Blood and Tissue

flagellates
 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
Outline
• General features of blood and tissue flagellates
• Summary of taxonomic classification of protozoa
• Blood and tissue flagellates
– For each species:
 Epidemiology , morphology, transmission life
cycle , clinical features, laboratory diagnosis
treatment, prevention& control
 Blood and Tissue flagellates
• Belonging to the family Trypomastidae

• Two are responsible to cause disease to man

Genus Leishmania
Genus Trypanosoma
 LEISHMANIA

EUGLENOZOA KINETOPLASTIDEA TRYPANOSOMATIDAE

PROTOZOA
TRYPANOSOMES
 General Characteristics of blood & tissue
flagellates
– Reproduces by simple longitudinal binary fission

– 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.
 The following are the main developmental forms

1. Amastigote (Leishmanial form)

 Rounded body, central nucleus and eccentric

kinetoplast visible

 No free flagellum,

 No undulating membrane,

 Theonly intracellular forms of all Leishmania

species and Trypanosoma cruzi. (in vertebrate
host)
2. Promastigote (Leptomonad form)
 Elongated body, central nucleus, anterior kinetoplast

 Single anterior flagellum arises from kinetoplast

 Found in the invertebrate host, and in culture media

(of all Leishmania species) and in man for
Tryponosoma cruzi
3.Epimastigote /crithidial/ forms
• Elongated body, single free flagellum, single
nucleus

• Has undulating membrane,

• kinetoplast is just anterior to the nucleus

• found in the invertebrate host and in culture

media (of Trypanosome species)
4. Trypomastigote
 Pleomorphic, it can be as “U” or “C” shaped,

 Central nucleus, posterior kinetoplast

 Single Flagellum arises posteriorly

 Has undulating membrane

 Found in the peripheral blood of vertebrates and is

the diagnostic stage of Trypanosome species.
Leishmaniasis
 Intoduction

• Leishmania is causative agent for

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
• Kingdom Protista
• Subkingdom Protozoa
• Phylum Sarcomastigophora
• Sub-phylum Mastigophora
• Class Zoomastigophora
• Order Kinetoplastida
• Suborder Trypanosomatina
• Genus Leishmania
 Classification

• 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;
– L. braziliensis complex
• L braziliensis
• L. Peruviana

– L. Guyanensis complex
• L. Guyanensis
• L. panamensis

– L. tropica; L. major & L. aethiopica

• Leishmaniasis can classified clinically as:

• Visceral leishmaniasis

• Cutaneous leishmaniasis

• Mucocutaneous leishmaniasis
• Cutaneous leishmaniasis(CL)
– L. tropica
– L. major
– L. aethiopica
– L. panamensis
– L. guyanensis
– L. peruviana

• Visceral leishmaniasis(VL)
– L. donovani
– L. infantum
– L. Chagasi
• Mucocutaneous leishmaniasis(MCL)

– L. panamensis

– L. guyanensis

– L. Brazilliensis
 Epidemiology

• 350 million people are at risk in 88 countries

around the world
72 of which are developing countries

• 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
 Global Status

new world: old 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
• Geographical distribution of leishmaniasis is

limited by:

– The distribution of the its vector (sand fly)

– Temperature, altitude
 Distribution in Ethiopia
• In Ethiopia

– Four species of Leishmania is found, namely,

• L. aethiopica,
• L.major
• L. tropica
• L. donovani
• L. donovani (Visceral leishmaniasis), Ethiopia
– 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 Konso Wereda

• The Omo river plains and

• The Metema and Humera plains in north western Ethiopia

• Cutaneous leishmaniasis (CL)- Ethiopia
– 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 Gamo Gofa with the highest rate in Ocholo
village in Gamo Gofa

– Major Reservoirs in Ethiopia: rock hyraxes (Procavia

habessinica) and tree hyraxes (Heterhyrax brucei ) for L.
aethiopica
 Vectors
• Sand flies (Diptera), Family-psychodidae,
subfamily-phlebotominae.
• About 30 spp of sand flies in the Genera
– Phlebotomus (Old World) and

– Lutzomyia (New World), which can transmit at

least 20 different species of leishmania parasites

• Adults: 2-4mm, with a yellowish hairy body.

• During the day, they rest in dark & sheltered places such
as burrows of rodents, bark of old tries, in ruined
buildings, cracks in house walls, and in house hold
rubbish.
• They are active at dusk & during the night.
• Both sexes feed on plants,
• But females also need a blood meal before they are able
to lay eggs
• Vectors In Ethiopia

• CL
– Phlebotomus longipes, Phlebotomus pedifer

• VL
– Phlebotomus orientalis, Phlebotomus martini,
Phlebotomus celiae
 Reservoirs
• The primary reservoir hosts of
Leishmaniasis are:

– Sylvatic mammals such as forest

rodents, hyraxes and wild canids,

– Domestic animals; Mainly dogs

 Transmission and life cycle

• Common mode of transmission.

– Bite of sand fly
• Genera Phlebotomus in Old
world
• Lutzomyia in New world
• Uncommon modes of
transmission:
– Congenital transmission,
– Blood transfusion,
– Rarely, inoculation of cultures.
 Life cycle
Two Life-cycle stages

Promastigote
Amastigote
 Elongated, with flagella
 Round (3-7 µm diameter)
(10-20 µm long)
 Occurs intracellularly, during
 Occur extracellularly in the nsect
mammalian stage
midgut & in artificial culture
 Non-motile
 Motile
General life cycle of Leishmania species

• Female sandflies inject the

infective stage/promastigotes
during blood meals
• Promastigotes are
phagocytized by macrophages
& transform into intracellular
amastigotes.
• 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) around sand fly bite
• 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
 Host-Parasite Interactions
 

• Entrance into the host and establishment of

infection by leishmanias is enhanced by saliva
from the vector

• The promastigotes are engulfed & form

phagosome
• phagosome + lysosome = phagolysosome

 As the promastigotes transform into amastigotes,

which produce compounds that counter lysosomal
enzymes

Leishmania can survive acidic environment of

lysosmes by regulating their internal PH.
 Clinical features and pathology

Cutaneous leishmaniasis (CL)

 most common form,

 Relatively benign self-healing skin

lesions (localized or simple CL)

• Local swelling with increased temperature
• Develop to small erythema then develops into a
papule, and the papule into a nodule,
• The nodule become ulcerates/lesion within
two weeks to six months,
• The ulcer is nodular with thick borders -
characteristic of the cutaneous leishmaniasis
• Such ulcers can last from 3–5 months to 15–20
years
• The ulcer is sometimes covered by a whitish
pseudo-membrane
• Not painful (if no secondary infection)

• Ulcers may be multiple

• Auto infection may occur in long contact

Mucocutaneous Leishmaniasis (MCL)

– Start with simple skin lesions that metastasize to

mucosa especially nose and mouth
region
– Tends to occur 1 to 5 years after cutaneous
leishmaniasis caused by these organisms has healed,
– But it can also be seen while skin lesions are still
present
• Initially it is similar with CL
• Then destructive inflammation of nasal
septum, and may be pharynx or larynx.
• Frequent nose bleeds can be an early sign.
• Cause severe disfigurement of the face, and
block the pharynx or larynx.
• In some cases, the genitalia may also be
involved.
• Mucocutaneous leishmaniasis does not heal
spontaneously
PICTURES
 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)
 Cutaneous
Leishmaniasis
• Chronic ulcerated, papular,
or nodular lesion
• Lesion is painless, non-
tender, non-pruritic and
usually clean

Chiclero Ulcer (L.

mexicana)
(L. aethopica)
 Characterized by one or more sores,
papules or nodules

occasionally
satellite lesions

ulcerated, papular, or
nodular lesions
 Cutaneous
Leishmaniasis

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

• Self-healing, months to
years
• Sores can leave significant
scars and be disfiguring if
they occur on the face
• Metastasis via blood or
lymphatic systems
• Especially, L. braziliensis
 CL
• 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
 Old World CL

L. Tropica (SW Asia, North

Africa
• Anthroponotic or dog
reservoir
• Dry urban oriental sore
• Dry painless lesion’
• 25-70mm diameter
 Old World CL
L. major
• In 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)
• Protect against reinfection
Ulcers are moist or open with
& also with L.tropica
seropurulent exudate
 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
Mucocutaneous Leishmaniasis

• Primarily by 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
 Mucocutaneous Leishmaniasis

L. braziliensis (espudia)
• Variable types and sizes of
lesions
• Chronic and painless
• Ulcerative type
• Rapid and extensive
mutilation
• Non-ulcerative type
• local edema (upper lip)
• 'tapir' nose
Visceral Leishmaniasis (Kala-azar)
 Caused by the Leishmania donovani complex,
Leishmania donovani
L. infantum
L. chagasi
• Affect the Reticuloendothelial system
• spleen, liver, bone marrow, lymph nodes
• Progressive disease
• 75-95% mortality if untreated
• death generally within 2 years
 In symptomatic cases, common visceral signs
include:
lethargy, weight loss, appetite loss, anemia,
splenomegaly, fever (accompanied by chills) and local
or generalized lymphadenopathy.

 Bleeding disorders, including epistaxis and

hematuria can also be seen
• Death usually occurs because of severe secondary
bacterial infections in advanced disease

• Pneumonia is the common complication

• Asymptomatic cases are common (host and parasite
factors)

• Patient history is very important (travel)

 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
• Profile view of a
teenage boy
suffering from
visceral
leishmaniasis. The
boy exhibits
splenomegaly,
distended abdomen
and severe muscle
wasting. 
• A 12-year-old boy
suffering from
visceral
leishmaniasis. The
boy exhibits
splenomegaly and
severe muscle
wasting
• Jaundiced hands
of a visceral
leishmaniasis
patient
• Enlarged spleen
and liver in an
autopsy of an infant
dying of visceral
leishmaniasis.
 Post Kala Azar Dermal leishmaniasis (PKDL)

 After recovery from L. donovani

 Characterized by macular, maculopapular, and
nodular rash (Lesion)
 5 to 60% of patients develop a dermatosis
(PKDL) during or after treatment.
Occurs in 1-3% of Indian and 50% of Sudanese VL
patients
 PKDL follows VL within 0–6 months in Sudan
and 2–3 years in India
Cause:
 The reality is still misery but the hypothesises are:
Immunological (patients start production of INF-γ,
inflammation)
Antimonial drugs - sodium antimony gluconate (SAG)
causes immunological advance for parasites
UV light contributes towards development of PKDL –
immunological advance for parasites
Reinfection or parasite persistence
Failure of tissue-specific T cell memory
Host genetic susceptibility factors
 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

 Low mortality but high stigma
Treatment:
 Use treatment guidelines
sodium antimony gluconate (SAG)
miltefosine and
amphotericin B
 It require expensive and prolonged treatment

 Easily cured with treatment (in contrast to

DCL)
 Leishmaniasis and HIV Infection
• Coexistence of leishmaniasis with HIV infection
is a serious concern

• Leishmaniasis is spreading in several areas of the

world because of the rapidly spreading epidemic
AIDS

• The immune deficiency has lead to increased

susceptibility to infections, including
leishmaniasis
• In patients infected with HIV, leishmaniasis
accelerates the onset of AIDS
– by cumulative immunosuppression and
– by stimulating the replication of the virus

• It may also change asymptomatic Leishmania

infections into symptomatic infections
 Diagnosis of CL and MCL
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: Novy –Nicolle-MacNeal(NNN),
3. Leishmainin test
4. Serology?
 1.Collection and examination of slit skin
smears for amastigotes
• Taken from the inflamed raised swollen edge
of an ulcer or nodule not from its base or
center

• if bacterial infection is present, examination

for Leishmania amastigotes is best delayed
until antimicrobial treatment has been
completed and the bacterial infection has
cleared.  
make incision in active part of lesion
scrape cells from incision
aspiration and culture
prepare Giemsa-stained smear
Amastigote
promastigotes in vitro culture/sandfly
 Leishmainin   or Montenegro test
•  The antigen is prepared from killed promastigotes
of L. braziliensis , L.mexicana or L.tropica with a
concentration of 10x106 parasites per ml

•  Positive reaction: when the area of indurations is

5mm in diameter or more
Delayed Hypersensitivity Skin Test
• leishmanin skin test, Montenegro reaction
• intradermal inoculation of leishmanin
• suspension of whole or
disrupted promastigotes
• preferably from local area
• include negative control
• induration ± erythema in 48-72 hours
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
 1.Demonstration of the parasite

 Finding of amasigote from aspirate

Aspirate %positive
Spleen …………………………………….95-98%
Bone marrow …………………………….64-86%
Enlarged lymph node …………………About 64%
Buffy coat (India) ………………………….67-99%
Buffy coat (Africa)……………….........About 50%
 prevention and control
1. Early detection by serological diagnosis (VL) and
treatment of infected persons

2. Personal protection from sand fly bites by:

– Using insect replants
– Avoiding endemic areas especially at times when sand flies 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
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
 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