Progress in DNA-sensing pathways in Epstein-Barr virus infection / 中华微生物学和免疫学杂志

Epstein-Barr virus (EBV) is generally susceptible in human beings and multi-organ systems can be involved in EBV infection, such as blood, respiratory, urinary, digestive and nervous systems. EBV infection also plays an important role in the pathogenesis of related tumors, autoimmune diseases and other diseases, posing a great threat to human health. As a DNA virus, EBV can be sensed by DNA recognition receptors to trigger a series of downstream immune responses. A DNA-sensing pathway consists of DNA sensors, adaptor molecules and downstream effector signals. Double-stranded DNA sensors mainly include absent in melanoma 2-like receptors (ALRs) and cyclic GMP-AMP synthase (cGAS). Adaptors were mainly stimulator of interferon genes (STING) and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). Downstream immune responses mainly involve typeⅠIFN, inflammasomes and proinflammatory cytokines. As a double-stranded DNA virus of the Herpesviridae family, EBV triggers complex innate and adaptive immune responses in the host, especially the sensing pathways mediated by a variety of DNA recognition receptors, which play a key role in host immune defense and pathogen immune evasion. This review made the DNA sensor as the clue to comprehensively summarize the progress in the activation, regulatory mechanism and clinical relevance of DNA-sensing pathways in EBV infection in recent years, aiming to achieve a better understanding of the host innate immune responses during EBV infection and provide an immunological basis for the prevention and treatment of EBV infection-related diseases.

Neospora caninum infection in cattle in the state of Amazonas, Brazil: seroprevalence, spatial distribution and risk factors / Infecção por Neospora caninum em bovinos no estado do Amazonas, Brasil: soroprevalência, distribuição espacial e fatores de risco

Abstract Livestock in the Amazon has grown significantly and, although neosporosis in cattle has been reported worldwide, there is no information about N. caninum in production systems in the state of Amazonas. The objective of this study was to determine the prevalence of anti-Neospora caninum antibodies in cattle, their spatial distribution and the risk factors associated with N. caninum infection in the state of Amazonas. Questionnaires were applied to farmers to assess risk factors associated with N. caninum infection. Blood samples were collected from 1,073 animals on 47 farms in 33 municipalities in the four Amazonian subpopulations. IgG anti-N.caninum antibodies were detected by the indirect fluorescence test, with a general prevalence of 30.2%, being seropositive in 43 farms (91.5%), with prevalence ranging from 2.2% to 69.2%. The highest number of high density points was found in subpopulation 3 (municipality of Apuí and other municipalities on the Madeira River and affluent). It was concluded that N. caninum is present with high seroprevalence values, when compared to other cattle producing states in the Amazon region of Brazil. The identified factors can be used as risk indicators so that control measures can be implemented to prevent infection by N. caninum in these herds.

Resumo A pecuária na Amazônia tem crescido significativamente e, embora a neosporose em bovinos tenha sido relatada em todo o mundo, não há informações sobre N. caninum nos sistemas de produção no estado do Amazonas. Objetivou-se determinar a prevalência de anticorpos anti-Neospora caninum em bovinos, sua distribuição espacial e os fatores de risco associados à infecção por N. caninum no estado do Amazonas. Questionários foram aplicados aos fazendeiros, para avaliar fatores de risco associados à infecção por N. caninum. Amostras de sangue foram coletadas de 1.073 animais em 47 fazendas, em 33 municípios das quatro subpopulações amazonenses. Anticorpos IgG anti-N.caninum foram detectados pelo teste de imunofluorescência indireta, com prevalência geral de 30,2%, com soropositividade em 43 fazendas (91,5%), com prevalência variando de 2,2% a 69,2%. O maior número de pontos de alta densidade foi encontrado na subpopulação 3 (município de Apuí e demais municípios do rio Madeira e afluentes). Concluiu-se que N. caninum está presente com altos valores de soroprevalência, quando comparado a outros estados produtores de gado na região amazônica do Brasil. Os fatores identificados podem ser usados como indicadores de risco, para que medidas de controle possam ser implementadas para prevenir a infecção por N. caninum nesses rebanhos.

Primera detección de Candidatus Rickettsia colombianensi en el departamento del Meta, Colombia / First detection of Candidatus Rickettsia colombianensi in the State of Meta, Colombia

RESUMEN Introducción: Las Rickettsias son un género de bacterias Gram negativas intracelulares obligadas causantes de muchas epidemias en el mundo y son transmitidas principalmente por garrapatas, pulgas, piojos y ácaros. Las rickettsiosis son enfermedades infecciosas reemergentes sin vigilancia epidemiológica en Colombia. Hasta hace pocos años Rickettsia rickettsi era la única Rickettsia transmitida por garrapatas presente en América; sin embargo, nuevas especies están siendo descritas, y aunque su patogenicidad no ha sido confirmada pueden ser consideradas patógenos potenciales. Objetivo: Determinar la presencia de rickettsias en garrapatas del departamento del Meta, Colombia. Material y Métodos: Se realizó un estudio descriptivo de corte transversal, fueron colectadas garrapatas y después de su identificación taxonómica, los genes gltA, ompA y ompB fueron amplificados y secuenciados. Resultados: Se obtuvieron un total de 169 grupos a los que se les realizó PCR de los cuales 2 grupos amplificaron para los genes gltA, ompA y ompB. Conclusiones: Los resultados demuestran la presencia de Rickettsia spp en garrapatas del departamento del Meta.

ABSTRACT Introduction: Rickettsia is a genus of Gram-negative obligate intracellular bacteria that cause several epidemics around the world and are transmitted mainly by ticks, fleas, lice and mites. Rickettsiosis is a re-emergent infectious disease without epidemiological surveillance in Colombia. Until few years ago, Rickettsia rickettsi was the only tick-borne Rickettsioses in America; however, new species are being described, and although their pathogenicity has not been confirmed, they should be considered as potential pathogens. Objective: The aim of this study was to determine the presence of rickettsial agents in ticks in the state of Meta, Colombia. Material and Methods: A descriptive cross-sectional study was conducted; ticks were collected and, after their taxonomic identification, gltA,ompAand ompB genes were amplified and sequenced. Results: A total of 169 pools were obtained to which PCR were carried out, of which 2 PCR were amplified for the gltA, ompA and ompB genes. Conclusions: These results demonstrate the presence of Rickettsia spp in ticks in the State of Meta.

Brotes emergentes de leptospirosis del Amazonas colombiano / Emerging leptospirosis outbreaks in the Colombian Department of Amazonas

Se describe por primera vez una serie de nueve casos con clínica indicativa de leptospirosis en el municipio Puerto Nariño en el departamento Amazonas, Colombia. Se muestran evidencias serológicas de exposición con Rickettsia del grupo de las fiebres manchadas. Los casos fueron clínicamente considerados como síndrome febril de origen desconocido. Se descartó infección por dengue y malaria. El diagnóstico de Leptospira se realizó mediante el método de reacción en cadena de la polimerasa en tiempo real. Igualmente, se detectó la presencia de anticuerpos contra rickettsias del grupo de las fiebres manchadas por inmunofluorescencia Indirecta. Finalmente, se realiza revisión del tema(AU)

A description is provided for the first time of a series of nine cases with a clinical examination suggestive of leptospirosis in the municipality of Puerto Nariño, Department of Amazonas, Colombia. Serological evidence is presented of exposure to Rickettsia, spotted fever group. The cases were clinically considered as febrile syndrome of unknown origin. Infection with dengue or malaria was ruled out. Diagnosis of leptospirosis was achieved by real-time polymerase chain reaction. Additionally, indirect immunofluorescence detected the presence of antibodies against rickettsia, spotted fever group. Finally, a review was conducted about the topic(AU)

Clinical outcomes in different target volume for cervical and upper-thoracic esophageal cancer in definitive chemoradiotherapy / 中华放射医学与防护杂志

Objective To investigate the prognostic effects and failure patterns of different clinical target volumes of IMRT in definitive chemoradiotherapy for cervical and upper-thoracic esophageal cancer,in order to provide a reference for radiotherapy target area delineation.Methods A retrospective analysis was performed on the clinical data of 132 patients with cervical and upper-thoracic esophageal cancer who received definitive IMRT and concurrent chemotherapy in our hospital from 2010 to 2014.Seventy-one patients received elective nodal irradiation (ENI) and the other 61 patients received involvedfield irradiation (IFI).The Kaplan-Meier method was used to calculate local control (LC),progressionfree survival (PFS) and overall survival (OS) rates.The significant difference was evaluated by the logrank test.The prognostic factors were determined by Cox univariate and multivariate analyses.Results The last follow-up time was December 2017,the median follow-up time was 59.5 (14.2-95.8) months.Follow-up rate was 99.2％.For the ENI and IFI groups,the 1-,3-,5-year LC were 77.5％,58.8％,48.8％ vs.64.3％,29.1％,26.2％ (x2=9.68,P=0.002),PFS were 68.6％,37.7％,25.9％ vs.47.5％,17.2％,3.6％ (x2=11.39,P=0.001),OS were 81.7％,53.9％,31.3％ vs.70.5％,31.9％,16.3％ (x2=7.70,P =0.006),respectively.In multivariate analysis,T stage,N stage,and RT field were independent factors for LC,PFS and OS(P＜0.05).The total failure rates,local-regional recurrent rate in ENI group were lower than those in IFI group (x2 =13.23,5.24,P＜0.05).No significant differences were found in acute radiation esophagitis,pneumonitis and myelosuppression (Grades ≥ 3) between the two groups(P＞0.05).Conclusions Compared with IFI,ENI can significantly reduce local-regional recurrence and distant metastasis and improve the long-term survival for cervical and upper-thoracic esophageal cancer patients who received definitive chemoradiotherapy.

Effects of interferon inducible protein 16 (IFI16) on viral protein and pro-inflammatory cytokine expression in human T-cell leukemia virus type 1 (HTLV-1)-positive T cells / 中华微生物学和免疫学杂志

Objective@#To investigate the effects of interferon inducible protein 16 (IFI16), a cytosolic DNA sensor, on the expression of human T-cell leukemia virus type 1 (HTLV-1) proteins and pro-inflammatory cytokines in adult HTLV-1-positive T cells.@*Methods@#IFI16 expression in different HTLV-1-positive T cell lines was detected by immunoblot assay. Specific siRNA targeting the IFI16 gene was constructed and the gene silencing efficiency was detected by immunoblot assay. Expression of HTLV-1 Tax protein at mRNA and protein levels was respectively detected by real-time PCR and immunoblot assay after knocking down the expression of IFI16 in HTLV-1-positive T cells with siRNA. Expression of interferon (IFN)-α, IFN-γ, tumor necrosis factor (TNF)-α, Tax and Env were detected by real-time PCR.@*Results@#Compared with the HTLV-1-negative T cell line Jurkat, IFI16 expression was enhanced in the HTLV-1-positive T cell lines MT2, MT4 and C8166. Tax expression was increased, while that of IFN-α, IFN-γ and TNF-α was decreased in MT2 and MT4 cells after silencing the expression of IFI16 with siRNA.@*Conclusions@#IFI16 expression was increased in HTLV-1-positive MT2 and MT4 cells. Meanwhile, IFI16 promoted the production of interferon and pro-inflammatory cytokines and inhibited the expression of HTLV-1 proteins.

Effects of interferon inducible protein 16 (IFI16) on viral protein and pro-inflammatory cytokine ex-pression in human T-cell leukemia virus type 1 (HTLV-1)-positive T cells / 中华微生物学和免疫学杂志

Objective To investigate the effects of interferon inducible protein 16 (IFI16), a cy-tosolic DNA sensor, on the expression of human T-cell leukemia virus type 1 (HTLV-1) proteins and pro-in-flammatory cytokines in adult HTLV-1-positive T cells. Methods IFI16 expression in different HTLV-1-positive T cell lines was detected by immunoblot assay. Specific siRNA targeting the IFI16 gene was con-structed and the gene silencing efficiency was detected by immunoblot assay. Expression of HTLV-1 Tax pro-tein at mRNA and protein levels was respectively detected by real-time PCR and immunoblot assay after knocking down the expression of IFI16 in HTLV-1-positive T cells with siRNA. Expression of interferon ( IFN)-α, IFN-γ, tumor necrosis factor ( TNF )-α, Tax and Env were detected by real-time PCR. Re-sults Compared with the HTLV-1-negative T cell line Jurkat, IFI16 expression was enhanced in the HTLV-1-positive T cell lines MT2, MT4 and C8166. Tax expression was increased, while that of IFN-α, IFN-γand TNF-α was decreased in MT2 and MT4 cells after silencing the expression of IFI16 with siRNA. Con-clusions IFI16 expression was increased in HTLV-1-positive MT2 and MT4 cells. Meanwhile, IFI16 pro-moted the production of interferon and pro-inflammatory cytokines and inhibited the expression of HTLV-1 proteins.

Comparative study of direct and indirect immunofluorescence for diagnosis of canine pemphigus foliaceus / Estudo comparativo da imunofluorescência direta e indireta para o diagnóstico do pênfigo foliáceo canino

Pemphigus foliaceus (PF) is the most common autoimmune skin disease in dogs. It is characterized by pustules, erosions, and crusts which occur due to the presence of autoantibodies that target intercellular adhesion. Histopathological examination is considered the gold standard pattern in the diagnosis, but may sometimes be inconclusive, especially when the characteristic findings are not identified. New diagnostic tests are continuously being developed and immunofluorescence assays, could be a valuable alternative diagnostic tool. This study aimed to evaluate the applicability of direct and indirect immunofluorescence (DIF and IIF) tests for the diagnosis of canine PF. Twenty eight dogs were divided into two groups: Group I with 14 dogs with PF and Group II (control) with 14 dogs with Superficial pyoderma (differential diagnoses of PF). All animals were submitted to skin biopsy to histopathological and DIF. Blood samples were collected to assess IIF. Comparing the DIF results against the histopathology test, there was an agreement of 75% (9/12) with a Kappa index of 0.77 (P<0.001). Considering IIF, the agreement was 100% (14/14), with a Kappa index of 1.0 (P<0.001). We conclude that DIF and IIF are highly effective and were useful and effective complementary examination tests for an improvement in the diagnosis of canine PF.(AU)

O pênfigo foliáceo (PF) é considerado uma das doenças tegumentares autoimunes mais frequentes em cães. Clinicamente, caracteriza-se pela presença de pústulas, erosões e crostas. O exame histopatológico é considerado o teste diagnóstico de eleição, porém pode se mostrar inconclusivo, sobretudo quando os achados característicos da doença não são observados. Novas ferramentas diagnósticas têm sido desenvolvidas e os testes de imunofluorecência são uma valiosa alternativa. Este estudo teve como objetivo avaliar a aplicabilidade das reações de imunofluorescência direta (IFD) e indireta (IFI) para o diagnóstico do PF canino. Vinte e oito cães foram divididos em dois grupos: grupo I com 14 cães com PF e grupo II (controle) com 14 cães com piodermite superficial (um dos principais diagnósticos diferenciais do PF). Todos os animais foram submetidos à biópsia cutânea, seguida de exame histopatológico e IFD. Amostras de sangue foram coletadas para realização da IFI. Comparando-se os valores de IFD com o histopatológico, obtiveram-se valores de concordância de 75% (9/12), com índice Kappa de 0,77 (P<0,001). Já na IFI, a concordância foi de 100% (14/14), com índice Kappa de 1,0 (P<0,001). Concluiu-se, então, que a IFD e a IFI apresentaram excelentes resultados e podem ser consideradas novas alternativas diagnósticas do PF canino.(AU)

An outbreak of caprine toxoplasmosis - investigation and case report / Surto de toxoplasmose caprina - investigação e relato de caso

ABSTRACT: The present study aimed to investigate an abortion outbreak in a dairy goat herd in the municipality of Arapoti, Parana, Brazil. At the beginning of the outbreak, blood samples were collected from 33 goats with clinical signs; later, of the whole goat herd, two cats and two dogs. Milk samples were collected from 78 lactating goats. Four environmental soil samples and four samples of feed residue from goat feeders were collected too. Immunofluorescence antibody test (IFA) was used for serodiagnosis, the molecular analysis was conducted by means of the polymerase chain reaction (PCR), for the isolation of the etiological agent the bioassay was used. The results of the IFA revealed that 76.53% (137/179) of the goats, two dogs and two cats were seropositive for Toxoplasma gondii. Bioassay revealed one buffy coat and two milk sample having viable T. gondii. In the PCR, 11 whole blood samples, eight milk, three feeder troughs, and all soil samples were positive. The findings of the present study confirmed an outbreak caused by environmental contamination (of soil and feed) with T. gondii oocysts that could have been shed by kittens that lived on the farm and had access to the stock of goat food, facilitating this contamination, which reinforces the need for veterinary assistance and good management practices on farms.

RESUMO: O presente estudo teve como objetivo investigar um surto de aborto em um rebanho de cabras leiteiras no município de Arapoti, Paraná, Brasil. No início do surto, foram coletadas amostras de sangue de 33 cabras com sinais clínicos; mais tarde, de todo o rebanho caprino, dois gatos e dois cachorros. Foram obtidas amostras de leite das 78 cabras em lactação. Quatro amostras ambientais de solo e quatro de resíduos de comedouro também foram coletadas. O teste de imunofluorescência (IFI) foi utilizado para o sorodiagnóstico, a análise molecular foi conduzida por meio da reação em cadeia da polimerase (PCR), para isolamento do agente etiológico utilizou-se o bioensaio. Os resultados da IFI revelaram que 76,53% (137/179) das cabras, todos os cães e gatos eram soropositivos para Toxoplasma gondii. O bioensaio revelou uma amostra de camada leucocitária e duas de leite contaminadas com T. gondii viável. Na PCR, 11 amostras de sangue total, oito de leite, três resíduos alimentares e todas as amostras de solo foram positivas. Os resultados do presente estudo confirmaram um surto causado por contaminação ambiental (de solo e alimentos) com oocistos de T. gondii que, provavelmente, foram eliminados por gatos que permaneceram na fazenda e tinham acesso ao estoque de alimento dos caprinos, reforçando a necessidade de assistência técnica veterinária e boas práticas de manejo.

Serosurvey of Borrelia in dogs, horses, and humans exposed to ticks in a rural settlement of southern Brazil / Soroprevalência e fatores associados a Borrelia em cães, equinos e humanos expostos a carrapatos em um assentamento rural do sul do Brasil

Abstract The aims of the present study were to serosurvey dogs, horses, and humans highly exposed to tick bites for anti-Borrelia burgdorferi s.l. antibodies, identify tick species present, and determine risk factors associated with seropositivity in a rural settlement of Paraná State, southern Brazil. Eighty-seven residents were sampled, along with their 83 dogs and 18 horses, and individual questionnaires were administered. Immunofluorescence antibody test (IFAT) was performed on serum samples and positive samples were subjected to western blot (WB) analysis. Anti-B. burgdorferi antibodies were found in 4/87 (4.6%) humans, 26/83 (31.3%) dogs, and 7/18 (38.9%) horses by IFAT, with 4/4 humans also positive by WB. Ticks identified were mostly from dogs and included 45/67 Rhipicephalus sanguineus, 21/67 Amblyomma ovale, and 1/67 A. cajennense sensu lato. All (34/34) horse ticks were identified as A. cajennense s.l.. No significant association was found when age, gender, or presence of ticks was correlated to seropositivity to Borrelia sp. In conclusion, although anti-Borrelia antibodies have been found in dogs, horses and their owners from the rural settlement, the lack of isolation, molecular characterization, absence of competent vectors and the low specificity of the commercial WB kit used herein may have impaired risk factor analysis.

Resumo Os objetivos do presente estudo foram realizar um levantamento sorológico de cães, cavalos e humanos altamente expostos a picadas de carrapatos para anticorpos anti-B. burgdorferi s.l., identificar as espécies de carrapatos presentes, e determinar os fatores de risco associados a soropositividade em um assentamento rural do Estado do Paraná, sul do Brasil. Oitenta e sete residentes foram amostrados junto com seus respectivos 83 cães e 118 cavalos e questionários individuais foram aplicados. O teste de imunofluorescência indireta (IFI) foi realizado nas amostras sorológicas e as positivas foram submetidas a análise por western blot (WB). Anticorpos anti-B. burgdorferi foram detectados em 4/87 (4,6%) humanos, 26/83 (31,3%) cães e 7/18 (38,9%) cavalos pela IFI, com 4/4 humanos também positivos pelo WB. Os carrapatos identificados foram em sua maioria de cães e incluíram 45/67 Rhipicephalus sanguineus, 21/67 Amblyomma ovale e 1/67 A. cajennense sensu lato. Todos (34/34) carrapatos dos cavalos foram identificados como A. cajennense s.l.. Não foram observadas diferenças estatísticas entre idade, sexo ou presença de carrapatos e soropositividade para Borrelia sp. Em conclusão, embora anticorpos anti-Borrelia tenham sido encontrados em cães, equinos e seus proprietários do assentamento rural, a ausência de isolamento, caracterização molecular, ausência de vetores competentes e baixa especificidade do kit comercial de WB utilizado podem ter limitado a análise de fatores de risco.

Frequency of Equine Monocytic Ehrlichiosis (EME) in Brazil / Relato epidemiológico da Erlichiose monocítica equina (EME) no Brasil

From a cross-sectional observational study with convenience samples, 347 blood samples from horses were collected from different physiographic regions, as follows: Santa Catarina Plateau (Santa Catarina State - SC), Médio Paraíba do Sul (São Paulo State - SP and Rio de Janeiro State RJ), Mountainous and Metropolitan regions (Rio de Janeiro State - RJ). Samples were tested for the presence of antibodies (IgG) anti Neorickettsia risticii by indirect immunofluorescence assay (IFA). The frequency obtained in this study corroborates with the ones obtained in the U.S.A., which refers to endemic regions. Fisher's exact test showed significant differences in the number of positive animals between regions, indicating that the probability of an animal becoming infected varies depending on the area. The CI 95% revealed no association between infection and geopolitical space. Moreover, Odds ratio test showed differences of an animal getting infected in different regions. This event could be influenced by the type of treatment used in each area, as the seasonal frequency of injury or even potential vectors. Therefore, there are seropositive animals for N. risticii in the studied areas, suggesting that this agent may be circulating in those regions. Future studies mainly based on molecular analyzes are needed to confirm these serological findings.

A partir de um delineamento observacional transversal com amostras de conveniência, 347 amostras de sangue foram coletadas de diferentes regiões fisiográficas: Planalto de Santa Catarina (Estado de Santa Catarina - SC), Região do Médio Paraíba do Sul (Estados de São Paulo - SP e Rio de Janeiro - RJ), Região Serrana e Metropolitana (ambas do Estado do Rio de Janeiro - RJ). As amostras foram testadas para a presença de anticorpos (IgG) anti-Neorickettsia risticii por imunofluorescência indireta (IFI). A prevalência obtida no presente estudo corrobora com demais resultados obtidos nos Estados Unidos da América. O Teste Exato de Fisher demonstrou diferença significativa no número de animais positivos entre as regiões, indicando assim que a probabilidade de um animal se infectar varia dependendo da região. O intervalo de confiança (IC 95%) revelou não haver associação entre a infecção e o espaço geopolítico, este evento pode ser influenciado pelo tipo de tratamento em cada área, como sazonalidade do agravo ou frequência de potenciais vetores. Assim, a soropositividade ora encontrada sugere a circulação de N. risticii nas áreas estudadas. Estudos futuros baseados, principalmente, em análises moleculares serão importantes para a confirmação dos achados sorológicos no presente trabalho.

Association between Interferon-Inducible Protein 6 (IFI6) Polymorphisms and Hepatitis B Virus Clearance

CD8+ T cells are key factors mediating hepatitis B virus (HBV) clearance. However, these cells are killed through HBV-induced apoptosis during the antigen-presenting period in HBV-induced chronic liver disease (CLD) patients. Interferon-inducible protein 6 (IFI6) delays type I interferon-induced apoptosis in cells. We hypothesized that single nucleotide polymorphisms (SNPs) in the IFI6 could affect the chronicity of CLD. The present study included a discovery stage, in which 195 CLD patients, including chronic hepatitis B (HEP) and cirrhosis patients and 107 spontaneous recovery (SR) controls, were analyzed. The genotype distributions of rs2808426 (C > T) and rs10902662 (C > T) were significantly different between the SR and HEP groups (odds ratio [OR], 6.60; 95% confidence interval [CI], 1.64 to 26.52, p = 0.008 for both SNPs) and between the SR and CLD groups (OR, 4.38; 95% CI, 1.25 to 15.26; p = 0.021 and OR, 4.12; 95% CI, 1.18 to 14.44; p = 0.027, respectively). The distribution of diplotypes that contained these SNPs was significantly different between the SR and HEP groups (OR, 6.58; 95% CI, 1.63 to 25.59; p = 0.008 and OR, 0.15; 95% CI, 0.04 to 0.61; p = 0.008, respectively) and between the SR and CLD groups (OR, 4.38; 95% CI, 1.25 to 15.26; p = 0.021 and OR, 4.12; 95% CI, 1.18 to 14.44; p = 0.027, respectively). We were unable to replicate the association shown by secondary enrolled samples. A large-scale validation study should be performed to confirm the association between IFI6 and HBV clearance.

Diagnóstico serológico y molecular de Ehrlichiosis humana en pacientes con sintomatologí­a clí­nica compatible con la enfermedad en el estado Zulia Venezuela 2004-2005 / Serological and molecular diagnosis of human Ehrlichiosis in patients with clinical manifestations associated with the disease in Zulia Venezuela 2004-2005

La Ehrlichiosis humana es una enfermedad zoonótica, transmitida al hombre por la picadura de garrapatas del perro y pocas veces del venado. E. chaffensis es el agente causal más relacionado con la Ehrlichiosis Humana, sin embargo, la ehrlichiosis en humanos puede ser causada por ehrlichiaspropias de los caninos como E. canis y E. ewingii. En 1992, en el estado Zulia se presenta el primer caso de Ehrlichiosis Humana, en una lactante de 17 meses de edad, en quien se detectaron anticuerpos frente a E. chaffensis. El objetivo de esta investigación fue estudiar la presencia de infección por Ehrlichia spp., en suero y sangre total de 30 sujetos con clínica sugestiva de esta patología. Se utilizaron las técnicas de IFI y ensayo nested PCR (Reacción en cadena de la polimerasa), usando primers derivados de la secuencia genética que codifica el ARN ribosomal 16S de Ehrlichia spp., que proporcionan información de género más no de especie, debido a que se necesitan primers de género que pusieran detectar Ehrlichias en cualquier huésped. Ninguna de las muestras fue positiva en la prueba IFI, no evidenciándose anticuerpos para Ehrilichia spp. El ensayo nested PCR de las muestras sanguíneas no mostró amplificación de las secuencias seleccionadas del gen del ARN ribosomal 16S en ninguna de las muestras. Los datos obtenidos no ponen en evidencia la infección por Ehrlichia spp. en los pacientes estudiados.

Human Ehrlichiosis is a zoonotic disease transmitted to humans through the bite of dog and occasionally, deer ticks. E. chaffeensis is the most common etiological agent related to human Ehrlichiosis; however, human Ehrlichiosis can be caused by canine ehrlichias such as E. canis and E. ewingii. In 1992, in the State of Zulia, the first case of human Ehrlichiosis appeared in a 17-month-old infant, in whom antibodies against E. chaffeensis were detected. The aim of this research was to study the presence of Ehrlichia spp. infection in the serum and whole blood of 30 persons with clinical symptoms associated with the disease. This was accomplished through techniques of indirect immunofluorescence and nested polymerase chain reaction assay (nested PCR), using primers derived from the genetic sequence that codify the ribosomal RNA 16S of Ehrlichia spp., which give information regarding gender but not species; due to this, gender primers are needed that could detect Ehrlichias in any host. None of the samples in the IFI assay was positive; no antibodies for Ehrilichia spp. were in evidence. The nested PCR test did not show amplification of the target sequence. The data did not support the evidence of Ehrlichia spp. infection in the patients studied.

Soroprevalência da Leishmaniose Tegumentar Americana (LTA) canina e fauna de Flebotomíneos (Diptera: Psychodidae) em Bela Vista do Paraíso, Paraná / Seroprevalence of Cutaneous Leishmaniasis (LC) in dogs and identification of vectors (Diptera: Psychodidae) in Bela Vista do Paraíso, Parana state

O trabalho teve como objetivo determinar a soroprevalência da Leishmaniose Tegumentar Americana (LTA) canina no município de Bela Vista do Paraíso, Paraná, comparar as técnicas de imunofluorescência indireta (IFI) e ensaio imunoenzimático (ELISA) e identificar as espécies de flebotomíneos presentes, possivelmente envolvidas no ciclo do parasito. Amostras de sangue de 489 cães foram submetidas à pesquisa de anticorpos anti-Leishmania sp. pela IFI e ELISA. Foram consideradas positivas as amostras que apresentaram título ≥ 40 na IFI e densidade ótica ≥ 0.174 no ELISA. Entre as amostras analisadas, 222 (45,4%) foram reagentes pela IFI e 189 (38,7%) pelo ELISA. Comparando-se os testes foram encontradas 176 amostras positivas (36,0%) e 254 negativas (51,9%) para ambas as técnicas. A sensibilidade do ELISA foi de 79,3% e a especificidade foi de 95,1%. O coeficiente global do teste foi de 87,0% com coeficiente Kappa de 0,75. A análise das variáveis para cães com sorologia positiva pela IFI demonstrou diferença significativa em relação à ausência de matas e ausência de convívio com outras espécies animais. Em cães sororeagentes pelo ELISA as variáveis que apresentaram diferença significativa foram o tipo de mata ciliar existente no ambiente, a ausência de lixo, o esgoto a céu aberto e lançado diretamente em rios ou córregos e do lixo lançado em terreno baldio, queimado ou enterrado. O resultado obtido com a captura dos flebotomíneos foi a predominância do Lutzomyia whitmani com 79,9% das espécies coletadas. Os resultados demonstraram que a LTA está amplamente disseminada na população canina do município de Bela Vista do Paraíso, e que tanto a IFI como o ELISA podem ser utilizados para o diagnóstico. Deste modo o cão apresenta-se como um elo entre o ciclo silvestre e o peridomiciliar da LTA, podendo tornar-se um sinalizador do agente no ecossistema da doença nesse ecossistema.

The aim of this study was to determine the seroprevalence of Cutaneous Leishmaniasis (CL) in dogs of Bela Vista do Paraiso, Parana state, compare the IFA and ELISA techniques and identify the vectors possibly involved in the cycle of the parasite. Were collected blood samples from 489 dogs that were subjected to detection of anti-Leishmania sp. by IFA and by ELISA. Were considered positive samples ≥40 titers in IFA and for ELISA ≥ 0.174 optical density. Among the samples analyzed, 222 (45.4%) were positive by IFA and 189 (38.7%) by ELISA. Comparing the tests were found 176 positive samples (36.0%) and 254 negative (51.9%) for both techniques. The sensitivity of ELISA was 79.3% and specificity was 95.1%. The global coefficient of the test was 87.0% with kappa coefficient of 0.75. Analysis of variables for dogs with positive serology by IFA showed significant differences regarding the absence of forests and lack of contact with other animal species. Positive sera by ELISA in dogs variables that showed significant differences were the type of riparian vegetation existing in the environment, the lack of garbage, open sewers and released directly into rivers or streams and garbage thrown on wasteland, burned or buried. The result obtained with the capture of sandflies was the predominance of Lutzomyia whitmani with 79.9% of the species collected. The result obtained with the capture of sandflies was the predominance of Lutzomyia whitmani with 79.9% of the species collected. The results showed that the LTA is widespread in the canine population of Bela Vista do Paraiso, and both the IFI and ELISA can be used for diagnosis. So the dog appears as a link between wild and peridomestic cycle of CL may become an amplifier of disease in this ecosystem.

Estabilidad enzoótica de babesiosis bovina en la región de Puerto Berrío, Colombia / Enzootic Stability of Bovine Babesiosis at Puerto Berrio Region, Colombia

En Colombia, la industria ganadera representa el tercer renglón de importancia económica después de la explotación petrolera y la agricultura. Este recurso económico se encuentra afectado por enfermedades hemoparasitarias como la babesiosis bovina. Su transmisión se encuentra determinada por la relación vector-parásito- hospedador y está condicionada por factores bióticos y abióticos. El estado de equilibrio entre el proceso infeccioso y la adquisición de inmunidad por parte de los hospedadores bovinos es conocido como estabilidad enzoótica. Para la determinación de la estabilidad enzoótica en la babesiosis bovina se ha utilizado como indicador anticuerpos tipo IgG específicos para cada especie de Babesia en bovinos entre 3 y 9 meses de edad. Así, una zona es estable para babesiosis cuando al menos 75% de los bovinos entre 3 y 9 meses de edad son serorreactivos para Babesia spp. Se diseñó un estudio descriptivo prospectivo de corte transversal, con una n de 282 bovinos, para evaluar el nivel de estabilidad enzoótica en nueve hatos ganaderos de la región de Puerto Berrío, a través de un muestreo probabilístico aleatorio simple, estratificado por género y pareado por edad. Se utilizó la técnica de inmunofluorescencia indirecta (IFI) para la detección de anticuerpos tipo IgG específicos contra Babesia bovis y Babesia bigemina. Se obtuvo un nivel de serorreactividad por hato para B. bovis superior al 75% en la población estudiada en cuatro de los nueve hatos. En los hatos con estabilidad enzoótica se encontró una relación positiva entre la frecuencia del tratamiento garrapaticida y la serorreactividad. En particular, cuando la frecuencia de baños es de 90 días o más, el nivel de serorreactividad es el doble frente a la frecuencia de baños de 60 días o menos.

In Colombia, cattle industry is the third level of economic importance after oil and agriculture exploitation systems. This economic resource is affected by hemoparasitic diseases like bovine babesiosis. Transmission of babesiosis is determined by the vector-parasitic-host relation and biotic and abiotic conditional factors. Equilibrium between infection and acquired immunity on bovine hosts is called enzootic stability. To determine enzootic stability levels of bovine babesiosis it has been used the detection of IgG specific antibodies for bovine babesias in calves between 3 and 9 months of age. Thus, a geographic region has enzootic stability when 75% or more of calves between 3 and 9 months of age are seroreactive for Babesia spp. A descriptive prospective cross sectional study was carried out. A sample of 282 calves was tested to determine enzootic stability level in cattle ranches of Puerto Berrio Region. A simple random probabilistic sampling was designed, stratified by sex and age. The sample was distributed in nine cattle ranches and was evaluated by indirect immunofluorescence antibodies technique to detection of IgG specific antibodies against Babesia bovis and Babesia bigemina. A level of seroreactivity higher than 75% to B. bovis in four of nine cattle farms was obtained. In the cattle farms with enzootic stability, a positive relation between acaricide treatment frequency and seroreactivity was found. Specifically, 90 days or more of acaricide treatment frequency induce a double level of seroreactivity than 60 days or less frequency.

Diagnostic value of (1,3)-β-D-glucan assay and galactomannan test for invasive fungal infection in patients of acute radiation sickness / 中华放射医学与防护杂志

Objective To investigate the diagnostic values of (1,3)-β-D-glucan (G) and galactomannan (GM) for invasive fungal infection (IFI) in patients of acute radiation sickness (ARS).Methods Samples of periogeral blood,pharyngeal secretion,urine,and feces were collected from 316patients with ARS and suspected to suffer from IFI,192 males and 124 females,aged 60.50(1-96),with the underlying diseases of blood or respiration systems.Platelia Aspergillus EIA kit was used to detect the plasma BG (G test),and ELISA was used to detect the serum GM (GM test).Fungal culture and bacterial culture were performed.Results The positive rates of G test,GM test,and fungal culture were 36.33%,35.84% ,and 34.18% respectively,but the positive rate of fungal culture of blood sample was 1/316 only.Pearson correlation analysis showed that G test,GM test and fungal culture test were positively correlated with IFI clinical diagnosis respectively (x2 = 0.564,0.357,0.727,P ＜ 0.05).Conclusions Easy to operate,rapid,and highly sencitive,G test and GM test can be used as adjunctive methods for early IFI diagnosis in ARS patients.

Estudio comparativo de pruebas serológicas para el diagnóstico de la leishmaniasis visceral canina: nota técnica / Comparative study of serological test for the diagnosis of canine visceral leishmaniasis: technical note

El objetivo del presente estudio consistió en comparar la concordancia entre los resultados obtenidos por las técnicas IFI, ELISA y Western Blot en 72 sueros caninos procedentes de siete municipios de la zona endémica de leishmaniasis visceral zoonótica (LVZ) del departamento del Tolima (Colombia). Se utilizó como antígeno la cepa colombiana de Leishmania infantum MHOM/CO/CL044B para IFI, ELISA y WB, y el antígeno rK39 para una prueba de ELISA disponible comercialmente. Se encontró que la concordancia entre las diferentes técnicas comparadas fue menor del 16 por ciento (k<16 por ciento), lo que sugiere que las pruebas no son consistentes y por lo tanto, no son aceptables como método de diagnóstico en el presente estudio. La baja asociación de las pruebas serológicas utilizadas en el diagnóstico de L. infantum sugiere que es necesario desarrollar estudios que permitan establecer un algoritmo de pruebas diagnósticas en el país para confirmar el estado real de la infección en los animales y de esta forma orientar eficientemente los recursos de salud pública destinados para el control de la enfermedad.

The goal of present study was to compare the agreement between the results obtained by ELISA, IFI and WB tests in 72 canine serums from the South of the Tolima Department (a visceral leishmaniasis endemic area). The Colombian Leishmania infantum MHOM/CO/CL044B strain was used as antigen for ELISA, IFI and WB test, and the rK39 antigen for the commercial ELISA test. The agreement among the compared techniques was smaller than 16 percent (k<16 percent), suggesting that the tests are not consistent and therefore not acceptable as a diagnostic tool in this study. The low association between the serological tests used in diagnosing Leishmania infantum suggested the need for further studies aimed at establishing an algorithm for diagnostic tests in Colombia for confirming the real state of the animals´ infection and thereby efficiently orientating public health resources allocated for controlling visceral leishmaniasis.

Anticuerpos antinucleares, imágenes y características obtenidas por inmunofluorescencia: Importancia de los isotipos IgA, IgM e IgG / Antinuclear antibodies, patterns and characteristics obtained by immunofluorescence: The importance of the IgA, IgM and IgG isotypes

La técnica de elección para el screening de anticuerpos antinucleares (ANA) es la inmunofluorescencia indirecta que utiliza como sustrato una línea de células epiteliales de carcinoma de laringe humano (IFI-HEp2), y como antisuero, anti-IgG o anti-Ig totales. Los ANA-IgG son los más importantes para el diagnóstico y monitoreo de las enfermedades del tejido conectivo (ETC), mientras los ANA-IgM son de menor relevancia clínica en estos pacientes. Sin embargo, poco se sabe de los ANA-IgA ya que estos Ac han sido menos investigados. El objetivo de este trabajo fue estudiar la prevalencia de los diferentes isotipos de inmunoglobulinas de anticuerpos antinucleares en los pacientes con ETC y evaluar la conveniencia de utilizar conjugados monovalentes o polivalentes. Se procesaron 100 sueros de pacientes con diversas ETC empleando IFI-HEp2, en los cuales se detectó 38% de ANA-IgA (títulos ≥ 1:80) y 12% de ANA-IgM (títulos ≤ 1:160). En 29 casos se detectó IgA en ausencia de IgM, en 3 casos IgM en ausencia de IgA. En todos los casos los ANA-IgG estuvieron presentes. En 6 sueros se observó un cambio de imagen con conjugado anti-IgA y en 3 con conjugado anti-IgM. Debido a la alta prevalencia de ANA-IgA detectada por IFI-HEp2, se destaca la conveniencia de utilizar conjugado anti-Ig totales en lugar de anti-IgG, mientras se desconozca la relevancia de los ANA-IgA en el diagnóstico, pronóstico y seguimiento de las enfermedades reumáticas sistémicas.

The indirect immunofluorescence with epitelial cell line from human laryngeal carcinoma as substrate (IIF-HEp2) and anti-IgG or anti-total Ig as antisera, is the technique currently used for the detection of antinuclear antibodies. The most important antibodies for the diagnosis and follow-up of connective tissue diseases (CTD) are the IgG-ANA, while the IgM-ANA have no clinical relevance. However the IgA-ANA have not been thoroughly investigated so far. The aim of this work was to study the prevalence of different ANA isotypes of Ig antibodies in CTD patients and to evaluate the convenience of the use of monovalent or polyvalent conjugate. We examined the sera of 100 patients with different CTD by IIF-HEp2 and detected a prevalence of 38% IgA-ANA (titles ≥ 1:80) and 12% IgM-ANA (titles ≤ 1:160). In twenty nine cases we detected IgA-ANA in absence of IgM-ANA, and in 3 cases IgM-ANA in absence of IgA-ANA. In all the cases IgG-ANA were present. In 6 sera a change in the immunofluorescence pattern was observed while using anti-IgA conjugate, whereas in 3 the change was observed with the use of anti-IgM conjugate. Because of the high prevalence of ANA-IgA detected by IIF-HEp2, we emphasize the convenience of employing anti-total Ig in spite of anti-IgG conjugated until the role of ANA-IgA is dilucidated in CTD patients, in order to establish its relevance in the diagnosis, prognosis and follow-up of systemic rheumatic diseases.

Clonagem, expressão, caracterização molecular da proteína de superfície MSP5 da amostra PR1 de Anaplasma marginale e sua aplicação em um teste de ELISA por competição / Cloning, expression, molecular characterization of the MSP5 protein from PR1 strain of Anaplasma marginale and its application in a competitive enzyme-linked immunosorbent test

No presente trabalho, um teste imunoenzimático por competição (cELISA) foi padronizado com a proteína recombinante de superfície rMSP5, clonada a partir do gene msp5 do isolado PR1 de A. marginale. O sequenciamento mostrou que o gene que codifica a rMSP5/PR1 tem 98 por cento de identidade com os isolados Flórida e Santa Maria, 97 por cento com isolados de Pernambuco, Brasil e Havana, Cuba e 91 por cento com A. centrale. O teste de cELISA-PR1 foi comparado com os testes de IFI e cELISA-USA. Para a padronização e validação do cELISA-PR1, foram utilizados 380 soros bovinos comprovadamente positivos ou negativos pelo cELISA-USA. Desse total, 245 soros positivos eram de animais de área endêmica e 135 soros eram negativos, de área livre de anaplasmose. Na padronização, foram utilizados 283 soros de bovinos, dos quais 135 eram negativos e 148 positivos. Os testes de cELISA-PR1 e IFI apresentaram especificidade de 100 e 99,3 por cento, sensibilidade de 100 e 98 por cento, com coeficiente kappa de 0,993 e 0,978, respectivamente. Na validação do teste, foram utilizados 245 soros de bovinos de áreas endêmicas para anaplasmose, testados pelo cELISA-PR1 e IFI e apresentaram especificidade de 96,7 e 69,1 por cento, sensibilidade de 98,9 e 96,3 por cento e coeficiente kappa de 0,956 e 0,699, respectivamente. Esses resultados permitem afirmar que o teste de cELISA-PR1 apresentou performance equivalente ao cELISA-USA, podendo também ser utilizado em estudos epidemiológicos, programas de controle e movimentação internacional de animais, enquanto a IFI, com os resultados menos precisos apresentados, não deve ser utilizada em situações que requerem maior rigor no diagnóstico.

A competitive enzyme-linked immunosorbent test using the PR1 recombinant major surface protein 5 (rMSP5-PR1-ELISA) of Anaplasma marginale was standardized and validated using sera from anaplasmosis free and endemic regions. The sequencing of the msp5 gene of PR1 isolate showed 98 percent of identity with the Florida and Saint Maries isolates, 97 percent with Brazil (Pernambuco) and Havana isolates; and 91 percent with A. centrale. The cELISA-PR1 test was compared to IFI and cELISA-USA. For the standardization and validation of the cELISA-PR1, 380 bovine sera were used, whereas 245 truly positives and 135 truly negatives sera tested by the cELISA-USA. In the standardization of the cELISA-PR1 135 negative and 148 positive bovine sera were used. The cELISA-PR1 and IFI tests showed 100 and 99.3 percent specificity, 100 and 98 percent, sensibility, and a kappa coefficient of 0.993 and 0.978, respectively. For test validation, 245 bovine sera from an anaplasmosis endemic area were analyzed by the cELISA-PR1 and IFI, which showed 96.7 and 69.1 percent specificity, 98.9 e 96.3 percent sensibility and kappa coefficient of 0.956 and 0.699, respectively. These results indicate that the cELISA-PR1, likewise the cELISA-USA, could sensitively and specifically detect cattle naturally infected with A. marginale and would be recommended for epidemiological studies, eradications program, and regulation of international cattle movement, while IFI, which presented lower specificity should not be used in situations that demand more specific diagnosis.

Comparação dos testes sorológicos de Imunofluorescência Indireta, Conglutinação Rápida, ELISA indireto e ELISA por competição para a detecção de anticorpos contra o Anaplasma marginale em soros de bovinos de diferentes áreas enzoóticas / Comparison of the serologic tests of Indirect Immunofluorescence,Rapid Conglutination, Indirect ELISA and Competition ELISA fordetection of antibodies against Anaplasma marginale in cattle sera fromdifferent enzootic areas

Os Testes Sorológicos de Conglutinação Rápida (TCR) Imunofluorescência Indireta (IFI) e Imunoenzimáticos Indireto (iELISA) utilizando ELISA por competição (cELISA), como padrão ouro, foram avaliados comparativamente para a detecção de anticorpos contra o Anaplasma marginale. Foram utilizadas 453 amostras de soros sangüíneos de bovinos vacinados e não-vacinados e de áreas de estabilidade e instabilidade enzoótica. O iELISA, IFI e TCR apresentaram respectivamente, índice kappa=0,77 (substancial), 0,57 e 0,49 (moderado), sensibilidade de 90,6, 90,2 e 73,7 e especificidade de 86,6, 62,8, e 79,3. O iELISA apresentou o melhor desempenho e maior especificidade, podendo ser indicado na avaliação do perfil sorológico de rebanhos, na detecção de animais persistentemente infectados e de animais submetidos a programas de vacinação. As técnicas de IFI e TCR, mesmo apresentando desempenho inferior, podem ser recomendadas para a realização de inquéritos epidemiológicos e para o monitoramento de animais em trânsito entre as diferentes regiões geográficas

The serological techniques Rapid Conglutination Test (RCT), Indirect ELISA (iELISA) and IndirectImmunofluorescent Assay (IFA), using the competition ELISA (cELISA) as gold test, were comparativelyevaluated to detect antibodies against Anaplasma marginale. A total of 453 sera from vaccinated andnon vaccinated cattle and, collected from enzootic stability and instability areas were tested. iELISA, IFAand TCR presented kappa index = 0.77 (substantial); 0.57 and 0.49 (moderate), sensibility of 90.6%;90.2% and 73.7% and specificity of 86.6%; 62.8%, and 79.3%, respectively. Therefore, iELISA presentedbetter specificity than IFA and RCT, and can be indicated for more detailed serological investigations,detection of persistently infected animals in cattle herds and for monitorating of vaccination programs.IFA and TCR can be used in prevalence studies and to monitor cattle movement between differentgeographical regions