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Contribuição para o estudo de resistência aos antimaláricos e análise de marcadores moleculares de Plasmodium falciparum e do hospedeiro humano, em Maputo, Moçambique
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A malária é de longe a doença parasitária mais importante em Moçambique, constituindo um grave problema de saúde pública no País. Apesar de se considerar que um diagnóstico atempado e um tratamento correcto são os elementos básicos para um programa de controlo da malária bem sucedido, nas últimas décadas, o controlo e tratamento têm sido bastante dificultados pelo aparecimento e disseminação da resistência parasitária aos antimaláricos mais utilizados. Os mecanismos que conferem ao parasita a capacidade de resistir à maioria dos antimaláricos disponíveis não se encontram completamente elucidados.
Deste modo, este trabalho teve como objectivo principal avaliar o envolvimento dos genes pfdhfr, pfdhps, pfcrt, pfmdr1 e pfATPase6 na resistência aos antimaláricos em Moçambique, recorrendo a populações naturais de parasitas em isolados colhidos de doentes com malária em Maputo, Moçambique. Neste contexto, foi efectuada a caracterização clínica dos pacientes com diferentes formas clínicas de malária e os perfis genotípicos relacionados com a resistência em P.
falciparum para quatro antimaláricos: cloroquina, sulfadoxina/pirimetamina, amodiaquina e artemisinina, através da técnica de PCR-RFLP, para os polimorfismos pfcrt K76T e N75E, pfdhfr
N51I, C59R, S108N e I164L, pfdhps A437G e K540E, pfmdr1 N86Y e N1246Y e pfATPase6, G1916 A, G110A, A2694T e G2306A.
Os dados genotípicos foram subsequentemente analisados estatísticamente, no intuito de detectar associações significativas entre a presença de um determinado marcador antes e depois do tratamento com os diferentes antimaláricos utilizados. Foram também avaliados os polimorfismos em dois marcadores moleculares (ICAM-1 e CD36) do hospedeiro relacionados com susceptibilidade/resistência à malária, em isolados de pacientes com e sem malária. Os polimorfismos da variante CYP-450 (CYP2C8), relacionada com o metabolismo dos fármacos
antimaláricos em pacientes com malária, foram também aqui analisados.
Os resultados demonstraram a gravidade do problema de resistência a antimaláricos, evidenciado pelas elevadas prevalências dos alelos mutantes antes e depois dos tratamentos efectuados. Foi aqui observada uma elevação significativa de amostras contendo o alelo mutado pfdhps 437G após do tratamento com Fansidar® e com Fansidar®+Amodiaquina.
Verificou-se existir uma correlação positiva entre o quíntuplo mutante e o número de isolados, depois do tratamento com Fansidar®. Estes resultados indicam reservas na utilização do Fansidar ® como uma componente da primeira linha de tratamento antimalárico no País e apontam o polimorfismo pfdhps 437G como um possível marcador para a monitorização da resistência a este fármaco.Apesar de não significativos, os resultados da análise do ICAM-1 mostraram a existência de uma possível associação entre a presença da mutação ICAM-1kilifi e a infecção malárica nas suas formas grave e não grave, enquanto para o CD36 foi notória a ausência da mutação T1264G no
grupo controle (sem malária). Os resultados da análise dos polimorfismos no gene CYP2C8 demonstraram alguma inconclusividade, tendo no entanto permitido a obtenção de conhecimentos para estudos que possam ser realizados no futuro.
Malaria is the most important parasitic disease and a public health problem in Mozambique. Though a correct therapy and diagnosis are basic for any control program, in the last decades, these have been hampered by the appearance and dissemination of drug resistance to most antimalarials. Mechanisms of drug resistance have not been clarified. This work had the objective to study the relation between a number of molecular markers pfdhfr, pfdhps, pfcrt, pfmdr1 and pfATPase6 in drug resistance in Mozambique, in parasite populations collected in Maputo. Clinical characterization of patients and their profiles associated to resistance in P. falciparum was carried out for 4 drugs, chloroquine, combined sulfadoxine/pyrimethamine, amodiaquine and artemisinin, through PCR-RFLP, for polymorphisms in pfcrt K76T and N75E, pfdhfr N51I, C59R, S108N and I164L, pfdhps A437G and K540E, pfmdr1 N86Y and N1246Y and pfATPase6, G1916 A, G110A, A2694T and G2306A. Statistical analysis was also carried out for a correct evaluation of presence of certain markers and response to treatment. Two other polymorphisms were also studied (ICAM-1 and CD36) in association to susceptibility to infection in isolates from people with or without malaria. Finally, polymorphisms of the gene CYP2C8, associated to drug metabolization in malaria patients was also investigated Results confirmed the severity of the problem of drug resistance in malaria, with very high presence and prevalence of mutant alleles in parasite populations before and after treatment. Pfdhps 437G after Fansidar® or Fansidar®+Amodiaquine treatment was significantly visible. There was also a positive correlation between a quintuple mutation association and number of isolates Fansidar® treatment. These results recommend reduction of the use of this drug as a component of first-line antimalarial treatment in Mozambique and indicate that pfdhps 437G can be used as a marker for resistance in clinical work. Though not significant, data from the studies with ICAM-1 showed a possible association between the presence of mutation of ICAM-1kilifi and malaria infection in their severe and non severe forms, while for CD36 it there was a negative association due to non presence of T1264G in the non malaria control group. The results regarding polymorphisms of the gene CYP2C8 were limiting in their interpretation, but nevertheless, contributed to the gathering of information that may be used in future studies.
Malaria is the most important parasitic disease and a public health problem in Mozambique. Though a correct therapy and diagnosis are basic for any control program, in the last decades, these have been hampered by the appearance and dissemination of drug resistance to most antimalarials. Mechanisms of drug resistance have not been clarified. This work had the objective to study the relation between a number of molecular markers pfdhfr, pfdhps, pfcrt, pfmdr1 and pfATPase6 in drug resistance in Mozambique, in parasite populations collected in Maputo. Clinical characterization of patients and their profiles associated to resistance in P. falciparum was carried out for 4 drugs, chloroquine, combined sulfadoxine/pyrimethamine, amodiaquine and artemisinin, through PCR-RFLP, for polymorphisms in pfcrt K76T and N75E, pfdhfr N51I, C59R, S108N and I164L, pfdhps A437G and K540E, pfmdr1 N86Y and N1246Y and pfATPase6, G1916 A, G110A, A2694T and G2306A. Statistical analysis was also carried out for a correct evaluation of presence of certain markers and response to treatment. Two other polymorphisms were also studied (ICAM-1 and CD36) in association to susceptibility to infection in isolates from people with or without malaria. Finally, polymorphisms of the gene CYP2C8, associated to drug metabolization in malaria patients was also investigated Results confirmed the severity of the problem of drug resistance in malaria, with very high presence and prevalence of mutant alleles in parasite populations before and after treatment. Pfdhps 437G after Fansidar® or Fansidar®+Amodiaquine treatment was significantly visible. There was also a positive correlation between a quintuple mutation association and number of isolates Fansidar® treatment. These results recommend reduction of the use of this drug as a component of first-line antimalarial treatment in Mozambique and indicate that pfdhps 437G can be used as a marker for resistance in clinical work. Though not significant, data from the studies with ICAM-1 showed a possible association between the presence of mutation of ICAM-1kilifi and malaria infection in their severe and non severe forms, while for CD36 it there was a negative association due to non presence of T1264G in the non malaria control group. The results regarding polymorphisms of the gene CYP2C8 were limiting in their interpretation, but nevertheless, contributed to the gathering of information that may be used in future studies.
Descrição
Palavras-chave
Malária Parasitologia Plasmodium falciparum Antimaláricos Hospedeiros Maputo Moçambique
Contexto Educativo
Citação
Editora
Instituto de Higiene e Medicina Tropical