Content Creator: Yasin Arafat, Lecturer, Dept. of Applied Laboratory Sciences, Bangladesh University of Health Sciences (BUHS), Dhaka-1216.

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Haemoflagellate
Leishmania
Yasin Arafat
Lecturer
Dept. of Applied Laboratory Sciences
Bangladesh University of Health Sciences

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Leishmania Parasites and Diseases
Species Disease
Leishmania donovani
Leishmania infantum
Leishmania chagasi
Visceral Leishmaniasis (VL) or Kala-azar
Leishmania tropica
Leishmania major
Leishmania aethiopica
Leishmania mexicana
Cutaneous Leishmaniasis (CL) or
Dermal Leishmaniasis
Leishmania braziliensis Mucocutaneous Leishmaniasis (MCL) or
Espundia

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Leishmaniasis
 Leishmaniasis is a vector-born disease that is transmitted
by female sand flies and caused by obligate intracellular
protozoa of the genus Leishmania.
 Transmitted among mammalian hosts by the bite of
infected female sand flies, Phlebotomus and Lutzomyia.
 Leishmaniasis is a zoonotic disease.
 Geographical Distribution: Leishmaniasis is prevalent
through out the tropical and sub-tropical regions Africa,
the Mediterranean, southern Europe (Old World) as well
as South and Central America (New World).
 In Bangladesh, India, Nepal and Sudan, 90% cases of VL
are reported.

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Lutzomyia sp.Phlebotomus sp.
Female Sand Flies

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Clinical Classification
of Leishmaniasis
1. Visceral Leishmaniasis (VL) or Kala-azar
2. Cutaneous Leishmanias (CL) or Dermal
Leishmanias
3. Muco-Cutaneous Leishmanias (MCL)

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Visceral Leishmaniasis
 This is the most severe form of leishmaniasis. It is caused
by L. donovani and L. infantum (L. chagasi). In endemic
areas, the disease is more chronic with young adults and
children being more commonly infected.
 In epidemics, all age groups are susceptible (except those
with acquired immunity), and the disease is often acute.
Without treatment, VL is usually fatal.
 Symptoms in chronic VL include irregular fever,
splenomegaly, hepatomegaly, and, or, lymphadenopathy,
loss of weight with wasting, diarrhoea, low white cell and
platelet counts, and anaemia. Skin changes are common.

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Visceral Leishmaniasis
 The local Indian name for VL, kala-azar (meaning ‘black
fever’ in Hindi) is a reference to the greyish colour (skin
pigmentation) which the patient’s skin becomes.
 In acute VL there is splenomegaly, high undulating fever,
chills, profuse sweating, rapid weight loss, fatigue, anaemia,
and leucopenia. Often there is epistaxis (nose bleed) and
bleeding from the gums.
 In the New World, VL is endemic or sporadic.
Asymptomatic infections and subclinical forms of the
disease are more common. Malnutrition and other infections
increase the risk of developing symptomatic VL.

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Visceral Leishmaniasis

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Cutaneous Leishmaniasis
The clinical forms of CL vary according to the species of
parasite, region, and response of the patient.
Post Kala-azar Dermal Leishmaniasis (PKDL)
In India and occasionally in East Africa, a cutaneous form of
leishmaniasis can occur about 2 years after treatment and
recovery from visceral leishmaniasis. This is referred to as
post kala-azar dermal leishmaniasis and affects about 20% of
patients in India.
Hypopigmented and raised erythematous patches can be
found on the face, trunk of the body, and limbs. Occasionally
there is ulceration of the lips and tongue. Amastigotes are
present in the papules and nodules.

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PKDL
 Post Kala-azar dermal leishmaniasis is so
called because it develops after kala-azar is
cured.
 Also called dermal leishmanoid.
 Prevalence:
– In endemic areas of Indian subcontinent
mostly in West Bengal and Bangladesh.
 Develops one or two years of treatment of
Kala-azar when Visceral infection cures but
skin infection persists.

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Post Kala-azar Dermal Leishmaniasis (PKDL)

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Cutaneous Leishmaniasis
Old World CL
L. tropica: Infection is often referred to as dry urban
oriental sore. Dry painless ulcers 25–70 mm in
diameter are produced which are self-healing
usually after 1–2 years but often leave disfiguring
scars. It can last many years and is difficult to treat.
Untreated LR is destructive and disfiguring.

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Cutaneous Leishmaniasis
L. major: Infection is often referred to as wet oriental sore. The
early papule is often inflamed and resembles a boil of 5–10
mm in diameter which rapidly develops into a large uneven
ulcer which is self-healing in as little as 3–6 months. Multiple
lesions may occur in non-immune persons.
L. aethiopica: A cutaneous lesion is produced that is similar to
typical oriental sore with healing in 1–3 years. L. aethiopica
however, can cause diffuse cutaneous leishmaniasis (DCL) in
patients with little or no cell mediated immunity against the
parasite. This is an incurable condition characterized by the
formation of disfiguring nodules over the surface of the body.
The nodules contain large numbers of amastigotes.

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Cutaneous Leishmaniasis
New World CL
L. mexicana: Causes chiclero’s ulcer or ‘bay sore’. Lesions of the
body tend to be self-healing but those on the ear may last up to 30
years and entirely destroy the pinna of the ear.
L. peruviana: Mainly infects children. The single or few lesions are
painless and usually heal spontaneously after about 4 months. It
occurs at high altitudes in dry valleys.
L. guyanensis: May give rise to painless dry single ulcers or
multiple lesions scattered all over the body. Spontaneous healing
is rare and relapses are frequent.
L. panamensis: Causes single or few skin ulcers which are not self-
healing. Lymphatic involvement is common, resulting in
secondary nodules.

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Cutaneous Leishmaniasis

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Mucocutaneous Leishmaniasis (MCL)
 Also known as ‘espundia’, MCL is caused by New
World Leishmania species, L. braziliensis, L.
panamensis and occasionally by L. guyanensis. In
immunosuppressed persons, mucosal lesions can be
caused by L. donovani, L. major, or L. infantum.
 MCL is the most severe and destructive form of
cutaneous leishmaniasis in South America. Lesions are
similar in development to those of oriental sore and the
resulting ulcers may become very large and long-
lasting.

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Mucocutaneous Leishmaniasis (MCL)
 Early in the infection, parasites may migrate to tissues
of the nasopharynx of the palate and sometimes after
many years when the first lesion has healed there
begins a slow continuous erosion of these tissues.
 Disfiguration is often extreme with complete
destruction of the nasal septum, perforation of the
palate, and damage to the tissues of the lips and larynx.

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Mucocutaneous Leishmaniasis (MCL)

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Leishmania donovani

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Leishmania donovani
 Leishmania donovani is a species of intracellular parasites
belonging to the genus Leishmania that cause the disease
leishmaniasis. It is a human blood parasite responsible for
visceral leishmaniasis or kala-azar, the most severe form of
leishmaniasis.
 Infection is transmitted by species of sandfly belonging to
the genus Phlebotomus in Old World and Lutzomyia in
New World.
 Named after it’s discoverers:
– William Boog Leishman (London, England; May,1903)
– Charles Donovan (Madras, India; July,1903).
 Disease: Kala-azar or Visceral leishmaniasis & PKDL.

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Leishmania donovani
 Geographical Distribution: Endemic in India, China,
Russia, Africa, Southern Europe & South America.
 Hosts:
 Definitive Host: Man
 Intermediate Host: Female Sand Fly
 Reservoir of Infection: Dog (China & Brazil), Man (India),
Rodents (Africa) and Jackals (Russia).
 Habitat: In reticulo-endothelial system (R.E. system) of
vertebrate hosts (Man, Dog & Hamster).
 Morphology: 2 Stages
 Amastigote stage
 Promastigote stage

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Leishmania donovani

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Amastigote Stage
 Aflagellar stage
 Habitat:
– Found in R.E. system of vertebrate host (Man, Dog
& hamster).
 Develops at 37O
C
 Shape: Round /Oval
 Size: 2-4 µm, along longitudinal axis.
 Has following components:
 Cell membrane
 Nucleus: <1 µm in diameter, round /oval & situated in
the middle of cell or along the side of the cell
membrane.

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Amastigote Stage

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Promastigote Stage
 Flagellar stage:
 Found in: Insect vector (sand fly) and
culture at 22O
C.
 Characteristics are as follows:
Shape and size:
Earlier ones, pear shaped (5-10 x 2-3 µm).
Fully developed ones, long slender spindle
shaped(15-20 x 1-2 µm).

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Promastigote Stage
 Nucleus situated centrally
 Kinetoplast, lies transversely near anterior end.
 Eosinophilic vacuole:
A light staining area lying in front of the
kinetoplast over which root of flagellum
runs.
 Flagellum:
One in number
Projecting from anterior end
5-10 µm Length of body

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Culture of Leishmania donovani
Culture Media
Solid Media
NNN medium (Novy-MacNeal-Nicolle medium)
Evan’s modified Tobie’s medium
Liquid Media
Schneider’s Drosophila medium
Grace’s insect tissue culture medium

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Culture of Leishmania donovani
Promastigotes as seen in artificial
culture medium
 Temperature:
22-25 O
C, body temperature of sand fly.
 Time of incubation :
1- 3 weeks.
Materials containing amastigote form is inoculated in
culture media where amastigote is converted to
promastigote form.

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Transmission
 By Vector
Female Sand Fly
Genus Phlebotomus (in old world)
Genus Lutzomyia (in new world)
Indian Kala-azar transmitted by species P. argentipes.
 Other methods
 Congenital infection (Transplacental)
Blood transfusion
Accidental inoculation from culture
Transmission during coitus

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Life Cycle of Leishmania donovani

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LifeLife Cycle ofCycle of LeishmaniaLeishmania
PromastigotePromastigote AmasitgoteAmasitgote
TransformationTransformation

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Cycle in Man
 Promastigote is the infective form.
 Introduced in to human body by bite of sand fly in
which the promastigote has been developed.
 Some of the promastigotes are destroyed by body’s
immune system. Some others take refuge inside the
cells of RE system, where they are changed into
amastigote form and undergo multiplication by binary
fission.

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Cycle in Man
 When the cells become packed with parasites, they
rupture releasing the parasites into circulation from
where they are again engulfed by or invade cells of RE
system.
 The cycle is repeated and entire RE system is
progressively infected.
 Some of the free parasites in circulation are engulfed by
neutrophils and monocytes (Macrophage). During
blood meal, the vector (sand fly) draws free as well as
those parasites inside the monocyte.

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Cycle in Sand fly
 In sand fly, the amastigotes are changed into
promastigotes and multiply enormously by binary
fission in the midgut and forwards to the pharynx and
buccal cavity. Between 6th
to 9th
day of infection, a
heavy pharyngeal infection is observed. This type of
development is known as anterior station
development.
 When this sand fly bites a man, transmission is
effected.

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Life Cycle at a Glance
 Life Cycle Stages: Amastigote and Promastigote
 Host: Two Hosts – Human (definitive) and Sand Fly
(intermediate)
 Vector: Sand Fly
 Reservoir Host: Dog (China & Brazil), Man (India), Rodents
(Africa) and Jackals (Russia).
 Infective Form: Promastigote
 Pathogenic Form: Amastigote
 Route of Infection: Penetration through skin by the bite of female
sand flies.
 Site of Localization: Mononuclear phagocyte system/ R.E.
system.

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Pathology
• Pathology of VL is due to blockage and destruction of
RE system most, marked in spleen, liver, lymph node,
bone marrow and intestine.
• Spleen: grossly enlarged and cells packed with
amastigotes; pulp greatly increased in amount and
friability.
• Liver: Enlarged, Kupffer cells packed with amastigotes,
has mottled brownish (nutmeg) appearance. In old
untreated cases, fibrosis and cirrhosis may occur.
• Bone marrow: appears dark red with depressed blood
cell production. Amastogotes present in R.E. system.

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Pathogenicity & Clinical Features
 Pathogenicity: Cause, Kala-azar (Visceral
Leishmaniasis) and PKDL.
 Incubation period: 3 – 6 months, sometimes up
to 2 years.
Pyrexia
Splenomegaly
Hepatomegaly
Lymphadenopathy

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Pathogenicity & Clinical Features
 Skin dry, rough and pigmented (darkened)
 General features:
Fever, Malaise, Weight loss, Anemia, Anorexia
Enlargement of abdomen
Epistaxis
 If untreated, patient dies within 2 years due to
complications.

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Laboratory Diagnosis of Kala-azar
• Direct Evidences
 Demonstration of L. donovani
• Indirect Evidences
 Serology and Blood count

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Direct Evidences
 Specimen:
 Aspirates from bone marrow, spleen & lymph
nodes.
 Blood.
 Smear and Staining (Leishman or Giemsa):
Findings: Amastigote form in monocytes.
 Culture in NNN media:
Incubation at 22◦ C.
Incubation time- 1 to 2 weeks.
Findings: Promastigote form of parasites are found.

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Indirect Evidences
 Serology:
 Specific:
For detection of antigen-
• ELISA
• RIA (Radio Immuno Assay)
For detection of antibody-
• IFAT (Indirect Fluorescent Antibody Test)
• DAT (Direct Agglutination Test)
• ICT (Indirect ChromatographicTest)
• CFT (Complement Fixation Test)

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Indirect Evidences
 Non-specific:
• Aldehyde test,
• Antimony test.
 Blood Count: Anaemia and leucopenia (neutropenia)
with lymphocytosis and monocytosis.
 Molecular Diagnosis: PCR (Polymerase Chain
Reaction).

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Laboratory Diagnosis of PKDL
Diagnosis is established by a microscopical
examination of leishman stained smear
prepared from the biopsy material obtained
from nodular lesions and demonstrating the
amastigote forms of leishmania parasites.

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Treatment & Prophylaxis
 Treatment
 Antimony compounds: Pentavalent antimony compounds,
Sodium antimony-gluconate.
 Synthetic non-metallic compound: Pentamidine isethionate,
Miltefosine.
 Prophylaxis
Attack on parasite (dogs serving as reservoirs of infection).
Attack on vector (sand fly).
Personal prophylaxis : Use of mosquito-net, voiding the
ground floor for sleeping purposes and periodic fumigation of
sleeping quarters.

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Prevention and Control of
Leishmaniasis
 Early detection and treatment of infected persons,
especially in areas where humans are the only or important
reservoirs of infection.
 Personal protection from sandfly bites by:
 Using insect repellants, although in hot and humid
conditions they are of limited use due to profuse sweating.
 Avoiding endemic areas especially at times when sandflies
are most active.
 Use of insecticide impregnated bednets and curtains.

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Prevention and Control of
Leishmaniasis
 Vector control by the use of light traps, sticky paper traps, or
residual insecticide spraying of houses and farm buildings
where this is practical, or alternatively using insecticide
paints in a slow-release emulsifiable solution.
 Destruction of stray dogs and infected domestic dogs in
areas where dogs are the main reservoir hosts.
 Elimination and control of rodents in areas where these are
sources of human infections.
 Whenever possible, siting human dwellings away from the
habitats of animal reservoir hosts where sandflies are known
to breed.

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