RESUMO
In 2010, a large outbreak of dengue occurred in Santos, Brazil. The detection of the NS1 antigen was used for diagnosis in addition to the detection of IgG, IgM, and RNA. A large number of NS1 false-negative results were obtained. A total of 379 RNA-positive samples were selected for thorough evaluation. NS1 was reactive in 37.7% of cases. Most of the cases were characterized as a secondary infection by dengue 2 virus. Sequencing of NS1 positive and negative isolates did not reveal any mutation that could justify the diagnostic failure. Use of existing NS1 tests in the Brazilian population may present a low negative predictive value, and they should be used with caution, preferentially after performing a validation with samples freshly obtained during the ongoing epidemic.
Assuntos
Técnicas de Laboratório Clínico/métodos , Vírus da Dengue/isolamento & purificação , Dengue/diagnóstico , Dengue/epidemiologia , Surtos de Doenças , Proteínas não Estruturais Virais/análise , Virologia/métodos , Anticorpos Antivirais/sangue , Brasil/epidemiologia , Vírus da Dengue/imunologia , Reações Falso-Negativas , Humanos , Imunoensaio/métodos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , RNA Viral/sangue , Sensibilidade e Especificidade , Proteínas não Estruturais Virais/imunologiaRESUMO
The present study aimed at standardizing a real-time quantitative polymerase chain reaction assay to evaluate the presence of GBV-C/HGV RNA. A "TaqMan" assay using primers and probe derived from the 5¢ NCR region was developed and validated. Two hundred and fifty-three plasma samples from HIV-infected women were tested for GBV-C viremia and antibody against the envelope protein 2. GBV-C RNA was detected in 22.5% of the patients whereas the antibody was identified in 25.3% of the cohort. Detection of viral RNA and of antibodies was mutually exclusive. Viral loads showed a mean of 1,777 arbitrary units / mL, being 1.1 and 13,625 arbitrary units / mL respectively the lowest and highest values measured. We conclude that the real-time quantitative polymerase chain reaction method developed is appropriate for the investigation of GBV-C RNA since it was shown to be highly specific and sensitive, as well as requiring few steps, preventing contamination and providing additional information as to the relative viremia of carriers, a parameter that must be included in studies evaluating the co-factors influencing the clinical outcome of HIV/AIDS.
Assuntos
Infecções Oportunistas Relacionadas com a AIDS/diagnóstico , Anticorpos Antivirais/sangue , Vírus GB C/genética , Hepatite Viral Humana/diagnóstico , Reação em Cadeia da Polimerase/métodos , RNA Viral/análise , Viremia/diagnóstico , Infecções Oportunistas Relacionadas com a AIDS/virologia , Contagem de Linfócito CD4 , Feminino , Vírus GB C/imunologia , Hepatite Viral Humana/virologia , Humanos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Viremia/virologiaRESUMO
The present study aimed at standardizing a real-time quantitative polymerase chain reaction assay to evaluate the presence of GBV-C/HGV RNA. A "TaqMan" assay using primers and probe derived from the 5¢ NCR region was developed and validated. Two hundred and fifty-three plasma samples from HIV-infected women were tested for GBV-C viremia and antibody against the envelope protein 2. GBV-C RNA was detected in 22.5 percent of the patients whereas the antibody was identified in 25.3 percent of the cohort. Detection of viral RNA and of antibodies was mutually exclusive. Viral loads showed a mean of 1,777 arbitrary units / mL, being 1.1 and 13,625 arbitrary units / mL respectively the lowest and highest values measured. We conclude that the real-time quantitative polymerase chain reaction method developed is appropriate for the investigation of GBV-C RNA since it was shown to be highly specific and sensitive, as well as requiring few steps, preventing contamination and providing additional information as to the relative viremia of carriers, a parameter that must be included in studies evaluating the co-factors influencing the clinical outcome of HIV/AIDS.
Este estudo teve como objetivo o desenvolvimento de método de PCR em Tempo Real para a determinação da viremia do vírus GBV-C. Ensaio baseado em primers e sonda "TaqMan" derivados da região 5' não-codificante deste vírus foi padronizado, validado e aplicado em uma série de 253 amostras de plasma de pacientes HIV+. Além do PCR em tempo real, as amostras foram submetidas a um ensaio imunoenzimático anti-E2 e a um nested-PCR. Das 253 amostras testadas, 64 foram positivas para o anticorpo anti-E2 (25,3 por cento), enquanto 57 amostras foram concordantemente RNA positivas pelo nested-PCR e PCR em tempo real (22,5 por cento), perfazendo um índice total de exposição de 48 por cento (25.3 + 22.5). A carga viral teve média de 1.777 UA/mL (13.625 - 1.1UA/mL). Foi obtida metodologia simples, rápida e de boa sensibilidade e especificidade, permitindo a quantificação do RNA do vírus GBV-C com reprodutibilidade. A metodologia permite a análise simultânea de grande número de amostras, sendo apropriada para estudos clínicos. A prevalência de exposição a este agente na população feminina HIV+ estudada é alta, provavelmente decorrente da via sexual comum de transmissão dos agentes.