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Kinetoplasta:

Leishmania
(Tissue Flagellates)
LESSON LEARNING OUTCOMES
Leishmanias are heteroxenous.
Vector/ IH Infected FEMALE sand fly (Phlebotomus sp & Lutzomyia sp).
*Promastigotes (in the midgut of the sand fly) will be injected in human while
blood meal takes place.
* found only in Leishmanias
Amastigotes (Leishman-Donovan (L-D) bodies) found in humans
macrophages (intracellular).
Vertebrates hosts are primarily mammals
Humans, dogs, and several species of rodents
Cause a complex of diseases called Leishmaniasis.

sandfly
Complex of diseases (Leishmaniasis)
epidemiology approach

Type of Type of Geographical


Leishmania Leishmania distribution
sis sp
Visceral L. donovani Worldwide but is predominantly
Leishmaniasis (L. chagasi) encountered in India, South
(Kala Azar or Dum (L infantum) America, Central Asia, the Middle
Dum Fever) East, and Africa
Cutaneous L. tropica West Central Africa, the Middle
Leishmaniasis L.major East, and Asia Minor to India.
L. mexicana Central America, Mexico, Texas,
possibly in Dominican Republic
and Trinidad.
Mucocutaneous L. braziliensis Central Mexico and Northern
Leishmaniasis Argentina
Life cycle
Promastigo
tes
(Metacyclic) PROBOS Promastigot
CIS
es
Amastigote (Metacyclic)
then multiply
by simple
division
Promastig
otes INFECTIV
(Procyclic)
MIDG E STAGE
UT

Amastigot
es
DIAGNOS
*sandfly ingests macrophages
TIC
infected by amastigotes STAGE
The sandflies inject the infective stage (promastigotes) from their
proboscis during blood meals.
Promastigotes that reach the puncture wound are phagocytized by
macrophages and other types of mononuclear phagocytic cells.
Progmastigotes transform in these cells into the tissue stage of the
parasite (amastigotes).
Amastigote then multiply by simple division and proceed **to infect
other mononuclear phagocytic cells.
Parasite, host, and other factors affect whether the infection
becomes symptomatic and whether cutaneous or visceral
leishmaniasis results.
Sandflies become infected by ingesting infected cells during blood
meals.
In sandflies, amastigotes transform into promastigotes (procyclic),
develop (metacyclic) in the gut (in the hindgut for leishmanial
organisms in the Viannia subgenus; in the midgut for organisms in
the Leishmania subgenus), and migrate to the proboscis.
Simplified view
Promastigot
es Promastigot
(Metacyclic) es
(Metacyclic)

Promastig
otes
(Procyclic)
Procyclics and Metacyclics
Promastigote
4.Metacyclic Promastigotes
(procyclic) differentiate
& replicate into
complement resistant
metacyclic promastigote
in the midgut
2. Procyclic

3. Replication
into
Metacyclic
1. Amastigotes
Ingest macrophages (contains
amastigotes)
Parasite interacts with the sand fly
midgut
Promastigotes attach
(procyclic) and detach to
the midgut (metacyclic)
epithelium.
(d
) Attachment helps them to
remain in the insect gut
when the blood meal is
passed.
(c
)
However, later they need to
detach to move to the
pharynx and proboscis for
infection
(b
)
Sand flies (Phlebotomus sp. &
Lutzomya sp.)
In the Old World (Africa, Asia & Europe), Phlebotomus
sand flies are primarily responsible for the transmission
of leishmaniasis.

While transmission in the New World (Western


hemisphere specifically America), is generally via sand
flies of the genus Lutzomyia.

Females are haematophagus.


most active at night (just like Reduviid bug)

Males are sap feeders.


Ecology of Old World
Leismaniasis
Close contact of humans and
their domestic animals can
provide optimal conditions for
sand flies and Leishmania
transmission (stables provide
good breeding ground for larvae).

In urban environments infection


is mostly human to human.

In rural areas Leishmaniasis can


be a zoonosis .

Infection in dogs is quite frequent


in the Mediterranean.
Ecology of New World
Leishmaniasis
In the new world most people get
infected while working or hunting
in the forest.

Here wild animals including


rodents, monkeys and sloths
provide a reservoir for the parasite.

A transmission pattern within a


population of wild animals that
result in occasional infection of
humans is called sylvatic (or
diseases or pathogens affecting
only wild animals).
Sand flies (Phlebotomus sp. &
Lutzomya sp.) cont
Family : Psychodidae
Subfamily : Phlebotominae
Adults are small sized about 1.53.0mm, yellowish in colour with
conspicuous black eyes, hairy body, wings and legs.

Phlebotomus Lutzomya
sp. sp.
14
15
16
Vector Control

Bednet protection from


sand flies 17
Important Vectors of Leishmaniasis

Visceral
Lutzomyia longipalpis Central and South America
Phlebotomus argentipes Middle East
Phlebotomus chinensis China
Cutaneous
Phlebotomus sergenti India
Phlebotomus papatasi Mediterranean
Lutzomyia verrucarum s.l. Central and South America
Mucocutaneous
Lutzomyia intermedia Central and South America
http://cipa.snv.jussieu.fr/index.html
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Cutaneous Leishmaniasis
L. tropica
L. major
L. mexicana
20
Leishmania tropica & L.major
Produce cutaneous ulcers variously known as:
Cutaneous leishmaniasis
Oriental sore
Jericho boil
Aleppo boil
Delhi Boil

Have similar life cycles BUT:


Found in different localities
Different reservoir and IH
Different lesions in human with vary in severity
according to age and other factors
Can be differentiated biochemically
Leishmania tropica & L.major
cont
Life Cycle

DH: Humans. Reservoir hosts include most mammals

IH: Phlebotomus sand flies.

Transmission: Infected female sand fly bites during


blood meal.
Leishmania mexicana
Infects several thousand persons a year.
Especially agricultural or forest laborers
The disease is on increase due to increased
clearing of forests and concomitant expansion of
farmland
Three clinical manifestation are found:
Cutaneous, nasopharyngeal mucosal, and visceral
Traditionally, the cutaneous form of disease has been called
chiclero ulcer because it is so common in chicleros, forest
dwelling people who glean a living by harvesting the gum of
chicle tree.
In Belize, it is called bay sore.
chiclero ulcers
Leishmania mexicana cont
Life Cycle

DH: Humans. The main reservoirs are rodents and those


that live or travel at ground level.

IH: Phlebotomus sp and several species of Lutzomyia


involved.

Transmission: Infected female sand fly bites


during blood meal.
Pathogenesis

Cutaneous leishmaniasis due to L.


mexicana usually heals spontaneously in a
few months:
Except when the lesions are in the ear
Ear cartilage is poorly vascularized so
immune responses are weak

Ear lesions may


persist for years,
destroying
cartilage
(chiclero ulcers)
Pathogenesis cont

Chronic lesions with a duration of 40 years


are known:
Considerable mutilation may result
Mucocutaneous and visceral
manifestations are rare

Diagnosis and Treatment


Same as for L. tropica.
Cutaneous
Leishmaniasis

Infection remains restricted to the initial


site of infection (the bite site)
Pathogenesis
Incubation period lasts from few days
to several month.

Small, red papule at the site of bite.

May disappear in few weeks, but


usually it develops a thin crust that
hides a spreading ulcer underneath.

Two/ more ulcers may coalesce to form


a large sore.

Uncomplicated ulcers will heal within


two months to a year, leaving a
depressed, unpigmented scar.

Oriental
sore
Diffuse Cutaneous
Leishmaniasis
Cutaneous
Leishmaniasis
30
Patients with diffuse
cutaneous
leishmaniasis develop
hundreds of papules,
nodules, and plaques
throughout the skin
in a clinical picture
that can be
reminiscent of
lepromatous leprosy.

This form of
leishmaniasis often is
resistant to therapy
and may assume a
chronic course.
L. tropica is found L. major is found in
in more densely sparsely inhabited
populated areas. regions.
Dry lesion, Its papule ulcerates
persist for month quickly, is of short
before ulcerating duration (open &
wet sore).
Has numerous
Contains few
amastigotes
amastigotes.
Diagnosis
Facilitated by finding amastigotes.
Scrapings from the side/ edge of an ulcer smeared on a slide and
stained with Wrights or Giemsas stain show the parasites in
endothelial cells and monocytes.
Cannot be found in blood circulation.
Culture should be made if amastigotes go undetected.
Treatment
Cutaneous leishmaniasis sometimes heals on its own and may not
require treatment.
Immune system shuts it down??
TREATMENT

Andy Crump WHO/TDR/Crump

A tube of Paromomycin cream, used for


topical application on lesions in the
treatment of cutaneous leishmaniasis
A doctor filling a syringe from a phial of glucantime, a pentavalent
antimonial which is used for the treatment of cutaneous leishmaniasis

34
Mucocutaneous
Leishmaniasis

L. braziliensis
Leishmania braziliensis
Produce disease in humans variously known as:
Mucocutaneous leishmaniasis
Espundia
Uta
Pian bois (in Venezuela & Paraguay)

The clinical manifestations of the disease vary along its range,


which has led to confusion regarding identity of the organisms
responsible.

Morphologically, cannot be differentiated from L. tropica, L.


mexicana and L.donovani.
Leishmania braziliensis cont

Life Cycle

DH: Humans. Reservoir hosts include dogs, cats, ant


eaters, sloths, and other mammals.

IH: Phlebotomus sp and several species of Lutzomyia


involved (** promastigotes reproduce in hindgut).

Transmission: Infected female sand fly bites


during blood meal.
Life Cycle cont

Promastigot
es
reproduce
in hindgut
Espundia or Mucocutaenous
Leishmaniasis

~20% of infected patients develop ulcers of the oral and nasal mucosa
Pathogenesis
Causes small, red papule on skin.

Itchy, ulcerated vesicle in 1-4


weeks (similar stage to oriental
sore).

Primary lesion heals within 6-15


months.

The parasites never causes a


visceral disease but often
develops a secondary lesion on
some region of the body.

Flat, ulcerated plaques that


remain open and oozing in
Mucocutaneous Venezuela & Paraguay (pian
leishmaniasis bois).
Pathogenesis cont
In southerly range, the
parasites have tendency to
metastasize, or spread directly
from the primary lesion to
mucocutaneous zones.

Secondary lesion may appear


before the primary has healed.

Secondary often involves the


nasal system and buccal
mucosa, causing degeneration
of the cartilaginous and soft
tissues.
Pathogenesis cont Necrosis and bacterial infection
are common.

Ulceration involves lips, palate,


and pharyngx deformity.

Invasion of trachea and larynx


destroys the voice.

Genitalia rarely become infected.

May last for many years, and


death may result from secondary
infection or respiratory
complications
Dogs can act as
reservoirs of
Leishmania
parasites.
They also exhibit
symptoms of
infection.
43
WHO/TDR/Mark Edwards
Dog
affected
by
Leishmani
asis
Diagnosis

Amastigotes (L-D bodies) in samples of infected tissue.

Need to eliminate possibility of leprosy, tuberculosis, syphilis, and


various fungal and viral disease that can cause same degeneration of
tissue.

Skin tests for occult (hidden) infection.

Culturing the parasites in vitro (L-D bodies cannot be demonstrated


in routine microscope preparation).
Treatment
Antimony-based drugs applied to lesions or injected intravenously or intramuscularly
(similar for kala azar and tropical sore).

Antibiotic treatment for secondary bacterial infection.

Chemotheraphy.

Relapse is common, but, once cured, a person usually has lifelong immunity.

If the infection is not cured but merely becomes occult, there may be a relapse with onset of
espundia many years later.
Prevention
Avoid bites of the sand fly.
Avoid outdoor activities, especially from dusk to dawn, when sand flies
generally are the most active.
When outdoors (or in unprotected quarters):
Minimize the amount of exposed skin. To the extent that is
tolerable in the climate, wear long-sleeved shirts, long pants,
and socks; and tuck your shirt into your pants.
Apply insect repellent to exposed (uncovered) skin and under the
ends of sleeves and pant legs.
The most effective repellents are those that contain the chemical
DEET (N,N-diethylmetatoluamide).
Visceral Leishmaniasis

L. donovani
In 1900 Sir William Leishman discovered L. donovani in splean smears
of a soldier who died of a fever at Dum-Dum, India.

Was known locally as Dum-Dum fever or kala-azar.

Leishman published his observation in 1903, the year that Charles


Donovan found the same parasite in a spleen biopsy.

The scientific name honors these men, as does the common name of the
amastigotes form (L-D bodies).
Geographic Distribution of L. donovani

Probably originated in Old World


Moved to New World with slave trade
Leishmania donovani

L. donovani amastigotes cannot be differentiated from other


Leishmania sp. on morphological basis.

Live within cells of the RE (reticuloendothelial) system,


including spleen, liver, mesenteric lymph nodes, intestine and
bone marrrow.

Amastigotes found in nearly every tissue and fluid of the


body.
Leishmania donovani
Life cycle

DH: Humans. Reservoir includes most mammals

IH: Phlebotomus sand flies.

Transmission: Infected female sand fly bites


during blood meal.
Leishmania donovani
Life cycle cont

Similar to L. tropica except L. donovani is primarily a


visceral infection.

Not all strains of L. donovani are adapted to all species and


strains of Phlebotomus.

Interestingly, amastigotes engulfed by neutrophils and


eosinphils are killed, but in untreated cases these PMN
leucocytes have little or no effect on the eventual outcome
of the disease.
Visceral Leishmaniasis or
Kala Azar

Systemic infection of reticulo-entdothelial cells (mostly


macrophages) throughout multiple internal organs and the blood
Amastigote is engulfed
by macrophage:
- Macrophage doesnt
kill amastigote.
-Neutrophils and
eosinophils will kill
amastigotes.

Multiplies, breaks out,


and each invades
Macrophage infected with another macrophage.
amastigotes of Leishmania
Leishmania sp.

Numerous ovoid to round small


(2-6 m) parasites in a bone
marrow aspirate.
In some parasites the typical
rod shaped kinetoplast is seen
besides the nucleus (May-
Grnwald-Giemsa stain).
Leishmania donovani promastigotes
(form present in sandfly host)

57
Leishmania donovani extracellular
amastigote
(form present in vertebrate host)
58
L. donovani tissue smear of the
spleen of an infected rodent.

The red arrows point to some of the


amastigotes which have broken out of
the splenic macrophages when the
smear was made.

The green arrows point to the


remains
of the nuclei of the host
macrophages.

Note the kinetoplast and nucleus


within each amastigote.
Pathology

Clinically, may range from asymptomatic to progressive, fully developed kala-azar.


Incubation period in humans between 10 days to 2-4 months.
Begins with low grade fever, malaise and is followed by progressive wasting and
anemia, protrusion of abdomen from enlarged liver and spleen
(hepatosplenomegaly).
Death (untreated cases) in 2-3 years.
In some cases, more acute onset, with chills, fever (up to 40oC/ 104oF) and vomiting,
death may occur within 6-12 months.
Edema of the face, bleeding of mucous membranes, breathing difficulties, and
diarrhea.
Immediate death is due to secondary pathogen that the body unable to combat.
Protrusion of abdomen
from enlarged liver and
spleen
(hepatosplenomegaly).
Pathology - cont

Skin condition known as post kala-azar dermal leishmanoid develops


in some cases.

Rare in Mediterranean and Latin America areas but develops in 5% to 10%


of cases in India.

Apparent about 1-2 years after inadequate treatment for kala-azar.

Marked by reddish, depigmented nodules that sometimes become quite


disfiguring.
Post kala-azar
dermal
leishmanoid
Diagnosis
Finding of L-D bodies (amastigotes) in tissues or secretions.
spleen punctures
blood or nasal smears
bone marrow
Immunodiagnostic are sensitive but cannot differentiate between species
of Leishmania and between current and cured cases.
IFA, ELISA tests (main limitation was a crossreacting positive with T.
cruzi).
Resolved by use of monoclonal antibody
Need to eliminate possibility of typhoid, paratyphoid, malaria, syphilis,
tuberculosis, dyssentery, and relapsing fever which cause similar
symptoms.
Andy Crump
WHO/TDR/Crump

TREATMENT

Injections of various antimony-based drugs


Promising oral drug - Miltefosine

A bottle of
capsules of
Miltefosine, the
first oral drug
for treating 65
Prevention

Related to human activities and sand flies biology.

Similar like other Leishmaniasis prevention steps.


Intracellular survival of
Leishmania
When Leishmania first enters the human body, it is in the promastigote form.
Promastigotes are engulfed by macrophages but are resistant to proteolysis and
degradation in the phagosome. Once inside the macrophage, the organism is termed
an amastigote.
Leishmania produces acid phosphatases on its surface which inhibit oxidative burst.
The macrophage uses an oxidative burst to destroy foreign material in the
phagosome. This process consists of an attack by superoxide and hydroxyl radicals on
the parasite.
The protozoa are resistant to acidic enzymes as well because it has a proton pump in
the surface which allows its intracellular pH to remain close to neutral.
Also, the protozoan molecule lipophosphoglycan (LPG) plays an active role by
inhibiting lysosomal enzymes. Macrophages often attempt to degrade parasites with
acidic enzymes which occur when lysosomes fuse with the phagosome.
Leishmaniasis/ HIV Co-
Infection

68
Visceral Leishmaniasis/ HIV Co-
Infection

69
Other Trypanosomatid
Parasites
Genus Leptomonas
Genus Herpetomonas
Genus Crithidia
Genus Blastocrithidia
Genus Phytomonas

Read the details from your textbook.


Before we end
Recap on Leishmaniasis

Infective stagemetacyclic
: promastigotes
Diagnostic stage:
amastigotes in cell/
tissues
Stages in Leishmania sp:
metacyclic promastigotes, procyclic
promastigotes & amastigotes (in cells of
infected tissue)
Recap on Leishmaniasis

1. Leishmania sp. stage


found in :
DH (macrophage) amastigotes

IH (Sand fly)
in midgut procyclic promastigotes
in proboscis metacyclic
promastigotes
EXAM JUNE 2014
Leishmania spp. are primarily zoonotic in nature, that are transmitted from host to host by the bite of
small blood sucking insects in the family Psychodidae.
a) Name the insect involved.
(2 marks)
Sandfly (1m), Phlebotomus and Lutzomyia (each Genus name, 0.5m)

a) Name the infective morphotype of the parasite.


(2 marks)
Metacyclic (1m) promastigotes (1m)
c) Explain what happens in human host that are invaded by the infective morphotype in (b).
(4 marks)

Metacyclic promastigotes that reach the wound are phagocytized by macrophages (1m) and other
types of mononuclear phagocytic cells (1m). Promastigotes transform into amastigotes (1m) inside
macrophages. Amastigotes multiply in macrophages of various tissues (1m).

d) Classify the clinical form of leishmaniasis.


(9 marks)
Cutaneous leishmaniasis (L. tropica, L. major & L. mexicana) (1m)
Cutaneous leishmaniasis is the most common form of the disease. It usually
produces ulcers (1m) on the exposed parts of the body, such as the face, arms
and leg. There may be a large number of lesions sometimes up to 200 which
can cause serious disability. When the ulcers heal, they invariably leave
permanent scars (1m), which are often the cause of serious social prejudice.
In mucocutaneous leishmaniasis, the lesions can lead to partial or total
destruction of the mucous membranes (1m) of the nose, mouth and throat
cavities and surrounding tissues (1m) This disabling form of leishmaniasis
can lead to the sufferer being rejected by the community.
Mucocutaneous leishmaniasis (L. braziliensis) (1m)

Visceral leishmaniasis (L. donovani) (1m)


Visceral leishmaniasis, also known as kala-azar or Dum-dum fever (1m), is
characterized by irregular bouts of fever, substantial weight loss, swelling of the
spleen and liver (1m), and anaemia (which may be serious). If the disease is not
treated, the fatality rate in developing countries can be as high as 100% within 2
years.
Post-kala-azar dermal leishmaniasis (PKDL)
Post-kala-azar dermal leishmaniasis (PKDL) is a complication of visceral
leishmaniasis (VL) in areas where Leishmania donovani is endemic; it is
characterized by a hypopigmented macular, maculopapular, and nodular rash
usually in patients who have recovered from VL . It usually appears 6 months to 1
or more years after apparent cure of of the disease but may occur earlier or even
concurrently with visceral leishmaniasis especially in the Sudan. PKDL heals
spontaneously in the majority of cases in Africa but rarely in patients in India. It is
considered to have an important role in maintaining and contributing to
transmission of the disease particularly in interepidemic periods of VL, acting as
a reservoir for parasites.
(**BONUS marks if the answer mention about PKDL)
Complex of diseases (Leishmaniasis)
epidemiology approach
Type of Type of Geographical
Leishmani Leishmani distribution
asis a sp
Visceral L. donovani Worldwide but is
Leishmaniasis predominantly encountered in
(Kala Azar or (L. chagasi) India, South America, Central
Dum Dum (L infantum) Asia, the Middle East, and
Fever) Africa
Cutaneous L. tropica West Central Africa, the Middle
Leishmaniasis L.major East, and Asia Minor to India.
L. mexicana Central America, Mexico,
Texas, possibly in Dominican
Republic and Trinidad.
Mucocutaneous L. braziliensis Central Mexico and Northern
Leishmaniasis Argentina
Summary for Kinetoplasta
KINETOPLASTA
Parasites Vectors Involved Diseases
T. brucei brucei Nagana
Tsetse fly
T. brucei (Glossina sp) African Sleeping
rhodiensie Sickness
T. brucei
gambiense
T. cruzi Reduviid bug Chagas Disease
(Triatomines)
L. tropica Phlebotomus sp. Cutaneous
L. major Leishmaniasis
L. mexicana Phlebotomus sp
&
Lutzomyia sp
L. braziliensis Phlebotomus sp Mucocutaneous
& Leismaniasis
Lutzomyia sp
Following lecture:

Other Flagellated Protozoa