Chapter 17
Treatment and Control of Leishmaniasis Using
Photodynamic Therapy
Debora P. Aureliano, Martha S. Ribeiro,
José Angelo Lauletta Lindoso, Fabio C. Pogliani,
Fábio P. Sellera, Dennis Song and
Mauricio S. Baptista
Additional information is available at the end of the chapter
https://dx.doi.org/10.5772/57456
1. Introduction
Leishmaniasis is a chronic disease affecting the skin, mucosal and/or internal organs, caused
by flagellate protozoa Leishmania of the Trypanosomatidae family. [1] It is among the six most
important disease in terms of its impact in public health. The world incidence of leishmaniasis
is very large with about half a million new cases per year. About 12 million people are infected
with Leishmania ssp parasites worldwide. New treatment alternatives are highly needed. Our
goal here is to critically revise the literature in order to show the potential of Photodynamic
Therapy in the treatment and comprehensive control of this disease. We have separated this
chapter in nine sections, besides this brief introduction, which are: Leishmaniasis: Background
and treatment strategies; Mechanisms in Photodynamic Therapy; Treatment of animals
infected with leishmaniasis using PDT; Vector control using PDT; PDT alternatives for Blood
purification; PDT on the treatment of Old World Tegumentary Leishmaniasis; PDT - In vitro
tests in species that cause Tegumentary Leishmaniasis; Conclusions; References.
2. Leishmaniasis — Background and treatment strategies
There are two main forms of leishmaniasis, visceral (VL) and tegumentary (TL) leishmaniasis,
which are also respectively called Kala Azar and Bauru ulcer. The later, received its name
because of the original high prevalence in Bauru, a city in the countryside of the State of São
© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons
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distribution, and reproduction in any medium, provided the original work is properly cited.
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Paulo, in Brazil. The tegumentary leishmaniasis is characterized by skin lesions (cutaneousCL) and mucocutaneous lesions (such as, nasal and mouth regions) [2].
Leishmaniasis is a common zoonosis, with domestic (dogs and cats) and wild (rodents,
marsupials, edentulous and wild canids) reservoirs. It is transmitted to humans by sand flies,
which comprise the genus Lutzomyia and Phlebotomus. Details of the etiology and patho‐
physiology of the disease are out of the scope of this chapter and we suggest that the reader
consult reviews that focus on these subjects [3].
The current scenario of leishmaniasis treatment is not promising. Therapeutic approaches
include systemic administrations of anti-parasitic medications, which often present serious
side effects. Few drugs are available in the clinic, mainly antimonials and amphotericin, and
the frequency of resistance development is rising. Therefore, there is an urgent need to establish
new and more effective treatments for both VL and TL. The treatment of TL (the focus of this
chapter) urges new drugs and new therapeutic forms, that allows for more effective and
conveniently administered treatments [4].
One of the promising approaches, and the one discussed in here, is photodynamic therapy
(PDT). The main expectation of this approach is that it treats lesions in a localized manner,
without damaging healthy tissues [5]. The few reports that are available in the literature have
validated this hypothesis. In addition, no sign of systemic toxicity is reported in PDT, elimi‐
nating one of the major health issues related to existing TL treatments.[6] These points will be
further discussed in this chapter.
The use of light as a therapeutic modality has gained strong impulse recently due to the
development of efficient and affordable light sources. Consequently, photo-activated drugs
(PhotoSensitizers-PS) play key roles in the present clinical portfolio, and more importantly,
are the major lead in the development of new drugs to treat a variety of diseases such as cancer,
microbial infections and tropical diseases. However, increasing the efficiency of PDT photo‐
sensitizers remains challenging [7-9].
The use of PDT in veterinary is much less common even considering the benefits that such
strategies could bring in the treatment of high-value reproducing animals, as well as, in the
treatment of animals that are reservoirs of human diseases [10].
In terms of developing effective treatments against leishmaniasis in endemic areas, it is
important to think of comprehensive strategies that could cause a quick decrease in the pool
of infected patients (Figure 1). It is also important to emphasize that leishmaniasis is a neglected
tropical disease and, therefore, it is highly relevant to consider low-cost strategies that would
serve as an alternative for public medicine in poor countries [9]. Developing efficient clinical
protocols that would cure/control the disease would not only favor the patient itself, but also,
would decrease the chance of this infection being transmitted to others by the vectors or by
blood transfusion. In the next sections, we will explain how PDT can be helpful in the treatment
of patients, as well as, of all the possible reservoirs and transmitting vectors that would favor
the parasite infection cycle (Figure 1). Some of this potential has been attained and some are
still in the step of hypothesis testing.
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Pool of infected
patients
Blood
transfusion
Vectors
PDT
Animal
Reservoir
Figure 1. Schematic representation of a comprehensive strategy to control leishmania disease in endemic areas by
using PDT. Besides treating patients and animals; killing vectors and disinfecting blood, should be considered in a PDT
strategy to control leishmaniasis. The star represents the multi-target characteristic of the PDT strategy.
3. Mechanisms in photodynamic therapy
PDT is a clinical modality based on the damage caused in biological tissues or in infecting
microorganisms by light-induced reactions, generically called photosensitization reactions.
Photosensitization occurs when PS absorb light and transfer its energy to neighboring
molecules, such that light converts into chemical reactivity [11-13]. After the end of a photocycle, PS returns to the ground state and may absorb another photon. The photophysical step
that allows the formation of an efficient PS is the intersystem crossing (ICS), that converts
singlet into triplet species, which are long lived and highly reactive (Figure 2) [13].
The photooxidation of biomolecules is responsible for changes in their structure and function.
It can occur by two main mechanisms: electron transfer reaction (excited states are stronger
oxidizing and reducing species than their respective ground states) catalyzing the formation
of various radical species, including the highly reactive hydroxyl radical. These reactions are
classified as type I. The photooxidation can also occur through energy transfer with molecular
oxygen, catalyzing the formation of singlet oxygen, a mechanism called type II (Figure 2) [14].
It is considered that type II mechanism is the most relevant effector of photooxidation, because
type I reactions usually lead to PS degradation [15]. However, in biological systems, there
usually is shifts between these two mechanisms (type I versus type II), for several reasons,
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including local concentrations of oxygen and of reducing species, interaction of PS with other
biomolecules and PS aggregation [17-21].
Free radicals and singlet oxygen have different reactivity towards biological targets, but both
can react with them [14,22]. Singlet oxygen mainly reacts by addition to double bonds (Figure
2). The efficiency of photo-induced cell killing seems to depend more on the amount of PS that
is located in the intracellular environment and on the specific intracellular location than on the
in-vitro photophysical efficiency of the PS [23-28].
1
PS
ICS
3
PS
O2
hν
Type I
FREE RADICALS
e-
Type II
PS
1
O2
Figure 2. Top scheme. Main mechanisms of photooxidation. PS, 1PS, 3PS: photosensitizer ground state, singlet and
triplet species, respectively. O2 and 1O2 correspond to oxygen in the ground state and the singlet excited state, respec‐
tively. hv represents light absorption at a specific wavelength and ICS is intersystem crossing between the singlet and
the triplet states. Bottom scheme: Reaction of singlet oxygen with a double bond forming a hydroperoxide, which is
the main reaction of singlet oxygen with lipid double bonds.
PDT combines three components to kill cells (eukaryotic and prokaryotic) and non-cellular
organisms such as virus: PS, light and oxygen. PS is applied either topically or systemically
and it must incorporate in the biological tissue to be treated, which is exposed to light in the
presence of oxygen. The PS needs to absorb efficiently the incident light and form triplet species
[14]. There are hundreds of PS molecules that have been synthesized and tested. In Figure 3
we present the chemical structures of few that are worth commenting in this chapter, because
they either have been involved on treatments of leishmania or have the potential to be.
Methylene Blue (MB) and Crystal Violet (CV) are positively charged and low-cost photosen‐
sitizers that enter cells and react mainly by type II and type I mechanisms, respectively. MB
has been used to treat several diseases including leishmania [27], while CV should be tested
since it has a great potential as a positively dye that mainly accumulates in mitochondria [28].
Riboflavin (RF, vitamin B2), is a natural PS that absorbs in the 400-500 nm region and has been
used for blood disinfection as well as in test-tube leishmania killing assays [29]. Hypericin is
another natural PS that is extract from St. John's wort and has been used in several PDT studies
[30]. ALA is the first compound in the porphyrin synthesis pathway. Protoporphyrin IX is
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formed intracellularly after the treatment with ALA and/or methyl ALA and is the most used
PS in leishmaniasis treatment [31-35]. Chlorophyll is the main pigment of photosynthesis and
their derivatives hold promising potential as low-cost PS [36].
A
D
B
E
C
F
7
3
1
ALA
III
II
Mg
20
N
N
I
17
Methyl-
O
MeOOC
ALA
8
N
N
14
IV
V
O
Ofitol
Figure 3. Molecular structure of relevant photosensitizers in PDT: (A) methylene blue; (B) crystal violet; (C) Riboflavin,
(D) Hypericin; (E) ALA, Methl ALA and Protoporphyrin IX; (F) chlorophyll.
The ability of PDT to act as an anti-microbial treatment, i.e., to treat fungi, bacteria and virus
infections, is well described in the scientific literature [37-39]. Many research groups have
developed experiments that prove the effectiveness of this therapy for a large number of
diseases, including certain parasitic diseases [40]. In vitro studies of photoinduced inactivation
of parasites have been used to unravel important aspects of the therapy including, the action
mechanisms, light dosimetry, structural-activity relationships, PS uptake and localization.
PDT has been used in the treatment of human and experimental murine leishmaniasis of the
Old and New Word. Despite the small number of cases related, literature highlights the ability
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of PDT to deliver better results compared to traditional treatments, emphasizing its better
effectiveness in leading to amastigote-free lesions in a shorter time periods, in addition to its
excellent esthetic results.
4. Treatment of animals infected with leishmaniasis using PDT
PDT has emerged in the treatment of cutaneous diseases among human and different animal
species [41]. Researchers have shown that PDT offers an effective alternative in the treatment
of CL indicating that it also has a great clinical potential in the treatment of this disease within
Veterinary Medicine [27]. The initial studies using PDT to treat leishmaniasis were performed
in humans and are further described on section 7 [31-35]. Although some animals, especially
mammals, constitute important reservoirs of the parasites, leishmaniasis also has clinical
importance because some species can develop injuries, become sick and die due to the disease
and its complications. Therefore, from this point of view, Veterinary Medicine has special
interest, not only to control the disease epidemiology, but also to treat infected and sick
animals.
The main vertebrate hosts (domestic and wild) described and classified as hosts of these
protozoan through natural and/or experimental infections, are: foxes, opossums, armadillos,
anteaters, sloths, rodents, cat, dog, goat, sheep, buffaloes, horses and primates [42-47]. While
the treatment of infected animals provides possibilities for partial or total removal of cutaneous
lesions, it is still not possible to guarantee the elimination of the infectious agents from the
carrier animal, remaining the possibility that it remains as a host reservoir. Therefore, there is
a great need to further investigate the treatment of domestic and wild animals with leishma‐
niasis, by using PDT.
Among all involved animals, the domestic dog and some rodents are the main sources of
human infection in America and in the Middle East, respectively; therefore, being the majors
urban reservoir hosts of leishmaniasis [44,48]. The proximity of this animal to humans
complicates the disease control. The lack of identification of infected animals becomes a
challenge, mainly due to the numerous generic clinical manifestations, and sometimes the
absence of pathognomonic lesions in the dogs [49]. The skin disorders are quite common in
animals, and include opaque hair coat, alopecia, depigmentation, hyperkeratosis of nasal plan
and digital cushions, mucocutaneous ulcers, intradermal nodules, onychogryphosis and
excessive flaking [50,51] but the most common presentation of the cutaneous disease is a
symmetrical alopecia accompanied by intense flaking with silvery appearance that often starts
on the head and spreads to other parts of the body [52]. However, these symptoms are
sometimes not correlated with leishmaniasis. Regarding the condition of the dogs as reservoir
hosts in the epidemiology of the disease, clinical treatment is not recommended so far [51],
making euthanasia of the infected animals mandatory in many countries [50] and keeping the
controversial discussion among public health authorities, animal protectors and veterinarians
[53,54]. Despite the importance of dogs in the epidemiology of the disease, the most used
animal model and the one that has shown success in the treatment of the cutaneous disease
are rodents, mainly mice and hamsters.
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Several studies demonstrated the possibility of using PDT in animal models, especially on
murines. In 2007 Akilov et al. reported an evaluation of the use of ALA (precursor of PpIX) in
TL caused by Old World species in ears of Balb/c mice [55]. Akilov et al. also highlighted the
action of ALA-PDT in murine with leishmaniasis compared to a control group treated with
ALA [56]. The results showed a significant reduction of 24.5 folds in the parasite load compared
with the control group. Nevertheless, they observed vascular damage in ears of the PDT-ALA
group probably caused by PDT. According to the authors, a wide inflammatory and immu‐
nologic response was noted in Balb/c ears of ALA-PDT group, which correlated with the
expressive decrease of parasite load and with the healing of the tissue.
Despite ALA, other classes of photosensitizers already widely used in PDT began to be tested.
The phenothiazine 3,7-bis(di-n-butylamino)phenothiazin-5-ium bromide (PPA904) was tested
by Akilov et al. in mice [57]. Ears of female Balb/c were infected with metacyclic parasites of
Leishmania sp. Following infection, mice were treated with PPA904 cream and irradiated with
a broad band light source. They tested the PS concentration, time of uptake and absorption
site in the ear. The results showed that PPA904 applied during at least 90 min in consecutive
sessions of PDT decreased parasite load around 5.2 log compared to the controls groups.
However, PPA904 application also lead to skin irritation. Another study was carried out with
female Balb/c infected with L. major parasites expressing green fluorescent protein (GFP) to
monitor the parasitic load and the efficacy of PDT [58]. PPA904 was applied in the ears of the
mice and the parasitic load was compared with control group (only infected). The fluorescence
of GFP parasite in the ear of mice after the PPA-PDT decrease significantly, about 80%,
compared to control group. The authors emphasized that this result was obtained after more
than one PDT session.
Peloi et al.chosen a different murine, which is also considered an appropriate model to develop
leishmaniasis caused by some New World Leishmania spp. Hamsters were used to investigate
the effectiveness of PDT with methylene blue (MB) photosensitizer [59]. A light-emitting diode
(LED) was chosen as light source. The footpads of hamsters were infected with Leishmania sp.
The control presented an increase in thickness throughout the treatment. An opposite reaction
occurred in the group A and B treated with oil/water lotion MB+LED and aqueous solution
MB+LED, respectively. Statistically significant reductions on the thickness of the footpad and
parasitic load were observed.
The scientific reports in PDT-treated animal models mentioned in this chapter show similar
results to those reported in humans. In other words, PDT is capable to treat infected wounds
reducing the parasitic load. In some cases, the complete disappearance of the parasite from
tissue is achieved. Other aspect to highlight is its ability to inactivate both Old and New world
Leishmania spp. Details of parameters from scientific studies using PDT on Old World and New
World TL in murine models are described in table 1. However, treatment conditions of infected
animals out of experimental controlled environment have not been described. Therefore, PDT
has to become a more common procedure to be used in the clinical practice of Veterinary
Medicine. It certainly has the unfulfilled potential to become a therapeutic alternative in
veterinary medicine, and to help controlling the parasitic cycle in humans.
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CG: control group / GA: group A / GB: group B
Table 1. Parameters used in PDT to the treatment of Old World tegumentary leishmaniasis and New World
tegumentary leishmaniasis in murine models
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5. Vector control using PDT
The field of insect photo-killing by administration of photosensitizer molecules and light
exposition (usually sun light) is one of the areas of possible PDT application that has received
small attention of the scientific community [60-63]. The few studies, which were mainly reported
by Jori and co-authors, sustain that there is indeed great potential on this area. There are reports
showing that the PS activity is a function of its log POW value and of its amphiphilic character
[62,63]. PDT was also shown to be efficient for Larva control of dengue vector Aedes aegypti [60].
However, there is no scientific report on the use of PDT to control the vector (Phlebotomus sand
flies) and its larva, which are responsible for the transmission the leishmania parasites. It is also
important to emphasize that the amount of information available concerning larva develop‐
ment of phlebotomine sand flies is much less than what is known for the mosquitoes whose
control have been studied by PDT. Nevertheless, for the matter of bringing new ideas to the
field of Leishmania treatment, the concentration of photosensitizers that are needed to neutral‐
ize larva and to kill those mosquitoes is several orders of magnitude smaller than the concentra‐
tions of chemical insecticides, which are currently used for vectors control, causing great
disturbance in the whole ecosystem. Therefore, it is up to our community to develop and test
strategies to control vectors of Leishmania parasites using PDT.
6. Blood purification
The purification of blood products is critical to avoid disease transmittance through blood
transfusion. Although this is not the main route of transmission of leishmaniasis, it is a possible
one, and cases have been reported in the literature [64]. The focus of the disinfection strategy
is to kill microorganisms without harming the cellular and plasma components. PDT offers
great potential to be successful in blood disinfection, because it is a multi–target strategy, i.e,
the reactive species that are formed (after light absorption and photosensitization reaction) are
effective against viruses, bacteria, fungi, and parasites [37-40]. This strategy has even been
proved effective to promote pathogen inactivation in the presence of fragile blood components,
such as stem cells from blood of embryo’s cord [65-68]. It is better than UV treatments, because
it does not cause direct damage to blood components. Several PS have been used for blood
disinfection including MB, CV and RF (Figure 2). Molecules that have intracellular targets such
as MB and CV can be used to treat plasma derivatives but not whole cell blood, because they
will cause extensive hemolysis. RF, however, is an aqueous based photosensitiser, which do
not enter cells and can be used to disinfect whole blood derivatives. RF reacts either by type I
or by type II mechanisms and is already in use. Several companies commercialize kits for blood
and plasma decontamination, like Macopharma, whose technology for plasma decontamina‐
tion is based on MB photosensitization (https://www.macopharmausa.com/). In the case of
leishmaniasis, parasites remain mostly in the intracellular environment, except when they are
in transit from a lysed cell to infect a macrophage or other phagocytic cell. We could think of
using PDT to remove parasites in the plasma or to develop strategies to target PSs to destroy
only infected cells of contaminated blood.
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7. Photodynamic therapy on the treatment of old world tegumentary
Leishmaniasis
There are several reports on the literature dealing with the treatment of leishmaniasis by PDT
[5,6,33,34]. The first report was conducted by Enk’s group in 2003 [5,6]. Both studies reported
the use ALA and MAL, combined with red light. These authors performed the treatment of 32
TL lesions from 11 Israeli patients. The diagnostic was accomplished by verifying the amasti‐
gote presence in direct smear from the lesions [5]. This work showed that about 96% of the
lesions healed, leaving some mild scars and pigment in place of the old lesions. Just one lesion
presented amastigotes forms after PDT. Gardlo et al. published the case of a patient, aged 34,
with CL confirmed by histology. According to the authors, the patient developed resistance
to the treatment with sodium stibogluconate and presented 10 lesions, which were treated five
times with PDT and five times with paromomycin sulfate ointment [6]. The result obtained is
similar to the previous work and showed that the five ulcers treated with PDT healed without
signs of amastigotes, while two ulcers treated with paromomycin partially responded to the
drug, one of them did not respond and two lesions were shown to have no amastigotes. The
ulcers that did not responded to paromomycin ointment were subsequently treated with PDT
successfully.
Asilian and Davami developed a placebo-controlled, randomized clinical trial that provided
definitive evidence of the efficacy of PDT in the treatment of CL [34]. 60 patients with confirmed
CL by clinical and parasitological diagnosis were separated in 3 groups with different
treatments. Group 1 was treated with PDT once a week, group 2 received twice daily para‐
momycin plus methylbenzethonium chloride ointment and in group 3 was used a paraffinbased ointment without active ingredients with same application time of the group 2. During
four weeks, the groups received the treatments described above. At the end of the study healing
was present in 93.5% of the patients of group 1, 41.2% of group 2 and 13.3% of group 3. At the
same time, 100%, 64.7% and 20% of the lesions had parasitological cure in group 1, 2 and 3,
respectively.
Other studies accomplished in Iran and German corroborated with the results described above.
According to the authors, PDT showed to have the capacity to treat wounds caused by Old
Word Leishmania species. We emphasize that most of the reports claim that this therapeutical
modality can achieve results above 90% healing of wounds, however, a caveat must be held
since some of these studies indicate that not all healed wounds become free of parasite [35,56].
The mechanism of ALA PDT in the case of leishmaniasis was shown to be due to the killing
of infected host-cell killing (macrophages) instead of direct parasite killing (see further
discussion about this issue on section 8).
One CL case of the New World leishmaniasis is described in the literature. Song et al. reported
the case of a Brazilian patient presenting cutaneous leishmaniasis confirmed by smear stained
by Giemsa. PDT was carried out using MB. In this specific case because of ethical concerns of
possible development of evolution to mucocutaneou disease, the patient received at the same
time a low dose of pentavalent antimony and PDT. The patient had two ulcers. One receive
PDT and the other was only being treated with the low-dose pentavalent [27]. The treatment
L. major
Photosensitizer
Photosensitazer
ALA *
Uptake
4h
Gardlo et al. 2003
Suspeita L.
donovani
MAL
5h
Asilian et al. 2006
L. major *
ALA
4h
Light source
Curelight,
Photocure
Curelight,
Photocure
Omnilux(visible
red light)
wavelength
570-670 nm
Irradiance
150 mW/cm²
Fluence
100 J/cm²
Treatment
sessions
Frequency
Patientes
Results
once weekly
until parasite
was not
detectable in
the direct
smear
11
96% cure
1 patiente with 1 lesion
presented parasite
1º Twice weekly (12
weeks) +
2º
once weekly (4
1º 12 weeks /
weeks)
2º 4 weeks
570-670 nm
150 mW/cm²
75 J/cm²
633 nm
#
100 J/cm²
once weekly
4 weeks
1
100% cure
20
93,5% cure
Ghaffarifar et al. 2006
L. major
ALA
4h
150 mW/cm²
100 J/cm²
once weekly
4 weeks
5
100% cure
L. tropica
MAL
3h
Red light
Waldman PDT
1200L
570-670 nm
Sohl et al. 2007
580-700 nm
#
100 J/cm²
once weekly
1 to 4 weeks
1
100% cure
Song et al. 2011
L. amazonensis
0.5 % Methilene Blue
#
RL 50 - LED
570-750 nm
35 mW/cm²
20 J/cm²
once weekly
4 weeks
1
100% cure
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Enk et al. 2003
Parasite
showed 100% of cure in both lesions, but the lesion treated with PDT presented a faster wound
recovery compared to the antimony alone (Table 2).
* First compound in the porphyrin synthesis pathway, precursor of Protoporphyrin IX (PpIX)
Table 2. Parameters used in PDT on the treatment of Old World and New World tegumentary leishmaniasis in
humans
Author/year
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This brief account of the use of PDT for the treatment of CL demonstrates the ability of
this therapeutic modality and encourages its use. It also stimulates research in the pur‐
suit of new protocols with new PS, which could ensure not only healing but also clinical
and parasitological cure of these patients.
Details of parameters from scientific studies using PDT on the treatment of Old World and
New World tegumentary leishmaniasis in humans are described in Table 2.
8. Photodynamic therapy — In vitro tests in species which cause
Tegumentary Leishmaniasis
The effectiveness of PDT on CL treatment was first conducted in humans and in animal models.
In vitro tests began less than ten years ago to allow testing of PDT parameters like the efficiency
of different types of photosensitizers, their respective uptakes and concentrations and
accumulation sites.
Sujoy Dutta et al. began in vitro studies with the New World specie, L. amazonensis in 2005 [69].
The first part of that work evaluated Leishmania transfectants expressing GFPs. The PS tested
was aluminum phthalocyanine chloride (AlPhCl) in different concentrations. The principal
factor tested was the light-mediated cytolysis when cells were in the presence or pre-incubated
with the AlPhCl. In the dark there was no phototoxicity for both promastigote and amastigote
forms of the parasite. The opposite effect occurred when the photosensitizer received red light
illumination, showing that promastigotes appear to be more sensible than amastigote forms.
In addition, the loss of fluorescence of the GFP parasites indicated cell death. On the second
part of the study, J774 cells (cell line immortalized murine Balb⁄ c monocyte ⁄ macrophage)
were tested at the same conditions reported above. The authors observed that they were 10-20
fold more resistant than promatigotes. According to the authors, the photosensitized Leishma‐
nia cells are susceptible to cytolysis, probably due to the generation of reactive oxidative species
after illumination, an indicative of inefficiency of their antioxidant mechanisms. ALA did not
induce protoporphyrin IX (PpIX) production in the Leishmania cells, because of a deficiency in
the heme biosynthetic pathway in this parasite [57, 70].
Tests with other phthalocyanines were developed by Pinto et al. using species of Old and New
world Leishmanias, L. major and L. braziliensis. The parasites were incubated with aluminum
phthalocyanine tetrasulfonate (AlPcS4) at different concentrations and irradiated with a
GaAlAs diode laser (λ= 659 nm, 40 mW). The experiments indicated a significant reduction of
viable parasites in both species compared to controls, however L. braziliensis demonstrated
higher mortality than L. major [71].
In Brazil, Song et al. performed tests to understand mechanism of action of PDT using MB in
a case report. Promastigotes of L. amazonensis were incubated with different concentrations of
MB, washed with PBS and illuminated using a home-built LED light source with a wavelength
of maximum emission at λ= 650 nm. After the irradiation, cell survival was determined with
MTT assay that detected cell toxicity after irradiation of light in the presence MB. There was
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an increase in the phototoxicity with the increase in the MB concentration indicating a
concentration-dependent response [27]. Differently from PPIX induced by ALA and MAL, MB
has parasite intracellular target. In fact, PS seems to be localized in mitochondria (Figure 4).
Figure 4. Top: Rhodamine 123 and Methylene blue fluorescences in promastigote parasites of L. amazonensis. Bot‐
tom: transmission image and colocalization of R123, MB and transmission images.
Other researchers have investigated the susceptibility of L. amazonensis regarding PDT. In
order to verify the lethality of phenothiazine’s derivatives on the promastigote forms, Barbosa
et al [72] tested TBO (toluidine blue O), MB and a TBO/MB solutions. Irradiation was performed
with a diode laser (λ= 660 nm, P= 40 mW). They tested different PS incubation time (5 and 60
min) and two energy densities (4.2 and 8.4 J/cm2). The results showed a representative decrease
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on the viability of L.amazonensis promastigotes for all treated groups in comparison to their
controls. The authors did not find statistical differences between the dyes, but reported that
the best result was observed with TBO.
Dutta et al. published an article that described the use of a combination of photosensitizers.
Uroporphyrin (URO1) and aluminum phthalocyanine chloride (AlPhCl) were used in
uroporphyrinogenic mutants of L. amazonensis (RAT/ BA/ 74 /LV78) 12-1 clone, transfected
with pX-alad and p6,5-PBGD [73]. This transfected Leishmania is able to absorb ALA and turns
it into URO 1. The authors evaluated the combination of both drugs into promastigotes with
and without irradiation of red light. Results showed photolysis of the irradiated parasites with
both photosensitizers whereas non-irradiated parasites showed no damage.
Hernández et al. published another study that compared encapsulated chloroaluminum
phthalocyanine (CLAlPc) in liposomes (UDL-CLAlPc) and free in solution. The experiments
were conducted with two species of New World Leishmania in promastigote and amastigote
forms and in THP1 cells. The experiments tried to verify the ability of the photosensitizer in
reaching the Leishmania inside THP1 host cell. According to the authors, the UDL-ClAlPc
photosensitizer was almost 10 times more photoactive than free ClAlPc on THP-1 cells as well
as on promastigotes and with intracellular amastigotes of L. chagasi and L. panamensis [74].
9. Conclusions
• First reports of cutaneous leishmaniasis using PDT were performed in humans;
• Treatments using porphyrin precursors, ALA and MAL, showed positive results on the cure
of patients with CL;
• The low-cost phenothiazine methylene blue and red light can be used to treat patients with
CL;
• More than one PDT session is necessary to achieve wound healing.
• Both New and Old World Leishmania can be treated with PDT.
• Murine models of infection such as Balb/c and hamster show to be appropriate for PDT
studies of CL treatment.
• In vitro tests demonstrate that Old and New world Leishmania species can be used to test
new photosensitizers and to establish structure/activity relationships.
• PDT also has the potential to control leishmaniasis transmission by the treatment of vectors
and infected animal reservoirs, although the development of these potentials will need
further investigation.
Treatment and Control of Leishmaniasis Using Photodynamic Therapy
https://dx.doi.org/10.5772/57456
Author details
Debora P. Aureliano1,2, Martha S. Ribeiro1, José Angelo Lauletta Lindoso2,3, Fabio C. Pogliani3,
Fábio P. Sellera4, Dennis Song3,5 and Mauricio S. Baptista5
1 Centro de Lasers e Aplicações (CLA)/ IPEN- CNEN, Brazil
2 Laboratório de Soroepidemiologia (LIM38-HC-FMUSP) e Instituto de Medicina Tropical
de São Paulo da Universidade de São Paulo, Brazil
3 Instituto de Infectologia Emilio Ribas-SES-SP, Brazil
4 Departamento de Clínica Médica, Faculdade de Medicina Veterinária e Zootecnia da Uni‐
versidade de São Paulo, Brazil
5 Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Brazil
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