Kobets T, Grekov I, Lipoldova M.: Leishmaniasis: prevention, parasite detection and treatment.

Abstract

Leishmaniasis remains a public health problem worldwide, affecting approximately 12 million people in 88 countries; 50 000 die of it each year. The disease is caused by Leishmania, obligate intracellular vector-borne parasites. In spite of its huge health impact on the populations in vast areas, leishmaniasis is one of the most neglected diseases. No safe and effective vaccine currently exists against any form of human leishmaniasis. The spectrum and efficacy of available antileishmanial drugs are also limited. First part of this review discusses the approaches used for the vaccination against leishmaniasis that are based on the pathogen and includes virulent or attenuated parasites, parasites of related nonpathogenic species, whole killed parasites, parasites' subunits, DNA vaccines, and vaccines based on the saliva or saliva components of transmitting phlebotomine vector. Second part describes parasite detection and quantification using microscopy assays, cell cultures, immunodetection, and DNA-based methods, and shows a progress in the development and application of these techniques. In the third part, first-line and alternative drugs used to treat leishmaniasis are characterized, and pre-clinical research of a range of natural and synthetic compounds studied for the leishmanicidal activity is described. The review also suggests that the application of novel strategies based on advances in genetics, genomics, advanced delivery systems, and high throughput screenings for leishmanicidal compounds would lead to improvement of prevention and treatment of this disease.

Full text

Page 1

Current Medicinal Chemistry, 2012, 19, 1443-1474 1443


Leishmaniasis: Prevention, Parasite Detection and Treatment
T. Kobets, I. Grekov and M. Lipoldová*
Laboratory of Molecular and Cellular Immunology, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic,
v.v.i., Prague, Czech Republic
Abstract: Leishmaniasis remains a public health problem worldwide, affecting approximately 12 million people in 88 countries; 50 000
die of it each year. The disease is caused by Leishmania, obligate intracellular vector-borne parasites. In spite of its huge health impact on
the populations in vast areas, leishmaniasis is one of the most neglected diseases. No safe and effective vaccine currently exists against
any form of human leishmaniasis. The spectrum and efficacy of available antileishmanial drugs are also limited. First part of this review
discusses the approaches used for the vaccination against leishmaniasis that are based on the pathogen and includes virulent or attenuated
parasites, parasites of related nonpathogenic species, whole killed parasites, parasites’ subunits, DNA vaccines, and vaccines based on the
saliva or saliva components of transmitting phlebotomine vector. Second part describes parasite detection and quantification using
microscopy assays, cell cultures, immunodetection, and DNA-based methods, and shows a progress in the development and application
of these techniques. In the third part, first-line and alternative drugs used to treat leishmaniasis are characterized, and pre-clinical research
of a range of natural and synthetic compounds studied for the leishmanicidal activity is described. The review also suggests that the
application of novel strategies based on advances in genetics, genomics, advanced delivery systems, and high throughput screenings for
leishmanicidal compounds would lead to improvement of prevention and treatment of this disease.
Keywords: Tropical disease, kala-azar, preventive medicine, animal model, therapy of visceral, cutaneous and mucocutaneous leishmaniasis,
estimation of pathogen load.
1. INTRODUCTION
Leishmaniasis is a vector-borne protozoan infection with a wide
clinical spectrum. About 21 Leishmania species are known to infect
humans; they are transmitted by approximately 30 species of
phlebotomine sand flies [1, 2]. Leishmania parasites have two basic
life stages: an extracellular motile stage (promastigote) inside an
invertebrate host and an intracellular non-motile stage (amastigote)
inside a vertebrate host [3]. In the vertebrate host organism,
Leishmania parasites infect so-called professional phagocytes
(neutrophils, monocytes and macrophages) [4], as well as dendritic
cells (DC) [5], immature myeloid precursor cells, sialoadhesin-
positive stromal macrophages of the bone marrow, hepatocytes and
fibroblasts [6]. Macrophages are supposed to be the main cellular
compartment for Leishmania in the mammalian host.
It has been assumed that leishmaniasis may have been
established 50 million years ago, during the Paleogene [7]. The
direct evidence that people suffered from this disease came from
samples 4 000 years old as DNA of L. donovani was found in
Egyptian mummies from a Middle Kingdom tomb [8]. The
presence of Leishmania was also detected in the facial lesions on
ancient skulls from the Atacama Desert in Chile [9].
Leishmania amastigotes have been first observed by in 1885 by
Cunningham in skin lesions of patients from India, but he suggested
that they were members of Mycetozoa (fungi) [10]. Protozoal nature
of Leishmania was first recognized in 1898 by Borovsky during his
study of skin lesions in Turkmenistan [11]. Leishman in 1903
discovered similar intracellular bodies in the visceral organs of fatal
cases of kala-azar from India, and established that they were
morphologically related to trypanosomes [12]. Similar observation
was made in the same year in India by Donovan [13]. The causal
relationship between Leishmania parasites and development of
cutaneous lesions was confirmed in 1908 by Martsinovsky who self
infected himself with the parasite cultures [14].
Leishmaniasis includes asymptomatic infection and three main
clinical syndromes. In the dermis, parasites cause the cutaneous
form of the disease, which can be localized or diffuse; in the
mucosa, they cause mucocutaneous leishmaniasis, and the
metastatic spread of infection to the spleen and liver leads to


*Address correspondence to this author at the Institute of Molecular Genetics,
Academy of Sciences of the Czech Republic, v.v.i., Vídeňská 1083, 14220 Prague 4,
Czech Republic; Tel: +(420)241063243; Fax: +(420)224310955;
E-mail: lipoldova@img.cas.cz

visceral leishmaniasis (also known as kala-azar or black fever).
Parasites can also enter other organs; such as lymph nodes, bone
marrow and lungs, and in rare cases, can even reach the brain [4].
Leishmaniasis is one of the most neglected diseases, along with
other like sleeping sickness or Chagas’ disease. Such diseases are
named “neglected” because they persist predominantly in
marginalized and poor communities [2, 15, 16]. The type of
pathology that is caused depends on the species of Leishmania, the
genotype and nutritional status of the host, the transmitting vector
and environmental and social factors [4]. The disease remains a
public health problem worldwide, affecting approximately 12
million people in 88 countries; 50 000 die of it each year.
Leishmaniasis is endemic in areas of tropics, subtropics, including
southern Europe, in setting ranging from rain forests in the
Americas to deserts in Asia. Leishmaniasis represents a major
public health problem in the Eastern Mediterranean Region. Based
on geographical distribution, the disease is divided into Old World
and New World leishmaniasis [1, 2, 15, 17].
Visceral leishmaniasis is endemic in more than 60 countries,
however, 90% of the 500 000 new cases that occur every year
concern six countries only – India, Bangladesh, Nepal, Brazil,
Ethiopia and Sudan. Visceral form of the disease is caused mainly
by L. donovani in the Indian subcontinent, Asia and Africa; L.
infantum in the Mediterranean basin [2], Central Asia and
Transcaucasia [18], and L. chagasi in South America [2]. In
Mediterranean countries, South America [2] and in Central Asia
and Transcaucasia [18], the disease is zoonotic and affects mainly
infants and young children. In these countries, stray and domestic
dogs are the main reservoir for the infection. In the Indian
subcontinent and Africa, visceral leishmaniasis is anthroponotic and
affects adults and children [2].
Cutaneous leishmaniasis is endemic in more than 70 countries,
with an estimation of 1.5-2 million new cases every year.
Afghanistan, Syria, and Brazil are the main foci. Cutaneous form of
the disease is caused mainly by L. tropica and L. major in the Old
World, and by L. braziliensis, L. guyanensis, L. panamensis, L.
peruviana, L. mexicana, L. amazonensis, and L. venesuelensis in the
New World. Mucosal leishmaniasis develops in a small number of
patients with New World cutaneous leishmaniasis, however its
course is chronic and may be life-threatening [2].
The disease is spreading because of risk factors that include
climate changes, population movements, long-distance tourism and
trade [15]. In the last decade, leishmaniasis expanded or emerged in
????-????/12 $58.00+.00 © 2012 Bentham Science Publishers

Page 2

1444 Current Medicinal Chemistry, 2012 Vol. 19, No. 10 Kobets et al.
several foci worldwide as the result of sand fly expansion due to
natural and human factors, such as urbanization and deforestation,
and global warming [2]. Cutaneous leishmaniasis is one of the top
10 diseases among tourists returning from tropical countries with
skin problems. Cases of leishmaniasis were also described in organ
transplant recipients [2, 16].
2. VACCINATION AGAINST LEISHMANIASIS
The aim of vaccination against an infectious agent is to provide
effective immunity by induction of clonal expansion in specific
memory T and/or memory B cells. Hence, a repeated encounter
with the same antigen(s) will induce secondary response, which
will be more rapid and more effective than the normal primary
response. A good vaccine must induce a protective level of
immunity at the appropriate site and this protective immune
response should have an adequate duration. It must be also safe to
administer, affordable by the population and suitable for the
pertinent population, as different populations might differ in
genetics of response. The majority of the most successful vaccines
have been developed empirically; recent advances in immunology
that are revealing key factors in protection against many diseases
will facilitate a more rational approach to vaccines design.
However, despite the considerable knowledge about the immune
response against Leishmania parasites [19, 20], no safe and
effective vaccine currently exists against any form of human
leishmaniasis. The only long time protection against cutanenous
leishmaniasis in humans is leishmanization – vaccination with the
virulent parasites that had to be abandoned for safety reasons [21,
22] (see Section 2.1.1.1.), and the only successful vaccine is
Leishmune®, which blocks the transmission of canine leishmaniasis
[23]. Vaccine CaniLeish® was recently registered for the veterinary
use in the European Union (see Section 2.1.2.1.). Probably not all
responses needed for protection against leishmaniasis have been
identified. Genetic analysis of susceptibility [24-30] that is
hypothesis free could speed the discovery of new important defense
mechanisms against leishmaniasis.
The following section and Table 1 give the examples of
approaches of the development of vaccines against leishmaniasis.
The comprehensive information can be found on the webpage:
http://www.leishvaccines.net/
2.1. Vaccines Based on Pathogen
2.1.1. Vaccination with the Whole Organism
2.1.1.1. Leishmanization – Inoculation of Virulent Leishmania
Parasites
Vaccination against cutaneous leishmaniasis using virulent
parasites has been used for centuries. Bedouin or some Kurdish
tribal societies traditionally exposed their babies’ bottoms to sand
fly bites in order to protect them from facial lesions. Another
ancient technique practicized in the Middle East was the use of a
thorn to transfer infectious material from lesions to uninfected
individuals [21]. Development of media for culturing Leishmania
parasites enabled controlled infections with live promastigotes.
Large-scale vaccination trials were carried in the past in the Soviet
Union [31, 32], Israel [21], and in Iran [22]. In Iran,
leishmanization of more than 160000 children was performed from
1982 to 1986; and leishmanization of 1 800 000 military personnel
and soldiers, 6000 war refugees and several thousands of
individuals from other population groups was carried until 1989
[22]. Several thousand persons were vaccinated during 60s in
Turkmenistan [31] and in Uzbekistan [32]. These trials led to a high
percentage of successful lesion development, but also faced
problems with the viability and infectivity of the injected
organisms, the development of large uncontrolled lesions, the
enhancement of other skin diseases, and immunosuppression and
were therefore abandoned [21, 22, 33]. Establishment of a suitable
condition to grow parasites and prepare stabilates [34, 35] could
prevent problems with the viability and infectivity of Leishmania
promastigotes. Leishmanization with well defined L. major
stabilates was used for evaluation of candidate vaccines against
leishmaniasis [34].
Although vaccination with live virulent parasites grown in
defined conditions leads to good protection against subsequent
infection [34], its use have been discontinued due to safety reasons.
Leishmania parasites persist in cured animals [36, 37] and could
cause serious problems in immunocompromised individuals [22].
Reactivation of persisting parasites might be especially dangerous
in areas with HIV infection [38]. In addition, leishmanization
cannot be applied for vaccination against visceral leishmaniasis.
Due to these reasons, researchers switched to development of other
types of vaccines.
2.1.1.2. Vaccination with Parasites of Related Nonpathogenic
Leishmania Species
Use of vaccinia virus, a species nonpathogenic for humans in
order to induce protective immunity against the related dangerous
smallpox virus was the first scientifically described case of
vaccination [39]. Breton and coworkers [40] applied this approach
to investigate the potential of lizard parasite L. tarentolae that is
nonpathogenic to humans and other mammals as a live candidate
vaccine. They found that a single intraperitoneal injection of L.
tarentolae elicits a protective immune response against the
subsequent infectious challenge with L. donovani in susceptible
BALB/c mice and led to decrease of a parasite burden in spleen and
liver. L. tarentolae expresses an Amastin-like gene, cysteine
protease B (cpb), lipophosphoglycan lpg3 and the leishmanolysin
gp63, genes that play role in parasite virulence in Leishmania
species pathogenic to humans. The degree of similarity varied from
59% and 60% for Amastin, 89% for lpg3 and 71% and 68% for cpb,
in L. major and L. infantum, respectively. The amastigote A2 gene,
expressed specifically by the L. donovani complex which promotes
visceralization, was absent in L. tarentolae [41]. Introduction of A2
gene into L. tarentolae increased ability of this species to survive in
liver of BALB/c mice [42]. The potential of L. tarentolae to protect
other species than mouse from pathogenic Leishmania species has
yet to be investigated.
2.1.1.3. Vaccination with Attenuated Parasites
An attenuated vaccine is a vaccine created by reducing the
virulence of a pathogen in order to induce a protective immune
response without causing the severe effects of the disease. Parasites
can be weakened through undefined or defined genome alteration.
Treatment of Leishmania parasites by γ -irradiation [43, 44],
chemical mutagenesis [45], chemical mutagenesis followed with
selection for temperature-sensitivity [46], by a long term in vitro
propagation [47], and by culturing in vitro under gentamicin
pressure [48, 49] leads to undefined genetic alterations and
parasites need to be further analyzed in order to establish the nature
of introduced mutation(s). These attenuated parasites induced a
protective immunity in several animal models. For example,
vaccination of BALB/c mice with L. major or L. mexicana
attenuated by gentamicin pressure led to decrease of lesions size
after a subsequent challenge with virulent parasites [48], and
vaccination with gentamicin-attenuated L. infantum protected dogs
against the wild type parasite [49]. Vaccination with irradiation-
attenuated L. major was shown to induce protective effect in CBA
mice [44].
Defined genome alteration was achieved by targeting
Leishmania genes such as dihydrofolate-reductase thymidylate
synthase (dhfr-ts) [50-53], cystein-proteinase (cpa, cpb) [54],
biopterin transporter (bt1) [55], lipophosphoglycan 2 (lpg2) [56],
silent information regulatory 2 (sir2) [57], phosphomannomutase
(pmn) [58] and centrin 1 (cen1) [59]. Vaccination of BALB/c mice

Page 3

Leishmaniasis: Prevention, Parasite Detection and Treatment Current Medicinal Chemistry, 2012 Vol. 19, No. 10 1445
Table 1.

Approaches to Vaccination Against Leishmaniasis
Type of vaccine Host Parasite Treatment Outcome Reference
Human,
160 000 children,
1 800 000
soldiers,
6 000 refugees
L. major Single injection,
2-3×105 promastigotes with BCG (Bacillus
Calmette-Guérin)
Protection, but in rare cases also
development of large uncontrolled
lesions and immunosuppression
[22]
Mouse,
C57BL/6,
6 mice per group
L. major Single injection,
10 µl to the ear,
40 µl to the foot pad;
16 weeks before infection
Protection after the repeated
injection to the foot pad, complete
clearence of parasites
[128]

Live virulent
parasites

Human,
28 males
L. major
2 injections of 5×105 promastigotes to the arm
at 18 month
All developed protection after the
1st injection, no lesion after 2nd
infection
[34]
Live non-virulent
parasites
Mouse,
BALB/c
5 animals per
group, 3 exp.
L. tarentolae
L. donovani
Single intraperitoneal injection of 5×106
L. tarentolae
promastigotes, challenge with 5×107 L.
donovani promastigotes six weeks after
Partial protection (less parasites in
spleen and liver)
[40]
Mouse,
CBA

L. major
(gamma-irradiated)
CBA one or two subcutaneous (intra-tail)
injections of 20x106 irradiated promastigotes
at ten week interval. Challenged three weeks
after the second immunization (different
doses and L. major substrains tested)
Partial protection in CBA mice
(smaller lesions), better protection
after two injections of irradiated
promastigotes

[44]
Mouse,
BALB/c,
C57BL/6,
CBA/Ca,
129Sv/Ev,
4-5 animals per
group
L. mexicana
(cystein proteinase
knockout)
Single injection of 5×106 promastigotes into
the rump 2 or 4 month before infection
Partial
protection (smaller lesion size)
[54]
Mouse,
BALB/c,
CBA/T6
4 per group, 2 or
3 exp.
L. major
(dihydrofolate
reductase-thymidylate
synthase knockout)

Subcutaneous, intramuscular or intravenous
injection 106-108 promastigotes 1 week before
the infection with 106 parasites
Partial protection (smaller lesions,
less parasites in lesion)
[51]
Mouse,
BALB/c
C57BL/6,
5 animals per
group
L. major
(dihydrofolate
reductase-thymidylate
synthase knockout)
L. amazonensis
Subcutaneous or intravenous injection of 104,
106 or 108 L. major promastigotes into the
footpad 1 week before infection with L.
amazonensis (106 or 5×106 promastigotes)
Partial protection (smaller lesions),
cross-protection
[52]
Mouse,
BALB/c

L. major
(lipophosphoglycan 2
knockout)
Injection of 5×106 into the footpad 10 weeks
before infection with 2x106 WT L. major
Strong, but not complete protection
(smaller lesions, less parasites)
[56]
Mouse,
BALB/c,
4 animals per
group, 2 exp.
L. infantum
(silent information
regulatory 2 single
knockout)
108 of promastigotes injected intraperitonealy
6 weeks before infection
Protection (elimination of parasites
70 days after infection)
[57]
Mouse,
BALB/c,
15-30 animals
per group
L. major
(phosphomannomutase
knockout)
Subcutaneous injection of 5×106
promastigotes 2, 4, 5, 6, or 12 weeks prior to
challenge with 106 of parasites
Partial protection (smaller lesions,
less parasites in lymph nodes). Best
protective effect observed in
vaccination 2 weeks before
challenge
[58]

Live
attenuated
parasites
Mouse,
BALB/c
14 animals per
group (L.
mexicana), 5
animals per
group (L. major)

L. major
L. mexicana
(gentamicin-attenuated)
5×106 promastigotes subcutaneously, and
5×106 promastigotes after 12 weeks
Partial
protection (smaller lesions)
[144]

Page 4

1446 Current Medicinal Chemistry, 2012 Vol. 19, No. 10 Kobets et al.
(Table 1). Contd…..
Type of vaccine Host Parasite Treatment Outcome Reference
Mouse,
BALB/c
SCID;
Golden hamster
(M. auratus)

L. donovani
(centrin gene deleted)
Intravenous inoculation of 3×106
promastigotes (mouse) or intracardial
inoculation of 107 promastigotes (hamster);
challenge with 3×106 parasites intravenously
(mouse) or 107 intracardially in 5 weeks
(hamster)
Significant protection (lower
parasite load in spleen and liver)
[59]
Monkey,Macaca
mulatta,
6-8 animals per
group
L. major
(dihydrofolate
reductase-thymidylate
synthase mutant)
108 of promastigotes inoculated in dermis
above the eye 18 weeks before infection
No influence [53]
German shepherd
dog,
6-8 animals per
group
L. infantum
(gentamicin-attenuated)
Intradermal or intravenous injection of 100 µl
of 108 promastigotes; challenge after 12
month
Protection (no clinical signs of the
disease)
[49]

Golden hamster
(M. auratus),
10-15 animals
per group
L. amazonensis
(δ-amino-
levulinate dehydratase
and porphobilinogen
deaminase transgenic)
L. donovani
Intradermal injection of 1-1.5×107 L.
amazonensis, porphyria induction, challenge
with 108 L. donovani 26 days after vaccination
Protection (no clinical signs of the
disease)
[63]
Human,
438 vaccinated,
406 control
males and
females
L. braziliensis
L. guayanensis
L. amazonensis
2 intradermal 100 µl doses of 7.2×108 killed
promastigotes per ml with bacille
Calmette – Guérin adjuvant; cases of
infection were estimated after 12-60 month
Partial protection (72.9%) after 12
month; no significant protection
after 24 month
[145, 146]
Human,
68 males and
females
L. amazonensis
2 intramuscular doses of 1.5 ml at 21 days, 32
received autoclaved vaccine, 36
nonautoclaved;
leishmanin skin test 40 days after vaccination
Partial immunogenic effect, 59%
after autoclaved,
83 % after nonautoclaved vaccine
[70]
Human,
750 vaccinated,
765 control
males and
females
L. amazonensis
2 doses of the intradermal injection of 100 µl
of Leishvaccin® with bacille
Calmette - Gue´rin adjuvant; cases of
infection were estimated after 26 month
No influence,
15 vaccinated and 10 control
subjects were infected and
developed the disease
[147]
Human,
1295 vaccinated,
1302 control
males and
females
L. amazonensis
Three injections of whole-cell killed parasite
vaccine at 20 days intervals; cases of infection
were estimated after 1 year
No influence,
101 vaccinated and 88 control
subjects developed leishmaniasis
[148]
Mouse,
BALB/c
5-6 animals per
group
L. major
107 formaline-killed parasites with adjuvants
(montanide ISA 720, alum, BCG) into the
rump; challenge with 106 promastigotes 2
weeks later
Partial protection (smaller lesions);
the best result achieved with
adjuvant montanide ISA 720)
[66]

Killed
parasites

Mouse,
BALB/c
7 animals per
group
L. major
3 subcutaneous immunizations at 3 week
intervals with nanospheres containing killed
parasites and cytosine-phosphodiester-
guanine oligodeoxynucleotide(CpG ODN);
infection with 5×106 promastigotes 3 weeks
after
Partial protection (smaller lesions) [71]
Protein
vaccines
Gp63 (63 kDa
glycoprotein)
derived peptides

Mouse,
BALB/c
8 animals per
group

L. major

Subcutaneous injection of 100 µg of the
peptide in a temperature-dependent sol gel
transition adjuvant, 8% Poloxamer 407; 6
weeks after vaccination, 2×104 promastigotes
injected in the hind quarters

Partial
protection (smaller lesion size)

[83]
Gp63 derived
peptide PT3
(residues 154-169)
Mouse,
BALB/c
8 animals per
group
L. major
Single subcutaneous injection of 100 µg in
PBS and 8% Poloxamer 407 adjuvant 7 weeks
after vaccination, subeutaneous injection of
2×104 promastigotes
Protection during at least 10 month
after vaccination (smaller lesion
size or no lesions)
[84]

Page 5

Leishmaniasis: Prevention, Parasite Detection and Treatment Current Medicinal Chemistry, 2012 Vol. 19, No. 10 1447
(Table 1). Contd…..
Type of vaccine Host Parasite Treatment Outcome Reference
Gp63
protein
Mouse,
CBA,
6-10 animals per
group
L. major
50 µl of gp63 with complete Freund’s
adjuvant into the tail or foot pads; 5×106
promastigotes injected subcutaneously
4-6 weeks after vaccination
Partial protection
(smaller lesion size)
[85]
Gp63 protein Mouse,
BALB/c,
10 animals per
group
L. major
3 inoculations of 5 µg of recombinant gp63 in
liposomes
in 3 week intervals;
106 promastigotes injected subcutaneously
4 weeks after immunization
Partial protection
(smaller lesion size, lower parasite
load in spleen)
[86]
LACK protein Mouse,
BALB/c
12 animals per
group
L. major
2 injections of 50 µg of LACK protein with or
without 1 mg of
IL-12 in phosphate buffer saline in 2 week
interval; 105 of promastigotes after 2 weeks
into the footpad
Partial protection in a case of the
combination of LACK and IL-12
(smaller lesion size, lower parasite
load in lymph nodes)
[91]
LACK,
LmPDI proteins

Mouse,
BALB/c,
C57BL/6,
PWK,
MAI
5 animals per
group, 2 exp.
L. major
3 injections of 25 µg of a protein with 30 µg
CpG oligonucleotide into the footpad at 4
week intervals; injection of 2×106
promastigotes into the footpad 30 days after
vaccination
Substantial protection (smaller
lesions, less parasites) with LACK
and LmPDI in PWK; partial
protection with LACK and LmPDI
in BALB/c; MAI mice - complete
protection with LmPDI, no
influence of LACK; in C57BL/6
mice none of the proteins conferred
protective response.
[135]
Promastigote
surface antigen 2
(PSA-2)

Mouse,
BALB/c-H2k,
C3H/He,
8-16 animals per
group
L. major,
L. mexicana
3 intraperitoneal injections of 1.5-2 µg of
PSA-2 with Corinebacterium parvum in 2
week intervals; infection with 105
promastigotes after 2 weeks
Partial protection
(smaller lesion size, lower parasite
load in lymph nodes)
[93]
Nucleoside
hydrolase
protein
Mouse,
BALB/c,
10 animals per
group
L. major
2 injections of 2.5-25 µg of the protein with or
without 1 µg of IL-12 in 2 weeks into the
rump; 105of promastigotes after 1 week into
the footpad
Partial protection after combination
of the protein and IL-12
(smaller lesion size, lower parasite
load in lymph nodes)
[94]
Nucleoside
hydrolase (NH36)
recombinant
proteins

Mouse,
BALB/c
4-8 animals per
group, 2 exp. (L.
chagasi),
5 animals per
group, 2 exp. (L.
amazonensis)
L. chagasi,
L. amazonensis
3 subcutaneous inoculations of 100 µg of the
protein with 100 µg of saponin; challenge
with 3×107 amastigotes 4 weeks after
Partial protection (lower parasite
load in liver); cross-protection
(reduced lesion and parasite load)
[81]
Leish-111f
polyprotein
Mouse,
BALB/c

L. major
3 subcutaneous injections of 10 µg of Leish-
111f with monophosphoryl lipid A plus
squalene (MPL–SE) and IL-12 into the
footpad; challenge with 2×105 promastigotes
or 104 amastigotes after 3 weeks
Partial protection
(smaller lesion size)
[98]
Leish-111f
polyprotein
45 beagle dogs L. infantum
2 courses including 3 injections of 45 µg of
Leish-111f with 50 µg MPL–SE or 1 µg
Adjuprime in 4 week intervals; natural
exposure to sand fly bites for infection
No effect [99]
Leish-111f
polyprotein
Mouse,
C57BL/6
(8 animals per
group),
golden Syrian
hamster
(5 animals per
group)
L. infantum
3 subcutaneous injections of 10 µg of the
protein in 3 week intervals into the foot pad;
intravenous injection of 5×106
promastigotes 4 weeks after immunization
Partial protection
(lower parasite load in spleen and
liver)
[95]
Leish-110f
polyprotein
Mouse,
BALB/c,
C57BL/6
3-5 animals per
group
L. major
L. infantum
3 subcutaneous injections of 0.5-10 µg of the
protein with 20 µg MPL–SE or 5 µg of
synthetic toll-like receptor 4 agonist (EM005)
in 2-3 week intervals; challenge with 103 L.
major promastigotes into the ear dermis 3-5
weeks later (BALB/c)
or 5 x 106 promastigotes of L. infantum
intravenously into the tail vein (C57BL/6)
Partial protection
(smaller lesion size, lower parasite
load in the ear – BALB/c after L.
major infection, and lower parasite
load in the liver after L. infantum
infection of C57BL/6)
[100]

Page 6

1448 Current Medicinal Chemistry, 2012 Vol. 19, No. 10 Kobets et al.
(Table 1). Contd…..
Type of vaccine Host Parasite Treatment Outcome Reference
KSAC polyprotein
(kinetoplastid
membrane protein
11, sterol 24-c-
methyltransferase,
A2 and cysteine
proteinase B)
Mouse,
BALB/c,
C57BL/6
5-6 animals per
group, 3 exp.
L. major,
L. infantum
3 subcutaneous injection of 10 µg of the
recombinant polyprotein or mixture of four
proteins (2.5 µg of each) with 20 µg of MPL-
SE in 3 week intervals into the rump;
C57BL/6 mice were challenged intravenously
with 5×106 of L. infantum promastigotes;
BALB/c mice were infected intradermally
with L. major 3 weeks after last immunization
Partial protection (smaller lesions,
lower parasite load)

[101]
Q polyprotein
(Lip2a, Lip2b, P0
and the histone
H2A proteins)
Mouse,
BALB/c,
5 animals per
group;
20 beagle dogs
L. infantum
3 intraperitoneal immunizations with 2 µg of
the protein Q and 5×105 of BCG (mice) or 4
µg/kg of the protein Q with 106 BCG (dogs) in
two week intervals; intravenous challenge
with 106 promastigotes after 2 weeks
Significant protection (lower
parasite load and lower
pathological changes in the inner
organs)
[102]
Q polyprotein 21 beagle dogs L. infantum
1 or 2 (in 3 weeks) subcutaneous 100 µg
doses of the protein Q; 5×105 promastigotes
intravenously 60 days after vaccination
Partial protection (lower parasite
load in spleen, lymph nodes and
skin)
[103]
LEISH-F1 + MPL-
SE peptides
Human,
34 vaccinated, 17
LEISH-F1
protein injected,
17 control males
and females
L. major
L. braziliensis
10 µg LEISH-F1 with 25 µg MPL-SE on days
0, 28, and 56
Safety trial,
the vaccine is considered as well
tolerable, immunogenic (estimation
of the antibody response)
[96]
Leishmune®
FML (fucose-
mannose ligand)
Dogs,
32 immunized,
40 controls
L. donovani 3 subcutaneous doses in 3 week intervals
Protection, 100% seropositivity to
FML and complete absence of the
disease in the vaccinated group
after 11 month
[23]
Leishmune® Dogs

Toxicity trials,
no parasite
administration
Three doses of 1.5 mg of fucose-mannose
ligand with 0.5 mg of saponin subcutaneously
in 21 days intervals
Well tolerable [149]
DNA vaccines
gp63 gene

Mouse,
CBA,
4-6 animals per
group

L. major

2 oral immunization with 5×109 of
Salmonella expressing gp63 in 2 week
intervals;
subcutaneous injection of 2×107
promastigotes 2 weeks after immunization
into the rump

Partial protection (smaller lesions)

[107]
gp63 gene Mouse,
BALB/c
6 animals per
group, 3 exp.
L. major
2 oral immunizations with 106 of Salmonella
expressing gp63 in 2 week intervals;
subcutaneous injection of 106 promastigotes 2
weeks after immunization into the rump
Partial protection (smaller lesions) [108]
LACK
(Leishmania
homolog of
receptors for
activated C-
kinase) gene
Mouse,
BALB/c
12 animals per
group
L. major
2 injections of 100 µg of the plasmid in
phosphate buffer saline in 2 week interval;
105 of promastigotes after 2 weeks into the
footpad
Partial protection
(smaller lesion size, lower parasite
load in lymph nodes)
[91]
LACK gene Mouse,
BALB/c
2-4 animals per
group, 3 exp.

L. chagasi
2 intramuscular or subcutaneous injections of
30 µg DNA in 2 week interval; intravenous
challenge with 107 promastigotes 4 or 12
weeks after immunization
No reduction of parasite load in
spleen and liver
[111]
pCI-neo-LACK
(LACK gene in
pCI-neo vector)
Mouse,
BALB/c,
6 animals per
group

L. chagasi
2 intranasal vaccinations wtith 30 µg DNA in
1 week interval; intravenous challenge with
107 promastigotes after 1 week
Significant protection (less
parasites in spleen and liver)
[110]
Parasite surface
antigen 2 (PSA-2)
Mouse,
BALB/c,
C3H/He
7-8 animals per
group

L. major
2 intramuscular injections containing 50 µg of
the PSA-2 and/or 50 µg of IL-12 plasmids in
2 weeks; 103or 105 of promastigotes after two
weeks to the rump
Partial protection in case of
administration of IL-12 or PSA-2
alone
(smaller lesion size)
[150]

Page 7

Leishmaniasis: Prevention, Parasite Detection and Treatment Current Medicinal Chemistry, 2012 Vol. 19, No. 10 1449
(Table 1). Contd…..
Type of vaccine Host Parasite Treatment Outcome Reference
L. major signal
peptidase
Mouse,
BALB/c,
10 animals per
group

L. major
2 injections of 30-100 µg of each plasmid in
phosphate buffer saline in 3 week interval;
3×105of promastigotes after 3 weeks into the
footpad
Partial protection
(smaller lesion size, lower parasite
load in lymph nodes)
[106]
Cocktail of
plasmids encoding
LACKp24,
TSA,
LmSTI1,
CPa
Mouse,
BALB/c
10 animals per
group
L. major
Intramuscular injection of 50 µg of each
plasmid in phosphate buffer saline; 5×105
promastigotes after 2 weeks into the footpad
or 500 promastigotes into the ear dermis
Complete protection only after
low-dose infection (78% of mice
with no lesions);
partial protection during the
standard infection
[109]
Cocktail of
plasmids encoding
cysteine
proteinases I, II
and III
Mouse,
BALB/c,
13 animals per
group
L. major
2 injections of 50 µg of each plasmid in solid
lipid nanoparticles in phosphate buffer saline
in 3 week intervals into the footpad; 3×106of
promastigotes after 3 weeks into the footpad
Partial protection
(smaller lesion size, lower parasite
load in lymph nodes)
[112, 151]
Combination of
TRYP
(tryparedoxin
peroxidase)
plasmid with
MVA (modified
vaccinia virus
Ankara) and TLR
1/2 agonist
Pam3CSK4
Mouse,
BALB/c,
8-10 animals per
group
L. panamensis
2 injections, priming immunization with of
100 µg of TRYP DNA with or without 10 µg
of Pam3CSK4, after 2 weeks, intraperitoneal
boosting with 3×106 of TRYP-expressing
MVA; 5×104 of promastigotes into the
footpad 6 weeks after boosting
Partial protection, requiring TLR
1/2 activation
(smaller lesion size, lower parasite
load in lymph nodes)
[152]
Sand fly saliva
Total saliva of
Phlebotomus
duboscqi

Mouse,
BALB/c,
6 animals per
group

L. major

Exposure to sand fly bites (30 sand flies per
mouse) in 4 groups: for 15 days and for 2
days 2 weeks before infection; for 15 days
and for 2 days immediately before infection;
challenge with 106 promastigotes into the ear
dermis

Protection only in case of short-
time immunization (2 days)
immediately before infection
(smaller lesions, lower parasite
load)

[123]
LJM11 salivary
protein gene
from Lutzomia
longipalpis
Mouse,
C57BL/6
5 animals per
group, 2 exp.
L. major
3 injections with 5 µg of plasmid DNA in 15
day intervals into the ear dermis; 500
parasites after 15 days into the ear dermis
Partial protection
(smaller lesion size, lower parasite
load in the ear dermis)
[125]
Synthetic
maxadilan (MAX)
of Lutzomyia
longipalpis
Mouse,
CBA,
5 animals per
group
L. major
First injection 25 µg of MAX subcutaneously
into the rump, second injection 25 µg of
MAX intraperitoneally 10 days later, third –
after 2 weeks; challenge with 106 L. major
after 3 days
Partial protection
(smaller lesions)
[124]
Protein SP15
(PpSP15) or
plasmid encoding
SP15 from
Phlebotomus
papatasi
Mouse,
C57BL/6
4-5 animals per
group
L. major
2 injections of 5-10 µg of plasmid DNA or 10
µl of the isolated salivary protein fraction into
the ear dermis in 2 week interval; infection
with 500 parasites 2 weeks later
Partial protection (smaller lesions) [126]
Plasmids encoding
PpSP15,
PpSP44 proteins
of Phlebotomus
papatasi
Mouse,
C57BL/6
10 animals per
group, 3 exp.

L. major
Injection of 5 µg of plasmid DNA into the ear
dermis; infection with 500 parasites 2 weeks
later
PpSP15-immunized mice were
partially protected (smaller lesions,
less parasites in ear)
[127]

with L. infantum sir+/- led to complete elimination of wild type
parasites after subsequent infection [57]. Complete protection
against L. donovani in liver and partial protection against this
parasite in spleens accompanied by a strong Th1 response was
achieved after vaccination of BALB/c mice with L. donovani cen1-/- .
The partially protective effect of this vaccine was observed also in
hamsters [59]. Several mouse strains (Table 1) vaccinated with L.
mexicana cpa/cpb-/- developed smaller lesion size after wild
parasite strain challenge in comparison with naïve animals [54].
Interestingly, BALB/c vaccinated with L. major lpg2-/- were
substantially protected against virulent L. major without developing
a strong Th1 response [56]. Partial protection was observed also
after vaccination of BALB/c mice with L. major pmn-/- [58].
Vaccination of BALB/c and CBA/T6 mice with L. major deficient
in dhfr-ts-/- led to substantial protection against L. major [51] and to
partial protection against L. amazonensis in BALB/c and C57BL/6
mice [52], but had no effect in monkeys [53]. Attenuated vaccines
usually have better effect when vaccination is performed shortly (1-
2 weeks) before the challenge [51, 58]. Some mutants, for example
L. major pmn-/- undergo a short life cycle [58], whereas L. major

Page 8

1450 Current Medicinal Chemistry, 2012 Vol. 19, No. 10 Kobets et al.
lpg2-/- can persist in mouse up to whole lifetime [60] and can revert
to virulence [61].
Suicidal Cassettes
Introduction into parasite genes, which will allow inducing
suicide in response to external signals, such as antibiotics [62] or
photoactivation [63] can prevent a long persistence and the possible
reversion to virulence. A double drug sensitive strain of L. major
was constructed by stably introducing into the chromosome a
modified HSV-1 thymidine kinase gene and a Saccharomyces
cerevisiae cytosine deaminase gene, conferring sensitivity to
ganciclovir and 5-fluorocytosine, respectively, which made it
possible to cure BALB/c mice infected by modified L. major using
these antibiotics [62]; the protective effect of this parasite has not
been tested. L. amazonensis, which episomally expressed human
gene δ-aminolevulinate dehydratase and rat gene porphobilinogen
deaminase, was intradermally
Administration of δ -aminolevulinate led to accumulation of
photosensitive uroporphyrin, which was excited by light to produce
leishmanolytic oxidative species. This treatment led to protection
against subsequent challenge with L. donovani [63]. Thus, these
experiments show an interesting alternative for development of safe
live vaccines.
2.1.1.4. Killed Parasites
A killed vaccine consists of pathogen, which is grown in culture
and then killed by heat [64, 65], formaldehyde [66], by repeated
cycles of freezing and thawing [67] or by autoclaving [65]. The
advantage of killed vaccines is safety and low cost. These should be
prepared in standardized conditions to have stable biochemical
composition and immunogenicity.
Some studies with killed Leishmania were reported from 1930s
and 1940s, but controlled trials were conducted only after 1970 [68,
69]. Noazin and coworkers [68] analyzed efficacy of vaccines
prepared using killed whole parasites that were used in Iran, Sudan,
Brazil, Colombia, and Ecuador and found that these vaccine
candidates do not confer significant protection against human
leishmaniasis [68]. Vaccines with killed parasites could be
ineffective due to several reasons. Autoclaving of parasites, which
is usually used in preparation of human vaccines lowers the
immunogenicity by destroying most of the proteins [70]; in
addition, inhibitory determinants might appear on surface.
Replication, metabolic activity and persistence of parasites might be
also necessary for the development of the protective immunity.
Comparison of killed and live vaccines in a mouse experiments has
shown that live parasites induce better immune response than the
dead ones [57, 59, 64].
New ways to deliver vaccines, such as nanovaccines might
improve the efficacy of whole-killed vaccines. Poly(D,L-lactide –
co-glycoside) nanospheres as an antigen delivery system and
cytosine-phosphodiester-guanine
ODN) as an adjuvant have been used to enhance the immune
response against autoclaved L. major in BALB/c mice. Highly
significant, although not complete protection against subsequent
infection was observed [71].
Although an efficient killed prophylactic Leishmania vaccine
has yet to be developed, killed parasites vaccines have been
successfully used to improve the chemotherapy. For example, the
combination of killed promastigotes of L. amazonensis with a half
dose regimen of the pentavalent antimonials was highly effective
for the treatment of American cutaneous leishmaniasis [72].
2.1.2. Vaccination with Parasite Subunits
The use of parasite subunits is based on the fact that pathogen
contains both the epitopes that enhance immune response against it
and those that aggravate the disease [73, 74]. So, the aim is to
selects proteins with epitopes enhancing host defense against the
injected into hamsters.
oligodeoxynucleotide (CpG
pathogen. This selection is based on the information about the
abundance and important roles of certain parasite proteins in
pathogen biology, and/or the entire genome sequence is used to
identify vaccine candidates. In the latter approach, which was
denominated “reverse vaccinology” a large number of candidate
antigens are expressed in the heterologous expression system,
purified, and used to immunize mice. As a result, the proteins
inducing beneficial antipathogen responses are selected [75, 76]. To
date, at least 30 vaccine subunits against leishmaniasis have been
tested [77, 78]. In most experimental systems that use pathogen-
derived proteins, adjuvants are essential to provoke protective
immunity. However, the most effective adjuvants generally cause
strong inflammation, which may be essential for adjuvanticity, but
may preclude their use in humans because of unacceptable side
effects [21].
2.1.2.1. Vaccination with Leishmania Fractions
Leishmune® consists of an affinity purified L. donovani
promastigote glycoprotein fraction, whose composition is not
completely defined, named fructose mannose ligand (FML) and the
adjuvant consisting of aldehyde-containing deacylated saponins of
Quillaja saponaria [79]. A fucose-mannose ligand (FML), also
present on Leishmania surface, is now commercially available and
was used for the treatment of dogs and proved to prevent
development of canine leishmaniasis and to block its transmission
[23, 79], which led to decrease of the incidence of human and
visceral leishmaniasis in Brazilian endemic areas [80]. Vaccination
of BALB/c mice with C-terminal domain of a secreted nucleoside
hydrolase, which is the dominant antigen in the FML, has been
shown to reduce parasite numbers in livers, parasite load in lesions,
and lesions size after challenge with L. chagasi and L. amazonensis,
respectively [81]. The first European canine Leishmania vaccine
CaniLeish® (LiESAp) was released in 2011 (http://ec.europa.eu/
health/documents/community-register/html/vreg.htm.txt). It con-
sists of excreted secreted proteins from Leishmania infantum
(LiESAp), the dominant antigen of which is the promastigote
surface antigen [82].
2.1.2.2. Vaccination with Defined Proteins
Range of early studies showed success in the use of peptide
vaccines in animal models [83, 84]. The most comprehensively
studied anti-leishmanial vaccine candidate is the surface- expressed
63 kDa glycoprotein (gp63), or leishmanolysin, tested for
development of vaccine against L. major [83-87]. This glycoprotein
metalloprotease is a parasite receptor for host macrophages and
functions in the receptor mediated uptake of promastigotes by
macrophages in the mammalian host. Hence, mutant parasites
lacking the protein have reduced virulence [86]. Some regimens of
vaccination with gp63 led to protection against leishmaniasis [83-
86], whereas others had no influence or even exagerated disease
[78, 85].
The LACK antigen (Leishmania homologue of receptor for
activated C kinase) 36 kDa is expressed by both promastigote and
amastigote form of the parasite [88]. The LACK antigen, which is
MHC class II associated, is an analogue of the mammalian receptor
for activated protein kinase C (RACK) [89]. It belongs to a protein
family containing the repeat motif termed WD 40 that is present in
a large number of eukaryotic genes [90]. Vaccination of BALB/c
mice with LACK 24kDa protein in combination with IL-12 led to
partial protection against L. major [88, 91].
PSA (promastigote surface antigen) is one of the major classes
of membrane-bound or secreted proteins of the parasitic protozoan
Leishmania, whose main signature consists of a specific LRR
(leucine rich repeats) sequence. All PSA genes found in the
genomes of three sequenced Leishmania species distribute into
eight subfamilies of orthologs. Seven of these subfamilies
correspond to basic functions related to parasite/host interactions,
the other PSA gene class, which include all so far experimentally

Page 9

Leishmaniasis: Prevention, Parasite Detection and Treatment Current Medicinal Chemistry, 2012 Vol. 19, No. 10 1451
studied PSA genes, could be involved in more specialized
adaptative functions [92]. Intraperitoneal vaccination of C3H/He
mice with PSA-2 with Corynebacterium parvum as an adjuvant
resulted in complete protection from lesion development after a
challenge infection with virulent L. major. Significant protection
was also obtained in the genetically susceptible BALB/c-H2k and
BALB/c mice [93]. Partial protection against L. major was achieved
also with vaccination of BALB/c mice with nucleoside hydrolase
protein [94]. Large number of other potential candidates for vaccine
design in this field is extensively studied [78]; however, this
approach has brought conflicting results in many cases due to
possible conformational changes and insufficient immunogenicity
of individual separated peptides, which could be effective only in
cocktails [21].
The LEISH-F1 + MPL®-SE vaccine is composed of the
recombinant Leishmania polyprotein LEISH-F1 (formerly known
as Leish-111f) antigen and the MPL®-SE adjuvant. The antigen
component of the vaccine includes three proteins derived from L.
major and conserved across various Leishmania species, including
L. donovani and L. chagasi, which causes Old World and New
World visceral leishmaniasis, respectively; and L. braziliensis,
which causes both cutaneous leishmaniasis and mucosal
leishmaniasis in the New World. The three proteins are: Leishmania
elongation initiation factor (LeIF), thiol-specific antioxidant (TSA),
and Leishmania major stress-inducible protein 1 (LmSTI1). The
adjuvant component is a potent TLR4 agonist - monophosphoryl
lipid A, which is derived from the lipopolysaccharide of Salmonella
enterica serovar Minnesota and formulated in the adjuvant
monophosphoryl lipid in a stable squalene oil-in-water emulsion
(MPL®-SE) [95-97]. Immunization trials in mice demonstrated that
Leish-111f was able to protect BALB/cByJ mice against L. major
and L. amazonensis infection [98] and to induce partial protection
against visceral leishmaniasis in C57BL/6 mice and golden Syrian
hamsters [95]. However, Leish-111f failed to protect dogs against
infection and did not prevent disease development in a Phase III
trial in dogs [99]. The Leish-111f polyprotein was modified in order
to eliminate a potential regulatory concern and an apparent
proteolytic hot spot was eliminated and the new 110 kDa construct
was named Leish-110f. Vaccination with this construct and MPL®-
SE adjuvant completely protected BALB/c mice against lesion
development after L. major infection and partially protected
C57BL/6 mice against visceral leishmaniasis after infection with L.
infantum [100].
KSAC is a polyprotein vaccine composed from fused proteins
KMP-11 (kinetoplastid membrane protein 11), SMT (sterol 24-c-
methyltransferase), A2 and CPB (cysteine proteinase B).
Administration of KSAC and MPL®-SE adjuvant strongly, but not
completely protected BALB/c mice against L. major infection and
C57BL/6 mice partially protected against L. infantum [101].
Another multi-component antigenic protein, named Q, was
formed by the genetic fusion of five fragments from the L. infantum
acidic ribosomal proteins Lip2a, Lip2b, P0 and the histone H2A
protein and administered with BCG adjuvant. This led to a strong
protection of BALB/c mice and dogs against L. infantum infection
[102]. Q protein protected dogs against L. infantum also when
administered without adjuvant [103].
2.1.3. DNA Vaccines
The concept of DNA vaccination was established by Wolff and
coworkers in 1990 [104]. DNA vaccines include cloned genes of
the vaccine candidates, which are injected directly into muscle
tissue or skin. A plasmid is taken up and expressed by the host
cells, revealing a properly folded protein of interest that causes
immunization [21, 105] and usually induces more effective
protective responses than vaccination with isolated protein [91,
106]. Among the first candidates for DNA vaccines was the gene
for gp63 [107, 108], mentioned above (section 2.1.2.2.). Vaccines
based on gp63 induced partial protection against L. major in CBA
[107] and BALB/c mice [108]. LACK (Leishmania homologue of
receptors for activated C kinase) is the most extensive DNA
vaccine studied so far against both cutaneous [91, 109] and visceral
leishmaniasis [110, 111]; partial protection was achieved in
BALB/c mice during challenge with L. major [91, 109]. Moreover,
a cocktail DNA vaccine containing LACKp24 (a truncated portion
of the LACK antigen), TSA (L. major homolog of the eukaryotic
thiol-specific-antioxidant) and CPa (cysteine proteinase A) was able
to induce complete protection after low-dose infection with L.
major in BALB/c [109]. Subcutaneous or intramuscular vaccination
of mice with plasmid encoding p36 L. infantum LACK protein
failed to protect of BALB/c from the subsequent infection with L.
chagasi although it induced strong IFN-γ production [111], whereas
intranasal vaccination with the same plasmid together with the
shortening the interval between last booster and infection (from 4 or
12 weeks to 1 week) led to strong, but not complete protection
[110]. Immunization of BALB/c mice with a cocktail DNA vaccine,
encoding cysteine proteinases type I, II, and III with solid lipid
nanoparticles, potentiated protective immunity against L. major
infection [112]. Knowledge of genome sequence of Leishmania
[113] enabled to test 100 candidate DNA vaccines against L. major.
Fourteen protective novel vaccine candidates were identified, seven
vaccines exacerbated disease. Two protective antigens were
identified as ribosomal proteins 60S ribosomal L22 and 40S
ribosomal S19, three other had significant matches to previously
identified proteins V-ATPase subunit F, dynein light chain, and
amastin-like protein. Functions of remaining nine protective
proteins are not known. The best novel protective antigen was an
amastin-like gene that conferred significant, but not complete,
decrease of footpad swelling [73].
2.2. Vaccines Based on Transmitting Phlebotomine Vector
2.2.1. Whole Saliva
The possibility of vaccine development using sand fly saliva
was also considered. Sand flies are bloodsucking insects that are
natural vectors of Leishmania parasites (see section 1). They
deposit parasites into the host skin along with saliva, which
contains immunomodulatory molecules [114, 115] that induce
species-specific humoral and cellular response in the host [116-
119]. Local inhabitants in endemic areas develop a specific
antibody response to salivary antigens, which correlates with
protection against visceral leishmaniasis [120], but not against
cutaneous leishmaniasis [121, 122]. The history of immunization
may also significantly change the character of anti-saliva immune
response with substantial consequences for development of
Leishmania. Using a mouse model, it was shown that the protection
took place only in a case of short-term exposure to bites of
Phlebotomus duboscqi (two days exposure to bites) right before the
challenge with L. major parasites, but not in cases of long-term
exposure (15 days) or short or long immunization periods followed
by a significant time interval before the infection occurred (Table 1)
[123]. This finding is in agreement with the field results from an
endemic area of cutaneous leishmaniasis caused by L. braziliensis
[122].
2.2.2. Saliva Components
Saliva components were isolated from both New World [124,
125] and Old World [126, 127] phlebotomine sandflies and their
prophylactic ability has been tested. Vaccination with Lutzomyia
longipalpis salivary protein maxadilan partially protected CBA
mice from L. major infection. The protective role of a LJM11 (a
yellow protein from saliva of Lutzomyia longipalpis) was proved by
immunization of C57BL/6 mice with peptide-encoding plasmids
and subsequent infection with L. major [125]. A DNA vaccine
coding Phlebotomus papatasi
(PpSP15/SP15) from the vector of conferred partial protection
15 kD salivary protein

Page 10

1452 Current Medicinal Chemistry, 2012 Vol. 19, No. 10 Kobets et al.
against L. major to C57BL/6 mice [126], which was accompanied
by the increase of IFN-γ RNA transcripts and decrease of IL-4
transcripts in the mouse ear 2 hours after immunization [127].
2.3. Problems of Vaccination Against Leishmaniasis and Novel
Vaccine Strategies
Despite the wide range of studies carried out during quite a long
period of time, there is no reliable vaccine against leishmaniasis
available at the present moment. The cost of developing a vaccine
has been estimated to be hundreds of millions of dollars; 60–80% of
this is allotted to preclinical and clinical development of the
vaccine. Except for a few cases, funding for human vaccine
development has been minimal until very recently. About 90% of
the burden of VL is present in five countries (India, Bangladesh,
Sudan, Ethiopia and Brazil). Those affected in these countries are
among the poorest of the poor, and hence there is not a large
enough market for pharmaceutical companies to invest a
considerable amount of time and money to develop a vaccine [69].
In addition, different research groups may obtain contradictory
results after testing the same candidate target for vaccine
development. This might be partly due to different conditions of the
experiments, as many factors influence immunogenicity and
efficacy of vaccination: type of the vaccine [86], the dose, the route
[128], and regimen [58, 123] of administration, type of adjuvant
[66, 86] and the mode of virulent challenge [109]. The same might
be true for differences in outcomes of experimental and human
trials. Moreover, few of the mouse experiments are completely
protective (Table 1) and usually the most efficient protection is
observed when challenge with virulent parasite is performed shortly
after vaccination. The short time protection might be beneficial for
tourists traveling to the endemic regions, but it is not suitable for
the local population.
The complexity of immune response to leishmaniasis is not yet
fully explored. Some vaccines induce Th1 type of immune
response, that is considered to be protective in leishmaniasis, but
have no or a limited influence on organ pathology [111, 129, 130].
This is in agreement with genetic studies in mouse, where some
genes influence both organ pathology and immune response
including cytokine levels, whereas the others determine only
cytokine levels [28, 131, 132]. Better definition of pathways that
are involved in control of pathology will help to prepare more
effective vaccines.
There is another important point that needs to be mentioned.
Majority of vaccination experiments are performed on the mouse
strain BALB/c [78] (Table 1). This is not representative of different
genotypes present in the outbred human population. Moreover, it
was shown that several patterns of the immune response are
observed after infection with L. major, depending on the host
genotype [133, 134]. Different regulatory pathways operating in
different genetic backgrouds could lead to different response to
vaccination. Indeed, Benhnini and coworkers [135] have shown that
capacity of L. major proteins LACK (Leishmania homolog of
receptor for activated C kinase) and LmPDI (L. major protein
disulfide isomerase) to confer protection to L. major infection
depended on the genotype of the mouse strain. In PWK and
BALB/c mice vaccination with both proteins led to substantial and
partial decrease of lesion size, respectively, whereas in C57BL/6
mice none of the proteins conferred protective response.
Vaccination of MAI mice with LmPDI led to complete protection,
while LACK had no influence on immunity. Thus, to more tightly
mimic the situation in the outbred human population, larger
spectrum of mouse strains needs to be tested. The antigen that
confers protection in the larger range of inbred strains may have
better chances to be also protective in outbred human population
and should be selected for clinical trials [135]. Hence, integration of
genetic studies with methods which will allow to select more
immunogenic candidate vaccine antigens by employing methods of
reverse vaccinology [73, 75, 76, 136], proteomics [137] and protein
engineering [138] together with the advanced delivery systems such
as nanoparticles [71, 112, 139], dendritic cells [140-142], and
dendritic cell-derived exosomes [141] can help to produce more
efficient vaccines against leishmaniasis (Table 1).
The lack of a safe vaccine or chemoprophylaxis limits the
options for prevention of leishmaniasis to elimination of reservoir
populations and some form of vector control, including barriers to
sand fly feeding [143]. Therefore the early pathogen detection and
identification that will be described in the following section has the
key importance for a timely disease treatment.
3. ASSAYS FOR PARASITE DETECTION AND QUANTI-
FICATION
As with any infectious disease, spread and load of the pathogen
during leishmaniasis is an important parameter, as well as the
precise diagnostics of Leishmania species. Until recently, this task
was a significant complication for practical doctors and researchers.
The optimal methods should be fast, simple and price-worthy.
Easy-to-use techniques for rapid pilot tests during field application
in the absence of the equipped laboratory are also highly necessary.
The main approaches for parasite detection include DNA-based
techniques, microscopy assays, cell cultures, and immunodetection
[153, 154].
3.1. Microscopy for Detection, Quantification and Histological
Studies
3.1.1. Classical and Fluorescent Microscopy
Microscopy assays were among the first methods for detection
and quantification of a wide range of pathogens, including
Leishmania parasites. This kind of methods allows detecting and
counting parasites directly. Histological examination of biopsies
can give information about parasite load, infiltration of different
cell types, formation of granulomas and other changes in
Leishmania invaded tissues,
Hematoxylin and eosin (H&E) or Giemsa are widely used for
routine staining of tissue smears and biopsies, for example, [40, 53,
63, 102, 122, 132, 153-165], as well as various fluorescent labels,
for example, [155, 157, 166-168]. Microscopy belongs to the gold
standard assays, being effective both for stand-alone studies [163,
167, 169] and validation of the novel methods [132, 154, 156, 158,
159, 161, 170]. Among the most significant drawbacks of the
microscopy assays is the fact that the direct detection of parasites
on stained smears from aspirates of bone marrow, lymph node or
spleen of patients, requires invasive procedures [171]. Microscopy
analysis of large numbers of samples is usually time consuming.
Moreover, quantification of parasites in certain samples may not
reflect the real parasite load, because parasites are distributed in
tissues unequally.
3.1.2. Electron Microscopy
Interactions between Leishmania parasites and macrophages
can be studied in details using electron microscopy. Scanning
electron microscopy has revealed that the phagocytosis of L.
amazonensis metacyclic promastigotes begins with the engulfment
of the parasite body and ends with the progressive internalization of
the flagellum. Transmission electron microscopy has revealed that
promastigotes are located in very long phagosomes, in which the
membrane tightly follows the outline of the parasite including that
of their flagellum. Immunogold cryosection electron microscopy
has been useful for improving the understanding regarding the
distribution and trafficking of major histocompatibility complex
class II molecules in macrophages parasitized by L. amazonensis
[169].
reflecting the pathogenesis.

Page 11

Leishmaniasis: Prevention, Parasite Detection and Treatment Current Medicinal Chemistry, 2012 Vol. 19, No. 10 1453
3.1.3. In-Vivo Imaging
The data obtained from imaging fixed samples gave a very
incomplete picture of the dynamic nature of the complex processes
Leishmania drives in both the insect and the mammalian host.
Emerging technologies using fluorescence and bioluminescence
imaging have been recently adapted for the study of host–
Leishmania interactions [166] including response to therapy [166,
167]. Bioluminescence imaging of mice inoculated with transgenic
Leishmania expressing the firefly luciferase provides an efficient
and reliable method for delineating the various phases of infectious
processes. Whole-body imaging with fluorescent parasites has also
recently been developed to monitor light emission without substrate
addition, making use of different colored fluorophores for multiplex
imaging. Because of its nondestructive and noninvasive nature, the
procedures for bioluminescent and fluorescent imaging can be
performed repeatedly, allowing each animal to be used as its own
control over time, overcoming the problem of animal–animal
variation [166, 167].
3.2. Cell Culture Based Methods
Cell cultures include isolation and cultivation of Leishmania
from cells and tissues. Viable parasites multiply and their numbers
are evaluated after several days of incubation in the nutrient
solution. This group of methods, along with microscopy assays,
belongs to the first techniques developed for the estimation of
parasite load in the infected host organism [36, 159, 161, 172, 173].
3.2.1. Limiting Dilution Assay
The majority of tissue culture techniques are based on limiting
dilution assay [172, 173]. These methods use a range of serial
dilutions to assess the ratio of cells containing viable parasites.
Limiting dilution assay, originally developed for Leishmania
detection and quantification, is a highly laborious and time
consuming technique. Detection of viable parasites is the main
advantage of this method. Since culture methods target viable cells,
they should be performed immediately after isolation of the tissues
and require sterile conditions.
3.2.2. Microculture and Miniculture
A microcapillary culture method (MCM) was developed for
diagnosis of cutaneous leishmaniasis. In contrast to traditional
culture method, MCM is more rapid, uses smaller sample, and has
higher sensitivity for detection of promastigotes. In comparative
studies, the average time period of incubation needed to detect
promastigotes was much shorter with the microcultures than the
conventional cultivation and required 2-7 days versus 2-30 days.
The high sensitivity of the MCM may be explained by the use of
capillary tubes, which concentrate the sample material and provide
microaerophilic conditions with high CO2 that is favorable for
transformation of amastigotes to promastigotes [161, 174].
Table 2.

Specific Targets Used in DNA-Based Assays for Detection and Quantification of Leishmania spp.
Target Description References
kDNA Minicircle kinetoplast DNA. Each parasite contains about 10 000 of minicircles. [154, 156, 159, 180, 181]
18S rRNA / ssu-
rRNA
The gene that encodes RNA included to the small ribosomal subunit. [171, 182-184]
mini-exon
A very small gene of 39 nucleotides, present in Kinetoplastida. An intron, adjacent to the mini-exon, is
conserved. It is followed by a sequence that varies in length considerably between species and is much less
conserved.

[184, 185]
ITS1 region
Internal transcribed spacer 1: the sequence located between the 18S ribosomal RNA and 5.8S ribosomal RNA
genes.
[184, 186]
ITS2 region
Internal transcribed spacer 2: the sequence located between the 5.8S ribosomal RNA and 28S ribosomal RNA
genes.
[165]
7SL RNA

7SL RNA together with six proteins forms a signal recognition particle which mediates protein translocation
across the endoplasmic reticulum in eukaryotes, including Leishmania.
[187]
gp63 The gene coding Leishmania surface protease; highly conserved. [188]
hsp70
The gene of 70kDa heat shock protein; highly conserved among eukaryotes, however, contains some variable
regions; used for phylogenetic studies.
[189, 190]
DNA/RNA
polymerases
Genes that encode DNA and RNA polymerases. Their sequences were used to reveal evolution of Leishmania. [191]
cyt b
Cytochrome b gene is present in the mitochondrial genome, encodes the central catalytic subunit of an enzyme
present in the respiratory chain of mitochondria; the gene was used for phylogenetic studies.
[192]
g6pdh The gene coding glucose-6-phosphate dehydrogenase. [193, 194]
icd The gene coding isocitrate dehydrogenase [193]
me The gene encoding cytosolic NADP-malic enzyme [193]
mpi The gene encoding mannose phosphate isomerase [193]
fh The gene encoding fumarate hydratase [193]
cpb A cluster of genes for cysteine protease B [195]
asat The gene encoding aspartate aminotransferase [176]
gpi The gene encoding glucose-6-phosphate isomerase [176]
nh1 The gene coding nucleoside hydrolase 1 [176]
nh2 The gene coding nucleoside hydrolase 2 [176]
pgd The gene coding 6-phosphogluconate dehydrogenase [176]

Page 12

1454 Current Medicinal Chemistry, 2012 Vol. 19, No. 10 Kobets et al.
Miniculture method, in which parasites are cultivated inside the
Eppendorf tubes, also showed better results than conventional
culture methods [174].
3.2.3. Multilocus Enzyme Electrophoresis (MLEE) for Parasite
Classification
MLEE is based on the isoenzyme analysis and also requires
preparation of parasite cultures. It is one of the most comprehensive
methods used for identification of Leishmania, particularly the
MON system, which was developed in Montpellier, France. MON
system is based on 15 enzymes (malate dehydrogenase, malic
enzyme, isocitrate dehydrogenase,
dehydrogenase, glucose-6-phosphate dehydrogenase, glutamate
dehydrogenase, NADH diaphorase,
phosphorylase, purine nucleoside phosphorylase, 2 glutamate-
oxaloacetate transaminases 1 and 2, phosphoglucomutase, fumarate
hydratase, mannose phosphate isomerase, glucose phosphate
isomerase) [175-177]. MLEE includes Leishmania parasite
isolation and cultivation, followed by preparation of enzyme
extracts from the promastigotes pellets. Extracts are analyzed by
electrophoresis. The distance that each reproducible enzyme band
migrates from the origin (anode) is measured. The obtained set of
bands defines the zymodeme, an electrophoretic profile for each
extract. Attribution of the species is made by comparison of the
profiles with the reference Leishmania strains [178, 179].
Isoenzymes have been used to generate phylogenetic trees and to
provide a basis of the current taxonomy of L. donovani complex,
which has three designated species, L. donovani, L. infantum and L.
archibaldi [176]. For diagnostic purposes, lesion aspirates from
patients were analyzed by MLEE [178, 179].
6-phosphogluconate
purine nucleoside
3.3. Assays that Detect Parasite DNA
In general, DNA-based methods were developed to detect
presence of different regions of parasite DNA in experimental or
clinical samples (Table 2). Techniques of this type serve two main
purposes. First, they can be used for detection of Leishmania
presence and measurement of parasite load in cells and tissues.
Second, species- or subspecies-specific methods help to identify the
parasite and make precise diagnosis.
3.3.1. Conventional Polymerase Chain Reaction (PCR), Nested
PCR, and Real-Time PCR (RT-PCR) for Parasite Detection and
Quantification
PCR is an extremely sensitive tool for detection of target DNA
from various sources, including Leishmania. Minicircle kinetoplast
DNA (kDNA) contains conserved regions that are widely used for
Leishmania detection and quantification. Each parasite contains
multiple copies of kinetoplast minicircles that makes kDNA a very
prominent PCR target. First PCR using primers specific to
conserved regions of kDNA was tested on seven Leishmania
species [180] (Table 3). Amplicon can be analyzed on agarose
electrophoresis or dot blot [159, 180]. Besides kDNA, a highly
conserved gene gp63 (encoding the surface protease), mini-exon (a
very small gene of 39 nucleotides, present in Kinetoplastida), and
ITS1 region (a sequence located between the 18S ribosomal RNA
and 5.8S ribosomal RNA genes) were proved to be good targets
both for classical PCR combined with electrophoresis and real-time
PCR, but their sensitivity was either comparable or lower than the
one obtained with kDNA [184, 188, 196-198]. Small ribosomal
subunit gene (ssu-rRNA) was among the first targets proposed for
the Leishmania-specific PCR assays development [171, 182].
Nested PCR is based on application of two pairs of primers and
includes two rounds. Additional internal primer set, specific for the
particular sequences of the first amplicon, can significantly increase
the sensitivity of the technique [156, 185]. For distinguishing
among Leishmania species, the first round PCR using universal
primers (for sequences that are conservative in all tested strains)
should be followed by the second round PCR with specific primers
that amplify the sequences that are unique for each tested strain
[165].
Real-time PCR (RT-PCR) allows analysis of formation of the
amplicon that makes it possible to quantify the initial number of
template molecules. RT-PCR assays for Leishmania detection are
based on the DNA polymerase gene, kinetoplast and ribosomal
DNA. Most of these tests are not species specific. In Real-time
PCR, the product is detected by the fluorescent labels [194, 196,
199, 200]. In addition to the detection and quantification, RT-PCR
served for Leishmania species typing. The test was developed using
a polymorphism in the glucose-6-phosphate dehydrogenase locus
and was performed with SYBR-green or TaqMan master mixes.
This enabled identification and quantification of wide range of
Leishmania species found in the Americas. Although any
amplification assay based on a single-copy target is less sensitive
than the one based on multi-copy targets, the method was effective
for identification and quantification of parasites in human biopsy
samples [194]. SYBR-green binds nonspecifically to any double-
stranded DNA what comprises the major limitation of this system.
Therefore, the highly specific primers and precise optimization of
the synthesis conditions are necessary. In addition, background
fluorescence may diminish both sensitivity and specificity [200].
The ITS1 region is commonly used target in many different
eukaryotic organisms for typing purposes [184, 201-203]. It has
enough conservation to serve as a PCR target but sufficient
polymorphisms to facilitate species typing. The sensitivity of this
RT-PCR was similar to the conventional PCR [184].
As mentioned above (Table 2), a variety of targets are used for
PCR. kDNA is one of the most reliable targets for Leishmania
detection and quantification since there are ~10 000 minicircles per
parasite. The sensitivity and specificity of this method were
compared with two other PCR assays (mini-exon and ITS1),
leishmanial culture and microscopic detection in order to validate
these techniques for molecular
leishmaniasis. The kDNA PCR was the most sensitive diagnostic
assay and should be employed when species identification is not
required. PCR using ssu-rRNA also showed lower sensitivity than
kDNA PCR [171, 182]. However, when further parasite
characterization is needed, the ITS1 PCR is both highly sensitive
and specific and enables the identification Leishmania species. PCR
is now the diagnostic method of choice for cutaneous and
mucocutaneous leishmaniasis, and kDNA PCR is the gold standard
against which all new techniques should be compared [186, 197,
204].
3.3.2. PCR-ELISA for Parasite Detection and Quantification
Important progress was achieved with application of PCR-
ELISA, which combines conventional PCR with enzyme-linked
immunosorbent assay (ELISA) for the detection of labeled product.
Conserved regions of kinetoplast minicircle appeared to be the
preferential target used for testing [153, 154, 200, 205-207]. PCR-
ELISA has sensitivity comparable to RT-PCR and allows detection
of small differences in parasite load. However, some steps of this
technique contain drawbacks that may influence the final result. If
the product is not purified, its hybridization with the biotinylated
[205, 206] or digoxigenin-labeled probes [200, 207] increases the
possibility of an incomplete or nonspecific binding of the probe to
the amplicon, or even DNA, and distortion of the result. Two
labeled primers can be used to omit the step of hybridization.
Highly optimized reaction with sensitive kDNA primers, originally
labeled with biotin and digoxygenin, excludes probability of
nonspecific effects, allows precise quantitative measurement of the
parasite load and a fast analysis of large number of samples [154].
The improved method has been already successfully applied in two
different experimental projects [123, 132].
diagnosis of cutaneous

Page 13

Leishmaniasis: Prevention, Parasite Detection and Treatment Current Medicinal Chemistry, 2012 Vol. 19, No. 10 1455
Table 3.

Methods for Leishmania Parasite Detection
Assay Leishmania
species
Target
Sensitivity (lowest
detectable parasite
load)
Samples Reference
Giemsa staining L. mexicana
L. major
parasite
cells
N/T skin biopsies [153]
Acridin orange staining L. donovani parasite
cells
N/T parasite culture [155]
H&E staining L. tropica parasite
cells
N/T skin biopsies [163]
Electron
microscopy
L. amazonensis
L. major
parasite
cells
N/T macrophages [169]
L. infantum parasite
cells
in vivo: 20000-
40000
amastigotes/mg
tissue;
ex vivo:
1000-6000
amastigotes/mg
tissue
whole
mouse
[166]
Microscopy
In-vivo
imaging

L. major parasite
cells
N/T whole
mouse
[167]
Limiting dilution assay L. major viable
parasites
N/T skin biopsies [173]
Microcapillary culture
method (MCM)
L. tropica viable parasites N/T skin biopsies [161]
Microcultures and
minicultures
Leishmania spp. viable parasites N/T lesion aspirates [174]
Culture-
based
methods
Multilocus
enzyme electrophoresis
(MLEE)
L. major
L. tropica
12 enzymes N/T lesion aspirates [178]
Polymerase chain reaction
(PCR)
L. braziliensis
L. mexicana
L. major
L. chagasi
L. donovani
L. aethiopica
L. enriettii
kDNA 0.1-10 fg parasite DNA,
skin biopsies
[180]
PCR + dot blot
hybridization
L. braziliensis
L. panamensis
L. peruviania
L. guyanensis
kDNA 0.1 fg parasite DNA,
skin biopsies,
lesion aspirates,
lesion scraping
[159]
PCR L. infantum
L. donovani
ssu-rRNA gene 10 parasites in 1 ml parasite DNA,
blood
[171, 182]
PCR L. braziliensis
L. amazonensis
kDNA N/T
Giemsa stained
slides
[181]
Nested PCR L. tropica kDNA 0.1 fg parasite DNA,
skin biopsies
[156]
Nested PCR L. donovani mini-exon 100 fg parasite DNA,
tissue aspirates
[185]
Nested PCR L. major
L. gerbilii
L. turanica
ITS2 N/T parasite DNA,
ear biopsies
[165]
DNA-based
methods
Real-time PCR (RT-PCR) L. major
L. donovani
L. amazonensis
L. mexicana

kDNA 100 fg parasite DNA [196]

Page 14

1456 Current Medicinal Chemistry, 2012 Vol. 19, No. 10 Kobets et al.
(Table 3). Contd…..
Assay Leishmania
species
Target
Sensitivity (lowest
detectable parasite
load)
Samples Reference
Real-time PCR (RT-PCR) L. braziliensis
L. peruviania
L. guianensis
L. panamensis
L. lainsoni
L. utingensis
L. amazonensis
L. mexicana
L. pifanoi
L. infantum
L. adleri
g6pdh
10 parasites in 0.2
ng of total DNA
parasite DNA,
skin biopsies,
mice footpad
[194]
Real-time PCR (RT-PCR) L. donovani
L. major
L. chagasi
L. mexicana
L. braziliensis
gp63 10 fg parasite DNA [188]

Real-time PCR (RT-PCR) L. donovani ITS1 10 fg parasite DNA [184]
PCR + enzyme-linked
immunosorbent assay
(PCR-ELISA)
L. braziliensis kDNA 0.5-0.6 pg parasite DNA,
blood
[205]
PCR-ELISA L. infantum kDNA 1 fg parasite DNA,
skin biopsies
[206]
PCR-ELISA L. major kDNA 0.3 fg
parasite DNA, lymph
nodes
[154]
Oligo-C-TesT L. guyanensis
L. braziliensis
18S
rRNA gene
N/T lesion scrapings [183]
18S
rRNA gene
100 fg

Mini-exon 50 fg
Restriction fragment
length polymorphism
(RFLP)
L. donovani
L. infantum
L. tropica
L. aethiopica
L. braziliensis
ITS1 10 fg
parasite DNA,
skin biopsies,
blood,
bone marrow
[184]
RFLP + reverse dot blot
(RDB)
L. donovani complex
L. major
L. tropica
L. aethiopica
7SL RNA gene 20 fg parasite DNA,
skin biopsies

[187]
Reverse line blot (RLB) L. donovani
L. major
L. tropica
L. aethiopica
L. infantum
ITS1 60 fg – 5 pg,
depending
on probe
parasite DNA,
skin biopsies

[186]

Sequencing

L. donovani
L. infantum
L. chagasi
L. major
L. tropica
L. arabica
L. aethiopica
L. mexicana
L. amazonensis
L. braziliensis
L. panamensis
L. hertigi
L. deanei
L. herreri
L. hoogstraali
L. adleri
L. gymnodactyli
L. tarentolae
Endotrypanum monterogeii
DNA polymerase a
catalytic
polypeptide
and RNA
polymerase II
largest subunit
genes
N/T

parasite DNA

[191]

Page 15

Leishmaniasis: Prevention, Parasite Detection and Treatment Current Medicinal Chemistry, 2012 Vol. 19, No. 10 1457
(Table 3). Contd…..
Assay Leishmania
species
Target
Sensitivity (lowest
detectable parasite
load)
Samples Reference
Multilocus
sequence
typing
L. donovani
L. infantum
L. archibaldi
asat
gpi
nh1
nh2
pgd
d6pdh
icd
me
mpi
fh
N/T parasite DNA [176]
[193]

Sequencing L. donovani
L. chagasi
L. infantum
L. archibaldi
L. hertigi
L. deanei
L. equatorensis
L. major
L. aethiopica
L. amazonesis
L. garnhami
L. mexicana
L. tropica
L. killiki
L. aristidesi
L. pifanoi
L. enrietti
L. braziliensis
L. guyanensis
L. panamensis
L. shawi
L. turanica
L. arabica
cyt b N/T parasite DNA,
clinical isolates
[192]
Sequencing L. donovani
L. infantum
L. tropica
L. aethiopica
L. braziliensis
mini-exon 50 fg parasite DNA,
tissue samples
[184]

Sequencing L. tropica
L. major
L. donovani
L. archibaldi
L. infantum
L. chagasi
L. mexicana
L. amazonensis
L. garnhami
L. braziliensis
L. peruviana
L. lainsoni
L. guyanensis
L. panamensis
L. naiffi
L. tarentolae
Trypanosoma
cruzi
hsp70 N/T parasite DNA [189]