Background
Of the five human malaria parasites,
Plasmodium vivax is the most prevalent in Asia, Melanesia, the Middle East, South and Central America, accounting for 70-80 million cases annually [
1], with 2.6 billion people at risk of infection [
2]. Despite modest gains in
Plasmodium falciparum control, the global burden of
P. vivax remains underestimated [
3] and emergence of drug resistant
P. vivax makes the control of vivax malaria more difficult than before [
4,
5]. In the Republic of Korea (South Korea), vivax malaria was eradicated once in the late 1970s [
6]. However, indigenous malaria has reemerged since the first infection of a soldier who had never been abroad was confirmed in 1993 in the Demilitarized Zone (DMZ) between South and North Korea [
7]. Thereafter, vivax malaria infection has spread to civilians and the number of cases from 2000 has fluctuated between 864 and 4,142 [
8]. Thus, vivax malaria has become endemic again and constitutes a serious public concern in South Korea. Meanwhile, in North Korea, vivax malaria is more prevalent than in South Korea and the number of cases has fluctuated between 7,436 and 296,540 from 2000 [
6,
9].
Genetic diversity and population structure of
P. vivax have a significant impact on malaria transmission, spread of drug-resistance and the acquisition of protective immunity against malaria. Studies of the parasite population diversity have practical significance for the strategic development and deployment of control measures [
10]. Limited studies on antigen-encoding genes such as merozoite surface protein-1 (MSP-1), circumsporozoite surface protein (CSP) and merozoite surface protein-3α (MSP-3α) in South Korea have previously shown that the diversity of those genes was low in 1990s, but increased after 2000 [
9,
11]. An increase in antigen diversity potentially affects the acquisition of protective immunity against vivax malaria. It is therefore important to determine whether the observed recent increase of the diversity of antigen-encoding genes is caused by human immune pressure or not. Since these antigens are exposed to the human immune system, diversity in the antigen-encoding loci can be attributed to not only to natural selection by immune pressure but also to the parasite's population history [
12]. Hence, it remains uncertain whether the low diversity observed in the antigen-encoding loci resulted from selective pressure imposed by the host immune system or due to a demographic change in the local parasite population. In contrast to polymorphic antigen genes, microsatellite markers are selectively neutral and highly polymorphic, and, thus, are suitable for the assessment of genetic diversity resulting from demography at the genome-wide level. Recently, microsatellite markers have been widely used to analyse genetic diversity of
P. vivax populations [
13‐
18]. In the present study, multilocus microsatellite analysis was conducted to elucidate genetic diversity and population structure of
P. vivax in South Korea. Results showed drastic changes in genetic diversity and population structure between 2000 and 2007.
Discussion
This study is the first to report microsatellite diversity of the
P. vivax population in South Korea. The present results obtained with 13 microsatellite markers across 12 chromosomes revealed a higher genetic diversity in 2007 than in 1997-2000. Consistent with previous limited studies showing a recent increase in antigen diversity, the study further support changes in
P. vivax isolates from South Korea. Analysis of South Korean isolates before 2000 using antigen-encoding genes such as Duffy-binding protein [
28], PvCSP [
9], PvMSP-3α [
11], and AMA-1 [
29] infers only two alleles whereas genetic diversity of these antigens remarkably increased after 2001 [
9,
11]. The present study shows only two major multilocus microsatellite genotypes that comprise the two major
P. vivax populations in 1997-2000, while numerous genotypes appeared in 2007. It is surmised that the recent increase in
P. vivax genetic diversity was displayed at the genome wide level, and suggest that increased antigen diversity was not driven, if any, by selective pressure on antigen genes. While it is of note that the
P. vivax genetic diversity in South Korea has increased in recent years (
HE = 0.55 in 2007), diversity is still low as compared to levels obtained from other geographic areas with the exception of Peru: thus,
HE = 0.71-0.86 in Brazil, Colombia, India, Laos, Thailand, Sri Lanka, Vietnam, Ethiopia, and Myanmar [
13‐
15,
17,
19],
vs HE = 0.44 - 0.69 in Peru (Table
3) [
18]. However, although the South Korean
P. vivax populations appear to have relatively low genetic diversity, direct comparisons of
HE with other parasite populations from different geographical areas must be treated with caution especially when the number of samples used is substantially different. In this study, sample number is somewhat small (
n = 58) as compared with other geographical areas (e.g., 159 in Peru [
18]; 140, 167 and 118 in Sri Lanka, Myanmar and Ethiopia, respectively [
14]). Further investigations using more samples should be performed to confirm a low genetic diversity in
P. vivax population in South Korea.
Table 3
Microsatellite expected heterozygosity (HE) of P. vivax populations from South Korea vs other geographic areas
Brazil | 1999-2005 | 0.71-0.80 | 13 |
Colombia | 2001-2003 | 0.79 | 15 |
India | 2003-2004 | 0.72 | |
Thailand | 1992-1998 | 0.76 | |
Laos | 2001-2003 | 0.75 | |
Sri Lanka | 2004-2005 | 0.79 | 19 |
Ethiopia | 2006-2008 | 0.752 | 14 |
Myanmar | 2007 | 0.845 | |
Sri Lanka | 2003-2008 | 0.861 | |
Vietnam | 1999-2000 | 0.86 | 17 |
Peru | 2006-2008 | 0.44-0.69 | 18 |
South Korea | 1997-2000 | 0.382 | this study |
| 2007 | 0.545 | |
Two mechanisms can be proposed to account for the observed increase in the genetic diversity of
P. vivax population in South Korea. One is the accumulation of microsatellite mutations in the population, and the other is migration of novel parasite populations from other geographic areas into South Korea. Since the mutation rate(s) of
P. vivax microsatellite is unknown, accumulation of mutation cannot be completely excluded. However, the present study does reveal the appearance of very many novel alleles at almost all the microsatellite loci examined. Previous studies have also reported rapid increase in genetic diversity in antigen-encoding genes [
9,
11]. It is very unlikely that in a short period (between 2000 and 2007, probably six generations of the parasite life cycle in South Korea), many novel alleles accumulate simultaneously at several microsatellite and antigen loci. The idea of migration of multiple different parasite genotypes from other geographic areas into South Korea is, therefore, favoured. Haplotype network analysis based on the parasite mitochondrial genome infers a genealogical origin in southern China for
P. vivax populations collected in 1999 and one in 2002 [
30]. Further investigations on genetic diversity of
P. vivax in neighbouring geographic areas can elucidate and verify the population mechanism.
The rate of inbreeding also contributes to the extent of genetic diversity in
P. vivax populations. In this study, the extent of inbreeding inferred from linkage disequilibrium was reduced in 2007, compared with that in 1997-2000; although overall linkage disequilibrium was significant in both sampling groups. These results indicate very strong linkage disequilibrium, leading to a high inbreeding rate in the parasite population in 1997-2000, whereas relatively weak linkage disequilibrium, leading to elevation of outbreeding levels in 2007. Analysis by STRUCTURE (Figure
2C and
2D) supports the increased outbreeding in 2007. It is thus concluded that the recent diversification of South Korean
P. vivax population resulted partly from an increase in the level of outbreeding among different parasite genotypes.
Moving forward, molecular tools and techniques can elucidate parasite genetic diversity and population structure that can help malaria control efforts and understand mechanisms of pathogenicity and drug resistance. It is generally believed in a parasite population with a high genetic diversity, protective immunity against malaria is slow to develop. There is accumulating evidence, which suggest that
P. vivax is more virulent than previously thought in tropical endemic areas [
5], where genetic diversity is generally high (Table
3). This raises the potential risk of severe malaria in South Korea if genetic diversity further increase to a level observed in tropical endemic areas. Recently, drug-resistant
P. vivax has been reported in South Korea [
31], which emphasizes increasing importance of assessing genetic diversity and population structure in this area. Since results obtained in this study strongly suggest an increase in the genetic diversity of
P. vivax population in South Korea, monitoring of the parasite genetic diversity in this country as well as in neighbouring endemic areas will provide valuable information for developing malaria control strategies in South Korea.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
HH performed experiments, data analyses and paper writing. JYK and WL coordinated the sampling. NMQP, TM, JYK and TH critically reviewed the manuscript. TM, JYK, WL and KT participated in acquisition of funding. KT made substantial contributions to conceive the study design, paper writing and reviewing. All authors read and approved the final manuscript.