Leishmaniasis - By Dr/ Faiz Al-Khawlani

MedicalParasitology

فایز الخوالني/ د

•The Most CommonParasitic Infections

In Yemen

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Tissue protozoa Leishmaniasis2014-2015

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Leishmaniasis

Leishmaniasis: is a vector-borne disease that transmitted by sandflies

and caused by obligate intracellular protozoa of the genus Leishmania.

About 21 of 30 species cause human infection. The different species are

morphologically indistinguishable, but they can be differentiated by

isoenzyme analysis, molecular methods, or monoclonal antibodies. The

following table summarizes the clinical diseases caused by the most

important Leishmania species.

Disease

Leishmania species

Visceral

leishmaniasis

L. donovani L. infantum L. chagasi

Cutaneous

leishmaniasis

L. tropica L. major L. aethiopica L. mexicana

Diffuse-cutaneous

leishmaniasis

(DCL)

L. aethiopica L. mexicana L. mazonensis

Muco-cutaneous

leishmaniasis

(MCL

L. braziliensis L. panamensis


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Epidemiology and Distribution

Leishmaniasis is endemic in 88 countries with an estimated 350 million

people at risk of infection. The overall prevalence of the leishmaniasis

estimated to be at 12 million cases with 0.5 million new visceral

leishmaniasis cases per year and 1.0–1.5 million new cutaneous

leishmaniasis cases per year. More than 90% of visceral leishmaniasis

occurs in Sudan, Bangladesh, Nepal, Brazil and India, and more than

90% of cutaneous leishmaniasis in Brazil, Peru, Afghanistan, Syria, Iran,

Yemen and Saudi Arabia. In recent years, there have been major

epidemics of visceral leishmaniasis in southern Sudan, eastern India,

Bangladesh, and Brazil. Increased infections and the spread of

leishmaniasis is related to environmental and behavioral changes and

development, conflict and war, bringing non-immune people into closer

contact with vectors and reservoir hosts.

Transmission and life cycle

□ Leishmania is mostly zoonotic (transmitted to humans from animals),

and humans become infected only when accidentally exposed to the

natural transmission cycle.

□ However, humans are the sole reservoir hosts when the transmission

occurs from human to human through the sand fly vector.


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□ Leishmania species are transmitted by the bite of an infected female

sandfly, belonging to the genus Phlebotomus in Africa, Asia and

Europe, and the genus Lutzomyia in the Americas.

□ About 30 species of sandflies act as vectors, infecting humans and

animal reservoir hosts.

Pattern of transmission

□ Human-to-human transmission: man is the only source and

reservoir of infection.

□ Dog-to-human transmission: The infection source is domestic dogs

and some rodents, which acts as a reservoir host.

Classification of leishmaniasis according to the location

Leishmaniasis

OLD WORLD

(Africa, Asia, Europe)

Leishmania species

NEW WORLD

(South America and Central

America)

Leishmania species

Visceral leishmaniasisL. donovani

L. infantum

L. chagasi

Cutaneous leishmaniasisL. tropicaL. major

L. aethiopica

L. guyanensisL. amazonensis

L. Mexicana

Diffuse cutaneous leishmaniasis L. aethiopicaL. Mexicana

L. amazonensis

Mucocutaneous leishmaniasis L. braziliensis

L. panamensis


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Sandfly vectors

The feeding, breeding and flight habits of sandflies are species specific.

Most sandflies feed mainly on plant juices, but female flies also require

blood meals for egg development. Most species feed at night, dusk or

dawn.

Morphology

The parasite exists in two forms:

1. Amastigote

2. Promastigote

Amastigote

Amastigotes are round in man and other vertebrate hosts. Amastigotes

live inside monocytes, polymorphonuclear leucocytes. They are small,

round to oval bodies measuring 2.9-5.9 μm in length (Fig below). They

are stained well with Giemsa’ or Wright’ stain. In the stained preparation,

the cytoplasm appears pale-blue and surrounded by a limiting membrane.

The nucleus relatively is large and stained red. The kinetoplast lies at

right angle to the nucleus. It is slender, rod-shaped and is stained deep

red. Axoneme arises from the kinetoplast and extends to margin of the

body. Vacuole, which is a clear unstained space, lies alongside the

axoneme.


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Leishmania. Amastigote

Promastigote

Promastigotes are excited in the digestive tract of sand fly (vector) and in

the culture media. The fully developed promastigotes are long, slender

and spindle-shaped. They measure 14.3 to 20μm in length and 1.5to 1.8

μm in breadth. A single nucleus lies at the center. The kinetoplast lies

transversely near the anterior end. The flagellum is single, delicate and

measures15-28 um. With Leishman stain, cytoplasm appears blue, the

nucleus pink and the kinetoplast blight red (Figure below).


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Fig. ( ) Leishmania species Promastigote

Life cycle in man

The life cycle of Leishmania species is summarized in Fig below.

It consists of two forms: amastigote, which presents in the human

macrophages and promastigote, which presents in the sandfly and

culture media.

Life cycle of Leishmania species involves two hosts: human host

(vertebrate host) and insect host (vector host, invertebrate host).

Because it is not identified the sexual stages of the parasite, the

definitive host is not recognized yet.

The life cycle begins with injection the infective stage metacyclic

promastigote into the human host at the time of taking blood meal by

the female sandfly.

The skin macrophages phagocytize the promastigotes by a process of

phagocytosis then transform into intracellular forms called

amastigotes.


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Amstigotes multiply within skin macrophages (in case of cutaneous

leishmaniasis), liberate from the macrophages, and infect new cells.

In visceral leishmaniasis, the amastigotes multiply in the

macrophages of the spleen, liver, bone marrow and lymph glands

of the reticuloendothelial system.

Blood monocytes are also infected.

Life cycle in sandfly

When intracellular and free amastigotes are ingested by a female

sandfly the life cycle is continued

After about 72 hours, the amastigotes become flagellated

promastigotes in the midgut of the sandfly.

They multiply and fill the lumen of the gut.

After 14–18 days (depending on species), the promastigotes move

forward to the head and mouth-parts of the sandfly.

Sandfly the leishmaniasis vector


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Injection of metacyclic promastigote the infective stage into the human skin

Life cycle of Leishmania


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Symptoms of Visceral leishmaniasis (VL)

□ This is the most severe form of leishmaniasis.

□ It is caused by L. donovani and L. infantum in the old world and L.

chagasi in the new world.

□ In the endemic areas, the disease is more chronic with young adults

and children being more commonly infected.

□ In epidemics, all age groups are susceptible (except those with

acquired immunity), and the disease is often acute.

□ Without treatment, VL is usually fatal.

□ Symptoms in acute VL, there is splenomegaly, high undulating fever

with two peaks in the day, chills, profuse sweating, weight loss,

fatigue, anaemia, and leucopenia.

□ Symptoms in chronic VL include irregular fever, massive

splenomegaly, hepatomegaly, and/or lymphadenopathy, marked loss

of weight with wasting, diarrhea, low white cell and platelet counts,

and anaemia.

□ The local Indian name for VL, kala-azar (meaning black sickness or

black fever) is a reference to the darken color of the infected patients.

□ Malnutrition and other infections increase the risk of developing

symptomatic VL.


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Massive splenomegaly in VL

Post kala-azar dermal leishmaniasis (PKDL)

□ In India and occasionally in Africa, a cutaneous form of leishmaniasis

can occur about 2 years after treatment and recovery from visceral

leishmaniasis.

□ This is referred to as post kala-azar dermal leishmaniasis and

affects about 20% of patients in India.

□ Hypopigmented and raised erythematous patches appear on the face,

trunk of the body, and limbs.

□ These may develop into nodules and resemble those of lepromatous

leprosy, fungal infections or other skin disorders.

□ Occasionally there is ulceration of the lips and tongue.

□ Amastigotes are present in the papules and nodules.


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Figure ( ) PKDL in sudan Papular and nodular PKDL

Figure ( ) PKDL affecting the earlobe

Figure ( ) PKDL: macular lesions, some are confluent.


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Immunity

□ Absence of Gamma Interferon IFN ᵧ and Interleukin 2 during Active

Visceral Leishmaniasis

□ Inhibition of parasitic Ag presentation by the antigen presenting cells

(APCs)-macrophage because lysis of intracellular amastigote is

blocked.

□ Leishmania proamastigote has surface inhibiter molecules called

lipophosphoglycan (LPG) that inhibits the toxic effect of macrophage

nitric oxide.

□ Nitric oxide or nitrogen mediators are a potent toxic oxidant that

destroys intracellular pathogens however, promastigote of leishmania

parasite has the ability of inhibition the nitric oxide-dependent killing

mechanism of the macrophage.

□ Because the acidic pH is an environment suitable for living and

multiplication of the amastigote, thus lysis of intra-phagosome

amstigote does not achieved by the macrophage lysosomes.

□ As a result, rupture of parasitized phagocyte occurs and releasing of

amastigotes that in turn infects other macrophages.

□ T lymphocytes are not activated unless recognizes the pathogen Ag

which must be expressed on the surface macrophage.


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□ Macrophages have the key role in an initiating the cell-mediated

immune response, and one of some immune cells that act as an

antigen presenting cells.

□ In normal phagocytosis, the phagocyte lysosomes destructs the

intracellular pathogen into small peptides, these peptides are then

expressed on the macrophage surface via the Major

Histocompatibility molecules class 1(MHC molecules).

□ After an antigen presenting, macrophage release a cytokines

molecules called Il-2 and INF ᵧ, which activate T lymphocytes.

□ The consequences of T lymphocytes activation is the direct killing of

infected cells and controlling the disease prognosis.


16Illustration of leishmania parasite during invasion of macrophage


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Development of the immune response to protozoan and helminth infection


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Cutaneous leishmaniasis (CL)

□ Cutaneous leishmaniasis is a potentially severe and disfiguring

disease in some people.

□ The clinical forms of CL vary according to the species of parasite,

region, and immune response of the patient.

□ People with cutaneous leishmaniasis have one or several long-lasting

lesions on the skin, usually without fever or general symptoms.

□ New cases are emerging in areas previously free of the disease.

□ Over 100 000 new cases of cutaneous leishmaniasis are reported

annually to WHO by countries in the Region, but the actual incidence

is estimated to be three to five times higher since many patients never


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seek medical attention and not all patients with a diagnosis of

cutaneous leishmaniasis are reported to health authorities.

Old world cutaneous leishmaniasis (CL)

Cutaneous leishmaniasis caused by L. tropica

□ Infection is often referred to as dry urban oriental sore.

□ Dry painless ulcers 25–70 mm in diameter are produced which are

self-healing usually after 1–2 years but often leave disfiguring scars.

□ The patient acquires immune to reinfection.

□ Rarely, multiple unhealed lesions may develop, often on the face.

□ It can last many years and is difficult to treat; this condition is known

as leishmaniasis recidivans (LR).

□ Untreated LR may leads to destruction and disfiguration of the

infected parts.

Initial brownish nodule of dry, urban type of cutaneous leishmaniasis


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Plaque lesion of dry, urban type of cutaneous leishmaniasis

Dry, urban and anthroponotic type of cutaneous leishmaniasis

Leishmaniasis recidivans due to (L. tropica) from Morocco.Note the healed scar

from which new lesions develop.


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Chronic cutaneous leishmanaisis of face with areas of scarring and reactivation

of disease (Leishmaniasis recidivans)

Leishmaniasis recidivans due to (L. tropica) from Afghanistan.

Note the healed scars from which new lesions develop.

Cutaneous leishmaniasis caused by L. major

□ Infection is often referred to as wet oriental sore.

□ The early papule is often inflamed and resembles a boil of 5–10 mm

in diameter which rapidly develops into a large uneven ulcer which is

self-healing in as little as 3–6 months.

□ Multiple lesions may occur in non-immune persons.

□ L. major infections show permanent immunity against reinfection.


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Wet ulcer in CL

Cutaneous leishmaniasis caused by L. aethiopica

□ A cutaneous lesion that is similar to typical oriental sore with healing

in 1–3 years.

□ Localized cutaneous lesions may spread to involve large cutaneous

area, forming a nodules often associated with scaling. This form is

known as diffuse cutaneous leishmaniasis.

□ The patients who have diffuse cutaneous leishmaniasis are more

likely of little or no cell -mediated immunity against the parasite.

Chronic localized cutaneous leishmanisis of face Non-healing chroniccutaneous leishmaniasis”


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Diffuse cutaneous leishmaniasis (DCL)

□ Both L. aethiopica (Old World) and L. amazonensis (New World) are

the causes of diffuse cutaneous leishmaniasis.

□ Skin lesions develop over a large area of the body.

□ The lesions on the eyebrows, nose and ears resemble those of

lepromatous leprosy.

□ At first, the lesions are smooth, and firm.

□ Later they become scaly and rough.

□ The nodules contain large numbers of amastigotes.

□ The lesions do not heal spontaneously and this is an incurable

condition characterized by the formation of disfiguring nodules over

the surface of the body.

□ DCL caused by L. amazonensis is resistant to treatment.

□ DCL caused by L. aethiopica, relapses occur after treatment.

Diffuse cutaneous leishmaniasis- Venezuela


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Diffuse Cutaneous Leishmaniasis (DCL) from Ethiopia. The patient originating

from the Highlands where CL and not VL is endemic; there is no previous history

of VL. Leishmania parasites were found in a skin scraping.

Mucocutaneous leishmaniasis (MCL)

□ In New World, both L. braziliensis and L. panamensis can cause

Mucocutaneous leishmaniasis (MCL).

□ In south America Mucocutaneous leishmaniasis (MCL) known as,

‘espundia’.

□ Rarely MCL is caused by L. tropica and L. aethiopica in the old

world.

□ MCL is the most severe and destructive form of cutaneous

leishmaniasis in South America.

□ Lesions are similar in development to those of oriental sore and the

resulting ulcers may become very large and long lasting.


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□ Disfiguration is extreme with complete destruction of the infected

part, such as nasal septum if the nose is the primary lesion and

damage to the tissues of the lips and ear cartilage.

□ Mucosal lesions do not heal spontaneously and severe secondary

bacterial infections can occur.

□ A Sudanese form of MCL is referred to as oro-nasal leishmaniasis.

Mucocutaneous leishmaniasis (MCL)

Treatment of Leishmaniasis

Most sores will heal spontaneously within one year.

Treatment of cutaneous and muco-cutaneous leishmaniasis is the

same while the latter needs more intensive treatment due to the more

severe and destructive complications.


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Pentavalent antimony:

Pentostam

Unfortunately, some cases of leishmaniasis may treated by topical

steroid preparation. This changes the clinical picture, deteriorates the

lesion, which becomes later more chronic and decreases its response

to the specific medications.

For adults, we give 6 cc of Pentostam I.M. daily for 10 days.

This usually gives very good results, causing rapid healing of the

ulcers.

The dose is adjusted according to the age (20 mg/kg of body weight).

Neostibosan

Neostibosan (Bayer): is also an effective medication.

The daily dose is 5mg/kg body weight .

A dose of 200-300 mg. can be given for older children and adults

daily for 16 days is proved to be effective.

Patients with diffuse cutaneous leishmaniasis require treatment for a

longer time.

Liposomal amphotericin-B (AmBisome®)

Is the drug of choice for VL.

It is given in a dose of 3 mg/kg per day on days 1-5, day 14 and day

21.


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Laboratory Diagnosis

Diagnosis of visceral leishmaniasis

The laboratory diagnosis of visceral leishmaniasis (VL) is by:

Finding amastigotes in:

o material aspirated from the spleen, bone marrow or an

enlarged lymph node,– nasal secretion.

o peripheral blood monocytes and less commonly in

neutrophils (buffy coat preparations).

Culturing aspirates and peripheral blood and examining cultures

for promastigotes.

Other tests

Formol gel (aldehyde) test. This is a non-specific screening test which

detects marked increases in IgG. Large amounts of polyclonal non-

specific immunoglobulin are produced by patients with active VL.

Haematological investigations including:

o measurement of haemoglobin,

o total and differential white cell (leukocyte) count,

o platelet (thrombocyte) count.

Detection of anti-leishmanial antibody

□ In visceral leishmaniasis, specific antibody as well as non-specific

polyclonal Ig G and Ig M are produced.


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□ Several techniques have been developed to detect and measure

specific anti-leishmanial antibodies in patients’ sera.

□ Those being used in district laboratories and field surveys include:

Direct agglutination test (DAT) or rK39 dipstick to detect anti-rK39

antibody.

Diagnosis of cutaneous and mucocutaneous Leishmaniasis

The laboratory diagnosis of CL and MCL is by:

Detecting amastigotes in smears taken from infected ulcers or

nodules. In MCL, the parasites are scanty and difficult to find in

smears.

Culturing ulcer material and examining cultures for promastigotes.

Serological diagnosis of CL and MCL

Because of the poor antibody response in CL, serological tests are

of little value in diagnosis.

Leishmanin skin test (Montenegro test)

It is a delayed hypersensitivity skin test. In this test, 0.2ml of

Leishmania antigen (containing 100,000,000 promastigotes of L.

donovani in l ml of 0.5% phenol saline) is injected intradermally.

The test is read after 48 to 72 hours.

A positive test shows an area of erythema and induration of 5 mm

in diameter or larger, which heals in 14-25 days.


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Positive reaction indicates prior exposure to leishmanial parasites.

In kala-azar, the skin test becomes positive usually only 6 to 8

weeks after cure from the disease, it is negative in active cases.

Culture of ulcer material

Culture is of value when cutaneous leishmaniasis is suspected and

parasites cannot be found in smears.

Measures to prevent and control leishmaniasis

□ Early detection and treatment of infected persons, especially in

areas where humans are the only or important reservoirs of infection.

□ Personal protection from sandfly bites by:

Using insect repellants, although in hot and humid conditions they are of

limited use due to profuse sweating.

Avoiding endemic areas especially at times when sandflies are most

active.

Use of insecticide impregnated bed nets and curtains.

□ Vector control by the use of light traps, sticky paper traps, or

residual insecticide spraying of houses and farm buildings where this

is practical, or alternatively using insecticide paints in a slow-release

emulsifiable solution.

□ Destruction of stray dogs and infected domestic dogs in areas where

dogs are the main reservoir hosts.


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□ Elimination and control of rodents in areas where these are sources

of human infections.

Leishmania amastigotes in Giemsa stained skin slit smear.

Leishmania amastigotes in monocyte in a Giemsa stained blood film.

Giemsa stained amastigotes of L.donovani. Right:

As seen in bone marrow. Left: As seen in splenic aspirate.


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Formol gel test showing positive (+) and negative ( - ) reactions.

Leishmaniasis - By Dr/ Faiz Al-Khawlani

Health & Medicine

Leishmaniasis - By Dr/ Faiz Al-Khawlani