Background
Annually, an estimated 219 million people are infected with malaria and the disease is endemic in 104 countries[
1].
Plasmodium falciparum malaria is associated with the highest mortality and is accordingly prioritized in research and control. Another species,
Plasmodium vivax, has gained less scientific attention despite being the most widely distributed malaria species endemic in tropical and subtropical countries worldwide, with an estimated 2.8 billion people currently at risk[
2‐
4]. It is estimated that at least 130 million people are infected with
P. vivax annually[
5], causing significant economic and financial burden to affected countries[
6].
In historic Europe,
P. vivax malaria was endemic in many countries, reaching as far as Finland and England in the north[
6,
7]. Malaria disappeared from Europe in the mid-20th Century, likely as a result of a combination of various factors, including improved housing conditions, better health care services, and the implementation of various malaria eradication programmes[
8,
9].
According to the World Malaria report in 2011, “The European Region has a real possibility of becoming the first to achieve the complete elimination of malaria within the next few years, and aims to do so by 2015”[
10]. Within the European Union (EU), all EU Member States are considered malaria-free[
11]. The likelihood of re-emergence of autochthonous
P. vivax malaria in southern Europe due to factors such as global warming, and even the ongoing economic crisis, has been debated. Various studies indicate an increased risk imposed by the presence of suitable vectors[
12] and the increased number of travellers and immigrants from endemic countries[
13,
14].
In Greece,
P. vivax malaria was a common disease before the Second World War, with epidemics involving thousands of cases every year. Following a national eradication programme between 1946 and 1960, Greece was declared free from malaria by the World Health Organization in 1974. Malaria is a mandatory notifiable disease in Greece. Accordingly, from 1974 to 2010, an average of 39 cases per year were reported, most of which were imported[
15,
16] presumably due to the large number of immigrants (estimated to be close to one million) that occasionally or permanently live in Greece[
17], or due to travellers returning from malaria-endemic countries. However, 17 sporadic autochthonous
P. vivax cases were detected during the years 1991, 1999, 2000, 2009, and 2010[
15,
16]. In Greece, the potential for re-emergence of malaria transmission is present due to the widespread occurrence of several anopheline vector species.
Anopheles sacharovi, Anopheles maculipennis, Anopheles superpictus and
Anopheles claviger are among the species identified in the country[
18,
19].
An. sacharovi was implicated as the vector species responsible for transmission of a majority of the malaria cases in Greece prior to the nationwide eradication in 1974[
20].
In 2011, a total of 40 autochthonous
P. vivax cases were reported, 34 of which were derived from a single region, Evrotas municipality in Southern Peloponnese[
16]. This outbreak in Evrotas calls for concern and should be explored further. One important aspect to investigate is the epidemiological pattern of occurrence and spread of autochthonous
P. vivax malaria in the affected areas. With this knowledge, it may be possible to determine at-risk populations and identify possible areas to focus interventions, which is crucial if similar
P. vivax epidemics emerge in the future. The use of molecular tools that genetically fingerprint
P. vivax parasites could provide a powerful tool as an adjunct to more classical epidemiological investigations.
Many molecular markers have been used to genotype
P. vivax. They can be classified into two categories, those that are under natural selection and those that are evolutionarily neutral or nearly neutral.
Plasmodium vivax circumsporozoite protein (
Pvcs), merozoite surface protein-1 (
Pvmsp-1), and merozoite surface protein-3α (
Pvmsp-3α) are genes that have been widely used for genotyping, as these are highly polymorphic and under natural selection[
21].
Recently, microsatellite markers (MS) have increasingly been used in studies of
P. vivax diversity. They are less laborious to perform, considered selectively neutral, and often increase the resolution compared to genetic markers such as
Pvcs,
Pvmsp-1 and
Pvmsp-3α[
22‐
26]. Three molecular markers were used (
Pvmsp-3α and the MS m1501 and m3502) to genotype
P. vivax isolates sampled from the majority of infected individuals during 2011 in Greece. With this approach it is most likely possible to substantiate whether the outbreak was caused by multiple
P. vivax re-introductions, and enables identification of areas with continuous transmission. These areas likely present locations of higher transmissibility of malaria, identification of which can aid in enhancing and expanding the findings of classical epidemiological investigations. Recording the genotypes involved in the 2011 outbreak is considered useful for future comparisons and will help guide implementation of precautionary control measures for potential future outbreaks of
P. vivax malaria in Greece.
Discussion
Malaria outbreaks in regions that eradicated malaria decades ago have been reported from countries such as Singapore (2009)[
29] and Korea (1993)[
30]. In the Republic of Korea, malaria was eradicated in the late 1970s, but a single case, which occurred in 1993, resulted in the re-introduction of
P. vivax malaria in the country in the years that followed[
30]. During the past 20 years, few confirmed autochthonous cases of
P. vivax malaria in the European Union have been reported in Italy (Maremma, 1997)[
31], France (Corsica, 2006)[
32] and Spain (Aragon, 2010)[
33].
In Greece, autochthonous cases have been scarce but have occurred annually since 2009[
34]. The outbreak in 2011, however, was characterized by an unusually higher number of malaria cases, and hence required a study, which was more thorough at the molecular level, in order to expand the epidemiological findings and the related applicability in the region.
The
Pvmsp-3α gene is one of the most variable polymorphic genes and has been widely used to study the diversity of
P. vivax and in the discrimination of multiple infections in epidemiological studies[
35‐
38]. The MS m1501 and m3502 have been shown to have high diversity among parasites from Southeast Asia[
22,
39,
40]. Consequently, the combined usage of these three molecular markers is expected to provide a good resolution to discriminate the
P. vivax strains.
The frequency of the A-genotype among the samples that produced conclusive results for the
Pvmsp-3α gene was high (0.86) which is in agreement with the high frequency reported from other areas studied[
41‐
43]. The diversity of the MS studied was higher, with 12 different MS m1501 alleles and seven MS m3502 alleles. Higher diversity of the m1501 locus has been reported mainly from Asian populations, while the opposite has been found with higher diversity of m3502 in South American and some Asian populations[
26,
40].
Pvmsp-3α and the MS combined showed multiple P. vivax haplotypes expressed in the autochthonous cases in various areas of Greece, which indicates the existence of multiple sources of infection. This is further supported by the haplotype diversity of the imported cases derived from the area of Athens and the unique haplotype of the 2009 sample. In Avlida, Orhomenos and Larisa, only a single autochthonous case was observed, suggesting that the region did not present suitable conditions to sustain continuous transmission (e g, low abundance of mosquito vectors and/or low number of Plasmodium carriers), or that the response of local health authorities disrupted the cycle of the parasite.
The two cases derived from East Attica (Marathon and Kalivia) had a common haplotype, indicating possible continuous local transmission, despite the long distance between the locations (approximately 30 km) or common exposure to an imported case. This may be also explained by frequent commuting of inhabitants between these two areas. Although examination of a higher number of molecular markers may increase the resolution of genotypic characterization and eventually separate the common haplotype into two distinct haplotypes, this area should be subject to careful monitoring of malaria transmission in the future. This recommendation is further substantiated by a 2004 entomological survey in the region of Marathon, which identified a high abundance of
An. saccharovi[
19], and recent reports of autochthonous cases in the area[
44].
In the municipality of Evrotas, common haplotypes were identified in up to 14 cases. Although this study did not have access to the
P. vivax genetic material from all infected individuals in Laconia, the data indicate relatively limited diversity and that the spread of only a few
P. vivax isolates was the main cause of the local outbreak. Additionally, limited autochthonous cases in Evrotas have been reported during the last four years, possibly rendering this an area of local transmission[
45,
46] culminating in the outbreak in 2011. The equal number of imported and autochthonous cases that share the same genotype indicates that a small number of cases, classified as imported, may have been locally infected. Misclassification was due to lack of documentation and unreliable information of the travel history.
Following the Evrotas outbreak in 2011, the Greek authorities enhanced vector control activities by large-scale spraying with insecticides, health information campaigns, enhanced surveillance and active case finding in affected areas. A possible effect of these activities is that only 20 autochthonous
P. vivax cases have been identified in four different regions in Greece since then. Of these, ten cases were from Evrotas[
46]. In 2013, no autochthonous cases have been reported as at 20 September, 2013.
Acknowledgements
The authors would like to thank Anastasia Mbimba for excellent technical assistance, and the Department of Immunology and Histocompatibility, School of Medicine, University of Thessally, for the sequencing. Additionally, Ulla Abildtrup at the Centre for Medical Parasitology at University of Copenhagen is thanked for excellent technical assistance on the microsatellites.
This work was partly supported by the “Integrated Surveillance and control programme for West Nile virus and malaria in Greece (MALWEST)”, which is implemented through Operational Programme entitled “Human Resources Development” of National Strategic Reference Framework (NSRF) 2007–2013. The programme is co- funded by Greece and the European Union- European Regional Development Fund.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
GS, MA, NCV, and ICB participated in the design of the study. GS, MLS, EP, NT, and HHH carried out the molecular genetic studies. CH, MT and JK provided most of the blood samples. GS, MA and MLS drafted the manuscript. ICB, NCV, CH, and JK critically revised and gave final approval of the manuscript. All authors read and approved the final manuscript.