RESUMO
ABSTRACT: This study detected Cryptosporidium spp. in cultivated oysters and the natural oyster stock of the state of Maranhão and determine the elective tissue(s) to examine this protozoan. For this purpose, 200 cultivated oysters were purchased from the municipality of Raposa and another 100 from Paço do Lumiar. Additionally, 100 oysters were extracted from the natural stock of the municipality of Primeira Cruz, thus making up a total of 400 oysters. They were grouped into 80 pools consisting of 5 oysters each. From each pool, the gills and visceral mass were removed to obtain 160 pools, 80 pools for the gill group and another 80 for the visceral mass group. Then, DNA was extracted from each pool using a commercial kit with modifications. Subsequently, the protozoan DNA was detected using nested polymerase chain reaction. With this technique, the DNA of the protozoan under investigation was detected in 2.5% (n = 2/80) of the pools containing gills, with 1.25% of the pools (n = 1/80) belonging to the cultivation group of oysters and the other 1.25% (n = 1/80) to the natural stock. With the results obtained in this study, it was concluded that the analyzed oysters of the genus Crassostrea, from cultivation and natural stock groups, found in the state of Maranhão, were contaminated by Cryptosporidium spp. and may become potential sources of infection in humans and other animals. In addition, the gills are the elective tissue for the study of Cryptosporidium spp. in oysters.
RESUMO: Objetivou-se com o estudo detectar Cryptosporidium sp. em ostras de cultivo e estoque natural no estado do Maranhão e determinar o(s) tecido(s) eletivo(s) para pesquisa desse protozoário. Para a realização do estudo foram adquiridas 200 ostras de cultivo do município de Raposa e 100 de Paço do Lumiar, além de 100 ostras extraídas de estoque natural do município de Primeira Cruz, totalizando 400 ostras. Estas foram agrupadas em 80 pools constituídos por cinco animais. De cada pool, as brânquias e a massa visceral foram removidas totalizando 160 pools, sendo 80 para o grupo das brânquias e 80 para o grupo de massa visceral. Na sequência, procedeu-se à extração de DNA de cada pool com a utilização de kit comercial com modificações. Posteriormente, realizou-se a detecção do DNA do protozoário por meio da técnica de Nested-PCR. Com a técnica utilizada, foi detectado o DNA do protozoário pesquisado em 2,5% (n=2/80) pools apenas de brânquias, sendo 1,25% pools (n=1/80) oriundos de cultivo e os outros 1,25% (n=1/80) de estoque natural. Com os resultados obtidos nesse estudo, conclui-se que as ostras analisadas do gênero Crassostrea sp., oriundas de cultivo e estoque natural no estado do Maranhão, estavam contaminadas por Cryptosporidium sp. e podem se reverter em fontes potenciais para seres humanos e outros animais. Para a pesquisa de Cryptosporidium sp. em ostras, as brânquias são o tecido eletivo.
RESUMO
This study detected Cryptosporidium spp. in cultivated oysters and the natural oyster stock of the state of Maranhão and determine the elective tissue(s) to examine this protozoan. For this purpose, 200 cultivated oysters were purchased from the municipality of Raposa and another 100 from Paço do Lumiar. Additionally, 100 oysters were extracted from the natural stock of the municipality of Primeira Cruz, thus making up a total of 400 oysters. They were grouped into 80 pools consisting of 5 oysters each. From each pool, the gills and visceral mass were removed to obtain 160 pools, 80 pools for the gill group and another 80 for the visceral mass group. Then, DNA was extracted from each pool using a commercial kit with modifications. Subsequently, the protozoan DNA was detected using nested polymerase chain reaction. With this technique, the DNA of the protozoan under investigation was detected in 2.5% (n = 2/80) of the pools containing gills, with 1.25% of the pools (n = 1/80) belonging to the cultivation group of oysters and the other 1.25% (n = 1/80) to the natural stock. With the results obtained in this study, it was concluded that the analyzed oysters of the genus Crassostrea, from cultivation and natural stock groups, found in the state of Maranhão, were contaminated by Cryptosporidium spp. and may become potential sources of infection in humans and other animals. In addition, the gills are the elective tissue for the study of Cryptosporidium spp. in oysters.
Objetivou-se com o estudo detectar Cryptosporidium sp. em ostras de cultivo e estoque natural no estado do Maranhão e determinar o(s) tecido(s) eletivo(s) para pesquisa desse protozoário. Para a realização do estudo foram adquiridas 200 ostras de cultivo do município de Raposa e 100 de Paço do Lumiar, além de 100 ostras extraídas de estoque natural do município de Primeira Cruz, totalizando 400 ostras. Estas foram agrupadas em 80 pools constituídos por cinco animais. De cada pool, as brânquias e a massa visceral foram removidas totalizando 160 pools, sendo 80 para o grupo das brânquias e 80 para o grupo de massa visceral. Na sequência, procedeu-se à extração de DNA de cada pool com a utilização de kit comercial com modificações. Posteriormente, realizou-se a detecção do DNA do protozoário por meio da técnica de Nested-PCR. Com a técnica utilizada, foi detectado o DNA do protozoário pesquisado em 2,5% (n=2/80) pools apenas de brânquias, sendo 1,25% pools (n=1/80) oriundos de cultivo e os outros 1,25% (n=1/80) de estoque natural. Com os resultados obtidos nesse estudo, conclui-se que as ostras analisadas do gênero Crassostrea sp., oriundas de cultivo e estoque natural no estado do Maranhão, estavam contaminadas por Cryptosporidium sp. e podem se reverter em fontes potenciais para seres humanos e outros animais. Para a pesquisa de Cryptosporidium sp. em ostras, as brânquias são o tecido eletivo.
Assuntos
Animais , Ostreidae/parasitologia , DNA de Protozoário , Cryptosporidium , BrânquiasRESUMO
Equine granulocytic anaplasmosis is caused by Anaplasma phagocytophilum, a gram negative, obligatory intracellular bacterium, member of Anaplasmataceae family, included in the Rickettsiales order. Little is known about the disease, transmission dynamics, genetic diversity and prevalence in Minas Gerais state, Brazil. This work aimed to do a serosurvey using indirect immunofluorescent assay (IFA) test and evaluation of buffy coat smears, and nested polymerase chain reaction (PCR) as diagnostic methods, to determine the disease situation in horses from two manga-larga marchador breeding farms located in the municipalities of Ataléia e São Vicente de Minas, in Minas Gerais state. It was found that 76% (131/172) of the animals were considered reactive for IFA test, and the total of 12.8% was positive at buffy coat smears analysis. At PCR analysis, it was found 1.94% of the samples positive to the infection. Those samples were sequenced and showed 96% of similarity to A. phagocytophilum from a Ixodes ricinus tick. There is a high frequency of animals with the evidence of contact to A. phagocytophilum on the two evaluated properties in this study, which was proved by positiveness in PCR analysis. New researches must be carried out to better understand the epidemiologic and clinical dynamic of the disease in the state of Minas Gerais.(AU)
A anaplasmose granulocítica equina é causada por uma bactéria gram-negativa, intracelular obrigatória, membro da família Anaplasmataceae, incluída na ordem Rickettsiales e denominada de Anaplasma phagocytophilum. Pouco se sabe sobre a doença, sua dinâmica de transmissão, diversidade genética e prevalência em Minas Gerais, Brasil. Este trabalho teve por objetivo realizar o levantamento sorológico utilizando a reação de imunofluorescência indireta, avaliação direta de capa leucocitária e nested reação em cadeia da polimerase (PCR) como métodos diagnósticos, a fim de avaliar a situação da doença em dois haras de criação de cavalos manga-larga marchador localizados nas cidades de Ataléia e São Vicente de Minas, no estado de Minas Gerais. Foi encontrada prevalência de 76% (131/172) de animais reativos para a reação de imunofluorescência indireta, quando todos os animais das duas propriedades e das duas coletas foram agrupados, e 12,8% dos animais foram positivos na avaliação da capa leucocitária. A reação de imunofluorescência indireta detectou 1,94% das amostras como positivas para o agente. Essas amostras foram submetidas ao sequenciamento de nucleotídeos, e foi observada similaridade de 96% com A. phagocytophilum proveniente de carrapatos Ixodes ricinus. Existe alta prevalência de animais positivos para a infecção por A. phagocytophilum, o que foi provado pela positividade dos animais à PCR. Novas pesquisas devem ser conduzidas a fim de entender a dinâmica epidemiológica e clínica da doença no estado de Minas Gerais.(AU)
Assuntos
Testes Sorológicos/métodos , Reação em Cadeia da Polimerase/métodos , Imunofluorescência/métodos , Cavalos , Anaplasmose , Ixodes , Bactérias Gram-Negativas/patogenicidadeRESUMO
Equine granulocytic anaplasmosis is caused by Anaplasma phagocytophilum, a gram negative, obligatory intracellular bacterium, member of Anaplasmataceae family, included in the Rickettsiales order. Little is known about the disease, transmission dynamics, genetic diversity and prevalence in Minas Gerais state, Brazil. This work aimed to do a serosurvey using indirect immunofluorescent assay (IFA) test and evaluation of buffy coat smears, and nested polymerase chain reaction (PCR) as diagnostic methods, to determine the disease situation in horses from two manga-larga marchador breeding farms located in the municipalities of Ataléia e São Vicente de Minas, in Minas Gerais state. It was found that 76% (131/172) of the animals were considered reactive for IFA test, and the total of 12.8% was positive at buffy coat smears analysis. At PCR analysis, it was found 1.94% of the samples positive to the infection. Those samples were sequenced and showed 96% of similarity to A. phagocytophilum from a Ixodes ricinus tick. There is a high frequency of animals with the evidence of contact to A. phagocytophilum on the two evaluated properties in this study, which was proved by positiveness in PCR analysis. New researches must be carried out to better understand the epidemiologic and clinical dynamic of the disease in the state of Minas Gerais.(AU)
A anaplasmose granulocítica equina é causada por uma bactéria gram-negativa, intracelular obrigatória, membro da família Anaplasmataceae, incluída na ordem Rickettsiales e denominada de Anaplasma phagocytophilum. Pouco se sabe sobre a doença, sua dinâmica de transmissão, diversidade genética e prevalência em Minas Gerais, Brasil. Este trabalho teve por objetivo realizar o levantamento sorológico utilizando a reação de imunofluorescência indireta, avaliação direta de capa leucocitária e nested reação em cadeia da polimerase (PCR) como métodos diagnósticos, a fim de avaliar a situação da doença em dois haras de criação de cavalos manga-larga marchador localizados nas cidades de Ataléia e São Vicente de Minas, no estado de Minas Gerais. Foi encontrada prevalência de 76% (131/172) de animais reativos para a reação de imunofluorescência indireta, quando todos os animais das duas propriedades e das duas coletas foram agrupados, e 12,8% dos animais foram positivos na avaliação da capa leucocitária. A reação de imunofluorescência indireta detectou 1,94% das amostras como positivas para o agente. Essas amostras foram submetidas ao sequenciamento de nucleotídeos, e foi observada similaridade de 96% com A. phagocytophilum proveniente de carrapatos Ixodes ricinus. Existe alta prevalência de animais positivos para a infecção por A. phagocytophilum, o que foi provado pela positividade dos animais à PCR. Novas pesquisas devem ser conduzidas a fim de entender a dinâmica epidemiológica e clínica da doença no estado de Minas Gerais.(AU)
Assuntos
Testes Sorológicos/métodos , Reação em Cadeia da Polimerase/métodos , Imunofluorescência/métodos , Cavalos , Anaplasmose , Ixodes , Bactérias Gram-Negativas/patogenicidadeRESUMO
Theileria equi infection prevalence was calculated from 1000 blood samples obtained from apparently healthy horses in western Mexico. Samples were sent to the Animal Biotechnology Laboratory of the University of Guadalajara (Mexico) for T. equi diagnosis. Nested polymerase chain reaction (nPCR) was used as a diagnostic method to detect pathogen DNA. Using primers for the merozoite antigen-1 (EMA-1) gene, 19.70±2.47% of the horses (95% CI, 17.23-22.17%) tested positive for T. equi. There was no significant association between gender and T. equi infection. However, prevalence was higher among stabled horses (25.81%) than that among grazing horses (15.02%). The positivity rate was also higher among Quarter Horse (24.70%), Lusitano (35.90%), and Costa Rican Saddle Horse (47.37%) breeds than that among the other seven breeds investigated in this study. The percentage of T. equi infection was higher among adult horses (≥ 4years old, 25.05%) than that among colts and fillies (2-4years old, 15.48%), yearlings (1-2years old, 10.49%), and foals (<1year old, 10.34%). This is the first study of T. equi infection prevalence among horses in Mexico by nPCR . The results indicate that the equine piroplasmosis (EP) caused by T. equi is enzootic in western Mexico.
Assuntos
Doenças dos Cavalos/diagnóstico , Doenças dos Cavalos/epidemiologia , Reação em Cadeia da Polimerase/veterinária , Theileria/isolamento & purificação , Theileriose/diagnóstico , Theileriose/epidemiologia , Animais , Babesiose/epidemiologia , Babesiose/parasitologia , Feminino , Doenças dos Cavalos/parasitologia , Cavalos/parasitologia , Masculino , México/epidemiologia , Prevalência , Theileria/genética , Theileriose/parasitologiaRESUMO
Abstract: INTRODUCTION : Molecular analyses are auxiliary tools for detecting Koch's bacilli in clinical specimens from patients with suspected tuberculosis (TB). However, there are still no efficient diagnostic tests that combine high sensitivity and specificity and yield rapid results in the detection of TB. This study evaluated single-tube nested polymerase chain reaction (STNPCR) as a molecular diagnostic test with low risk of cross contamination for detecting Mycobacterium tuberculosis in clinical samples. METHODS: Mycobacterium tuberculosis deoxyribonucleic acid (DNA) was detected in blood and urine samples by STNPCR followed by agarose gel electrophoresis. In this system, reaction tubes were not opened between the two stages of PCR (simple and nested). RESULTS: STNPCR demonstrated good accuracy in clinical samples with no cross contamination between microtubes. Sensitivity in blood and urine, analyzed in parallel, was 35%-62% for pulmonary and 41%-72% for extrapulmonary TB. The specificity of STNPCR was 100% in most analyses, depending on the type of clinical sample (blood or urine) and clinical form of disease (pulmonary or extrapulmonary). CONCLUSIONS: STNPCR was effective in detecting TB, especially the extrapulmonary form for which sensitivity was higher, and had the advantage of less invasive sample collection from patients for whom a spontaneous sputum sample was unavailable. With low risk of cross contamination, the STNPCR can be used as an adjunct to conventional methods for diagnosing TB.
Assuntos
Adolescente , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , DNA Bacteriano , Mycobacterium tuberculosis/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Tuberculose/diagnóstico , DNA Bacteriano/sangue , DNA Bacteriano/urina , Mycobacterium tuberculosis/genética , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
The purpose of this study was to investigate the occurrence of Cryptosporidium spp. and Giardia spp. in a public water-treatment system. Samples of raw and treated water were collected and concentrated using the membrane filtration technique. Direct Immunofluorescence Test was performed on the samples. DNA extraction using a commercial kit was performed and the DNA extracted was submitted to a nested-PCR reaction (n-PCR) and sequencing. In the immunofluorescence, 2/24 (8.33%) samples of raw water were positive for Giardia spp.. In n-PCR and sequencing, 2/24 (8.33%) samples of raw water were positive for Giardia spp., and 2/24 (8.33%) samples were positive for Cryptosporidium spp.. The sequencing showed Cryptosporidium parvum and Giardia duodenalis DNA. In raw water, there was moderate correlation among turbidity, color and Cryptosporidium spp. and between turbidity and Giardia spp.. The presence of these protozoans in the water indicates the need for monitoring for water-treatment companies.
O objetivo deste estudo foi investigar a ocorrência de Cryptosporidium spp. e Giardia spp. em um sistema público de tratamento de água. Amostras de água bruta e tratada foram coletadas e concentradas, utilizando-se a técnica de filtração em membranas. Foi realizada a técnica de Imunofluorescência Direta nas amostras. A extração de DNA foi realizada, utilizando-se um kit comercial, e o DNA extraído foi submetido a uma reação de nested-PCR (n-PCR). Na imunofluorescência, 2/24 (8,33%) amostras de água bruta foram positivas para Giardiaspp.. Na n-PCR, 2/24 (8,33%) amostras de água bruta foram positivas para Giardia spp., e 2/24 (8,33%) amostras foram positivas para Cryptosporidium spp.. O sequenciamento demonstrou DNA de Cryptosporidium parvum e de Giardia duodenalis. Na água bruta, houve correlação moderada entre turbidez, cor e Cryptosporidium spp. e entre a turbidez e Giardia spp.. A presença desses protozoários na água indica a necessidade de monitoramento pelas empresas de tratamento de água.
Assuntos
Água/parasitologia , Purificação da Água , Cryptosporidium/isolamento & purificação , Giardia/isolamento & purificação , BrasilRESUMO
The purpose of this study was to investigate the occurrence of Cryptosporidium spp. and Giardia spp. in a public water-treatment system. Samples of raw and treated water were collected and concentrated using the membrane filtration technique. Direct Immunofluorescence Test was performed on the samples. DNA extraction using a commercial kit was performed and the DNA extracted was submitted to a nested-PCR reaction (n-PCR) and sequencing. In the immunofluorescence, 2/24 (8.33%) samples of raw water were positive for Giardia spp.. In n-PCR and sequencing, 2/24 (8.33%) samples of raw water were positive for Giardia spp., and 2/24 (8.33%) samples were positive for Cryptosporidium spp.. The sequencing showed Cryptosporidium parvum and Giardia duodenalis DNA. In raw water, there was moderate correlation among turbidity, color and Cryptosporidium spp. and between turbidity and Giardia spp.. The presence of these protozoans in the water indicates the need for monitoring for water-treatment companies.(AU)
O objetivo deste estudo foi investigar a ocorrência de Cryptosporidium spp. e Giardia spp. em um sistema público de tratamento de água. Amostras de água bruta e tratada foram coletadas e concentradas, utilizando-se a técnica de filtração em membranas. Foi realizada a técnica de Imunofluorescência Direta nas amostras. A extração de DNA foi realizada, utilizando-se um kit comercial, e o DNA extraído foi submetido a uma reação de nested-PCR (n-PCR). Na imunofluorescência, 2/24 (8,33%) amostras de água bruta foram positivas para Giardiaspp.. Na n-PCR, 2/24 (8,33%) amostras de água bruta foram positivas para Giardia spp., e 2/24 (8,33%) amostras foram positivas para Cryptosporidium spp.. O sequenciamento demonstrou DNA de Cryptosporidium parvum e de Giardia duodenalis. Na água bruta, houve correlação moderada entre turbidez, cor e Cryptosporidium spp. e entre a turbidez e Giardia spp.. A presença desses protozoários na água indica a necessidade de monitoramento pelas empresas de tratamento de água.(AU)
Assuntos
Cryptosporidium/isolamento & purificação , Giardia/isolamento & purificação , Água/parasitologia , Purificação da Água , Brasil , Reação em Cadeia da PolimeraseRESUMO
AIM: To study the frequency of congenital cytomegalovirus (CMV) infection in newborns admitted to the Division of Neonatology, using nested polymerase chain reaction (PCR) and DNA to detect differences in blood and urine specimens. METHODS: The study was carried out for eight months. Newborns (n = 520) hospitalized in five hospitals in Campo Grande, Mato Grosso do Sul, Brazil, were checked for CMV by analysing blood and urine samples. RESULTS: Cytomegalovirus was PCR positive in 13 urine and 10 blood samples. Of the 13 positive urine patients, three (23%) had no clinical signs suggestive of CMV, and another three (23%) patients admitted to the neonatal intensive care unit (NICU) had no definite findings of bacterial infection, with negative blood culture and some clinical signs consistent with CMV as cholestasis, hepatomegaly and eosinophilia. Three patients were on mechanical ventilation and showed improvement after prescription of ganciclovir. One CMV positive child progressed to death. CONCLUSION: Cytomegalovirus detection in urine was slightly more efficient than in blood, and showed better sensitivity than in serological analysis (p < 0.01) therefore, boiled urine may be a better and easier specimen tool for CMV diagnosis in neonatal infection. The findings of the present research suggest that patients admitted to the NICU, especially premature infants, whose laboratory results are not compatible with bacterial infection, and exhibiting signs suggestive of CMV infection should have PCR done on urine for confirmation.
RESUMO
The aims of this study were to assess in vitro if bovine oocytes and oviductal epithelial cells from slaughterhouses for in vitro fertilization use may be infected with bovine herpesvirus 1; to analyze whether the treatment with trypsin according to the International Embryo Transfer Society guideline is efficient to inactivate the bovine herpesvirus 1; to morphologically study the virus-oocyte interaction through optical microscopy. In this study, Madin Darby Bovine Kidney (MDBK) cells that were co-cultured with oocytes matured in vitro and exposed to bovine herpesvirus 1 showed a cytopathic effect. The nested polymerase chain reaction for the supernatant was positive for the bovine herpesvirus 1, thus suggesting that the cytopathic effect observed in the MDBK monolayer was seen due to virus replication and not because of any culture toxicity. It was also observed cytopathic effect and positive nested polymerase chain reaction in MDBK cells co-cultured with in vitro maturated oocytes free of virus, but that were co-cultured in uterine epithelial cells pre-infected with bovine herpesvirus 1 and washed or not with trypsin, demonstrating an oocyte contamination by the virus. When trypsin-washing efficacy was evaluated, we could observe that the trypsin treatment was not able to eliminate the bovine herpesvirus 1 of the oocytes, and it was not observed any morphological difference in the infected oocytes.(AU)
Os objetivos do presente estudo foram avaliar in vitro se oócitos bovinos e células epiteliais de oviduto provenientes de abatedouros para uso em fertilização in vitro podem ser infectados com o herpesvírus bovino tipo 1; analisar se o tratamento com tripsina padronizado pelo International Embryo Transfer Society é eficiente para inativar o herpesvírus bovino tipo 1; estudar morfologicamente a interação vírus e oócito pela microscopia óptica. Neste estudo, as células Madin Darby Bovine Kidney (MDBK), que foram cocultivadas com oócitos maturados in vitro e expostos ao herpesvírus bovino tipo 1, apresentaram efeito citopático. A reação em cadeia da polimerase aninhada ao sobrenadante foi positiva para o herpesvírus bovino tipo 1, sugerindo que o efeito citopático observado na monocamada MDBK foi em função da replicação do vírus, mas não devido a qualquer toxicidade da cultura. Também foram mostrados efeito citopático e reação em cadeia da polimerase aninhada positivos em células MDBK cocultivadas com oócitos maturados in vitro isentos de vírus, porém que foram cocultivados em células epiteliais uterinas previamente infectadas com herpesvírus bovino tipo 1, que se lavou ou não com tripsina, demonstrando uma contaminação pelo vírus do oócito. Quando foi avaliada a eficácia de lavagem com a tripsina, foi possível notar que este tratamento não foi capaz de eliminar o herpesvírus bovino tipo 1 dos oócitos, e não foi observada qualquer diferença morfológica nos oócitos infectados.(AU)
Assuntos
Animais , Bovinos , Oócitos , Tripsina/uso terapêutico , Fertilização in vitro , Herpesvirus Bovino 1 , Células Epiteliais , Reação em Cadeia da Polimerase/veterináriaRESUMO
The aims of this study were to assess in vitro if bovine oocytes and oviductal epithelial cells from slaughterhouses for in vitro fertilization use may be infected with bovine herpesvirus 1; to analyze whether the treatment with trypsin according to the International Embryo Transfer Society guideline is efficient to inactivate the bovine herpesvirus 1; to morphologically study the virus-oocyte interaction through optical microscopy. In this study, Madin Darby Bovine Kidney (MDBK) cells that were co-cultured with oocytes matured in vitro and exposed to bovine herpesvirus 1 showed a cytopathic effect. The nested polymerase chain reaction for the supernatant was positive for the bovine herpesvirus 1, thus suggesting that the cytopathic effect observed in the MDBK monolayer was seen due to virus replication and not because of any culture toxicity. It was also observed cytopathic effect and positive nested polymerase chain reaction in MDBK cells co-cultured with in vitro maturated oocytes free of virus, but that were co-cultured in uterine epithelial cells pre-infected with bovine herpesvirus 1 and washed or not with trypsin, demonstrating an oocyte contamination by the virus. When trypsin-washing efficacy was evaluated, we could observe that the trypsin treatment was not able to eliminate the bovine herpesvirus 1 of the oocytes, and it was not observed any morphological difference in the infected oocytes.
Os objetivos do presente estudo foram avaliar in vitro se oócitos bovinos e células epiteliais de oviduto provenientes de abatedouros para uso em fertilização in vitro podem ser infectados com o herpesvírus bovino tipo 1; analisar se o tratamento com tripsina padronizado pelo International Embryo Transfer Society é eficiente para inativar o herpesvírus bovino tipo 1; estudar morfologicamente a interação vírus e oócito pela microscopia óptica. Neste estudo, as células Madin Darby Bovine Kidney (MDBK), que foram cocultivadas com oócitos maturados in vitro e expostos ao herpesvírus bovino tipo 1, apresentaram efeito citopático. A reação em cadeia da polimerase aninhada ao sobrenadante foi positiva para o herpesvírus bovino tipo 1, sugerindo que o efeito citopático observado na monocamada MDBK foi em função da replicação do vírus, mas não devido a qualquer toxicidade da cultura. Também foram mostrados efeito citopático e reação em cadeia da polimerase aninhada positivos em células MDBK cocultivadas com oócitos maturados in vitro isentos de vírus, porém que foram cocultivados em células epiteliais uterinas previamente infectadas com herpesvírus bovino tipo 1, que se lavou ou não com tripsina, demonstrando uma contaminação pelo vírus do oócito. Quando foi avaliada a eficácia de lavagem com a tripsina, foi possível notar que este tratamento não foi capaz de eliminar o herpesvírus bovino tipo 1 dos oócitos, e não foi observada qualquer diferença morfológica nos oócitos infectados.
Assuntos
Animais , Bovinos , Células Epiteliais , Fertilização in vitro , Herpesvirus Bovino 1 , Oócitos , Tripsina/uso terapêutico , Reação em Cadeia da PolimeraseRESUMO
The aims of this study were to assess in vitro if bovine oocytes and oviductal epithelial cells from slaughterhouses for in vitro fertilization use may be infected with bovine herpesvirus 1; to analyze whether the treatment with trypsin according to the International Embryo Transfer Society guideline is efficient to inactivate the bovine herpesvirus 1; to morphologically study the virus-oocyte interaction through optical microscopy. In this study, Madin Darby Bovine Kidney (MDBK) cells that were co-cultured with oocytes matured in vitro and exposed to bovine herpesvirus 1 showed a cytopathic effect. The nested polymerase chain reaction for the supernatant was positive for the bovine herpesvirus 1, thus suggesting that the cytopathic effect observed in the MDBK monolayer was seen due to virus replication and not because of any culture toxicity. It was also observed cytopathic effect and positive nested polymerase chain reaction in MDBK cells co-cultured with in vitro maturated oocytes free of virus, but that were co-cultured in uterine epithelial cells pre-infected with bovine herpesvirus 1 and washed or not with trypsin, demonstrating an oocyte contamination by the virus. When trypsin-washing efficacy was evaluated, we could observe that the trypsin treatment was not able to eliminate the bovine herpesvirus 1 of the oocytes, and it was not observed any morphological difference in the infected oocytes.
RESUMO Os objetivos do presente estudo foram avaliar in vitro se oócitos bovinos e células epiteliais de oviduto provenientes de abatedouros para uso em fertilização in vitro podem ser infectados com o herpesvírus bovino tipo 1; analisar se o tratamento com tripsina padronizado pelo International Embryo Transfer Society é eficiente para inativar o herpesvírus bovino tipo 1; estudar morfologicamente a interação vírus e oócito pela microscopia óptica. Neste estudo, as células Madin Darby Bovine Kidney (MDBK), que foram cocultivadas com oócitos maturados in vitro e expostos ao herpesvírus bovino tipo 1, apresentaram efeito citopático. A reação em cadeia da polimerase aninhada ao sobrenadante foi positiva para o herpesvírus bovino tipo 1, sugerindo que o efeito citopático observado na monocamada MDBK foi em função da replicação do vírus, mas não devido a qualquer toxicidade da cultura. Também foram mostrados efeito citopático e reação em cadeia da polimerase aninhada positivos em células MDBK cocultivadas com oócitos maturados in vitro isentos de vírus, porém que foram cocultivados em células epiteliais uterinas previamente infectadas com herpesvírus bovino tipo 1, que se lavou ou não com tripsina, demonstrando uma contaminação pelo vírus do oócito. Quando foi avaliada a eficácia de lavagem com a tripsina, foi possível notar que este tratamento não foi capaz de eliminar o herpesvírus bovino tipo 1 dos oócitos, e não foi observada qualquer diferença morfológica nos oócitos infectados.
RESUMO
The aims of this study were to assess in vitro if bovine oocytes and oviductal epithelial cells from slaughterhouses for in vitro fertilization use may be infected with bovine herpesvirus 1; to analyze whether the treatment with trypsin according to the International Embryo Transfer Society guideline is efficient to inactivate the bovine herpesvirus 1; to morphologically study the virus-oocyte interaction through optical microscopy. In this study, Madin Darby Bovine Kidney (MDBK) cells that were co-cultured with oocytes matured in vitro and exposed to bovine herpesvirus 1 showed a cytopathic effect. The nested polymerase chain reaction for the supernatant was positive for the bovine herpesvirus 1, thus suggesting that the cytopathic effect observed in the MDBK monolayer was seen due to virus replication and not because of any culture toxicity. It was also observed cytopathic effect and positive nested polymerase chain reaction in MDBK cells co-cultured with in vitro maturated oocytes free of virus, but that were co-cultured in uterine epithelial cells pre-infected with bovine herpesvirus 1 and washed or not with trypsin, demonstrating an oocyte contamination by the virus. When trypsin-washing efficacy was evaluated, we could observe that the trypsin treatment was not able to eliminate the bovine herpesvirus 1 of the oocytes, and it was not observed any morphological difference in the infected oocytes.(AU)
Os objetivos do presente estudo foram avaliar in vitro se oócitos bovinos e células epiteliais de oviduto provenientes de abatedouros para uso em fertilização in vitro podem ser infectados com o herpesvírus bovino tipo 1; analisar se o tratamento com tripsina padronizado pelo International Embryo Transfer Society é eficiente para inativar o herpesvírus bovino tipo 1; estudar morfologicamente a interação vírus e oócito pela microscopia óptica. Neste estudo, as células Madin Darby Bovine Kidney (MDBK), que foram cocultivadas com oócitos maturados in vitro e expostos ao herpesvírus bovino tipo 1, apresentaram efeito citopático. A reação em cadeia da polimerase aninhada ao sobrenadante foi positiva para o herpesvírus bovino tipo 1, sugerindo que o efeito citopático observado na monocamada MDBK foi em função da replicação do vírus, mas não devido a qualquer toxicidade da cultura. Também foram mostrados efeito citopático e reação em cadeia da polimerase aninhada positivos em células MDBK cocultivadas com oócitos maturados in vitro isentos de vírus, porém que foram cocultivados em células epiteliais uterinas previamente infectadas com herpesvírus bovino tipo 1, que se lavou ou não com tripsina, demonstrando uma contaminação pelo vírus do oócito. Quando foi avaliada a eficácia de lavagem com a tripsina, foi possível notar que este tratamento não foi capaz de eliminar o herpesvírus bovino tipo 1 dos oócitos, e não foi observada qualquer diferença morfológica nos oócitos infectados.(AU)
Assuntos
Animais , Bovinos , Oócitos , Células Epiteliais , Fertilização in vitro , Herpesvirus Bovino 1 , Tripsina/uso terapêutico , Reação em Cadeia da PolimeraseRESUMO
The purpose of this study was to investigate the occurrence of Cryptosporidium spp. and Giardia spp. in a public water-treatment system. Samples of raw and treated water were collected and concentrated using the membrane filtration technique. Direct Immunofluorescence Test was performed on the samples. DNA extraction using a commercial kit was performed and the DNA extracted was submitted to a nested-PCR reaction (n-PCR) and sequencing. In the immunofluorescence, 2/24 (8.33%) samples of raw water were positive for Giardia spp.. In n-PCR and sequencing, 2/24 (8.33%) samples of raw water were positive for Giardia spp., and 2/24 (8.33%) samples were positive for Cryptosporidium spp.. The sequencing showed Cryptosporidium parvum and Giardia duodenalis DNA. In raw water, there was moderate correlation among turbidity, color and Cryptosporidium spp. and between turbidity and Giardia spp.. The presence of these protozoans in the water indicates the need for monitoring for water-treatment companies.
O objetivo deste estudo foi investigar a ocorrência de Cryptosporidium spp. e Giardia spp. em um sistema público de tratamento de água. Amostras de água bruta e tratada foram coletadas e concentradas, utilizando-se a técnica de filtração em membranas. Foi realizada a técnica de Imunofluorescência Direta nas amostras. A extração de DNA foi realizada, utilizando-se um kit comercial, e o DNA extraído foi submetido a uma reação de nested-PCR (n-PCR). Na imunofluorescência, 2/24 (8,33%) amostras de água bruta foram positivas para Giardiaspp.. Na n-PCR, 2/24 (8,33%) amostras de água bruta foram positivas para Giardia spp., e 2/24 (8,33%) amostras foram positivas para Cryptosporidium spp.. O sequenciamento demonstrou DNA de Cryptosporidium parvum e de Giardia duodenalis. Na água bruta, houve correlação moderada entre turbidez, cor e Cryptosporidium spp. e entre a turbidez e Giardia spp.. A presença desses protozoários na água indica a necessidade de monitoramento pelas empresas de tratamento de água.
RESUMO
The purpose of this study was to investigate the occurrence of Cryptosporidium spp. and Giardia spp. in a public water-treatment system. Samples of raw and treated water were collected and concentrated using the membrane filtration technique. Direct Immunofluorescence Test was performed on the samples. DNA extraction using a commercial kit was performed and the DNA extracted was submitted to a nested-PCR reaction (n-PCR) and sequencing. In the immunofluorescence, 2/24 (8.33%) samples of raw water were positive for Giardia spp.. In n-PCR and sequencing, 2/24 (8.33%) samples of raw water were positive for Giardia spp., and 2/24 (8.33%) samples were positive for Cryptosporidium spp.. The sequencing showed Cryptosporidium parvum and Giardia duodenalis DNA. In raw water, there was moderate correlation among turbidity, color and Cryptosporidium spp. and between turbidity and Giardia spp.. The presence of these protozoans in the water indicates the need for monitoring for water-treatment companies.
O objetivo deste estudo foi investigar a ocorrência de Cryptosporidium spp. e Giardia spp. em um sistema público de tratamento de água. Amostras de água bruta e tratada foram coletadas e concentradas, utilizando-se a técnica de filtração em membranas. Foi realizada a técnica de Imunofluorescência Direta nas amostras. A extração de DNA foi realizada, utilizando-se um kit comercial, e o DNA extraído foi submetido a uma reação de nested-PCR (n-PCR). Na imunofluorescência, 2/24 (8,33%) amostras de água bruta foram positivas para Giardiaspp.. Na n-PCR, 2/24 (8,33%) amostras de água bruta foram positivas para Giardia spp., e 2/24 (8,33%) amostras foram positivas para Cryptosporidium spp.. O sequenciamento demonstrou DNA de Cryptosporidium parvum e de Giardia duodenalis. Na água bruta, houve correlação moderada entre turbidez, cor e Cryptosporidium spp. e entre a turbidez e Giardia spp.. A presença desses protozoários na água indica a necessidade de monitoramento pelas empresas de tratamento de água.
RESUMO
The aims of this study were to assess in vitro if bovine oocytes and oviductal epithelial cells from slaughterhouses for in vitro fertilization use may be infected with bovine herpesvirus 1; to analyze whether the treatment with trypsin according to the International Embryo Transfer Society guideline is efficient to inactivate the bovine herpesvirus 1; to morphologically study the virus-oocyte interaction through optical microscopy. In this study, Madin Darby Bovine Kidney (MDBK) cells that were co-cultured with oocytes matured in vitro and exposed to bovine herpesvirus 1 showed a cytopathic effect. The nested polymerase chain reaction for the supernatant was positive for the bovine herpesvirus 1, thus suggesting that the cytopathic effect observed in the MDBK monolayer was seen due to virus replication and not because of any culture toxicity. It was also observed cytopathic effect and positive nested polymerase chain reaction in MDBK cells co-cultured with in vitro maturated oocytes free of virus, but that were co-cultured in uterine epithelial cells pre-infected with bovine herpesvirus 1 and washed or not with trypsin, demonstrating an oocyte contamination by the virus. When trypsin-washing efficacy was evaluated, we could observe that the trypsin treatment was not able to eliminate the bovine herpesvirus 1 of the oocytes, and it was not observed any morphological difference in the infected oocytes.
RESUMO Os objetivos do presente estudo foram avaliar in vitro se oócitos bovinos e células epiteliais de oviduto provenientes de abatedouros para uso em fertilização in vitro podem ser infectados com o herpesvírus bovino tipo 1; analisar se o tratamento com tripsina padronizado pelo International Embryo Transfer Society é eficiente para inativar o herpesvírus bovino tipo 1; estudar morfologicamente a interação vírus e oócito pela microscopia óptica. Neste estudo, as células Madin Darby Bovine Kidney (MDBK), que foram cocultivadas com oócitos maturados in vitro e expostos ao herpesvírus bovino tipo 1, apresentaram efeito citopático. A reação em cadeia da polimerase aninhada ao sobrenadante foi positiva para o herpesvírus bovino tipo 1, sugerindo que o efeito citopático observado na monocamada MDBK foi em função da replicação do vírus, mas não devido a qualquer toxicidade da cultura. Também foram mostrados efeito citopático e reação em cadeia da polimerase aninhada positivos em células MDBK cocultivadas com oócitos maturados in vitro isentos de vírus, porém que foram cocultivados em células epiteliais uterinas previamente infectadas com herpesvírus bovino tipo 1, que se lavou ou não com tripsina, demonstrando uma contaminação pelo vírus do oócito. Quando foi avaliada a eficácia de lavagem com a tripsina, foi possível notar que este tratamento não foi capaz de eliminar o herpesvírus bovino tipo 1 dos oócitos, e não foi observada qualquer diferença morfológica nos oócitos infectados.
RESUMO
BACKGROUND: Hepatitis A virus (HAV) has shown intermediate endemicity in Argentina, but notification of clinical cases has decreased since the introduction of the vaccine in 2005. OBJECTIVES: In order to get insight into the local circulation of this virus after four years of the official introduction of the vaccine, the aims of this study were to provide information on HAV immune status of the adult population of Córdoba city and to conduct environmental surveillance of HAV in sewage and river samples in the same region. STUDY DESIGN: The prevalence of anti-HAV was determined by EIA in 416 samples of people (without prior vaccination) from Córdoba city (2009-2010). Spline regression models were estimated under generalized additive models. Environmental surveillance was conducted in river and sewage samples collected in the same period. Viral detection was performed by RT-Nested PCR of the 5'UTR. RESULTS: In Córdoba, the global prevalence of anti-HAV was 73.5%. It increased with age (p<0.0001) and it was associated with the low-income population (OR: 1.14; 95% CI 1.05-1.25). This prevalence decreased in younger age groups, especially in the high-income population. Environmental monitoring revealed the presence of HAV (IA) in 20.8% and 16.1% of wastewater and river samples, respectively. CONCLUSIONS: As a consequence of a decrease in HAV circulation due to improvements in immunization, socio-economic and hygienic conditions, young adults are becoming increasingly susceptible to HAV infections. Environmental monitoring demonstrated that HAV circulates in the local population; therefore, health care systems should consider the implementation of preventive measures for susceptible adults in order to reduce the risk of HAV infection.
Assuntos
Monitoramento Ambiental , Anticorpos Anti-Hepatite A/sangue , Vírus da Hepatite A Humana/imunologia , Vírus da Hepatite A Humana/isolamento & purificação , Hepatite A/epidemiologia , Adolescente , Adulto , Idoso , Argentina/epidemiologia , Feminino , Vírus da Hepatite A Humana/classificação , Vírus da Hepatite A Humana/genética , Humanos , Técnicas Imunoenzimáticas , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Viral/genética , Estudos Retrospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rios/virologia , Análise de Sequência de DNA , Estudos Soroepidemiológicos , Esgotos/virologia , Adulto JovemRESUMO
La asociación EPOC- neumocitosis está descrita y existe la necesidad de optimizar la diferenciación entre enfermedad y colonización. Demostrar la presencia del Pneumocistys Jirovecci, como patógeno y/o colonizador. Estudio descriptivo, analítico, de cohorte de pacientes con diagnóstico de EPOC del Hospital General del Oeste (Caracas, Venezuela) durante el periodo de abril julio 2012 con seguimiento hasta julio de 2013 y aplicación de la técnica de inmunofluorescencia directa (IFD) y/o PCR anidada (PCRa) en muestra de esputo (espontáneo inducido) durante los periodos asintomáticos o durante la exacerbación del EPOC en seguimiento de un año. Se incluyeron 20 pacientes en el reclutamiento, con seguimiento al primer control de 5 pacientes; de estos solo 2 cumplieron la medición de esputo. Para la tercera evaluación una paciente había fallecido y la otra no cumplió con el seguimiento. Se demostró IFI+ en 10% de los reclutados, todos con clínica de exacerbación de la EPOC. La PCRa se demostró en 45%, 2 con exacerbación y el resto sin exacerbación. De los dos pacientes de seguimiento, una fue positiva para PCRa y no tenía exacerbación, la otra negativa por ambos métodos. Se demostró infección por Pj en los pacientes con EPOC exacerbado a través de IFI y la PCRa señala su positividad en infección pero también en aquellos sin infección o exacerbación documentando así la colonización y potencial fuente de infección para neumocistosis. Se demostró infección por Pj en paciente con exacerbación y colonización a través de la evidencia del genoma del hongo en pacientes sin exacerbación.
Pneumocistosis and COPD association is described and there is a need to differenciate between disease and colonization. To document the presence of Pnemocistys jirovecci as pathogenic or colonizer by direct immunofluoresencence technique (DIF) and/or nested polymerase chain reaction (nPCR) in sputum (spontaneous-induced) during asymptomatic periods or exacerbation of COPD during a year of follow-up. This is a a descriptive, analytic cohort of patients with COPD of the Hospital General del Oeste (Caracas, Venezuela) . They were studied during April - July 2012, with follow-up until July 2013. 20 patients were included. The first control follow up was in 5 patients with only two measures of IFI - PCRa. For the third evaluation one patient had died and the other did not comply with control. IFI + was demonstrated in 10 % of the recruits, all had COPD, exacerbation. PCRa + was demonstrated in 45%, 2 with exacerbation and all other without exacerbation. From the two followed patients one was positive for PCRn and had no exacerbation, the other was negative by both methods. Pj infection was demonstrated in patients with exacerbated COPD by IFI+ and the PCRa positivity in infection but also in those without infection or exacerbation documenting the colonization and potential source of infection for Pj. Pj infection wasdiagnosed in patients with exacerbation COPD and colonization through the evidence of the genome of the fungus in patients without exacerbation.
Assuntos
Humanos , Masculino , Feminino , Doença Pulmonar Obstrutiva Crônica/diagnóstico , Pneumonia por Pneumocystis/diagnóstico , Reação em Cadeia da Polimerase/métodos , Técnica Indireta de Fluorescência para Anticorpo/métodosRESUMO
AIM: To investigate the association between human papillomavirus (HPV) and esophageal squamous cell carcinoma (ESCC) in southern Brazil. METHODS: We studied 189 esophageal samples from 125 patients from three different groups: (1) 102 biopsies from 51 patients with ESCC, with one sample from the tumor and another from normal esophageal mucosa distant from the tumor; (2) 50 esophageal biopsies from 37 patients with a previous diagnosis of head and neck squamous cell carcinoma (HNSCC); and (3) 37 biopsies from esophageal mucosa with normal appearance from 37 dyspeptic patients, not exposed to smoking or alcohol consumption. Nested-polymerase chain reaction (PCR) with the MY09/11 and GP5/6 L1 primers was used to detect HPV L1 in samples fixed in formalin and stored in paraffin blocks. All PCR reactions were performed with a positive control (cervicovaginal samples), with a negative control (Human Genomic DNA) and with a blank reaction containing all reagents except DNA. We took extreme care to prevent DNA contamination in sample collection, processing, and testing. RESULTS: The histological biopsies confirmed the diagnosis of ESCC in 52 samples (51 from ESCC group and 1 from the HNSCC group) and classified as well differentiated (12/52, 23.1%), moderately differentiated (27/52, 51.9%) or poorly differentiated (7/52, 13.5%). One hundred twenty-eight esophageal biopsies were considered normal (51 from the ESCC group, 42 from the HNSCC group and 35 from dyspeptic patients). Nine had esophagitis (7 from the HNSCC and 2 from dyspeptic patients). Of a total of 189 samples, only 6 samples had insufficient material for PCR analysis: 1 from mucosa distant from the tumor in a patient with ESCC, 3 from patients with HNSCC and 2 from patients without cancer. In 183 samples (96.8%) GAPDH, G3PDH and/or ß-globin were amplified, thus indicating the adequacy of the DNA in those samples. HPV DNA was negative in all the 183 samples tested: 52 with ESCC, 9 with esophagitis and 122 with normal esophageal mucosa. CONCLUSION: There was no evidence of HPV infection in different ESCC from southern Brazil.
Assuntos
Carcinoma de Células Escamosas/virologia , DNA Viral/análise , Neoplasias Esofágicas/virologia , Testes de DNA para Papilomavírus Humano , Papillomaviridae/genética , Infecções por Papillomavirus/virologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia , Brasil/epidemiologia , Carcinoma de Células Escamosas/epidemiologia , Carcinoma de Células Escamosas/patologia , Neoplasias Esofágicas/epidemiologia , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Infecções por Papillomavirus/diagnóstico , Infecções por Papillomavirus/epidemiologia , Reação em Cadeia da Polimerase , Valor Preditivo dos Testes , Fatores de Risco , Adulto JovemRESUMO
OBJECTIVE:To evaluate the effectiveness of nested polymerase chain reaction (PCR) for diagnosis of extrapulmonary tuberculosis (ETB), as well as the impact of PCR results on clinical management. MATERIALS AND METHODS:We conducted a study of nested PCR tests in 45 patients and a review of patient hospital files, calculating sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). RESULTS:PCR was positive in 51% of cases; PCR sensitivity for diagnosing TB was 86%, specificity was 79%, PPV was 76%, and NPV was 88%. When solely analyzing urine samples, sensitivity and NPV increased to 100%. PCR exerted an influence on management in 27% of patients. CONCLUSIONS:PCR for rapid diagnosis of extrapulmonary TB has an adequate effect, which improves when performed on urine. The results of PCR exerted an acceptable impact on the clinical management of these patients.
OBJETIVO:Evaluar la eficacia de la reacción en cadena de la polimerasa (PCR) anidada para el diagnóstico de tuberculosis extrapulmonar, así como el impacto de sus resultados en el manejo clínico. MATERIAL Y MÉTODOS: Se realizó PCR anidada en 45 pacientes y se llevó a cabo la revisión de expedientes. Se calculó sensibilidad, especificidad, valor predictivo positivo (VPP) y valor predictivo negativo (VPN). RESULTADOS:La PCR fue positiva en 51% de los casos, la sensibilidad fue de 86%, la especificidad de 79%, el VPP de 76% y el VPN de 88%. Al analizar solamente las muestras de orina, la sensibilidad y VPN se incrementaron a 100%. La PCR influyó en el manejo de 27% de los pacientes. CONCLUSIONES:La PCR para el diagnóstico rápido de TB extrapulmonar tiene una eficacia adecuada, la cual mejora cuando se realiza en orina. El resultado de la PCR tuvo un impacto aceptable en el manejo clínico de estos pacientes.