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Leishmaniasis in Libya

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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

1
 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.

2
 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

3
 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.

4
 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

5
 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

6
 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

7
CHAPTER ONE

Literature review

8
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].

9
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.

10
 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

11
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].

12
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

13
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].

14
 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].

15
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.

16
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

17
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].

18
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].

19
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].

20
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].

21
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].

22
 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].

23
 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].

24
 1.7. Treatment of Leishmaniasis:

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].

25
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].

26
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].

27
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].

28
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79- Nylén and D. Sacks, Interleukin-10 and the pathogenesis of human visceral leishmaniasis,
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34
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].

35
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].

36
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].

37
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].

38
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].

39
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].

40
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].

41
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].

42
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].

43
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].

44
Table 1: Susceptibility or resistance of different mouse strains to Leishmania spp.

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

R—resistant; S—susceptible.

45
 Table 2: Selected treatment regimens for cutaneous and mucosal leishmaniasis.

Species Treatment regimen* Cure rate at 3 months

Cutaneous
disease

L major Observation alone 60–70%

Old Sb: IL ≥ 1 mL per lesion; qod ×8–15 injections or every 1–2 wk×3–8 injections
world ≥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 L. tropica Sb: IL(as above) weekly×8-11( average) ≥ 90%

world Sb: IM/IV 20 mg per kg per day×10 days Not well documented
L. mexicana Observation alone > 75%
Sb: IM/IV 20 mg per kg per day×10 days 90%
L. panamensis 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%

L. braziliensis 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%
Other species

Sb: IM/IV 20 mg per kg per day×20 days 80–90%‡

Mucosal
disease Any species
New Sb: IM/IV 20 mg per kg per day×28 days Mild disease 75%
Severe disease 10–67%
world
Amphotericin B: IV 1 mg per kg qod×20 infusions 75%

46
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].

47
CHAPTER TWO

Leishmaniasis in Libya

48
 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.

49
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].

50
 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].

51
 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].

52
 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].

53
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.

54
 References

1- El-Buni, A.A., I. Jabeal, and A.T. Ben-Darif, Cutaneous leishmaniasis in the Libyan Arab
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.

Ann Trop Paediatr, 1992. 12(2): p. 159-63.

4- El-Buni, A. and A. Ben-Darif, Cutaneous leishmaniasis in Libya: epidemiological survey in

Al-Badarna. Parassitologia, 1996. 38(3): p. 579-80.

5- Fathy, F.M., F. El-Kasah, and A.M. El-Ahwal, Emerging cutaneous leishmaniasis in Sirte-
Libya: epidemiology, recognition and management. J Egypt Soc Parasitol, 2009. 39(3): p.
881-905.

6- El Buni AA, Edwebi H, Ben Darif AL, Prospective study among cutaneous leishmaniasis
cases in Tripoli Central Hospital, Tripoli, Libya, Arch Inst Pasteur Tunis,1997. 74(1-2): P 3-4

7- El-Buni AA, Ben-Darif AL, Taleb I, Refai A, Cutaneous leishmaniasis in Al-Badarna: a

prospective study among school children, Arch Inst Pasteur Tunis,1998. 75(1-2): P19-20.

8- Pratlong F, Dereure J, Ravel C, Lami P, Balard Y, Serres G, Lanotte G, Rioux JA, Dedet JP,
Geographical distribution and epidemiological features of Old World cutaneous
leishmaniasis foci, based on the isoenzyme analysis of 1048 strains,Trop Med Int Health,
2009. 14(9): P1071-85

9- Report of The Consultative Meeting on cutaneous leishmaniasis WHO, 2007.

10- Albert Kimutai, Peter Kamau Ngure, Willy Kiprotich Tonui, Michael Muita Gicheru and
Lydia Bonareri Nyamwamu, Leishmaniasis in Northern and Western Africa , Afr. J. Infect.
Di, 2009.3(1):P14-25.

11- Elkasah F., Dow M., Elahwel A. Leishmaniasis in Libya, Euro Cong, 2008 2(4) P 5-6.

12- http://www.raywilsonbirdphotography.co.uk/Galleries/Invertebrates/vectors/sand_fly.html.

Gallery

55
13- http://www.biolib.cz/en/image/id29925/Gallery

14- Geneva, World Health Organization Headquarters, April to 2 May. 2007.

56
Figure 1. Meriones libycus, a reservoir host of Leishmania major and L. killicki in Libya [13].

57
Figure 2. Phlebotomus papatasi, a vector of Leishmania major and L. killicki [12].

58
Figure 3. Phlebotomus sergenti, a vector of Leishmania major and L. killicki [12].

59
 Figure 5. Geographic distribution of leishmaniasis in Libya, 2007 [14].

60

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