Of the 122 samples, we genotyped 104 successfully; 14 had 1 or 2 of the 9 markers fail to amplify, and 4 were excluded because <7 markers amplified. These 118 samples were obtained from 114 separate patients (4 patients had P. vivax infection and were tested in 2011 and 2012). This set comprised a substantial proportion of all the P. vivax cases recorded during 2009–2013 (Table). The samples were divided into 3 subsets by their geographic location (Figure 1): the first 2, Laconia (with 68 samples) and Kardhítsa (with 12 samples), were foci of transmission where multiple cases were recorded over time in a relatively restricted area, and the third encompassed the samples from the rest of Greece (with 38 samples).

Figure 2

Figure 2. Frequency distribution of Plasmodium vivax allelic variants and lengths or subtype of genetic markers, by geographic location, Greece, 2009–2013. The frequencies for microsatellite markers MS1 (A), MS5 (B), MS7 (C), MS8...

The extent of allelic diversity observed differed for the main genetic markers (range 5–23 distinct variants/marker; Technical Appendix 1 Tables 1–3), and the corresponding allelic frequency distributions differed by collection site (Figure 2, panels A–I). Mixed clone infections were detected in 18 samples, in which only 2 allelic variants were observed; among these 36 variants, 27 were the same 2 variants (12 of 1 variant and 15 of the other). Thus, most P. vivax cases diagnosed in Greece appeared to harbor monoclonal infections.

Figure 3

Figure 3. Temporal representation of the occurrence of Plasmodium vivax haplotypes, Greece, 2009–2013. Data for each region (Laconia, Kardhítsa, and rest of Greece) are depicted. Samples are indicated by shapes: in Laconia, squares...

A total of 67 distinct haplotypes were observed when all 9 genetic markers were considered (Technical Appendix 1 Table 1), of which 53 were unequivocal and 14 corresponded to samples in which 1 or 2 of the markers failed to amplify. Of the 68 samples from the Laconia outbreak, we observed 28 unique P. vivax haplotypes only once, and we detected only 1 of these haplotypes (family Greece 4 [Gr4]) in samples collected from another region of Greece. Fourteen of the haplotypes from Laconia (observed in 51 of 68 samples) fell into 4 families: 1) family Laconia 1 (La1), which was observed in 4 samples collected in 2009; 2) La4, which had 8 subtypes and was observed in 32 samples (24 of which were subtype La4–2); 3) La5, which had 2 subtypes, each observed only once; and 4) La6, which had 3 subtypes observed in 13 samples (11 of which were subtype La6–1). One fourth (17/68) of the samples from Laconia had unique haplotypes that did not belong to these 4 haplotype families. Of the 35 haplotypes recorded outside Laconia, 1 haplotype of the family Kardhítsa (Ka) was observed solely in the 12 samples (from 3 Greece residents and 9 migrants) in regional unit Kardhítsa. Two related haplotypes (of family Gr1) were observed in the same person who had a relapse in February 2012 seven months after the first episode, and another 2 haplotypes (Gr31 and Gr32, also related) were found in 2 Greece residents from regional unit Évros who had symptoms 1 month apart from each other. Finally, 2 haplotypes of the Gr4 family were observed in 6 persons in the east of regional unit Attica (5 Greece residents and 1 migrant). The remaining 28 haplotypes were each observed only once in samples obtained throughout the rest of Greece (Figure 3; Technical Appendix 2 Figures 1, 2).

To obtain a dynamic picture of the P. vivax isolates that were circulating in Greece throughout the episodes of reintroduction, we displayed the haplotypes in their order of temporal appearance (Figure 3). The dispersal pattern of P. vivax parasites, in particular those of the major haplotype families (Gr4 in the eastern part of Attica; Ka in Kardhítsa; and La1, La4, La5, and La6 in Laconia), could be discerned. The genetic analysis of the parasites from Laconia, the main focus of reintroduced P. vivax, showed that the parasite haplotype found in Greek residents in 2011 and 2012, when most cases occurred, represented a restricted subset of the haplotypes found in the migrants. All but 3 of the samples acquired from 31 Greek residents (34 samples) belonged to 3 haplotype families: La4, La5, and La6. Fifteen of the 27 samples acquired from migrants during the same years also belonged to families La4–La6, with the remaining 12 samples from migrants each harboring a unique but unrelated parasite haplotype.

In Attica, 6 of the cases recorded during August 2011 and summer 2012 among Greek residents from 4 different locations were the same haplotype (Gr4); thus, Attica can be considered an additional focus of local transmission. We observed the same haplotype in an Afghani migrant who had P. vivax in early July 2012 in Laconia. Finally, in regional unit Kardhítsa, Thessalía, all 12 P. vivax samples (11 from 2012 and 1 from 2013) collected from a single village (6 of which were from a focus investigation) shared the same haplotype. Eleven of these samples were from patients of different nationalities (7 Afghani, 2 Bangladeshi, and 2 Romanian) who lived in close proximity and sought treatment within 2 months of each other.

Given that the differences between the haplotypes within each of these 6 families were minor, the corresponding samples were subjected to genotyping with 4 additional genetic markers: 3 microsatellite loci MS10, MS16, and Pv3.27, and the polymorphic F3 domain of PvMSP1. This analysis revealed that the Gr4 family comprises 4 subtypes that differ with respect to 1 or 2 of the additional markers. We were able to distinguish 4 subtypes of haplotype family Ka and 3 subtypes of haplotype family La4 with 1 of these 4 additional loci. The members of the La6 family were homogeneous at all these loci (Technical Appendix 2 Figures 1, 2).

Using archived blood samples, we determined the genetic anatomy of many of the P. vivax isolates introduced into Greece during 2009–2013 and investigated the dynamics of the transition of a location from a source of imported cases to an area with established, sustained autochthonous transmission. These transitions are increasingly likely to occur in areas where malaria control efforts have eliminated or will eliminate the parasite. This fact is of particular concern for P. vivax, whose ability to form hypnozoites substantially enhances its potential to transmit over extended periods. Standard approaches of screening and treatment are inapplicable for P. vivax because screening for hypnozoite carriage is not possible. Moreover, the toxicity of primaquine phosphate, the only hypnozoitocidal drug available, precludes its use without prior onerous testing for glucose-6-phosphate dehydrogenase deficiency. Thus, the often-asymptomatic or clinically mild relapses that might occur in migrants over time provide additional opportunities for local anopheline mosquitoes to transmit the parasite locally.

A total of 1,320 cases of malaria were reported in Greece during 1975–2008, a mean of 39 (range 16–79) cases/year; 475 of these cases were P. vivax diagnosed in visitors from the Indian subcontinent. Over this period only 13 autochthonous cases (2 in 1975, 3 in 1976, 5 in 1991, 1 in 1999, and 2 in 2000) occurred in diverse regions of Greece (8). In contrast, during 2009–2013, P. vivax malaria was diagnosed in 139 migrants or travelers from endemic countries and 76 Greece residents (Table). Most likely, the substantial increase after 2006 in numbers of Indian subcontinent migrants, with many employed over the spring and summer months as agricultural workers, led to the reintroduction of P. vivax in some rural areas. The focus of introduced P. vivax malaria was considered to be in regional unit Laconia because 53 of the 76 cases in Greece residents were from 6 villages in Laconia (municipality Evrótas) within a 2.5-km radius, with the remaining 23 being from diverse and distant regions of Greece. The identification of the same haplotypes as in an earlier study, in which a combination of only 3 markers were used to analyze samples from 2 consecutive years (2011 and 2012), suggested that sustained transmission in the area was possible and further measures were needed to control the disease. Thus, mass drug administration with the immigrant population in Laconia was decided on and performed as a supplementary measure (12).

Our detailed spatiotemporal genetic analysis, in which we identified and compared the P. vivax haplotypes in patient samples, helped shed light on the epidemiology of reintroduced P. vivax episodes. Some of the infections in migrants in Laconia in 2011–2012 were most probably acquired locally and should consequently be reclassified as autochthonous. Careful analysis of the genotyping data uncovered 2 additional foci of local transmission and confirmed the need to reevaluate the classification of cases as autochthonous and imported. Despite the high number of cases, the focus in Attica was unsuspected because cases were scattered throughout the region. Genotyping revealed that parasites from 6 unrelated Greece residents (8 samples, 2 in 2011 and 6 in 2012) living within a 15-km radius of each other shared the same haplotype (Gr4). This haplotype was also found in 2012 in 1 migrant living in Laconia, but further investigation did not reveal any epidemiologic connection between this case and those from Attica.

The cluster of 11 cases recorded in 2011 in Kardhítsa was not thought of as a focus of local transmission because 9 of these cases were in migrants from the Indian subcontinent. However, genotyping revealed that the parasites from all 11 migrants were of the same haplotype, as were those from a Greece resident with no history of malaria or travel in 2013. This infection might have been acquired during the previous months from an asymptomatic carrier, or the infection might represent a delayed primary attack after inoculation by an infected mosquito the previous year. Thus, Kardhítsa represents a third focus of local transmission, where many of the migrant cases classified as imported malaria by using classic epidemiologic criteria should be reclassified as locally acquired.

From an epidemiologic point of view, P. vivax parasites have a high potential for initiating and maintaining local transmission within a short period of time from carriers reaching receptive areas hitherto free of malaria. In such areas, the nonimmune status of the local population will facilitate rapid detection of cases, considering that infected, previously malaria-naive residents would generally have clinical symptoms that are sometimes severe. However, in regions where P. vivax malaria has been eliminated or brought to very low incidence, the imported parasite strains might persist as subpatent infections because of residual levels of acquired immunity in the local population, which would enhance the likelihood of wider dissemination. Nonetheless, data from the reintroduction episodes we present suggest that the potential for establishing transmission might differ by parasite strain, considering that relatively few P. vivax strains that were introduced into Greece during the 5 years of our analysis led to clusters of sustained local transmission. The difference in transmission might be attributable to biologic or environmental factors or a combination of both. The local mosquito populations might be inefficient at transmitting some of the imported strains (17), and variation in the vector midgut bacterial biota might also restrict transmission (18). However, the 3 foci we identified involved genetically diverse parasites (Technical Appendix Figure 2), which could be explained by the parasites having differential transmission efficacies for the local anopheles populations. Finally, the degree of human–anopheline contact also plays a crucial role, such that the parasites in carriers residing in a rural area with a high mosquito density are more likely to be transmitted than parasites in carriers living in an urban environment with a low mosquito density.

The reintroduction of P. vivax malaria into Greece during 2009–2013 should serve as a warning to those engaged in efforts to eliminate malaria (19) in countries where P. vivax predominates, especially for those in countries close to elimination and in malaria-free areas neighboring P. vivax–endemic regions. The prompt and flexible countermeasures brought to bear by the Greek public health infrastructure were effective in containing and then extinguishing the foci of transmission (12). In our genotypic analyses, we highlight 2 epidemiologic features: first, the high potential for imported P. vivax to establish sustainable transmission within a short time; and second, the difficulty in predicting the locations where such transmission might occur. P. vivax is a recognized obstacle for malaria elimination (20). Maintaining vigilance will be crucial to ensure that this parasite will not threaten those countries that achieve it.