RESUMO
CRISPR-Cas12a (also called Cpf1) has been commonly used for genomic editing, based on its ability to generate precise double-stranded DNA (dsDNA) breaks. Recently, it was demonstrated that Cas12a exhibits unspecific ssDNAse activity upon target recognition. This feature allows CRISPR-Cas to be coupled with a ssDNA reporter and generate a fast, accurate and ultrasensitive molecular detection method. Here, we demonstrate that Cas12a was able to detect DNA target sequences corresponding to carbapenemases resistance genes such as KPC, NDM and OXA. Also, with the addition of a reverse-transcription step, we were able to detect viral RNA sequences from DENV, ZIKV and HANTV genomes. In all cases, assay run time was less than two hours. Additionally, we report attomolar levels of detection. This methodology was validated using clinical samples from patients infected with Dengue virus. Reactions were visualized by detection of a fluorescent signal, as well as by the use of a simple lateral flow strip. These results indicate that Cas12a is able to detect both DNA and RNA targets, making it an appropriate and convenient tool to detect all types of pathogens.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Farmacorresistência Bacteriana/genética , Endodesoxirribonucleases/genética , Edição de Genes/métodos , Vírus de RNA/genética , beta-Lactamases/farmacologia , DNA de Cadeia Simples/genética , Dengue/virologia , Vírus da Dengue/genética , Corantes Fluorescentes , Vírus Hantaan/genética , Humanos , Técnicas de Diagnóstico Molecular , Vírus de RNA/patogenicidade , RNA Viral/genética , Zika virus/genéticaRESUMO
An important aspect of virological surveillance is the identification of the detected viruses. Broad surveillance, that typically employs deep sequencing of collected tissue samples, provides the investigator with many sequence files constructed from overlapping stretches of DNA sequences. Directed surveillance for viruses of a specific taxonomic group provides the investigator with sequence files from cDNA amplified using specific primers to conserved viral regions. Here we will describe general approaches to identify hemorrhagic viral agents through phylogenetic analysis of cDNA sequences obtained during surveillance activities.
Assuntos
Vírus Hantaan/genética , Filogenia , Biologia Computacional , DNA Complementar/genética , HumanosRESUMO
Among the members of the Arenaviridae family, Junín virus and Lassa virus represent important human health threats generating annual outbreaks of severe human hemorrhagic fever (HF) in endemic areas of Argentina and Western Africa, respectively. Given the lack of a specific and safe chemotherapy, the search for effective antiviral compounds is a continuous demanding effort. During the last two decades, academic research studies originated important results identifying novel molecules to be considered for further in vivo characterization. This chapter summarizes experimental in vitro approaches used to determine the possible mechanism of action of these antiviral agents.
Assuntos
Antivirais/farmacologia , Arenavirus/efeitos dos fármacos , Arenavirus/fisiologia , Vírus Hantaan/efeitos dos fármacos , Vírus Hantaan/fisiologiaRESUMO
ANTECEDENTES Y OBJETIVO El Virus Hanta es una infección viral que se transmite principalmente por el contacto con roedores que se encuentran fundamentalmente en zonas rurales. Para prevenir su transmisión, una alternativa es realizar campañas comunicacionales con información relevante. En este contexto el Subsecretario de Salud Pública solicita esta síntesis de evidencia con el objetivo de estudiar el impacto de estas campañas comunicacionales en la prevención de este virus, y de esta manera informar la toma de decisiones respecto del impacto de las campañas comunicacionales para prevenir el virus Hanta en la población. METODOLOGÍA Se formuló una estrategia de búsqueda para ser utilizada en las bases de datos Epistemonikos, Health Systems Evidence, Health Evidence, la Biblioteca Cochrane y PubMed con el objetivo de identificar revisiones sistemáticas del tema. que abordaran la pregunta formulada. Al no encontrarse, se buscaron estudios primarios en PubMed y CENTRAL. Se utiliza la metodología de la certeza de la evidencia GRADE. Consultando con expertos en el área, se decidió excluir todos los artículos que contemplaran campañas comunicacionales para otro tipo de enfermedades. RESULTADOS Se utiliza 1 estudio primario, del cual se obtiene los siguientes resultados. -No se puede concluir sobre el impacto de las campañas comunicacionales para promover conductas de prevención del virus Hanta, porque la certeza en la evidencia es muy baja. -Se podría considerar evaluar formalmente el impacto que las campañas comunicacionales ya realizadas han tenido sobre la conducta de la población, y la incidencia del virus en nuestro país.
Assuntos
Orthohantavírus , Vírus Hantaan , Publicidade , Chile , Comunicação , Promoção da SaúdeRESUMO
BACKGROUND: Currently in Chile, due to the frequent clinical suspicion of Hantavirus disease and the high public health impact that this causes, it is necessary to strengthen the criteria for clinical and epidemiological suspicion in the health team. OBJECTIVE: To analyze the information contained in the reports of suspected Hantavirus infection versus the confirmatory diagnosis with the reference technique, IgM capture ELISA anti-hantavirus. Material andMethods: Correlation between the information provided in notifications versus the result of confirmation was analyzed by calculating diagnostic accuracy. RESULTS: 3.4% of 1,566 patients studied (53 cases) was confirmed as SCPH. 58.6% of the analyzed notifications was incomplete. The percentage of positivity of the reference technique associated with fever, myalgia and headache was 80-85%. The presence of immunoblasts (> 10%) showed 25% sensitivity, 98% specificity, 37% PPV, 97% NPV. Thrombocytopenia exhibited 98% sensitivity, 74% specificity, 16% PPV, 100% NPV. CONCLUSION: It is necessary to reinforce the importance of comprehensive data reporting at the health system level. The presence of thrombocytopenia and immunoblasts (> 10%) is highly sensitive and specific, respectively, for detecting patients with SCPH. There is a need to develop training programs in order to optimize the suspicion of Hantavirus infection and appropriate use of health resources.
Assuntos
Notificação de Doenças/normas , Vírus Hantaan/isolamento & purificação , Síndrome Pulmonar por Hantavirus/diagnóstico , Febre Hemorrágica com Síndrome Renal/diagnóstico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antivirais/sangue , Criança , Pré-Escolar , Chile , Ensaio de Imunoadsorção Enzimática , Feminino , Síndrome Pulmonar por Hantavirus/sangue , Febre Hemorrágica com Síndrome Renal/sangue , Humanos , Imunoglobulina M/sangue , Lactente , Masculino , Pessoa de Meia-Idade , Padrões de Referência , Valores de Referência , Sensibilidade e Especificidade , Estudos Soroepidemiológicos , Testes Sorológicos/métodos , Trombocitopenia/sangue , Trombocitopenia/diagnóstico , Adulto JovemRESUMO
Antecedentes: Actualmente en Chile, debido a la elevada sospecha clínica de enfermedad por hantavirus y el alto impacto en salud pública que esto provoca, se hace necesario reforzar al equipo de salud, los criterios de sospecha clínica y epidemiológica de hantavirosis. Objetivo: Analizar la información contenida en las notificaciones de sospecha de infección por hantavirus versus la técnica de referencia para el diagnóstico confirmatorio de casos sospechosos, ELISA IgM de captura anti-hantavirus. Material y Método: Mediante cálculo de precisión diagnóstica se analizó la correlación que existe entre la información entregada en las notificaciones versus el resultado de la confirmación mediante la técnica de referencia. Resultados: De 1.566 pacientes estudiados 3,4% (53 casos) fue confirmado para SCPH. De las notificaciones analizadas 58,6% estaban con datos incompletos. Los porcentajes de positividad de la técnica de referencia asociada a fiebre, mialgia y cefalea, fueron de 80-85%. Destaca que la presencia de inmunoblastos (> 10%), presenta: S: 25%, E: 98%, VPP: 37%, VPN: 97%. Paratrombocitopenia se obtuvo: S: 98%, E: 74%, VPP: 16%, VPN: 100%. Conclusión: Se hace necesario reiterar a nivel del sistema sanitario chileno la importancia de contar con datos completos en los formularios de notificación. La presencia de trombocitopenia e inmunoblastos (> 10%) fue altamente sensible y especifica, respectivamente, en la detección de pacientes con SCPH. Con el fin de optimizar la sospecha de infección por hantavirus, según la definición de caso sospechoso, se plantea la necesidad de desarrollar programas de capacitación para la sospecha clínica y lectura de parámetros de laboratorio, tales como presencia de inmunoblastos en el hemograma, así como incluir un algoritmo con el fin de optimizar la sospecha y el uso adecuado de los recursos sanitarios.
Background: Currently in Chile, due to the frequent clinical suspicion of Hantavirus disease and the high public health impact that this causes, it is necessary to strengthen the criteria for clinical and epidemiological suspicion in the health team. Objective: To analyze the information contained in the reports of suspected Hantavirus infection versus the confirmatory diagnosis with the reference technique, IgM capture ELISA anti-hantavirus. Material andMethods: Correlation between the information provided in notifications versus the result of confirmation was analyzed by calculating diagnostic accuracy. Results: 3.4% of 1,566 patients studied (53 cases) was confirmed as SCPH. 58.6% of the analyzed notifications was incomplete. The percentage of positivity of the reference technique associated with fever, myalgia and headache was 80-85%. The presence of immunoblasts (> 10%) showed 25% sensitivity, 98% specificity, 37% PPV, 97% NPV. Thrombocytopenia exhibited 98% sensitivity, 74% specificity, 16% PPV, 100% NPV. Conclusion: It is necessary to reinforce the importance of comprehensive data reporting at the health system level. The presence of thrombocytopenia and immunoblasts (> 10%) is highly sensitive and specific, respectively, for detecting patients with SCPH. There is a need to develop training programs in order to optimize the suspicion of Hantavirus infection and appropriate use of health resources.
Assuntos
Humanos , Masculino , Feminino , Lactente , Pré-Escolar , Criança , Adolescente , Adulto , Pessoa de Meia-Idade , Idoso , Idoso de 80 Anos ou mais , Adulto Jovem , Vírus Hantaan/isolamento & purificação , Síndrome Pulmonar por Hantavirus/diagnóstico , Notificação de Doenças/normas , Febre Hemorrágica com Síndrome Renal/diagnóstico , Padrões de Referência , Valores de Referência , Trombocitopenia/diagnóstico , Trombocitopenia/sangue , Imunoglobulina M/sangue , Ensaio de Imunoadsorção Enzimática , Testes Sorológicos/métodos , Estudos Soroepidemiológicos , Chile , Sensibilidade e Especificidade , Síndrome Pulmonar por Hantavirus/sangue , Febre Hemorrágica com Síndrome Renal/sangue , Anticorpos Antivirais/sangueRESUMO
Hantavirus pulmonary syndrome (HPS) is a severe disease characterized by a rapid onset of pulmonary edema followed by respiratory failure and cardiogenic shock. The HPS associated viruses are members of the genus Hantavirus, family Bunyaviridae. Hantaviruses have a worldwide distribution and are broadly split into the New World hantaviruses, which includes those causing HPS, and the Old World hantaviruses [including the prototype Hantaan virus (HTNV)], which are associated with a different disease, hemorrhagic fever with renal syndrome (HFRS). Sin Nombre virus (SNV) and Andes virus (ANDV) are the most common causes of HPS in North and South America, respectively. Case fatality of HPS is approximately 40%. Pathogenic New World hantaviruses infect the lung microvascular endothelium without causing any virus induced cytopathic effect. However, virus infection results in microvascular leakage, which is the hallmark of HPS. This article briefly reviews the knowledge on HPS-associated hantaviruses accumulated since their discovery, less than 20 years ago.
Assuntos
Genoma Viral , Vírus Hantaan/fisiologia , Síndrome Pulmonar por Hantavirus/virologia , Febre Hemorrágica com Síndrome Renal/virologia , Pulmão/virologia , Orthohantavírus/fisiologia , Insuficiência Respiratória/virologia , Choque Cardiogênico/virologia , Vírus Sin Nombre/fisiologia , Animais , Antivirais/administração & dosagem , Cricetinae , Europa (Continente) , Orthohantavírus/patogenicidade , Síndrome Pulmonar por Hantavirus/complicações , Síndrome Pulmonar por Hantavirus/tratamento farmacológico , Síndrome Pulmonar por Hantavirus/epidemiologia , Síndrome Pulmonar por Hantavirus/patologia , Febre Hemorrágica com Síndrome Renal/tratamento farmacológico , Febre Hemorrágica com Síndrome Renal/epidemiologia , Febre Hemorrágica com Síndrome Renal/patologia , Humanos , Pulmão/patologia , América do Norte , Filogeografia , Insuficiência Respiratória/tratamento farmacológico , Insuficiência Respiratória/epidemiologia , Insuficiência Respiratória/etiologia , Insuficiência Respiratória/patologia , Ribavirina/administração & dosagem , Choque Cardiogênico/tratamento farmacológico , Choque Cardiogênico/epidemiologia , Choque Cardiogênico/etiologia , Choque Cardiogênico/patologia , Vírus Sin Nombre/patogenicidade , América do SulAssuntos
Vírus Hantaan , Infecções por Hantavirus/diagnóstico , Anticorpos Antivirais/sangue , Criança , Surtos de Doenças , Transmissão de Doença Infecciosa , Vírus Hantaan/isolamento & purificação , Infecções por Hantavirus/epidemiologia , Infecções por Hantavirus/terapia , Infecções por Hantavirus/virologia , Humanos , América do Norte/epidemiologia , Estudos Soroepidemiológicos , América do Sul/epidemiologiaRESUMO
Since 1995 when the first case of hantavirus pulmonary syndrome (HPS) was reported in Patagonia, there have been more than 400 cases of HPS reported in five countries in South America. The first case of HPS was associated with Andes (AND) virus. In this study, we report on the genetic diversity, geographical distribution, and serological features of hantavirus infection in six countries in South America based on 87 HPS cases from Argentina, Bolivia, Chile, Paraguay, and Uruguay. An early immunoglobulin M (IgM), IgA, and IgG humoral response was observed in almost all HPS cases. The IgM response appears to peak 1 or 2 days after the onset of symptoms. Peak IgG antibody titers occur mostly after the first week. Low IgG titers or the absence of IgG was associated with higher mortality rates. The IgA response peaks around day 15 and then rapidly decreases. The results of phylogenetic analysis based on partial M-fragment G1- and G2-encoding sequences showed that HPS cases from the five countries were infected with viruses related to AND or Laguna Negra (LN) virus. Within AND virus-infected persons, at least five major genetic lineages were found; one lineage was detected in Uruguayan and Argentinean cases from both sides of the Rio de la Plata river. Two Paraguayan patients were infected with a virus different from LN virus. According to the results of phylogenetic analyses, this virus probably belongs to a distinct lineage related more closely to the AND virus than to the LN virus, suggesting that there is probably an Oligoryzomys-borne viral variant circulating in Paraguay. These studies may contribute to a better understanding of hantavirus human infection in South America.