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  • Case report
  • Open Access

First report of a phylogenetic analysis of an autochthonous Plasmodium vivax strain isolated from a malaria case in East Attica, Greece

  • 1,
  • 2,
  • 3,
  • 2,
  • 3,
  • 3,
  • 4,
  • 5 and
  • 2Email author
Malaria Journal201312:299

https://doi.org/10.1186/1475-2875-12-299

  • Received: 21 June 2013
  • Accepted: 18 August 2013
  • Published:

Abstract

Malaria has become an emerging infection in Greece, which is the doorstep to Europe for thousands of immigrants. With increasing immigration, cases with evidence of domestic transmission (autochthonous) are being reported. In the present study, an isolate of Plasmodium vivax from an autochthonous clinical case was subjected to phylogenetic analysis of the genes encoding the merozoite surface protein 1 (MSP-1) and the circumsporozoite protein (CSP). In the MSP region, the strain was related with strains from Brazil, South Korea, Turkey and Thailand, whereas in the CSP region, with strains from Brazil, Colombia and New Guinea. The present study establishes for the first time in Greece the basis for the creation of a database comprising genotypic and phylogenetic characteristics of Plasmodium spp.

Keywords

  • Malaria
  • Greece
  • Plasmodium vivax
  • CSP
  • MSP-1
  • Phylogenetic analysis

Background

In 1974, Greece was officially declared as “malaria free” and until 2008 only imported cases were notified. However in 2009, with increasing immigration, isolates with evidence of domestic transmission (autochthonous) were reported from Hellenic Center for Disease Control and Prevention[1]. This study presents a recent autochthonous case of Plasmodium vivax malaria in a Greek woman from East Attica. The isolated Plasmodium strain was phylogenetically analysed in order to establish a database as a tool for further epidemiological monitoring.

Case presentation

Clinical data

The patient, a 44 year-old woman, and a permanent resident in Markopoulo, a city in East Attica (Figure 1), was admitted on 11 July, 2012 to the outpatient department of the private hospital Athens Medical Center- Peristeri, with high fever (39°C) for the previous ten days, starting with rigour. There was no history of previous travel abroad, blood transfusion, tissue organ transplantation, intravenous drug abuse or a prolonged febrile illness over the last year. Heart and abdomen examinations were normal. Blood cultures (six) were negative. The haematological examination revealed a progressively aggravating pancytopaenia. The serological viral and bacterial investigations for EBV, CMV, adenovirus, parvovirus, coxsackie, HIV, Rickettsiae, Chlamydia and spirochetes were negative. Chest computer tomography (CT) was normal. CT of the lower abdomen showed a mild splenomegaly (about 15 cm). Transthoracic echocardiograph, breast ultrasound (required because of the family history reporting breast cancer of her mother and sister) and MRI (including angiography) of the brain were normal and could not justify the fever attacks. PCR for Leishmania was negative. Microscopic examination of thick and thin blood smears, performed eight days after admission, was positive for P. vivax. The result was also confirmed by PCR, and then several blood samples were sent to special laboratories for further molecular processing.
Figure 1
Figure 1

Map of Attica showing the city of Markopoulo where the autochthonous malaria case was isolated.

The patient was successfully treated, initially with atovaquone/proguanil (Malarone®, 1 g atovaquone /400 mg proguanil total daily dose, referring to four tablets as a single dose) for three consecutive days and then for 14 days with primaquine (30 mg daily dose, after examination for G6PDH deficiency).

Consent

Conclusions

Phylogenetic analysis in the MSP region showed that the investigated strain was related with strains from Brazil, South Korea, Turkey and Thailand (Figure 2A and B), whereas in the CSP region it clustered with strains from Brazil, Colombia and New Guinea (Figure 3A and B).
Figure 2
Figure 2

Neighbour-joining tree of Plasmodium vivax merozoite surface protein-1. A. MSP-1 gene sequences from the strain obtained from Greece, as well as from the GenBank. B. Subtree including the Greek strain and depicting its phylogenetic relationship with other strains. Bullets represent >75% bootstrap value (1,000 replicates).

Figure 3
Figure 3

Neighbour-joining tree of Plasmodium vivax circumsporozoite protein. A. CSP gene sequences from the strain obtained from Greece, as well as from the GenBank. B. Subtree including the Greek strain and depicting its phylogenetic relationship with other strains. Bullets represent >75% bootstrap value (1,000 replicates).

Taking into consideration previously published studies[2, 6, 811, 14], the amino acid sequence was in the MSP and CSP regions with reference isolates. Regarding the MSP region, the strain belonged to the Belem type, whereas with respect to the CSP region, to the VK210 type (alignment not shown). The obvious discrepancy between the results of the phylogenetic analysis in the MSP and CSP regions is due to the absence of adequate Plasmodium sequence data at the GenBank database and to the absence of adequate number of paired sequences from the same strain[14]. However, using msp1 and csp as genetic markers, the close proximity of the strain in both regions with strains from South America in both trees (Brazil, Colombia) denotes a possible origin of the Plasmodium strain from South America. Further studies, including isolates of autochthonous cases, will be required in order to obtain a clearer picture of Plasmodium diversity in Greece.

The present study establishes, for the first time in Greece the basis for the creation of a database comprising genotypic and phylogenetic characteristics of Plasmodium spp. Malaria has become an emerging infection in Greece, which is the doorstep to Europe for thousands of immigrants. Therefore clinicians should be aware of considering malaria early in their differential diagnosis. The enrichment in the future with new strains of imported and autochthonous cases will be an invaluable tool for the epidemiological monitoring of malaria in Europe.

Abbreviations

MSP-1: 

Merozoite surface protein 1

CSP: 

Circumsporozoite protein

EBV: 

Epstein-Barr Virus

CMV: 

Cytomegalovirus

MEGA: 

Molecular evolutionary genetics analysis.

Declarations

Authors’ Affiliations

(1)
Department of Nursing, Faculty of Human Movement and Quality of Life Sciences, University of Peloponnese, Sparta, Greece
(2)
Department of Biopathology and Clinical Microbiology, Athens Medical School, Aeginition Hospital, Vass. Sophias av. 72-74, Athens, 115 28, Greece
(3)
Athens Medical Centre - Peristeri, Peristeri, Greece
(4)
Department of Hygiene, Epidemiology and Medical Statistics, Medical School, University of Athens, Athens-Goudi, Greece
(5)
Department of Microbiology, Medical School, University of Thessalia, Larissa, Greece

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