminate phase of the disease, which is marked by an extremely low parasitemia and no sequelae. This stage can last for 10 to 30 years, after which a
significant percentage of patients develop the chronic manifestations of Chagas' disease (cardiopathy, megacolon, and/or megaesophagus). While traditional methods of parasite detection (hemoculture and xenodiagnosis) are time-consuming and of low sensitivity, PCR amplification of nuclear (32, 40) or kinetoplast (3,
43) DNA was shown to be very sensitive (10, 46).
However, at present, PCR is not feasible for blood bank screening, and
the best way of diagnosing an indeterminate or chronic T. cruzi infection is the serologic detection of antibodies directed
against the parasite. Serologic assays include the indirect
immunofluorescence assay (IFA), indirect hemagglutination, complement
fixation, the radioimmunoprecipitation assay, the enzyme-linked
immunosorbent assay (ELISA), and Western blots. Antigen preparations
employed in these tests range from crude parasite extracts and
subcellular fractions to cloned antigens and synthetic peptides
(24, 27-30, 34-36, 41, 44, 45). Some of these tests are
available commercially, while others are in-house assays being used
only in research settings. In Brazilian blood banks today, the
screening of donors for Chagas' disease by at least two tests
based on different methodologies is obligatory. Although IFAs and
hemagglutination often lead to false-positive or -negative test results
due to subjective interpretation, both assays are still widely used in
blood bank screening and epidemiological surveys, and the results are
generally confirmed by an ELISA.
T. cruzi is polymorphic, and different parasite strains
circulate in different areas (21). While to date no definite
correlation between infecting strain and clinical manifestation has
been demonstrated, survey studies in regions in which the disease is
endemic show differences in antibody titers found in the patients and
in the degree of the clinical manifestations in the chronic phase of the disease (21). Since the infected donor populations
encountered in the large urban centers of Brazil migrated from many
different regions of the country in which the disease is endemic, in
this study, we compared the performances of three commercial enzyme immunoassays (EIAs) by using panels of sera obtained from patients and
healthy residents of four Brazilian areas where Chagas' disease is
either endemic or emergent.
Study population and description of areas in which Chagas'
disease is endemic.
Sera were obtained from patients and healthy
residents from the following areas: the state of Minas Gerais in the
south-central region of Brazil (municipality of Virgem da Lapa,
n = 261; 12.6% seroprevalence), where the cardiac and
digestive forms of the disease are common (4, 5); the
hinterlands of the northeastern states of Paraíba
(n = 466; 9.5% seroprevalence) and Piauí
(n = 253; 5.9% seroprevalence), where the
indeterminate form of the disease is common (7, 16, 17); and
the Amazon state in the north of Brazil (municipality of Barcellos,
n = 85; 13.2% seroprevalence), where Chagas' disease
is emergent (15, 18, 19).
Indirect immunofluorescence.
All sera were tested at a final
dilution of 1/40 in in-house tests according to the method of Camargo
(12) with T. cruzi Y epimastigotes as
antigen and fluorescein isothiocyanate-conjugated goat anti-human
immunoglobulin G (IgG) (Cappel Biomedical Inc., Malvern, Pa.).
In-house ELISA.
The cytosolic fraction of T. cruzi Y epimastigotes was used as antigen. Briefly, Nunc
microtiter plates were sensitized overnight at 4°C with 100 µl of
antigen solution in 0.05 M sodium bicarbonate buffer (pH 9.6) at a
concentration of 200 ng/ml. Sera were diluted 1/200 in
phosphate-buffered saline-Tween 20 (PBST) (0.3%)-fetal calf serum
(5.0%), and 100 µl of the mixture was added to the wells. After 30 min at 37°C, the plates were washed eight times with PBST, anti-human
IgG-peroxidase conjugate (Cappel Biomedical Inc.) was added to the
wells at a dilution of 1/10,000 in PBST-fetal calf serum, and the wells
were incubated at 37°C for 30 min. After eight additional washes, the
immune complexes were developed with tetramethylbenzidine-H2O2 (Sigma), and the
absorbances were read at 450 nm. Cutoff values were calculated by
dividing the difference of the average absorbances of two positive and
three negative controls by three.
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Evaluation of Three Commercial Enzyme-Linked Immunosorbent Assays
for Diagnosis of Chagas' Disease
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
TABLE 1.
Consensus classifications of sera from four regions
of Brazil by in-house ELISAs and IFAs
Commercial EIAs. Three commercial EIAs were evaluated in this study: the Abbott Chagas antibody EIA (Abbott Laboratórios do Brasil, Sãn Paulo), the BIOELISACRUZI kit (Biolab-Mérieux, Rio de Janeiro, Brazil), and the BIOZIMA Chagas kit (Polychaco S.A.I.C., Buenos Aires, Argentina). Each EIA was carried out strictly according to the instructions provided by the manufacturer. Calculations of the cutoff values and evaluation of the test results were performed as described in the respective sections of each manual.
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RESULTS |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Evaluation of the EIAs. The results of the evaluation of the three kits are presented in Tables 2 and 3 for each study area and for the study population as a whole. Due to the lack of a serologic "gold standard" for the indeterminate and chronic phases of Chagas' disease (see also Discussion), the sera employed in the evaluation were characterized by matched IFA and in-house ELISA results. Of a total of 1,025 sera, 520 were consensus positive and 505 were consensus negative. Performances of the commercial tests were expressed as relative sensitivity, relative specificity, and accuracy (11).
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DISCUSSION |
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Materials & Methods
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Discussion
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In the present study, we compared the performances of the Abbott Chagas antibody EIA, the Biolab-Mérieux BIOELISACRUZI kit, and the BIOZIMA Chagas kit, which are routinely employed in Brazilian blood banks for the detection of antibodies against T. cruzi.
The BIOZIMA Chagas and BIOELISACRUZI kits are 96-well ELISAs, while the Abbott Chagas antibody EIA employs coated beads as the solid matrix. The Brazilian prices (in U.S. dollars) for a single test are $2.17, $1.59, and $3.51, respectively. The total test incubation times varied from 50 to 120 min, with the BIOZIMA Chagas kit being the fastest, providing a result by visual reading after a little over 1 h. In addition, it was the most easily performed, with controls, conjugate, and substrate supplied in dropper bottles as ready-to-use solutions.
All three assays gave satisfactory results with sera which were obtained in four Brazilian areas and classified as either consensus positive or negative by matched in-house IFA and in-house ELISA results. Relative assay sensitivities and specificities varied depending on the area under investigation (Table 3) and ranged for the total population from 98.6 to 100% and 94.7 to 99.8%, respectively. The observed area-dependent differences may in part be attributed to the disproportional fractions of positive and negative sera obtained in each area (e.g., 3 positive versus 75 negative sera from the Amazon and 202 positive versus 44 negative sera from Piauí [Table 2]). However, for the total population, we employed 520 (50.7%) consensus-positive and 505 (49.3%) consensus-negative sera. Consequently, assay performances calculated for the four panels as a whole should reflect interassay differences more precisely.
The ELISAs yielded conflicting results for a number of sera, but the same sera were not necessarily problematic for each of the three kits evaluated in this study. Thus, for 21 of 520 (4.0%) positive and 42 of 505 (8.3%) negative sera, the results obtained with at least one of the three kits were not in agreement with the consensus. These findings corroborate the results obtained by others (1, 26). A chemiluminescent ELISA for the diagnosis of active infection by T. cruzi (1) was evaluated with sera which yielded inconclusive results in eight conventional serologic tests. Depending on the combination of test results, the percentage of inconclusive results varied between 18 and 78%. In another study (26), the Abbott Chagas antibody EIA, the Biolab-Mérieux BIOELISACRUZI kit, and the Chagas IgG ELISA (Gull Laboratories, Salt Lake City, Utah) were evaluated with 60 sera obtained at a blood bank. The authors defined a combined assay performance in which a serum was considered positive if at least two of the three ELISAs to be evaluated plus a confirmatory IFA were positive. Using the combined assay performance results as the gold standard, ELISA sensitivities were reported to be 100% and specificities varied from 87 to 97%. Carvalho et al. (13) compared the performances of an in-house recombinant-antigen ELISA and four commercial ELISAs (Abbott, Biolab-Mérieux, Gull Laboratories, and Ortho Diagnostic, Buenos Aires, Argentina) with sera obtained in Virgem da Lapa, Minas Gerais, and at the state blood bank of São Paulo. The authors report for the commercial tests specificities ranging from 95.0 to 98.0% and sensitivities from 99.0 to 100.0%.
The observed variation of sera that were problematic for a given assay is not surprising since the antigen preparations employed in each of the evaluated kits are obtained by different procedures. Furthermore, the T. cruzi Y epimastigotes are cultivated according to different protocols in various culture media. As previously reported (37), extraction procedures influence drastically the epitopes retained on antigenic molecules. Furthermore, binding of these molecules to solid surfaces hides or exposes epitopes that have different affinities for both specific and nonspecific antibodies present in the sera, thus accounting for conflicting results.
The Abbott Chagas antibody EIA was also evaluated in two studies published earlier (9, 33). Pan et al. (33) reported a sensitivity of 93.48% and a specificity of 99.48% with 1,392 sera from Brazil and Argentina which had been previously characterized by a commercial indirect hemagglutination assay.
Brashear et al. (9) used the Abbott Chagas antibody EIA to screen 13,309 sera from a potentially high-risk U.S. donor population and calculated a specificity of 99.98% and positive and negative predictive values of 81.25 and 99.99%, respectively.
The sera employed in our study were obtained in Minas Gerais, Paraíba, Piauí, and the Amazon, regions where disease manifestation, circulating parasite strains, and parasitemia vary (6, 7, 16, 18, 19). As a consequence of the sampling technique, in which houses and dwellings were first investigated for the presence of triatomid bugs and then, in a second step, blood samples were drawn from the residents and their neighbors (Minas Gerais, Piauí, and Paraíba), the panels we used did not reflect the overall prevalences of T. cruzi infection described in serologic surveys for the populations in the study areas. However, in the Amazon region, triatomid bugs are not found in houses, and people get infected while working in the rain forest. The Amazon panel utilized in this study consists of a small part of the samples obtained during the serologic survey (19).
In the particular case of Chagas' disease, no serologic gold standard for the definition of the disease status exists, since detection of T. cruzi-specific antibodies depends on the patient's immune status and since cross-reactivity of T. cruzi antigens with antibodies raised against other coendemic parasites (Leishmania and Trypanosoma rangeli) is frequent (2, 25, 42). Nevertheless, despite its drawbacks, IFA is the most commonly used serologic test for Chagas' disease and, as a result, is widely accepted as the gold standard (22). Therefore, as a first step we determined the status of the sera according to the results obtained in an in-house IFA and an in-house ELISA (Table 1). The sera were considered either positive or negative if IFA and ELISA results were concordant and indeterminate if the two results were discrepant. However, while no indeterminate serum was found in the panel from Minas Gerais, 26 (5.6%) of the 466 sera from Paraíba were found to be indeterminate, as were 7 (2.8%) of the 253 sera from Piauí and 7 (8.2%) of the 85 sera from the Amazon. These findings can be explained by the epidemiological characteristics and circulating parasite strains in the different areas. In Virgem da Lapa, Minas Gerais, the cardiac and digestive forms of Chagas' disease are frequent, and the circulating T. cruzi strains generally cause a high-titer immune response in the patients (6). Furthermore, this area is not one in which Leishmania spp. (5), which can cause false-positive results in Chagas' disease serology (14), is endemic. On the other hand, in the states of Paraíba and Piauí, the indeterminate form of the disease predominates, and patients show mostly moderate or weak immune responses to the infection (7). Also, in these areas leishmaniasis is frequent. As far as the Amazon is concerned, cross-reactions with Leishmania spp. may account for the high seroprevalence reported for this region (15, 19), and infections with the nonpathogenic parasite T. rangeli have been demonstrated (18). Taken together, these facts are likely to account for the indeterminate classification of some sera by our in-house tests. In addition, we cannot rule out the possibility that some of the discrepancies observed between the in-house consensus results and those obtained with the three kits were due to a misclassification of the sera by our in-house assays. However, the use of in-house tests for the characterization of serum panels and subsequent evaluation of a commercial kit has been reported by others (35).
This study shows that the Abbott Chagas antibody EIA, the Biolab-Mérieux BIOELISACRUZI kit, and the BIOZIMA Chagas test are well suited for the detection of IgG antibodies against T. cruzi. Nevertheless, when used for routine diagnoses and blood bank screening, problems can occur if the patients or donors come from areas in which the epidemiology of Chagas' disease is complex. Therefore, confirmatory tests with higher specificities need to be developed, and good candidates for such tests are those that include a combination of T. cruzi-specific cloned antigens and/or synthetic peptides (13, 28, 35, 36).
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ACKNOWLEDGMENTS |
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This work was supported in part by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Financiadora de Estudos e Projetos (FINEP), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Conselho de Ensino para Graduados de Universidade Federal do Rio de Janeiro (CEPG-UFRJ).
We are deeply indebted to Carmen Nogueira from the blood bank of the University Hospital Clementino Fraga Filho, Rio de Janeiro, Brazil, for permission to use the Commander Dynamic Incubator and Quantum II reader with the Abbott Chagas antibody EIA and to Carlos A. B. de Souza for excellent technical assistance.
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FOOTNOTES |
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* Corresponding author. Mailing address: Departamento de Imunologia, Instituto de Microbiologia, UFRJ-CCS, Ilha do Fundão, 21941-590 Rio de Janeiro, Brazil. Phone: 0055-21-270 0990. Fax: 0055-21-560 8028. E-mail: IMIMWAL{at}MICROBIO.UFRJ.BR.
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REFERENCES |
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Materials & Methods
Results
Discussion
References
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Journal of Clinical Microbiology, September 1998, p. 2423-2427, Vol. 36, No. 9
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