Analysis of polymorphisms in Plasmodium falciparum genes related to drug resistance: a survey over four decades under different treatment policies in Brazil

Abstract

BackgroundAnti-malarial resistance in Plasmodium falciparum remains an obstacle for malaria control. Resistance-associated genes were analysed in Brazilian samples over four decades to evaluate the impact of different treatment regimens on the parasite genetic profile.MethodsSamples were collected on filter paper from patients infected in the Amazon region from 1984 to 2011. DNA was extracted with Chelex® 100 and monoinfection confirmed by PCR. SNPs in the pfcrt, pfmdr1, pfdhfr and pfdhps genes were assessed by PCR-RFLP. The pfmdr1 copy number was estimated using real time quantitative PCR with SYBR® Green. Parasite response was assessed ex vivo with seven concentrations of each anti-malarial. Patients were treated according to Brazilian guidelines: quinine plus tetracycline or mefloquine in period 1 and ACT in period 2.ResultsAll 96 samples presented the pfcrt 76T mutant throughout the assessed periods. In addition, all isolates showed ex vivo chloroquine resistance. The pfmdr1 86Y was detected in 1.5% of samples in period 1, and in 25% in period 2. All samples presented the pfmdr1 1246Y. The analysis of pfmdr1 copy number showed amplification in 37.3% in period 1 and in 42% in period 2. Mutations in pfdhfr were shown as follows: 51I in all samples in period 1 and in 81.2% in period 2; 59R in 6.4% in period 2. The pfdhfr 108N and the pfdhps 437G were seen in all samples along time; the pfdhps 540E in 93.7% in period 1 and in 75% in period 2.ConclusionsThe 76T mutation associated to chloroquine resistance is still present in the parasite population, although this anti-malarial was withdrawn from the chemotherapy of P. falciparum in Brazil in the mid-1980s. All isolates assayed ex vivo for chloroquine showed resistant phenotype and 76T. No association was observed between pfmdr1 mutations and resistance to quinine, mefloquine and artemisinin derivatives. Additionally, the pfdhfr 108N mutation was detected in all samples throughout the evaluated periods, demonstrating fixation of the mutant allele in the parasite population. Changes in Brazilian national guidelines for the malaria chemotherapy in the last 27 years yielded a discreet genetic impact in the parasite population.

Assessment of copy number variation in genes related to drug resistance in Plasmodium vivax and Plasmodium falciparum isolates from the Brazilian Amazon and a systematic review of the literature

“…The fixation of the 76T allele is in agreement with other studies in Brazil [43, 55, 56], despite the withdrawal of CQ from national treatment guidelines in the mid-1980s [57]. The presence of the 76Y allele is in accordance with the description of isolates resistant to CQ in the same regions analysed here [43, 55]. The K76T mutation has been proven essential to CQ resistance, as demonstrated by transfection experiments and it was suggested that this mutation in the pfcrt gene confers resistance to CQ by reducing the amount of drug in the digestive vacuole of the parasite [33, 34, 58, 59].…”

“…1). The fixation of the 76T allele is in agreement with other studies in Brazil [43, 55, 56], despite the withdrawal of CQ from national treatment guidelines in the mid-1980s [57]. The presence of the 76Y allele is in accordance with the description of isolates resistant to CQ in the same regions analysed here [43, 55].…”

Genetic polymorphisms in Plasmodium falciparum chloroquine resistance genes, pfcrt and pfmdr1, in North Sulawesi, Indonesia

“…Similar observations have been reported from other African countries [12, 14]. However, 76T remains highly prevalent in Brazil [18], the Thai–Myanmar border [19], and Pakistan [20]. …”

Prevalence of crt and mdr-1 mutations in Plasmodium falciparum isolates from Grande Comore island after withdrawal of chloroquine

“…In Malawi [ 15 , 20 , 21 ], Gambia [ 18 ], Kenya [ 19 ], Ethiopia [ 22 ], and Tanzania [ 23 ], the withdrawal of CQ chemotherapy led to a decline in the frequency of mutant pfcrt and pfmdr - 1 alleles and the return of CQ sensitive (CQS) malaria. In the contrary, high frequencies of CQR strains were still detected in some endemic regions despite the withdrawal of CQ from national treatment guidelines [ 17 , 24 , 25 ].…”