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Chagas disease (CD), caused by the protozoan Trypanosoma cruzi, is an important public health problem, occurring mainly in Latin America. The disease has a major social and economical effect, negatively impacting the life of the infected individuals, and bringing great costs to public health. An early and accurate diagnosis is essential for administration of early treatment. In addition, prognostic tests may aid disease management, decreasing hospitalization costs. However, the serological diagnostic scenario for CD still faces several challenges, making the development of new diagnostic kits a pressing matter. Facing this scenario, several researchers have expanded efforts in developing and testing new antigens, such as recombinant proteins and recombinant multiepitope proteins, with promising results. These recombinant antigens offer several advantages, such as improved sensitivity and specificity, in addition to facilitated scaling. Also, it has been possible to observe a rising number of studies using ELISA and point-of-care platforms, employing these antigens in the past few years. Among them, recombinant proteins were the most applied antigens, demonstrating great capacity to discriminate between positive and negative samples. Although fewer in number, recombinant multiepitope proteins also demonstrated an improved diagnostic performance. Indeed, a great number of studies employing these antigens showed sensitivity and specificity values above 90%, greatly impacting diagnostic accuracy. Nevertheless, despite the good results found, it is still possible to observe some bottlenecks in the development of new antigens, such as the scarcity of tests with sera from the acute phase and the variability of results in different geographic areas. In this sense, aiming to contribute to control and health programs, the continuous search for a more accurate serological diagnosis is essential, both for the acute and chronic phases of the disease.

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COVID-19 laboratory diagnosis primarily relies on molecular tests, highly sensitive during early infection stages with high viral loads. As the disease progresses, sensitivity decreases, requiring antibody detection. Since the beginning of the pandemic, serological tests have been developed and made available in Brazil, but their diagnostic performance varies. This study evaluated the IBMP ELISA IgA/IgM/IgG COVID-19 kit performance in detecting SARS-CoV-2 antibodies. A total of 90 samples, including 64 from COVID-19 patients and 26 pre-pandemic donors, were assessed based on time post symptom onset (0-7, 8-14, and 15-21 days). The kit showed 61% sensitivity, 100% specificity, and 72% accuracy overall. Sensitivity varied with time, being 25%, 57%, and 96% for 0-7, 8-14, and 15-21 days, respectively. Similar variations were noted in other commercial tests. The Gold ELISA COVID-19 (IgG/IgM) had sensitivities of 31%, 71%, and 100%, while the Anti-SARS-CoV-2 NCP ELISA (IgG) and Anti-SARS-CoV-2 NCP ELISA (IgM) showed varying sensitivities. The IBMP ELISA kit displayed high diagnostic capability, especially as the disease progressed, complementing COVID-19 diagnosis. Reproducibility assessment revealed minimal systematic and analytical errors. In conclusion, the IBMP ELISA IgA/IgM/IgG COVID-19 kit is a robust tool for detecting anti-SARS-CoV-2 antibodies, increasing in efficacy over the disease course, and minimizing false negatives in RT-PCR COVID-19 diagnosis.

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Recombinant multiepitope proteins (RMPs) are a promising alternative for application in diagnostic tests and, given their wide application in the most diverse diseases, this review article aims to survey the use of these antigens for diagnosis, as well as discuss the main points surrounding these antigens. RMPs usually consisting of linear, immunodominant, and phylogenetically conserved epitopes, has been applied in the experimental diagnosis of various human and animal diseases, such as leishmaniasis, brucellosis, cysticercosis, Chagas disease, hepatitis, leptospirosis, leprosy, filariasis, schistosomiasis, dengue, and COVID-19. The synthetic genes for these epitopes are joined to code a single RMP, either with spacers or fused, with different biochemical properties. The epitopes' high density within the RMPs contributes to a high degree of sensitivity and specificity. The RMPs can also sidestep the need for multiple peptide synthesis or multiple recombinant proteins, reducing costs and enhancing the standardization conditions for immunoassays. Methods such as bioinformatics and circular dichroism have been widely applied in the development of new RMPs, helping to guide their construction and better understand their structure. Several RMPs have been expressed, mainly using the Escherichia coli expression system, highlighting the importance of these cells in the biotechnological field. In fact, technological advances in this area, offering a wide range of different strains to be used, make these cells the most widely used expression platform. RMPs have been experimentally used to diagnose a broad range of illnesses in the laboratory, suggesting they could also be useful for accurate diagnoses commercially. On this point, the RMP method offers a tempting substitute for the production of promising antigens used to assemble commercial diagnostic kits.

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Syphilis is a sexually transmitted infection (STI) caused by the spiral bacterium Treponema pallidum. Diagnosis is based on epidemiology, clinical and serology, but serodiagnosis is challenging because distinct clinical forms of the infection may influence serological performance. Several recombinant Treponema pallidum-proteins have already been tested for syphilis diagnosis and they are critical to achieve high accuracy in serological testing. A total of 647 samples were included in the study: 180 T. pallidum-positive samples, 191 T. pallidum-negative samples and 276 sera from individuals infected with unrelated diseases. The diagnostic potential was validated by analysis of ROC curves. For the indirect ELISA, TpN17 (100%) and TmpA (99%) showed excellent AUC values. Sensitivity values were 97.2% for TpN17 and 90.6% for TmpA, while specificity was 100% for both molecules. According to the clinical phase, TmpA ranged from 84% to 97%, with the highest value for secondary syphilis. TpN17 was 100% sensitive for the primary and secondary stages and 93.2% for recent latent syphilis. All clinical phases achieved 100% specificity. Accuracy values showed that TmpA (> 95%) and TpN17 (> 98%) presented high diagnostic accuracy for all clinical stages of syphilis. Cross-reactivity was only observed in one sample positive for Chagas disease (1.5%), when TpN17 was evaluated. On the other hand, TmpA showed reactivity for two samples positive for Chagas disease (3.1%), one sample positive for HBV (1.25%), two samples positive for HIV (9.5%) and one sample positive for HTLV (1.6%). The TmpA antigen's performance was evaluated in multiple studies for syphilis diagnosis, corroborating our findings. However, TpN17 sensitivity values have ranged in other studies. According to clinical stages of the infection, our findings obtained close performance values.

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BACKGROUND: The high structural similarity between the Zika virus (ZIKV) and other flaviviruses, such as Dengue Virus (DENV), complicates the identification of the infecting virus due to the occurrence of cross-reactions in serological assays. This phenomenon has increased the demand for more specific antigens for immunodiagnostic applications. METHODS: The present work aimed to identify specific regions of ZIKV and produce unique antigens through computational methods, molecular and microbiological techniques. RESULTS: Based on the computational analysis we successfully expressed two recombinant proteins derived from specific regions of the ZIKV. Through serological assays using characterized sera, we observed that the region 146-182 of ZIKV's E protein, expressed in tandem, was not reactive despite the predictive sensitivity and specificity observed by computer analyses. On the other hand, the non-denatured fraction 220-352 of ZIKV's NS1 showed greater specificity to IgG+ sera of ZIKV by dot blot and western blot, which highlights its properties as a possible tool in the diagnosis of ZIKV. CONCLUSION: These findings demonstrate that ZIKV NS1 fraction 220-352 is a potential tool that may be applied in the development of serological diagnosis. We also provided data that suggest the non-applicability of the region 146-182 of ZIKV's protein E in serological assays despite previous indications about its potential based on computational analysis.

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Leishmaniasis is a neglected disease and more than 1 billion people live in endemic areas with the risk of infection worldwide. Although it is an important epidemiological issue, the gold standard method of diagnosis requires invasive sample collection and is accompanied by a high level of sensitivity variation in results. The present study aims to conduct a patent prospection of immunodiagnostic methods for human tegumentary leishmaniasis in the last 10 years, focused on those with high sensitivity and specificity, and simple usability. We searched seven patent databases: The LENS, WIPO, EPO, USPTO, Patent Inspiration, Google patents, and INPI. Eleven patents were found that satisfy our search criteria, with six of them being registered in 2017. Most patents were registered in Brazil. The information obtained here covers the main characteristics of the immunodiagnostic methods evaluated. Moreover, our prospective study reveals the latest biotechnological advancements achieved in the immunodiagnosis of tegumentary leishmaniasis, especially in Brazil, which holds the majority of patents in this subject. However, no patent for immunodiagnostic methods was found in the last three years, which raises concerns about the present and future trends of leishmaniasis diagnosis.

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Chagas disease remains a neglected disease that is considered to be a public health problem. The early diagnosis of cases is important to improve the prognosis of infected patients and prevent transmission. Serological tests are the method of choice for diagnosis. However, two serological tests are currently recommended to confirm positive cases. In this sense, more sensitive and specific serological tests need to be developed to overcome these current diagnosis problems. This study aimed to develop a new recombinant multiepitope protein for the diagnosis of Chagas disease, hereafter named rTC. The rTC was constructed based on amino acid sequences from different combinations of Trypanosoma cruzi antigens in the same polypeptide and tested using an enzyme-linked immunosorbent assay (ELISA) to detect different types of Chagas disease. rTC was able to discriminate between indeterminate (IND) and cardiac (CARD) cases and cross-reactive diseases, as well as healthy samples, with 98.28% sensitivity and 96.67% specificity, respectively. These data suggest that rTC has the potential to be tested in future studies against a larger serological panel for the diagnosis of Chagas disease.

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A cinomose é uma enfermidade causada pelo vírus Canine Distemper Virus (CDV). Essa doença afeta principalmente cães, mas também acomete outras espécies domésticas e selvagens. A imunidade do animal está relacionada ao grau que a esse patógeno vai atingir o organismo do indivíduo. Ela afeta a respiração do animal, pode causar vômito, diarreia, convulsões, podendo levar o animal à óbito. O objetivo do presente trabalho foi padronizar um teste ELISA indireto com antígeno de superfície para o diagnostico cinomose utilizando amostras de soro canino. Para padronização da técnica, fez-se necessário o estudo da diluição do antígeno para identificar a melhor concentração para sensibilização da placa. O teste foi aplicado primeiramente com diferentes diluições do antígeno para detecção do melhor desempenho do antígeno. Feito isso, foi testado em um banco de soro de 45 animais comprovadamente positivos no teste ELISA comercial e em soro de 45 animais comprovadamente negativos no teste ELISA comercial, posteriormente foi calculado o ponto de corte, especificidade e sensibilidade do teste. O teste ELISA indireto se mostrou com excelência como um teste de diagnóstico para a cinomose canina, obtendo-se ponto de corte de densidade óptica de 0,229, sensibilidade de 95,5% e especificidade de 84,4%.

Distemper is a disease or the disease by the CDV virus, Distemper Virus. This disease mainly affects dogs, but also affects other domestic and wild species. The animal's immunity is related to the degree to which it will reach the individual's organism. It affects the animal's breathing, can cause vomiting, diarrhea, convulsions, and can lead to death. The aim of the present work test was to standardize an indirect ELISA for distemper diagnosis in experiments using a surface antigen. For the study of technical identification, it was necessary to specify the antigen for the best concentration of plaque sensitization. The test was initially applied with different dilutions of the antigen to detect the best performance of the antigen. This was tested in a serum bank of 45 animals proven positive in the commercial ELISA test and in the serum of 45 animals proven negative in the commercial ELISA test, later it was tested on the cut-off point, specificity and sensitivity of the test. The indirect ELISA test proved to be excellent as a diagnostic test for canine distemper, with an optical density cut-off of 0.229, sensitivity of 95.5% and specificity of 84.4% being obtained.

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BACKGROUND: Cat-transmitted sporotrichosis (CTS) caused by Sporothrix brasiliensis has emerged as an important zoonosis in Brazil and neighbouring countries. OBJECTIVES: Evaluate the performance of a lateral flow assay (LFA) for the detection of anti-Sporothrix antibodies in human sera. METHODS: A LFA for the detection of anti-Sporothrix antibodies (Anti-Sporo LFA) in human sera, developed by IMMY, was evaluated using 300 human sera collected prospectively at the Hospital de Clínicas, Federal University of Paraná (HC-UFPR), in Curitiba, Brazil. These specimens included 100 sera from patients with CTS. CTS cases were classified as follows: 59 lymphocutaneous, 27 fixed cutaneous,13 ocular, and one mixed form. One-hundred specimens from patients with other mycoses, including cryptococcosis (n = 32), candidemia (n = 27), paracoccidioidomycosis (n = 14), aspergillosis (n = 10), histoplasmosis (n = 9), fusariosis (n = 4), lobomycosis (n = 1), chromoblastomycosis (n = 1), mucormycosis (n = 1) and trichosporonosis (n = 1). And 100 specimens from apparently healthy volunteers (AHV). RESULTS: The Anti-Sporo LFA showed a global sensitivity of 83% (95% confidence interval [CI] = 74%-90%), a global specificity of 82% (95% CI = 76%-87%), and accuracy of 82% (95% CI = 77%-86%). By clinical form sensitivity was as follows: Mixed form 100%, ocular 92%, lymphocutaneous 83% and fixed cutaneous 78%. False-positive results were observed in 11 specimens from people with other mycoses and 26 specimens from AHV. CONCLUSION AND DISCUSSION: This study presents the results of the evaluation of the first lateral flow assay for the detection of anti-Sporothrix antibodies in human sera. The findings here show evidence that IMMY's Anti-Sporo LFA is a promising tool for the rapid diagnosis of CTS.

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Using microscopy and/or immunodiagnosis, the authors analyzed 284 fecal samples from the Brazilian rock cavy, Kerodon rupestris, that were collected between 1984 and 2015 in Serra da Capivara National Park for the presence of helminths and protozoa. Fourteen morphospecies of helminth eggs of the following taxa were found: Trematoda, Nematoda, Strongylidae, Lagochilascaris sp., Strongylida, Trichuris (2 species), Oxyuridae (3 species), Ancylostomatidae (2 species), and Ascarididae (2 species), along with 3 protozoan taxa: Coccidia, Cryptosporidium sp., and Balantidium sp. During the last 30 yr, the population of K. rupestris has increased in the region as a consequence of the creation and management of the National Park, and data from this study show a concurrent increase in the diversity of intestinal parasites in this host, including new reports. Some of these species have zoonotic potential, which suggests that K. rupestris may be in contact with domestic farm animals and/or human feces. These results show the importance of integrating different diagnostic approaches for the identification of protozoa in the region and indicate that further methods need to be employed to increase recovery. This work highlights the usefulness of parasite studies in assessing the health of ecosystems, especially in protected areas, which should be considered by park managers and health agencies.

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