44 The Leishmanioses

Leishmanioses, a large group of parasitic diseases, range over the intertropical zones of America and Africa and extend into temperate regions of South America, southern Europe and Asia. The clinical aspects of the disease is wide ranging from a simple, self-resolving cutaneous lesion to the potentially fatal visceral leishmanioses, known as kala-azar. In numerous underdeveloped countries, leishmanioses remain a major public health problem representing one of the most neglected diseases. Among 15 well-recognized Leishmania species known to infect humans, 13 have definite zoonotic nature, which include agents of visceral, cutaneous and mucocutaneous forms of the disease in both the Old and New Worlds. Mammal reservoir hosts belong to the marsupalia, edentata, carnivora, hyracoidea, and rodentia, maintaining sylvatic zoonotic foci in the deserts of Africa and Asia, the forests of South and Central America, as well as synanthropic foci in the Mediterranean basin and much of South America. Although the known vectors are all phlebotomine sand flies, these have a wide range of specific habits and habitats. The complexity of this group of infections has only recently been appreciated and is still being worked out. Currently, leishmanioses show a wider geographical distribution than previously known, with increased global incidence of human disease. Environmental, demographic and human behavioural factors contribute to the changing leishmaniosis landscape, which basically include increasing risk factors for zoonotic cutaneous leishmanioses, and new scenarios associated with the zoonotic entity of visceral leishmaniosis. In comparison with the anthroponotic entities of leishmaniosis, limited progresses were made for the control of the zoonotic ones, consisting mainly in new tools developed for the control of L. infantum in the canine reservoir.

Leishmanioses are protozoan diseases caused by members of the genus Leishmania, parasites infecting numerous mammal species, including humans, and transmitted by the bite of phlebotomine sand flies. Human leishmanioses have diverse clinical manifestations. Visceral leishmaniosis (VL), caused by Leishmania donovani in the Old World and L. infantum in both the Old and New Worlds, is the most severe form which, if left untreated, invariably leads to death. A number of different species of Leishmania cause cutaneous (CL) or mucocutaneous (MCL) leishmanioses which, if not fatal, are responsible for considerable morbidity of a vast number of people in endemic foci. The impact of leishmanioses on human health has been grossly underestimated for many years and it has now been classified by the World Health Organization (WHO) as one of the most neglected tropical diseases. At present, an estimated 12 million people are infected in 66 Old World and 22 New World endemic countries (72 developing and 16 developed) with an estimated yearly incidence of 1–1.5 million cases of CL forms and 500,000 cases of VL forms (Desjeux 1996). Incidence of leishmanioses is not uniformly distributed in endemic areas: about 90% of CL cases occur in 7 countries only (Afghanistan, Algeria, Brazil, Iran, Peru, Saudi Arabia, and Syria), whereas some 90% of VL cases occur in rural and suburban areas of 5 countries (Bangladesh, India, Nepal, Sudan, and Brazil). At least 60,000 people succumb to VL each year and a loss of 2.4 million disability-adjusted life years (DALYs) has been calculated (Hotez et al. 2004). These figures are much probably underestimated, as official data are often obtained through passive case detection.

During the last decade, it appears that the global incidence of human leishmanioses is higher than before, although it is difficult to differentiate between real and artificial increase, due to progress in diagnosis, case detection, improved reporting, and accessibility to treatment. For example, in Brazil, CL cases passed from 21,800 in 1998, to 40,000 in 2002; VL cases recorded in the same periods were 1,840 and 6,000, respectively; in Kabul, Afghanistan, CL cases were 14,200 in 1994 and 65,000 in 2002 (Desjeux 2001; 2004). Undoubtedly, human and animal leishmanioses show a wider geographical distribution than previously known. Autochthonous Leishmania transmission has been recently recorded in traditionally non-endemic areas, as in western Upper Nile, Sudan (Desjeux 2001), a number of U.S. states and Canada provinces (Duprey et al. 2006), Australia’s Northern Territory (Rose et al. 2004), and in some parts of Europe (Gramiccia and Gradoni 2005). It is widely accepted that leishmanioses are dynamic diseases and the circumstances of transmission are continually changing in relation to environmental, demographic and human behavioural factors. Changes in the habitat of the natural host and vector, immunosuppressive conditions (e.g. HIV infection or organ transplantation-associated therapies) and the consequences of conflicts, all contribute to the changing leishmaniosis landscape.

Lesions suggestive of CL have been known in Egypt since 2000 BC and in Assyria since 650 BC. The earliest clear reference to CL is by Ibn Sina (Avicenna) who wrote of Balkh sore; actually, 10 centuries later Balkh Province in northern Afghanistan is still suffering annual outbreaks of CL caused by Leishmania major. As for the New World, texts from the Inca period in the fifteenth and sixteenth centuries mention the risk run by seasonal agricultural workers who returned from the Andes with skin ulcers (CL) called ‘valley sickness’ or ‘Andean sickness’. Later, the disfigurements of the nose and mouth become known as white leprosy to the resemblance to the lesions caused by leprosy.

Evidence for VL has been found recently in ancient Egyptian mummies from the Middle Kingdom period (2050–1650BC). Clinically, this disease was probably masked for centuries by the overlapping malaria, however several decades before the discovery of the aetiological agent, Mediterranean infantile VL was known as

infantile infectious splenic pseudo-leukemia.

In the first few years of the twentieth century, in a rush of discovery, it was found that this different set of diseases, oriental sore, kala-azar, infantile splenomegaly, and espundia, were all caused by indistinguishable organisms. During the same period Nicolle, working in Tunis, found dogs to be infected with similar parasites, and postulated the zoonotic origin of infantile kala-azar. The term leishmaniosis was initially applied only to the canine disease and only became widely used for the human diseases in the 1930s. Reconstruction of the history of this disease has been facilitated by the collection of DNA and amplification of nucleic acids (PCR) to identify protozoan material from paleontological fossils (Tuon et al. 2008). The definition of a digenetic parasite makes it difficult to consider the emergence of the current genus Leishmania before the emergence of two adequate hosts, one of them a winged insect vector. Considering Leishmania as an evolutionary form of a primitive protozoan, the first host could have been a primitive water-dwelling animal and it appeared around the Proterozoic when the Earth was covered by water with a lower concentration of oxygen. The theory of digenetic life goes back up to the Ordovician. The separation of primitive winged insects within the Diptera occurred during the Triassic, more than 200 millions of years ago (Mya). Flagellates could be transmitted to a vertebrate, thus establishing a continuing cycle between vectors and vertebrates, during the Paleocene before the appearance of placental mammals. It was after this that the current vector of Leishmania (Phlebotomus) appeared. The vector, mammal host and fossils suggest that leishmanioses may have been established during the Paleogene, around 50 Mya. While the subgenus L. (Leishmania) can be reasonably considered as originated in Paleoartic, the origin of subgenus L. (Viannia) is controversial, some authors considering that it originated independently from the Neotropic, others from the Paleoartic or Neoartic. Apart from its origin, the dissemination of Leishmania followed the migration of vectors and hosts together.

Leishmania (Kinetoplastida: Trypanosomatidae) are dimorphic protozoa characterized by the intracellular presence of the kinetoplast, a network of maxi- and mini-circles of mitochondrial DNA found close to the base of the flagellum. Leishmania shows two principal morphological stages:

Leishmania are alternatively hosted by the sand fly and by mammals hosts. When a female sand fly takes a blood meal from a Leishmania-infected mammal, amastigotes are ingested and, following at least one cycle of binary division, they transform into motile promastigotes which escape through the peritrophic membrane enveloping the blood meal. The promastigotes multiply intensively inside the intestinal tract of the sand fly. This development occurs in the midgut (Leishmania subgenus) or in the hindgut and the midgut (Viannia subgenus). Whatever the multiplication site, the parasites subsequently migrate to the foregut (anterior cardia area and/or pharynx and proboscis) where they change into metacyclic forms. The time requested to complete the parasite cycle in the sand fly is variable, depending on both Leishmania and phlebotomine species, but is about 5 days on average.

Once the metacyclic promastigotes have been deposited in the mammal’s dermis by the bite of the sand fly, they are rapidly phagocytosed by cells of the mononuclear phagocyte system. The ingested parasites change into the non-motile amastigote stage. The surviving amastigotes divide by mitosis within the macrophage’s phagolysosome and the infection can spread in the mammalian host when heavily parasitized macrophages burst and amastigotes are ingested by other macrophages. The outcome of exposure to infection may not necessarily be overt disease and, in any case, the complex of parasite-cell biochemical interactions affects the course of the disease. The life cycle of Leishmania is completed when a female sand fly takes a blood meal containing Leishmania-infected cells. The inoculation of metacyclic promastigotes through the sand fly bite is the usual method of leishmaniosis transmission, other routes (e.g. transfusion or congenital transmission) being exceptional. The life cycle is illustrated in Fig. 44.3.

Since the creation of the genus Leishmania by Ross in 1903, the classification and nomenclature of the species and subspecies has for a long time and, sometime still is, a contentious matter. They show a homogeneous protozoan group mostly morphologically undistinguishable, for which the initial taxonomic criteria, like the human clinical picture and geographical distribution, were not enough for a correct taxonomical identification. Since the 1980s considerable efforts have been made to base the taxonomy of the genus Leishmania on scientific footings. The technique of multilocus enzyme electrophoresis (MLEE) has been applied from more than 25 years on several thousand parasite strains and still represents the current gold standard for Leishmania identification and taxonomy. Strains are characterized by their enzymatic profiles and grouped into homogeneous taxonomic units, the zymodemes. Phylogenetic classification of zymodeme complexes reveals a parental relationship between different Leishmania taxa (Table 44.1).

MLEE shows some limitations mainly due to the need for live parasite cultures and time-consuming procedures, therefore DNA genotyping methods have been investigated as alternative techniques, e.g. multi-locus microsatellite typing (MLMT) and PCR-restriction fragment length polymorphism (RFLP) (Schönian et al. 2008). Phylogenies based on nucleotide polymorphisms in different genomic targets have largely confirmed the taxonomy of the genus Leishmania by MLEE with some exceptions, e.g. the intra-zymodeme genetic polymorphism of L. infantum Montpellier (MON)-1, or the taxonomic status of the L. donovani complex in east Africa (Kuhls et al. 2007; Lukeš et al. 2007).

The establishment of metacyclic parasites in the dermis of the mammalian skin is facilitated by the sand fly saliva, which enhances Leishmania infectivity. After phagocytosis by macrophages, amastigotes have the capacity to resist intracellular digestion as a result of several parasite and host cell factors.

When the intracellular development of the amastigotes remains localized at the inoculation site, various cytokines are released and cell reactions are generated, resulting in the development of a CL localized lesion. In other instances, the parasites spread to organs rich in mononuclear phagocytes, giving rise to VL. Amastigotes may also spread to other cutaneous sites, as in diffuse CL (DCL), or to facial mucosae in the case of MCL. The localization of parasites in various tissues and organs is dependent on both intrinsic parasite tropism of a given Leishmania species and the immunological status of the host, resulting in the clinical expression of the disease.

Two species are usually responsible for VL: L. donovani in the Indian sub-continent, east-Africa and Arabic peninsula, and L. infantum in the Mediterranean, Middle East, central Asia and the Americas (Fig. 44.4). The incubation period is generally 2–6 months, but can range from 10 days (exceptional) to years (more common). A classical VL syndrome includes fever, asthenia, weight loss, anaemia, splenomegaly, hepatomegaly, and sometimes adenopathy. An intermittent and irregular fever is the major symptom. Splenomegaly appears early and is almost invariably present; anaemia is responsible for an extreme paleness of skin and mucosa. In India, patient skin has a greyish pigmentation which gives rise to the local name of the disease (kala-azar). If left untreated, VL is fatal in more than 90% of patients. VL caused by L. donovani shows frequently a dermal manifestation known as post kala-azar dermal leishmaniosis (PKDL) occurring after an apparent VL cure or recovering. Begining as depigmented maculae, the PKDL lesions can extend to the whole body, playing an important role in the sand fly transmission.

The world distribution of all tegumentary leishmanioses is shown in Fig. 44.5. CL consists of one or more localized skin lesions (depending on the infecting bites) without mucosal involvement nor evidence of dissemination. Lesions occur on exposed parts of the body accessible to sand fly bites. All anthropophilic Leishmania species, including the viscerotropic ones, can be responsible for a localized CL, which presents as a mild self-healing infection. The incubation period varies between a week and several months. The mature lesion is well defined, generally round or oval with variable dimensions ranging 0.5–10 cm in diameter. The most common clinical feature is the ulcerative lesion with sloping sides and central ulcer. A ‘wet’ type is typical of zoonotic CL lesions caused by L. major, L. mexicana, L. peruviana and L. braziliensis. A ‘dry’ type, showed as papulo-nodular lesions covered by scales, is the usual form of the anthroponotic CL caused by L. tropica. The clinical evolution of CL is chronic and leads to spontaneous cure in a time varying from few months (L. major, L. mexicana, L. peruviana) to few years (L. aethiopica, L. infantum, L. tropica, L. guyanensis, L. panamensis). The spontaneous cure always results in a disfiguring scar, while early treatment with pentavalent antimony salts can prevent such condition.

DCL is a severe form caused by a few Leishmania species, L. aethiopica in the Old World and L. amazonensis (rarely L. mexicana) in the New World, in patients who defect in cell-mediated immunity. The primary lesion is a non-ulcerated nodule rich in parasites. The nodules become numerous, disseminate to the whole body mimicking the presentation of lepromatous leprosy. The severity of DCL is shown by its resistance to anti-leishmanial drugs and it never cures spontaneously.

MCL, known also as ‘espundia’, is a severe clinical entity caused by L. braziliensis and occasionally L. panamensis. Following a primary CL lesion, secondary mucosal involvement occurs in a period between several weeks to many years. The mucosal involvement usually starts from the cartilaginous part of the nasal septum, which is rapidly destroyed. Mouth and lips mucosa is affected at a later stage of the disease which, in the advanced stage, leads to severe tissue necrosis and disfigurement. Death can occur following pulmonary superinfections. When treated and cured, MCL patients show disfiguring, sometimes retractile scars.

Leishmanioses range over the intertropical zones of America and Africa, and extend into temperate regions of South America, southern Europe and Asia. Their extension limits are latitude 45° North and 32° South. About 30 sand fly species are proven vectors. Each parasite species circulate in natural foci of infection where susceptible phlebotomine species and mammals coexist. Few human VL cases have been reported as congenital and blood transfusion transmission. However, direct transmission by sexual contact and exchange of syringes has been incriminated to explain the high prevalence of L. infantum-HIV co-infection in drug users in southern Europe. In CL cases contact with the active lesion is innocuous.

There are two main epidemiological leishmanioses entities:

There is no consensus about the named Leishmania species causing disease in humans. The New World species L. chagasi is now widely accepted to be a synonym of L. infantum; some authors describe L. archibaldi and L. killicki as species distinct from the close related species L. donovani and L. tropica, respectively. Finally, the taxonomic status of the New World species L. colombiensis is still controversial.

Among the 15 well-recognized Leishmania species known to cause disease in humans, 13 have zoonotic nature (Gramiccia and Gradoni 2005). Futhermore, for the only two species considered as having an exclusive or predominant anthroponotic transmission pattern, i.e. L donovani and L. tropica, the presence of animal reservoir hosts has been indicated in several endemic settings, such as eastern Sudan for L. donovani, and Morocco, northern Israel and Iran for L. tropica.

Finally, a number of Leishmania species have been recorded in animal hosts but not in humans: L. gerbilli, L. turanica and L. arabica, from Old World rodents; L. equatoriensis from arboreal mammals in Ecuador; Leishmania sp. from red kangaroo, Macropus rufus.

It is the most widespread entity of zoonotic leishmanioses caused by a single species complex, L. infantum. In the acute disease the agent multiplies in the macrophages of the reticuloendothelial system, resulting in generalized signs and symptoms like fever, splenomegaly and pancytopenia (Fig. 44.6). The disease occurs in several countries of Central and South America (especially in Brazil), Mediterranean basin, and Central Asia. Sporadic cases of localized CL due to dermotropic L. infantum strains are found in the same endemic areas (Fig. 44.7).

The principal vector in the New World is Lutzomyia longipalpis but in the Old World several species are involved, mainly belonging to the subgenus Phlebotomus (Larroussious), e.g. P. perniciosus, P. ariasi, P. neglectus, P. perfiliewi and P. kandelakii (Fig. 44.8). Dogs are the main domestic reservoirs, and foxes, jackals and wolfs are thought to be the sylvatic ones. With the combined use of serological and molecular diagnostic techniques, the prevalence rates of canine infections in different endemic settings were found to be much higher than previously thought. Asymptomatic L. infantum infections are common in healthy populations. Known risk factors for clinical disease are age below 2 years, malnutrition and immunosuppression (e.g. HIV patients or transplant recipients).

The main foci of ZCL are found in Africa, Asia, and in most Latin American countries. The Leishmania species involved are:

The usual clinical CL feature consists in localized nodulo-ulcerative lesions. Rarely, it may evolve toward a DCL, with multiple, not ulcerating nodules distributed over large areas of the skin, or to MCL, with the involvement of oronasopharyngeal mucosa and cartilages.

The parasite is widely distributed in arid and savannah rodents from the Old World. In humans the parasite causes localized, self-healing CL, often presenting as multiple lesions associated with numerous bites by infected sand flies (Fig. 44.9).

Several rodents species are the reservoir hosts:

All the proven vectors belong to the subgenus Phlebotomus (Phlebotomus): P. papatasi, the principal one, and the related species P. salehi and P. dubosqi. Well described stable zoonotic systems are the associations between the parasite and P. obesus/P.papatasi in north Africa and Middle East, and R. opimus/P. papatasi in central Asia, Afghanistan, and Iran. Unstable systems are the parasite associations with any Meriones spp./Phlebotomus spp. found in areas from Morocco to India, where population surges of Meriones may cause CL outbreaks in humans (Elfari et al. 2005). Migrations and fluctuations of rodent populations were the cause of several epidemic phenomena occurred in the Maghreb countries in the 1980s. Despite this widespread geographical distribution and the involvement of different rodent species, L. major appears genetically uniform, with large genetic groups corresponding to the Asian, Middle Eastern and African parasite populations.

L. major cases are increasingly reported from sub-Saharan African countries, e.g. Mali, Senegal, Camerun, Niger, Nigeria, and Burkina Faso, with some cases associated with HIV infection.

The parasite shows a geographical distribution limited to the highlands of Ethiopia, and to similar biotopes in Kenya. Suspected human cases were also reported from Uganda and Yemen. It is a classical parasite of the Hyracoidea, such as Procavia capensis and Heterohyrax brucei, which live in a wide altitudinal range up to 4,000 m. They always require restricted habitat as large rock outcrops with deep crevices for shelter. The vectors are two highland species of the subgenus Larroussius, P. longipes and P. pedifer. Recently L. aethiopica was isolated from a squirrel (Xerus rutilus) and from P. (Paraphlebotomus) sergenti at lower altitudes in Ethiopia (Gramiccia and Gradoni 2005). The caused clinical form results in a spectrum from uncomplicated localized, to DCL (Fig. 44.10).

American CL forms are originally sylvatic zoonoses. Some of them have shown a remarkable potential to adapt to human modifications of the environment and can show a synanthropic distribution, as in tropical rain forest, secondary forest or peri-urban areas.

This parasite is a Central American species with a geographical distribution restricted to the Mexican peninsula of Yucatan and the northern part of Guatemala, Belize, and Honduras, although strains of this species were described from Peru and Ecuador. L. mexicana has been found infecting various species of sylvatic ground-dwelling rodents in which produce generally small cutaneous lesions. The primary reservoir is commonly considered the tree rat Ototylomys phyllotis, with diverse forest rodents as secondary reservoir. The principal vector is Lu. olmeca olmeca, a sand fly highly attracted by rodents but also anthropophilic, biting during the day. The clinical form is a classical localized CL, although some cases of DCL have been recently described.

Originally described in the Brazilian Amazon region, L. amazone nsis shows a vast geographical distribution in different states of Brazil, Bolivia, Colombia, Ecuador, Peru, French Guiana, Panama and Venezuela. The primary reservoirs are generally considered forest rodents which develop subpatent infections. Secondary hosts are marsupial species where the parasite was isolated. The principal vector is Lu. flaviscutellata. The main clinical forms are localized or DCL, with frequent anergic or borderline anergic features.

This parasite was firstly described in 1980 in human cases of localized CL from Barquisimeto, Lara state, Venezuela. This species was subsequently identified in several urban centres of that state. So far vectors remain unknown, while domestic cats were suspected as the reservoir host.

This species shows the widest geographical distribution among the American CL agents. L. braziliensis spreads from the majority of countries of South America (Argentina, Bolivia, Brazil, Colombia, Ecuador, Paraguay, Peru, and Venezuela), to Central America (Belize, Guatemala, Nicaragua, Costa Rica, Honduras, Panama) and Mexico. Until now, the life cycle(s) is not completely known due to the wide dispersion of the species that covers multiple biotopes, with different reservoir and vector interactions. The disease appears originally as a wild zoonoses of primary rain forest areas that has been adapted to the human environments resulting from deforestation and agriculture extension. Various mammals have been found infected by the parasite including carnivores, rodents, and perissodactyls. Dogs have been recently found infected in the absence of other infected mammal hosts, in Guatemala (Ryan et al. 2003). Although L. braziliensis was identified in several Lutzomyia species, Lu. wellcomei is considered the most efficient vector. The usual clinical form caused by the parasite is a localized CL, with variable evolution toward a severe MCL in 30–80% of patients (Fig. 44.11).

This parasite is localized in the northern part of the Amazon Basin, especially in Guyana and some Brazilian states. The life cycle has long been elucidated, with a primary reservoir in a sylvatic edentate, the sloth, and the principal vector in Lu. umbratilis. The clinical form is a localized CL and human exposure to L. guyanensis results from occasional intrusions into the forest.

This species occurs in the rain forest of the northern part of the Para state of Brazil, Bolivia, and Peru. The life cycle is known since 1987, showing its reservoir in the rodent Agouti paca and the vector in Lu. ubiquitalis, a sand fly species abundant in the endemic area. The clinical form caused is a localized CL.

This species was firstly described in 1989 as a parasite of the armadillo Dasypus novemcinctus in Amazon Brazil. The uncertain taxonomical position was clarified by Thomaz-Soccol et al. (1993), who demonstrated L. naiffi as a Viannia parasite in an intermediate state between L. braziliensis and L. guyanensis. Although being a prevalent parasite in armadillos, it was found as a cause of human localized CL in Brazil, French Guiana, Ecuador and Peru (Pratlong et al. 2002).

This parasite is distributed in Central America countries as Panama, Costa Rica, Nicaragua, Honduras, Guatemala, and in Pacific regions of Colombia and Ecuador. Epidemiological investigations showed the reservoir in different sloths and the possible vector in Lu. trapidoi. As in the case of L. guyanensis, its life cycle is closely associated to the forest biotope. In humans, clinical forms vary from localized CL to MCL in 2–5% of patients.

This agent is the only non-sylvatic species responsible for American CL known prior to the conquest by Europeans. Its geographical distribution is limited to the Peruvian Andes, confined to the arid valleys of the western slopes between 1,200 and 3,000 metres altitude. The life cycle was elucidated only recently by the identification of the natural reservoir of L. peruviana in dogs (Llanos-Cuentas et al. 1999). The principal vectors are Lu. verrucarum and Lu. peruensis, even if other anthropophilic Lutzomyia species were found infected. The clinical form, locally called ‘uta’, is a typical localized ulcerative CL.

This is a sylvatic Leishmania species parasitizing various arboreal mammals in the rain forest of the Para state of Brazil, and causing localized CL in humans. It was originally discovered in the monkey Cebus apella and then isolated from different wild mammals as monkeys (Chiropotes satanas), sloths (Choloepus didactylus and Bradypus tridactylus) and coatis (Nasua nasua). In the forest of Para state, Lu. whitmani was found infected by L. shawi.

Over the past few years, there have been a number of eco-epidemiological situations which resulted in changing patterns of leishmanioses transmission. Not all factors underlying such changes were identified however both climatic modifications associated to global warming and human behavioural factors probably play a major role. The distribution and seasonality of vector-borne diseases are likely to be affected by climate changes, especially in temperate zones where increased average temperatures allow extension of the breeding seasons of the endemic vector species, or the de novo introduction of exogenous vectors, permitting pathogen transmission in areas where low temperatures had prevented their over-wintering.

Risk factors for ZCL are strongly associated to human exposure depending by the presence of the vectors and their activity cycles. In the Old World, urbanization is a major risk factor: new human settlements or suburbs located in the outskirts of the towns, when intruded on the terrain formerly inhabited by P. papatasi and the rodent P. obesus, have led to an increased transmission of L. major to humans, as observed in several areas of north Africa, Middle East, and Central Asia. Also, the building of dams with new irrigation schemes and crops may cause a rapid change in the rodent reservoir populations, followed by epidemics (Desjeux 2001).

In the New World, industrial and commercial projects have produced relevant immigration of workers in the Brazilian Amazon, resulting in the construction of several suburbs on the border of the primary forest. Newly urbanized areas brought the new population in contact with the zoonotic cycle of L. guyanensis and the incidence of new localized CL cases rapidly increased. In the Andean countries, the development of new projects (road building, mining, tourism etc.) and the subsequent new settlements and deforestation, has frequently caused a domestication of zoonotic transmission cycles with an increase of the peri and intra-domiciliary transmission (Desjeux 2001).

Several re-emerging issues are associated with the zoonotic entity of VL due to L. infantum. Spreading of infections in different territories have been monitored and recorded by means of investigations carried out among susceptible domestic dogs, which act as suitable sentinel hosts.

Being previously confined to coastal Mediterranean biotopes, ZVL incidence has been increased in Italy in human and dogs since 1990s (Maroli et al. 2008). During 2002–2009, the northward spread of the disease was monitored through human, canine and entomological surveys performed in northern continental regions at the border with France, Switzerland, Austria and Slovenia. Results showed that the most competent L. infantum vector, P. perniciosus, was widespread in these territories, associated with P. neglectus in the sub-Alpine and with P. perfiliewi in sub-Apennines territories. The large Padana valley was apparently found free from sandfly colonization, probably acting as natural barrier. CanL investigations confirmed the ongoing northward spread of ZVL, with a mean seroprevalence of 1.8% found in sub-Alpine sites, and an increase from 2 to 4% in sub-Apennines sites during the survey period. Despite the presence of a competent vector, Bolzano-South Tyrol province at the border with Austria was found still free from autochthonous CanL. Both VL and CL autochthonous human cases due to L. infantum have been recorded in the newly endemic regions. These findings demonstrate conclusively that northern continental Italy became focally endemic for ZVL after 1990s.

In Germany, the detection of leishmaniosis cases in humans and animals (dog, cat, horse) that never travelled abroad, has led to the hypothesis of a recent establishment of autochthonous transmission in that country (Naucke et al. 2008). A northward L. infantum expansion was suggested, although entomological surveys did not provide solid evidence for the presence of competent vector species.

The description of a novel CanL focus, with P. perniciosus and P. ariasi acting as local vectors, has been recently reported in a territory of French Pyrenees outside the traditional endemic range of leishmanioses in southern France (Dereure et al. 2009). During a period of 13 years, seroprevalence rates in foothill villages increased by 10 folds as a probable consequence of the 1°C increase in the mean annual temperature.

The progressive increase in CanL seroprevalence rate was also reported at elevated altitudes of 600–900m a.s.l. in the Alpujarras region of southeastern Spain, climbing from 9.2% in 1984, to 15.4% in 1991 and 20.1% in 2006 (Martin-Sanchez et al. 2009).

In the former USSR, ZVL was prevalent in republics of Central Asia and in the Caucasus. Due to intensive control programs, disease incidence decreased dramatically and was almost forgotten by local doctors. However, persistence of stable but sporadic foci in the Namangan and Fergana regions of Uzbekistan has been documented. In 1987–1999, nineteen VL cases were recorded in three villages of Namangan region. Clinical and epidemiological features suggested that the disease was zoonotic, with L. infantum as putative agent and P. longiductus as possible vector. Subsequent VL surveillance during 2004–2007 revealed an epidemic trend with a total of 34 human cases, of which 15 recorded in 2007. The pathogen was then identified from both Uzbek and neighbouring Tajik territories, and assigned to a distinct L. infantum genetic cluster different from Europe, Middle East and Africa ones. This suggests the local origin of the parasite in Uzbekistan and Tajikistan, rather than a newly introduction by human or reservoir migration. (Alam et al. 2009).

Human VL is rarely reported in USA, occasionally diagnosed in persons returning from endemic countries. An unexpected outbreak among foxhounds recently suggested endemic transmission of the disease (Duprey et al. 2006). In a New York state hunt club, in the summer 1999 some foxhounds developed severe illness characterized by typical CanL signs. A serosurvey revealed a high prevalence among foxhounds (but not in other breeds) and L. infantum MON-1 was isolated by infected animals. Serological screenings in other states revealed a widespread infection in a vast region of eastern North America extending from Florida state northward to Ontario province, Canada, and from the Eastern coastal regions to Kansas and Oklahoma in the West, for a total of 21 US states and 2 Canadian provinces. The transmission routes in these dogs are still unclear. Some epidemiological characteristics support only partially a classical vectorial transmission, whereas suggest possible dog-to-dog transmission both vertical (transplacental and transmammary) and horizontal (by direct contacts). No cases of autochthonous human VL were reported from the affected areas.

Only 14 autochthonous human VL cases were reported in Argentina from 1925 to 1989, interspersed with L. braziliensis CL cases. Lu. longipalpis, the most competent vector of L. infantum, was reported only twice in Misiones province, on the border with Brazil. The risk for ZVL transmission in Argentina changed dramatically since 2000s when, despite rapid human and canine disease spreading in the neighbour Paraguay state, no preventive CanL surveys were carried out in Misiones until autochthonous human VL cases have appeared. Both CanL infections and Lu. longipalpis vector were found widespread following active surveys. This is the first autochthonous ZVL focus reported in Argentina, representing the southernmost focus in Latin America (Salomon et al. 2008).

Among recent reports on newly identified or unusual animal hosts recurrently found infected with different Leishmania species, those regarding domestic cats and equines deserve attention for the obvious implications in public health.

Leishmaniosis in cats has been described since 1912. Afterwards several scattered reports on feline Leishmania infections have appeared in southern America (Brazil), Europe (Portugal, Spain, France, Italy, Switzerland, Germany) and Middle East (Israel, Iran) (Gramiccia and Gradoni 2005; Maia et al. 2008, Nasereddin et al. 2008; Hatam et al. 2009). Recently, the seroprevalence of infection has been estimated in feline populations of southern Europe, which showed antibody titres usually lower than in CanL. Seroprevalences ranged 0.6–59.1% in different endemic settings. Five Leishmania species have been identified in feline cases: L. mexicana, L. venezuelensis, L. braziliensis and L. amazonensis in the New World, and L. infantum in both the New and Old Worlds. Polymorphic cutaneous forms are frequent, including localized nodular, ulcerative, crusty or papular lesions, or generalized dermatits, alopecia and scaling. Systemic disease was described less frequently. The first evidence of transmissibility of feline parasites to a proven vector was recently provided in Italy, suggesting that cats may represent a secondary reservoir host for L. infantum (Maroli et al. 2007).

Since early 1980s Leishmania infections have been reported in domestic equines from different regions of Latin America (Brazil, Venezuela, Argentina and Puerto Rico) (Gramiccia and Gradoni 2005). Epidemics due to L. braziliensis were described in donkeys, and sporadic cases in horses and mules. The clinical forms observed consisted of nodular or ulcerated cutaneous lesions, occasionally disseminating without visceralization. Spontaneous regression of the lesions was also reported. In the past few years, there have been sporadic cases of horse leishmaniosis in Europe caused by L. infantum. Like for the New World forms, infections consisted of self-resolving cutaneous lesions. Domestic equines seem to display clinical and immunological responses of resistant type, and much probably they represent an incidental host.

Parasitological, molecular and serological assays are routine methods available for the diagnosis of leishmanioses. However, the direct demonstration of parasite is the only way to confirm the disease conclusively. Isolation and identification of the parasite from biopsies (skin, lymph node, bone marrow, and spleen aspirate) is performed by slide smear microscopy and/or culture in appropriate blood-agar based media. In VL, because sensitivity of standard parasitological methods may not be high enough, immunodiagnostic and molecular tests are also recommended. Serological tests include indirect immunofluorescent antibody test, ELISA, Direct Agglutination Test and Western-blot assay. More recently, immunochromatographic dipsticks using recombinant antigen k39 have been developed. Direct parasitological diagnosis is necessary for confirmation of CL (by means of scraping, punch biopsy or needle aspiration of the lesions) as neither clinical examination nor serology are adequate. Several protocols are now available for the PCR amplification of Leishmania DNA from biopsy material for both diagnosis of different clinical forms, and the identification of the agent.

Indian kala-azar has been included by the WHO in the list of neglected tropical diseases targeted for elimination by 2015 (WHO 2009). Since there is no vaccine in clinical use, control relies almost exclusively on chemotherapy. Treatment depended on arsenicals and tartar emetic until the efficacy of pentavalent antimony compounds was discovered in the 1920s. Since then, treatment with pentavalent antimonial compounds represents the mainstay therapy for all forms of leishmaniosis. Following the increasing incidence of Leishmania-HIV co-infections and the acquired antimonial resistance in India, amphotericin B formulations have joined the antimonials as treatment choice. Alternative drugs as pentamidine and paromomycin (aminosidine) were included in the VL and CL treatment. Since 2006 the new oral agent miltefosine has been introduced especially in the areas with reported antimonial resistance. In response to concerns about preserving the currently available antileishmanials, especially in regions with anthroponotic parasite transmission, there is growing interest on combinations regimens.

In Europe, treatment of CanL in pets has followed the same historical process. Nowadays, veterinary brands include pentavalent antimony, miltefosine and aminosidine (Solano-Gallego et al. 2009).

Two closely related pentavalent antimonials are currently used, sodium stibogluconate (Pentostam^®) and meglumine antimoniate (Glucantime^®). It is generally accepted that pentavalent antimonials (SbV) are the prodrug and that they should convert to trivalent antimonials (SbIII) in order to demonstrate their antileishmanial activity. Recent evidence indicates that antimonials kill leishmanias by a process of apoptosis involving thiol metabolism and trypanothione activity. SbIII inhibits trypanothione reductase in vitro, inducing the loss of intracellular thiolsan, a lethal imbalance in thiol homeostasis, leading to accumulation of reactive oxygen species. In spite of numerous side effects attributed to antimonials, the scarcity of reported accidents allows their continued use. Initially antimonial were given at 10mg/kg for 6–10 days with 90% cure rates, however after the first treatment failure in India twenty years ago, higher doses and prolonged schemes (up to 20mg/kg for 30 days) were gradually introduced and in parallel with the increasing rates of antimony unresponsiveness. During the last decade antimonial resistance reached epidemic dimension in Bihar, India that abandoned the antimonials use. Low rates of antimonial resistance have been reported in Sudan. This aspect is not an emerging problem in Mediterranean areas (Gradoni et al. 2008). Low cost is the main advantage of antimonials.

Conventional amphotericin B (AmB) is a polyene antibiotic isolated in 1955 and currently used in the treatment of systemic fungal infections. Since 1960s it was used as a second-line treatment for VL. Its target is ergosterol-like sterols, which are the major membrane sterols of Leishmania as well as fungi. Lipid formulations of AmB improved highly antileishmanial activity and safety profile of this drug. Lipid formulations are taken selectively by the reticulo-endothelial system, and exhibit a highly localized enhanced anti-leishmanial action. There are three lipid formulations of amphotericin B:

Currently, liposomal formulations of AmB are the first treatment choice in southern Europe endemic countries as well as in other developed countries, because of their rapid and up to 100% cure rates with 3–5 days schemes, improved convenience for the patient included immunocompromised patients, and reduction of health care costs. However, in poor countries even short courses of liposomal formulations are unaffordable, and the selection of anti-leishmanial treatment turns more to a question of cost than of efficacy or toxicity. Short-course treatment is now currently used, including five daily injections of 3–4mg/kg, plus a further same dose on the 10th day.

It is alkilphospholipid (hexadecylphosphocoline) oral antineoplastic agent. This is the first oral administered drug for VL and the latest to enter the market. This agent is associated with high efficacy rates including cases unresponsive to antimonials. So far, miltefosine is licensed in India, Germany, and Colombia. The scheme of miltefosine is 100 mg/kg/day for 28 days in adults weighing ≥50 kg, 50mg/kg/day in adults <50 kg, and 2.5mg/kg/day in children (maximum dose: 100 mg/day). Major concerns for the wide use of miltefosine include its teratogenic potential and its long half-life (approximately 150 hours) which may facilitate the emergence of resistance. Miltefosine is strictly forbidden in women of child-bearing age who may become pregnant up to two months following drug discontinuation. In India, miltefosine is available over the counter, a fact that may expose this drug to misuse and emergence of resistance. The exact anti-leishmanial mechanism of miltefosine remains largely unknown. The intracellular accumulation of the drug appears to be the critical step for its action. It includes the following steps: binding to plasma membrane, internalization in the parasite cell and intracellular targeting and metabolism. It has been found that miltefosine induces an apoptosis like cell death in L. donovani, by producing numerous defects.

Paromomycin (aminosidine) is an aminoglycoside with antileishmanial activity. In a phase IV study of VL in India, this drug was associated with 94.6% cure rates, similar to amphotericin B. Adverse effects were more frequent and high in the paromomycin-treated group compared with the amphotericin B-treated group; paromomycin-related adverse effects included elevated hepatic transaminases, ototoxicity, and pain at injection-site. Paromomycin is inexpensive but requires daily intramuscular injections for 21 days. Paromomycin inhibits protein synthesis and modifies membrane fluidity and permeability and in vitro study showed that mitochondria are the targets of the drug. Since paromomycin is an aminoglycoside, it is possible that resistance will emerge rapidly if used as monotherapy.

Pentamidine is an aromatic diamine. The isethionate salt (Pentacarinat^®) is the only form now available for human use and is restricted to systemic CL treatment. The drug inhibits the synthesis of parasitic DNA by blocking thymidine synthase and fixation of transfer RNA. It can be responsible for immediate and side effects which severity depends on the dose. Pentamidine is given in doses of 4mg/kg per injection for a short course of four doses on alternate days.

The rational for using combination regimens with different resistance mechanisms over monotherapy relies on the expected enhanced efficacy, shorter treatment duration, less toxicity, improved compliance, reduced likelihood of emergence of resistance, and reduced costs. A combination policy for VL is supported by the fact that anti-leishmanial drugs belong to different chemical classes. Recent studies have investigated this option. In a retrospective study conducted among Sudanese patients with VL, it was found that combination of sodium stibogluconate and paromomycin administered for 17 days was associated with higher cure and survival rates compared to sodium stibogluconate. Combinations of miltefosine with amphotericin B, paromomycin or pentavalent antimonials have been evaluated in an in vivo model and revealed that the combinations of miltefosine with amphotericin B or paromomycin were efficacious. Recent studies indicate that a single dose of liposomal amphotericin B followed by 7–14 days of miltefosine is active against Indian VL.

VL should be treated as soon as diagnosis is completed. Treatment requires confirmed first line drugs, principally antimonials or various AmB formulations. Clinical response is slow, the patient become afebrile within 4–5 days, other clinical symptoms and biological parameters slowly regressing and evolving to normal. VL occurring in HIV-infected patients appears generally as non-responsive to the classical anti-leishmanial drugs with incomplete cure and frequent relapses. Similar poor responses are observed in VL following organ transplantation or generalized immunosuppression, such as that caused by prolonged corticosteroid therapies.

With regard to CL and MCL, most of the Old World forms consist of simple lesions in contrast with the variety of the New World forms. The drugs employed are basically the same as for VL, with different applications and regimens according to the type and character of lesions. A variety of physical treatments, such as heat-, cryo-, laser- and radiotherapy, is commonly employed for topical use in highly CL endemic countries. Topical treatment, consisting of local infiltrations of pentavalent antimonials 2 or 3 times/week, is mainly employed for the Old World CL. Systemic treatment is recommended for the New World CL and MCL, using pentavalent antimonials at similar dosages used for VL, or pentamidine with a course of 4–5 injections on alternate days.

The treatment of MCL should be as early as possible, in order to avoid extension of lesions and subsequent mutilations. The cure rate is variable according to Leishmania (Viannia) species/strain involved and the evolution of the lesions. Antimonials and AmB are the first line drugs, while oral miltefosine is still under study. Drug treatments are not efficacious in DCL conditions once established.

The first line pharmaceutical protocol for CanL is the combination of meglumine antimoniate and allopurinol, but many different therapeutic protocols are suggested (Solano-Gallego et al. 2009). Recently a combination of miltefosine with allopurinol was proposed as an alternative to the association antimonial-allopurinol. However, although most dogs recover clinically after therapy, complete elimination of the parasite is usually not achieved and infected dogs may relapse.

Over the past few years, international health agencies have increased efforts to improve methodologies for the surveillance and control of leishmanioses characterized by a predominant anthroponotic transmission pattern (Desjeux 2004). In the field of zoonotic leishmanioses, no significant advances were made in the CL control in both Old and New Worlds. The exophilic habit of the phlebotomine vectors and the sylvatic nature of the mammal reservoir hosts would require expensive environmental management difficult to implement and sustain, such as forest cleaning or destruction of rodent burrows around human dwellings.

On the other hand, new tools have been developed for the surveillance and control of zoonotic VL, based on the control of CanL. Culling of infected dogs is not considered an acceptable measure, for both ethical considerations and the low efficacy. Several canine vaccine candidates are under study (Gradoni 2001); one of them has been recently registered in Brazil for veterinary use. A number of insecticide-based preparations have been specifically registered for dog protection against sand fly bites, which include deltamethrin impregnated collars and topical (‘spot-on’) permethrin. Laboratory and field studies have shown elevated efficacy of these preparations for both individual and mass protection. The impact of this type of control measure can be limited if not integrated with stray dog control.

With the exception of the compulsory reporting of cases, which applies in an increasing number of countries following WHO recommendations (Desjeux 1991), legislation appears inappropriate for most of leishmanioses endemic countries. Governmental control programmes against VL are in course in Brazil, which is based on CanL control, and in the Indian sub-continent (some states of India, Nepal, and Bangladesh), which is mainly addressed to interrupt the anthroponotic transmission of L. donovani.