Genotyping Plasmodium vivax isolates from the 2011 outbreak in Greece

Abstract

Background: Plasmodium vivax malaria was common in Greece until the 1950s with epidemics involving thousands of cases every year. Greece was declared free of malaria by the World Health Organization in 1974. From 1974 to 2010, an average of 39 cases per year were reported, which were mainly imported. However, in 2009 and 2010 six and one autochthonous cases were reported culminating with a total of 40 autochthonous cases reported in 2011, of which 34 originated from a single region: Laconia of Southern Peloponnese. In this study the genotypic complexity of the P. vivax infections from the outbreak in Greece during 2011 is described, to elucidate the possible origin and spread of the disease.

Methods: Three polymorphic markers of P. vivax were used; Pvmsp-3α and the microsatellites m1501 and m3502 on P. vivax isolates sampled from individuals diagnosed in Greece. Thirty-nine isolates were available for this study (20 autochthonous and 19 imported), mostly from Evrotas municipality in Laconia region, in southern Greece, (n = 29), with the remaining representing sporadic cases originating from other areas of Greece.

Results: Genotyping the Evrotas samples revealed seven different haplotypes where the majority of the P. vivax infections expressed two particular Pvmsp-3α-m1501-m3502 haplotypes, A10-128-151 (n = 14) and A10-121-142 (n = 7). These haplotypes appeared throughout the period in autochthonous and imported cases, indicating continuous transmission. In contrast, the P. vivax autochthonous cases from other parts of Greece were largely comprised of unique haplotypes, indicating limited transmission in these other areas.

Conclusions: The results indicate that several P. vivax strains were imported into various areas of Greece in 2011, thereby increasing the risk of re-introduction of malaria. In the region of Evrotas ongoing transmission occurred exemplifying that further control measures are urgently needed in this region of southern Europe. In circumstances where medical or travel history is scarce, methods of molecular epidemiology may prove highly useful for the correct classification of the cases.

Figure 1

Map of Greece indicating the locations of the cases included in the study. The size of the dot is roughly proportional to the number of the samples.

Figure 2

Representation of the Pvmsp-3α results for a subset of Plasmodium vivax samples. The photographs shown are RFLP patterns obtained after digestion of Pvmsp-3α products with Alu I (upper photo) and Hha I (lower photo) restriction endonucleases. The characterization of each pattern is indicated at the top.

Figure 3

The combined Pvmsp-3α, microsatellite (MS) m1501- m3502 haplotypes derived from the cases in Laconia during 2011. The number of cases as a function of month of sampling are shown where each P. vivax isolate is combined into Pvmsp-3α MS m1501-m3502 haplotypes. #: denote unknown genotype at one or more of the markers.