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Performance Assessment of Four Chimeric Trypanosoma cruzi Antigens Based on Antigen-Antibody Detection for Diagnosis of Chronic Chagas Disease.

ABSTRACT: The performance of serologic tests in chronic Chagas disease diagnosis largely depends on the type and quality of the antigen preparations that are used for detection of anti-Trypanosoma cruzi antibodies. Whole-cell T. cruzi extracts or recombinant proteins have shown variation in the performance and cross-reactivity. Synthetic chimeric proteins comprising fragments of repetitive amino acids of several different proteins have been shown to improve assay performances to detect Chagasic infections. Here, we describe the production of four chimeric T. cruzi proteins and the assessment of their performance for diagnostic purposes. Circular Dichroism spectra indicated the absence of well-defined secondary structures, while polydispersity evaluated by Dynamic Light Scattering revealed only minor aggregates in 50 mM carbonate-bicarbonate (pH 9.6), demonstrating that it is an appropriate buffering system for sensitizing microplates. Serum samples from T. cruzi-infected and non-infected individuals were used to assess the performance of these antigens for detecting antibodies against T. cruzi, using both enzyme-linked immunosorbent assay and a liquid bead array platform. Performance parameters (AUC, sensitivity, specificity, accuracy and J index) showed high diagnostic accuracy for all chimeric proteins for detection of specific anti-T. cruzi antibodies and differentiated seropositive individuals from those who were seronegative. Our data suggest that these four chimeric proteins are eligible for phase II studies.

SUBMITTER: Santos FL

PROVIDER: S-EPMC4982698 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Performance Assessment of Four Chimeric Trypanosoma cruzi Antigens Based on Antigen-Antibody Detection for Diagnosis of Chronic Chagas Disease.

Santos Fred Luciano Neves FL Celedon Paola Alejandra Fiorani PA Zanchin Nilson Ivo Tonin NI Brasil Tatiana de Arruda Campos Tde A Foti Leonardo L Souza Wayner Vieira de WV Silva Edmilson Domingos ED Gomes Yara de Miranda Yde M Krieger Marco Aurélio MA

PloS one 20160812 8

The performance of serologic tests in chronic Chagas disease diagnosis largely depends on the type and quality of the antigen preparations that are used for detection of anti-Trypanosoma cruzi antibodies. Whole-cell T. cruzi extracts or recombinant proteins have shown variation in the performance and cross-reactivity. Synthetic chimeric proteins comprising fragments of repetitive amino acids of several different proteins have been shown to improve assay performances to detect Chagasic infections ...[more]

PMID: 27517281

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Project description:Chagas disease (CD) is among the top 10 causes of inability to blood donation. Blood donation centers screen for anti-Trypanosoma cruzi antibodies using highly sensitive immunoenzymatic (ELISA) or chemiluminescent methods, which can lead to false positive results. Since positive samples cannot be used, to avoid the loss of valuable blood donations, it is necessary to improve specificity without reducing the sensitivity of the tests used for blood screening. For this purpose, our group has developed four chimeric proteins (IBMP-8.1, IBMP-8.2, IBMP-8.3, and IBMP-8.4) that have been evaluated in phase I and II studies with high performance and low cross-reactivity rates. The study included a panel of 5,014 serum samples collected from volunteer blood donors at the Hematology and Hemotherapy Foundation of the State of Bahia (Brazil). They were subjected to the detection of anti-T. cruzi antibodies, using all four IBMP antigens individually and latent class analysis (LCA) as a reference test, since there is no gold standard test for this purpose. Considering the sample size analyzed, LCA classified 4,993 (99.6%) samples as T. cruzi-negative and 21 (0.42%) as T. cruzi-positive. Sensitivity values ranged from 85.71% for IBMP-8.1 and 90.48% for IBMP-8.2-95.24% for IBMP-8.3 and 100% for IBMP-8.4, while specificity ranged from 99.98% for IBMP-8.3 and IBMP-8.4-100% for IBMP-8.1 and IBMP-8.2. Accuracy values ranged from 99.4 to 99.98%. The pretest probability for the molecules was 0.42, whereas the positive posttest probability ranged from 95.24 to 99.95% and the negative posttest probability ranged from 0.00001 to 0.0006% for all antigens. The higher odds ratio diagnosis was found for IBMP-8.4, which has been shown to be a safe single antigen for serological screening of CD in blood samples. The use of chimeric IBMP antigens is an alternative to reduce the number of bags discarded due to false-positive results. These molecules have high diagnostic performance and were shown to be suitable for use in screening CD in blood banks, isolated (IBMP-8.4) or in combination; and their use in blood banks could significantly reduce unnecessary disposal of blood bags or the risk of T. cruzi transmission.

Project description:BackgroundLaboratory diagnosis of chronic Chagas disease is a troubling factor due to lack of reference tests. The WHO suggests the use of two distinct commercial serological tests in parallel. The performance of commercial immunoassays might fluctuate depending on the antigenic matrices and the local strains of T. cruzi in different geographical settings. The use of antigenic matrices based on chimeric proteins can solve these limitations. Here, we evaluated the diagnostic performance of two chimeric T. cruzi antigens (IBMP-8.1 and -8.4) to diagnose chronic Chagas disease in individuals from endemic South American countries.Methodology/principal findingsIBMP-8.1 and IBMP-8.4 chimeric antigens were expressed as soluble proteins in E. coli and purified using chromatography methods. Reactivity of IBMP-8.1 and IBMP-8.4 was assessed using an in-house ELISA with sera from 122 non-infected and 215 T. cruzi-infected individuals from Argentina, Bolivia, and Paraguay. Cut-off values were based on ROC curves and performance parameters were determined using a dichotomous approach. Area under the curve values were > 99.7% for both IBMP-8.1 and IBMP-8.4 antigens. IgG levels in T. cruzi-positive and negative samples were higher for IBMP-8.4 than IBMP-8.1. Both IBMP-8.1 and -8.4 were 100% specific, while IBMP-8.4 were 100% sensitive compared to IBMP-8.1 (95.3%). Admitting RI values of 1.0 ± 0.10 as the inconclusive interval, 6.2% of the samples tested using IBMP-8.1 and 2.1% using IBMP-8.4 fell inside the grey zone. Based on accuracy and diagnostic odds ratio values, IBMP-8.4 presented the best performance. Differences in sensitivity and IgG levels among the samples from Argentina, Bolivia, and Paraguay were not significant.Conclusions/significanceOur findings showed a notable performance of IBMP-8.1 and -8.4 chimeric antigens in diagnosing chronic Chagas disease in individuals from endemic South American countries, confirming our hypothesis that these antigens could be used in geographical areas where distinct T. cruzi DTUs occur.

Project description:BackgroundDogs are considered sentinels in areas of Trypanosoma cruzi transmission risk to humans. ELISA is generally the method of choice for diagnosing T. cruzi exposure in dogs, but its performance substantially depends on the antigenic matrix employed. In previous studies, our group has developed four chimeric antigens (IBMP-8.1, 8.2, 8.3, and 8.4) and evaluated their potential for diagnosing T. cruzi exposure in humans. For human sera, these chimeric antigens presented superior diagnostic performances as compared to commercial tests available in Brazil, Spain, and Argentina. Therefore, in this study we have evaluated the potential of these antigenic proteins for detection of anti-T. cruzi IgG antibodies in dog sera.Methodology/principal findingsThe IBMP-ELISA assays were optimized by checkerboard titration. Subsequently, the diagnostic potential was validated through analysis of ROC curves and the performance of the tests was determined using double entry tables. Cross-reactivity was also evaluated for babesiosis, ehrlichiosis, dirofilariosis, anaplasmosis, and visceral leishmaniasis. Best performance was shown by IBMP-8.3 and IBMP-8.4, although all four antigens demonstrated a high diagnostic performance with 46 positive and 149 negative samples tested. IBMP-8.3 demonstrated 100% sensitivity, followed by IBMP-8.4 (96.7-100%), IBMP-8.2 (73.3-87.5%), and IBMP-8.1 (50-100%). The highest specificities were achieved with IBMP-8.2 (100%) and IBMP-8.4 (100%), followed by IBMP-8.3 (96.7-97.5%) and IBMP 8.1 (89.1-100%).Conclusions/significanceThe use of chimeric antigenic matrices in immunoassays for anti-T. cruzi IgG antibody detection in sera of infected dogs was shown to be a promising tool for veterinary diagnosis and epidemiological studies. The chimeric antigens used in this work allowed also to overcome the common hurdles related to serodiagnosis of T. cruzi infection, especially regarding variation of efficiency parameters according to different strains and cross-reactivity with other infectious diseases.

Project description:BackgroundEnzyme-linked immunosorbent assays (ELISA) are generally the chosen test for Chagas disease (CD) diagnosis; however, its performance depends on the antigen preparation adsorbed to the solid phase, which may lead to false-positive results and cross-reactions. The use of chimeric recombinant antigens can overcome this limitation. Four chimeric antigens from Trypanosoma cruzi (IBMP-8.1, IBMP-8.2, IBMP-8.3 and IBMP-8.4) were developed and evaluated in phase I, II and III studies using indirect ELISA as diagnostic platform. However, peroxidase-labeled secondary anti-human IgG antibody, which is employed in indirect ELISAs, limits its use for the detection of species-specific and class-specific antibodies. To overcome this limitation, peroxidase-labeled antigens can be utilized, diagnosing both acute or chronic infection, in a species and immunoglobulin class-independent manner, through the use of a double-antigen sandwich ELISA (DAgS-ELISA). We aimed to evaluate and validate the diagnostic performance of the chimeric antigens IBMP-8.1, IBMP-8.2, IBMP-8.3 and IBMP-8.4 in the DAgS-ELISA platform.Methodology/principal findingsDAgS-ELISA was optimized by checkerboard titration. In phase I study, 207 positive and 205 negative samples were evaluated. Cross-reactivity to other infections was also assessed using 68 samples. The selected conditions for the tests utilized 25 ng of antigen per well and the conjugate diluted at 1:2,000 for all molecules. In the phase I study, the areas under the curve of IBMP-8.1, IBMP-8.2, IBMP-8.3 and IBMP-8.4 were 98.7%, 99.5%, 98.6% and 98.8%, respectively. Among the positive samples, IBMP-8.1 antigen classified 53 (25.6%) as false negative, IBMP-8.2, 27 (13%), IBMP-8.3, 24 (11.6%) and IBMP-8.4, 43 (20.8%), giving sensitivities of 74.4%, 87%, 88.4% and 79.2%, respectively. The only antigen that did not reach 100% specificity was IBMP-8.3, with 96.6%. IBMP-8.3 was also the only molecule to show cross-reactivity with HTLV.Conclusions/significanceDAgS-ELISA is a promising tool for immunodiagnosis, and despite the high AUC values, the performance of this assay was different from the values obtained by our group when using these antigens in the indirect ELISA, for this reason, improvements are being considered to increase the sensitivity of the DAgS-ELISA.

Dataset Information

Performance Assessment of Four Chimeric Trypanosoma cruzi Antigens Based on Antigen-Antibody Detection for Diagnosis of Chronic Chagas Disease.

Publications

Performance Assessment of Four Chimeric Trypanosoma cruzi Antigens Based on Antigen-Antibody Detection for Diagnosis of Chronic Chagas Disease.

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