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Aplicação da tecnologia de sequenciação da nova geração (NGS), para o estudo da resistência aos antirretrovirais na infeção por vírus da imunodeficiência humana tipo 2
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Resumo(s)
O vírus da imunodeficiência humana tipo 2, um dos agentes etiológicos da SIDA, foi identificado
em 1985. Trata-se de uma infeção zoonótica, com origem nos símios sooty mangabeys. Quando
comparada com a infeção pelo vírus da imunodeficiência humana tipo 1, esta infeção possui menor
taxa de infecciosidade, menor virulência, transmite-se mal sexualmente assim como verticalmente
e consequentemente apresenta menor diversidade genética. Caracteriza-se ainda por ter um período
assintomático, na maior parte dos casos, igual ou superior a 20 anos. No entanto, se não for tratada
pode evoluir para SIDA e morte.
A epidemia por HIV-2 é endémica nalguns países da África Ocidental e encontra-se também fora
do continente africano, em países que possuem ligações a essas regiões. Portugal, pelas suas
relações de colonização com alguns desses países, apresenta o maior número de pessoas com HIV
2, na Europa.
As orientações da DGS, INSA e APECS recomendam a monitorização destes indivíduos após o
diagnóstico, e sempre que haja carga viral detetável, a realização de testes de resistência aos
antirretrovirais para o HIV-2, para facilitar a escolha do melhor regime terapêutico para cada caso.
Devido à ausência de testes comerciais, tornou-se imperativo investir, investigar e desenvolver
metodologias in house, por forma a contribuir para a melhoria da qualidade de vida destes
indivíduos.
O principal objetivo do estudo aqui apresentado foi desenvolver uma metodologia, por
sequenciação de nova geração (NGS-Ion Torrent), capaz de identificar mutações nas regiões de
interesse do gene pol (protéase, transcriptase reversa e integrase), principais alvos terapêuticos para
os antirretrovirais, atualmente disponíveis. O segundo objetivo foi comparar os resultados obtidos
por NGS, com a metodologia utilizada até à data (método Sanger), e por último integrar a nova
metodologia na prática clínica.
Utilizaram-se primers desenhados para as três regiões do gene pol, e recorreu-se à plataforma
Sentosa® para a extração de ácidos nucleicos e criação de bibliotecas. O passo seguinte foi a
realização de PCR por emulsão. Por fim a sequenciação foi realizada por Ion Torrent. Os ficheiros
BAM obtidos após a sequenciação foram tratados e editados para conversão num ficheiro fasta, que
permitisse utilizar um algoritmo de interpretação para identificação de mutações associadas a
resistência aos antirretrovirais.
Neste trabalho, por NGS, foi possível sequenciar com sucesso 92% das amostras para a região da
protéase e 91% para a região da transcriptase reversa. Na região da integrase o sucesso foi menor
(49%), indicando que será necessário continuar a otimizar esta região. No estudo comparativo entre
os dois métodos, obteve-se uma concordância de p<0,001 estatisticamente significativa para as três
regiões, embora por NGS se tenham detetado mais mutações
Apesar da necessidade de continuar a otimizar a região da integrase, a realização do teste de
resistência aos antirretrovirais para o HIV-2, pela metodologia NGS-Ion Torrent mostrou dar
resposta adequada e consequentemente encontra-se em condições de ser utilizada na prática clínica.
Abstract Human immunodeficiency virus type 2, one of the etiological agents of AIDS, was identified in 1985. It is a zoonotic infection that originated in the sooty mangabey monkey. Compared with human immunodeficiency virus type 1, this infection has a lower infectivity rate, is less virulent, is poorly transmitted both sexually and vertically, and consequently has less genetic diversity. It is also characterised by an asymptomatic period of 20 years or more in most cases. However, if left untreated, it can progress to AIDS and death. The HIV-2 epidemic is endemic in some West African countries and is also found outside the African continent in countries with links to these regions. Portugal, due to its colonial links with some of these countries, has the highest number of people living with HIV-2 in Europe. The DGS, INSA and APECS guidelines recommend that these people be monitored after diagnosis and, whenever there is a detectable viral load, that they be tested for resistance to HIV-2 antiretrovirals in order to facilitate the choice of the best therapeutic regimen for each case. Due to the lack of commercial tests, it has become imperative to invest, research and develop in house methodologies to improve the quality of life of these individuals. The main objective of the study presented here was to develop a methodology using next-generation sequencing (NGS Ion Torrent) capable of identifying mutations in the regions of interest of the pol gene (protease, reverse transcriptase and integrase), the main therapeutic targets of currently available antiretroviral drugs. The second objective was to compare the results obtained by NGS with the previously used method (Sanger method) and, finally, to integrate the new method into clinical practice. Primers designed for the three regions of the pol gene were used, and the Sentosa® platform was used to extract nucleic acids and create libraries. The next step was emulsion PCR. Finally, sequencing was performed using Ion Torrent. The BAM files obtained after sequencing were processed and edited to convert them into a fasta file that would allow an interpretation algorithm to be used to identify mutations associated with antiretroviral resistance. In this work, NGS was able to successfully sequence 92% of the samples for the protease region and 91% for the reverse transcriptase region. In the integrase region, the success rate was lower (49%), indicating the need for further optimisation of this region. In the comparative study between the two methods, a statistically significant agreement of p<0.001 was obtained for the three regions, although more mutations were detected by NGS. Despite the need for further optimisation of the integrase region, the antiretroviral resistance test for HIV-2 using the NGS Ion Torrent method proved to be sufficiently sensitive and can therefore be used in clinical practice.
Abstract Human immunodeficiency virus type 2, one of the etiological agents of AIDS, was identified in 1985. It is a zoonotic infection that originated in the sooty mangabey monkey. Compared with human immunodeficiency virus type 1, this infection has a lower infectivity rate, is less virulent, is poorly transmitted both sexually and vertically, and consequently has less genetic diversity. It is also characterised by an asymptomatic period of 20 years or more in most cases. However, if left untreated, it can progress to AIDS and death. The HIV-2 epidemic is endemic in some West African countries and is also found outside the African continent in countries with links to these regions. Portugal, due to its colonial links with some of these countries, has the highest number of people living with HIV-2 in Europe. The DGS, INSA and APECS guidelines recommend that these people be monitored after diagnosis and, whenever there is a detectable viral load, that they be tested for resistance to HIV-2 antiretrovirals in order to facilitate the choice of the best therapeutic regimen for each case. Due to the lack of commercial tests, it has become imperative to invest, research and develop in house methodologies to improve the quality of life of these individuals. The main objective of the study presented here was to develop a methodology using next-generation sequencing (NGS Ion Torrent) capable of identifying mutations in the regions of interest of the pol gene (protease, reverse transcriptase and integrase), the main therapeutic targets of currently available antiretroviral drugs. The second objective was to compare the results obtained by NGS with the previously used method (Sanger method) and, finally, to integrate the new method into clinical practice. Primers designed for the three regions of the pol gene were used, and the Sentosa® platform was used to extract nucleic acids and create libraries. The next step was emulsion PCR. Finally, sequencing was performed using Ion Torrent. The BAM files obtained after sequencing were processed and edited to convert them into a fasta file that would allow an interpretation algorithm to be used to identify mutations associated with antiretroviral resistance. In this work, NGS was able to successfully sequence 92% of the samples for the protease region and 91% for the reverse transcriptase region. In the integrase region, the success rate was lower (49%), indicating the need for further optimisation of this region. In the comparative study between the two methods, a statistically significant agreement of p<0.001 was obtained for the three regions, although more mutations were detected by NGS. Despite the need for further optimisation of the integrase region, the antiretroviral resistance test for HIV-2 using the NGS Ion Torrent method proved to be sufficiently sensitive and can therefore be used in clinical practice.
Descrição
Palavras-chave
Ciências biomédicas Biologia molecular Microbiologia HIV-2 Sida Resistência aos retrovírus NGS