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Otimização do protocolo de deteção do CMV em Dried blood spots em diferentes materiais
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Resumo: O Citomegalovírus humano (HCMV) é responsável pela infeção congénita mais comum a nível mundial, afetando entre 0,2% a 2% dos recém-nascidos. Os dados da literatura indicam que a seroprevalência da mulher grávida nos países em desenvolvimento é alta, enquanto nos países desenvolvidos é ligeiramente inferior. A infeção congénita pode ser diagnosticada após o nascimento, através da cultura ou analise por PCR, utilizando amostras de urina e saliva colhidas nas primeiras três semanas de vida. A utilização de Dried blood spots(DBS) permite o diagnóstico tardio, quando surgem sintomas sugestivos de uma infeção congénita não diagnosticada anteriormente. Esta abordagem oferece ainda benefícios para as localidades remotas longes dos centros de testagem. No entanto, a implementação da sua utilização é dificultada pela variação da sensibilidade associada às técnicas utilizadas para a sua análise. O presente estudo procurou avaliar o desempenho de três tipos de papel de filtro: Whatman 903 (W903), Whatman 4 (W4) e Filtro de café (Fc), com o objetivo de diminuir os custos associados à deteção de HCMV em DBS, e deste tipo de amostras poder ser utilizado nos países com poucos recursos económicos. Secundariamente, pretendeu-se otimizar o método de extração de DNA de HCMV dos DBS e estudar a estabilidade de DNA de HCMV em DBS em diferentes papéis de filtro. Foram avaliados cinco métodos de extração, dois métodos de choque térmico, dois que requerem kits e uma com fenol-clorofórmio, e a técnica de amplificação utilizada foi a nested PCR. Foram analisadas amostras com cargas virais de 100 000 UI/mL e 2 000 UI/mL. Os dois métodos de choque térmico, demonstraram maior sensibilidade de deteção, sendo também mais práticos e economicamente mais viáveis. A extrações através do kit ExtractaTM DBS, do QIAamp DNA investigator Kit, e com o fenol-clorofórmio, resultaram numa baixa sensibilidade. O método de choque térmico adaptado foi selecionado, por demonstrar maior sensibilidade de deteção de amostras com 2 000 UI/mL, tendo-se registado também benefícios associados a tempos superiores de eluição e o tampão utilizado. A estabilidade de DNA de HCMV foi testada armazenando os DBS a diferentes temperaturas: 4°C, 25°C e 37°C, durante dois meses e meio e seis meses. Neste ensaio foram analisados DBS com carga viral de 10 000 UI/mL e 2 000 UI/mL. Verificou-se uma conservação da estabilidade de DNA a 4°C e a 37°C e uma perda de estabilidade a 25°C. O Fc apresenta um desempenho semelhante aos restantes papéis de filtro testados, apesar de não ser concebido para a recolha de amostras clínicas. Constitui uma opção significativamente mais económica, contudo as suas propriedades para a recolha de amostras, não demonstraram ser as ideais. O W4 demonstrou ser uma opção económica e apresentou um melhor desempenho global nos parâmetros avaliados, maior estabilidade do DNA e níveis de deteção similares ao papel de filtro de referência (W903).
Abstract: Human cytomegalovirus (HCMV) is responsible for the most common congenital infection worldwide, affecting between 0.2% and 2% of newborns. Literature data indicates that the seroprevalence in pregnant women in developing countries is high, while in developed countries it is slightly lower. Congenital infection can be diagnosed after birth, through culture or PCR analysis, using urine and saliva samples taken in the first three weeks of life. The use of Dried blood spots (DBS) allows for late diagnosis when symptoms suggestive of a previously undiagnosed congenital infection appear. This approach also offers benefits for remote locations far from testing centres. However, the implementation of its use is hampered by the variation in sensitivity associated with the techniques used to analyse it. This study sought to evaluate the performance of three types of filter paper: Whatman 903 (W903), Whatman 4 (W4) and coffee filter (Fc), with the aim of reducing the costs associated with detecting HCMV in DBS, and for this type of sample to be used in countries with few economic resources. Secondly, the aim was to optimise the method for extracting HCMV DNA from DBS and to study the stability of HCMV DNA in DBS on different filter papers. Five extraction methods were evaluated, two heat shock methods, two requiring kits and one with phenol chloroform, and the amplification technique used was nested PCR. Samples with viral loads of 100 000 IU/mL and 2 000 IU/mL were analysed. The two heat shock methods showed greater detection sensitivity and were also more practical and economically viable. Extractions using the ExtractaTM DBS kit, the QIAamp DNA investigator Kit and phenol-chloroform resulted in low sensitivity. The adapted heat shock method was selected because it showed greater sensitivity in detecting samples with 2 000 IU/mL, and there were also benefits associated with longer elution times and the buffer used. The stability of HCMV DNA was tested by storing the DBS at different temperatures: 4°C, 25°C and 37°C, for two and a half and six months. This test analysed DBS with a viral load of 10 000 IU/mL and 2 000 IU/mL. DNA stability was preserved at 4°C and 37°C and stability was lost at 25°C. Fc performs similarly to the other filter papers tested, despite not being designed for the collection of clinical samples. It is a significantly cheaper option, but its properties for sample collection have not proved to be ideal. W4 proved to be an economical option and showed better overall performance in the parameters assessed, greater DNA stability and detection levels similar to the reference filter paper (W903).
Abstract: Human cytomegalovirus (HCMV) is responsible for the most common congenital infection worldwide, affecting between 0.2% and 2% of newborns. Literature data indicates that the seroprevalence in pregnant women in developing countries is high, while in developed countries it is slightly lower. Congenital infection can be diagnosed after birth, through culture or PCR analysis, using urine and saliva samples taken in the first three weeks of life. The use of Dried blood spots (DBS) allows for late diagnosis when symptoms suggestive of a previously undiagnosed congenital infection appear. This approach also offers benefits for remote locations far from testing centres. However, the implementation of its use is hampered by the variation in sensitivity associated with the techniques used to analyse it. This study sought to evaluate the performance of three types of filter paper: Whatman 903 (W903), Whatman 4 (W4) and coffee filter (Fc), with the aim of reducing the costs associated with detecting HCMV in DBS, and for this type of sample to be used in countries with few economic resources. Secondly, the aim was to optimise the method for extracting HCMV DNA from DBS and to study the stability of HCMV DNA in DBS on different filter papers. Five extraction methods were evaluated, two heat shock methods, two requiring kits and one with phenol chloroform, and the amplification technique used was nested PCR. Samples with viral loads of 100 000 IU/mL and 2 000 IU/mL were analysed. The two heat shock methods showed greater detection sensitivity and were also more practical and economically viable. Extractions using the ExtractaTM DBS kit, the QIAamp DNA investigator Kit and phenol-chloroform resulted in low sensitivity. The adapted heat shock method was selected because it showed greater sensitivity in detecting samples with 2 000 IU/mL, and there were also benefits associated with longer elution times and the buffer used. The stability of HCMV DNA was tested by storing the DBS at different temperatures: 4°C, 25°C and 37°C, for two and a half and six months. This test analysed DBS with a viral load of 10 000 IU/mL and 2 000 IU/mL. DNA stability was preserved at 4°C and 37°C and stability was lost at 25°C. Fc performs similarly to the other filter papers tested, despite not being designed for the collection of clinical samples. It is a significantly cheaper option, but its properties for sample collection have not proved to be ideal. W4 proved to be an economical option and showed better overall performance in the parameters assessed, greater DNA stability and detection levels similar to the reference filter paper (W903).
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HCMV DBS Sensitivity Stability