Genetic polymorphisms in Plasmodium falciparum chloroquine resistance genes, pfcrt and pfmdr1, in North Sulawesi, Indonesia
© The Author(s) 2017
Received: 7 October 2016
Accepted: 24 March 2017
Published: 4 April 2017
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.
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%).
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.
KeywordsChloroquine Genetic polymorphism Indonesia Malaria Multiplex sequencing North Sulawesi PCR–RFLP pfcrt pfmdr1 Plasmodium falciparum
Malaria caused by Plasmodium falciparum remains a major health concern, particularly in tropical and subtropical regions. P. falciparum uses anopheline mosquitoes as a vector and spreads rapidly in the tropics and subtropics, where 3.3 billion people are at risk of contracting the parasite . According to a 2016 World Health Organization report, >212 million cases of malaria were reported annually and approximately 429,000 people died from malaria worldwide . In Southeast Asia, 15 million cases of malaria (7% global cases) and 26,000 deaths (6% global deaths) are reportedly associated with malaria each year .
Malaria is curable and preventable; however, the disease has not yet been completely eradicated. P. falciparum develops resistance against available medication, which explains the failed eradication of malaria; medication plays an important role in malaria control programs [2, 3]. Reports suggest that P. falciparum has now developed resistance to most antimalarial drugs, including chloroquine and its derivatives, sulfadoxine–pyrimethamine, mefloquine, and artemisinin [2–6]. Indeed, chloroquine was the standard antimalarial drug; however, chloroquine-resistant P. falciparum emerged in the late 1950s and spread worldwide . It is widely accepted that several polymorphisms play important roles in chloroquine-resistant P. falciparum, particularly a threonine substitution at codon 76 in the P. falciparum chloroquine-resistant transporter (pfcrt) and a tyrosine substitution at codon 86 in P. falciparum multidrug-resistant protein (pfmdr1) [7–11].
Chloroquine resistance is widely distributed; however, reports from some African countries indicate a decline in the resistant parasite population after chloroquine discontinuation [12–16], although this reduction in resistance varied between countries. A study in Malawi demonstrated a marked decrease in the prevalence of the chloroquine-resistant marker from 85 to 15% 13 years after chloroquine discontinuation , where its incidence was only 1/685 in 2009 . Similar observations have been reported from other African countries [12, 14]. However, 76T remains highly prevalent in Brazil , the Thai–Myanmar border , and Pakistan .
Blood samples were collected from 95 malaria patients clinically diagnosed at hospitals in Manado and Bitung, North Sulawesi, Indonesia (Fig. 1), from August to December 2010. The collected blood samples were diagnosed via Giemsa staining and microscopic analysis by medical staff members at each hospital and collected on FTA Elute cards (GE Healthcare Life Sciences, Little Chalfont, UK).
Polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analysis
Primers used in the PCR–RFLP analyses
2nd amplification for PCR–RFLP
2nd amplification for NGS amplicon sequencing
2nd amplification for PCR–RFLP
2nd amplification for NGS amplicon sequencing
Multiplex amplicon sequencing with MiSeq and genotyping
The primary PCR reaction was performed as described above. The second nested PCR analyses used 2 μL of the 100× diluted PCR product from the first amplification step as template, with added tab nucleotides (Table 1). For the third PCR, with added dual indices, 2 μL of the 100× diluted PCR product from the second amplification step and TruSeq® DNA HT and RNA HT Sample Prep Kits (Illumina) were used. The amplicons were quantified with an Agilent 2200 TapeStation (Agilent), and then mixed to achieve the same molecular number. The samples were subjected to MiSeq (Illumina), and then sequenced with a MiSeq v3 Reagent Kit v3 (600-cycle). The short read sequences obtained were mapped onto the pfcrt and pfmdr1 coding sequences extracted from P. falciparum genome version 13, which was obtained from plasmoDB  by bowtie2 . To identify genetic polymorphisms, the mapped results were analyzed with GATK .
Regional differences in the genotypes were examined with Fisher’s exact test.
Results and discussion
Detection of the frequencies of the mutant genotypes of pfcrt and pfmdr1
We conducted PCR–RFLP assays of the pfmdr1 gene in parallel (Fig. 3; Additional file 1: Table S1). We obtained clear results from 51/59 samples. We also detected P. falciparum genomes in 10 cases initially diagnosed as P. vivax and 3 cases without any medical records. Among the 51 samples, the 86Y and 86N genotypes were identified in 45 (88.2%) and 6 (11.8%) cases, respectively. We conducted amplicon sequencing of these samples on MiSeq, which confirmed the correct genotype results in all cases. Based on these results, we can make the following conclusions. First, conventional Giemsa staining is occasionally imprecise and further confirmation is needed to precisely detect the infecting parasite species. Second, PCR–RFLP is a convenient and accurate method, although further sequencing analysis can provide extensive information associated with the surrounding bases. Finally, and most importantly, the dominant population of P. falciparum in this area still has a chloroquine-resistant genotype. We also collected and analyzed blood samples from two neighboring regions, i.e., Manado and Bitun, in North Sulawesi, but found no significant differences between the areas in terms of the prevalence of 76T and 86Y mutants (p = 1.000 and 0.218, respectively).
Comparison of the observed prevalence of pfcrt genotype with that in previous studies
Several previous studies have investigated the prevalence of the mutant genotypes of P. falciparum. A study that covered all of Indonesia in 2004 based on PCR–RLFP analyses showed that most of the genotypes in North Sulawesi were 76T . However, compared to previous studies, we found a slight increase in the prevalence of 76T mutation (from 94.4 to 100%), despite the lack of clear statistical significance (p = 0.290).
Another previous study showed that in Lombok, Indonesia, codons 72–76 comprised SVMNT in most cases (87.5%) and only a few isolates possessed the Southeast Asia type of CVIET (10.4%) during 2002 . The high prevalence of 76T was a common feature of all our observations, but we observed no cases of the CVIET haplotype in North Sulawesi. It is known that SVMNT is dominant in the Pacific region, such as Indonesia, Papua New Guinea, and the Philippines in Southeast Asia. By contrast, CVIET is dominant in Thailand, Vietnam, and Myanmar [27, 28], and the CVIET haplotype is assumed to have been spread from there to Africa . Therefore, our finding that SVMNT occurred exclusively in North Sulawesi may reflect the distinct geological origins of the drug-resistant genotypes to some extent.
Intriguingly, the results of our study were very different from those obtained in previous studies conducted in Malawi and other African countries. In Malawi, the prevalence of the pfcrt 76T genotype decreased significantly from 85% in 1992 to 13% in 2000 [16, 30]. Subsequently, it was only present in 1/685 (0.1%) cases in 2009 . In agreement, a field study showed that the susceptibility rate of the parasites to chloroquine increased from 50% in 1993 to 99% in 2005 . Similar reductions in the prevalence of the pfcrt 76T genotype have also been reported from Kenya, Senegal, and other countries that are mainly located in Africa, although their remission rates were less drastic than that in Malawi [12, 14, 31]. Based on these studies, it is suggested that the mutants are fitter than the wild type under the selective pressure of chloroquine. In contrast, the advantage should disappear without the drug. Thus, the corresponding mutation genotypes may have disappeared after a certain time following the drug’s withdrawal.
To rationalize the observed reduction, it should also be hypothesized that the mutants are less fit than the wild type without the drug pressure. It seems to be true in CVIET, which is a major mutated haplotype around the 76T genotype and observed in the regions where the reduction of 76T was reported. In contrast, the SVMNT haplotype, which we found was dominant in North Sulawesi, is likely to be no less fit than the wild type . Indeed, the fixed prevalence of 76T was also reported even after chloroquine withdrawal in Venezuela, where the SVMNT haplotype is similarly dominant . Thus, differences in the biological features of the CVIET and SVMNT haplotypes with respect to their fitness may contribute to the distinct variations in their prevalence. Another even more confusing factor is the administration policy for the use of antimalarial drugs, where incomplete drug withdrawal and unauthorized chloroquine usage may have exposed the parasite to subtherapeutic concentrations, thereby contributing to the fixed mutant genotype. It has been shown that SVMNT is also involved in resistance to amodiaquine, which is used in a combination of artesunate–amodiaquine (AA) . In Indonesia, AA is used despite the current policy recommending dihydroartemisinin piperaquine (DHP). Therefore, suboptimal administration of AA is potentially involved in the sustained SVMNT haplotype, and the inverse effect by strict substitution from AA to DHP may be expected. In addition, misdiagnosis may contribute to the confusion. Hence, further biological studies combined closely with epidemiological and possibly social science studies may yield a more thorough understanding of the prevalence dynamics of the mutant genotypes and their controls.
Prevalence of the genotypes of pfmdr1
Prevalence rates of pfcrt 76T and pfmdr1 86Y polymorphisms in this and previous studies
Flores (East NusaTenggara)
Armopa (West Papua)
Minahasa (North Sulawesi)
Mamuju (South Sulawesi)
Nias (North Sumatra)
Kokap (Central Java)
Kutai (East Kalimantan)
Upper southern part of Thailand
Lower southern part of Thailand
West part if Thailand
In this study, using samples obtained from malaria patients from North Sulawesi, Indonesia, we examined the chloroquine resistance polymorphisms in P. falciparum, i.e., 76T in pfcrt and 86Y in pfmdr1, by PCR–RFLP followed by multiplex amplicon sequencing. We emphasized the power of employing the multiplex amplicon sequencing method as well as confirming the convenience of using the conventional RFLP–PCR method. The cost of sequencing is decreasing rapidly; thus, a more comprehensive overview of the changes in genotypes throughout the world can be obtained by targeted sequencing of a larger number of genes in parasites or even whole genome sequencing. The results obtained in the present study showed that the prevalence rates of the mutant genotypes were 100 and 88.2% for 76T in pfcrt and 86Y in pfmdr1, respectively. We consider that the fixation of 76T mutations can be explained by the incomplete withdrawal of the drug, the short time interval since the withdrawal of chloroquine, and/or the possible equal fitness costs of the susceptible and resistant SMVNT genotypes. This suggests that re-emergence of the use of chloroquine is by no means guaranteed and that national health authorities should consider the results of continuous molecular surveillance when formulating their malaria treatment policies. To obtain definitive conclusion and rationalize these assumptions, the size of our cohort is not comprehensive enough and more detailed epidemiological information might be required. The multiplex amplicon sequencing system we applied is, however, supportive to this end.
- P. falciparum :
- P. vivax :
polymerase chain reaction–restriction fragment length polymorphism
- pfcrt :
P. falciparum chloroquine-resistant transporter
- pfmdr1 :
P. falciparum multidrug-resistant protein
YS, AEM, SMW, JY, and JT conceived and designed the study. AEM and JT conducted sample collection. PR, VV, IS, IHD, JAK, GEJ, LRR, YE, RM, and JY performed experiments. PR and JY conducted the literature search, performed data extraction and analysis, and interpreted the results. PR drafted and wrote the manuscript. YE, RM, and SMW critically reviewed the manuscript for important intellectual content and revised the manuscript. All authors read and approved the final manuscript.
We are grateful to F. Minamoto and M. Imada for their positive coordination of the Japanese and Indonesian research communities. The authors also gratefully acknowledge support from the Asia-Africa S & T Strategic Cooperation Promotion Program from the Special Coordination Funds for Promoting Science & Technology and a Grant-in-aid for Scientific Research on Innovative Areas “Genome Science” (221S0002) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and JSPS KAKENHI Grant Number 15H05272.
The authors declare that they have no competing interests.
Availability of data and materials
All data were included either in the main text or the supplemental materials except MiSeq raw reads as they were summarized in the Additional file 1: Table S1.
Ethics approval and consent to participate
Samples were collected with written informed consent or verbal consent, confirmed by doctors at Sam Ratulangi University, for participants who had difficulties reading the form. Records of these agreements were kept by the doctors. The study protocol was approved by the local ethical committees of Sam Ratulangi University and the University of Tokyo (approved number 10-49).
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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