Leishmania species (Leishmaniasis)

PARASITOLOGY

Leishmaniasis is caused by protozoan parasites of the genus Leishmania. The genus Leishmania has 2 subgenera: Leishmania and Viannia. In Europe, Africa and Asia (the "Old World") parasites belonging to the subgenus Leishmania are found; in America (the "New World") parasites belonging to both genera occur. About 20 species are known to cause disease in man. Several molecular techniques are being used to identify Leishmania resulting in different numbers of species (275). A phylogenetic analysis of the heat-shock protein 70 gene suggests that there may be eight medically relevant species: Leishmania (Leishmania) donovani, L.(L.) major, L.(L.) tropica, L.(L.) mexicana, L.(Viannia) lainsoni, L.(V.) naiffi, L.(V.) guyanensis and L.(V.) braziliensis (113). In several species, subspecies are recognized but a definite revised nomenclature is not yet decided upon. L. infantum is grouped within the L.donovani complex, L aethiopica within L. tropica complex, L. amazonensis within L. mexicana complex, L. panamensis within L. guyanensis complex and L. peruviana within L. braziliensis complex (275). In recent years a new species, L. siamensis has been identified in patients In Thailand and Myanmar. In patients with and without HIV-infection and infected with L. siamensis, all manifestations of leishmaniasis, see below, have been reported in patients. Apparently identical parasites have been found in cutaneous lesions of cows and horses in Europe and the USA (233, 301).

Human disease can be divided into three major clinical syndromes:

1. Visceral leishmaniasis (VL), also called kala azar, caused by L. donovani, and L. infantum (L. chagasi = L. infantum) and rarely by other species.

2. Cutaneous leishmaniasis (CL), also known under many local names like oriental sore, Bagdad sore, chiclero, a.o., caused by L. major, L. tropica and L. aethiopica, and parasites belonging to the L. mexicana complex (L. mexicana, L. amazonensis), the L. braziliensis complex (L. braziliensis and L. peruviana) and L. guyanensis complex (L. guyanensis and L. panamensis). L. infantum may cause cutaneous leishmaniasis as well.

3. Mucocutaneous leishmaniasis (ML), also known as espundia, caused by parasites of the L. braziliensis complex. Mucocutaneous leishmaniasis may also be caused by L. panamensis, L. amazonensis and, rarely, by L. guyanensis.

Leishmania parasites are transmitted by the bite of female sand flies. In the human host parasites survive intracellularly in mononuclear cells, as amastigotes, rounded to oval bodies without a flagellum. In the vector and in culture media they grow and multiply as promastigotes, slender, elongated and flagellated organisms. Both amastigotes and promastigotes multiply asexually.

EPIDEMIOLOGY

The WHO Leishmaniasis Control Team reported that in 2012 endemic leishmaniasis occurred in 98 countries and 3 territories on 5 continents with a reported total of more than 58,000 cases of visceral leishmaniasis and 220,000 cases of cutaneous leishmaniasis per year. Assessing for underreporting, it was thought that approximately 0.2 to 0.4 million visceral leishmaniasis cases and 0.7 to 1.2 million cases of cutaneous leishmaniasis occurred (11). More than 90% of visceral leishmaniasis cases occur in six countries: India, Bangladesh, Sudan, South Sudan, Brazil and Ethiopia. One third of the cases of cutaneous leishmaniasis occur in each of three regions, the Americas, the Mediterranean Basin and western Asia from the Middle East to Central Asia. Seventy to 75% of the global estimated cutaneous leishmaniasis incidence occurs in ten countries: Afghanistan, Algeria, Colombia, Brazil, Iran, Syria, Ethiopia, Sudan, Costa Rica and Peru. Mortality data are sparse; reported data are from hospitals. If the case fatality rate would be 10%, about 20,000 to 40,000 patients would die of leishmaniasis per year. Due to lack of data estimates of post-kala azar dermal leishmaniasis (PKDL), mucocutaneous leishmaniasis and other less frequent forms of leishmaniasis were not made (11).

CLINICAL MANIFESTATIONS

Visceral Leishmaniasis

After an incubation period of 3-4 months (with variation from 7 days to many years) disease develops that is characterized by fever, lymphadenopathy, hepato-splenomegaly, wasting, pancytopenia and ultimately secondary infection of the respiratory and gastro-intestinal tract, bleeding tendency, and death. In endemic areas disease generally extends over months to some years with initial periods of fever, then gradual enlargement of liver and spleen, and wasting. Laboratory features include pancytopenia, high ESR and CRP, low albumen and high gammaglobulin levels due to specific, but non protective and non-specific antibodies. Assessment of cure after treatment is difficult. Initial cure is defined as improvement or, ideally, disappearance of clinical and laboratory signs and symptoms while definite cure is generally defined by disappearance of signs and symptoms and no re-appearance of any of these ("relapse") up to 6 months after end of treatment. This period of 6 months is challenged recently in studies from India (53) and Nepal (252) because of several percentages of relapses between 6 and 12 months. In particular for the assessment of new treatments, follow up for at least 12 months is advocated (53). A systematic review on biomarkers for monitoring treatment of visceral leishmaniasis recently appeared (158). Patients co-infected with HIV may present with atypical manifestations, especially lesions of the gastro-intestinal tract (88). Post-kala azar dermal leishmaniasis (PKDL), a condition of unknown pathogenesis, occurs during or after treatment of visceral leishmaniasis. Some cases are not known to have had clinical visceral leishmaniasis (362). In India, post-kala azar dermal leishmaniasis occurs in up to 10% of patients, generally after a long interval of several years after treatment (55, 246, 329). In East Africa it is more frequent (in East Sudan in some series up to 50% of cases) and occurs earlier, between 3 and 6 months after treatment (259). In Ethiopia, post-kala azar dermal leishmaniasis is rare (Hailu, personal communication). Clinical manifestations are macules, papules, nodules and infiltrative plaques (98). Post-kala azar dermal leishmaniasis plays a role in transmission (329).

In endemic areas many persons become infected without developing clinical manifestations and disease; some may develop mild constitutional symptoms (28). Once (cellular) immunity wanes these persons may develop full-blown visceral leishmaniasis.

Immunocompromised patients with visceral leishmaniasis were known before the advent of HIV/AIDS. These included, amongst others, patients with a renal transplant, lymphoma, corticosteroid use (18, 112, 244). HIV/AIDS has given a new dimension to this: Leishmania/HIV co-infection became a particular problem in southern European countries and has now been reported from 35 countries (12). In Ethiopia the number of cases of co-infection has increased dramatically in recent years (12). HIV-infection and leishmaniasis attack similar immune cells; together they exert a synergistic effect. HIV increases risk of developing symptomatic visceral leishmaniasis, modifies and aggravates clinical manifestations, reduces efficacy of treatment and increases risk of relapses and Leishmania promotes viral replication and aggravates clinical progression of HIV. The Leishmania-HIV co-infection epidemic of the 1980s-1990s in southern Europe dramatically waned after the introduction of HAART in 1997. Since then the problem has spread to all major foci of leishmaniasis. Thirty five countries have reported cases of co-infection (12). In general clinical manifestations of visceral leishmaniasis in immunocompromised patients are like in immunocompetent patients but gastro-intestinal involvement, diarrhea, is more frequently reported. The most prominent differences are the reduced response to treatment, increased toxicity of drugs, in particular antimony, and occurrence of relapses, even in patients on HAART and despite "secondary prophylaxis", see below under Immunocompromised Patients Including Leishmania/HIV Co-infection

Cutaneous Leishmaniasis

At the site of the sand fly bite a papule, subsequently a nodule develops, that ulcerates, forms a crust and heals after months to years, leaving a scar. Lesions may appear as indurated plaques, scaly nodules, or ulcers; they may be single or multiple and lymph vessels and lymph nodes may be involved.

In the Old World, cutaneous leishmaniasis is caused by L. tropica, L. major and L. aethiopica. L. aethiopica causes simple cutaneous leishmaniasis and also diffuse cutaneous leishmaniasis in Ethiopia and Kenya. In Burkina Faso patients with Leishmania (L. major) –HIV co-infection demonstrated many unusual clinical manifestations, in one visceralization occurred with L. major parasites in a bone marrow aspirate (12). L. donovani and L. infantum may also cause cutaneous leishmaniasis. L. tropica is related to urban leishmaniasis. It is a disease of man with dry, crustated, single lesions that heal spontaneously in about one to 2 years. L. major, endemic in rural areas with a reservoir in rodents, causes exudative, often large ulcers that may be multiple. Lymph node involvement may occur. Spontaneous healing is within 6 to 12 months. After healing of cutaneous leishmaniasis there is strain specific immunity.

In the New World, cutaneous leishmaniasis is caused by parasites of the L. mexicana complex, the L. guyanensis complex and L. braziliensis complex. Parasites of the L. mexicana complex generally produce chronic ulcers with a tendency to heal within 6 to 12 months. Parasites of the L. guyanensis complex may involve lymph vessels and lymph nodes and the parasites of the L. braziliensis complex have the reputation of not healing spontaneously. Spontaneous cure does occur however (67, 129, 142, 187, 188, 218) but we do not know how often and under which circumstances. In 2-5% of cases, L. braziliensis infection may be followed by mucosal disease (South American mucocutaneous leishmaniasis, mucocutaneous leishmaniasis (MCL) (227). As mentioned, mucocutaneous leishmaniasis may also be caused by L. panamensis, L. amazonensis and L. guyanensis.

Disseminated leishmaniasis is seen in Latin America and defined by ≥ 10 mixed-type lesions like acneiform, papular, nodular, and/or ulcerated lesions, located in ≥ 2 body parts (head, trunk, arms and legs). These patients have a strongly positive Leishmanin Skin Test (LST), in South America also called Montenegro Skin Test (MST) a delayed type hypersensitivity reaction (338).

Diffuse cutaneous leishmaniasis is a rare, anergic manifestation of cutaneous leishmaniasis related to L. aethiopica in East Africa, L. amazonensis or L. mexicana in Central and South America, or an unnamed Leishmania species in the Dominican Republic. The primary nodular lesion does not ulcerate but persists for months to years and spreads producing many nodules, plaques and hypopigmented macules. In these patients the leishmanin skin test is negative.

Leishmaniasis recidivans, also called relapsing or lupoid leishmaniasis, caused by L. tropica, is a chronic manifestation with slowly enlarging and or spreading lesions that heal centrally but continue to crop up as nodules that ulcerate, heal and reappear.

Mucocutaneous Leishmaniasis

In a minority of patients infected with L. braziliensis parasites (< 5%) the mucosa of nose, mouth, pharynx and larynx may get involved with granulomatous lesions that mutilate and may be so destructive as to cause death because of malnutrition, wasting, superinfections and aspiration pneumonia. Involvement of the naso-pharyngeal mucosa occurs months to years after healing of the primary cutaneous lesion, in the majority of cases within 2 years and often after inadequate or no treatment (227). However, concomitant early mucosal and cutaneous leishmaniasis does occur (46).

LABORATORY DIAGNOSIS

Leishmaniasis is diagnosed by demonstration of parasites in aspirates of spleen, lymph nodes or bone marrow in visceral leishmaniasis, of edges of ulcers in cutaneous leishmaniasis and biopsies of mucosa in mucocutaneous leishmaniasis. Aspirates and biopsies may be stained with stains according to Giemsa, Field or Wright and examined microscopically at 1000x magnification. Parasites are seen intracellular in macrophages, and extracellular because of rupture of the macrophages during processing. Aspirates may be cultured in several media; mice and Syrian hamsters are suitable animals for isolation and culturing. In Leishmania/HIV co-infected patients, parasites are found in peripheral blood (microscopy and culture) in as many as 50% (189).

In mucocutaneous leishmaniasis, parasites are often difficult to demonstrate, and may not be found by direct methods.

Many serological tests are available with variable sensitivity and specificity in different endemic areas, and different populations. In visceral leishmaniasis, subclinical infections and antibodies persisting long after cure, may confuse the interpretation. An ELISA and a dipstick test based on a recombinant (r) antigen rK39 which consists of 39 amino acid repeats of a kinesin-like gene of L. chagasi proved very satisfactory in India, Brazil, the Mediterranean Basin and Sudan (254). Serologic diagnosis is less reliable in HIV patients (270). In cutaneous leishmaniasis in the Old World, serology is generally not useful. In the New World ELISA and IFA with L. amazonensis antigen proved useful in L. braziliensis and L. guyanensis infections (263). In mucocutaneous leishmaniasis, diagnosis often has to rely on the clinical and histopathological picture together with results of serological tests and of the leishmanin skin test (MST) and technologies of immunohistochemistry or PCR (259).

Nucleic acid hybridization and PCR technologies with genus- and species-specific probes are increasingly used. They demonstrate and identify parasites in aspirates, biopsies and cultures and are major advances in diagnosis and for species-directed treatment (143, 212).

A positive LST (MST) is evidence of infection; it is negative during symptomatic visceral leishmaniasis. How many infected persons effectively clear parasites and thus will never develop disease is not known. Probably many remain infected without signs and symptoms but harboring parasites that may give rise to clinical leishmaniasis once immunity diminishes due to disease (e.g. HIV, lymphoma) or immunosuppressive treatment.

PATHOGENESIS

The outcome of infection with Leishmania parasites depends on the complex, only partially understood interaction between parasites and immune response of the host, the latter dominated by macrophage reaction and cellular and cytokine response. Studies in mice and man indicate that IFN-γ, IL-10 and IL-12 are important. In endemic areas many persons are infected without developing disease.

SUSCEPTIBILITY IN VITRO AND IN VIVO

Data on in vitro and in vivo susceptibility and resistance to drugs, on minimum inhibitory concentrations (MIC's) and on mechanisms of drug action and resistance are relatively limited. Reasons are: the complex nature of the parasite with different life stages as extracellular, free living promastigotes and intracellular amastigotes; the many different species and various clinical manifestations; and difficulties in obtaining field isolates in areas where the diseases are endemic. Data on pharmacokinetics and pharmacodynamics of the available drugs are extremely limited.

Methods to establish drug sensitivity and resistance are limited to research laboratories. Clinical impressions of reduced drug sensitivity cannot easily be confirmed in the laboratory. Clinical studies of drug response are tedious because of the long duration of treatment and are hampered by the lack of a "test of cure". The combination of clinical, hematological and biochemical improvement, disappearance of parasites and no recurrence of disease and parasites during follow up of 6, preferably 12 months, indicates "cure". Recent information from, India and Nepal indicates that the till recently accepted follow-up of 6 months, is no longer adequate (61, 252). There is no single marker of cure (223). The systems to test drugs for leishmaniasis may not or only partly be adequate. Animal models for visceral leishmaniasis and cutaneous leishmaniasis exist and are useful but at best mimic the conditions in man. In vitro studies of promastigotes in culture and of amastigotes in cell lines have given important information but validation to what is actually happening in man is limited. Methods to assess drug activities available up to 1987 were reviewed by Neal who noted that promastigotes of L. donovani, L. tropica and L. braziliensis were not affected by antimonials but that amastigotes in macrophages were susceptible at clinically achievable concentrations (219). Development of the radiospirometric test (RAM) (145), the semi-automated microdilution technique (128), and models of macrophage and axenic amastigotes have given more insight (72, 102, 172). In France (107), India (172) and Colombia (260) a strong correlation was found between clinical response and Sb sensitivity in vitro in intracellular amastigotes. A recently developed simple resazurin- based promastigote assay was validated for the monitoring of miltefosine susceptibility of clinical isolates of L.donovani (164). In Colombia, a novel approach to in vitro drug susceptibility assessment using infected U-937 human macrophages proved to be an efficient and reliable strategy (109).

Mechanism of Action and Resistance

Apoptosis

Apoptosis may play an important role in leishmanial infections and the antileishmanial drugs antimony, amphotericin B, pentamidine and miltefosine all induce apoptosis in leishmania parasites (320). Ca 2+ and cytosolic Ca 2+ are important in this process (280).

Antimony

The mechanism of action of antimony (stibium, Sb) involves activation of several components of the intracellular signaling pathway resulting first in Reactive Oxygen Species (ROS)- dependent killing of parasites and subsequently in strong Nitric oxide (NO)- dependent killing (34). It is likely that interaction with sulphhydryl groups of the parasite plays a role. Trivalent antimony (Sb^III) was the first antimonial drug to be used in the treatment of leishmaniasis. It proved to be very effective but also toxic, reason why pentavalent antimonials were developed that proved effective and less toxic. Sb^III interferes with trypanothione metabolism both in amastigotes and promastigotes. The pentavalent antimonial sodium stibogluconate (Sb^v) has the same effects in axenically cultured amastigotes (359). Goodwin and Page already in 1943 suggested that Sb^v was converted to Sb^III, the active substance, intracellularly and thus was a prodrug for Sb^III (359). Antimonials target cardiac calcium channels which explains the cardiotoxicity that manifests itself in particular at high and repeated doses (166). More recently developed analytical methods of measurement of Sb^v and Sb^III and of intracellular antimony make comparison with older studies difficult. Macrophages were demonstrated to accumulate and retain antimony for at least 3 days after a 4-hour exposure which may give rise to an effect similar to the post-antibiotic effect (258). Moderate drug resistance may be present in nature and drug sensitivity decreases after subcurative treatment (126,128); Sb resistant promastigotes can be created in the laboratory by drug pressure and these promastigotes retain Sb resistance once transferred into amastigotes (42, 102,127). A mechanism of development of resistance may be selection; patients are infected with several "strains" of parasites with different susceptibilities to the drug and parasites may survive suboptimal treatment. Amplification of P-glycoprotein genes does occur in Leishmania parasites but a role for these genes in antimony resistance is unlikely. Increased efflux of drug has been demonstrated in arsenite-resistant cells. Transfection experiments with L. major and the ltpgpA-gene which is associated with Sb-resistance in wild type L. tarentolae isolates, did not confer resistance. Until recently drug resistant clinical isolates had not been used in models of resistance of Leishmaniae (264); since then isolates of Indian, Sudanese and French patients have been used in such studies (172). Rojas et al.(260) in Colombia found primary resistance and demonstrated that even a single cycle of antimony treatment can select for resistant Leishmania parasites (secondary resistance). From Iran, primary antimony resistant L. tropica field isolates were reported (132).

Complicating factors are the differences between batches of the commonly available antimonial drugs, both in actual Sb content and in percentage of Sb^III. Sodium stibogluconate (Pentostam®) is supposed to be a 30-34% solution but often is a 25-30% solution (145); it should contain 100 mg Sb^v/ml but was shown to have 125 and 123 mg/ml in 2 samples, 12.5 respectively 15.4% of this being trivalent (114). Four lots in India, supposedly containing 100 mg/ml, actually contained between 114 and 121.5 mg/ml (305). Samples of glucamine antimonite (Glucantime®), supposed to contain 85 mg Sb^v/ml, contained 91.6 to 128.6 mg Sb/ml, 10.5 to 15.8% of the antimony being trivalent (114). Apparently, nowadays the content is 81 mg/ml (357). Another complicating factor may be the preservative chlorocresol. In a promastigote system of L. panamensis the leishmanicidal activity of sodium stibogluconate appeared to be due to the preservative. Preservative free sodium stibogluconate had a very high IC50 of >4000 mg Sb/ml but in an amastigote-macrophage system sodium stibogluconate itself showed antileishmanial activity at levels achievable in serum during treatment (102, 257). Axenic L. donovani amastigotes and promastigotes were susceptible to chlorocresol. Resistance to Pentostam® was induced in promastigotes and this manifested itself as resistance to chlorocresol in promastigotes and amastigotes and as resistance to sodium stibogluconate in amastigotes (102). In the study in Colombia mentioned above that demonstrated primary and secondary resistance, preservative free antimony was used (260).

The variability of commonly used drugs and the presence of variable amounts of trivalent antimony might at least in part be responsible for differences in treatment results, toxicity and susceptibilities in different studies and these variables cast doubts on the comparability and generalized in vitro- and in vivo-studies, to some extend also of pharmacokinetic studies. Interest in Sb^v renewed when effectiveness in experimental models of cancer, hepatitis C and AIDS was shown. Research is ongoing on development of simple synthetic methods, of liposome-based formulations and cyclodextrin-based formulations to promote oral delivery (117).

Mode of Action of Other Drugs

Miltefosine

Miltefosine is an alkylphospholipid oral antineoplastic agent that was not further developed for oncological purposes because of toxicity at the doses required. It proved to be effective in visceral leishmaniasis in lower, less toxic doses (148, 245, 255, 308, 309). Its mode of action is probably by perturbation of the alkyl-phospholipid metabolism and biosynthesis of alkyl-anchored glycolipids and glycoproteins and by induction of apoptosis (72, 351).

Amphotericin B

Amphotericin B binds to ergosterol, the predominant membrane sterol of Leishmaniae. It induces apoptosis. Until recently its use in leishmaniasis was limited because of toxicity and the effectiveness of antimony. Because of increasing antimony resistance, especially in India, amphotericin B and its lipid formulations are now more and more studied and used. There are no reports on resistance yet.

Aminosidine (paromomycin)

Aminosidine, now generally called paromomycin, is an aminoglycoside that has activity against Leishmania parasites with different sensitivity for different species (71, 221). Its mode of action is unknown..

Pentamidine

Pentamidine, an aromatic diamidine drug, acts by damaging the kinetoplast-DNA-mitochondrial complex (48) and induces apoptosis. Results of in vitro and in vivo tests with pentamidine were disappointing; activity at high concentration was found against promastigotes but activity against amastigotes varied with the cell source. The human monocyte appeared to be the most susceptible model (219).

Azole drugs

Antifungal agents like ketoconazole, fluconazole and itraconazole inhibit ergosterol synthesis and are cytostatic in vitro (71).

Allopurinol

Allopurinol, an analogue of hypoxanthine, inhibits xanthine-oxidase and is in clinical use amongst other conditions in gout. It proved to form aberrant nucleotides and to interfere in nucleic acid synthesis in vitro in several species of Leishmania and Trypanosoma cruzi (71).

Sitamaquine

Sitamaquine,an oral 8-aminoquinoline analoque, was effective in animal models of leishmaniasis (71, 72) but clinical development is stopped because of the too small therapeutic index.

Imiquimod

Imiquimod is an antiviral agent, an immunomodulator that stimulates local immune response at the site of application by inducing production of cytokines and nitric oxide in macrophages (71).

New Agents

Studies of the biology, metabolism and biochemistry of parasites may lead to new compounds: polyamine analogues, phospholipids and the flavanoid licochalcone (71) and other compounds of plants (72). Advances are slow. Nitroimidazole compounds seem quite promising, one of those DNDI-visceral leishmaniasis-2098 may soon enter clinical trial (131).

ANTIPARASITIC THERAPY

Problems with the literature on treatment of the various manifestations of leishmaniasis are the limited numbers of randomized controlled trials, absence of parasitological confirmation, lack of species typing, small sample sizes, lack of clearly defined entrance criteria, lack of information regarding beginning of complaints and disease manifestations in relation to entrance into the study, lack of clearly defined endpoints, of criteria of cure and of follow-up. A particular problem in studies of self-limiting diseases (cutaneous leishmaniasis) is the lack of placebo control. There are very few well-designed and well- executed comparative studies of various drugs, with different dosing and duration schedules, very few studies reporting the actual dose of the given drug per kg (mean, range) (143). Cochrane Reviews on the treatment of Old World cutaneous leishmaniasis (120) and on the treatment of American cutaneous and mucocutaneous leishmaniasis (121) concluded that "most trials had been designed and reported poorly resulting in a lack of evidence for potentially beneficial treatments of current information" and called for "an international strategy to improve the quality and standardization of future trials for a better evidence-based strategic approach". For visceral leishmaniasis, so far, only a protocol of the Cochrane Collaboration is available (13). An answer to these pleas for better studies is the paper on "Methodology of clinical trials aimed at assessing interventions for cutaneous leishmaniasis" (230).

Visceral Leishmaniasis

The treatment of visceral leishmaniasis in a particular area depends on the susceptibility for drugs of the parasites circulating locally. Pharmacokinetics play an important role in dose, interval and duration of treatment and are dealt with in a chapter in volume II. Pharmacodynamic studies were difficult to perform in leishmaniasis as they required assessment of disappearance of parasites from spleen or bone marrow (60, 64, 151, 328). Quantitative PCR or QT-NASBA can be used to follow disappearance of parasites as demonstrated in a visceral leishmaniasis patient treated with miltefosine (82) and a simple resazurin-based promastigote assay is proposed for routine monitoring of miltefosine treatment (164). As a rule pharmacodynamic studies do not play a role in the choice of treatment but this may, and probably should change in the near future because of the need to assess new treatments, new dosing and duration schedules and new treatment combinations. Molecular tools as biomarkers are likely to play an important role in pharmacodynamic studies (158).

Treatment of visceral leishmaniasis is with pentavalent antimony, amphotericin B, miltefosine, paromomycin and rarely with pentamidine (Table 1). Other drugs that have been tried include allopurinol and the azole drugs. They may be recommended in special circumstances. Where parasites are sensitive to antimony (Mediterranean Basin and Middle east up to Central Asia, East Africa, South America), antimony is often still first choice of the clinician because of availability, costs, ease of administration (can be given on an outpatient basis), habits, the toxicity of amphotericin B deoxy-cholate (classic amphotericin B, c-AMB) and non-availability of alternatives like lipid associated amphotericin B (in particular liposomal amphotericin B, LAMB), paromomycin and miltefosine. The costs of liposomal amphotericin B, if not subsidized, are prohibitive in most endemic areas. Otherwise, it would be first choice. In India, amphotericin B displaced antimony as first choice because of development of Sb resistance. Antimonials are not FDA approved but they are available. After CDC approval, locally Institutional Review Board (IRB) permission is also required.

Pentavalent Antimonials

Pentavalent antimonials are still widely used and often are first choice of the clinician. In Bihar antimony should no longer be used (307) and in Nepal (251) and France (107) resistance is increasing. Also in other countries the position of antimony as first line treatment is challenged especially in patients with HIV co-infection (79, 255). In Italy liposomal amphotericin B has replaced Sb (87, 125). In Eastern Sudan the high post-kala azar dermal leishmaniasis rate (362) and relatively high relapse rate (234) suggest that Sb treatment is no longer satisfactory.

Trivalent antimony, first used in 1912 in a case of mucocutaneous leishmaniasis was replaced by the much less toxic pentavalent compounds in the 1920s. Although trivalent antimony is probably responsible for the antileishmanial activity, trivalent compounds are of historical interest only. The products currently available are sodium stibogluconate (Pentostam®, Glaxo-Wellcome, UK) and meglumine antimoniate (Glucantime®, Specia, France) were developed in the 1940s. In India and China sodium stibogluconate (SSG), also called sodium antimony gluconate (SAG) is produced by several companies. Sodium stibogluconate produced by Albert David Ltd, Calcutta, was tested for quality by the International Dispensary Association, IDA, Amsterdam, The Netherlands, who also inspected the manufacturing process. Randomized trials of generic SSG from this producer and branded sodium stibogluconate (Pentostam®) have been performed by Médecins sans Frontières (MSF) in Sudan (348), Ethiopia (253) and Kenya (202). Apart from these trials no formal comparisons have been made between the different products for effectiveness and toxicity in visceral leishmaniasis patients. In South America meglumine antimoniate and generic products from China (SSG, BP 80®, ShadongXinhan, China) and India (SSG, Albert David Ltd, Calcutta) were compared in patients with cutaneous leishmaniasis (270, 293).

Sodium stibogluconate and meglumine antimoniate are complex compounds of pentavalent antimony (Sb^v) complexed in carbohydrates, the exact structures of which are actually unknown. The solution consists of numerous moieties with molecular weights of 100 to 4,000 and an osmolarity of 800 mOsmol, a solution that may be described as "an unknown number of uncharacterized complexes of Sb with carbohydrates derived from gluconic acid" (39). Differences in osmolarity (305), in Sb^v and Sb^III contents between batches have been mentioned already (114, 145).

Dose and duration of the antimonials varied in the respective endemic areas and were more or less found by trial and error. Doses have varied from 10 mg Sb/kg (stibogluconate) to 28 mg Sb/kg (meglumine antimoniate) with variable maximum doses being applied while durations varied from 6 to 15 days ("a course of treatment"" ). After a rest pause of 7 to 10 days, courses were generally repeated (48). The clinical impression that meglumine antimoniate was both more effective and more toxic may at least partly be explained by the much higher dose given. Both sodium stibogluconate and meglumine antimoniate can be given by intramuscular and intravenous (slow) injection. Many studies performed in India between 1980 and 2004 are discussed and summarized by Olliaro et al. (229). Based on randomized comparative studies of patients with visceral leishmaniasis (59, 61, 229), with cutaneous leishmaniasis (32) and with mucocutaneous leishmaniasis (186) the dose of antimony was increased from 10 to 20 mg/kg/day, first with a maximum of 850 mg per day, but without a maximum since 1992 (141). The most recent WHO advice (2010) is Sb^v 20 mg/kg once per day for 28-30 days (357).

Sodium stibogluconate (Pentostam®) comes in vials of 100 ml with 100 mg Sb^v per ml. Dosing is relatively easy: a person of 55, 60 or 65 kg receives 11, 12 respectively 13 ml. Meglumine antimoniate (Glucantime®) comes in ampoules of 5 ml with 85 mg Sb^v per ml, 425 mg Sb per ampoule. The recently mentioned 81mg/ml, 405 mg per ampoule (357, 307) complicates making comparisons between studies. Dosing with Glucantime® is more complicated: at 55 kg one should receive 2.6 ampoules (of the product with 85 mg per ml), at 60 kg 2.8 and at 65 kg 3 ampoules. There might well be and in practice there is, a tendency to give full ampoules thus under- or overdose (294). Even in trials a dose of 20 mg/kg/d is routinely reported, not the actual dose per kg given with the mean and range.

Once Daily versus Multiple Daily Administration: Traditionally antimony is administered once per day. Because of the short elimination half-life of about 2 h (63) twice or thrice daily administration would seem logical which, in fact already had been practiced in the past although to limited extend (48). Small studies on multiple daily dosing in Kenya showed quicker disappearance of parasites from splenic aspirates, and good tolerance but they were too small to address efficacy (61), Other studies were reported in conferences and referred to by Nyakundi (223). Thrice daily administration of 20 mg/kg to patients with "antimony resistant" disease was helpful in a few (2/10 cured) but was too toxic (extreme lethargy, cardiotoxic death) to be recommended (49, 61). Macrophages accumulate antimony during a 4-h exposure and retain it for at least 3 days and amastigotes within macrophages have a higher Sb content 6 days after exposure than immediately after exposure, comparable with the so-called "post-antibiotic effect" (258). Clinically, Sb is effective at once daily dosing. The duration of exposure is better reflected by the terminal elimination half-life of 76 h than the initial half-life of 2 h. In reviews and advice (40, 141, 211, 357) multiple dosing per day is not mentioned.

Toxicity: In visceral leishmaniasis assessment of toxicity is difficult because abnormalities may be due to the disease, its treatment, or the interaction between the two. Toxicity of antimonials did not frequently lead to discontinuation of treatment in various regions of the world (48, 124, 229), even not in severely debilitated patients in South Sudan (278). In patients with Leishmania/HIV co-infection a high frequency of serious toxicity due to antimony is reported (58, 79,168, 255): acute pancreatitis, acute renal failure and leucopenia. In a study in Spain, eight of 19 Leishmania/HIV co-infected patients discontinued Sb treatment because of toxicity (4 pancreatitis, 1 renal failure; 3 died) (168).

Treatment of cutaneous leishmaniasis may give a better view of toxicity of Sb. Variable percentages of abnormalities in various degrees of severity are reported in the treatment of cutaneous leishmaniasis and mucocutaneous leishmaniasis. These studies at best give an indication of toxicity because of differences in size, populations, disease syndromes, variations in treatment regimes and collection of data (225). Frequently reported complaints during Sb treatment are arthralgia and myalgia (about 60-80% of patients), gastro-intestinal disturbances ( 30-70%), headache (50-70%), fatique (15-40%), mostly in the second and third week of treatment (21, 79, 126, 293). Elevation of levels of amylase and lipase, "chemical pancreatitis", with or without abdominal pain but without the clinical syndrome of acute pancreatitis and without hypo- or hyperglycemia during Sb treatment of visceral leishmaniasis, cutaneous leishmaniasis and mucocutaneous leishmaniasis commonly occurs and severe, acute pancreatitis and deaths have been reported (119, 168, 192). In patients treated with Sb, who experience abdominal pain or nausea and vomiting, amylase levels should be assessed and if found increased treatment should be interrupted. After a few days treatment can generally be resumed without further problems (21). The cardiotoxicity of trivalent antimony is well known (138). Long term exposure to Sb^v with conversion of Sbv to Sb^III may lead to increase in cardiac calcium currents and toxicity (166). High doses of pentavalent antimony are toxic: once daily 30 mg/kg or 3 x 20 mg/kg per day led to QT-prolongation, tachyarrhythmia's and death (49). A dose of 20 mg/kg/day up to 30 days is considered safe (62) although ECG recording once or twice per week seems advisable. A dose of 3 x 10 mg/kg/day, toxic in India (324) was well tolerated in small studies in Kenya (61). In India 32 of 320 patients treated with a standard course of Sb^v, experienced cardiotoxicity, fatal in 20 (307). Severe cardiotoxicity, with mortality, was seen during treatment with generic products (SAG) from India (250, 305). A generic SSG product from China also showed high toxicity (270). Again, the variability in Sb^v and Sb^III content of the different products and different lots, makes comparison difficult. In a review of 67 consecutive travelers with NWCL, Wise et al. concluded that Sb could safely be administered on an outpatient basis among patients aged below 65 years and without comorbidity. At 3 weeks of treatment 59.6% of patients experienced myalgia, 29.8% malaise and reversible reductions of mean lymphocyte and platelet numbers and elevations of hepatic enzymes and of lipase occurred in a few (356).

Efficacy: General conclusions on efficacy are difficult to draw; the dose and duration have to be adapted to the region and to the sensitivity pattern of the local parasite. When given in the proper dose and for a duration adequate for the area and the given parasite, antimony should be efficacious treatment with cure rates above 90% in areas where secondary resistance is not widespread. Rates of death, of non-response and of relapse should be low, a few percentages each (48). An overview of treatment results in the Mediterranean region up to 1995 mentions good treatment results with unresponsiveness and relapse in a few cases only (124) but in Italy, since 1995, meglumine antimoniate was gradually replaced by lipid amphotericin B (LAMB). Since 2001 almost all patients are treated with liposomal amphotericin B(87). Reasons were toxicity of antimony drugs, increase in failure rates, ease of administration and cost-effectiveness of liposomal amphotericin B, mainly because of much shorter hospitalization (125). In South Sudan where due to the war follow up was impossible, 2562 of 3076 (83.3%) patients treated with Sb were "cured" at the end of treatment; 335 (10.9%) died and 79 (3%) were known to have relapsed (278). Definite "cure" should probably be defined as clinical, hematological and biochemical response and no relapse at 6 months follow up, as mentioned, a period that should be extended to 12 months, at least in the Indian continent (52). Post-kala azar dermal leishmaniasis (PKDL complicates the matter. In Sudan, PKDL is seen frequently during or shortly after treatment. It often does not require treatment and generally is cleared in 6 months (362). In India, PKDL may occur many years after treatment of visceral leishmaniasis. It requires treatment, in the past this was with many courses of Sb but now, with increasing Sb resistance with alternative treatment, see below (334).

Combination treatment with paromomycin. This is reported under Paromomycin section below.

Pregnancy. For treatment during pregnancy, see section titled Special Situations, Pregnancy.

 

Summary: Pentavalent antimony is given as intramuscular or intravenous injection in a dose of 20 mg Sb^v per kg body weight once daily for 28 days (186, 211). Weekly assessment of haematological and biochemical parameters (liver enzymes, amylase) and a weekly ECG are recommended. Treatment may have to be interrupted because of severe thrombocytopenia (very rare), increase of liver enzyme or amylase levels to more than 5 times normal values (rare), QTc prolongation and dysrhythmias (rare) or severe arthralgia. In immunocompetent patients in the Mediterranean region where resistance is not yet a big problem, 90 - 95 % of patients are expected to be cured, a few die and a few relapse. Elsewhere the cure rate may be lower but firm data are lacking for many regions (11). In Ethiopia, with many severely debilitated, malnourished patients, with or without HIV-infection, Sb^v treatment comes with severe toxicity, reduced efficacy and increased case-fatality rate (up to 20%) (253, 255). At the end of the 20th century in Bihar 35% (95% Confidence Interval (CI) 28 – 42%) of patients only were cured with Sb treatment; elsewhere in India antimony response was still 86% (95% CI 79 – 93%) (307). In India up to 10 to 20 % develop PKDL after several years (329). In Sudan, post-kala azar dermal leishmaniasis occurs earlier and is more frequent (362).

Amphotericin B and Lipid Associated Amphotericin B

Amphotericin B deoxycholate (c-AMB) is a polyene antibiotic used in systemic fungal infections It is very active in vitro and in vivo against Leishmania parasites. It is given by intravenous infusion and administration is often accompanied by adverse effects ranging from infusion related fever and chills, to toxic effects of severe anemia, hypokalemia and renal failure (40, 146). These can be reduced by adequate hydration and infusion over several, even 24 hours (103). Administration requires hospitalization. Until the early 1990s its use in leishmaniasis was almost exclusively restricted to mucocutaneous leishmaniasis (40, 48) for which it is effective treatment (186). Because of the toxicity of antimony, the increase of antimony resistance and the relatively high toxicity and mortality rate in Leishmania/HIV co-infection, amphotericin B was increasingly studied and the advent of new formulations has accelerated these studies. For these reasons and because of failure and toxicity of pentamidine and non-availability of alternatives, amphotericin B became first line treatment of visceral leishmaniasis in Bihar, India in the early 2000s (229, 310).

In search of less toxic, possibly more effective formulations, lipid associated formulations of amphotericin B were developed. Liposomal amphotericin B (Ambisome®, Gilead Pharmaceuticals, Foster City, CA. USA) (LAMB) is the most widely used and studied product. It is FDA approved for treatment of visceral leishmaniasis, it is recommended by WHO (357) and is available at reduced WHO-negotiated price for selected countries with limited resources. Another product, amphotericin B lipid complex (formerly ABLC, now Abelcet®, Liposome Company, Princeton, New Jersey, USA) has been studied to some extent and on a third preparation, amphotericin B colloidal (or cholesteryl) dispersion (formerly Amphocil®, now Amphotec® Intermune, Sequus Pharmaceuticals, Menlo Park, California, USA) only few reports are available. Because of the high price of liposomal amphotericin B, a drug unaffordable in India, local preparations were produced, amongst these amphotericin B in various lipids, "heated amphotericin B" (47, 316, 320, 326) and premixed amphotericin B deoxycholate with lipid emulsion (ABLE), licensed in India and recently formally compared with liposomal amphotericin B (320).

Pharmacokinetics. Since the early clinical studies of liposomal amphotericin B in patients with fungal infections and visceral leishmaniasis (39,63,64), studies of pharmacokinetics, of tissue penetration, excretion, safety and tolerance have appeared (1, 36, 353, 359). Dosages up to 15 mg/kg/d followed non-linear saturation-like kinetics, were well tolerated and effective in severe fungal infections (353). A first dose of 5 mg/kg to reach a certain level of drug in the body may well be advisable. " Higher initial doses ( ≥ 5 mg/kg) provide better penetration and longer tissue persistence than do frequent low doses", cited from (44).

Historical note on visceral leishmaniasis treatment in India. Pentavalent antimony (SSG) was the first line treatment of visceral leishmaniasis within the National Program in India, with amphotericin B deoxycholate as second line drug (229). From about 2005 SSG was gradually replaced by miltefosine as first line treatment but after some years of use, efficacy declined to 90% and relapse rates increased to 7% (319). This, together with the known teratogenicity of miltefosine and problems with compliance related to gastro-intestinal adverse effects and long, 28 days, course of treatment led to increased use of the second line drug amphotericin B, both classic and liposomal.

Dose, Duration, Efficacy of Amphotericin B deoxycholate: c-AMB is very effective both in patients not yet treated and in those not cured after treatment with antimony or pentamidine. Cure rates of 98 - 100 % are obtained (229, 332, 335). A dose of 1 mg/kg bodyweight daily, infused in 2 h for 20 days or on alternate days for 30 days is recommended (310) also for children and pregnant women (335). Febrile reactions (80 - 100 %), loss of appetite (up to 30 %) and thrombophlebitis (up to 18 %) are frequent but severe toxicity is rare (229, 332, 335). The incidence of adverse reactions was not dose dependent (332) and not different in a daily or alternate day schedule and incremental doses were not necessary (229, 332). Several patients died of cardiac complications when amphotericin B was given immediately after a course of Sb, not when an interval of 10 days was allowed for (327). C-AMB has to be used regularly in India for lack of other drugs. One of the schedules is a dose of 0.75 mg/kg administered in 15 daily infusions (319).

Dose, Duration, Efficacy of Lipid-Associated Amphotericin B: LAMB has been shown to be effective and non-toxic in the treatment of immunocompetent and immunocompromised patients (40, 43, 47, 73, 74, 123,197, 242, 310, 333, 336). Early studies used doses of 2 - 4 mg/kg/d over 10 days to a total dose of about 20 mg/kg with extension to 14 days in severely ill patients (43, 74, 277). FDA approved liposomal amphotericin B for treatment of visceral leishmaniasis at a dose of 3 mg/kg/d on days 1 - 5, 14 and 21 (196). This schedule, based on early, limited data, was cumbersome, not practical and was challenged from the beginning (41). These low doses are probably of historical interest only; a minimum of 5 mg/kg is to be advised (44). Infusion of liposomal amphotericin B, 10 mg/kg once per day for 2 days cured 40 of 41 children in Greece (321). This high dose (10 mg/kg/dose) liposomal amphotericin B for 2 days for children has become general practice in southern European countries, in particular in Italy (87,125). In France for adults 20 mg/kg total dose ( 5mg/kg/d, 4 days) is advised and 10mg/kg/d, 2 days has been suggested, and probably is practiced but has not been studied formally (50,197). It is remarkable that 37 different regimes of liposomal amphotericin B were applied among 153 of 166 visceral leishmaniasis patients treated in 15 centers in Italy between 2004 and 2008 (87). No further information is given about the regimes; it would be of interest to know how many adults were treated with the schedule of 10 mg/kg/d for 2 days. Although in this observational, retrospective study doses and duration were not significantly associated with outcome (cure), it is about time to agree on standardization of this treatment. In East Africa (Sudan, South Sudan, Ethiopia) liposomal amphotericin B is less effective than in the Mediterranean Basin and in India (154, 256). This may be due to the host, the parasite or a combination of both. Many patients in East Africa are extremely ill, due to the disease, to malnutrition to co-infection with HIV and other diseases. Intrinsic differences in susceptibility for the drug between parasites from East Africa and India have not been examined. In both regions anthroponotic L. donovani is the causative organism; in the Mediterranean Basin it is the zoonotic L. infantum. Médecins sans Frontières is involved in combating leishmaniasis in East Africa since several decades (253, 254, 255, 256, 276, 277, 278, 348). Because of unacceptable high mortality related to SSG treatment in Leishmania-HIV co-infected patients and in severely ill patients, alternative treatments had to be introduced. In 2006 liposomal amphotericin B was introduced in Ethiopia. Médecins sans Frontières had already used this drug in southern Sudan in the early 90s (277). Severely ill patients, patients with known intolerance for SSG, with visceral leishmaniasis relapse and HIV-positive patients were treated with liposomal amphotericin B, total dose 30 mg/kg, 6 infusions of 5mg/kg on alternate days. Patients failing liposomal amphotericin B treatment received rescue treatment with SSG, 20 mg/kg/d, 30 days for primary failures and 40 days for relapse patients (256). Of 94 HIV negative severely ill patients treated between January 2007 and January 2009, 93% had initial cure and 6% died. Of 195 HIV positive patients (79 of these relapsed visceral leishmaniasis patients), 60% had initial cure, 7% died and 32% failed treatment. Of the 79 HIV positive relapse patients, 56% failed, 38% showed initial cure and 5% died, in contrast to 116 HIV positive primary patients: 74% initial cure, 8% mortality and 16% failure. SSG rescue treatment increased the cure rate of all HIV positive patients from 60% to 83% but 9 of the 59 patients (16%) died, mainly due to SSG toxicity. Combining liposomal amphotericin B with another drug and avoiding SSG in HIV positive patients was advised (256). In a multi-centre study in Ethiopia and Sudan, single doses of liposomal amphotericin B up to 10 mg/kg and 7 doses of 3 mg/kg (days 1-5,14 and 21) in non-severely ill visceral leishmaniasis patients proved suboptimal: 85% cure at 6 months (95%CI 73-93%) for the multiple dose, total 21 mg/kg treatment (n=63) and 58% (95%CI 41-73%) for 10mg/kg single dose treatment (n=40). In southern Ethiopia no failures occurred but numbers were small (154).

In India many studies on amphotericin B, both in classic and in lipid-associated formulation have been performed (229). Locally produced lipid-associated products have been used in small numbers of patients (47, 228, 242, 316, 320, 326). Liposomal amphotericin B is generally well tolerated, can be given in short courses of high doses but is very expensive and unaffordable for many in India despite the WHO-negotiated reduced price. A study on single dose liposomal amphotericin B at 10 mg/kg with 95.7% cure at 6 months (95%CI 93.4-97.9%) (317) led to the advice of WHO to adopt this regimen as first line treatment for visceral leishmaniasis in SE Asia (357). This advice is based on a single study that was well designed and performed but on 300, not severely ill patients only, without comorbidities within a research setting and with follow-up of 6 months. A second study in real life with follow-up to at least 12 months and preferably comparing 10 mg/kg single dose versus 10 mg/kg on two consecutive days would have given a more solid foundation for an advice. The single dose advice is practical and economical but the cure rate will certainly be lower in real life situations and is that acceptable, also in view of the elimination attempt? Even after 20 mg/kg total dose of liposomal amphotericin B, rates of relapses and of patients with pkdl at 12 months are considerable , see below (53, 54, 55). Together with the increasing numbers of patients with HIV co-infection , they may threaten the elimination campaign (53, 345). The largest studies on liposomal amphotericin B came from centers of Médecins sans Frontières in cooperation with the Rajendra Memorial Research Institute in Bihar, India (54). Between 2007 and 2012 8749 patients were treated with liposomal amphotericin B 20 mg/kg total dose, 5 mg/kg/d over 4 to 10 days, in PHC centers (1396 patients), in hospital (7189 patients) and a treatment camp (164 patients). Twenty % (1767/8749) received the drug on 4 consecutive days, the others over 7 to 10 days. 161 (1.8%) were co-infected with HIV. Initial cure rate was 99.3% (8692/8749); 26 patients defaulted after ≥ 1 dose and 31 (0.4%) died during treatment. The Case Fatality Rate of HIV co-infected patients was 2.5% (4/161). The relative risk of mortality of HIV patients was 7.9 (95%CI 2.8-22.3%) times higher than of non HIV infected patients. Passive follow up rates were low: 53.2% at 3, 38.1% at 6 and 1.5% at 12 months. In March 2012 an attempt was made to follow up all those treated between September 2010 and December 2011. Of 984 immunocompetent patients, 827 (84.0%) were traced. Twenty patients (2.4%) had relapsed: at 6 months 2 of 767 (0.3%), at 12 months 14 of 383 (3.7%) and at 15 months 4 of 164 (2.4%). Fourteen of the 20 relapses (70%) occurred from 6 to 12 months with a mean ± SD time to relapse of 9.6 ± 3 months. The outcome of treatment of 49 pregnant women was good, both for the mothers and the children. The stage of pregnancy and long term outcomes were not recorded routinely (54). Risk factors for relapse in this large cohort were male sex, age < 5 and > 45 year, a smaller decrease of splenomegaly at discharge and a shorter duration of symptoms prior to seeking treatment. Authors pleaded for a one year follow up in future studies on treatment of visceral leishmaniasis (53). Post-kala azar dermal leishmaniasis (PKDL) was confirmed in 24 (0.3%) patients who returned passively with skin complaints. Most, 89%, had macular lesions that appeared 1.2 (median, IQR 0.8-2.2) y following treatment. Risk factors for development of PKDL could not be identified. The time to developing post-kala azar dermal leishmaniasis was shorter after liposomal amphotericin B treatment than after SSG treatment (54).

Other lipid-associated amphotericin B formulations. Locally produced ABLE (n=376 patients) was compared with liposomal amphotericin B (n=124) both at 15 mg/kg as single infusion, in a multi-centre, open label study (320). The authors concluded that this treatment offered advantages "considering the demographic profile of the population" but the definite cure rate at 6 months was 85.9% only. Where no other treatment is available (affordable), this may be acceptable but aspirations should be for a better result. Amphotericin B lipid complex (Abelcet®) )and amphotercin B colloidal dispersion (ABCD, Amphocil®) were tried in a few studies in India, not in recent years (303, 319).

Studies comparing antimony and amphotericin B and of pentamidine versus amphotericin B have become of historical interest (229). Amphotericin B remained as the single effective agent until the advent and availability of miltefosine (229, 310). Head to head comparisons of amphotericin B and the different liposomal products are rare and unlikely to be performed in the future. Comparison of amphotericin B , liposomal amphotericin B and amphotericin B lipid complex showed comparable cure rates from 92 to 96%. Two patients treated with classic amphotericin B died of severe anaemia and hypokalaemia (310).

Amphotericin B colloidal dispersion (ABCD, Amphocil®) 2 mg/kg/d for 7 days was used successfully in Brazil in 2 small studies (83, 84). In an older study in Spain in Leishmania/HIV co-infected patients efficacy of c-AMB and meglumine antimoniate was similar (167) but both were highly toxic. In a more recent randomized multicentre trial in co-infected patients, ABLC 3 mg/kg/d, for 5 and 10 days and Sb 20 mg/kg/d for 28 d were compared (168). Parasitological cure rates were low: 33% (95%CI,13-59%) for ABLC 5 d, 42% (95%CI,16-62%) for ABLC 10 d and 37% (95%CI,16-62%) for Sb. Treatment had to be discontinued in 8 of 19 Sb treated patients versus in 1 in the combined ABLC groups.

Summary: Amphotericin B (c-AMB) and liposomal amphotericin B (LAMB) are highly effective in the treatment of visceral leishmaniasis, both for newly diagnosed patients and for antimony and pentamidine resistant cases. Cure rates close to 100% are to be expected. The generally advised dose of c-AMB is 1 mg/kg/d, infused over 2 h on alternate days for 15 doses (310, 335) but currently, in India, 075 mg/kg/d on 15 consecutive days is also practiced (319). Liposomal amphotericin B is the preferred treatment for both immunocompetent and immunocompromised patients. For immunocompetent patients the total dose is 20 mg/kg, for immunocompromised patients the total dose is 40 mg/kg (326). The minimal dose should probably be 5 mg/kg. Children in the Mediterranean region are treated with 10 mg/kg/d, 2 consecutive days (321). It is regularly stipulated that this should be studied for adults also but thus far formal studies are lacking (50). Advise for the Mediterranean region extends to south America where the same zoonotic L. infantum is the cause of visceral leishmaniasis. In India a single infusion of 10 mg/kg is advised to be the standard treatment (317) and this is adopted and advised for visceral leishmaniasis in SE Asia by the WHO (357). It is based on a study of 300 patients with 6 months follow up, in a study setting. How this will work out in daily practice in field circumstances remains to be seen. It is based on follow up of 6 months which is insufficient; 1 year is advised.

The FDA approved treatment schedule is cumbersome and based on early, relatively limited data. Since then, studies on pharmacokinetics, safety and tolerance and efficacy have indicated that higher doses, probably at 5 mg/kg are to be advised. Formal studies comparing this dose with lower doses to support this advice are not available and unlikely to be performed. Unfortunately it is unlikely that a dossier will be submitted to the FDA to adapt the treatment schedule.

In East Africa, liposomal amphotericin B seems less effective (256), dose and duration may have to be adapted. Other options of combination treatments are being studied.

Data on amphotericin B lipid complex and amphotericine colloidal dispersion are limited.

For Leishmania/HIV co-infected patients and immunocompromised patients in general, the total dose of liposomal amphotericin B is a minimum of 40 mg/kg. For these patients treatment may have to be extended to 14 days and longer as needed and "secondary prophylaxis" with bi-weekly or monthly doses of an appropriate drug (in most circumstances antimony or liposomal amphotericin B) is required until CD4 counts are 350 per mm3, preferably for at least 3 months. See below.

 

Pentamidine

Pentamidine isethionate (Pentacarinat®, Aventis, France) is no longer used in the treatment of visceral leishmaniasis because of low efficacy and severe toxicity. If no other drug is available, it may still be used as a last resort. It is the preferred drug for the treatment of L. guyanensis infections, see below. Pentamidine may be used as "secondary prophylaxis" after visceral leishmaniasis treatment in selected Leishmania/HIV co-infected patients; see below. For details on mechanism, dose , duration and toxicity see volume II.

Paromomycin (Aminosidine)

Paromomycin (PM), formerly aminosidine, is an aminoglycoside antibiotic. Its use as an antibiotic has been abandoned. It proved effective against Leishmania, Entamoeba and Cryptosporidium. It was developed as a product for endemic countries in cooperation between OneWorld Health (SFLA, California, USA) and Drugs for Neglected tropical Diseases initiative (DNDi). It is produced by Gland Pharma Ltd, Hyderabad, India. In in vitro and animal studies activity against the various Leishmania species varied with highest activity against L. major and L. tropica and for the New World species, highest activity against L. panamensis and lowest against L. mexicana. L. donovani species showed variable sensitivity. Combination of antimony and aminosidine had an additive effect in vivo in mice infected with Ethiopian L. donovani and variable synergy in vitro against Indian and Ethiopian L. donovani (72, 221). Combination with miltefosine potentiates the activity of miltefosine (279). The mode of action is not known. After early experiences in India (229), Kenya (65) and Sudan (246), in 2002 Médecins sans Frontières introduced combination treatment of PM 15mg/kg/d equivalent to 11 mg base, by daily intramuscular injection plus SSG 20 mg/kg also by daily intramuscular injection, both for 17 days in its treatment program in warn torn southern Sudan. From 2002 to 2005, 3085 patients were treated with the combination treatment and 1178 patients during the same period with the standard SSG treatment of 20 mg/kg/d for 30 days (retrospective field evaluation, not a randomized, controlled trial). The initial cure rate in the combination treatment group was better than the standard SSG group, respectively 97.0% and 92.4% and odds for dying were consistently lower for the combination treatment from year to year (193). A randomized controlled trial in India of PM 11 mg base/kg/d intramuscularly, 21 d versus c-AMB 1mg/kg/d iv, 15 injections over 30 d showed that PM was not inferior (final cure 94.6% and 98.9% respectively) but there were more PM related adverse events. A big advantage of PM is its costs; its costs are the lowest of all visceral leishmaniasis treatments (314). Shortening this regimen to 14 days was not a good option: definite cure 82% versus 92% in the 21 day group (283). In East Africa monotherapy with PM 15 mg/kg (salt, 11 mg base) proved less effective than the same treatment in India. In a multi-country study in Sudan, Ethiopia, and Kenya, PM, 11 mg base /kg/d, im, 21 days, SSG 20mg/kg/d. intramuscularly or intravenously, 30 days and PM+SSG in the mentioned doses for 17 days were compared. PM monotherapy results were first reported. PM showed variable results by region with low efficacy in eastern Sudan, somewhat better result in southern Ethiopia and Kenya but nowhere satisfactory. Overall cure rate was 63.8%, an unacceptable result (134). A follow up study in one centre in Sudan comparing PM 15 mg/kg/d for 28 days and PM 20 mg/kg/d for 21 days showed higher cure rates at 6 months, respectively 80% and 81%, than the earlier 15 mg/kg/d for 21 days treatment (about 60%) but was still not satisfactory. A small pharmacokinetic study within this trial suggested that Sudanese visceral leishmaniasis patients may have different PK characteristics with lower plasma levels, from Sudanese and American healthy volunteers (213). In a multi-country study (Sudan, Ethiopia, Kenya and Uganda) 2 comparisons were studied: PM, 20 mg/kg/d, 21 days versus SSG 20mg/kg, 30 days and PM 15 mg/kg/d + SSG 20 mg/kg/d both 17 days versus SSG 20 mg/kg, 30 d. At 6 months, efficacy of SSG was 94% and of PM was 84%, a significant difference. For SSG versus SSG + PM no difference was noted ( respectively 94 and 91% (ITT analysis). Approximately 3% of patients experienced an adverse event; one death occurred in each arm in the SSG versus PM study. During the SSG+ PM versus SSG comparison study there were 3 deaths and one person died of unknown cause in the SSG group during follow up. Disabling hearing impairment (195) (paromomycin is an aminoglucoside antibiotic) was reported in one patient in the PM and one patient in the combination arm of the study at the end of treatment, both had resolved at 6 month follow up. Thirty-six patients had audiometric shift (195) at the end of treatment, 11 in the SSG arm, nine in the PM arm, 16 in the SSG + PM arm This had not resolved at 6 months follow up in three of the SSG, four of the PM and eight of the combination patients. Authors concluded that the results together with the results of the earlier large Médecins sans Frontières study in South Sudan (193) support the use of this combination treatment. The treatment course is shorter than the SSG treatment (17 versus 30 days) which reduces the total costs (drug costs for PM and SSG are about the same). Moreover development of resistance may be reduced. The actual status of paromomycin in the treatment of visceral leishmaniasis is not clear at the time of writing (early 2015).

Dose, Duration, Efficacy, Toxicity: Monotherapy at doses of 16 to 20 mg salt/kg/d for a duration of 21 days showed efficacy of 93-97% (65, 147, 229, 314); combination with antimony was synergistic (229, 276). Assessment of toxicity has been limited in these small studies. Aminosidine being an aminoglycoside has the potential of oto- and nephrotoxicity. Phase III trials are ongoing in India and East Africa (231) (http://www.clinicaltrials.gov/ct2/search).

Summary: Aminosidine at a dose of 16-20 mg/kg/d for 21 days seems a safe and effective drug for the treatment of visceral leishmaniasis. It acts synergistically with Sb and may help delaying further development of Sb resistance. It is likely to be a good candidate for combination treatment with miltefosine.

Miltefosine

Miltefosine (Impavido®), Paladin Labs. Inc. Canada), hexadecylphosphocholine, was developed as an oral antineoplastic agent but for this purpose abandoned because of gastro-intestinal toxicity. It was found to have anti-leishmanial activity in vitro and in vivo (71,163, 339). Its mode of action in leishmaniasis is not yet fully determined; induction of apoptosis seems plausible (351). Leishmania species vary in sensitivity to miltefosine in the following order from very sensitive to least sensitive: L. donovani, L. infantum (=chagasi), L aethiopica, L. tropica, L. panamensis, L. mexicana, L. major (105). L. braziliensis complex parasites from patients in Peru were insensitive with the exception of L. (V.) lainsoni parasites (360).

Miltefosine is registered in India, Bangladesh, Nepal and Germany for the treatment of visceral leishmaniasis and in five South American countries for treatment of visceral leishmaniasis and cutaneous leishmaniasis. Phase IV studies are ongoing in India. It is the first oral drug of proved efficacy against visceral leishmaniasis. It is not FDA approved but can be obtained through an IND application and a local IRB approval.

Dose, Duration, Efficacy: After pilot and dose finding studies (229), a randomized, comparative phase III study comparing miltefosine and c-AMB reported an efficacy of miltefosine (MF) of 94% (ITT analysis) and 97% (per protocol analysis). Severely ill patients and those ≤ 12 y were excluded. The dose was 50 mg per day for those < 25 kg and 100 mg per day for those weighing ≥ 25 kg, both for 28 days (actual dose not reported). Vomiting (38%) and diarrhea (20%), reversible ASAT (17%) and creatinine increase (16%) were noted. One patient developed Stevens Johnson syndrome (309). Eighty not severely ill children, 2 – 11 y old, with a mean weight of 18 kg, range 10 – 30 kg, were treated with 2.5 mg/kg/d, 28 days. One patient died of pneumonia, the other 79 were apparently cured at the end of treatment: up to 6 months 3 relapsed and 1 was lost. In the evaluable patients (75 of 78) cure rate was 96%, in ITT analysis it was 93.8% (45). The duration of 28 days had been chosen in conformation with the duration of antimony treatment, not based on studies. Different durations of 14, 21 and 28 days (18 Indian patients in each group) gave cure rates of 89% in the 14 day group and 100% in both other groups (308). Twenty one day treatment seemed sufficient yet a 28 day course of miltefosine became the first line treatment in the elimination campaign of visceral leishmaniasis in India, Bangladesh and Nepal. Miltefosine became available in the public and private sector in India. Fears of problems with compliance due to outpatient treatment with an oral drug that was to be taken twice a day during 4 weeks and of development of resistance due to the long half-life led to an appeal to restrict distribution of the drug to the public sector, under supervision, free of charge, like for tuberculosis drugs (130, 311). Since 2006 miltefosine is only available in public hospitals in India through the National Visceral Leishmaniasis Control Program. The continued use of miltefosine monotherapy was questioned. In Bangladesh miltefosine was introduced in a phase IV trial in real world conditions; an open label, single group, outpatient treatment study, performed in 2006-2007 (245). Of 977 patients who started treatment, 36 stopped (adverse events, opted out) thus 941 were evaluated at the end of treatment; 865 were initially cured, 24 failed and 52 were lost. At 6 months 701 were cured, 95 failed and 69 were lost. The final cure rate was 92%, or 85% if a low Hb at 6 months was considered a failure (245). In Nepal in a study of 120 patients, the initial cure rate was 95.8% (95%CI, 92.2-99.4%), the relapse rate at 6 months 10.8% (95%CI, 5.2-16.4%), at 12 months 20.6% (95%C,I 12.8-27.2%) and the final cure rate at 12 months was 73.3% (at 6 months 82.5%). Reinfection, parasite resistance and drug noncompliance were unlikely as assessed by appropriate methods (kDNA fingerprinting, resazurin-based promastigote assay and pharmacokinetic studies). These results call for a chance in the treatment regimen of the elimination program and for a 12 months follow up period (252). A small study of the Médecins sans Frontières group in Bihar supports the advice to extent follow up to 12 months; of 124 patients treated with miltefosine, one died, four defaulted and 119 achieved initial cure. After 6 months, 111 were cured (94%), 7 relapsed and 1 was lost and at 12 months 108 were cured (92.3%), 1 relapsed and died and 2 were lost. In total there were 9 relapses, 7.6% (54). Of the 16 patients in this study younger than 12 y, five (31%) relapsed, all within 6 months after treatment, supporting the conclusions of a study by Dorlo et al. that the standard dose of treatment results in substantially lower miltefosine levels in children (92). In Ethiopia miltefosine and SSG monotherapy were compared in a randomized, unblinded study of 580 adult men, 375 of whom agreed in HIV testing. HIV seroprevalence was 29%. Six months after treatment 60% of the 290 miltefosine treated patients were cured and 65.2% of those treated with SSG. The relapse rate was 10% in the miltefosine group and 2% in the SSG group and the mortality was 6% in the miltefosine, 12% in the SSG group. The relapse rate among HIV-infected patients treated with miltefosine was 25% whereas it was 5% in the non HIV-infected patients. Initial treatment failure (end of treatment) was more frequent in the miltefosine group: 7.9% versus 0.7% (OR 12.4; p,.0001). Failed and later relapsed patients were treated with SSG (30 d) and final cure rates were comparable: 68.3% miltefosine group and 66.2%, SSG group. HIV infection influenced the outcome: there were more initial failures in HIV infected patients (17.5% versus 4.6%; OR 4.41, p=.044) and there was a higher seroprevalence in those who failed than in those who were cured: 63.3% versus 26.0%;OR 4.89, p=.0001). Multivariate logistic regression showed that SSG treatment (OR 6.53; 2.53-16.89), HIV infection or unknown HIV status (OR 3.54; 1.25-10.06) and vomiting (OR 2.97; 1.28-6.87) were independent risk factors for death. Excluding those who were lost to follow up (per protocol analysis), cure rates in non HIV infected patients were comparable: 93.4% (miltefosine) and 94.6% (SSG). Miltefosine was considered an acceptable alternative to SSG in this population with many severely ill patients with massive splenomegaly, anemia, malnutrition, inability to walk unaided and HIV co-infection. The high death rate in this study is likely to be a reflection of the severity of disease. In the miltefosine studies in India severely ill patients and HIV infected patients were excluded, and the death rate was low, < 0.2%. It was concluded that miltefosine is equivalent to SSG in non HIV co-infected patients and probably safer, but less effective in HIV co-infected patients. However, a "standard" course of treatment for 28 days was followed; optimal dose and duration of miltefosine for African visceral leishmaniasis with and without HIV infection were still to be established in 2006 (255), and they still are at the time of writing, early 2015.

Toxicity: Vomiting and diarrhea occur frequently and are dose related. In pilot and dose finding pilot studies in India a dose of 200 mg/d (about 4 mg/kg/d) appeared to be the maximum tolerable dose; one of five patients treated with 250 mg/d died of nephrotoxicity and of the five patients treated with 200 mg/d and three of the five receiving 250 mg/d discontinued treatment because of vomiting. In two patients receiving 200 mg/d considerable increases in creatinine levels occurred (reversibility not reported). There were no indications of ophthalmologic toxicity as seen in animal studies (229). Animal studies showed embryotoxic, fetotoxic and teratogenic effects and, in male rats, also reproductive toxicity. Pregnancy is a contraindication and women of reproductive age should use effective contraception at least 3 months after treatment. In the phase III trial in India 48 healthy babies were born to partners of 80 male patients (309). Spermiograms in Colombian patients treated with miltefosine for cutaneous leishmaniasis appeared normal so it was concluded that there was no concern for male reproductive toxicity (228). In the larger studies from India 26-38% of patients vomited, 20-25% had diarrhea and ASAT levels rose by 17-19% in the first week (45, 309). In the Ethiopian study vomiting was common: 54.8 % in the miltefosine group vs 32.15 % in the SSG group (p =.0001). It was of longer duration in the miltefosine group but less severe, leading less often to interruption of treatment than in the SSG group. Incidence and duration of diarrhea were similar in both groups, occurring in about 50%, but diarrhea was more common in HIV infected patients (255).

Summary: After more than ten years of use the following picture arises: Miltefosine as the first oral treatment of visceral leishmaniasis is a useful drug with originally high, but now declining efficacy in the Indian continent and with lower efficacy in East Africa. Miltefosine is effective treatment of visceral leishmaniasis in immunocompetent patients in India at a dose of about 2.5 mg/kg/d and duration of treatment of 28 days. Dose and duration for visceral leishmaniasis in other areas and for immunocompromised patients have not yet been established. Toxicity may preclude daily doses higher than 150-200 mg; efficacy of doses < 2 mg/kg/d is not assessed. A total dose needed has not been established. Pharmacokinetic and pharmacodynamic studies are limited (92, 93). Failure rates in children were higher and steady-state concentrations appeared lower than those in adults. A new allometric dosing algorithm was proposed for optimal exposure to miltefosine in both adults and children (92). Disadvantages, other than the embryotoxicity and adverse gastro-intestinal events, are the costs of the drug, the long duration of treatment, and the long elimination half life that may contribute to development of resistance. Regulation of distribution and prescription is needed and combination treatment is advocated and should be studied. Paromomycin is likely to be a good candidate for this combination.

Sitamaquine

Sitamaquine, an oral 8-aminoquinoline showed promising anti-leishmanial activity in small, phase II clinical studies in Kenya (283), Brazil (85) and India (149) but was nephrotoxic. Clinical development has been stopped.

Allopurinol

Allopurinol, an analogue of hypoxanthine is metabolized by Leishmania spp and Trypanosoma cruzi whereby aberrant nucleotides are formed that, incorporated into the RNA of the protozoa, interfere with normal protein synthesis (71). Allopurinol showed anti-leishmanial activity in in vitro cultures and in animal models but results in vivo have been variable, both in visceral leishmaniasis and cutaneous leishmaniasis. Most reports are about non-controlled trials, mostly in combination treatments with Sb of patients non responsive to standard Sb treatment (48, 64, 80, 86, 150, 151) but also with the antifungal azole drugs keto-, flu, and itraconazole, in the treatment of visceral leishmaniasis and as secondary prophylaxis in patients co-infected with Leishmania/HIV (123, 337). Its use in the latter condition before the advent of HAART seemed reasonable. Allopurinol is not mentioned in the WHO report on the leishmaniasis (357) and in Cochrane Reviews on the treatment of the leishmaniases (120, 121). A search of the literature results in 51 references involving allopurinol and leishmaniasis. Allopurinol is widely used in canine leishmaniasis in monotherapy and various combinations (184).

Dose, Duration: In the dose used in leishmaniasis, 7 mg/kg x 3 per day for prolonged periods of time, adverse effects are more likely than with the dose used for gout. Adverse effects include rash, itching, fever, eosinophilia, hepatic granulomas, interstitial nephritis and vasculitis, bone marrow depression and exfoliative dermatitis. There is no room for allopurinol monotherapy in visceral leishmaniasis. Its place in combination therapy remains undecided.

Other Treatments: The antifungal azoles ketoconazole, fluconazole, itraconazole and posaconazole have been tried in all manifestations of the leishmaniasis, visceral leishmaniasis, cutaneous leishmaniasis, mucocutaneous leishmaniasis and PKDL, without well designed, properly controlled comparative trials and a sufficient number of patients. They have shown variable results probably due to variation in sensitivity of species (40, 71, 304, 306), possibly also due to too low doses given and or too short duration of treatment (203, 299). Study of a role in the secondary prophylaxis in HIV-infection seems warranted (40, 123, 306). Ketoconazole is banned by the FDA and European Medicines Agency (EMA) because of hepatotoxicity.

Bryceson summarized data on other treatments, mostly of unproved value (48). Atovaquone showed some activity in experimental leishmaniasis (209). Its effects in vivo, both alone and in combination with fluconazole were limited but warrant further study in combination with other antileishmanials and in the secondary prophylaxis in Leishmania/HIV co-infection (306). Several plant products are in various stages of research and development: licochalcone A from the Chinese liquorice plant Glycyrrhiza, PX-6581 from the Vietnamese plant Maesa balansae (several quinolines, one from Galipea longiflora from Bolivia (72). No product has reached clinical study yet. Immunochemotherapy is studied in several models and experimental animals and includes induction or enhancement of IFN-γ or IL-12 production, T-cell stimulation to strengthen Th1 response or down regulate suppressive mechanisms, influencing prostaglandin mediated actions and granulomas, and other (210, 211).

Combination therapy

Combination treatments have been introduced because of reduced sensitivity, even resistance to antimonial drugs, to prevent or delay development of resistance, to shorten treatment courses and to reduce treatment costs. In Kenya, in the 1980s, combination of antimony and allopurinol (64) and of antimony and paromomycin (PM) (65) were studied. During the epidemic of visceral leishmaniasis in southern Sudan in the 1980s-90s, Médecins sans Frontières established the efficacy of SSG/PM, given during 17 days (276) and subsequently used this combination from 1991 onwards for patients who relapsed after standard SSG treatment. Because of the heavy patient load SSG/PM was introduced as first line treatment from 2002. A retrospective analysis showed an initial cure rate of 97% (standard treatment 92.4%) and a lower death rate (3%) than for SSG (7.6%). This was not a randomized trial but indications were that the combination had advantages of reduction of costs, hospital stay, and possibly deaths (193). A later analysis showed that the combination treatment was associated with two-fold higher odds of relapse than the 30 day SSG monotherapy. The possibility was raised that those patients who were saved from dying by SSG/PM were those who relapsed (122). In randomized controlled trials in 6 centers and in 4 countries (Sudan, Ethiopia, Kenya and Uganda), efficacy and safety of 20 d PM versus 30 d SSG and of 17d combination of PM + SSG versus 30 d SSG were compared Cure rates were (ITT analysis) PM 84.3%, SSG 94.1%, SSG/PM 93.3% and SSG 91.4%. During SSG treatment there were four deaths and another death occurred during follow up: cause unknown. In the PM only arm there was one death and in the combination arm there was one treatment period death and one unrelated death during follow up. The safety profile of the different treatments was comparable. It was concluded that the 17 day combination treatment was suitable for treatment of visceral leishmaniasis in East Africa (195). For comparison with the field study in southern Sudan performed in war time conditions (283), it may be remarked that this study was performed in hospitals with patients from 4 to 60 years of age, not severely ill and not having contraindications (195). An ongoing study will assess safety and efficacy of miltefosin alone versus liposomal amphotericin B + miltefosine and liposomal amphotericin B + SSG (231).

The need for combination treatment and a proper research agenda has been emphasized (130, 343) In vitro and in vivo studies showed that amphotericin B and paromomycin potentiated activity of miltefosine but no significant interaction was observed of the combination of miltefosine and sodium stibogluconate (279). In India several studies of combination of paromomycin and antimony were performed (229, 239) and more recently of liposomal amphotericin B with miltefosine. Three combinations of liposomal amphotericin B, 5mg/kg single dose plus miltefosine,7 days, liposomal amphotericin B5mg/kg single dose plus paromomycin, 10 days and miltefosine plus paromomycin, both 10 days were all three not inferior to standard treatment with c-AMB. They were effective ( respective cure rates 97.5%, 97.5% and 98.7%), better tolerated and less toxic than c-AMB (318). This study was performed in a research setting and severely ill patients were excluded. Before these regimes can be applied at large in the countryside several considerations arise: conditions at the facilities, presence of means like infusion sets, of transport and storage capacity ≤ 25^oC, needed for liposomal amphotericin B and costs. Where, for these reasons liposomal amphotericin B is not feasible, miltefosine plus paromomycin for 10 days might well be the treatment of choice. But miltefosin can not be used by pregnant women who should agree to long term contraception, until 3 months after end of treatment. If this can not be guaranteed, liposomal amphotericin B plus paromomycin becomes treatment of choice (318). The miltefosine- paromomycin combination was the most cost-effective at US$91 per death averted, followed by liposomal amphotericin B plus paromomycin ($652) (194). Combination treatments are cost-effective, safe and efficacious and should be applied widely in an attempt to prevent development of resistance, or halt further spread of already existing resistance.

From the Mediterranean region and South America no studies on combination treatment are available.

Cutaneous Leishmaniasis

Assessment of treatment of cutaneous leishmaniasis (cutaneous leishmaniasis) is hampered by several factors in published reports: lack of parasite characterization; lack of, or difference in, diagnostic criteria and criteria of cure; lack of comparisons and of controls, especially of placebo treatment in a disease with spontaneous healing tendency. There are relatively few comparative, randomized trials, and these are rarely (double-) blind. Cochrane Reviews on Old World cutaneous leishmaniasis (120) and New World cutaneous leishmaniasis and mucocutaneous leishmaniasis (121) highlight the poverty of information and stress upon the need of properly designed and performed studies. Cutaneous leishmaniasis is not a life-threatening condition and severe complications are rare, yet its consequences may be grave, socially and psychologically because of disfiguring lesions, scars and other sequels. For each individual patient the health care worker has to consider the balance between risks and benefit of the envisaged therapy and this has to be discussed with the patient who then can take an informed decision. For immunocompetent patients with up to five lesions with a size of less than 5 cm in diameter, without potentially disfiguring or disabling lesions in the face, over joints, fingers or toes, local wound care and "watchful wait and see" would well be an option, in particular for patients with suspected, preferably proved, L. major infection and if follow-up seems likely (357). The evolution, the history of the lesion, also contributes to the decision whether to treat or not; if the lesion is already improving it is better to wait for self-cure. A report by the Cutaneous Leishmaniasis French Study Group on 135 travelers with proved cutaneous leishmaniasis due to 10 Leishmania species acquired in 29 countries endorses this approach (204).

Treatment of Cutaneous Leishmaniasis

Treatment options are:

• wound care and watchful " wait and see"
• local treatment
  • physical methods

    • excision, curettage

    • bipolar-high frequency electro-cauterization

    • cryotherapy

    • thermotherapy

    • photodynamic therapy
  • topical treatment

    • paromomycin ointment

    • paromomycin-gentamycin ointment

    • other ointments (imiquimod a.o.)
  • intralesional treatment
    • Sb
  • systemic treatment
    • oral agents

      • antifungal drugs (azoles)

      • miltefosine

      • other
    • parenteral agents

      • antimony

      • amphotericin B

      • paromomycin (aminosidine)

Cutaneous Leishmaniasis of the Old World

Wound care and watchful "wait and see"

Observational studies and placebo arms of comparative studies have shown that most- L. major and L.tropica lesions self cure within 6 to 12 respectively 12 to 24 months (10, 23, 38, 96, 102).

Local Treatment

Physical Methods: Excision and curettage of small lesions (papules, nodules) have been reported but not in comparative studies and data on successes and recurrences are unreliable. They are not recommended because of high relapse rates (96). Results of a randomized controlled trial in the field in Afghanistan with bipolar-high frequency electro-cauterization (HF-EC) with 0.045% pharmaceutical chlorite (DACN-055) moist wound treatment proved promising (300). The HF-EC device is robust, requires practically no maintenance and can be run with a car battery. Further trials are warranted (300).

Cryotherapy has been applied by itself or combined with intralesional antimony (il Sb^v). Assessment of results of cryotherapy is difficult because of variations in techniques, numbers and intervals of applications, differences in follow-up and lack of reporting of parasite species. Reports of efficacy vary from 68% (96) to more than 90 % (25, 26). One session of cryotherapy was sufficient in 90% of 461 cutaneous leishmaniasis patients (L. tropica area) in Turkey (340). Localized cutaneous leishmaniasis due to L. aethiopica responds to cryotherapy (221). Combination of cryotherapy with il Sb^v was consistently more effective (≥ 90%) in comparative studies (24, 25, 97, 272).

Thermotherapy with the FDA approved device Thermo Med® (Thermosurgery Technologies, Inc, Phoenix AZ, USA) proved effective in randomized, controlled trials in Afghanistan (L. tropica; heat versus il Sb^v and systemic Sb^v) (247), in the USA (L. major from Iraq and Kuwait; heat versus Sb^v 20 mg/kg/d, 10 d) (22), and India (L. tropica; heat versus il Sb^v 2 x per week, seven injections and with follow-up till 18 months) (50). The latter study with the long follow-up concluded that the single heat application was safe, caused less adverse effects, less scaring and pigmentation than il Sb^v and was thus cosmetically more acceptable. In another retrospective, observational study from Rajasthan, India (L. tropica), thermotherapy cured 13 of 14 patients (3).

Photodynamic therapy (PDT), used for premalignant and malignant skin lesions, is effective for cutaneous leishmaniasis but is expensive, time consuming and requires special equipment. Daylight-activated PDT (DA-PDT) abolishes the need for special light sources, shortens the time of the procedure and is virtually pain free. The procedure is: the skin around the lesion is protected against sun burn, the crust is removed from the lesion, a thick layer of 16% methyl aminolevulinate cream (Metvix ®, Golderma, La Défense, Cedex, France) is applied, then covered with dressing and a light impermeable aluminum foil for 30 minutes. After removal of the cover the lesion is exposed to day light for 2.5 h. Hereafter the lesion is covered to avoid further day light exposure. These sessions are repeated weekly until clinical and parasitological cure. In a proof of concept study among patients with L. major and L. tropica infections, 24 of 27 evaluable patients were cured (89%). Further studies are needed and warranted. An advantage of this approach is that it can be applied in field circumstances in endemic areas (101).

Topical treatment with paromomycin 15% in methylbenzethonium 12% (PMB cutaneous leishmaniasis) showed variable results, probably related to differences of parasites (not always characterized), of dose, duration and way of application. A meta-analysis of 14 RCTs, 8 from the Old World, 6 from the New World, published up to September 2007 and comparing PMB cutaneous leishmaniasis versus placebo, PMB cutaneous leishmaniasis versus il Sb^v and parenteral paromomycin and parenteral Sb^v, concluded that PMB cutaneous leishmaniasis was better than placebo in old world and new world cutaneous leishmaniasis, that there was no difference between PMBcutaneous leishmaniasis and il Sb^v in Old World cutaneous leishmaniasis but that PMBcutaneous leishmaniasis was inferior to parenteral Sb^v in New World cutaneous leishmaniasis and that there was no difference between parenteral PMBcutaneous leishmaniasis and to parenteral Sb^v. Systemic side effects were fewer with topical or parenteral paromomycin than with parenteral Sb^v (156). A new formulation of paromomycin 15% plus gentamycin 0.5% in a complex base to aid drug penetration, manufactured under GMP conditions (WR 279,396, Teva Pharmaceuticals, Sellersville, PA, USA) was compared with paromomycin without gentamycin dissolved in the same cream in the treatment of L. major cutaneous leishmaniasis in Tunesia in a randomized, vehicle controlled phase 3 trial (38). There was no difference in efficacy of the cream with or without the gentamycin in contrast to results of a smaller phase 2 study in Panama (L. panamensis) (286). Topical treatment with miltefosine (274) and ethanolic lipid amphotericin B (316) deserve formal study. A nitric-oxide generating cream was not effective in Syrian patients with L. tropica lesions (75).

Intralesional Treatment: Both sodium stibogluconate and meglumine antimoniate have been studied for intralesional treatment (il Sb^v) in observational and comparative studies. About 0.5-1ml, up to 5 ml, Sb is injected in the wall of a lesion until "blanching". Various schedules are applied: once a week for 3 up to 8 weeks or twice or thrice a week for 2 to 3 weeks. Cure rates from about 40 to 55 % (25) up to >90% (7, 24, 108, 156, 271, 322) are reported. Uzun et al. report large series of patients treated with il Sb^v: 2329 (340) and 1030 (341) patients with cure rates >90%. They gave weekly injections of 0.2-1 ml up to 20 injections, mean 11 and 8.5 respectively in the 2 reports. Adverse events were pain and transient erythema and edema. Fears that il Sb^v might lead to increase of chronic cases and reduced sensitivity to Sb have been expressed but have not been substantiated (95). Intralesional and intramuscular antimony were equally effective in a study in Saudi Arabia (mainly L. major) (7) but in an area in Iran where L. major and L. tropica are found il Sb^v was less effective than ketoconazole (72 versus 89%, p<0.05) (271). Intralesional injections of triamcinolone, bleomycine, mepacrine, emetine, hypertonic sodium chloride and zinc sulfate 2% have been applied in various studies in the past (96, 160, 282).

Note: there is no IND protocol for intralesional injection of antimonials in the USA.

Systemic Treatment

In general, systemic treatment is nowadays discouraged because of relative toxicity, of lack of evidence of better efficacy than of local therapies and of availability of new modalities of topical treatment as discussed. In patients with extensive or multiple lesions, with mucosal involvement and lesions on the face, in the eye brows and other places where local therapy will be very painful or may be disfiguring, systemic therapy may be required.

Oral Therapy: evidence for efficacy of azole drugs is limited. Fluconazole, effective in L. major infection in Saudi Arabia (10) was less effective in L. major infections from other regions (203). The dose might have been too low (299). Ketoconazole, banned from use in the USA and European Union, was effective in L. major-, not in L. tropica- and L. aethiopica infection and itraconazole seemed promising, both in L. major - and in L. tropica infection (90, 160, 200). Itraconazole was not better than placebo in a randomized double-blind placebo-controlled clinical trial in Iran (L. major area) (216). Data on drug resorption and blood levels are lacking in these studies. Recently a patient with L. infantum cutaneous leishmaniasis was successfully treated with posaconazole, 400 mg b.i.d., 14 days (240). Miltefosine was prescribed to 34 Dutch soldiers with L. major infection acquired in northern Afghanistan after failure of combined cryo- and il Sb^v therapy. Six weeks after treatment with miltefosine (1.3-2.1 mg/kg/d, 28 d) 30 were cured and three received a few Sb^v injections and 1 was treated with iv Sb^v for 28 days because of reactivation of lesions. At 6 months 31 were cured, at 12 months all 34. Treatment was well tolerated (346).

Antibiotics and Chemotherapeutics: Many drugs have been tried. Dapsone 2 x 100 mg/d for 6 weeks, proved effective in a double blind study of 120 patients in India (89). Rifampicin has given equivocal results (cure rates from 0 to 80%), addition of INH did not improve efficacy (159, 160). Metronidazole and co-trimoxazole are probably not of use (160). Azithromycin, a macrolide antibiotic that concentrates in macrophages, showed activity against L. major in vitro and in vivo (BALBc mice) and deserves further study (162). Many antimalarial drugs have some antileishmanial activity in vitro but have not been shown to be of use in vivo, may be with the exception of cycloguanil (48) and atovaquone (209, 306).

Parenteral Therapy: Antimonials are not first line therapy for Old World cutaneous leishmaniasis because of the tendency to spontaneous cure, the costs, side effects and the parenteral administration. A placebo controlled study of L. major infections in Algeria showed no benefit of systemic antimony treatment (37) in contrast to an uncontrolled study in Sudan (89). The comparative studies of intralesional and intramuscular Sb^v treatment referred to above (7, 24, 25) are not in favor of intramuscular administration. If indicated, parenteral antimony is generally advised for 20 days.

Dffuse Cutaneous Leishmaniasis

Treatment studies in this condition are rare and evidence based treatment advice cannot be given. In the past, pentamidine, 3-4 mg/kg intramuscularly once weekly, 4 months longer than needed to eliminate parasites was the recommended treatment for cutaneous leishmaniasis caused by L. aethiopica (48). In a small double blind trial in 14 patients with L. aethiopica infection (4 diffuse, 10 localized leishmaniasis) itraconazole (200 mg/d for 4 weeks) was not more effective than placebo (4). Other treatments with Il-2, liposomal amphotericin B and combination of paromomycin and antimony need further study (5, 323).

Leishmaniasis Recidivans

This condition is rare and comparative trials of its treatment are lacking. Up to 5% of patients with cutaneous leishmaniasis, especially due to L. tropica may develop recurrent leishmaniasis (95, 199). Combination treatment with Sb (20 mg/kg/d x 15 d) and allopurinol (20 mg/kg/d x 30 d) cured 24 of 25 Iranian patients (199).

Cutaneous Leishmaniasis of the New World

It is important to differentiate between parasites of the 3 complexes of L. braziliensis, L. mexicana and L. guyanensis because of the differences in natural history, healing tendency, drug sensitivity and possibility of mucosal involvement. Different species in the respective complexes have different sensitivity patterns to the available drugs (261). In many studies parasites are not characterized to the species level. Localized cutaneous leishmaniasis is characterized by a single or few lesions, a cellular immune response (LST or MST), spontaneous healing tendency and response to treatment. Lcutaneous leishmaniasis may be caused by parasites of all 3 complexes. Diffuse cutaneous leishmaniasis is caused by parasites of the L. mexicana complex and shows numerous non ulcerating lesions, no manifestations of cellular immune response, no spontaneous healing tendency and unsatisfactory response to chemotherapy. Intermediate forms include mucocutaneous disease and patients with extensive lesions and verrucous and vegetative forms. There is exacerbated delayed type hypersensitivity but apparently incomplete protective immunity. It is associated with L. braziliensis infection. Disseminated leishmaniasis is characterized by ≥ 10 mixed-type lesions in at least 2 body parts. There is a strong cellular immune response (LST).

Leishmania mexicana Infections

Parasites of the L. mexicana complex usually cause solitary lesions without lymphatic involvement and without later mucosal spread. L. mexicana infections of the ear may involve the cartilage ("chiclero") and may become chronic.

Treatment of L. mexicana Infection

Wound care and watchful wait and see

It is generally held that simple L. mexicana lesions heal spontaneously in 3 to 6 months and do not need treatment (354) but this is disputed (349). In Guatemala 22 of 25 L. mexicana lesions completely re-epithelialized without treatment by 14 weeks (median) and 17 (68%) were cured when assessed 6 months later while only 7 (22%) of 32 L. braziliensis lesions re-epithelialized by 13 weeks (median) and 2 were definitely cured (142). Other studies have shown spontaneous cure rates from 38% (L. mexicana) (Guatemala) (218) to 75% (mainly L. braziliensis species) (Ecuador) (129). Simple L. mexicana lesions might be left alone to self cure (129, 142, 218, 354) a notion challenged in Mexico (349).

Local Therapy

Thermotherapy: In a placebo-controlled study of local heat and systemic Sb^v treatment with 22 patients in all 3 groups, 16 patients (73%) were cured in both treatment groups and 27% in the placebo group. Fifty-three isolates were characterized: 40 were L. braziliensis and 13 L. mexicana. L. braziliensis results were: cure with Sb^v 11 of 14 (79%), heat 9 of 14 (64%) and placebo 0 of 11. For L. mexicana the numbers were small: 1 of 4cure with Sb^v, 2 of 2 with heat and 5 of 7 with placebo (217). Treatment with ThermoMed®) of 201 patients with cutaneous leishmaniasis in Mexico resulted in 90% cure (191 patients seen 8 weeks after treatment) (349). Thermotherapy was less effective than im antimony in a randomized trial in Colombia but side effects of Sb^v treatment, its higher costs, problems with compliance and the low risk of mucocutaneous leishmaniasis in Colombian cutaneous leishmaniasis at about 0.5% led authors to advice thermotherapy as first line treatment (178). Thermotherapy, applied once (149 adult patients) and miltefosine 3x50mg/d (145 adults) were compared in another Columbian trial. Efficacy of miltefosine (ITT 59%, PP 70%) and thermotherapy (ITT 58%, PP 64%) were comparable. Also for the respective parasite species, L. braziliensis and L. panamensis, there were no differences. Single session Thermomed is not recommended (177).

Topical Therapy: Only few studies of topical treatment of new world cutaneous leishmaniasis with characterized parasite species are available. In the above mentioned meta-analysis (156) PMBcutaneous leishmaniasis was more effective than placebo but inferior to parenteral Sb^v in 6 RCTs about NWCL. The new formulation of paromomycin15% and gentamycin 0.5% in a complex base (WR 279,396) (see under topical therapy of new world cutaneous leishmaniasis) was compared with paromomycin alone in the same base in a study in Panama (L. panamensis) The cure rate for the combination (86.7%) was higher than for paromomycin alone (60%). Adverse events were experienced by all and were limited to mild local skin reactions (286).

Oral Treatment:

Azoles: Ketoconazole (no longer approved by FDA and EMA) was effective in 8 of 9 (89%) L. mexicana infections while Sb cured 4 of 7 (57%) (218).

Miltefosine: In a study of miltefosine treatment in cutaneous leishmaniasis in Colombia and Guatemala of 14 confirmed L. mexicana infections, 9 (60%) were cured (291).

Other Treatment: Antituberculous drugs (rifampicin, isoniazid and para-aminosalicylic acid) have no proved efficacy (160) although animal studies (241) and a case report on concurrent mucosal leishmaniasis and pulmonary tuberculosis treated and cured with antituberculous therapy alone suggest otherwise (104).

Parenteral Treatment Antimony treatment at 20 mg/kg/d for 20 days is effective but may be over-treatment in L. mexicana infections; 10 day treatment cured 90% and has less adverse events (19).

Leishmania braziliensis Infection

Most infections due to parasites of the L. braziliensis complex (L. braziliensis and L. peruviana) lead to a single lesion or a limited number of skin lesions. Mucosal disease may develop in less than 3 to 5% of L. braziliensis infected patients, months to years after cure of the cutaneous lesion (186, 226); concurrent cutaneous and mucosal disease does occur (46). It was generally held that L. braziliensis infections should be treated with systemic drugs because of the risk of later mucosal development and that placebo treatment or no treatment are unnecessary or unethical (48, 160, 190, 267). The picture is complicated because the risk of mucosal disease depends on the species, may be even the strain involved and may be less than 3% (226). Moreover, L. braziliensis lesions, both cutaneous and mucosal, may heal spontaneously (67, 187, 188). Many trials lack controls and long follow up which makes drawing firm conclusions difficult. The greatest risk of developing mucocutaneous leishmaniasis is probably in Bolivian L. braziliensis infection.

Treatment of L. braziliensis Infection

Wound care and watchful wait and see

As mentioned, both cutaneous and mucosal lesions due to L. braziliensis may heal spontaneously but treatment, local or systemic, is advised (48, 160, 267).

Local Therapy

Cryotherapy: In his review of 1987, Bryceson mentioned that cryotherapy was not useful in L. braziliensis infections (48,). Results of a recent placebo-controlled study in Bolivia, with il Sb^v and cryotherapy were: cure with il Sb^v 70% (95%CI, 52%-83%), with cryotherapy 20% (8%-42%) and with placebo 17% (7%-34%) (295). In the French study on treatment of travelers with cutaneous leishmaniasis two of the eight patients with L. braziliensis infection were cured after cryotherapy plus il Sb^v (204).

Thermotherapy: In the above named placebo-controlled study in Guatemala of local heat and systemic Sb^v treatment with 22 patients in each group, 16 patients (73%) were cured in both treatment groups and 27% in the placebo group. Results of cure for proved L. braziliensis infections were 11 of 14 (79%) for Sb, 9 of 14 (64%) for heat and 0 of 11 for placebo (217). In a comparative study of thermotherapy and meglumine antimoniate of L. braziliensis infected patients, thermotherapy led to the same systemic cytokine response as intravenous antimony treatment and untouched lesions would also be cured (176). All heat treated patients were also treated with antimony at day 28 (at that time 75% of lesions had healed or were healing versus 90% in the antimony group, p=0.13) so conclusions on efficacy cannot be made. Additionally it was mentioned that 7 patients who had not responded to long courses of antimony, 2 pregnant women and a patient allergic to Glucantime®, excluded for the comparative trial, all responded well to heat therapy (176). In Peru a low cost heat technology was developed named Hand-held Exothermic Crystallization Thermotherapy for Cutaneous Leishmaniasis (HECT-cutaneous leishmaniasis). It is based on commercially available Heat Pads used as hand warmers for hikers and backpackers and for treatment of injuries in athletes. It is a supersaturated sodium acetate solution and a flexible metal disc in a plastic pouch that by flexing the disc gives rise to an exothermic liquid-to solid phase change reaction that produces a reliable maximum temperature of 52 ± 2^oC. Boiling restores the liquid phase. A pilot study on this device in 25 patients was performed with application on each lesion during 3 minutes daily, seven days. Three of six L. braziliensis infected patients were cured, nine of 16 infected with L. peruviana and all three infected with L. guyanensis (60% definite cure rate). Eleven of 13 additional patients, all with L. peruviana infection, who had contra-indications for systemic treatment or who had failed to previous treatment, were also cured after this Hand-held Exothermic Crystallization Thermotherapy for cutaneous leishmaniasis treatment (84%). One patient had re-ulceration and another patient developed subcutaneous satellite nodules despite complete re-epithelialization of the treated ulcer. The treatment was safe, well tolerated, reversible second-degree burns occurred in two patients and bacterial superinfections did not occur. Further study of this low cost therapy is recommended (342).

Topical treatment: A meta-analysis of 6 RCTs on PMBCL treatment in NWCL concluded that topical paromomycin was inferior to parenteral Sb^v (156). The new formulation of paromommycin plus gentamycin showed promising results in Panama (L. panamensis) (286).

Intralesional Therapy: Several studies reported on il Sb^v treatment in small numbers of patients infected with L. braziliensis (33, 48, 76). Eighty-two lesions in 74 patients living around Rio de Janeiro were treated with il Sb^v repeated after 15 days if there was no improvement. Fifteen of the 35 positive cultures were characterized and found to be L. braziliensis. At 12 weeks, 59 patients (79.2%) had healed, 47after 1 dose, 12 after 2 doses and 12 (16.2%) had failed. Three were lost. Fifty-six were followed for at least 2, up to 10 years, 43 for 5 years or more. No relapses and no mucosal development occurred (227). The authors concluded that this treatment was effective, had no toxicity and could be applied in the field at low cost. In a more recent study in Rio, 20 of 24 patients with contra-indications for systemic therapy were treated with il Sb^v and cured. Three required treatment with amphotericin B and one with systemic Sb. No patient developed mucosal lesions up to 60 months post treatment (77). In Bolivia, 21 of 30 patients treated with il Sb^v were cured (70%, 95% CI 52%-83%) in a randomized controlled trial with cryotherapy (20% cure; 95%CI, 8%-42%) and placebo (17% cure; 95%CI, 7%-34%). The authors concluded that if a decision is made to treat single Bolivian cutaneous leishmaniasis lesions with local therapy, this treatment with 3 intralesional injections at days 1, 3 and 5, is attractive and effective (295). The decision on local treatment of Bolivian cutaneous leishmaniasis remains complex; a point of consideration remains the possibility of follow up, of good information of the patient and his/her possibilities of returning as soon as new manifestations appear.

Azoles: The importance of speciation of the parasite and of a control group was shown in a study in Guatemala: Sb^v cured 24 of 25 patients (96%) infected with L. braziliensis and 4 of 7 (57%) L. mexicana infections; ketoconazole cured 7 of 23 L. braziliensis infections (30%) and 8 of 9 (89%) L. mexicana infections. One of 15 L. braziliensis infections (7%) and 5 of 16 (38%) L. mexicana infections were cured in the placebo group. The conclusion that Sb was effective in L. braziliensis infections but not better than placebo in L. mexicana infections and that ketoconazole deserved further study is reasonable but numbers are small (218). In a study in Colombia comparing Sb^v, pentamidine, itraconazole and no treatment (total 92 patients) itraconazole was not better than no treatment. The parasites in this study were not characterized, the majority would be L. panamensis, the rest L. braziliensis and L. mexicana (298). Treatment of L. mexicana lesions with ketoconazole or itraconazole seems justified but L. braziliensis infections need other treatment. A systematic review with 10 new RCTs on the treatment of American cutaneous leishmaniasis and mucocutaneous leishmaniasis (248), an update on the Cochrane review of 2009 (121) does not mention use of azole drugs.

Miltefosine: In an open phase I/II trial in 72 soldiers in Colombia miltefosine at 50-100 mg/d for 3 weeks cured 21 of 32 patients (66%) and at 133-150 mg/d for 3-4 weeks 30 of 32 patients (94%). The historic cure rate for Sb for the area is 93% (L. braziliensis parasites mainly). Dose related motion sickness was experienced by 40% (291). In further studies with miltefosine 2.5 mg/kg/d for 28 days in Colombia (mainly L. panamensis in the area) the per-protocol cure rate for miltefosine was 91%, for placebo 38%, comparable to historic values for Sb^v and placebo. In Guatemala, in an area where both L. braziliensis and L. mexicana were found, the per-protocol cure rate for miltefosine was 53% and for placebo 21% while the historic cure rate for Sb was > 90%. Forty-six of 60 parasite isolates were characterized: 29 were L. braziliensis (cure rate 33%), 17 L. mexicana (cure rate 60%) (292). Miltefosine did not seem to be useful against L. braziliensis in this study. The already mentioned systematic review with 10 RCTs (248) included 5 studies comparing miltefosine and antimony, four of these with cutaneous leishmaniasis due to various parasite species and one study with L. braziliensis infections only. The analysis of the four studies with various Leishmania species found no significant difference between miltefosine and Sb^v in cure rates. Two studies with mostly L. panamensis and L. guyanensis infections showed higher cure rates for miltefosine at 6 months (248). In the L. braziliensis study (182) miltefosine was more effective than standard Sb^v (miltefosine 75% cure, Sb^v 53.3%; difference 21.7%, 95%CI 0.08-42.7%, p=0.04). There was no difference in cure rates between Sb^v (70%) and miltefosine (68%) in children but in patients > 12 years old miltefosine was superior to Sb^v: respectively 79% and 45%. Explanations include the small number of children, the reduced efficacy of Sb^v in adults and differences in pharmacokinetics and need for higher doses in children as pointed out by Dorlo et al. (92). Incidence of adverse events was similar, but the miltefosine treated patients experienced more gastrointestinal problems of vomiting, nausea and abdominal pain whereas those treated with Sb^v complained of arthralgia, myalgia and fever (182).

Other Treatment: In an uncontrolled study of 24 patients in an area of L. braziliensis, azithromycin used in various dosages and different durations, showed promising results: 17 of 20 patients who completed the study were cured: six patients in 60 days, seven in 90 days and four in 120 days without recurrences in 14 months follow-up (243). Studies on allopurinol are limited and of poor quality but indicate that allopurinol is not effective in this infection (91, 129, 187).

Parenteral Treatment: For L. braziliensis infections systemic treatment with a pentavalent antimonial at 20 mg/kg/d is commonly recommended for cutaneous disease 20 days, for mucosal disease 28 days (48, 160). Treatment failure is more frequent in children; the dosing in children may not be adequate (238). In Central and South America treatment normally is with Glucantime® that comes in ampoules of 5 ml. As mentioned, there will be a tendency to administer full ampoules in order not to waste drug. Studies reporting the actual dose per kg given (mean and range) are extremely rare. Results of Sb^v treatment are variable: 79-96% for adults in Guatemala and in Peru with L. braziliensis infections (75, 217, 218), 51% for L. braziliensis infections in Brazil (261) and only 25 % in children in Colombia, 5-15 year old (238). On the other hand, Arana et al (19) in Guatemala in a comparative study of 10 and 20 days Sb^v and Sb^v plus interferon-gamma for 10 days, concluded that 10 days Sb^v was sufficient treatment for L. braziliensis infection. A low dose of Sb^v Sb, 5 mg/kg/d but with a longer course of 30 days cured 84% of 159 patients (226). Of the 120 healed patients, 98 could be followed for at least one year, 75 for 5 years or more and 15 up to 10 years. In these 98 there was no mucosal development. Side effects were few in this study and treatment costs were relatively low compared with high dose Sb^v for 30 days (226). However, this 30 day treatment duration is long and cumbersome and costly for the patient especially in comparison to a 10 day treatment that may be sufficient also (19). In a campaign in the nineties on diagnosis and treatment of cutaneous and mucocutaneous leishmaniasis in rural areas of Bolivia results were as follows: of 1667 patients with cutaneous infection (L. braziliensis) treated with Sb^v (20mg/kg/d for 20 days), 1553 (93.2%) were cured, 68 (4.1%) had to interrupt treatment because of side effects and 21 (1.3%) failed. They were further treated. Only 11 patients, 0.7%, had further re-activation and 24 patients could not be followed. Patients with mucocutaneous leishmaniasis were treated with amphotericin B, see below (78). Note that this was reported in 1995. In a study from Bolivia reported in 2004, comparing several brands of Sb^v in 45 patients, cure rates were 86-94% in a per protocol analysis and 75-85% in an intention to treat analysis; parasites were not characterized but supposedly were L. braziliensis (293). In Peru, in a comparative study of 80 patients with L. braziliensis infection, of 40 treated with Sb^v (20 mg/kg/d, 20d), 31 (78%) were cured, 6 (15%) failed and 3 were lost while in 40 treated with pentamidine (2mg/kg/d, 7 injections) 14 (35%) were cured, 23 (58%) failed and 3 were lost (17). Clinical and parasite specific risk factors for Sb^v treatment failure were studied in 127 patients with cutaneous leishmaniasis in Peru. One hundred nineteen patients were infected with the three most frequent parasites, 63 with L. peruviana , 29 with L. braziliensis and 27 with L. guyanensis. Treatment was with Sb^v, 20 mg/kg/d, 20 days. In total 29 patients (24.4%; 95%CI, 16.5-32.1%) failed treatment (up to 12 months). Most failures (94.6%) had already occurred at 3 months follow-up. Failure rates at 6 months were 7.4% (95%CI; 2.3-23.5%) for L. guyanensis, 28.6% (95%CI; 18.9-40.7%) for L. peruviana, and 31.0% (95%CI; 17.3-49.4%) for L. braziliensis. Risk factors for failure were age, duration of stay in endemic region, duration of disease, number of lesions and parasite species. Failed patients were younger (16.0 versus 31.2 years) and were more likely to have stayed <72 months in the area. Diagnosis and treatment within 5 weeks of infection increased the risk of failure to 47%. Risk of failure was higher with L. peruviana and L. braziliensis (no difference between those two) and L. guyanensis (177). Most studies lack long observations. Netto et al. (222) reported on 4 year follow up in 79 patients with L. braziliensis infections treated with Sb^v, 62 with cutaneous and 17 with mucosal disease. Relapse was seen in 6 of the 62 cutaneous patients (10%) and in 2 mucosal manifestations developed (3%). Two of the 17 with mucosal disease had later mucosal relapse (17%). A review of 151 patients with L braziliensis infection, treated between 1967 and 1982 revealed that 45 had received continuous Sb^v treatment from 25 up to 116 days and that 94 had intermittent treatments with rest intervals. The antimony dose varied from 3.9 to 28.7 mg/kg/d (47.5% of patients). There were no significant differences in time to healing between adults and children, between intermittent and continuous therapy or high and low antimony dose. Fifty-one patients could be reassessed 5 to 14 years after treatment and no one showed evidence of disease (81). Although an interesting and relatively reassuring report that invites for non-dogmatic thinking and properly conducted clinical trials, extrapolation to the present time with changed epidemiology and sensitivity patterns, remains difficult if not impossible. Indications are that sensitivity for antimony is decreasing yet antimony is everywhere available and used and is the standard of care for cutaneous leishmaniasis in Latin America (175, 182, 261, 266).

Amphotericin B is considered the drug of second choice (40) but formal comparative studies are lacking. Lipid associated amphotericin B cured 2 immunocompromised patients: one HIV-infected patient with lesions due to L.(V.) guyanensis or L.(V.) shawi (not possible to distinguish between the 2) was cured after a total dose of 1500 mg amphotericin B colloidal dispersion. The other patient had developed cutaneous leishmaniasis (parasites not isolated) while on hemodialysis and was cured after a total of 600 mg liposomal amphotericin B (14).These authors refer to 3 other case reports, 2 successful, 1 failure. Paromomycin (aminosidine) gave unsatisfactory results in L. panamensis infection in Colombia (288). In Belize 10 of 17 patients were cured with aminosidine compared to 15 of 17 treated with Sb^v. Amosidine seemed less good in L. braziliensis infection: 3 of 9 patients with proved L. braziliensis infection were cured (7 of 7 with Sb^v) while 3 of 3 with L. mexicana infection were cured, both by aminosidine and Sb^v. The numbers are small though (139). In the systematic review update of RCTs performed between 2008 and 2012 (248) amphotericin B and paromomycin are not mentioned.

Immunotherapy: Immunotherapy is standard treatment in Venezuela for patients with Lcutaneous leishmaniasis due to L. braziliensis. Data for the period 1990-1999 were reviewed (66). Immunotherapy is by intradermal injection of a combined vaccine containing heat-killed L. (L.) amazonensis promastigotes and BCG. One or 2 additional injections were given with 6 to 8 weeks interval if clinical activity persisted. Of 11 532 patients treated, 5341 could be evaluated. Healing varied from 91.2%-98.7% (average 95.7%); adverse reactions were associated with BCG vaccination. Of the 143 failures, 54.5% were with localized ulcers and 45.5% with non-mucosal intermediate cutaneous leishmaniasis, meaning extensive chronic lesions. Less than 2% of all patients were intermediate cutaneous leishmaniasis patients, so immunotherapy should not be used as monotherapy in this group (66). In Brazil another L. amazonensis vaccine was studied in a controlled, double blind trial in comparison with antimony. The vaccine was administered subcutaneously together with Sb^v 8.5 mg/kg/d intramuscularly, both daily for 10 days. The comparison group received a daily s.c. injection of placebo and the same Sb^v injection, both for 10 days. After 10 days patients were evaluated and if not cured the same cycle was repeated up to a maximum of 4 cycles. Of 102 patients entered, 3 were wrong inclusions, 3 were lost. Forty seven of the 47 on immunotherapy were cured and 4 of 49 in the placebo group. Time to cure was 43 days in the treatment group and 102 days in the control group. The vaccine seems to be effective but the whole procedure is quite cumbersome (183).

L. guyanensis and L panamensis Infections

L. guyanensis and L panamensis belong to this complex. Studies on L. panamensis mostly come from Colombia, those on L guyanensis from French Guyana and Brazil. Mucosal leishmaniasis may be caused by both parasites but is rare. L. guyanensis regularly comes with lymphatic involvement.

Treatment of L. panamensis infection

Wound care and watchful wait and see

A spontaneous cure rate of 75% was reported in L. panamensis infection in Ecuador (129).

Local Therapy

Topical treatment with paromomycin/methylbenzethonium chloride ointment (PMBcutaneous leishmaniasis) gave good results in an uncontrolled study of L. panamensis infection in Ecuador: 72% cure at day 50 (161) where a spontaneous cure rate of 75% had been reported (129). In Colombia (mainly L. panamensis infections) PMBCL in combination with 7days parenteral Sb^v gave a 90% cure rate) (289). RCTs and observational studies on il Sb^v, on cryotherapy and the combination of both, are not available (143).

Oral Treatment: Ketoconazole (400 - 600 mg/d, 4 - 8 weeks) cured 16 of 22 (73%) patients with L. panamensis infection while 13 of 19 (68%) treated with Sb (dose 13 mg/kg/d) were cured. In a subsequent placebo study of 11 patients no one was cured after one month (268). As self cure may become apparent much later, this observation period is too short. In a study in Colombia comparing Sb, pentamidine, itraconazole and no treatment (total 92 patients) itraconazole was not better than no treatment, respectively 25 and 36% effect. Parasites were not characterized, the majority would be L. panamensis, the rest L. braziliensis and L. mexicana (298). Dapsone (235) and mefloquine (137) were not effective in L. panamensis infections. In a small, uncontrolled study of 18 patients with L. panamensis infection, 5 of 9 (56%) patients treated with allopurinol riboside together with probenecid and 3 of 9 treated with allopurinol alone were cured without relapse in 1 year; in a separate study Sb cured 59% but 22% relapsed within 1 year (267). Allopurinol plus Sb and Sb alone cured 74% (26 of 35) respectively 36% (12 of 35) of patients with L. panamensis infection in Colombia. There was no cure in 17 who refused to be treated and were followed, and 80% cure in 25 patients who refused antimonial treatment and who received allopurinol alone (190). A comment focused on the low Sb cure rate (139) but cure rates around 30 to 50% are no exception (267, 289). Another study in Colombia (mainly L. panamensis) comparing Sb alone and Sb plus allopurinol, gave similar cure rates: 39% for Sb and 71% for the combination (190). In a randomized controlled trial in Colombia with allopurinol and placebo given double blind, 33% of 55 patients on allopurinol were cured, 37% of 46 on placebo and 93% of 56 treated with Sb (350). Of the 187 patients 84% were infected with L. panamensis, 16 % with L. braziliensis. The authors concluded that allopurinol monotherapy was unlikely to be beneficial in American cutaneous leishmaniasis. Well-designed studies should establish if combination therapy might be useful. These have not been published since.

Parenteral Treatment: Sb^v 20 mg/kg/d for 20 d and pentamidine 2mg/kg/d for 7 injections cured ≥ 90% of patients (298, 350). More recently several brands of Sb^v showed cure rates from 75 to 90% (per protocol analysis) and from 70-87% (intention to treat analysis) (293). In a randomized, non inferiority clinical trial in Colombia among children 2-12 year old miltefosine was proved not to be inferior to Sb^v: ITT analysis failure rate for miltefosine17.2% (98%CI,5.7-28.7&) and for Sb^v 31% (98%CI, 16.9%-45.2%) (difference p=.04). Efficacy, low toxicity profile, oral administration favor use of miltefosine among children with cutaneous leishmaniasis needing systemic treatment (266). Four injections of pentamidine 3mg/kg per dose on alternate days cured 96% of 51 patients (287). Aminosidine alone gave unsatisfactory results in a study of different dosages and duration in Colombia (L. panamensis) (288).

Treatment of L. guyanensis infection

Watchful wait and see approach and local therapy

No RCT and observational studies on these approaches are available (143).

Systemic treatment

Oral treatment

Miltefosine. In a randomized trial in Manaus, northern Brazil, comparing miltefosine and Sb^v, efficacy of miltefosine was 71.4% (95%CI 57.8-82.7%), of Sb^v 53.6% (95% CI, 33.9-72.5%) (59). A systematic review of 4 RTs comparing miltefosine and Sb^v in South American cutaneous leishmaniasis concluded that miltefosine was not different from meglumine ammoniate in the cure rate at 6 months overall (584 patients) but when Leishmania species were considered, the cure rate for miltefosine was significantly better than for antimony for L. guyanensis and L. panamensis infections (248).

Parenteral treatment (antimony and pentamidine): Comparison of pentamidine treatment (4 mg base/kg on day 1 and 3) of 205 French soldiers treated in French Guiana and 32 treated in Marseille revealed cure rates of 95% and 75% respectively. The failure rate in Marseille was considered to be related to delay in treatment (170). Two injections of pentamidine 4 mg base/kg, 48 hours apart cured 87% of 198 consecutive patients and 80% of the failures responded to a second course. Satellite papules (p=0.01;OR 3.5 (95%CI 1.3-11.1) and presence of more than 3 lesions (p=0.01) were associated with failure (214). Another retrospective study of 281 patients treated between 1996 and 2000 with pentamidine 7 mg/kg per dose (4 mg base), 2 injections 48 hours apart and 137 patients treated from 2000 – 2003 with a single injection at the same dose showed comparable results: 78.8% and 83.6% cure respectively for 1 and 2 injections (patients lost to follow up assumed to be cured) and 67.4% and 69.7% respectively, excluding those lost to follow up (152 respectively 89 evaluable) (265). In British Guyana of 110 patients treated with pentamidine 120 mg per injection (up to 2 mg/kg) for 7 injections once daily or on alternate days, 19 relapsed. The relapsed cases were given another course, 10 needed a third course. All were cured; duration of follow up is not mentioned. Parasites were not cultured but supposed to be L. guyanensis (180). Also in Surinam, pentamidine is considered the drug of choice for treatment of L. guyanensis infection (169). Pentamidine at two injections of 7 mg (salt)/kg (4 mg base),48 hours apart, seems appropriate therapy for infection with L. guyanensis in the Guyana's, possibly also for infection with L. panamensis Treatment with 4 mg salt/kg/dose for 4 injections, given on alternate days is also practiced and is likely as effective.

In Brazil results of treatment with S^v were not satisfactory: in 2001 Romero et al. reported for Sb treatment (20 mg/kg/d, 20 days) an ITT cure rate of 50.8% for L. braziliensis (61 patients) and of 26.3% for L. guyanensis (57 patients). Parasite species was the most important factor predicting outcome (261). In a randomized controlled trial of treatment of L. guyanensis infections in the area of Manaus, northern Brazil, performed from January 2009 to February 2010, result of Sb^v treatment at 15 mg/kg/d, 20 d was: cure per ITT analysis, 55.5% (95% CI, 42.4 – 68.1%) , cure P P analysis 60.3% (95%CI, 46.7 – 72.9%) and of pentamidine treatment at 4 mg/kg administered 3 times with interval of 72 hrs, ITT 58.1% (95%CI 44.8 – 70.5%), PP 62.1% (95% CI, 48.4 – 74.5%). The planned comparison with c-AMB failed as most patients refused this treatment because of the long hospitalization of at least 20 days. Analysis was performed on L. guyanensis infected patients only. Pentamidine and Sb^v had similar efficacy in L. guyanensis infection in the area but pentamidine had fewer adverse effects and had the advantage of the shorter period of administration which is one of the problems with antimony treatment. But results of both treatments were not satisfactory (236). The dose of 15 mgSb^v /kg was low and if the pentamidine was given at 4 mg salt per kg (not uncommon but data not given), this was also low. In a randomized clinical trial among 90 patients with L. guyanensis infection in Manaus, 60 were treated with miltefosine and 30 with Sb^v. ITT analysis showed a cure rate of 71.4% (95% CI,57.8-82.7%) for miltefosine and of 53.6% (95%CI, 33.9-72.5%) for Sb^v. In the age group of 2-12 y there was no difference in efficacy between miltefosine and Sb^v (cure rate respectively 63% and 55%) but in the 13-65 y age group efficacy of miltefosine was higher. Speciation of parasites, variations between the same species from different regions,existence of antigenically distinct populations of L. guyanensis, differences of response to treatment in different age groups are emphasized. The cure rate of antimony for Cl due to L. guyanensis from the Brazilian Amazon is low, miltefosine is a better option (59). Pharmacokinetics of miltefosine and also of antimony in children are different but rarely taken into account as emphasized in the above mentioned study among Colombian children (266). Two further studies from Colombia highlight the importance of studying population, geographic and epidemiologic differences of parasite species and strains for monitoring susceptibility to various drugs. The importance of alternative and combination treatments is stressed (110). The other paper, a large scale evaluation of drug susceptibility of clinical strains and species provides the first evidence of emergence of miltefosine resistance during treatment (224).

Diffuse Cutaneous Leishmaniasis

As mentioned, this is a difficult to treat condition. Many therapies have been applied; in early disease they may be helpful but in later, chronic disease they are not. iin the beginning most therapies produce improvement but after suspension of treatment in most cases a relapse follows. Applied treatments have included the standard anti-Leishmania drugs, Sb^v, pentamidine, thermotherapy, IFNγ + pentamidine+ allopurinol, immunotherapy with Leishmania promastigotes and BCG, itraconazole, metronidazole, nifurtimox and antimalarial drugs like mefloquine and artesunate (35, 57, 361). Prolonged treatment with miltefosine, up to 218 days, resulted in impressive improvement but was followed by relapse once treatment was stopped. Only one of 16 patients, the only one to develop a positive LST, remained free of lesionss during follow up of one year. Eleven cultures of L. amazonensis and 2 of L. mexicana were obtained (361). Case reports of patients with extensive, chronic cutaneous leishmaniasis due to L. mexicana infection ( 57, 232), one with extensive involvement of oropharynx, epiglottis and glottis, mention impressive improvements after miltefosine treatment but relapses after suspension of treatment. Treatment with miltefosine was better tolerated than treatment with Sb or pentamidine and initial results were impressive but the end result was deeply frustrating for the patients and the attending health care workers.

Mucocutaneous (Mucosal) Leishmaniasis of the New World

Spontaneous cure is rare in this condition but has been reported (188). In a review, Marsden in 1986 reported that with Sb^v , 20 mg/kg/d, 30 days the relapse rate should be low and that c-AMB was the alternative. He mentions a few cases of pentamidine treatment (186). Pentavalent antimony is still first line treatment, preferably plus pentoxyfilline (181) followed by amphotericin B, preferably in its liposomal form (15, 16). Pentamidine has a limited role. Miltefosine may be a good alternative but comparative trials are not available and eagerly awaited (118, 248, 295).

Antimony: Differences in cure and relapse rates depend on extensiveness and duration of disease and of treatment given and follow up. Geographical differences, differences in parasite species and strains are involved. In Peru, six of eight patients with mild (nose only) to moderate (nose with septal perforation) disease (L. braziliensis) were cured by standard Sb^v treatment but only 2 of 21 with severe disease (involvement of nose and oral cavity) (115). Extending this treatment from 20 to 40 days was of no benefit (116) but in the latter study the cure rate with Sb^v for 28 days for severe disease was 63%. In a study in Panama of 16 patients with mild mucosal L. panamensis infection (nose involvement only, 1 with septal perforation) 28 days Sb^v treatment resulted in cure without relapse during 12 months in 10 of 13 who completed treatment, 3 relapsed. The other 3 who discontinued treatment because of toxicity did not heal. The overall cure rate was thus 63%, but in those who completed treatment 77% (269). Llanos-Cuentas et al., using different definitions of moderate disease (more than 2 mucous membranes involved with "mild or no respiratory distress") and severe disease (moderate disease plus severe respiratory distress) compared Sb^v alone versus Sb^v plus allopurinol in 22 patients with severe and 59 patients with moderate disease due to L. braziliensis. The failure rate was high in the group with severe disease, 80% versus 100% for Sb^v alone respectively Sb^v plus allopurinol, and in the moderate disease group the cure rate was 75% for the Sb^v alone group, 63% for Sb^v plus allopurinol, p=0.389. Addition of allopurinol was of no clinical benefit (173).

Amphotericin B, liposomal amphotericin B: Amato et al. (15) present a retrospective cohort study of 140 Brazilian patients. Sixty four patients with no contraindications for antimony treatment (cardiac, renal, hepatic, previous experience) were treated with Sb^vs. Remaining patients without contraindications for pentamidine were treated with this drug (22 patients) and those with contraindications to both these drugs were treated with c-AMB (17 patients). In the presence of contraindications for amphotericin B itraconazole was prescribed (15 patients) and for those who had contraindications for this drug, lipid formulations of amphotericin B were considered ( 9 colloidal dispersion and 4 liposomal amphotericin B). Cure was defined as total healing (otorhinolaryngeal and fiberoptic examination) until one year after treatment, failure was absence of improvement or return of lesions before 1 year and recurrence was defined as new lesions or return of lesions after one year of therapy. Results were: Sb^v cure 91%, recurrence 22%; pentamidine 91% cure, 25% recurrence; itraconazole 73% cure and 18% recurrence; c-AMB cure 63%, no recurrence, lipid amphotericin B, both formulations, cure 100%, 1 patient with recurrence after liposomal amphotericin B. All patients were followed for at least 1.5 year. Systemic arterial hypertension and HIV infection (7 patients in this cohort) were associated with failure of treatment (15). In a large observational study of 211 patients with mucosal disease in Bolivia, 186 patients were cured (88%) after treatment with a total of 2.25 g c-AMB, 22 had to interrupt treatment (10.4%), but failures and further reactivations were very few (78). Results of treatment with liposomal amphotericin B in small series of patients have been analyzed retrospectively. Fourteen of 16 HIV-negative patients (88%) were cured, 2 failed, after liposomal amphotericin B, at a dose of mean 2.5 mg/kg/d, total dose 30-35 mg/kg with a mean duration of treatment of 13.7 d, median 12 d (range 4-40). Five patients experienced renal failure, two restarted treatment and were cured, one was cured and two were considered failures. The authors advise on a total dose of 30 – 35 mg.kg (259), less than the dose advised by WHO of 40 – 60 mg/kg (357) and in the range of the advice of Murray (25- 35mg/kg) (211). Amato et al. retrospectively reviewed 8 patients, one HIV-positive, who were treated with liposomal amphotericin B, 3-5mg/kg/d to a total dose of 35 mg/kg (mean, range 24 – 40) (14). All were cured without recurrence during follow-up to a mean of 25 months, range 7 - 40. Six patients experienced side effects of chills, myalgia, back pain, nausea and headache and in two reduction of glomerular filtration rate and hypokalemia were observed, both reversible. The ideal dose has not yet been established, the authors mention, they suggest a total dose of 25 – 35 mg/kg, and daily doses of 3 – 5 mg/kg/d. The costs would be US$ 8,000 to 12,000 while the cost of Sb^v treatment is US$ 176.55 (16). A Leishmaniasis/ HIV co-infected patient with dysphagia, a penile ulcer and extensive lesions of palatum, larynx and epiglottis who had been diagnosed with mucocutaneous leishmaniasis 12 years earlier and who had received cycles of Sb without cure was treated with pentamidine. The penile lesion cured, the mucosal lesions persisted and he developed diabetes mellitus. After a total dose of 12,800 mg liposomal amphotericin B, all lesions were cured (14).

Paromomycin: In Peru a randomized open trial with paromomycin (14mg/kg/d, 21 days) and Sb^v (20mg/kg/d, 28 days) was stopped after enrollment of 38 patients as there was no response in the paromomycin group. Cure rates were 0/21 in the paromomycin group and 8/17 (47%, 95%CI 23-71%) in the Sb^v group. Paromomycin alone should not be used for treatment of new world mucocutaneous leishmaniasis or for cutaneous leishmaniasis in areas where the parasites are still susceptible to Sb^v or where L. braziliensis is the main parasite (174).

Miltefosine: Data are limited. In Bolivia, of 36 patients with mild mucosal leishmaniasis (L. braziliensis), treated with miltefosine, 2.5 mg/kg/d, 28 days and followed 12 months 83% were cured while only 58% of 36 patients with more extensive disease were cured (overall cure rate 71%). These results are comparable to those of antimony treatment. Prior to availability of miltefosine, c-AMB, 45 mg/kg over 90 days cured 7 of 14 patients. As miltefosine is an oral agent, is better tolerated than antimony and amphotericin B, it could become the treatment of choice (294). Wondering if the overall 71% cure rate would be diminished by extending the follow-up period or would be higher by increasing the dose of miltefosine, or the duration of treatment, a further study was performed. Increasing the dose was not possible because of the small therapeutic index of miltefosine: at higher doses gastrointestinal adverse events become unbearable. Prolonging the treatment period to 6 weeks and the follow-up to two years did not make essential differences in cure rate and number of relapses (296).

Other Treatment: Sb^v plus γ-IFN. Combined treatment of Sb^v with γ-IFN in 13 patients (4 cutaneous, 9 mucosal disease) who had failed previous Sb^v treatment and who were first treated by the authors with Sb^v 20mg/kg/d for 29 days, resulted in complete healing in 11 (amongst these all four with cutaneous leishmaniasis). Ten were followed for 6 months. One patient stopped prematurely because of side effects but seemed cured 4 months later. One patient with severe disease failed.treatment. Thus, overall efficacy 91% (106). A patient in Colombia with extensive mucosal leishmaniasis (parasite proved) and pulmonary tuberculosis was treated with streptomycine, rifampicine, isoniazide and pyrazinamide for 2 months, followed by rifampicine and INH for 7 months, without antileishmanial treatment and was cured of both diseases (104).

Summary: there are no comparative trials of the newer treatments of liposomal amphotericin B and miltefosine versus the older ones of antimony, pentamidine and c-AMB,in any combination. All considerations that have been mentioned already of efficacy, toxicity, comfort for the patient, accessibility, need of hospitalization or possibility of out-patient administration, compliance, costs, availability, are to be considered. In practice antimony is most widely available and has a reasonable efficacy of around 70-80% but comes with considerable adverse events. Pentoxifylline improves results but may not be available everywhere. Results of treatment with pentamidine are about the same but pentamidine has many side effects, some very serious. It is not regularly used for this indication. Efficacy of miltefosine is about 70 % in Andean mucocutaneous leishmaniasis (Bolivia, Peru), comparable to antimony treatment. Advantages of miltefosine are the oral administration and the lower toxicity. Disadvantages have been mentioned several times. Liposomal amphotericin B is a good alternative but data are rather limited and it is very expensive. A recent review ends with "Mucosal disease is the more neglected form of cutaneous leishmaniasis and a multicenter trial should be urgently considered" (248).

Special Situations

Post-Kala Azar Dermal Leishmaniasis

Post-kala azar dermal leishmaniasis (PKDL) is a sequel after apparent successful treatment of visceral leishmaniasis and is the result of immune (re-)constitution (365). PKDL is mainly related to L. donovani infection so occurs in South Asia (India, Nepal, Bangladesh) and in East Africa (in particular in Sudan (98, 362), also in Ethiopia and rarely in Kenya (180). Post-kala azar dermal leishmaniasis does also occur after L. infantum infection. Manifestations of post-kala azar dermal leishmaniasis are macular, papular and nodular lesions of the skin. There are no constitutional symptoms of fever and other symptoms of visceral leishmaniasis, it is not life threatening but is stigmatizing. Manifestations may be extensive, may include lips and bordering mucosa and, eyelids. Uveitis may occur. Sand flies may become infected by bites from the parasite rich lesions and PKDL plays a role in transmission of leishmaniasis. There are differences in the manifestations between the two main geographic areas of occurrence. In South Asia post-kala azar dermal leishmaniasis occurs generally in young adults, from about 2 up to 10 years after cure of visceral leishmaniasis. Manifestations are dominated by macules/patches together with papules and nodules. Spontaneous clearing of the lesions does occur but takes long in the Indian continent; 48 of 98 (49%) untreated patients were cleared with a median time of 19 months (144). In Sudan, post-kala azar dermal leishmaniasis (PKDL) occurs within a few months after treatment, also during treatment (para-kala azar), it is more frequent in children and papular and nodular lesions dominate. The time to clearing is shorter (98, 362). The etiology and pathophysiology of post-kala azar dermal leishmaniasis are not elucidated. A nice and clear description and discussion of post-kala azar dermal leishmaniasis, hypotheses of etiology, immunology, host, parasite, drugs interactions can be found in a review by Mukhopadhyay et al. (207). An extensive and thorough review of PKDL and other dermal lesions in Leishmania/HIV co-infected patients from the Old and the New World was compiled by Zijlstra (365). The review presents the literature and a lot of clinical and immunological data on patients with visceral leishmaniasis, cutaneous leishmaniasis and mucocutaneous leishmaniasis and HIV-infection with skin manifestations that often become severe, overlap, disseminate or visceralize. The author proposes a new terminology based on infecting parasite species and region in relation to visceral leishmaniasis which basically is: disseminated cutaneous leishmaniasis, -without, - preceding, -concomitant with or -after visceral leishmaniasis, and for each with various Leishmania species. For details see the paper (365). An instructive Atlas with clinical images was recently published (364).

In the Indian subcontinent the policy is to treat patients with post-kala azar dermal leishmaniasis (PKDL) because of the long time to spontaneous resolution and the role in transmission, and this in view of the control and elimination program that is on-going. In a large epidemiological, clinical and immunological study in Bangladesh between 2002 and 2010, 1,002 visceral leishmaniasis and 185 PKDL patients occurred. The median age of the post-kala azar dermal leishmaniasis patients was 12 years and 9% had no history of treatment of visceral leishmaniasis. The cumulative incidence of PKDL among visceral leishmaniasis patients was 17% by 5 years. visceral leishmaniasis patients younger than 15 years were more likely to develop PKDL than older patients. visceral leishmaniasis and PKDL were treated with SSG, respectively 28 and 120 days according to National Guidelines at the time. Fifty percent of the treated post-kala azar dermal leishmaniasis patients were cured by the end of treatment and 1 year after initiation of treatment, 91 % were cured. Spontaneous resolution was more frequent among those younger than 15 years (144). From 2010 Médecins sans Frontières took over care for visceral leishmaniasis and post-kala azar dermal leishmaniasis patients in this area and, as the optimal treatment for PKDL is not established by clinical trials, they followed the advice of WHO on the safest and most effective treatment , which is liposomal amphotericin B (357). Post-kala azar dermal leishmaniasis (PKDL) patients were treated with a total cumulative dose of 30 mg liposomal amphotericin B per kg, divided into six doses of 5mg/kg over a period of 3 weeks, on an outpatient basis. From October to December 2011, 6 confirmed patients with hypokalaemia-induced rhabdomyolysis were encountered, together with another presumed case (185). A retrospective search in the records of the 1292 PKDL patients treated with liposomal amphotericin B revealed 19 patients with symptoms consistent with rhabdomyolysis. Rhabdomyolysis due to liposomal amphotericin B had been reported very rarely before. Drug quality was excluded as possible cause as were temperature during transport and storage. Six of the seven cases were female, 11 to 18 years old, and 18 of the 19 patients found in retrospect were female, 15 were 11-26 years old. No patient <11 years developed rhabdomyolysis. Electrolytes and creatinine were not routinely measured. Manifestations of this toxicity appeared at the fifth or sixth dose, at 25 – 30 mg/kg total dose. Authors recall experience with 20 clinical trials with 2293 treated patients who received total doses from 3.75 to 76 mg/kg, daily dose from 1 to 15 mg/kg without report of rhabdomyolysis and severe hypokalaemia. Micronutrient deficiencies among adolescent girls are a known problem in Bangladesh. Authors consider the possibility of magnesium deficiency, aggravated by liposomal amphotericin B induced Mg wasting as a risk factor. As PKDL treatment is mainly provided for public health reasons (transmission reduction), a lower dose of liposomal amphotericin B and monitoring of potassium levels are recommended (185). Antimony and amphotericin B need long courses (246, 334). Pentamidine was not effective, ketoconazole (800 mg/d for 9 months) was effective but toxic and is no longer advised; rifampicin and allopurinol have been used in an uncontrolled way (246). Miltefosine has been used to limited extend, long courses are needed and its place still has to be defined (246, 313).

In East Africa, post-kala azar dermal leishmaniasis often clears by itself or is more amenable to treatment (205, 362) although long Sb treatment courses may be needed (98). Combination of terbinafine and itraconazole was not satisfactory in a small study in Sudan (153).

Immunocompromised Patients Including Leishmania/HIV Co-infection

Visceral leishmaniasisis

Pentavalent antimony is still widely available and used but is not effective in India (Bihar) and Nepal. In immunocompromised patients it is less effective, comes with serious toxicity leading to discontinuation of treatment and early death (281). Pancreatitis and cardiotoxicity are among the serious adverse events (21, 225, 356). C-AMB is widely used, its efficacy in HIV co-infected patients is comparable to that of Sb^v whereas in non HIV infected patients it is more effective than Sb^v. Major problem with c-AMB is nephrotoxicity. This and other side effects are more severe in co-infected patients (12). Liposomal amphotericin B (LAMB) is the preferred drug because of efficacy, reduced toxicity, shortness of treatment course (44) but even at the reduced, subsidized nonprofit price of $20 (US currency) per vial, it is too expensive for many endemic regions. Dose and duration are still debated; they may not be the same in the various regions with different parasite species. There are arguments to give a daily dose of at least 5 mg/kg/d and, in immunocompromised patients a total dose of at least 25-35, preferably up to 40 mg/kg (326). Experience with miltefosine in co-infected patients is limited (255, 284); there is no published experience with paromomycin to date. Pentamidine is a candidate for secondary prophylaxis (12). HIV-1 protease inhibitors inhibit Leishmania parasites in in-vitro studies. This is an extra argument to start anti-retroviral treatment (ART) in co-infected patients, apart from the arguments of treating HIV-infection, reducing viral load, improving CD4 numbers and reducing relapse rates (344). Despite antileishmanial treatment and ART, relapses do occur and secondary prophylaxis is strongly advised (198). Evidence based advice on secondary prophylaxis is lacking. Sb^v, 20 mg/kg/dose or liposomal amphotericin B 2-5 mg/kg/dose, once every two weeks for the first few months, thereafter once per month are good options. Pentamidine at 4 mg base per kg per dose, 300 mg for an adult, is another possibility. At this dose with single injections, severe adverse events are unlikely (12, 357).

A systematic review with indirect comparisons with literature up to October 2012 including 17 studies, 12 from southern European countries, 4 from Ethiopia, 1 from India, concluded that liposomal amphotericin B was superior to Sb^v and that clinical improvement and cure rates were higher with both formulations of amphotericin B than with Sb^v. The adverse events rates of standard dose Sb^v (23.3%, 95% CI 17.4-30.4%) and liposomal amphotericin B (9.5%, 95% CI 3.5-25.3%), and the early discontinuation rate, respectively 10.8% (95% CI 3.8-27.4%) and 4.2% (95% CI 1.1-14.9%) overlapped but seem relevant. The mortality rate was about 3 times higher with Sb^v (18.4%; 95%CI 13.3-25%), indirectly compared to liposomal amphotericin B (6.1%; 95%CI 3.9-9,4%). Relapse rates in 10 studies without secondary prophylaxis were 26-50% with follow-up of 5 to 14 months. Authors concluded that amphotericin B, in particular liposomal amphotericin B is first choice treatment for Leishmania-HIV co-infected patients (69). Predictors for relapse were a. absence of increase of CD4 cells; b. lack of secondary prophylaxis; c. previous history of relapse. CD4 counts below 100 cells/mm³ were indicative of later relapse. In five observational studies with patients on ART, there was no reduction of relapses but three studies with secondary prophylaxis, 2 with Sb^v, 1 with liposomal amphotericin B, reported reduction of relapses and one study with pentamidine reported a trend (p=0.08). A threshold for discontinuing secondary prophylaxis has not been established, 200 or 350 cells/mm³ for at least 3 to 6 months are mentioned (68).

In India HIV prevalence is still relatively low, about 0.3%, but in Bihar, with many migratory laborers and a high burden of leishmaniasis, prevalence is rising (285). Of 55 co-infected patients, all ART naïve, treated between 2007 and 2010 with liposomal amphotericin B, three died during treatment and the others responded to treatment. liposomal amphotericin B was with total dose of 20 mg/kg, 4 doses on d 1,2 5 and 10 each. ART with stavudine, lamivudine, nevirapine, generic fixed dose combination tablets was started after visceral leishmaniasis treatment. CD4 counts were measured at 6 months interval. Eight patients relapsed at a median of 1.3 years; no relapse occurred within the first 6 months. With CD 4 counts > 250 at 6 months, the risk of relapse was very low (exact number not given). Four of the eight relapsed patients, all four with CD 4 counts < 100 cells/mm³ at 6 months relapsed again after treatment with higher dose (25 mg/kg total) liposomal amphotericin B. Probability of relapse was 8.1% at 12 and 26.5% at 24 months. The 24 months mortality rate was 14.5% (285). Due to ambiguity in guidelines, definitions and guidelines on HIV/AIDS respectively leishmaniasis (56), HIV testing is not required and thus not regularly performed in patients with visceral leishmaniasis in India. In the Médecins sans Frontières supported treatment set up in Bihar (54) from 2011 HIV counseling and testing were offered to all visceral leishmaniasis patients ≥ 14 years. Up until 2013, 2077 patients were tested, of whom 117 (5.6%) were HIV positive. Of these, 49 (2.4%) were newly diagnosed and 68 were known cases. In the male age group of 35-40 years, the prevalence was 5.4%; together with the known cases, in the age group of 35-40 years of age, 12.8% of the men and 6.1% of the women were co-infected. Burza et al. call for systematic screening for HIV in all visceral leishmaniasis patients in India and for visceral leishmaniasis screening in non-endemic areas of HIV patients who spent time in endemic areas, in particular migrant laborers (56). This was endorsed in an Editorial by van Griensven (345) who referred to experience in Brazil where visceral leishmaniasis-HIV co-infection was recognized as a problem early and routinely testing was implemented. Visceral leishmaniasis is reported from 12 Latin American countries; 96% of the cases are in Brazil. From 2001 to 2011, 38,808 patients were reported; underreporting is estimated at 11 – 40%. Leishmaniasis spreads from rural areas to towns and HIV is spreading from urban to rural areas. In 2001, 0.7% of the visceral leishmaniasis patients was HIV positive and in 2012, 8.5% (171). Treatment has been variable, no RCT form these countries is available. The 17 studies (1990-2012), 15 from Brazil, one each from Venezuela and Mexico included in a review on visceral leishmaniasis-HIV co-infection in Latin America consist of 8 case reports, 8 case series and one review paper. Authors acknowledge that there is only a weak basis for advice on treatment and prophylaxis and call for collaboration across centers and countries. They advise that ART, that should include a protease inhibitor, be introduced as soon as anti-leishmania treatment is tolerated, generally in the 2^nd week of treatment (171). Since September 2013 liposomal amphotericin B is advised both for treatment and secondary prophylaxis (70). Before this date visceral leishmaniasis treatment was with c-AMB, as antimony was avoided for visceral leishmaniasis-HIV co-infected patients. Liposomal amphotericin B was reserved for patients older than 50 years, renal transplant patients and those with renal dysfunction. After treatment, all patients with CD4 counts below 350 cells/mm³ received secondary prophylaxis every two weeks with c-AMB or those with creatinine clearance of 50ml/min with liposomal amphotericin B. Secondary prophylaxis was always with liposomal amphotericin B after September 2013. Prophylaxis was continued at least 6 months and was discontinued when two consecutive CD4 counts were more than 350cells/mm³. Authors describe results of treatment of 46 co-infected patients ,in comparison with 44 HIV negative patients treated concurrently, before the liposomal amphotericin B policy change (2011-2013). Six patients, 2 HIV-, 4 HIV+, died within 30 days of start of treatment (no significant difference). One of the HIV – patients relapsed within 6 months, 14 of the HIV+ patients, despite secondary prophylaxis (37% of the 38 evaluable patients). Clinical cure was achieved in 91% of the HIV- patients and 40% of the HIV+ group. Only 16 patients took ART regularly. HIV infection, bleeding and previous visceral leishmaniasis episodes were independent predictors of unfavorable outcome (69). A review on leishmaniasis in organ transplant recipients (79 cases) concluded that liposomal amphotericin B is the first line therapy in transplant recipients (18).

Cutaneous leishmaniasis and mucocutaneous leishmaniasis

Clinical aspects have been discussed above. Evidence based advice on treatment based on RCTs, large comparative or observational trials is not available. Lesions of patients under immunosuppressive treatment will generally respond to treatment, though this may have to be extended. It may be considered to stop the immunosuppressive therapy if possible. HIV infected patients with cutaneous leishmaniasis and mucocutaneous leishmaniasis should be assessed for visceral leishmaniasis. Treatment is as for HIV – patients, may be prolonged. Recurrences are more frequent.

Pregnancy and Breastfeeding

Amphotericin B and the lipid formulations can safely be used during pregnancy (157). Detailed descriptions were published on treatment of 5 pregnant women with c-AMB (335) and 5 with liposomal amphotericin B (237). Treatment was effective without harm to the fetus and mother. Two studies from eastern Sudan report results of case series, respectively 39 and 42 patients (2, 207). Médecins sans Frontières workers retrospectively analyzed data about 23 pregnant women treated with Sb^v, 20 mg/kg/d, 30 d., four women with severe disease treated with liposomal amphotericin B, 3-7 mg/kg/d, 4 doses, followed by a standard course of Sb^v and 12 women treated with liposomal amphotericin B, 3-7 mg/kg/d, 6 doses which was new policy from October 2014. Of 11 women treated with Sb^v during the first trimester, 8 aborted, none of the 4 treated with the combination and none of the 2 with liposomal amphotericin B. Of the eight women treated with Sb^v in the second trimester, 5 aborted. No abortion occurred in the women treated with the combination (2 women) or liposomal amphotericin B (6 women). Thus of 19 women treated with Sb^v in the first and second trimester, 13 aborted and there were no abortions in 14 women in the two other treatment groups. One patient, treated with liposomal amphotericin B delivered a premature baby who died one week later. The mother reportedly died 2 weeks after discharge in another hospital of unknown cause. One woman in the Sb^v group had a spontaneous abortion before treatment and one mother in this group delivered a healthy baby while in hospital. One woman in the Sb^v group defaulted, the others were discharged cured. There was no follow-up, so nothing is known of long-term effects (207). A prospective cohort study also from Sudan, from June 2006 through January 2009 reports on 42 pregnant women treated with Sb^v, 9 in the first (2 abortions), 21 in the second and 12 in the third trimester (both no abortions). Four women died of hepatic encephalopathy between the 22^nd and 30^th week of pregnancy. A 47 year old woman gave birth to a child with Down syndrome and another to a baby with a myelomeningocele in the 32^nd week. This baby died after two hours; the mother had received Sb^v in the first trimester. One baby was diagnosed with visceral leishmaniasis two months after birth and died in the first week of treatment (2). Antimony must be avoided in pregnancy, in particular in the first and second trimester but if no other drug is available it has to be used as visceral leishmaniasis during pregnancy has to be treated. Miltefosine (teratogenic), paromomycin (aminoglycoside) and azole drugs (157) are not to be used during pregnancy and lactation.

In summary, amphotericin B, preferably liposomal amphotericin B, is the treatment for visceral leishmaniasis during pregnancy.

For cutaneous leishmaniasis and mucocutaneous leishmaniasis during pregnancy there is no evidence based advise on treatment. For cutaneous leishmaniasis preference is for topical treatment with wound care. Cryo- or thermotherapy seem good options as safety of il Sb^v and paromomycin ointment are not known. Liposomal amphotericin B is the best option if systemic treatment is needed.

Monotherapy or Combination Therapy

Combination therapy is advocated for several reasons: a. to increase efficacy; b. to reduce length of treatment courses; c. to reduce costs; d. to prevent development of resistance or, where already present, to prevent further spread. Monotherapy is still the normal practice in antileishmanial therapy. Resistance to antimony can be created in the laboratory under drug pressure (127) and is created in dogs after several courses of treatment (126). The Sb resistance now prevailing in Bihar (172, 307) is likely to be due to the widespread misuse of Sb drugs with low dosing, interrupted treatment courses, short duration of treatment and possibly also the quality of drugs. Fears about development of resistance to miltefosine have been expressed (311). In order to prevent further deterioration of the situation, and to protect newly introduced drugs from development of resistance, combination therapies like in tuberculosis, leprosy AIDS and malaria are advocated. The armamentarium against Leishmania parasites is limited but oral miltefosine and parenteral paromomycin are now available and these drugs should be studied for combination with each other and with the other available drugs, appropriate for the various regions (279). For the spreading epidemic of Leishmania/HIV co-infection in Ethiopia and the impending, or already ongoing epidemic in India, plans, funds and action are urgently needed (56, 345). In East Africa several studies are ongoing (231). Investments in drugs and studies will have to be made, in order to gain later. But where will the money come from?

ADJUNCTIVE THERAPY

Visceral Leishmaniasis

Old remedies of cauterization, still practiced in traditional medicine, blister provoking herbal medicines, injections of turpentine (215) which sometimes might have been beneficial, may find a scientific justification in provoking an "acute phase response", a cytokine response and or a leukocytosis. Strategies for immune intervention in visceral leishmaniasis based on animal models were reviewed (152, 210, 211). Interferon gamma IFN-γ, a potent activator of macrophages has been used alone and in addition to antimonials in previously not treated patients and in patients with relapsing or drug-unresponsive visceral disease (29, 30, 302). Its role in Leishmania/HIV co-infection is not clear. IFN-γ has side effects (flu-like illness), and is expensive; it may have a limited role in combination with other antileishmanial drugs in difficult to treat cases. Human granulocyte-macrophage colony stimulating factor (GMCSF) was beneficial in experimental leishmaniasis (208). Recombinant human GMCSF was well tolerated, reversed neutropenia, eosinopenia and monocytopenia and reduced the number of secondary infections. Costs of treatment combined with antimony were slightly more than with antimony alone ($850 versus $744), assuming reduction in hospital costs (10 versus 20 days of hospitalization), and reduction of complications and treatment thereof (31). IL-12 restored IFN-γ production and lymphocyte proliferation in in vitro cultures of peripheral blood mononuclear cells of patients with visceral leishmaniasis (27). Study of its use as an immunotherapeutic adjuvant is of interest but these additional interventions offer no hope for the people in the endemic areas where the real problem is.

Splenectomy may cure visceral leishmaniasis in exceptional cases. In recent years splenectomy was performed in several HIV co-infected patients after multiple relapses (9). Further drug treatment is required but may then be effective (8, 48). The operation carries morbidity and mortality, and splenectomized patients run risks of septicaemias and malaria (48). In January 2015, PubMed search using the terms splenectomy and kala-azar found 13 references from 1945 to 1984 of splenectomy and leishmaniasis. Five other references were cited from 1947 to 2012 and nine more within the last year (9).

Cutaneous Leishmaniasis of the Old World

In a review, Koff and Rosen (160) report that intralesional IFN-γ was of no benefit but that IL-12 was useful in the treatment of some patients with disseminated disease. Perilesional injections of Sb were effective and by far superior to IFN-γ injections in L. tropica lesions (136). In a preliminary study in Iran Leishmania-antigen preparations showed promise (201), but apparently this was not followed further. A child with L. infantum cutaneous leishmaniasis was treated with topical paromomycin, oral fluconazole, monthly il Sb^v without improvement. After referral and species identification, he was treated with two courses of liposomal amphotericin B respectively of 39 mg/kg and 18 mg/kg total dose, the second course together with azithromycin. Because of persistence of the lesion cryotherapy followed by photodynamic therapy was applied but this was poorly tolerated. Then topical imiquimod, 3 times per week was applied for 8 weeks and re-epithalization was observed 1 month after end of treatment. There was no recurrence up to 12 months (140). What tipped the balance? The time scale?

Cutaneous Leishmaniasis of the New World

As mentioned, immunotherapy with killed L. amazonensis promastigotes with BCG, with or without Sb^v became standard therapy for cutaneous leishmaniasis and Dcutaneous leishmaniasis in Venezuela (66). Intralesional injection of IFN-γ gave reduction of lesion size in 12 of 13 L. guyanensis lesions (only 4 were free of parasites) (135). A patient with Dcutaneous leishmaniasis due to L. amazonensis infection was cured after prolonged combined treatment with Sb^v and IFN-γ, interspersed with amphotericin B. Results of 5 more patients remain unknown (30). IFN-γ contributed to the side effects but not to the results of treatment in patients with ( mainly) L. braziliensis lesions, treated with Sb^v alone and Sb^v with IFN-γ (19). Combination of Sb^v with GMCSF led to earlier clearance of lesions (reportedly L. braziliensis in the area) than Sb^v alone (8). Application of S-nitroso-N-acetylpenicillamine, a NO generating compound, during 10 days cured 11 patients with L. b. braziliensis lesions in Ecuador without recurrence up to 8 months (179). Imiquimod cream, an FDA approved immune response-activating compound, proved useful in combination with Sb^v (90% cure at 6 months) in 12 patients with cutaneous leishmaniasis (L. peruviana) who had not responded to Sb^v. Nine of 10 showed no relapse at 6 months (20).

ENDPOINTS FOR MONITORING THERAPY

Visceral Leishmaniasis

Generally immunocompetent patients respond quickly to treatment; they feel better within 5 to 7 days and the temperature is normal by this time. Hematological parameters improve gradually and the spleen regresses in size. With a satisfactory clinical, hematological and biochemical response to treatment and likelihood of follow up, there is no need to assess "parasitological cure" at the end of treatment: "parasitological cure" meaning not finding parasites in aspirates of lymph node, bone marrow or spleen. A patient who has had a favorable clinical, hematological and biochemical response at the end of treatment and who is parasitologically cured, will be labeled "initial cure". In the absence of satisfactory response or when follow up seems unlikely, parasitological assessment is advisable and this may lead to further or alternative treatment. Not finding parasites at the end of treatment does not guarantee "cure" (definite cure). Follow up at 6 weeks, 3 and 6 months is recommended; longer follow up was considered not necessary (223) but recent information from India and Nepal on relapses and post-kala azar dermal leishmaniasis (PKDL) up to two years after treatment (52, 252) indicates that longer follow-up is required. This may not be practical in daily practice but should definitely be considered in studies that are meant to influence policy and give advice on treatment. At 6 months the patient should have no symptoms, no fever, and should have gained weight. The spleen should be smaller, not necessarily non palpable and hematological parameters should be normal. Gammaglobulin levels may still be high and serological tests may remain positive for a long period of time (133). The role of molecular tests is not yet established. If all these criteria are fulfilled, the patient will be discharged as "definite cure". In the presence of symptoms or signs, and a still palpable spleen, a search for parasites must be undertaken. This is the more pressing if further follow up is not guaranteed. In immunocompromised patients the same criteria apply but without "maintenance therapy" or "secondary prophylaxis" relapse seems inevitable. ART does not seem to change this until CD4 counts have risen to levels of 350 per mm3 (12, 38, 123, 198, 352).

Cutaneous Leishmaniasis

In cutaneous leishmaniasis endpoints are complete re-epithelialization of the lesion (all lesions) and no residual wound inflammation and no reactivation or recurrence in six months.

Mucocutaneous Leishmaniasis

Assessment of treatment in mucocutaneous leishmaniasis is difficult; clinical and histological criteria apply. After medical treatment for leishmaniasis and secondary bacterial infections, plastic surgery is often required.

VACCINES

There is no effective vaccine available for any of the leishmaniases. The fact that many people in the endemic areas become naturally resistant to disease and that others may have immunity to disease indicates that immunization should be possible. There is no vaccine for human use in trial. A review by Kumar and Engwerda presents what is known about host immunity after Leishmania infection. It discusses past endeavors at vaccination and recent advances in development of leishmaniasis vaccines (165). A veterinary vaccine is licensed and there are several promising developments but there is no vaccine ready for human testing yet.

PREVENTION

Antiparasitic Drugs

Antiparasitic drugs are not used for the prevention of leishmaniasis.

Prevention of Insect Bites

Sand flies become active in the evening and night; they may bite in daytime, when disturbed. Repellents based on N,N-diethyl-m-toluamide (DEET) and soap based on DEET do have repellent activity (6). Sand flies can pass wide-meshed bed nets but bed nets as used for malaria protection, impregnated with insecticides are effective protection against sand fly bites (99). A controlled trial of wearing uniforms impregnated with permethrin by Colombian soldiers showed protection against leishmaniasis and malaria (290). In Kabul, Afghanistan, a 65% protective efficacy for insecticide treated bed nets (ITNs) and also for chaddars (sleeping dress), impregnated with insecticide was shown, as well as a 40 % protective efficacy for house spraying. ITNs were most popular (249). In Brazil, diagnosing leishmaniasis in the dog reservoir and killing all serologically positive dogs, is considered an essential element in the control of visceral leishmaniasis (239). Dogs can be protected by collars impregnated with insecticide (155). A veterinary vaccine is available (165). Bougainvillea glabra is noxious for sand flies and affords local protection against sand fly bites, at least in Israel (273). This seems an attractive way of protection.

COMMENTS AND CONTROVERSIES

Urgent questions are the small number of available drugs and the thread of development of resistance to these few drugs, in particular to miltefosine.

Only few drugs are available to treat visceral leishmaniasis; antimony, amphotericin B in various formulations, miltefosine, paromomycin and pentamidine. Pentamidine is hardly used apart from use in L. guyanensis cutaneous leishmaniasis, because of low efficacy and serious toxicity. There is only one oral drug , the other drugs are parenteral and expensive, apart from paromomycin. Antimony and amphotericin B have considerable toxicity, need long treatment courses and have to be given by intramuscular or intravenous injection. Liposomal amphotericin B is the most effective drug for most indications, is much better tolerated than antimony and conventional amphotericin B, needs a much shorter course of treatment but is very expensive, without subsidies prohibitively expensive in endemic areas. Miltefosine, an oral drug that also needs a long course of 28 days, is effective although efficacy seems to decline in India and Nepal, where late relapses and PKDL after 6 months of follow-up are now reported (53, 54, 252). Its long elimination half-life is a risk for development of resistance. In animal studies miltefosine is embryotoxic and teratogenic and its use is contraindicated in pregnancy and during 3 months after treatment. Women of fertile age should use/practice effective contraception during treatment and during 3 months thereafter.

Miltefosine should ideally be combined with a second drug; paromomycin is a good candidate. Combination with liposomal amphotericin B should also be studied. In Bihar combination with Sb^v is no longer beneficial because of the widespread resistance to this drug but elsewhere this combination may still be useful.

Liposomal amphotericin B is extremely expensive but hospitalization is shorter with liposomal amphotericin B. In Mediterranean visceral leishmaniasis in children, possibly also adults, treatment is only two days. In the full analysis one should consider costs of complications, treatment thereof and of relapses. Outpatient treatment is often possible. A patient who quickly responds to antimony and other treatment may be discharged to continue treatment on an out-patient basis. So, making the balance will not always be easy. The increasing and spreading Sb^v resistance will probably shift the balance. Experience with miltefosine may further increase the possibility of treatment of visceral leishmaniasis on an outpatient basis but this comes with the problem of a long course of treatment with drugs to be taken several times per day, unobserved. Combination treatment should be advocated but at present the choice is limited. Combination of miltefosine and paromomycin is of proved benefit in in vitro and in vivo studies. A disadvantage of paromomycin is that it has to be given by intramuscular injection. Dose and duration of miltefosine for the different endemic areas and for Leishmania/HIV co-infected patients have still to be worked out and there is concern about its teratogenic potential. We do not know if allopurinol would help in retarding development of resistance. In recent years more information has become available on treatment of intralesional Sb administration, on cryo- and thermotherapy for cutaneous leishmaniasis both of the Old and of the New World. In view of the increase of patients with Leishmania/HIV co-infection in East-Africa and in India, there is an urgent need to know more about "secondary prophylaxis", or "maintenance treatment". What drugs to use, at what dose and frequency?

Pharmacokinetic and pharmacodynamic studies are urgently needed especially among children and pregnant women.

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Tables

Table 1.  Drugs for the Treatment of Visceral Leishmaniasis

	Dose	Duration (days)	Route
Proved efficacy and available
Antimony	20 mg/kg/day	28 or >	i.m., i.v.
Amphotericin B	1 mg/kg/day	20	i.v. (slow)
Liposomal amphotericin B	3-4 mg/kg/day	D 1-5 +10	i.v.
Lipid associated amphotericin B	2-3 mg/kd/day	5	i.v.
Miltefosine	<25 kg 50mg/d >25 kg 100-150 mg/d	28	Oral
Of proved efficacy but not (yet) available)
Aminosidin1	16-20 mg/kg/day	21-28	i.m.
Efficacy less certain
Pentamidine	3-4 mg/kg/day	15 inj.	i.m., i.v.

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Deresinski, S. In The Literature.  Di Masi F, et al.  Visceral Leishmaniasis in Italy: Treatment With Liposomal Amphotericin B.  Clin Infect Dis 2014:58(1May):iii.

Deresinski, S. In The Literature. Rocio C, et al. Treatment of New World Mucosal Leishmaniasis With Liposomal Amphotericin B.  Clin Infect Dis 2014:58(1May):iii.

Hervás JA, et al.  Old World Leishmania infantum Cutaneous Leishmaniasis Unresponsive to Liposomal Amphotericin B Treated With Topical Imiquimod.  Pediatr Infect Dis J. 2012;31:97-100.

Paniz Mondolfi AE, et al. Successful Treatment of Old World Cutaneous Leishmaniasis Caused by Leishmania infantum with Posaconazole. Antimicrob Agents Chemother 2011;55:1174-1776.

Kim DH, et al. Is Paromomycin an Effective and Safe Treatment against Cutaneous Leishmaniasis? A Meta-Analysis of 14 Randomized Controlled Trials. PLoS Negl Trop Dis 2009;3(2): e381.

Wasan KM, Wasan EK, et al. Highly Effective Oral Amphotericin B Formulation against Murine Visceral Leishmaniasis. J Infect Dis. 2009 Aug 1;200:357-360.

Potera C.  Heart Drug Helps to Beat Chagas, Leishmania Parasites.  Microbe 2009;4:490-491.

Antinori S, Cascio A, et al. Leishmaniasis Among Organ Transplant Recipients. Lancet Infect Dis. 2008 Mar;8:191-9.

Pedras  MJ, et al. Comparative evaluation of direct agglutination test, rK39 and soluble antigen ELISA and IFAT for the diagnosis of visceral leishmaniasis. Trans R Soc Trop Med Hyg. 2008 Feb;102(2):172-8. Epub 2007 Dec 26.

Arevalo J, et al.  Influence of Leishmania (Vianna) Species on the Response to Antimonial Treatment in Patients with American Tegumentary Leishmaniasis. JID 2007;195:1846-1851.

Sundar S, Jha TK, Thakur CP, Sinha PK, Bhattacharya SK.  Injectable paromomycin for visceral leishmaniasis in India.  New Engl J Med 2007;356:2571-2581.

Sundar S, Chakravarty J, Rai VK, Agrawal N, Singh SP, Chauhan V, Murray HW.  Amphotericin B Treatment for Indian Visceral Leishmaniasis: Response to 15 Daily versus Alternate-Day Infusions. Clin Infect Dis 2007;45:556-561.

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