Essay: Development of tools to determine the phenotype and genotype of Leishmania donovani

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  • Development of tools to determine the phenotype and genotype of Leishmania donovani
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Sandflies (Diptera: Psychodidae and Phlebotominae) are abundantly found throughout the tropical and temperate region. In contrast to their name given, they are not found on sand. The sand fly larvae usually need organic matter, heat and humidity for the development and most commonly found in household rubbish, bark of old trees and cracks in house walls. The transmission of different Leishmania infections are carried out by specific sand fly found in different geographical regions. About 30 species of sand fly are proven as vector for Leishmania transmission [15]. The sand flies of genus Phlebotomus transmit Leishmania parasite in Europe, Africa, Middle-east and Asia (also called Old world). The transmission of Leishmania parasites in American continent are carried out by different sandflies of genus Lutzomyia.
Some of sandfly transmit species specific Leishmania (specific vectors) while others can transmit several species (permissive vectors). For example, Phlebotomus papatasi and Phlebotomus sergenti are specific vectors that can transmit L. major and L. tropica respectively. On the other hand, Phlebotomus argentipes, the natural vector of L. donovani in the Indian sub-continent has shown to support the development of L. tropica and L. amazonensis at Laboratory condition [16,17]. Likewise, Lutzomia longipalpis, the natural vector of L. infantum was also found susceptible for the full development of L. amazonensis and L. major [18,19].
Lipophosphoglycan (LPG) expression at the surface of Leishmania promastigotes are considered as the major determinant in Leishmania-sandfly species specific relationship. LPG of Leishmania promastigotes protects from digestive enzymes released into the mid-gut and support the attachment of parasite to the mid-gut epithelium [20]. The receptors on the vector midgut are highly specific to the Leishmania species. The lectin-like components at the surface of parasite are pivotal for attachment; it binds conserved GalNac (an amino derivative of galactose) on the microvillar border of the sand fly mid-gut [21].
1.2.2. Molecular phylogeny of genus Leishmania
Phylogenetics is the study of evolutionary relatedness among various groups of organisms such as species or populations, through morphological data or molecular sequencing data. Modern phylogenetic studies with different molecular datasets have been used to trace out position of Leishmania genus on evolution and, consequently, taxonomy.
The phylogenetic trees of the genus Leishmania were based on Multilocus enzyme electrophoresis (MLEE) analysed by phenetic and cladistic techniques [22,23]. These analyses confirmed, at the time, the monophyletic origin of the genus and its subdivision into two subgenera:
(a) L. (Leishmania) comprises all old world species including L. mexicana and L. amazonensis complexs from the New World.
(b) L. (vianna) consist of only the New World species.
In fact, the SSU rRNA gene and mitochondrial gene sequences can be used for the species identification within genus Leishmania. . The variation in the SSU rRNA gene was, however, insufficient to resolve any of the groups in Leishmania [24], and the extensive editing of some mitochondrial genes in Leishmania [25] were major problems in phylogenetic studies.
A recent phylogenetic analysis of the genus Leishmania, based on the hsp70 gene, has included by far the greatest number of taxa [26]. The phylogenetic trees were rooted using hsp70 sequences of the two most closely related Trypanosoma species Figure 1.2. 2. The resulting phylogeny supported the existence of three monophyletic groups (exception to Sauroleishmania) representing the subgenera L. (Leishmania), L. (Sauroleishmania), and L. (Viannia).

Figure 1.2. 2 Neighbour-joining phylogeny of hsp70 sequences of 52 strains representing 17 Leishmania and two Trypanosoma species, based on an alignment of 1,380 nucleotide. Distances were estimated using the Kimura 2-parameter model. Bootstrap support of the branches was inferred from 2,000 replicates and given percentage at the internodes were exceeding 70%. Old World clusters are indicated by a dot (•) on the branch leading to the cluster, while a square () is used for the New World groups. Numbers between brackets following the strain names indicate the amount of ambiguous nucleotide in sequences (Fraga et al. 2010).

1.2.3. Transmission and geographical distribution
The host of Leishmania could be different, and depend on the species of Leishmania; human or other mammals can be the reservoir of parasite as depicted in Table 1. 1. Three types of transmission are considered for Leishmaniasis:
i. Zoonotic transmission: wild mammals are assumed to be the only regular hosts for the parasite and human are considered accidental hosts. For instance, in Amazonian forest, rodents, sloths and opssums infected with L. braziliensis do not show major symptoms. When human (wood cutters, farmers, tourists) become infected, symptoms of muco-cutaneous Leishmaniasis developed.
ii. Anthropo-zoonotic transmission: both humans and other mammals are regular hosts in the parasite life cycle. For instance, L. mexicana circulates between rodents and humans causing cutaneous Leishmaniasis in human. Likewise, L. infantum in the Mediterranean basin which causes visceral Leishmaniasis in children and immuno-compromised adults, while domesticated dogs are the important reservoir (who can also suffer from disease).
iii. Anthroponotic transmission: humans are assumed as the only mammal host in the life cycle of parasite. For instance, the visceral Leishmaniasis caused by L. donovani in the Indian sub-continent. However, some domestic animals such as goat in Nepal have recently found to contain Leishmania as well which conflict the current assumption of transmission [27–33]. Whether or not these animals have also active involvement in the transmission cycle is not yet clear.
1.3. Visceral leishmaniasis in Nepal

Figure 1.3. 1 Map of Nepal indicating the location of the VL endemic regions in the low land (Terai) with Koshi river of Nepal and bordering Bihar in the Gangetic plain of India.

1.3.1. Epidemiology
More than 68% of estimate annual 50,000 deaths due to VL occur in Indian sub-continent, including Bangladesh, India and Nepal due to diseased caused by L. donovani [1,34]. The geographical location of VL edemicity at Indian subcontinent is shown in Figure 1.3. 1. The historic burden of VL in Indian sub-continent, greatly fall after the DDT spraying for the malaria eradication in late 1960s [35]. In Nepal, the historic burden of VL is more difficult to determine due to the lack of an organized public health system before 1956 [36]. However, the first official Nepalese VL case were reported in 1980 which was spread to bordering districts in Nepal through migration during late 1970s [37]. The current estimated incidence of 12,600 VL cases per year is mainly located in the central and eastern low land area (called as Terai) where there are approximately 6 million people at risk [35]. The current trend of VL incidence in Nepal is depicted in Figure 1.3. 2. A few VL cases were also found in western region of Nepal that might be the new focus of research on VL [38,39].

Figure 1.3. 2 Number of cases and Incidence rate of VL per 10,000 population at Nepal from 1998 to 2014 (Source: WHO [40])

VL is a poverty-related disease in the Indian subcontinent (ISC) including India, Nepal, and Bangladesh [41]. VL affects people who have lower socioeconomic status and neglected from political attention [42]. Hence, VL is said to affect the poorest of the poor [43,44]. However, there is clear link between VL cases and economic strength; it is difficult to determine whether poverty is the cause or more consequence of VL [45,46]. The VL patient, also prevent from executing their normal daily tasks, which consequence into serious loss of income and other household member must take care. The estimated economic burden of the VL cases on the household is more than the annual per capita income of that household [45,47,48]. Thus, VL not only force people into poverty, but also act as a poverty trap [49] and make their life more difficult.
1.3.2. Clinical profile
The major clinical symptoms in VL are prolonged fever, fatigue, malaise, loss of appetite and weight loss, and these symptoms are persistent in systemic infection. Swelling of the liver and spleen (Hepatosplenomegaly) is often occurred in VL infection which is caused by the accumulation of infected mononuclear cells in visceral area of body. The other name in Nepali language for VL is “Kala-azar”, meaning black fever as darkening of the skin in advanced stages of the disease. Together with hepatosplenomegaly swelling of the lymph nodes, anaemia, leukopenia, thrombocytopenia and/or hypergamma-globulinemia can occur as well. Immuno-compromised condition such as HIV co-infection cases may shows other additional symptoms due to atypical localisation of Leishmania and many secondary infections due to the poor cellular immunity. The untreated VL cases could result into serious cachexia, multisystem failure, internal bleedings and/or an increased susceptibility to several secondary infections and eventually progress towards the death of patient [50–53].
However, the patient is successfully treated (clearance of clinical symptoms), the parasites are sometimes still exist in the visceral organs or niches where the drugs are inaccessible, such as skin. There could be the possibility that the parasites are present in sub-clinical densities, clinically cured VL patients will always have a risk of redeveloping disease later on, i.e. relapse. This can occur due to the same immunosuppressive conditions that cause asymptomatic cases to develop active disease [54].
After the infection with L. donovani, typical skin rash (macular, maculopapular or nodular) can develop in patient (mostly face, also may occur on trunk, arms and/or legs) called as post Kala-azar dermal Leishmaniasis (PKDL). The prevalence of PKDL is less common in Indian subcontinent often around 1 to 5 years after VL-cure but mostly within 6 months after treatment in Africa [55,56].
Furthermore, it has been said that majority of L. donovani infected humans do not show clinical symptoms owing to the low infection level and are referred as asymptomatic VL [57]. The precise factor of asymptomatic VL is still being a research question. The virulent potency of parasite might be another factor for development of disease symptom since it has direct relation with development of symptoms in host. Indeed, immune-compromised host condition such as malnutrition, HIV or any sort of immunosuppressive disease/disorder has profound impact on development of active VL [58–60].
1.3.3. Diagnosis
a) Microscopy
The diagnosis of VL with direct visualisation of amastigotes (LD bodies) in clinical samples (such as lymph node, bone marrow and spleen aspirates) is the gold standard technique. Reading smears from spleen aspirates shows the greatest sensitivity i.e. >95% [54,61] and requires only microscope and technical skills for the correct detection of Leishmania amastigotes. The sampling itself, however, is highly invasive and extremely painful for the patient and requires considerable technical expertise. Complications rarely occur but can be very severe up to death due to haemorrhages [62]. Therefore, there is a need for less invasive diagnostic tools with a high sensitivity and specificity.
b) Serological diagnosis
Leishmania infection can be directly determined by either antigen or antibody detection in clinical specimen. This antigen based technique is more specific in determining the treatment efficacy, detecting sub-clinical infections and the diagnosis of immuno-compromised patients with decreased antibody response e.g. Katex urine latex agglutination test [63–66]. The salient feature of this technique is simple and non-invasive. But, major drawbacks in this technique are the necessity to boil the urine to increase specificity, and lower sensitivity in case of short duration of fever, small spleen size and low parasitemia. Hence, the Katex urine test does not meet the criteria for an standard diagnostic test, it could be the more appropriate when antibody detection tests fail due to the low antibody response such as HIV co-infected patients [64,67].
The direct agglutination test (DAT) is one of the sensitive antibody detection test to diagnose VL. It is based on colour coated promastigotes that are agglutinated by antibodies from the serum of the patient. It is more feasible as it is highly sensitive (95-98%) and acceptable specificity (90-94%) [68–72]. It’s also approved as the excellent test for the epidemiological purposes, since the dried blood samples on filter paper can be used, which is suitable for epidemiological study in high transmission foci in Indian subcontinent [73,74]. However, Antibody detection tests are more sensitive than antigen detection test, the immunocapacity of the patient is crucial for the antibody detection method.
The rapid diagnosis test such as rK39-dipstick test increase the rate of VL detection. It detects anti-K39 antibodies in the blood. In fact, K39 is a conserved antigen (39 amino acids) of L. infantum which is fixated on a paper-strip in recombinant freeze-dried form. It is highly feasible than the invasive technique (tissue aspirates) since a finger pricked blood is sufficient to determine VL infection. In symptomatic VL-cases in ISC, this technique has shown a high sensitivity 87-99% and specificity 87-97% [64,68–72]. It is highly reliable, and generate the result within 10-20 minutes. Therefore, it is considered as the best first line diagnostic tool currently available for the diagnosis of VL in field conditions in the Indian subcontinent [65,72]. In contrast, this test is not recommended in Africa since it has a shown lesser sensitivity in African countries due to the insufficient titres of anti-K39 antibodies in African VL patients than Indian subcontinent [75].
c) PCR
The detection of Leishmania DNA in clinical specimens, using PCR technique is one of the most sensitive and specific diagnostic tool to determine VL infection. It can be done on all sort of samples including bone marrow aspirates, spleen aspirates, blood, urine [76] and buccal swaps [77]. Various Leishmania DNA targets that have high copy number in Leishmania (such as kinetoplast DNA, ribosomal DNA or mini-exon genes) and highly specific for Leishmania species (GP63, ITS1, HSP70, Cysteine proteinases) can be employed for diagnosis and identification of Leishmania species. The ability of PCR assays to determine the species of Leishmania present in the clinical sample is highly advantageous over the other techniques. However, it has extreme sensitivity to determine Leishmania infection, PCR results should always be interpreted in the context of the patient’s clinical history since it is not considered as the sole diagnostic tool. The PCR technique required extremely clean lab for DNA extraction by using highly expensive chemicals, and it could be limited to well-equipped clinics [78,79]. Although, PCR is a reliable diagnosis tool [80], more development is required to use PCR in basic laboratory at field [81]. Recently, the Loop-mediated isothermal amplification (LAMP) assay has been developed for poor laboratory setup [82] and has already optimized in field conditions for increasing the diagnostic accuracy [83,84].
1.3.4. Treatment
VL always consequence to death of patient if not treated. VL classified under neglected tropical disease, since it received little attention in research of drug exploration. Before 30 years ago, antimonial was the only drug to treat VL. But now a days, a few more drug regimens are available to treat the VL and some are still in clinical trial, as shown in Figure 1.3. 3.
a) Pentavalent antimonials
Before 1950s, Ureum stibamate and solustibosan (pentavalent antimonials) were used for the treatment of VL. But, after 1950, sodium stibogluconate (SSG) and meglumine antimonials are recommended as the standard first line VL-treatment throughout the world [78,85,86].
The SSG treatment have several drawbacks such as (i) patient need to be admitted in hospital during treatment, (ii) the painful intramuscular injection to administer drug and (iii) cardiotoxic side-effects that consequence in death of the patient [87]. But, the increasing treatment failure rate up to 65% was reported in India that urged the health authorities to withdraw SSG as the first line treatment for VL [88]. However, an alteration of drug policy, a study indicated that it is still being used in some regions in Bihar due to lack of drugs [89].
b) Amphotericin B
The drug Amphotericin B (AmB) is originally discovered against the fungal infection, but later on it has also found leishmanicidal effect, as it binds on 24-substituted sterols resulting in the formation of aqueous pores in the membrane that triggers the alter in membrane permeability and finally kills the parasite [90]. Amphotericin B is available in its conventional form AmB deoxycholate and in various lipid formulas with a lower toxicity.
c) Miltefosine
Miltefosine, MIL (Impavido as trade name) is the first registered oral treatment against VL. It is derivative product of alkylphospholipid that was originally developed as an anti-neoplastic drug [90]. Clinical trials have shown that more than 95% of the patients are cured when treated with MIL for 28 days with a systematic follow up until 6 months and it is also effective in SSG-treatment failures [91–93]. The mechanism of action of miltefosine can be a direct anti-parasitic action against the parasite by impairing the lipid metabolism [94] and also causing parasite apoptosis [95]. Miltefosine has also shown to act at the host cell level stimulating the production of inducible nitric oxide synthetase 2 (iNOS2) that catalyses the generation of nitric oxide (NO) to kill the parasite within the macrophage [96]. MIL has been used as the first line treatment of VL in Indian subcontinent since 2005 and also in Nepal from 2007 in order to minimize the risk of SSG resistance from bordering districts with India. But, it has been given in combination with other drugs from 2012 [97]. Common side-effects with MIL treatment are vomiting (40%) and mild diarrhoea (15-20%). Since, there is proven teratogenicity nature, pregnant women are not recommended for the treatment. Unsupervised oral intake can be linked to a greater risk for the emergence of resistance.
d) Paromomycin
Paramomycin (PMM), an aminoglycoside antibiotic has already reported to possess the anti-Leishmanial activity in 1960s [98,99]. But, the study at clinical level was done during 1980s and found efficient to treat VL in Kenya and UK [100,101]. The clinical trials conducted at Indian subcontinent shows an efficacy of approximately 95% and is relatively cheap. Its safety and tolerability is also favourable with 55% experiencing pain at the injection site, 1% renal dysfunction and 2% ototoxicity [102]. The in vitro induced resistant strains showed that a reduced uptake of the drug instead of mutations in the ribosomal subunits as seen in PMM-resistant bacteria [103,104]; however the exact mechanism of PMM resistance is still unknown. Since, there are other treatment regimens, it is unlikely that PMM monotherapy will ever become a standard treatment for VL in Indian subcontinent. There are combination therapy in which PMM could be major component for treatment.
e) Liposomal Amphotericin B
Liposomal amphotericin B (L-AmB) is a formulation of amphotericin B in which the drug is packaged with cholesterol and other phospholipids within a small unilamellar liposome. In Liposomal Amphotericin B, deoxycolate is replaced with other lipids leading to less exposure of the free drug to organs. Thus the tolerance is greatly improved and adverse effects including hypokalemia and nephrotoxicity are greatly reduced. It is approved as an empirical therapy for presumed fungal infection in febrile neutropenic patients, treatment of cryptococcal meningitis in HIV-infected patients, treatment of patients with Aspergillus species, Candida species and/or Cryptococcus species infections refractory to amphotericin B deoxycholate, or in patients where renal impairment or unacceptable toxicity precluding the use of amphotericin B deoxycholate. The mechanism of leishmanicidal action is thought to be drug-binding to parasite ergosterol precursors, such as lanosterol, causing disruption of the parasite membrane.
In 1995, the clinical trial of L-AmB (AmBisome) has shown the high treatment efficacy [105] and study from India also suggests that L-AmB (Ambisome) cause substantially lower rates of toxicity than conventional amphotericin B [106,107]. WHO has also recommended 15 or 10 mg/kg of L-AmB is adequate to treat VL in Indian subcontinent [108]. L-AmB is currently used as a first line therapy for VL treatment in Nepal [109].
f) Combination therapy
The combination therapy has an advantageous over monotherapy, as the patient benefits from the anti-Leishmania activity of two different drugs, and it reduces the chance of the emergence of drug-resistant parasites. The combined therapy has significant impact on field, where the emergence of resistance to SSG significantly reduces the treatment options. The treatment regimen duration can be shortened compared to monotherapy without affecting treatment efficacy. The shorten regimen will not only increase treatment compliance, but also decrease toxicity, hospital costs and other indirect costs for patients. The combination include one fast acting drug to quickly reduce the parasite burden and another slow acting drug mode of action that can kill the remaining parasite [78]. The first combination therapy for VL in India had an efficacy above 90% in which SSG and PMM were administered for 21 days. However, cure rates were above 92%, the cured rates for combination of PMM and SSG were similar to PMM mono-therapy. There is no such strong advantage of using SSG+PMM combination, since SSG was highly toxic and reduced efficacy [110]. The efficacy of several combinations with liposomal AmB, MIL and PMM have been determined recently [111], the cure rate was above 97.5% and causing fewer adverse events compared to the control groups that was treated with AmB deoxycholate. However, the combination therapy shows promising for VL treatment, the feasibility of these combinations need to further assessed with criteria of patient recruitment and with a long term follow-up period to determine the accuracy in treatment outcome and adverse events. The feasibility of combination therapy must be studied before any implementation into control programmes [112]. Given the good efficacy of all combination that were recently tested, the actual choice of drugs to combine depends on various factors such as cost, ease of administration, adverse events [113]. For example, MIL and PMM is easier to administer, but the teratogenicity of MIL can hampers woman of childbearing age. The cost effectiveness of MIL+PMM is much feasible than combination of liposomal AmB and PMM [114].

Figure 1.3. 3 Chemical profile of drugs for VL treatment (Croft & Coombs 2003) [115]

1.3.5. Treatment failure
In general, the treatment failure is the broad term that ranges from non-responder/unresponsive, to relapse several months after an apparent clinical cure. In fact, several factors can be the cause of the treatment failure such as host factors, parasite factors, drug quality, etc. Among the host factors such as clinical profile, immunological response, host genetics are the crucial determinant for the treatment outcome. Parasite related factors: (a) drug resistance (b) infectivity are the also the contributor for the treatment outcome.
1.3.6. Monitoring treatment efficacy
In general, the treatment efficacy is considered to be associated with the emergence of drug resistant parasite. The cellular assays are developed to analyse the drug susceptibility in parasite. But these assays are labour intensive and need more skilful handling and hence rapid, easy-to use tools have the priority to monitor treatment efficacy. Several Leishmania specific genes associated with antimonial resistance were reported, but a few genes for MIL resistance had been reported such as LdMT and LdMT ros3. Antimony resistance genes reported are:
(a) AQP1 transporter mediate the antimony (SbIII) entry into the cell. Reduced expression lead to the development of antimony resistance [116,117].
(b) Overexpression of trypanothione reductase activity cause the antimony resistant isolates[118]
(c) Decrease the activation of the prodrug form of SbV to active form of SbIII. TDR1, thiol-dependent reductase belonging to glutathione S-transferase [119] and LmACR2, have active role in activation of SbV [120].
(d) Efflux of drug by ABC transporter MRPA gene [121,122]
(e) SNPs in antimony drug resistant genome [123]
1.4. Kala Azar Elimination Programme (KAEP)
The major pillars to control the VL in Indian subcontinent are: (a) vector control, (b) early diagnosis and (c) complete treatment of VL. The goal of the KAEP is to reduce the incidence of kala-azar 1 in 10,000 population at upzilla (Bangladesh), sub-district (India) and district (Nepal) levels by 2015 [124]. Three countries India, Nepal, and Bangladesh had committed to implement the KAEP strategy which was prepared by Regional Technical Advisory Group on Kala-azar elimination to accomplish the goal of kala-azar elimination from Indian sub-continent. The strategies of the KAEP are:
(a) Early diagnosis and complete treatment
(b) Integrated vector management and vector surveillance through indoor residual spraying (IRS), insecticide treated nets (ITN) and environmental management
(c) Effective disease surveillance through passive and active case detection
(d) Social mobilization and building partnerships
(e) Clinical and operational research
However, the regional commitment has been made, the goal of KAEP is bleak because of several challenges ahead such as bed nets prove no effective [125], low performance of insecticide spraying [126], increased MIL-treatment failure etc. More flexible strategies could guide the success of KAEP. Furthermore, the VL elimination might be affected by understanding complex connection between poverty and Leishmaniasis, as it is diseases of poor in Indian sub-continent [43].

Chapter 2 : Miltefosine treatment and Visceral Leishmaniasis
2.1 Action mechanism of MIL
MIL is a member of alkylphosphocholine drug, which has the phosphocholine esters of aliphatic long-chain and analogous to phosphocholine of cell membrane. It is considered an inhibitor of Akt also known as protein kinase B, PKB. Akt/PKB is a crucial protein within the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) intracellular signalling pathway, which is involved in cell survival [1] by preventing from the apoptotic like death mechanism. Therefore, apoptotic killing of cell is considered to be major action mechanism of MIL since it induce to activate the apoptotic signalling pathway of cell.

Figure 2.1. 1 Cellular inhibition of phagocytised Leishmania in macrophage.
(Source: Dorlo et al 2012, Oxford University Press license number: 4251230988921)

2.1.1 Interaction between MIL and L. donovani
MIL has demonstrated similar activity in both Leishmania parasite and neoplastic cells. It inhibits the cell in two ways: (a) interruption of lipid-dependent cell signalling pathways and (b) apoptosis.
The in vitro and in vivo anti-leishmaniasis activity of MIL was first described by Croft, 1987 [2]. Several experiments show that the MIL susceptibility in Trypanosomatid parasites are species specific. Various pitfalls in susceptibility complicate the interpretation and comparison of in vitro results for the screening of anti-leishmanial drug activity. Intracellular amastigote model is considered the most appropriate in vitro drug susceptibility model because it is more similar to the host infection [3]. Although, interpretation of the results is complicated by variability in parasite infectivity for macrophage cell [4], in drug activity dependent on the type of macrophage host cell used [5], but also in the intrinsic susceptibility of laboratory strains and clinical isolates [6]. Among the intracellular amastigotes of L. donovani, L. aethiopica, L. tropica, L. panamensis, L. mexicana and L. major laboratory strains, L. donovani was the most susceptible to MIL, with IC50 values of 3.3 to 4.6 µM, while L. major was the least susceptible, with IC50 values of 31.6 to 37.2 µM [7].
Several potential hypothesis for the anti-leishmanial mechanism of action of MIL have described and are schematically depicted in Figure 2.1. 1; even though no mechanisms has been clearly determined yet. The contradictory proposed several mechanism indicate that MIL has multiple molecular mode of action [8].
2.1.2. Clinical activity profile of MIL
The study on Indian adults treated with 100 mg of MIL for 28 day, showed a median maximal concentration of 70µg/ml at day 23 of treatment and indicated an elimination half-life of between 150 and 200 hours (approximately 7 days). In case of children, treated with 2.5mg/kg, the reported mean pre-dose concentration between days 23 and 28 of treatment was 24 µg/ml, with an elimination half-life of 180 hours [9].
Several cases reports on MIL plasma concentrations in CL [10] and VL [11] were published. A study on CL shows that absorption of MIL is slow, with an absorption rate of 0.36 per hour. Drug clearance and the volume of distribution are rather constant, as indicated by estimated between-subject variation. MIL keeps accumulating until the end of treatment (day 28) and depending on the exact daily dosage and the individual’s body weight, steady-state is reached in a subset of patients in the last week of treatment. The remarkable slow elimination of MIL is due to prolong half-life elimination. A pharmacokinetic study [12] revealed that primary elimination half-life is 7.05 days and a terminal half-life of 30.9 days.
2.2 Miltefosine regimen for the VL treatment
a) Historical treatment with SSG
In 1920, urea stibamine, the first stable form of pentavalent antimonial was discovered, and found successful to treat VL. It was a promising drug to treat VL patients during 1920s [13]. After 1940s, two organic pentavalent antimonial compounds: sodium stibogluconate (SSG) and meglumine antimoniate (Glucatime) were used to treat VL patients [14]. SSG in dosage of 10mg/kg/day intravenous/intramuscular for 6-10 days (600mg maximum) was used in Indian subcontinent to treat VL until first outbreak of 30% failed treatment in 1977 [13]. Although the treatment coursed was increased to 20 days after the recommendation from expert committee, the cure rate (87%) was not markedly improved [15]. In 1984, the WHO recommended SSG at a higher dose of 20mg/kg/day for 20 days for new VL cases (850mg maximum) and a second identical regimen for patients who failed the first SSG-regimen [16,17]. In 1990, the WHO again changed its recommendation for SSG-treatment to 20 mg/kg/day for 28 days for all VL patients [18–20]. Unfortunately recommended regimen also fail to be a promising VL treatment since the high SSG-treatment failure rates continued in endemic regions of Bihar (up to 65%) [18,21,22] and Nepal (24%) [23]. However, the emergence of SSG resistant L. donovani was correlated with increased level of treatment failure in Indian patients [24,25], there was no direct relation established in Nepalese patients [26].
b) MIL treatment for VL
In early 1980s, there was independent discovery of the antiprotozoal and antineoplastic activities of MIL and related alkylphosphocholine drugs [27]. Coincidentally, the compound was synthesized by two different research groups who were studying anti-inflammatory properties in UK and similarly for their anti-tumour activity in Germany. Previously, MIL was studied in a local treatment for breast cancer and eventually receives the approval of a topical formulation of MIL [28]. The oral formulation of MIL in the treatment of solid tumours was also evaluated in several phase II studies with different tumour types [29,30], but was eventually abandoned due to gastrointestinal disorder in cancer patients [31].
Interestingly, the study in mouse model found superior activity of oral MIL over standard intravenous SSG [32] to treat Leishmania infection. In 2002, MIL (hexadecylphosphocholine) was registered in India as a first line drug to treat VL , since the phase III trial of oral MIL to treat Indian VL patient had promising results of 94% cure rate [33,34]. The successful treatment of MIL led to the development of a unique public-private collaboration between Zentaris, WHO Special Programme for Research and Training in Tropical Diseases, and the Government of India [35]. In Nepal, the SSG treatment was substituted with MIL from 2007 in order to increase the treatment efficacy in Nepal because of increasing threat of SSG-resistant infection from bordering districts with India [36].
2.3 MIL treatment failure
In general, the treatment failure can range from non-responding, to relapse several months after an apparent clinical cure. The evaluation of parasites’ drug susceptibility in treatment failure provides the parasite’s tolerance to the treatment. The emergence of drug resistant parasite is considered to be the possible causes of treatment failure.
2.3.1 Factors for treatment failure
The clinical study of miltefosine treatment shows that the risk of relapse is highest in between 6 to 12 months after treatment [37]. The possible cause for the risk of relapse after successful treatment could be the persistence of parasite in host. In fact, none of drug can eliminate all parasites from all tissues, also other forms of Leishmaniasis [38,39]. The studies on parasite clearance after the treatment does not shows complete clearance of parasite from host and still presumed to harbour quiescent parasites. Similarly, asymptomatic VL also carry the quiescent parasites but lack of any signs and symptoms. Furthermore, host immunological responses [40], host genetics could also play a role in development of disease [41].
The existence of quiescent parasite in cured patients seems the endogenous reservoir for the recurrence of symptomatic VL. The parasites’ capacity to manipulation of host specific T-cell response result into the development of reservoir which could consequence into prospective treatment failure [42–45]. In fact, emergence of drug resistance in parasite could be associated the treatment failure where fitness cost in parasite deals with more lethal infection [46].
The treatment outcome is depended on the available concentration of drug, treatment compliance [47]. In addition, metabolism of drug also drive the treatment outcome since degree of parasite killing effect is determined by the available drug [48,49].
2.3.2 In vitro induced MIL-resistance
MIL resistance or less drug susceptibility, had been successfully induced in laboratory setting with promastigote, although it had not been characterized in vivo amastigote. The study on drug resistant induction shows that promastigote of L. donovani are resistant up to 40 µM i.e. 16.3 µg/mL [50]. These resistant parasites have 15-fold less susceptible to MIL [51]. Indeed, the induced resistant strains also retain the resistant phenotype in vivo since it resist miltefosine treatment up to 30mg/kg in the murine experiment. Therefore, there was strong correlation between in vitro promastigotes and in vivo intracellular amastigotes [52].
The transport of MIL through the parasite cell membrane is facilitated by a putative L. donovani MIL transporter (LdMT) and the protein subunit LdRos3. Previous studies indicated that decreased MIL accumulation and defective inward translocation was the main determinant of decreased susceptibility [51], and it was mediated through inactivation of LdMT and LdRos3 [53–55]. LdMT is a novel inward-directed lipid translocase that belongs to the P4 subfamily of P-type ATPase and LdRos3 is a non-catalytic subunit of this membrane protein related to the Cdc50 family, which together have crucial role in maintaining the phospholipid asymmetry of the parasite membrane [56]. LdMT-associated MIL resistance could be transferred to amastigote stage, with no apparent loss of infectivity, and also in vivo [52]. In contrast, the clinical isolates of L. donovani from Indian subcontinent, low expression of the LdMT-LdRos3 complex was not correlated to the less-susceptibility to MIL [57].
Indeed, increased efflux of MIL including endogenous phospholipids have also been implicated in MIL resistance, facilitated through the overexpression of an ABC transporter: the Leishmania P-glycoprotein-like transporter such as Leishmania LtrMDR1 [58,59]. In addition, the overexpression of two Leishmania-specific ABC subfamily G-like transporters (LiABCG6 and LiABCG4 half-transporter) conferred resistance to MIL as well as aminoquinolines [60,61].
The recent whole genome sequencing showed that MIL resistance in L. major mutants can be both genetically and phenotypically highly heterogeneous. Two of the three identified markers of MIL resistance in this study were involved in drug susceptibility: the previously described P-type ATPase and pyridoxal kinase [62]. Pyridoxal kinase plays a vital role in the formation of pyridoxal-5’-phosphate (vitamin B6).
2.3.3 MIL resistance in clinical isolates
A clinical decreased drug susceptibility of parasites to MIL has not yet been determined [37,63], however several relapse cases MIL treatment have been reported also from elsewhere [64]. A recent studies on MIL efficacy in Indian subcontinent, after a decade of registration in India, revealed a 90.3% final cure rate at 6 month follow-up. The cure rate had decreased from 94% final cure rate that was achieved in Indian Phase III trials a decade before [63], but was still higher than the most recent reported cure rate in other countries such as Nepal (80%) [37], Bangladesh (85%) [65], where the MIL was recently introduced.
The previous study shows that the induced MIL-resistant promastigotes also transform to MIL-resistant amastigotes. These MIL-resistant parasites were infective to macrophages in vitro as well as BALB/c mice in vivo. The MIL-resistance phenotype was strongly correlated in both promastigote and intracellular amastigote. Moreover, MIL-resistant parasite also retain the resistance phenotype in vivo, hepatic burden in BALB/c mice was stable following MIL treatment up to 30 mg/kg. This result indicates that the MIL-resistant parasite retain MIL-tolerance phenotype nevertheless the life stage of parasite [52]. Hence, the transmission of such MIL-resistant parasite in endemic areas clearly endanger the treatment efficacy as these parasite phenotype could be capable of resist the current treatment regimen.

Chapter 3 : Rationale & objectives of study
3.1 Rationale
Visceral leishmaniasis (VL) is endemic in more than 80 countries and is also one of the neglected, poverty related disease. Moreover, 80% of VL patients are living in Indian subcontinent including Bangladesh, India and Nepal. The treatment failure is a major issue in infectious disease targeted for the elimination. Miltefosine treatment failure is a reappearance of disease symptoms and/or microscopy positive cases even though initial cured with miltefosine treatment. However, the miltefosine treatment failure has already reported from India [1] and Nepal [2], the huge gap of knowledge require to be filled that could track the problems in miltefosine treatment. In case of miltefosine treatment failure, a role of L. donovani parasite is not yet clearly identified.
Since, parasite phenotype has not yet determined in clinical isolate from miltefosine relapse, we assume that the change in parasite phenotype is contributing the consequence of miltefosine relapse in Nepal. Therefore, we have developed the simple and improved tools that can identify the responsible phenotype of infecting parasite and it attempt to correlate with miltefosine relapse. On the other hand, the Single Locus Genotyping (SLG) assay has been developed to identify the L. donovani genotype that are already identified by Whole Genome Sequencing (WGS) [3]. Therefore, our work aimed to develop the simplified tools to understand the biological characteristics of infecting L. donovani in anthroponotic VL treatment failure in Nepal.
3.2 General Objectives

4.3.2 In vitro MIL susceptibility and treatment outcome

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