1. Leishmania
Dr N P Singh
Professor
Deptt. Of Microbiology

2. Leishmania
Phylum Sarcomastigophora
Order Kinetoplastida
Family Trypanosomatidae
Genus Leishmania
• Transmitted to the mammalian hosts by the bite of
infected sandflies, Phlebotomus and Lutzomyia

3. • Currently, leishmaniasis occurs in 4 continents and is
considered to be endemic in 88 countries, 72 of which are
developing countries:
 90% of all VL: Bangladesh, Brazil, India, Nepal and
Sudan
 90% of all MCL: Bolivia, Brazil and Peru
 90% of all CL : Afghanistan, Brazil, Iran, Peru, Saudi
Arabia and Syria
• Annual incidence: 1- 1.5 million cases of CL
: 500,000 cases of VL
• Prevalence: 12 million people
• Population at risk: 350 million
(WHO, 2010)

4. SITUATION IN INDIA
• 40-50% of global burden
(Bora 1999, Natl Med J India)
• Surveillance being done by
NVBDCP
• INDIA: 15538 cases and 47
deaths by VL (2010)
• Endemic states in Eastern
India: Bihar, Jharkhand, West
Bengal, Uttar Pradesh
• Estimated 165.4 million
population at risk in 4 states
(NVBDCP, 2010)

5. • On the basis of development, divided in two genera:
Subgenus Leishmania: in the anterior part of alimentary
tract of sandfly
Subgenus Viannia: midgut and hindgut of sandfly
Leishmania L. major complex (L. major) Old
L. tropica complex (L. tropica, L. killicki) World
L. aethiopica complex (L. aethiopica)
L. donovani complex (L. donovani, L. infantum)
L. donovani complex (L. chagasi) New
L. mexicana complex World
(L. mexicana, L. venezuelensis, L. garnhami,
L. amazonensis, L.pifanoi)
Viannia L. braziliensis complex (L. braziliensis, L. peruviana, L.
columbiensis, L. lainsoni)
L. guyanensis complex (L. guyanensis, L. panamensis)

6. Leishmania Parasites and Diseases
SPECIES Disease
Leishmania tropica
Leishmania major Cutaneous leishmaniasis
Leishmania aethiopica (CL)
Leishmania mexicana
Leishmania braziliensis Mucocutaneous
leishmaniasis (MCL)
Leishmania donovani
Leishmania infantum Visceral leishmaniasis
Leishmania chagasi (VL)

8. Digenetic Life Cycle
Promastigote Amastigote
– Insect – Mammalian stage
– Motile – Non-motile
– Midgut – Intracellular

9. INFECTION
Sub-clinical or inapparent infection
Recovery Death
Immune to reinfection Concurrent
infection
PKDL

10. LEISHMANIASIS
• Vector borne disease
• A wide clinical spectrum
 VISCERAL
 PKDL
 DIFFUSE CUTANEOUS
 CUTANEOUS
 MUCOCUTANEOUS

11. VL - Clinical Manifestation
• Variable - Incubation 3-100+ weeks
• Lowgrade fever
• Hepato-splenomegaly
• Bone marrow hyperplasia
• Anemia, Leucopenia & Cachexia
• Hypergammaglobulinnemia
• Epistaxis , Proteinuria, Hematuria

13. • Most severe form of the disease, may be fatal if
left untreated
• Usually associated with fever, weight loss, and an
enlarged spleen and liver
• Anemia (low RBC), leukopenia (low WBC), and
thrombocytopenia (low platelets) are common
• Lymphadenopathy may be present
• Visceral disease from the Middle East is usually
milder with less specific findings than visceral
leishmaniasis from other areas of the world

14. Post Kala Azar Dermal
Leishmaniasis
• Normally develops <2 years after recovery
• Recrudescence
• Restricted to skin
• Rare but varies geographically

15. Cutaneous Leishmaniasis
• Most common form
• Characterized by one or more sores, papules or
nodules on the skin
• Sores can change in size and appearance over
time
• Often described as looking somewhat like a
volcano with a raised edge and central crater
• Sores are usually painless but can become painful
if secondarily infected
• Swollen lymph nodes may be present near the
sores (under the arm if the sores are on the arm
or hand…)

17. Cutaneous Leishmaniasis
• Most sores develop within a few weeks of the sandfly
bite, however they can appear up to months later
• Skin sores of cutaneous leishmaniasis can heal on their
own, but this can take months or even years
• Sores can leave significant scars and be disfiguring if
they occur on the face
• If infection is from L. tropica it can spread to
contiguous mucous membranes (upper lip to nose)

18. Mucocutaneous Leishmaniasis
• Occurs with Leishmania species from Central and South
America
• Very rarely associated with L. tropica which is found in the
Middle East
- This type occurs if a cutaneous lesion on the face spreads
to involve the nose or mouth
- This rare mucosal involvement may occur if a skin
lesion near the mouth or nose is not treated
• May occur months to years after original skin lesion
• Hard to confirm diagnosis as few parasites are in the lesion
• Lesions can be very disfiguring

19. DIAGNOSIS

20. Direct evidence:
Demonstration of Leishmania
Specimens that may be collected
• Splenic aspirate and biopsy
• Liver biopsy
• Bone marrow (Sternum or iliac crest)
• FNAC and biopsy
• Blood buffy coat
• Tegumantary leishmaniasis- dermal scrapings, sections
from skin biopsy
DEMONSTRATION OF Leishmania
AMASTIGOTES/ L.D. BODIES

21. MICROSCOPY

22. CULTURE
Culture media for axenic culture
• SOLID MEDIUM
 NNN medium
 Evan’s modified Tobie’s medium
• LIQUID MEDIA
 Schneider’s Drosophila medium
 Grace’s insect tissue culture medium
DEMONSTRATION OF Leishmania PROMASTIGOTES
Animal inoculation
• Golden hamsters inoculated intraperitoneally

23. Promastigotes as seen in artificial culture medium

24. IMMUNOLOGICAL METHODS
• Aldehyde (formol gel) Test (Napier)
• Antimony test (Chopra)
• Complement fixation test with W.K.K Ag

25. Indirect Fluorescent
Antibody test
• Detection of anti-leishmanial
antibody using fixed promastigotes
• Demonstrated in the very early
stages of infection and
undetectable six to nine months
after cure
• Titers >1:20 are significant and
above 1:128 are diagnostic
• Cross reaction with trypanosomal
sera (overcome by
using Leishmania amastigotes as
the antigen instead of the
promastigotes)

26. Direct Agglutination Test
• Use of whole, stained • Relative long incubation time
promastigotes either as a of 18 hours
suspension or in a freeze-dried • Need for serial dilutions of
form. serum
• The freeze-dried form is heat • No prognostic value
stable • Remain positive for several
• Utilized for field purposes years after cure

27. Modifications of DAT
• Fast Agglutination Screening Test
(Schoone et al, 2001)
 Need of only 1 serum dilution
 Rapid: results available in less than 3 hours
• EasyDAT method
(Gomez-Ochoa et al, 2003, Clin Diagn Lab Immunol)

28. ELISA BASED ASSAYS

29. Many antigens have been explored for the diagnosis of
leishmaniasis:
• Whole soluble antigens (Ld-ESM—Excretory, secretory and
metabolic antigen by L.donovani)
• Purified antigens such as fucose- mannose
• Defined, synthetic peptides
• Recombinant antigens
 rGBP (L.major protein encoding a hydrophilic protein)
 rORFF (L. infantum)
 gp63
 rK39
 rK26, rK9
 rKE16

30. rK39
• Rapid dipstick test
• Based on the recombinant k39 protein, a 39-amino acid
cloned in Escherichia coli, from the C terminus of the
kinesin protein of Leishmania major in India

31. • Case definition for enrolling a subject
“A case presenting to a clinician with a fever of more
than two weeks duration, with splenomegaly and not
responding to the full course of anti-malarials”
• Sensitivity-100%, Specificity-97% (NVBDCP, 2010)
• Not to be used in the following cases:
 Kala-azar relapses
 In cases of kala-azar re-infection
 Kala-azar and HIV co-infection
• This test has been incorporated in NVBDCP for the
diagnosis of VL in India.

32. Prevention
• Suppress the reservoir: dogs, rats, gerbils, other small
mammals and rodents
• Suppress the vector: Sandfly
• Critical to preventing disease in stationary troop populations
• Prevent sandfly bites: Personal Protective Measures
• Most important at night
• Sleeves down
• Insect repellent w/ DEET
• Permethrin treated uniforms
• Permethrin treated bed nets

33. Treatment
Cutaneous and Mucocutaneous
• Antimony (Pentostam®, Sodium stibogluconate) is the drug
of choice
• Given under an experimental protocol at Walter Reed Army Medical
Center (WRAMC)
• 20 days of intravenous therapy
• Available at WRAMC for all branches of the military
• Requires patient to come to WRAMC
• Fluconazole may decrease healing time in L. major infection
• Biopsy and culture to determine species is required
• Six weeks of therapy is needed

34. Visceral Leishmaniasis
• Liposomal amphotericin-B (AmBisome®) is the drug of
choice
• 3 mg/kg per day on days 1-5, day 14 and day 21
• Pentostam® is an alternative therapy
• 28 days of therapy is required
• Although AmBisome® is widely available, the difficulty of
accurate diagnosis and the potential severity of visceral
infection suggest possible patients be referred to the
Leishmania Treatment Center at WRAMC for maximal
diagnostic efficiency

35. Vaccine
• There is as yet no effective vaccine for prevention
of any form of leishmaniasis.
• first generation vaccine was prepared using
whole killed parasites combined or not with BCG.
• Live: including new genetically modified
constructs
• 1st generation vaccines: whole killed parasite
with/without adjuvants or fractions of the
parasite
• 2nd generation vaccines: recombinant proteins,
DNA vaccines & combinations