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

BACKGROUND: Malaria still poses one of the major threats to human health. Development of effective antimalarial drugs has decreased this threat; however, the emergence of drug-resistant Plasmodium falciparum, a cause of Malaria, is disconcerting. The antimalarial drug chloroquine has been effectively used, but resistant parasites have spread worldwide. Interestingly, the withdrawal of the drug reportedly leads to an increased population of susceptible parasites in some cases. We examined the prevalence of genomic polymorphisms in a malaria parasite P. falciparum, associated with resistance to an antimalarial drug chloroquine, after the withdrawal of the drug from Indonesia.

RESULTS: Blood samples were collected from 95 malaria patients in North Sulawesi, Indonesia, in 2010. Parasite DNA was extracted and analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for pfcrt and pfmdr1. In parallel, multiplex amplicon sequencing for the same genes was carried out with Illumina MiSeq. Of the 59 cases diagnosed as P. falciparum infection by microscopy, PCR-RFLP analysis clearly identified the genotype 76T in pfcrt in 44 cases. Sequencing analysis validated the identified genotypes in the 44 cases and demonstrated that the haplotype in the surrounding genomic region was exclusively SVMNT. Results of pfmdr1 were successfully obtained for 51 samples, where the genotyping results obtained by the two methods were completely consistent. In pfmdr1, the 86Y mutant genotype was observed in 45 cases (88.2%).

CONCLUSIONS: Our results suggest that the prevalence of the mutated genotypes remained dominant even 6 years after the withdrawal of chloroquine from this region. Diversified haplotype of the resistance-related locus, potentially involved in fitness costs, unauthorized usage of chloroquine, and/or a short post-withdrawal period may account for the observed high persistence of prevalence.