University of Gondar

College of Medicine and Health Sciences

School of Biomedical Laboratory Sciences
Department of Medical Parasitology

MODULE TITLE: HAEMATOLOGY MODULE

MODULE CODE: HEMA-2032
TOPICS: BLOOD AND TISSUE FLAGELLATES

Aberham Abere (BSc, MSc)

 Learning objective
At the end of this unit the students will be able to:

• Describe the epidemiological aspects of blood & tissue flagellates

• Discuss the general characteristics of blood & tissue flagellates

• Discuss the characteristics of each blood & tissue flagellates

• Explain the life cycle of each blood & tissue flagellates

• Apply the necessary laboratory procedures for the detection and identification
of blood & tissue flagellates

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 Blood and Tissue flagellates
• belonging to the family Trypomastidae

• six genera but only two of them are responsible to

cause disease to man
Genus Leishmania
Genus Trypanosoma LEISHMANIA

PROTOZOA EUGLENOZOA KINETOPLASTIDEA TRYPANOSOMATIDAE

TRYPANOSOMES

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General Characteristics blood & tissue flagellates

• Leishmaniasis is a common NTD/neglected disease in

tropical, subtropical, and arid parts of the world

• Transmission occurs through biological insect vectors as

intermediate hosts & human as definitive host

• The species are morphologically indistinguishable, but

they can be differentiated on the basis of on their
clinical features, geographical distribution, serologic tests,
immunological tests, etc.
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• The different developmental forms are differentiated on the basis of
a)Presence or absence of free flagellum
b) Presence or absence of undulating membrane
c) Position of the kinetoplast relative to the nucleus.

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 anterior
Flagellum
Kinetosome Flagellum
Kinetoplast Kinetosome
Nucleus Kinetoplast
Nucleus

posterior

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Developmental forms

1. Amastigote /Leishmanial form)

2. Promastigote /Leptomonad
3. Epimastigote /crithidial
4. Trypomastigote
5. Metacyclic Trypomastigote /Trypanosomal

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 Anterior

Undulating
membrane

Posterior

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• Causative agent of Leishmaniasis

• obligate intracellular protozoa of the genus Leishmania

• Named after Leishman, who first described it in London in May 1903

• In the human host, Leishmania are intracellular parasites that infect the
mononuclear phagocytes

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• Human infection is caused by about 21 of 30 species that infect
mammals.  These include:
• L. donovani complex with 3 species
L. donovani,
 L. infantum,
 L. chagasi

• L. mexicana complex with 3 main species

L. mexicana
 L. amazonensis
 L. venezuelensis

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• L braziliensis complex
• L braziliensis
• L pruviana

• L guyanensis complex
• L guyanensis
• L panamensis

• L tropica; L major & L aethiopica

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• Leishmaniasis can easily classified clinically as

•Visceral leishmaniasis
•Cutaneous leishmaniasis
•Mucocutaneous leishmaniasis
•Diffuse cutaneous leishmaniasis

• These different forms of the disease is caused by the different species of

Leishmania

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 Epidemiology

• 350 million people are at risk in 88 countries around the world

72 of which are developing countries

• an estimated 12 million cases world wide ;1.5 to 2 million new cases occur every
year
CL form representing 50 to 75% of all new cases

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• Most of the affected countries are in tropics and sub tropics
• 90% of all VL cases occur in Bangladesh, Brazil,
India, Nepal and Sudan;

• 90% of all MCL cases occurs in Bolivia, Brazil and

Peru

• 90% of all CL cases occur in Afghanistan, Brazil,

Iran, Peru, Saudi Arabia and Syria

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 Global Status

old world:
new world: Asia, Africa, Europe
south and central America

L. infantum
( L. chagasi )
L. donovani
L.mexicana
L. infantum
L.brazilliensis
L. tropica
L. peruriana
L. major
L.panamensis
L. aethiopica
L.guyanensis
L.amzonensis
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• Geographical distribution of leishmaniasis is limited by:
• The distribution of the sandfly,

• Its tendency to take blood from humans or

animals only, and

• Its capacity to support the internal

development of specific species of leishmania

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 • The incidence of leishmaniasis is increasing, mainly because of:
• Man-made environmental changes

• Poverty and malnutrition

• Movement of susceptible populations into

endemic areas

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Distribution in Ethiopia

• In Ethiopia
• Four species of Leishmania is found, namely,
• L. aethiopica,
• L. major
• L. tropica
• L. donovani

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• Visceral leishmaniasis (VL)
• Occurs mainly in arid and semiarid lowlands below
1,300 m altitude

• Important endemic foci include

• Gelana focus at lake abaya,
• the segen valley (Aba- Roba focus) in Knoso Wereda
• the Omo river plains and
• the Metema and Humera plains in north western
Ethiopia

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• Cutaneous leishmaniasis
• Endemic at altitudes between 1400 and 2700 m in
most administrative regions

• Prevalence rates of 5.5 – 40% were reported from

villages in Shewa , Wello and G.Gofa with the highest
rate in Ocholo village in G. Gofa

• Rock hyrax (Procavia habessinica) and tree

(Heterhyrax brucei ) hyraxes serving as reservoir host
for L. aethiopica

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 Transmission and life cycle
• Common mode of transmission.
• Bite of sandfly
• Genera Phlebotomus in Old
world
• Lutzomyia in New world
• Uncommon modes of
transmission:
• Congenital transmission,
• Blood transfusion,
• Rarely, inoculation of cultures.

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 Life cycle
Two Life-cycle stages
ProPromastigote
AmastigoAmastigote
-Elongated, with flagella
(10-20 µm long) -Round (3-7 µm diameter)

-Occur extracellularly in the -Occurs intracellularly,

-insect midgut & in artificial culture during mammalian stage

-Motile -Non-motile

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Mammalian R. Hosts
• Sloths
• Rodents
• Primates
• Gerbils
• Dogs
• Hyraxes
• Foxes
• Bats
• Anteaters
• Porcupines
• Opossums

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General life cycle of Leishmania species

• female sandflies inject the infective

stage, promastigotes, during blood
meals
• about 30 species of sand flies acts
a vector

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 Promastigotes are phagocytized by macrophages &
transform into intracellular amastigotes form

Amastigotes multiply by binary fission, rapture from

macrophages , and infect new cells
 In VL the amastigotes are carried through blood
circulation , then invade and multiply in the macrophages
of spleen, liver, Bone marrow, lymph glands , etc.
In Cl , MCL – the amasigote multiply in skin
macrophages (histocytes)

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• Sandflies become infected during blood meals when they ingest
macrophages infected with amastigotes

• the host cell break down and releasing the amasigotes which is then
transform to promastigotes

• multiply , fill the lumen of the gut and migrate to the proboscis

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 Host-Parasite Interactions
 
• Entrance into the host and establishment of infection by leishmanias is
enhanced by saliva from the vector

• Two substances were involved

Maximum dilation molecule/maxadilin: keeps the
capillary bed open
 Salivary immunosuppressive protein/SIP: restrains
the immune system’s early efforts to eliminate the
parasites

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 Infective promastigotes entering the blood of the vertebrate are covered by two
key molecules:
the protein gp 63 and
 lipophosphoglycan (LPG)
 Both mediate the uptake of promastigotes by
macrophages

• The promastigotes are engulfed & form phagosome

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• phagosome + lysosome to form a phagolysosome

 As the promastigotes transform into amastigotes,

which produce compounds that counter lysosomal
enzymes

 The gp 63 molecule inactivates proteolytic enzymes

 LPG protects against other enzymes

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 Clinical features and pathology
Cutaneous leishmaniasis (CL)
 most common form,
 Relatively benign self-healing skin
lesions (localized or simple CL)
Leishmaniasis recivida: rare hypersensitive dermal

 Diffuse cutaneous leishmaniasis (DCL)

 Rare cutaneous infection with non- ulcerating
Nodules resembling lepromatous leprosy

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Mucocutaneous Leishmaniasis (MCL)
- simple skin lesions that metastasize to
mucosae especially nose and mouth
region

Visceral Leishmaniasis (VL)

 generalized infection of the reticuloendothelial
system, high mortality response

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Cutaneous Leishmaniasis
Causative agents

Leishmania tropica
Leishmania major
Leishmania aethiopica
Leishmania mexicana
Leishmania peruriana
Leishmania panamensis
Leishmania guyanensis

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 Cutaneous
Leishmaniasis
• incubation period: 2 weeks
to several months
• Initially, the lesion is a
small, red papule up to 2
cm in diameter
• change in size and
appearance over time

Chiclero Ulcer (L.

mexicana)
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 Cutaneous
Leishmaniasis
• chronic ulcerated,
papular, or nodular lesion
• lesion is painless, non-
tender, non-pruritic and
usually clean

Chiclero Ulcer (L.

mexicana)
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 Characterized by one or more sores,
papules or nodules

occasionally
satellite lesions

ulcerated, papular, or
nodular lesions

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 Cutaneous
Leishmaniasis
• chronic ulcerated,
popular, or nodular
lesion
• lesion is painless, non-
tender, non-pruritic and
usually clean

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 Cutaneous
Leishmaniasis

• self-healing, months to
years
• Sores can leave significant
scars and be disfiguring if
they occur on the face

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 • metastasis via blood or
lymphatic systems
• especially L. braziliensis

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 Cutaneous
Leishmaniasis
Often described as looking
somewhat like a volcano with a
raised edge and central crater
• occasionally palpable lymph
nodes

The clinical forms of CL vary

according to
-The species of parasite
-Region and
-Response of patient
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 Old World CL
L. Tropica (SW Asia, N.Africa
• Anthroponotic or dog
reservoir
• dry urban oriental sore
• 'dry painless lesion’
• 25-70mm diameter

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 Soldier in
Afghanistan with
Officer holding Iraqi child Leishmania tropica
with Leishmania tropica on hand
on face
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 DRY, CRUSTED/SCABBED APPEARANCE

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 Old World CL
• Are self-healing,1-2yrs
• Often leave disfiguring
scars
• Immune to re-infection
• Rarely develop multiple
un-healing lesion known
as leishmaniasis
hyper-pigmentation of scar recidivans (LR)

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 Leishmaniasis Recidivans

• multiple unhealing lesions, often on

the face
• Relapsing leishmaniasis
• Often due to inadequate treatment
or allergic state
• Nodular lesions or rash around
central healing
•Can persist for decades
•Variable expression
• Not easily cured

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 Old World CL
L. major
• (central Asia, middle East,
Africa)
• rural (rodent reservoir)
• wet oreintal sore.
• Wet lesion
• Early papules is
inflamed(5-10mm)
• Develop to large uneven
ulcer
• Self-healing (3-6mths)
ulcers are moist or open with • Protect against
seropurulent exudate reinfection & also with
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L.tropica 59
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 Old World CL

L. infantum
• Mediterranea, Europe
• dermotrophic strains
recently recognized
• L. aethiopica
• highlands of Kenya and
Ethiopia
• Similar to oreintal sore
• Self-heal 1-3 yrs
• Can cause DCL

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 Diffuse Cutaneous
Leishmaniasis
• Lesion develop over
large areas of the body
• Scaly, not ulcerated,
nodules
• Chronic and painless
• Numerous parasites in
lesions
• Seldom heal despite
treatment
L. aethiopica
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 Diffuse Cutaneous
Leishmaniasis
• scaly, not ulcerated,
nodules
• chronic and painless
• numerous parasites in
lesions
• seldom heal despite
treatment

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 Diffuse Cutaneous
Leishmaniasis

• scaly, not ulcerated,

nodules
• chronic and painless
• numerous parasites in
lesions
• seldom heal despite
treatment

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New World (sp?) 64
 Mucocutaneous Leishmaniasis
• primarily L. braziliensis
(espudia)
• two stages
• simple skin lesion
• 2o mucosal involvement
• metastasis via blood or
lymphatic systems
• can occur long after primary
lesion (up to 16 years)
• frequently in naso-pharyngeal
mucosae
• junction of skin and mucosa
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 Mucocutaneous
Leishmaniasis

L. braziliensis (espudia)
• variable types and sizes
of lesions
• chronic and painless

• ulcerative type
• rapid and extensive
mutilation

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 Mucocutaneous
Leishmaniasis
• L. braziliensis (espudia)
• variable types and sizes
of lesions

• ulcerative type
• rapid and extensive
mutilation

• non-ulcerative type
• local edema (upper lip)
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• 'tapir' nose 67
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 Visceral Leishmaniasis
 caused by the Leishmania donovani complex,
 Leishmania donovani
L. infantum
L. chagasi
• reticuloendothelial system affected
• spleen, liver, bone marrow, lymph nodes

• onset is generally insidious

• progressive disease
• 75-95% mortality if untreated
• death generally within 2 years
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• Death usually occurs because of severe secondary bacterial infections in
advanced disease

• Pneumonia is the common complication

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 Clinical Presentation
• incubation period
•generally 2-6 months
•can range 10 days to years
• fever, malaise, weakness
• wasting despite good appetite
• spleno- and hepatomegaly,
enlarged lymph nodes
• depressed hematopoiesis
•severe anemia
•leucopenia
•thrombopenia  petechial
hemorrhages in mucosa
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• Profile view of a teenage boy
suffering from visceral
leishmaniasis. The boy exhibits
splenomegaly, distended
abdomen and severe muscle
wasting. 

73
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• Jaundiced hands of a
visceral leishmaniasis
patient

74
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Post Kala azar Dermal leishmaniasis (PKDL)
characterized by hypo pigmented and raised erythematous
patches on the face, trunk of the body and limbs

 may develop in to nodules and resembles those of lepromatous

leprosy, fungi infections or other skin disorders
Occasionally there is ulceration of lips and tongue
occurs in 1-3% of Indian and 50% of Sudanese VL patients
It require expensive and prolonged treatment
easily cured with treatment (in contrast to DCL)

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 General Laboratory diagnosis of parasitic
diseases
1. Morphological diagnosis (Macroscopic /Microscopic diagnosis)

2. Immunological diagnosis (Antibody and antigen detection

4. Molecular diagnosis

5. Culture

6. Animal inoculation

7. Xenodiagnosis

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 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

1. Demonstration of parasite
amastigotes (scrapings, biopsy,
aspirates)
2. culture from ulcer material
3. Leishmainin test
4. serology?
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 DIAGNOSIS Visceral leishmaniasis
(i) Demonstration of parasite in tissues by
 light microscopic examination of the stained
specimen,
 culture
 animal inoculation
(ii) Detection of parasite DNA in tissue samples

(iii) immunodiagnosis by detection

 Antigen detection
 antibody detection

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 Treatment
Treatment

• Sodium stibogluconate (Pentostam)

• Pentamidine isethionate

• amphotericin B

• cryotherapy and thermotherapy

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 prevent and control

1. Early detection by serological diagnosis (VL) and treatment of infected

persons

1. Personal protection from sandfly bites by:

• Using insect replants
• Avoiding endemic areas especially at times when sandifies are most active
• Use of pyrethroid impregnated bed nets and curtains

3. Vector control by the use of light traps, sticky paper traps, or residual
insecticide spraying of houses

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4. Destruction of stray dogs and infected domestic dogs

5. Elimination and control of rodents

6. sitting human dwellings away from the habitats of animal reservoir

hosts where sandifies are known to breed

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 Summary

• Discuss the general charcterstics of blood and tissue flagelates

• List the laboratory diagnosis approaches for Cl DCL, MCL and VL

• List the general control and prevention methods used for

leishmaniansis

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