This presentation include detail of Leishmania donovani along with its current treatment.

1. LEISHMANIA DONOVANI
PRESENTED BY: SHUBHI RANA
MSc. Molecular medicine
(211/PMM/005)

2. DISCOVERY
• 1900,Calcutta - Sir William Boog
Leishman discovered L.donovani
in spleen smear of a solider who
died of fever at Dum-Dum, India.
• The disease was locally known as
dum-dum or kala-azar.

3. • Charles Donovan, Irish doctor at
Madras Medical college of India
also reported same ovoid bodies in
other kala-azar patients and
published his discovery a few
weeks after Leishman.
• After examining the parasite using
Leishman’s stain these amastigotes
were known as Leishman –Donovan
bodies.

4. • Leishmaniasis is a globally important but neglected disease.
• Largely affects individual of low socio-economic level, mainly in
developing countries and can affect people of all age group.
• Currently, Leishmaniasis occur in 4 continents (endemic in 88 countries, 72
developing countries)
 90% of all VL: Bangladesh, Brazil, India, Nepal & Sudan
 90% of all MCL: Bolivia, Brazil & Peru
 90% of all CL: Afghanistan, Brazil, Iran, Peru, SA & Syria
• Prevalence: 12 million people
• Population at risk: 350 million

5. • Overall infection is caused by
more than 20 species of
Leishmania parasites, which are
spread by approx. 30 species of
phlebotomine sand flies.
• Sand fly vectors generally most
active after evening, in the night
hours.
• Climate and geographical factors
also play role.
Recently, Nepal reported 5 new cases in Kalikot. The district is located above 650m above
from sea level, thereby supporting the hypothesis that sand fly can survive high altitude
regions. With this development, it may become much difficult to contain this disease.

6. CLINICAL MANIFESTATION & PERSPECTIVE
• 3 main clinical manifestations of the disease leishmaniasis
• D/f species contribute to these 3 types
• 3 types of Leishmaniasis are:
• Visceral leishmaniasis (VL) most lethal & severe type
• Cutaneous leishmaniasis (CL)
• Mucocutaneous leishmaniasis (MCL)
• 2 other rare types
• Diffuse cutaneous leishmaniasis (DCL)
• Post kala-azar dermal leishmaniasis (PKDL)

8. Leishmania Donovani
• Hemoflagellate protozoan
• Intracellular parasite
• Insect vector: Sand fly
• Belonging to Phlebotomus genus in old world
Leishmania.
• Lutzomyia genus for new world Leishmania species
• Causes fatal vector – borne disease called
leishmaniasis (kala-azar).
• Second largest parasitic killer in the world,
after malaria.
• Life cycle: digenetic (alternates b/w 2 hosts)
LD Bodies

9. Habits and Habitat of Leishmania donovani
• It is an intracellular parasite of man
and other mammalian hosts.
• In man, intracellular amastigote
forms are found in
reticuloendothelial cells of the
spleen, bone marrow, leucocytes or
cells of the liver, intestinal mucosa,
and mesenteric lymph nodes.
• The promastigote form is found in
the midgut of sandfly or in artificial
culture.

10. Morphology of Leishmania donovani
Leishmanial or Amastigote stage
• + in reticuloendothelial cells / blood cells , vertebrates.
• microscopic, rounded, or oval in shape measuring 2-4 micrometer in
length.
• no free flagellum, it is greatly reduced, fibril-like, and lies embedded
in the cytoplasm.
• The nucleus is central or eccentric.
• The cell membrane is delicate
• Kinetoplast is rod-shaped or dot-like and lies at the right angle to
the nucleus.
• The axoneme is a delicate filament extending from the kinetoplast
to the margin of the body. It represents the foot of the flagellum.
• stained well with Giemsa or Wright stain.
• In a Giemsa stained preparation, the cytoplasm surrounded by a
limiting membrane appears pale blue. The nucleus relatively is
larger and stained red. The kinetoplast stained deep red.
• Amastigote divides by binary fission at 37°C.
Leptomonad or promastigote stage
• It is found in the midgut of the invertebrates host or sandfly.
• elongated, slender, and spindle-shaped measuring 15-20µ in length
and 1-2µ in width.
• A flagellum is long measures 15-28µ and free and arises from a minute
basal body situated near the anterior end.
• The flagellum does not curve around the body of the parasite and
therefore there is no undulating membrane.
• The nucleus is centrally placed.
• The kinetoplast lies transversely near the anterior end.
• A vacuole is present near the root of the flagellum
• With Leishman stain, the cytoplasm appears blue, the nucleus pink or
violet, and the kinetoplast bright red.
• Promastigote multiplies by binary fission at 27°C.

11. LIFE CYCLE
Hosts
• Leishmania is a digenetic parasite that requires 2 hosts for completion
of its life cycle.
• The primary host is a vertebrate or man, in which the parasite feeds
and multiplies asexually.
• The secondary host or vector is invertebrates or blood-sucking
insects or sand-fly, belonging to the genus Phlebotomus.
• Some mammals like dogs, jackals, gerbils, and squirrels also serve as
reservoir hosts in which the parasite does not undergo any change
but simply waits for its introduction into the human host.

12. (I) Life cycle in Man
• The parasite has two stages in its life cycle:
• Amastigote form occurs in humans and mammals.
• Promastigote form occurs in sandfly.
• L. donovani is transmitted to humans or other vertebrates by the bite of blood-sucking
sandfly Phlebotomus argentipes.
• The parasites introduced by sandfly into the human body are in the promastigote form.
• Some of the promastigote entering the blood circulation directly become destroyed.
• while those entering the reticuloendothelial system(liver, spleen, bone marrow, and lymph nodes )
change into amastigote or leishmanial forms.
• The amastigotes multiply by simple binary fusion inside the Reticuloendothelial system to form a
large number of amastigotes.
• When the number of parasite reaches 50 to 200 or even more, the host cell rupture.
• The liberated parasites are taken up by new host cells and the multiplication cycle is repeated so
that the reticuloendothelial system becomes progressively infected.
• Some of the free amastigotes are phagocytized by the neutrophils and monocytes(macrophages)
in the bloodstream.
• These heavily parasitized cells wander through the general blood circulation leading to a general
infection.

13. (II) Life cycle in sandfly
• When the sandfly sucks the blood of an infected person, it obtains free
amastigotes as well as the parasitized neutrophils and monocytes along with the
blood-meal.
• The parasite begins a process of transformation and the amastigotes change to
procyclic promastigotes and then to metacyclic promastigotes in the midgut of
the sandfly.
• These promastigotes multiply by longitudinal binary fusion and produce large
numbers of promastigotes completely filling the lumen of the gut.
• In 6 to 9 days, the number of parasites becomes enormous and heavily spread
into the pharynx and buccal cavity. The salivary glands are not infected.
• Transmission into a new host occurs when such a heavily infected sandfly bites
the host.

15. Survival in host
• Phagolysosome of macrophages
are the niche of parasitic
protozoan Leishmania and causes
human leishmaniasis.
• Leishmania turned the extremely
harsh environment inside the host
macrophage into a shelter to most
likely hide from the host immune
system
• Once it identifies its location inside
phagolysosome, Leishmania starts
to transform to the intracellular
form, the amastigote.
• To block parasite invasion,
macrophages activate release of
(ROS) and synthesis of cytotoxic
nitric oxide (NO) by NO synthase.
• The latter requires a massive
conversion of arginine to NO, which
exhausts intracellular arginine.
• Parasites developed sensing
mechanism that monitor arginine
in the phagolysosome, a
mechanism that is essential for
their survival and ability to develop
into amastigotes.

16. • During phagocytosis of uninfected macrophages, it takes about 30 min for
lysosome markers to appear in a phagosome, whereas it takes more than
2h for lysosome markers to appear in promastigote-infected phagosomes.
• Lipophosphoglycan (LPG), a major component of the promastigote
surface.
• It plays a key role in the infective pathway
• by interfering with the pro-inflammatory host cell responses via binding of Toll-like
receptor (TLR) 2 and 4 on macrophages and NK cells
• delays the appearance of vacuolar ATPases in phagosome membranes until they fuse
with primary lysosomes.
• delays phagosome acidification and maturation.
• Sensing acidic pH is one of the two cues that signal parasite to enter the
phagolysosome and subsequently initiate promastigote differentiation
into amastigotes.
• Protein Kinase A Is the Differentiation Gatekeeper

17. ROLE OF HSP23
• Change of temperature during the transmission from sandflies to mammals
is both a key trigger for the progression of their life cycle and for elevated
synthesis of heat shock proteins, which have been implicated in their
survival at higher temperatures.
• HSP23 protects L. donovani against cell stress, elevated temperature,
unfolded protein stress and redox stress.
• Loss of HSP23 causes increased sensitivity to chemical stressors and
renders L. donovani non-viable at 37°C.
• HSP23-null mutants are non-infectious to primary macrophages in vitro
• Thus, HSP23 expression is a prerequisite for L. donovani survival at
mammalian host temperatures and a crucial virulence factor.

18. SYMPTOMS
Phase 1
• enlargement of liver
and spleen along with
acute fever.
Phase 2
• acute enlargements of
liver and spleen along
with low fever
Phase 3
• cachexia (irreversible
weight loss & muscle
wasting) with no
observed fever
 Kala azar = reticuloendotheliosis
 Additional Symptoms: night sweating, anemia, headache, fatigue, swollen lymph
nodes.
 Skin become dry, rough & darkly pigmented.
 PKDL, is a rare reoccurrence of VL, in some instance even after 20 years of primary
infection.
 Also co-infection of Leishmaniasis along with HIV due to immune- compromised state,
can be seen.

19. PATHOGENICITY
• SPLEEN – most affected, enlarged, cut section is red or chocolate in color due to
dilated and engorged vascular spaces.
• LIVER – enlarged, but functioning not affected, nutmeg appearance.
• BONE MARROW – parasitized macrophages
• LYMPH NODES – lymphadenopathy
• Fatal after 2-3 years if not treated
• Immediate cause of death is usually an invasion of a secondary
pathogen that the body is unable to combat. (pancytopenia)

20. PREVENTION
• Suppress the reservoir: dogs, rats, gerbils, other small mammal and
rodents
• Suppress the vector: Sand fly
(Critical to preventing disease in stationary troop population)
• Prevent Sand fly bites: personal protective measures
• Importantly at night
• Sleeves down
• Repellent
• Permethrin treated nets and clothes

21. TREATMENT
• Without anti- parasitic treatment, the case fatality rate of clinical VL
(kala-azar) is estimated to be >90%.
• Mortality is often due to hemorrhagic or infectious complications.
• Liposomal amphotericin B (L- AMP) only drug US FDA approved
• Miltefosine - orally administered phosphocholine
• Paromomycin - injectable aminoglycoside, has been shown to be effective in
Indian VL. Approved by India govt. in 2006.
• Combination therapies have the potential advantages of shortening
the duration and cost.
L-AMB + Miltefosine

22. HOW THESE DRUGS WORKS?
Liposomal amphotericin B (L- AMP)
• The activity mechanism involves
the high affinity binding of
amphotericin B to 24-substituted
sterol, ergosterol.
• Ergosterol is the major component
of leishmanial cell membrane,
which in turns leads to formation
of aqueous pores.
• Pores increased permeability of
monovalent cations, anions and
other small metabolites and
ultimately leads to cell death
Miltefosine
• This drug was developed as an
antineoplastic agent against MBC*
& treatment with this result in 95%
& 91% cure rate of VL & CL.
• The activity mechanism involves
the alteration of GPI* synthesis,
ether lipid metabolism & signal
transduction.
• It also induce nitric oxide (NO) ,
which leads to killing of parasite
within the macrophages.
Paromomycin
• It is an aminoglycoside,
relatively new broad spectrum
drug.
• It inhibits the leishmanial
protein synthesis by promoting
ribosomal subunit association &
low Mg2+ conc. induced
dissociation is known.
• Studies also shown that strain of
L. donovani developing
resistance against it.
MBC*- metastatic breast cancer
GPI* – glycosylphosphatidylinositol anchor