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Leishmaniasis in Libya
Thesis · August 2012
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2. 1
To the University of Wyoming:
The members of the Committee approve the thesis of Wahid H.S Dakhel presented on
8/3/2012.
Dr. Chaoqun Yao, Chairperson
Dr. Paul Ludden, External Department Member
Dr. Gerry Andrews
APPROVED:
Dr. William Laegreid, Department Chair, Veterinary Sciences
Dr. Frank Galey, College Dean, Agriculture and Natural Resources
3. 2
Dakhel, Wahid H.S, Leishmaniasis in Libya, Master, Veterinary Sciences, August, 2012.
Abstract
Leishmaniasis is a disease caused by Leishmania spp. protozoa. The latter are transmitted to
humans by sand fly vectors. Some Leishmania spp. cause chronic lesions in skin and mucus
known as cutaneous (CL) and mucocutanous leishmaniasis (ML), respectively. Others,
disseminating to internal organs such as the liver, the spleen, and the bone marrow, cause
visceral leishmaniasis (VL). Twelve million people are affected by leishmaniasis with 59,000
annual deaths, and 350 million people in 88 countries around the world are at risk. Anti-
leishmanial immune response is shown to be host genotype dependent in murine models so that
some inbred strains of mouse are susceptible while others are resistant. However, it is not so
clean-cut in humans. Diagnosis of leishmaniasis is made by 1) direct identification of the
parasite, 2) positive culture of the parasite, or 3) detection of parasite DNA by PCR. There is no
approved vaccine for human use against leishmaniasis. Chemotherapy is effective, but current
drugs are toxic, expensive and parasite resistance has occurred. Many strategies are efficient in
controlling leishmaniasis. These include, vector control, reservoir reduction, adjusting human
activity to avoid sand flies, and treating patients. Leishmaniasis is endemic in Libya, a country in
North Africa. The VL cases have been recorded in the Eastern and Southern Libya since 1980,
whereas CL is mainly found in Western Libya.
4. 3
LEISHMANIASIS IN LIBYA
By
Wahid H.S Dakhel
A thesis submitted to the Department of Veterinary Sciences
and the University of Wyoming
in partial fulfillment of the requirements
for the degree of
MASTER
in
VETERINARY SCIENCES
Laramie, Wyoming
August / 2012
5. 4
ACKNOWLEDGMENTS
I would like to thank my advisor, Dr. Chaoqun Yao, for his knowledge, assistance, and
continuous motivation. Without his support I would not have completed this thesis. I would also
like to thank my committee members, Dr. Paul Ludden for this knowledge, experience and
encouragement, and Dr. Gerry Andrews for his knowledge, support and leadership. I would also
like to give special thanks to my wife, all my family members in Libya, and give special thanks
to all people in Libya who did the revolution for freedom.
6. 5
TABLE OF CONTENTS
Acknowledgments…………………………………………………………………….....4
Chapter 1- Literature Review...........................................................................................8
1.1-Introduction…………………………………………………………………….........9
1.2- Leishmaniasis ………………………………………………………………….…...10
1.3- Transmission of Leishmania ……………………………………………….….…...13
1.4- Epidemiology of Leishmaniasis ………………………………………………..…..15
1.5- Immunity to leishmaniasis………………………………………………….....……16
1.6- Diagnosis of Leishmaniasis …………………………………………..………….…23
1.7- Treatment of Leishmaniasis ………………………….………………………..…...25
1.8- Control of Leishmaniasis ……………………………...…………………………...27
1.9- References …………………………………….………………………..…………..29
Chapter 2-Leishmaniasis in Libya…. ..……………………………………………..…..48
2.1- Pathogens and Vector …..…………………………………………………..………50
2.2- Epidemiology……………………………………………………………………….52
2.3.- Concluding Remarks……..………………………………………………………...54
2.4.- References……………………………………………………………………….....55
7. 6
LIST OF TABLES
1- Susceptibility or resistance of different mouse strains to Leishmania spp…45
2- Selected treatment regimens for cutaneous and mucosal leishmaniasis……46
8. 7
LIST OF FIGURES
- Chapter One
1- Classification of Leishmania………………………………………………………..35
2- Development of Leishmania species in the sand fly vector………………………...36
3- Life cycle of Leishmania spp………………………………………………………..37
4- Clinical manifestations of cutaneous leishmaniasis…………………………………38
5- Mucocutaneous leishmaniasis…………………………………………….…………39
6- Visceral leishmaniasis…………………………………………………………….…40
7- Geographical distribution of the leishmaniasis………………………………..…….41
8- Worldwide distribution of visceral leishmaniasis……………………………….…..42
9- Micrographs showing cutaneous leishmaniasis……………………………………...43
10- Diagnosis of visceral leishmaniasis………………………………………………….44
- Chapter Two:
11- Meriones libycus……………………………………………………………………..57
12- Phlebotomus papatasi…………………………………………………..…………....58
13- Phlebotomus sergenti……………………..…………………………………..…...…59
14- Geographic distribution of leishmaniasis in Libya…………………………………..60
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1.1. Introduction:
Many Leishmania spp. infect mammals including humans. The infections may lead to
leishmaniasis, a significant but largely neglected disease. The disease is widely distributed
throughout the world, mainly in tropical and subtropical regions such as south and central
America, Africa, Middle East, India and Asia [1].
Leishmaniasis is divided into three major categories according to the clinical
manifestations: cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (ML), and visceral
leishmaniasis (VL), which is also called Kala-azar [2]. In terms of significance, 350 million
people in 88 countries are at risk of infection and the annual number of new cases is estimated
between 1.5 and 2 million with 59,000 deaths [3]. Moreover, the disease has emerged and re-
emerged dramatically in the last decade due to environmental risk factors such as urbanization
into the endemic areas [3].
Although leishmaniasis is prevalent worldwide, there are no vaccines approved for
human use, and the medicine is cumbersome to use with unacceptable side effects [4]. The
disease is naturally transmitted by sand fly vectors. The main sand fly species include
Phlebotomus papatasi in the Old World and Lutzomyia longipalpis in the New World [5].
Leishmania spp. propagate as promastigotes in the midgut of the sand fly vectors and
amastigotes in macrophages of the mammalian hosts. These two life-cycle stages can be readily
distinguished by light microscopy [6].
11. 10
1.2. Leishmaniasis:
Leishmaniasis is caused by many Leishmania species (Figure 1). In general, there is a
correlation between clinical manifestations and parasite species. For example, members of
Leishmania mexicana complex, which consists of L. mexicana, L. amazonensis, and L.
venezuelensis, cause localized cutaneous lesions that generally self-heal, resulting in lifelong
immunity [7]. CL is endemic in both the Old World and the New World. In the Old World, such
as Spain and the Mediterranean and Caspian Sea area the main reservoir of CL is dog [8, 9]. CL
in the Old World is caused by L. major, L. tropica and L. aethiopica [10], whereas in the New
World members of L. mexicana complex, and L. braziliensis, L. peruviana , L. panamensis in
Viannia subgenus are the causative agents, so are L. chagasi and L. major [10].
Both adults and children in the Old World can be affected by CL. Usually a papule starts
at the place where a sand-fly bite is located followed by development of a nodule and ulceration
in 1-3 months (Figure 2). The common lesions of CL are ulcerative lesions [15].
The second form of leishmaniasis, ML, is caused by the species of Vianna subgenus.
Infections are localized in the mucosal membranes of the nose and the mouth, resulting in
damage of the organs and permanent disfiguring [7]. The causative agents of ML are the
members of L. braziliensis complex including L. braziliensis, L. panamensis, L. guyanensis and
L. peruviana.
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Mucosal spread of these New World species occurs in 1-10% infections, and may happen
many years after CL has healed. Usually ML starts with erythema and ulceration at the nasal
septum followed by destroying inflammatory lesions. It may extend from the nose and mouth to
the pharynx and larynx (Figure 3) [16]. ML is occasionally reported outside of the Latin
America, which is acquired by traveling to the endemic region [17].
Leishmania donovani complex consists of three species, i.e., L. donovani, L. infantum in
the Old World, and L. chagasi in the New World [7]. L. chagasi is now considered the same
species as L. infantum that was brought into the New World by the colonists. Infection by L.
donovani, and L. infantum may result in VL with the parasites mainly distributed in the liver, the
bone marrow, and the spleen. VL is always fatal without medical treatment [11]. It is caused by
parasites in L. donovani complex in India, Asia, and Africa, by L. infantum or L. chagasi in
Mediterranean areas, Southwest and central Asia, and South America [2].
Visceral leishmaniasis is caused by the parasite that grows inside the reticuloendothelial
cells [2]. Reticuloendothelial cells are used as a collective term for cells of the immune system,
and primarily include macrophages and monocytes in their tissue-resident forms (e.g.,
histiocytes) [12]. Patients show clinical symptoms such as fever, weakness, fatigue, anorexia,
and weight loss over weeks and months [19]. In addition, enlarged lymph nodes, spleen, and
liver are a result of parasitic invasion of the blood and reticuloendothelial system [20]. Enlarged
lymph nodes can clearly be found in Sudanese VL patients, as well as hyperpigmentation, hence
kala-azar (black fever in Hindi). As the disease progresses, there is an increase of abdominal
13. 12
extension and pain, resulting from splenomegaly and hepatomegaly (Figure 4). Co-infection with
bacteria may lead to diarrhea, pneumonia or even deaths [19].
In addition to the above mentioned three major forms other less commonly found
manifestations exist. One such example is post –Kala-azar dermal leishmaniasis (PKDL). PKDL
may appear after successful treatment of VL and is characterized by macular, maculopapular,
and nodular rash in a patient who has recovered from VL. Usually the rash begins around the
mouth from where it diffuses to other parts of the body depending on severity of infection [13].
14. 13
1.3. Transmission of Leishmania:
Leishmania spp. are transmitted by the female phlebotomine sand flies. They may be
transmitted from human to human, from human to animal or from animal to human. Accordingly
two types of VL transmission occur: zoonotic through which VL can be transmitted from animal
to human, and anthroponotic through which VL can be transmitted from human to human [18].
Sand flies belonging to two different genera are vectors. These are Phlebotomus in the Old
World and Lutzomyia in the New World [28].
When sand flies take a blood meal, they saw into the skin, and make a small wound
where a pool of blood is formed from the broken superficial capillaries. During blood meals sand
flies ingest macrophages laden with amastigotes, the intracellular form of the parasite. The
transferring from mammalian hosts to the midgut of a sand fly results in a dramatic change in the
parasite’s environments, which include an elevated pH and a lowered temperature. As a result,
amastigotes transform into motile procyclic promastigotes [23]. Procyclic promastigotes in the
gut of the female sand fly attach to the midgut epithelium cells, where they divide by binary
fission. Development of promastigotes from procyclic to metacyclic promastigotes through a few
intermediate stages in sand flies takes 8-20 days. Promastigotes move forward to the pharynx
where they produce a partial or complete blockage of the sucking system. Metacyclic
promastigotes are the infectious stage to mammalian hosts [24] (Figure 3).
When the sand fly takes another blood meal it inoculates the metacyclic promastigotes
into the dermis of a mammalian host where promastigotes are phagocytized by host macrophages
that is rapidly recruited to the bite site. Leishmania spp. are resistant to the acidic pH and
15. 14
hydrolytic enzymes present in the phagolysosomes of macrophages that destroy many other
organisms [14]. Metacyclic promastigotes transform to non-motile amastigotes in the
parasitophorous vesicle, thus establishing infections [22]. Amastigotes replicate by binary fission
and eventually lead to disruption of macrophages, resulting release of more amastigotes that are
phagocytized by macrophages. The cycle repeats in the infected hosts, perpetuating infections
(Figure 3).
Metacyclogenesis: Promastigotes in the sand fly vector are extracellular, flagellated, and
spindle-shaped. They develop and multiply within the midgut of sand fly vector. Development
from procyclic promastigote to metacyclic promastigotes is termed as metacyclogenesis. In
addition to morphological changes as showed in Figure 2 the parasites undergone biochemical
modifications. One such change is surface molecules. The surface of promastigotes is covered by
glycoconjugates, one of which is lipophosphoglycan (LPG). LPG consists of “polymer of
repeating phosphorylated di-, tri-, and tetra-saccharide (subunits depending on the species)
linked via a phosphosaccharide core to a glycophosphatidylinositol anchor” [25]. L. major LPG
of the non-infectious procyclic promastigotes is shorter than that of the infectious metacyclic
promastigotes. The former is capped with a terminal galactose, which binds to the lectins such as
peanut agglutinin (PNA). In contrast the terminal cap of metacyclic promastigote LPG is
arabinose which does not bind to PNA [27]. It has been suggested that promastigotes may be
attached to the inner surface of the female sand fly’s gut and this attachment may be abridged by
LPG [55, 26]. The female sand fly must have suitable ligands on the cell of the gut to ensure the
promastigotes attachment. Blood meals also stimulate the lectin secretion into the midgut [27].
16. 15
1.4. Epidemiology of Leishmaniasis:
Different species of Leishmania are found in different geographic areas (Figure 7). The
diseases they cause are endemic at both the Old World such as Asia, and Africa, and the New
World, such as America. In the Old World leishmaniasis may be caused by many species at
various geographic areas. For instance, L. infantum, L. aethiopica, L. tropica, and L. major cause
CL in Spain, Italy, and France; L. donovani and L. infantum are two species that cause VL [8]. In
the New World, VL is caused by L. chagasi. However, CL can be caused by L. mexicana
complex, L. braziliensis complex in Peru, Brazil, Mexico, and Argentina [8].
There are around 0.5 million new VL cases resulting in 59,000 deaths annually around
the world [30]. India, Brazil, Bangladesh, Ethiopia, and Nepal account for more than 90% of
cases [30]. Between 1984 and 1994 in Southern Sudan, VL killed around 100,000 among a
population of 280,000 [67]. VL is usually more common in poor countries or the low income
countries such as India because of the high cost of disease diagnosis and treatment [31].
Bangladesh, India, and Nepal harbor around 67% of the global VL disease burden [32].
17. 16
1.5. Immunity to leishmaniasis:
Leishmania protozoan is an intracellular parasite as amastigotes inside macrophages of
the mammalian host. This situation has necessary implications for the immunological response
of the host to the parasite because the intracellular parasite is not always involved in the humoral
immunity resistance. The protective immunity for leishmaniasis is cell-mediated response such
as DTH-delayed type hypersensitivity. The conclusion is drawn based on the following
observations: 1) Most of the animal studies indicated that infection had not been affected by B-
cells. 2) Inhibition of antibodies with anti IgM has no effect. 3) Even though there are high
amounts of IgM and IgG, they don’t provide protection against infection [70]. In addition, CD-8
subtype T–cells plays critical roles in controlling Leishmania infections. As mentioned earlier
CL can be self-healing, suggesting protective immunity responses to infections exist.
Nevertheless, parasites are not entirely cleared out from hosts. When a host becomes immune
compromised, such as with HIV infections, or immunodepressed such as with immune
suppressive medication in cases of organ transplantations, parasites re-emerge and causes clinical
manifestations.
18. 17
1.5.1. L. major infections in mice:
Association of Th1 development with control of infection, and Th2 development with
progressive disease is well established in murine models of Leishmania infections [71]. Active
Th1 cell response leads to production of macrophage activating cytokines, especially interferon-
γ, which is needed for control of parasite replication. Different strains of mice are genetically
either susceptible or resistant to leishmaniasis, and their responses to leismanial infection are
different, which is briefly summarized in Table 1 [69]. ‘‘In both genetically resistant and
susceptible mice has been identified key effector cytokines that must be present at the time of
initial priming of T cells in order to affect the CD4 switch phenotype’’. Th1 expansion in
resistant mice (e.g. C3H) is initiated by interleukin (IL)-12 and IFN- γ, resulting resolution of
lesions caused L. major [69]. Susceptible strains of BALB/c background are associated with
development of Th2 cell responses with production of IL-4, IL-10 and IL-13. Th2 is unable to
mediate macrophage activation and inhibits actions of Th1-type cytokines [69].
Resolution of leishmanial infections or development of leishmaniasis mainly depends
upon distinct CD4 T cell subsets, i.e., IFN-γ and IL-4 producing Th1 and Th2 cells, respectively.
James and colleague nicely show Th1/Th2 paradigm of healing and non-healing lesions with L.
major infections [72]. Th1 is potent protective immune response against L. major infections. IL-
12 produced by antigen presenting cells, macrophages and dendritic cells was probably
augmented by other cytokines such as IL-18 and IL-23. The exquisite sensitivity of BALB/c
mice is related to the lack antigen dependent expansion of vß 4 Vα 8 CD4 T cells producing IL-
4, which rendered T cells unresponsive to IL-12 by suppression of IL-12Rß2. Therefore, a strong
19. 18
case for the predominant role of IL-4 and Th2 response in non-healing disease was clearly
established [72].
1.5.2. L. major infections in human:
Susceptibility to leishmanial infection in human is associated with production of Th2
cytokines, for instant, IL-4, IL-5, and IL-10 [73]. One antigen located on the surface of
Leishmania promastigotes is promastigote surface antigen-2 (PSA-2), which stimulates Th1-
mediated immune responses. The assessment of PSA-2 as human vaccine revealed that Th1
responses to PSA-2 were characteristic in individuals immune to CL [74]. Mononuclear blood
cells from Sudanese with a history of self-healing CL grow rapidly and respond actively to PSA-
2 of L. major, resulting in a high level of IFN-γ, and little amount of IL-4. However, the antigen
did not stimulate cells from presumably unexposed Danish people [74]. Cytometric analysis
indicated that PSA-2 induced blastogenesis of CD3 positive lymphocytes that produce IFN-γ
during L. major infections [74]. The studies showed that ‘‘Th1- such as cells recognizing PSA-2
are expanded during infection by L. major, and that they maintain their Th1-like cytokine profile
upon reactivation in vitro’’ [74]. Further, individuals with immunity acquired during natural
infections and/or cured infections maintain Th1-type memory immune cells [74].
20. 19
1.5.3. L. amazonensis infections in mice:
Different from what had just described in the section 1.5.1 with L. major C3H and C57BL/6
mice that are infected with L. amazonensis develop chronic skin lesions with persistent parasite
loads. In addition, these lesions develop in the absence of IL-4, indicating that host exposure to
this parasite may not stimulate Th2 responses. At the same time, presence of IL-10 could account
for decreasing level of IL-12 and lack of cell-mediated immunity towards the parasite [75].
Jones DE and colleagues showed that IL-10 plays important role in downmodulating host
Th1 response during L. amazonensis infection [75]. They specifically demonstrated early
enhanced Th1 responses in IL-10 deficient C57BL/6. However, Th1 response was
downregulated in the chronic stage of the disease. Nevertheless, at the time of chronic infection
these deficient mice had improved delayed-type hypersensitivity response, which meant Th1
type cells were presented in vivo at the late stage of lesion. The results show that in acute
infection stage, IL-10 has a critical role in restrict of the Th1 response, whereas in chronic
infection phase, Th1 response can be limited by other immunomodulatory factors [75]. Even
though there were 1-2 log declines in the parasite load inside the lesion in IL-10 deficient mice,
the parasite persisted for a long period of time, and the chronic lesion was very close in the size
with the chronic infection [75].
21. 20
1.5.4. L. amazonensis infections in human:
Dendritic cells (DCs) paly a very important role in initiating immune responses and may affect
pathogen survival. Infected DCs with parasite cannot function well, resulting in impaired
immune responses [76]. Favali C and colleagues assessed the influence of L. amazonensis on the
differentiation and maturation of human monocyte- monocyte-derived DCs.
Co-culturing DC with live L. amazonensis promastigotes led to dramatic decreases in
cluster of differentiation CD80 protein found on activated B cells and monocytes that provides a
stimulatory signal necessary for T cell activation and survival, and cluster of differentiation CD1
which is glycoproteins expressed on the surface of various human antigen-presenting cells.
CD86 expression was increased. CD86 is a protein found on activated B cells and monocytes that
provides a costimulatory signal necessary for T cell activation and survival [76]. Afterwards,
levels of secreted IL-6 were decreased days after DC differentiation. Nevertheless, they found
little amount of IFN-γ compared with control DCs. DC differentiation and maturation were the
same in the presence of heat-killed parasite [76].
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1.5.5. Murine Visceral Leishmaniasis:
Information on host immunity to VL is insufficient. Th1/Th2 dichotomy in visceral infections
of murine leishmaniasis is not clean-cut. In general, resistance is associated with CD4 and CD8
T cells, and IFN-γ. IL-12 is linked to transforming growth factor (TGF) - ß production. However,
IL-10, and B cells involves in the susceptibility in absence of IL-4 [77]. ‘‘Mice infected with
agent of visceral leishmaniasis (L. donovani, L. chagasi) are susceptible if they don’t develop a
Th1- type (IFN-γ), response, but they appear not to expand cells producing IL-4’’[78].
In VL infection associated with disease, the damaged functions of cellular immunity may
result from the inhibitory influence of IL-10 independent of the IFN-γ amount. In contrast in
healing cases of VL (both in murine and human) Th1 cytokines (IL-21, IL-31, IL-38, and IL-54)
mainly contribute to resolute infections. IL-12 and IFN-γ are important in development of host
immunity and control of parasite growth whereas IL-10 restrains host immunity and assist
parasite survival [78].
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1.5.6. Human Visceral Leishmaniasis:
In leishmaniasis, host defense against intracellular Leishmania is cell mediated, which is
associated with Th1 responses due to T-cells primed primarily by dendritic and macrophage cells
producing IL-12 [72]. A clear dichotomy between Th1-mediated protection (mediated by major
cytokines IFNγ, IL-2, and TNF) and Th2-mediated disease progression (mediated by major
cytokines IL-10, IL-4) has been presented in mice against cutaneous leishmaniasis [72].
Nevertheless, this Th1/Th2 dichotomy is not as clear in visceral infection of mice and even
less in human visceral leishmaniasis [79]. ‘‘The immune response and pathology of visceral
leishmaniasis are complicated, involving a number of genetic and cellular factors in the process
of susceptibility or resistance to parasites’’ [80].
In fact, the immune responses to visceral leishmaniasis depending on form of disease.
Peripheral blood mononuclear cells (PBMCs) from individuals with subclinical manifestation
infection respond to leishmania with release of IL-2, IFN-γ and IL-12[69].
24. 23
1.6. Diagnosis of Leishmaniasis:
CL can be diagnosed by identifying amastigotes in impression smears, skin scrapings or
skin biopsy by microscopy (Figure 9). Biopsies are often taken from the edge of skin lesions.
Impression smears are fixed in 95% ethanol and stained with Giemsa. Skin scrapings are taken at
the center or margin of the skin lesions. The latter is most commonly used due to its simplicity
although its sensitivity is only around 70% [63, 14].
Cultures can be applied to the biopsy samples and promastigotes are recovered [14]. A
needle aspiration is another method to diagnosis CL. This can be done by injecting 0.9% saline
in the edge of a skin lesion and aspirating small amount of tissue that can be used to prepare a
smear [64]. In addition to identifying the presence of amastigotes in a sample, immunologic tests
may be used to detect parasite antigens. PCR (Polymerase chain reaction) is used to detect
Leishmania DNA. It works even weeks before the development of clinical manifestations. PCR
is more sensitive than many other methods, particularly in the cases that negative results are
obtained microscopically [63, 64]. It’s also very useful for the speciation of Leishmania
parasites, thus the correct treatment can be administered [14]. Its specificity is 100% and
sensitivity is improved by 20% to 30% in localized CL. Its sensitivity is 55% to 70% in ML [63,
64]. However, due to limited resources both cultures and PCR are not widely adapted yet in the
developing countries [34].
25. 24
Diagnosis of VL is complicated because parasites are harbored in internal organs. They
may be found in a splenic aspirate, liver biopsy or bone marrow biopsy. Splenic aspirate and
liver biopsy can be dangerous due to fragility of these organs and the procedures may lead to
uncontrollable bleeding [56]. The ‘‘gold standard” of VL diagnosis is viewing amastigotes under
microscope in clinical samples. The test is 100% specific (Figure 10) [14]. However, different
sensitivity does occur due to the origin of biopsy samples. “Sensitivity for the spleen, bone
marrow, and lymph node aspiration smears is >95%, 55-97%, and 60%, respectively” [35].
‘‘PCR is particularly sensitive, and can be used to detect Leishmania spp. DNA in blood, lymph
nodes, bone marrow and conjunctival swabs’’ [14]. Patients with VL produce large amounts of
specific IgG that can be used in serological diagnosis. Currently the most widely used serological
tests are Indirect-immuno Fluorescent Antibody Test (IFAT), Enzyme Linked Immunosorbent
Assay (ELISA), and Direct Agglutination Test (DAT) [56]. Immunodiagnosis can be made by
detecting antigens in blood, urine or tissue specimen [68].
26. 25
1.7. Treatment ofLeishmaniasis:
Many factors need to be considered in selecting a regimen against leishmaniasis. First is
the disease form, whether it is cutaneous, mucocutanous or visceral; Second is affordability,
whether patients can carry the financial burden; Third is route of administration, i.e., oral versus
intravenous injection; Fourth is side effect, whether it is too severe to be tolerated by patients.
CL usually heals on its own and may not need treatment, while all VL patients need to be treated
with the most effective and affordable regimens by individuals.
CL in the Old World, such as those caused by L. major can be self-healed in 2-4 months,
cases of L. tropica may take 6-15 months to resolve [14]. CL of the New World caused by L.
mexicana tend to heal themselves after 3 months, lesions caused by L. braziliensis or L.
panamensis are slower in healing, taking a few more months. That had been said, CL has to be
treated to decrease scarring and quicken the healing, especially in the face area, and also to avoid
dissemination in the case of ML [36, 37].
Treatment is usually provided for a long period of time, more than 6 months in case of
large lesions or lesions at joints or faces [38]. Otherwise standard regimens are applied (Table 2).
Several medicines have been used. One treatment is the injection of pentavalent antimony which
gives excellent results in treating CL in all regions (Table 2). However, if infection lesion is large
or multiply lesions exist, antimony can be given for 10 days [38, 39]. Besides, topical
paromomycin can be applied for 20 days. Nevertheless, the effectiveness is variable [40].
27. 26
Lesions caused by L. tropica are slow in self-healing and are difficult to treat. Miltefosine had
been tested in Afghanistan. However, the test was ended earlier than scheduled due to severe
side effects such as abdominal pains [41].
CL in the New World such as those caused by L. panamensis or L. braziliensis can be
treated by antimony for 20 days [42, 43]. Further, ML can be treated with parenteral antimony
for 28 days as treatment duration to decrease inflammation, while amphotericin B can be given
as a rescue therapy [16, 45]. Topical paromomycin was effective with lesions caused by L.
braziliensis or L. mexicana in the geographic areas that do not have a high risk of mucosal
infections, such as Guatemala [44]. Miltefosine had shown excellent results for the curing CL
caused by L. panamensis, in more than 90% of cases in Columbia [37].
Pentavalent antimonial sodium (stibogluconate) and meglumine antimoniate have been
used in treating VL for more than 70 years [46]. Antimonials are toxic with side effects such as
irregular pulses, pancreatic inflammations, severe malnutrition, or even death [47]. Conventional
amphotericin B is considered to be first line of drug in treatment of VL in India, especially in
Bihar State, in replacement of antimonials due to widespread of parasite’s resistance to the latter.
Chills and rigor are side effects of conventional amphotericin B. Moreover, this medicine is
much more expensive than antimonials and is needed in a complicated regimen of ‘‘15 slow
infusions on alternate days’’ [2]. Paromomycin is another affordable drug that can be used as an
antileishmanial drug due to its effectiveness [49]. Sitamaquine, an oral 8-aminoquinoline, has
high efficacy against VL and has been used for more than 20 years [50]. Miltefosine is the
number one drug used against the VL that is taken orally [48]. European patients have taken
liposomal amphotericin B, which is considered the first-line treatment for VL, and produces high
efficacy in short periods of treatment [48].
28. 27
1.8. Control of Leishmaniasis:
Leishmaniasis is a neglected disease of great importance worldwide. Effective control
measures will certainly decrease case numbers and reduce the exposure risks of people living in
endemic areas. Experience has shown integrated measures are superior to single-handed ones.
However, control measures are discussed one by one for the convenience of description. These
include control of sand fly vectors, reduction of reservoir hosts, and individual protection.
1.8.1. Vector control:
Controlling sand fly vectors in many endemic areas of leishmaniasis is often integrated
with controlling vectors of other diseases. For instance, in Bangladesh and India, this is usually
associated with controlling mosquito vectors of malaria. In Brazil, it is often combined with
malaria and Chagas’ disease control [52]. Spraying chemicals such as malathion, fenitrothion,
propoxur and diazinon is very effective although expensive. Thus, it should be continued to keep
the vector populations low in endemic areas [51]. Indoor residual spraying is a simple and cost
effective method for controlling vector. It should cover walls as well as roofs of all houses and
domestic animal shelters. Crack and cervices where sand flies breed should be covered too [57].
All individuals involving the task should be trained. They should know basic information such as
stroke of spraying machines, handling pumps, and total area of coverage by a given amount of
insecticide. For example, “DDT should be mixed in the proportion of 3.3 pounds/3 gallons’’ [57,
58].
29. 28
1.8.2. Reduction of reservoir hosts:
In anthroponotic foci of VL detection and treatment of infected individuals, whether with
or without clinical manifestations, are necessary. Studies in Israel have demonstrated that the
asymptomatic or sub-clinical cases are 4 to 30 times more common than those with VL
syndromes [59]. Dogs are the primary reservoir hosts in many endemic regions such as Brazil,
Northern Europe, the Mediterranean basin, and Americans [60]. It has been demonstrated that
deltamethrin-impregnated dog’s collars protect dogs from sand fly bites and reduce L. infantum
infection by 86% [58]. Diagnosis and treatment of domestic animals, particularly dogs, using
various tests such as IFAT, DAT and ELISA, are highly recommended [53]. Further, elimination
of stray dogs may be carried out by shooting and by using poisoned baits impregnated with
strychnine [53, 61].
1.8.3. Individual protection:
Individual protection is necessary and there are many methods and products
commercially available to prevent individuals’ from being bitten by sand flies. One is repellent
diethyltoluamide (DEET), which can be applied to the exposure parts of the body and clothing
[54, 58]. DEET is highly effective against hematophagous insects, and has been used for more
than 50 years against Leishmania vectors [58]. Second one is physical barriers. Fine mesh
screens (less than 16-mesh) may be used on doors and windows to prevent sand flies from
entering into human dwellings. Bed nets that are impregnated with insecticides, such as
permethrin and deltamethrin may be used to prevent individuals from sand fly biting. Due to
smaller body size than mosquitoes, sand flies can enter through the small holes of bed nets, so
standard mosquito nets do not provide humans with good protection against sand fly biting [51].
30. 29
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79- Nylén and D. Sacks, Interleukin-10 and the pathogenesis of human visceral leishmaniasis,
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36. 35
Figure 1. Classification of Leishmania illustrating three subgenera. The list of named species is
not comprehensive; over 30 species have been named in the genus, including many that are non-
pathogenic or of minor medical importance. Parasites in the subgenera Leishmania and Viannia
infect mammals, whereas the Sauroleishmania infect reptiles. “Reprinted from The Lancet, Vol
37. Bates PA, Transmission of Leishmania metacyclic promastigotes, 10, Copyright (2007), with
permission from Elsevier’’ [23].
37. 36
Figure 2. Development of Leishmania species in the sand fly vector. (a) Morphology of
amastigotes and promastigotes. Each form has a nucleus (N), kinetoplast (K) and flagellum (F).
(b) Developmental sequence of the five major promastigote form i.e., procyclic, nectomonad,
leptomonad, haptomonad and metacyclic promastigotes. ‘‘Reprinted from The Lancet, Vol 37.
Bates PA,Transmission of Leishmania metacyclic promastigotes, 10, Copyright (2007), with
permission from Elsevier’’. [23].
38. 37
Figure 3. Life cycle of Leishmania spp. ‘‘Reprinted from The Lancet, Vol 7. Richard Reithinger
R et al., Cutaneous lesihmaniasis,16, Copyright (2007), with permission from Elsevier’’ [65].
39. 38
Figure 4. Clinical manifestations of cutaneous leishmaniasis. A. A red raised border and a
depression in the middle is shown. B. A clear appearance of circle scars on the face is shown.
‘‘Reprinted from The Lancet, Vol 7. Richard Reithinger R et al., Cutaneous lesihmaniasis, 16,
Copyright (2007), with permission from Elsevier’’ [65].
40. 39
Figure 5. Mucocutaneous leishmaniasis involving mucus membrane of the mouth and the nose.
‘‘Reprinted from The Lancet, Vol 7. Reithinger R et al., Cutaneous lesihmaniasis, 16, Copyright
(2007), with permission from Elsevier’’ [65].
41. 40
Figure 6. Visceral leishmaniasis showing enlarged liver and spleen. ‘‘Reprinted from The
Lancet, Vol. 366, Murray HW et al., Advances in leishmaniasis, 17, Copyright (2005), with
permission from Elsevier’’ [14].
42. 41
Figure7. Geographical distribution of leishmaniasis. ‘‘Reprinted from The Lancet, Vol 7.
Reithinger R et al., Cutaneous lesihmaniasis, 16, Copyright (2007), with permission from
Elsevier’’ [65].
43. 42
Figure 8. Worldwide distribution of visceral leishmaniasis. ‘‘Reprinted from The Lancet, Vol 10.
van Griensven J et al., Combination therapy for visceral leishmaniasis, 11, Copyright (2010),
with permission from Elsevier’’ [66].
44. 43
Figure 9. Micrographs showing cutaneous leishmaniasis. Haematoxylin and eosin-stained skin
lesions and Giemsa-stained lesions are shown. Arrows indicate amastigotes. Biopsies show
amastigotes in (A) L. major and (B) L. mexicana infection. (C) Smear of lesion scraping in
probable L. panamensis infection. (D) Impression smears in L. mexicana infection. Original
magnification ×500, except in (A), ×1000. ‘‘Reprinted from The Lancet, Vol. 366, Murray HW
et al., Advances in leishmaniasis, 17, Copyright (2005), with permission from Elsevier’’ [14].
45. 44
Figure 10. Diangosis of visceral leishmaniasis. A. Giemsa-stained splenic aspirate smear,
showing clumped mononuclear cells and numerous amastigotes. Original magnification ×500. B.
Serodiagnosis of kala azar by anti-K39 antibody detection in immunochromatographic strip test.
Right-hand strip is positive by a second pink band (arrow). Left-hand strip is negative.
‘‘Reprinted from The Lancet, Vol. 366, Murray HW et al., Advances in leishmaniasis, 17,
Copyright (2005), with permission from Elsevier’’ [14].
46. 45
Table 1: Susceptibility or resistance of different mouse strains to Leishmania spp.
R—resistant; S—susceptible.
Strain L. donovani/chagasi/infantum L. major
A/Jax R R
C57BL/6 S R
C57BL/10 S R
BALB/c S S
DBA/2 R S
CBA R R
47. 46
Table 2: Selected treatment regimens for cutaneous and mucosal leishmaniasis.
Species Treatment regimen* Cure rate at 3 months
Cutaneous
disease
Old
world
L major Observation alone 60–70%
Sb: IL ≥ 1 mL per lesion; qod ×8–15 injections or every 1–2 wk×3–8 injections
≥75%
Sb: IM/IV 20 mg per kg per day ×10 days In evaluation
Fluconazole: oral 200 mg once-daily ×6 weeks 90%
Paromomycin: topical, bid×2 weeks In evaluation In evaluation
New
world
L. tropica
L. mexicana
L. panamensis
L. braziliensis
Other species
Sb: IL(as above) weekly×8-11( average) ≥ 90%
Sb: IM/IV 20 mg per kg per day×10 days Not well documented
Observation alone > 75%
Sb: IM/IV 20 mg per kg per day×10 days 90%
Sb: IM/IV, 20 mg per kg per day×20 days 80–90%
Miltefosine: oral, 2·5 mg per kg per day× 28 days 80-90%
Pentamidine: IM 2 mg per kg qod_7 injections 60–95%
Sb: IM/IV 20 mg per kg per day×20 days 80–90%
Pentamidine: IM 2 mg per kg qod×7 injections < 50%
Sb: IM/IV 20 mg per kg per day×20 days 80–90%‡
Mucosal
disease Any species
Sb: IM/IV 20 mg per kg per day×28 days Mild disease 75%
Severe disease 10–67%
Amphotericin B: IV 1 mg per kg qod×20 infusions 75%
New
world
48. 47
Sb=pentavalent antimony; IL=intralesional; IM/IV=intramuscular/intravenous; bid=twice-daily;
qod=every second day. *Sb available in branded (sodium stibogluconate [Pentostam],
meglumine antimoniate [Glucantime], and generic form. Limiting the maximum daily dose to
850 mg is no longer recommended. Miltefosine is available in 10 mg and 50 mg capsules; for
bodyweight 25 kg given as single daily dose, for >25 kg in two divided doses. In pregnant
women, Sb is not recommended and miltefosine is contraindicated (see text). Efficacy of
intralesional Sb in L. tropica (vs L. major) infection may reflect longer treatment duration. Cure
rates for L. guyanesis infection may be lower than those reported here. ‘‘Reprinted from The
Lancet, Vol. 366, Murray HW et al., Advances in leishmaniasis, 17, Copyright (2005), with
permission from Elsevier’’ [14].
50. 49
Libya is bordered by the Mediterranean Sea to the north, Egypt to the east, Sudan to the
southeast, Chad and Niger to the south, and Algeria and Tunisia to the west. With an area of
almost 1.8 million square kilometers (700,000 sq. mi), Libya is the fourth largest country in
Africa by area, and has the 17th largest city in the world. Its capital Tripoli is home to 1.7 million
of 6.4 million Libyans. Libya plays an important role in the region in economy and society and
has become more urbanized and modernized in the last two decades. Urbanization has expanded
city limits significantly into the previous countryside leading to leishmaniasis outbreaks in
various regions. The consequence of climate change increases the vector species [10]. In many
Libyan towns, both development of water supply and the transition from a desert environment to
agricultural farmland have increased the reservoirs of Leishmania protozoa. One example is
Sebha, a city located at southern Libya. In addition travelers from adjacent countries to Libya
bring a new form of leishmaniasis, which is caused by L. Killiki.
51. 50
2.1. Pathogens and vectors:
Both cutaneous (CL) and visceral leishmaniasis (VL) have been reported in Libya. CL is
endemic in the northwestern region that is bordered with Tunisia as well as in southern Libya. Its
causative agents are L. major and L. killicki [1, 2]. In addition to human beings, small rodents
Meriones libycus (Figure 1) and M. shawi are found infected with these protozoa. They may
serve as reservoirs for CL [1].
VL is endemic in eastern and southern Libya [3]. It was proposed that L. donovani was
the causative agent of VL in Libya. Nevertheless, the parasite was identified solely by finding
amastigotes in the bone marrow of infected patients, which does not have the capacity to species
identification at all [10]. It might be L. infantum instead provided that it is the only species found
in the adjacent areas and neighboring countries [10].
The most common manifestations of CL patients include red rashes at the biting site
followed by development of the ulcerative and papulo-nodules. The lesions often scatter at face,
legs and arms [6]. Interestingly CL caused by L. major is self-healing without treatment, usually
within a couple of months to half a year. In contrast, the one caused by L. killicki is chronic,
lasting for many years [1]. Most of VL patients show such symptoms as fever, anorexia, body-
weight loss, anemia, and hepatosplenomegaly [3].
52. 51
In Libya the major vectors of CL versus VL are different. Whether this is due to the difference
in geographic distribution is not clear at present. Several sand fly species have been recorded in
Libya according to Walter Reed Biosystematics Unit (WRBU). These include Phlebotomus
papatasi, P. longicuspis, P. neglectus, P. perniciosus, P. chabaudi, and P. sergenti. P. papatasi
(Figure 2) and P. sergenti (Figure 3) are the vectors of CL, whereas P. longicuspis is the vector
of VL [1, 9].
53. 52
2.2. Epidemiology:
CL is endemic in the north-western region of Libya, especially in Nafusa Mountains. The
earliest report of two cases was in 1930 [1]. Later 40 cases were found in the area close to the
Tunisia border [1]. In a study carried out in a small village called Yafran in Nafusa Mountains
between February 1991 and December 1992, 445 CL cases were diagnosed by skin biopsy and
microscopy. The peak season of infection was in November 1991 and December 1992. The
causative agent was L. major, and it was more common in male than female with a ratio of male
to female 1.9:1.0. The highest incidents were among the people between 11 and 20 years old [1].
The same study also showed that the most common vector was P. papatasi (Figure 2) followed
by P. sergenti, (Figure 3) and that Meriones libycus (Figure 1) and M. shawi were reservoirs in
the area [1].
Another survey covered 199 CL patients from 24 localities in North–west Libya between
September and December 1994. Most of the skin lesions were distributed on the uncovered parts
of the body, and the size of the skin scares ranged from 1 to 5 cm in diameter. 65.3% of infected
individual are between ages 1-30 years old, and patients responded well to sodium stibagluconate
treatment [5]. Elkasah and colleagues found 84 CL patients including 78 adults and 6 children in
Surt between October 2006 and February 2007. Among the infected individuals 26 were female
and 58 were male with most in the age 17- 30 years old [5, 11]. 54.4% of cases were in Elhish
[11]. 32.1% were in Tawargha, a small village 200 km away from Elhish [11].
54. 53
VL cases recorded in Libya over past 80 years were all from the east of the country in
Green Mountain Region (Figure 4) [9]. Mehabresh and el-Mauhoub [3] reviewed the records of
21 patients that were referred to Benghazi Children Hospital from March 1982 to May 1990. All
cases presented fever, hepatosplenomegaly, and anorexia followed by body-weight loss and
abdominal distension. 86% of patients were positive for L. donovani in bone marrow biopsy,
whereas all patients had positive results by the indirect haemagglutination test [3]. All patients
were treated by sodium stibogluconate at a dose of, 10mg/kg/day. They all were cured without
showing any side effects.
Cases have also been reported in the south part of the country in Sebha since 1985
(Figure 4) [9]. The environment of this area has changed and encouragement to grow the
agriculture since 1999 due to water supply projects. This might be the reason for presenting
reservoirs to live and increase in their numbers in that area [10].
55. 54
Concluding remarks:
Leishmaniasis is considered as one of the most important vector-borne protozoan
diseases in humans. The disease is caused by many species of Leishmania, with some of species
targeting the skin while others the internal organs. It is widespread throughout the world. The
disease is transmitted by sand fly vectors. Leishmaniasis can be diagnosed by 1) direct
observation of infected macrophages with amastigotes in biopsy tissues; 2) culture of the
promastigotes from biopsy; and 3) PCR to detect parasite. There is no vaccine approved for
human use. Current medicines are not optimal. They are cumbersome to apply with severe side
effects. In addition parasites resistant to common regimens have occurred and have a tendency
to spread.
Leishmaniasis can be effectively controlled by a comprehensive approaches using
combination of methods such as sand fly control, insect precautions, treating patients, and
control of the animal reservoirs.
Leishmaniasis is considered as an important public health problem in North Africa
including Libya. Most of the cutaneous leishmaniasis has been recorded in the West region,
whereas visceral leishmaniasis is found in the East and South regions of Libya. The World
Health Organization assessed in 2008 that an urgent action and assistant would be required to
control the disease in the newly affected area near to Tripoli.
56. 55
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Jamahiriya: a study of the Yafran area. East Mediterr Health J, 2000. 6(5-6): p. 884-7.
2- Jaouadi, K., et al., First detection of Leishmania killicki (Kinetoplastida, Trypanosomatidae)
in Ctenodactylus gundi (Rodentia, Ctenodactylidae), a possible reservoir of human cutaneous
leishmaniasis in Tunisia. Parasit Vectors. 4: p. 159.
3- Mehabresh, M.I. and M.M. el-Mauhoub, Visceral leishmaniasis in Libya--review of 21 cases.
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