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‘What treatments are currently available for leishmaniasis, what

are the challenges of these and how is new research helping to

address these?’

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Contents

Abstract.................................................................................................................................. iii
1.1 Introduction...................................................................................................................... 1
1.2 Manifestations of Leishmaniasis.......................................................................................1
1.3 Current diagnosis strategy................................................................................................3
1.4 Current leishmaniasis treatments.....................................................................................5
1.4.1 Pentavalent antimonials.............................................................................................5
1.4.2 Amphotericin B (AmB)...............................................................................................7
1.4.3 Miltefosine.................................................................................................................. 9
1.4.4 Paromomycin........................................................................................................... 10
1.4.5 Pentamidine............................................................................................................. 12
1.4.6 Azoles...................................................................................................................... 12
2.1 How is new research helping to address these issues?.................................................14
2.1.1 Improvements to current chemotherapies................................................................14
2.1.2 CO2 laser administration and thermotherapy............................................................16
2.1.3 Drug repurposing.....................................................................................................16
2.1.4 Improving diagnostics..............................................................................................18
2.1.5 New potential drug targets.......................................................................................18
2.1.6 Vaccines.................................................................................................................. 20
3.0 Conclusion...................................................................................................................... 22
Reference List...................................................................................................................... 23

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Abstract

Leishmaniasis is a neglected tropical disease caused by several different species of

protozoan parasites of the genus Leishmania that can take on many manifestations, with

visceral leishmaniasis (VL) being the most severe. Current strategies to control this disease

are mainly based on chemotherapy. Despite chemotherapy being available for the last 70

years, toxicity and resistance is synonymous with almost all available drugs. To that effect,

the medical-scientific community is implementing vast amounts of research to help

overcome these problems. Despite this research outlining several possible candidates for

the betterment of leishmaniasis treatment, including several vaccine candidates, there are

still numerous challenges to overcome; the requirement for clinical trials, cost, lack of

funding from the ‘modern world’ and time constraints all pose as obstacles. Since

modification/repurposing of already established treatments mitigates a lot of these issues;

drug repurposing and combination therapy both appear to be the most attractive avenues of

research to help address issues with current treatments.

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1.1 Introduction

Leishmaniasis is a tropical and subtropical disease caused by an intracellular protozoan

parasite from more than twenty Leishmania species (CDC., 2020). WHO regards

leishmaniasis as one of the seven most important tropical disease with between 12 and 15

million people already infected and, 350 million at risk of infection (WHO., 2021). Clinical

features of a leishmaniasis are diverse, depending on the species involved and the immune

response of the host (Andrade-Narváez et al., 2001). Traditionally, leishmaniasis is classified

into three different clinical forms according to the parasite tropism (Monzote., 2009):

Cutaneous (CL), Mucocutaneous (MCL) and Visceral (VL/ kala-azar), with the latter being

responsible for the majority of the 70,000 annual deaths (Bravo and Sanchez., 2003)-

though there are numerous other manifestations (Table 1). Transmission of the disease to

humans is caused the bite of infected female hematophagous.

1.2 Manifestations of Leishmaniasis

CL manifests itself, usually, withing several weeks of a sand fly bite with skin lesions,

nodules, and plaque-like lesions developing on exposed areas of the body, particularly the

face, ears, and extremities (Eiras et al., 2015) (Figure 1). Typically, CL is resolved within six

months without the need for therapy (Eiras et al., 2015). MCL can be characterised by the

destruction of oral-nasal and pharyngeal cavities, usually beginning with mild nasal

inflammation and congestion with perforation of the septum slowly developing- in extreme

cases, lesions can extend to the soft palate, pharynx, and face (Goto and Lindoso., 2010)

(Figure 1). VL, also known as Kala-Azar is regarded as the most severe forms of

leishmaniasis, symptoms are typified by fever, splenomegaly, hepatomegaly,

hypergammaglobulinemia and weight loss. If left untreated, VL is frequently fatal (Sundar

and Rai., 2002).

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Table 1. Leishmaniasis species and their clinical manifestations.

Adapted from Kaye et al., 2020

Main Leishmania species Manifestation Pathophysiology Main Reservoir Countries or Regions Found

L. donovani VL, PKDL Fatal within 2 years (VL). PKDL lesions India, Bangladesh, Ethiopia,
may follow drug treatment Humans Sudan, South Sudan
L. tropica CL, LR, rarely VL Often self-healing after 12 months East Mediterranean, Middle
Humans East, North Africa
L. aethiopica CL, DCL, DsCL Self-healing (except DCL) within 5 years
Hyraxes Ethiopia, Kenya
L. major CL Self-healing within 12 months North Africa, Middle East,
Rodents Central Asia, West Africa
L. infantum VL, CL Can be fatal (VL) China, South Europe, Brazil,
Dogs, humans South and Central America
L. braziliensis CL, MCL,DCL,LR May self-heal within 6 months < 5%
progress to MCL. Dogs, humans, rodents South America
L. amazonensis CL,DCL,DsCL Little information avaliable Marsupials, rodents South America
L. guyanensis CL,DsCL, MCL May self-heal within 6 month Marsupials South America
L. mexicana CL,DCL,DsCL Often self-healing within 4 months Rodents, marsupials South America

VL, Visceral leishmaniasis; PKDL, post-kala-azar dermal leishmaniasis; CL, cutaneous leishmaniasis;
MCL, mucocutaneous leishmaniasis; DCL, diffuse cutaneous leishmaniasis; DsCL, disseminated
cutaneous leishmaniasis; LR, leishmaniasis recidivans

i ii

Figure 1. Clinical Presentation of Cutaneous Leishmaniasis and severe Mucocutaneous

Leishmaniasis. Image i. shows CL: A) Face lesion beginning to show ulceration, caused by L. infantum. B)
Ulcerated L. major infection on the inside of the leg, C) Nodules on the skin caused by L. major. D)
Disseminated L. infantum infection. Image ii: Severe Mucocutaneous Leishmaniasis. The image depicts an
individual with disfiguring lesions of the oral-nasal and pharyngeal cavities
Images taken from Eiras et al., 2015 (i); AFPMB., 2015 (ii).
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1.3 Current diagnosis strategy

Leishmaniases are characterised by clinical pleomorphism and so confirmation based solely

on clinical grounds is very challenging (Kaye et al., 2020). The current gold standard for

leishmaniasis diagnosis is parasite demonstration via microscopy of Giemsa-stained tissue

smears where amastigotes can be identified (Reimão et al., 2020). This technique is not

always accessible, can be invasive, and has variable sensitivity. Therefore, in some areas,

conformation rates of CL have been reported to be as low as 5% causing patients to be

administered with potentially toxic drugs without receiving accurate confirmation of infection

(Kaye et al., 2020). VL requires invasive tissue sampling for diagnosis, including biopsies

from the bone marrow, spleen, or lymph nodes (Sundar and Rai et al., 2002). Naturally, this

requires expertise and special facilities to be performed safely and effectively, thus making

diagnosis extremely difficult, considering that leishmaniasis is primarily isolated to poverty-

stricken areas in which education and medical equipment is rarely available (Okwor and

Uzonna., 2016), though other diagnostic methods are implemented also.

VL is characterised by polyclonal hypergammaglobulinaemia with a notable increase in in

serum IgG levels (Ghose et al., 1980). Consequently, identification of anti-Leishmania

antibodies can play a key role in the diagnostic procedure of visceral leishmaniasis. In Latin

America and the Indian subcontinent, rapid diagnostic tests (RDTs), utilising the recombinant

antigen rK39, are widely used with sensitivity and specificity exceeding 95% (Boelaert et al.,

2014). However, the efficacy of these test in eastern Africa has shown to waver, the

specificity remains similar (91.1%), but sensitivity drops to 85.3% (Boelaert et al., 2014).

Subsequently, in eastern Africa it is necessary to include a direct agglutination test (DAT)

within the diagnostic algorithm (Figure 2). The DAT cannot be defined as an RDT as

laboratory skills and overnight incubation is required (Chappuis et al., 2007). rk39 based

RDTs utilise a kinesin sequence, derived from an American strain of L. infantum and

consequently is deemed responsible for the poorer performance of these RDTs in eastern

African, where rK39 homologous sequences show great diversity among localised and

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regional specific L. donovani strains (Bhattacharyya et al., 2013). Early, rapid, and efficient

diagnosis is essential to proper clinical treatment of leishmaniasis and effective control

of Leishmania transmission (Jesus et al., 2021). To that effect, new research is helping to

address the current diagnostic problems, thus indirectly working to improvement treatment of

the disease.

Figure 2. Eastern Africa VL diagnostic algorithm.

Adapted from national guidelines from Ethiopia, Kenya , Sudan & Uganda.
*Parasitological diagnosis may also be performed
**In Sudan rK28 RDT’s are being investigated to replace rK28 RDT’s (See 2.1.4 Improving diagnostics)

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1.4 Current leishmaniasis treatments

1.4.1 Pentavalent antimonials

Due to their low cost and abundance, pentavalent antimonials, the generic sodium

stibogluconate (100 mg Sbv+/mL) and branded meglumine antimoniate (85 mg Sbv+/mL),

have been implemented to treat leishmaniasis for over five decades and they still remain a

first-line treatment in areas of limited antimonial resistance (Singh et al., 2012). But, in recent

years, the use of pentavalent antimonials has become restricted due to numerous reports of

treatment failure (Table 2), notably endemic areas of India, particularly Bihar where

resistance exceeds 65% (Menezes et al., 2015).

The drug is usually administered via intramuscular injection. Doses and durations of

treatment with antimonials has observed constant changes in the years since

implementation (Monge-Maillo and López-Vélez., 2013). Current recommended dosage of

antimonials is variable and dependent on numerous factors, ranging from 10 mg/Kg/day for

21 days in the case of VL patients to 20 mg/kg/day for at least 120 days in Indian PKDL

(WHO., 2010). These doses, intra-muscularly, are painful, though this issue can be

ameliorated via IV administration, which comes with inherent problems as this requires

hospitalisation (Datta et al., 2021).

The precise mechanism of action of pentavalent antimonials is unknown, but there are

several proposed mechanisms, principle of which is the theory of the conversion of

pentavalent antimonials (Sb5+) to the more toxic trivalent form (Sb3+) via As5+ reductase

(Datta et al., 2021). As5+ reductase is an enzyme present in the intracellular amastigote

stage within the phagolysosome of the macrophage. Sb3+ interacts with sulfhydryl-containing

molecules, notably, thiol groups and proteins to exerts its effect via inducing efflux of

trypanothione and glutathione, and inhibition of trypanothione reductase (Datta et al., 2021),

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further increase susceptibility to oxidative stress by altering the thiol-redox potential in both

promastigotes and amastigotes (Ameen., 2007). Hence leading to its associated

leishmanicidal action. However, both forms of pentavalent antimonials (SbV and SbIII ) are

known to have an effect on leishmania (Singh et al., 2012).

The other form, SbV, is considered a pro-drug and, and is similarly known to also inhibit

trypanothion reductase (Singh et al., 2012). Trypanothion reductase is responsible for the

enzymatic protection of the parasite from host reactive oxygen and nitrogen species, hence

this inhibition can result in the Leishmania becoming susceptible to oxidative stress. (Wyllie

et al., 2004).

Despite the popularity of these drugs for Leishmaniasis treatment, they are synonymous with

several challenges, principle of which is the increasingly common acquired resistance and

consequent treatment failure, resulting in variable efficacy (Table 2). Indiscriminate use of

this chemotherapy due to its low cost and over the counter availability in endemic areas;

coupled with loss of drug activation by the parasites, are the greatest causatives of

resistance (Datta et al., 2021). Furthermore, numerous studies have identified several genes

(P229 & γ-glutamylcysteine synthetase heavy chain genes) to be associated with antimonial

unresponsive Leishmania (Carter et al., 2006; Choudhurry et al., 2008), hence implying that

the mechanism of resistance is multifactorial. Resistance aside, the use of antimonials

results in large accumulations of the drug in tissues, often the liver, thyroid, and heart (Datta

et al., 2021). This accumulation is synonymous with adverse effects: nausea, abdominal

pain, myalgia and cardiac arrythmia- which frequently cause a cessation of therapy (Takur et

al., 2004; Monzote., 2009). In addition, some bad batches have caused fatal cardiotoxicity in

numerous patients (Croft et al., 1987).

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Study Sample Previous Clinically Therapy Relapse or

Leishmania species Treatment
source size treatment healed failed Recurrence
Almeida et L. braziliensis MA* 5 Yes 100%
0% after 0% after 12
al., 2005 12 months
months
Arevalo et Leishmania spp. MA 7 No 57%  43% after 0% after 3
al., 2007 3 months months
Van Thiel L. major SSG 118 No 55.10% 20.3% 15.3% after
et al., 2010 after 6 6 months
months
Brito et al., L. braziliensis MA 82 N/A 45% 55% after N/A
Table 2. Tabulated variability in Pentavalent antimonials efficacy as shown by primary research
2017 6 months
papers

1.4.2 Amphotericin B (AmB)

AmB is a polyene antifungal drug derived from the filamentous bacteria Streptomyces

The efficacy of treatment

nodusus (Dattacan be seeing
et al., to increase
2021). AmB in more
is available recent years.
in numerous 100%
forms: healing
plain, rate in Almeida et al’s.,
cholesteryl
2005 down to 45% in Brito and colleagues 2017 investigation.
sulphated, lipid complex and liposomal (Bulbake et al., 2017). In its classic form, AmB
MA, meglumine antimoniate; SSG, intralesional sodium stibogluconate
deoxycholate, there are a broad range of adverse reactions, making treatment and patient
* Combined with granulocyte macrophage colony-stimulating factor
compliance particularly challenging (Singh et al., 2012). The liposomal variation is preferred

as there are minimal adverse effects associated with its use and is currently the most

desirable treatment option for PKDL (Datta et al., 2021), especially in cases of antimonial

resistance (Bern et al, 2006). Conversely, in nonendemic areas, it acts as a second line

drug.

Amb interacts with sterols, with a particularly high affinity for ergosterol of Leishmania (Singh

et al., 2012). At concentrations exceeding 0.1M, AmB induces the formation of aqueous

pores on the promastigotes cell membrane, resulting in osmotic changes due to increased

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permeability and leakage of anions and cation (Ramesh et al., 2016); ultimately resulting in

cell lysis (Ramos et al, 1996). A further mechanism of action of Amphotericin B is the

generation of reactive oxygen free radicals which damage the parasite to an extent which

often causes cell death (Purkait et al., 2012).

The efficacy of all forms of AmB is well documented. In an open-label, randomised trial, Das

et al (2017) compared the efficacy of two frequencies of traditional AmB dosing in 50 PKDL

patients. One group received administration of AmB daily, and the other a dose on alternate

days. The results of the investigation show the dosing method to have comparable efficacy,

both indicated a cure rate over 88%. Despite this, nephrotoxicity remained an issue in both

regimens. Similarly, Den Boer et al (2018) recorded a 78% cure rate and 89.7% clinical

improvement rate in their 280-patient investigation using single-dose liposomal AmB,

although 10.3% of the patients failed to show any response to treatment.

One of the largest drawbacks of AmB use is dose-limiting nephrotoxicity (Menezes et al.,

2005), as demonstrated by Das et al (2017). Since AmB interacts with sterols, the drug has

a high affinity for not only ergosterol of Leishmania but also cholesterol of host cells,

particularly renal tubular epithelial cells. Subsequently, at high concentration, AmB induces

the formation of pores in cell membranes which facilitates the entry of Na+, leading to the

activation of mitogen-activated protein kinases and elevation of intracellular

Ca2+ concentration (Yano et al., 2009)- a mechanism is synonymous with apoptosis in

eukaryotic cells (Singh et al., 2012), hence nephrotoxicity. Resistance against AmB is

currently extremely rare but the relatively nonspecific mechanism of action of the drug may

be a factor contributing to likely future resistance (Douanne et al., 2020). Despite resistance

against AmB being uncommon, amplification of Amphotericin B-resistant Leishmania

tarentolae mutant cell lines by Singh and Ouellette (2001) indicated that AmB resistance can

be associated with TarII 64.4 and tarII 512.2 genes. Similarly, genetic mutations effecting

sterol C5-desaturase and Sterol C24-methyltransferase have been linked to resistance

(Pountain et al., 2019). Thus, reiterating the possibility of the emergence of resistant. Other

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challenges of AmB use include the requirement for administration via slow intravenous

infusion (Menezes et al., 2015) and heat instability (Meheus et al., 2010). Furthermore,

hypokalemia-induced rhabdomyolysis has been reported in some studies using liposomal

amphotericin B (Marking et al., 2014).

1.4.3 Miltefosine

Originally developed as a cancer treatment drug, Miltefosine is an alkylphosphocholine

(hexadecylphosphocholine) moiety (Croft and Coombs., 2003). Administered orally, it is the

first drug of its kind used for the treatment of VL to depict extremely high efficacy; to that

effect, miltefosine has provided a significant breakthrough in anti-leishmanial chemotherapy

due to ease of administration and subsequent increased patient compliance (Datta et al.,

2021).

The exact mode of antileishmanial action is unknown however, Paris et al (2004) have

observed that Miltefosine causes apoptosis like processes in amastigotes of Leishmania

donovani after intracellular accumulation, but exactly how this occurs is undetermined.

Miltefosine also reduces the lipid content in promastigotes membrane whilst promoting

phosphatidylethanolamine (Singh et al., 2012). Therefore, suggesting

phosphatidylethanolamine-N-methyltransferase inhibition leading to a reduction in parasitic

proliferation (Loiseau and Bories., 2006). Other proposed mechanisms mode of action

include disruption to lipid-dependent signalling pathways (Lira et al., 2001) and inhibition of

cytochrome c oxidase (Luque-Ortega and Rivas., 2007). More recently, interference of the

intracellular Ca2+ homeostasis has shown to be an important object for the action of

Miltefosine against leishmaniasis, particularly as the drug has shown to activate the plasma

membrane Ca2+ channel in L. donovani (Pinto-Martinez et al., 2018).

Miltefosine is well documented as an efficacious drug. In an open-label single-arm study by

Ramesh et al (2015), 86 patients were treated with 50 mg miltefosine three times daily, over

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the course of 60 days. A cure rate of 96% resulted. Despite the high efficacy depicted by the

study, 15% relapsed after 18 months, thus indicating the requirement for alternate

drugs/combination therapy with miltefosine. Meta-analysis by Pijpers et al (2019) indicated

miltefosine to have an initial cure rate of 92.5% and a definite cure rate of 90% in 324 Post

Kala-azar dermal leishmaniasis patients from eight studies- reiterating the high efficacy

depicted by Ramesh et al.

Although currently there are few cases of clinical resistance reported, there are several

challenges associated with the likelihood of potential resistance. Since the half-life of

Miltefosine in the body is approximately 152 hours, it is likely to encourage the development

of resistance, particularly when coupled with likely improper use in highly endemic countries

as a consequence of the oral administration (Singh et al., 2012). Perez-Victoria et al (2001)

have reported a strong association between the over expression of the MDR1 gene, which

encodes a glycoprotein, and anti-Leishmania drug resistance. In a similar, more recent

study, Vacchina et al (2016) proposed several theories to explain the development of

Miltefosine resistance, including mention of Perez-Victoria et al’s study. In this literature,

additional theories included single-point mutation in LdMT and LDRos3 genes, reduction in

unsaturated phospholipid alkyl chains in Leishmania cell membrane as well as alterations in

the mitochondrial HSP70. Other problems with Miltefosine include the teratogenic and

abortifacient nature of the drug makes it unsuitable for use in pregnancies (Singh et al.,

2012). Generally, treatment with miltefosine is devoid of extreme adverse effects, but the

majority of published studies report gastrointestinal symptoms including nausea, vomiting,

diarrhoea and raised bilirubin levels (Tayyebi et al., 2021).

1.4.4 Paromomycin

Clinical trials were first conducted using Paromomycin during the 1990s in India and Africa

(Monge-Maillo and López-Vélez., 2013). This chemotherapy is chemically an

aminoglycoside antibiotic, extracted from the bacterial spp. Streptomyces rimosus var.

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paromomycinus and has both antileishmanial and antibacterial activity (Singh et al., 2012).

Paromomycin’s curative role for VL and CL is already established and recently investigations

are being made into its potential use against post kala-azar dermal leishmaniasis also (Datta

et al., 2021). Paromomycin is amongst the cheapest of all current leishmania treatments with

costing ≈ $10–15 (Menezes et al., 2015).

The mechanism of action is still vague, though it is probable that paromomycin acts by

inhibiting the Leishmanial cytochrome C enzyme (Chawla et al., 2011). Additionally, the

inhibition of protein synthesis is regarded as a potential action- translocation (translocation

initiation factor-3) and recycling of cytoplasmic and mitochondrial ribosomes are all inhibited

by the drug via interaction with 30S and 50S subunits within the ribosome (Hirokawa et al.,

2007), thus inhibiting protein synthesis.

One of the benefits of paromomycin is reduced treatment duration. One study by El-On et al

(1992) using CL patients showed that the drug eliminated Leishmania within 10 days of

treatment. Moreover, numerous trials in Indian (Sundar et al., 2009), Sudan (Musa et al.,

2010), Tunisia and France (Singh et al., 2012) have indicated that Paromomycin is

synonymous with high efficacy and excellent tolerability and performance comparable to

AmB (Sundar et al., 2009). On the contrary, in an open-label single-arm study by Sundar et

al (2014), paromomycin at doses of 11 mg/kg over a 45-day Despite the unacceptably low

efficacy, the study showed there to be no serious adverse effects of the treatment.

Generally, paromomycin has few adverse effects, the most commonly reported ones mainly

being pain at injection site, reversible nephrotoxicity and occasional liver complications

(Sundar et al., 2016). As a consequence of its limited use, resistance has not yet been

reported (Datta et al., 2021). However, in in vitro applications Leishmania donovani and

Leishmania tropica have reported resistance to the drug (Jhingran et al., 2009). The

potential rapid emergence of resistance against paromomycin cannot be disregarded (Singh

et al., 2012) and so caution should be implemented with its use.

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1.4.5 Pentamidine

Pentamidine is an aromatic diamine that is rarely used in an attempt to cure leishmaniasis as

a second line drug (Singh et al., 2012). Since the drug was first synthesized in the 1940’s, its

anti-Leishmania properties have been studied, mainly in cases of antimonial resistant

Leishmania in India (Jha et al., 1991). However, declining efficacy and increasing resistance,

coupled with extreme toxicity led to abandonment of its use. More recently, monthly use of

pentamidine in doses of 4 mg/kg for 12 months has been recommended as a second line

treatment for VL patients with a HIV coinfection in Ethiopia (Diro et al., 2019). Pentamidine

has also shown to be effective to curtail CL infections caused by L. panamensis and L.

guyanensis (Soto et al., 1994; Romero et al., 2001).

Again, the precise mechanism of action of pentamidine is unknown. Studies have reported

that the drug enters promastigotes via the arginine and polyamine transporters (Basselin et

al., 2000; Kandpal and Tekwani et al., 1997). Furthermore, pentamidine is shown to

accumulate in parasitic mitochondria which enhances the efficacy of mitochondrial

respiratory chain complex II inhibitors; thus, inferring that the leishmanicidal ability of the

drug can be accounted to a reduced mitochondrial membrane potential (Singh et al., 2012).

Basselin et al (1999) have similarly identified an inhibition of mitochondrial topoisomerase II

associated with pentamidine.

Since Pentamidine is currently infrequently used as a consequence of rapid appearance of

resistance cases (Bray et al., 2003), high toxicity, and often low efficacy (Jain et al., 2013), it

is only recommended as part of a combination therapy approach (Croft et al., 2003).

1.4.6 Azoles

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Azoles, notably Ketoconazole, fluconazole, and itraconazole are antifungal agents have

been identified to harbour antileishmanial properties. They act by inhibiting the cytochrome

enzyme that is responsible for the conversion of lanosterol to ergosterol; the main sterol in

Leishmania cell membranes (McCall., 2015).The depletion of ergosterol damages the cell

membranes and ultimately results in cell death. The efficacy of azole treatments against

leishmaniasis has shown to be variable. In the case of Emad et al (2011) CL oral fluconazole

therapy, cure rates have been shown to be around 60% with substantial increases in

effectiveness when the drug concentration was doubled. Though to the detriment of this

treatment, hepatotoxicity and cardiotoxicity were also shown to be alarmingly common in the

same study (Emad et al., 2011). Moreover, high dose oral fluconazole therapy in L.

braziliensis-infected patients has depicted greater efficacy with a cure rate of 75–100%

(Sousa et al., 2011). However, a more recent randomised clinical trial using oral fluconazole,

in a similar therapeutic scheme to Sousa et al, for the CL treatment caused by L.

braziliensis, exhibited only 22% cure rate (Prates et al., 2017). A systematic meta-analysis

performed by Galvão et al (2017) outlined that azole are not effective for any form of

leishmaniasis and their use should only be associated when used in combination with other

drugs.

In spite of the magnitude of chemotherapies currently available, all of the drugs have

challenges predominately, cost, efficacy, toxicity, and a high likelihood of the development of

resistance (Table 3).

Drugs Advantages Disadvantages Resistance Price Concluding Remark

Pentavalent antimonials Availability; low cost Varying quality prolonged Very common $50–198 First-line drugs but with high
treatment; toxicity; well- (>65% in Bihar, incidences of resistance; variable
documented resistance India) response (species dependent)

Amphotericin B (AmB) No current resistance Requires slow IV infusion; Likely to develop ~$21–100 Toxicity; need for prolonged
Table 3. Tabulatedissynopsis
known of established
dose-limitingLeishmaniasis chemotherapy. hospitalization; first-line drug for
nephrotoxicity; needs to be VL where there is antimonial
chilled resistance
Liposomal AmB Very good efficacy; Extremely expensive; Not documented $280–3000 Cost
reduced toxicity requires slow IV infusion;
compared to AmB needs to be chilled
Miltefosine Effective; oral Price; likelihood of Demonstrated in $70–150 Effective orally, long half-life may
administration developing resistance; poor vitro encourage emergence of
patient compliance resistance, not suitable in
pregnancy
Paromomycin Effective; well- Variable performance; Demonstrated in $10–15 Low cost; extremely varied
tolerated; and low likelihood of developing vitro performance
cost resistance
Pentamidine Reduced treatment Variable performance Not documented N/A 13
Extremely varied performance;
course only suitable for certain
applications
Azoles Effective in Not effective in Not documented N/A Not effective in monotherapy;
combination therapy monotherapy requires combination treatment
with antimonials
19020181

2.1 How is new research helping to address these issues?

Clearly, current anti-Leishmania chemotherapy leaves a lot to be desired. To that effect, vast

amounts of research is being conducted into the betterment of treatment options for

Leishmaniasis, both looking at entirely new methods and also improving established

treatments.

2.1.1 Improvements to current chemotherapies

Combination therapy using current antileishmanial drugs, has resulted in a favourable

reduction in individual doses, toxicity, treatment duration, cost, and occurrence of drug

resistance when compared to traditional monotherapy (Menezes et al., 2015; Sundar et al.,

2011). Seifert and Croft (2006) examined in vitro and in vivo interactions between a

combination of antileishmanial drugs as candidates to replace pentavalent antimonials in

cases of VL resistant to pentavalent antimonials. The study findings showed there to be no

signs of toxicity with any combination of drugs. The in vivo findings depicted a favourable

combination of miltefosine and amphotericin B or paromomycin, though; considering toxicity

and cost of the partner drug, paromomycin would perhaps be more desirable than

amphotericin B. Similarly, Sundar et al (2011) compared the efficacy and safety of a

combination of liposomal AmB, miltefosine, and paromomycin, to those of monotherapy with

traditional AmB in a study conducted in India using 634 VL patients. The multidrug regimes

all showed to be non-inferior to the monotherapy with decreased duration of therapy.

Patients in the combination groups also had fewer adverse events. Hence, both of these

research papers indicate that combination treatment for leishmaniasis is efficacious and safe

with potential to reduce treatment duration and increase treatment adherence, consequently

reducing the likelihood of resistance to treatment developing. Therefore, acting as a vey

desirable options to improve current therapy. Moreover, the findings also suggest rapid, low-

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cost implementation as the drugs are already in use, negating the necessity for large scale

clinical trials.

Alternatively, functionalised carbon nanotubes have been investigated as potential drug

carriers against leishmaniasis (Menezes et al., 2015). In animal models, Prajapati et al

(2011) showed AmB attached to carbon nanotubes to have superior leishmanicidal activity

than conventional AmB in in L. donovani-infected Mesocricetus auratus, though this only

acts as proof-of-concept in animal models. Similarly, using Amb conjugated to chitosan-

chondroitin sulphate nanoparticles, Ribeiro et al (2014) indicated that when compared to

traditional AmB, the nanoparticle formulation demonstrated sufficient anti-leishmanial

activity, and a high capacity to treat infected macrophages, whilst also being devoid of

toxicity that is usually observed when mice or human cells are administered with pure AmB.

Therefore, combination therapy in place of monotherapy using established treatments as

well as the use of additional drug carriers shows great potential to ameliorate a magnitude of

current issues however additional research is necessary.

2.1.2 CO2 laser administration and thermotherapy

CO2 laser and thermotherapy are ways in which to deliver heat to external tissues, causing

damage to areas which display parasitism, consequently damage/destroying Leishmania.

(Asilian et al. 2004; Valencia et al. 2013). Studies have demonstrated that thermotherapy

showed better results in terms of cure rate when compared with traditional chemotherapy

using antimonials, with similar or reduced side-effects (Sadeghian et al. 2007; Aronson et al.

2010). In a prospective, randomized open trial study performed by Shamsi et al (2011) the

efficacy of CO2 laser for leishmaniasis treatment, compared with a combination treatment

comprised of cryotherapy and intralesional antimonials was investigated. The CO2 laser

thermotherapy cohort demonstrated a superior cure rate, 93.7% compared to a 78% in the

combination therapy cohort and a reduced healing time, 6 weeks compared to 12 weeks. In

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terms of adverse events, both groups were remarkably similar. Despite this, the laser group

did experience have a larger number of sporotrichoid, raised papular lesions and persistent

erythema. The findings of this investigation therefore demonstrate that the CO2 laser

methodology proposes a promising alternative to current chemotherapies. The emergence of

resistance is very unlikely, with high efficacy, although this technique is only applicable to the

typically self-healing CL. Thus, this research mitigates only a few challenges of current

treatment and not those associated with the more severe leishmaniasis manifestations.

2.1.3 Drug repurposing

Drug repurposing, often referred to as drug reprofiling or repositioning, is a modern strategy

implemented to develop new uses for existing drugs, often for the treatment of neglected

diseases (Ashburn and Thor., 2004). Advantages of the strategy include reduced chance of

failure, reduced development time, lower cost and the protocol can also reveal new

pathways and targets for future exploration (Pushpakom et al., 2019). In the case of

leishmaniasis, drug repurposing utilises a computational approach. These computational

techniques perform systematic analysis of data, most commonly bioinformatics, gene

expression, chemical structure, and genotype data (Roatt et al., 2020).

Molecular docking is an example of this computational strategy. Molecular docking works by

predicting binding sites between the ligands (in this case a drug) and the target (Leishmania)

(Bustamante et al., 2019). Conventional docking is another variation of molecular docking. In

this variant one receptor/protein target is selected and multiple drugs are then tested against

this target. This helps to build knowledge about the target/protein, hence making selection of

a possible ideal drug with a greater chance of success in subsequent trials easier

(Pushpakom et al., 2019). Another hugely important tool is signature matching. Signature

matching works on the premise of the comparison between the characteristic (signature) of a

drug/molecule compared with that of alternative drug/molecule (Roatt et al., 2020). This

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analysis is performed using metabolomic, proteomic, transcriptomic, or chemical structures.

Both molecular docking and signature matching are the most used drug repurposing

strategies against leishmaniasis currently (Roatt et al., 2020). In a recent study, Bustamante

et al., (2019) used a bioinformatic, molecular docking approach to predict and detect drugs,

with in vitro validations and pharmacokinetic simulations, for repurposing in leishmaniasis

treatment. Bioinformatic predictions were used to detect potential homologs between target

proteins by approved drugs and other proteins of the Leishmania (Roatt et al., 2020). Their

findings identified 33 potential drugs for repurposing including perphenazine (an anti-

psychotic medicine) and rifabutin (an antibiotic used in the treatment of tuberculosis). Due to

the previously mentioned inherent advantages, steps towards drug repurposing could be the

future in the search for leishmanicidal drug candidates (Chaurasia and Jain., 2022).

2.1.4 Improving diagnostics

The diagnostic process for leishmaniasis is synonymous with a magnitude of limitations

leading to inaccurate or delayed diagnosis. To effectively control leishmaniasis early

diagnosis and consequently early treatment is incredibly important (Elmahallawy et al.,

2014). Hence, in an attempt to resolve these issues, a new RDT has been developed. The

test is based upon a recombinant antigen rK28, a synthetic polyprotein that contains rK39

repeats derived from a L. donovani strain (Pattabhi et al., 2010). These rK39 repeats are

flanked by HASPB1 repeats and the HASPB2 open reading frame, taken from a L. donovani

strain (Pattabhi et al., 2010). Preliminary investigations indicate that these rK28 RDTs show

superior diagnostic capabilities in eastern Africa over the traditional rK39 antigen RDT’s.

Some of the more recent literature by Mukhtar et al (2018) concludes that K28 RDT has

great potential to be used first in the diagnostic algorithm for VL. However, in their

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investigation, samples were provided from well-characterized controls and cases confirmed

by parasitology, leaving the specificity and sensitivity of the assay under actual conditions in

the clinical still unknown. Additionally, a comparison of efficacy of the proposed rK28 RDT

with rK39 RDT IT-Leish (Bio-Rad) would be ideal as rK39 RDT IT-Leish (Bio-Rad) is

recommend in the guidelines by the majority of eastern African governments for VL

diagnosis (Kaye et al., 2020). In order to definitively evaluate if rK28 RDT can become the

new gold standard, point-of-care diagnostic procedure, prospective large-scale evaluation is

required; AfriKADIA have planned to perform this sort of investigation using a consortium in

Ethiopia, Kenya, Sudan, and Uganda (Kaye et al., 2020). The result were expected by late

2020, however the results appear to have not yet been published.

2.1.5 New potential drug targets

There is a great incentive to research and design superior drugs with greater efficacies and

lower toxicities than current chemotherapies; new novel potential drug targets are thus a

promising candidate to help achieve this goal.

The Leishmania parasite utilises various enzymes in its metabolic pathways, which are often

genus specific; hence acting as an attractive drug target (Ramu and Singh., 2022). The thiol

metabolism pathway, which is unique to trypanosomatids (a group of protozoan parasites of

the class Kinetoplastida, which Leishmania belongs to (Kaufer et al., 2017)), has a heavy

involvement in both the maintenance of redox homeostasis and protection of the parasite to

oxidative stress (Mansuri et al., 2020). Once Leishmania enters the body during a blood

meal by the vector, an immune response is initiated, and macrophages engulf the parasite

within the host. Various reactive oxygen and nitrogen species are produced to initiate

oxidative damage. This occurs whilst the parasite proliferates and survives within the

hazardous environment of macrophage, remaining safe though the use of various reductant

enzymes to maintain redox homeostasis (Mansuri et al., 2020). One of the most significant

compounds responsible for this safety is trypanothione (TSH), a low molecular mass dithiol

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containing thiol groups that act as redox buffers to maintain intracellular redox state of

enzymes (Mansuri et al., 2020). TSH is the primary compound, universally found in

Trypanosoma and Leishmania that acts to regulate oxidative stress (Mesías et al., 2019).

Therefore, it is clear that TSH is crucial for parasite survival. Hence, enzymes of the

trypanothione generation pathway act as attractive future drug targets for new leishmanicidal

drug discovery.

Kinetoplast DNA (kDNA) is the mitochondrial genome of trypanosomatids containing over

5,000 topologically linked DNA minicircles (Onn et al., 2004), whose replication is controlled

by universal minicircle sequence-binding protein (UMSBP) (Mansuri et al., 2020). UMSBP is

a participant in the reaction mechanism of the components involved in Thiol Metabolic

Pathway (Mansuri et al., 2020). The elimination or alteration of the gene coding for UMSBP

in L. donovani has shown to result in programmed cell death like reactions, increase reactive

oxygen species and reduce parasitic survival due to inactivation of Complex 3, which is a

key contributor to the electron transport chain (Milman et al., 2007; Maldonado et al., 2021).

Therefore, UMSBP is clearly presented as a therapeutic target for visceral leishmaniasis.

Various other components of the trypanothione generation pathway that have promise as

targets for future targets including trypanothione synthetase (Ariyanayagam et al., 2005),

trypanothione reductase (Matadamas-Martínez et al., 2019), and tryparedoxin (Schlecker et

al., 2007). Drugs that act upon these thiol biosynthesis pathway targets are considered the

most promising drug target against leishmaniasis (Mansuri et al., 2020). Research needs to

be conducted to explore potential novel inhibitors against leishmaniasis through various in-

vitro approaches and ultimately largescale clinical trials. These new drug targets have great

potential to resolve some of the challenges associated with current leishmaniasis treatment,

most notably resistance/treatment failure and, due to the thiol metabolism pathway being

unique to trypanosomatids, likely toxicity also. However, the development of new drugs is

time consuming and costly.

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2.1.6 Vaccines

No current leishmaniasis vaccines are available for human use (Volpedo et al., 2021).

However, a vast amount of encompassing research has been conducted for numerous forms

of leishmaniasis in animal models including antigen identification via computational

prediction, utilising serological data (Schroeder and Aebischer., 2011) and almost all

possible vaccine delivery strategies (Majumder et al., 2014). Despite such scrupulous

research efforts, there are numerous factors hindering the successful development of a

vaccine; lack of translational funding, overuse, dependence on animal models, and the

absence of a coherent programme of advocacy for example (Kaye et al., 2020).

Nevertheless, there has been various breakthroughs. Potential vaccine candidates include

LEISHF3+ GLA-SE, a recombinant fusion derived protein with strong Th1-inducing

adjuvants (Cecilio et al., 2017); LeishDNAvax, a multi-epitope DNA vaccine (Das et al.,

2014); ChAd63-KH, a modified adenovirus-based vaccine (Osman et al., 2017); and L.

major/L. donovani centrin, a live genetically modified vaccine (Gannavaram et al., 2017).

Each of these acts as a proof of concept and has taken alternative approaches to vaccine

antigen identification and vaccine delivery methods. Hence, providing huge opportunity for

further research trials to help aid in understanding the determinants of vaccine-induced

Leishmania immunity.

In recent literature, Zhang et al (2020) built upon the afore mentioned Gannavaram et al’s

findings and used a genetically modified L. major strain, LmCen−/− in a vaccination protocol.

LmCen−/−  is a genetically engineered centrin gene knock-out mutant strain. Zhang and

colleagues’ findings indicate that CRISPR methods can facilitated the development of

Leishmania genetic mutants with precise deletions that mimic the immunogenicity and

protection effect associated with non-mutant variant L. major whilst being pathology devoid.

Therefore, indicating that a combination of old technology, leishmanization (leishmanization

being the practice of inoculation with live Leishmania to build immunity, preventing

reinfection (Mohebali et al., 2019)), and new technology (CRISPR gene editing) has great

20
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potential for major antileishmanial vaccine advancements. Moreover, preclinical trials by

Karmakar et al (2021) identified the importance of combining old and new technology. Their

study shows that genetically modified live attenuated Leishmania can promote protection

against VL. The results of Karmakar and colleagues study demonstrate that a single

intradermal injection can provide robust and durable protection against otherwise lethal

visceral leishmaniasis in Mesocricetus auratus models. Hence, current research shows

potential for a safe and effective pan-Leishmania vaccine, therefore potentially elevating

most of the challenges associated with current treatment as successful vaccination would

limit the need for treatment. Despite this, the problem of cost is still a recurring theme as is

the need for additional research and in the case of Karmakar et al’s findings, human clinical

trials also.

3.0 Conclusion

Currently, countless studies are investigating potential treatment options for leishmaniasis in

order to help improve the challenges with established treatments or to propose new avenues

of therapeutics. Though there are many promising studies, as outlined throughout this paper,

almost all require additionally research which in itself brings the challenges of loss of time

and expense. Consequently, the most attractive options for leishmaniasis treatment would

appear to be the modification of already established treatments. Hence, drug repurposing is

of particular interest as is combination therapy. These methodologies work well under time

constraints as it negates the need for extensive clinical trials testing safety due to using

already implemented drugs, thus indirectly reducing cost. However, to the detriment of drug

repurposing, there is still a need for confirmation of the effectiveness of these hypothesised

chemotherapies. The ultimate goal in leishmaniasis treatment would appear to be the

21
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development of a successful vaccine. But again, despite the promise of an effective pan-

Leishmania vaccine being developed in years to come, several considerations must be

satisfied before this is a possibility; the need for clinical trials, cost and lack of funding from

the ‘modern world’ all propose obstacles (Ngere et al., 2020).

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