- Open Access
Evaluation of single nucleotide polymorphisms of pvmdr1 and microsatellite genotype in Plasmodium vivax isolates from Republic of Korea military personnel
- Dong-Il Chung†1,
- Sookwan Jeong†2,
- Sylvatrie-Danne Dinzouna-Boutamba1,
- Hye-Won Yang1,
- Sang-Geon Yeo3,
- Yeonchul Hong1Email author and
- Youn-Kyoung Goo1Email author
© Chung et al. 2015
- Received: 5 January 2015
- Accepted: 11 August 2015
- Published: 4 September 2015
Chloroquine has been administered to the soldiers of the Republic of Korea as prophylaxis against vivax malaria. Recent increase in the number of chloroquine-resistant parasites has raised concern over the chemoprophylaxis and treatment of vivax malaria.
To monitor the development of chloroquine-resistant parasites in the Republic of Korea, analyses of single nucleotide polymorphisms (SNPs) of pvmdr1 and microsatellite markers were performed using samples collected from 55 South Korean soldiers infected with Plasmodium vivax.
Four SNPs, F1076L, T529, E1233, and S1358, were identified. Among these, S1358 was detected for the first time in Korea. The microsatellite-based study revealed higher genetic diversity in samples collected in 2012 than in 2011.
Taken together, the results indicate that P. vivax with a newly identified SNP of pvmdr1 has been introduced into the Korean P. vivax population. Therefore, continuous monitoring for chloroquine-resistant parasites is required for controlling vivax malaria in the Republic of Korea.
- Plasmodium vivax
- Multidrug resistant-1 gene
- Microsatellite marker
- Genetic diversity
Malaria caused by Plasmodium vivax is the most common human malaria infection, affecting 40 % of the world’s population [1, 2]. In the Republic of Korea, vivax malaria had been successfully eliminated by the late 1970s by an effective World Health Organization (WHO) programme. However, this infectious disease has re-emerged, since a soldier was diagnosed with P. vivax infection in 1993 [3, 4]. Since then, vivax malaria has been the only type of malaria detected in the Republic of Korea, accounting for 18,052 cases reported from 1994 to 2013 .
Vivax malaria endemic regions in the Republic of Korea are concentrated near the demilitarized zone that separates South Korea from the Democratic People’s Republic of Korea (DPRK or North Korea). Thus, military personnel and residents living in the demilitarized zone are under high risk of contracting vivax malaria. Military personnel accounted for 25.3 % (1029/4063) of all malaria cases reported from 2008 to 2010 . This percentage rose to 44.7 % (1811/4063) when military personnel diagnosed with vivax malaria following discharge from the service were counted . In this regard, mass chemoprophylaxis using chloroquine and primaquine has been administered to soldiers since the year 1997 to control vivax malaria infection. For the chemoprophylaxis, 300 mg chloroquine is administrated weekly to military personnel from June to August (for 12 weeks), and then, 30 mg primaquine is administrated daily for 2 weeks.
Chloroquine has been used to not only kill P. vivax in asexual blood stages and gametocytes, but also to prevent the spread of malaria in low-risk areas. However, its massive use in the treatment of vivax malaria and continuing long-term chemoprophylaxis could facilitate the acquisition of resistance to chloroquine . Since the first report of chloroquine-resistant P. vivax in 1989 in Papua New Guinea, the number of chloroquine-resistant cases has increased in several countries, including Indonesia, Southeast Asia, India, and Central and South America [9–15]. Although cases of chloroquine-resistant malaria infections have been confirmed recently, chloroquine is still used as the therapeutic and chemoprophylactic drug for P. vivax infections in the Republic of Korea . Thus, the administration of chloroquine to soldiers stationed near the demilitarized zone has raised the concern of accelerating the development of drug-resistant P. vivax.
Monitoring the genetic polymorphism that confers chloroquine resistance to malaria provides useful information regarding the efficacy of drugs in treating malaria. However, compared to P. falciparum, previous studies using genetic markers for chloroquine-resistant P. vivax did not conclude a strong correlation between the genetic markers and chloroquine-resistant phenotype in P. vivax, because the molecular mechanisms of chloroquine resistance in P. vivax is still elusive . Among the genetic markers for chloroquine-resistant P. vivax, the multidrug resistance-1 gene of P. vivax (pvmdr1) has been identified as a possible genetic marker of chloroquine resistance with in vitro characterization of isolates . In Southeast Asia and Papua New Guinea, the Y976F mutation (TAC→TTC) in pvmdr1 has been shown to be correlated with chloroquine resistance [19–21]. In addition, the association of severe malaria and expression levels of pvmdr1 with chloroquine resistance was reported by showing 2.4-fold increase in pvmdr1 expression levels in parasites from patients compared to the susceptible group of vivax malaria in the Brazilian Amazon . This chloroquine resistance appears to be the result of a two-step mutation pathway, in which the F1076L mutation is followed by the Y976F mutation [22, 23]. The F1076L mutation is found in all Korean samples tested, and is unlikely to result in chloroquine treatment failure , while the Y976F mutation has not yet been reported in the Republic of Korea . However, considering that the South Korean military has been performing mass chemoprophylaxis for more than 15 years, there likely is strong evolutionary pressure for selection of the double mutant.
Recently, the genetic diversity as well as intra- and inter-population relationships of P. vivax isolates obtained from the Republic of Korea (from 1994 to 2008) were analysed [26, 27]. By using microsatellite markers, this analysis provided an explanation for the genetic diversity observed among strains. In this study, the prevalence of five common non-synonymous single-nucleotide polymorphisms (SNPs) and four synonymous SNPs at the pvmdr1 locus, including the Y976F and F1076L, was examined in 55 P. vivax isolates obtained from military vivax malaria patients who had taken chemoprophylaxis near the demilitarized zone of the Republic of Korea. The population structure of these isolates was analysed using the microsatellite method with 10 microsatellite markers.
This study was approved by the ethics committee of the Army Forces Medical Command (Approval No. AFMC-13-IRB-053, July 2011). An approval form was used to obtain written informed consent from each participant and all participants provided their informed consent for collecting a 5-mL blood sample.
Blood samples and DNA extraction
Identification of single nucleotide polymorphism (SNP) in the pvmdr1 gene
Primers and cycling condition of a nested PCR for amplifying pvmdr1
Annealing temperature (°C)
Size of PCR product (bp)
Analysis of ten microsatellite markers
To determine the relationships between the different pvmdr1 genotypes of P. vivax, 10 microsatellite markers were typed for 28 samples collected in the year 2011 and 27 collected in the year 2012. The 10 microsatellite markers used for this assay were as follows: MS1, MS3, MS5, MS8, MS10, MS12, MS16, MS20, Msp1F7, and Pv3,27. The primer sets and amplification conditions used for the PCRs have been described elsewhere [30, 31]. The fluorophore-labelled PCR products were quantified using an Applied Biosystems 3730 DNA Analyzer with the GeneMapper software Version 4.0 (Applied Biosystems, USA). In order to reduce potential artifacts from background noise or stutter, an arbitrary fluorescent intensity threshold of 50 relative florescence units was applied for peak detection. All electropherogram traces were additionally inspected manually. For each isolate, at each locus, the predominant allele, the highest intensity peak and any additional alleles with a peak height of at least one-third of the height of the predominant allele were scored . Genotyping success was defined as the presence of at least one allele at a given locus in a given sample.
Population genetic analyses and statistical treatments
The major alleles of each locus were used for our population genetic analysis. The level of genetic diversity of the P. vivax population in Republic of Korea was assessed by allele number per locus (A) and expected heterozygosity (He). The He values for each locus were calculated using the formula He = [n/(n − 1)] [1 − ∑p i 2 ], where n is the number of isolates examined and p i is the frequency of the ith allele. The statistical significance of the differences in these values was evaluated by Welch’s t test.
Multilocus linkage disequilibrium (LD) was assessed using LIAN v3.6 based on the allelic data for the 10 microsatellite DNA loci . This program computed the standardized index of association (I A S ), a measure of genotype-wide linkage. The P-values were determined by a Monte Carlo simulation process, performing 100,000 iterations. Only those samples for which a complete set of microsatellite alleles were scored were used for this analysis. Additionally, the multilocus genotypes found in multiple isolates were only counted once in the analysis .
Microsatellite genotypes of the isolates were determined based on a combination of the allelic data of the 10 loci. The relationships between the genotypes were determined by eBURST analysis .
In the Republic of Korea, an extensive malaria chemoprophylaxis campaign using chloroquine and primaquine has been conducted annually since the year 1997. The cumulative numbers of the soldiers receiving this treatment exceeded approximately 1.8 million by 2011. Although this chemoprophylaxis has contributed to the containment of vivax malaria, the possibility of the emergence of chloroquine-resistant P. vivax strains has been a concern. Indeed, chemoprophylaxis failure has been reported in several cases, despite the attainment of sufficiently high plasma concentrations of hydroxychloroquine [6, 16]. Therefore, monitoring for chloroquine-resistant P. vivax is important for the control of malaria in Republic of Korea.
Distribution of pvmdr1 mutations among the P. vivax isolates obtained from the South Korean soldiers
Year % of mutated isolates (no. of isolates with mutation/total no. of isolates)
K1393 N (AAG/AAC)
Following the identification of SNPs, the multiple clone infection pattern, genetic diversity as well as inter- and intra-population differences between the pvmdr1 groups was evaluated using 10 loci. Different alleles sizes observed in a single locus were classified as a multiplicity of infection (MOI). The MOI referred to multiple clone infection. Multiple clone infection was observed in some of the microsatellite loci in 24 of the 55 isolates (49.1 %). Multiple clone infection occurred more frequently in samples from 2011 (60.7 %, n = 17) than in those from 2012 (37.0 %, n = 10). The number of MOI loci per sample was also examined. The highest number of MOI loci per isolate was four, which we observed in a single isolate.
Genetic diversity and multilocus linkage disequilibrium in P. vivax populations
I A S
5.23 ± 0.76
0.52 ± 0.29
6.49 ± 0.49
0.72 ± 0.14
Thirty genotypes of the P. vivax population in the Republic of Korea
SNP of pvmdr1
In conclusion, the pvmdr1 gene was analysed in samples collected from South Korean soldiers. The results showed that an isolate with a new SNP (S1358) of pvmdr1 has been introduced into the Korean P. vivax population and that the genetic diversity of the Korean P. vivax population is likely to be greater in 2012 than in 2011. Therefore, further continuous monitoring for the presence of chloroquine resistant parasites using molecular markers is needed for the control of vivax malaria in the Republic of Korea.
DIC, YH, and YKG designed the study; SJ, SDDB and HWY conducted the experiments; DIC, YH, and YKG were involved in data analysis; SJ provided the materials; YH and YKG contributed to writing the manuscript. All authors read and approved the final manuscript.
This work was supported by the Korean Military Medical Research Project funded by the Republic of Korea Ministry of National Defense (ROK-MND-2012-KMMRP-15) and Kyungpook National University Research Fund, 2012. We thank the medical officers in the Republic of Korea Armed Forces Medical Command for providing the patient data.
Compliance with ethical guidelines
Competing interests The authors declare that they have no competing interests.
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