Recent advances in

Leishmaniasis

Dr.Pinaki Chattopadhayay

17/02/2011
 INTRODUCTION
• Leishmania is a blood and tissue flagellates

• Disease caused by this parasite is called Leishmaniasis

• Of two types : Old world Leishmaniasis and New world Leishmaniasis

• Old world leiashmaniasis : Cutaneous and Visceral

• New world leishmaniasis : Cutaneous , Mucocutaneous and Visceral

 Taxonomy

• Domain: Eukaryota

• Phylum: Euglenozoa

• Class: Kinetoplastida

• Order: Trypanosomatida

• Genus: Leishmania

• Species: Leishmania species (more than 20)

 Species, disease and their geographical distribution

Old world Form of disease Geographical distribution

Leishmaniasis
L. donovani Visceral leishmaniasis,Kala- Indian subcontinenet, Africa,
azar, PKDL Middle east ,Kenya
L.infantum VL, China ,Mediterranian coast ,
Middle East, Africa ,China
L.archibaldi VL, Ethiopia, Sudan, Kenya
old world cutaneous East Africa , Sudan
leishmaniasis
L. tropica Old world cutaneous Central Asia, Middle east ,Africa,
leishmaniasis, Oriental sore Indian subcontinent
L.major Old world cutaneous Indian subcontinent, Central
leishmaniasis Asia, Africa,Middle east
L.aethiopica Old world cutaneous Ethiopia, Kenya
leishmaniasis, diffuse
cutaneous leishmaniasis
New world Leishmaniasis Form of disease Geographical distribution

L.braziliensis complex
L. braziliensis, Mucosal leishmaniasis(Espundia) South America,Brazil
L. panamensis Mucosal leishmaniasis Panama
L. guyanensis Cutaneous leishmaniasis Guyana
L.peruviana Cutaneous leishmaniasis (Uta) Peru, Western Andes

L. mexicana complex
L. mexicana, Cutaneous leishmaniasis Central America , Mexico,
Diffuse cutaneous leish maniasis
L. venezuelensis Cutaneous leishmaniasis,Diffuse Venezuela
cutaneous leishmaniasis
L. amazonensis Cutaneous leishmaniasis,Diffuse Amazonian basin
cutaneous leishmaniasis

L.chagasi American visceral leishmaniasis Latin America

 HISTORY

• Descriptions of conspicuous lesions similar to cutaneous

leishmaniasis (CL) 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.

• Muslim physicians including Avicenna in the 10th century AD gave

detailed descriptions of what was called Balkh sore.

• In 1756, Alexander Russell, after examining a Turkish patient, gave

one of the most detailed clinical descriptions of the disease.

• Physicians in the Indian subcontinent would describe it as Kala-azar

(pronounced kālā āzār, the Urdu, Hindi and Hindustani phrase for
black fever, kālā meaning black and āzār meaning fever or disease).

• As for the new world, evidence of the 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 AD.
 History contd…
• 15th and 16th century texts from the Inca period and from Spanish
colonials mention "valley sickness", "Andean sickness", or "white
leprosy", which are likely to be CL.

• Who first discovered the organism is somewhat disputed. It is

possible that Surgeon major Cunningham of the British Indian army
saw it first in 1885 without being able to relate it to the disease.

• Peter Borovsky, a Russian military surgeon working in Tashkent,

conducted research into the etiology of oriental sore, locally known
as Sart sore, and in 1898 published the first accurate description of
the causative agent, correctly described the parasite's relation to host
tissues and correctly referred it to Protozoa.
 History contd….
In 1901, Leishman identified certain organisms in smears taken from the spleen of a
patient who had died from "dum-dum fever" (Dum Dum is an area close to Calcutta) and
proposed them to be trypanosomes, found for the first time in India.

A few months later Captain Charles Donovan (1863–1951) confirmed the finding of what
became known as Leishman-Donovan bodies in smears taken from patients in Madras,
India.

But it was Ronald Ross who proposed that Leishman-Donovan bodies were the
intracellular stages of a new parasite, which he named Leishmania donovani.

The link with the disease kala-azar was first suggested by Charles Donovan, but was
conclusively demonstrated by Charles Bentley's discovery of Leishmania donovani in
patients with kala-azar.

The disease was a major problem for Allied troops fighting in Sicily during the Second
World War, and it was then that research by Leonard Goodwin showed that pentostam was
an effective treatment.
 Classification
• The ideal classification for an asexual organism at the level of species combines the
maximum biological homogeneity within each species , but separates biologically different
organism .

• Among the techniques , isoenzyme analysis have been found to describe strains at a level
that allows the construction of phenograms (taxonomic relationships among organisms)
and cladograms (ancestral relations between organisms).

• Using a panel of c.12 enzymes ,it is possible to identify and distinguish most, but not all of
the currently accepted species .

• The group of strains that share a given pattern of enzymes electrophoretic mobilities are
known as “zymodeme”. A given species may contain a number of zymodemes indicating
subspecies.

• The main disadvantage of isoenzyme analysis is the lack of standardisation between

laboratories
 Classification

• Of the other techniques , the resolution of of restriction fragment length

polymorphism (RFLP) and karyotype analysis has been found to be too fine except for
studies at the population genetic level .

• Microsatellite DNA analysis is the latest technique being used with Leishmania and
seems well suited to resolve taxonomic issues .DNA and ribosomal RNA sequence
information hold much promise and have given valuable result s at the generic level .

• Monoclonal antibody and DNA probes , with or with out PCR amplification , are of
value in identification of stocks that have been well classified .
 Vector and the Host

• Leishmania -two host

• Invertebrate, i.e the sandflies, develops in gut and tranmission via mouth

• Vertebrate hosts i.e mammals( man and also dog in some areas ) in which
parasite resides within the phagolysosomal system of mononuclear phagocytic
cells ,typically macrophages.

• It is mainly a zoonosis , man is the incidental host , sometimes human –vector –

human transmission also occur in some part of the world .

• The invertebrate host hosts are all phlebotomine sandflies of two genera
;Phlebotomus( P.argentipes, P.orientalis, P.martini) in the Old world and
Lutzomiya(L.longipalpis, L. vetulus ) and Psychodopygus in the New world.

• These are small ,hairy ,dipteran flies of the family Psychodidae , in which only the
females feed on blood and transmit disease
Phlebotomus Lutzomiya
 Different forms of the parasite

• Amastigote:
• it is found in man and other mammalian hosts .
• Found inside the monocyte , polymorpho nuclear leucocytes or endothelial cells
• Small, round to oval bodies measuring 2-3µm in length
• Also known as LD bodies
• Stained well with Giemsa or Wright

Amastigotes of Leishmania donovani

are seen in an impression smear. The
nuclei of L. donovaniis are clearly
visible (Giemsa stain, 1000x).
 Promastigote

• It is found in the digestive tract of sand fly (vector) and in the culture
media
• Long , slender and spindle shaped
• 15-25 µm in length and 1.5-3.5 µm in breadth
• Single nucleus situated in the centre
• The kinetoplast lies transversely near the anterior part , flagellum is single
• With leishman stain , cytoplasm appears blue , the nucleus pink, ,
kinetoplast bright red

•Promastigote in culture, Giemsa

stained
•Elongated shaped, with free
flagellum at the anterior end
•No undulating membrane
•A conspicuous rod-shaped
kinetoplast
Morphological forms

They possess a single

nucleus,
A single kinetoplast consists
of parabasal body and an
adjacent dot like
blepharoplast ( connected
with delicate fibrils) and
Singe flagellum .
The portion of the flagellum
extending from the
blepharoplast to the surface
of the body of the parasite is
known as axoneme
LIFE CYCLE
 Types of presentation
• Visceral leishmaniasis (VL), also known as kala azar, is the most severe form of the disease,
which, if untreated, has a mortality rate of almost 100%. It is characterized by irregular
bouts of fever, substantial weight loss, swelling of the spleen and liver, and anaemia.

• Mucocutaneous leishmaniasis (MCL), or espundia, produces lesions which can lead to

extensive and disfiguring destruction of mucous membranes of the nose, mouth and throat
cavities.

• Cutaneous leishmaniasis (CL) can produce large numbers of skin ulcers—as many as 200 in
some cases—on the exposed parts of the body, such as the face, arms and legs, causing
serious disability and leaving the patient permanently scarred.

• Diffuse cutaneous leishmaniasis (DCL) never heals spontaneously and tends to relapse
after treatment. The cutaneous forms of leishmaniasis are the most common and represent
50-75% of all new cases.
 Pathogenesis of VL

• Sand fly liberates biologically active substances , which promote infectivity of promastigotes by
partially deactivating fixed macrophages in the skin.
•
• The promastigote activate complement by alternate pathway producing activated products of
complement such as C3b or C3bi.

• These activated products bind with two specific receptors present on the outer membrane of
promastigotes (63 kDa mw glycoprotein and lipophosphoglycan) . These receptors bind with
complement receptors present on the surface of macrophages either directly or through bound
C3b or C3bi receptors .

• Promastigotes phagocytosed by macrophages are transformed into amastigotes and multiply by

binary fission within phagolysosomes of the macrophages .
• Amastigotes subsequently invade throughout the reticuloendothelial system of the spleen, liver ,
bone marorw , and lymph nodes and multiply in large numbers .

• Increased number of macrophages produce progressive hypertrophy of the organs, replace

lymphoid follicles in the spleen and also haemopoetic tissue in the bone marrow .

• Proliferation and destruction of RE cells of the internal organs and heavy parasitisation of external
organs by parasitised cells are the characteristic pathological changes in VL
Visceral leishmaniasis
 Leishmania virus

• It is a double stranded RNA virus

• Persistently infects some strains of Leishmania

• Possibility that the presence of this virus may be able to alter the
parasite phenotype and may affect disease pathogenesis.

• The virus has been detected in cultured parasites and also been
detected in human biopsy samples prior to manipulation in culture
 Host immune response

• Leishmaniasis is a disease in which the clinical diversity reflects a

complex interplay between the virulence of the infecting species
and the host's immune response.

• At one extreme, localized cutaneous disease demonstrates a

vigorous immune response, with most cases resolving without
intervention.

• This form of disease exhibits a helper T-cell subtype 1 (TH1) immune

response, with interleukin 2, interferon g, and interleukin 12 as the
prominent cytokines that induce disease resolution.

• At the other extreme, with visceral or diffuse cutaneous disease,

patients exhibit relative anergy to the Leishmania organism and
have a prominent helper T-cell subtype 2(TH2) cytokine profile.
 Leishmaniasis and The Immune System

• In order to develop a successful parasitic relationship with its host, the leishmania
must evade both the innate and adaptive immune responses.

• When leishmania first enters the human body, it is in the promastigote form.
Promastigotes are engulfed by macrophages but are resistant to proteolysis and
degradation in the phagosome.

• Once inside the macrophage, the organism is termed an amastigote. By continuing

to live inside the macrophage, leishmania effectively avoids the humoral branch of
the immune system.

• During each of the steps described, the protozoa evade and at times manipulate
the human immune system and avoid digestion.
 Leishmaniasis and The Immune System

• After being engulfed, the leishmania must endure harsh conditions inside the
phagosome. For example, the macrophage uses an oxidative burst to destroy
foreign material in the phagosome. This process consists of an attack by
superoxide and hydroxyl radicals on the parasite.

• Leishmania produces acid phosphatases on its surface which inhibit this burst.

• In addition to the oxidative burst, macrophages often attempt to degrade

parasites with acidic enzymes. This occurs when lysosomes fuse with the
phagosome. The protozoa is resistant to this attack as well because it has a proton
pump in the surface which allows its intracellular pH to remain close to neutral.

• Also, the protozoan molecule lipophosphoglycan (LPG) plays an active role by

inhibiting lysosomal enzymes.
 Recent studies
Cellular and Humoral Immunity in Leishmaniasis

• Because Leishmania manipulates the complement system in order to expedite

phagocytosis, humoral immunity is largely ineffective against the organism.

• This has been proven in many experiments. For example, the course of the
infection is unaltered in animals that have been depleted of B-cells.
• Thus, the current reasoning is that cell mediated immunity is critical in
producing immunity to Leishmania.

• This idea was further explored in Reiner and Locksley's seminal experiments in
the 80's.

• In a key experiment, they showed that athymic mice developed Leishmaniasis

whereas control mice did not. Thus, Reiner and Locksley proved that
immunity to the disease was regulated by T cells.
 Recent studies

• A further set of experiments were key not only in furthering

knowledge about the disease but also resulted in the development
of the TH1/TH2 paradigm of immunity, showed that various strains
of mice differed in their susceptibility to the disease.

• For example, C57/BL6 mice recovered after a L. major challenge,

whereas Balb/C mice did not.

• Cytokine studies showed that the serum from the resistant C57/BL6
mice contained high levels of Interferon Gamma and IL-2, but low
levels of IL-4 and IL-10.

• Conversely, the susceptible BALB/C mice had low levels of Interferon

Gamma and IL-2, but high levels of IL-4 and IL-10. (Locksley, 1991)
 Recent studies
• In further studies it was clearly demonstrated that these cytokines are responsible for biasing
the nature of the immune response.

• It was found that treating a BALB/C susceptible mouse with anti-IL-4 after infection with
Leishmaniasis results in recovery. Therefore, inhibiting IL-4 leads to clearing of the pathogen.

• Resistant C57/BL6 mice were treated with anti-Interferon Gamma after infection with
Leishmaniasis and then died.

• Therefore, Interferon Gamma is essential in eliminating Leishmania. These studies suggested

that IL-2 and Interferon Gamma are key cytokines in directing an immune response toward
intracellular pathogens.

• Conversely, IL-4 and IL-10 gear the immune response toward the clearance of extracellular
pathogens.

• Further experiments with this model led to further developments of the TH1/TH2 paradigm.
(Reiner, 1995)
 Signs and symptoms
• Delayed hypersensitivity reaction as determined by leishmanian skin test and
invitro lymphocyte response to leishmanial antigen is completely absent during
infection .

• The delayed hypersensitivity , however develops again after successful treatment .

• The intact CMI confers protection agaist the infection .

• Profound hyperglobulinemia-production of large volume of polyclonal non-specific

immunoglobulins especially IgG and also specific antileishmanial antibody .

• The circulating antibodies are not protective

 Signs and symptoms

• Persons who have recovered from kala-azar are immune from re-infection

• Anorexia ,wasting seen in the disease possibly mediated by cytokines

such as TNF and Interleukines.

• Onset of disease is gradual or sudden ( incubation period 3-6 months )

• Presents with fever, hepatosplenomegaly , haematological abnormalities

etc.

• Dry skin , dull hairs, skin on hands, mouth ,fore-head becomes grayish
and dark coloured ( Kala-azar, India)
 Leishmania/HIV Co-infection
• Leishmania/HIV co-infection is emerging as an extremely serious, new disease and it
is increasing.

• Leishmania/HIV co-infections are considered a real threat, especially in south-

western Europe where between 25% and 70% of adult VL cases are related to HIV
and where 1.5%to 9% of AIDS cases suffer form newly acquired or reactivated VL.

• Intravenous drug users have been identified as the main population at risk.

•  It is anticipated that the number of Leishmania/HIV co-infections will continue to

rise in the coming years and there are indications that cases are no longer restricted
to endemic areas.

• The overlapping geographical distribution of VL and AIDS is increasing due to two

main factors: the spread of the AIDS pandemic in suburban and rural areas of the
world, and the simultaneous spread of VL from rural to suburban areas.

• There are important clinical, diagnostic, chemotherapeutic, epidemiological and

economic implications of this trend.
 Leishmania/HIV Co-infection

• Although people are often bitten by sandflies infected with Leishmania

protozoa, most do not develop the disease.

• However, among persons who are immunosuppressed (e.g. as a result

of advanced HIV infections, immunosuppressive treatment for organ
transplants, haematological malignancy, auto-immune diseases), cases
quickly evolve to a full clinical presentation of severe leishmaniasis.

• AIDS and VL are locked in a vicious circle of mutual reinforcement.

• On the one hand, VL quickly accelerates the onset of AIDS (with

opportunistic diseases such as tuberculosis or pneumonia) and
shortens the life expectancy of HIV-infected people.

• On the other hand, HIV spurs the spread of VL. AIDS increases the risk
of VL by 100-1000 times in endemic areas.
 Leishmania/HIV Co-infection

• This duo of diseases produces cumulative deficiency of the immune response

since Leishmania parasites and HIV destroy the same cells, exponentially
increasing disease severity and consequences.

• VL is considered a major contributor to a fatal outcome in co-infected patients.

Lately, however, use of tritherapy, where it is available, has improved the
prognosis for Leishmania/HIV cases.

• Leishmaniasis can be transmitted directly person to person through the sharing

of needles, as is often the case among intravenous drug users.

• This group is the main population at risk for co-infection.

• Based on a study done in Spain, Leishmania spp were detected by PCR

technique, in 65 (52%) of 125 syringes collected in southern Madrid,  in 1998,
and in 52 (34%) of 154 collected in southwestern Madrid in 2000-01.
 Leishmania/HIV Co-infection

• Specific Problems
• Leishmania/HIV co-infections impose specific difficulties in terms of diagnosis and treatment.
The usual clinical features (fever, weight loss, liver and spleen enlargement, inflammation of
the lymph nodes) are not always present.

• The clinical diagnosis can also be made difficult by associated diseases such as cryptosporidium,
disseminated cryptococcosis, cytomegalovirus infection or mycobacterial infection.

• The serological diagnosis is falsely negative in 42.6% of co-infected patients. HIV-positive

patients have difficulty in producing antibodies against new infectious agents, especially at a
late stage or during relapses.
• Consequently, there is a need to use two or more serological tests and antigens freshly
prepared in the laboratory to increase sensitivity.

• Although multiple localizations are frequent (blood, skin, digestive tract, lungs, central nervous
system), parasitological diagnosis can be difficult and has to be repeated to orient the
treatment.

• Bone marrow aspirate (BMA) remains the safest and most sensitive technique, but spleen
aspirate and liver biopsy are also used. When BMA cannot be performed, the search for
Leishmania can be conducted in peripheral blood samples.
 Leishmania/HIV Co-infection
• Treatment for co-infected patients is aimed at clinical and parasitological cures and prevention of relapses.
Unfortunately, in such patients treatment failure, relapses due to drug resistance and drug toxicity are very
common.

• In south-western Europe, follow-up studies using pentavalent antimonials, the same first-line drug used
to treat classic leishmaniasis, show a positive response in 83% of cases. However, 52% of patients relapse
within a period of one month to three years, with the number of relapses ranging from one to four.

• The main alternative drugs include pentamidine, amphotericin B and amphotericin B encapsulated in
liposomes. This encapsulation reduces the occurrence of side-effects, but relapses still occur and the drug
remains extremely expensive.
Epidemiological Changes :
• Leishmania/HIV co-infections can lead to epidemiological changes which modify the traditional patterns of
zoonotic VL. Co-infected patients harbour a high number of Leishmania in their blood so there is also a
risk of them becoming reservoirs of the disease (that is, infective for the sandfly vector) as in
anthroponotic foci in Bangladesh, India, Nepal and East Africa. Consequently, there is an increased risk of
future epidemics.

• Experimentally, sandflies can be infected through a blood meal containing a very small quantity of blood
from co-infected patients. The quantity may be less than the content of a needle. As 71.1% of co-infected
patients in south-western Europe are intravenous drug users, transmission of Leishmania has occurred
through the sharing of syringes in this population group.
 PKDL

• It is a non-ulcerative lesion of the skin – seen after the completion of the

treatment of the kala-azar .

• Develops in 10% cases in India & 3% in Africa.

• Characterised by multiple , hypopigmented , erythematous macules

involving the face and the trunk.

• Gradually progressed to non tender plaplaques and nodules .

• In Indian form PKDL appears after a latent period of 2 yrs,and may even
persist upto 20 yrs.
PKDL
 Epidemiology
• Leishmaniasis found in 88 countries ( 16 developed countries and 72 developing coutries ) and
in 5 continents of Asia , Africa, South America, North America and Europe .

• Annual incidence of the disease 600,000 cases per year .

• An estimated 12 million people are infected , and 350 million people are at risk, Of these
approx. 25% are of VL

• India: Kala-azar primarily seen in endemic form in Bihar and West Bengal and parts of Uttar
Pradesh. Sporadic cases have been reported from Tamilnadu, Pondicherry,Assam, Orissa,
Gujrat .
• Annual kala-azar incidence peaked at 85 cases per 10,000 person-years in 2004 and fell to 46
cases per 10,000 person-years in 2007.

• There has been a resurgence of kala-azar in India , due to failure in successful implementation
of the control programme and emergence of resistance of the parasite to commonly used
antimonial drugs.

• In India , Kala-azar restricted to eastern part , whereas Cutaneous leishmaniasis restricted to

western states and Rajasthan .
 Leishmania tropica complex

• It includes L.tropica, L.major, and L.aethipica –all these causes Old world cutaneous
leishmaniasis

• L.tropica causes oriental sore (classical form), also known as Aleppo button, ericho boil,
bouton de Biskra, Delhi boil etc.

• Typically these lesions always ulcerates and heal spontaneously .

• Habitat :L.tropica and other species are found in humans ( Amastigote form in monocytes
and polymorpho nuclear leucocytes ) and promastigote in sand fly and culture.
• They are not found in the peripheral blood or in the internal organs
• Morphology : similar to L.donovani
• Life cycle : similar to L.donovani except that the amastigotes resides in the large
mononuclear cells of skin .
• Pathogenesis:bite of sand fly causes deposition of promastigotes on surface of the skin , at
the site of inoculation promastigotes are phagocytosed by macrophages and transformed
into amastigotes .
• Papule- in the early stage of the disease , and Ulcer –single or multiple , key pathological
lesion in the skin ( oriental sore) .
Oriental sore
 Types of Cutaneous leishmaniasis

• Two polar forms are found:

 Diffuse cutaneous leishmaniasis – occurs in an anergic host with poor immune response
,papule without ulceration , satellite lesion around the papule ,mainly in face &
extremities , chronic form lasts for 20 yrs or more .

• Leishmania skin test is negative .

 Leishmaniasis recidivans : occur years after localised lesion healed , due to reactivation
of dormant parasites or following new infection from a different species .

• usually in face , also causing destruction of nose .

• Skin test strongly positive , suggestive of exaggerated delayed hypersensitivity

reaction .

• The condition is chronic and even persists for 20 years.

 New world cutaneous leishmaniasis

• Caused by : L.braziliensis comple x( L. braziliensis, L. panamensis, L.

guyanensis, L.peruviana)
• L. mexicana complex( L. mexicana,L. venezuelensis,L. amazonensis)

• Habitat : These are intracellular parasites of man .

• Found inside the macrophages of the skin and the in the mucous memb.
of the nose and buccal cavity .

• Absent in either in the inernal organ or in the peripheral blood

• Morphology: similar to that of L.donovani

•
• Life cycle : similar to L.donovani , except that amastigotes are found in
RE cells and lymphatic tissue of the skin but not in the internal organs .
 Pathogenesis
• Pathogenesis of Oriental sore and Espundia are same except :
• Lesion in espundia has a tendency to expand rapidly , with distinct margins

• Mucosal metastasis occurs in the mucosal leishmaniasis by L.braziliensis, it

invades mucous memb . of mouth , nose , pharynx, and larynx either by direct
extension or by blood
• Host immunity : development of marked cell mediated hypersensitivity
reaction
• The suppression of cell mediated immunity causes amastigotes to multiply and
spread in the skin causing diffuse cutaneous leishmaniasis
 Different cutaneous manifestations

• L.braziliensis complex :
• Espundia : most severe and destructive form of cutaneous leishmaniasis,
single or multiple ulcers are found in lower extremity
• Pian bois: by L.guyanensis : ( forest jaws) single or multiple , painless, dry
persistent ulcers all over the body
• Uta : by L.peruviana: single or multiple ulcers in the face, nasopharynx
not affected , heal spontaneously within 3 months to year

• L.mexicana complex :
• Chiclero ulcer : (bay sore ) by L.mexicana : ulcers in the hand or head,
heals spontaneously within 6 months
• Indolent nodular lesion : by L.venezuelensis
• Cutaneous single sore type : by L.amazoniansis , sometimes causing DCL
 New world VL (AVL)

• Caused by L.chagasi in tropical South America (AVL)

• Mammalian hosts include human and domestic dog

• Lutzomiya vetulus is the vector

• Affected primarily the children ( similarity with L.infantum), potentially fatal if not
treated

• Life cycle & morphology: same as L.donovani , amastigote can be found throgh out
the body ( bone marrow, spleen, liver)and are not confined to the skin
macrophages .

• Clinical disease: AVL is associated with malnourished children>5 yrs and young
adults .
 Laboratory Diagnosis
• Parasitic diagnosis: demonstration of Leishmania in different specimens confirms the diagnosis .

• Specimens:

• Spleen, bone marrow, liver, lymph node, and peripheral blood smear ,

• margin of the skin ulcer either by puncture of the raised nodule or aspiration of the outer edge

of the ulcer , Full thickness skin biopsy from margin of the ulcer,

• Slit skin smear

• Methods of examination :

• Microscopy

• Culture

• Animal Inoculation

• Immunodiagnosis
 Lab. diagnosis
• Microscopy;

• The amastigotes of L.donovani ( LD bodies) can be demonstrated in the smears of

spleen, bone marrow, liver, lymph node, peripheral blood smear stained with
Leishman, Giemsa, or Wright stain

• Brown –Hopps staining is a recent method shown to have a higher sensitivity

compared to other staining techniques
• Splenic aspiration: is the most sensitive ( but there is a risk of haemorrhage)
• Bone marrow aspiration : Sternum/ Iliac crest , safer
• Liver biopsy:- not so safe

• LN biopsy: Positive in 60 % cases

• Peripheral blood smear : LD bodies can be seen in monocytes and neutrophils,

Buffy coat
 Brown-Hopps Method

• Fixation
• 10% Buffered Neutral Formalin
• Section
• Paraffin, @ 6 microns
• Staining Procedure
• Deparaffinize and hydrate to distilled water.
• Place slides on staining rack and pour on Crystal Violet Solution, 1%, Aqueous for two minutes. Rinse in
distilled water.
• Mordant in Gram's Iodine for five minutes. Rinse in distilled water.
• Differentiate in Cellosolve, until blue color no longer streams away from the section (approximately 5-10
seconds).

• Quickly rinse in distilled water.

• Stain in Basic Fuchsin Solution, 0.5%, Aqueous for five minutes. Rinse in distilled water.
• Gallego's Solution , for five minutes. (Differentiates and "fixes" the Basic Fuchsin).
• Rinse thoroughly in distilled water and blot lightly to remove excess water (not to dryness).
• Stain for three seconds in Tartrazine Solution . Immediately blot away excess, but not to dryness).
• Cellosolve three changes, for six quick dips in each.
• Xylene, three changes, for 10 dips each. Slides may remain in Xylene until ready for mounting.
• Mount with Permount
Amastigote stage of Leishmania donovani. Showing Leishman-Donovan (LD) bodies in
spleen smear of experimentally infected hamster. Many intracellular and extracellular
LD bodies are seen. x400
 Culture:

• Tissue samples and aspirates are inoculated in:

• NNN( Novy ,McNeal,Nicole) media ( 2 parts of salt agar & One part of
defribrinated rabbit blood) ,

• Schneider drosophila media( Insect cell culture media) ( 20 % foetal calf

serum) , better than NNN

• Grace’s, and Mituhasi –Maramorosch media for demonstration of

promastigotes ( Insect cell culture media)

• Incubated at 22°C-26°C for 1-4 wks.

• At the end of each week a drop of culture fluid is examined for

promastigotes
 Animal inoculation

• Intraperitoneal inoculation of chinese and Golden hamster may reveal parasites

• In a positive case amastigote can be demonstrated

 Immunodiagnosis
• The hallmark of visceral leishmaniasis is hyperimmunoglobulinaemia, while in case of
cutaneous and mucocutaneous leishmaniasis, the humoral immune response is
extremely poor.

• Exploiting this host-parasite interaction, for the diagnosis of visceral leishmaniasis a

number of antibody detection methods have been developed from time to time.

• Some of these tests include:

• Non- specific tests and specific tests

• Indirect haemagglutination (IHA),

• Counter current immunoelectrophoresis (CCIEP),

• Immunodiffusion (ID) etc.

• These tests are cumbersome and lack sensitivity and specificity and hence not
commonly used
 Non- specific serological tests

• These test detects the hypergammaglobulinemia:

• Aldehyde test of Napier

• Antimony test of Chopra

• Aldehyde test :done with pt’s serum , positive result shows jellification of a milky white opacity
within 2-20 mins.

• Disadvantage – it shows false positive with sera from Schistosoma japonicum, African
trypanosomiasis, multiple myeloma, cirrhosis of liver.

• It is always negative in cutaneous leishmaniasis

• Antimony test : a positive test is indicated by development of a white flocculent precipitate on

addition of urea stilbamine solution to the patient serum
•
 Specific immunological tests :
Fluorescent antibody test
• The indirect fluorescent antibody (IFA) test is one of the commonly used tests for anti-
leishmanial antibody detection using fixed promastigotes.

• The test is based on detecting antibodies, which are demonstrated in the very early
stages of infection and are undetectable six to nine months after cure.
• Titres above 1:20 are significant and above 1:128 are diagnostic. However, there is a
possibility of a crossreaction with trypanosomal sera .

• The sensitivity of these tests varies extremely from as low as 28.493 to 86.6 per cent. This
can be overcome by using Leishmania amastigotes as the antigen instead of the
promastigotes.

• To detect the antigen (amastigotes) in the tissue sections or smears, fluorescent dye
conjugated antibodies can be used as tracers. This test is known as direct fluorescent test.

• The direct fluorescence test is more useful in the diagnosis of CL, MCL and PKDL. In place
of fluorescence, horse radish peroxidase (HRP) can be used to tag the antibody. This will
not require fluorescence microscope and the stained slides can be stored for long time.
 Direct agglutination test:
• The direct agglutination test (DAT) is a highly specific and sensitive test.
• It is cheap and simple to perform making it ideal for both field and laboratory use.
• DAT in various studies has been found to be 91-100 per cent sensitive and 72- 100
per cent specific.
• The method uses whole promastigotes either as a suspension or in a freeze dried
form. The freeze-dried form is heat stable and facilitates the use of DAT in the field
• However, the major disadvantage of DAT is the long incubation time of 18 h and
the need for serial dilutions of blood or serum. Also the DAT has no prognostic
value for evaluating the parasitological cure of the disease, as the test may remain
positive for several years after cure.

• Recently, Schoone et al. have developed a fast agglutination-screening test

(FAST) for the rapid detection (<3 h) of anti-Leishmania antibodies in serum
samples and on blood collected on filter paper.

• The FAST utilizes only one serum dilution leading to qualitative results. The FAST
offers advantages over the DAT as it uses freeze-dried antigen, which gives more
antigen stability, reproducibility, specificity and sensitivity.
 Enzyme linked immunosorbent assay (ELISA):

• ELISAis a valuable tool and one of the most sensitive tests for the serodiagnosis of visceral
leishmaniasis. The test is useful for laboratory analysis or field applications and to screen a large
number of samples at a rapid pace.

• An excretory, secretary and metabolic antigens released by L. Donovan promastigotes (Ld-ESM)

into a protein free medium was used for the serodiagnosis of VL by ELISA. This antigen has been
reported to be 100 per cent specific and sensitive, the PPV was 99.99 per cent and NPV was 95.45
per cent.

• The protein encoded by L. donovani gene B homologue (Ld-rGBP) , performing ELISA by using this
antigen is reported to be specific for L. donovani infections only.

• Another recombinant protein rORFF of L. infantum origin has been developed by Raj et al. for
diagnosis of VL in India.

• The ORFF protein is encoded in the LD1 locus of chromosome 35 of L. infantum. An ELISA based
test using this antigen was found to be highly sensitive and specific.

• In addition, rK39 ELISA, has a high predictive value for detecting VL in immunocompromised
persons, like AIDS patients78. This antigen is now commercially available in the form of antigen-
impregnated nitrocellulose paper strips adapted for use under field conditions
 ELISA

• Very recently, cloning and characterization of a recombinant antigen

from an Indian isolate of L. donovani strain KE16 is done .

• The antigen (rKE16) is found to be 100 per cent sensitive and specific.

• In fact it has better sensitivity than rK39 which showed 98 per cent
sensitivity for the diagnosis of Indian kala-azar and PKDL.

• It also showed 100 per cent concordance with rK39 in sera from
leishmaniasis patients from China, Pakistan, and Turkey.

• This antigen has now been commercialized and has got tremendous
potential for the serological diagnosis of VL worldwide.
 Rapid antibody detection method

• In most of the Leishmania endemic areas resources are limited in

terms of poor or non-availability of electricity, poor laboratory set up
and lack of equipment.

• Therefore, need of rapid, simple and easy to perform tests has always
been felt. With this objective two rapid tests have been developed,
one by InBios (USA) which uses Lc-rK39 antigen and the other one is
by Span Diagnostic Limited (India) which uses Ld-rKE-16 antigen.

• Both are commercially available and are based on membrane filtration

technology.
 Antigen detection

• Antigen detection test would, in principle provide better means of

diagnosis of active leishmaniasis.

• Since antigen levels are expected to theoretically correlate with the

parasite load, the antigen detection may be an ideal test in
immunocompromised patients, where antibody response is very poor.

• Recently, a latex agglutination test (KATEX) for the detection of

leishmanial antigens in the VL patients’ urine has been developed.

• The test had 100 per cent specificity and sensitivity between 68-100
per cent.
 Leishmanin skin test (LST): Montenegro test

• 0.2 ml of leishmania antigen ( containing 10^8 promastigotes of L.donovani in 1ml of

0.5% phenol saline ) is injected intradermally. Test is read after 48 to 72 hrs .

• Positive – area of erythema & induration of ≥5mm in D ,which heals in 14-25 days .It
indicates prior exposure to leishmanial parasites .

• In kala-azar , it becomes positive usually only 6-8 wks after cure from the disease.

• Delayed hypersensitivity is an important feature of cutaneous forms of human

leishmaniasis and can be measured by the Leishmanin test, also known as the Montenegro
reaction.

• No cross-reaction occurs with Chagas’ disease, but some cross-reactions are found with
cases of glandular tuberculosis and lepromatous leprosy.

• LST is used as an indicator of the prevalence of cutaneous and mucocutaneous

leishmaniasis in human and animal populations and successful cure of the visceral
leishmaniasis.
 Molecular methods
• A variety of nucleic acid detection methods targetingDNA and RNA genes have
been developed.

• However, amongst all the molecular advances gene amplification techniques

have been most rewarding as far as diagnosis and disease management is
concerned.

• Polymerase chain reaction (PCR): Amongst the molecular methods used for
clinical diagnosis, PCR has been proved to be most sensitive and specific
technique, but limited to tertiary care hospitals and research laboratories.

• The important gene targets are 18S-rRNA, small subunit rRNA (SSU rRNA), a
repetitive genomic sequence of DNA, the miniexon (spliced ladder) gene repeat,
the b-tubulin gene region, gp63 gene locus, internal transcribed spacer (ITS)
regions; micro-satellite DNAs such as maxi- and minicircles of kinetoplast DNA
 Molecular methods

• Recently Martin-Sanchez et al. using PCR-ELISA found 24% asymptomatic

individuals carrying Leishmania kDNA in their blood.

• It was pointed out that these individual could be potential source of

transmitting the infections in the community.

• These authors also found a good correlation between the antibody titres, skin
test positivity and PCR positivity.
• Bone marrow, lymph node aspirates, skin biopsy, skin scrape/exudates and
blood samples have been used for PCR in several studies.

• The specificity of PCR on bone marrow aspirates has been reported up to 100%
and sensitivity 80-93.3% as compared to 50-60% sensitivity of smear and
culture examination.

• Also a modified form of PCR such as nested PCR has proved its predictive
values in diagnosis of PKDL.
 Molecular methods
• The chronic CL patients are greatest diagnostic challenge In such cases,
PCR has been proved to be the most important tool for diagnosis. The
sensitivity of PCR in CL has been reported 100 per cent.

• A recent development in PCR technology (fluorogenic probes and

automation) takes care of non-specific amplification, speed and inter-
operator variability.

• The real-time PCR is used qualitatively and quantitatively, as the

fluorescence is directly proportional to the number of amplicons, or in
other words, the parasite load in the given specimen.

• The automization of real-time PCR with comprehensive portable units

has made these tools field friendly.

• The multiplex PCR can be used whenever, double or mixed infections

are suspected as in AIDS patients.
 Summary of the Diagnosis

• Direct visualisation of amastigotes in clinical specimens is the diagnostic gold standard in

regions where tissue aspiration is feasible and microscopy and technical skill are available.

• Diagnostic sensitivity for splenic, bone marrow, and lymph node aspirate smears is 95%, 55–
97%, and 60%, respectively

• Elsewhere, including epidemic settings,serum antileishmanial immunoglobulin G in high titre is

the diagnostic standard, primarily with direct agglutination tests or other laboratory-based
serological assays.

• Freeze-dried antigen (refrigeration not needed) and rapid detection of anti-K39 antibody with
fingerstick blood in an immunochromatographic strip test have advanced field serodiagnosis.

• In symptomatic patients, anti-K39 strip-test sensitivity is high (90–100%)and while specificity

might vary by region this test can safely substitute for invasive diagnostic procedures in Indian
visceral leishmaniasis and is useful in PKDL.

• Testing urine for leishmanial antigen or antibody is a new approach.

• In a central laboratory, clinical samples can be cultured for parasite isolation and leishmanial
DNA is readily detectable by PCR testing, including in peripheral blood and serum.
Giemsa-stained splenic
aspirate smear, showing clumped
mononuclear cells and numerous
amastigotes . Original
Magnification X500.
Serodiagnosis of kala azar by anti-K39 antibody detection
in immunochromatographic strip test. 5 min after
application of one drop of fingerprick blood and buffer,
right-hand strip shows second pink band (arrow)
indicating presence of anti-K39. Left-hand strip shows a
negative result (no second band).
 TREATMENT

• The drugs of choice today are the same compounds that were used in the early 1900s and their
mechanisms of function are not completely understood. They are all extremely toxic, and the
patient must be monitored closely during treatment.

• Pentavalent antimony coumpounds, derived from the heavy metal antimony (Sb), are the drugs
of choice for treating cutaneous and visceral Leishmaniasis. The recommended dosages follow:
Disease Dose Duration
• Cutaneous leishmaniasis 20 mg Sb/kg/day 20 days
Mucocutaneous leishmaniasis 20 mg Sb/kg/day 28 days
Visceral leishmaniasis 20 mg Sb/kg/day 28 days
•
 
 Studies have shown that longer courses prove more effective in preventing relapse, however
they also increase the risk of drug-related toxicity. Further complicating the issue is that parasite
resistivity seems to be increasing and  relapse rates in some areas have been reported as high as
50%-70% with mucocutaneous leishmaniasis and 10% with visceral leishmaniasis.

• Side effects include: Cardiotoxicity, reversible renal insufficience, pancreatitis, anemia, leukopenia, rash,
headache, abdominal pain, nausea, vomiting, arthralgia, myalgia, thrombocytopenia, and transaminase
elevation.
 
 Treatment
• Amphotericin B has been shown to be more effective than pentavalent antimony in vitro,
but has not been used extensively in the past due to extreme toxicity.

• However, more recent developments have made Amphotericin B less toxic and more
useful. It has also become useful in treating strains of visceral leishmaniasis causing
organisms that are resistant to pentavalent anitmony.

• Amphotericin B cannot be used in the treatment of mucocutaneous leishmaniasis because

very large doses are required.

• Side effects include: fever nausea, vomiting malaise, anemia, phlebitis, hypokalemia, hypomagnesemia, and
nephrotoxity.

• Pentamidine isothionate is a general anti-parasitic drug that has demonstrated some

success with the treatment of viceral leishmaniasis. Unfortunately the cure rate has
declined from the early 1980s to the 1990s due to resistance of Leishmania to pentamide.
Nonetheless, it can be used as a second-line agent in the event that the pentavalent
antimony compounds fail.

• Side effects include: nausea, vomiting, abdominal pain, and headache.

 Treatment

• Other Agents:

• The following drugs and therapeutic approaches seem promising and are
currently being studied, but have not been approved for general use.

• Cytokine therapy: Cytokines such as interferon gamma and granulocyte

macrophage colony-stimulating factors are being used in combination and with
pentavalent antimony in attempts to enhance reponse.

• Miltefosine: An oral drug for visceral leishmaniasis is showing progress in studies.

Apparently it is less toxic and exhibits higher cure rates than traditional drugs.
• Furthermore, a cheap, orally administered treatment would be extremely
beneficial for the people most affected by leishmaniasis
 VACCINES:

• Currently there are no drugs or vaccines for the prevention of Leishmaniasis.

• Leishmaniasis is an intracellular infection.

• Ideally a vaccine would illicit a strong Th1 response as a Th2 response has been
implicated in chronic, non-healing disease.

• Live-attenuated candidates have been proven to create this kind of response,

but the dangers associated with them have hindered their use.

• With the help of adjuvants and cytokines, whole-killed vaccines seem show
great promise.
 Vaccines

• Whole-killed Leishmaniasis: Currently, the most successful vaccine attempts in humans has
been achieved with whole-killed leishmania promastigotes.

• Phase III clinical trials in the Middle East and South America with an autoclaved Leishmania and
BCG have proven to be effective at reducing the incidence of cutaneous leishmaniasis.

• Reported efficacy rates range from 18% to 78% (Sharifi, 1998; Antunes, 1986). Evidence also
suggests that the Leishmania major creates a certain degree of cross-protection to Leishmania
donovani, the parasite implicated in the deadly visceral leishmaniasis (O'Daly JA, 1993).

• The vaccine seems to boost IFN-gamma and T-cell production, inducing a strong Th1 response.
This strong Th1 response may also explain cross-protection to another intracellular parasite
Trypanosoma cruzi (Araujo, 1999) and it may account for its proven therapeutic qualities for
individuals and mice infected with cutaneous Leismaniasis.

• It has been shown to have similar cure rates as standard antimonial regimens with fewer side
effects (Convit, 1987).

• Unfortunately, the autoclaved parasite shows decreasing efficacy with time. Studies of efficacy
with thiomersal-preserved and non-autoclaved preparations have shown to keep better (De-
Luca, 1999).
 Vaccines

• Live-attenuated:
Historically, protection from cutaneous leishmaniasis was achieved by taking the
pus from a healing lesion and innoculating a naive recipient in an inconspicuous
location.

• This crude version of a live-attenuated vaccine conferred protection upon the

host, however serious disease was occasionally a complication.

• Both protection and disease stem from the parasites ability to get inside the cells
and stimulate a cell-mediated T-cell response.

• Radioattenuated promasitigotes and biochemically altered Leishmanial parasites

have proven to confer very good protection in mice and hamsters without
adjuvant (Rivier, 1999).

• Unfortunately, live cultures of Leishmaniasis are hard to sustain and there is

always the concern of reversion to a virulent strain.
• Synthetic Peptide:

A single synthetic T-cell epitope from gp63 administered with Th1 stimulating
poloxamer 407 conferred protection against Leishmania major in BALB/c mice
(Spitzer, 1999).

• Other:
Infection of mice with adenovirus expressing IL-12 skews the immune
response in a Th1 direction preventing leishmaniasis in susceptible mice
(Gabaglia, 1999).

• Antibodies produced against Liposphoglycan (LPG) may prove to be effective

in limiting the transmission of cutaneous leishmaniasis in its vector
Phlebotomus dubosqi (Tonui, 1999).
• To date, several different approaches to antileishmanial vaccine have been
tested.

• First generation vaccines composed of whole killed parasites have been

proposed as both prophylactic and therapeutic vaccines.

• In theory, these vaccines should be easy to produce at a low cost in

endemic countries; however, standardization of cultured parasite-derived
vaccines is a drawback in the way to their registration.

• In general, the whole-cell, killed vaccines have been rather poorly defined
and variable in potency, hence they have rendered inconclusive results.

• Nevertheless, the trials completed so far demonstrated their good safety

profile, and despite poor prophylactic outcomes, showed encouraging
results as therapeutic vaccines in South America and Sudan.
• Most of the vaccine studies concentrate on the second generation vaccines
consisting of recombinant proteins, poly-proteins, DNA vaccines or dendritic cells
loaded with peptides derived from leishmanial antigens.

• A variety of different molecules has been tested to date, and these included
antigens such as surface expressed glycoprotein leishmaniolysin (gp63) delivered
by a plethora of immunization regimens, however, promising findings from animal
models were overshadowed by mostly negative T cell responses in humans.

• Another vaccine candidate has been a GPI-anchored membrane protein gp46 or

Parasite Surface Antigen 2 (PSA-2), that belongs to a gene family present in all
Leishmania species except L. braziliensis.

• Immunization with the native polypeptides derived from promastigotes protected

mice against infection,but vaccination with a recombinant protein derived from
either promastigotes or amastigotes protein showed lack of protective efficacy.
• Similarly, DNA vaccination conferred protection in mice when used as either
prophylactic or therapeutic vaccines.

• Another extensively tested antigen is the Leishmania homologue for receptors

of activated C kinase (LACK) that is expressed throughout leishmanial life cycle.

• Immunization with LACK appears to promote the expansion of IL-4 secreting T

cells skewing the response towards detrimental Th2 responses.

• However, susceptible BALB/c mice immunized with LACK had the ability to
control a subsequent infection with L. major.

• To date, the protective efficacy of LACK has been mainly demonstrated in the
L. major model, and LACK failed to protect against visceral leishmaniasis.
• Several other antigens from different species have been tested in animal models.

• These include amastigote cysteine proteases (CP),cysteine proteinase A2 and

amastigote membrane proteins P4 and P8, kinetoplastid membrane protein-11 (KMP-

11), amastigote LCR1, hydrophilic acylated surface protein B1 (HASPB1), leishmanial

antigen ORFF, acidic ribosomal protein P0, paraflagellar rod protein 2 (PRP-2), NH36, a

main component of the fucose-mannose ligand and proteophosphoglycan (PPG).

• In addition, molecules such as ATP synthase alpha chain, beta-tubulin and heat shock
70-related protein 1 precursor have been recently identified as novel vaccine
candidates.
 Vaccines
• To date, only one second generation vaccine, Leish-111f, has been assessed in clinical trials.

• Leish-111f is a single polyprotein composed of three molecules fused; the L. major

homologue of eukaryotic thiol-specific antioxidant (TSA), the L. major stress-inducible
protein-1 (LmSTI1) and the L. braziliensis elongation and initiation factor (LeIF).

• Initial immunisation trials in mice demonstrated that Leish-111f was able to protect mice
against L. major and L. amazonensis infection.

• There is some evidence that the Leish-111f vaccine can also induce partial protection against
visceral leishmaniasis in animal models, however, Leish-111f failed to protect dogs against
infection and did not prevent disease development in a recent Phase III trial in dogs.

• A slightly improved version of the original construct, Leish-110f, has also been tested in dogs
as a therapeutic vaccine in combination with chemotherapy and led to reduced number of
deaths and higher survival probability.

• Human Phase I and II clinical trials (safety and immunogenicity) of Leish-111f have been
completed over the past few years in Brazil, Peru and Columbia, and Phase I trial has been
conducted in India (http://clinicaltrials.gov).
 Reference centre
• RAJENDRA MEMORIAL RESEARCH INSTITUTE OF MEDICAL SCIENCES
PATNA - 800007 (Established in 1981)
•   Phone: 0612 -2631565 Fax: 0612 -2634379 Email : rmrims@rmrims.org.in,
dirrmris@sancharnet.in Website :  http://www.rmrims.org.in
• Scope of Activities:
• Situated at Patna in Bihar State, the Rajendra Memorial Research Institute of Medical Sciences
(RMRIMS) conducts research on various aspects of Visceral Leishmaniasis, also known as Kala-
azar, and HIV/AIDS. Tuberculosis is now being included. RMRIMS is one of the designated ICMR
centres for bio-medical informatics.
• Thrust Areas:
• PCR based diagnosis
• Studies on VL-HIV co-infection
• Clinical trials
• Cost effective integrated vector management
• Leishmania repository
• Role of cytokines in responsive and unresponsive patients
• Immuno-pathology of PKDL patients
• Innate immunity in malnutrition
• Database design of leishmania parasite
• Routine Biochemical and Hematological Diagnosis and Treatment of VL/PKDL/HIV
• HLA Typing of VL Patients
• Studies on Drug Resistance Mechanism in Genomic and Proteomic Level
THANK
YOU
 Refferences

• Toplay & Wilson’s Parasitology 10th edn.

• Diagnostic medical parasitology –Garcia 4th edn.
• Parasitology by K.D.Chatterjee-12th edn.
• Text book of medical parasitology –S.C .Parija-3rd edn.
• Medical parasitology –D.R.Arora,B.Arora- 2nd edn.
• Internet sources
 Vaccines

• Leishmania parasites are transmitted from one host to another during the sand fly
bite as a suspension in sand fly saliva.

• Sand fly saliva contains an array of molecules able to interfere with the host immune
responses,

• therefore, immunity against saliva components may indirectly enhance anti-

leishmanial immunity.

• Prior exposure of mice to bites of uninfected sand flies conferred protection from L.
major infection.

• Immunization with molecules present in saliva, such as maxadilan or a 15 kDa

protein, SP15 also induced protection against cutaneous leishmaniasis.

• More recently, it has been shown that vector salivary proteins, in particular LJM19,
protect hamster from VL,and immunization of dogs with salivary antigens led to the
development of high IgG2 antibody levels and significant IFN-γ production.