Η γεωγραφική κατανομή της λεϊσμανίασης στην Ελλάδα: βάση του είδους και του ζυμοδέματος των στελεχών που απαντώνται στις διάφορες περιοχές, της κλινικής εικόνας που παρουσιάζουν και της αντοχής των στελεχών στα αντιλεϊσμανιακά φάρμακα

The aim of the current thesis was to study leishmaniasis in Greece, in man and in the dog:the geographical distribution of the disease, the species and zymodemes of the parasite andtheir resistance to drugs, as well as the geographic spread of vectors-hosts of the parasite.Leishmaniasis is a parasitic disease that is endemic in tropical and subtropical areas suchas in southern European countries (including Greece). It constitutes the second disease ofprotozoal origin worldwide, with a wide geographical spread. Until recently it was ranked amongthe most "neglected" diseases. The disease is caused by the intracellular protozoan Leishmaniainfantum and presents a wide range of clinical presentations, depending on the species ofprotozoa and the immune status of the host. If is characterized by a variety of clinicalsyndromes (visceral, cutaneous and mucocutaneous form), with the most severe being visceralleishmaniasis.The survey was designed and conducted in order to identify the areas with the highestrates of seropositivity in dogs, that is the areas with the greatest risk for the transmission ofleishmaniasis in humans by: a) creating a network of physicians and veterinarians in thecountry, b) sending questionnaires for the study of leishmaniasis in veterinary private and publicsector (840 questionnaires), c) the epidemiological study in dogs by examining 8316 samplescorresponding to 5772 animals from 13 regions of the country (almost all prefectures of thecountry) and mapping the results using GIS, d) processing the results of the epidemiologicalstudy in dogs and correlating to answers provided by veterinarians into the questionnaires e) thestudy of the disease in the human population with laboratory testing of patient samples fromCrete and other regions of the country (153 patients) (and use of data from the Center forDisease Control and Prevention, g) the isolation, cultivation and standardization of theprotozoan parasite Leishmania from dogs and patients, recording the species / strains of theparasite detected in dogs and people in Greece and comparing with strains detected in the Mediterranean, i) creating a parasite bank and j) studying the resistance of Leishmania strainsisolated against antileishmanial drugs using flow cytometry and mapping the results using GIS.A network was created consisting of doctors in government hospitals in the country,notably Crete and Athens (where biological samples were obtained from 153 patients withsuspected leishmaniasis), and veterinarians from across the country (who collected 8316received biological samples from 5772 randomly selected dogs). 840 questionnaires were sentto veterinarians in the private and public sector. The analysis of the questionnaires showed thatthe vast majority of veterinarians believe that an increase in incidence of the disease in dogs isobserved at their areas and that the measures taken to fight bites of sandflies have not beensuccessful in dogs. The correlation of the results of the questionnaires and of theepidemiological study in 5772 randomly selected dogs from all regions of the country showedthat a) the questionnaire developed by the European project FP6 EDEN - Leish subgroup, is areliable epidemiological tool and when the results are analyzed and mapped using GIS system itcan be used to draw useful results in identifying high-risk areas and areas of new input of thedisease (Ntais et al., 2013 c).The epidemiological study in dogs demonstrated the seropositivity rate of dogs all over thecountry and the mapping results revealed the high risk areas for humans. The climatic and geographical features of each area were associated with seropositivity rate of dogs, with thepresence of human cases and the presence of species midges playing the role of transmitterhost of the Leishmania parasite (Ntais et al., 2013 a).The laboratory diagnosis and process of biological samples from patients and dogsresulted in the isolation of 212 strains of the parasite that were typed with the gold standardmethod of analysis of an enzyme profile of each strain. This analysis revealed that in Greecethe encountered species of the parasite are Leishmania infantum MON-1 and MON-98,responsible for visceral leishmaniasis and L. tropica MON-300, MON-58, responsible forcutaneous leishmaniasis (Ntais et al., 2013 a). Leishmania tropica MON-300 was recorded onlyin Greece (Crete) and MON-58, although being an anthroponotic species, was recorded in manand in a dog in Crete. This zymodeme was reported in only six patients in Afghanistan and theidentification of Crete in young refugee from Afghanistan (which plagued his country) and a dogis an alarming fact towards the installation of a new pathogen in a local midge population anddevelopment of new epidemiological cycle of the parasite in man and dog (Ntais et al., 2013 b).Until today, L. tropica was considered a human disease in Greece.A parasite bank was created to use in future studies from the laboratory of Parasitology and in collaboration with other laboratories in Greece and abroad. The spread of strains in the country was mapped and they were correlated with the species of gnat-vector host found in therespective areas. In the continuity, we compared the Greek strains with strains detected in otherMediterranean regions.The results of the study of the resistance of the parasite to antileishmanial drugs, whichhad been conducted in the laboratory from previous studies, were mapped with a GIS systemand concluded in the regions of Greece with resistant parasites, which enhances the degree ofrisk area to humans and enhances the need for action to combat the disease and the vector atthese areas. Of the 212 strains studied in the laboratory, only 11 strains (two from humans and9 from dogs) revealed drug resistance.The survey results are very important for understanding the magnitude in terms of publichealth in Greece. Clearly leishmaniasis has spread across the country and drug-resistantparasites have developed and spread all over the country. Greece is located at the crossroadsof Asia, Africa and Europe, while the changes arising from globalization and global warming,favor the appearance of new species and strains o Leishmania in our country. The large numberof species of midges (13) occurring in Greece, enables the import, installation and dispersion ofthese new pathogens with unknown implications on public health. This study provides data onthe basis of which the competent institutions need to intervene to combat the problem in the high-risk areas for humans.Σκοπός της παρούσας διατριβής ήταν η μελέτη της λεϊσμανίασης στηνΕλλάδα, στον άνθρωπο και στο σκύλο: η γεωγραφική κατανομή της νόσου,των ειδών και ζυμοδεμάτων του παράσιτου και της αντοχής τους σταφάρμακα, όπως και της γεωγραφικής διασποράς των διαβιβαστών ξενιστώντου παράσιτου.Η λεϊσμανίαση είναι μια παρασιτική νόσος η οποία ενδημεί σε τροπικές καιυποτροπικές περιοχές όπως και σε χώρες της Νοτίου Ευρώπης (συμπεριλαμβανόμενηςκαι της χώρας μας). Αποτελεί τη δεύτερη ασθένεια πρωτοζωικής προέλευσηςπαγκοσμίως, με ευρεία γεωγραφική εξάπλωση. Μέχρι πρότινος κατατασσόταν στις πλέον«παραμελημένες» νόσους. Η ασθένεια προκαλείται από ενδοκυτταρικό πρωτόζωοLeishmania infantum και παρουσιάζει ένα μεγάλο εύρος κλινικών εκδηλώσεων, ανάλογαμε το είδος του πρωτόζωου και την ανοσιακή κατάσταση του ξενιστή. Χαρακτηρίζεται απόμια ποικιλία κλινικών συνδρόμων (σπλαχνική, δερματική και βλεννογονοδερματική μορφή),με σοβαρότερο τη σπλαχνική λεϊσμανίαση (ΣΛ, Visceral leishmaniasis).Η έρευνα σχεδιάστηκε και πραγματοποιήθηκε έτσι ώστε να εντοπιστούν οι περιοχέςμε τα υψηλότερα ποσοστά οροθετικότητας στα σκυλιά, δηλαδή οι περιοχές με τονμεγαλύτερο κίνδυνο για τη μετάδοση της λεϊσμανίασης στον άνθρωπο με: α) τη δημιουργίαδικτύου γιατρών και κτηνιάτρων στη χώρα, β) την αποστολή ερωτηματολογίων για τη μελέτη της Λεϊσμανίασης σε κτηνίατρους του ιδιωτικού και δημόσιού τομέα (840ερωτηματολόγια), γ) την επιδημιολογική μελέτη στα σκυλιά, με την εξέταση 8316δειγμάτων τα οποία αφορούσαν σε 5772 ζώα από τις 13 περιφέρειες της χώρας (σχεδόναπό όλους τους νομούς της χώρας) και την χαρτογράφηση των αποτελεσμάτων με τοσύστημα GIS, δ) την επεξεργασία των αποτελεσμάτων από την επιδημιολογική μελέτη στασκυλιά και τη συσχέτιση τους με τα αποτελέσματα από τις απαντήσεις των κτηνιάτρων σταερωτηματολόγια, ε) την μελέτη της νόσου στον ανθρώπινο πληθυσμό με την εργαστηριακήεξέταση δειγμάτων ασθενών από την Κρήτη και άλλες περιοχές της χώρας (153 ασθενείς)(και αξιοποίηση στοιχείων από το Κέντρο Ελέγχου και Πρόληψης Νοσημάτων(ΚΕΕΛΠΝΟ), ζ) την απομόνωση, καλλιέργεια και τυποποίηση του πρωτόζωου παράσιτουLeishmania από σκυλιά και ασθενείς, η) την καταγραφή των ειδών/στελεχών τουπαράσιτου που απαντώνται σε σκυλιά και ανθρώπους στην Ελλάδα και τη σύγκρισή τουςμε στελέχη που απαντώνται στη Μεσόγειο, θ) τη δημιουργία τράπεζας στελεχών τουπαράσιτου και ι) τη μελέτης της αντοχής των στελεχών Leishmania που απομονώθηκανστα αντιλεϊσμανιακά φάρμακα με τη χρήση της κυτταρομετρίας ροής (Flow Cytometry) καιτη χαρτογράφηση των αποτελεσμάτων με το σύστημα GIS

Will the introduction of Leishmania tropica MON-58, in the island of Crete, lead to the settlement and spread of this rare zymodeme?

International audienceThe rare zymodeme, Leishmania tropica MON-58, was isolated from a young Afghan refugee with a facial cutaneous lesion who had come to live in Crete early 2008. The same zymodeme variant was isolated from a local dog that had never travelled outside the island, with symptoms of visceral leishmaniasis, which stayed in the area where the patient worked during the summer months. This is the first record of L. tropica in a host, other than human, in Greece and another example of introduction of a vector borne pathogen in a focus where local vector/s can sustain it, with the risk of initiation of new transmission cycle/s.Keywords

Erratum to: Frequency of MDR 1-related p-gp overexpression in Greek Leishmania isolates

International audienc

Seroprevalence of Sandfly-Borne Phleboviruses Belonging to Three Serocomplexes (Sandfly fever Naples, Sandfly fever Sicilian and Salehabad) in Dogs from Greece and Cyprus Using Neutralization Test.

Phleboviruses transmitted by sandflies are endemic in the Mediterranean area. The last decade has witnessed the description of an accumulating number of novel viruses. Although, the risk of exposure of vertebrates is globally assessed, detailed geographic knowledge is poor even in Greece and Cyprus where sandfly fever has been recognized for a long time and repeatedly. A total of 1,250 dogs from mainland Greece and Greek archipelago on one hand and 422 dogs from Cyprus on the other hand have been sampled and tested for neutralising antibodies against Toscana virus (TOSV), Sandfly fever Sicilian virus (SFSV), Arbia virus, and Adana virus i.e. four viruses belonging to the 3 sandfly-borne serocomplexes known to circulate actively in the Mediterranean area. Our results showed that (i) SFSV is highly prevalent with 71.9% (50.7-84.9% depending on the region) in Greece and 60.2% (40.0-72.6%) in Cyprus; (ii) TOSV ranked second with 4.4% (0-15.4%) in Greece and 8.4% (0-11.4%) in Cyprus; (iii) Salehabad viruses (Arbia and Adana) displayed also substantial prevalence rates in both countries with values ranging from 0-22.6% depending on the region and on the virus strain used in the test. These results demonstrate that circulation of viruses transmitted by sand flies can be estimated qualitatively using dog sera. As reported in other regions of the Mediterranean, these results indicate that it is time to shift these viruses from the "neglected" status to the "priority" status in order to stimulate studies aiming at defining and quantifying their medical and veterinary importance and possible public health impact. Specifically, viruses belonging to the Sandfly fever Sicilian complex should be given careful consideration. This calls for implementation of direct and indirect diagnosis in National reference centers and in hospital microbiology laboratories and systematic testing of unelucidated febrile illness and central and peripheral nervous system febrile manifestations

Re-Emergence of Visceral and Cutaneous Leishmaniasis in the Greek Island of Crete

International audienceLeishmaniases are vector-borne diseases transmitted by phlebotomine sand flies. Three species of Leishmania are found in the Mediterranean basin: Leishmania infantum, the most common species responsible for both visceral (VL) and cutaneous leishmaniasis (CL); Leishmania major, found in North Africa and Middle East causing CL; Leishmania tropica with a limited presence in Europe, causing CL. During the last 25 years, Crete has become an endemic zone for L. infantum with a high number of infected dogs and an increasing number of human cases every year; in the last 4 years, the incidence has reached an average of seven VL patients per year in a population of 600,000. At the same time, CL has re-emerged in Crete due to L. tropica, with an average of three CL cases per year in the last 4 years. Isolates were typed as L. infantum MON-1 and MON-98 and L. tropica MON-300, a zymodeme not reported before. Both VL and CL have spread to the whole of the island during the last 25 years, primarily in semi-urban and urban areas with altitudes of 0-50 m. The prevailing Phlebotomus species were Phlebotomus neglectus (proven vector of L. infantum) and Phlebotomus similis (suspected vector of L. tropica)

Characteristics of dogs tested in the study.

<p>Characteristics of dogs tested in the study.</p

Seroprevalence of 1,250 dog sera from Greece.

<p>Seroprevalence of 1,250 dog sera from Greece.</p

Seroprevalence of Sandfly‐Borne Phleboviruses Belonging to Three Serocomplexes (<i>Sandfly fever Naples</i>, <i>Sandfly fever Sicilian</i> and <i>Salehabad</i>) in Dogs from Greece and Cyprus Using Neutralization Test - Fig 1

<p>Geographic distribution of neutralising antibodies against Toscana virus (panel A), Sandfly fever Sicilian virus (panel B), Arbia virus (panel C) in Greece, using ArcGIS 10). Panel D represent the localisation of regions listed in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005063#pntd.0005063.t002" target="_blank">Table 2</a>.</p