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Malária por Plasmodium falciparum em Angola: estudos genéticos associados a combinações terapêuticas à base de artemisinina
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A falência terapêutica aos antimaláricos constituí um problema relevante para os pacientes, um obstáculo e desafio para os programas de controlo da malária. Com base nos estudos de eficácia terapêutica e de acordo com as recomendações da OMS, em 2006, Angola substituiu a cloroquina pela combinação Artemeter + Lumefantrina (AL) como fármaco de 1ª linha, no tratamento de malária não complicada. Sendo a falência terapêutica uma ocorrência multifatorial, realizamos um estudo integrado, que incluiu fatores inerentes ao parasita e, os associados à metabolização de fármacos no hospedeiro humano. Foram incluídos 103 pacientes com malária não complicada, selecionados entre 2011 e 2013, tratados com AL, seguidos durante 28 dias, de acordo com o protocolo da OMS para o estudo de eficácia terapêutica e colhidas amostras de sague para exames moleculares e HPLC. A extração de DNA das amostras sanguíneas em papel de filtro foi realizada com Fenol-Clorofórmio. A identificação de espécies de Plasmodium e a genotipagem do pfmsp2 foram efetuadas por Nested-PCR; os polimorfismos nos genes pfmdr1 e pfatp6 de P. falciparum e os SNPs em genes das famílias MDR1 e CYP450 no hospedeiro humano foram analisados por PCR-RFLP e RT-PCR, tendo os SNP’s do pfK13 sido processados por sequenciação. A taxa de cura foi de 91,3%; 12,6% (n = 13) dos 103 pacientes apresentavam parasitémia no D3; 73,4% das amostras eram portadoras do alelo selvagem pfmdr1 86N, tendo o alelo mutante pfmdr1-1246Y sido identificado em apenas uma amostra; 11% das amostras apresentaram aumento do número de cópias do pfmdr1; não foram identificadas amostras portadoras de mutação no SNP 769 do gene pfatp6, nem de mutações nos codões estudados do gene pfK13. Relativamente aos polimorfismos nos genes do hospedeiro humano, as variantes que codificam para proteínas de atividade alterada apresentaram as seguintes frequências: MDR1 3435TT (6,9%), MDR1 129CC (1%); CYP1A1 * 2B (0%); CYP2C8 * 2 (4%), CYP2C8 * 3 (0%); CYP3A4 * 1B (54,5%) e CYP3A5 * 3 (4,85%). Os resultados deste estudo sugerem uma alteração do perfil do P. falciparum no que concerne a genética e à suscetibilidade à Artemeter + Lumefantrina em comparação com os dados de 2004; a resposta clínica após tratamento com AL não foi associado com os SNPs analisados no parasita; a taxa de falência encontrada (8.7%) não justificam uma mudança de política terapêutica do país, de acordo com as diretrizes da OMS, mas reforçam a necessidade de uma monitorização regular do fármaco. Em relação aos polimorfismos em genes humanos, os SNP’s analisados apresentaram frequências alélicas semelhantes às reportadas noutros estudos em populações africanas. Não foram observadas associações significativas entre os SNP’s analisados nos genes CYP3A4/5 e a resposta terapêutica à AL; no entanto, foi observada uma associação significativa entre a variante mutante ABC1 3435TT e pacientes com parasitémia recrudescente no período de tratamento, o que sugere um envolvimento deste transportador na resposta ao tratamento com Artemeter + Lumefantrina. Foram constatadas frequências relevantes das variantes alélicas dos genes CYP1B1 e CYP2C8, com atividade enzimática associada à lenta metabolização da amodiaquina, o que sugere a existência na população analisada, de risco de efeitos adversos ao tratamento contendo amodiaquina.
Antimalarials therapeutic failure is a relevant problem for patients, an obstacle and challenge to the malaria control programs. Based on therapeutic efficacy studies and in accordance with WHO recommendations, in 2006, Angola replaced chloroquine by Artemether + Lumefantrine (AL) combination as first line drug for the malaria uncomplicated treatment. Considering the therapeutic failure as a multifactorial event, we developed an integrated study, which includes factors inherent to the parasite susceptibility as well as those associated with the metabolism of drugs in the human host. 103 patients with uncomplicated malaria were include between 2011 and 2013, treated with Artemether + Lumefantrine, followed for 28 days, according to the WHO Protocol for the therapeutic efficacy study and blood samples were collected for molecular analyses and HPLC. DNA extraction from blood samples on filter paper was processed by Phenol-Chloroform method. Plasmodium species identification and pfmsp2 genotyping were performed by Nested-PCR; polymorphisms in the pfmdr1 and pfatp6 of malaria P. falciparum genes and those in CYP450 and MDR1 families genes of the human host was analyzed by RFLP-PCR and RT-PCR and the pfK13 SNP's were performed by sequencing. The cure rate was 91.3%; 12.6% (n = 13) of 103 patients had parasitémia in the D3. The wild type pfmdr1 86N allele was found in 73.4% of the samples; mutant pfmdr1-1246Y was identified in only one sample; 11% of samples showed an increase in the pfmdr1 number of copies. No samples had mutation in the 769 SNP of the pfatp6 gene, as well as in the studied codons of the pfK13 gene. Regarding to the human host genetic polymorphisms, the allelic variants encoding for proteins with altered activity showed the following frequencies: MDR1 3435TT (6,9%), MDR1 129CC (1%); CYP1A1 * 2B (0%); CYP2C8 * 2 (4%), CYP2C8 * 3 (0%); CYP3A4 * 1B (54,5%) e CYP3A5 * 3 (4,85%). The results of this study suggest some change of the P. falciparum regarding the susceptibility to AL combination and to the genetics profile in comparison with 2004 data. The clinical outcome after AL treatment was not associated with any allelic status of the analyzed SNPs of the parasite; the failure rate found (8.7%) do not justify a therapeutic policy change according to WHO guidelines, but reinforce the need for regular drug monitoring. In relation to the human host genetics, the analyzed SNP's displayed allele frequencies similar to those usually reported in other studies in African populations. No significant associations were detected between the analyzed SNP's of CYP3A4/5 genes and AL treatment outcomes; however, it was observed a significant association between carriers of the ABC1 3435TT mutant variant and malaria recrudescence, which suggests some involvement of this allele in the AL treatment response. There were observed relevant frequencies of mutant variants of CYP1B1 and CYP2C8 genes encoding to enzymes associated with low metabolizing activity for amodiaquine, that suggests the existence in the studied population a potential risk of adverse effects to treatment containing amodiaquine.
Antimalarials therapeutic failure is a relevant problem for patients, an obstacle and challenge to the malaria control programs. Based on therapeutic efficacy studies and in accordance with WHO recommendations, in 2006, Angola replaced chloroquine by Artemether + Lumefantrine (AL) combination as first line drug for the malaria uncomplicated treatment. Considering the therapeutic failure as a multifactorial event, we developed an integrated study, which includes factors inherent to the parasite susceptibility as well as those associated with the metabolism of drugs in the human host. 103 patients with uncomplicated malaria were include between 2011 and 2013, treated with Artemether + Lumefantrine, followed for 28 days, according to the WHO Protocol for the therapeutic efficacy study and blood samples were collected for molecular analyses and HPLC. DNA extraction from blood samples on filter paper was processed by Phenol-Chloroform method. Plasmodium species identification and pfmsp2 genotyping were performed by Nested-PCR; polymorphisms in the pfmdr1 and pfatp6 of malaria P. falciparum genes and those in CYP450 and MDR1 families genes of the human host was analyzed by RFLP-PCR and RT-PCR and the pfK13 SNP's were performed by sequencing. The cure rate was 91.3%; 12.6% (n = 13) of 103 patients had parasitémia in the D3. The wild type pfmdr1 86N allele was found in 73.4% of the samples; mutant pfmdr1-1246Y was identified in only one sample; 11% of samples showed an increase in the pfmdr1 number of copies. No samples had mutation in the 769 SNP of the pfatp6 gene, as well as in the studied codons of the pfK13 gene. Regarding to the human host genetic polymorphisms, the allelic variants encoding for proteins with altered activity showed the following frequencies: MDR1 3435TT (6,9%), MDR1 129CC (1%); CYP1A1 * 2B (0%); CYP2C8 * 2 (4%), CYP2C8 * 3 (0%); CYP3A4 * 1B (54,5%) e CYP3A5 * 3 (4,85%). The results of this study suggest some change of the P. falciparum regarding the susceptibility to AL combination and to the genetics profile in comparison with 2004 data. The clinical outcome after AL treatment was not associated with any allelic status of the analyzed SNPs of the parasite; the failure rate found (8.7%) do not justify a therapeutic policy change according to WHO guidelines, but reinforce the need for regular drug monitoring. In relation to the human host genetics, the analyzed SNP's displayed allele frequencies similar to those usually reported in other studies in African populations. No significant associations were detected between the analyzed SNP's of CYP3A4/5 genes and AL treatment outcomes; however, it was observed a significant association between carriers of the ABC1 3435TT mutant variant and malaria recrudescence, which suggests some involvement of this allele in the AL treatment response. There were observed relevant frequencies of mutant variants of CYP1B1 and CYP2C8 genes encoding to enzymes associated with low metabolizing activity for amodiaquine, that suggests the existence in the studied population a potential risk of adverse effects to treatment containing amodiaquine.
Descrição
Palavras-chave
Parasitologia médica Malária Plasmodium falciparum Terapêutica Farmacogenética Angola
Contexto Educativo
Citação
Editora
Instituto de Higiene e Medicina Tropical