[page 11↓]

1. Introduction

The following passages will give a general overview on leishmaniasis worldwide and specifically in Israel and the West Bank. The disease will be presented in its various clinical forms, in relation to the different causative Leishmania species. This will provide a background for understanding why sensitive and species-specific diagnosis is important. Consecutively, the facts about the biology and ecology of the parasite will be presented, which will convey a more profound understanding of the complexity of the disease. This is intended to illustrate that improved diagnostic tools are required in order to understand the epidemiology of the disease, which is a prerequisite for effective control measures. In the review of diagnostic methods the most important methods from past to present will be presented, including comparative aspects. By understanding the benefits and limitations of each of the methods presented, the selection of the specific diagnostic tools for this study will be comprehensible.

1.1. General facts about leishmaniasis:

1.1.1. Epidemiology:

Leishmaniasis is a vector borne disease caused by various members of the genus Leishmania , a protozoic parasite. The clinical presentation ranges from simple cutaneous lesions to life threatening visceral forms. The disease is endemic in many tropical and subtropical countries around the world (more than 80 countries according to the WHO). Leishmaniasis is not only widely distributed in warm countries, but it is also prevalent in very different topographic areas. It is endemic in rain forests (Bolivia, Brazil), deserts (Middle East, North Africa), in the countries bordering the Mediterranean Sea and also in elevations of several thousand meters (Peruvian Andes, Ethiopian highlands).

According to WHO estimates 350 million people are at risk world wide and 12 million people are affected. The annual incidence is estimated at 1-1.5 million new cases of cutaneous leishmaniasis (CL) and 0.5 million cases of visceral leishmaniasis (VL). The disease is greatly underreported, with only 600,000 officially declared cases annually. In most of the endemic countries leishmaniasis is not a reportable disease (http://www.who.int/emc/diseases/ leish/leisdis1.html). The incidence worldwide is on the rise. New endemic foci have emerged over the past decades, epidemics are not controlled and endemic areas are spreading due to development and population shifts (Desjeux, 1999). In western countries the incidence is increasing due to HIV- Leishmania coinfection and tourism. In recent years coinfection with HIV became a serious threat in south-western Europe with 1.5-9.5% of AIDS patients being affected. [page 12↓] For the following passages on the parasite, the clinical picture, the transmission and control of leishmaniasis Manson’s Tropical Diseases (1987; 1996) served as the main source of information, if not cited otherwise.

1.1.2. The parasite:

The Leishmania parasite is a protozoa belonging to the order Kinetoplastida and the family of Trypanosomatidae . The genus Leishmania includes more than 20 species. The parasite exists in two morphological forms: the nonflagellated amastigote (3-5 m m in diameter) living intracellular in macrophages of the mammalian host, and the flagellated promastigote (15-30 m m in length, plus the flagella), living extracellular in the intestinal tract of the sandfly-vector. In the macrophages the amastigotes are able to survive and multiply within the acidic phagolysosomes of the host cells (reviewed by Alexander et al ., 1999). After multiplication in the host cell the amastigotes are released. Subsequently other macrophages are infected and the infection spreads (reviewed by Peters and Killick-Kendrick, 1987; Rittig and Bogdan, 2000).

The parasite contains two prominent organelles, the nucleus and the kinetoplast. The kinetoplast is found in all protozoa of the order kinetoplastidae (eg. Leishmania, Trypanosoma, Crithidia ). It is a rod-shaped mitochondrial structure consisting of a DNA network of about 10,000 minicircles and about 50 maxicircles, the kinetoplast-DNA (kDNA). The function of the kinetoplast has not been clear until recently. It was found that maxicircles encode for mitochondrial ribosomal RNAs. The minicircles play a role in the editing process of these mRNAs (reviewed by Shlomai, 1994). Figure 1a and 1b show amastigotes in Giemsa stained smears, Figure 2 shows promastigotes in the sandfly gut (electron micrograph).


[page 13↓]

Figure 1: Amastigotes in Giemsa stained smears

Figure 2: Promastigotes in sandfly gut

Electron micrograph by Alon Warburg

1.1.3 Life cycle:

The transmission cycle is maintained between the vector and the reservoir. Depending on the species of Leishmania the transmission is either zoonotic or anthroponotic, involving either animals or humans as reservoir. The parasite is transmitted by the bite of female sandflies of the genus Phlebotomus and Lutzomyia. During the blood meal Leishmania infected macrophages are ingested by the vector. In the gut of the sandfly the intracellular amastigotes develop into flagellated promastigotes at an ambient temperature of 24-28 ° C. During another blood meal the mature promastigotes are inoculated into a mammalian host. Macrophages ingest the parasites, which then transform into the amastigote form. The life cycle is shown in Figure 3.

1.1.4. Clinical forms of leishmaniasis:

The three distinct forms, cutaneous (CL), visceral (VL) and mucocutaneous leishmaniasis (MCL) are classically caused by a spectrum of different Leishmania species each. Even though there is a clear correlation between the causative species and the clinical presentation many variations are seen. Depending on the specific characteristics of species/strains and also on the immunocompetence of the host the clinical manifestation may vary to a great extent. Species causing typically CL may visceralize and visceral species may show dermatotropism. In many endemic areas of the world a few Leishmania species are prevalent simultaneously so that a species specific diagnosis can not rely on clinical findings alone. Species specific diagnosis is necessary for adequate treatment.


[page 14↓]

Figure 3:


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1.1.5. Immunology:

Apart from the characteristics of species and strains, the host immunology influences the severity of the disease to a great extent. Genetically determined immunological patterns of reaction, the present health condition and the nutritional status play a decisive role in the course of the disease. The events responsible for resistance or susceptibility occur early in infection. Resistance depends on a Th1-type response resulting in the production of g -interferon (IFN- g ) and nitric oxide (NO). A Th2-type response is related to susceptibility (Blackwell, 1996; reviewed by Alexander et al ., 1999; reviewed by Solbach and Laskay et al ., 2000). The severity of the disease depends as well on factors related to the causative agent itself and the sandfly vector. The antigenicity is variable among strains of the same species, leading to atypical clinical presentations (eg. visceralizing L.tropica strains and dermatotrophic L.d.infantum and L.d.chagasi strains). Apart from this also the composition of the sand fly saliva influences the severity of the infection (reviewed by Kamhawi, 2000).

1.1.5.1. Cutaneous Leishmaniasis:

Old World:

In the Old World, cutaneous leishmaniasis (CL) is also known as “Oriental Sore”, “Baghdad boil”, “the little sister” (Aljebori and Evans, 1980), “Delhi boil” or “Rose of Jericho”. CL is usually caused by L.major, L.tropica and L.aethiopica (see Table 1). CL presents usually as a self-limited, ulcerative skin lesion. The uncovered parts of the body, especially the face, are affected. After an incubation time of several weeks to several months a papule develops at the site of the sandfly bite, which later evolves into an ulcer. The size of the ulcer can reach a diameter of several centimeters. It lasts normally for a few (3-4) months but it can also persist for more than one year before it resolves spontaneously. The lesion is usually painless unless a secondary bacterial infection is present. A slightly depressed and often hypopigmented scar remains. If left to self-cure, life-long immunity is usually acquired against the same species. L.tropica infections tend to be more severe than those of L.major and have a potential to recidivate and also to visceralize (Magill et al ., 1993; 1994; Sacks et al ., 1995). As an unusual causative agent L.d.infantum has been reported to cause CL, eg. in Tunisia (Gramiccia et al ., 1991), in Lebanon (Nuwayri-Salti et al ., 1994) and in Iran (Hatam et al. 1997).

New World CL:

In the New World, CL is caused by species of the L.mexicana and the L.braziliensis complexes (see Table 1) . L.mexicana infections are normally uncomplicated and self-limited. If the ear is infected, a so-called Chiclero's ulcer may develop which is characterized by destruction of the [page 16↓] cartilage and disfigurement of the ear. The infections caused by species of the L.braziliensis complex primarily present as CL. Especially the L.braziliensis infections are related to a high risk for later progression to mucocutaneous disease.

An atypical form of CL has been reported from Honduras (Ponce et. al. , 1991; Noyes et al ., 1997) and from Nicaragua (Belli et al ., 1999) caused by L.d.chagasi, which normally causes VL. In this atypical presentation the symptoms are confined to the skin. The lesions are nodular and long-lasting, resembling leprosy.

Chronic forms of CL:

Leishmaniasis recidivans or lupoid leishmaniasis is one chronic form of CL caused by L.tropica (Salman et al . 1999). Self-cure is not completed and new eruptions evolve at the border of the scarring lesion over many years. The appearance resembles lupus vulgaris. Parasites are scarce and the chronic skin lesion is mostly due to a hyperreactive immune response.

Diffuse CL (DCL) is another chronic form of CL. The infection spreads over large areas of the body. It represents a condition of anergy with a failure of cell-mediated immune response (negative Leishmanin test) and an abundance of amastigotes. In the Old World it is associated with L.aethiopica infections and is mostly seen in Ethiopia. It resembles lepromatous leprosy, which often led to misdiagnosis in the past. As a result, DCL patients were often kept in leprosaria by mistake. In the New World DCL is caused mainly by L.amazonensis (30% of the infections result in DCL). DCL occurs also in immunodeficient patients with no species-specific relation; coinfection with HIV is the most common cause (Ramos-Santos et al ., 2000; Gillis et al ., 1995).

1.1.5.2. Visceral leishmaniasis (kala azar):

Visceral leishmaniasis (VL) or kala azar is usually caused by species belonging to the L.donovani complex ( L.donovani, L.d.infantum, L.d.chagasi , see Table1). L.donovani is the causative agent in the highly endemic areas of India and Sudan. In Middle Eastern and Mediterranean countries L.d.infantum prevails. In this region of the world mostly infants and immunocompromised adults suffer from the disease. In the New World L.d.chagasi is the only known species causing VL.

VL is a severe systemic disease presenting with fever, splenomegaly and cachexia. Typical laboratory findings are pancytopenia, albumin deficiency and hypergammaglobulinaemia. If not treated patients usually die after approximately two years. Often super-infections, typically tuberculosis, pneumonia or bacterial dysentery, are the lethal causes. Most infections are subclinical, only 3% of infections lead to the full picture of the kala-azar syndrome.


[page 17↓]

Rarely VL can be caused also by L.tropica . In India L.tropica has been isolated from 4 patients suffering from classical kala azar (Sacks et al ., 1995). Visceral infections caused by L.tropica have been reported from veterans of Operation Desert Storm. The onset of symptoms was more acute, presenting with fever, abdominal pain, fatigue and cough (Magill et. al., 1993). No cutaneous manifestations were seen and the disease was described as being viscerotropic but not identical with kala azar (Kreutzer et. al., 1993).

Cutaneous manifestations related to VL:

The site of inoculation is often symptomatic and is known as leishmanioma. A small lesion appears about 3-4 months before the onset of kala azar, which should not be mistaken for CL (Adler, 1964).

Post kala azar dermal leishmaniasis (PKDL) is a form of CL occurring in some patients after treatment and cure of VL, usually after a latent phase of several years. PKDL presents as a rash, which may last for many years and may spread over large parts of the body. It can appear either as a macular, maculo-papular or nodular rash (Zijlstra and El-Hassan 2001). Often it begins with depigmented patches, which later turn to be nodular. PKDL is a serious condition which often is disfiguring the patient (social problem); it has to be differentiated from lepromatous leprosy and other skin diseases. It is mostly seen in the north-east of India (Bengal, Bihar) where 20% of the VL patients are affected. According to Manson’s Tropical Diseases, (1996) East African PKDL occurs in only 2-5% of the VL cases and it does not exist in any other endemic area of VL. Recently it was reported that PKDL affects a much higher percentage of VL patients in Sudan. Zijlstra and El-Hassan (2001) found that 55% of the VL patients in Sudan developed PKDL. In the African form of PKDL the rash appears much earlier than in the patients from India, either during active kala azar or within 6 months after cure.

1.1.5.3. Mucocutaneous Leishmaniasis (Espundia):

Mucocutaneous leishmaniasis (MCL) or Espundia is almost exclusively seen in Central and South America. It is a chronic and very serious condition, developing years after self-cure of cutaneous lesions. The infection causes a progressive destruction of the mucosa, the cartilage and bones of nose and pharynx, leading to a severe mutilation of the face. MCL is mainly caused by Leishmania species belonging to the L.braziliensis complex (see Table 1), predominantly by L.braziliensis. The risk of developing MCL after cure of CL is estimated to be up to 40%. MCL can be lethal, often by aspiration pneumonia. Infections caused by L.panamanensis and L.guyanensis rarely result in MCL, whereas L.peruviana is not associated with MCL. L.guyanensis infections are also known as ‘pian bois’, typically presenting with multiple lesions. [page 18↓] L.peruviana infections are known as ‘uta’ and are usually self-healing within a few months. Infections caused by the L.braziliensis complex are often associated with lymphadenopathy, especially infections with L.braziliensis (Barral et al ., 1992). In Sudan a less invasive form of MCL is seen in patients infected with L.donovani . In contrast to the American form, mucosal involvement usually precedes the clinical signs of kala azar (El-Hassan and Zijlstra, 2001).

1.1.5.4. Leishmaniasis and HIV-coinfection:

Most cases of coinfection with HIV and Leishmania have been reported in south-western Europe, with L.d.infantum being the predominant causative species. In conditions of immune suppression the clinical picture of leishmaniasis is often atypical. The typical affiliation between species and clinical presentation does not exist in many cases. Not only can cutaneous Leishmania species lead to systemic visceral disease, but also parasites infiltrate tissues and organs which are normally not affected. VL in HIV-positive patients has also been reported to be caused also by species which normally do not visceralize, eg. L.braziliensis (Hernandez et al ., 1993), L.major (Gillis et al ., 1995), L.mexicana (Ramos-Santos et al ., 2000). Likewise, visceral disease ( L.d.infantum ) can be accompanied with unusual cutaneous lesions (Postigo et al ., 1997). Alvar et al ., (1997) have reported that L.d.infantum - HIV coinfections can present with all forms of leishmaniasis including MCL, PKDL and disseminated CL. In disseminated forms of leishmaniasis parasites have been repeatedly found in the digestive tract, namely in the colon (Sebastian et al ., 1997), in the duodenum (Hamour et al ., 1998) and in an anal ulcer (Perez-Molina et al ., 1997). A retrospecive study in 91 coinfected patients suggests the presence of Leishmania amastigotes in atypical locations to be related to the immunological status (Rosenthal et al ., 2000). The following Table 1 gives an overview on the different Leishmania species , the most common clinical manifestations, the geographical distribution as well as the specific host and vector species.

1.1.6. Treatment:

Simple CL due to L.major is mostly left to self-cure. It is even preferable not to treat, so that long-term immunity can be acquired. This especially applies to patients living in endemic areas. Treatment is necessary when cosmetically or functionally important sites are involved. L.tropica infections generally require treatment. For local treatment, intralesional Pentostam (sodium-stibogluconate), ketoconazol, cryotherapy with liquid nitrogen or heat can be applied. For systemic treatment, pentavalent antimonal compounds (Sb) eg. Pentostam and Glucantime are used (Herwaldt, 1999 a; Norton et. al ., 1992).


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Table 1 : Species of Leishmania, clinical manifestation, geographical distribution , reservoir and vector:

Complex

Species

Clinical

manifestation

Geographical distribution

Reservoir

Vector

Old World:

 

 

 

 

 

 

L.major

CL

Western and Central Asia

Middle East, Africa, India

rodents: Fat sand rat ( Psammomys.obesus ), gerbils ( Meriones, Rhombomys )

Phlebotomus paptasi

 

L.tropica

CL, L.recidivans

 

Western and Central Asia

Middle East, North Africa, Sub-Saharan Savanna, India

humans, dog infections reported, hyraxes suspected

P. sergenti

L.donovani

L.donovani

VL, PKDL

India, Sudan, Kenya

humans

P.argentipes

L.donovani

L.d.infantum

VL, (CL), CVL

Mediterranean basin

Middle East, China

dogs, wild canids (foxes, jackals)

P.perniciosus, P.ariasi

 

L.aethiopica

CL, DCL

Highlands of Ethiopia and Kenya, Sudan

hyraxes

P.longipes, P.pedifer

New World:

 

 

 

 

 

L.braziliensis

L.braziliensis

CL, MCL

Central and South America

forest rodents

Lutzomyia ssp.

L.braziliensis

L.panamanensis

CL

Central America, Columbia

sloths

Lu. sp.

L.braziliensis

L.guyanensis

CL

Guyana, Brazil

sloths

Lu .sp.

L.braziliensis

L.peruviana

CL

Peru, Argentine

dogs, humans?

Lu sp.

L.mexicana

L.mexicana

CL, Chiclero's ulcer

Central and South America, Texas

forest rodents

Lu. sp.

L.mexicana

L.amazonensis

CL, DCL

Brazil, Venezuela,

mostly north of the Amazon

forest rodents, Opossums

Lu .sp.

L.donovani

L.d.chagasi

VL, atypical CL, CVL

Central and South America

CL in Honduras, Nicaragua

dogs, wild canids

Lutzomyia longipalpis

Others: (Old World)

 

 

 

 

 

 

L.gerbilli

human infection unknown

Eastern Russia, Mongolia

Great Gerbil (Rhombomys opimus)

 

 

L.turanica

human infection unknown

Central Asia

R. opimus

 

 

L.arabica

human infection unknown

Saudi Arabia

Psammomys obesus

 

 

L.killicki

 

Tunisia

 

 

This table is an extract from 3 different sources: Manson's Tropical Diseases, 19th edition (1987); in New Generation Vaccines: Vaccines against leishmaniasis (Eisenbeger and Jaffe, 1997); the leishmaniases as emerging and reemerging zoonoses (Ashford, 2000). CL -cutaneous leishmaniasis, VL -visceral leishmaniasis, PKDL-post-kala-azar dermal leishmaniasis, CVL -canine visceral leishmaniasis, DCL -diffuse cutaneous leishmaniasis MCL -mucocutaneous leishmaniasis, L.- Leishmania, L-leishmaniasis


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Infections caused by species of the L.braziliensis complex generally require treatment with pentavalent antimonal drugs. In cases of MCL early diagnosis is essential for effective treatment. Simple CL caused by the L.mexicana complex does not necessarily require treatment. For L.braziliensis infections, sodium-stibogluconate is the drug of choice. Relapses are not uncommon and require a second course of treatment, possibly also with another drug (eg. Amphotericin B). In contrast to L.braziliensis infections, L.mexicana infections respond only poorly to antimonal compounds, but do instead respond well to ketoconazol. Reversely, ketoconazol is not efficient for the cure of L.braziliensis infections (Navin et al ., 1992).

For the treatment of kala azar, pentavalent antimonial drugs have been the therapy of choice since 1916. Due to its toxicity the treatment has to be monitored carefully. Heart and liver are typically affected (Hepburn, 2000). Resistance against pentavalent antimonals has become a serious problem, especially in India. As an alternative drug Amphotericin-B has proved to be effective too. Less toxic lipid-associated formulations of Amphotericin can be administered as well. The combinations of Paromomycin (Aminosidine) with Sb and interferon- g with Sb are also efficient and may help to reduce side effects of Sb compounds (less Sb is required) (reviewed by Berman, 1997).

The treatment of VL and MCL requires hospitalization for several weeks and the drugs are expensive. New hope for an oral treatment came with the discovery of Miltefosine, which appears to be highly efficient, less toxic, and can be administered orally. If clinical trials are successful, this drug will be very beneficial, especially in India and Sudan (Herwaldt, 1999 b).

1.1.7. Transmission:

The presence of leishmaniasis depends on a variety of ecological and biological factors. Since the various Leishmania species depend as much on specific reservoir as on specific vector species, a Leishmania focus can only exist if suitable ecological conditions are present for both the host animal species and the sandfly species. The topography and the climate are essential for the maintenance of the life cycle. Only if the reservoir and the vector live close enough together is the transmission of the parasite possible and the infectious cycle maintained.

CL:

CL occurs either as a zoonotic or an anthroponotic infection. Zoonotic CL is caused by L.major, with rodents serving as the reservoir. These rodents live usually in colonies and are commonly found in vast uninhabited areas. The rodent burrows provide excellent breeding places for the sandflies. Through the coexistence of rodents and sandflies in the same habitat the natural transmission cycle of L.major is maintained. In zoonotic leishmaniasis humans are only [page 21↓] accidental hosts. New endemic foci of CL can emerge when developmental changes take place (eg. building, agriculture). In the Middle East and North Africa the fat sand rat ( Psammomys obesus ) is the main reservoir. Also jirds ( Meriones sp.) were found to host the parasite (Schlein et al ., 1982; 1984). In Central Asia, Iran and Afghanistan the great gerbil ( Rhombomys opimus ) was identified as being the reservoir of L.major (Strelkova, 1996; Yaghoobi-Ershadi and Javadian, 1996). This gerbil species also hosts nonpathogenic species of Leishmania ( L.gerbilli and L.turanica ). L.major is transmitted predominantly by sandflies of the species Ph.papatasi .

Anthroponotic CL has been attributed to L.tropica . Major urban centers of the Middle East (Aleppo, Damascus, Baghdad, Tashkent, Teheran and Kabul) were known to be highly endemic for L.tropica . Humans were the only known reservoir. The infections were so numerous as to maintain the transmission cycle. In recent years it became increasingly obvious that the classical transmission pattern of L.tropica infections has changed. Epidemics in densely populated areas became less common. A shift from an urban to a rural distribution took place. In some of the mentioned Middle Eastern cities, CL has been quite efficiently eradicated by systematic and repeated spraying of houses with insecticides and also as a result of spraying within anti-malaria campaigns. In the past decade the outskirts of cities and villages have been affected predominantly, and infections have occurred more sporadically. These characteristics suggest the existence of an animal reservoir and a zoonotic transmission of L.tropica . Until today the animal reservoir of L.tropica has not been identified, but several animal species are suspected to function as reservoir. L.tropica has been isolated from a hyrax ( Procavia ssp.) in Kenya (Sang et al ., 1992), from dogs in Morocco (Dereure, 1991) and from a rat (Aljeboori and Evans, 1980). Hyraxes are the proven reservoir of L.aethiopica in Ethiopia (Ashford et al. , 1973) . In Jordan hyraxes are abundant in endemic areas of L.tropica and are therefore suspected to be the reservoir (Saliba et al ., 1997). This has been discussed also by Ashford, (2000). L.tropica is predominantly transmitted by sandflies of the species Ph.sergenti.

VL:

VL occurs either in a typical zoonotic or an anthroponotic pattern, depending on the species involved. L.donovani depends on interhuman transmission (Thakur, 2000). It occurs in epidemics in densely populated areas of India (Bengal, Bihar) and in Sudan. Subclinical infections are thought to exist at such a high number that this alone would be sufficient to maintain the infectious cycle. Besides, PKDL patients are suspected as serving as a reservoir, especially bridging long-term intervals between epidemics. The homophilic nature of the vector [page 22↓] of Indian kala azar, Ph.argentipes , supports the hypothesis that humans are the only reservoir in India.

In other regions of the World (Mediteranean countries, the Middle East, Central Asia and China) VL is a zoonosis. The causative agent is L.d.infantum, which is transmitted by phlebotomine sandflies mainly of the subgenus Larroussius . Canids, predominantly dogs, serve as a reservoir (Adler, 1962). Dogs suffer from canine visceral leishmaniasis (CVL), a disease closely related to human VL. In addition, dogs suffer typically from various dermal symptoms. The distribution of the disease is typically rural. Wild canids (eg. foxes and jackals) seem to be an important factor for the distribution of the parasite over larger geographical areas (Baneth et al ., 1998). The epidemiology of L.d.chagasi in the New World is very similar, causing VL in humans and CVL in canids.

MCL:

CL and MCL of the New World are zoonotic, with small forest rodents and sloths serving as reservoir. Forest workers, hunters and settlers on cleared forest land are especially at risk. Sandfly species of the genus Lutzomyia and Psychodopogus are the vectors.

1.1.8. Antiepidemic measures:

Control measures target the interruption of the transmission cycle. For efficient control, the ecology and epidemiology of the disease have to be understood (differences depending on species and local ecology). Depending on the circumstances (eg. zoonotic or anthroponotic transmission), the control of either the reservoir or the vector is advisable. The human link in the transmission cycle can be controlled by taking personal precautions.

Prevention of human infections:

As sandflies are only active at night, inhabitants of endemic areas or travellers in endemic areas can diminish the risk of exposure to sandfly bites by long sleeve clothing during evening hours, application of insect repellents and usage of bed nets. Fine mesh screens for windows are advisable as well. In the case of anthroponotic leishmaniasis, early case detection and treatment are the most important control measures. Despite various attempts to develop a vaccine no efficient immunization is yet available against leishmaniasis (reviewed by Eisenberger and Jaffe, 1997).

[page 23↓]Control of the reservoir:

One effective measure for the control of zoonotic CL is the deep ploughing of the area around houses, in order to destroy the rodent burrows. In the northern Jordan Valley CL cases became rare in recent years due to extensive agriculture and development which had resulted in the destruction of the natural habitat of the Psammomys (personal communication with Dr. Alon Warburg). In order to control VL, different measures have been employed. It has been shown in a study in Brazil that the removal of infected dogs led to a lower incidence but did not eradicate the disease (Ashford et al ., 1998). Vaccination of dogs might be an alternative approach for the future (Tesh, 1995). Another control measure has been presented by Killick-Kendrick et al ., (1997), who found that deltamethrin impregnated collars protect dogs very efficiently from sandfly bites; biting activity was reduced by 96%.

Vector control:

One important goal is the destruction of breeding places. This involves the closure of cracks in walls and the removal of rubble. A common measure is the spraying of houses with insecticides (Alexander et al ., 1995; Tayeh et al ., 1997 a). Biological control measures seem to be effective as well (Robert et al ., 1997). In India kala azar had been almost eradicated after an anti-malaria campaign. Since the spraying of houses has been stopped, the disease has returned.

1.1.9. Leishmaniasis in the Middle East:

The Middle East is endemic for CL and VL. In most countries of the Middle East the three Old World species, L.major , L.tropica and L.d.infantum are prevalent. Even though urban L.tropica infections became less frequent the overall picture is that CL and VL are emerging diseases in the region. Several new outbreaks were reported during the last decade. Besides developmental changes, the political instability is an important factor: non-immune population groups as military troops, workers and also refugees enter endemic areas. More than 50 nations provide peacekeeping forces in the Middle East. In some of the home countries of these multinational forces, leishmaniasis has never been diagnosed before and therefore might be misdiagnosed. An endemic focus of L.major emerged in the northeastern Sinai among the multinational peace- keeping forces stationed in this area (Fryauff et al. 1993). Norton et al ., (1992) reported about 23 Fijian members of an international observer force who had acquired CL. Visceral infections caused by L.tropica have been reported from veterans of Operation Desert Storm (Magill et al., 1993).

In recent years L.tropica infections have been reported in rural areas in the Middle East and North Africa. Either new endemic foci or a higher incidence in already known endemic areas [page 24↓] were reported in Jordan (Saliba et al . 1993; 1997), Morocco (Dereure et al., 1991), Oman (Scrimgeour et al., 1999), an Afghan refugee camp in Pakistan (Rowland et al., 1999) and in Aleppo in Syria (Ashford et al ., 1993; Tayeh et al ., 1997 b).

L.major, L.tropica and L.donovani ssp. have been reported in Iraq, Iran and Saudi Arabia (Aljeboori and Evans, 1980; Hatam et. al ., 1997; Momeni and Aminjavaheri, 1994; Peters et. al ., 1985). VL has been reported in Jordan (Qubain et al ., 1997), in Tunisia as an emerging disease (Ben Salah et al ., 2000), and new foci were reported in Libya (Mehabresh, 1994). L.donovani infantum has been reported in Lebanon and Tunisia (Nuwayri-Salti et. al ., 1994; Gramiccia et al ., 1991) causing CL. In Iran, CL is endemic in many parts of the country. L.major and L.tropica are prevalent, partly in the same regions. In 1989, 15,757 cases of CL were reported, in some areas up to 70% of the population have scars from past infections, the incidence being 1.4/1,000 in the Isfahan province of Iran (Momeni and Aminjavaheri, 1994). The disease is common in the cities as well as in the rural areas. Leishmaniasis is endemic also in Turkey ( Uzun et al ., 1999).

Leishmaniasis in Israel and the West Bank:

CL has first been reported in the area of Jericho in the beginning of the 20th century (Huntemueller, 1914; Canaan, 1916). Settling and development of arid and semiarid areas has taken place over the last few decades, which gave rise to the emergence of new foci of CL (see map, Figure 4). According to reports from residents of Jericho, the disease was not seen in the years before the Six Days War in June 1967. This was due to spraying against malaria with DDT. A survey of Israeli soldiers who had been in the greater Jericho area during the war, revealed that the disease was still hyperendemic, with an incidence of 50% during an average exposure time of 1 month (Naggan et al ., 1970). The recent epidemiology of CL in the Jericho district has been profoundly studied by Jawabreh (2000). The epidemiology of leishmaniasis in Israel has been reviewed by Greenblatt et al. , (1985). Leishmania major accounts for the majority of infections with Leishmania in the country. It is endemic in the Jordan Valley, the Jericho area, along the Dead Sea, in the Arava and the Negev. CL caused by L.major has been thoroughly studied over many years, and the reservoir animal species ( P.obesus, M.crassus ) as well as the vector species ( Ph.papatasi ) have been identified (Schlein et al., 1982; 1984).

Leishmania tropica is endemic in a number of semiarid hilly areas in central and northern Israel as well as in the northern West Bank (Samaria), the Jenin district being a major endemic area (Abdeen et al ., 2000, in press). An outbreak has been reported in Salfit (Blum, 1978), an Israeli settlement north of Nablus. The species has never been finally confirmed, but the hilly environment was highly suggestive of a focus of L.tropica . Another L.tropica focus has been [page 25↓] reported by Klaus et al., (1994): since 1989, CL had been diagnosed in 19 residents of Kfar Adumim, a village between Jerusalem and the Dead Sea. In 6 patients the cultivation of promastigotes succeeded and L.tropica was identified. Oren et al. , (1991) reported about a 21 year old Israeli suffering from VL caused by a L.tropica variant who came from the area south of the Sea of Galilee. Hyraxes were found to be abundant in the environment of several L.tropica foci. They are highly suspected as being the reservoir. According to Sawalha (2001) more than 700 cases of CL have been reported from many single foci in the northern West Bank over the last 10 years. The causative species has not been identified, but the geographical area suggests L.tropica.

VL caused by L.d.infantum is an emerging disease in the northern West Bank. More than one hundred (127) human VL cases have been recorded over the past 10 years. Most cases were reported in the Jenin district, followed by Hebron, Tulkarem and Ramallah. Predominantly children between 1 and 6 years of age were affected (Abdeen et al ., 2000, in press, Greenblatt et al. , 1985, Qubain et al. , 1997). A serological survey in an Arab village in an endemic area of VL in western Galilee showed a prevalence of 10% (Ephros et al ., 1994). CVL has been diagnosed in dogs in central Israel. In a village close to Tiberias (Wadi Hamam), 4% of the dogs were seropositive and L.donovani sensu lato was identified from cultured parasites (Jaffe et al ., 1988). Wild canids are suspected of playing a major role in distribution of the disease over greater distances. More than 5% of the foxes and jackals have been found to be positive by serology. In two villages west of Jerusalem the prevalence was about 10% of the domestic dog population (Baneth et al ., 1998). Some dogs showed severe signs of CVL. In the northern West Bank, where human VL is endemic, the L.tropica foci are adjacent or even overlapping.

Leishmaniasis is by law a reportable disease in Israel, but nevertheless the disease seems to be greatly underreported. The number of cases reported to the Israeli Ministry of Health over the last few years did not exceed the number of patients seen in the Hadassah Hospital, which is only one of several hospitals where patients with CL are treated. The Bedouins of the Negev and the Judean Desert are familiar with the disease . Since the disease is well-known among the inhabitants in endemic areas, simple cutaneous lesions are often diagnosed by the patients themselves and are left for self cure. As many young Israelis tour Central and South America after military service, New World leishmaniasis is increasingly diagnosed among returners (Zlotogorski, 1998).

Figures 4 and 5 show the distribution of CL and VL over the last decade (1990-2000) in Israel and the West Bank.


[page 26↓]

Figure 4
[page 27↓]

Figure 5:


[page 28↓]

1.2. Review of the diagnostic methods for leishmaniasis:

1.2.1 Direct identification by classical routine methods:

Microscopy:

Classically the diagnosis of leishmaniasis relies on direct microscopical identification of amastigotes. Its sensitivity is limited to about 60% (Rodriguez et al ., 1994), which has been found by many others too. No species-specific diagnosis can be achieved. Smears from skin lesions, tissue biopsies or aspirates from bone marrow, spleen or lymph nodes are used. Microscopy is performed on Giemsa stained smears or sections of the named specimens. Typically round to oval amastigotes are seen inside of macrophages. Sometimes they are also found extracellular. The densely stained (violet) rod-shaped kinetoplast should be always identified. Biopsies have shown a greater sensitivity than smears for the diagnosis of CL (Rodriguez et al ., 1994). Andresen et al ., (1996) identified amastigotes in 76% of histological sections, but only in 55% of smears taken from CL lesions. To improve the microscopical sensitivity of smears it has been recommended that a superficial slit be made radially into the lesion and that some tissue be scraped off with a surgical blade (Herwaldt, 1999 a; Hepburn 2000). This collection method is much less invasive than taking biopsies. For the microscopical diagnosis of VL, splenic aspirates proved to have the highest detection rate (96.4%, 84 out of 88 patients), followed by bone marrow aspiration (70.2%) and lymph node aspiration (58.3%) as shown by Zijlstra et al ., (1992) and also reported by others. Bleeding complications are feared, but are low (0.6%), if applied exclusively to patients with coagulation parameters within the normal range. In peripheral blood the detection rate by microscopy is very low.

Culturing:

Cultures are obtained from aspirates or biopsies from the above-named sources. Cultures are usually grown in NNN (Nicolle-Novy-McNeal) and in Schneider's Drosophila medium. Culturing of promastigotes is required for a number of diagnostic methods such as isoenzyme analysis, and some of the serological and DNA-based methods. The sensitivity of culturing is variable and depends on various factors, as for example the viability of collected parasites, the strain, the media (different requirements for different species), the presence of a superinfection, and the expertise of the investigator. Different success rates have been reported, ranging between 4% in Nicaragua (6 of 143) (Belli et al ., 1999) and 95% (61 of 64) in Switzerland (Grimm et al ., 1996). Mostly a sensitivity of 40-50% was found (Rodriguez et al ., 1994). Culturing is expensive and time-consuming as cultures sometimes become positive only after two or more weeks (Gramiccia et al., 1991). It depends on adequate facilities, which are often not available in less-developed countries. Furthermore, cultures are susceptible to contamination. In cases of double [page 29↓] infections one strain usually outgrows the other, so that misdiagnosis can occur (Klaus et al ., 1994).

1.2.2. Indirect diagnosis:

1.2.2.1. Cellular immunity:

Leishmanin skin test (LST) or Montenegro skin test:

The LST is widely used for the screening of the exposure to Leishmania parasites within endemic areas. Analogous to the tuberculin test, leishmanial antigen (killed promastigotes) is applied intradermally and a delayed hypersensitivity reaction is measured after 48-72 h. A reaction is seen in people with previous contact to the antigen who have developed cellular immunity. Conversion occurs after several weeks in CL, and in VL usually only after treatment and cure (Peters and Killick-Kendrick, 1987). Present and past infections can not be differentiated.

Lymphocyte proliferation assay (LPA):

Alternatively a lymphocyte proliferation test can be used to measure the cellular response (Alvarado et al. 1989). In contrast to the LST one patient contact is sufficient, which may facilitate larger field studies. On the other hand the LPA is more labour intensive.

1.2.2.2. Serological methods:

Serology is suitable for mass screening within epidemiological surveys on VL or CVL. It is limited in its use for the diagnosis of leishmaniasis since present, subclinical and past infections generally not be discriminated. It is not reliable in CL since antibodies are often not detectable (Herwaldt, 1999). Cross reactions often complicate the interpretation (Wilson, 1995). Another disadvantage of serology is the fact that it is generally not reliable in immuno-compromised patients (Pizzuto et al ., 2001; Agostoni et al ., 1998).

Enzyme linked immunosorbent assay (ELISA):

The ELISA technique has been adapted especially to the diagnosis of VL by Hommel et al ., (1978). Monoclonal Antibodies have been developed and are widely used for the ELISA, Western Blot and immunofluorescence assays (Jaffe and McMahon-Pratt, 1983; 1987; Jaffe et al., 1984; Jaffe and Sarfstein, 1987; Baneth et al ., 1998). A highly specific and sensitive ELISA was developed by Jaffe and McMahon-Pratt (1987) for the diagnosis of VL. The assay was based on specific antibodies competing with serum antibodies. This competitive assay proved to be superior to direct binding tests, since no cross reactivity was reported, but it is not generally [page 30↓] used. In a comparative study on different serological methods conducted on 49 patient sera from India, the ELISA using a recombinant antigen proved to be more sensitive (100%) than the regular ELISA (77%), and was more sensitive than the DAT (90%) (Raj et al ., 1999).

Immunofluorescence assay (IFA):

Immunofluorescence assays can be employed to detect either antibodies in patient sera or to identify the antigen. The IFA has been adapted to detection of Leishmania antibodies in patient sera by Walton et al ., (1972). Rachamim et al ., (1991) found in a comparative study in dog sera that the ELISA yields the same qualitative diagnosis as the IFA. It was concluded that the ELISA can replace the IFA due to its better practicability and its advantage in examining many more sera simultaneously. Mancianti et al ., (1995) found a slightly higher sensitivity with the ELISA (99.5%) than with the IFA (98.4%), but the IFA was more specific (100%) than the ELISA (97.1%). The study was conducted with 290 dogs (186 Leishmania infected and 104 control animals).

Direct agglutination test (DAT):

The DAT is a comparatively simple and reliable screening test suitable for field work. It is based on the agglutination of positive sera with stained promastigotes and can be performed in microtiter plates (Allain and Kagan, 1975; Harith et al., 1986; Zijlstra et al ., 1991; 1992). In a study on treated VL patients the DAT was reported to be to 100% sensitive and to 99.3% specific. Oskam et al ., (1999) used a freeze-dried antigen which is more stable and therefore improves the usefulnes of the DAT for field studies.

Western Blot:

The Western Blot relies on the recognition of Leishmania antigens by anti-leishmanial antibodies. Antigenic proteins of the parasites are separated by electrophoresis and are incubated with patient sera. In the Western Blot several different antigenic products can be recognized by different antibodies simultaneously in one assay. It was found that the sera of PKDL patients consistently recognize a specific antigen which is not recognized in VL patients without PKDL. This could be utilized for the identification of PKDL patients (Salotra et al ., 1999).

A recent improvement in serological diagnosis is the development of the rK39 dipstick test. One antigen (rK39) with high antigenicity has been selected and is recognized by the serum of kala azar patients. It is successfully employed in the diagnosis of VL in Nepal. It was found to be 100% sensitive and 100% specific when tested in 14 newly diagnosed VL patients and 113 [page 31↓] controls (Bern et al ., 2000). It is comparatively inexpensive and results are obtained within minutes.

1.2.3. Identification of Leishmania species and subspecies:

Isoenzyme analysis:

The analysis of isoenzymes is a generally accepted reference method for the species-specific diagnosis of Leishmania strains. The electrophoretic mobility of soluble enzymes is examined on starch or cellulose acetate gels. Patterns of single or multiple bands are seen. A consistent profile of isoenzyme patterns is found in closely related strains, which is also referred to as zymodemes. Between 10-20 enzymes have to be examined in order to classify new strains into their zymodemes (Miles et al ., 1980; Aljeboori and Evans, 1980; Peters et al ., 1985; Oren et al. , 1991; Barral et al ., 1991; Kreutzer et al ., 1993). Gramiccia et al ., (1992) found that viscerotropic and dermatotropic L.d.infantum strains differed in their zymodemes. Isoenzyme analysis is work- intensive, time-consuming and not feasible for a regular laboratory. It is usually performed in specialized laboratories which are also internationally acknowledged to label new strains with codes according to their zymodemes (eg. LON- for London School of Hygiene and Tropical Medicine, or MON- for University of Montpellier).

Excreted Factor (EF):

This method allows species specific diagnosis of Leishmania strains (some species can be distinguished, some not). The EF refers to soluble antigenic substances (glycoconjugate compounds) which are present in the medium of growing promastigotes. Culture media is used in a gel diffusion test in which the EF compounds are precipitated by antileishmanial antibodies of the same species (Schnur et al ., 1972).

1.2.4. DNA based methods:

1.2.4.1. Classical methods: hybridization and restriction:

Efforts have been made to develop hybridization methods. Before the introduction of PCR, Southern Blotting, using radiolabelled probes for hybridization, was one of the most sensitive detection methods. Smith et al., (1989) were able to routinely detect 10,000 promastigotes. Washed promastigotes were blotted on nitrocellulose filters and hybridized with radiolabelled, cloned fragments of minicircles (das Gupta et al ., 1991). Gramiccia et al ., (1992) have employed a kinetoplast probe specific for L.d. infantum . Laskay et al. , (1991) introduced a dot blot hybridization method using squashed sandflies on nylon membranes and L.aethiopica DNA as a probe. The hybridization detected every infected sandfly and reached the same level of [page 32↓] sensitivity as microscopy on infected sandflies. A weak signal was obtained from a minimum of 100 promastigotes. Lambson et al. , (2000) developed a probe based on a cloned fragment of a minicircle which hybridized with DNA from the L.donovani complex only. Dot blots of promastigotes with this radiolabelled kDNA probe confirmed that the detection limit ranges between 10 3 –10 4 parasites, as others had shown previously.

Also the digestion of purified kDNA with analysis of the restricted fragments (RFLP-analysis) was used for diagnosis (Jackson et al., 1984). The sensitivity of these methods is not sufficient for direct diagnosis in clinical material.

1.2.4.2. Modern methods based on the polymerase chain reaction (PCR):

The polymerase chain reaction (PCR) provides an excellent tool for the diagnosis and characterization of various infectious agents. It proved to be very useful for the diagnosis of leishmaniasis too. The method is based on the enzymatic amplification of selected DNA sequences, which are made visible by gel electrophoresis. PCR has a high potential for sensitivity, up to the detection of single parasites (Rodgers et al ., 1990; Lopez et al ., 1993; Noyes et al ., 1998; Harris et al ., 1998). In many studies PCR was found to be more sensitive than microscopy (Osman et al ., 1997 a), and it proved to be more sensitive (100%) when compared to serology (63%) (Ashford et al ., 1995). So far, several different PCR approaches have been developed for the diagnosis of leishmaniasis.

Depending on the objective, different targets for amplification are chosen: for diagnosis multi-copy sequence repeats are usually selected, implying the potential for high sensitivity. For this purpose the sequences need to be highly conserved either within the genus or within the species, depending on the specific aim. For genetic characterization of individual strains, more variable regions are selected, which usually do not appear in high copy numbers and are therefore less sensitive targets. Culturing of promastigotes is usually required for genetic characterization. Sensitivity and specificity can be enhanced by the hybridization of PCR products (southern blotting). The specificity can be improved by restriction fragment analysis of the PCR-amplificates.

Targets for diagnostic PCR: Kinetoplast DNA (kDNA)

The targets with the highest copy number (10,000) are the kDNA minicircles. The minicircles have a size of about 800 bp with specific differences in sizes among some of the Leishmania species. They divide into subclasses of different sequences, but all of them share the same conserved sequence of 120 bp. This fact has been utilised for a genus-specific diagnostic PCR approach introduced by Rodger s et al ., (1990) and later employed by others (Rodriguez et al., [page 33↓] 1994; Ashfor d et al., 1995; Laskay et al., 1995 ; Reale et al., 1999). The major part of the minicircles consists of variable sequences. The different subclasses show variations, which to some extent are shared among species and to another are even different among clones of the same strain . This can be explained by a fast rate of sequence evolution of the minicircles. In contrast, some minicircle classes are well preserved within the species, even from geographically distant origins. Gutiérrez-Solar et al., (1995) showed that minicircle sequences from a well preserved minicircle class can be almost identical, even if the strains are isolated in China or in Europe. These facts make the design of species-specific oligonucleotides and probes problematic. Nevertheless, several approaches were undertaken, which have proved to be useful for species-specific and comparatively sensitive diagnosis. One pair of oligonucleotides was introduced by Smyth et al ., (1992) for the diagnosis of kala-azar patients, which amplified whole minicircles of different species. Bhattacharyya et al ., (1996) developed genus specific kDNA primers able to amplify whole minicircles. Eresh et al., (1993) introduced primers with the purpose of amplifying the species from the Old World and specifically to differentiate between L.major and L.tropica . These oligonucleotides were applied to purified DNA, but the sensitivity has never been studied (Klaus et al ., 1994).

2. Nuclear targets:

Two genomic targets have been preferably selected: The small subunit ribosomal RNA (ssu rRNA) and the spliced leader sequence or mini-exon gene. Both targets are present in about 200 copies. Due to the fact that these gene repeats consist of both conserved and variable regions, which are often species-specific, these targets are suitable for the development of more specific diagnostic approaches. The ssu rRNA gene has been successfully used as a target (van Eys et al ., 1992; Campino et al ., 2000). The internal transcribed spacer (ITS) within the ribosomal operons (ssurRNA) is another target which has been selected by Guevara et al ., (1992), Cupolillo et al ., (1995) Schönian et al ., (2000) and El Tai et al ., (2000). The latter showed that the PCR was highly sensitive using clinical material. Combined with restriction fragment analysis of the amplified ITS region all species complexes could be identified. Also the mini-exon gene was successfully used as a target (Fernandes et al ., 1994, Ramos et al ., 1996; Harris et al , 1998). Harris et al , (1998) developed a multiplex PCR which was able to discriminate between the New World complexes of Leishmania and which was also very sensitive.

PCR and hybridization:

In order to enhance the sensitivity and also to confirm the specificity, PCR has been combined with hybridization. In recent years non-radioactive detection methods have been introduced. [page 34↓] Lopez et al., (1993) were able to increase the sensitivity of a kDNA based PCR 10 fold by hybridizing the PCR product to membrane-bound oligonucleotides. The PCR product was biotinylated and colorimetrically detected by a streptavidin-alkaline phosphatase (SAP). Rodriguez et al., (1994) developed kDNA probes able to distinguish between L.mexicana and L.braziliensis infections. The combination of PCR with primers 13A/13B and subsequent hybridization proved to be highly sensitive as well as highly specific. 233 biopsies of skin lesions suggestive of CL were examined. PCR products were seen in 226 (97%) of the patients. After hybridizing the 120 bp product, 117 samples hybridized to a L.mexicana specific probe and 109 hybridized to a L.braziliensis specific probe. The results reflected the epidemiological situation in Venezuela very well, when the origin of the patients was compared to the results. Furthermore, the results were confirmed by restriction patterns found after digestion with Msp I on purified kDNA from 60 positive cultures. Nuzum et al ., (1995) evaluated the usefulness of PCR on peripheral blood for the diagnosis of kala-azar. Peripheral blood mononuclear cells (PBMC) were separated and lysed. The PCR product was biotinylated and bound to a specific oligonuctleotide probe in a microtiter plate, followed by an enzymatic reaction, which could be detected by an ELISA reader . Laskay et al. , (1995) chose a similar approach, combining PCR with primers 13A/13B and Southern Blotting using L.aethiopica kDNA as a probe to confirm the specifity. The hybridization enhanced the sensitivity 10 fold so that DNA corresponding to 0.01 promastigote was detectable. Also digoxigenin (DIG) labelled probes were used and hybridization was visualized by a chemiluminescent signal (Rodriguez et al., 1999; Reale et al ., 1999). The latter found the genus specific kDNA-PCR (13A/13B), to be sensitive up to 0.1 fg of purified DNA (corresponding to 0.01 parasite), when combined with hybridization.

1.2.4.3. Molecular strain typing of Leishmania:

For genetic characterization as well as for species-specific diagnosis several other PCR-based methods have been developed:

Randomly amplified polymorphic DNA analysis (RAPD) or arbitrarily primed polymerase chain reaction (AP-PCR):

For RAPD/AP-PCR non-specific oligonucleotides are employed, which yield highly polymorphic products (Bhattacharyya et al .,1993; Schönian et al ., 1996). Characteristic patterns for PCR products of different sizes are obtained. Mimori et al. , (1998) used single products obtained by the AP-PCR to develop species-specific primers for five Leishmania species of the New World. Eisenberger and Jaffe, (1999) employed a Leishmania- specific oligonucleotide in combination with a non-specific permissive oligonucleotide. Banding patterns were [page 35↓] characteristic for each species complex, occasionally showing also regional differences within the species. RAPD/AP-PCR-methods are generally not suitable for direct diagnosis: the sensitivity is not sufficient and the conditions need to be highly standardized. The patterns show alterations if the PCR conditions or the amount of template is changed. Due to the characteristics mentioned these methods are limited to the use of purified DNA (Noyes et al. , 1996; Eisenberger and Jaffe, 1999).

Restriction fragment length polymorphism (RFLP) or Schizodeme analysis:

Amplified minicircle DNA is restricted enzymatically into fragments, which are visualized by gel electrophoresis. The fragment patterns or schizodemes are specific, either for species or for individual strains. The polymorphism of patterns indicates differences in sequences. Schizodeme analysis is a valuable tool for epidemiology and for species-specific diagnosis, usually on the basis of purified kDNA (Noyes et al ., 1998; Qubain et al ., 1997). For L.d.infantum it was shown that the known zymodeme groups around the Mediterranean could be subclassified by schizodeme analysis into various other groups (Noyes et al. , 1998). Also this method is sensitive to minor changes in the conditions. Apart from that, point mutations may change the pattern due to changed restriction sites. This is desirable for genetic characterization, but for direct diagnosis from clinical material the method is neither sensitive nor consistent enough.

1.3. Objectives of the study:

At the onset of this work, microscopy, serology (ELISA, IFA, EF) and PCR-based on cultured parasites (Eisenberger and Jaffe, 1999) were the standard diagnostic methods for leishmaniasis at the Kuvin Center, which is the most advanced and specialized center for the study of leishmaniasis in Israel and the West Bank. The methods followed in this institute therefore reflect the diagnostic standard of the whole country. No sensitive direct (without culturing and not serological) diagnostic method had been established so far. For the diagnosis of patients as well as for epidemiological studies on reservoir animal species there was need for a field applicable sensitive and specific PCR as a routine method.

The prevalence of three Leishmania species in Israel and the West Bank, with overlapping clinical pictures and endemic areas, increasing numbers of patients and unidentified reservoir animal and vector species in some areas made the development of improved laboratory tests an urgent priority. In recent years new L.tropica foci are emerging, and L.d.infantum has been increasingly identified among canids in central Israel (see maps, Figures 4 and 5). Also the fact that some reservoir animals, eg. dogs, may harbour different species of Leishmania emphasizes the need of a directly applicable diagnosis method at the species level (Baneth et al., 1998; [page 36↓] Dereure et al. 1991). Furthermore, New World CL is a serious problem in the country, as many Israelis travel to Central and South America (Dan et al ., 1985). There are a number of cases seen annually (~10 in the Tel Hashomer Hospital). These patients need to be diagnosed at least at the complex level, since only infections due to the L.braziliensis complex require a three-weeks course of antimonal treatment. Specifically L.braziliensis infections need to be distinguished from L.mexicana infections. Species of both complexes are prevalent in large parts of Central and South America and have an overlapping geographical distribution and clinical presentation (Eresh et al., 1994). Differential diagnosis within the New World species of Leishmania has not been performed in the country to date. Old World infections need to be excluded, since the infections might as well have been contracted in Israel either before or after the journey. Due to immigration from Ethiopia, infections caused by L.aethiopica also need to be considered, as a differential diagnosis of other skin diseases. Presumably in no other country is such a variety of causative agents of leishmaniasis seen on a regular basis as in Israel.

Over the last decade many efforts have been made in many parts of the world to improve PCR diagnosis of leishmaniasis. It was emphasized in a recent article on leishmaniasis in the Lancet that field applicable, diagnostic methods including species identification are desparately needed (Herwaldt, 1999). Several sensitive PCR methods have been developed by various research groups, using extracted DNA directly from clinical material. It has been shown that clinical material, eg. dermal lesion scrapings (CL) or peripheral blood (VL) could be collected and preserved on filter paper (Osman et al ., 1997 a; Harris et al ., 1998; Färnert et al ., 1999; Campino et al ., 2000). PCR appeared to be the method of choice, for achieving sensitive and species-specific diagnosis of leishmaniasis in Israel and in the West Bank.

Specific aims:

  1. To establish direct PCR diagnosis of leishmaniasis in Israel and the West Bank. This included: testing for the most effective DNA extraction method, developing a sampling strategy (which samples to be collected), optimization of the PCR approach, achievement of the highest possible sensitivity and adaption for routine use.
  2. To differentiate between the endemic species L.major , L.tropica and L.d.infantum by direct PCR diagnosis.
  3. To establish direct PCR diagnosis of New World leishmaniasis, especially of the L.braziliensis complex.
  4. To establish PCR methods for the screening of reservoir animals and sandfly vectors.


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