Clinica Chimica Acta 454 (2016) 39–45

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Applicability of a novel immunoassay based on surface plasmon resonance for the diagnosis of Chagas disease João G.G. Luz a,b, Dênio E.P. Souto c, Girley F. Machado-Assis d, Marta de Lana e, Rita C.S. Luz f, Olindo A. Martins-Filho g, Flávio S. Damos f, Helen R. Martins b,⁎ a

Instituto de Ciências Exatas e Naturais, Universidade Federal de Mato Grosso (UFMT), Rondonópolis, MT, Brazil Departamento de Farmácia, Universidade Federal dos Vales do Jequitinhonha e Mucuri (UFVJM), Diamantina, MG, Brazil Instituto Nacional de Ciência e Tecnologia em Bioanalítica, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, Brazil d Laboratório de Parasitologia, Universidade Federal de Juiz de Fora (UFJF), Campus Governador Valadares, MG, Brazil e Laboratório de Doenças de Chagas, Núcleo de Pesquisas em Ciências Biológicas (NUPEB), Universidade Federal de Ouro Preto (UFOP), Ouro Preto, MG, Brazil f Departamento de Química, Universidade Federal do Maranhão (UFMA), São Luís, MA, Brazil g Laboratório de Biomarcadores de Diagnóstico e Monitoração, Centro de Pesquisas René Rachou, Fundação Oswaldo Cruz (CPqRR-FIOCRUZ/MG), Belo Horizonte, MG, Brazil b c

a r t i c l e

i n f o

Article history: Received 5 November 2015 Received in revised form 14 December 2015 Accepted 18 December 2015 Available online 28 December 2015 Keywords: Chagas disease Diagnosis Surface plasmon resonance Immunoassay Applicability

a b s t r a c t Background: We defined the methodological criteria for the interpretation of the results provided by a novel immunoassay based on surface plasmon resonance (SPR) to detect antibodies anti-Trypanosoma cruzi in human sera (SPRCruzi). Then, we evaluated its applicability as a diagnostic tool for Chagas disease. Methods: To define the cut-off point and serum dilution factor, 57 samples were analyzed at SPRCruzi and the obtained values of SPR angle displacement (ΔθSPR) were submitted to statistical analysis. Adopting the indicated criteria, its performance was evaluated into a wide panel of samples, being 99 Chagas disease patients, 30 noninfected subjects and 42 with other parasitic/infectious diseases. In parallel, these samples were also analyzed by ELISA. Results: Our data demonstrated that 1:320 dilution and cut-off point at ΔθSPR = 17.2 m° provided the best results. Global performance analysis demonstrated satisfactory sensitivity (100%), specificity (97.2%), positive predictive value (98%), negative predictive value (100%) and global accuracy (99.6%). ELISA and SPRCruzi showed almost perfect agreement, mainly between chagasic and non-infected individuals. However, the new immunoassay was better in discriminate Chagas disease from other diseases. Conclusion: This work demonstrated the applicability of SPRCruzi as a feasible, real time, label free, sensible and specific methodology for the diagnosis of Chagas disease. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Chagas disease (CD) is caused by the hemoflagellate protozoan Trypanosoma cruzi, included in order Kinetoplastida and family Trypanosomatidae [1]. It is endemic to a wide region of Latin America, where occurs in 21 countries and affects approximately 7 million people, being responsible for a high burden of morbidity and mortality, including sudden death events [2]. However, in the last decades, T. cruzi infections have been increasingly found in non-endemic areas, such as the USA, Canada, many European and some Western Pacific countries, mainly due to the increase of infected population movements [3]. It is well referenced that the conduits for the diagnosis of CD are related to the phases of the infection that a patient present. Thereby, in acute phase, period that comprehends 1 to 4 months following initial ⁎ Corresponding author at: Departamento de Farmácia, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 39100-000 Diamantina, MG, Brazil. E-mail address: [email protected] (H.R. Martins).

http://dx.doi.org/10.1016/j.cca.2015.12.025 0009-8981/© 2015 Elsevier B.V. All rights reserved.

infection, laboratorial diagnosis is carried out by the detection of trypomastigote forms directly in the blood [4]. On the other hand, during the successive chronic phase, which is a lifelong phase, the number of parasites in the peripheral blood is low and subpatent. Consequently, due to the low sensitivity of the parasitological methods, at this phase the laboratorial diagnosis is usually performed by indirect serological methods for detection of IgG antibodies anti-T. cruzi, which are present in different concentration in approximately 98% of the infected patients [5]. For this purpose, the serological tests currently employed are indirect hemagglutination assay (IHA), indirect immunofluorescence (IIF) and enzyme-linked immunosorbent assay (ELISA), so that a conclusive result is achieved when at least two different methodologies are used [6]. In this sense, according to the Brazilian Consensus on Chagas Disease, the diagnosis during the chronic phase should be carried out using a test with high sensitivity, i.e. ELISA with total antigen or semipurified fractions of the parasite, IIF or IHA, in combination with another test with high specificity, i.e. ELISA employing T. cruzi-specific recombinant antigens [7].

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Although these serological tests are widely used in routine, they present some methodological limitations, including frequent crossreactivity with other infectious agents also observed in endemic areas, e.g. trypanosomatids as Leishmania spp., and problems with sensibility and specificity, which lead to inconclusive results usually observed in clinical practice and screening of blood donors [8]. In addition, they also require a long pre-analytical and analytical time to be performed, skilled labor, refrigeration for reagents and expensive supplies [9]. Therefore, the searches for alternatives serological tests which combine robustness, simplicity, portability with adequate specificity and sensitivity have been explored in order to overcome such problems [10]. Surface plasmon resonance (SPR) has been applied by several authors as an innovative method for the evaluation of interfacial events and, consequently, the development of immunoassays, since it combines great selectivity and sensitivity to recognize biomolecular interactions in label free and real time analysis [11,12]. Recently our team proposed a novel immunosensor based on SPR for the detection of antibodies anti-T. cruzi (IgG) in human serum [13]. On that occasion, we also optimized the operational parameters of the new immunoassay — termed SPRCruzi, which employs crude soluble antigens of T. cruzi covalently immobilized on a SPR sensor gold chip modified with an organic mixed self-assembled monolayer (SAM). The direct addition of a serum sample infected with T. cruzi generates a displacement of the SPR angle (ΔθSPR), which is derived from the occurrence of antigen-antibodies interactions on the sensor surface, and is proportional to the amount of antibodies anti-T. cruzi present in the sample. On the other hand, a null or smaller SPR angle variation is obtained if a non-infect sample is injected. Although we previously demonstrated that SPRCruzi represents a promising diagnostic tool for CD diagnosis, once it allowed clear segregation of infected and non-infected groups in simple and fast analysis, still is necessary to establish and confirm the methodological criteria for interpretation of the immunoassay, and conduct a thorough and wide evaluation of its applicability.

committee of the René Rachou Research Center, under the process number 007/2002. The human sera with HBV, HTLV, HIV, ATX and CTX were kindly provided by the Ezequiel Dias Foundation, Belo Horizonte, Minas Gerais. Samples from CL and VL patients were provided by the biorepository of the Laboratory of Biomarkers for Diagnosis and Monitoring, René Rachou Research Center, Oswaldo Cruz Foundation, FIOCRUZ, Minas Gerais, Brazil. The diagnosis of other diseases samples was confirmed according to the recommended for each disease. 2.2. Preparation of crude soluble antigens The crude soluble antigens used in SPRCruzi and ELISA were obtained by alkaline extraction from epimastigotes of T. cruzi Y-strain harvested in axenic culture in liver infusion tryptose (LIT) medium and maintained at 28 °C. Briefly, the parasites were submitted to lysis with NaOH 0.15 mol/l overnight in an ice bath under agitation. The addition of HCl 0.15 mol/l was carried out aiming the neutralization of the material. Then, the suspension was centrifuged at 15,000 rpm for 30 min at 4 °C and the supernatant was submitted to protein determination according to Lowry et al. [14]. The concentration was adjusted to 1000 μg ml−1 and antigen aliquots were frozen at −80 °C until analysis. Posteriorly, dilutions of antigen for the use in the methodologies were carried out with HBS-EP pH 7.4 buffer solution. 2.3. SPR immunoassay (SPRCruzi) The immunoassay based on SPR for the detection of anti-T. cruzi antibodies in tested serum samples was performed as optimized and described by Luz et al. [13]. In order to obtain the parameter ΔθSPR from the injection of samples, and consequently detect the occurrence of interactions between immobilized antigen and antibodies present in serum, an SPR analyzer from Autolab Springle (Eco Chemie B.V., Netherlands) was used. 2.4. Analysis for standardization of methodological criteria of SPRCruzi

2. Population, materials and methods 2.1. Study population Two groups of human serum samples were applied to achieve the proposed investigations of the present study. The first group (Group 1) was used during the standardization of the methodological criteria of SPRCruzi. It included 57 sera, being 20 of these from non-infected individuals (NI) and 37 from CD patients (CH). The latter were divided into three groups according to the reactivity index (RI) defined by ELISA: high (n = 12) (2.9 ≤ RI ≤ 3.5), medium (n = 13) (2.0 ≤ RI ≤ 2.2) and low reactivity (n = 12) (1.0 ≤ RI ≤ 1.3). The RI was calculated by dividing the absorbance value of reading from each sample assayed by the cut-off value adopted in the analysis. The second population group (Group 2) was used for the evaluation of the applicability of SPRCruzi in the diagnosis of CD. It included 171 human sera samples, being 99 from CD patients (CH), 30 negative control sera from non-infected individuals (NI) and 42 from individuals not infected with T. cruzi but infected with other relevant parasitic/ infectious diseases usually find in endemic areas for CD (other diseases), which were subdivided as patients with Visceral Leishmaniasis (VL) (n = 7), Cutaneous Leishmaniasis (CL) (n = 6), Acute Toxoplasmosis (ATX) (n = 6), Chronic Toxoplasmosis (CTX) (n = 6), as well as human T lymphotropic virus (HTLV) (n = 8), hepatitis B virus (HBV) (n = 4) and human immunodeficiency virus (HIV) (n = 5) infections. All the serum samples CH and NI referred above were collected from individuals from the municipality of Berilo, located in the Jequitinhonha Valley, northeast of Minas Gerais State, Brazil, an important endemic region for CD. The diagnosis of all samples was previously confirmed by three serological tests, including ELISA, IIF and IHA. The collection of these samples from endemic area was approved by the research ethics

Aiming to ascertain the best serum dilution and cut-off point to be adopted for interpretation the values of Δ θSPR provided by SPRCruzi, and thus identify the CD patients with assurance, the samples of Group 1 were diluted with HBS-EP buffer at pH 7.4 into three potential serial dilutions: 1:160, 1:320 and 1:640. The samples were first analyzed as a pool of sera in triplicate and then individually. Other dilution factors were not chosen because they did not provide a good separation between CH and NI individuals, as previously verified [13]. The results of SPR analysis were expressed as ΔθSPR, and the obtained data were analyzed by receiver operating characteristic (ROC) curve analysis, a statistical tool that indicates the more appropriate cut-off point to discriminate negative from positive results. Moreover, the area under the curve (AUC) also provided the global accuracy of the test, which can be classified as low (0.51–0.61), moderate (0.62–0.81), elevated (0.82–0.99) and outstanding (1.0) [15]. The ROC curve analysis was performed using MedCalc 14.8.1. 2.5. Evaluation of the applicability of SPRCruzi For the evaluation of the applicability of the immunoassay, the samples of Group 2 were analyzed individually with the most adequate serum dilution and cut-off previously defined. From the obtained data, the performance indexes were also calculated to determine how much SPRCruzi is possible of application for the diagnosis of CD in endemic areas. 2.6. Comparison of SPRCruzi and ELISA In parallel, the detection of antibodies in sera from Group 2 was also evaluated by ELISA performed according to Voller et al. [16] using the

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same crude soluble antigen. The concurrence between the results provided by both immunoassays was assessed by the K index, which range from “0” to “1”. The degree of agreement was determined according to Landis and Koch [17] interpretation criteria. 3. Results and discussion

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that can represent possible sources of erroneous results. Negrette et al. [22] and Lana et al. [23] performed different ELISA techniques for monitoring cure after etiological treatment of chronic CD patients, and also demonstrated that 1:320 is a promising dilution for the serological diagnosis of T. cruzi infections, once that it allowed a greater segregation between infected individuals before and after treatment, which suggested a therapeutic efficacy due to a significant decrease in IgG levels.

3.1. Standardization of methodological criteria of SPRCruzi: serum dilution and cut-off point

3.2. Evaluation of the applicability of SPRCruzi in a diversified population

The diagnosis of chronic phase of CD is usually performed by epidemiological features combined with serological methods, due to the low parasitemia and the constant presence of antibodies observed in infected individuals [4]. However, the methods currently used in clinical practice present several diagnostic and operational limitations [8]. In the past years, our group has been working toward developing of new immunosensors employing SPR for the serological diagnosis of infectious diseases [12,13,18]. Herein, we report an essential evaluation of the applicability of SPRCruzi for the diagnosis of CD front of a large panel of serum samples [19]. However, firstly it was necessary to standardize and confirm the methodological criteria, i.e. serum dilution and cut-off point, to be adopted at the assay. Fig. 1A represents the average values of ΔθSPR obtained for pools of samples from Group 1 evaluated at different dilutions. It is graphically apparent that CH samples in all three dilutions showed behavior similar to the RI previously determined by ELISA, so that the average values of ΔθSPR differentiated the groups already classified as high, medium and low reactivity. This fact was expected, since the RI is directly related to the amount of antibodies anti-T. cruzi present in the tested serum [20]. Thus, due to the presence of a higher amount of antibodies, high reactivity samples probably generated more linkages between these biomolecules (antibodies) and T. cruzi antigens immobilized on sensor surface, which in turn result in higher SPR angle variations [21]. Similar associations can be made for the medium and low reactivity samples. In addition, it is important to highlight that the discrete values of ΔθSPR found for NI samples, are related to the presence of unspecific ligands present in serum that supposed to bound to immobilized T. cruzi antigens [13]. It was verified that the values of ΔθSPR found for the NI and CH individuals have different profiles, particularly in 1:320 dilution factor, where a higher variation between groups was achieved. By fixing the cut-off point at Δ θSPR = 17.2 m°, as appointed by ROC curve analysis for 1:320 dilution, an area under the curve equal to 1.0 was observed, which suggests perfect accuracy of the method without false-positive or false-negative results, and is desirable for a serological method [15]. Indeed, when the individual values of ΔθSPR are represented in a scatter graph, it can be noted a clear segregation between NI and CH samples at 1:320 dilution, including that sera with low reactivity detected by ELISA, which in turn are samples that present lower amounts of antibodies anti-T. cruzi and that could be misclassified as a false-negative diagnosis (Fig. 1C). On the other hand, the cut-off point determined for 1:160 dilution (Δ θSPR = 53.9 m°) classified 13.5% (5/37) of CH samples as falsenegative results, mainly those with low reactivity. Concomitantly, 25.0% (5/20) of the NI sera were misclassified as positive (Fig. 1B). For the cut-off point fixed at Δ θSPR = 3.7 m° for 1:640 dilution, the presumed high specificity of the immunoassay due to the absence of false-positive diagnostics is notable. However, such dilution accused negativity for 37.0% (10/37) of the CH samples, especially among the low reactivity sera (Fig. 1D). Corroborating ROC curve analysis, the performance indexes demonstrated that at 1:320 dilution factor SPRCruzi immunoassay presented maximum values of performance, which was considerably larger than what was found for 1:160 and 1:640 (Table 1). Therefore, the serum dilution at 1:320 and the cut-off at ΔθSPR = 17.2 m° are the most promising experimental condition to better discriminate CH and NI subjects by SPRCruzi, including those that present low levels of serum antibodies

After the establishment of methodological criteria, the applicability of SPRCruzi for the real diagnosis of CD was evaluated, which represents a pioneer investigation in the field of immunosensors based on SPR. For this purpose, a varied panel of serum samples, including 99 samples from CD patients, 30 healthy control samples and 42 samples with other parasitic and infectious diseases was tested by the immunoassay adopting such criteria, where samples with values of Δ θSPR higher than 17.2 m° were considered positive (CH), while values less than or equal to 17.2 m° corresponded to negative (NI) sera. Fig. 2A demonstrates the ΔθSPR values obtained after the analysis of samples from Group 2 at SPRCruzi. Considering each subgroup of sera, the found values ranging between 90.2–18.2 m° for CH samples, 0–16.9 m° for NI samples and 0–32.1 m° for other diseases samples. The set of the results suggests a high sensitivity of the assay, since all CH sera were diagnosed as positive without false-negative results. This fact is probably attributed to the high sensitivity and reliability of SPR signal transduction, which allows the detection of small molecules in complex matrices, e.g. human serum, with very low detection limits [11]. In addition, the high capacity of the SPRCruzi in discriminate CH and NI individuals is noticeable, since only 4.8% (2/42) of the patients considered as negative for CD but positive for VL (n = 1) and CL (n = 1) were appointed as false-positive (Fig. 2B). The occurrence of crossreactivity between infections by T. cruzi and several pathogens is an important limitation of the conventional serological methodologies used in diagnosis of CD [8]. This phenomenon is especially noted with another protozoan from Trypanosomatidae family, such as Leishmania spp., and is probably related to the sharing of antigenic epitopes between these parasites [24]. Thus, due to the occurrence of false-positive results, it is possible that several epidemiological data may be over- or underestimated, and that individual diagnoses may be wrongly defined in areas where multiple endemics coexist. According to Bottino et al. [25], this cross-reactivity can be reduced by the use of recombinant antigens or synthetic peptide for the generation of more specific diagnostic tools. However, such antigen preparations require slower and expensive synthesis processes. In this sense, using a crude soluble antigen of T. cruzi that is easily obtained, our immunoassay exhibits low cross-reactivity than ELISA, which is a current need in the diagnosis of CD due to the differences in clinical and epidemiological management of patients infected with T. cruzi or Leishmania spp. [26]. Thus, the results indicate that SPRCruzi would be subject to application in CD diagnosis. To substantiate this observation, we determined the general levels of performance of the new immunoassay, which are widely applied in literature for the evaluation of serological methodologies [27,28]. The obtained data are listed in Table 2 and demonstrate that SPRCruzi had a satisfactory performance in discriminating NI and CH patients, presenting high sensitivity (100.0%), specificity (97.2%), positive predictive value (98.0%), negative predictive value (100.0%), global accuracy (99.6%) and Youden index (97.2%). Corroborating the observations of Fig. 2A, the maximum sensitivity indicates the total absence of false-negative results, while the found specificity indicates a false-positive rate of only 2.8%. These results demonstrated that SPRCruzi presented more sensitivity than other diagnostic methods also applicable in the chronic phase of CD, such as xenodiagnoses (9.0 to 87.5%), hemoculture (0.0 to 94.0%) [29], polymerase chain reaction (50.0 to 90.0%) [30], IHA

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Fig. 1. Establishment of methodological criteria (serum dilution and cut-off point) for SPRCruzi immunoassay that better discriminates Chagas diseases patients (CH) from non-infected (NI) individuals. The values of SPR angle variation (ΔθSPR) expressed here are equivalent to the amount of antibodies anti-Trypanosoma cruzi present in sera samples. (A) represents titration curves with the promising serum dilutions to be adopted at SPRCruzi immunoassay (1:160, 1:320 and 1:640) and the average ± standard deviation (SD) values of ΔθSPR obtained after triplicate analysis from a pool of CH sera with high (▲), medium (●) and low (■) reactivity in ELISA, in addition to a control pool of NI subjects (○). The dotted box represents the dilution that better distinguishes the samples. (B), (C) and (D) represent the dispersion of individual values of ΔθSPR obtained for each sample described above at dilutions 1:160, 1:320 and 1:640, respectively, associated with the cut-off point (dotted line) selected in the ROC curve analysis (represented below that). The dotted box selects the serum dilution and cut-off point that show the highest segregation between CH and NI clinical groups.

(66.0 to 74.0%) [31] and flow cytometry immunofluorescence assay (82.0% to 98.1%) [32,33]. Furthermore, it has great clinical relevance, once that sensibility and specificity represent basic parameters in the

evaluation of a diagnostic test, once that they are considered stable properties and do not undergo variations according to the prevalence of a disease in one population.

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Table 1 Performance index of SPRCruzi immunoassay for different serum dilutions. Performance indexa

Cut-off value Sensitivity Specificity Positive predictive value Valor Preditivo Negativo Global accuracy Youden index Likelihood ratio positive Likelihood ratio negative

Serum dilution 1:160

1:320

1:640

53.9 m° 83.8% (68.0–93.8%) 75.0% (50.9–91.3%) 86.1% (70.5–95.3%) 71.4% (47.8–88.7%) 83.0% (70.7–91.6%) 58.8% (35.1–74.2%) 3.35 0.22

17.2 m° 100.0% (90.5–100%) 100.0% (83.2–100%) 100.0% (90.5–100%) 100.0% (83.2–100%) 100.0% (93.7–100%) 100.0% (100–100%) +∞ 0.00

3.7 m° 72.9% (55.9–86.2%) 100.0% (83.2–100%) 100.0% (87.2–100%) 66.7% (47.2–82.7%) 86.5% (74.8–94.1%) 73.0% (54.1–83.8%) +∞ 0.27

a The results are expressed as percentage (95.0% confidence interval) established for serum dilutions at 1:160, 1:320 and 1:640 with the associated cut-off point determined by ROC curve analysis. The negative and positive Likelihood ratios are expressed in chances.

The positive and negative predictive values also have biomedical importance, once that it reflects the contribution of a test in proper clinical decisions. Thus, the values found in our investigation suggest the great capacity of SPRCruzi in the confirmation of individuals who are really positive for CD, as well as the proportion of individuals with negative results that do not have the disease. The detected percentage in the evaluation of the global accuracy indicated by the ROC curve analysis reflects that 99.6% of the results presented by SPRCruzi are correct, which reinforces the excellent performance of the methodology. Moreover, even the assessment of the accuracy by Youden index, which gives equal weights to correct and incorrect test results, was high [34]. Regarding the determination of likelihood ratios, the methodology also was satisfactory, once that the values of LRPos and LRNeg were 36.0 and 0.0, respectively. These results demonstrate that for a positive result provided by SPRCruzi, the probability of a value of ΔθSPR greater than 17.2 m° found in an individual with CD is about thirty-six times greater than the chance of it being found in non-infected individuals. On the other hand, is zero the chance of a negative outcome provided by SPRCruzi come from a CD patient when compared to non-chagasic individuals. Furthermore, according to Jaeschke et al. [35], while LRPos N10 practically confirms the diagnosis of CD, LRNeg b 0.1 practically excludes diagnosis of CD. 3.3. ELISA and SPRCruzi Aiming to compare the performance of SPRCruzi with the results obtained by a conventional serological method for CD diagnosis, we evaluated the serum samples of Group 2 by ELISA method employing Table 2 Global performance indexes of SPRCruzi immunoassay for the diagnosis of Chagas disease in a study population including other parasitic/infectious diseases.

Fig. 2. Evaluation of the applicability of SPRCruzi immunoassay on a broad panel of serum samples from a real endemic area for Chagas disease. (A) represents the dispersion of individual values of SPR angle variation (Δ θSPR) for samples from Chagas disease patients (CH) (■) (n = 99), non-infected subjects (NI) (○) (n = 30) and individuals not infected with T. cruzi but infected with other parasitic/infectious diseases usually found in endemic areas for Chagas disease (other diseases) (▲) (n = 42). For the last group, the values of ΔθSPR are discriminated in (B) for samples from patients with Visceral Leishmaniasis (●) (n = 7), Cutaneous Leishmaniasis (◆) (n = 6), Acute Toxoplasmosis (▼) (n = 6) Chronic Toxoplasmosis (★) (n = 6) as well as HTLV (△) (n = 8), Hepatitis B virus (◇) (n = 4) and HIV (□) (n = 5) infections. All the analyses represented here were carried out adopting the standardized methodological criteria, i. e., 1:320 dilution factor and cut-off point at ΔθSPR = 17.2 m°, which is represented as a dotted line.

Performance indexa

SPRCruzi immunoassay

Cut-off value Sensitivity Specificity Positive predictive value Valor Preditivo Negativo Global accuracy Youden index Likelihood ratio positive Likelihood ratio negative Kappa index

17.2 m° 100.0% (96.3–100%) 97.2% (90.3–99.7%) 95.2% (93.0–99.8%) 100.0% (94.8–100%) 99.6% (97.0–100%) 97.2% (91.7–98.6%) 36.0 0.0 0.939

a Data are expressed as percentage (95.0% confidence interval). These values were determined from a panel of human sera consisting of 171 samples, being 99 from Chagas disease patients, 30 from non-infected subjects and 42 from patients with other parasitic/infectious diseases, including Visceral Leishmaniasis (n = 7), Cutaneous Leishmaniasis (n = 6), Acute Toxoplasmosis (n = 6) Chronic Toxoplasmosis (n = 6) as well as HTLV (n = 8), hepatitis B virus (n = 4) and HIV (n = 5) infections.

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Fig. 3. Comparison between the performance of ELISA (A) and SPRCruzi (B). Both immunoassays were applied in 171 serum samples from study group 2, being 99 from Chagas disease patients (CH), 30 from non-infected individuals (NI) and 42 from not infected with Trypanosoma cruzi but positive for other parasitic/infectious diseases (other diseases). The results are expressed as percentage of results classified as negative (−) and positive (+) in both methods. Statistical analysis performed by K index demonstrated high agreement between ELISA and SPRCruzi.

the same T. cruzi antigen preparation (Fig. 3A). Our findings demonstrated that the analysis by ELISA displayed 16.7% (7/42) of falsepositive results among the subgroup of other diseases, being six for LV (6/7) and one for CL (1/5). On the other hand, SPRCruzi led to a significant decrease in the number of erroneous results for these samples, displaying only 4.8% of false-positive results (Fig. 3B). Additionally, the K index observed between both immunoassays was 0.939, which suggested that the results obtained by the new immunoassay SPRCruzi displayed almost perfect agreement with those obtained by ELISA [17]. However, it is important to highlight that SPRCruzi demonstrated a better ability in distinguish NI and other diseases samples from CH sera. Another major advantage of the new immunoassay against ELISA lies in the fact that it provides a rapid diagnosis of CD, which is achieved in about 20 min in a simple and direct analysis, dispensing sample pretreatment, labeling and refrigeration of reagents and supplies. Associated with the high diagnostic sensitivity and low occurrence of crossreactivity, these benefits approach SPRCruzi to what is desired by WHO as an ideal tool for the diagnosis of chronic CD [6]. In that way, SPRCruzi presents a great option for screening purposes in epidemiological studies, bank blood analysis, experimental evaluations or individual diagnosis of infections by T. cruzi, which needs urgent alternative methodologies [8–10,36]. However, it is important to note that despite the analysis presented here have been performed in a SPR analyzer of laboratory scale, portability solutions have already been described for this type of device, besides other analyzers that allow the execution of multiple and automated analysis [37,38]. Thus, after some adaptation steps, SPRCruzi could be easily deployed in such systems depending on the analytical requirement. In addition, although high costs may represent a limitation for the immediate use of immunosensors based on SPR, the possibility of regeneration of the sensing surface allows the realization of multiple analyses in a single modified SPR sensor chip. Indeed, studies have reported the possibility of running more than 1500 analytical cycles in a single regenerated sensor [39], which associated with real time, sensible and specific analysis, demonstrates that SPRCruzi is a promising serological method for the diagnosis of CD. Taken together, these facts are encouraging our group to further investigate the application of the immunoassay in the post-therapeutic monitoring of CD, which is still greatly lacking [22,23].

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Applicability of a novel immunoassay based on surface plasmon resonance for the diagnosis of Chagas disease.

We defined the methodological criteria for the interpretation of the results provided by a novel immunoassay based on surface plasmon resonance (SPR) ...
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