Chagas disease (CD) is endemic in 21 continental American countries. The migratory movement of people has increased in recent decades due to push factors, such as poverty and unemployment, and pull factors, including better employment conditions and family reunification [1]. As a result, CD is no longer exclusively a rural problem specific to Latin America [2–4].
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The World Health Organization (WHO) estimates that 70 million people worldwide are at risk of being infected by Trypanosoma cruzi, the parasite that causes CD. Furthermore, it is estimated that 6 million people are infected with T. cruzi, with two thirds of these individuals currently living in urban areas; however, just 10% of these infected individuals have been diagnosed, preventing them from receiving appropriate care [5–7].
According to the WHO (), it is estimated that there are 1,350,000 individuals infected with T. cruzi in Argentina. The Argentinian – National Strategic Plan for Chagas disease aimed (a) to interrupt the transmission of T. cruzi and (b) to reduce the morbidity and mortality due to CD and the subsequent socioeconomic impact [8].
The discussion of CD as a public health issue should not be limited simply to estimates of the number of infected individuals and reported numbers of cases. Individuals living in vulnerable situations or who are members of marginalized populations are often more susceptible to T. cruzi infection and associated morbidity, due to a variety of factors including a widespread lack of access to formal education, timely healthcare, and adequate living conditions. The reasons for such deprivation are complicated, but ultimately result in the persistence of inequalities in communities affected by CD in Latin America [9].
Pharmacologic treatment in its acute stage of CD can cure the disease and prevent progression and disrupts the transmission cycle in the case of future infected pregnant women [10]. However, treatment exhibits reduced efficacy during the chronic stage of the disease and requires a long period of administration, which frequently results in undesirable adverse reactions to the drugs used [11].
Currently, access to diagnosis is the main barrier to receiving appropriate treatment for CD. In the absence of a gold standard, the diagnosis of chronic CD is a complex process, involving an algorithm that includes at least two laboratory determinations. According to the Pan American Health Organization (PAHO), the diagnostic standard for patients with suspected chronic CD is the combination of two serological tests that are based on different principles and antigens e.g., an enzyme-linked immunosorbent assay (ELISA), indirect hemagglutination (IHA), or indirect immunofluorescence (IIF), with a third serological test if the results are discordant [12,13].
A healthcare system that offers timely access to reliable diagnosis is the basis for the implementation of policies that guarantee access to appropriate treatment [12]. As long ago as , WHO proposed the need to validate diagnostic systems in primary healthcare centers that allow the rapid detection of T. cruzi infection as a strategy to reduce morbimortality due to CD [14]. Since then, some studies have been carried out to evaluate the performance of lateral flow assays, also known as rapid diagnostic tests (RDTs), in different population groups and with different sample types. The advantages of RDTs compared with the diagnostic standard proposed by PAHO include that RDTs do not require highly skilled or trained operators to use them, do not require refrigeration of the reagents used, have a short time from sampling to results, and allow the use of different sample types; these features mean that RDTs are also useful as field screening assays [15].
Recent field and laboratory studies, as well as systematic reviews, have found that RDTs demonstrate very good sensitivity and specificity (with 95% confidence interval (CI) ranges from 95–100%), with RDTs being proposed as an alternative to the current diagnostic standards in some regions [16–20]. However, a high risk of bias was detected in some publications in relation to the design of clinical trials and reference standards used [20]. Furthermore, the diagnostic performances of RDTs for CD have shown high variability, with sensitivity values ranging from 33 to 100% and specificity values ranging from 94 to 99.9% [13,21]. It is necessary to evaluate commercially available RDTs in various epidemiological scenarios and in different clinical settings where medical care for CD is provided. However, before conducting prospective trials, it is necessary to establish the performance of the tests under controlled laboratory conditions.
The primary objective of this study was to evaluate, under controlled laboratory conditions, the sensitivity and specificity of four commercially available T. cruzi RDTs, using the current CD diagnostic algorithm as a reference standard method and following the guidelines of both the Argentina’s Ministry of health and PAHO/WHO. As a secondary objective, we evaluated the agreement between the results of RDTs and the reference standard.
A flowchart depicting the study design and flow of participants samples, in conformity with the Standards for Reporting of Diagnostic Accuracy Studies (STARD) guidelines [25] is shown in Fig 1.
In , 5,929 individuals attended INP, in Buenos Aires, Argentina, to perform T. cruzi serology. Considering the eligibility, the first 200 negative samples and the first 200 positive samples with a volume greater than 500 μl, in good preservation condition, and with available reference standard results from INP, were conveniently selected (Fig 1). Regarding the 400 samples, the median age of the individuals was 49 years (range 18–84). The place of birth of these individuals was Argentina (253/400, 63.2%), Bolivia (99/400, 24.7%), Paraguay (24/400, 6%), Uruguay (2/400, 0.5%), Peru (1/400, 0.2%), and Venezuela (1/400, 0.2%); place of birth data was not available for 20/400 individuals (5%). All individuals whose samples were included had permanent residence in Argentina and were therefore considered to be users of the national health system.
During the process of verifying the recorded data, an acceptable error of 1.7%, in 5% of the critical fields, was obtained, including the images stored on the TeleSpot cloud platform and the results reported in OpenClinica Enterprise, compared with the original documents.
The capacity of the four index tests to detect antibodies anti-T. cruzi was evaluated with a serological external quality assurance panel. All index tests were able to detect anti-T. cruzi antibodies in human sera samples from regions where DTUs TcI and TcII are endemic, i.e., Mexico and Brazil respectively.
Table 2 shows true-positive, false-negative, true-negative and false-positive results and the estimated statistical parameters for the four RDTs evaluated. The SD Chagas Ab Rapid (Standard Diagnostic, Korea) showed the highest sensitivity (100%, 95%CI: 98.2–100%), followed by WL Check Chagas (Wiener Lab, Argentina) (99%, 95%CI: 96.4–99.9%), and Accu-tell (98%, 95%CI 95.0–99.5%). The Chagas Rapid First Response (Lemos Laboratories, Argentina) showed lowest sensitivity (92.5%, 95%CI: 87.9–95.7%). The Chagas Rapid First Response (Lemos Laboratories, Argentina) showed highest specificity (96%, 95%CI: 92.3–98.3%) while SD Chagas Ab Rapid (Standard Diagnostic, Korea) had the lowest specificity (76%, 95%CI: 69.5–81.7%).
No invalid tests were reported from 3/4 RDT, only WL Check Chagas (Wiener Lab, Argentina) had one invalid test result this sample was repeated.
Table 2 shows the Kappa index for overall agreement between the results of each RDT and the results of the reference standard, with 95% CI. The level of concordance with the reference standard of three of the four RDTs was 0.89 to 0.92 (for the WL Check Chagas by Wiener Lab, Argentina, Chagas Rapid First Response by Lemos Laboratories, Argentina, and ACCU-TELL Chagas Cassette by AccuBiotech Co. Ltd, China), i.e., an almost perfect concordance. The SD Chagas Ab Rapid by Standard Diagnostic, Korea (κ = 0.76) indicated a substantial agreement. There were no statistically significant differences in the kappa index for any of the RDTs (Table 2, overall agreement between each RDT and the reference standard).
The likelihood ratios were highly relevant for the WL Check Chagas (Wiener Lab, Argentina) and ACCU-TELL Chagas Cassette (AccuBiotech Co. Ltd, China) and Chagas Rapid First Response (Lemos Laboratories, Argentina (Table 2). Positive diagnoses results were missed by the following proportions for each test: WL Check Chagas (Wiener Lab, Argentina), 1%; SD Chagas Ab Rapid (Standard Diagnostic, Korea), 0%, Chagas Rapid First Response (Lemos Laboratories, Argentina) 7.5%; and ACCU-TELL Chagas Cassette (AccuBiotech Co. Ltd, China), 2%.
The three-way analysis showed that, in terms of sensitivity, all pairs of tests were statistically significantly different to each other, except for the WL Check Chagas (Wiener Lab, Argentina) compared with the SD Chagas Ab Rapid (Standard Diagnostic, Korea) (p = 0.); and the WL Check Chagas (Wiener Lab, Argentina) compared with the ACCU-TELL Chagas Cassette (AccuBiotech Co. Ltd, China) (p = 0.) (Table 3).
In terms of specificity, all pairs of tests were statistically significantly different except for the WL Check Chagas (Wiener Lab, Argentina) compared with the Chagas Rapid First Response (Lemos Laboratories, Argentina) (p = 0.); and WL Check Chagas (Wiener Lab, Argentina) compared to ACCU-TELL Chagas Cassette (AccuBiotech Co. Ltd, China); as well as Chagas Rapid First Response (Lemos Laboratories, Argentina) compared to ACCU-TELL Chagas Cassette (AccuBiotech Co. Ltd, China) (p = 0.) (Table 3).
Qualitative data, relating to the operating and storage requirements of each index test, were obtained from the IFU provided by the manufacturers (Table 1). All RDTs required the same operating temperature (15–30°C), and similar ranges of transport / storage temperatures (1–30°C), and in-use stability range (between 15–35 minutes following the addition of buffer). In terms of sample type, all of the RDTs worked with plasma, serum and whole blood. However, the SD Chagas Ab Rapid only worked whole blood obtained by venipuncture, while the other three tests allow the use of fingerstick blood. The sample volume of whole blood sample volume required ranged between 5 and 100 μl.
The usability score was estimated for each test, by all laboratory technicians (S2 Table). The score was calculated by considering: appearance of the background in the device after testing, test and control band intensity, quality of package insert, ease of reading, sample dispenser included in the kit, lancet included in the kit. The laboratory technicians all assigned a usability score higher than the average [11] for all RDTs.
To reduce and ultimately eliminate CD as a public health problem it is necessary to increase diagnostic coverage [26]. In this context, although RDTs are still only recommended for screening, and more evidence of its diagnostic utility is needed, RDTs have emerged as an option for the rapid and conclusive diagnosis of T. cruzi infection, assisting provision of treatment, improved adherence and contributing to the prevention of vertical transmission [16]. In our study, the four RDTs evaluated each displayed very good performance, both in terms of sensitivity and specificity, with similar values to those reported previously for these tests [13,19,26].
The SD Chagas Ab Rapid by Standard Diagnostic (Korea), WL Check Chagas by Wiener Lab (Argentina), and ACCU-TELL Chagas Cassette by AccuBiotech Co. Ltd (China) showed high sensitivity. These tests were able to correctly classify more than 196 out of 200 infected samples, individually tested by each RDT. The Chagas Rapid First Response by Lemos Laboratories (Argentina), WL Check Chagas by Wiener Lab (Argentina), and ACCU-TELL Chagas Cassette by AccuBiotech Co. Ltd (China) showed high specificity. These tests were able to correctly classify between 186 and 192 out of 200 non-infected samples, individually tested by each RDT.
In our study, Cohen’s Kappa for the majority of the RDTs showed an almost perfect concordance with the reference standard, except for the SD Chagas Ab Rapid by Standard Diagnostic (Korea), that showed a substantial concordance given that this test displayed a higher proportion of false-positive results compared with the other tests. However, none of the RDTs had statistically significant different overall agreement with the reference standard (Kappa index ≥ 0.8). Our findings are in agreement with the increasing recent evidence suggesting that the performance of RDTs is comparable to that of laboratory-based tests [18,27,28].
Interestingly, in another laboratory evaluation with a similar study design to our study [18], sponsored by FIND, the authors assessed WL Check Chagas by Wiener Lab (Argentina), SD Chagas Ab Rapid by Standard Diagnostic (Korea), and Chagas Rapid First Response by Lemos Laboratories (Argentina), using samples from Colombian individuals and the diagnostic algorithm in Colombia as reference. These RDTs displayed lower sensitivity (94%; 86.7% and 81.8%, respectively) and higher specificity (98.9%; 99.6% and 98.6%, respectively) compared to our study. Also, the diagnostic performance of the tests could be influenced by other factors such as regional differences in parasite antigenicity due to different DTUs.
In terms of usability, all of the RDTs we evaluated required the same operating conditions. Regarding sample type, only the SD Chagas Ab Rapid by Standard Diagnostic (Korea) requires whole blood obtained by venipuncture rather than fingerstick and a larger sample volume (0.1 ml), more than twice the volume required for the other tests. This might limit this test’s potential usefulness under real-world conditions with capillary fingerstick samples. According to the users, all of the tests were easy to handle and interpret (S2 Table).
Chagas disease, along with human immunodeficiency virus (HIV), syphilis, and hepatitis B virus, is included in the Framework for Elimination of Mother-to-Child Transmission (EMTCT Plus) initiative of the PAHO member states. The use of RDTs for HIV and syphilis would allow treatment and care to be initiated with no unnecessary delays, favoring immediate adherence, and reducing the risk of transmission [8]. Screening with RDTs is currently used for the other three diseases in the EMTCT Plus framework, but not yet for CD. In light of the results obtained by Eguez et. al. () (with Chagas Stat-Pak, by Chembio, and Chagas Detect Plus, by Inbios) [16] and Lopez Albizu et. al. () (with SD Chagas Ab Rapid by Standard Diagnostic and WL Check Chagas by Wiener Lab) [21], and in our study, with any of the RDTs that work with a small volume of serum as a sample, RDTs are comparable to the reference laboratory-based tests, and could potentially be used not only as screening tests but also as a secondary confirmatory tool, similar to what is already the case for diagnosing HIV infections in perinatal care centers. Together, these results show that RDTs on their own are not sufficient as stand-alone tests, but they could be particularly useful in remote areas that lack basic equipment, such as a centrifuge, where it is difficult to follow recommendations for diagnosis [27], but further evidence about performance and economic impact in different real clinical scenarios is further needed.
Truyens et. al. () have demonstrated that a combination of two RDTs showed reactivity very similar to that of the combination of two ELISAs, based on a cohort of T. cruzi-infected women and including blood/plasma samples from Argentina, Honduras, and Mexico, thus confirming the usefulness of RDTs for screening [29]. This strategy could potentially also reduce healthcare costs, given that RDTs are considered to be a cost-effective strategy compared with laboratory-based diagnostic methods, if the logistical costs are included, such as the cost of facilities and of training personnel in the handling of samples and interpretation of the results [16].
Although many RDTs for CD have been described, not all are available for purchase by health structures in CD-endemic countries, so it is important that from the various RDTs each performs well. Our initial objective was to compare seven RDTs, but this was not possible because all were not all available for procurement in Argentina.
It will be necessary to confirm the optimal performance of the RDTs we evaluated in a field evaluation study using capillary fingerstick samples, along with the analysis of usability and cost-effectiveness, to be able to recommend their use in different scenarios and with different sample types. However, given the sensitivity and specificity values obtained under controlled laboratory conditions in our study, our hypothesis is that the use of RDTs could strengthen local health structures. The accuracies of all RDTs evaluated in this study, conducted by the national reference laboratory for diagnosis of CD, are considered sufficiently good to recommend their use in Argentina to increase diagnosis rates at the primary healthcare level and reduce the time to diagnosis, once their optimal performance has been demonstrated in real-world settings.
Trypanosoma cruzi (Chagas, ), the etiologic agent of Chagas disease (CD), is a flagellate protozoan of the Trypanosomatidae family that is widely distributed in nature, occurring from the southern United States to southern Argentina and Chile. It is capable of infecting almost all tissues of hundreds of species of mammals to which it is transmitted by hematophagous hemiptera of the subfamily Triatominae, known as kissing bugs (barbeiros, vinchuca, bicudos and many other regional terms in Latin America) (Galvão et al., ; Noireau et al., ; Jansen et al., ). T. cruzi is a taxon that presents expressive intraspecific diversity whose interpretation has been challenging scientists since Carlos Chagas. Currently, seven genotypes or discrete typing units (DTUs) are recognized in the species, namely, TcI-TcVI, in addition to a genotype described as Tcbat. The latter, initially associated with Chiroptera (Marcili et al., ), has already been detected in humans (Guhl et al., ; Ramírez et al., a).
Trypanosomiasis by T. cruzi is primarily a wild enzooty. Its transmission in nature takes place within a complex trophic network, in which each animal species plays a different role, in space and time, regarding its maintenance and infective competence (Jansen et al., ; Jansen et al., ), resulting in distinct enzootic scenarios. In other words, each region is unique and has a specific transmission network, which needs to be understood and known so that one can recognize areas of epidemiological risk and correctly guide health agents and local residents (Roque et al., ).
Dogs represent the first domestic T. cruzi hosts studied by Carlos Chagas and are among the first experimental models used by him (Jansen et al., ). Among the different species of mammals, Canis lupus familiaris (domestic dog) has a significant role in the epidemiology of T. cruzi because it can act as a bioindicator. This is because this taxon is demonstrably capable of acting as a competent sentinel, signalizing the transmission of the parasite among wild mammals (Travi, ). Unlike other countries, domestic dogs in Brazil rarely present high parasitemia; that is, they display low infective potential (Xavier et al., ; Brandão et al., ). A proposal from our group that has received increasing attention from the Brazilian Health Authorities is the longitudinal serological survey of domestic and peridomestic mammalian species to determine the prevalence and/or incidence of T. cruzi infection (Jansen et al., ).
Serological diagnosis is the method of choice to assess the spread of the wild transmission cycle of T. cruzi and, consequently, define areas where the risk of human disease occurs. However, serological tests such as the indirect immunofluorescence reaction test (IFAT) and enzyme immunoassay (ELISA) often use a complex mixture of parasitic antigens, which can be related to false negative or false positive results (Santos et al., ). This fact is due to the possibility of cross-reactions of T. cruzi antigens with those other trypanosomatids, such as Leishmania sp. or other Trypanosoma species because they share common epitopes. With the development of recombinant DNA technology, several bacterial and eukaryotic gene expression systems allow the production of parasite antigens in large quantities, with a high degree of purity and standardized quality (Silveira et al., ; Foti et al., ). Thus, studies have shown that to improve diagnostic accuracy, the selection of more specific antigenic fragments for T. cruzi would be effective (Silveira et al., ; Camussone et al., ; Marchi et al., ; Santos et al., ; Santos et al., ; Peverengo et al., ; Santos et al., ; Daltro et al., ; Del-Rei et al., ; Leony et al., ; Silva et al., ). Chimeric proteins consist of antigenic sequences of the parasite, presenting a series of epitopes, which increases the diagnostic sensitivity (Santos et al., ). The IBMP proteins (IBMP-8.1, IBMP-8.2, IBMP-8.3 and IBMP-8.4) were evaluated their potential for diagnosing T. cruzi. The T. cruzi proteins whose antigenic regions (IBMP muti-epitope antigens) were used to construct the chimeric antigens are described in the [Supplementary Material of the study by Santos et al. ()].
Silva et al. () showed that the lateral flow immunochromatographic rapid test (LFRT) for the diagnosis of T. cruzi in humans, using the chimeric proteins IBMP-8.1 and IBMP-8.4, is extremely efficient, and our idea was to expand the use of this test for the diagnosis of this parasite in domestic dogs and others species of wild canids in its original format. The use of two chimeric proteins increases the diagnostic potential of the rapid test as it increases the amount of available T. cruzi epitopes. This is because the two chimeric proteins used have different epitopes due to their different compositions. The rapid test results are quick, easy to perform and interpret, do not require laboratory infrastructure, and use a small amount of biological material (serum, plasma or whole blood). Additionally, there is no need for equipment and specific knowledge to carry it out. The use of the test will allow rapid preventive actions to be taken in places with or without notification of Chagas disease (Sánchez-Camargo et al., ). Consequently, it will bring benefits mainly to locations where access to a more complex laboratory test is limited.
The recombinant antigens were formatted in a rapid immunochromatographic test using either Staphylococcus aureus protein A or Streptococcus pyogenes protein G as the gold-labeled reagents for the visualization of the precipitin band formed between the immunoglobulin (Ig) G-specific antibodies and the recombinant antigen immobilized on the nitrocellulose stripe used in the test. Despite the fact that most rapid immunochromatographic tests are formatted with protein A, the rationale to test both protein A and protein G was based on the fact that these microbial molecules bind with different affinities and specificities to immunoglobulins of various species, including dogs (Frohman et al., ; Nilsson et al., ; Bjorck and Kronvall, ; Costa et al., ).
Here we need to consider that: 1) establishing a diagnosis based on only one serological test is not secure enough. Two tests are always required, one being confirmatory; 2) the screening of dogs (not to mentioning wild animals) in the field is extremely advantageous, due to the speed of the response of the test, which already allows the immediate adoption of some mitigation measures. Biological material is a difficult to be maintained in field conditions, and bad storage conditions may even result in its loss. Also, the subsequent communication of results to the examined community constitutes a challenge. There are still many places in Brazil without internet access or mail making the communication of the results a difficult task.Insufficient access to diagnosis is yet a major issue to diagnose T. cruzi infection. Therefore, there is a need for more practical diagnostics and/or diagnostic algorithms that better suit the demands and field conditions and are indispensable to prevent the reemergence of CD in vulnerable regions of Brazil with similar epidemiological characteristics. Such diagnostics should be made available not only to easily detect T. cruzi infection, including different DTUs and mixed infection but also to avoid crossed reaction by other trypanosomatid infections (Gállego et al., ). In this study, it was evaluated the performance of recombinant chimeric antigens (IBMP-8.1 and IBMP-8.4) for the detection of anti-T. cruzi IgG antibodies in dog sera using Chagas-LFRT.
All procedures performed with wild and domestic animals received authorization from IBAMA (wild canids) and followed protocols approved by FIOCRUZ’s Animal Use Ethics Committee (P-99; P-03; P/06; L-07; L-050/; LW-81/12).
Serum samples from species of the order Carnivora were submitted to the Protein A affinity test to validate the use of the Chagas/Bio-Manguinhos Lateral Flow Immunochromatographic Rapid Test (Chagas-LFRT) for domestic and wild canids. The species tested were Canis lupus familiaris (n = 4), Canis lupus (n = 2), Chrysocyon brachyurus (n = 1), Lycalopex vetulus (n = 2), Lycaon pictus (n = 1), Speothos venaticus (n = 1) and Cerdocyon thous (n = 3). These samples were submitted to the Bio-Manguinhos Canine Visceral Leishmaniasis DPP LVC® Rapid Test kit, which also uses protein A, and the intensity in the control band marking was used to classify the strength of the interaction between the antibodies of the dog species and this protein, regardless of the marking of diagnostic bands. The affinity of the interaction with Protein A of these samples was classified as strong (++++), medium (+++), low (++) and very low (+).
We used 253 serum samples from Canis lupus familiaris and nine serum samples from six species of wild canid to detect T. cruzi infection: European wolf, Canis lupus (n = 2), Cerdocyon thous (n = 2), Lycalopex vetulus (n = 2), Lycaon pictus (n = 1), Speothos venaticus (n = 1) and Chrysocyon brachyurus (n = 1). C. l. familiaris samples were collected in distinct scientific expeditions to different regions of Brazil by the Laboratory of Trypanosomatids Biology (LABTRIP) and by the Laboratory of Wild Mammals Reservoir Biology and Parasitology, both from the Oswaldo Cruz Institute – FIOCRUZ/Rio de Janeiro between and . Wild canid samples were obtained from the Zoo Park Foundation of São Paulo and from studies conducted in the southeastern Goiás state municipality of Cumari (Brandão et al., ) (Table 1 and Figure 1). This material is kept at -20°C in the LABTRIP serum bank. Sera were aliquoted and each aliquot thawed only once.
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TABLE 1FIGURE 1Serum samples with previous serological diagnosis (IFAT and ELISA) were selected to compose the panel of positive (n = 133) and negative (n = 129) samples. The serologically positive samples were those with positive IFAT (titer ≥ 1/40) and ELISA (optical density ≥ 0.200). Within the panel of positive samples, samples with positive blood cultures (n = 20), including T. cruzi isolates characterized by different DTUs TcI (n = 8), TcII (n = 1), TcI/TcII (n = 2), TcIII (n = 2), TcIV (n = 1), and TcIII/TcV (n = 6), were included to validate the test based on the recognition of the different genotypes of the parasites deposited in the Coleção de Trypanosoma de Mamíferos Silvestres, Domésticos e Vetores, and COLTRYP/Fiocruz (Oswaldo Cruz Foundation, Rio de Janeiro, RJ, Brazil). To compose the panel of serum samples with different parasitic infections (n = 19), we used samples diagnosed as Leishmania infantum (n = 10), Trypanosoma caninum (n = 3), Trypanosoma rangeli (n = 1), Crithidia mellificae (n = 1), Anaplasma platys (n = 1), Dirofilaria immitis (n = 1), Ehrlichia sp. (n = 1) and a sample with mixed infection by Ehrlichia sp./Dirofilaria immitis (n = 1) (Table 1 and Figure 1). All samples of dogs serum used in the cross-reaction panel were subjected to immunofluorescence antibody test (IFAT) and ELISA to diagnose T. cruzi and Leishmania infantum infection and those positive for T. cruzi were excluded from the analysis.
The Chagas rapid test works with lateral flow immunochromatographic labeling, employing a combination of a protein conjugated with colloidal gold particles and T. cruzi antigens (recombinant proteins IBMP-8.1 and IBMP-8.4) bound to a solid phase (nitrocellulose membrane) (Silva et al., ). The sample to be analyzed is applied to a specific area of the plastic holder (cassette), followed by the addition of a running buffer. The buffer provides lateral flow of released components, promoting the binding of antibodies to antigens. Four different formulations of running buffer with and without blocking were tested, with modifications to the buffer formulation: high, medium or low blocking of nonspecific reactions in the tests and TR DPP LVC® canine visceral leishmaniasis from Bio-Manguinhos kit buffer.
To reveal the reaction, a protein A system conjugated to colloidal gold was used. The use of this type of conjugation basically privileges reactions with IgG because it makes use of the extraordinary affinity of protein A with the Fc region (crystallizable fragment) of IgG. Antibodies present in the sample bind to specific proteins conjugated to colloidal gold. In case a sample is positive, the “immunoconjugate” complex migrates on the nitrocellulose membrane, being captured by the fixed antigens in the test area and producing two purple/pink lines. In the absence of specific anti-T. cruzi antibodies, the purple/pink lines do not appear in the test area. In all cases, the sample continues to migrate across the membrane, producing a purple/pink line in the control area, which demonstrates proper functioning of the reagents (Figure 2A).
FIGURE 2The test was performed by applying 5 µL of sample (serum, plasma or whole blood) to the sample well followed by the addition of three drops of running buffer using a dropper bottle with a measuring nozzle. This mixture migrates by capillarity, eluting the conjugate (protein A conjugated to colloidal gold) to the test area where there are the antigens obtained through collaboration with the Proteomics and Protein Engineering Laboratory of the Carlos Chagas Institute - ICC/Fiocruz/PR, which provided two recombinant chimeras named IBMP-8.1 (21 KDa) and IBMP-8.4 (40 KDa). These chimeric proteins were expressed in Escherichia coli and purified by chromatography (Santos et al., ; Santos et al., ). Excess nonreaction-related components migrate to the other end of the tape where they are retained. The reading was carried out between 10 and 15 minutes, counted with the addition of running buffer, and incubated at room temperature. All inputs used in standardization are from the Chagas Rapid Test/Bio-Manguinhos (https://www.bio.fiocruz.br/index.php/br/produtos/reativos/testes-rapidos/teste-rapido-chagas) for Chagas disease produced by Bio-Manguinhos (Silva et al., ).
Visual reading of the recognition densities of the test and control bands was performed by two observers, as well as with a rapid lateral flow test reader to define the sensitivity and specificity parameters of the test to equate the inherent subjectivity of the human eye when reporting results in samples with low titers of antibodies. This type of device was developed in collaboration with the Carlos Chagas Institute-ICC/PR, Paraná Molecular Biology Institute-IBMP/PR and the Paraná Technological University-UTFPR/PR. Visual reading was standardized as follows: N1= strong negative; N2=weak negative; P1= positive of weak intensity; P2= positive of medium intensity and P3= positive of strong intensity (Figure 2B). The automated reader expresses the results quantitatively (ranging from 0 to 100) in optical density (OD), performing the reading for both diagnostic bands and the control band. These values were used to define the cutoff by the ROC curve.
A statistical summary of the data, a box-plot graph and a cluster analysis were performed to determine the Chagas rapid test cutoff point for classification of data collected by the automated reader of the IBMP-8.1 and IBMP-8.4 chimeric antigens by the matrix of confusion, which is intended to evaluate the performance of a binary classifier. The test performance (sensitivity and specificity) was evaluated format in single or duplex using the IBMP-8.1 and IBMP-8.4 proteins, separately and together to the serological diagnosis of T. cruzi infection wild and domestic Canids. The cut off, sensitivity, specificity and accuracy values were established by determining the largest area under the ROC curve (receiver operating characteristic) (Swets, ). The ROC curve is a statistical and graphical method for determining the best cutoff point for a diagnostic test. The highest point on the curve, corresponding to the upper left angle of the graph, represents 100% sensitivity and 0% false positives (d = 0); in this case, the ideal value for a diagnostic test, called the gold standard. Samples were separated by IFAT serological titers to generate the ROC curve and calculate sensitivity, specificity and cutoff values. Cohen’s kappa (K) analysis was used to determine the strength of agreement between the reference standard tests (IFAT and ELISA) and the Chagas LFRT, which was interpreted as weak agreement (< 0), slight agreement (0.01-0.20), fair agreement (0.21-0.40), moderate agreement (0.41-0.60), substantial agreement (0.61-0.80), and near perfect agreement (0.81-0.99) (Cohen, ). All data analyses were performed using RStudio software version 1.2. and R version 3.6.3, and a p-value under 5% (p < 0:05) was considered significant.
In the Protein A affinity test of samples from different species, all canids (wild and domestic), a strong interaction was demonstrated for the species Canis lupus familiaris, Canis lupus, Chrysocyon brachyurus, Lycalopex vetulus, Lycaon pictus, Speothos venaticus and Cerdocyon thous. This result allowed the choice of Canis lupus familiaris for the standardization of the Chagas rapid test for T. cruzi, in addition to this animal being considered an important sentinel of infection in different areas of study. Among the four formulations of running buffer with different types of blocking tested, the buffer selected was the original of the commercial kit of Chagas Rapid Test (with blocking) because it presented greater intensity in the labeling of the chimeric proteins IBMP-8.1 and IBMP-8.4. The selected buffer showed a high intensity recognition profile, visually classified as P3 for the two chimeric proteins and for the test control band.
Figure 3 shows the mean optical density values (OD 1.61) in serum samples from dogs with negative serological diagnosis. The mean of the optical density values of the samples from dogs infected by T. cruzi was 45.36 against the chimeric antigens IBMP-8.1 and IBMP-8.4. Among the positive samples considering the cutoff point OD ≥ 4.8, the IBMP-8.1 antigen was positive in 68% and the IBMP-8.4 antigen in 60% of the samples tested, and an agreement of 45% was observed between the IBMP-8.1 and 8.4 antigens in the positive samples against T. cruzi infection. This result highlights the importance of combining two antigens in a single test to increase the test sensitivity in the diagnosis of T. cruzi in dogs. Among the negative samples, only one (OD 5.1) reacted against the IBMP-8.1 antigen and means (OD 1.49), and four were false positive against the IBMP-8.4 antigen (OD ≥ 4.8 and ≤ 10.9) and the mean of the negative samples (OD 1.18).
FIGURE 3The optical densities obtained by the negative and positive samples were directly proportional to the serological titer by IFAT. The automated reading of the Chagas rapid test showed a pattern in which the optical density increased as the IFAT serological titer increased, mainly demonstrated by the means (Figure 4).
FIGURE 4Visual analysis showed high accordance with the band intensities of the IBMP-8.1 and IBMP-8.4 chimeric antigens according to the automated reader device. Negative titers showed total absence of color in 93.8%, classified as N1 and N2. Among the panel of negative samples by IFAT (titer ≤ 1/20), the observer rated was 78 (N1) and 13 (N2), 2 (N2/P1) and 4 (P1) false positive samples. In the panel of positive samples (titer ≥ 1/40), the observer classified as follows: 13 (P1), 15 (P2), 15 (P3) and 10 samples were classified as false negatives: 3 (N1) and 7 (N2). The intermediate titers (1/40 to 1/320) had a medium intensity in the test zone lines, mainly diagnosed as P1 (10), P2 (9) and P3 (1), as the higher titers (1/640 to 1/) showed the strongest intensities in the marking of chimeric proteins, with the most prevalent visual diagnosis being positive with strong intensity P3 (14), P2 (6) and P1 (3). The visual classification did not present a significant difference in the classification based on the optical densities of the automated reader in relation to the cut off 4.8 for positive and negative samples; that is, the visual classification can replace the reader’s classification by Kappa analysis (Figure 2B and Table 2).
TABLE 2Of the samples with positive blood cultures, 90% (18/20) had a positive result through visual and automated reading in the Chagas-LFRT. Samples from wild and domestic canids infected with the DTUs TcI, TcII, TcIII, TcIII/TcV and TcIV were considered positive by the test. Only two samples from domestic dogs with positive blood cultures (DTU TcI), with titers of 1/40 and 1/80, had a negative result in Chagas-LFRT (Figure 2B).
Regarding the samples infected with other trypanosomatids and other parasites from domestic canids, all samples from dogs infected by L. infantum, T. rangeli, T. caninum and D. immitis presented negative results in the Chagas-LFRT. However, positive samples for the parasites Anaplasma platys, Crithidia mellificae, Ehrlichia sp. and a sample with a mixed infection of Ehrlichia sp. and D. immitis showed weak positive results for T. cruzi in the rapid test (P1). The test was able to evaluate 78.9% (15/19) of the samples infected by other dog parasites as negative (Table 1).
When we only use one single of the chimeric proteins the sensitivity and specificity was lower (BMP-8.1: sensitivity 92.5% and specificity 85.6% or IBMP-8.4: sensitivity 75% and specificity 83.3%), already the duplex test format (with two chimeric proteins, IBMP 8.1 and IBMP8.4). The chimeric proteins IBMP-8.1 and IBMP-8.4 using Chagas-LFRT had a sensitivity of 94% and a specificity of 91%, increasing the diagnostic power of the test when used the two chimeric proteins to the serological diagnosis of T. cruzi infection wild and domestic Canids. Five cutoff points were tested and evaluated (2.3, 3.7, 4.8, 5.9 and 8.7), and the cutoff point was considered an optical density of 4.8 and an area under the curve (AUC) value of 92.6% (Figure 5A and Supplementary Figure S1). The ROC curve presented an AUC of 97.3% for the cutoff point by IFAT of >1/20 and 96.8% for samples >1/40 in IFAT (Figure 5B). In the Kappa test, the coefficient was 0.84 (elevated), accuracy was 0.90, predictive positive values was 0.96 and negative values was 0.87 between the IFAT and the automatic reader of the Chagas rapid test (Table 3 and Supplementary Figure S1).
FIGURE 5TABLE 3One of the main bottlenecks in monitoring parasitosis in wild and even domestic animals is the small number of simple and feasible diagnostic methods to be applied in the field. In this sense, the test that we are proposing fills an important gap both in the feasibility and in the spectrum of animal taxa in which trypanosomatid infection can be diagnosed. Establishing access to diagnostics for wild animals species is of utmost epidemiological and epizootiological importance as the health of all animals (including humans) is profoundly interdependent and this awareness is always increasing as clearly stated in the One Health perspective (Jansen et al., ).
At present, most Chagas disease occurs in a new epidemiological profile in which Trypanosoma cruzi transmission occurs independently of the domiciliation of triatomines. A new approach to control measures is necessary, as the measures previously applied, in this case, the use of insecticides to control species of intradomiciliary triatomines, does not fit the current epidemiological reality. Targeted control of reemerging transmission can be achieved by improved understanding of T. cruzi in canine populations. Castillo-Neyra et al. () suggest that dogs may be useful sentinels to detect reinitiation of transmission following insecticide treatment. A correlation between dogs with positive serology to T. cruzi and proximity to an infected triatomine (distance ≤50 m) was observed, suggesting that dogs may be useful sentinels to detect reinitiation of transmission following insecticide treatment (Castillo-Neyra et al., ).
The use of sentinel dogs has proven to be appropriate for any area that wants to assess the risk of human disease regardless of biome or cultural habits. Diagnosis using rapid tests is absolutely suitable for field conditions. It will enable large-scale diagnosis of areas of transmission of T. cruzi and, consequently, of risk for human disease. This work is perfectly aligned with the National Agenda of Priorities in Health Research, which textually recommends the development of new models and approaches for the surveillance of adverse health events and emerging diseases and the development of new technologies for the epidemiological surveillance of health problems, including specific forms of monitoring and scenario studies. Integration of canine T. cruzi blood sampling into existing interventions for zoonotic disease control (e.g., rabies vaccination programs) can be an effective method of increasing surveillance and improving the understanding of disease distribution (Castillo-Neyra et al., ).
The classical bottleneck has always been to preserve biological samples in field conditions that are often rather precarious besides transporting samples to the central level, as well as dispose of personnel sufficiently trained to carry out conventional methods of serological diagnosis on a large scale. Fast and easy tests to be performed have been increasingly sought after in regard to diagnosis. In this study, we performed an evaluation of the performance of the rapid lateral flow immunochromatographic test (Chagas-LFRT) for the serological diagnosis of T. cruzi infection in Canis lupus familiaris using two chimeric proteins (IBMP-8.1 and IBMP-8.4) as antigens. In this way, a quick decision-making process that will shape the epidemiological surveillance actions associated with the transmission of the parasite is made possible. Point-of-care tests are applicable in different sectors and bring short-term results in healthcare facilities and remote locations to complex diagnostic methods (Silva et al., ). Here, the use of a rapid test for dogs was validated, which has already been tested and approved for the diagnosis of T. cruzi infection in humans (Silva et al., ), optimizing its production and expanding its application in the field. Our results show that the test was sensitive to detect seroconversion, enhancing its application as a first measure to monitor the presence of the parasite, which indicates active transmission in the area, attested by seroconversion events, despite the low rate of positive parasitological tests in dogs from Brazil (Xavier et al., ; Brandão et al., ).
Dogs are animals that live together and accompany humans since they are still in the hunter-gatherer stage. In an evolutionary trade-off, dogs guaranteed safety, and humans, in turn, guaranteed dog food and shelter. In several cultures, dogs were used for different purposes, including prewarming the feet, hunting, guarding, adorning, sports and even a source of protein. Additionally, dogs are very common domestic animals and are easily traceable, and annual rabies vaccination campaigns as well as leishmaniasis control programs have resulted in the training of health service personnel in handling and collecting blood from these animals (Xavier et al., ; Daltro et al., ).
Dogs participate in the transmission cycle of T. cruzi in different ecoepidemiological scenarios, in addition to being a common animal in all areas, regardless of environmental and climatic conditions, which are not limiting for its occurrence. In Brazil, the pattern of T. cruzi infection in dogs is more related to the maintenance of the parasite in low/absence parasitemia, which results in low importance for the transmission of the parasite (Xavier et al., ). The plasticity of T. cruzi in nature ensures that it is transmitted in multiple environmental characteristics that vary in space and time, which is supported by the richness and diversity of wild mammals and vectors. In these multifactorial scenarios, the dog can be used as a sentinel for T. cruzi presence. Thus, the diagnosis of T. cruzi in domestic dogs is extremely important, as the dog’s role in the transmission of T. cruzi has been described as a dead-end host and sentinel of infection in the wild environment (Roque and Jansen, ). Areas where the dog’s infection is ≥ 30% signal the presence of a wild transmission cycle occurring close to the dog’s circulation areas, indicating the risk of Chagas disease cases (Xavier et al., ). As this test proved to be highly viable for the diagnosis of the parasite in dogs in our study, its use for detecting T. cruzi infection in dogs will allow area monitoring in real time, signaling areas with transmission of the parasite in the wild environment and perhaps also undetected human cases. Thus, accurate situational diagnosis is still in the field.
As T. cruzi is a parasite with high genetic variability, there is a significant difference in the performance of commercial serological tests related to the antigens used (Dopico et al., ). Therefore, the serological diagnosis of T. cruzi infection is quite complex due to the lack of reference methods. Furthermore, methods using parasite DNA are viable only in the initial phase of infection and are not capable of attesting to parasite viability (Daltro et al., ; Leony et al., ; Silva et al., ). Due to this situation, the World Health Organization (WHO) recommends the use of two tests to complete the diagnosis of T. cruzi infection (WHO Expert Committee, ). The diagnosis of the parasite is even more complicated in regard to wild and domestic canids due to the lack of validated tests. The antigenic matrices currently used can lead to low specificity and a high rate of cross-reaction with other trypanosomatids and other dog parasites (Leony et al., ).
Costa et al. () evaluated both IgG binding proteins in rapid immunochromatographic test kits for the diagnosis of canine visceral leishmaniasis. The tests were assembled with either Leishmania infantum recombinant antigens K39 or K26 and with either gold-labeled Staphylococcus aureus protein A or Streptococcus pyogenes protein G. The test using recombinant antigens K39 or K26 produced results that were only slightly superior to protein A than to protein G (no significant differences were observed), possibly because this minor difference in sensitivity between these 2 tests is due to the slightly higher affinity that protein A has over protein G for dog IgG (Miele and Krakowka, ; Peng et al., ). Additionally, our test that relies on gold-labelled Staphylococcus aureus protein A conjugate that may be easily adapted to other wild species thus, optimizing large-scale surveys. This is especially important in investigation of disease outbreaks. Therefore, our test is opening the possibility of the diagnosis not only of T. cruzi in dogs but also of other trypanosomatids species infections in other species that domestic dogs.
Chimeric antigens provide the diagnosis with a greater number of parasite epitopes, thus reducing false-negative results when compared to the use of nonrecombinant antigens (Dopico et al., ). The capacity of the rapid test to detect all T. cruzi DTUs that circulate in Brazil, also suggested by Santos et al. () and Dopico et al. (), is an important characteristic. In fact, in Brazil, there is a predominance of TcI and TcII DTUs, which are also the most frequent DTUs in wild carnivores and dogs (Rocha et al., ; Jansen et al., ; Brandão et al., ), but other DTUs may also be observed at minor rates. As an example, DTUs TcIII and TcIV have already been described in infected dogs in Brazil (Brandão et al., ). The chimeric antigens IBMP-8.1 and IBMP-8.4 in the rapid test platform are presented as tools capable of overcoming limitations imposed on other antigens and are also successful in other platforms investigated in previous studies, such as indirect ELISA and liquid microarray (Santos et al., ; Santos et al., ; Santos et al., ; Del-Rei et al., ; Leony et al., ; Silva et al., ).
Leony et al. () used three reference strains (Colombian, Y and Berenice), known to have TcI and TcII DTUs, for experimental infection of domestic dogs with T. cruzi. This study was successful in diagnosing the parasite using the chimeric proteins through ELISA. Here we emphasize that, in addition to expanding the recognition panel of DTUs different from those analyzed by Leony et al. (), our study showed the possibility of using a rapid test platform with chimeric proteins to detect T. cruzi in naturally infected domestic and wild dogs.
The breadth of the serological profile of the samples used in our study, which included samples with recent and older infections and with different serological titers, indicates that the cutoff point found in our study for dog samples was 4.8, indicating the high diagnostic accuracy of the chimeric proteins IBMP-8.1 and IBMP-8.4 for dog and wild carnivore infections. A sample of serum from a domestic dog from Guarapari (Espírito Santo) was included in two panels: panel of negative samples in the serological diagnosis (IFAT and ELISA) with titer of 1/20 in the RIFI, below the cut-off point and panel of positive samples in the parasitological examination. This type of phenomenon is not uncommon and suggests an initial infection, when the immune response is not yet established.
Leony et al. () found high sensitivity for four chimeric proteins, including two tested here (IBMP-8.1, IBMP-8.2, IBMP-8.3 and IBMP-8.4), in the diagnosis of T. cruzi infection in domestic dogs using ELISA. However, Leony et al. () found cross-reactivity to IBMP-8.1 for at least one of all tested parasites (anaplasmosis, babesiosis, dirofilariosis and ehrlichiosis), with the exception of other trypanosomatids. IBMP-8.1, in Chagas-LFRT, also cross-reacted with other parasites, with positive results for anaplasmosis, ehrlichiosis and Crithidia mellificae. However, unlike the study by Leony et al. (), this antigen did not react with dirofilariosis in the rapid test Chagas. The chimeric protein IBMP-8.4 did not cross-react on the ELISA platform, but in the rapid test, it reacted with Ehrlichia sp. This can be explained by the difference number of epitopes provided by IBMP-8.4 compared to IBMP-8.1 (Del-Rei et al., ). Herein, we propose the use of Chagas-LFRT for the serological diagnosis of T. cruzi infection in dogs as a screening test in the field to monitor the area and that a second confirmatory test (IFAT and/or ELISA) has to be used for individual diagnosis.
We did not observe cross-reactions with the main trypanosomatids described infecting dogs (L. infantum, T. caninum and T. rangeli), and these results show that the rapid test for T. cruzi also proves to be a useful and viable tool in places where there is spatial and temporal overlapping of transmission by other trypanosomatids. Daltro et al. () evaluated the cross-reactivity of chimeric proteins in the diagnosis of T. cruzi through ELISA in human samples with American Tegumentary Leishmaniasis and Visceral Leishmaniasis, finding practically negligible reactivity. Daltro et al. () state that the use of chimeric parasitic antigens can reduce the cost of diagnosis, since there would be no need to repeat tests on the same sample, suggesting their use where Leishmania sp. and T. cruzi are coendemic.
There are hundreds of wild mammal species in which T. cruzi is capable of infecting and sampling, and diagnosing infection in free-living wild fauna is still a huge challenge. One of the biggest obstacles is the lack of diagnostic improvement. As a result of the anthropocentric view of health, only products for serological diagnosis of humans and animals of economic interest are commercially found. Thus, the potential to perform the diagnosis through Protein A or G linked to colloidal gold in a rapid test will multiply the application, since a specific antibody is not necessary to reveal the antigen-antibody reactions as in conventional serological tests. It will bring benefits mainly for the diagnosis of wild mammal fauna.
We propose here the spatiotemporal monitoring of infection in domestic dogs as an environmental diagnostic tool through Chagas-LFRT to be used as a first measure in the identification of areas where there is a potential risk of transmission of T. cruzi and the presence of the vector. The sole use of Chagas-LFRT can be used for an environmental diagnosis, while a second confirmatory test has to be employed for the individual diagnosis. Domestic dogs are animals that are easy to handle, in addition to having spatial and temporal traceability. The collection of blood from these animals does not require large infrastructure and cost, especially with the use of the rapid test for T. cruzi, a point-of-care technology that is fast and easy to handle. From this, Chagas-LFRT proved to be sensitive for use as a first environmental diagnostic tool for the presence of T. cruzi for early monitoring of the risk of new human cases.
Our main contribution was to validate and expand the use of the rapid Chagas test, which was developed in Bio-Manguinhos for the diagnosis of Chagas disease in humans (Silva et al., ), for field diagnosis of T. cruzi infection in domestic dogs and to evaluate its potential of application to wild canid species. Our motivation was to implement in the field work routine, a quick test that is easy to perform (point-of-care exam), which does not require technical training and is not dependent on a complex laboratory infrastructure for its execution, as in the conventional diagnostic tests available (ELISA and IFAT). One of the great advantages is to have one same type of test that can be used under the same conditions for the diagnosis of T. cruzi infection in humans, domestic and wild dogs.
The surveillance system as a whole will benefit because it will have in its hands a specific and reliable rapid test for “in loco” diagnosis of infected dogs and proximity or presence of infected triatomines, that is, the signalling of an expressive enzootic cycle of T. cruzi transmission near human dwellings. Chagas-LFRT will allow rapid diagnosis in a safer, reliable and low-cost manner without the need for more complex laboratory tests. The detection of “hot areas” of enzootic transmission detected by the test will streamline the decision-making process by allowing quick mapping of target areas for implementation of prevention measures.
The datasets presented in this article are readily available. Requests to access the datasets should be directed to Samanta Xavier,c2FtYW50YUBpb2MuZmlvY3J1ei5icg==.
The animal study was reviewed and approved by IBAMA (wild canids) and followed protocols approved by FIOCRUZ’s Animal Use Ethics Committee (P-99; P-03; P/06; L-07; L-050/; LW-81/12). Written informed consent was obtained from the owners for the participation of their animals in this study.
Conceptualization, SX, AJ, ES, and NR. Formal analysis, SX, ER, NR, AJ, and ES. Funding acquisition, SX. Investigation, SX, AJ, and AR. Methodology, SX, ER, GS, MS, JB, AB, RD, NR, ES, and AJ. Project administration, SX, NR, AJ, and ES. Resources, AB, RD, ES, and NR. Supervision, SX, ES, NR, and AJ. Visualization, SX, AJ, AR, NR, and ES. Writing original draft, ER, SX, JB, AJ, NR, and AR. Writing—review and editing, ER, SX, AJ, and AR. All authors have read and agreed to the published version of the manuscript.
SCCX has received financial support from CNPq (MCTIC/CNPq No. 28/ - Universal, process number /-2) and from Faperj ARC_ - Auxílio ao Pesquisador Recém-contratado (E-26/010./).
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
The new TR Chagas development project, led by Bio-Manguinhos, is the result of the collaboration of several Fiocruz units, such as the team of Dr. Nilson Zanchin from the Carlos Chagas Institute (ICC/Fiocruz-PR), who developed the proteins and improved the processes of expression and purification, and the Ageu Magalhães Institute (IAM-Fiocruz-PE), Gonçalo Moniz Institute (IGM-Fiocruz-BA), Institute of Molecular Biology of Paraná (IBMP) and Oswaldo Cruz Institute (IOC-Fiocruz- RJ). We thank Dr. Julio Israel Fernandes and the staff of the Veterinary Hospital of Universidade Federal Rural do Rio de Janeiro (UFRRJ) for serum samples of dogs infected by Anaplasma platys, Dirofilaria immitis and Erlichia sp. We want to thank LABTRIP, Fundação Parque Zoológico de São Paulo and Programa de Conservação Mamíferos do Cerrado (PCMC), Brazil; Cristiane Varella for the COLTRYP isolate characterization and to the Scientific Initiation Scholars of the PIBITI Program of the Oswaldo Cruz Institute, Fiocruz: Matheus Santos Luquez, Victoria Brigido Lamim and Ana Carolina Ferreira de Carvalho for the contributions during this study.
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10./fcimb../full#supplementary-material
Supplementary Figure S1 | Receiver operating characteristic (ROC) curve, area under the curve (AUC) and statistical summary of cutoff point values (2.3, 3.7, 4.8, 5.9 and 8.7).