Presented by:

Mohammed Adil Sarvar

LEISHMANIA

Leishmania is a genus of trypanosomatid protozoa, which causes a fatal vector-borne parasitic disease called Leishmaniasis .

It is spread by the bite of sandflies of the genus Phlebotomus in the Old World, and of the genus Lutzomyia in the New World.

Leishmaniasis is the second-largest parasitic killer in the world (after malaria) and is endemic in many parts of Africa, Asia and South America.

INTRODUCTION

The parasite was named by Ronald Ross in 1903 after the Scottish pathologist William Boog Leishman.

In 1901, Leishman identified the organism in smears taken from the spleen of a patient who had died from "dum-dum fever“.

HISTORY

Protista

Sarcomastigophora

Protozoa

Mastigophora

zoomastigophora

Kinetplastida

Leishmania

donovani, tropica, mexicana, braziliensis, etc.

Kingdom

Subkingdom

Phylum

Subphylum

Class

Order

Genus

Species

CLASSIFICATION

IMPORTANT SPECIES

• L. donovani• L. tropica• L. mexicana• L. braziliensis

• L.major • L.guyanensis• L.lainsoni• L.naiffi• L.aethiopica, etc

L.donovani

L. tropica L.mexicana

L. braziliensis

Parasites of

Visceral organs

Skin Skin Skin and mucus membrane of nose and buccal cavity

Amastigote form found in

HumanReticuloendothelial cells of •spleen,• bone marrow ,• liver •intestinal mucosa

Human•Reticuloendothelial cells of skin

Human•Reticuloendothelial cells of skin

Human•Macrophage of skin•Mucous membrane of nose and buccal cavity

Promastigote form found in

Sand fly and culture

Sand fly and culture

Sand fly and culture

Sand fly and culture

HABITAT OF OTHER SPECIES

The parasite exists in 2 forms;-

1. Amastigotes – aflagellar stage

2. Promastigotes- flagellar stage

MORPHOLOGY(same in all species)

Amastigotes Promastigotes

Flagellar stage

Occurs in the sand fly

divides by binary fission at 27oC.

They are spindle shaped ;15-20 µm in length & 1-2µm in width.

Nucleus smaller and situated in the middle of the cell or along the side of cell-wall.

Kinetoplast lies transversely near the anterior end.

Aflagellar stage

Occurs in the vertebrate host

divides by binary fission at 37oC.

There are round or oval ;2-4µm along longitudinal axis.

Nucleus relatively larger and situated centrally.

Kinetoplast situated right angle to nucleus.

Morphological Differences

Life cycle of other species of Leishmania are similar to L.donovani except that

In L.tropica• amastigotes reside in the large

mononuclear cells of the skin

In L.mexicana• Amastigotes found in

reticuloendothelial cells and lymphatic tissues of skin

In L.braziliensis • amastigotes are found in

reticuloendothelial cells and lymphatic tissues of skin and mucus membrane

L.donovani L.tropica L.mexicana L.braziliensis

Reservoir Man, rodents, foxes, dogs

Man,Dog

Sloth, ant eater, rat, dog

Sloth, ant eater, rat, dog

Vector Sand flyPhlebotomus argentipus

Sand fly Phlebotomus argentipus

Sand fly Lutzomyia spp.,

Sand fly Lutzomyia spp.,

Mode of transmission

•Bite of sand fly•blood transfusion•Congenital infection •sexual intercourse

Bite of sand fly

•Bite of sand fly, •Bite of ticks ,•autoinfection

•Bite of sand fly, •Bite of ticks ,•autoinfection

Individual at risk

Males are affected more

Adolescents and young adults

Persons working at the edge of forest and in the people staying in rural areas.

Persons working at the edge of forest and in the people staying in rural areas.

Reservoir, vector and transmission of other species

VECTOR (Sand fly)

• Phlebotomas • Lutzomyia

1. Pyrexia

2. Spleen enlargement

3. Lymphadenopathy

4. Darkening of the skin (KALA AZAR, MEANING “BLACK FEVER” IN HINDI, BECAUSE OF ITS TENDENCY TO DISCOLOR ITS VICTIM’S COMPLEXION DURING ADVANCED STAGES)

5. Others:- kala-azar with HIV co-infection

Post kala-azar dermal leishmaniasis(PKDL)

Complications:- pneumonia, TB, dysentery, uncontrolled haemorrhage

Prognosis:- With an early treatment, cure rate >90%

If not treated, death occurs within 2 years.

CLINICAL MANIFESTATIONS

Leishmaniasis is divided into clinical syndromes according to what part of the body is affected most.

TYPES OF LEISHMANIASIS

Visceral Leishmaniasis(VL)

Cutaneous Leishmaniasis(CL)

Mucocutaneous leishmaniasis(MCL)

1. Visceral Leishmaniasis (VL)

or Kala-azar

caused by L.donovani

part of the body affected most is internal organs

Continued....

Spleenomegaly

2. Cutaneous Leishmaniasis(CL)

( most common type)

a) Old world CL:- caused by L.tropica, L. aethiopica

b) New world CL:- caused by L.mexicana, L.braziliensis, L.guyanensis

c)Dermal leishmanoid or Post kala-azar dermal leishmaniasis(PKDL):- caused by L.donovani

Part of the body most affected is skin

Continued....

....continued

3. Mucocutaneous leishmaniasis(MCL)

Caused by L. braziliensis and occasionally by L.panamensis

Part of the body affected most is skin and mucous membrane of nose and pharynx

LABORATORY DIAGNOSIS

Direct Evidences

Peripheral blood by thick film method.(Amastigote form)

Blood culture in N.N.N. Medium.

(Promastigote form)

Biopsy material obtained by lymph

node puncture, sternal or iliac crest

puncture(marrow) and spleen

puncture(spleen pulp) only for L. donovani

Indirect evidences

Blood count

Serum Tests

Other methods

Animal inoculatio

n

Leishmanin or

Montenegro Test

Adler’s test

Romanowsky stains are universally employed for routine staining of blood films.

It depends on two components, namely, azure B(trimethylthionin) and eosine Y(tetrabromo-fluorescein).

The original Romanowsky combination was polychrome methylene blue and eosine.

The presence of basic grouping on the haemoglobin molecule results in its affinity for acidic dyes and its staining by eosin. The nucleic acids uptake of the basic dye azure B.

STAINING

Types of Romanowsky stains LEISHMAN’S STAIN

• Leishman staining is used for staining blood in microscopy and its purpose is to both identify and differentiate trypanosomas, leucocytes and malaria parasites.

GIEMSA STAIN

• The most dependable stain for blood parasites, particularly in thick films, is Giemsa stain containing azure B

COMPONENTS:

1.Methanol : fixes cells to slide

2.methylene blue stains RNA,DNA

blue-grey color

3.Eosin stains hemoglobin, eosin granules

orange-red color

pH value of phosphate buffer is very important

Leishman's stain

COMPONENTS:

1. METHYLENE BLUE

2. AZURE B

3. SODIUM SALT OF EOSIN Y

GIEMSA stain

Buffy coat : Finding Leishmania parasites in blood is sometimes possible in patients with Kala-azar from India.

Blood in anticoagulant is centrifuged at 2000 g for 10min and the cells from the buffy coat removed and used to prepare smears and inoculate cultures.

Amastigotes can be found in and around macrophages.

DETECTION OF LEISHMANIA IN PERIPHERAL BLOOD SMEAR

Looking for parasites in the spleen and liver is one of the most accurate methods available to determine Leishmania infections.

Splenic aspirate is preferred to liver biopsy.

Ninety percent of active cases show parasites in splenic aspirates.

Part of the splenic aspirate can be used to make smears for direct microscopic examination and the rest should be cultured.

Splenic aspirate and Liver biopsy

Bone marrow aspiration is a safer

method than splenic aspirate or liver

biopsy.Lymph node puncture gives

positiveresults in 60% of cases

where lymph nodes are enlarged.

Bone Marrow Aspirate and Lymph Node Puncture

LEISHMANIA – HIV CO INFECTION

The diagnostic principles remain essentially the same as those for non-HIV-infected patients.

The presence of amastigotes may be demonstrated in buffy coat preparation.

Sometimes the presence of amastigotes in unusual sites may be demonstrated (e.g., amastigotes may be present in specimens from bronchoalveolar lavage, pleural fluid, or biopsy specimens from the gastrointestinal tract).

Drugs

Sodium stibogluconate solution

Amphotericin B

Pentamidine

Miltefosine

Interferon

TREATMENT

PREVENTION AND CONTROL(.....contd.)