DR. SANJAY SINGH NEGIASSISTANT PROFESSOR, DEPARTMENT OF MICROBIOLOGY, AIIMS, RAIPUR

LEISMANIASIS

kala azar, black fever, sandfly disease, Dum-Dum fever and espundia.

SYNONYMS

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.

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PROMASTIGOTESAMASTIGOTES

Leishmania

Phylum

Order

Family

Genus

Sarcomastigophora

Kinetoplastida

Trypanosomatidae

Leishmania

• Transmitted to the mammalian hosts by the bite of infected sandflies, Phlebotomus and LutzomyiaIn India: Phlebotomus argentipus

• 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)

Leishmania distribution (all species)

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, Assam, Orissa, Tamil Nadu, Uttar Pradesh

• Estimated 165.4 million population at risk in 4 states

(NVBDCP, 2010)

Vector: Phlebotomus

procyclics and metacyclics• 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

(attached)

(detached)

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

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

Pathogenicity of Leishmania donovani(Visceral leishmaniasis• 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%

Hepatosplenomegaly and emaciation.

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

Post Kala Azar Dermal Leishmaniasis• 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

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 distributionIncubation 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 dogVL 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

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: DogsClinical 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)

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

• Promastigote– InsectInsect

– MotileMotile

– MidgutMidgut

• Amastigote– Mammalian stageMammalian stage

– Non-motileNon-motile

– IntracellularIntracellularFlagella

Kinetoplast

Golgi

Nucleus

Cytoskeleton

Leishmania donovani Leishmania donovani MorphologyMorphologyDigenetic Life CycleDigenetic Life Cycle

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

• A macrophage filled with Leishmania amastigotes.

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

Susceptible Animal: Dog naturally infected with L.donovani

Common laboratory animals mice, rats and guinea pigs not susceptible.

Hamster very susceptible.

Leishmania infects and thrives in macrophages

• 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 ...

Leishmania parasites exist Amastigote & Pro mastigote)

• The parasite lives in the digestive tract of sandflies as extracellular promastigote

• In the mammalian host parasites multiply as intracellular amastiogotes

LIFE CYCLE

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.

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.

DIAGNOSIS

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

MICROSCOPY

CULTURECulture media for axenic culture• SOLID MEDIUM

NNN medium (Novy, MacNeal & Nicolle)

(2ml of patient blood + 10ml of Citrated saline kept at 220 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

Promastigotes as seen in artificial culture medium

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)

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)

Direct Agglutination Test

• 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

• Relative long incubation time of 18 hours

• Need for serial dilutions of serum

• No prognostic value• Remain positive for several

years after cure

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)

ELISA BASED ASSAYS

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

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

GOAL OF NATIONAL HEALTH POLICY (INDIA) 2002

ELIMINATION OF KALA AZAR 2010

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.

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