Hemoflagellates

Leishmania
Dr. Talal Saeed Alwajeeh

‫المحاضرة العاشرة‬
♦ Hemoflagellates are the flagellated protozoa that are found in peripheral blood
circulation.

♦ They complete their life cycle in two hosts, i.e. vertebrate host and insect vector.

♦ Hemoflagellates of medical importance belongs to the genus of Leishmania and

Trypanosoma.

♦ Leishmaniasis is caused by the obligatory intracellular protozoa of the genus

Leishmania. Primarily it affects the reticuloendothelial system of the host.

♦ Leishmania species produce widely varying group of clinical syndromes ranging

from self-healing cutaneous ulcers to fatal visceral disease.

♦ Leishmaniasis is mainly a zoonotic disease affecting dogs, foxes, jackals and

rodents.

♦ Animal reservoir plays a major role for transmission; except in some area where it
is anthropophilic affecting only humans.

♦ The parasite is transmitted by bite of the female sand fly vector.

♦ Three different diseases are caused by various species of genus Leishmania. These
are:

I. Visceral leishmaniasis: The species L. donovani complex infecting internal

organs (liver, spleen and bone marrow) of human is the causative parasite.

2. Cutaneous leishmaniasis: The species L. tropica complex, L. aethiopica, L.

major and L. Mexicana complex are the causative parasite.

3. Mucocutaneous leishmaniasis: It is caused by the L. braziliensis complex.

Distribution
♦ L. donovani (Old world
leishmaniasis) causes visceral leishmaniasis or kala azar
(a hindi term meaning “black fever”) which is a major public health problem in
many parts of the world. It also causes the condition, Post-kala-azar dermal
Leishmaniasis (PKDL).

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♦ Leishmania tropica and L. major are found in Middle East, India, Afghanistan,
Eastern Mediterranean countries and North Africa.
♦ L. aethiopica occurs in Ethiopia, Uganda and Kenya.
♦ Leishmania braziliensis complex and L. mexicana complex cause new world
leishmaniasis in Central and South America.

Habitat
♦ In human, the amastigotes of L. donovani is found in the reticuloendothelial
system. They are found mostly within the macrophages in the spleen, liver, bone
marrow and less often in other locations such as skin intestinal mucosa and
mesenteric lymph nodes.
♦ The amastigote forms of Leishmania tropica Complex reside in reticuloendothelial
cells of skin (they do not migrate to viscera), whereas promastigote forms are seen
in sand fly vector.
♦ The amastigote form of Leishmania braziliensis complex and L. mexicana
complex is seen inside the macrophages of skin and mucous membrane of the nose
and buccal cavity. The promastigote form occurs in vector species.

Morphology
The parasite exists in 2 forms:
1. Amastigote form is found in humans and other mammals.
2. Promastigote form is found in the sand fly and in culture.
Amastigote form
• The amastigote form is an ovoid or rounded cell, about 2–4 μm in size.
• It is intracellular, found inside macrophages, monocytes, neutrophils, or
endothelial cells and is the infective stage to vector, sand fly.
• There is no external flagellum and it is nonmotile.
Nucleus: It measures less than 1 μm, oval to round, located in center or side of the
cell.
Kinetoplast: Consists of copies of mitochondrial DNA. It lies at right angle to the
nucleus.
Axoneme: It represents the intracellular portion (root) of flagellum.

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 Amastigote form of L. donovani

Promastigote form
• Promastigote is an extracellular form, infective stage to humans.
• It is a flagellate (motile) and is present in sand fly and in culture.
• It is long, spindle shaped, 15–25 μm in length and 1.5–3.5 μm in breadth.
• Nucleus is situated centrally
• kinetoplast is placed near the anterior end transversely.
• Axoneme represents the intracellular portion of flagellum.

Promastigote form of L. donovani

Life cycle
• L. donovani completes its life cycle in two hosts:
1. Definitive host: Man, dog and other mammals.
2. Vector: Female sand fly (Phlebotomus species)
Infective form: Promastigote form present in midgut of female sand fly.
Mode of transmission:
• Humans acquire by bite of an infected female sand fly.

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• It can also be transmitted vertically from mother to fetus, by blood transfusion and
accidental inoculation in the laboratory.
Incubation period: Usually 2 - 6 months.
In vertebrate hosts, including humans:
• Promastigotes are directly injected from foregut (proboscis) of the female sand
fly into the skin of the vertebrate host.
• Promastigotes are phagocytosed by the skin macrophages and transform into
amastigote forms within 12–24 hours.
• The amastigote forms inside the macrophages multiply further causing cell
rupture and release into the circulation.
• Amastigotes are carried out in the circulation to various organs like liver, spleen
and bone marrow and invade the reticuloendothelial cells like macrophages,
endothelial cells, etc.
In sand fly:
• Sand fly taken amastigotes during the blood meal, the amastigotes are ingested
and transformed into promastigote forms in the insect midgut.
• Promastigotes multiply by longitudinal fission and pass through various stages
such as:
Amastigote → procyclic promastigote → nectomonad promastigote →
haptomonad promastigote → leptomonad promastigote → metacyclic
promastigote.
• The metacyclic promastigotes multiply in the midgut of vector and a small
proportion migrates to the foregut (proboscis). They infect a new host during
another blood meal.
• The duration of the life cycle in sand fly varies from 4 to 18 days depending on
the species.

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Pathogenesis and Clinical Features
• Leishmania donovani causes visceral leishmaniasis or kala azar. The parasitized
macrophages disseminate the infection to all parts of the body.
• It causes hepatosplenomegaly and lymphadenopathy.
• The bone marrow is heavily infiltrated with parasitized macrophages causing
pancytopenia (Severe anaemia, Leukopenia with marked neutropenia and
thrombocytopenia).
• Severe anaemia may occur in kala azar, as a result of infiltration of the bone
marrow as well as by the increased destruction of erythrocytes due to
hypersplenism.
• Patients usually present with fever and weight loss.
• Some patients with visceral leishmaniasis in endemic areas may develop post
kala azar dermal leishmaniasis (PKDL), about a year or 2 after recovery from
the systemic illness.

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• PKDL is seen mainly in India and East Africa and is a nonulcerative lesion of
skin.

(A) (B)
Real images showing clinical features
(A) Splenomegaly seen in visceral leishmaniasis
(B) Hypopigmented skin changes in early PKDL

Extensive facial nodular lesions in late PKDL

Cutaneous leishmaniasis
• It is caused by L. tropica complex.
• This condition is also known as “Oriental sore”.
• Oriental sore usually occurs on face and hands.
• It begins as papule, becomes nodular and finally it ulcerates.
• The margins of the ulcers are raised, painless and indurated.

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 Real images showing clinical features of Cutaneous leishmaniasis

Leishmaniasis recidivans
• It is a granulomatous response occurs years after healing of primary sore due to
L. tropica.
• Characterized by new lesions formed on the face, usually scaly, erythematous
papules and nodules develop in the center or periphery of a previously healed sore.

Real images showing clinical features of Leishmaniasis recidivans

Diffuse cutaneous leishmaniasis

• It is a rare form of leishmaniasis, non-ulcerative and often diffuse lesions caused
by L. aethiopica in Ethiopia and Kenya (old World).

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 Real images showing clinical features of Diffuse cutaneous leishmaniasis

• Leishmania mexicana complex causes cutaneous leishmaniasis which closely

resembles the old world cutaneous leishmaniasis. However, a specific lesion
caused by L. mexicana is chiclero ulcer which is characterized by ulcerations in
pinna.

Real image showing chiclero ulcer in pinna

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• L. braziliensis complex causes both cutaneous and mucocutaneous
leishmaniasis. It causes the most severe and destructive form of cutaneous
lesion. It involves the nose, mouth and larynx.

Real image showing mucocutaneous leishmaniasis

Laboratory Diagnosis

1. Microscopic examination

♦ Demonstration of amastigotes in blood smears, tissue aspirates (bone marrow,

spleen, lymph nodes), and from indurated edge of the lesions of skin and mucous
membrane is the gold standard for diagnosis.

♦ Fixed smears can be stained with Leishman, Giemsa, or Wright's stains to

demonstrate amastigotes.

Leishmania amastigotes

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2. Culture

♦Aspiration from the ulcers, tissue specimens or blood are cultured in Novy-
MacNeal-Nicolle (NNN) medium for the isolation of promastigote forms.

NNN medium Promastigote forms

3. Serological diagnosis

♦ By Detection of antigen & antibodies in the serum or other body fluids by IFA
test and ELISA.

4. Skin test : Leishmanin skin test (Montenegro test)

♦ It is a delayed hypersensitivity skin test to a suspension of killed L. donovani

promastigote injected intradermally

♦ A Positive leishmanin skin test indicates delayed hypersensitivity reaction to the

parasite by induration of more than or equal to 5 mm in 72 hours.

♦ Positive test indicates prior exposure to Leishmania antigens.

5. Animal inoculation

♦ Intranasal inoculation of specimens to golden hamsters yields amastigotes after

several months.

6. Biopsy
♦ Skin biopsy from the margin of nodular lesions or from the margin of ulcer to
demonstrate amastigotes.

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Blood picture:
♦ Complete blood count (CBC) to detect pancytopenia.
♦ Leukocyte count reveals leukopenia accompanied by a relative increase of
lymphocytes and monocytes.
Nonspecific tests:
♦ Serum shows hypergammaglobulinemia.
♦ Liver function tests show mild elevations of liver enzymes.
♦ Erythrocyte sedimentation rate (ESR) is elevated.

Treatment
1. Pentavalent antimonial compound
♦ It is the drug of choice in most endemic regions of the world.
♦ Two pentavalent antimonial preparations are available:
►Sodium stibogluconate (100 mg)
► Meglumine antimoniate (85 mg)
Dosage: The daily dose is 20 mg/kg by rapid intravenous (IV) infusion or intramuscular (IM)
injection for 20 - 30 days.
2. Amphotericin B is the best alternative drug.
Dosage: 0.75 - 1.0 mg/kg on alternate days for a total of 15 infusions.
3. Paromomycin
Dosage: It is given in a dose of 11 mg/kg daily for 21 days.
4. Miltefosine
Dosage: 50 mg daily for 28 days for patients weighing less than 25 kg, and twice
daily for patients weighing more than 25 kg.
Prevention and Control

1. Early detection and treatment of cases.

2. Insecticide spraying.
3. Control of animal reservoir hosts.
4. Use of protective clothing, bed nets, window mesh or insect repellants.

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