A cluster of four cases of cutaneous leishmaniasis by Leishmania donovani in Cyprus: a case series

Introduction: Leishmaniasis is endemic in more than 95 countries and is the only tropical/subtropical vector-borne disease that has been endemic in Southern Europe for decades. To the best of our knowledge, this is the first case of cutaneous leishmaniasis by Leishmania donovani in a child and the first cluster with adult cases reported in Europe. Case presentation: We describe a familial cluster of four cutaneous leishmaniasis cases among Greek Cypriots caused by L. donovani in a Paphos village, in Cyprus. A 6-year-old boy (Case number 1) had a persistent lesion in the left angle of his upper lip, a 60-year-old woman (Case number 2) presented with a 2cm-diameter glabella lesion on her forehead, a 60-year-old man (Case number 3) developed a lesion on his moustache area and a 40-year-old woman (Case number 4) had a lesion on her neck. In Case number 3 the lesion was self-cured; the other cases recovered after surgical resection followed by liposomal amphotericin B (Case numbers 1 and 4) or thermotherapy and liposomal amphotericin B (Case number 2). Conclusions: This familial cluster of cutaneous leishmaniasis, due to the anthroponotic L. donovani, shows that the sand fly species responsible for transmitting this parasite species is found in the area around the three neighbouring houses involved. The factors favourable for the survival, spread and contact of the vector with people could be assessed in this area for the establishment of preventative measures to safeguard public health

Spread of Vector-borne Diseases and Neglect of Leishmaniasis, Europe

Exotic vector-borne diseases are gaining attention at the expense of leishmaniasis

Epidemiological investigation of leishmaniasis in Cyprus: control and surveillance of the disease

Leishmaniases are diseases caused by members of the genus Leishmania, protozoan parasites infecting numerous mammal species including humans, and transmitted by the bite of phlebotomine sandflies. In Cyprus, leishmaniasis has been considered exclusively a veterinary problem. The aim of the study is the epidemiological investigation of leishmaniases in Cyprus, in dogs and humans, for the control and surveillance of the disease. The survey conducted to resolve this was based on the assumption that if there were to be VL cases, they had to be in the areas with the highest dog seroprevalence. To locate these areas, an epidemiological study was carried out on the dog population. A map of Cyprus was divided into 82 equal squares of which 30 were chosen randomly as the study area. Dog seroprevalence revealed the areas with the highest risk for humans. In such two areas, as well as in one with zero dog seroprevalence, a seroepidemiological study was conducted on the human population and sandfly collections were done to compare species in these three areas. The Government controlled part of the island, southern Cyprus (Fig.1), covering 5,896 km2 and comprising five prefectures, was divided into 82 equal arbitrary squares on a map. The squares were given numbers and 30 numbers were drawn from a ballot to select the squares to be included in the study. One or more villages falling in each of the 30 squares, according to dog population size, were chosen so as to have at least 200 dogs per square. A total of 900 dogs (379 males and 521 females; 719 hunting, 87 guard, 94 companion) living in the 30 areas were sampled (30 dogs per area) during 2005 and 2006. Owned dogs, living permanently in the area irrespective of race, age color or health status were included in the study. To select the dogs, the houses in each village in the study area were given numbers and drawn from a ballot; visited with a government veterinarian and the dog owners were informed about leishmaniasis and asked to participate in the study by providing a written consent. The dogs were examined clinically, peripheral blood (900 samples) and lymph node aspirates when enlarged lymph nodes were observed (22 samples) were collected and personal, epidemiological and clinical data were registered in questionnaires for each dog. Severe leishmaniasis symptoms were diagnosed in 18 dogs, the disease was confirmed by serology and PCR, the dogs were euthanized with the consent of the owner and the spleen provided. In addition, 2,056 dog sera were provided by veterinarians throughout the island for routine testing for leishmaniasis. ...................Οι λεϊσμανιάσεις είναι μολυσματικές ασθένειες που μεταδίδονται μέσω αιματοφάγου διαβιβαστή ξενιστή, τη σκνίπα και είναι ανθρωπονόσοι ή/ ζωονόσοι. Στην Κύπρο, ένα νησί στο όριο της Ευρώπης με την Ασία και την Αφρική, η ΣΛ θεωρείτο μέχρι πρότινος ασθένεια των σκύλων, καθώς δεν υπήρχε επίσημη αναφορά ανθρώπινων περιστατικών μέχρι και το 2005. Σκοπός αυτής της διατριβής ήταν η επιδημιολογική διερεύνηση της λεϊσμανίασης στην Κύπρο, στο σκύλο και στον άνθρωπο, για τον έλεγχο και την επιτήρηση της νόσου και η ανάπτυξη ενός ικανού συστήματος ενεργού επιτήρησης της ασθένειας, με σκοπό το φραγμό μετάδοσης της στον άνθρωπο και την παρεμπόδιση της εξάπλωσης της στο σκύλο. Η έρευνα σχεδιάστηκε και εκτελέστηκε έτσι ώστε να εντοπιστούν οι περιοχές με το υψηλότερο ποσοστό κρουσμάτων στα σκυλιά, οι περιοχές δηλαδή με τον μεγαλύτερο κίνδυνο για τη μετάδοση της λεϊσμανίασης στον άνθρωπο και οι περιοχές με το χαμηλότερο ποσοστό ή αρνητικές, περιοχές δηλαδή με χαμηλό κίνδυνο για τη μετάδοση της λεϊσμανίασης στον άνθρωπο. Σε αυτές τις περιοχές πραγματοποιήθηκε οροεπιδεμιολογική έρευνα στον άνθρωπο και έγιναν συλλήψεις Φλεβοτόμων με σκοπό την σύγκριση των ειδών που παρατηρούνται στις τρεις περιοχές. Για τον σκοπό αυτό, οι ελεύθερες περιοχές της Κύπρου, έκτασης 5,896 τετραγωνικών χιλιόμετρων, χωρίστηκαν σε χάρτη σε 82 ίσα τετράγωνα. Στο κάθε τετράγωνο δόθηκε αριθμός 1-82 και με κλήρωση επιλέχθηκαν τυχαία 30 τετράγωνα. Μια περιοχή-τετράγωνο μπορούσε να περιλαμβάνει 1,2,3 ή και περισσότερα χωριά ή πόλεις. Από αυτά επιλέχθηκαν, με κλήρωση, τα χωριά στα οποία πραγματοποιήθηκαν οι δειγματοληψίες στα σκυλιά και η επιλογή του αριθμού των χωριών σε κάθε περίπτωση ήταν ανάλογη του πληθυσμού των σκυλιών ώστε να περιλαμβάνει τουλάχιστο 200 ζώα ανά τετράγωνο. Από σκυλιά που ζούσαν στα 30 τετράγωνα ελήφθησαν συνολικά δείγματα από 900 ζώα (30 σκυλιά ανά τετράγωνο). Για το σκοπό αυτό, οι οικίες που θα συμπεριλαμβάνονταν στη μελέτη, από τα χωριά των 30 τετραγώνων, επιλέγηκαν μετά από κλήρωση. Τα σκυλιά εξετάστηκαν κλινικά και ελήφθησαν δείγματα περιφερικού αίματος (900 δείγματα) και πολφός λεμφογαγγλοίων στις περιπτώσεις που διαπιστώθηκε διόγκωση των λεμφαδένων (22 δείγματα). Για κάθε σκυλί συμπληρώθηκε ερωτηματολόγιο με όλα τα προσωπικά, επιδημιολογικά και κλινικά του στοιχεία. Σοβαρά κλινικά συμπτώματα διαγνώστηκαν σε 18 σκυλιά, στα οποία έγινε ευθανασία μετά από επιβεβαίωση σπλαχνικής λεϊσμανίασης με ορολογικές και μοριακές εξετάσεις και προσκομίστηκε η σπλήνα για καλλιέργεια, μετά από τη σύμφωνη γνώμη του ιδιοκτήτη. Επιπρόσθετα εξετάστηκαν 2056 δείγματα ορών αίματος από σκυλιά από όλες τις ελεύθερες περιοχές της Κύπρου, που ελήφθησαν από ιδιώτες κτηνίατρους από ζώα για σκοπούς ελέγχου ρουτίνας. ..................

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

Detection of Pgp 170 by Western Blotting in the 10 <i>Leishmania</i> isolates.

<p>The Pgp 170 molecules (130-200 kDa) were evident in the “resistant” (high Rhod-123 efflux) but not in the “susceptible” (low Rhod-123 efflux) isolates. The “resistant” human isolate (H2) presented lower signal compared to the dog “resistant” isolates. For the evaluation of the Pgp expression in each isolate, the C219 monoclonal antibody and exponential phase promastigotes, at a concentration of 10⁷ parasites, were used.</p

Treatment with Glucantime resulted in an increase in the number of Pgp molecules.

<p>Mean Fluorescent Intensity (MFI) of the 10 <i>Leishmania</i> isolates, measured by Flow Cytometry, showed an increase in the number of Pgp molecules, in all isolates, if they had previously been treated with the drug Glucantime compared to the untreated controls. This increase was not linear (r₂ = 0.27).</p

Number of Pgp molecules in a ''resistant'' and a ''susceptible'' isolate before/after exposure to Glucantime.

<p>The number of Pgp 170 molecules was found in higher numbers in the “resistant” compared to the “susceptible” isolate. This number increased, in both isolates, after exposure to Glucantime.</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

Detection of Pgp 170 molecules, by Transmission Electron Microscopy, after immunogold labeling.

<p><i>Leishmania</i> amastigote (inside a THP-1 infected cell): cytoplasm of the THP-1 cell (a); <i>Leishmania infantum</i> body (b). Black spots (as indicated by white arrow) show Pgp 170 molecules (gold granules after immunogold labeling using the C219 monoclonal antibody).</p

Rate of efflux of Rhod-123, in the 10 <i>Leishmania</i> isolates, estimated by Flow Cytometry.

<p>Efflux, both in the absence (slope “α”) and presence (slope “β”) of Verapamil hydrochloride was found to be correlated to the influx (the amount of drug that entered the parasite body: Mean Fluorescent Intensity on baseline (MFI) (c = 0.63 and c = 0.58, respectively). Efflux was blocked by Verapamil hydrochloride in all isolates except in isolate D2, with the lowest slope “α”, for which slope “β” had the same value (0.16)*.</p