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Aspergillus species can colonize and infect immunocompetent and immunocompromised hosts. Conventional fungal identification depends on microscopic analysis and microorganism medium growth. Other diagnostic methods, non-growth dependent, to invasive fungal infections, are the biomarkers that detect circulating polysaccharides, for example, 1-3-ß-d-Glucan and galactomannan. Both are polysaccharides present on the external layer of fungi cell wall and can be detected in clinical samples during the growth of the fungus in the patient. This study aimed to compare the galactomannan detection of Lateral Flow Assay and Enzyme Immunoassay methods in Bronchoalveolar Lavage Fluid. The galactomannan antigen in Bronchoalveolar Lavage Fluid was measured using Enzyme Immunoassay according to the manufacturer's instructions (PLATELIA ASPERGILLUS™ BioRad) and, using a Lateral Flow Assay according to the manufacturer's instructions (Galactomannan LFA IMMY©). The 71 samples were Bronchoalveolar Lavage Fluid of patients hospitalized at Unicamp Clinical Hospital between 2019 and 2021; of these samples 12/71 (16.9 %) resulted in positive Galactomannan-Lateral Flow Assay. In contrast, Galactomannan-Enzyme Immunoassay resulted as positive in 9/71 (12.6 %) samples, a difference that showed not significant statistically (p-value = 0.36) Comparing both assays' results identified 8 divergences between them, about 11 % of the total sample. The Sensitivity (73.3 %), Specificity (92.35 %), Positive Predictive Value (62.85 %) and Negative Predictive Value (95.15 %) of Lateral Flow Assay were calculated using the Galactomannan Enzyme Immunoassay as standard. The Lateral Flow Assay demonstrated good results when compared with the Enzyme Immunoassay.

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Coccidiomycosis is a potentially life-threatening fungal infection endemic to certain regions of Argentina. The infection is caused by Coccidioides spp. and is primarily diagnosed by Coccidioides antibody (Ab) detection. Access to rapid, highly accurate diagnostic testing is critical to ensure prompt antifungal therapy. The sona Coccidioides Ab Lateral Flow Assay (LFA) performs faster and requires less laboratory infrastructure and equipment compared with other Ab detection assays, potentially providing a substantial improvement for rapid case screening in coccidioidomycosis-endemic regions; however, validation of this test is needed. Thus, we aimed to evaluate the analytical performance of the sona Coccidioides Ab (LFA) and compare agreement with anti-Coccidioides Ab detection assays. A total of 103 human sera specimens were tested, including 25 specimens from patients with coccidioidomycosis and 78 from patients without coccidioidomycosis. The sona Coccidioides Ab Lateral Flow Assay (LFA) was performed with a sensitivity of 88%, and specificity and accuracy of 87%. Furthermore, the Coccidioides Ab LFA had good agreement with other anti-Coccidioides Ab detection assays. Our findings suggest the sona Coccidioides Ab LFA has satisfactory performance and may be useful for diagnosing coccidioidomycosis in endemic regions.

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The POC-CCA test is subject to variations in reading interpretations depending on the intensity of its results, and trace test reading have implications for determining prevalence. The aim of this study was to assess whether the readings obtained from the POC-CCA tests, conducted using a semi-quantitative scale (the G-score classification for test determination), exhibited concurrence with the direct visual interpretation (positive, negative, or trace) performed by two distinct analysts, using photographs from previously performed POC-CCA test carried out in the municipality of Maruim, in the state of Sergipe-Brazil, a region of high endemicity. The devices used to read the photographs were smartphones, so as to simulate field usage, and a desktop, a tool with higher image quality that would help the researchers in the evaluation and establishment of the final result at a later. In direct visual interpretation of the POC-CCA photographs, the most discordant results occurred in the identification of the trace response (T). The Kappa index established for the direct visual interpretation between the two analysts, in which T is considered as positive, in the desktop was κ=0.826 and in the smartphone, κ=0.950. When we use the G-score as a reading standardization technique and classify the results according to the manufacturer, with trace being evaluated as positive, the highest level of agreement was obtained. Some disagreement remains between the direct visual interpretation and the G-score when performed on the desktop, with more individuals being classified as negative in the direct visual interpretation, by both analysts. However, this result was not statistically significant. The use of the G-score scale proved to be an excellent tool for standardizing the readings and classifying the results according to the semi-quantitative scale showed greater concordance of results both among analysts and among the different devices used to view the photographs.

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We report on an analytical and biological validation of a commercial cortisol enzyme immunoassay (EIA) to measure glucocorticoids (GC) in feces of Geoffroy's spider monkeys (Ateles geoffroyi). Validation of endocrinological methods for each sample matrix and study species is crucial to establish that the methods produce reliable results. For the analytical validation of the EIA, we assessed parallelism, accuracy, and precision. We carried out a biological validation based on three well-studied GC patterns with the following predictions: (1) increased fecal GC metabolite (fGCM) concentrations after veterinary intervention; (2) increased fGCM concentrations during early morning hours; and (3) higher fGCM concentrations during gestation than in other female reproductive states. For the first prediction, we sampled feces of two zoo-housed females 2 days before, the day of, and 2 days after a veterinary intervention. For the second prediction, we analyzed 284 fecal samples collected from 12 wild males using a linear mixed model (LMM). For the third prediction, we analyzed 269 fecal samples of eight wild females using an LMM. Analytical validation revealed that the EIA showed parallelism, was accurate, and precise within each assay. However, there was elevated variation in between-assay precision. The biological validation supported all predictions: (1) the two zoo-housed females showed a substantial increase in fGCM concentrations 2.5 and 11 h after veterinary intervention; (2) there was a negative effect of sample collection time on fGCM concentrations (i.e., higher concentrations during early morning); (3) gestating females had significantly higher fGCM concentrations than lactating females. Thus, we analytically validated the commercial EIA and, despite between-assay variation, we were able to find three biologically relevant GC signals in captive and wild settings, and in males and females. We are therefore confident that the method can be used to noninvasively address behavioral endocrinology questions in Geoffroy's spider monkeys.

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Abstract Aspergillus species can colonize and infect immunocompetent and immunocompromised hosts. Conventional fungal identification depends on microscopic analysis and microorganism medium growth. Other diagnostic methods, non-growth dependent, to invasive fungal infections, are the biomarkers that detect circulating polysaccharides, for example, 1-3-β-d-Glucan and galactomannan. Both are polysaccharides present on the external layer of fungi cell wall and can be detected in clinical samples during the growth of the fungus in the patient. This study aimed to compare the galactomannan detection of Lateral Flow Assay and Enzyme Immunoassay methods in Bronchoalveolar Lavage Fluid. The galactomannan antigen in Bronchoalveolar Lavage Fluid was measured using Enzyme Immunoassay according to the manufacturer's instructions (PLATELIA ASPERGILLUS™ BioRad) and, using a Lateral Flow Assay according to the manufacturer's instructions (Galactomannan LFA IMMY©). The 71 samples were Bronchoalveolar Lavage Fluid of patients hospitalized at Unicamp Clinical Hospital between 2019 and 2021; of these samples 12/71 (16.9 %) resulted in positive Galactomannan-Lateral Flow Assay. In contrast, Galactomannan-Enzyme Immunoassay resulted as positive in 9/71 (12.6 %) samples, a difference that showed not significant statistically (p-value = 0.36) Comparing both assays' results identified 8 divergences between them, about 11 % of the total sample. The Sensitivity (73.3 %), Specificity (92.35 %), Positive Predictive Value (62.85 %) and Negative Predictive Value (95.15 %) of Lateral Flow Assay were calculated using the Galactomannan Enzyme Immunoassay as standard. The Lateral Flow Assay demonstrated good results when compared with the Enzyme Immunoassay.

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Resumen Objetivo: Establecer un inmunoensayo semicuantitativo para la detección de anticuerpos contra el dominio de unión al receptor de la proteína de espícula del coronavirus del síndrome respiratorio agudo grave tipo 2 y la evaluación de su desempeño como herramienta de apoyo diagnóstico. Métodos: Se generó una proteína recombinante del dominio de unión a receptor de la proteína de espícula del coronavirus del síndrome respiratorio agudo grave tipo 2. Dicha proteína se empleó como sustrato antigénico en la estandarización de dos ensayos semicuantitativos por inmunoadsorción ligados a enzima para la detección de inmunoglobulinas M e inmunoglobulinas G humanas. Se utilizó un conjunto de muestras de suero positivas (n=129), provenientes de donantes voluntarios con infección previa por el virus SARS-CoV-2, confirmada mediante reacción en cadena de la polimerasa con transcriptasa reversa, y tomadas entre agosto de 2020 y noviembre de 2021. Además, se empleó un panel de muestras prepandémicas negativas (n=196) obtenidas antes de diciembre de 2019 para la evaluación del desempeño de los ensayos; se recibieron muestras múltiples seriadas de 99 donantes voluntarios para examinar la respuesta de la prueba ante la seroconversión y se estudió la posible asociación entre las seropositividades por coronavirus del síndrome respiratorio agudo grave tipo 2 y por el virus del dengue para la evaluación de reacciones cruzadas inespecíficas. Resultados: El ensayo de detección de inmunoglobulina G mostró 81.4 % de sensibilidad, 86.2 % de especificidad y valores predictivos positivos y negativos de 79.5 % y 87.6 % respectivamente. Por su parte, el ensayo de detección de inmunoglobulina M mostró solamente 72.1 % de sensibilidad, 54.1 % de especificidad y valores predictivos positivos y negativos de 25.6 % y 89.8 % respectivamente. No se encontraron diferencias significativas entre las mediciones semicuantitativas según sexo ni correlación lineal entre esta variable y la edad. Los valores obtenidos para el inmunoensayo presentaron diferencias significativas según el autorreporte de presencia o ausencia de síntomas compatibles con COVID-19. No se encontró correlación entre las seropositividades contra el coronavirus del síndrome respiratorio agudo grave tipo 2 y el virus del dengue. El ensayo de detección de inmunoglobulina G generó valores inferiores pero constantes en muestras de donantes voluntarios que autorreportaron no haber tenido contacto con el virus SARS-CoV-2. En contraste, las muestras de donantes expuestos al virus SARS-CoV-2 mostraron valores elevados pero variables en magnitud. Además, se observaron valores elevados y variables en muestras de voluntarios vacunados o con infección previa. Conclusiones: Nuestro ensayo de detección de inmunoglobulina M presenta escaso valor diagnóstico. Por el contrario, el ensayo de detección de inmunoglobulina G muestra un rendimiento satisfactorio y se apega al comportamiento reportado para este tipo de prueba según las características demográficas y clínicas de los usuarios; por lo tanto, este ensayo podría ser empleado como herramienta fiable y práctica en aplicaciones clínicas y como apoyo al diagnóstico. Es necesario desarrollar más estudios sobre reacciones cruzadas entre los anticuerpos contra el coronavirus del síndrome respiratorio agudo grave tipo 2 con aquellos de otras entidades de interés clínico, sobre todo las presentes en países tropicales como el nuestro.

Abstract Aim: To establish a semiquantitative immunoassay for antibody detection against the RBD of the severe acute respiratory syndrome coronavirus 2 spike protein and to evaluate its performance to be used as a diagnostic supporting tool. Methods: A recombinant severe acute respiratory syndrome coronavirus 2 spike protein was produced. This protein was used as antigenic substrate in two semiquantitative enzyme-linked immunoassays for the detection of human immunoglobulins M and immunoglobulins G. A set of serum samples (N=129) from patients with prior viral infection confirmed by reverse transcription polymerase chain reaction, processed between August 2020 and November 2021, were used as positive samples. A panel of pre-pandemic samples (N=196), obtained prior to December 2019, were used as negative samples to evaluate the assay performance. Multiple samples from 99 volunteers were used to examine test response to seroconversion. The interference between seropositivity against severe acute respiratory syndrome coronavirus 2 and dengue virus was also evaluated. Results: The immunoglobulin G detection assay showed 81.4% sensitivity, 86.2% specificity, and positive and negative predictive values of 79.5% and 87.6% respectively. The immunoglobulin M detection assay yielded 72.1% sensitivity, 54.1% specificity, and positive and negative predictive values of 25.6% and 89.8% respectively. No significant differences were found between the measurements according to sex or linear correlation between this variable and age. The values presented significant differences according to the condition of self-reported presence or absence of COVID-19 like symptoms. No correlation was found between seropositivity for severe acute respiratory syndrome coronavirus 2 and dengue virus. The immunoglobulin G detection assay generated lower but constant values on samples from voluntary donors who reported not having any contact with the virus compared to samples from donors exposed to it, and high but variable values in magnitude on samples from vaccinated volunteers or those with previous severe acute respiratory syndrome coronavirus 2 infection compared to samples from donors without exposure to the viral antigen. Conclusions: Our established immunoglobulin M detection assay presents poor diagnostic value. On the other hand, the immunoglobulin G detection assay shows satisfactory performance, and coheres to the behavior reported for this type of test according to the demographic and clinic characteristics of the volunteer, so it could be used as a reliable and practical tool in clinical applications and as diagnostic complement. It is necessary to develop more studies on cross-reactions of antibodies against severe acute respiratory syndrome coronavirus 2 with other entities of clinical interest and present in our tropical area.

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The simultaneous capture and detection of biomolecules is crucial for revolutionizing bioanalytical platforms in terms of portability, response time and cost-efficiency. Herein, we demonstrate how the sensitivity to external stimuli and changes in the local electronic environment of silver clusters lead to an advantageous biosensing platform based on the fluorometric response of bioactive luminescent silver clusters (BioLuSiC) confined in faujasite X zeolites functionalized with antibodies. The photoluminescence response of BioLuSiC was enhanced upon immunocomplex formation, empowering a wash-free and quick biodetection system offering optimal results from 5 min. Proteins and pathogens (immunoglobulin G and Escherichia coli) were targeted to demonstrate the biosensing performance of BioLuSiC, and a human serum titration assay was also established. BioLuSiC will pave the way for innovative bioanalytical platforms, including real-time monitoring systems, point-of-care devices and bioimaging techniques.

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Chikungunya (CHIK) is a debilitating mosquito-borne disease with an epidemiology and early clinical symptoms similar to those of other arboviruses-triggered diseases such as dengue or Zika. Accurate and rapid diagnosis of CHIK virus (CHIKV) infection is therefore challenging. This international study evaluated the performance of the automated VIDAS® anti-CHIKV IgM and IgG assays compared to that of manual competitor IgM and IgG ELISA for the detection of anti-CHIKV IgM and IgG antibodies in 660 patients with suspected CHIKV infection. Positive and negative agreements of the VIDAS® CHIKV assays with ELISA ranged from 97.5% to 100.0%. The sensitivity of the VIDAS® CHIKV assays evaluated in patients with a proven CHIKV infection confirmed reported kinetics of anti-CHIKV IgM and IgG response, with a positive detection of 88.2-100.0% for IgM ≥ 5 days post symptom onset and of 100.0% for IgG ≥ 11 days post symptom onset. Our study also demonstrated the superiority of ELISA and VIDAS® assays over rapid diagnostic IgM/IgG tests. The analytical performance of VIDAS® anti-CHIKV IgM and IgG assays was excellent, with a high precision (coefficients of variation ≤ 7.4%) and high specificity (cross-reactivity rate ≤ 2.9%). This study demonstrates the suitability of the automated VIDAS® anti-CHIKV IgM and IgG assays to diagnose CHIKV infections and supports its applicability for epidemiological surveillance and differential diagnosis in regions endemic for CHIKV.

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Phenytoin (DFH), is an anticonvulsant widely used for the treatment of different types of seizures.(1) Therapeutic monitoring (TDM) is required for DFH due to its narrow therapeutic range and nonlinear pharmacokinetics, among other characteristics. Monitoring is frequently done on plasma or serum (total drug) through immunological methods. DFH can also be monitored in saliva, which shows a good correlation with plasma. The concentration of DFH in saliva reflects the concentration of free drug and due to the simplicity in its collection, it leads to a less stressful process for the patient. The aim of this study was to validate the immunological method of kinetic interaction of microparticles in solution (KIMS) for the determination of DFH using saliva as biological matrix. Linearity, precision, detection and quantification limit, accuracy and interference were analyzed. Infostat 8.0 student version software was used for statistical analysis. The method was linear in a range between 0.41 and 5ug/ml. The detection and quantification limits were 0.14 and 0.45ug/ml, respectively. The equation of the straight line obtained based on the method comparison between KIMS and HPLC-UV was DFHKIMS= 0,81* DFHHPLC ­ 0,03.  The KIMS method proved to have the necessary analytical characteristics to be applied as a useful and practical tool for the follow-up of those patients with difficult venous access and/or children with chronic DFH treatments.

La Fenitoína (DFH), es un anticonvulsivante ampliamente utilizado para el tratamiento de distintos tipos de convulsiones.(1) El monitoreo terapéutico (TDM) es requerido para la DFH debido a su estrecho rango terapéutico y farmacocinética no lineal, entre otras características. Los monitoreos se realizan frecuentemente en plasma o suero (droga total) a través de métodos inmunológicos. También se puede monitorear DFH en saliva, la cual presenta una buena correlación con el plasma. La concentración de DFH en saliva refleja la concentración de droga libre y debido a la simplicidad en su recolección, conlleva a un proceso menos estresante para el paciente. El objetivo del trabajo fue validar el método inmunológico de interacción cinética de micropartículas en solución (KIMS) para la determinación de DFH usando como matriz biológica saliva. Se analizó linealidad, precisión, límite de detección y cuantificación, exactitud e interferencia. Se utilizó el software Infostat 8.0 versión estudiantil para el análisis estadístico. El método fue lineal en un intervalo entre 0,41 y 5ug/ml. Los límites de detección y cuantificación fueron 0,14 y 0,45ug/ml respectivamente. La ecuación de la recta obtenida en base a la comparación de métodos entre KIMS y HPLC-UV fue DFHKIMS= 0,81* DFHHPLC - 0,03.  El método KIMS demostró tener las características analíticas necesarias paran ser aplicada como herramienta útil y práctica para el seguimiento de aquellos pacientes de difícil acceso venoso y/o niños con tratamientos crónicos con DFH.

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Immune cells play a major role in the development of cancer, from being able to inhibit it by secreting pro-inflammatory mediators, to assist in its development by secreting growth factors, immunosuppressive mediators, and ECM-modifying enzymes. Therefore, the ex vivo analysis of the secretion function of immune cells can be employed as a reliable prognostic biomarker in cancer. However, one limiting factor in current approaches to probe the ex vivo secretion function of cells is their low throughput and the consumption of large quantities of sample. Microfluidics provides a unique advantage, by being able to integrate different components, such as cell culture and biosensors in a monolithic microdevice; it can increase the analytical throughput and leverage it with its intrinsic low sample requirement. Furthermore, the integration of fluid control elements also allows this analysis to be highly automatable, leading to increases in consistency in the results. Here, we describe an approach to analyze the ex vivo secretion function of immune cells using a highly integrated microfluidic device.

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Introducción: Las cianobacterias son microrganismos fotosintéticos, con capacidad de sintetizar una gran diversidad de metabolitos secundarios de interés para la industria, pero también han llamado la atención en las últimas décadas las toxinas denominas cianotoxinas, metabolitos que causan distintas alternaciones fisiológicas hasta llegar ocasionar la muerte de diferentes especies. Metodología: La determinación del estado de arte para el tema de cianobacterias se basó en una búsqueda bibliográfica en la base de datos especializada como Elservier, Springer, Google académico y MDPI basadas en palabras clave en español e inglés "microcistinas", "degradación de MC" y "cuantificación y detección de MC". Resultados: En la presente revisión considera dos áreas de caracterización de la microcistinas (MCs) las propiedades fisicoquímicas y propiedades biológicas, para entender su comportamiento e importancia tóxica en los sembradíos agrícolas y en la salud humana. Además de comprender alternativas para su degradación, por métodos fisicoquímicos como fotocatálisis y la gradación biológica por bacterias. Finalmente se mencionará algunos métodos actuales y en desarrollo, para la detección y cuantificación de estas MCs en ambientes acuáticos. Conclusiones: Las MCs tienen el potencial contaminar fuentes de agua como ríos y lagunas, causando daños a la salud humana y a las plantas agrícolas, tienen la capacidad de tolerar distintos cambios drásticos en factores fisicoquímicos y biológicos. Entre las alternativas reportadas la degradación bacteriana promete ser la más confiable. Finalmente, entre los distintos métodos para la detección de MCs, entre los métodos más aplicados son los inmunoensayos, debido a su versatilidad y estabilidad del método.

Introduction: Cyanobacteria are photosynthetic microorganisms, with the capacity to synthesize a great diversity of secondary metabolites of interest to the industry, but toxins called cyanotoxins have also attracted attention in recent decades, metabolites that cause different physiological alterations until they cause the death of different species. Methodology: The determination of the state of the art for the subject of cyanobacteria was based on a bibliographic search in specialized databases such as Elservier, Springer, Google Scholar and MDPI based on keywords in Spanish and English "microcystins", "MC degradation " and "quantification and detection of MC". Results: In the present review, two areas of characterization of microcystins are considered: the physicochemical properties and biological properties of microcystins (MC), to understand their behavior and toxic importance in agricultural crops and in human health.In addition to understanding alternatives for their degradation, by physicochemical methods such as photocatalysis and biological grading by bacteria.Finally, some current and developing methods will be mentioned for the detection and quantification of these MCs in aquatic environments. Conclusions: MCs have the potential to contaminate water sources such as rivers and lagoon, causing damage to human health and agricultural plants, they have the ability to tolerate different drastic changes in physicochemical and biological factors. Among the reported alternatives, bacterial degradation promises to be the most reliable. Finally, among the different methods for the detection of MCs, among the most applied methods are immunoassays, due to their versatility and stability of the method.

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Glucocorticoids mediate physiological processes to obtain energy, presenting daily variation in basal levels that may be related to behavioural activity pattern. Identification of plasticity in the secretion of these hormones is essential to understand their effects on physiology and behaviour of wild birds and, therefore, their success in their natural or artificial environment. Serial endocrine evaluations are facilitated by implementing non-invasive methodologies that minimize possible effects of manipulation on the animal's physiological variables. However, non-invasive endocrine-behavioural studies in nocturnal birds, such as owls, are immature. The present work aimed to validate an enzyme immunoassay (EIA) to quantify glucocorticoid metabolites (MGC) in Megascops choliba as well as to evaluate differences in their production at the individual, sexual or daily level. We recorded the behaviour of nine owls during three continuous days to establish activity budget under captive conditions and aiming to correlate with daily MGC variation. The EIA proved to be effective in analytical assays and in pharmacological testing with synthetic ACTH, validating this immunoassay for the species. Additionally, individual differences in MGC production were confirmed in relation to the time of day, especially at 1700 and 2100, but not in relation to sex. During night hours, the owls showed greater behavioural activity, positively related to MGC values. Higher MGC concentrations were significantly related to greater expressions of active behaviours, such as maintenance, while lower MGC concentrations were recorded during moments of higher alertness and resting. The results presented show daily MGC variation to be inversed in this nocturnal species. Our findings can aid future theoretical studies of daily rhythm and evaluations of challenging and/or disturbing situations that result in changes in behaviour or hormonal cascades of these changes in ex situ populations of owls.

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Zika virus (ZIKV) is a flavivirus transmitted by infected Aedes genus mosquitoes. An infected person may be asymptomatic or present symptoms such as fever, arthralgia, and in pregnancy it may lead to neurological disorders in the fetus, such as microcephaly. Based on the high dissemination potential of ZIVK and its similar antigen composition to other arboviruses, new approaches for selective virus detection are urgently needed. This work reports the development of an electrochemical immunoassay for detection of anti-ZIKV antibodies, using magnetic beads functionalized with recombinant protein derived from the non-structural protein 1 (ΔNS1-ZIKV) and anti-IgG antibodies labeled with horseradish peroxidase (HRP) enzyme. The magneto-immunoassay uses disposable microfluidic devices for detection of anti-ZIKV in serum samples. A linear response was obtained for a wide concentration range from 0.01 to 9.80 × 105 pg mL-1 (r2 = 0.982), with a limit of detection of 0.48 pg mL-1. The proposed immunoassay proved to be highly efficient for the detection of anti-ZIKV antibodies in serum, offering promising perspectives for the development of fast, simple, and affordable point-of-care diagnosis devices for ZIKV.

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Zika virus has spread around the world with rapid pace in the last five years. Although symptoms are typically mild and unspecific, Zika's major impact occurs during pregnancy, generating a congenital syndrome. Serology plays a key role in its diagnosis. However, its use is limited due to the uncertainty caused by the cross-reaction of antibodies elicited in response to other flavivirus infections when tested in direct immunoassays. Using a panel of previously generated anti-Zika non-structural protein 1 (NS1) nanobodies, a set was selected that only recognizes epitopes present in Zika and is immunogenic to humans. A proper arrangement of these nanobodies was made and conditions were optimized in order to develop a novel serology assay. This new ELISA relies on the inhibition of the binding of a set of selected nanobodies to Zika-immobilized NS1 when previously incubated with Zika convalescent sera. Using the developed blocking of binding assay, it was possible to discriminate between Zika-specific and cross-reactive antibodies in serum samples from infections with Zika and other flaviviruses.

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Aquatic biota is increasingly being exposed to chemical pollutants due to human activities and the relationship between the level of environmental pollution and fish reproduction is a continuously ongoing issue. The vitellogenin (Vtg) protein synthesis can be induced in the liver of juvenile and male fish after stimulation of the estrogen receptor and therefore, Vtg has been used as a biomarker of xenoestrogen exposure in several fish species. The current study reported the first physicochemical characterization of Vtg from Oreochromis niloticus. Adult male fish were exposed to 17α-ethinylestradiol for Vtg induction. Purified vitellogenin from plasma showed low stability at 25 and 4 °C in saline conditions, and good stability in acidic (low pH) or in heated conditions. The 3D modeling provided useful information on the structure of O. niloticus Vtg showing conserved structural features. According to bioinformatics and experimental results, there are important structural differences between the two chemical forms of Vtg (VtgAb and VtgC) in a phylogenetic context. The present results add information about the development of ecotoxicological immunoassays to study the endocrine disruption in O. niloticus improving the Vtg performance as a biomarker of reproduction in fish.

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Ultrasensitive electroanalytical monitoring of interleukin-6 levels in serum samples has emerged as a valuable tool for the early diagnosis of inflammatory diseases. Despite its advantages, there is a lack of strategies for the label-free voltammetric determination of cytokines. Here, a novel chitosan/genipin modified fluorine tin oxide electrode was developed providing an in-situ hydrogel formation (FTO/CSG). This platform was applied for the detection of interleukin-6, a major pro-inflammatory cytokine. Transmission electron microscopy (TEM), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) indicated genipin serves as an efficient green cross-linker to build the immunosensing platform (FTO/CSG/anti-IL-6). EIS showed an increase in charge transfer resistance from 326 to 1360 kΩ after the immobilization of anti-IL-6 antibodies. By square wave voltammetry, this method achieved a detection limit of 0.03 pg mL-1 with a wide linear range of 0.05-1000 pg mL-1. Additionally, it displayed a high selectivity index when tested in the presence of three inflammatory cytokines as interfering proteins: IL-12, IL-1ß, and TNF-α. The sample matrix effect showed a peak current variation lower than 5 %. The novel method was applied for the quantification of IL-6 in serum samples of septic mice. No statistical differences were observed between the standard ELISA and the proposed method using a confidence level of 95 %.

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BACKGROUND: In low-risk populations, variability in the sensitivity of current serological tests for Hepatitis C virus (HCV) blood donor screening may lead to the presence of false-positive results. This contributes to the unnecessary loss of blood donor samples as well as to difficulty in accurate donor counselling. The present study determined the optimal cut-off value of a chemiluminescent immunoassay for identification of HCV-reactive blood donors. STUDY DESIGN AND METHODS: In a retrospective cross-sectional analysis of 193 973 blood donations, 578 samples that were positive for HCV antibody in a chemiluminescent immunoassay and/or RNA screening tests were identified. Blood from 379 of these positive samples was available for retesting by a second confirmatory HCV immunoassay followed by a receiver operating characteristic (ROC) curve analysis. Donors were also recalled for a new analysis. RESULTS: Only 71 (18.7%) blood samples remained HCV-positive upon retesting, while 233 (61.5%) now tested negative and 75 (19.8%) yielding indeterminate results. A signal to cutoff ratio ≥4.32 was determined as the best differential threshold between a positive and negative result, increasing the positive predictive value from 27.3% to 66.7%. CONCLUSION: Using a higher threshold for an HCV-positive blood sample enhances the chemiluminescent immunoassay screening test´s accuracy and helps to improve donor counselling and notification processes.

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Zika virus (ZIKV) is a flavivirus transmitted by infected Aedes genus mosquitoes. An infected person may be asymptomatic or present symptoms such as fever, arthralgia, and in pregnancy it may lead to neurological disorders in the fetus, such as microcephaly. Based on the high dissemination potential of ZIVK and its similar antigen composition to other arboviruses, new approaches for selective virus detection are urgently needed. This work reports the development of an electrochemical immunoassay for detection of anti-ZIKV antibodies, using magnetic beads functionalized with recombinant protein derived from the non-structural protein 1 (ΔNS1-ZIKV) and anti-IgG antibodies labeled with horseradish peroxidase (HRP) enzyme. The magneto-immunoassay uses disposable microfluidic devices for detection of anti-ZIKV in serum samples. A linear response was obtained for a wide concentration range from 0.01 to 9.80 × 105 pg mL−1 (r2 = 0.982), with a limit of detection of 0.48 pg mL−1. The proposed immunoassay proved to be highly efficient for the detection of anti-ZIKV antibodies in serum, offering promising perspectives for the development of fast, simple, and affordable point-of-care diagnosis devices for ZIKV.

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A anemia infecciosa equina é uma importante enfermidade que acomete os equídeos em todo o mundo, se apresentando de forma aguda, crônica e assintomática causando grandes prejuízos para a economia tanto para criadores que vivem do trabalho desses animais quantos aos criadores que investem no melhoramento das raças, impedindo o acesso ao mercado tanto nacional quanto internacional. O Ministério da Agricultura, Pecuária e Abastecimento considera o IDGA como teste oficial para diagnóstico dessa enfermidade, porém essa técnica é demorada e muita vez acaba sendo subjetiva, dependendo da experiencia particular de cada Laboratorista. Além de não conseguir detectar animais no início da infecção. Logo, a necessidade de se buscar novas técnicas como o ELISA indireto que aperfeiçoem o tempo de análise dos resultados, facilita a automação e obtém resultados confiáveis. O estudo realizado teve como objetivo padronizar uma técnica de ELISA indireto utilizando uma proteína de envelope viral GP90 como antígeno para diagnóstico da anemia infecciosa equina. Avaliando o desempenho do teste a partir da sensibilidade, especificidade e valores preditivos positivo e negativo. Os valores obtidos foram: 91,11%, 93,33%, 91,11% e 93,33% respectivamente. Concluiu-se que o teste apresenta bom desempenho, além da possibilidade de detectar amimais positivos no início da infecção.

Equine infectious anemia is an important disease that affects horses all over the world, presenting in an acute, chronic and asymptomatic way, causing great damage to the economy, both for breeders who live off the work of these animals and for breeders who invest in the improvement of breeds, preventing access to both national and international markets. The Ministry of Agriculture, Livestock and Food Supply considers AGID to be the official test for diagnosing this disease, but this technique takes time and often ends up being subjective, depending on the particular experience of each laboratory worker. In addition to not being able to detect animals at the beginning of the infection. Therefore, the need to seek new techniques such as indirect ELISA that improve the time of analysis of results, facilitate automation and obtain reliable results. The aim of this study was to standardize an indirect ELISA technique using a GP90 viral envelope protein as an antigen for the diagnosis of equine infectious anemia. Evaluating test performance based on sensitivity, specificity and positive and negative predictive values. The values obtained were 91.11%, 93.33%, 91.11 and 93.33 respectively. It was concluded that the test performs well, in addition to the possibility of detecting positive animals at the beginning of the infection.

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RESUMO

The World Health Organizations declaration of the COVID-19 pandemic was a milestone for the scientific community. The high transmission rate and the huge number of deaths, along with the lack of knowledge about the virus and the evolution of the disease, stimulated a relentless search for diagnostic tests, treatments, and vaccines. The main challenges were the differential diagnosis of COVID-19 and the development of specific, rapid, and sensitive tests that could reach all people. RT-PCR remains the gold standard for diagnosing COVID-19. However, new methods, such as other molecular techniques and immunoassays emerged. Also, the need for accessible tests with quick results boosted the development of point of care tests (POCT) that are fast, and automated, with high precision and accuracy. This assay reduces the dependence on laboratory conditions and mass testing of the population, dispersing the pressure regarding screening and detection. This review summarizes the advances in the diagnostic field since the pandemic started, emphasizing various laboratory techniques for detecting COVID-19. We reviewed the main existing diagnostic methods, as well as POCT under development, starting with RT-PCR detection, but also exploring other nucleic acid techniques, such as digital PCR, loop-mediated isothermal amplification-based assay (RT-LAMP), clustered regularly interspaced short palindromic repeats (CRISPR), and next-generation sequencing (NGS), and immunoassay tests, and nanoparticle-based biosensors, developed as portable instruments for the rapid standard diagnosis of COVID-19.

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