INTRODUCTION

1 INTRODUCTION

Parasites are living organisms that use other living creatures like our body for food and a

place to live. We can get them from contaminated food or water, a bug bite, or sexual

contact. Parasitic diseases can cause mild discomfort or be deadly. Parasites range in size

from tiny, one-celled organisms called protozoa to worms tl13t can be seen with the naked

eye. Parasitic diseases were always a heavy burden for humanity. Protozoan parasites are

responsible for several important diseases that threaten the lives of nearly one quarter of

the human population world-wide. Leishmaniasis is one of the six major parasitic

diseases identified by WHO for extensive research for its control and eradication

(http://www.who.int/leishmaniasis/en). With more than 70,000 deaths per year from

visceral leishmaniasis, it is second only to malaria for de[!ths associated with parasitic

infection. Leishmaniasis is widely recognized as one of the major neglected diseases

associated with poverty (Alvar et aI., 2006). The disease is spreading in several areas, as

a consequence of massive rural urban migration and its association with AIDS.

Leishmania I HIV association is indeed considered by the WHO as a real threat especially

in Spain and Mediterranean countries in south Western Europe (www.who.int/inf­

fs/en/fact.html.factsheet-116, 2000).

1.1 Historical background

The disease has a long history; description of conspicuous lesions has been discovered on

tablets from King Ashurbanipal from the 7th century BC, some of which may have been

derived from even earlier texts from 1500 to 2500 BC. Arab physicians including

Avicenna in the 10th century gave detailed description of what was called Balkh sore

(Cox, 2002). As for the new world, evidence of cutaneous form of the disease was found

in Ecuador and Peru in pre-Inca potteries depicting skin lesions and deformed faces

dating back to the first century CE (http://www.who.intitdr/diseases/leish).InI901.

Leishman identified certain organisms in smears taken from the spleen of a patient who

had died from "dum-dum fever" (Dhum dhum is an area c:ose to Calcutta) and in 1903

Captain Charles Donovan (1863-1951) described theni as being new organism

INTRODUCTION

(http://www.who.intltdr/diseases/leish). Eventually Ronald Ross established the link with

the disease and named the organism Leishmania donovani.

1.2 The Leishmania Parasite: Classification

Evolutionarily Leishmania are closely related to the trypanosomes and have probably

evolved from the lower insect trypanosomatids (McGhee & Cosgrove, 1980). The genus

Leishmania can be classified as:

Phylum

Sub. Phylum

Class

Order

Sub-order

Family

1.3 Geographical Distribution

Sarcomastigophora

Mastigophora

Zoomastigophora

Kinetoplastida

Trypanosomatina

T rypanosomatidae

The geographical distribution of leishmaniasis is restricted to tropical and temperate

regions, the living area of the sandfly. The leishmaniasis is considered to be endemic in

88 countries (16 developed countries and 72 developing countries) on four continents

(Figure. I. 1 ). Ninety per cent of visceral leishmaniasis cases are found in Bangladesh,

Brazil, India, Nepal and Sudan. The disease is completely absent from western

hemisphere except the eastern parts of Brazil.

Figure 1.1 World wide geographical distributions of Visceral and Cutaneous I Mucocutaneous leishmaniasis (http://www.who.int/ctdihtrnllleis.html).

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INTRODUCTION

To date, the greatest prevalence of LeishmaniafHIV co-infection has been in the

Mediterranean basin. Of more than 2,000 cases notified to the WHO, 90 per cent of them

come from Spain, Italy, France and Portugal (Figure 1.2).

Figure 1.2 Countries with endemic leishmaniasis and with Leishmania-HlY co-infection . • global distribution of leishmaniasis; • countries that have reported co-infection (Desjeux & Alvar, 2003).

1.4 Leishmaniasis in India

In the Indian sub-continent, the most common endemic form of the dieses is visceral

leishmaniasis (VL or Kala-azar) and this is mainly found in the eastern parts of the

country viz. Bihar, West Bengal, eastern districts of Uttar Pradesh, Assam, foothills of

Sikkim and to a lesser extent in Tamil Nadu and Orissa (Marin kelle, 1980).

Uttar Pradesh

MAP OF utAR SHOWIIO ENoa.cm OF KAlA-AZAIt H.rttW;-.; 0 _~ D LOW BIIIEIE

NEPAL

West Bengal

Figure 1.3 Map of Bihar State, India, showing distribution of resistance to pentavalent antimonials in kala-azar-endemic areas (Simon et ai., 2006).

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INTRODUCTION

The Bihar state has witnessed two major epidemic outbreak of Kala-azar in the year IlJ78

and 1992. Presently 12 districts, out of total 38 in Bihar are badly affected with it (Figure

1.3). In 2005 the health ministers of three Member States of WHO's South-East Asia

Region, India, Nepal and Bangladesh, had signed a Memorandum of Understanding

pledging to collaborate to eliminate VL from their countries.

1.5 Clinical manifestations and Pathology of the disease

The clinical spectrum of Leishmaniasis encompasses subclinical (inapparent), localised

(skin lesions), and disseminated infection (cutaneous, mucosal, or visceral) (Table 1.1)

(Chang and Fong, 1983).

Table 1.1 Summary of clinical manifestations and geographical distribution of Leishmania -~

I Species Clinical Manifestation Ceographical Distribution

, L. ama::onensis Visceral,cutaneous,mucocutaneous, New World: South and Central America L. bra::iliensis Cutaneous. mucocutaneous New World: Throughout South America L. chagasi Visceral New World: South and Central America L. donovani Visceral (kala-azar) Old World: China, India, Bangladesh L. inian/llm Visceral. cutaneous Old World: North Central Asia, Northwc~t

I China, Uzbekistan, Middle East

IL. major Cutaneous Old World: Africa, Middle East,Nol1hern A~ia

IL. mexicana Cutaneous, mucocutaneous New World: Southern Mexico, Belize, nOl1hern Guatemala, southern Texas I

1.5.1 Cutaneous Leishmaniasis (CL): Cutaneous leishmaniasis (CL) is the 1110st

common form and also known as oriental sore, an infection characterized by ulcerative

skin lesion in the face, on arms or legs (Figure l.4a). CL is principally caused by L.

major, L. lropica and L. l71exicana. Although the lesions ',viII persist and disseminate.

cutaneous leishmaniasis is generally a self-healing disease. However, it's most severe

form, recidivisms Leishmaniasis, is very difficult to treat, long lasting, destructive and

disfiguring.

1.5.2 Diffuse cutaneous Leishmaniasis (DCL): Diffuse cutaneous leishmaniasis

(DCL) is a mutilating disease caused by L. mexicana. The infection disseminates with

chronic skin lesions resembling those of leprotamous leprosy, :ll1d is difficult to treat.

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INTRODUCTION

Figure 1.4 (a) Cutaneous Leishmaniasis (CL) (www3.baylor.edu); (b) Mucocutaneous leishmaniasis (MCL) or espundia. Active lesions in nasal mucosal tissue with septal perforation and disfigurement of the nose and swelling of upper lip. (Calvopina et ai, 2006).

1.5.3 Mucocutaneous Leishmaniasis (MeL): Also called 'espundia' In South

America. It is mostly related to Leishmania species of the New World such as L.

braziliensis, L. panamensis and L. guyanensis, but mucosal lesions have been reported in

the Old World due to L. donovani, L. major and L. in/anlum in immunosupresed patients

(Desjeux, 1996). The parasite invades the mucocutaneous region of the body and spread

to the oronasal/pharyngeal mucosa. The soft tissues and cartilage of the

oronasal/pharyngeal cavity undergo progressive erosion (Figure l.4b). In contrast to

cutaneous Leishmaniasis, these lesions do not heal spontaneously. Suffering and

mutilation are severe and death occurs as a result of bronchopneumonia or malnutrition .

1.5.4 Visceral Leishmaniasis (VL) : It is also known as 'kala-azar'. It is caused by L.

donovani. Initially, the disease is characterized by high fever, headache, chill, malaise,

dizziness, anorexia, and vomiting and weight loss (Desjeux, 1996; WHO, 1996 & 1998).

In chronic stage the disease IS followed by hepatomegaly, splenomegaly,

Iymphoadenopathy, occasional acute abdominal pain, emaciation, anemia, leucopenia,

and blackness of skin (Figure 1.5a&b) hence the name given Kala-azar or Black fever. It

is the most severe form of Leishmaniasis and is usually fatal if left untreated. The

incubation period can be months or years and, unlike the cutaneous forms of

Leishmaniasis, it involves the internal organs.

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Figure 1.5 (a) A young Sudanese boy with visceral leishmaniasis (b) splenomegaly (enlargement of spleen) and hepatomegaly (enlargement of liver) in visceral leishmaniasis (A CRUMP, TOR, HO/SCIENCE PHOTO LIBRARY).

1.5.5 Post kala-azar dermal Leishmaniasis (PKDL): Post kala-azar dermal

Leishmaniasis is a sequel to the infection with L. donovani. It is a type of non-ulcerative

cutaneous lesion, developed in about 10% of kala azar patients generally one or two years

after completion of antimonial treatment (Zij Istra et aI., 1991).

1.6 Leishmania life cycle and morphology

Leishmania parasite leads digenetic life cycle between invertebrate sand fly and

vertebrate host and differentiate into two morphologically distinct stages, insect-stage

flagellated promastigotes and mammalian-stage intracellular amastigotes.

1.6.1 Promastigote or Leptomonad stage: They are long, slender cells and measure

approximately 15-20 ~m by \.5-3.5 ~m with a ] 5-28 ~ anterior flagellum which

functions in locomotion and attachment to the insect gut wall (Figure 1.6a) (Herwaldt,

1999; Bogitsh & Cheng, 1990). The surface membrane has binding sites molecules such

as glycoproteins and mannose receptors that are important for uptake of promastigotes by

macrophages.

1.6.2 Amastigotes or Leishman stage: Amastigotes are smaller than promastigotes

and appear non-flagellated. They are ovoid in shape and measure approximately 2-4 J..lm

in diameter. This stage is found in parasitophorus vacuole of macrophages. The outer

membrane has polysaccharide component but there is no surface coat (Figure 1.6b)

(Herwaldt, 1999).

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INTRODUCTION

Figure 1.6 (a) Morphological features of L. donovani promastigotes and (b) axenically-cultured amastigotes (Habtemariam, 2003).

1.6.3 Establishment of infection: Sandflies of the genera Phlebotomus in the case of

Old World Leishmaniaspecies, or Lutzomyia for New World Leishmania species are

infected with the Leishmania parasite mainly from natural reservoir hosts such as canines

and rodents (Figure 1.7).

Sandfly Stag-. HumanStagn

Figure 1.7 Life Cycle and Morphological Stages of Leishmania

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INTRODUCTION

After ingestion by the fly, procyclic promastigotes reproduce in the lumen of the

digestive tract by longitudinal binary fission. Approximately four days after ingestion, the

promastigotes undergo a developmental transition into metacyclic promastigotes through

a process called metacyclogenesis. Metacyclic promastigotes are longer and narrower,

have a longer flagellum, and display changes in surface coat proteins which together

result in a form of the parasite that is adapted to infect macrophage cells (Spath &

Beverley, 2001). Metacyclic promastigotes migrate anteriorly to the esophagus and

pharynx of the fly, and when the sandfly next feeds, some are dislodged and deposited on

the skin of the host through the sandfly's proboscis. The cells are then phagocytosed by

macrophages. The parasite begins a transformation inside the endocytotic

parasitophorous vacuole and change into amastigote form. These cells divide by binary

fission and accumulate in the macrophage until it ruptures and releases its amastigotes

which are then free to infect other macrophages. This cycle continues and results in one

of the clinical symptoms of leishmaniasis with different speci.:::s having different tropisms

for macrophages in particular organs in the host (Bogitsh & Cheng, 1990).

1.7 Diagnosis of the disease

The clinical and epidemiological findings in various forms of leishmaniases are non

pathognomonic and these can mimick several other conditions. Hence a laboratory

diagnosis is required to confirm the clinical suspicion. The diagnostic tools used for each

leishmanial syndrome viz. visceral, cutaneous, and mucocutaneous form vary but the

gold standard in each case remains to be the demonstration and isolation of the parasite

from appropriate tissues. VL is usually diagnosed by demonstrating the parasite in aspirates

from the spleen, bone marrow or lymph nodes, but this method is unsuitable in field settings.

Quick differential diagnosis of leishmaniasis (Sundar and Rai, 2002) can be achieved with

the direct agglutination test (DA T), which is quantitative, and uses a freeze-dried antigen

(Oskam et at., 1998), the urine antigen-detection test (dipstick K39) that is particularly

useful in immunocompromised patients and to evaluate treatment efficacy (Attar et aI.,

2001; Sundar and Rai, 2002). For CL, parasitological diagnosis is based on skin smears. For

MCL cases, diagnosis relies on serological tests. This is however, not helpful in most cases

because the antibody levels are too low, whereas manifestations of cell-mediated immunity

e.g. Skin-test reactivity, usually develop during an active infection (Herwaldt, 1999).

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INTRODUCTION

Table 1.2 Epidemiological traits of leishmaniasis: distribution, reservoirs and clinical syndromes Species Geographic distribution Reservoir Clinical

syndrome L. donovani Africa, India, Bangladesh, East Asia, Humans, rodents VL,PKDL (Old world) China L. infantum S. Europe, Mediterranean, Eastern China, Dogs, foxes, ackals VL,CL (Old world) Central Asia L. major Middle East, Indian, Northwestern China, Humans, rodents CL (Old world) Africa L. tropica Middle East, Indian, Western Asia Humans, rodents CL (Old world) L. aethiopica East Africa Hyraxes DCL,MCL (Old world) L. chagasi Central and South America, Dogs, foxes VL,CL,PKDL (New world) L. amazonensis South America Forest rodents CL, VL,PKDL (New world) MCL,DCL L. braziliensis Central and South America Forest rodents CL,MCL (New world) L. mexicana Central America Forest rodents CL, DCL (New world)

1.8 Prevention Leishmaniasis

Control measures target the interruption of the transmission cycle. 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.

1.8.1 Vector control: One important goal is the destruction of breeding places of

sandtly. 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 aI., 1995;

Tayeh et al ., 1997). Biological control measures seem to be effective as well (Robert et

aI., 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.

1.8.2 Control of the reservoir: One effective measure for the control of zoonotic. 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. 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 aI., 1998). Vaccination of dogs

might be an alternative approach for the future (Tesh, 1995).

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INTRODUCTION

1.8.3 Vaccine against Leishmaniasis : There is no vaccine against Leishmania in

routine use anywhere in the world, however many vaccine candidates are currently being

tested. These include the use of killed or attenuated parasites, protein or synthetic

peptides, and even naked DNA plasm ids - all of which have shown the limited protection

from infection in animal models (Handman et aI., 2001). Some of the genes or gene

products that have conferred protection in vaccine testing display developmental gene

regulation (Almeida et ai., 2002; Saravia, 2005; Walker, 1998). Several vaccine

preparations are in more or less advance stage of testing which are summarized in the

table 1.3.

Table 1.3 Summary of vaccination studies in humans and experimental models

I Antigen I Mode ~~~~;~~ization I Protection IIHost I~==============~~============~

ILive promastigotes I Prophylactic (Russia, Dependent on virulence IHumans

1~·==================~·~Is=ra=e=I)============~:~==============~~. ========~

IKilled promastigotes I Prophylactic Ivariable IIHumans

1~·==================~·~(M==id=d=le=E=a=s=t,=B=r=az=i=I)====~~. ==============~.~. ========~I IKilled promastigotes with BCG IITherapeutic (Brazil) IIHigh cure rate IIHumans I~==============~~============~ ~======~I Killed promastigotes with BCG IProPhYlactic (Iran) I N? prot~ction, transient IHumans

. . stImulatIOn . ~==============~:~============~ ~======~I Killed promastigotes with IL-12lproPhYlactic IIGOOd 11~~i;ates, mice,

I~================~~==============~ IIrradiated promastigotes IIProphylactic IIGood IIMice I I~==============~~============~ ILive attenuated promastigotes IIProphylactic IIGood IIMice I

~============~~============~~======~I Recombinant or native gp63 and IProPhYlactic IIGOOd I Mice, primates synthetic peptides. .. .

~============~~============~~======~I Recombinant or native IProPhYlactic I Excellent but .dependent Dice gp46/M2/PSA-2 on conformatIon and

adjuvant ~============~~======~==~

IRecombinant LACK IIProphylactic IIGood, enhanced by IL-1211Mice i====~

IA2, P4, and P8 IIProphylactic IIGood IIMice

1~IF=la=g=el=la=r=a=nt=ig=e=n=L=C=R=l======I:I~p=ro=P=h=y=la=ct=ic========~IIGood 1~IM=ic=e======~ II~~~~ DNA gp63, PSA-2, and Prophylactic or therapeutic IGOOd IIMice

1.9 Treatment of Leishmaniasis

The only effective way to control Leishmania infections currently is chemotherapy. The

treatment of choice for all types of leishmaniasis is based on pentavalent antimonials

despite of its cardiac and renal toxicity and difficulty of administration (Bryceson et ai,

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INTRODUCTION

1985; WHO, 1990). Pentavalent antimonials are being used for treatment for more than

50 years. Currently recommended dose of Sb V is 20 mg/kg/day (MKD) for 30 days

(Herwaldt and Berman, 1992). The mode of action of SAG is shown to be the inhibition

of glycolytic pathway and fatty acid oxidation pathway in the parasite (Berman et al.,

1987). However, in recent years, a large scale increase in clinical resistance to

pentavalent antimonials has been reported (Lira et al., 1999). In India as many as 65% of

the previously untreated patients failed to respond promptly or relapse after therapy with

antimony drugs due to development of drug resistance (Sundar et a!., 2000). Further, HIV

co-infected individuals often relapse after traditional antimonial treatment. Pentamidine

(Second line drug) was the drug of choice for some time but high toxicity and resistance

to parasite had stopped its use. Amphotericin B is now becoming the treatment of choice

for visceral leishmaniasis, especially in cases refractory to Sb v and/or pentamidine but

severe side effects and high cost limit their use (Mishra et a!., 1992; Bryceson, 1996;

Herwaldt, 1999). Ongoing research on drugs for treating leishmaniasis includes

Miltefosine, the first oral agent, currently undergoing trials. Miltefosine, registered for

use in India in 2002, is an alkylphosphocholine for treatment of VL. Phase IV clinical

trials that are aimed at evaluating the level of compliance were recently concluded.

Owing to its teratogenic potential, the drug cannot be administered to females of

childbearing age unless contraception is taken. With this exception, trials in India have

confirmed the safety and efficacy of the drug (Eibl, 2000). Paramomycin, an

aminoglycoside, is also currently undergoing Phase III clinical trials in India, so far it has

shown great promise and it has the potential for multidrug therapy (Thakur, 2000). Other

types of drugs are also being developed that target the immune system rather than the

parasite itself. These 'immune modulators' enhance natural immunity and lead to more

rapid cures. One such drug, which targets a primitive recognition system in phagocytic

cells (involved in initial immune system response and wound healing) is Imiquimod, a

cream used in the treatment of skin cancer. Proven to have benefit in mouse models, this

is now being tested in patients and initial studies show some promise, particularly in

combination with other drugs. Local injections of interferon-y can increase cellular

immunity, but immunotherapy has so far proved unsuccessful (Awasthi et a!., 2004;

Bryceson, 1996; Herwaldt, 1999).

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INTRODUCTION

Thus, drugs available for the treatment of Leishmaniasis are either inadequate, or

compromised by the development of resistance. This has led to research on the basic

studies to evaluate the significant differences between host and parasite which will lead

to the development of logical approaches to chemotherapy. Inspite of significant

progress in the past few decades, much remains to be discovered on the biochemical­

molecular mechanisms governing important properties of the parasite such as virulence,

drug resistance, genetic polymorphism, regulation of gene expression, kinetoplast DNA

function etc. Addressing these questions will lead in the identification of new drug targets

or vaccine molecules.

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