Variability in indigenous Pakistani tomato lines and worldwide reference collection for Tomato Mosaic Virus (ToMV) and Tomato Yellow Leaf Curl Virus (TYLCV) infection / Variabilidade em linhagens indígenas de tomate do Paquistão e coleção de referência global para infecção por vírus do mosaico do tomateiro (ToMV) e vírus da folha amarela do tomate (TYLCV)

Local and exotic germplasm of tomato remains a major source for genetic improvement. Assessment of such lines for biotic stresses particularly viral diseases are the most important criteria for selection in Pakistan, where Tomato Yellow Leaf Curl Virus (TYLCV) and Tomato Mosaic Virus (ToMV) are the major diseases/viruses. A set of 40 accessions (including indigenous Pakistani lines and exotic germplasm from Europe, the United States, and Asia) were evaluated for their resistance/infection response to ToMV with artificial inoculation under greenhouse conditions. Infection response was quantified through disease scoring and DAS-ELISA test (for ToMV). A subset of 24 lines, was further screened for TYLCV using disease scoring and TAS-ELISA. The tested lines showed significant variability for resistance to ToMV. Only one accession (Acc-17878) was resistant to the ToMV whereas seven accessions i.e. Acc-17890, AVR-261, CLN-312, AVR-321, EUR-333, CLN-352, and CLN-362 expressed resistance to TYLCV. Correlation between phenotypic evaluation was confirmed by the ELISA results in both diseases, although both tools complemented to assess the viral infection status. In future, tomato breeding programs must consider breeding for ToMV and TYLCV resistance (using identified germplasm in our study) so as to deliver virus resistant tomato varieties.

O germoplasma local e exótico do tomate continua sendo uma importante fonte de melhoramento genético. A avaliação de linhagens para estresses bióticos, particularmente as doenças virais, é o critério mais importantes para seleção no Paquistão, onde o vírus da folha amarela do tomate (TYLCV) e o vírus do mosaico do tomateiro (ToMV) são as principais doenças/vírus. Um conjunto de 40 acessos (incluindo linhagens indígenas do Paquistão e germoplasma exótico da Europa, dos Estados Unidos e da Ásia) foi avaliado quanto à resistência/resposta à infecção ao ToMV com inoculação artificial em casa de vegetação. A resposta à infecção foi quantificada por meio de pontuação da doença e de teste DAS-ELISA (para ToMV). Um subconjunto de 24 linhas foi posteriormente rastreado para TYLCV usando pontuação de doença e TAS-ELISA. As linhas testadas apresentaram variabilidade significativa para resistência ao ToMV. Apenas um acesso (Acc-17878) foi resistente ao ToMV, enquanto sete acessos (Acc-17890, AVR-261, CLN-312, AVR-321, EUR-333, CLN-352 e CLN-362) expressaram resistência ao TYLCV. A correlação entre a avaliação fenotípica foi confirmada pelos resultados do ELISA nas duas doenças, embora ambas as ferramentas tenham se complementado para avaliar o estado da infecção viral. No futuro, os programas de melhoramento de tomate devem considerar aperfeiçoamentos para resistência ao ToMV e TYLCV (usando germoplasma identificado em nosso estudo) de modo a fornecer variedades de tomate resistentes a vírus.

Using guinea pig as a model for evaluation of equine influenza vaccine

Equine influenza is a highly contagious viral disease, specially among 1-5 years old naive horses. Vaccination is considered the best way to control the disease spread and outbreaks. Although foals are the main animal used for evaluation of equine influenza vaccines, guinea pigs were chosen as an alternative model in the present work, as they have a negligible antibody titer against equine influenza virus and are cheaper and easier to handle than foals. Five equine influenza vaccine batches were evaluated in two animal models, foals and guinea pigs, by injection of two doses/animal with 4 weeks apart using 2 mL/animal/dose and evaluation of immune responses by hemagglutination inhibition test and enzyme-linked immunosorbent assay. On the 7th week post vaccination, equine influenza antibodies titers reached maximum values of 9-10.2 and 8.7-10 hemagglutination inhibition units for foals and guinea pigs, respectively; sample/negative ratios were 0.126-0.464 and 0.128-0.445 for both animals, respectively. The use of guinea pigs as an animal model for the evaluation of equine influenza vaccines could be recommended instead of foals.

La gripe equina es una enfermedad viral muy contagiosa, especialmente entre los caballos jóvenes de 1 a 5 años de edad. La vacunación se considera la mejor forma de controlar la propagación y los brotes de la enfermedad. Aunque los potros son el principal animal utilizado para la evaluación de vacunas contra la gripe equina, en el presente trabajo se eligieron cobayos como modelo alternativo, ya que tienen un título insignificante de anticuerpos contra el virus de la gripe equina y son más baratos y fáciles de manejar que los potros. Se evaluaron cinco lotes de vacunas contra la gripe equina en dos modelos animales, potros y cobayos, mediante la inyección de dos dosis/animal con 4 semanas de intervalo utilizando 2 mL/animal/dosis y la evaluación de las respuestas inmunitarias mediante la prueba de inhibición de la hemaglutinación y el ensayo inmunoenzimático. En la 7ª semana posvacunación, los títulos de anticuerpos contra la gripe equina alcanzaron valores máximos de 9-10,2 y 8,7-10 unidades de inhibición de la hemaglutinación para potros y cobayos, respectivamente; las relaciones muestras/negativos fueron de 0,126-0,464 y 0,128-0,445 para ambos animales, respectivamente. Podría recomendarse el uso de cobayos como modelo animal para la evaluación de vacunas contra la gripe equina, en lugar de potros.

Variability in indigenous Pakistani tomato lines and worldwide reference collection for Tomato Mosaic Virus (ToMV) and Tomato Yellow Leaf Curl Virus (TYLCV) infection

Abstract Local and exotic germplasm of tomato remains a major source for genetic improvement. Assessment of such lines for biotic stresses particularly viral diseases are the most important criteria for selection in Pakistan, where Tomato Yellow Leaf Curl Virus (TYLCV) and Tomato Mosaic Virus (ToMV) are the major diseases/viruses. A set of 40 accessions (including indigenous Pakistani lines and exotic germplasm from Europe, the United States, and Asia) were evaluated for their resistance/infection response to ToMV with artificial inoculation under greenhouse conditions. Infection response was quantified through disease scoring and DAS-ELISA test (for ToMV). A subset of 24 lines, was further screened for TYLCV using disease scoring and TAS-ELISA. The tested lines showed significant variability for resistance to ToMV. Only one accession (Acc-17878) was resistant to the ToMV whereas seven accessions i.e. Acc-17890, AVR-261, CLN-312, AVR-321, EUR-333, CLN-352, and CLN-362 expressed resistance to TYLCV. Correlation between phenotypic evaluation was confirmed by the ELISA results in both diseases, although both tools complemented to assess the viral infection status. In future, tomato breeding programs must consider breeding for ToMV and TYLCV resistance (using identified germplasm in our study) so as to deliver virus resistant tomato varieties.

RESUMO O germoplasma local e exótico do tomate continua sendo uma importante fonte de melhoramento genético. A avaliação de linhagens para estresses bióticos, particularmente as doenças virais, é o critério mais importantes para seleção no Paquistão, onde o vírus da folha amarela do tomate (TYLCV) e o vírus do mosaico do tomateiro (ToMV) são as principais doenças/vírus. Um conjunto de 40 acessos (incluindo linhagens indígenas do Paquistão e germoplasma exótico da Europa, dos Estados Unidos e da Ásia) foi avaliado quanto à resistência/resposta à infecção ao ToMV com inoculação artificial em casa de vegetação. A resposta à infecção foi quantificada por meio de pontuação da doença e de teste DAS-ELISA (para ToMV). Um subconjunto de 24 linhas foi posteriormente rastreado para TYLCV usando pontuação de doença e TAS-ELISA. As linhas testadas apresentaram variabilidade significativa para resistência ao ToMV. Apenas um acesso (Acc-17878) foi resistente ao ToMV, enquanto sete acessos (Acc-17890, AVR-261, CLN-312, AVR-321, EUR-333, CLN-352 e CLN-362) expressaram resistência ao TYLCV. A correlação entre a avaliação fenotípica foi confirmada pelos resultados do ELISA nas duas doenças, embora ambas as ferramentas tenham se complementado para avaliar o estado da infecção viral. No futuro, os programas de melhoramento de tomate devem considerar aperfeiçoamentos para resistência ao ToMV e TYLCV (usando germoplasma identificado em nosso estudo) de modo a fornecer variedades de tomate resistentes a vírus.

Homologous Assay Development for Detection of Dexamethasone using HRP as Label Enzyme

Background: Dexamethasone is a synthetic corticosteroid similar to cortisol produced naturally by the adrenal glands. As an anti- inflammatory and immunosuppressive agent, it is used in many diseases such as rheumatoid arthritis and allergic anaphylactic shock, and its suppression test to diagnose Cushing's syndrome. Its further use includes its administration before antibiotics in bacterial meningitis, antitumor treatment, for treatment of glucocorticoid resistance, Addison抯 disease, and congenital adrenal hyperplasia. The drug is abused by using it in animal husbandry as a growth promoter and in horse sports to enhance their performance. Methods: In this study, the development of homologous ELISA using Dexamethasone-21-hemisuccinate (DEX-21-HS)-Bovine serum albumin antiserum and Dexamethasone-21-hemisuccinate (DEX-21-HS)-Horseradish peroxidase enzyme conjugate has been done. The n-hydroxysuccinimide ester method was used to prepare the immunogen and enzyme conjugate. Results: The sensitivity 0.25 ng/mL, affinity 2.8x10-8 L/mol and ED50 4.98 ng/mL of the assay were found. The cross-reactivity of the assay was checked and found with three steroids (Corticosterone- 1.13%, Progesterone- 2.25% and Prednisolone- 6.3%) out of 48 structurally related steroids. Then, analytical variables of the developed assay were studied, such as recovery (98.55% to 105.08%), precision (Inter and Intra- assay coefficient of variation <9.28%), correlation (R2= 0.98) by utilizing a commercially available Dexamethasone kit for comparison. Conclusion: This study concluded that low-cost indigenous ELISA for Dexamethasone had been developed, which can give results within 75-80 minutes.

Vigilancia serológica de la zoonosis parasitaria en 13 regiones de la sierra del Perú: Periodo 2016-2019 / Serological surveillance of parasitic zoonoses in 13 highlands regions of Peru: Period 2016-2019

RESUMEN Objetivos. Determinar la seropositividad a anticuerpos anti-IgG por infección de Echinococcus granulosus, Fasciola hepatica y cisticerco de Taenia solium y describir las características de los infectados en 13 regiones de la sierra peruana entre 2016 y 2019. Materiales y métodos. Estudio observacional transversal, que analizó 7811 fichas epidemiológicas de la vigilancia basada en laboratorio de las zoonosis parasitarias del periodo 2016-2019. El diagnóstico se realizó mediante la detección de anticuerpos tipo IgG anti E. granulosus, F. hepatica y cisticerco de T. solium utilizando antígenos nativos mediante el ensayo inmunoabsorbente ligado a enzimas (ELISA) e Inmunoblot. La diferencia en la frecuencia de casos de estas zoonosis según características identificadas se realizó mediante la prueba chi-cuadrado de Pearson y prueba exacta de Fisher. Resultados. Se determinó una seropositividad de 7,9% para fascioliasis, 4,9% para equinococosis quística, y 2,3% para cisticerco de T. solium. Estas frecuencias fueron mayores en Cerro de Pasco para equinococosis quística (24,5%), en Ayacucho para cisticerco de T. solium (4,5%) y en Puno para fascioliasis (40,6%). Entre las características sociodemográficas, se encontró una diferencia estadísticamente significativa en la frecuencia de casos para todas las zoonosis según grupo etario, ocupación, y región de residencia. Además, se encontró diferencia con el consumo de verduras en emolientes, y entre las características clínico-epidemiológicas con tener antecedentes familiares de las zoonosis parasitarias. Conclusiones. A partir de las 7811 muestras evaluadas, se encontró que estas zoonosis parasitarias están distribuidas en 13 regiones de la sierra del Perú, ocasionando un problema de salud importante, con frecuencias que varían según diversas características.

ABSTRACT Objectives. To determine seropositivity to anti-IgG antibodies against Echinococcus granulosus, Fasciola hepatica and Taenia solium cysticercus infection and to describe the characteristics of the infected patients in 13 regions of the Peruvian highlands between 2016 and 2019. Materials and methods. Cross-sectional, observational study, in which we analyzed 7811 epidemiological records of laboratory-based surveillance of parasitic zoonoses from 2016 to 2019. Diagnosis was established by detecting IgG type anti-E. granulosus, F. hepatica and T. solium cysticercus antibodies using native antigens by enzyme-linked immunosorbent assay (ELISA) and Immunoblot. We evaluated the difference in the frequency of the cases according to identified characteristics using Pearson's chi-square test and Fisher's exact test. Results. Seropositivity was 7.9% for fascioliasis, 4.9% for cystic echinococcosis, and 2.3% for T. solium cysticercus. These rates were higher in Cerro de Pasco for cystic echinococcosis (24.5%), in Ayacucho for T. solium cysticercus (4.5%) and in Puno for fascioliasis (40.6%). Regarding the sociodemographic characteristics, we found a statistically significant difference in the frequency of cases for all zoonoses according to age group, occupation, and region of residence. We also found a difference with the consumption of vegetables in emollients, and between clinical-epidemiological characteristics and having a family history of parasitic zoonoses. Conclusions. From the 7811 samples, we found that these parasitic zoonoses are distributed in 13 regions of the Peruvian highlands, and represent a major health problem, with frequencies that change according to different characteristics.

Utility of serum anti-phospholipase A2 receptor antibody as a biomarker in membranous nephropathy: An experience from a tertiary care center of Eastern India

Background: Membranous nephropathy (MN) is a pattern of glomerular injury. Exact categorization into primary membranous nephropathy (PMN) or secondary membranous nephropathy (SMN) is essential for treatment. An endogenous podocyte antigen, M-type phospholipase A2 receptor (PLA2R) has been discovered to be involved in the pathogenesis of PMN. Aims and Objectives: In this article, we aimed to analyze renal tissue PLA2R and serum anti-PLA2R antibodies in MN cases and determined the diagnostic utility. Materials and Methods: The study was of prospective type carried out from March 2019 to August 2020. Analysis of cases of MN was performed with PLA2R paraffin immunoflourescence and serum anti-PLA2R antibody ELISA. Results: Overall sensitivity, specificity, PPV, and NPV of serum anti-PLA2R ELISA for PMN was 91.3%, 80%, 75%, and 93.3%, respectively, and of tissue PLA2R staining for PMN was 91.67%, 81.08%, 75.86%, and 93.75%, respectively. There was strong concordance between two methods. In the patients that were followed up, we found baseline serum anti-PLA2R antibody was less in complete remission group than that in non-remission group and the reduction in serum anti-PLA2R antibody was more in complete remission group than that in non-remission group. Conclusion: Routine light and immunofluorescence examination are incapable of giving exact categorical opinion regarding PMN and SMN. Serum anti-PLA2R antibody detection and renal tissue PLA2R analysis are sensitive and specific in detecting PMN. Baseline serum anti-PLA2R antibody and anti-PLA2R antibody quantification trends are related to prognosis of PMN. So they can be incorporated as additional biomarker.

Modulation of mucin secretion using combined polyethylene glycol–propylene glycol topical formulation in a hyperosmotic stress-based explant model

Purpose: Ocular surface discomfort and dry eye disease are caused by a dysfunctional tear film. The efficacy of lubricating eye drops on the human eye is known, but the compositions may show differential effects on rescuing the tear film. Mucins form a critical layer of the tear film, a reduction of which may be causative for ocular surface conditions. Therefore, it is essential to develop relevant human?derived models to test mucin production. Methods: Human corneoscleral rims were obtained from a healthy donor (n = 8) post?corneal keratoplasty and cultured in DMEM/F12 media. Hyperosmolar stress mimicking dry eye disease was induced by exposing the corneoscleral rim tissues to +200 mOsml NaCl?containing media. The corneoscleral rims were treated with polyethylene glycol–propylene glycol (PEG–PG)?based topical formulation. Gene expression analysis was performed for NFAT5, MUC5AC, and MUC16. Secreted mucins were measured by enzyme?linked immunosorbent assay (ELISA) (Elabscience, Houston, TX, USA) for MUC5AC and MUC16. Results: The corneoscleral rims responded to hyperosmolar stress by upregulating NFAT5, a marker for increased osmolarity, as observed in the case of dry eye disease. The expression of MUC5AC and MUC16 was reduced upon an increase in hyperosmotic stress. The corneoscleral rim tissues showed induction of MUC5AC and MUC16 expression upon treatment with PEG–PG topical formulation but did not show significant changes in the presence of hyperosmolar treatments. Conclusion: Our findings showed that PEG–PG?based topical formulation slightly alleviated hyperosmolar stress?induced decrease in MUC5AC and MUC16 gene expression that is encountered in DED

Presença do alérgeno de soja Gly m 1 no ar em Maringá - PR / Levels of airborne soy allergen Gly m 1 in Maringá, PR, Brazil

Introdução: O conhecimento da aerobiologia local é fundamental para o alergista. Os aeroalérgenos são capazes de sensibilizar e levar ao desenvolvimento de doenças respiratórias alérgicas, portanto devem ser monitorados rotineiramente, tendo em vista possíveis mudanças locais conforme alterações climáticas, poluição e atividades agroindustriais. Objetivo: Verificar a presença e concentração do alérgeno principal da poeira da casca da soja (Gly m 1) na atmosfera da cidade de Maringá-PR e possíveis associações aos fatores climáticos. A escolha da soja deve-se a alta prevalência desta cultura no Brasil e nesta região do país. Até o presente momento, há apenas um estudo piloto feito por este mesmo grupo avaliando a presença deste alérgeno no Brasil. Métodos: Foram realizadas coletas de material atmosférico, durante o período de março de 2017 a março de 2018, durante 24 ou 48 horas distribuídas no decorrer do período, totalizando 70 amostras, das quais 10 foram excluídas por problemas técnicos de coleta. As amostras foram avaliadas pelo método ELISA (Enzyme linked immunosorbent assay ) para Gly m 1, sendo que todas as amostras apresentaram níveis detectáveis do alérgeno. Resultados: A mediana de concentração de Gly m 1 foi de 4,89 ng/m3. Os valores encontrados variaram de 0,66 ng/ m3 a 1826,1 ng/m3. Das 60 amostras analisadas, 23% delas apresentaram valores superiores a 90 ng/m3, sendo os meses de junho/2017 e março/2018 com concentrações mais elevadas. Houve correlação positiva das concentrações de Gly m 1 com as temperaturas máxima, média e mínima, umidade relativa, vento e insolação. Conclusão: Os dados evidenciam exposições constantes da população ao alérgeno do Gly m 1, por vezes em níveis elevados possivelmente capazes de gerar sensibilização e sintomas.

Introduction: Knowledge of local aerobiology is essential for allergists. Because airborne allergens can sensitize the population and lead to allergic respiratory diseases, they must be routinely monitored for the effects of climate change, pollution, and agroindustry. Objective: To verify the airborne presence and concentration of the main soy hull dust allergen (Gly m 1) in Maringá, PR, Brazil and possible associations with climatic factors. Soybeans were selected due to the high prevalence of this crop in this region. To date, only 1 pilot study (conducted by our group) has evaluated this allergen's presence in Brazil. Methods: Atmospheric material was collected between March 2017 and March 2018 in 24- or 48-hour intervals, totaling 70 samples, of which 10 were excluded due to technical problems. The samples were tested for Gly m 1 using enzyme-linked immunosorbent assay, and all samples showed detectable levels of the allergen. Results: The median concentration of Gly m 1 was 4.89 ng/m3, with values ranging from 0.66 ng/m3 to 1826.1 ng/m3. Of the 60 samples, 23% showed values > 90 ng/m3, with June 2017 and March 2018 having the highest concentrations. There was a positive correlation between Gly m 1 concentration and maximum, mean, and minimum temperatures, relative humidity, wind, and insolation. Conclusion: The data show that the population is constantly exposed to the Gly m 1 allergen, sometimes at high levels, which may lead to sensitization and symptoms.

LEPTOSPIROSIS: A NEGLECTED DISEASE FROM NORTH INDIA

Introduction: Leptospirosis is a zoonotic disease caused by Leptospira interrogans and has been reported from various countries worldwide. As very few studies were conducted on leptospirosis from north India, this study was conducted to know the status of this disease in this region. This retrospective hospital Material & Methods: based study was conducted in the Department of Microbiology of a tertiary care super specialty teaching institute from north India for a period of two consecutive years. Blood specimens from acute febrile illness cases were tested for presence of IgM antibodies against Leptospira interrogans by rapid card (Leptocheck from TULIP) testing and ELISA (Leptospira IgM ELISA from PanBio). Out of total 216 samples Results: collected and included in this study, 40 were found to be positive for presence of IgM antibodies against Leptospira interrogans. Seropositivity for leptospirosis was observed to be 19%. Maximum number of patients were from economically productive age groups, 31-40 years of age group followed by 21-30 and 41-50 years of age groups. CONCLUSION: Leptospirosis was found to be a major cause of acute febrile illness from north India. It is neglected and under reported from most of the regions of India due to lack of clinician's suspicion. More studies with more samples are required on leptospirosis from this region to reach on final conclusion.

Investigation of Chikungunya virus genotype at tertiary care center in Western Maharashtra, India

Background & objectives: Chikungunya is a reemerging arbovirus infection. Laboratory diagnosis can be done by Classical test involving Rapid Immunochromatography, Enzyme-Linked Immunosorbent assay and Molecular methods. The present study was undertaken to know the genotype of the Chikungunya virus (CHICKV) among patients suspected of CHICKV and investigated by virus culture, partial sequencing, Rapid Immunochromatography, and Enzyme-linked Immunosorbent assay (ELISA). To understand different techniques used in Chikungunya diagnosis viz., virus culture, partial sequencing along with Immunochromatography and ELISA. Methods: This is a prospective, laboratory-based study at a tertiary care center. Lateral flow chromatography and ELISA was carried out on serum samples. All 50 samples were cultured and indirect Immunofluorescence was performed on positive samples at Interactive Research School for Health Affairs (IRSHA), Bharati Vidyapeeth Medical College Pune, Maharashtra, India. Virus isolates were subjected to partial sequencing for identification of genotype after confirmation by PCR. Statistical Package of Social Science (SPSS) version 22.0 software was used to calculate the Receiver operating curve (ROC) for different tests. Results: Out of 50 samples, 20 were positive by Immunochromatography, 23 by ELISA, and 3 by culture, PCR confirmed CHIKV isolates and sequencing identified genotypes as East Central South African type. Interpretation & conclusion: CHIKV culture isolates of East Central South African type lineage were predominantly found in the present study. These are also common genotypes present in Asia including India.

The first serological detection of West Nile virus infection among residents living in northern Turkey

Background & objectives: West Nile virus (WNV) is transmitted by a mosquito-borne virus whose natural reservoir is birds. Humans and horses are considered accidental hosts. Even if the vast majority of WNV infections in humans have asymptomatic or mild disease settings, serious neurological disorders with lethal outcomes can also be observed in around 1% of the cases. We aimed to serologically investigate the presence of WNV in humans living in Black sea of Turkey, and to obtain epidemiological data that will contribute to the implementation of public health policies to control and prevent potentially other life-threatening arboviral infections. Methods: In the current study, a total of 416 human sera were collected from native patients of Samsun and its boroughs attending Samsun Training and Research Hospital; these sera were tested for WNV with pooling method, using anti-IgM and IgG ELISA commercial kits. All pools that were found positive for both IgM and IgG were individually retested for the detection of positive WNV sera. After that, all positive samples were tested using realtime PCR to detect the presence of WNV-RNA particles. Results: Total seropositivity rates of WNV in terms of IgM and IgG were found as 0.96% and 0.72%, respectively. No presence of WNV-RNA could be detected in positive samples. Interpretation & conclusion: According to the data, further studies should be conducted to better understand the epidemiological dynamics of WNV in Turkey. It is recommended that other antigenically related flaviviruses which can give cross-reaction with WNV should also be investigated.

Evaluación de tres pruebas serológicas para la detección de anticuerpos contra el virus linfotrópico T humano tipo 1 (HTLV-1)

Introducción: El Perú es endémico al virus linfotrópico T humano tipo 1 (HTLV-1), por esas razones es importante conocer la fiabilidad de las pruebas diagnósticas que se usan en el país, con la finalidad de continuar o no su uso. El objetivo fue evaluar el rendimiento de tres pruebas serológicas ELISA Murex, ELISA Wantai e IFI INS-Perú para la detección de anticuerpos anti HTLV-1 frente a muestras peruanas. El estudio. Las tres pruebas fueron evaluadas frente a 382 sueros: 215 positivos y 167 negativos a HTLV-1 (Gold Standar: inmunoblot). Hallazgos. IFI no presentó falsos positivos, Wantai tuvo más falsos negativos (siete) y Murex más falsos positivos (ocho). Las tres pruebas mostraron resultados superiores a 95% para los parámetros estimados de exactitud diagnóstica. Conclusiones. IFI INS-Perú y ELISA Murex tuvieron buen rendimiento diagnóstico para la detección de anticuerpos contra HTLV-1 y son buenos candidatos para continuar siendo usados en Perú.

Background: Peru is endemic to the human T-lymphotropic virus type 1 (HTLV-1), for these reasons it is important to know the reliability of the diagnostic tests used in the country, in order to continue their use or not. The objective was to evaluate the performance of three serological tests ELISA Murex, ELISA Wantai and IFI INS-Peru for the detection of anti-HTLV-1 antibodies against Peruvian samples. The study. The three tests were evaluated against 382 sera: 215 positive and 167 negative for HTLV-1 (Gold Standard: immunoblot). Findings. IFI had no false positives, Wantai had more false negatives (seven) and Murex more false positives (eight). The three tests showed results above 95% for the estimated parameters of diagnostic accuracy. Conclusions. IIF INS-Perú and ELISA Murex had good diagnostic performance for the detection of antibodies against HTLV-1 and are good candidates to continue being used in Peru.

Prevalence and association of mycoplasma infection in the development of coronary artery disease / Prevalência e associação de infecção por micoplasma no desenvolvimento de doença arterial coronariana

Abstract Coronary heart disease (CHD) has been associated with significant morbidity and mortality worldwide. Although remain controversial, several studies have demonstrated the association of M. pneumoniae infections with atherosclerosis. We evaluated the possible association of mycoplasma infections in patients diagnosed with atherosclerosis by ELISA and PCR methods. Atherosclerotic tissue samples and blood samples were collected for the detection of mycoplasma antibodies (IgA) by ELISA from the 97 patients with coronary artery disease (CAD). M. pneumoniae specific IgA, IgG and IgM were measured by using the Anti-M. pneumoniae IgA/IgG/IgM ELISA. Detection of M. pneumoniae targeting the P1 adhesion gene was performed by PCR Acute infection of M. pneumoniae was diagnosed in 43.3% (42) of patients by PCR. The M. pneumoniae specific antibodies were detected in 36.1% (35) of patients. Twenty-five (25.8%) cases had IgG antibodies, 15 (15.5%) cases had IgM antibodies, 3 (3.1%) cases had IgA antibodies, 10 (10.3%) cases had both IgM + IgG antibodies and 1 (1%) case of each had IgM + IgA and IgG + IgA antibodies. None of the cases was positive for all three antibodies. A Pearson correlation coefficient analysis revealed an excellent correlation between the PCR and the serological results (r=0.921, p<0.001). A majority (17, 40.5%) of the M. pneumoniae positive patients are within the 41-50 years of age group, followed by 10 (23.8%) patients in the age group of 61-70 years and 2 (4.8%) patients were >70 years of age. Our study reported an unusually higher prevalence of M. pneumoniae by serological tests (36.1%) and PCR (43.3%). Although the hypothesis of the association of M. pneumoniae and CAD is yet to be proven, the unusually high prevalence of M. pneumoniae in CAD patients indicates an association, if not, in the development of atherosclerosis.

Resumo A doença coronariana (DCC) tem sido associada a significativa morbidade e mortalidade em todo o mundo. Embora ainda sejam controversos, vários estudos têm demonstrado a associação de infecções por M. pneumoniae com aterosclerose. Avaliamos a possível associação de infecções por micoplasma em pacientes com diagnóstico de aterosclerose pelos métodos ELISA e PCR. Amostras de tecido aterosclerótico e amostras de sangue foram coletadas para a detecção de anticorpos contra micoplasma (IgA) por ELISA de 97 pacientes com doença arterial coronariana (DAC). IgA, IgG e IgM específicos para M. pneumoniae foram medidos usando o Anti-M. pneumoniae IgA / IgG / IgM ELISA. A detecção de M. pneumoniae visando o gene de adesão P1 foi realizada por PCR. A infecção aguda por M. pneumoniae foi diagnosticada em 43,3% (42) dos pacientes pela PCR. Os anticorpos específicos para M. pneumoniae foram detectados em 36,1% (35) dos pacientes. Vinte e cinco (25,8%) casos tinham anticorpos IgG, 15 (15,5%) casos tinham anticorpos IgM, 3 (3,1%) casos tinham anticorpos IgA, 10 (10,3%) casos tinham anticorpos IgM + IgG e 1 (1%) caso de cada um tinha anticorpos IgM + IgA e IgG + IgA. Nenhum dos casos foi positivo para os três anticorpos. A análise do coeficiente de correlação de Pearson revelou uma excelente correlação entre o PCR e os resultados sorológicos (r = 0,921, p < 0,001). A maioria (17, 40,5%) dos pacientes positivos para M. pneumoniae está na faixa etária de 41-50 anos, seguida por 10 (23,8%) pacientes na faixa etária de 61-70 anos e 2 (4,8%) pacientes tinham > 70 anos de idade. Nosso estudo relatou uma prevalência incomumente maior de M. pneumoniae por testes sorológicos (36,1%) e PCR (43,3%). Embora a hipótese da associação de M. pneumoniae e DAC ainda não tenha sido comprovada, a prevalência incomumente alta de M. pneumoniae em pacientes com DAC indica uma associação, se não, no desenvolvimento de aterosclerose.

Impact of nitrogen and phosphorus fertilizers on Cry1Ac protein contents in transgenic cotton / Impacto de fertilizantes de nitrogênio e fósforo nos conteúdos de proteína Cry1Ac em algodão transgênico

Abstract Application of different fertilizers to check the efficiency of expression of Bt (Bacillus thuringiensis) gene in one of the leading commercialized crops (cotton) against Lepidopteran species is of great concern. The expression of Cry protein level can be controlled by the improvement of nutrients levels. Therefore, the myth of response of Cry toxin to different combinations of NP fertilizers was explored in three Bt cotton cultivars. Combinations include three levels of nitrogen and three levels of phosphorus fertilizers. Immunostrips and Cry gene(s) specific primer based PCR (Polymerase Chain Reaction) analysis were used for the presence of Bt gene that unveiled the presence of Cry1Ac gene only. Further, the ELISA (enzyme-linked immunosorbent assay) kit was used to quantify the expression of Cry1Ac protein. Under various NP fertilizers rates, the level of toxin protein exhibited highly significant differences. The highest toxin level mean was found to be 2.3740 and 2.1732 µg/g under the treatment of N150P75 kg ha-1 combination while the lowest toxin level mean was found to be 0.9158 and 0.7641 µg/g at the N50P25 kg ha-1 level at 80 and 120 DAS (Days After Sowing), respectively. It was concluded from the research that the usage of NP fertilizers has a positive relation with the expression of Cry1Ac toxin in Bt cotton. We recommend using the N150P50 kg ha-1 level as the most economical and practicable fertilizer instead of the standard dose N100P50 kg ha-1 to get the desired level of Cry1Ac level for long lasting plant resistance (<1.5). The revised dose of fertilizer may help farmers to avoid the cross-resistance development in contradiction of insect pests.

Resumo A aplicação de diferentes fertilizantes para verificar a eficiência da expressão do gene Bt (Bacillus thuringiensis) em uma das principais culturas comercializadas (algodão) contra espécies de lepidópteros é uma grande preocupação. A expressão do nível de proteína Cry pode ser controlada pela melhoria dos níveis de nutrientes. Portanto, o mito da resposta da toxina Cry a diferentes combinações de fertilizantes NP foi explorado em três cultivares de algodão Bt. As combinações incluem três níveis de nitrogênio e três níveis de fertilizantes de fósforo. A análise de PCR (reação em cadeia da polimerase) específica para o gene (s) Immunostrips e Cry (s) foi usada para a presença do gene Bt que revelou a presença do gene Cry1Ac apenas. Além disso, o kit ELISA (ensaio de imunoabsorção enzimática) foi usado para quantificar a expressão da proteína Cry1Ac. Sob várias taxas de fertilizantes NP, o nível de proteína de toxina exibiu diferenças altamente significativas. A média do nível mais alto de toxina foi de 2,3740 e 2,1732 µg / g sob o tratamento da combinação N150P75 kg ha-1, enquanto a média do nível mais baixo de toxina foi de 0,9158 e 0,7641 µg / g no nível de N50P25 kg ha-1 em 80 e 120 DAS (dias após a semeadura), respectivamente. Concluiu-se com a pesquisa que o uso de fertilizantes NP tem relação positiva com a expressão da toxina Cry1Ac no algodão Bt. Recomendamos o uso do nível de N150P50 kg ha-1 como o fertilizante mais econômico e praticável em vez da dose padrão N100P50 kg ha-1 para obter o nível desejado de nível de Cry1Ac para resistência de planta de longa duração (<1,5). A dose revisada de fertilizante pode ajudar os agricultores a evitar o desenvolvimento de resistência cruzada em contradição com as pragas de insetos.

Prevalence of active HCV infection and genotypic distribution among the general population of district Mardan, Pakistan / Prevalência de infecção HCV ativa a distribuição genotípica entre a população geral do distrito de Mardan, Paquistão

Abstract Hepatitis C virus (HCV) is the serious global public health burden of liver disease. Approximately 170 million people in the world are infected with (HCV). In Pakistan, where the disease has high occurrence rate. The present study envisages an up-to-date prevalence of HCV and genotypic distribution in the general population of Mardan District, Khyber Pakhtunkhwa (KP), Pakistan. The blood samples from 6,538 individuals including 3,263 males and 3,275 females were analyzed for hepatitis C surface antigen by Immuno-chromatographic test (ICT), Enzyme-linked immunosorbent assay (ELISA), and reverse transcription-polymerase chain reaction (PCR). It was found that 396 (12.13%) out of 3263 individuals contained antibodies in their blood against HCV, while among the different age groups, the highest incidences of HCV antibodies were found in the 31-40 age group (11.01%). The ICT positive samples were further screened by nested PCR to determine the existence of active HCV-RNA. It was identified that 7.11% (3263) of the total population (6538) tested was positive, among which the 461 (14.07%) females possessed antibodies in their blood against HCV. Our data showed total HCV infection in the investigated population was 5.78%. Higher percentage of HCV prevalence was detected in males than females in the age group 31-40 and 41-50. To compare the prevalence of HCV genotypes age-wise in male and female genotype 3a was found most prevalent genotype followed by 1a, 2a and 3b, respectively.

Resumo O vírus da hepatite C (HCV) é o grave problema de saúde pública das doenças hepáticas. Aproximadamente 170 milhões de pessoas no mundo estão infectadas com HCV; no Paquistão, a doença tem alto índice de ocorrência. O presente estudo prevê uma prevalência atualizada do HCV e distribuição genotípica na população geral do distrito de Mardan, Khyber Pakhtunkhwa (KP), Paquistão. As amostras de sangue de 6.538 indivíduos, incluindo 3.263 homens e 3.275 mulheres, foram analisadas para o antígeno de superfície da hepatite C por teste imunocromatográfico (ICT), ensaio imunoenzimático (ELISA) e reação em cadeia da polimerase de transcrição reversa (PCR). Verificou-se que 396 (12,13%) de 3.263 indivíduos continham anticorpos no sangue contra o HCV, enquanto entre as diferentes faixas etárias as maiores incidências de anticorpos anti-HCV foram encontradas na faixa etária de 31 a 40 anos (11,01%). As amostras positivas para ICT foram posteriormente rastreadas por nested PCR para determinar a existência de HCV-RNA ativo. Identificou-se que 7,11% (3.263) do total da população (6.538) testada foram positivos, dentre os quais 461 (14,07%) mulheres possuíam anticorpos no sangue contra o HCV. Nossos dados mostraram que a infecção total pelo HCV na população investigada foi de 5,78%. Maior porcentagem de prevalência de HCV foi detectada em homens do que em mulheres nas faixas etárias de 31-40 e 41-50. Para comparar a prevalência de genótipos de HCV com relação à idade no genótipo masculino e feminino 3a foi encontrado o genótipo mais prevalente seguido por 1a, 2a e 3b, respectivamente.

Molecular detection of Mycoplasma gallisepticum in different poultry breeds of Abbottabad and Rawalpindi, Pakistan / Detecção molecular de Mycoplasma gallisepticum em diferentes raças de aves de Abbottabad e Rawalpindi, Paquistão

Abstract The poultry sector in Pakistan is contributing mainly in bridging gap between demand and supply for protein. Mycoplasma gallisepticum is an emerging bacterium causing serious problems in poultry industry of Pakistan. A cross-sectional study was conducted to evaluate the M. gallisepticum load in poultry populated regions of Pakistan. Total 600 serum and 600 swab samples were collected, 200 from each broiler, layers and breeders poultry in Rawalpindi and Abbottabad districts. Serum samples were analyzed through ELISA for seroprevalence. Swabs were cultured on Frey's medium followed by PCR and partial mgc2 gene sequencing. Results of seroprevalence of M. gallisepticum showed that layers (75%, n=150) are more positive as compared to breeders (70%, n=140) and broilers (50%, n=100). Typical colonies of the M. gallisepticum were observed in breeder (26.5%), followed by layer (21%) and broilers (9%). A total of 37.1% (n=42) samples were identified positive through PCR out of total 113 cultured based positive samples. A total of six M. gallisepticum isolates of current study showed 98-99 percent similarity with previously reported isolates on the basis of mgc2 gene partial sequencing. The M. gallisepticum was found highly prevalent in different poultry breads. Results of this study would add into basic data and provide a direction for livestock sector to strengthen a control strategy for mycoplasmosis in poultry farms.

Resumo O setor avícola do Paquistão está contribuindo principalmente para preencher a lacuna entre a demanda e a oferta de proteína. Mycoplasma gallisepticum é uma bactéria emergente que causa sérios problemas na indústria avícola do Paquistão. Um estudo transversal foi conduzido para avaliar a carga de M. gallisepticum em regiões de avicultura do Paquistão. Um total de 600 amostras de soro e 600 amostras de esfregaço foi coletado, 200 de cada frango de corte, poedeiras e aves reprodutoras nos distritos de Rawalpindi e Abbottabad. Amostras de soro foram analisadas por ELISA para soroprevalência. As zaragatoas foram cultivadas em meio Frey, seguido de PCR e sequenciação parcial do gene mgc2. Os resultados da soroprevalência de M. gallisepticum mostraram que as poedeiras (75%, n = 150) são mais positivas em comparação com matrizes (70%, n = 140) e frangos de corte (50%, n = 100). Colônias típicas de M. gallisepticum foram observadas em reprodutoras (26,5%), seguidas de poedeiras (21%) e frangos de corte (9%). Um total de 37,1% (n = 42) das amostras foi identificado como positivas por PCR de um total de 113 amostras positivas baseadas em cultura. Um total de seis isolados de M. gallisepticum do estudo atual mostrou 98-99% de similaridade com isolados relatados anteriormente com base no sequenciamento parcial do gene mgc2. O M. gallisepticum foi encontrado com alta prevalência em diferentes pães de aves. Os resultados deste estudo acrescentariam dados básicos e forneceriam orientação para o setor pecuário fortalecer uma estratégia de controle da micoplasmose em granjas avícolas.

Preparation and evaluation of formalized bivalent Newcastle and Salmonella poultry vaccine

This study was conducted to prepare and evaluate the potency of different inactivated vaccine formulations that protect chickens against Salmonella Enteritidis and Newcastle disease virus using Montanide as adjuvant. Protection and the humoral immune response of prepared vaccines against Salmonella Enteritidis and Newcastle disease virus was evaluated and compared to imported vaccine. In this study, different formulae of Salmonella Enteritidis and Newcastle disease vaccines were prepared and compared with the imported one by measuring the antibody titer against Newcastle disease virus by hemagglutination inhibition test and the antibody titer against Salmonella Enteritidis using Enzyme Linked Immunosorbent Assay. On the other hand, the protection percentages against Newcastle disease and Salmonella Enteritidis were recorded to determine the best effective formula. The highest hemagglutination inhibition antibody level against NDV at first week was recorded for the prepared combined Newcastle disease and Salmonella Enteritidis vaccine (4.2 log2) followed by the prepared monovalent Newcastle disease (3.4 log2); the lowest antibody level (3.1 log2) was obtained with the imported vaccine. A gradual increase was observed in all groups to 7.1 log2, 6.8 log2 and 6.4 log2 at fourth week post vaccination, respectively. The antibody titer against Salmonella Enteritidis was 552 for the prepared combined Salmonella Enteritidis and Newcastle disease, followed by the prepared monovalent Salmonella Enteritidis (477) at first week post vaccination; the antibody titer obtained for the imported vaccine was 477. There was a gradual increase to 1456, 1406 and 1130 at fourth week post vaccination, respectively. Prepared combined vaccines gave the highest protection percentage, followed by prepared monovalent types and finally imported vaccines. Vaccination by the prepared combined Salmonella Enteritidis and Newcastle disease vaccine may be a way to increase the resistance of birds to Salmonella and Newcastle and to decrease the shedding rate(AU)

Este estudio se llevó a cabo para preparar y evaluar la potencia de diferentes formulaciones de vacunas inactivadas que protegen a los pollos contra Salmonella Enteritidis y el virus de la enfermedad de Newcastle utilizando Montanide como adyuvante. Se evaluó la protección y la respuesta inmune humoral de las vacunas preparadas contra Salmonella Enteritidis y el virus de la enfermedad de Newcastle y se comparó con la vacuna importada. En este estudio se prepararon diferentes fórmulas de vacunas contra Salmonella Enteritidis y la enfermedad de Newcastle y se compararon con la importada midiendo el título de anticuerpos contra el virus de la enfermedad de Newcastle mediante la prueba de inhibición de la hemaglutinación y el título de anticuerpos contra Salmonella Enteritidis mediante ELISA. Por otra parte, se registraron los porcentajes de protección contra la enfermedad de Newcastle y Salmonella Enteritidis para determinar la fórmula más eficaz. El mayor nivel de anticuerpos inhibidores de la hemaglutinación contra el virus de la enfermedad de Newcastle, en la primera semana, se registró con la vacuna combinada preparada contra la enfermedad de Newcastle y Salmonella Enteritidis (4,2 log2), seguida de la vacuna monovalente preparada contra la enfermedad de Newcastle (3,4 log2); el menor nivel de anticuerpos (3,1 log2) se obtuvo con la vacuna importada. Se observó un aumento gradual en todos los grupos hasta alcanzar 7,1 log2, 6,8 log2 y 6,4 log2 en la cuarta semana tras la vacunación, respectivamente. El título de anticuerpos contra Salmonella Enteritidis fue de 552 para la vacuna combinada preparada contra la Salmonella Enteritidis y enfermedad de Newcastle, seguida por la vacuna monovalente preparada contra Salmonella Enteritidis (477) en la primera semana después de la vacunación; el título de anticuerpos obtenido con la vacuna importada fue de 477. Hubo un aumento gradual hasta 1456, 1406 y 1130 en la cuarta semana después de la vacunación, respectivamente. Las vacunas combinadas preparadas dieron el mayor porcentaje de protección, seguidas por los tipos monovalentes preparados y, por último, por las vacunas importadas. La vacunación con la vacuna combinada preparada contra la Salmonella Enteritidis y la enfermedad de Newcastle puede ser una forma de aumentar la resistencia de las aves a la Salmonella y Newcastle y de disminuir la tasa de excreción(AU)

Niveles de protección contra Haemophilus influenzae tipo b en niños, adolescentes y adultos cubanos / Levels of protection against Haemophilus influenzae type b in Cuban children, adolescents and adults

La concentración de los anticuerpos contra el polisacárido capsular polirribosilribitol fosfato del Haemophilus influenzae tipo b se considera un buen indicador serológico para evaluar protección contra la enfermedad invasiva. Existen pocos reportes que estudien la inmunidad serológica en Cuba. El objetivo general de este estudio fue determinar los niveles de protección séricos contra Haemophilus influenzae tipo b en niños, adolescentes y adultos cubanos, en una muestra de 575 individuos. Se cuantificó la concentración de IgG anti-polirribosilribitol fosfato de Haemophilus influenzae tipo b mediante un inmunoensayo enzimático estandarizado y validado en el laboratorio de inmunología del Centro Nacional de Genética Médica, La Habana, Cuba. Se determinaron las concentraciones medias geométricas de anticuerpos y los niveles de protección frente a la enfermedad invasiva por Haemophilus influenzae tipo b. La concentración media geométrica de IgG anti-polirribosilribitol fosfato fue de 1,94 μg/mL (IC95 por ciento 1,80; 2,08) y fue mayor en el grupo de 16 a 22 años. El porcentaje con protección de larga duración fue mayor para el sexo femenino que para el masculino (82,2 por ciento vs 71,4 por ciento; p=0,0339) entre los que poseían inmunidad natural. El grupo de sujetos nacidos en el periodo en que se vacunó con la vacuna conjugada cubana QUIMI-HIB® presentó concentraciones medias geométricas superiores (2,75 μg/mL, IC95 por ciento 2,00; 3,79). El 99,1 por ciento de los participantes presentó protección frente a la enfermedad invasiva por Haemophilus influenzae tipo b, el 19,8 por ciento a corto plazo y el 79,3 por ciento protección de larga duración. El inmunoensayo validado para la cuantificación de IgG anti-polirribosilribitol fosfato podría emplearse en estudios de seroprevalencia. En los sujetos estudiados, se encontró un predominio de elevadas concentraciones de IgG anti- polirribosilribitol fosfato del Haemophilus influenzae tipo b que confieren protección de larga duración(AU)

The levels of antibodies directed against the capsular polysaccharide polyribosylribitol phosphate of Haemophilus influenzae type b are considered a good serological indicator to assess the immunity against invasive disease. In Cuba, there are few reports that study serological immunity. The general objective was to determine serum protection levels against Haemophilus influenzae type b in Cuban children, adolescents and adults, in a sample of 575 Cuban individuals. The concentration of IgG against Haemophilus influenzae type b was quantified by means of an indirect ELISA standardized and validated in the immunology laboratory of the National Center of Medical Genetics, Havana, Cuba. The geometric mean concentration of IgG anti- polyribosylribitol phosphate and the levels of protection against invasive Haemophilus influenzae type b disease were determined. The geometric mean concentration of IgG anti- polyribosylribitol phosphate was 1.94 μg/mL (95percentCI 1.80;2.08) and the group from 16 to 22 years old presented the highest. Among those with natural immunity, the percentage with long-term protection was higher for females vs. males (82.2percent vs. 71.4percent; p=0.0339). The group of subjects born in the period in which they were vaccinated with the Cuban conjugate vaccine QUIMI-HIB® presented higher geometric mean concentration (2.75 μg/mL, CI95percent 2.00; 3.79). The 99.1percent of the participants had protection against invasive Haemophilus influenzae type b disease, 19.8percent short-term and 79.3percent long-term protection. The ELISA for the quantification of anti- Haemophilus influenzae type b IgG antibodies, developed and validated, could be used in seroprevalence studies. In the subjects studied, there was a predominance of high IgG anti- Haemophilus influenzae type b polyribosylribitol phosphate concentration values that confer long-term protection(AU)

Estandarización de un ELISA para la cuantificación de anticuerpos IgG humanos contra células enteras de Bordetella pertussis / Standardization of an ELISA for the quantification of human IgG antibodies against whole cells of Bordetella pertussis

Bordetella pertussis es un patógeno exclusivo de humanos que causa la tos ferina, enfermedad respiratoria aguda que afecta principalmente a la población pediátrica. Existen dos tipos de vacunas comercializadas contra este patógeno: celulares y acelulares. Las vacunas celulares han sido extensamente utilizadas y siguen teniendo gran relevancia. El presente trabajo tuvo como objetivo la estandarización de un ELISA para la cuantificación de anticuerpos IgG contra células enteras de Bordetella pertussis. Para ello se determinó la concentración de recubrimiento, el rango lineal de la curva, los parámetros de precisión intra e interensayo, la especificidad, el valor de corte y el límite de detección. Se determinó como concentración de recubrimiento 0,5 UO/mL de células enteras. La curva estándar utilizando un suero de referencia internacional presentó un buen ajuste a una función polinómica en un intervalo entre las diluciones 1/100 y 1/24.300 con un coeficiente de correlación R2≥0,98. Los coeficientes de variación en los ensayos de precisión intra e interensayo estuvieron en los intervalos establecidos para cada uno (≤10 por ciento, ≤20 por ciento respectivamente). Los resultados obtenidos avalan el empleo de este ELISA cuantitativo para la evaluación de la respuesta a células enteras de Bordetella pertussis en ensayos clínicos(AU)

Bordetella pertussis is a pathogen exclusive to humans that causes pertussis, an acute respiratory disease that mainly affects the pediatric population. There are two types of vaccines commercially available against this pathogen: cellular and acellular. Cellular vaccines have been widely used and continue to be of great relevance. The aim of the present work was to standardize an ELISA for the quantification of IgG antibodies against whole cells of Bordetella pertussis. For this purpose, the coating concentration, the linear range of the curve, the intra- and inter-assay precision parameters, the specificity, the cut-off value and the detection limit were determined. The coating concentration was determined as 0.5 UO/mL of whole cells. The standard curve using an international reference serum presented a good fit to a polynomial function in a range between dilutions 1/100 and 1/24,300 with a correlation coefficient R2≥0.98. The coefficients of variation in the intra- and inter-assay precision tests were in the intervals established for each (≤10percent, ≤20percent respectively). The results obtained support the use of this quantitative ELISA for the evaluation of whole-cell response to Bordetella pertussis in clinical trials(AU)

Desenvolvimento e avaliação de uma proteína quimérica recombinante para o diagnóstico e prognóstico das leishmanioses tegumentar e visceral / Development and evaluation of a recombinant chimeric protein for the diagnosis and prognosis of tegumentary and visceral leishmaniasis

As leishmanioses são doenças tropicais negligenciadas com alta endemicidade e que afetam milhares de pessoas no mundo. Sua infecção é causada por parasitos protozoários do gênero Leishmania. A diversidade biológica entre as espécies é quem permite determinar as manifestações clínicas, sendo elas na forma de leishmaniose visceral (LV) ou leishmaniose tegumentar (LT). Dentre estas manifestações, a LV é considerada a mais grave, devido sua alta letalidade e grande emergência em indivíduos com a infecção provocada pelo vírus da imunodeficiência humana (HIV). Atualmente, as medidas de controle e prevenção adotadas pela Organização Mundial da Saúde (OMS), baseiam-se em uma combinação de estratégias de intervenção contra a infecção, uma vez que o diagnóstico eficaz e precoce é indispensável para que se possa intervir com o tratamento adequado, diminuindo índices de mortalidade e a evolução de complicações clínicas. Entretanto, os testes sorológicos utilizados apresentam sensibilidade e especificidade prejudicadas em pacientes com leishmanioses e/ou coinfectados LV/HIV, devido a baixos ní-veis de anticorpos antileishmanial ou pela presença de doenças que causem reação cruzada, levando a resultados falso-positivos. A sensibilidade torna-se também variável em pacientes tratados, uma vez que a sorologia pode manter-se positiva por meses ou anos após o fim do tratamento e cura da doença. Buscando resolver tal problemática, a identificação de novos antígenos, por meio de análises de bioinformática associadas à imunoproteômica, tem permitido a detecção de novas proteínas com potencial aplicação diagnóstica. Em estudos anteriores, as proteínas hipotéticas LiHyT, LiHyD, LiHyV e LiHyP foram encontradas em espécies de Leishmania spp, e avaliadas em suas versões recombinantes por meio de ensaios de ELISA, obtendo resultados satisfatórios para a detecção da LV humana e canina. Com base nessas informações, o presente trabalho teve como objetivo desenvolver uma proteína quimera recombinante base-ada na predição de epítopos lineares específicos de células B das quatro proteínas antigênicas de L. infantum citadas e avaliar o potencial diagnóstico, assim como dos peptídeos individuais que a constituíram, frente à leishmaniose humana, bem como com a coinfecção com HIV, além de testar os antígenos como marcadores prognóstico após o tratamento da LV e LT. As sequências de aminoácidos das proteínas foram avaliadas e oito epítopos de células B foram preditos e utilizados na construção de uma nova proteína quimérica. A proteína foi expressa, purificada e avaliada como antígeno recombinante em ELISA para o diagnóstico de LV, LT, coinfecção LV/HIV e prognóstico em amostras de pacientes tratados de LV e LT. Os epítopos de células B usados na construção da quimera foram sintetizados e também testados em ELISA frente às mesmas amostras, assim como um extrato antigênico solúvel de Leishmania braziliensis (SLA). Os resultados mostraram que a proteína quimera apresentou sensibilidade e especificidade de 100% para diagnosticar a LV, LT e LV/HIV, enquanto os peptídeos sintéticos apresentaram sensibilidade variando entre eles de 9,1% a 90,9% para amostras de LT e 76,8% a 99,2% para amostras de LV e LV/HIV, já os valores de especificidade atingiram 98,3% a 99,1% para LT e 67,1% a 95,7% para LV e LV/HIV. O SLA apresentou sensibilidade e especificidade de 18,2% e 98,3% para LT, e 56,8% a 69,5% para amostras de LV e LV/HIV, respectivamente. Uma avaliação prognóstica preliminar mostrou ainda que os anticorpos anti-quimera diminuíram em níveis significativos, quando comparada a reatividade sorológica antes e seis meses após o tratamento, sugerindo um possível papel prognóstico da quimera para as leishmanioses. O presente estudo, mostrou-se eficaz na construção e avaliação de novos candidatos, que demonstram ter um bom desempenho na detecção diagnóstica e prognóstica para as leishmanioses e dos casos de coinfecção LV/HIV.

Leishmaniasis are neglected tropical diseases with high endemicity that affect thousands of people in the world. Infection is caused by protozoan parasites of the genus Leishmania. The biological diversity between species is what allows determining the clinical manifestations, either in the form of visceral leishmaniasis (VL) or tegumentary leishmaniasis (TL). Among these clinical manifestations, VL is considered the most serious, due to its high lethality and great emergence in individuals with infection caused by the human immunodeficiency virus (HIV). Currently, the control and prevention measures adopted by the World Health Organiza-tion (WHO) are based on a combination of intervention strategies against the infection, since an effective and early diagnosis is essential to intervene with the appropriate treatment, decrea-sing mortality rates and evolution of clinical complications. However, the serological tests used show impaired sensitivity and specificity in patients with leishmaniasis and/or coinfected with VL/HIV, due to low levels of anti-leishmanial antibodies or the presence of diseases that cause cross-reaction, leading to false-positive results. The sensitivity also becomes variable in treated patients, since the serology can remain positive for months or years after the end of the trea-tment and cure of the disease. Seeking to solve this problem, the identification of new antigens, through bioinformatic analysis associated with immunoproteomic, has allowed the detection of new proteins with potential diagnostic application. In previous studies, the hypothetical proteins LiHyT, LiHyD, LiHyV and LiHyP were found in species of Leishmania spp, and evaluated in their recombinant versions through ELISA assays, and satisfactory results were obtained for the detection of human and canine VL. Based on this information, the present work aimed to develop a recombinant chimera protein through on the prediction of specific linear epitopes of B cells derived from these four antigenic proteins of L. infantum and to evaluate its diagnostic potential, as well as the individual peptides that constitute it, against human leishmaniasis, as well as co-infection with HIV, in addition to testing them as possible prognostic markers of patients after VL and TL treatment. The amino acid sequences of the proteins were evaluated and eight B cell epitopes were predicted and used in the construction of a new chimeric protein. The protein was expressed, purified and evaluated as a recombinant antigen in ELISA for the diagnosis of VL, TL, VL/HIV co-infection and prognosis in samples from patients treated for VL and TL. The B cell epitopes used in the construction of the chimera were synthesized and also tested in ELISA against the same samples, as well as a soluble Leishmania braziliensis antigenic extract (SLA). The results showed that the chimera protein apresented sensitivity and specificity of 100% for diagnosing VL, TL and VL/HIV, while the synthetic peptides showed sensitivity ranging from 9.1% to 90.9% for TL samples and 76.8 % to 99.2% for VL and VL/HIV samples, while the specificity values reached from 98.3% to 99.1% for TL and 67.1% to 95.7% for VL and VL/HIV. The SLA showed sensitivity and specificity of 18.2% and 98.3% for TL, and 56.8% to 69.5% for VL and VL/HIV samples, respectively. A preliminary prog-nostic evaluation also showed that anti-chimera antibodies significantly decreased when com-pared to serological reactivity before and six months after treatment, suggesting a possible prognostic role of the antigen for leishmaniasis. The present study proved to be effective in the construction and evaluation of new candidates, who demonstrate good performance in diagnos-tic and prognostic detection for leishmaniasis and VL/HIV co-infection.