title: Genetic polymorphisms in Plasmodium falciparum chloroquine resistance genes, pfcrt and pfmdr1, in North Sulawesi, Indonesia.
abstract: 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.
authors.affiliation: Department of Medicine, Sam Ratulangi University, Kampus Unsrat, Bahu, Manado, 95115, Indonesia.
authors.forename: Patrick
authors.initials: P
authors.lastname: Reteng
authors.affiliation: Department of Medicine, Sam Ratulangi University, Kampus Unsrat, Bahu, Manado, 95115, Indonesia.
authors.forename: Visia
authors.initials: V
authors.lastname: Vrisca
authors.affiliation: Department of Medicine, Sam Ratulangi University, Kampus Unsrat, Bahu, Manado, 95115, Indonesia.
authors.forename: Inka
authors.initials: I
authors.lastname: Sukarno
authors.affiliation: Department of Medicine, Sam Ratulangi University, Kampus Unsrat, Bahu, Manado, 95115, Indonesia.
authors.forename: Ilham Habib
authors.initials: IH
authors.lastname: Djarkoni
authors.affiliation: Department of Medicine, Sam Ratulangi University, Kampus Unsrat, Bahu, Manado, 95115, Indonesia.
authors.forename: Jane Angela
authors.initials: JA
authors.lastname: Kalangi
authors.affiliation: Department of Medicine, Sam Ratulangi University, Kampus Unsrat, Bahu, Manado, 95115, Indonesia.
authors.forename: George Eduardo
authors.initials: GE
authors.lastname: Jacobs
authors.affiliation: Department of Medical Genome Sciences, University of Tokyo, Kashiwa, Chiba, 277-8562, Japan.
authors.forename: Lucky Ronald
authors.initials: LR
authors.lastname: Runtuwene
authors.affiliation: Faculty of Medicine, Oita University, Yufu, Oita, 879-5593, Japan.|Faculty of Tropical Medicine, Mahidol University, Bangkok, 10400, Thailand.|Research Center for Zoonosis Control, Hokkaido University, North 20, West 10 Kita-ku, Sapporo, Hokkaido, 001-0020, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Eshita
authors.affiliation: Department of Human Sciences, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, 080-8555, Japan.
authors.forename: Ryuichiro
authors.initials: R
authors.lastname: Maeda
authors.affiliation: Department of Medical Genome Sciences, University of Tokyo, Kashiwa, Chiba, 277-8562, Japan.
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Suzuki
authors.affiliation: Department of Medicine, Sam Ratulangi University, Kampus Unsrat, Bahu, Manado, 95115, Indonesia.
authors.forename: Arthur Elia
authors.initials: AE
authors.lastname: Mongan
authors.affiliation: Department of Medicine, Sam Ratulangi University, Kampus Unsrat, Bahu, Manado, 95115, Indonesia.
authors.forename: Sarah Maria
authors.initials: SM
authors.lastname: Warouw
authors.affiliation: Research Center for Zoonosis Control, Hokkaido University, North 20, West 10 Kita-ku, Sapporo, Hokkaido, 001-0020, Japan. junya@czc.hokudai.ac.jp.|Global Station for Zoonosis Control, GI-CoRE, Hokkaido University, North 20, West 10 Kita-ku, Sapporo, Hokkaido, 001-0020, Japan. junya@czc.hokudai.ac.jp.
authors.forename: Junya
authors.initials: J
authors.lastname: Yamagishi
authors.affiliation: Department of Medicine, Sam Ratulangi University, Kampus Unsrat, Bahu, Manado, 95115, Indonesia.
authors.forename: Josef
authors.initials: J
authors.lastname: Tuda
mesh_terms: D000962:Antimalarials / Q000627:therapeutic use; D002738:Chloroquine / Q000627:therapeutic use*; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics; D004351:Drug Resistance / Q000235:genetics*; D018432:Drug Resistance, Multiple / Q000235:genetics; D005787:Gene Frequency; D005838:Genotype; D005843:Geography; D006239:Haplotypes; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D007214:Indonesia; D016778:Malaria, Falciparum / Q000188:drug therapy* / Q000469:parasitology; D026901:Membrane Transport Proteins / Q000235:genetics*; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics*; D009154:Mutation; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D012150:Polymorphism, Restriction Fragment Length; D015800:Protozoan Proteins / Q000235:genetics*
chemical_list: D000962:Antimalarials; D002738:Chloroquine; D016054:DNA, Protozoan; D026901:Membrane Transport Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D015800:Protozoan Proteins; C513498:Mdr1 protein, Plasmodium falciparum; C416369:PfCRT protein, Plasmodium falciparum
keywords: Chloroquine; Genetic polymorphism; Indonesia; Malaria; Multiplex sequencing; North Sulawesi; PCR–RFLP; Plasmodium falciparum; pfcrt; pfmdr1