European Journal of Pharmacology 910 (2021) 174436

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European Journal of Pharmacology

journal homepage: www.elsevier.com/locate/ejphar

Advancement in leishmaniasis diagnosis and therapeutics: An update

Diksha Kumari a, b, Summaya Perveen a, b, Rashmi Sharma a, b, Kuljit Singh a, b, *
a
Infectious Diseases Division, CSIR- Indian Institute of Integrative Medicine, Jammu, 180001, India
b
Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, 201002, India

A R T I C L E I N F O A B S T R A C T

Keywords: Leishmaniasis is regarded as a neglected tropical disease by World Health Organization (WHO) and is ranked
Amphotericin B next to malaria as the deadliest protozoan disease. The primary causative agents of the disease comprise of
Diagnosis diverse leishmanial species sharing clinical features ranging from skin abrasions to lethal infection in the visceral
Drug repurposing
organs. As several Leishmania species are involved in infection, the role of accurate diagnosis becomes pivotal in
Host-directed therapy
Leishmaniasis
adding new dimensions to anti-leishmanial therapy. Diagnostic methods must be fast, reliable, easy to perform,
Nanotechnology highly sensitive, and specific to differentiate among similar parasitic diseases. Herein, we present the conven­
tional and recent approaches impended for the disease diagnosis and their sensitivity, specificity, and clinical
application in parasite detection. Furthermore, we have also elaborated various new methods to cure leish­
maniasis, which include host-directed therapies, drug repurposing, nanotechnology, and combinational therapy.
This review addresses novel techniques and innovations in leishmaniasis, which can aid in unraveling new
strategies to fight against the deadly infection.

manifestation of each differs in the degree of severities, among these VL

is the most life-threatening with the highest mortality rate (Burza et al.,
1. Introduction 2019).
CL is clinically identified as prominent ulcers or lesions on the skin
Leishmaniasis, a broad range of parasitic diseases, is caused by areas (like nose, hands, forearms, legs) exposed to insect bites. Global
various Leishmania species, with clinical challenges ranging from small annual reports of CL is about 0.6–1.0 million new cases, and mainly
skin ulcers to lethal systemic infection in visceral organs, particularly >90% of the total CL cases are developed in Afghanistan, Brazil,
bone marrow, liver, and spleen. The disease is prevalent in more than 90 Colombia, Iraq, Peru, Sudan, Saudi Arabia, and Syria (Singh et al., 2016;
countries belonging to tropical and subtropical areas, including Asia, Thakur et al., 2020). The primary causative agents of CL are
Africa, America, and Europe (Steverding and vectors, 2017). Globally, L. aethiopica, L. infantum, L. tropica, and L. major in the old world or
regarded as a neglected tropical disease, this fatal parasite has infected L. amazonensis, L. braziliensis, L. chagasi, and L. mexicana in the new
more than 12 million of the population worldwide (Inceboz, 2019). world (Inceboz, 2019). In MCL, metastatic lesions occur, damaging
Yearly, 0.7–1.2 million new cases are identified and nearly 350 million mucous or soft tissues of the mouth, nose, larynx, and pharynx.
of the global population are in danger of developing this infection (Burza L. braziliensis, L. panamensis, and L. guyanensis species cause MCL, and it
et al., 2019). Leishmania is a unicellular protozoan parasite carried by is reported predominantly in Bolivia, Colombia, Ecuador, Peru, and
female Phlebotomus (old world) and Lutzomyia (new world) sand fly Paraguay (Strazzulla et al., 2013). On the other hand, VL, commonly
vectors. Around 21 different Leishmania parasite species and 30 diverse called kala-azar is a systemic infection caused by three different species
species of sand fly vector are known to transmit the infection to humans of Leishmania (L. chagasi, L. donovani, and L. infantum). It is a fatal
(Alvar et al., 2012; Sundar and Singh, 2018b). This parasite is dimorphic clinical condition, adversely affecting major visceral organs of the
in nature, which alternates between promastigote (motile form) in human host. Epidemically, VL is predominantly present in three regions:
vector and amastigote (non-motile form) residing in the infected human (A) Asia- Bangladesh, India, and Nepal; (B) Africa- Ethiopia, Kenya,
hosts (Inceboz, 2019; Torres-Guerrero et al., 2017). Clinically, this Sudan, Somalia, and Uganda; and (C) Latin America, including Brazil
infection is differentiated into four major categories, namely cutaneous and nearby states (Singh and Sundar, 2015; Sundar and Singh, 2018b).
leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), visceral leish­ As reported by WHO, 0.2–0.4 million new cases of VL were reported
maniasis (VL), and post kala-azar dermal leishmaniasis (PKDL). Clinical

* Corresponding author. Infectious Diseases Division, CSIR- Indian Institute of Integrative Medicine, Jammu, 180001, India.
E-mail address: singh.kuljit@iiim.res.in (K. Singh).

https://doi.org/10.1016/j.ejphar.2021.174436
Received 25 May 2021; Received in revised form 10 August 2021; Accepted 16 August 2021
Available online 21 August 2021
0014-2999/© 2021 Elsevier B.V. All rights reserved.
D. Kumari et al. European Journal of Pharmacology 910 (2021) 174436

Abbreviations used NO Nitric oxide

NASBA Nucleic acid sequence-based amplification
Amp B Amphotericin B NF-κB Nuclear factor-kappa
AFLP Amplified fragment length polymorphism PCR Polymerase chain reaction
CL Cutaneous leishmaniasis PI3K Phosphoinositide 3-kinase
DAT Direct agglutination test PKDL Post kala-azar dermal leishmaniasis
ELISA Enzyme-linked immunosorbent assay ROS Reactive oxygen species
ERK1/2 Extracellular-regulated kinase RT-PCR Real-time-PCR
HDT Host-directed therapy rK39 Recombinant Kinesin-39 antigen
HIV Human immunodeficiency virus RFLP Restriction fragment length polymorphism
ICT Immuno-chromatographic test SSU rRNA Small subunit of ribosomal RNA gene
IgG Immunoglobulin G TB Tuberculosis
IFN-ү interferon-gamma TNF Tumor necrosis factor
IL Interleukin Th1/2 T-helper cells type 1 and 2
LAMP Loop-mediated isothermal amplification VL Visceral leishmaniasis
MCL Mucocutaneous leishmaniasis WHO World Health Organization
NPs Nanoparticles

globally annually (Singh et al., 2016). However, in developing coun­ adopted as a way forward to fight the infection caused by Leishmania.
tries, including Bangladesh, India, and Nepal, the number of cases and
fatality rate may be more due to under-reporting (Alvar et al., 2012). In 2. Diagnosis of leishmaniasis
India, particularly Bihar and neighboring states are responsible for
80–90% of the total VL cases (Singh et al., 2016). PKDL is a The initial, simple, accurate, and reliable diagnosis is decisive for the
post-treatment complication of VL and is prevalent in India and East appropriate treatment, cure, and eradication of any disease worldwide.
Africa. It is marked by the presence of skin rashes and polymorphic le­ For the diagnosis of leishmaniasis, many diverse methodologies,
sions on the human body (Garg et al., 2018; Zijlstra et al., 2019). including parasitological, immunological, and molecular, have been
To limit the rising cases of leishmaniasis, a fast and appropriate developed (De Brito et al., 2020; Sundar and Singh, 2018b). However,
diagnosis of the infection is pivotal in defining the dosage as well as in none of them has proved to be an idealistic approach due to various
the proper management of the disease. The diagnostic methods for issues such as a broad spectrum of clinical features and varieties of
leishmaniasis should be effective in analyzing the clinical form of the species involved in the disease development, misapprehended clinical
disease, detecting the asymptomatic or co-infected cases, and suitable to features with other identical infections, along with increasing
differentiate individuals infected with other parasitic diseases (Burza co-infected cases of Tuberculosis (TB) and Human immunodeficiency
et al., 2019; De Brito et al., 2020). Conventional diagnostic methods for virus (HIV) (Hurissa et al., 2010; Lindoso et al., 2016). The snapshot of
leishmaniasis include parasitological, molecular, and immunological various challenges involved in the diagnosis of leishmaniasis is pre­
ones. In parasitological methods, the detection of Leishmania infection is sented in Fig. 1. However, with the advancement of technology various
performed by direct microscopy, histopathology, and also by parasite new approaches have developed, the following section discusses
culturing. However, parasitological methods are no longer preferred as different diagnostic methods from ancient to modern age.
they are time-consuming, require skilled technical expertise and a so­
phisticated laboratory (Maurya et al., 2010; Sundar and Singh, 2018b). 2.1. Conventional diagnostic methods
On the other hand, for detecting leishmaniasis, various polymerase
chain reaction (PCR) based molecular methods are employed, which 2.1.1. Parasitological methods
have high sensitivity and specificity (Akhoundi et al., 2017). Various The parasitological diagnosis methods detect the amastigote form of
immunological assays like direct agglutination test (DAT), the parasite in tissues (microscopic examination), isolating the pro­
enzyme-linked immunosorbent assay (ELISA), and immune chromato­ mastigote form in cultures (culturing parasites), or inoculating in mice
graphic test (ICT) also have a wide variety of applications in e diag­ model (experimental model inoculation) (Sakkas et al., 2016; Thakur
nosing Leishmaniasis (Elmahallawy et al., 2014; Singh and Sundar, et al., 2020). These methods are mainly used due to the low cost, easy
2015). After the appropriate diagnosis of infection, current procedure, and the ability to detect parasites at the genus but not the
anti-leishmanial chemotherapy includes antimonials, amphotericin B species level. In the microscopic examination method, the amastigote
(Amp B), miltefosine (first oral drug), paromomycin, pentamidine, and a form of the parasite is obtained from skin ulcers for CL. Aspirates from
combination of these drugs (Uliana et al., 2018). However, treatment of bone marrow, spleen, or lymph nodes are used for VL diagnosis
leishmaniasis infection remains a global challenge due to severe issues (Elmahallawy et al., 2014), which are further subjected to staining using
like side effects, toxicity, reduced therapeutic arsenal, and drug resis­ dyes such as Giemsa, Panotic, and Wright (De Brito et al., 2020). These
tance (Mandal et al., 2017; Ponte-Sucre et al., 2017). Thus, to combat stained amastigotes are seen as round or ovoid bodies, also called as
treatment failure globally, it is requisite to explore different new stra­ Leishman-Donovan bodies, with a diameter of 2–4 μm, along with
tegies and alternative treatments. prominent nuclei and kinetoplasts (Fig. 2). In the case of CL and MCL,
In the present review, we collated and discussed the conventional as direct visualization of amastigotes from skin lesions isolated by dermal
well as recent advances in diagnosis and treatment strategies against scrapping and biopsied material from tissue (histopathology) is a com­
leishmaniasis. First, we have shed light on the new diagnostic methods mon practice (de Vries et al., 2015). In one study, 40 biopsied materials
focusing on their specificity and sensitivity, and clinical application in were evaluated histopathologically from CL patients, recognized unique
parasite detection. Next, we have evaluated the recent developments in patterns containing macrophages and granuloma at different stages
anti-leishmanial chemotherapy. This study would give an insight into (Handler et al., 2015). For MCL, in a case study, microscopic examina­
the current diagnostic approaches with the available treatment options. tion revealed the presence of amastigotes in patient with oral leish­
It would instill discussion on the alternative novel strategies to be maniasis. Finally, the diagnosis was confirmed by

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D. Kumari et al. European Journal of Pharmacology 910 (2021) 174436

Fig. 1. Various challenges involved in the diagnosis of leishmaniasis.

Fig. 2. Visualization of Leishman-Donovan bodies (amastigotes) using microscopy.

immuno-histochemical analysis (Almeida et al., 2016). However, sam­ infection. The sensitivity in peripheral blood smears is even lower
ple extraction by aspirates often becomes invasive and lethal; therefore, (51–53%), especially in individuals with low parasitic load in the blood
press-imprint-smear and tape strip disc methods can be a better alter­ (Elmahallawy et al., 2014). However, these methods are cost-effective
native to conventional painful sampling techniques. In a study, when but require well-trained technicians, laboratories, facilities for blood
press-imprint-smear, was compared with histopathological based sam­ transfusion, nursing surveillance, and surgery. However, due to deadly
pling, press-imprint-smear displayed a positivity in ~85% cases sus­ after effects, researchers search for sampling methods that are less
pected for CL, whereas positivity of ~44% was recorded in the case of painful, patient-friendly, and more effective. In this regard, Taslami’s
histopathology making the former being more sensitive approach for the group (Taslimi et al., 2017) in their study, employed a non-invasive tape
diagnosis of CL (Sousa et al., 2014). Additionally, flinders technology strip sampling method that is quick (within a minute), painless, and
associates filter paper cards have also been proved to be a successful budget-friendly. They collected samples from both infected and recov­
non-invasive sampling approach for diagnosis of CL which further uti­ ering dermal lesions of the patient having CL and other related in­
lizes PCR amplification for the analysis of CL and MCL infections fections. Further using PCR amplification, a 100% sensitivity rate was
(Miranda et al., 2012). reported for the diagnosis of CL. Comparative analysis of diagnostic
For diagnosis of VL, these aspirate samples are highly specific accuracy of cultured parasite obtained from biopsied material and
(100%) but their sensitivity varies depending upon the organ from sample extracted from tape strip disc displayed the sensitivity of 51%
which the sample is acquired (53–99%) (Singh and Sundar, 2015). and 100%, respectively. Therefore, the study concluded that the
Various detection methods, including the source of the test sample, test non-invasive tape strip assay presents several advantages including
time, sensitivity, and specificity, are summarized in Table 1. Spleen higher sensitivity rate, avoiding use of needles, syringes, least require­
sample being the most sensitive of all is used as the gold standard in ment of skilled expertise and efficient monitoring of treatment progress.
significant parts of the endemic areas. It can, however, result in deadly To further increase the sensitivity, the clinical sample can be inoc­
hemorrhages in patients if done by unskilled persons (Sakkas et al., ulated in the culture medium such as Schneider’s insect medium and
2016; Sundar and Singh, 2018b). Bone marrow aspirates isolated from diphasic culture medium (Evans modified Tobie’s or Novy-McNeal-
the iliac crest or sternal puncture are safer but require painful precision. Nicolle medium) along with 5–10% fetal bovine serum (Sakkas et al.,
The sample collection from lymph nodes is comparatively more acces­ 2016). Amastigotes are transformed into promastigotes by vector-like
sible but has lower sensitivity (53–65%) than spleen and bone marrow environmental conditions provided by highly enriched media. Promas­
(de Vries et al., 2015; Singh and Sundar, 2015). togotes were grown in 7–21 days by incubating the parasite culture at
Moreover, the result shown may not be good enough in case of less 24–26 ◦ C in the incubator. These cultured parasites can be used further

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D. Kumari et al. European Journal of Pharmacology 910 (2021) 174436

Table 1
Various diagnostic methods for visceral leishmaniasis along with the source of the test sample, test time, sensitivity, and specificity percentages.
Methods Sample source Test Sensitivity Specificity Inference REF.
time (%) (%)

Parasitological Spleen aspirates Spleen Hours 93–99% 100% A cheap method, the parasite can be (Elmahallawy
methods directly visualized, most sensitive, and et al., 2014; Singh
remains gold standard but require proper and Sundar, 2015)
expertise to take a sample
Bone marrow Bone marrow Hours 60–85% 100% Moderate sensitivity and also associated (De Brito et al.,
aspirates with a high risk of hemorrhage 2020; Thakur et al.,
2020)
Lymph nodes Lymph Hours 53–65% 100% Lower sensitivity percentage and requires Sakkas et al. (2016)
aspirates skilled health worker for sampling
Buffy coat from the Spleen or bone Days 51–53% 100% Restricted to endemic areas and requires (de Vries et al.,
peripheral blood marrow sophisticated laboratories and skilled 2015; Singh and
expertise Sundar, 2015)
Immunological Montenegro test Lesion (as a result of Days 75% 100% Can detect asymptomatic cases but is Srivastava et al.
methods hypersensitivity) time-consuming and require technique (2011)
experts
DAT Serum/Plasma Hours 96% 95% Semi-quantitative, rapid, cost-effective Mohebali et al.
method but cannot differentiate present (2020)
and post-infection or treatment relapse
Fluorescent Serum/Plasma Hours 91% 81% Fast and sensitive approach but requires Sarkari et al.
antibody test proper facilities and fluorescent (2014)
microscope
Indirect Serum/Plasma Hours 90–100% 86% High sensitivity percentages but may vary Sakkas et al. (2016)
hemagglutination rendering to immune response
assay
ICT Blood/Saliva 10–15 91.5% 89% Rapid, easily accessible strip test with Herrera et al.
min minimum analysis time but however lack (2019)
the differentiating capability between
current infection and clinical relapse
KAtex (Antigen Urine Hours 47–95% 82–100% An easy, cost-effective approach can (Akhoundi et al.,
detection) detect the present and the post-infection 2013, 2017)
but has lower sensitivity as compared to
other methods
ELISA Crude Serum/Plasma/ Hours 80–100% 84–94% Fast, sensitive approach and can screen Ryan et al. (2002)
soluble Saliva large population. Also, the use of a vast
antigen variety of antigens made this technique
ESM Hours 71–89% 91% highly sensitive in endemic and non- (Soares et al., 2015;
antigen endemic regions of the world. However, it Thakur et al., 2020)
rKLO8 Hours 98% 96% is technically demanding thus cannot be (Abass et al., 2013;
antigen used in a field setting, and cannot detect Sakkas et al., 2016)
rK39 Hours 98% 97% clinical relapse cases Salam et al. (2021)
antigen
rK28 Hours 98% 94% Salam et al. (2021)
antigen
rK9 Hours 78–80% 84% (Mohapatra et al.,
antigen 2010; Sakkas et al.,
2016)
rA2 Hours 91.5% 87% dos Santos Maciel
protein et al. (2019)
LiHypA Hours 100% 98% Carvalho et al.
(2018)
Molecular Conventional PCR Blood/serum/buffy Hours 70–100% 60–100% A highly sensitive and specific diagnostic Lemrani et al.
Methods coat cells/buccal method with early and easy results (2009)
Nested and semi- swab/urine Hours 97% 100% Increased sensitivity and specificity as (Gedda et al., 2021;
nested PCR compared with conventional PCR and Salam et al., 2010)
reduce non-specific binding. But it is a
non-quantitative approach and expensive
RT-PCR and Hours 90–100% 83–100% A highly sensitive and specific method (Cota et al., 2013;
Multiplex PCR with reduced risk of contamination. Can Moreira et al.,
amplify several targets at the same time 2018)
using multiple primers
NASBA -PCR Hours 60–95% 70–100% Rapid analysis technique having optimum (Saad et al., 2010;
sensitivity and specificity. But it is unable van der Meide
to differentiate between different parasite et al., 2005)
species
RFLP and AFLP Hours 92–100% 100% Highly reliable and reproducible (Mohammadi et al.,
techniques effective to identify the 2017; Restrepo
genetic variation in the strain. However, et al., 2013)
is costly and time taking
LAMP Hours 80–96% 86–100% Less time required, budget-friendly, (Nzelu et al., 2019;
highly specific, and effective Prajapati and
amplification Mehrotra, 2013)

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D. Kumari et al. European Journal of Pharmacology 910 (2021) 174436

for molecular identification of species, as an antigens source for immune from 12 to 18 h (Silva et al., 2005). With the high range of sensitivity and
techniques, animal injection, and high-throughput screening of drugs. specificity values of 91–95% and 70–88%, respectively, the method is
But parasite culturing is monotonous, time-consuming, has a high risk of used to screen the larger population at one go, reducing cost per patient
contamination, and requires sophisticated laboratories and technical (Hailu et al., 2006).
expertise (de Vries et al., 2015; Kumar et al., 2020). Therefore, this A fluorescent antibody test or immuno-fluorescence assay is a fast
technique is not an ideal approach for field diagnosis, and it mainly opts and precise method. A fluorescent probe is attached to an antibody that
for the research laboratories. Laboratory animals such as mice, golden binds to the target and forms the resultant molecule. As reported,
hamsters, guinea pigs, or rodents can also be used to diagnose Leish­ sensitivity and specificity values of this assay for CL are 91% and 81%,
mania parasite infection (De Brito et al., 2020; Thakur et al., 2020). In respectively (Sarkari et al., 2014). It is also a vital method to distinguish
this method, parasites isolated from clinical specimens are injected in between present infection and clinically cured patients (Reimão et al.,
the tail base, nose, or footpad of the model organism for diagnosis of CL, 2020). This assay has a unique ability to spot the occurrence of anti­
or intravenously or intraperitoneally in mice or golden hamsters for bodies (low titers) in the serum of VL patients in case of treatment
diagnosis of VL (Reimão et al., 2020). This technique is rarely used for relapse. However, trypanosomal serum cross-reactivity can be mini­
diagnostic purposes as it may require several days or months to examine mized using promastigotes as antigens by replacing amastigotes (Rezvan
the resultant infection in the animal model. et al., 2017). Immuno-fluorescence assay can be performed directly
when the labeled antibody (primary) binds to the target (antigen) or
2.1.2. Immunological methods indirectly where the secondary polyclonal antibody binds to the patient
Immunological methods are rapid, non-invasive, and detect Leish­ serum (Thakur et al., 2020). The need for proper facilities and a fluo­
mania antigens or antibodies in human patients. Herein, we will be rescent microscope are major pitfalls of this test.
discussing various immunological methods used for the diagnosis of Sensitized human erythrocytes derived from Leishmania antigens are
parasite infection along with their applications, sensitivity, and speci­ used in sensitive indirect hemagglutination assay, a susceptible method
ficity ranges. Leishmania skin test or Montenegro test relies on the idea of used for detecting anti-leishmanial antibodies (Reimão et al., 2020).
type IV hypersensitivity or delay hypersensitivity in cutaneous and This approach sensitivity and specificity percentages range from 90 to
mucocutaneous infections. Infections in epidemiological areas with 100% and 86%, respectively (Kumar et al., 2020) but may vary
greater sensitivity than that of serological techniques highly favor the depending on human host immune responses (Sakkas et al., 2016). In­
detection of asymptomatic patients (Kumar et al., 2020). However, direct hemagglutination assay can be implemented to diagnose VL pa­
glandular tuberculosis and lepromatous leprosy showed some tients in non-endemic areas, where the VL cases are limited (Iqbal et al.,
cross-reactivity (Singh, 2003). Leishmania antigen is injected within the 2002). But this assay is not suggested as the solitary screening approach
layers of the skin to perform the Montenegro test, which consists of a for VL as even after recovery the clinical samples show high titer (Kumar
suspension of attenuated parasites or disordered promastigotes in a et al., 2020).
pyrogen-free phenol saline solution (Bettaieb et al., 2020). Greater than ELISA is one of the primary diagnostic approaches for major in­
5 mm induration of skin is considered positive, and size less than 5 mm is fections because of its highly sensitivity and specificity. In the serodi­
considered negative after 48 h of injection. It is an easy, budget-friendly agnosis of leishmaniasis, ELISA plays a crucial role in screening vast
test with high sensitivity (86–100%) and specificity (>90%) (De Brito samples by using different antigens (Deepachandi et al., 2019). How­
et al., 2020). The test shows negative results for VL during early infec­ ever, the type of antigen used describes the sensitivity of the assay. Many
tion and later turned positive after treatment, thus indicating the effi­ antigens are being used, crude soluble antigen being the conventional
cacious clinical cure (Reimão et al., 2020). It also shows the occurrence one prepared by freeze-thawing Leishmania promastigotes in
of CL and MCL forms but not for PKDL as a result may not be associated phosphate-buffered saline. As reported, this method’s sensitivity and
with the presence of infection. However undistinguishable nature be­ specificity values using crude soluble antigens range from 80 to 100%
tween the present and past disease is still the major downside. and 82–95%, respectively (Kumar et al., 2020). Although,
DAT is an easy, rapid, cost-effective, and semi-quantitative assay for cross-sensitivity in some patients with tuberculosis, trypanosomiasis,
detecting VL. As reported, high sensitivity and specificity values asso­ and toxoplasmosis has also been observed (Elmahallawy et al., 2014;
ciated with this assay make it perfect for fieldwork and laboratory Herrera et al., 2019; Kumar et al., 2020). Though immense studies have
studies (do Vale et al., 2020). In a recent meta-analysis, 2928 records been reported for its high sensitivity, its application is still limited
from 2004 to 2019 reported that diagnostic sensitivity and specificity because of the requirement of proper equipment, well-established lab­
rates of DAT were 96% and 95%, respectively (Mohebali et al., 2020). oratories, and trained technicians. Moreover, the ability to differentiate
Antigen-antibody interactions assays are functional, where promasti­ between present infection and possible recovery is still lacking.
gotes treated with trypsin, stained with Coomassie brilliant blue are ICT using recombinant Kinesin-39 antigen (rK39) is a non-invasive
used as antigens, and the patient’s whole blood/serum/plasma is rapid screening test for VL. It is useful for field purposes in endemic
checked for the presence of antibodies. The assay is performed in a and non-endemic regions due to its low cost and minimum analysis time.
microtiter plate and the existence of a specific antibody in the collected In this strip test, rK39 immobilized on nitro-cellulose paper in a band
sample leads to agglutination (do Vale et al., 2020; Humbert et al., form along with colloidal gold is used for detection purposes. Serum or
2019). Previously, the used liquid antigen sample in this test had various blood from the suspected patient and buffer is smeared on the strip, and
issues such as short life span, thermolabile, and required cold chain the color development can be visualized in 10–15 min (Karimi Kakh
treatment (Kumar et al., 2020). Nowadays, to improve the quality of et al., 2020). A pictorial representation of the ICT approach is shown in
antigen, a heat-resistant freeze-dried form is developed, which is stable Fig. 3. The sensitivity and specificity values have shown huge variation
at room temperature for two years (Elmahallawy et al., 2014). The among the different epidemiological populations. A study by Bangert’s
major problem associated with this method is that it remains positive group (Bangert et al., 2018) showed that in the Mediterranean region,
even after a year of recovery, thus it is questionable for the diagnosis or sensitivity with using the rK39 strip test was found to be 78% in all VL
cure in treatment relapse cases (Srivastava et al., 2011). Also, multiple patients, and immuno-compromised individual’s sensitivity rates were
pipetting during serial dilutions, unabridged antigen, and a long incu­ further reduced to 67.3%. In another study by Herrera’s group (Herrera
bation period (12–18 h) are some of the significant disadvantages of the et al., 2019), sensitivity and specificity rates of rK39 strip test performed
DAT technique (Kumar et al., 2020). To overcome the drawbacks of in endemic areas of Colombia were reported to be 91.5% and 89.2%,
DAT, an advanced form of DAT i.e., fast agglutination screening test has respectively. A comparative study of rk39 strip test, ELISA, and indirect
been established. This method involves one-time serial dilution and an fluorescent antibody test for the serodiagnosis of VL in Morocco (North
incubation period of 3 h compared to the conventional time ranging Africa) reported the highest sensitivity of 95.5% for rk39 strip test,

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D. Kumari et al. European Journal of Pharmacology 910 (2021) 174436

Fig. 3. Representation of various components of

ICT strip. An immuno-chromatographic strip com­
prises a sample pad, nitrocellulose membrane,
conjugate pad, and an absorbent pad. The blood
sample is loaded onto the sample pad then a buffer
solution is added for the smooth flow of the sample.
The membrane of the strip is loaded with the anti­
gen/protein (rK39 or rK16 or rK26) on the test line.
As the blood sample flows through the membrane, if
antibodies are present in the patient sample, we will
observe a colored line on the test lane. A colored
line must be kept in the control lane for validation
purposes. The absorbent pad acts as a sponge to
absorb the sample entering the strip.

followed by indirect fluorescent antibody test (87.5%) and ELISA with specificity along with the possibility of analysis of hundreds of samples
the lowest sensitivity rate of 75% (Mniouil et al., 2018). In the Indian simultaneously (Akhoundi et al., 2017). Numerous molecular methods
subcontinent, another rapid test was developed using rKE16 antigen as a for identification detection, quantification, and ethnological analysis
base from the Indian strain of L. donovani. It showed the sensitivity range have been developed (Mouttaki et al., 2014). The blood sample (general
92–100% in the Indian population, but their sensitivity range (36–92%) population) was of higher sensitivity and specificity, whereas there was
significantly altered in individuals from East Africa and Brazil (Abass minimal variation in efficacy among buffy coat, whole blood, or bone
et al., 2013). Recently, numerous recombinant antigens, including marrow samples. Therefore, an easily extracted blood sample would
rKRP-42, rK26, rK9, rK26, and rKE16, have been used for serodiagnosis serve the purpose instead of more invasive bone marrow samples (De
of VL, but recent WHO reports suggest that the rK39 antigen strip test is Ruiter et al., 2014). Although many molecular diagnostic techniques
the most effective one (Kumar et al., 2020). Maude’s group (Lévêque have been developed, PCR-based assay constitutes the gold standard of
et al., 2020) evaluated six marketed kits for the immunological analysis molecular diagnosis for scientists and health professionals (Akhoundi
of VL in Mediterranean regions. It concluded that TruQuick is the most et al., 2017). We will enhance our understanding of the conventional
efficient commercial kit having 90.1% sensitivity and 95.7% specificity. molecular method to the most recent molecular approaches (in the
But like most of the serodiagnosis techniques, the ICT approach also current advances section).
lacks in differenciating between current infection and cured cases, Conventional PCR is a quick, sensitive, and multipurpose in vitro
which makes it doubtful to be used as an ideal diagnostic procedure. approach for amplifying the defined target of DNA or RNA through re­
Latex agglutination test (KAtex) is a non-invasive method of petitive cycles (Sundar and Singh, 2018b). The readily available data­
detecting antigens in the urine of VL patients. It can be used as an bases with genome sequencing of most of the Leishmania species,
alternative approach to antibody detection methods where antibody consisting of overall conservation of gene order, chromosome number,
production is lower, such as immuno-compromised individuals (Leish­ and structure, and distinct variability in gene content, made it easier to
mania and HIV co-infection) (Rezvan et al., 2017). This test uses a boiled execute the available molecular techniques efficiently and to develop
sample of urine that detects thermal resistance, low molecular weight more rapid diagnosis devices for field works (Sundar and Singh, 2018b).
(5–20 kDa) carbohydrate-based leishmanial antigen-specific to VL pa­ A study done to compare conventional parasitological methods with that
tients (Gedda et al., 2021). The test is also helpful for examining the of PCR using a small subunit of ribosomal RNA gene (SSU rRNA)
treatment progress as the antigen can be detected from one to six months revealed that PCR based approach reported higher sensitivity (84.6%) in
after treatment. It can also be beneficial for recognizing preclinical comparison with direct microscopy (69.2%) and in vitro culture (69.2%)
infection (Chappuis et al., 2007). As reported, the sensitivity and spec­ techniques (Lemrani et al., 2009). The main disadvantages of conven­
ificity of KAtex range from 47 to 95% and 82-10%, respectively (Sakkas tional PCR include the requirement of skilled expertise, contamination
et al., 2016). However, in the case of immuno-compromised individuals, risk, and unable to differentiate between present infection and treatment
improved sensitivity (85–100%) and specificity (96–100%) values were relapse (Sakkas et al., 2016).
observed (Reimão et al., 2020). This method is easy, cost-effective, avoid The use of nested and semi-nested PCR has significantly increased
cross-reactivity and can differentiate between current infection and sensitivity and specificity by reducing the non-specific amplification of
clinical cure but major drawback includes moderate sensitivity and false DNA. It is mainly advantageous for detecting a low level of parasitemia
results if the samples are not boiled properly (Chappuis et al., 2007; (Deepachandi et al., 2019). In this method, two primers sets are being
Elmahallawy et al., 2014). employed in two PCR reactions, first followed by the second reaction. In
the primary reaction protocol, one primer set binds to generate a DNA
2.1.3. Molecular methods product with non-specific amplicons and the intended target. The other
The current immunological methods pose several limitations, such as primer amplifies the secondary DNA target-present within the first PCR
low antibody titers, low sensitivity, inability to diagnose asymptomatic product (Hossain et al., 2017). Salam’s group (Salam et al., 2010), in
patients and detect treatment relapse, etc. (Kitano et al., 2021; Sagi their studies for the diagnosis of VL using SSU rRNA, reported superior
et al., 2017; Varani et al., 2017). With the advent of molecular biology sensitivity (97%) and specificity (100%) of nested PCR compared to the
practices, the diagnosis of leishmaniasis has also been revolutionized. conventional parasitological assays. But unfortunately, nested PCRs are
Molecular biology techniques are an effective alternative to conven­ prone to contamination risks and thus, require well-established
tional parasite detection. It comes up with higher sensitivity and laboratories.

6
D. Kumari et al. European Journal of Pharmacology 910 (2021) 174436

Real-time-PCR (RT-PCR) is the modified PCR amplification method of target sequence using random short primers without having prior
that amplifies and simultaneously quantifies the target DNA molecule. It knowledge of the genome to be analyzed (Akhoundi et al., 2017). In the
is a rapid quantitative method with high sensitivity and reduced risk of recent study, an unusual CL and ocular leishmaniasis case was reported
contamination as there is no requirement to open the reaction tubes whose causative agent was L. major, which was identified by using this
(Galluzzi et al., 2018). In this method, the amplified product can be approach (Doroodgar et al., 2017). Since it has poor reproducibility and
visualized employing dyes or fluorescent probes. A dye mainly used is requires pure Leishmania DNA, it cannot be used in field of diagnosis
SYBR Green I, which non-specifically binds to the target DNA. Alter­ (Akhoundi et al., 2017). In the Restriction fragment length poly­
natively, a sequence-specific fluorescence probe that specifically binds morphism (RFLP) method, an amplified DNA from the sample undergoes
to the internal region of the PCR product is used, such as a TaqMan digestion using restriction endonuclease and generates species-specific
probe. The TaqMan is degraded during amplification, releasing a re­ size fragments. These are then analyzed on an agarose gel electropho­
porter that emits fluorescence which can further be analyzed (Paiva-­ resis. The resultant PCR-RFLP banding patterns confirm the presence of
Cavalcanti et al., 2010). The performance of qPCR is influenced by Leishmania species in the respective sample (Montalvo et al., 2010).
sample source (bone marrow, blood, skin, or splenic fluids), DNA PCR-RFLP shows superior sensitivity compared to other parasitological
extraction procedures (manual or kits), primer sets, and SYBER Green or methods, particularly for samples with low parasitic load (Blaizot et al.,
TaqMan probes (Sundar and Singh, 2018b). Ghosh’s group (Ghosh et al., 2021). Ribosomal DNA locus (ITS1 and ITS2), hsp70 gene, mini exon for
2018) evaluated the PKDL in the endemic region of Bangladesh using the nuclear DNA, and minicircles for Kinetoplast DNA are the prime targets
RT-PCR approach showed a high sensitivity of 91.2% as compared to for this approach (Reimão et al., 2020). Mohammadi’s group (Moham­
microscopy with a lower sensitivity of 50.6%. American tegumentary madi et al., 2017) in a comparative study using different PCR-based
leishmaniasis validated in patients by RT-PCR assay targeting HSP70 approaches, concluded that PCR-RFLP can discriminate between
and 18S rDNA. The assay employing HSP70 showed increased speci­ various leishmanial species efficiently. Similarly, Espada’s group
ficity, while the 18S rDNA target has higher sensitivity. When RT-PCR (Espada et al., 2018), using heat shock protein 70 (Hsp 70) described the
assays were analyzed combinedly, results revealed 100% net speci­ importance of the PCR-RFLP method in the identification of species and
ficity and ~98% net sensitivity (Filgueira et al., 2020). However, this spotting intra-species difference ((L. (Viannia) and L. (L.) amazonensis
technique can only be beneficial for identification and quantification in species)) in the clinical sample from Brazil. Another highly reliable and
general but not for the identification of species in particular (Galluzzi reproducible PCR technique extensively available for DNA finger­
et al., 2018). printing of Leishmania species is amplified fragment length poly­
Multiplex PCR amplifies several different DNA targets simulta­ morphism (AFLP). It synergizes RFLP with the applicability of
neously by engaging multiple primer sets and a temperature-regulated PCR-based tools by ligating primer sequence to the target DNA. Here,
DNA polymerase in a thermo-cycler (Giantsis et al., 2017). It com­ Amplification of specific restriction fragments occurs using particular
prises several primers combined in a single PCR mixture and produced primer set (Thakur et al., 2020). The PCR product is segregated and run
varied length amplicons pertaining to specific DNA sequences. It is to be on gel electrophoresis to check the polymorphisms. It is an effective
noted that there should be optimization of annealing temperatures for identification tool to explore genetic variation in closely related species
the primers for efficient working in one reaction, and the amplicon sizes and different strains of Leishmania (Kumar et al., 2010). It allows an
diversity must be considered to visualize them by gel electrophoresis accurate genomic analysis of the various genus of the Leishmania para­
(Akhoundi et al., 2017). In their studies using the multiplex PCR site, distinguishing major groups and extremely close species like
method, Cassia-Pires’s group (de Cássia-Pires et al., 2017) recognized L. guyanensis and L. panamensis (Restrepo et al., 2013).
the presence of Kinetoplast DNA and GAPDH gene in the tissue isolates
from diverse mammalian species. The presence of the mammalian 2.2. Recent advances in diagnostic methods
GAPDH gene in the reaction is considered an internal control. However,
the reproducibility was tested using different tissue aspirates from 106 2.2.1. Fluorescence-activated cell sorting
wild mammals which reported that 51 samples tested positive while the Flow cytometry is a practical approach for the assessment of the
other 55 were tested negative. Although the multiplex PCR approach has diagnosis and effective VL treatment. This serological method has
improved the sensitivity as compared to the conventional PCR but due to become incredibly beneficial for its clinical applicability in health cen­
its high run cost it is not recommended in routine diagnosis (Sundar and ters and research laboratories for being quick, precise, and reproducibile
Singh, 2018b). (Ker et al., 2013). This assay is convenient for evaluating particular
Nucleic acid sequence-based amplification (NASBA) is a PCR-based protein expression, cell viability, apoptotic cell death, cell to cell in­
technology that utilizes the function of an RNA polymerase to make teractions, and cell enhancement which makes it suitable for screening
RNA and reverse transcriptase to form DNA from the RNA templates. and diagnosis (Abraham et al., 2016). Pedral-Sampaio’s group
The NASBA products can be identified using colorimetric assay, gel (Pedral-Sampaio et al., 2016) evaluated the flow cytometry technique to
electrophoresis, and fluorescence probes (Gill et al., 2008). It has two detect the Immunoglobulin G (IgG) employing polystyrene globules
variants, namely quantitative-NASBA and oligo-chromatography- sensitized with soluble Leishmania antigen in the human sample and
NASBA. Meide’s group (van der Meide et al., 2005) in their studies equated (compared) the result with the ELISA using similar antigen
used the quantitative-NASBA method to quantify parasites of Leishmania preparation for assay validation. The study concluded flow cytometry to
in samples of biopsied skin from CL patients, targeting SSU rRNA as a be highly sensitive (100%) but have low specificity as compared to
gene of interest. Interestingly, this approach was able to detect 0.1 ELISA (Pedral-Sampaio et al., 2016). Many scientists foresee flow cy­
parasites per amplification reaction compared with conventional PCR tometer as a novel tool in serology to be widely used for common pur­
having 10 parasites per amplification limit. Thus, it can be concluded poses in the near future (Kumar et al., 2020).
that quantitative-NASBA is more sensitive than conventional PCR.
Saad’s group (Saad et al., 2010) in their studies analyzed the diagnostic 2.2.2. Proteomics
accuracy of different clinical forms of leishmaniasis (CL, VL, PKDL) in The analysis of the whole protein complexes expressed in an or­
Sudan using oligo-chromatography-NASBA and OligoC-TesT. Both the ganism, tissue, or cell in a determined state of conditions is called pro­
test displayed high sensitivity (>90%) in blood, lymph and tissue teomics (Yu et al., 2010). These complex protein complements are
scraping samples but the specificity of the quantitative-NASBA approach generally analyzed by mass spectrometry-based technology. It allows
for VL diagnosis was significantly higher (>95%). However, this tech­ the analysis of protein abundance, post-translational modifications,
nique is unable to differentiate various Leishmania species. protein interactions, structure, and distribution of proteins within the
Randomly amplified polymorphic DNA is a method of amplification cell. The proteomics analysis strategy comprises extracting, separating,

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D. Kumari et al. European Journal of Pharmacology 910 (2021) 174436

identifying, and quantifying the protein. The separated proteins or electrophoresis of several intracellular enzymes called isoenzyme (Cec­
peptides are subsequently ionized by Matrix-assisted Laser Desorption carelli et al., 2018). With the help of a suitable set of isoenzymes, a
Ionization - Time of Flight Mass Spectrometry (Capelli-Peixoto et al., typical mobility pattern that is strain-specific or species-specific is ach­
2019). This approach is a quick and reliable platform for parasite ieved called electro-morphs. The unique profile of electro-morphs pro­
identification. The process starts with the ionization of a clinical spec­ duced for each organism’s strain is called an electro-morphic type
imen in a particular acidic solution by using laser beams. The ions are (Caierão et al., 2016). According to WHO, this is the preferred method of
further desorbed from the solution/matrix and fast-tracked at a fixed choice for Leishmania species typing (Van der Auwera and Dujardin,
potential via a vacuum flight tube that contains a system of detection, 2015). However, various species and subspecies of Leishmania parasite
measuring the time of flight for particular ions. The Time of Flight value are distinguished and identified by multilocus enzyme electrophoresis,
corresponds to the molecular weight of the molecule ionized which is an efficient and robust method. Its use is limited due to high
(Caraballo-Guzmán et al., 2021; Culha et al., 2014). Based on the time of cost, need for a large amount of protein and culture, less distinction
flight, a unique characteristic pattern is generated and compared with an power, and requirement of equipped laboratories (Moffatt, 2016; Sun­
existing mass spectra database for identification. Hence, it allows a dar and Singh, 2018b; Tsokana et al., 2014).
high-resolution evaluation by employing multiple samples at once. Multilocus sequence typing is a promising technique that can accu­
Jaiswal’s group (Jaiswal et al., 2020) detected PKDL disease-specific rately identify various subgenera, species complexes, and species of the
glycated protein biomarkers, exploiting the major liquid genus (Lauthier et al., 2020). In this typing process, multiple unlinked
chromatography-mass spectrometry platform, proving a good method to housekeeping genes (6–8 genes) are amplified using the PCR amplifi­
identify and quantify protein biomarkers specific to a particular infec­ cation technique and subsequently analyzed by DNA sequencing
tion. Similarly, plasminogen and vitronectin proteins were identified as (Akhoundi et al., 2017). The rationale behind the process is to recognize
possible promising biomarkers for PKDL diagnostics. These novel iden­ internal nucleotide sequences (approximately 400–500 bp) in these
tified glycoproteins that may be employed in forthcoming as housekeeping genes. Exclusive sequences (unique) named alleles are
immuno-chromatographic based methods in effective disease diagnosis allocated a random mathematic integer. A unique pair of alleles at each
(Jaiswal et al., 2020). Immuno-proteomics is a technique used to detect locus is termed as allelic profile, which signifies the sequence type
the subset of proteins allied with the infection that induce immune re­ (Larsen et al., 2012). A comparative study using seven different markers
sponses. It is performed using various methods such as protein separa­ with a multilocus sequence typing approach reported that L. panamensis
tion, immunological detection (western blotting), and Mass has lower genetic diversity than L. braziliensis. The concluded results are
Spectroscopy (Ohyama and Kuroda, 2013). In this method, protein based on haplotype variations pertaining to different genes (Herrera
extracted from different cells is separated by 2-D electrophoresis. Then et al., 2017). Similarly, multilocus sequence typing successfully reported
the segregated proteins are immobilized on a nitrocellulose membrane genetic variability between L. tropica and L. major strains isolated from
by western blotting technique. The patient serum is then applied to the CL patients in Iran (Hosseini et al., 2020). Though this technique is
blot. The antibody specific to the disease will bind to the respective prevalent but it’s expensive, requiring expertise and proper laboratory
antigenic protein and the corresponding secondary antibody, which is analysis. Moreover, the sequence is conserved in housekeeping genes,
enzyme-labeled, will bind to the primary antibody (patient serum). The thus reducing the discriminatory power to differentiate various Leish­
bounded spots are cut out and undergo in-gel digestion by protease. MS mania strains (Hosseini et al., 2020). Furthermore, the development in
then analyzes the formed fragments and peptide fingerprinting for molecular techniques will provide ample opportunities to scientists for
immunogenic proteins (Tjalsma et al., 2008). Machado’s group improved diagnostic and treatment methods.
(Machado et al., 2020) presented a study to identify a novel protein
target for the diagnosis of VL and immuno-compromised individuals 2.2.4. Nano-diagnosis
employing immuno-proteomics assay in L. infantum antigenic extracts. Nanotechnology has become a promising technique for the diagnosis
Similarly, Caraballo-Guzmán’s group (Caraballo-Guzmán et al., 2021) and drug delivery system nowadays. In diagnosing leishmaniasis, the
identified the alleged protein 3-oxoacil (acyl transporter proteins) combination of nanoscience and nanotechnology has become the pre­
reductase from L. panamensis strain, which is explicit for MCL. Further, it vailing molecular method (Moffatt, 2016). The system such as quantum
will be explored as a potential disease-specific diagnostic marker. dots, magnetic beads, metal nanoparticles (NPs), nanodiscs, stable lipid
NPs, polymer NPs, and inorganic compounds has found optimistic out­
2.2.3. Advanced molecular methods comes in detecting the Leishmania promastigotes in vivo as well as in
The conventional method has helped in diagnosis; meanwhile, the vitro. NPs-based biosensors have also proven to be budget-friendly in
advancement in science has led to improved methods applying the basic identifying pathogens both biologically and in medical samples (Gedda
concepts of molecular technology. Loop-mediated isothermal amplifi­ et al., 2021). Using DNA based nano-diagnostic approach Toubanaki’s
cation (LAMP) is one such methods. It is a fast-performed and innovative group (Toubanaki et al., 2016) employed Kinetoplast DNA for the
method based on isothermal conditions (using heat block or water bath). species-specific amplification product detection of Leishmania species in
Therefore, it does not require thermocyclers (Bezerra et al., 2020). The canine samples. They developed the nucleic acid-lateral flow biosensor
amplified fragments can be perceived visually by changing color, fluo­ in association with gold NPs. The intense red color in the strip indicates a
rescence, and turbidity (Sriworarat et al., 2015). In the VL sample from positive signal showing the hybridization of the target DNA (poly dA
Ethiopia; LAMP test using whole blood, PBMC, and buffy coat showed probe) with the embedded gold NPs (oligo dT). The experiment showed
the sensitivity value of 92.3%, 88.5%, and 92.3% and specificity values optimum sensitivity as a positive test zone is only observed in the case of
100%, 95.8%, and 95.8%, respectively (Adams et al., 2018). Dixit’s infected dogs (both with symptoms of infection and lacking infectious
group (Dixit et al., 2018), in their studies used the LAMP test, reported symptoms). Nano-diagnostic approach based on RNA was utilized by
sensitivity of 96.9% and specificity of 100% for the VL diagnosis in the Bose’s group (Bose et al., 2016) using salt-triggered gold NPs detection
endemic areas of India. For the immune-compromised individuals, the by colorimetric assay. They used SSU rRNA of L. donovani amastigote as
LAMP test reported good sensitivity (60–100%) and specificity a particular marker. Binding between the target and isolated RNA leads
(85–100%) values (Nzelu et al., 2019). However, the need for GC con­ to instability of gold NPs from salt-triggered aggregation resulting in the
tent (non-extreme), the probability of secondary DNA structures for­ production of blue color. Otherwise, in case of no complementarity, the
mation, and the requirement of appropriate temperature value limit its red color is retained. Thus the blue color indicates the presence of
use (Akhoundi et al., 2017). L. donovani amastigote RNA, whereas red color restoration indicates its
The protein-based multilocus enzyme electrophoresis technique is absence. It is a cost-effective, fast, non-PCR, and non-culture-based
helpful in describing parasites by the comparative mobilities under diagnostic approach. To develop a protein-based nano-diagnostic

8
D. Kumari et al. European Journal of Pharmacology 910 (2021) 174436

method, Martins’s group (Martins et al., 2020) used screen-printed 3.1. Conventional anti-leishmanial therapy
carbon electrodes with functionalized gold NPs to identify VL. The
crude antigen of L. infantum was immobilized on a surface modified with 3.1.1. Pentavalent antimonials
gold NPs, permitting recognition of a particular epitope in the sera of For the past several years, the pentavalent antimonials (first intro­
suspected patients. Similarly, a signal generation mechanism was duced in 1945) is used as a front-line drug for the clinical cure of all the
developed using plasmonic ELISA for recognizing IgG antibodies against major types of leishmaniasis. Two major formulations of pentavalent
Leishmania infection with the unaided eyes. This assay has been very antimonials are available now, namely meglumine antimonate (Glu­
effective in diagnosing canine leishmaniasis by employing indirect cantime) or sodium stibogluconate (Pentostam) (Pospíšil et al., 2021;
ELISA to produce red color (unaggregated gold NPs) or blue color Rajkhowa et al., 2021). Fig. 6 shows the chemical structures of
(aggregated gold NPs) for the indication of IgG antibodies absence or anti-leishmanial drugs. It is believed that pentavalent antimonial is an
presence simultaineously (dos Santos Maciel et al., 2019). inactive drug reduced to its trivalent form for its effective functionality
Parasitological methods remain the gold standard for diagnosing in the biological system (Singh et al., 2016). Moreover, questions such as
Leishmania infection but it is accompanied by hemorrhage risk and where the actual reduction takes place, whether amastigotes or macro­
bleeding in patients. On the other hand, serological techniques are phages and the mode of reduction, whether non-enzymatic or enzy­
simple, rapid, and non-invasive. The major drawbacks of this technique matic, are still unanswered. The mechanism of action is postulated
include cross-reactivity between different parasitic infections and lower differently by several researchers but there is no standard pathway re­
sensitivity detection of asymptomatic cases (Singh and Sundar, 2015; ported. The possible mechanism of antimonials are: (a) pentavalent form
Thakur et al., 2020). The recent approaches such as proteomics is either reduced to trivalent form by thiols or thiol dependent reductase,
methods, flow cytometry, nano-diagnosis, and advanced molecular causing inhibition of trypanothione (virulence factor of parasite) which
techniques proved to be revolutionary in diagnosing parasite infection in turn cause oxidative stress (b) pentavalent form can be directly
due to their high sensitivity and specificity outcomes (Akhoundi et al., involved in ADP and GDP reduction (No, 2016) (c) It can obstruct major
2017; Sundar and Singh, 2018b). Various conventional and modern energy-driven metabolic pathways namely, fatty acid-oxidation,
diagnostic methods for leishmaniasis are listed in Fig. 4. glycolysis and ADP phosphorylation (d) It can induce oxidative stress
by continues efflux of thiols through a multidrug-resistant protein A
3. Treatment of leishmaniasis using ABC transporter. This alteration in thiol level imbalances their
anti-oxidant defense mechanism (Ponte-Sucre et al., 2017) (e) It can also
The mainstay for chemotherapy against leishmaniasis relies upon cause DNA fragmentation and apoptotic cell death (Roatt et al., 2020).
pentavalent antimonials, liposomal Amp B, miltefosine (only oral drug), For VL, the recommended dosage regimen is 20 mg/kg/day for about
and paromomycin (Table 2). But the treatment of this deadly disease is one month, either as an intravenous or intramuscular injection, and 850
still a global challenge due to a limited drug regimens, the emergence of mg as the highest limit for a single dose (Table 2). Due to its effectiveness
resistance, toxicities issues, and co-infection cases (Roatt et al., 2020; and reduced cost, it is still used as a gold standard in some old world and
Singh et al., 2016). New strategies must be explored and impended to new world countries (Sundar and Singh, 2018a). In phase II clinical
combat this neglected tropical disease to overcome the drawbacks of trials for CL conducted in Brazil, 53 patients were treated with intrale­
conventional drugs. In the following section, we present insights into the sional meglumine antimoniate displayed a cure rate of 87% (Ramalho
conventional anti-leishmanial therapy followed by applications of et al., 2018). Similarly, a case study of 12 patients suffering from CL was
innovative strategies such as combination therapy, drug repurposing, performed using intralesional administration of meglumine anti­
nanotechnology, host-directed therapy (HDT), and anti-microbial pep­ moniate. After the completion of the study, a cure rate of >90% was
tides as a way forward against leishmaniasis in the future. An overview observed with minimal after-effects (Arboleda et al., 2019). Thus, the
of conventional therapies and recent advances in the treatment of use of an intralesional approach for the chemotherapy of CL is a safe and
leishmaniasis is shown in Fig. 5. low-cost treatment option. However, in India usage of antimonials for
the cure of leishmaniasis is limited, due to emerging drug resistance and
toxicity issues (such as hepatotoxicity, nephrotoxicity, stiffness in joints,

Fig. 4. Various diagnostic approaches for leishmaniasis.

9
D. Kumari et al. European Journal of Pharmacology 910 (2021) 174436

Table 2
Overview of the drugs currently used to treat leishmaniasis.
Parameters Chemical Mechanism of Mode of Dosage Clinical Advantages Disadvantages REF.
class action administration regimen application

Sodium Pentavalent Inhibition of Intramuscular or 20 mg/kg Used as first-line Budget-friendly, Long treatment (Arboleda et al.,
Stibogluconate antimonials glycolysis, fatty intravenous or daily for anti-leishmanial easily available, course, pain at the 2019; Mansuri
acid oxidation, intralymphatic or 20–30 therapy in South and site of injection, et al., 2020;
ATP and GTP intralesional days America, East intralesional cardiotoxicity, Ponte-Sucre
synthesis Africa, and the route for CL hepatotoxicity, and et al., 2017;
Middle East with a showed reduced nephrotoxicity Ramalho et al.,
cure rate of after-effects 2018; Singh
35–95%. However, et al., 2016)
developed
resistance in India
Liposomal Polyene Pore formation in Intravenous 10–30 Indicated after Highly effective Rigors and chills (Kaur and
Amphotericin antibiotic membrane or mg/kg treatment failure with decreased during slow Rajput, 2014;
B bind to total dose to antimonials. toxicity and a infusion along with Nagle et al.,
ergosterol of cell (single Valuable short course of anaphylaxis, 2014; No, 2016;
membrane dose 3–5 therapeutics treatment nephrotoxicity, Roatt et al.,
halting its mg/kg/ against VL in India hypokalemia 2020)
synthesis dose) with an effective
rate of cure >90%
Miltefosine Alkyl Modulate the cell Oral 1.5–2.5 Recovery rate Only oral drug Long half-life, (Nagle et al.,
phospholipid surface receptors mg/kg >90% in India and available and teratogenic, 2014; No, 2016;
and alters sterol daily for 60–90% in Africa effective in gastrointestinal Sinha et al.,
and phospholipid 28 days but not effective as antimonials disorders, 2011)
composition a single dose resistant cases nephrotoxicity
and hepatotoxicity
Paromomycin Amino- Obstruct the Topical (CL) or 20 mg/kg Used for both CL Cost-effective Pain at the site of (Hussain et al.,
glycoside machinery of intramuscular (17 days) and VL treatment. and used in injection, 2014; Singh
protein synthesis (VL) or 15 mg/ Cure rate >90% in combination hepatotoxicity, et al., 2016;
as it binds to 30S kg (21 India and 45–85% therapy nephrotoxicity, and Sundar and
smaller subunit days) in Africa ototoxicity Singh, 2018a)
of the ribosomal
complex

Fig. 5. Conventional therapies and recent advances in the treatment of leishmaniasis.

and cardiotoxicity) (Mansuri et al., 2020). Drug resistance could be due leaking major components within the cells, leading to cell death (No,
to decreased activation of prodrug from pentavalent to trivalent form, 2016; Sangshetti et al., 2015). Other mechanisms like auto-oxidation
reduced drug uptake due to overexpression of aquaglyceroporin, and and reactive oxygen species (ROS) production also halt the parasite
down-regulation of calcineurin (calcium-mediated protein phosphatases survival (Singh et al., 2016). Though it has shown excellent response in
required in cellular activities) (Kaur and Rajput, 2014; Singh et al., many antimonials resistant individuals, Amp B deoxycholate come up
2016). with major side effects such as nephrotoxicity, hypokalemia, chills
during slow infusion along with anaphylaxis and thus, patients need to
3.1.2. Amphotericin B be hospitalized for nearly a month (Kaur and Rajput, 2014; Roatt et al.,
Amp B deoxycholate is an antifungal antibiotic having a high affinity 2020; Shirzadi and medicine, 2019). To counteract these limitations, a
for ergosterol (fungal membrane) and low affinity towards cholesterol liposomal formulation of Amp B was developed. Broadly, liposomal Amp
(host membrane) (Autmizguine et al., 2014). The pioneer in vitro activity B (AmBisome), Amp B lipid complex, and Amp B cholesterol dispersion
of Amp B was reported in 1960 as a valuable anti-leishmanial candidate are three formulations available for the cure of VL. Among them,
(Nagle et al., 2014). After the emergence of antimonials resistance in AmBisome is extensively used in India and is also approved by the US
India, Amp B becomes the first preferred drug for the cure of VL. It binds Food and Drug Administration (Sundar et al., 2014). This formulation
24-substituted sterol (ergosterol) of the biosynthetic pathway, by not only reduces its toxicity and targets drug delivery but also enhances
altering the membrane fluidity forming macropores and micropores its pharmaco-kinetics and assimilability (Freitas-Junior et al., 2012).

10
D. Kumari et al. European Journal of Pharmacology 910 (2021) 174436

Fig. 6. Chemical structures of currently used drugs against leishmaniasis.

The intravenous dosage regimen may vary following the geographic resistance may be due to the alteration in the working of the membrane
location, such as 30–50 mg/kg, 24–35 mg/kg, and18-21 mg/kg for transporter and the protein LdRos3, leading to the decreased influx of
Sudan, Ethiopia, and South Africa respectively. A dosage regimen of the drug (Dorlo et al., 2012; Sánchez-Cañete et al., 2009). Though
3.75 mg/kg can efficiently cure >85% of patients with VL in India miltefosine has shown good efficacy, little toxicity of hepatic, gastric,
(Tamiru et al., 2016). A comparative study in Bihar to figure out the and renal systems has also been reported (Sundar and Chakravarty,
efficacies of Amp B deoxycholate with dosage regimen 1 mg/kg alter­ 2015). The major reason that limits its clinical use is the teratogenicity
nate days for 1 month and AmBisome with the dose of 2 mg/kg for five that restricts its use for pregnant females and breastfeeding women
days resulted in a similar rate of cure (96%) at the end of 6 months in VL (Loureiro et al., 2018). Although, use of miltefosine in combination
patients (Sundar et al., 2003). In recent years, resistance to Amp B in the therapy with others drugs have shown great success. In a study, Rebel­
clinical isolates have been reported due to altered membrane composi­ lo’s group (Rebello et al., 2019) reported that combination strategy of
tion, increased mRNA levels of MDR1 gene (ABC transporter), and lopinavir and miltefosine resulted in marked reduction in parasite load
up-regulated expression of trypanothione biosynthesis pathway proteins in Leishmania-infected BALB/c mice and also significantly decrease the
(Equbal et al., 2014; Purkait et al., 2012; Suman et al., 2016). However, cytotoxicity as compared with miltefosine alone. Thus, indicating
the combinational treatment approach with other drugs has substantial combination therapy as a better alternative to combat increased
benefits overpowering the increasing clinical resistance cases. The resistance.
combination of Amp B and doxorubicin (anti-parasitic) loaded within
the mannosylated polymeric nano-micelles has improved the thera­ 3.1.4. Paromomycin
peutic efficacy of the drug as an anti-leishmanial agent comparative to Paromomycin is an antibiotic with a wide range of clinical applica­
Amp B alone (Wei et al., 2020). bility and is isolated from Streptomyces rimosus. It belongs to the ami­
noglycoside class and is found highly beneficial against infections such
3.1.3. Miltefosine as amoebiasis, cryptosporidiosis, giardiasis, and also against numerous
Miltefosine (hexadecyl-phosphocholine) is the “only oral drug” gram-negative and gram-positive bacteria (Matos et al., 2020; Singh
available for chemotherapy against leishmaniasis (Sundar and Chakra­ et al., 2016; Taslimi et al., 2018). It was recognized for its characteristics
varty, 2015). It is an amphipathic drug belonging to class alkyl phos­ as an anti-leishmanial agent in the 1960s and has been proved to be an
phocholine containing a polar phosphocholine group and long alkyl effective therapy against both CL and VL (Goto and Lindoso, 2010).
chain (Loureiro et al., 2018). Originally administered for breast cancer, Paromomycin mainly acts by impeding protein synthesis machinery,
it shows great potential as anti-leishmanial agents (1980s), especially in targeting ribosomal complex by binding to its 30S smaller subunit. This
antimonials resistant cases (dos Santos Nogueira et al., 2019). Miltefo­ binding leads to stabilization of the complex, and thus the ribosome
sine mainly acts by (a) altering plasma membrane composition by cannot be recycled, which further halts the formation of desirable pro­
inhibiting phospholipid metabolism and alkyl lipid metabolism (Pin­ tein. It can also disrupt membrane potential by changing the membrane
to-Martinez et al., 2018) (b) damaging mitochondria (depolarization), fluidity and affecting lipid metabolism (Mansuri et al., 2020; No, 2016).
and obstructing cytochrome c oxidase, causing programmed cell death The government approved paromomycin for VL treatment in India in
(Ponte-Sucre et al., 2017) (d) increasing expression of nitric oxide syn­ 2006 as it displayed 94% cure rates in phase IV clinical trials (Sinha
thetase 2 (iNOS2) in host macrophages that in turn generate nitric oxide et al., 2011). It is administered daily at 11 mg/kg body weight of dose for
(NO), which is toxic for the parasitic survival (Ortega et al., 2017). With around 21 days via the intramuscular route (Singh et al., 2016). Previ­
the successful phase II and III clinical trials in India with 50–100 mg/day ously reported resistance cases (Andrade-Neto et al., 2021) were mini­
dose for a month, in 2002, it was recommended for use in India (Nagle mized in using these specific compounds in many developing countries
et al., 2014). 28 days of miltefosine administration (1.5–2.5 mg/kg/day) because of their post-treatment effects. These effects include pain at the
effectively cured >90% of VL patients (Freitas-Junior et al., 2012; Sinha site of infection, hepatotoxicity, renal toxicity, and ototoxicity (Hussain
et al., 2011). However, for PKDL, a dosage regimen of 50 mg thrice daily et al., 2014).
for 60 days or twice daily for 75 days is recommended (Sundar and
Singh, 2018a). It is also observed that inadequate use and long shelf life
3.2. Recent advances in therapeutics of leishmaniasis
have increased the resistance cases of miltefosine, leading to treatment
relapse and which significantly declined its clinical application. Clinical
3.2.1. Multi-drug therapy
relapse (10–20%) is reported in few countries, including Bangladesh,
The escalating incidences of drug resistance and rate of treatment
India, and Nepal (Monge-Maillo and López-Vélez, 2015). The increased
relapse worldwide pertaining to conventional treatment therapy for

11
D. Kumari et al. European Journal of Pharmacology 910 (2021) 174436

leishmaniasis have made researchers critically hunt for a better alter­ 2020) reported successful use of multi-drug therapy in curing patients
native with improved effectivity and efficiency. Multidrug therapy is an suffering from PKDL infection. In this study, 32 patients were enrolled;
approach to study the collective effect of drugs. The main objectives of 16 patients were administrated with miltefosine (100 mg/day) for three
the combinational or multi-drug strategy are to reduce treatment course, months and the remaining 16 with the combination of miltefosine (100
relapse rate, dosage, lethal side effects, and overcome drug resistance mg/day) for 1.5 months and three infusions of liposomal Amp B (5
issues (Sotgiu et al., 2015; Sundar and Singh, 2018a). This strategy has mg/kg). The combinational approach resulted in a rapid decline (within
already been proved to be a valuable success story in treating TB, Ma­ two weeks) of the infection rate, leading to a 100% cure rate. No relapse
laria, and HIV (Larkins-Ford et al., 2021; Stellbrink et al., 2020; Tindana cases were observed until the follow-up period of 18 months. However,
et al., 2021). in the case of miltefosine monotherapy signs of recovery were observed
The anti-leishmanial efficacy against the VL model of infection in after 3–4 weeks and 25% of relapsed cases were also reported after the
BALB/c of mice was evaluated by multi-drug therapy approach by follow-up period of 18 months. A similar, combinational approach of
various drugs like itraconazole (anti-fungal), ezetimibe (hypercholes­ liposomal Amp B, allopurinol, and IFN-γ led to the complete recovery
terolemic curing drug), and miltefosine (Andrade-Neto et al., 2021). The from VL infection (Khodabandeh et al., 2019). Thus, all of the above
monotherapy approach reported an IC50 of 0.89 μM, 11.46 μM, and 0.63 studies indicate the crucial role of multi-drug therapy in the treatment of
μM for itraconazole, ezetimibe, and miltefosine, respectively. However, leishmaniasis.
combinations of (itraconazole + ezetimibe) and (itraconazole + ezeti­ In the recent era, the combination therapeutic approach is exten­
mibe + miltefosine) showed average fractional inhibitory concentration sively exploited against leishmaniasis and other contagious diseases due
index of 0.56 μM and 0.81 μM, respectively, indicating additive effect in to its ability to embrace the existing drug efficacy and reduce drug dose
total for these combinations. Furthermore, these two combinations were and treatment durations. Combining a drug with pre-existing clinically
also influential in significantly (>90%) reducing parasite load in the approved drug opens up a wide spectrum of reliable combinations to
liver and spleen. Trinconi’s group (Trinconi et al., 2018) evaluated the treat leishmaniasis. Thus, novel drug combinations can be further
leishmanicidal efficacy of tamoxifen in combination with meglumine explored for an ideal treatment regime for leishmaniasis.
antimoniate using ex vivo and in vivo study models. During ex vivo
studies, this combinational approach displayed an average fractional 3.2.2. Drug repurposing
inhibitory concentration index of 1.05 μM against intracellular amasti­ With the dawn of new innovative research, medical experts are
gotes of L. amazonensis, indicating the additive effect of two drugs. In working to discover efficient, cost-effective, and reliable therapeutics,
vivo studies also demonstrated a prominent reduction in lesion size and which can be easily accessible to all. Drug repurposing (also termed as
parasite load in Leishmania-infected BALB/c mice. Similarly, Caridha’s reprofiling, drug repositioning, or re-tasking) is a unique approach that
group (Caridha et al., 2021) drug combination study revealed that a determines new uses of approved clinical drugs pioneered for another
sub-therapeutic dosage regimen of liposomal Amp B and tretazicar medical cure (Pushpakom et al., 2019). This approach is associated with
(anti-cancer) successfully reduced skin abrasion in L. major infected a reduced time frame and investment cost for drug development as the
BALB/c mice within 18 days. On the other hand, a complete cure of drugs have already been through preclinical trials, lower risk of devel­
infection with a monotherapy approach was observed in 64 days. Recent oping resistance, reduced side effects and relapses, along with the
reports revealed that the synergistic effect of terbinafine and ketoco­ emergence of new potential targets that can be exploited in the future
nazole in inhibiting the growth of L. braziliensis (promastigote form) and (Breckenridge and Jacob, 2019). This technique involves in-silico ap­
L. amazonensis (amastigote form) (Bezemer et al., 2021). These studies, proaches, where molecular interactions of the drug with suitable/target
as mentioned earlier, confirmed the more pronounced anti-leishmanial proteins are performed (Leishmania - our parasite of interest) (Busta­
efficacy with the use of combination therapy as compared to mante et al., 2019). For leishmaniasis, many drugs have been repur­
monotherapy. posed so far and have displayed good results. Drug candidates such as
Moving back towards the 19th century, the two or more drugs in Amp B, miltefosine, azoles (fluconazole, itraconazole, and pos­
combination were started to be administered for the cases with resis­ aconazole) acting on multiple targets are of great value in drug repur­
tance against antimonials. Infected individuals treated for 20 days in the posing strategy (Braga, 2019). Many anti-depressants like Ketanserin,
mix of paromomycin (12 mg/kg/day) and sodium stibogluconate (20 imipramine, clomipramine, nitroimipramine, sertraline, etc. have
mg/kg/day) for about 20 days of treatment revealed a recovery rate of shown good potential as an anti-leishmanial repurposed drugs (da Silva
80–90%. However, this regimen is no more recommended in the Indian Rodrigues et al., 2019; Lima et al., 2018). A recent report revealed that
subcontinent but is still followed in African countries for VL treatment the mechanism of the leishmanicidal effect of sertraline on Leishmania
(Singh et al., 2016; van Griensven et al., 2010). For VL patients in India, parasites using transmission electron microscopy and metabolomics
a single dose of miltefosine in combination with liposomal Amp B (5 platforms (Lima et al., 2018). Sertraline inhibited the growth of pro­
mg/kg) has shown the efficacy of >95% in the treatment (Sundar et al., mastigotes and amastigotes of L. infantum with an IC50 value of ~2.0 μM
2008). Thus, the rationale use of multi-drug therapy depends upon the and ~4.0 μM, respectively. Sertraline also induced bioenergetic collapse
resistance strains endemic to that areas. In a randomized phase II clinical in L. infantum parasites by decreasing ATP synthesis and disrupting
trial for VL conducted in Sudan and Kenya, evaluation of miltefosine as mitochondrial potential. Metabolomics studies using liquid
monotherapy (2.5 mg/kg/day for 28 days), 10 mg/kg single dose of chromatography-mass spectrometry and capillary electrophoresis-mass
liposomal Amp B combined with miltefosine (2.5 mg/kg daily) for 10 spectrometry confirmed remarkable alternations in the levels of phos­
days and10 mg/kg single dose of liposomal Amp B in combination with pholipids, amino acids, trypanothione, and polyamine biosynthesis in­
Sodium stibogluconate (20 mg/kg daily) for 10 days regimen was termediates. Microscopy studies showed that sertraline-treated
assessed. Among the three studied treatment regimens, liposomal Amp B promastigotes had round vesicular mitochondria morphology and
and Sodium stibogluconate combination having maximum recovery rate condensed DNA inside the nucleus as compared to the untreated pro­
of 87% (Wasunna et al., 2016). Multi-drug therapy consisting of mastigotes. Thus, with the multi-target mechanism of sertraline, it can
AmBisome and miltefosine was investigated in immuno-compromised become a valuable clinical candidate against VL infection.
patients (HIV-VL co-infected) (Mahajan et al., 2015). In this investiga­ Bustamante’s group identified 33 drugs (Bustamante et al., 2019) in
tion, for 14 days, 102 enrolled patients were administrated with 30 an in-silico study (interaction between drug and protein) as the probable
mg/kg (AmBisome) bifurcated into 6 infusions doses along with milte­ hits to potentially kill the Leishmania parasite. Out of these, six drugs,
fosine (50 mg/day). As reported, this combinational approach proved to namely cladribine, lamivudine, metformin, perphenazine, rifabutin, and
be safe, well-tolerated, and efficacious in reducing mortality and VL tenofovir were assessed for their leishmanicidal properties against
relapse rate. In an interesting study, Ramesh’s group (Ramesh et al., L. braziliensis and L. panamensis. As reported, rifabutin at the

12
D. Kumari et al. European Journal of Pharmacology 910 (2021) 174436

concentration of 12.3 μg/mL led to growth regression of intracellular clearance of splenic parasite load. Interestingly, nifuratel was also
amastigotes of L. braziliensis and L. panamensis by ~95% and ~65%, effective against cutaneous infection caused by L. major parasites.
respectively. Interestingly, the use of perphenazine at the concentration Intralesional administration of nifuratel to the footpads of L. major
of 1.4 μg/mL also successfully inhibited the growth rate of L. braziliensis infected-BALB/c mice led to a marked decrease in the lesions. Thus, this
and L. panamensis parasites by ~55% and ~45%. However, the efficacy study represents the potential role of nifuratel in curing both cutaneous
of the other four drugs was poor against these two species of Leishmania. and visceral infections caused by the Leishmania parasites. Similarly,
Further, in vivo studies on an animal model for CL using rifabutin and Tabrez’s group (Tabrez et al., 2021) virtually screened an FDA-approved
perphenazine should be performed to consider them as good candidates drug library (containing 1355 compounds) against the L. donovani sterol
for topical formulation. Tunes’s group (Tunes et al., 2020) recently C-24 methyltransferase enzyme. This enzyme plays a vital role in the
evaluated gold (I)-derived complexes (originally showing anti-cancer sterol biosynthesis pathway in the parasite. Out of 1355 drugs, 10 were
activities) for their leishmanicidal activities against amastigotes of shortlisted based upon binding energy (− 10.1 to − 10.6 kcal/mol).
L. braziliensis and L. infantum. As reported, gold (I)-derived complexes Anti-viral drug (simeprevir) with minimum binding energy − 10.6
hindered the proliferation of L. braziliensis and L. infantum amastigotes kcal/mol was effective in killing L. donovani promastigotes and amas­
with low IC50 values (0.5–5.5 μM). These complexes impaired trypa­ tigotes with an IC50 value of 51 μM and 98 μM, respectively. Flow
nothione reductase activity and cause ROS-mediated cell death in the cytometry analysis also revealed that the increasing concentration of
parasite. Further, in vivo experimentation in BALB/c mice model of CL simeprevir led to a progressive rise in oxidative stress, possibly leading
infected with L. amazonensis or L. braziliensis confirmed a 60–100% to cell death. Thus, the above studies signify the importance of drug
reduction in parasitic load and lesion size. In a similar study, Mesquita’s repurposing strategy in discovering new anti-leishmanial agents and
group (Mesquita et al., 2020) performed a preclinical evaluation of reprofiling of known drugs already approved for clinical safety. Such
topical cream-based formulation (triclosan) against CL infection caused drugs can provide novel, safe, and cost reliable therapies for leishman­
by L. amazonensis. Triclosan inhibited the progressive growth rate of iasis in upcoming times.
promastigotes and amastigotes of L. amazonensis with an IC50 of 51.5 μM
and 16 μM, respectively. Mechanistic studies reported that incubation of 3.2.3. Nanotechnology
promastigote with triclosan leads to the loss of membrane permeability Nanomedicines is the branch of science (nanoscience) dealing with
and depolarization of the mitochondrial membrane potential. Ultra­ the particles at the nanoscale, making it desirable for drug delivery and
structural studies using transmission electron microscopy confirmed the drug designing (Gharpure et al., 2020). The conventional delivery sys­
pore formation in the cell membrane and damage to the organelles. tem possesses several limitations: low permeability, insolubility, painful
Further, the use of 1% triclosan topical cream in Leishmania infected injections, prolonged hospitalization, and adverse side effects. In search
mice significantly reduced the parasite load by ~89%. of an ideal cure for leishmaniasis, researchers have encountered an
Khadir’s group (Khadir et al., 2018) evaluated the clinical efficacy of NPs-based approach as a ‘state-of-the-art’ to combat the ever-increasing
rapamycin (potential mTOR inhibitor) against L. major. Rapamycin resistance of parasites for traditional drugs and to overcome other
effectively inhibited the growth of L. major promastigotes and intracel­ drawbacks. NPs are used for drug delivery because of their biocompat­
lular amastigotes with an IC50 of 10 μg/mL and 12 μg/mL, respectively. ibility, improved drug solubility, on-point drug delivery to the target
Microscopic studies using scanning and transmission electron micro­ organ, immuno-compatibility, and increased possibility of various
scopy revealed that the use of rapamycin in a dose-dependent manner administration routes (Karuppusamy et al., 2017). Metallic, inorganic,
led to shrinkage in cell size, the roundness of cells, and the appearance of organic, and polymeric nanomaterials, including carbon nanotubes,
cell debris. In vivo studies in L. major infected-BALB/c mice also reduced dendrimers, liposomes, micelles, are commonly employed as
footpad swelling and parasitic load after treatment with rapamycin. target-mediated drug delivery systems (Patra et al., 2018). Since the
Similarly, the same group (Khadir et al., 2019) assessed the efficacy of target organ in leishmaniasis is the phagocytic cell, particularly mac­
rapamycin as monotherapy and its combinatorial effect with the front rophages, the most employed NPs for drug delivery are liposomal de­
line available drug Amp B and Glucantime against L. tropica. As re­ rivatives and polymeric NPs. However, metal NPs and natural
ported, rapamycin alone significantly inhibited the proliferation of product-derived nanostructures (chitosan, Alginate) are equally poten­
promastigotes and intracellular amastigotes of L. tropica with an IC50 of tiated (Ahmad et al., 2021; Nafari et al., 2020). Various mechanisms
8.2 μg/mL and 7.6 μg/mL, respectively. Ultrastructural studies responsible for leishmanicidal effects of NPs encapsulated drugs/mole­
confirmed that rapamycin causes cell distortion with dense cytoplasm cules are higher ROS generation, increase in immuno-modulatory
and vacuole formation (indicating stress) in L. tropica promastigotes. response, DNA damage, disruption of mitochondrial membrane poten­
The use of rapamycin (10.2 μg/dose) alone also efficiently reduced the tial and electron transport chain, and inhibiting trypanothione reductase
parasite load in lymph nodes of L. tropica infected-BALB/c mice. In enzyme vital for anti-oxidation process in Leishmania parasite (Kumar
contrast, the combination therapy of rapamycin with Amp B or Glu­ et al., 2019b; Ovais et al., 2017) Fig. 7.
cantime did not present a satisfactory result indicating the least syner­ To cure CL, research groups have exploited various types of nano-
gistic effect of the three drugs. In both the above-mentioned studies entities tailored to improve medicines and transport. A recent study
(Khadir et al., 2018, 2019), the use of rapamycin against L. major and L. by Bahraminegad’s (Bahraminegad et al., 2021) concluded that using
tropica elicited host immuno-stimulatory response; which proved to be chitosan/CdO core-shell nanodots is efficient in hindering the propa­
crucial in the polarization of T-helper cells type 1 (Th1) response, gation of L. major promastigotes (IC50 0.6 μg/mL) but also proved to be
leading to parasite clearance. less toxic to the macrophages. At different concentration of nanodots
Recently, Dominguez-Asenjo’s group (Domínguez-Asenjo et al., 0.25, 0.5, and 1 mg/mL, the percentage cell death was reported to be
2021), used ex vivo phenotypic screening approach to test 1769 com­ 23%, 31%, and 56%, respectively. Gene expression analysis showed
pounds from a repurposed drug library, against L. infantum infected significant over-expression of Th1 response (iNOS2 and IL-12) heading
mouse splenocytes. Out of screened 1769 drugs, 12 hits were identified. towards disease regression. Flow cytometry analysis also revealed that
Based on the selectivity index 4 drugs (nifuratel, salinomycin, paclitaxel, increasing concentration of nanodots is directly proportional to the
and docetaxel) were further evaluated for in vivo anti-leishmanial percentage of apoptosis, possibly leading to parasite killing by pro­
studies against L. donovani infected BALB/c mice model. Nifuratel grammed cell death. Recently, Almayouf’s group (Almayouf et al.,
administered with an oral dose of 50 mg/kg bw/day for about one and 2020) reported the protective efficacy of silver NPs containing Fig and
half weeks displayed a significant reduction (75–90%) of the parasite Olive extracts in curing CL caused by L. major. BALB/c mice infected
load in the thymus bone marrow, liver, and spleen of the infected mice. with the parasite showed significantly reduced skin lesions, along with
Moreover, when given twice a day, a similar dosage showed >90% improved anti-inflammatory and anti-oxidant activities. Lalatsa’s group

13
D. Kumari et al. European Journal of Pharmacology 910 (2021) 174436

Fig. 7. Promastigote cell of parasite depicting

the various mechanisms of action and targets of
nanoparticles mediated drug delivery. (A) It
causes depolarization of the mitochondrial
membrane, inhibiting ATPases, which further
block the electron transport chain and ATP pro­
duction (B and C). An increase in oxidative and
nitrosative stress by nanoparticles is lethal to
parasite survival. (D) Acts as a Trypanothione
reductase inhibitor, an enzyme responsible for
anti-oxidative effect in parasitic. (E) It binds or
degenerate the smaller subunits of ribosomes and
in turn halt protein synthesis machinery. (F) It
enhances drug influx inside the cell for better
drug release effects. (G) Metal ions released from
metallic nanoparticles act as an intercalating
agent between bases, causing DNA damage (H) It
also leads to cell cycle arrest inhibiting cell
proliferation.

(Lalatsa et al., 2020) also elucidated the potential role of nano-hydrogels Meanwhile, these simple downsides can be overcome by the advanced
loaded with buparvaquone in curing ~95% parasite burden and scientific technologies available in today’s era. We cannot ignore the
reducing skin inflammation in mice models infected with L. amazonensis. fact that nanotechnology-mediated drug delivery will revolutionize
In another study by Awad’s group (Awad et al., 2021a), silver NPs pharmaceutical discoveries in the future ahead.
developed using Commiphora molmol (myrrh) successfully cured skin
lesions in the Leishmania-infected BALB/c mice after 21 days. Thus, the 3.2.4. Host-directed therapy
above studies signify the importance of nanotechnology in curing Despite the severity of neglected tropical disease leishmaniasis,
cutaneous infection caused by Leishmania parasite. current treatment cannot be considered the gold standard due to pro­
Using NPs several attempts are also made for curing the systemic longed and painful medications, increased resistance, and toxic effects.
infection (VL) caused by the protozoan parasite. Parvez’s group (Parvez In this context, HDT is one of those potential approaches in which the
et al., 2020a) prepared cyclodextrin-based NPs containing Amp B and host immunology is targeted and orchestrated for the clinical cure rather
paromomycin as an oral drug delivery approach against L. donovani than incorporating drugs in the parasite. This makes it a successful novel
infection. As confirmed by confocal laser scanning microscopy, NPs strategy (Zahid et al., 2019; Zumla et al., 2016). HDT utilizes any
were incorporated entirely in the cellular compartment within 24 h. molecule (repurposed drug, immuno-modulator, synthetic nucleic acids,
Cytotoxicity assay in J774A.1 macrophage cell line and Swiss albino cytokines, cellular therapy, and micronutrients) that can amplify the
mice assured that the developed formulations are safe and biologically host body’s immune system or modulate the inflammatory response.
favorable. Ex vivo experiments confirmed the ability of NPs to inhibit the Both result in reduced mortality and morbidity with long-lasting re­
proliferation of amastigotes of L. donovani efficiently with an IC50 of covery (Novais et al., 2021; Varikuti et al., 2018).
0.013 μg/mL and potentially cleared the parasitic load (70–90%) in An insight into the immune response against the Leishmania parasite
Leishmania-infected BALB/c mice. In a similar study, chitosan-based NPs enlightened us with the major fact that the existence of infection de­
containing Amp B and paromomycin showed ~92% growth inhibition of pends on successful regression of the Th1 immune response along with
intracellular amastigote of L. donovani in the J774A.1 macrophage cell up-gradation of the T-helper cells type 2 (Th2) responses. Leishmania
line (Parvez et al., 2020b). In an interesting study, Gedda’s group parasite enters within the host’s body as promastigotes which are
(Gedda et al., 2020) reported the role of amine-functionalized nanotube phagocytosed by macrophages and are transformed to amastigotes in
appended with Amp B in inhibiting the growth of L. donovani parasites in the phagocytic cells and reside there. The primary immune escape is
J774A.1 cell line and golden hamster animal model by 90–98%. when it survives the macrophages oxygen burst and acidic enzymes by
Recently, Ray’s group (Ray et al., 2020) developed guar gum NPs con­ producing acidic phosphatases and activating proton pumps, respec­
taining piperine (natural alkaloid as bioenhancer) and Amp B drug tively (Sharma and Singh, 2009). When it comes to humoral response,
suitable for oral administration. This NPs formulation improved the antibody titer is elevated in the case of VL compared with an antibody
drug bioavailability and inhibited the growth of the L. donovani parasite concentration of CL. Moreover, IgG is unable to protect from infection,
by 2–3 folds times higher as compared to Amp B alone. In vivo studies in and it is even adverse to the situation. The cell-mediated response
the golden hamster animal model reported a ~95% reduction in parasite mainly revolved around the Th1/Th2 paradigm in which Th1 secrete
load. Similarly, Kumar’s group (Kumar et al., 2019a) observed ~5 folds interferon-gamma (IFN-ү) along with ROS and NO in the infected
enhanced leishmanicidal property of gold NPs conjugated with Amp B macrophages that cause infection control. At the same time, a Th2
compared to Amp B alone. Various drugs/molecules with potent leish­ produces Interleukin (IL)-4 and IL-10 harmful to the host and favors
manicidal effects identified through new approaches are discussed in disease progression (Singh et al., 2019). Many molecules lined up for the
Table 3. treatment of leishmaniasis following the HDT approach to manipulate
However, despite endless advantages, the utilization of nanoscience and modify the host immune system in an efficient way (Fig. 8). Thus, in
in practical life is restricted because they are biologically sensitive and the following section, molecules that have been report for
may cause considerable systemic toxicity in some cases (Nargund et al., immuno-modulatory responses leading to disease protection against
2021). The preparation and manipulation require specialized and leishmaniasis are discussed.
engineered products, making it a cost intensive task unsuitable for poor Oleuropein is a phenolic compound extracted from Olea europaea, a
tropical diseases like leishmaniasis (Omkar S. Sangar, 2021). plant from the olive family, that stimulates ROS and NO production in

14
D. Kumari et al. European Journal of Pharmacology 910 (2021) 174436

Table 3
Drugs/compounds with anti-leishmanial effects identified through new approaches.
Strategy Drugs/compounds Disease Target organism Model Outcomes REF.

Drug repurposing Sertraline VL L. infantum BALB/c mice Significantly inhibited the growth of the Lima et al. (2018)
parasite. Oxidative damage along with
altered expression of essential metabolic
pathways
Cladribine, Lamivudine, CL L. braziliensis and L. U-937 cell line Out of these six, two drugs i.e., rifabutin Bustamante et al.
Metformin, Perphenazine, panamensis and perphenazine, showed better parasitic (2019)
Rifabutin, and Tenofovir inhibition during in vitro studies
Gold (I) triphenyl- CL L. amazonensis and BALB/c mice The anti-leishmanial activity was reported Tunes et al. (2020)
phosphine and triethyl- L. braziliensis with IC50 0.5–5.5 μM. It impairs
phosphine based trypanothione reductase activity and
complexes causes ROS-mediated cell death
Triclosan CL L. amazonensis BALB/c mice Inhibited the growth of the parasite along Mesquita et al. (2020)
with loss of membrane permeability and
damage to mitochondria
Simeprevir VL L. donovani THP-1 cell line Leishmanicidal properties against Tabrez et al. (2021)
promastigotes and amastigotes along with
ROS-mediated cell death
Rapamycin CL L. major and BALB/c mice Marked inhibition in the parasite (Khadir et al., 2018,
L. tropica proliferation. Reduced parasite load in 2019)
mice along with immuno-stimulatory
responses towards Th1 polarization
Nifuratel CL and L. major, BALB/c mice Oral and intralesional administration led to Domínguez-Asenjo et al.
VL L. infantum, and the cure of both cutaneous and visceral (2021)
L. donovani infections in mice model
Nanotechnology Chitosan/CdO core shell CL L. major THP-1 cell line It worked efficiently at IC50 0.6 μl/mL Bahraminegad et al.
nanodots inhibiting the promastigotes proliferation (2021)
along with up-regulation of Th1 immune
response
Silver NPs containing Fig CL L. major BALB/c mice Significantly reduced skin lesions and Almayouf et al. (2020)
and Olive extracts improved anti-oxidant capacity
Nano-hydrogels loaded CL L. amazonensis BALB/c mice Significantly decreased parasitic burden by Lalatsa et al. (2020)
with buparvaquone ~95% in BALB/c mice infected can be a
possible cure option in future
Silver NPs with CL L. major BALB/c mice Dose-dependent growth inhibition along (Awad et al., 2021a,
Commiphora molmol with complete healing of skin lesions 2021b)
within 3 weeks
Cyclodextrin NPs VL L. donovani J774A.1 cell Obstructed the growth of the parasite Parvez et al. (2020a)
containing Amp B and line and Swiss efficiently (IC50 0.013 μg/mL) and reduced
paromomycin albino mice the parasitic burden by 70–90%
Chitosan NPs containing VL L. donovani J774A.1 cell Inhibitory activity of NPs is better than Parvez et al. (2020b)
Amp B and paromomycin line Amp B alone and reported parasite
clearance >90%
Nanotube appended with VL L. donovani J774A.1 cell Graphene-carbon nanotube composite Gedda et al. (2020)
Amp B line and golden showed a significant decrease >90% in
hamster parasitic proliferation in comparison to
AmB alone proving it safe cure
Guar gum NPs containing VL L. donovani Golden 2–3 folds higher inhibition compared to Ray et al. (2020)
and Amp B hamster drug alone along with ~95% reduction in
parasitic burden
Host-directed Oleuropein VL L. donovani J774A.1 cell An immuno-stimulatory response along Kyriazis et al. (2016)
therapy line and BALB/ with up-regulated expression of IFN-γ and
c mice IL-12
Leptin VL L. donovani THP-1 cell line Promote Th1 response and increase in NO Dayakar et al. (2016)
production causing parasite killing
Mahanine VL L. donovani J774A.1 cell Modulates Th1 cytokines and suppresses Roy et al. (2017)
line and BALB/ Th2 cytokines (IL10)
c mice
Imatinib CL L. amazonensis C57BL/6 mice Reduced abrasion in mice along with Wetzel et al. (2012)
enhanced phagocytosis process
AS101(tellurium-based VL L. donovani BALB/c mice Increased production of ROS and NO. Vishwakarma et al.
compound) Activation of NF-κB and mitogen-activated (2018)
protein kinase pathways
Simvastatin CL L. major BALB/c mice Inhibited the cholesterol biosynthesis and Parihar et al. (2016)
reduce promastigotes attachment to host
cells
Lovastatin VL L. donovani J774A.1 cell Cause reduction in adherence of the Kumar et al. (2017)
line parasite to host macrophage critical for
parasitic survival
Eugenol CL L. amazonensis BALB/c mice Th1 immune response is generated by the Raja et al. (2017)
release of IL-12 and IFN-γ
Phospholipase A2 CL L. amazonensis BALB/c mice Activates NF-κB in macrophages and TNF-α de Barros et al. (2018)
production (increased Th1 immune
response) and NO is also increased
Fucoidan VL L. donovani Sharma et al. (2014)
(continued on next page)

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D. Kumari et al. European Journal of Pharmacology 910 (2021) 174436

Table 3 (continued )
Strategy Drugs/compounds Disease Target organism Model Outcomes REF.

RAW 264.7 Enhances DCs maturation and stimulates

cell line and Th1 cytokines and suppresses Th2
BALB/c mice cytokines
Artemisinin VL L. donovani BALB/c mice Higher expression of pro-inflammatory Want et al. (2015)
cytokines and clampdown of Th2 cytokines
Berberine VL L. infantum BALB/c mice Increase in ROS and NO production. IL-12 (Calvo et al., 2020; De
production is enhanced while IL-10 release Sarkar et al., 2019)
is decreased
Ibrutinib CL L. major BALB/c mice Th1 polarized immune response is up- Dubovsky et al. (2013)
regulated
Naloxonazine VL L. donovani THP-1 cell line Increased activity of vacuolar ATPase De Muylder et al. (2016)
proton pump leading to rapid vacuolar
acidic flux
Anti-microbial Cathelicidin CL L. major J774A.1 cell Up-regulation of cathelin-related anti- Asadi et al. (2020)
peptides line and BALB/ microbial peptide expression leading to
c mice reduced parasitic load confirmed by ex vivo
and in vivo studies
Brevinin 2R CL L. major BALB/c mice Significant reduction in parasitic load in Zahedifard et al. (2019)
promastigotes and intracellular
amastigotes along with an increase in IFN-γ
release
CXCL-10 CL L. major BALB/c mice A marked decrease in parasite burden in Abdossamadi et al.
mice. Increased production of IFN-γ and (2017b)
NO (Th1 polarization)
Human neutrophilic CL L. major BALB/c mice Inhibition of parasite proliferation along Abdossamadi et al.
peptide-1 with up-regulation of Th1 response and (2017a)
down-regulation of Th2 response

Fig. 8. Mechanisms of host-targeting against Leishmania parasite infection.

vitro (J774A.1 cell line) and in vivo (BALB/c mice) models of VL. tyrosine kinase directly involved in initiating the phagocytosis driven by
Oleuropein treatment also elevated the expression of immune genes (IL- actin cytoskeleton rearrangement. Wetzel’s group (Wetzel et al., 2012)
12, IFN-γ, and transforming growth factor-β1) and transcription factors demonstrated the reduced abrasion in the C57BL/6 mice model of CL
(transacting T-cell specific transcription factor 3 and T-box transcription when treated with imatinib due to impairment of parasite invasion. 6–16
factor 21) which produced prominent Th1 mediated cellular immunity folds reduced parasite burden was reported in imatinib-treated mice as
against established L. donovani infection in mice (Kyriazis et al., 2016). compared to the control groups. Likewise, phosphoinositide 3-kinase
Leptin (adipose hormone) shows a similar expression, which phos­ (PI3K) facilitates the cell engulfing process by cytoskeletal remodel­
phorylates protein kinase B and extracellular-regulated kinase ing. Vishwakarma’s group (Vishwakarma et al., 2018) reported that
(ERK1/2), leading to a protective response against L. donovani infection. AS101 (tellurium-based compound) repressed the growth of promasti­
Leptin treatment increased ROS levels and induced macrophages gotes and amastigotes of L. amazonensis with an IC50 of 26.9 μM and
phagocytic activity, causing oxidative damage to the parasite (Dayakar 10.6 μM, respectively. Mechanistic studies revealed that AS101 acts as
et al., 2016). Mahanine is a natural indole alkaloid (Micromelum minu­ an immuno-modulating agent by producing ROS and NO, hindering the
tum), which affects the JAK/STAT pathways by impeding the tran­ PI3K pathway, activating nuclear factor-kappa (NF-κB) and
scription factors (JAK1 and Src), which thereafter promote the STAT3 mitogen-activated protein kinase pathways in host immune cells.
degradation leading to clampdown of Th2 immune response (decrease Since cholesterol is of paramount importance for the promastigotes
in IL-10 concentration (Roy et al., 2017). Imatinib is an inhibitor of to adhere to macrophages, several research groups have explored the

16
D. Kumari et al. European Journal of Pharmacology 910 (2021) 174436

beneficial role of statins in depleting cholesterol levels (Kumar et al., in the field of leishmaniasis (Cao et al., 2019; Zahedifard et al., 2020).
2017; Parihar et al., 2016; Singh et al., 2019). Topical application of Asadi’s group (Asadi et al., 2020) assessed the role of murine cath­
simvastatin in L. major infected BALB/c mice significantly reduced elicidin or cathelin-related anti-microbial peptide against infection
inflammation, ulceration, and parasite load in the ear region. In the mice caused by L. major. In vitro results displayed an enhanced expression of a
model, systemic administration (intraperitoneal route) of simvastatin cathelin-related anti-microbial peptide in the J774A.1 cell line
also reduced parasite load in lymph nodes and footpads. Further studies compared to control. Similarly, up-regulated expression of the
revealed that simvastatin inhibited the cholesterol biosynthesis pathway cathelin-related antimicrobial peptide was recorded at the inoculation
and induced phagosome maturation in macrophages, increasing parasite site of the dermis layer of L. major infected BALB/c mice, unlike the
clearance (Parihar et al., 2016). Similarly, Kumar’s group (Kumar et al., control group. Further, protein assay revealed the increase in the level of
2017) also reported the vital role of lovastatin in reducing parasite a cathelin-related anti-microbial peptide in both Leishmania-infected
internalization and infectivity in the J774A.1 macrophage cell line due macrophages and mice model confirming the role of the cathelin-related
to cholesterol depletion. Eugenol (Syzygium aromaticum) and its de­ anti-microbial peptide as a defensive immune response against L. major.
rivatives are described as promising agents for displaying Thus, cathelicidin can be considered a novel candidate for vaccine
immuno-stimulatory responses desirable to the host. Raja’s group (Raja designing in the future. Zahedifard’s group (Zahedifard et al., 2019)
et al., 2017) reported that eugenol derivative obstructed the growth of evaluated the leishmanicidal potential of Brevinin 2R (belonging to the
promastigotes and amastigotes of L. amazonensis with an IC50 of 20.3 μM class of defensins) alone and in conjugation with lauric acid against
and 4.25 μM, respectively. In vivo experimentation in mice model of VL L. major parasites. An in vitro analysis of anti-leishmanial activity indi­
suggest that this natural compound elicited an immuno-stimulatory cated a prominent decrease in parasitic proliferation. Further, scanning
response by increasing NO production and up-regulating Th1 response electron microscopy confirmed distortion in cell shape and size of the
(IL-12 and IFN-γ), resulting in splenic and hepatic clearance of the L. major promastigotes. Though the cytokines production and footpad
parasite. Similarly, Phospholipase A2 (lipid mediator) and Pentalinon­ swelling in treated groups did not show impressive outcomes, the study
sterol (Pentalinon andrieuxii) activate the NF-κB signaling pathway with explored that the desirable manipulation of anti-microbial peptides will
significant up-regulation of tumor necrosis factor (TNF)-α and IL-12 serve good potential in drug discovery against various infections.
generation in phagocytic cells (de Barros et al., 2018; Oghumu et al., Montakhab-Yeganeh’s group (Abdossamadi et al., 2017b) perform a
2017). Fucoidan obtained Undaria suringar (Phaeophyceae) is sulfur and study to assess the therapeutic efficacy of recombinant L. tarentolae
fucose containing polysaccharide with great potential as an (non-pathogenic) expressing CXCL-10 (chemokines) in BALB/c mice
anti-coagulant, viricidal and anti-parasitic agent (Jin et al., 2014). It infected with L. major. In vivo experiment demonstrated that recombi­
boosts the maturation of dendritic cells and releases IFN-γ, IL-6, IL-12, nant L. tarentolae expressing CXCL-10 significantly reduced the lesion on
and TNF-α. It up-regulates NF-κB signaling by the primary activation of footpads and the parasitic load in lymph nodes of mice. Besides this, the
p38 and ERK1/2, which further regulates IL-2 and TNF-ɑ, respectively elevated IFN-γ/IL-4 ratio and NO generation were also reported, indi­
(Sharma et al., 2014). Want’s group (Want et al., 2015) reported that the cating Th1 polarized response, whereas a reduced IL-4 and IL-10 along
treatment of Leishmania-infected BALB/c model with Artemisinin with decreased arginase activity specifies the disease regression condi­
(extracted from Artemisia annua) lead to an 80–85% decrease in the tion. Abdossamadi’s group (Abdossamadi et al., 2017a) assessed the
parasitic burden in the spleen and liver. Further, the use of artemisinin immunotherapeutic potency of transgenic L. tarentolae secreting human
restored the immune responses in mice with elevated expression of neutrophilic peptide-1 against L. major infection. The results from in vivo
pro-inflammatory response (IFN-γ and IL-2) and suppression of Th2 analysis demonstrated that this peptide successfully reduced the para­
cytokines (IL-4 and IL-10). Similarly, Berberine and Ibrutinib also sitic proliferation in the lymph nodes as compared to the control group.
showed potential anti-leishmanial activity by up-regulating the expres­ Also, the cytokine profile was indicative of a favorable immune
sion of Th1 immune responses in Leishmania-infected animal models response. Thus, in both the above study (Abdossamadi et al., 2017a,
(Calvo et al., 2020; De Sarkar et al., 2019; Dubovsky et al., 2013). An 2017b), the transgenic L. tarentolae efficiently induce cytokine saga to
opioid-receptor antagonist Naloxonazine (Moretti et al., 2018) improves combat the infection caused by L. major along with significantly
the vacuolar ATPase proton pump function, which further promotes decreasing parasitic persistence. As a way forward, in the course of drug
more acidic vacuoles in the cell. Since promastigotes undergo destruc­ discovery pipeline and vaccine designing for leishmaniasis,
tion in an acidic medium, increased expression of vacuolar ATPase anti-microbial peptides can serve the novel therapeutics pertaining to
proton pump in the phagocytes before metamorphosis of parasite from their excellent leishmanicidal and immuno-modulatory potential.
promastigotes to amastigotes can be fatal for parasite survival. De
Muylder’s group (De Muylder et al., 2016) established the potential role 4. Conclusions and future perspectives
of naloxonazine in vacuolar remodeling, leading to inhibition of intra­
cellular growth of the parasite in the THP-1 cell line. In a nutshell, we Leishmaniasis is a neglected tropical disease and is regarded as a
can conclude that the immuno-modulatory compounds can prove to be global health issue by WHO. Recurring failures in the diagnosis of
potentially valuable candidates and foot forward in treatment thera­ leishmaniasis due to conventional approaches is the root cause of delay
peutics of leishmaniasis. in the chemotherapy and eventually causing death in the major endemic
areas. Hence, accurate and timely disease diagnosis plays a crucial role
3.2.5. Anti-microbial peptides in identifying asymptomatic patients, co-infected cases, and differenti­
Anti-microbial peptides are small cationic molecules, amphipathic ating between healthy and cured individuals. However, recent diag­
with a wide spectrum of activities against viruses, bacteria, fungi, and nostic procedures such as advanced molecular techniques, flow
parasites (Marr et al., 2016; McGwire and Kulkarni, 2010). Membrane cytometry, proteomics, and nano-diagnosis have proved to be revolu­
depolarization, disruption of plasma membrane permeability, and pro­ tionary in the prognosis of this deadly infection. Similarly, to tackle the
grammed cell death are some major microbicidal mechanisms of action emergence of drug resistance to conventional therapy, new strategies,
employed by these peptides (Browne et al., 2020; Zahedifard and Rafati, including multi-drug and host-directed therapies, have yielded fruitful
2018). Moreover, these anti-microbial peptides are considered first-line outcomes in the treatment of leishmaniasis. Conversely, the drug
immune defense systems that instigate the production of specific cyto­ repurposing approach has led to exploring new drug interventions, and
kines molecules, leading to host immuno-modulatory responses (Marr the use of nanotechnology has also speeded up the process of efficient
et al., 2016; Robles-Loaiza et al., 2021). drug delivery to specific tissues. Potential molecules described in Table 3
Mammalian anti-microbial peptides are also called host defense of this review may prove as valuable assets and can act as a break­
peptides and have gained immense attention from researchers working through in the roadmap leading to improved anti-leishmanial therapy. A

17
D. Kumari et al. European Journal of Pharmacology 910 (2021) 174436

better understanding of drug resistance and immuno-stimulatory Autmizguine, J., Guptill, J.T., Cohen-Wolkowiez, M., Benjamin, D.K., Capparelli, 2014.
Pharmacokinetics and pharmacodynamics of antifungals in children: clinical
mechanisms will enhance the drug discovery process against leish­
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with HDT may prove beneficial for immuno-compromised and co- S., 2021a. Antileishmanial effect of silver nanoparticles: green synthesis,
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Bahraminegad, S., Pardakhty, A., Sharifi, I., Ranjbar, M., 2021. The assessment of
CRediT authors contribution statement apoptosis, toxicity effects and anti-leishmanial study of Chitosan/CdO core-shell
nanoparticles, eco-friendly synthesis and evaluation. Arabian Journal of Chemistry
14, 103085.
Diksha Kumari: Writing - original draft, tables and figures. Summaya Bangert, M., Flores-Chávez, M.D., Llanes-Acevedo, I.P., Arcones, C., Chicharro, C.,
Perveen & Rashmi Sharma: Writing - review & editing. Kuljit Singh: García, E., Ortega, S., Nieto, J., Cruz, 2018. Validation of rK39
Conceptualization, Writing - review & editing, Supervision. All authors immunochromatographic test and direct agglutination test for the diagnosis of
Mediterranean visceral leishmaniasis in Spain. PLoS Neglected Trop. Dis. 12,
have read and approved the final version of the manuscript. e0006277.
Bettaieb, J., Toumi, A., Ghawar, W., Chlif, S., Nouira, M., Belhaj-Hamida, N., Gharbi, A.,
Ben-Alaya, N., Laouini, D., Louzir, 2020. A prospective cohort study of Cutaneous
Declaration of competing interest Leishmaniasis due to Leishmania major: dynamics of the Leishmanin skin test and its
predictive value for protection against infection and disease. PLoS Neglected Trop.
The authors confirm that this article has no conflict of interest. Dis. 14, e0008550.
Bezemer, J.M., van der Ende, J., Limpens, J., de Vries, H.J., Schallig, H.D., 2021. Safety
and efficacy of allylamines in the treatment of cutaneous and mucocutaneous
Acknowledgment leishmaniasis: a systematic review. PloS One 16, e0249628.
Bezerra, G.S.N., Júnior, W.L.B., Leal, N.C., De Medeiros, 2020. Application of loop-
mediated isothermal amplification (LAMP) assay for detection of Leishmania
Diksha Kumari (UGC-JRF) and Summaya Perveen (UGC-JRF) are infantum strain from Brazil. Iran. J. Parasitol. 15, 155.
highly thankful to University Grants Commission (UGC), New Delhi for Blaizot, R., Simon, S., Ginouves, M., Prévôt, G., Blanchet, D., Ravel, C., Couppie, P.,
fellowship assistance. The institutional manuscript communication Demar, M., Nabet, 2021. Validation of swab sampling and SYBR green-based real-
time PCR for the diagnosis of cutaneous leishmaniasis in French Guiana. J. Clin.
number is CSIR-IIIM/IPR/00309. Microbiol. 59.
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