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Evolução da resistência in vivo aos inibidores da transcriptase reversa em mães multíparas infectadas pelo vírus da Imunodeficiênca Humana tipo 1 que cumpriram regimes terapêuticos para prevenção da transmissão do vírus aos seus filhos
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A transmissão mãe-filho (TMF) é a principal causa de infecção por HIV-1 em crianças nascidas de mães infectadas nos países em vias de desenvolvimento. A elevada diversidade genética do HIV-1 tem um impacto negativo na eficácia do tratamento. A identificação de mutações no gene pol do HIV-1 descrita na literatura sugere que a resistência a alguns anti-retrovíricos pode ocorrer rapidamente, principalmente em situações de baixa adesão à terapêutica. O presente estudo pretendeu caracterizar a diversidade genética da região da transcriptase reversa (RT) do gene pol numa população de mães multíparas infectadas por HIV-1, avaliar a proporção de mutações de resistência aos inibidores da transcriptase reversa (RTI) em função dos regimes de terapia anti-retrovírica (TAR) de prevenção da TMF administrados entre 1999 e 2008. Um grupo de 68 amostras colhidas pós parto a 34 mães multíparas infectadas por HIV-1 foram submetidas a amplificação de uma região da RT por nested PCR, seguindo-se a sequenciação dos produtos amplificados e classificação molecular das sequências obtidas por análise filogenética. A proporção de mães que cumpriram TAR de prevenção da TMF aumentou de 85,3% (1ª gravidez) para 94,1% (2ª gravidez). A prevenção em regime de terapia tripla (TT) ocorreu em 66,2% dos casos, a terapia dupla (TD) em 8,8% dos casos (administrada apenas até 2003) e a prevenção por HAART em 14,7% dos casos (administrada só depois de 2005). Associados à ausência de prevenção foram diagnosticados 2 casos de TMF do HIV-1. Foi obtida uma sensibilidade de 97% para a amplificação da região RT e um sucesso de 89,4% na sequenciação das amostras amplificadas. A classificação das sequências revelou uma prevalência de 42,4% do subtipo G, de 28,8% do subtipo B, de 13,6% do subtipo C, de 3,4% do subtipo A, de 1,7% do subtipo D, e ainda, foram identificadas duas formas recombinadas CRF02_AG e CRF14_BG/CRF02_AG. Observou-se um aumento na proporção de mutações de resistência aos RTI de 32,1% para 38,7%, partindo do grupo de 1as amostras (1º filho) para o grupo de 2as amostras (2º filho) analisadas. As mutações M184V e L74V (associadas a elevados níveis de resistência aos NRTI) e as mutações K103N, Y181C e Y188L (associadas a elevados níveis de resistência aos NNRTI) foram identificadas no grupo de sequências analisadas. Foram encontradas mutações de resistência aos RTI (T215Y, K219Q e V179D) em mães que não transmitiram o vírus e não fizeram prevenção da TMF. De um modo geral, constatou-se maior prevalência de mutações associadas aos NRTI, nomeadamente ao AZT. A avaliação da proporção de mutações de resistência aos RTI revelou um incremento na sua proporção e diversidade, a partir do grupo de sequências associadas a regimes de TD para o grupo de sequências associadas a regimes de TT. Contrariamente ao descrito na literatura, uma percentagem significativa de mutações de resistência de 22,2% foi encontrada no grupo de sequências derivadas de mães que cumpriram prevenção da TMF por HAART. Estudos de maior amostragem e alargados a outras regiões genómicas do HIV-1 podem conduzir a uma melhor avaliação clinica sobre a influência de mutações de resistência na eficácia dos regimes terapêuticos para tratamento e prevenção da TMF e também melhorar a classificação dos vírus.
The mother-to-child transmission (MTCT) is the leading cause of HIV-1 infection in children born to infected mothers in developing countries. The high genetic diversity of HIV-1 has a negative impact on the effectiveness of the treatment. The identification of mutations in the pol gene of HIV-1 described in the literature suggests that resistance to antiretroviral drugs can occur rapidly, especially in situation of low adherence. The present study aimed to characterize the genetic diversity of the reverse transcriptase (RT) in a population of multiparous mothers infected with HIV-1, assess the proportion of mutations conferring resistance to reverse transcriptase inhibitors (RTI) according to the fulfillment of antiretroviral therapy regimens (ART) for preventing MTCT between 1999 and 2008. A group of 68 samples collected postpartum to 34 multiparous infected mothers were submitted to nested PCR, followed by sequencing of the RT region. The molecular classification of sequences was performed by phylogenetic analysis. The prevention of MTCT was increased from 85.3% (first pregnancies) to 94.1% (second pregnancies) in the group of multiparous mothers studied. The regimens for prevention under triple therapy (TT) have occurred in 66.2% of cases, dual therapy (DT) in 8.8% of cases (administered only until 2003) and HAART prevention in 14.7% of cases (administered only after 2005). Two cases of TMF of HIV-1 were associated to absence of prevention during pregnancy. Sensitivity of 97% for the amplification assay and a success of 89.4% for sequencing the RT region were achieved by the methodology implemented. The classification of the sequences obtained from the group studied revealed the prevalence of 42.4% for subtype G, 28.8% for subtype B, 13.6% for subtype C, 3.4% for subtype A, 1.7% for subtype D, and additionally, two putative recombinants CRF02_AG and CRF14_BG/CRF02_AG. Among the analyzed groups, from the first to the second maternal samples, we observed an increase in the proportion of resistance mutations to RTI from 32.1% to 38.7%. The mutations L74V and M184V (associated with high levels of resistance to NRTIs) and the mutations K103N, Y181C and Y188L (associated with high levels of resistance to NNRTIs) have been identified. In absence of prevention of MTCT, resistance mutations to RTIs like, T215Y, K219Q and V179D, were found in mothers who did not transmitted the virus to their children. In general, there was a higher prevalence of mutations associated with NRTIs, specifically to AZT. An increase in the proportion and diversity of resistance mutations to RTIs was observed from the group that fulfilled TD regimen to the group that fulfilled TT regimen. However, contrary to data described in literature, a significant proportion of resistance mutations of 22% was found in the group of sequences derived from infected mothers in which was fulfilled the HAART regimen for preventing MTCT of HIV-1. Enlarged studies, comprising more samples and analysis of other genomic regions of HIV-1, may lead to a better clinical evaluation of the influence of resistance mutations and efficacy of the regimens for treatment and prevention of MTCT and also to improve the classification of viruses.
The mother-to-child transmission (MTCT) is the leading cause of HIV-1 infection in children born to infected mothers in developing countries. The high genetic diversity of HIV-1 has a negative impact on the effectiveness of the treatment. The identification of mutations in the pol gene of HIV-1 described in the literature suggests that resistance to antiretroviral drugs can occur rapidly, especially in situation of low adherence. The present study aimed to characterize the genetic diversity of the reverse transcriptase (RT) in a population of multiparous mothers infected with HIV-1, assess the proportion of mutations conferring resistance to reverse transcriptase inhibitors (RTI) according to the fulfillment of antiretroviral therapy regimens (ART) for preventing MTCT between 1999 and 2008. A group of 68 samples collected postpartum to 34 multiparous infected mothers were submitted to nested PCR, followed by sequencing of the RT region. The molecular classification of sequences was performed by phylogenetic analysis. The prevention of MTCT was increased from 85.3% (first pregnancies) to 94.1% (second pregnancies) in the group of multiparous mothers studied. The regimens for prevention under triple therapy (TT) have occurred in 66.2% of cases, dual therapy (DT) in 8.8% of cases (administered only until 2003) and HAART prevention in 14.7% of cases (administered only after 2005). Two cases of TMF of HIV-1 were associated to absence of prevention during pregnancy. Sensitivity of 97% for the amplification assay and a success of 89.4% for sequencing the RT region were achieved by the methodology implemented. The classification of the sequences obtained from the group studied revealed the prevalence of 42.4% for subtype G, 28.8% for subtype B, 13.6% for subtype C, 3.4% for subtype A, 1.7% for subtype D, and additionally, two putative recombinants CRF02_AG and CRF14_BG/CRF02_AG. Among the analyzed groups, from the first to the second maternal samples, we observed an increase in the proportion of resistance mutations to RTI from 32.1% to 38.7%. The mutations L74V and M184V (associated with high levels of resistance to NRTIs) and the mutations K103N, Y181C and Y188L (associated with high levels of resistance to NNRTIs) have been identified. In absence of prevention of MTCT, resistance mutations to RTIs like, T215Y, K219Q and V179D, were found in mothers who did not transmitted the virus to their children. In general, there was a higher prevalence of mutations associated with NRTIs, specifically to AZT. An increase in the proportion and diversity of resistance mutations to RTIs was observed from the group that fulfilled TD regimen to the group that fulfilled TT regimen. However, contrary to data described in literature, a significant proportion of resistance mutations of 22% was found in the group of sequences derived from infected mothers in which was fulfilled the HAART regimen for preventing MTCT of HIV-1. Enlarged studies, comprising more samples and analysis of other genomic regions of HIV-1, may lead to a better clinical evaluation of the influence of resistance mutations and efficacy of the regimens for treatment and prevention of MTCT and also to improve the classification of viruses.
Descrição
Tese de mestrado. Biologia Humana e Ambiente. Universidade de Lisboa, Faculdade de Ciências, 2013
Palavras-chave
HIV-1 Resistência aos antivirais Transmissão mãe-filho Teses de Mestrado - 2013