Otimização de uma estratégia baseada em PCR em tempo-real para detecção e quantificação do DNA do Herpesvirus Humano 6 (HHV-6) em pacientes submetidos a transplante de células progenitoras hematopoiéticas
Data
2014
Autores
Ueda, Miriam Yurika Hiramoto 
Orientadores
Granato, Celso Francisco Hernandes
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
O Herpesvirus humano 6 (HHV-6) pode causar graves complicações em pacientes submetidos a transplante de células progenitoras hematopoiéticas (TCPH), como encefalite, atraso na enxertia plaquetaria e agravamento da doença do enxerto contra o hospedeiro (DECH). Monitorar esse vírus e prover diagnóstico precoce, rápido e preciso das doenças associadas a ele constitui medida essencial para tentar melhorar os resultados do transplante. Até a presente data, não ha estudo prospectivo sobre a incidência e características clinicas das infecções causadas pelos herpes vírus em receptores TCPH no Brasil. Além disso o impacto da maioria das infecções por Herpesvirus após o transplante permanece obscuro. Nosso objetivo foi padronizar uma técnica de detecção rápida, baseada em PCR em tempo real para detectar e quantificar o HHV-6 em amostras de plasma. O teste foi baseado na tecnologia TaqMan® utilizando sonda de hidrolise. As sequencias de primers e sondas previamente descritas foram otimizadas para avaliação da eficiência, sensibilidade e reprodutibilidade. Foi utilizado como controle interno o controle comercial Exogenous Internal Positive Control (IPC) Reagents (Life Technologies) e como controle positivo células infectadas com HHV-6. O DNA foi extraído em robo semi-automatizado (QIAcube); as reações foram preparadas no robo de pipetagem de alta precisão (QIAgility) e todas as reações foram processadas no equipamento 7900HT (Life Technologies). A eficiência obtida foi de 91,5%; range de linearidade de 10 a 106 copias/reação; limite de detecção após análise dos resultados das amostras de plasma foi de 5 copias/reação ou 250 copias/mL de plasma e o valor do range da variação entre ensaios (reprodutibilidade) foi 0,78 a 2,82. Não foram detectados resultados falsos positivos e nem reações cruzadas com outros vírus. O teste foi aplicado a 1.082 amostras de plasma, coletados de 98 receptores de TCPH. O DNA do HHV-6 foi detectado em 18 (2%) amostras de 12 pacientes. A carga viral mais alta detectada foi de 69.163 copias/mL. A técnica de PCR quantitativa padronizada se mostrou simples, rápida, capaz de detectar uma ampla variação da carga viral. Devido ao formato do teste de alta produção, este método pode ser aplicado na monitoração dos pacientes submetidos ao TCPH. O teste parece ser uma boa ferramenta e um potencial meio de fazer o diagnóstico das infecções causadas pelo HHV-6 e realizar pesquisa clínica na área.
Human Herpesvirus 6 (HHV6) may cause severe complications after Hematopoietic Stem Cell Transplantation (HSCT) as encephalitis, worsening of GVHD and delayed platelet engraftment. Monitoring this virus and providing precise, rapid and early diagnosis of related clinical diseases constitute an essential measure to improve outcomes. A prospective survey on the incidence and clinical features of herpes virus infections after HSCT has not yet been performed in Brazilian patients. Additionally, the impact of most of this infection on the HSCT outcome is still unclear. We aim to standardize a fast test based on realtime PCR (qPCR) to screen and quantify clinical samples for HHV6. The detection step was based on reaction with TaqMan® hydrolysis probes. One set of primers and probes previously described for HHV6 have been tested in order to evaluate efficiency, sensitivity and reproducibility analysis. Exogenous Internal Positive Control (IPC) Reagents (Life Technologies) has been used as an internal control and infected cell culture has been used as HHV6 control. In a semiautomated workflow, the DNA was extracted from plasma sample in the QIAcube robot and the PCR reactions were set up using QIAgility robot for highprecision pipetting and all qPCR reactions have been performed in a 7900HT (Life Technologies). The target efficiency obtained was 91,5%; linearity ranging from at least 10 to 106 copies per reaction; limit of detection after sample results analyzing was 5 copies per reaction or 250 copies/ mL of plasma and the interassay range variation was 0.782.82. No crossreaction or false positive results were detected. We applied this qPCR assay to 1,082 plasma samples collected from 98 HSCT recipients. HHV6 DNA was detected in 18 (2%) plasma samples of 12 patients. The highest viral load detected was 69,163 copies/ plasma mL. The qPCR assay developed here was simple, rapid to perform and sensitive allowing the detection of a wide range of HHV6 viral loads Furthermore, by its highthroughput format, this method is well suited for monitoring this virus after HSCT. It therefore appears to be of potential value in clinical investigation and diagnosis of HHV6 infection.
Human Herpesvirus 6 (HHV6) may cause severe complications after Hematopoietic Stem Cell Transplantation (HSCT) as encephalitis, worsening of GVHD and delayed platelet engraftment. Monitoring this virus and providing precise, rapid and early diagnosis of related clinical diseases constitute an essential measure to improve outcomes. A prospective survey on the incidence and clinical features of herpes virus infections after HSCT has not yet been performed in Brazilian patients. Additionally, the impact of most of this infection on the HSCT outcome is still unclear. We aim to standardize a fast test based on realtime PCR (qPCR) to screen and quantify clinical samples for HHV6. The detection step was based on reaction with TaqMan® hydrolysis probes. One set of primers and probes previously described for HHV6 have been tested in order to evaluate efficiency, sensitivity and reproducibility analysis. Exogenous Internal Positive Control (IPC) Reagents (Life Technologies) has been used as an internal control and infected cell culture has been used as HHV6 control. In a semiautomated workflow, the DNA was extracted from plasma sample in the QIAcube robot and the PCR reactions were set up using QIAgility robot for highprecision pipetting and all qPCR reactions have been performed in a 7900HT (Life Technologies). The target efficiency obtained was 91,5%; linearity ranging from at least 10 to 106 copies per reaction; limit of detection after sample results analyzing was 5 copies per reaction or 250 copies/ mL of plasma and the interassay range variation was 0.782.82. No crossreaction or false positive results were detected. We applied this qPCR assay to 1,082 plasma samples collected from 98 HSCT recipients. HHV6 DNA was detected in 18 (2%) plasma samples of 12 patients. The highest viral load detected was 69,163 copies/ plasma mL. The qPCR assay developed here was simple, rapid to perform and sensitive allowing the detection of a wide range of HHV6 viral loads Furthermore, by its highthroughput format, this method is well suited for monitoring this virus after HSCT. It therefore appears to be of potential value in clinical investigation and diagnosis of HHV6 infection.
Descrição
Citação
UEDA, Miriam Yurika Hiramoto. Otimização de uma estratégia baseada em PCR em temporeal para detecção e quantificação do DNA do Herpesvírus Humano 6 (HHV6) em pacientes submetidos a transplante de células progenitoras hematopoiéticas. 2014. 120 f. Dissertação (Mestrado em Infectologia) – Escola Paulista de Medicina, Universidade Federal de São Paulo, 2014.