BLOOD AND

TISSUE
F L A G E L L AT E S
Hemoflagellates
 This are parasites which inhabits the
tissue and the blood of human with the
aid of vectors.
Member species
 Leishmania spp.
 Trypanosoma brucei gambiense
 Trypanosoma brucei rhodesiense
 Trypanosoma cruzi
Different stages of hemoflagellates
Structural parts
• Blepharoplast
– basal body in certain flagellated protozoans that consists of a
minute mass of chromatin embedded in the cytoplasm at the base
of the flagellum.
• Kinetoplast
– is a disk-shaped mass of circular DNAs inside a large
mitochondrion that contains many copies of the mitochondrial
genome
Structural parts
• Undulating membrane
– a locomotory organelle of certain flagellate
(trypanosome and trichomonad) parasites,
consisting of a finlike extension of the limiting
membrane with the flagellar sheath; wavelike
rippling of the undulating membrane produces a
characteristic movement
 Stage of development
Amastigote
“Leishman Donovan Body”
“Leishmanial form”
PARAMETER DESCRIPTION

Size: 5 by 3 μm
Shape: Round to oval

Nucleus: One, usually off center

Other features Kinetoplast present, consisting of
dotlike blepharoplast from which
emerges a small axoneme
Parabasal body located adjacent to the
H
blepharoplast
Nonflagellate, intracellularly seen
in bone marrow or tissue samples

DIAGNOSTIC STAGE found in human samples

N
K 10 mm
 Stage of development
Promastigote
“Leptomonal form”
PARAMETER DESCRIPTION

Size: 9-15 μm long

Shape: Long and slender

Nucleus: One, located in or near center

Other features Kinetoplast, located in
anterior end
Single free flagellum, K
extending from anterior end
usually an extracellular phase
as in the insect intermediate N
host (or in culture) of
leishmania parasites
 Stage of development
Epimastigote
“Crithidial form”
PARAMETER DESCRIPTION

Size: 9-15 μm long

Shape: Long and slightly wider
than promastigote form
Nucleus: One, located in posterior end
Other features Kinetoplast located anterior
to the nucleus
Undulating membrane, N
K
extending half of body length U
Free flagellum, extending from
anterior end
Found in the intestine of the
vectors
10 mm
 Stage of development
Trypomastigote
“Trypanosomal form”
PARAMETER DESCRIPTION
Size: 12-35 μm long by 2-4 μm wide
Shape: C, S or U shape often seen in
stained blood films
Nucleus: One, located anterior to the
kinetoplast U
Other features Kinetoplast located in the N
posterior end F
Undulating membrane, K
extending entire body length
Free flagellum, extending from
anterior end when present

DIAGNOSTIC STAGE found in human samples 10 mm

Trypanosoma cruzi
 Trypanosoma cruzi
• Disease: Chagas disease or American
Trypanosomiasis
• Carlos Chagas: found trypanosome on the intestine of
a triatomid bug were the same parasite found in a child
suffering from fever and enlargement of lymph nodes.
• An intracellular parasite
• Exhibits all four stage of development: amastigote,
promastigote, epimastigote, trypomastigote
BIOLOGICAL VECTOR
Common Names
• Triatomine bugs
• Reduviid bugs
• Assassin bugs
• Conenose bugs
• Kissing bugs

Genera
• Triatoma
• Rhodnius
• Panstrongylus

Reservoir: domestic animals, armadillos, raccoons, rodents, marsupials, primates

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 Modes of Transmission
Source
natural transmission by triatomine bugs through
Vector-borne blood meal/contamination with infected feces

a prevalent mode of transmission in urban area.

Transfusion Gentian violet (24hr) eliminates parasite in
blood

occurs during any stage of T. cruzi infection. Can

Congenital result in premature labor, abortion or neonatal
defects
 Types of Biologic Vector
Salivarian Stercoralian
hind gut station: acquired from
transmission via mouth parts feces or eating the vector

very efficient inefficient

infection rate in vector is low infection rate

Tsetse fly (T. brucei complex) Triatomine bug (T. cruzi)

Pathogenesis
• Acute Phase
- active infection (with chagoma and/or Romaña’s sign)
- 1-4 months duration
- most are asymptomatic (children most likely to be
symptomatic)
• Indeterminate Phase/Latency phase
- Can last up to 10-40 years of latency
- relatively asymptomatic with no detectable parasitemia
- Seropositive
• Chronic Phase
- 10-30% of infected exhibit cardiomyopathy or
megasyndromes (megaesophagus, megastomach, megacolon)
Acute Phase Features
• 1-2 weeks incubation period
• local inflammation
• Romaña’s sign: edema of the
eyelid and conjunctiva
• Chagoma: inflammation at the site
of inoculation
• Symptoms can include: fever, malaise,
lymphadenopathy,
hepatosplenomegaly, nausea, diarrhea
• Acute, often fatal, myocarditis
develops in a few individuals
o high parasitemias in myofibrils
Chronic Chagas' Cardiomyopathy
• Long latency characterized by seropositivity and no
parasitemia
• Higher prevalence of ECG abnormalities in asymptomatic
seropositive persons
• Progressive development of abnormalities
o right bundle branch block
o left anterior hemiblock
• Clinical presentations include:
o arrhythmias and conduction defects
o congestive heart failure
o thromboembolic phenomenon
Pathology
• Cardiomegaly
• Apical aneurysm (left ventricle)
• Extensive fibrosis*
• Hypertrophy*
•  Cellular infiltration
*correlates best with cardiac symptoms
Amastigotes of Trypanosoma cruzi

20 mm

• Pseudocyst in a section of heart muscle

• Note necrosis in upper right corner.
Amastigotes of Trypanosoma cruzi

• Spleen smear
• Note the absence of an
H undulating membrane or
emergent flagellum, the
kinetoplast (K) is more darkly
stained than the nucleus (N),
and the parasite’s cytoplasm is
unstained
• Amastigotes of T. cruzi would
N be indistinguishable from
K 10 mm those of L. donovoni
 Diagnosis
• History of living in infested house
• Bug bite, Chagoma, Romaña's sign

• Cardiac or gastro-intestinal
symptoms
• Imaging

• Detection of parasite (acute stage)

• Serology (chronic stage)
 Diagnosis
• Parasite detection
• Direct examination
• Blood Smears
• Trypomastigotes: Giemsa blood smear
K • Free flagellum, moderately long undulating membrane
10 mm • Posterior location of larger size of the kinetoplast (K)
• Characteristic “C”-shape of several cells

• In Vivo culture (Inoculation Into Mice )

• In Vitro Culture
• Xenodiagnosis - allow trypanosome-free
triatomine bugs to feed on patient and look in
the bug’s intestine for flagellates. Examined
monthly over a period of 3 months.
• PCR
Diagnosis
• Indirect examination
• Serological tests – detection of antibody against T. cruzi
• Hemagglutination
• Immunofluorescence
• ELISA
• Complement fixation
Prevention and Control
• Improvement of human dwellings
• Separation of animal stalls from house
• Health education
• Insecticides
• Synthetic Pyrethroids
• Gentian violet in blood for transfusions
 Treatment
• acute stage
–nifurtimox (8-16 mg/kg/day, 60-90 days)
–benzidazole (5-7 mg/kg/day, 30-120 days)
–allopurinol (experimental)
–azole antifungal agents (experimental)

• chronic stage
–treat symptoms
Trypanosoma rangeli
 Trypanosoma rangeli
• Asymptomatic illness
• Vector: Reduviid bug (Rhodnius sp.)
(but transmitted via saliva compared to T. cruzi )

• Reservoir: wild rodents

• Life cycle: Same as T. cruzi
• Diagnostic tests: Same as T. cruzi
• Diagnostic stage: Trypomastigote
• Specimen: Blood
Tr y p a n o s o m a b r u c e i c o m p l e x

( 1 ) Tr y p a n o s o m a b r u c e i g a m b i e n s e

( 2 ) Tr y p a n o s o m a b r u c e i r h o d e s i e n s e
Trypanosoma brucei
• Disease: Human African Trypanosomiasis
• It is caused by two subspecies of Trypanosoma brucei, namely:
– Trypanosoma brucei rhodesiense: East Africa, wild and
domestic animal reservoirs, East African/Rhodesian
sleeping sickness
– Trypanosoma brucei gambiense: West and Central Africa,
mainly human infection, West African/Gambian sleeping
sickness
• forms exhibited: Epimastigote, Trypomastigote
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 Transmission
• Through the bite of the tsetse
fly (Glossina spp.), the
metacyclic trypomastigotes will
be inoculated to the blood of
the host
– Glossina morsitans
• (Trypanosoma brucei rhodesiense)

– Glossina palpalis
• (Trypanosoma brucei gambiense)
 Pathogenesis of Gambain Trypanosomiasis

1. Initial symptoms (w/in 2-3 days)

 Chancre (earliest sign)
2. Acute phase/Stage I (1-6 months)
 Hemolymphatic phase
• Fever, Headache, Joint And Muscle Pain,
Weakness, And Lymphadenopathy.
• Winterbottom’s sign: enlarged,
non-tender posterior cervical lymph
nodes with a consistency of ripe
plums
• Edema of Arms and Legs
 Pathogenesis of Gambain Trypanosomiasis
3. Late phase/Stage II (3-10 months)
 Meningoencephalitic phase
• Kerandel’s sign: CNS invasion, more
severe headache, increased mental
dullness and apathy, tremors,
hyperesthesia
• Behavioural changes, Progressive
meningoencephalitis, Somnolence
• Eventually, the patient has convulsions, lapses
into a coma, and dies
Rhodesian trypanosomiasis
More rapid and fatal. CNS involvement appear early and neurologic
deterioration is rapid. (< 9 months)
 Diagnosis
• Demonstration of trypomastigotes in
the Giemsa stained blood, lymph
node aspirate and/or CSF
• Serologic techniques:
Lymph node aspirate
Card Agglutination test for
Trypanosoma (CATT), indirect
hemagglutination, ELISA,
immunofluorescence

CSF
Trypanosoma brucei Trypanosoma brucei
gambiense rhodesiense

indistinguishable from one another

 Diagnosis
• Quantitative Buffy coat (QBC) concentration method
 Sample: Buffy coat
 Capillary tube precoated with Acridine orange and potassium oxalate
 Cylindrical float is inserted to enlarge the layers.
Lymph node aspirate

Sample collection  Centrifugation  Examination under Ultraviolet microscope

Positive result: fluorescing trypomastigote among the non-fluorescing RBC

Utility: Trypanosomes, Malaria and Babesia parasites, Microfilaria parasites

CSF
 Treatment
• Effective when begun early in the course of the disease
(Hemolymphatic phase)
• Pentamidine and suramin
• Melarsoprol or tryparsamide (late stage-CSF)
• DL-alpha-diflouoromethylornithine (DFMO, Eflornithine) is an
ornithine decarboxylase inhibitor that is highly effective in
early and late phase of Gambain Trypanosomiasis
o Eflornithine: not very effective against Rhodesian
sleeping sickness
 Prevention and control
• Reduction of contact with tsetse flies
– Traps, screen, insecticides
• Diagnosis and treatment of infected individuals
• Tsetse belt: endemic area extending over third of Africa
Leishmania spp.
Leishmania spp.
• Leishmania have two morphological forms:
a. amastigote
b. promastigote

Causative agent Disease

Leishmania tropica Baghdad boils, Oriental sore
Leishmania mexicana Bay sore, Chiclero ulcer,
Leishmania donovani Dum dum fever, Kala-azar
Leishmania braziliensis Espundia, Uta
Leishmania guyanensis Forest yaws, Pian bois
 Promastigote
• INFECTIVE STAGE to humans
• have single free flagellum arising from
kinetoplast at the anterior end
• Promastigote is in the proboscis of the
insect vector and the one that grows
in artificial media.
 Amastigotes
• Lives intracellularly in monocytes,
polymorphonuclear leukocytes and
endothelial cells of the vertebrate host.
Life cycle of Leishmania
 Transmission
•The reservoir hosts are rodents, dogs,
foxes and jackals
•The infection is usually transmitted by
the bite (blood feed) of the female
sandfly, genus Phlebotomus and
Lutzomyia
•Human infection has been reported
from blood transfusion, congenital
transmission, contamination of
bite wounds.
Pathogenesis
• The multiplying amastigotes inside phagocytes cause
destruction of the host cells.
• Macrophages with amastigote forms in their cytoplasm are
set free in the circulation, i.e from the skin to the
viscera.
• Amastigotes are released and are taken-up by the fixed
macrophages in the spleen, liver, bone marrow, and other
centers of reticuloendothelial activity.
Pathogenesis
• The host cellular defense is stimulated resulting to
proliferation of macrophages in the bone marrow,
which comprises the production of red cells and
granulocytes.
• The end-effects are granulocytopenia and anemia.
• The spleen, liver and lymph nodes are enlarged
whereby the spleen may end up into hypersplenism that
causes more destruction of the red blood cells.
Pathogenesis
• The host immune reaction is also stimulated resulting to
increase production of globulin that may result to
reversal of the albumin-globulin ratio.
• The infection is therefore, regarded as a form of
“reticuloendotheliosis”.
 Types of Leishmaniasis Pathogen Location Diagnosis
CUTANEOUS
LEISHMANIASIS L.. major, L.. tropica, Cutaneous infections are most common in
(localized and diffuse) infections
appear as obvious skin reactions.
L.. aethiopica, and L. Afghanistan, Brazil, Iran, Peru, Saudi Arabia Skin biopsy
mexicana and Syria.
The most common is the
ORIENTAL SORE
MUCOCUTANEOUS
LEISHMANIASIS
Mucocutaneous infections are most
(ESPUNDIA) infections will start Skin biopsy,
common in Bolivia, Brazil and Peru, in
off as a reaction at the bite, and can L. braziliensis Karamay, China Xinjiang Uygur
Membrane
go via metastasis into the biopsy
Autonomous Region.
mucous membrane and become
fatal.
VISCERAL LEISHMANIASIS
infections are often recognized by Found in tropical and subtropical areas of
fever, swelling of the liver and L. donovani complex all continents except Australia. Blood,
spleen, and anemia. They are (L.. donovani, Visceral infections are most common in Bone marrow,
known by many local names, DUM L. infantum syn. L. Bangladesh, Brazil, India, Nepal and Sudan, in Liver/Spleen
DUM FEVER, DEATH FEVER chagasi). part of China, such as Province and Xinjiang biopsy
and Uygur Autonomous Region.
KALA AZAR.
Clinical types of cutaneous leishmaniasis
Leishmania major Leishmania tropica
found in sparsely inhabited areas found in more densely populated regions

Zoonotic Cutaneous leishmaniasis Anthroponotic Cutaneous leishmaniasis

Rural Oriental Sore Urban Oriental Sore

Moist lesions with severe reaction Dry lesions with minimal ulceration

rapid ulceration; few amastigotes Many amastigotes; persists for months

Cutaneous leishmaniasis
Leishmania tropica
• Disease: cutaneous leishmaniasis, Old World
cutaneous leishmaniasis, oriental sores,
Delhi boils, Baghdad boils, dry or urban
cutaneous leishmaniasis.
• incubation period: 2 weeks to several months
• skin ulcer: elevated and indurated
• lesions are painless but with subcutaneous
nodules
Leishmania mexicana
• Disease: New World cutaneous leishmaniasis,
Chiclero Ulcer, Bay Sore
• North Central America, Mexico, Texas and possibly the
Dominican Republic and Trinidad
• Cutaneous form, increasing in numbers of infected
• 3 clinical manifestastions
– Cutaneous – Chiclero-ulcer
– Nasopharyngeal mucosal – rare manifestation
– Visceral – rare manifestation
• main reservoir are rodents
 Uncommon types
Leishmania aethiopica
– Diffuse Cutaneous
Leishmaniasis (DCL):
caused by, diffuse nodular
non-ulcerating lesions.
Low immunity to
Leishmania antigens
(anergic), numerous
parasites.
*DCL is sometimes referred to as Anergic Leishmaniasis
 Uncommon types
• Leishmaniasis recidiva
– Lupoid leishmaniasis:
severe immunological
reaction to leishmania
antigen leading to persistent
dry skin lesions, few
parasites.
Cutaneous Leishmaniasis
• Clean the surface of the lesion with 70% alcohol.
• At the margin of the lesion, aspiration, scraping, or punch
biopsy is done.
• The collected samples are smeared and stained with Giemsa or
Wright’s stain.
• Physicians send biopsy samples to Histopathology section.
Leishmania braziliensis
• Causes Espundia, Uta or Mucotaneous
Leishmaniasis/American Leishmaniasis
• Found in Central Mexico and Northern Argentina
• Find LD bodies in tissues
• Once cured, lifelong immunity; if dormant – may re-occur
Mucocutaneous Leishmaniasis

 Has a clinical picture dominated by great

destruction of the nasal mucosa,
sometimes with respiratory complications
Mucocutaneous leishmaniasis is the most
feared form of leishmaniasis because it
produces destructive and disfiguring
lesions of the face (Tapir nose)
Espundia: metastatic spread to the
oronasal and pharyngeal mucosa
 Diagnosis: Cutaneous and
Mucocutaneous Leishmaniasis
Diagnosis:
• Smear: Giemsa stain – microscopy for
LD bodies (amastigotes)
• Biopsy: microscopy for LD bodies or
NNN medium culture in NNN medium for
promastigotes
• Serologic techniques: IFA
 Diagnosis: Cutaneous and
Mucocutaneous Leishmaniasis
• Montenegro intradermal skin test
– highly specific and of great use in cutaneous
leishmaniasis, although the test may be negative in the
disseminated form.
– Cellular immunity depends on T-lymphocytes and
becomes positive 24-48 hours after infection.
– Positive: Redness with induration
Leishmania donovani
• Disease: Visceral leishmaniasis, kala-azar, dum dum fever
• There are geographical variations.
• Leishmania infantum mainly affect children
• Leishmania donovani mainly affects adults

• Samples: Blood sample, Bone marrow, Liver/Spleen biopsy

Clinical manifestation
• Fever: twice daily elevations
• Splenomegaly, hepatomegaly,
hepatosplenomegaly
• Weight loss
• Anemia
• Epistaxis
• Cough
• Diarrhea
• Loss of weight
• Lymphadenopathy
• pancytopenia
• Hypergammaglobinemia
• darkening of the skin
• Untreated disease can be fatal
• After recovery it might produce
a condition called post kala-
azar dermal leishmaniasis
(PKDL) that resembles
histioid type of leprosy

PKDL
Diagnosis • Microscopy of clinical samples
(amastigote)
• Culture in NNN medium of the
following specimen:
 Bone marrow aspirate
 Splenic aspirate
 Lymph node
 Tissue biopsy
Diagnosis
• Specific serologic tests: Direct Agglutination Test (DAT), ELISA,
IFAT, Complement fixation test
• Skin test (leishmanin test) for survey of populations and follow-
up after treatment.
• Non specific detection of hypergammaglobulin by formaldehyde
(formol-gel) test or by electrophoresis.
• Antibody titers
– low in cutaneous, high in mucocutaneous and very high in
disseminated cutaneous or visceral leishmaniasis.
Treatment
• Similar medications used for leishmanisis but modes
of administration and dosages may vary.
 First-line therapy (Antimonials): SbV, Pentavalent
antimonials include sodium stibogluconate and methyl-
glucamine antimonite.
 Second line theraphy: Amphotericin B,
pentamidine(for kala-azar), metronidazole, nifurtimox.
 Liposomal AMB (L-AMB) is less toxic than AMB.
It has been effective in the primary treatment of VL
in both immunocompetent and
immunocompromised patients
Prevention and Control
• improvement of human dwellings
• separation of animal stalls from house
• health education
• insecticides
• synthetic pyrethroids
• gentian violet in blood for transfusions