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Genotipagem do vírus citomegálico humano na infeção congénita
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O CMV é o principal agente de infeção congénita, atingindo cerca de 0,2 a 2,2% de todos os recém-nascidos. Este agente exibe uma imensa variabilidade genética principalmente nos genes que codificam glicoproteínas estruturais do invólucro. A mais relevante é a glicoproteína B (gB) do vírus, codificada pelo gene UL55, sendo um
importante alvo do sistema imunitário do hospedeiro humano. Com base nas variações de sequências deste gene, o vírus pode ser classificado em pelo menos, 4 genótipos distintos (gB1-4).
O objetivo do presente estudo foi determinar quais os genótipos para o UL55, presentes em amostras de casos de infeção congénita e/ou perinatal utilizando a técnica de PCR em tempo real. A confirmação dos resultados foi realizada por técnicas de sequenciação (Sanger e Next-Generation Sequencing) e por análise do polimorfismo do tamanho dos fragmentos de restrição (RFLP). No total, foram analisadas 36 amostras de urina e 20 amostras de líquido amniótico (LA), colhidos entre 2009 e 2016.
Das 35 amostras de urina genotipadas, em 29 foi detetado um genótipo (13 gB1; 7 gB2; 6 gB4; 3 gB3), sendo em 6 detetado mais do que um genótipo. Das 19 amostras de LA, em 17 foi detetado um genótipo (5 gB1; 5 gB2; 5 gB3; 2 gB4) e em 2 foi detetado mais do que um genótipo. Não houve amplificação em 2 amostras (1 urina e 1 LA).
Recorrendo às diferentes técnicas de confirmação, não foi possível confirmar a presença de infeções mistas nem de 5 gB4 detetados em amostras de urina.
A nível nacional, o gB1 parece ser o genótipo mais frequente na infeção congénita, em concordância com o descrito na literatura. Corroborámos também a ideia de que todos os genótipos estão envolvidos neste tipo de infeção.
Por PCR em tempo real foi possível assinalar a existência de infeções mistas, no entanto, estes dados devem ser alvo de melhor análise, dadas as contradições, principalmente em comparação com a sequenciação.
Cytomegalovirus (CMV) is the main congenital infection agent, affecting about 0.2 to 2.2% of all newborns. This pathogen exhibits extensive genetic variability mainly in structural genes encoding envelope glycoproteins. The most relevant is the CMV glycoprotein B (gB), encoded by the UL55 gene, is an important target of the imune system of the human host. On the basis of sequence variation of this gene, the virus can be classified, at least into 4 gB genotypes (types 1-4). The aim of this study was to determine the genotypes for UL55, present in samples from cases of congenital and/or perinatal infection using real-time PCR. Confirmation of the results was performed by sequencing techniques (Sanger and Next-Generation Sequencing) and restriction fragment length polymorphism(RFLP). Overall, we analyzed 36 urines and 20 amniotic fluids (LAs), collected from 2009 to 2016.Thirty-five urine samples could be assigned a gB genotype, in 29 was detected single genotype (13 gB1; 7 gB2; 6 gB4; 3 gB3). The presence of mixed genotypes was also detected in 6 urines. Of the 19 LAs, 17 has detected a single genotype (5 gB1; gB2 5, 5 gB3; two gB4). In 2 was detected mixed genotypes. No amplification was detected in other 2 samples (1 urine and 1 LA). Resorting to differenttechniques of confirmation was not possible to confirm the presence of mixed infections, neither 5 gB4 detected in urine samples. At the national level, the gB1 seems to be the most common genotype in congenital infection, consistent with that described in the literature. Also we corroborate the notion that all genotypes can be involved in this type of infection.For real-time PCR was also possible to note the existence of mixed infections, however, these date should be subjected to further analysis, given the apparent contradiction, in comparison to sequencing techniques.
Cytomegalovirus (CMV) is the main congenital infection agent, affecting about 0.2 to 2.2% of all newborns. This pathogen exhibits extensive genetic variability mainly in structural genes encoding envelope glycoproteins. The most relevant is the CMV glycoprotein B (gB), encoded by the UL55 gene, is an important target of the imune system of the human host. On the basis of sequence variation of this gene, the virus can be classified, at least into 4 gB genotypes (types 1-4). The aim of this study was to determine the genotypes for UL55, present in samples from cases of congenital and/or perinatal infection using real-time PCR. Confirmation of the results was performed by sequencing techniques (Sanger and Next-Generation Sequencing) and restriction fragment length polymorphism(RFLP). Overall, we analyzed 36 urines and 20 amniotic fluids (LAs), collected from 2009 to 2016.Thirty-five urine samples could be assigned a gB genotype, in 29 was detected single genotype (13 gB1; 7 gB2; 6 gB4; 3 gB3). The presence of mixed genotypes was also detected in 6 urines. Of the 19 LAs, 17 has detected a single genotype (5 gB1; gB2 5, 5 gB3; two gB4). In 2 was detected mixed genotypes. No amplification was detected in other 2 samples (1 urine and 1 LA). Resorting to differenttechniques of confirmation was not possible to confirm the presence of mixed infections, neither 5 gB4 detected in urine samples. At the national level, the gB1 seems to be the most common genotype in congenital infection, consistent with that described in the literature. Also we corroborate the notion that all genotypes can be involved in this type of infection.For real-time PCR was also possible to note the existence of mixed infections, however, these date should be subjected to further analysis, given the apparent contradiction, in comparison to sequencing techniques.
Descrição
Palavras-chave
Microbiologia médica Virologia Infeção congénita
Contexto Educativo
Citação
Editora
Instituto de Higiene e Medicina Tropical