1. LEISMANIASIS
DR. SANJAY SINGH NEGI
ASSISTANT PROFESSOR, DEPARTMENT OF MICROBIOLOGY, AIIMS, RAIPUR

2. SYNONYMS
kala azar, black fever, sandfly disease,
Dum-Dum fever and espundia.
1903 – Sir William Leishman discovered L.
donovani in spleen smears of a soldier who died
of fever at Dum-Dum, India. The disease was
known locally as Dum-Dum fever or kala-azar.
1903 – Charles Donovan found same parasite
in a spleen biopsy.
•Cultured by Rogers(1904)
•Amastigote and promastigote forms were
described by Batton(1907)
•Phlebotomus argentipes identified as vector.

7. AMASTIGOTES
PROMASTIGOTES

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

9. • 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, India(Central & Western India)
• 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)

10. Leishmania distribution
(all species)

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

12. Vector: Phlebotomus

13. procyclics and metacyclics
(detached)
(attached)
• Infected macrophages are
taken up with the blood
meal and amastigotes are
released by digestion,
transform into procyclic
promastigotes and attach to
the midgut epithelium
• Attached promastigotes
divide rapidly (procyclics
are not infective to
mammals)
• Metacyclic (infective)
promastigotes cease
replication, detach and pass
forward into the pharynx
from where they are
regurgitated into the bite
site

14. TYPES OF LEISMANIASIS
• VISCERAL LEISHMANIASIS or Kala-azar ( Middle east,
Africa, Bangladesh, Brazil, India,China, South America, Europe,
Nepal and Sudan)
•
Post kala azar dermal leishmaniasis (Endemic to India and the
Sudan)
• India(Bihar, West bengal, Orissa, Assam, Tamil Nadu, Gujarat,
Punjab & Jammu)
• Species responsible: L.donovani
• Vector: P.argentipes
• Resvoir: Man

15. Other species causing Visceral Leishmaniasis
Leishmania infantum: Cause Zoonotic visceral leishmaniasis
(ZVL) in Mediterranean areas, Middle east, and China
Reservoir: Dogs, foxes and jackals
Leishmania chagasi: Zoonotic visceral leishmaniasis(ZVL) in
New World.
Reservoir: Dogs and foxes

16. Pathogenicity of Leishmania donovani(Visceral
leishmaniasis
•
•
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Weeks to months incubation period.
May exceed one and sometimes two years.
Clinical features.
High fever. Fever often oscillates with a
peak every second day
The lead symptom is abdominal swelling
due to hepato- and splenomegaly
Lymphadenopathy
Progressive drastic weight loss (kachexia).
Epistaxix(presenting symptom)
In fully developed cases, emaciation and
anaemia become noticeable.
Darkening of the skin
Mortality of untreated disease 75-95%

17. Hepatosplenomegaly and emaciation.

18. • Enlarged spleen and liver in an autopsy of an
infant dying of visceral leishmaniasis.

19. Post Kala Azar Dermal Leishmaniasis
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Non ulcerative cutaneous lesion prevalent in
endemic areas of kala azar in India.
In 10 % treated cases of Kala azar, Normally
develops <2 years after recovery(When Visceral
infection disappears but skin infection persists).
Clinical feature:
Depigmented macules: earliest lesion, trunk,
extremities and face
Erythematous patches: Nose, cheeks and chin
Yellowish pink nodules: Nodules mostly on face
& are soft, painless granulomatous growth of
varying sizes(Absences of ulceration is a
noticeable feature)
Do not heal spontaneously.
Recrudescence
Restricted to skin
Extensive nodular
lesion, resembling
lepromatous leprosy.
A very resistant case
cured after long
continued treatment

20. CUTANEOUS LEISHMANIASIS (Oriental Sore /Old World
Cutaneous Leishmaniasis PKDL):
(Middle east, Mediterranean areas, N.Africa, N.W. India and Pakistan)
Cause dry type of cutaneous lesion(non-ulcerating type)
Urban distribution
Incubation period (2 months to > year)
Lesion usually facial. Ulcer starts as small itching papule covered with
fine whitish scale which subsequently becomes thick, dark and finally
falls off. The lesion may be found on the face, feet, legs and arms.
Children are usually affected.
Reservoir: man, domestic dog
VL in exceptional cases
Species: Leismania tropica
Leishmania major: Cause a moist type local cutaneous lesion(Ulcerating
type).
Ulcer found on extremities with regional lymphadenitis. Incubation period
2-6 weeks

21. Leishmania tropica (Cutaneous Leishmaniasis or Oriental Sore or
Tropical Sore)
Life Cycle: Same as L.donovani except
amastigote
form resides
in
large
mononuclear cells of the skin and not in the
viscera.
Reservoir: Dogs
Clinical features: Cutaneous lesion begins
as a raised nodule, its ulcerates, heal
spontaneously taking about 6 months or
more by Ulcer filled up by granaluation
tissues and a depressed white scar is often
left.
Laboratory Diagnosis:
Smear made from specimen obtained by
puncture of indurated edges of the sore and
stained by Leishman method.
Leishmanin reaction
Oriental Sore on the face)

22. DIFFUSE CUTANEOUS LEISHMANIASIS / MUCO
CUTANEOUS LEISHMANIASIS / New world Cutaneous
Leishmaniasis OR ESPUNDIA
Geographical Distribution: Central and South America
(Bolivia, Brazil,Argentina, Columbia,Mexico, panama,
Paraguay, Venezuela and Peru.
Species: Leishmania braziliensis
Clinical feature: Two stages primary cutaneous lesion
followed by secondary mucosal involvement which occurs
after a variable time of latency of primary cutaneous lesion
Nasal mucous membrane, pharynx, larynx & upper lip are
involved.
mouth
and throat cavities
Develops in 5 % patients suffering from primary cutaneous lesion.
Diagnosis by demonstrating amastigote forms of L.brazilensis in skin
and mucocutaneous lesions

23. Leishmania donovani
Morphology
• Promastigote
–
–
–
Insect
Motile
Midgut
Digenetic Life Cycle
Flagella
Kinetoplast
Golgi
Nucleus
Cytoskeleton
• Amastigote
– Mammalian stage
– Non-motile
– Intracellular

24. • Leishmania
(Leishman-Donovan
or LD bodies). Lying
in macrophage cells
from liver. Giemsa.
×12000. Enlarged by
9.6.

25. • A macrophage
filled with
Leishmania
amastigotes.

26. • Amastigotes (*)
of Leishmania
donovani in the
cells of a spleen.
The individual
amastigotes
measure
approximately 1
µm in diameter.

27. Susceptible Animal: Dog naturally infected with L.donovani
Common laboratory animals mice, rats and guinea pigs not
susceptible.
Hamster very susceptible.

28. Leishmania infects and thrives in
macrophages
•
•
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Macrophages are important
“microbe killers”, however
several pathogens have found
ways to escape killing
Trypansoma cruzi -- induces
phagocytosis but then escapes
into the cytoplasm
Toxoplasma -- active invasion,
parasitophorous vacuole is
never part of the endocytic
pathway
Mycobacterium tuberculosis -induce phagocytosis and block
lysosomal maturation
Leishmania ...

29. Leishmania parasites exist Amastigote & Pro
mastigote)
•
The parasite lives in the
digestive tract of sandflies as
extracellular promastigote
• In
the
mammalian
parasites
multiply
intracellular amastiogotes
host
as

30. LIFE CYCLE

31. 1. Leishmaniasis is transmitted by the bite of female
phlebotomine sandflies. The sandflies inject the
infective stage, promastigotes, during blood meals.
2. Promastigotes that reach the puncture wound are
phagocytized by macrophages.
3.They transform into amastigotes.
4. Amastigotes multiply in infected cells and affect
different tissues.
5. Sandflies become infected during blood meals on
an infected host when they ingest macrophages
infected with amastigotes.
6. In the sandfly's midgut, the parasites differentiate
into promastigotes.
7. They multiply and migrate to the proboscis.

32. Other method of transmission
Congenital infection of a child in utero
Transmission by blood transfusion
Transmission by inoculation of cultures of L.donovani
Possibly transmission during coitus.

33. DIAGNOSIS

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

37. MICROSCOPY

38. CULTURE
Culture media for axenic culture
• SOLID MEDIUM
 NNN medium (Novy, MacNeal & Nicolle)
(2ml of patient blood + 10ml of Citrated saline kept at 22 0 C
overnight and deposit inoculated into the water of
condensation of NNN medium and incubated at 220 C for
1 to 4 wks
 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

39. Promastigotes as seen in artificial culture medium

40. IMMUNOLOGICAL METHODS (Indirect Evidence)
• Leishmanin test: 0.1 to 0.2 ml of a suspension(having 6 to 10 million
promastigotes/ml) injected intradermally. Positive reaction after 72
hours in cured kala azar cases 6 to 8 wks after recovery.
• Blood Count: Leucopenia(Neutropenia)
• Aldehyde (formol gel) Test (Napier)(To test the rise of gamma globulin):
1 to 2 ml of a serum + one or two drop of 40% formalin: Jellification of
milk white opacity
• Complement fixation test with W.K.K Ag.(Not used nowadays).
• Other Serological tests: CIEP, IHA, IFA(Most Commonly Used), ELISA,
Direct agglutination test and Latex particle agglutination test)
• Molecular(PCR)

41. 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)

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

43. 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)

44. ELISA BASED ASSAYS

45. 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

46. 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

47. GOAL OF NATIONAL HEALTH POLICY
(INDIA) 2002
ELIMINATION OF KALA AZAR
2010

48. TREATMENT
• SODIUM ANTIMONY COMPOUND: SODIUM ANTIMONY
GLUCONATE(SAG 600mg daily for 6-10 days IV route)
• PENTAMIDINE ISTHIONATE
• AMPHOTERICIN-B
• Phase III Trials with a first-generation vaccine (killed
Leishmania organism mixed with a low concentration of
BCG as an adjuvant) have also yielded promising results
•
Leishmania major mixed with BCG have been successful
in preventing infection with Leishmania donovani.

49. 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
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Most important at night
Sleeves down
Insect repellent w/ DEET
Permethrin treated uniforms
Permethrin treated bed nets