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Leishmaniasis Dr.Pragati MDDr.Rubina MD

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What is Kala-Azar• Kala-azar means dark pigmentation which is

characteristic of cases of visceral leishmaniasis. It is caused by Leishmania donovani bodies and may be present either in endemic, epidemic or sporadic forms. It is widely prevalent in India in epidemic form in states of Bihar, Assam and Bengal. Kala azar found in East and North Africa is a disease of young children and young adults, being more common in males as compared to females.

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World distribution of Visceral Leishmaniasis

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kala azar, black fever, sandfly disease, Dum-Dum fever

SYNONYMS

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KALA AZAR• Leishmaniasis is a disease caused by protozoan

parasites of to the genus Leishmania and is transmitted by the bite of sand fly.

• Human infection is caused by about 21 of 30 species that infect mammals. These include the L. donovani complex with three species (L. donovani, L. infantum, and L. chagasi

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Pathogenesis• Infections range from asymptomatic to progressive,

fully developed kala-azar. • Incubation period is usually 2 – 4 months. • Symptoms – Begins with low-grade fever and

malaise, followed by progressive wasting, anemia, and protrusion of the abdomen from enlarged liver and spleen.

• Fatal after 2 – 3 years if not treated.• In acute cases with chills, fevers up to 104⁰ F and

vomiting; death may occur within 6 – 12 months. • Immediate cause of death is usually an invasion of a

secondary pathogen that the body is unable to combat.

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Leishmaniasis is Neglected Disease

• Leishmaniasis is a globally important but neglected disease, affecting approximately two million people every year. For most people, infection results in a slow-to-heal skin ulcer. In others, however, the parasite targets the liver, spleen and bone marrow, leading to over 70,000 deaths annually.

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The Parasite• Phylum

• Order

• Family

• Genus

Sarcomastigophora

Kinetoplastida

Trypanosomatidae

Leishmania

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Leishmania Parasites and DiseasesDisease SPECIES

Cutaneous leishmaniasisLeishmania tropica*Leishmania major*

Leishmania aethiopicaLeishmania mexicana

Mucocutaneous leishmaniasis Leishmania braziliensis

Visceral leishmaniasisLeishmania donovani*Leishmania infantum*

Leishmania chagasi

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CLASSIFICATION

Old world leishmaniasis

• Leishmania donovani• L.infantum• L.tropica• L.major• L.aethiopica

New world leishmaniasis

• L.braziliensis• L.mexicana complex• L.peruviana• L.chagasi

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• ALL the leishmania species are morphologically identical to each other.

• These are distinguished by• Intrinsic characters• Biochemical characters• Immunological characters

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Morphology

• Promastigote • Amastigote

Flagella

Kinetoplast

Golgi

Nucleus

Cytoskeleton

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Promastigote & Amastigote of Leishmania

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Morphology

• Promastigote– Insect– Motile– Midgut

• Amastigote– Mammalian stage– Non-motile– Intracellular

Digenetic Life Cycle

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

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Morphology and Life Cycle• Amastigotes

measure 2-3 micrometers, with a large nucleus and Kinetoplast.

• Amastigotes mainly live within cells of the RE system, but have been found in nearly every tissue and fluid of the body.

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Life cycle• The organism is transmitted by the

bite of several species of blood-feeding sand flies (Phlebotomus) which carries the promastigote in the anterior gut and pharynx. It gains access to mononuclear phagocytes where it transform into amastigote and divides until the infected cell ruptures.

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Sand fly- Vector

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Life cycle• The released organisms infect other

cells. The sand-fly acquires the organisms during the blood meal, the Amastigote transform into flagellate Promastigote and multiply in the gut until the anterior gut and pharynx are packed. Dogs and rodents are common reservoirs.

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Different stages of Haemoflagellates

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VL - Clinical Manifestation

Variable - Incubation 3-100+ weeksLow grade fever

Hepato-splenomegalyBone marrow hyperplasia

Anemia , Leucopenia & CachexiaHypergammaglobulinemia

Epistaxis , Proteinuria, Hematuria

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Hepatosplenomegaly in visceral leishmaniasis

• Pallor in hands of a visceral leishmaniasis patient.

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Laboratory diagnosis

• Blood counts: Anaemia, neutropenia, thrombocytopenia• A/G ratio reversed (n-4.5:2, in kala-azar-2.8:4)• Parasitological Diagnosis• PBF• Needle biopsy/aspiration• Culture• Animal inoculation• Immunological tests• Non-specific tests• Aldehyde tests• Antimony test• A:G Complement fixation test with W.K.K antigen

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• Immunological tests• Non-specific test• Aldehyde test• Antimony test• Complement fixation test with W.K.K antigen• Specific tests• Direct agglutination test(DAT)• Indirect haemagglutination test (IHA)• Indirect fluorescent antibody test(IFAT)• ELISA

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Parasitological diagnosis: Specimen :Bone marrow aspirate Splenic aspirate Lymph node aspirationTissue biopsy

Microscopy – PBF, Buffy coat smearCultureAnimal inoculation- golden hamester

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Bone marrow aspiration

Bone marrow amastigotes

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L. donovani bodies L. donovani bodies may be

demonstrated in buffy coat preparations of blood and bone marrow aspirate.

PBF is made with straight leucocytic edge

Aspirates taken from enlarged lymph nodes show parasites in 60 percent of cases.

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Culturing of the Parasite

• Organisms can be cultured in Nicolle-NovyMcneal (NNN media) media from clinical specimens obtained from splenic or bone marrow aspirates.

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Cultivation

• NNN medium-Biphasic media- 2 parts of salt agar+1 part of defibrinated rabbit blood, melted & cooled to 48⁰C ice, inoculated into water of condensation, incubated at 22-25⁰C for the 21 days.

• Hockmeyer medium- Insect cell culture medium+ foetal calf serum+ penicillin+ streptomycin( detected after 2-3 d)

• Schneider Dorsophila Medium

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Promastigote in culture in NNN medium (magnification 100×)

NNN culture medium

Promastigotes growing in culture medium Bone marrow aspirate -“rosette” of extra-cellular Promastigotes (Giemsa stain).

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Immunological Diagnosis:

• Specific serologic tests:• Direct Agglutination Test (DAT), • ELISA,• IFAT,• CFT for detection of antibodies• Rapid immunochromatic test(ICT): By using

rk39Ag• Molecular Methods: PCR & RT-PCR.

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Direct agglutination test • Direct agglutination test

(DAT) based on agglutination of the trypsinized whole promastigotes is useful in endemic regions. Its sensitivity ranges from 91-100% and specificity from 72 to 100%.

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ELISA• ELISA is an important

sero diagnostic tool for leishmaniasis. It is a highly sensitive test and its specificity depends upon the antigen used.

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Chromatographic strip test • A ready to use immuno

chromatographic strip test based on rk 39 antigen has been developed as a rapid test for diagnosis of kala azar. An important limitation of this test is the presence of antibodies in healthy controls hailing from endemic regions.

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• Non specific serological tests for hypergammaglobinemia

• Napier’s Aldehyde test: Patient’s sera is mixed with a drop of 40% formalin in a test tube. Positive test is indicated by jellification.

• Chopra’s antimony test: Positive test is by formation of profuse flocculation when patient ‘ sera is mixed with 4% urea stilbamine solution.

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Skin test ( leishmanin test or Montenegro test) It is a delayed hypersensitivity skin test for survey of populations and follow-up after treatment - 0.2 ml(6-10 million/ml of killed promastigotes in 0.5% phenol saline) injected—erythema ≥ 5mm→ +ve after 6-8 weeks of cure.

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The leishmanin skin test (LST) or MST (Montenegro skin test)

PROBLEMS:• Reference standards -antigens & performance (reading)- not developed. • Most antigens, crude extracts of parasites, neither sensitive nor specific.• Positivity not demonstrable until 5 months after the acute phase of VL-20%• May or may not be positive for PKDL.• Persistence of the lesions has been frequently associated with non reactivity in the

LST and high levels of anti-leishmanial antibodies.

A positive leishmanin skin test

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Treatment: • Pentavalent antimony (Pentostam)• Amphotericin B• Miltefosine• Interferon

Treatment of complications:• Anemia• Bleeding• Infections etc.

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Control

• Vector control• Reservoir control• Treatment of active cases• Vaccination

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Management of Kala-azar Patients

• It includes both supportive and curative. All patients of Kala azar should preferably be hospitalized. Any infection complicating the disease be treated by use of proper antibiotics. Nutrition must be maintained. Cases with severe anemia may require blood transfusions. Pentavalent antimony compounds are the drug of choice. Sodium antimony gluconate (Pentostam) is the most commonly used drug.

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Kala-azar prevention:

• Multipronged approach is needed.• Sand-flies are extremely sensitive to

insecticides & vector control through insecticide spray is very important.

• Mosquito nets or curtains treated with insecticides will keep out the tiny sand-flies.

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Kala-azar prevention:

• In endemic areas with zoonotic transmission, infected or stray dogs should be destroyed.

• In areas with anthroponotic transmission, early diagnosis & treatment of human infections, to reduce the reservoir & control epidemics of VL, is extremely important.

• Serology is useful for screening of suspected cases in the field.

• No vaccine is currently available .

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• NLCP-National leishmania control programme is to eradicate leishmania by 2015.

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Post-kalazar dermal leishmaniasis (PKDL)

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Post Kala Azar Dermal Leishmanoid

Normally develops <2 years after recoveryRecrudescence

Restricted to skinRare but varies geographically(10% of cases

in India and 3% of cases in Africa)

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PKDL•L.donovani•Non ulcerative skin lesin.•Late sequale of VL.•1-2 years after recovery fron VL.•Currently, PKDL is reported to occur in only 1% of Indian VL patient.

•3 major representations-(i) Erythematous indurated lesions on the butterfly area of face; (ii) Multiple symmetrical hypopigmented macules with irregular margins that may coalesce, having generalized distribution to the extremities and trunk; and (iii) Combination of papules, nodules and plaques.

Nodular skin lesions in PKDL

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Fig. Clinical presentation in PKDL (A) PKDL nodular and popular lesions on face and hypopigmented macules on neck in a patientwith polymorphic presentation (B) hypopigmented macules on trunk in a patient with macular PKDL. (C) Erythematous and induratedlesions over the butterfly area of face.

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Complications of PKDL

1. Blindness due to corneal involvement. 2. Recently, nerve involvement in Indian PKDL reportd.

• Nowadays – reported from South America, Europe & India.

• Recurrence of VL following PKDL reported.

• LD bodies rich nodular skin lesions -sole reservoir to disseminate in absence of zoonotic transmission.

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Grey areas in PKDL1. The factors of parasite/host origin -drive the parasite to shift from viscera to dermis. --It is

not known it is the residual parasite after VL infection, attributed to altered immune status or is introduced upon re-infection by sandfly vector.

Antimony therapy for PKDL patients needs to be continued for much longer duration than for VL patients (4 months instead of 4 wk for VL).

• Recently-High interleukin-10 (IL-10) levels in skin & peripheral blood. -High level of C reactive protein in plasma of patients with VL Predictive of the subsequent development of PKDL.

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THANKS