Leishmaniasis

Leishmaniasis • a variety of disease manifestations

• focal distribution throughout world,

especially tropics and subtropics • new world: southern Texas to northern

Argentina

• old world: Asia, Africa, middle east,

Mediterranean

• transmitted by sand flies • new world: Lutzomyia • old world: Phlebotomus

• 350 million at risk

• 12 million infected • 1.5-2 million clinical

cases/year

Some Leishmania Species Infecting Humans

New World Cutaneous,Mucocutaneous, and

Diffuse Leishmaniasis

Old World Cutaneous,Recidivans, and

Diffuse LeishmaniasisVisceral

Leishmaniasis

Mexicana ComplexL. mexicanaL. amazonensis

Braziliensis ComplexL. braziliensisL. panamensisL. guyanensis

L. tropica

L. major

L. aethiopica

L. infantum*

L. donovani

L. infantum*

L. chagasi**

*Both dermotrophic and viscerotrophic strains exist.**L. chagasi (Americas) may be the same as L. infantum (Mediteranean)

Geographical Distribution

Currently the leishmaniases, prevalent in four continents, are considered to be

endemic in 88 countries, 72 of which are developing countries:

90% of all visceral leishmaniasis cases occur in Bangladesh, Brazil, India,

Nepal and Sudan;

90% of mucocutaneous leishmaniasis occurs in Bolivia, Brazil and Peru;

90% of cutaneous leishmaniasis cases occur in Afghanistan, Brazil, Iran, Peru,

Saudi Arabia and Syria.

Clinical Spectrum of Leishmaniasis

Cutaneous Leishmaniasis (CL) most common form, relatively benign self-healing

skin lesions (aka, localized or simple CL)

Mucocutaneous Leishmaniasis (MCL) simple skin lesions that metastasize, especially to

nose and mouth region

Visceral Leishmaniasis (VL) generalized infection of the reticuloendothelial

system, high mortality

Leishmaniasis is transmitted by the bite of female phlebotomine sandflies. The sandflies inject the infective stage, promastigotes, during blood meals. Promastigotes that reach the puncture wound are phagocytized by macrophages and transform into amastigotes. Amastigotes multiply in infected cells and affect different tissues, depending in part on the Leishmania species. This originates the clinical manifestations of leishmaniasis. Sandflies become infected during blood meals on an infected host when they ingest macrophages infected with amastigotes. In the sandfly's midgut, the parasites differentiate into promastigotes, which multiply and migrate to the proboscis .

Sandfly Vector

Human Host Animal Reservoir

Macrophage

Promastigote Amastigote

Skin

1) metacyclic

promastigotes

2) phagocytosis by

macrophage

amastigote

3) replication within

macrophage

4) release and

phagocytosis of

amastigotes

The symptoms and pathology associated with leishmaniasis

result from the amastigotes killing the host's cells.

There are many different "diseases" caused by Leishmania. In

some diseases the amastigotes do not spread beyond the site

of the vector's bite. This results in a "cutaneous

leishmaniasis" (oriental sore, Jericho boil, Aleppo boil, or

Dehli boil) that often heals spontaneously.

In other instances the amastigotes may spread to the visceral

organs (liver, spleen), resulting in "visceral leishmaniasis"

(kala-azar or Dum-Dum fever) or to the mucous membranes

of the mouth and nose, resulting in "mucocutaneous

leishmaniasis" (espundia or uta). Left untreated, these latter

diseases result in high rates of mortality.

CUTANEOUS LEISHMANIASIS:

Old World Cutaneous Leishmaniasis (CL)

L. major causes a moist, cutaneous, ulcerlike lesion at the site of the bite; it starts as a

papule that runs an acute course (1-3 weeks) with early ulceration and a

surrounding zone of inflammation, that usually heals in two months to a year

leaving a depressed unpigmented scar, and lasting immunity.

It is transmitted by Phlebotomus spp. from gerbil, dogs or rodents to human or

human-to-human. It is generally found in sparsely populated rural areas.

New World Cutaneous Leishmaniasis

L. mexicana

The ulcer usually heals spontaneously in a few months. However, when the bite

occurs on the ear it results in chronic lesions known as chiclero's ulcer. Because

the cartilage of the ear pinna is poorly vascularized the immune response is weak,

and in 40% of cases the result is mutilation of the pinna. Found principally in

Central America and Mexico where it occurs in the forest dwelling people who

harvest latex from the chicle trees to be used in the manufacture of chewing gum.

In Belize it is also known as bay sore.

cutaneous leishmaniasis, is usually localized to the site in which the

sandfly bite occurs. The amastigotes multiply in the reticuloendothelial

system of the skin. The incubation period spans from a period of day up

to several months. If kept clean the sores will heal spontaneously within

2 months to 1 year. However, these sores often are the sites of

secondary infections and can result in permanent disfiguration.

Once the infection has been cleared the host is immune to reinfection.

In some regions, residents inoculate their children in a site normally not

visible to protect the child from getting disfiguring scars later in life.

TYPICAL VS. ATYPICAL

CUTANEOUS LEISHMANIASIS

TYPICAL CUTANEOUS

LESION

ATYPICAL CUTANEOUS

LESION

L. braziliensis Inoculation of promastigotes by the bite of a sandfly results in a small,

red, skin papule that is itchy and ulcerates in 1-4 weeks like cutaneous

leishmaniasis; this ulcer usually heals spontaneously within 6-15 months.

However, there is metastatic spread of the promastigotes from the site of

the bite via the lymphatics. Disease is called espundia; the metastatic

lesion involves the nasal and buccal mucosa causing destruction and

malformations of the cartilage and soft tissues. The ulcerations can

involve the nose, pharynx, palate and lips. Invasion of the larynx may

result in a loss of speech. In Brazil 1/3 of the espundia cases are in

children. May take many years for it to spread from the initial site of bite.

Death may occur from secondary infections or respiratory complications.

Muco-cutaneous leishmaniasis

VISCERAL LEISHMANIASIS

L. donovani causes the classic type found in India. This is a metastatic disease.

Rarely is a lesion seen at the site of bite and parasites are only occasionally seen

in blood, but are present in the spleen and lymph nodes. The incubation period is

1-4 months. Disease is characterized by fever, anemia, splenomgegaly, wasting,

imbalance of serum proteins and hyperpigmentation of the skin. The death rate is

very high if left untreated. L. infantum causes the Mediterranean form of kala azar

and has dogs, jackals and foxes as reservoirs. Humans are accidental hosts.

L. donovani group

This visceral disease has a new and old world form: particularly Brazil,

and Mediterranean Europe, North Africa, East Africa, India and China.

The amastigote forms are found within the reticulo-endothelial cells of the

viscera, ie the spleen, lymph nodes, liver and intestine.

The incubation time of 10 days-a year. The symptoms are a slow

developing low grade fever, and general malaise, a progressive wasting

of the patient with anaemia. Other classic symptoms as the disease

progresses is the protrusion of the abdomen, hepatospenomegaly. If

untreated death will occur within 2-3 years of contracting the infection.

In acute forms-the disease can run its course within 6- 12 months.

Clinical signs include edema, particularly of the face, bleeding mucus

membranes, breathing difficulties and diarrhea.

The epidemiology is extremely diverse: 20 Leishmania species are pathogenic for humans, and 30 sandfly species

are proven vectors.

Old World: Phlebotomous spp.

New World: Lutzomyia spp

There are two main epidemiological entities:

Zoonotic, where animal reservoir hosts are involved in the

transmission cycle

Urban endemic: mostly human-human

Diagnosis of CL, MCL, DCL

• suspected because of:

• geographical presence of parasite

• history of sandfly bite

• + skin lesion: • chronic, painless, ‘clean’ ulcer

• nasopharyngeal lesions

• nodular lesions

• demonstration of

parasite

• delayed hypersensitivity

skin test

• serology?

• amastigotes

(scrapings, biopsy,

aspirates)

• in vitro culture

(promastigotes)

• inoculate into

hamsters

make incision in active part of lesion

A, B: Leishmania tropica amastigotes from an impression smear of a biopsy

specimen from a skin lesion. In A, an intact macrophage is practically filled

with amastigotes (arrows), several of which have a clearly visible nucleus and

kinetoplast; in B, amastigotes are being freed from a rupturing

macrophage. Patient had traveled to Egypt, Africa, and the Middle East.

C: Three Leishmania amastigotes, each with a clearly visible nucleus and

kinetoplast

VL Diagnosis

• suspected because of:

• geographical presence of parasite

• history of sandfly bite

• prolonged fever, splenomegaly,

hepatomegaly, anemia, etc.

• amastigotes in bone marrow

aspirates

• in vitro culture of aspirates

• serological tests

• direct agglutination

• ELISA dipstick (39 kDa Ag)

Treatment

• Kala azar is treated today essentially as it was in 1940. The major drug is Sodium

stibogluconate or Pentastam, a derivative of antimony, which was developed in

1930! Severe reactions including death occur in 10% of those treated. The

recommended one month treatment costs around $150. Drug resistance has also

developed. Up to 70% of infected patients in India have drug-resistant parasites.

• Amphotericin is used with or after an antimony compound for visceral

leishmaniasis unresponsive to the antimonial alone.

•.

Pentamidine isotionate has been used in antimony-resistant visceral

leishmaniasis, but although the initial response is often good, the

relapse rate is high and it is associated with serious side-effects.

Recently a new drug was developed, miltefosine. This is a membrane

signaling pathway inhibitor. This can be taken orally and is very

effective against visceral leishmaniasis. Clinical trials have a 95% cure

rate!

Pentamidine is also used for New World cutaneous leishmaniasis, but

it usually heals spontaneously.

There is no treatment for muco-cutaneous leishmaniasis

Control and

Epidemiology

• depends on local

transmission

• avoid sandfly bites

• bed nets

• insecticides

• destruction of dog

reservoir

• ‘tropica vaccine’ • historical inoculation

in covered areas

• risk of recidiva or VL

New World Dermal • zoonosis (arboreal

mammals = reservoir)

• lowland forest

• occupational

Old World Dermal • urban = dog reservoir

• rural = rodent reservoir

Visceral • India (Ld): human-fly-

human

• Africa (Ld): rodent

reservoir

• others: dogs (with lesions)

are usual reservoir

Major problems and challenges for disease control

Technical, managerial, financial and political constraints.

There is no recognized cost-effective control package. Although tools for control have

been considerably improved in recent years, they are still far from perfect:

Parasitology: existing tools (spleen, bone marrow [BM], lymph node aspirates) are either

invasive methods and difficult to decentralize (spleen, BM) or of low sensitivity (lymph

nodes). Treatment of VL cases by first-line drugs (Sb5+) is long expensive (US$ 120-150).

Poor compliance often leads to increased unresponsiveness.

Vector control by spraying houses with insecticide is not sustainable.

Animal reservoir control through environmental management is expensive and

difficult to implement; the efficacy of dog culling is questionable.

Other constraints: lack of well-trained technical personnel, and weak delivery

systems. Political and financial commitments are low and the level of

implementation is frequently poor.

Notification of leishmaniasis is compulsory in only 33

countries. Leishmaniasis is a disease of poverty: patients

are the poorest among the poor (India: 88% of patients

have a daily income of less than US$ 2), with poor

socioeconomic environment and low educational level;

they live either in remote rural areas or poor suburbs.

Problems with Control programs

Enzootic foci in wild animals: humans were rarely infected

(central Asia)

Rural endemic foci: as humans colonized areas parasite moved

into domesticated dogs, and cases in humans became more

common (Asia, Russia, China, Mediterranean)

Urban endemic foci: wild animal populations are reduced in

importance of life cycle- dogs become reservoirs and frequent

transmission to humans (Central Asia, Eastern China)

Endemic / epidemic foci: few reservoirs- transmission

principally man-man (India today)

Each of these different types of situation requires different

approaches to control

Control of vectors

Protection vs bites

Treatment if infected persons

Elimination of reservoirs

Vaccination

When an infected Phlebotomid sand fly takes a blood meal it infects the

vertebrate host with promastigote metacyclic forms. Within a short time

the promastigotes are taken up by macrophages, the first line of defense

of the immune system. During the process of uptake by the macrophage,

the promastigote loses its flagella and transforms into the amastigote

form. Once internalized in a phagosome the macrophage lysosome fuses

with the phagosome to from a phagolysosome containing the parasite.

How does the parasite survive this hostile environment?

Leishmania-Macrophage Interactions

• attachment and entry • involves CR3 and surface molecules

on parasite (Lipophosphoglycan)

• entry is typical phagocytosis

• phagosome fuses with lysosome

SURVIVAL IN THE MACROPHAGE PHAGOLYSOSOME

Respiratory burst products:

H2O2, superoxide hydroxyl radicals

Cell Biology of Promastigote Entry into Macrophages

Promastigotes are taken up by phagocytosis and the parasites

differentiate into amastigotes inside the phagosome. This is a very

hostile environment where cells taken up by the macrophage are

normally destroyed by:

• Oxidative burst - Phagocytosis of a foreign body activates an NAD(P)H

oxidase in the plasma membrane. This produces the highly reactive

superoxide and hydroxyl radicals at the site of engulfment. These

radicals react with the pathogen's phospholipid membrane and also with

its macromolecules

•

Acidification - After fusion of the phagosome with the

endosome, the vesicle is acidified by a proton ATPase. The

low pH (4.5-5.0) causes denaturation of proteins, which

become susceptible to acid hydrolases.

Digestion - The endosome fuses with primary lysosomes,

and acid hydrolases are released which degrade DNA, RNA,

proteins and carbohydrates.

Defense strategies:

a) Detoxification with antioxidant enzymes:

Trypanothione peroxidase is a unique antioxidant enzyme which

detoxifies hydrogen peroxide.

Superoxide dismutase - removes the reactive superoxide molecule.

b) Down-regulation - Lipophosphoglycans on the surface of the

promastigote interfere with signal transduction pathways, specifically

inhibits protein kinase C, which trigger the respiratory burst in

response to the parasite.

c) Hydrolytic enzymes:

Leishmania possess surface glycoproteins that are refractory to

host lysosomal enzymes and may also destroy them.

Life inside the Macrophage and Virulence Factors

Oxidative Burst

Trypanothione is an unusual form of glutathione

containing two molecules of glutathione joined by a

spermidine linker. It is found in parasitic protozoa such

as Leishmania and the trypanosomes. It is unique to the

Kinetoplastida and not found in other parasitic protozoa

such as Entamoeba histolytica. Since this thiol is absent

in humans and is essential for the survival of the

parasites, the enzymes that make and use this molecule

are targets for the development of new drugs.

Trypanothione-dependent enzymes include reductases,

peroxidases, glyoxalases and transferases. The first discovered

molecule, Trypanothione-disulfide reductase (TyrR) is a

flavoenzyme that reduces trypanothione disulfide. TryR is essential

for survival of these parasites in dealing with oxidative stress.

Interference with polyamine synthesis also affects trypanosomes

because spermidine is required for the synthesis of

trypanothione.

Many of the newer anti trypanosome drugs interfere with the

trypanothione pathway and thereby increase sensitivity towards

oxidative stress.

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