Phlebotomine sand fly–borne pathogens in the Mediterranean Basin: Human leishmaniasis and phlebovirus infections

Pathogens transmitted to humans by phlebotomine sand flies are neglected, as they cause infectious diseases that are not on the priority list of national and international public health systems. However, the infections caused by protozoa of the Leishmania genus and viruses belonging to the Phlebovirus genus (family Phenuiviridae)—the most significant group of viruses transmitted by sand flies—have a relevant role for human pathology. These infections are emerging in the Mediterranean region and will likely spread in forthcoming decades, posing a complex threat to human health. Four species and 2 hybrid strains of Leishmania are pathogenic for humans in the Mediterranean Basin, with an estimated annual incidence of 239,500–393,600 cases of cutaneous leishmaniasis and 1,200–2,000 cases of visceral leishmaniasis. Among the phleboviruses, Toscana virus can cause neuroinvasive infections, while other phleboviruses are responsible for a typical “3-day fever”; the actual incidence of Phlebovirus infections in the Mediterranean area is unknown, although at least 250 million people are exposed. Here, we reviewed the current literature on epidemiology of sand fly–borne infections in the Mediterranean Basin, with a focus on humans. Our analysis indicates the need for increased public health activities directed to determine the disease burden of these infections as well as to improve their surveillance. Among the emerging challenges concerning sand fly–borne pathogens, the relationships between sand fly–borne protozoa and viruses should be considered in future studies, including epidemiological links between Leishmania and phleboviruses as well as the conditional capacity for these pathogens to be involved in interactions that may evolve towards increased virulence.

Introduction Pathogens transmitted to humans and animals by phlebotomine sand flies are relatively neglected, as they cause infectious diseases that are not on the priority list of national and international public health agencies in Europe. However, these diseases are emerging in southern Europe [1]. Among sand fly-borne pathogens, protozoa of the Leishmania genus and viruses belonging to the Phlebovirus genus (order Bunyavirales, family Phenuiviridae)-the most significant group of viruses transmitted by sand flies-contribute significantly to human pathologic disease. Four recognized species and 2 hybrid strains of Leishmania are known to be pathogenic for humans in the Mediterranean region, with an estimated annual incidence of 239,500-393,600 cases of cutaneous leishmaniasis (CL) and 1,200-2,000 cases of visceral leishmaniasis (VL) [2]. Among the phleboviruses, Toscana virus (TOSV) can cause neuroinvasive infections, while other phleboviruses are responsible for a typical "3-day fever" or "pappataci fever;" the actual incidence of Phlebovirus infections in the Mediterranean area is unknown. However, at least 250 million people are potentially exposed [3]. The aim of this study is to provide an overview of the epidemiology of sand fly-borne infections in the Mediterranean Basin, with particular focus on humans.

Methods
A comprehensive literature search was conducted on the distribution of Leishmania and Phlebovirus species responsible for human infection in the Mediterranean region. An additional search was dedicated to identifying the reservoir(s) of Leishmania and Phlebovirus. We analyzed all articles listed in the PubMed database in English, French, Italian, and Spanish between 1980 and December 2016, when the final search was conducted. The search terms used included leishmanial species, phlebovirus species, reservoir species, and countries included in the study. Titles relevant to the scope of this review were obtained in full text and selected for inclusion. Unpublished data were not considered. Data on the presence of Leishmania and/or Phlebovirus detection were reported on a geographic information system (GIS) at country level. Maps were created in the open source GIS software, QGIS 2.12 (www.qgis. org).

Characteristics of phlebotomine sand fly-borne pathogens (Leishmania and Phlebovirus) Leishmania
The genus Leishmania is composed of protozoa belonging to the Trypanosamatidae family, order Kinetoplastida. Within the genus, there are 2 subgenera of medical importance that were created on the basis of the parasite developmental patterns within the sand fly gut: Leishmania (all development anterior to the pylorus) and Viannia (developing in the hindgut and pylorus) [4]. On the basis of multilocus enzyme electrophoresis (MLEE), from which the current Leishmania taxonomy originates, leishmanial species are grouped within each subgenus into socalled "species complexes" or "complexes," which are distributed in countries with a subtropical climate [5].
These protozoa have a digenetic life cycle, requiring a susceptible vertebrate host and a permissive insect vector, which allow their transmission [4]. The blood-feeding females of phlebotomine sand flies are considered the only natural vectors of leishmanial species. The genus Phlebotomus includes species responsible for the transmission of Leishmania in the Old World, while Lutzomyia species are responsible for the transmission of the parasite in the New World [4].
The morphological forms of these protozoa are: (1) an extracellular flagellated promastigote that may be found in sand fly gut and (2) an obligate intracellular nonflagellated amastigote, typically residing in monocyte macrophages of the mammalian host [4].
About 20 Leishmania species are known to be pathogenic for humans [6] and are differently distributed in the New and Old Worlds [5]. The species circulating in the Mediterranean region include L. donovani and L. infantum, causing VL, and L. tropica and L. major, which, together with dermotropic L. infantum strains and L. infantum/L. donovani hybrid, cause CL [4,[7][8][9]. A classification of human pathogenic Leishmania species circulating in the Mediterranean region is presented in Table 1.
Survival of the Leishmania parasites depends on successful zoonotic or anthroponotic transmission and on suitable environment and climate. Although the areas in which different leishmanial species are diffused appear well defined, the risk for introduction of exotic species through travel or migration should be considered. The probability that new species could enter into a local transmission cycle depends on various factors, including the competence of sand flies for different leishmanial species; the presence of a suitable reservoir; and the contact between sand flies, reservoirs, and humans [1]. Considering these aspects, the introduction of zoonotic L. major to Europe is regarded as unlikely, as the fat sand rats and the gerbils-the natural animal reservoirs of L. major-live in the desert or semidesert areas of the African and Asian continents not found on the European continent [ The reproduction mode (clonality versus sexuality) of the Leishmania parasite is still under debate, but the existence of natural hybrid strains suggests that the exchange of genetic material does occur. Recently, whole genome sequencing demonstrated that the çukurova strain is the progeny of a single outcrossing event between 1 parent related to L. infantum and an asyet-unidentified parent belonging to the L. donovani complex [9]. Moreover, 2 hybrid strains produced by L. infantum and L. major were isolated in Portugal from immunocompromised patients suffering from VL, indicating that close phylogenetic association is not a necessary prerequisite for genetic exchange [20]. In addition, molecular techniques show that these chimeric strains contain the complete genome of both L. infantum and L. major. Genetic exchange may have potential implications such as emergence of virulent strains or increasing fitness [21,22]. Indeed, L. infantum/L. major hybrid is capable of replicating in P. papatasi [22], while L. infantum/L. donovani hybrid replicates in P. tobbi and P. perniciosus [15], suggesting that hybrid strains may circulate in infected sand flies.

Phlebovirus
The genus Phlebovirus belongs to the family Phenuiviridae, order Bunyavirales [3]. Phleboviruses are enveloped, spherical viruses. The genome is a negative-sense, single-stranded RNA. The 3 RNA segments designated as large (L), medium (M), and small (S) encode the RNAdependent RNA polymerase, the envelope glycoproteins, and the nucleoprotein, respectively. The single-stranded RNA segments have high mutation rates due to the lack of proofreading activity of the viral polymerase, which may result in genetic drift following point mutation, while the 3 genome segments are involved in reassortment/recombination, leading to the generation of new viruses [3].
The genus Phlebovirus includes 9 species; 70 antigenically distinct viruses are segregated into the 9 species, while 33 viruses are not yet attributed to one of these species [23].
In the Mediterranean area, phleboviruses transmitted by sand flies belong to 1 of the following groups: (i) 2 International Committee on Taxonomy of Viruses (ICTV)-recognized species, Sandfly fever Naples (SFNV) and Salehabad, (ii) 2 tentative species (Sandfly fever Sicilian virus [SFSV] and Corfou virus) [23]. Furthermore, Karimabad virus was erroneously listed in the Sandfly fever Naples species as demonstrated by Palacios et al. [24] and should be listed as a tentative species. A large number of new sand fly-borne phleboviruses were recently described, all of them belong to species/groups listed as (i) or (ii) ( Table 2). A phlebovirus phylogeny reconstruction is depicted in Fig 1. SFSV and SFNV were first isolated from the sera of sick soldiers in Egypt in 1943 and Naples in 1944, respectively [3]. TOSV was first isolated in the Tuscany region of central Italy from P. perniciosus and P. perfiliewi in 1971 and 12 years later was detected in the central nervous system (CNS) of patients with meningitis [3]. Because of the geographic spread of TOSV in southern Europe and surrounding countries and the growing number of human cases [36], TOSV is recognized as an emerging pathogen and one of the most common causes of summer meningitis in the Mediterranean Basin. On the basis of sequence analysis of the M RNA segment, different clusters of TOSV were clearly distinguished and named TOSV lineages A, B, and C (S1 Table).

Reservoirs of Leishmania
The ecological system in which Leishmania is maintained is composed of vector and vertebrate reservoirs [4]. Usually, there is 1 principal reservoir host, which differs on the basis of the Leishmania infecting species and the infection focus (Table 3). Other susceptible mammals may become infected as minor or incidental hosts. Minor hosts may play a role in parasite maintenance, while incidental hosts are infected mammals without this role.
Humans. Humans are directly involved as principal reservoir hosts in VL caused by L. donovani and CL caused by L. tropica [4]. While the role of patients with clinical symptoms as reservoirs is clear, the role of asymptomatically infected individuals in the transmission of infection is currently unknown. Asymptomatic carriers exhibit low parasitemia, thus being less infective than symptomatic individuals [4]. Nevertheless, the huge numbers of asymptomatically infected individuals constitute a possible source of infection [37]. Animals. Wild or domestic animals are the main reservoirs of L. infantum and L. major. Recently, some animal species have been implicated as reservoirs for L. tropica [4,6,[38][39][40] ( Table 3)  L. major mainly causes a zoonotic infection, with infected wild rodents regarded as reservoirs [54] (Table 3). In the Mediterranean Basin, the main reservoir species is the fat sand rat (Psammomys obesus), a rodent that is very abundant in arid pre-Saharan areas, from Mauritania to the Middle East. In addition, gerbils (Meriones spp.) play a role in the maintenance of the circulation of L. major. Among the others, Meriones shawi is the reservoir host of L. major in some parts of Tunisia, Morocco, and Algeria [13], while M. tristrami and M. guentheri are suspected as the main reservoir hosts of L. major in a focus of human CL in Israel [46]. The epidemiology and control strategies of human leishmaniasis largely depend on the distribution and abundance of these rodents, which in turn are influenced by agricultural development and ecological changes. Migratory habits of some rodent species (i.e., Meriones spp.) could also contribute to the spread of L. major from endemic foci towards neighboring areas [13]. In addition, other mammals can be involved in the maintenance of the transmission cycle of L. major; natural infection in 2 species of hedgehogs (Ateleris algirus and Paraechinus aethiopicus) was recently reported from Algeria, suggesting their involvement in the parasite cycle [47].

Reservoirs of phleboviruses
No reservoir host has been defined with certainty for phleboviruses. Concerning TOSV infection, it is unlikely that humans play this role because of the transient viremia occurring in infected patients [36]; competent sand fly species might act as reservoirs in the viral cycle [6,55,56]. In support of this hypothesis, male sand flies were found to be infected by TOSV in nature, while transovarial transmission was experimentally demonstrated as well as venereal transmission from infected P. pernicious males to uninfected females. However, the progressive decrease in viral infection rates observed from generation to generation in sand fly colonies suggests that TOSV cannot be maintained indefinitely by vertical or venereal transmission [6]. Consequently, the existence of additional reservoirs should be considered.
Only a few studies were conducted on animals to verify their role as reservoirs of phleboviruses. In 1984, TOSV was isolated from a bat in Italy, while the high frequency of viral RNA and specific antibodies for TOSV in the canine population in Tunisia Table. The clinical spectrum of human leishmaniasis ranges from asymptomatic infection to 3 main clinical syndromes: VL, CL, and mucosal leishmaniasis (ML) [4]. Clinical manifestations are the result of the relationship between the leishmanial species and the host and are partially dependent on strain differences, making some species more adapted to target the skin and others to invade visceral organs [62]. VL is the most severe form of the disease, and it is fatal if untreated; the target organs of VL are the spleen, the liver, the bone marrow, and the lymph nodes, where parasites proliferate in the cells of the mononuclear phagocytic system [4,63]. CL is characterized by single or multiple ulcerative or nodular lesions in exposed areas of the body; L. tropica mainly causes a single lesion, while L. major is often characterized by multiple lesions [5,64]. ML pathophysiology in the Mediterranean region remains unclear as it is usually not associated with previous CL [65]. The most frequent localizations are larynx, pharynx, mouth, and nose, and the lesions are polypoid, infiltrative, ulcerative, or nodular [65][66][67][68][69].

Human infection caused by phlebotomine sand fly-borne pathogens in the Mediterranean Basin
SFSV and SFNV cause a typical "3-day fever" or "pappataci fever," while TOSV displays a strong neurotropism responsible for acute meningitis and meningoencephalitis [36]. Nevertheless, most cases of TOSV infection present as asymptomatic cases or as a flu-like syndrome [70]. In addition, an outbreak of febrile syndrome was observed in Greek soldiers stationed in Cyprus in 2002; molecular investigation confirmed the presence of a new phlebovirus provisionally named Sandfly fever Cyprus virus [71]. Finally, Adria virus was detected in the blood of a Greek child with febrile seizure [72] and Sandfly fever Turkey virus was isolated from the cerebrospinal fluid of a Turkish patient with encephalitis [73].  Table 4 report the distribution of human infections caused by Leishmania and phleboviruses in the Mediterranean region.

Effect of global changes on phlebotomine sand fly-borne pathogens
Environmental aspects such as climate change, increasing urbanization, economic development, and population movements for economic, social, or political reasons are causing changes in the epidemiology of vector-borne infections, as they lead to alteration in the range of vectors, reservoirs, and pathogens [4].
Man-made ecological changes including urbanization, spread of agriculture into semiarid lands, and the introduction of artificial irrigation systems can affect the incidence of sand flies [4,110,111]. Other human activities, such as tourism and pilgrimages from or to endemic areas, may contribute to the spread of exotic pathogens to areas that are already endemic for other sand fly-borne pathogens.
In disadvantaged areas, other factors contribute to the diffusion of sand fly-borne diseases. For example, poor domestic and peridomestic sanitary conditions may attract anthropophilic sand flies and may increase breeding sites of the vectors, while malnutrition and high incidence of HIV infection may result in inadequate immune response and higher clinical expression of infections.
In conflict areas, lack of or destruction of healthcare infrastructures may occur as well as massive population displacement, the latter potentially causing the introduction of nonimmune people into areas with endemic/enzootic disease or the emergence of pathogens in areas where infected individuals are displaced and where reservoirs and vectors coexist. As the most recent example, the Syrian refugee crisis has precipitated a catastrophic outbreak of CL, leading to hundreds of thousands of CL-affected people living in refugee camps in Lebanon or trapped in conflict zones [112]. The exodus of migrants from Syria has led to a dramatic increase of CL cases in southern Turkey and to the introduction of new leishmanial species, such as L. major [50]. Similar situations may also be unfolding in other conflict areas, such as Libya and Yemen [112].
Although different factors may influence the diffusion of sand fly-borne diseases, the range and density of vectors are considered the most important risk factors for the emergence of leishmaniasis and phlebovirus diseases in Mediterranean and continental Europe [14,113]. The recent geographical expansion of phlebotomine vectors in the Mediterranean region has been attributed to ongoing climate changes and investigation on seasonal dynamics of vectors for L. infantum in Mediterranean Europe confirmed that temperature is a major determinant for the seasonal interval of sand fly activity [114]. Thus, global warming can affect the diffusion of sand flies and sand fly-borne diseases, as shown by the detection of sand flies in continental Europe, including Germany, Austria, Switzerland, and Slovakia [113,[115][116][117] and by the northward spread of leishmaniasis in Italy, respectively [6]. In addition, large areas of continental Europe that are unsuitable for sand fly species today are projected to change towards a climate that will support the survival and replication of these vectors [113].
Factors other than vector density can influence the presence and diffusion of sand flyborne pathogens, including the replication rate of the pathogen, the vector biting rate, and host and reservoir availability [113]. Climate changes, indeed, can also influence the development cycle of Leishmania promastigotes in sand flies; increases in minimum temperature accelerate the parasitic life cycle [113,118].

Conclusions
Sand fly-borne pathogens such as Leishmania spp. and phleboviruses are currently emerging in southern Europe, posing a complex threat to human health. A number of factors can influence the epidemiology of sand fly-transmitted infections, including environmental and climate changes, poverty, mass migration, and conflicts. Concerning the spreading of Leishmania, changes in infected animal reservoirs are also important; the northward spread of human leishmaniasis in Italy following the detection of infected dogs in the northern part of the country as well as the recent outbreak in Spain with hares as reservoirs show that hosts play a major role in transmission cycle under changing conditions.
Our review highlights the fact that, despite their emergence, several aspects of the epidemiology of phlebotomine sand fly-borne infections are poorly studied. Among others, there is an urgent need for increased public health activities to ameliorate the surveillance of sand flyborne infections, considering that underreporting of VL and CL is observed in countries with a compulsory notification system for leishmaniasis [119] and Phlebovirus infection are not subjected to notification. Diagnostic tools should also be implemented, focusing on molecular tests for rapid pathogen detection, while studies on the potential role of domestic and wild mammals as reservoir hosts should be carried out as well as continuous surveillance of canine leishmaniasis. There is also a need to continuously monitor vector distribution in endemic as well as nonendemic countries-activities that are so far scarce and scattered-with in-depth investigation into the vectorial capacity of the sand fly species identified in different areas. Among the emerging challenges concerning sand fly-borne pathogens, the relationships between sand fly-borne protozoa and viruses should be considered in future studies, including epidemiological links between Leishmania and phleboviruses as well as the conditional capacity for these pathogens to be involved in interactions that may evolve towards increased virulence.

Key learning points
• Pathogens transmitted to humans by phlebotomine sand flies such as Leishmania spp. and phleboviruses are currently emerging in southern Europe.
• Evidence of Leishmania hybrid strains suggests that the exchange of genetic material can occur in these parasites.
• Genomic data on newly detected sand fly-borne phleboviruses are available, which allows the development of novel molecular tests to evaluate the role of these viruses in human pathology.
• A number of environmental factors such as climate change, increasing urbanization, economic development, and population movements are causing changes in the epidemiology of sand fly-borne infections as the result of altering the accessible range of vectors, reservoirs, and pathogens.
• Massive population displacement from conflict areas can cause the introduction of nonimmune people into areas with endemic/enzootic disease or the emergence of pathogens in areas where infected individuals are displaced and where reservoirs and vectors coexist.