Leishmaniasis in Cyprus: Where Are We Now?

Leishmaniasis in Cyprus: Where are we now?

Summary

(500 words)

Little is known about the distribution and prevalence of leishmaniasis in Cyprus, as well as the distribution of the potential sand fly vectors.

Leishmaniasis is a considerable global public health problem that presents an expanding concern during the last decade (Alvar, Vélez et al. 2012).

Monitoring medically important phlebotomine sand fly species is of a huge significance for the prediction of potential transmission cycles of Leishmania parasite in Cyprus.

Introduction and Existing knowledge

Leishmaniases are neglected protozoan diseases transmitted to humans and other animals by phlebotomine sand flies (Diptera, Psychodidae). Leishmaniases are caused by different species of the genus Leishmania (Kinetoplastida, Trypanosomatidae), that are obligate protozoan parasites. These diseases are endemic in more than 98 countries, including the Mediterranean basin, threatening a total of 350 million people, while 12 million people are currently affected by leishmaniasis (Alvar, Vélez et al. 2012, Akhoundi, Kuhls et al. 2016) (einai review). The majority of people infected by Leishmania parasites do/does not develop any symptom at all during their life, and therefore have a silent infection without any symptoms (World Health Organization 2017a) (+CDC fylladio). Leishmaniasis will be developed in only a small fraction of those infected individuals (World Health Organization 2017b). However, leishmaniases are a major public health problem with a wide spectrum of clinical symptoms, including/resulting in 3 main forms of leishmaniases; visceral leishmaniasis (VL), also known as kala-azar, cutaneous leishmaniasis (CL), and mucosal or mucocutaneous leishmaniasis (ML/MCL) (WHO website). VL is the most aggressive form of the disease and is associated with serious morbidity and mortality. If left untreated, it can be life-threatening. The incubation period varies from 10 days to more than 1 year, and the beginning of the disease is progressive (World Health Organization 2010). VL is characterised by prolonged fever, enlargement of the spleen (splenomegaly), enlargement of the liver (hepatomegaly), weight loss, pallor, and anaemia (Berman 1997, Daher, Evangelista et al. 2008, World Health Organization 2010). CL is the most common form of leishmaniasis (WHO website) and has a tendency to be self-healing (Den Boer, Argaw et al. 2011). The incubation period is generally 2-8 months. CL causes skin sores, dry ulcers of the skin, on exposed parts of the body, leaving permanent scars and causing serious disability (World Health Organization 2010). Some people may have swollen glands, if the skin lesions are near glands (Centers for Disease Control and Prevention 2013)(CDC fylladio). MCL can occur when the infection spreads from the skin to the mucous membranes of the mouth, nose, or throat, causing sores. This could happen from delayed healing of the primary CL (World Health Organization 2010). Annually, there are an estimated 0.2-0.4 million new cases of VL and 0.7-1.2 million new cases of CL worldwide (Alvar, Vélez et al. 2012, Akhoundi, Kuhls et al. 2016).

Leishmania parasites

The parasite Leishmania was named after the Scottish pathologist William Boog Leishman who discovered the parasite in the spleen of a dead soldier. He is credited together with the Irish doctor Charles Donovan for the discovery of the parasite that caused visceral leishmaniasis (Steverding 2017). The term Leishmania donovani was suggested by Ross in 1903 (Ross 1903).   

Leishmania parasites are unicellular elongated or spherical organisms characterised by the presence of a flagellum, that is intracellular or extracellular, and a kinetoplast that is mitochondrial DNA (reviewed by (Lukes, Guilbride et al. 2002, Waller, McConville 2002, Bates 2007)). The life cycle of Leishmania is characterised as a complex life cycle, involving both invertebrate and vertebrate hosts; a female sand fly and mammals, including humans and dogs. These parasites have two developmental stages: promastigotes (flagellated), the proliferative form found in the lumen of the female sand fly, and amastigotes (non-flagellated), the proliferative form found inside several types of mammalian host cells (Handman, Bullen 2002, Bates 2007). Promastigotes are weakly elongated motile forms with a short flagellum at the anterior end of the cell, while amastigotes are small (3-5 μm) immotile rounded forms of the parasite (Murray, Berman et al. 2005, Dostálová, Volf 2012). The infection initiates when a female sand fly bites an infected mammalian host for blood feeding (ref) (Killick-Kendrick 1999, Bates 2007). Amastigotes are found in phagolysosomes (phagosome fused with lysosome) of macrophages and other phagocytes of the mammalian host (Handman, Bullen 2002). The sand fly ingests blood containing macrophages infected with amastigotes, and becomes infected and hence, the development of the parasite begins in the vector. Due to the change in conditions moving from the mammalian host to the sand fly midgut, the transformation of amastigotes into procyclic promastigotes is triggered (Kamhawi 2006, Bates 2007). This is the first stage in the vector and is called a procyclic promastigote, as it is a weakly motile proliferative form that replicates in the bloodmeal. During this phase, the parasites are located at the posterior end of the midgut and development goes on until metacyclic promastigotes are situated in the anterior of the gut (Gossage, Rogers et al. 2003, Bates 2007). These metacyclic promastigotes are infective Leishmania parasites that are delivered to the skin of the new mammalian host when the infected sand fly feeds again, leading to the transmission of disease (Sacks, Perkins 1985, Bates 2007, Dostálová, Volf 2012). Then, these are taken up by macrophages and transform into intracellular amastigotes (Handman, Bullen 2002). Amastigotes multiply in phagolysosomes of macrophages and later, burst out of (released from-parousiasi) macrophages, infecting new macrophages and other phagocytes of the host (Rittig, Bogdan 2000, Handman, Bullen 2002). Leishmania parasites need approximately 6-9 days to complete their development in the sand fly (the approximate time needed-parousiasi, des paper Kamhawi 2006). However, the time needed for the completion of their development depends on several factors, such as Leishmania species, sand fly species, and environmental temperature (Kamhawi 2006). Apart from phlebotomine sand flies, transmission of the parasite and therefore infection can arise from blood transfusion, when contaminated needles are being used, or through transplacental spread, and organ transplantation (Murray, Berman et al. 2005). Therefore, an infected mother can transmit the parasite to her child, and an infected female dog to its puppy (European Centre for Disease Prevention and Control 2017).

Leishmania parasites are classified into three distinct subgenera; Leishmania (Leishmania), Leishmania (Viannia), and Leishmania (Sauroleishmania). These subgenera differ based on the sand fly’s gut parts that the parasites colonise (Bates 2007). Parasites belonging to the subgenera Leishmania and Viannia infect mammals, while those belonging to the Sauroleishmania infect reptiles (Bates 2007). For the members of the subgenus Leishmania, the parasite development takes place in the midgut and foregut of the sand fly, whereas for the members of the subgenus Viannia, the development takes place also in the hindgut. The subgenus Leishmania is present in both the Old and the New Worlds, while the subgenus Viannia is only present in the New World (Lainson, Ward et al. 1977).  Currently, 31 Leishmania species are identified as parasites of mammals, and 18 of these are known to be pathogenic for human beings (Akhoundi, Kuhls et al. 2016, Espinosa, Serrano et al. 2016, Steverding 2017). Leishmania strains are characterised by their enzymatic profiles and organised into groups of homogenous taxonomic units, the zymodemes (Rioux, Lanotte et al. 1990).

VL is highly endemic in the Indian subcontinent and in East Africa. Furthermore, in the Mediterranean basin, VL is the main form of the disease, which occurs in areas where Leishmania parasites live primarily on dogs (WHO website).  Visceral leishmaniasis is caused by L. infantum in the Mediterranean, Middle East, Central Asia (Old World), and Americas (New World), and L. donovani in India, East Africa, and the Arabian Peninsula (Old World). Cutaneous leishmaniasis of the Old World is caused by L.infantum, which is the most common cause of CL in Southern Europe, L. donovani, L. tropica, L. major,and L. aethiopica. CL of the New World is caused by various species of the two subgenera, Leishmania and Viannia, such as L. mexicana, and L. infantum, belonging to the subgenus Leishmania, and L. braziliensis, and L. panamensis, belonging to the subgenus Viannia. Mucocutaneous leishmaniasis is a disease of the New World and is caused primarily by L. braziliensis, and L. panamensis (World Health Organization 2010). L. infantum was initially found in children with infantile splenic anaemia and hence was named like this (World Health Organization 2010).

Currently, there are 3 different ways of Leishmania transmission (different epidemiological cycles); zoonotic, anthropozoonotic, and anthroponotic. In the zoonotic cycle, Leishmania parasites circulate between animals via sand flies, causing zoonotic CL and VL, while humans are infected accidentally. The reservoir hosts are wild or domestic animals (Bañuls, Hide et al. 2007, World Health Organization 2010). In Southern Europe, zoonotic CL and VL are caused by L. infantum, with dogs being the main reservoir host (Gradoni 2013, Koliou, Antoniou et al. 2014). In anthropozoonotic cycle, parasites circulate between animals and humans via sand flies (prepei na pw eidos parasitou logika opws stis alles dyo katigories) (Bañuls, Hide et al. 2007). In anthroponotic cycle (transmissible from human to human), parasites circulate between humans via anthropophilic sand flies and therefore, the reservoir host is human (Bañuls, Hide et al. 2007, World Health Organization 2010). In Greece and eastern Mediterranean countries, anthroponotic CL is caused by L. tropica and it happens sporadically (Gradoni 2013, Koliou, Antoniou et al. 2014). In Cyprus, anthroponotic CL and VL are caused by L. donovani (Antoniou, Haralambous et al. 2008).

Dogs are reservoir hosts of VL caused by L. infantum and develop canine leishmaniasis (CanL) more frequently than humans developing leishmaniasis caused by L. infantum. (reference). A dog can become infected through the bite of a sand fly that became infected after feeding on an infected dog (an dagkwsei infected mammal pali mporei na to metadosei sto dog?-an exei sand fly p dagkwnei k dog k mammal tote yes). Humans become infected by L. infantum accidentally (Moreno, Alvar 2002). CanL is characterised by a broad range of clinical symptoms. These include weight loss, skin abnormalities such as exfoliative dermatitis and ulcers, enlargement of the lymph nodes, onychogryphosis, and renal failure (Ciaramella, Oliva et al. 1997).  Nevertheless, many infected dogs are asymptomatic (Oliva, Scalone et al. 2006, Otranto, Paradies et al. 2007). Both, symptomatic and asymptomatic infected dogs constitute a risk for humans because they are major reservoir of the parasite and can transmit the parasite through phlebotomine sand fly vectors (Dujardin, Campino et al. 2008). It is estimated that 2.5 million dogs are infected with Leishmania in the Mediterranean basin only (Moreno, Alvar 2002, Akhoundi, Kuhls et al. 2016). In Cyprus, leishmaniasis (VL) has been considered to be a disease exclusively widespread in dogs, as no human cases were officially reported until 2006 (Antoniou, Haralambous et al. 2008). This may be attributed to the non-recording of human cases, although they did exist/This is because human cases were not recorded, although they did exist, and also to the fact that before 1945 (in former years-paper tou Deplazes1998), CanL was extensively spread in Cyprus (prepei na vrw to paper) (Minter, Eitrem 1989). In 1985 and 1987, two human VL cases have occurred in Cyprus (prepei na vrw to paper) (Minter, Eitrem 1989, Deplazes, Grimm et al. 1998). In 2006, six autochthonous human cases, four of CL and two of VL, caused by L. donovani were detected on the island (Antoniou, Haralambous et al. 2008).

Little is known about the distribution and prevalence of leishmaniasis in Cyprus, as well as the distribution of the potential sand fly vectors.

It is well-known that L. infantum is culpable for VL and CL in the Mediterranean basin (Dujardin, Campino et al. 2008, World Health Organization 2010) (paper 23), and it is also responsible for canine leishmaniasis in Cyprus and has been previously detected in sand flies (Deplazes, Grimm et al. 1998, Mazeris, Soteriadou et al. 2010).

In 2005-2006, CanL, caused by L. infantum, was found to be extensively distributed across the island, and seroprevalence was found to be increased by 9 times since 1996 (Deplazes, Grimm et al. 1998), indicating that the parasite was actively transmitted in the country by autochthonous sand fly species (Mazeris, Soteriadou et al. 2010). It was the first attempt of the study of leishmaniasis in Cyprus, but, since 2006, no studies have been conducted in Cyprus in order to see what the current status of Leishmania is in the country. According to the study conducted between 2005-2006 by Mazeris, Soteriadou et al. (2010), in the Republic of Cyprus there is a paradox regarding Leishmania infection, since two distinct transmission cycles appear to run in parallel. The first transmission cycle occurs in dogs, with L. infantum causing CanL (Deplazes, Grimm et al. 1998). Seropositivity of dogs for L. infantum reached 33.3% in some areas. However, there were no human CL or VL cases, caused by L. infantum,between Greek Cypriots (Mazeris, Soteriadou et al. 2010). The second transmission cycle occurs in humans, with L. donovani causing CL or VL (Antoniou, Haralambous et al. 2008). This unique situation occurring in Cyprus is not the case (is not applied in other) for other Mediterranean countries (World Health Organization 2010), such as Greece (Mono stin ellada? pou allou? Kai tyxaia paei ston anthrwpo?), where L. infantum causes CanL and human CL or VL cases (Ntais, Sifaki-Pistola et al. 2013).

VL of the Old World is caused by

Leishmaniasis is a considerable global public health problem that presents an expanding concern during the last decade (Alvar, Vélez et al. 2012).           

Sand flies

Phlebotomine sand flies are vectors of several pathogens and therefore, this has an important impact for public health (paper 24). Leishmaniasis, in general, is classified as the second most common cause of parasite-related death after malaria (Den Boer, Argaw et al. 2011, Al-Kamel 2016) (prepei na vrw kalytero citation).  Leishmania parasites are transmitted to mammals through the bite of infected female sand flies (Murray, Berman et al. 2005, Bates 2007, Ready 2013). Sand flies pass through four developmental stages; egg, larva, pupa, and adult (Killick-Kendrick 1999, Claborn 2010) (einai review). Shortly after emergence from the pupal stage, both male and female adult sand flies feed on plant nectar and sugary secretions that provide adequate nourishment for them to survive and fly (Schlein, Warburg 1986, Killick-Kendrick 1999, Claborn 2010, Ready 2013). Female sand flies need protein in order to produce eggs and therefore seek bloodmeal. Proteins are important nutrients for reproductive fitness and egg production (Killick-Kendrick 1999). When they feed on mammalian hosts, infected by Leishmania parasites, they can become infected too. Infected sand flies can then transmit the infection to more mammalian hosts when they feed again (Murray, Berman et al. 2005, Bates 2007). Most sand fly species are active at dusk, at night and in the early morning, when humidity increases and temperature decreases (Maroli, Feliciangeli et al. 2013) (They are nocturnal) (ecdc.europa website-paper 2from email). The feeding activity of sand flies can be affected by humidity, air movement, and temperature as well (Maroli, Feliciangeli et al. 2013, Ready 2013). During daylight, they rest in cool and humid places including fissures in walls, houses, caves, tree holes, and dense vegetation (Killick-Kendrick 1999). The breeding sites of sand flies are characterised by humidity, cool temperature, and organic matter. These environmental conditions are important for the eggs, as they require high humidity in order to survive, and later for the larvae that need to be fed on organic matter (Killick-Kendrick 1999). The fluctuations in sand fly numbers and hence in sand fly population densities depend on the local climate, and mostly on temperature and precipitation (Maroli, Feliciangeli et al. 2013) (paper 2from email). Climate changes contribute to the increase of the sand fly activity, and therefore to the increase of the disease incidence (Antinori, Schifanella et al. 2012). In recent years, the geographical range/distribution of phlebotomine sand flies in the Mediterranean region has increased and this geographical expansion has been attributed to ongoing climate changes (Fischer, Moeller et al. 2011, Moriconi, Rugna et al. 2017). According to the findings of (Alten, Maia et al. 2016), it is confirmed that temperature is a major determinant for the activity of sand flies. Moreover, climate changes can affect the developmental cycle of Leishmania in sand flies (Bounoua, Kahime et al. 2013, Medlock, Hansford et al. 2014). Therefore, global warming can influence the dispersal of sand fly vectors and leishmaniasis, as well (Medlock, Hansford et al. 2014, Moriconi, Rugna et al. 2017). So far, approximately 900 sand fly species are known, 70 of those have been identified as vectors in leishmaniasis transmission (Ready 2013) (isws na mpei akoma ena paper from ruby), and 98 of those are proven or suspected vectors of human leishmaniases (Maroli, Feliciangeli et al. 2013, Steverding 2017). Two genera of sand flies, Phlebotomus (Old World) and Lutzomyia (New World), are the only proven vectors of Leishmania species pathogenic for humans and therefore are of medical importance (Killick-Kendrick 1999, Murray, Berman et al. 2005, Bates, Depaquit et al. 2015). In 2016, (Alten, Maia et al. 2016)presented that seven out of eight L. infantum vectors, found in the Mediterranean Basin (Phlebotomous species), appeared to be of Larroussius subgenus. Apart from P. tobbi (Larroussius Subgenus), (Alten, Maia et al. 2016)have collected P. galilaeus (Larroussius Subgenus), P. sergenti (Paraphlebotomus Subgenus), and P. papatasi (Phlebotomus Subgenus). According to (Léger, Depaquit et al. 2000), P. tobbi is the proven L. infantum vector in Cyprus. However, this vector may also be responsible for the transmission of L. donovani in Cyprus, but its role in the transmission of L. donovani needs to be determined (Antoniou, Haralambous et al. 2009, Antoniou, Gramiccia et al. 2013). Regarding a specific vector of L. donovani in Cyprusis still unknown (WHO 2017), but candidate vectors could be P. galilaeus, and P. alexandri (Léger, Depaquit 2008, Antoniou, Haralambous et al. 2009).    

(na mpei paperMazeri gia to poia eidi sknipas vrikan tote).

Evidence indicates the spread of sand flies, as well as the emergence of autochthonous cases of CanL and VL into new regions in Europe that were not previously affected by the parasite, such as northern Italy and southern Germany (Bogdan, Schonian et al. 2001, Maroli, Rossi et al. 2008) (paper 23&24). Therefore, it is really important that epidemiological studies are often conducted to observe the expansion of the disease in dogs.

Scientific Hypothesis and Specific Objectives

Leishmaniasis is a major global public health problem that presents an expanding concern during the last decade (Alvar, Vélez et al. 2012). However, little is known about the distribution and prevalence of leishmaniasis in Cyprus, as well as the distribution of the potential sand fly vectors. Knowing that many neighbouring countries of Cyprus have recently reported leishmaniasis cases (CL or VL), it can be concluded that the island is at a considerable risk. The Syrian Arab Republic, a neighbouring country of Cyprus, is among the 10 countries that hold more than 70% of the global number of CL cases (World Health Organization 2016).

There is the need for updating the epidemiological evidence base of the country in order to design effective strategies for the control of leishmaniasis. Therefore, detailed information on epidemiology and geographical distribution of leishmaniasis for Cyprus need to be known.

Moreover, the climate conditions in Cyprus permit the proliferation of sand flies throughout the year with some seasons of intense activity. However, studies investigating potential future distribution of sand fly species in Cyprus, considering a changing climate are lacking (paper combining climatic projections). Any insight into the distribution of the sand fly species in the country is essential in defining the potential change in Leishmaniasis distribution in Cyprus (paper-a summary of the evidence).

Furthermore, in the Mediterranean basin three Leishmania species seem to be involved causing VL and/or CL. These include Leishmania infantum, which is the most common species in the Mediterranean basin and is responsible for CanL, as well as, VL and CL cases in humans, Leishmania major, which causes CL in the Middle East and North Africa, and Leishmania tropica, which causes CL and is present in Greece (World Health Organization 2010, Ntais, Sifaki-Pistola et al. 2013).

Taking all the above into account, it can be assumed that a new transmission cycle that involves L. infantum causing VL or CL cases in humans may be involved in the epidemiology of the Leishmania parasite in Cyprus. If this is the case, then the possibility of human infection in areas where infected dogs reside increases immediately, as the possibility of human infection typically increases with the number of infected dogs in an area. Furthermore, new Leishmania species may have entered the country, causing the disease to humans, or dogs, or both. To oti exoume immigrants ktl k taxidevoun se mas. In neighbouring countries of Cyprus, such as the Syrian Arab Republic, Iraq, Islamic Republic of Iran, Egypt, Libya, and Saudi Arabia, L. major and L. tropica are responsible for CL (World Health Organization 2014). Because of the Syrian civil war, many Syrian refugees migrate/travel to Cyprus and other parts of Europe pursuing the safety they need. Apart from that, many people from neighbouring countries or even around the world other countries where leishmaniasis is found travel to Cyprus to work and live a better life. Therefore, the migration of people from areas with transmission of Leishmania to areas previously unaffected or affected by other Leishmania species poses a risk of spreading the disease and introducing new Leishmania species. A recent example is the migration of Syrian citizens that has precipitated to the introduction of L. major in southern Turkey, causing an increase of CL cases (Koltas, Eroglu et al. 2014, Özbilgin, Çulha et al. 2016, Moriconi, Rugna et al. 2017) (prepei na vrw to turkiko paper).

(In a few words), The purpose of this future work will be the study of the present status of leishmaniasis in Cyprus. The study will include the geographical distribution of sand fly species in Cyprus, the infection rate in sand fly populations, an investigation into whether L. infantum causes VL and/or CL in humans, whether other Leishmania species are involved in leishmaniasis in Cyprus, and last but not least, the identification of areas in Cyprus that are presently at high risk.

First Objective

Geographical distribution of sand fly species

Establishing the geographical distribution of sand fly species is one of the objectives of this work. It is well-known that the phlebotomine sand flies are serious vectors of leishmaniases (isws na mpei kapoio reference). The potential distribution of several main Phlebotomous spp. found in Cyprus, which are of medical importance, will be predicted by performing ecological niche modelling. This will give information on vector species distribution on the island and will help in predicting potential disease risk and analysing the relationship between climate and sand flies’ distribution. More specifically, the resulting models will be used to associate environmental factors with the distribution of these species, and leading to a risk analysis for leishmaniasis in Cyprus identifying and understanding the distribution of these sand fly species will lead to a better understanding of the epidemiology of leishmaniasis in Cyprus.

Fifth Objective

Infection rate in sand flies

The final objective of this future work is the estimation of the infection rate in sand fly populations in Cyprus. Leishmaniases are one of the most diverse vector-borne diseases in their ecology and epidemiology, as they include more than 20 Leishmania species, and can infect (a spectrum of reservoir hosts and sand fly vector species) some reservoir hosts and several sand fly vector species (Bañuls, Hide et al. 2007, World Health Organization 2010). Knowing/Estimating the percentage of positive sand flies is a benefit and power for controlling leishmaniasis in areas where endemicity is high. Moreover, detecting naturally infected sand flies is important for the identification of a sand fly species as a vector of Leishmania parasite. According to a study previously conducted in Northern Cyprus and published in the journal Parasites and Vector, there was an ongoing activity of Leishmania parasite, and more specifically of L. infantum, that was detected in Phlebotomus Tobbi species (Ergunay, Kasap et al. 2014). This indicates that the same scenario is applied across the island.

Second Objective

Seroepidemiological study in dogs

Another objective of this future work is the identification of the areas in Cyprus that are in a high risk for leishmaniasis. It is important for the public health to be aware of areas where the risk for leishmanial infection is high. The identification of these areas can be achieved by performing seroepidemiological study in the dog population in the island for a start, and then in the human population. The possibility of a human infection typically increases with the number of infected dogs in an area. A comparison between a study conducted in 2005-2006 in dogs in Cyprus (Mazeris, Soteriadou et al. 2010) and this future study should be made in order to find out whether the risk is increased or decreased in the areas. Also, it is important to find whether there are any changes in climate/habitat that occurred all these years since 2005-2006 and that have influenced the current results. The proposed study would contain the areas in Cyprus that were found to have the greatest risk in 2005-2006, such as Pafos and Limassol prefectures (Mazeris, Soteriadou et al. 2010), among others. The estimation of leishmaniasis in dogs is essential since they are the reservoir host of the disease.

Third Objective

An important objective of this work is to investigate whether there are human cases of Leishmania infection caused by L.infantum in Cyprus. According to the study conducted between 2005-2006 by Mazeris, Soteriadou et al. (2010), there were no human CL or VL cases between Greek Cypriots, caused by L.infantum and there are two distinct transmission cycles in the Republic of Cyprus that appear to run in parallel. Specifically, there is a transmission cycle that occurs in dogs, with L.infantum causing CanL (Deplazes, Grimm et al. 1998), and another transmission cycle that occurs in humans, with L.donovani causing CL or VL (Antoniou, Haralambous et al. 2008). In other Mediterranean countries (World Health Organization 2010), such as Greece, there is a transmission cycle occurring in dogs and humans, with L.infantum causing CanL, and CL or VL, respectively (Ntais, Sifaki-Pistola et al. 2013). This was not the case for Cyprus 10 years ago, when Mazeris, Soteriadou et al. (2010) reported that there were no human CL or VL cases between Greek Cypriots, caused by L.infantum. (So, what will happen if this is the case in Cyprus now?) If this scenario is applied in Cyprus nowadays then the risk for Leishmania infection in humans is extremely high as the chance of getting Leishmania infection for humans generally increases with the number of infected dogs in an area. Also, another reason that the risk for Leishmania infection will be high if the aforementioned scenario is currently applied in Cyprus is that there are human VL cases caused by L.infantum in all areas in the Mediterranean basin where CanL is high prevalent (Alvar, Canavate et al. 1997) (isws prp na mpei k prosfato paper p na leei ayto to pragma).

Fourth Objective

Identification of Leishmania parasites in dogs and sand flies

It’s really important to know whether other types of Leishmania parasite are present in the dog population in Cyprus, as well as in sand fly species that are distributed across the island. Apart from L.infantum in dogs, are there any other types of the parasite in the island? Apart from L.infantum in dogs, the possibility of existence of any other types of the parasite in the island should be investigated. The existence of new Leishmania types is of a huge importance as in countries near to Cyprus (na vrw paper gia deadly type Indias) there are other types which are of medical importance and most probably are deadly types. According to World Health Organization (WHO) (http://www.who.int/mediacentre/factsheets/fs375/en/), Leishmania infection occurs in areas where migrations of people caused by many socioeconomic reasons, such as war, take place. Therefore, the risk for Leishmania infection in Cyprus exist and is quite high as many migrants from Syria, India and other countries arrive in the island for a better future. Since 2005-2006, when the last survey of human leishmaniasis was conducted in Cyprus, it has been over a decade, and hence the risk is high as changes in the climate should have occurred that influence the existence of Leishmania parasites.

Importance and Innovation

Leishmaniases are a considerable global public health problem that presents an expanding concern during the last decade (Alvar, Vélez et al. 2012, Akhoundi, Kuhls et al. 2016). They have a high impact on individuals with a wide spectrum of clinical symptoms and the potential for spreading further. Therefore, surveillance of leishmaniasis in Cyprus is crucial for public health. It is thus essential to identify areas in Cyprus that are at high risk for leishmaniasis. It is important for the public to be aware of areas where the risk for leishmanial infection is high. Predicting a potential disease risk and analysing the relationship between climate and sand flies’ distribution, will contribute to a better understanding of the epidemiology of leishmaniasis in Cyprus. Risk areas of leishmaniasis will be predicted using GIS, as this, based on statistical associations between environmental data and previous knowledge on the distribution of the disease, makes it possible to produce predicted-risk maps of the disease. This will lead to the development of vector-control programmes and strategies for the controlling and surveillance of Leishmania infection. All the resulted facts that will come out of this proposed work make it really important and requisite/necessary for Cyprus.

It has been over a decade since the first and last survey of human and dog leishmaniasis conducted in Cyprus. During this time, no studies were performed on the island to test leishmaniasis infection. It is not known whether other types of Leishmania parasite are present in the human and dog population in Cyprus or whether there are human cases of Leishmania infection caused by L.infantum in Cyprus, as in Greece. Hence, such a study, described in this proposal is really important to be conducted in Cyprus to give answers to the aforementioned questions and show us what is the prevailing/current situation regarding leishmaniasis on the island.

Surveillance of the disease in dog population is of a huge importance, as the number of dogs infected with Leishmania in an area indicates/determines the local risk of human infection (paper 23) (Dujardin 2006). Therefore, in order to be able to determine the risk of human VL infection in an area, it is important to be aware of the geographical distribution and prevalence of the leishmaniasis disease in dogs (paper 23).

Furthermore, with this work done, the Leishmania infection problem can be diagnosed/detected/identified, if there is any, in its initial stages. This will make possible the prevention and deterrence of a severe problem that could have happened in the public health of Cyprus. Moreover, the size of the problem will be recorded by performing this work and if the Leishmania infection problem in the country is at a large scale, then all competent authorities of Cyprus will be alarmed and awaken, and notified to take appropriate measures. These include Ministry of Health, Ministry of Agriculture, and Veterinary Services. All medical doctors including dermatologists, paediatricians, pathologists will become aware of the problem and therefore, they will take into account leishmaniasis during diagnosis.

Knowing that many other countries face cases of Leishmania infection, the work proposed here is worth the research effort, as many immigrants arrive in Cyprus from countries in which new CL or VL cases occur. According to World Health Organization (website-kamil), in 2014 more than 90% of reported new cases occurred in Brazil, India, Ethiopia, Somalia, South Sudan, and Sudan (http://www.who.int/leishmaniasis/burden/en/). Moreover, the majority of CL cases occur in Afghanistan, Algeria, the Islamic Republic of Iran, Pakistan, Peru, Saudi Arabia, and the Syrian Arab Republic. This information ought to spark Cypriots into action, as waves of immigrants arrive on the island for several reasons, putting residents in danger of infection with Leishmania parasite.

According to a study previously conducted in Northern Cyprus and published in the journal Parasites and Vector, there was an ongoing activity of Leishmania parasite, and more specifically of L. infantum, that was detected in Phlebotomus Tobbi species (Ergunay, Kasap et al. 2014). This indicates that the same scenario or worse is probably applied across the island. Therefore, it is essential that the work proposed here is carried out to find out the distribution of sand fly species in Cyprus, the areas in Cyprus that are in an elevated risk, whether there are other types of Leishmania parasite present in sand flies and dog population, and the estimation of the infection rate in sand fly population. This study will provide critical information for evaluating potential public health threat, and establishing ideal strategies on the prevention of transmission.

but, since 2006, no experiments/studies were performed/conducted in Cyprus in order to see in what situation is the country.

Since 2005-2006, when the last survey of human leishmaniasis was conducted in Cyprus, it has been over a decade, and the importance is high, with possible changes in climate and habitat may have occurred that influence the prevalence of Leishmania parasites.

Materials and Methods

Geographic/Spatial distribution of sand-fly species in the country  

Species distribution modelling to predict

In order to predict the distribution and understand the geography (geographical range) of phlebotomine sand fly species in (the Republic of) Cyprus (government controlled area of Cyprus), ecological niche modelling will be performed. The study area for the construction of the model will consist of the entire island/Republic of Cyprus/the Southern Cyprus. Cyprus is the third largest island in the Mediterranean Sea and covers an area of 9,251 km² (Constantinides 2002). Since 1974, Cyprus is divided into two parts; the Northern part that is under Turkish occupation and the Southern Greek-Cypriot part (The United Nations 2017). Therefore, this proposal covers only the southern part of the island which is government-controlled. Southern Cyprus covers an area of 8997 km² and has five main districts: Nicosia, which is the capital, Famagusta, Limassol, Larnaca, and Paphos. The study area will consist of those five districts.

In general, the climate in Cyprus is Mediterranean. Cyprus has hot dry summers from mid-May to mid-September and mild winters from November to mid-March. Weather conditions change during short autumn and spring seasons that separate summers and winters. Average annual rainfall is approximately 480 mm. The air temperature is affected by altitude, which reduces temperature by 5 ºC per 1,000 m, and marine influences, which make summers cooler and winters warmer. Mean soil temperatures change seasonally from about 10 ºC in January to 33 ºC in July at 10 cm depth and from 14 ºC to 28 ºC at one metre. The elevation above mean sea level in the Troodos mountain range, which is the biggest mountain range of Cyprus, can reach 1952 m (http://www.justaboutcyprus.com/cyprus_geography.html ).

http://www.moa.gov.cy/moa/ms/ms.nsf/DMLcyclimate_en/DMLcyclimate_en?OpenDocument

For the construction of the species distribution model, occurrence data of phlebotomine sand fly species will be obtained from the Joint Services Health Unit in Cyprus (ekei p doulevei I Kelly) for the previous 10 years. Therefore, the phlebotomine occurrence data will correspond to captures conducted between 2005-2018. Also, species point occurrences will be obtained from samples that will be taken in several rural and urban areas across Southern Cyprus. Sand fly collection should be performed during the active sand fly seasons. Therefore, sand fly collection will be performed from the beginning of April through the end of November 2018. Phlebotomine sand flies will be trapped alive using CDC light traps, which consist of a light source, a small fan, and a space, where sand flies are trapped, covered by a hardware cloth, (that excludes) excluding larger insects. The traps must be placed in areas surrounding homes and near to animal shelters and livestock housing, where there is humidity and animal manure which are vital for sand flies for one or more consecutive nights at intervals of 10 days. The traps will be set from the dusk until early in the morning. The species identification of sand flies will be performed based on their morphological characteristics. Of all these presence records that will be obtained by performing sampling, some will be randomly selected for model development. More specifically, 75% of the presence records for each species will be randomly selected as training points and used in model development and calibration. The remaining 25% of the presence records for each species will be test points and used in model validation.

Distribution models of three sand fly species are going to be created/produced. These include Phlebotomus Papatasi, Phlebotomus Tobbi, and Phlebotomus Galilaeus, which are the main Phlebotomous spp. caught in Cyprus. MaxEnt software will be used for model development. It is a program for modelling species distributions from presence-only species records (Elith, Phillips et al. 2011). Maxent requires presence-only records and a set of predictive environmental variables. Therefore, all the species records must be imported into the MaxEnt software for model development. The software must be set to exclude duplicate presence records within the same pixel, before model development. Each distribution model will be produced with more than 20 species records to allow for more accurate modelling (Stockwell, Peterson 2002, Hernandez, Graham et al. 2006). All coordinates can be converted to the decimal degrees format.

For the modelling, predictive environmental variables from the WorldClim database need to be used, on which the models will be based (Hijmans, Cameron et al. 2005) (istoselida tou worldclim). This database provides climate layers at a spatial resolution of 1 km². Nineteen bioclimatic variables will be used/selected, based on my assessment that they would likely have relevance for the species that will be modelled, and these include annual mean temperature (BIO 1), mean diurnal range (BIO 2), isothermality (BIO 3), temperature seasonality (BIO 4), maximum temperature of warmest month (BIO 5), minimum temperature of coldest month (BIO 6), temperature annual range (BIO 7), mean temperature of wettest quarter (BIO 8), mean temperature of driest quarter (BIO 9), mean temperature of warmest quarter (BIO 10), mean temperature of coldest quarter (BIO 11), annual precipitation (BIO 12), precipitation of wettest month (BIO 13), precipitation of driest month (BIO 14), precipitation seasonality (BIO 15), precipitation of wettest quarter (BIO 16), precipitation of driest quarter (BIO 17), precipitation of warmest quarter (BIO 18), precipitation of coldest quarter (BIO 19). These data will be obtained from the WorldClim database and are derived from interpolations of climatic data from 1950 to 2000, obtained from approximately 50000 weather stations allocated around the world (Hijmans, Cameron et al. 2005). Additionally, topographic variables, such as elevation, slope, and aspect need to be obtained from… (paper2+14). All these 22 environmental variables will be used for the construction of the species distribution model.

Ecological niche models will be constructed using the Maxent software package (Version ….). This software develops models of species distribution, based on environmental variables, using the principles of the maximum entropy distribution (Phillips, Anderson et al. 2006, Phillips, Dudík 2008). All environmental variables are entered into the model building process. The Maxent software allows predictive models to be fitted to future climate projections. In case there are duplicate presence records within the same pixel, they will be removed by the Maxent software prior to model development.  The Maxent software has been shown to be a powerful tool for developing species distribution models from presence-only data (P Anderson, Dudík et al. 2006). When Maxent is applied to presence-only distribution modelling, the study area is partitioned into a grid of pixels and the pixels of the study area make up the space on which the Maxent probability distribution is defined (Phillips, Anderson et al. 2006). Pixels with known species occurrence records constitute the sample points (Phillips, Anderson et al. 2006). Seventy-five percent of the presence points for each species, will be randomly selected to construct the models and 25% will be used to test the models (Pawar, Koo et al. 2007). Following published recommendations (Phillips, Dudík 2008), the Maxent model output will be set to logistic, that gives an estimate of probability of presence for a given location between the values of 0 and 1, where 0 indicates no probability of species presence, and 1 indicates species is certain to be present. All other parameters can be set to the default settings. Maxent’s default is to allow all feature types (Elith, Phillips et al. 2011). Features in Maxent are derived from continuous environmental variables and categorical environmental variables (Phillips, Dudík 2008). Linear (L), quadratic (Q), product (P), and threshold (T) features are derived from continuous variables and were introduced in (Phillips, Anderson et al. 2006). Hinge (H) features are, also, derived from continuous variables and were introduced in (Phillips, Dudík 2008). Since the environmental variables that will be used in these models are continuous, then linear, quadratic, product, threshold, and hinge features can be enabled for the construction of the models. To test the amount of variability in the model, a number of sub-sample replicates can be ran. To avoid overfitting and collinearity in the environmental variables, an initial explorative analysis can be done by choosing a subset of potential predictors from the full set (Signorini, Cassini et al. 2014). This will lead to the fitting of several models using a single environmental layer at a time and comparing the predictive power of each variable as measured by the area under the curve (AUC).

The model should be validated using both threshold-dependent and threshold-independent methods. The AUC of the receiver operating characteristic (ROC) analysis is a threshold-independent method of evaluating model quality. This technique calculates the total area under the curve produced by plotting sensitivity against the fractional predicted area for the species (Phillips, Anderson et al. 2006, Pearson, Raxworthy et al. 2007, Phillips, Dudík 2008). The threshold-dependent evaluation will be the minimum training presence in which the probabilities are converted to binomial values with 0 being absent and 1 being present (Phillips, Anderson et al. 2006, Pearson, Raxworthy et al. 2007, Phillips, Dudík 2008). Using this technique, all pixels with a probability of presence equal to or greater than that of the training point with the lowest probability of presence are classified as present and all pixels with a lower probability of presence are classified as absent. A one-tailed binomial test can be performed to determine whether a model predicts the test points significantly better than random (Phillips, Anderson et al. 2006) The null hypothesis for this test can be that the model does not predict the test points better than random.

In order to evaluate the relative importance of each environmental variable in the model and decide which variables are most important to model development/contribute most to the model development, the Maxent software must be set to calculate jackknife tests of variable importance. These tests help to identify the variables with important individual effects (Elith, Phillips et al. 2011). The jackknife procedure generates three diverse types of models. These include models generated with one variable at a time excluded and all other variables included, models generated with only one variable included, and a model generated with all variables (Phillips, Anderson et al. 2006, Pearson, Raxworthy et al. 2007, Phillips, Dudík 2008). Variables that contribute the most to model development are those that decrease the training gain when excluded from the model and show gain when the model is developed with only this one variable.

Whether there are human cases caused by L. infantum in Cyprus

Which areas are at high risk- Seroepidemiological study on dog population

Whether there are inserted new Leishmania parasites in dogs

To find whether there are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL) cases in humans caused by L. infantum in Cyprus nowadays, a survey must be conducted in areas where dog seroprevalence is high,

It is really important to know in which areas in Cyprus dogs are most infected with Leishmania parasites, as the possibility of human infection typically increases with the number of infected dogs in an area. In addition, human VL cases caused by L. infantum are known in all areas in the Mediterranean basin with high prevalence in canine VL (Alvar, Canavate et al. 1997). Therefore, a seroepidemiological study needs to be carried out on the dog population, in order to find the areas with the highest risk for humans. The government-controlled part of the island, southern Cyprus, which covers 5,896 km², will be divided into equal squares, which will be given numbers. Each square can have one or more villages or towns. The squares to be included in the study can be chosen randomly. Then, the villages will be chosen according to dog population, so as to have at least 200 dogs per square.

The study will cover a large geographical area. The study area will include regions with preliminary data as well as regions that were not previously examined. Dogs living in the chosen areas, irrespective of race, colour, age, or health status, will be sampled. The sampling of dogs in the different areas will be performed with the help of private veterinarians that will be asked to be part of this project and will be employed. Of course, the veterinarians that will be employed are practicing of Veterinary Medicine according to the Law on Veterinary Practice and the Registration of Veterinarians Law of 1990 (Law 169/1990) as all the legal veterinarians in Cyprus. For the permission of doing the sampling with the way mentioned above, I will send a letter to the Principal of the Veterinary Services in Cyprus, stating that I intend to perform this research study on dogs and I would like your permission to be able to do this. Both, the study and the protocols that will be used, will be approved by the Veterinary Services in Cyprus, which is the Ethical Scientific Committee for animals in Cyprus. Dog owners will be approached with the help of a government or private veterinarian and will be asked to participate in the study by providing a written consent (see appendix). For the selection of the dogs, the houses in each village, or town will be chosen randomly. For each dog, a personal questionnaire with epidemiological and clinical data will be completed (see appendix). Blood samples will be taken from the dogs and tested for leishmaniasis. All the samples will be stored at…. And serum will be isolated from each blood sample using centrifugation. (HOW??). Also, dog sera maybe can be provided from veterinarians for routine testing for leishmaniasis. All dog sera will be examined for the presence of Leishmania IgG antibodies using the ELISA method (Enzyme-Linked Immunosorbent Assay). Seroprevalence measures will be derived entirely from the ELISA result, in order to be comparable with previous publications in Cyprus (Deplazes, Grimm et al. 1998) (den vriskw ayto to paper). All positive samples will be further tested by the Indirect Immunofluorescence Test (IFAT-Immunofluorescence Antibody Test), which is one of the most frequently used techniques for detection of anti-Leishmania antibodies (Solano-Gallego, Koutinas et al. 2009). This technique is suggested by World Organisation for Animal Health (OIE) as the reference serological method (Oie 2008) (den vriskw ayto to paper). IFAT will be performed using anti-dog anti-IgG antibodies. For this technique, a series of 2-fold serum dilutions starting from 1/50 will be performed, and cut-off titers ≥ 1/200 for dogs will be considered as positive. Furthermore, all positive samples derived from the ELISA result will be also tested by Electrosyneresis (ES) test which is a very sensitive serological test and can identify the acute stage of the disease (and confirm an active infection) (reference). For each one of these 3 techniques, sera from dogs with parasitologically proven leishmaniasis will be used as positive-controls, and sera from Leishmania-free dogs living in areas with no proven leishmaniasis will be used as negative-controls. Dog seroprevalence will reveal the areas with the highest risk for humans. For all these laboratory techniques, I will be trained at the Veterinary Services in Cyprus by qualified