Extensive microsatellite diversity in the human malaria parasite Plasmodium vivax

Abstract

The population structure of Plasmodium vivax remains elusive. The markers of choice for large-scale population genetic studies of eukaryotes, short tandem repeats known as microsatellites, have been recently reported to be less polymorphic in P. vivax. Here we investigate the microsatellite diversity and geographic structure in P. vivax, at both local and global levels, using 14 new markers consisting of tri- or tetranucleotide repeats. The local-level analysis, which involved 50 field isolates from Sri Lanka, revealed unexpectedly high diversity (average virtual heterozygosity [H_E], 0.807) and significant multilocus linkage disequilibrium in this region of low malaria endemicity. Multiple-clone infections occurred in 60% of isolates sampled in 2005. The global-level analysis of field isolates or monkey-adapted strains identified 150 unique haplotypes among 164 parasites from four continents. Individual P. vivax isolates could not be unambiguously assigned to geographic populations. For example, we found relatively low divergence among parasites from Central America, Africa, Southeast Asia and Oceania, but substantial differentiation between parasites from the same continent (South Asia and Southeast Asia) or even from the same country (Brazil). Parasite relapses, which may extend the duration of P. vivax carriage in humans, are suggested to facilitate the spread of strains across continents, breaking down any pre-existing geographic structure.

Introduction

Plasmodium vivax is a major public health challenge in Central and South America, the Middle East, Central, South and Southeast Asia, Oceania and East Africa, where 2.6 billion people are currently at risk of infection (Guerra et al., 2006) and 70–80 million clinical cases are reported each year (Mendis et al., 2001). This human pathogen is re-emerging in some Asian countries where eradication efforts had been successful in the 1960s, such as Uzbekistan (Severini et al., 2004), Azerbaijan (Leclerc et al., 2004b) and the Republic of Korea (Lim et al., 2000). In addition, P. vivax has recently surpassed P. falciparum as the main cause of malaria morbidity across the Amazon Basin of Brazil (Loiola et al., 2002). Little is currently known about the population structure of this major human pathogen.

The extensive polymorphism found in surface antigens of P. vivax (Cui et al., 2003) reflects the combined effects of the parasite's population history and selective constraints imposed by the host's immunity (Escalante et al., 2004), being little informative about the population structure of this species. The current markers of choice for large-scale population genetic studies of eukaryotes, the highly polymorphic and short (1–6 base pair-long) tandem repeats known as microsatellites (Schlotterer, 1998), are not as abundant across the genome of P. vivax (Feng et al., 2003) as they are in that of P. falciparum (Joy et al., 2004), and appear to be little polymorphic in worldwide isolates (Leclerc et al., 2004a). The recent availability of P. vivax genome sequence data (Carlton, 2003), however, leads to the identification of additional arrays of highly variable dinucleotide microsatellites (Gómez et al., 2003, Imwong et al., 2006), which may represent promising tools for population-level studies of genetic variation in this species (Russell et al., 2006).

Here we examine the genetic diversity of P. vivax with a new set of highly polymorphic tri- and tetranucleotide microsatellites (Karunaweera et al., 2007), which are expected to yield more accurate allele scoring than dinucleotide markers (Anderson et al., 1999). First we describe the genetic structure and diversity of a sample of 50 field isolates from Sri Lanka. Next we present a microsatellite-based analysis of 132 field isolates (including those from Sri Lanka) and 32 monkey-adapted strains of P. vivax originated from four continents. Extensive microsatellite variation is observed at both the local and global levels, without clear clustering according to major geographic regions. The high levels of genetic diversity and the high prevalence of multiple-clone infections in natural populations of P. vivax do not translate into frequent outcrossing and multilocus linkage equilibrium, with interesting evolutionary and epidemiologic implications.