RESUMO
The direct rapid immunohistochemical test (dRIT) has been recommended for laboratorial diagnosis of rabies, especially in developing countries. The absence of commercial primary antibodies, however, still represents a major limitation to its wider use in testing. We describe here the development of a biotinylated polyclonal antibody against Rabies lyssavirus (RABV) ribonucleoprotein (RNP) and its use as a primary reagent in dRIT. Anti-RNP polyclonal horse IgG was purified by ionic exchange chromatography followed by immunoaffinity column chromatography, and its affinity, diagnostic sensitivity, and specificity were evaluated. CNS samples (120) of suspected rabies cases in different animal species were tested by dRIT, with the positive (n = 14) and negative (n = 106) results confirmed by direct fluorescence antibody testing (dFAT). Comparing the results of dRIT and dFAT, we found that the biotinylated anti-RNP IgG delivered 100% diagnostic specificity and sensibility for rabies diagnosis. Our findings show that the biotinylated anti-RNP polyclonal IgG can be produced with the quality required for application in dRIT. This work represents an important step in efforts to diagnose rabies in developing countries.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos Virais/imunologia , Imunoglobulina G/imunologia , Vírus da Raiva/imunologia , Raiva/imunologia , Ribonucleoproteínas/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/metabolismo , Biotinilação , Encéfalo/imunologia , Encéfalo/virologia , Gatos , Bovinos , Quirópteros , Cães , Técnica Direta de Fluorescência para Anticorpo/métodos , Cavalos , Imunoglobulina G/metabolismo , Imuno-Histoquímica/métodos , Primatas , Raiva/diagnóstico , Raiva/virologia , Sensibilidade e Especificidade , Especificidade da Espécie , SuínosRESUMO
The direct-fluorescent antibody test (dFAT) is considered the "gold standard" assay to diagnose rabies. However, it is crucial to develop molecular techniques, such as RT-PCR and RT-qPCR, since many laboratories lack the needed supplies for performing complementary methods (viral isolation, for example). For this purpose, diagnostic techniques must be specific and sensitive to guarantee accuracy. This present investigation aimed to detect rabies virus (RABV) in 126 clinically suspected cattle in Brazil using different diagnostic tests [dFAT, mouse inoculation test (MIT), immunohistochemistry (IHC), RT-PCR and RT-qPCR] and to compare those results obtained under routine laboratory conditions. The results of the present investigation demonstrate that the molecular techniques are more sensitive and may detect low viral load, even though the non-homogeneous viral distribution caused a false-negative result in dFAT. We also observed a usual alteration in antigens distribution among regions of the central nervous system (CNS). By both dFAT and IHC assays, the most reliable CNS structures were thalamus and midbrain. Although this investigation demonstrated diagnostic sensitivity and specificity close to 100 % in all laboratory techniques employed, a dFAT auxiliary test is required for bovine specimens, such as molecular techniques, when there are poor sampling conditions (low viral load combined with unavailability of brainstem structures).
Assuntos
Doenças dos Bovinos/diagnóstico , Técnicas de Laboratório Clínico/métodos , Testes Imunológicos/métodos , Raiva/diagnóstico , Raiva/veterinária , Animais , Brasil , Bovinos , Doenças dos Bovinos/virologia , Modelos Animais de Doenças , Técnica Direta de Fluorescência para Anticorpo/métodos , Imuno-Histoquímica/métodos , Camundongos , Raiva/imunologia , Raiva/virologia , Vírus da Raiva/imunologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sensibilidade e Especificidade , Carga ViralRESUMO
Pemphigus herpetiformis is an autoimmune bullous disease, that combines clinical features of dermatitis herpetiformis and linear IgA bullous dermatosis and immunological characteristics of pemphigus, which makes this disease peculiar and this diagnosis rarely suspected in the first evaluation of the patient. The reported case is of a patient with clinically bullous disease similar to dermatitis herpetiformis, whose multiple biopsies were inconclusive, and only after direct immunofluorescence with a pemphigus pattern (intraepidermal intercellular pattern) the confirmation of the diagnosis was possible.
Assuntos
Dermatite Herpetiforme/patologia , Técnica Direta de Fluorescência para Anticorpo/métodos , Pênfigo/patologia , Adulto , Biópsia , Eritema/patologia , Feminino , HumanosRESUMO
We present the case of a sixty five year old woman with two months history of pruritus and hyperpigmented annular lesions on the trunk, buttocks and upper extremities. In addition, she presents vesicles with healthy skin on the basis, in the flexor aspect of wrists. No evidence of mucosal involvement. Histological study showed subepidermal vesicular dermatitis with inflammatory infiltrate of neutrophils and eosinophils. Direct immunofluorescence evidenced linear and continuous deposition of immunoglobulin A in basement membrane zone, compatible with linear immunoglobulin A disease.
En este texto se presenta el caso de una paciente de sesenta y cinco años con sintomatología de dos meses de evolución consistente en prurito y lesiones hiperpigmentadas anulares en tronco, glúteos y extremidades superiores. En el área flexora de las muñecas presenta vesículas sobre base eritematosa y piel sana, sin evidencia de compromiso mucoso. Al estudio histológico se constata dermatitis vesicular subepidérmica con infiltrado inflamatorio de neutrófilos y eosinófilos. La inmunofluorescencia directa muestra depósito lineal y continuo de inmunoglobulina A en zona de membrana basal, compatible con dermatosis por inmunoglobulina A lineal.
Assuntos
Imunoglobulina A/imunologia , Dermatose Linear Bolhosa por IgA/diagnóstico , Prurido/imunologia , Idoso , Membrana Basal/imunologia , Eosinófilos/metabolismo , Feminino , Técnica Direta de Fluorescência para Anticorpo/métodos , Humanos , Dermatose Linear Bolhosa por IgA/imunologia , Neutrófilos/metabolismoRESUMO
Abstract Pemphigus herpetiformis is an autoimmune bullous disease, that combines clinical features of dermatitis herpetiformis and linear IgA bullous dermatosis and immunological characteristics of pemphigus, which makes this disease peculiar and this diagnosis rarely suspected in the first evaluation of the patient. The reported case is of a patient with clinically bullous disease similar to dermatitis herpetiformis, whose multiple biopsies were inconclusive, and only after direct immunofluorescence with a pemphigus pattern (intraepidermal intercellular pattern) the confirmation of the diagnosis was possible.
Assuntos
Humanos , Feminino , Adulto , Dermatite Herpetiforme/patologia , Pênfigo/patologia , Técnica Direta de Fluorescência para Anticorpo/métodos , Biópsia , Eritema/patologiaRESUMO
INTRODUCCIÓN: la toxoplasmosis es la enfermedad parasitaria más difundida en el mundo que afecta al hombre, descrita hace poco más de 100 años y producida por Toxoplasma gondii. Una de las formas que el hombre adquiere la enfermedad es a través de la placenta, de órganos trasplantados y por transfusiones de sangre; la forma infectante del parásito denominada "taquizoitos" es la responsable de este tipo de infección y ocurre durante la fase hematógena del mismo en un individuo seropositivo a T gondii. En Cuba al igual que en otras partes del mundo se ha demostrado la presencia de este parásito; en la provincia de Holguín su circulación se ha confirmado en receptores de trasplante renal. OBJETIVOS: conocer la seroprevalencia en donantes de sangre que motivó el interés para la realización de este trabajo. MÉTODOS: se evaluaron 892 muestras de sueros de donantes de los 14 municipios de la provincia de Holguín, el comportamiento serológico se determinó por la Técnica de Inmunofluorescencia Indirecta. RESULTADOS: la seropositividad para Inmunoglobulina G, anti Toxoplasma gondii de un 38,2 %, CONCLUSIONES: los donantes de la provincia de Holguín, están expuesto al Toxoplasma gondii, donde existe endemicidad del parásito en todo sus municipios y queda demostrado que los individuos procedentes de áreas rurales, tienen mayor incidencia de seropositivos al Toxoplasma gondii que los de áreas urbanas.
INTRODUCTION: Toxoplasmosis is a parasitic disease caused by Toxoplasma gondii. First described a little over 100 years ago, it is the most widely distributed parasitic disease affecting humans. The disease may be acquired from the placenta, from transplanted organs or from blood transfusions. The infecting form of the parasite, known as "tachyzoite", is responsible for this type of infection, which occurs during the hematogenous stage in a T. gondii-seropositive individual. The presence of this parasite has been demonstrated both in Cuba and in other regions of the world. In the province of Holguín its circulation has been confirmed in renal transplant recipients. OBJECTIVES: The purpose of the present study was to determine the seroprevalence of toxoplasmosis among blood donors. METHODS: An evaluation was conducted of 892 serum samples from donors from the 14 municipalities in the province of Holguín. Serological behavior was determined by indirect immunofluorescence technique. RESULTS: Seropositivity for anti-Toxoplasma gondii Immunoglobulin G was found to be 38.2 %. CONCLUSIONS: Blood donors from the province of Holguín are exposed to Toxoplasma gondii. The parasite is endemic in all municipalities, and it has been shown that Toxoplasma gondii seropositivity is higher in rural areas than in urban areas.
Assuntos
Humanos , Toxoplasma/patogenicidade , Doadores de Sangue , Toxoplasmose/etnologia , Técnica Direta de Fluorescência para Anticorpo/métodosRESUMO
We evaluated the presence of DNA of Giardia, Toxoplasma, and Cryptosporidium by PCR, and of Giardia and Cryptosporidium genera by immunofluorescence antibody test (IFAT), in water samples, before, during, and after plant treatment for drinkable water. We applied this method in 38 samples of 10 l of water taken from each of the water treatment steps and in 8 samples taken at home (only for Toxoplasma PCR) in Quindio region in Colombia. There were 8 positive samples for Cryptosporidium parvum (21 %), 4 for Cryptosporidium hominis (10.5 %), 27 for Toxoplasma gondii (58.6 %), 2 for Giardia duodenalis assemblage A (5.2 %), and 5 for G. duodenalis assemblage B (13.1 %). By IFAT, 23 % were positive for Giardia and 21 % for Cryptosporidium. An almost perfect agreement was found between IFAT and combined results of PCR, by Kappa composite proportion analysis. PCR positive samples were significantly more frequent in untreated raw water for C. parvum (p = 0.02). High mean of fecal coliforms, high pH values, and low mean of chlorine residuals were strongly correlated with PCR positivity for G. duodenalis assemblage B. High pH value was correlated with PCR positivity for C. parvum. Phylogenetic analysis of DNA sequences was possible, showing water and human clinical sequences for Toxoplasma within the same phylogenetic group for B1 repeated sequence. PCR assay is complementary to IFAT assay for monitoring of protozoa in raw and drinkable water, enabling species identification and to look for phylogenetic analysis in protozoa from human and environmental sources.
Assuntos
Cryptosporidium parvum/isolamento & purificação , Água Potável/parasitologia , Giardia lamblia/isolamento & purificação , Toxoplasma/isolamento & purificação , Purificação da Água , Animais , Sequência de Bases , Colômbia , Criptosporidiose/parasitologia , Cryptosporidium parvum/classificação , Cryptosporidium parvum/genética , DNA de Protozoário/genética , Fezes/parasitologia , Técnica Direta de Fluorescência para Anticorpo/métodos , Giardia lamblia/classificação , Giardia lamblia/genética , Giardíase/parasitologia , Humanos , Filogenia , Reação em Cadeia da Polimerase/métodos , Proteínas de Protozoários/genética , Toxoplasma/classificação , Toxoplasma/genética , Toxoplasmose/parasitologiaRESUMO
OBJECTIVE: There are few reports on the migration of CLA+ T cells through E-selectin in cutaneous lichen planus, with only one study on oral lichen planus (OLP). This study aimed to analyze CLA expression and assess whether there is a correlation with E-selectin (CD62E) in OLP lesions. MATERIAL AND METHODS: Biopsies were performed on 11 patients including two areas: one without clinical and histopathological features of OLP [perilesional group (PLG)] and the other with clinical and histopathological features of OLP [OLP group (OLPG)]. The specimens obtained were divided into two: One was fixed in formalin for routine analysis (H&E), and the other was frozen for CD3, CD4, CD8, CLA, and CD62E immunofluorescence markers. RESULTS: More CD4+ (median 1409, range 860-2519), CD8+ (median 1568, range 654-3258), and CLA+ T cells (median 958, range 453-2198) and higher CD62E expression (median 37, range 27-85) were identified in OLPG (P = 0.003; P = 0.003; P = 0.004; P = 0.003, respectively) than those in PLG. The median prevalence analysis was also significantly higher for CLA+CD8+ T cells in OLPG (OLPG = 39.4%, range 18.4-64.2; PLG = 29.4%, range 12.1-47.1) (P = 0.026). None of the correlations between CD3+ or CLA+ T cells and CD62E in OLPG and in PLG were significant. CONCLUSION: The significant presence of CLA+ T cells and E-selectin expressions in the OLPG suggests their involvement in the etiopathogenesis of OLP; however, only a weak correlation between CLA+ T cells and E-selectin was observed.
Assuntos
Antígenos de Diferenciação de Linfócitos T/biossíntese , Selectina E/biossíntese , Líquen Plano Bucal/metabolismo , Glicoproteínas de Membrana/biossíntese , Antígenos CD/imunologia , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/imunologia , Antígenos de Diferenciação de Linfócitos T/metabolismo , Selectina E/imunologia , Selectina E/metabolismo , Técnica Direta de Fluorescência para Anticorpo/métodos , Humanos , Líquen Plano/imunologia , Líquen Plano/metabolismo , Líquen Plano/patologia , Líquen Plano Bucal/imunologia , Líquen Plano Bucal/patologia , Linfócitos/imunologia , Linfócitos/metabolismo , Linfócitos/patologia , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Mucosa Bucal/imunologia , Mucosa Bucal/metabolismo , Mucosa Bucal/patologia , Prevalência , Linfócitos T/imunologiaRESUMO
La campilobacteriosis genital bovina es una enfermedad reproductiva que afecta la producción bovina. Es causada por las subespecies de Campylobacter fetus, C. fetus fetus (Cff) y C. fetus venerealis (Cfv). El objetivo de este estudio fue identificar la presencia de C. fetus en fluidos genitales mediante cultivo bacteriológico e inmunofluorescencia directa (IFD) y comparar los resultados. Se conformaron 2 grupos de 6 vaquillonas y 5 toros cada uno. Uno se infectó con Cff (grupo Cff) y el otro con Cfv (grupo Cfv). Dos vaquillonas y 2 toros sin infectar conformaron el grupo control. Periódicamente se tomaron muestras de mucus cervicovaginal y fluido prepucial, las que se procesaron por cultivo e IFD. En el grupo Cff se infectó el 100 % de las vaquillonas y el 80 % de los toros, mientras que en el grupo Cfv se infectó el 50 y el 60 %, respectivamente. Los valores de concordancia (Kappa) obtenidos al comparar las técnicas diagnósticas fueron de 0,57 para las vaquillonas del grupo Cff y 0,52 para las del grupo Cfv, y para los toros fueron de 0,17 y 0,27, respectivamente. En las vaquillonas, la IFD arrojó más resultados positivos que el cultivo, un 5,6 % más para el grupo Cff y un 7,4 % más para el grupo Cfv. El menor porcentaje de resultados positivos por IFD en los toros, un 40 % menos que por cultivo para el grupo Cff y un 5,3 % menos para el grupo Cfv, podría deberse a un muestreo incorrecto. Los valores de Kappa indican una concordancia moderada en las vaquillonas y baja en los toros.
Bovine genital campylobacteriosis is a reproductive disease that affects cattle production. It is caused by Campylobacter fetus subspecies, C. fetus fetus (Cff) and C. fetus venerealis (Cfv). The aim of this study was to identify the presence of C. fetus in genital fluids by bacteriological culture and direct immunofluorescence (DIF) and to compare the results. Two groups of 6 heifers and 5 bulls, one infected with Cff (Cff group) and the other with Cfv (Cfv group) were formed. Two heifers and 2 bulls, all of them uninfected, made up the control group. Samples of cervicovaginal mucus and preputial fluid were processed by culture and DIF. In the Cff group, 100 % of the heifers and 80 % of the bulls were infected, while in the Cfv group, 50 % of the heifers and 60 % of the bulls were infected. The degree of agreement (Kappa values) from benchmarking diagnostic techniques were 0.57 for heifers in the Cff group and 0.52 for heifers in the Cfv group, whereas the values for bulls were 0.17 and 0.27, respectively. Heifers yielded more positive results in the DIF assay than in the culture, exhibiting 5.6 % increase in the Cff group and 7.4 % in the Cfv group. The lowest percentage of positive results for DIF in bulls, 40 % less for the Cff group and 5.2 % for the Cfv group, could be due to improper sampling. Kappa values showed moderate agreement for the heifers and low for the bulls.
Assuntos
Animais , Campylobacter fetus/isolamento & purificação , Infecções por Campylobacter/veterinária , Doenças dos Bovinos/prevenção & controle , Campylobacter fetus/crescimento & desenvolvimento , Infecções por Campylobacter/prevenção & controle , Técnicas Bacteriológicas/métodos , Técnica Direta de Fluorescência para Anticorpo/métodosRESUMO
Introduction: The specific diagnosis of influenza A infection makes it possible to control its spread, decreases the unnecessary use of antibiotics, clinical procedures and laboratory test, and allows early recognition of outbreaks. Different technologies are currently available in Chile for this purpose. Objective: The study presented here compares the sensitivity for influenza A virus detection of immunocromatography (RIDT), direct fluorescent antibodies-DFA and DFA with cytocentrifugation against the gold standard, RT-PCR. Material and Methods: In 175 nasal swab samples influenza A RIDT and RT-PCR were performed. Another 1689 nasal swab samples were tested by DFA and RT-PCR for influenza A. Finally, 29 nasal swab samples confirmed as Influenza A positive by RT-PCR were tested by DFA with cytocentrifugation. Results: The RIDT, DFA and DFA + cytocentrifugation sensitivity was 47,3%, 57,2% and 72,4%, respectively. Discussion and Conclusion: Their lower cost and faster turnaround time when compared to PCR make RIDT and DFA the tests of choice in diagnostic laboratories in Chile. However, their low sensitivity and NPV, especially during low season, makes more sensitive diagnostic tools necessary to confirm the results. In our study cytocentrifugation increased DFA sensitivity from 57% to 72%.
Introducción: El diagnóstico específico de influenza permite controlar la diseminación de la enfermedad, disminuir el uso de antimicrobianos, procedimientos clínicos y exámenes, e identificar rápidamente brotes. Diferentes tecnologías están actualmente disponibles en Chile para este propósito. Objetivo: Comparar la sensibilidad diagnóstica para la infección por el virus influenza A de las técnicas inmunocromatografía, inmunofluorescencia directa-IFD e IFD con citocentrifugado contra el estándar de oro, RPC-TR. Materiales y Método: En 175 muestras de hisopado nasofaríngeo se realizó inmunocromatografía y RPC-TR para influenza A. Otras 1.689 muestras de hisopado nasofaríngeo fueron procesadas mediante IFD y RPC-TR para influenza A. Finalmente, en 29 muestras de hisopado nasofaríngeo, confirmadas positivas para influenza A mediante RPC-TR, se realizó IFD con citocentrifugado. Resultados: La sensibilidad de la inmunocromatografía, IFD e IFD + citocentrifugado fue de 47,3%, 57,2% y 72,4%, respectivamente. Discusión y Conclusión: El menor costo y tiempo de respuesta de las técnicas rápidas (inmunocomatografía e IFD) en relación a la RPC-TR hacen que se mantengan como exámenes de rutina en los laboratorios diagnósticos del país. Sin embargo, su baja sensibilidad y VPN, especialmente durante períodos de baja prevalencia, obligaría a confirmar los resultados negativos con técnicas más sensibles. En nuestra comparación la citocentrifugación mejoró la sensibilidad de la IFD de 57% a 72%.