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Title: Laboratory DIangosis of Trypanosomiasis and Leishmaniasis
Description: This is an overview of the biology and laboratory diagnosis of Trypanosomiasis and Leishmaniasis, which are the most clinically important human hemoflagellates.

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HEMOFLAGELLATES

Members of the group Kinetoplastea

Two medically important genera:
o Leishmania
o Trypanosoma

Kinetoplastid – complex of abundant circular DNA within
mitochondrion
o visible with Giemsa stain under light microscopy
o Assumes different morphology depending on presence of
hosts or insect vector

Transported via insect vector
1
...

Cruzi or Leishmania – multiply intracellularly
exclusively

2
...
Brucei
Old world/African
30 ɥm
Graceful curves

Smaller kinetoplast
Leishamnia amastigote





Promastigote






Epimastigote






Trypomastigote




Elongated, slender
Central nucleus
Anterior kinetoplast and axoneme
Free flagellum extending from anterior
end
Occurs in insect vectors of Leishmania
(tsetse fly); Stage detected in cultures,
not seen in humans
not detected in clinical specimens unless
there is a substantial delay in processing
Kinetoplast found closer to the nucleus
Small undulating membrane → free
flagellum
All human-infecting Trypanosoma
assume epimastigote stage in in cultures
and vector, not seen in humans
Kinetoplast found on the posterior end
Flagellum extends along entire length as
undulating membrane + free flagellum at
the anterior end
Occurs predominantly in bloodstream of
mammalian host infected with
Trypanosoma
Metacyclic trypomastigote - infectious
stages found in appropriate insect
vectors; can undergo binary fusion

T
...
Rangeli

Has been described in humans in the Americas

does not cause clinical illness

T
...
Brucei
T
...
Brucei Gambiense

East African Trypanosomiasis
Acute Disease

West African Trypanosomiasis
Chronic Disease

2% of cases
Vector: G
...
Palpalis
Humans are primary reservoir
“African Sleeping Sickness”
Chronic course
Intermittent fevers, night
sweats, malaise
Winterbottom sign – posterior
CLAD
Confusion, fatigue → stupor
and coma

Area of initial bite of Tsetse fly → local multiplication → transient
chancre at bite site

Reduvid bug



Diagnosis

Based on geography and clinical findings

CSF
o ↑ total IgM
o Pleiocytosis: 50 to 500 mononuclears/ɥL of CSF

Identification of parasites via:
o (1) thick and thin smears
o (2) buffy coat preparations
o (3) lymph node and bone marrow aspirate
o (4) centrifuged CSF with Giemsa stain

Culture, Animal inoculation

Molecular Methods

Life Cycle:
o Inoculation
o Enters nearby tissue cells → becomes dividing amastigotes
o Infected cells filled with amastigotes → transformation to
trypomastigote
o Cell rupture → trypomastigote are released into the
peripheral blood → reach distant tissues

Life Cycle

Acute Manifestations

Most common: children <5 y/o

Malaise hills fever, hepatosplenomegaly and myocarditis

Romana’s Sign – swelling of tissues around the eyes

American Trypanosomiasis
Trypanosoma Cruzi: Chaga’s Disease

Transmission: “kissing bugs” - Reduviidae
o Reduviid bugs defecate at time of feeding (contains the
trypomastigotes)
o Rubbing and scratching → enters the body or though intact
mucosa of the mouth or conjunctiva
o Can also be transmitted by blood transfusion




Chagoma – swelling around bite site of reduviid bug
Disease is milder and more asymptomatic in older individuals –
remains infected throughout life

Chronic Disease

Megaesophagus, megacolon, hear conduction problems

Related to destruction of the effector cells of the
parasympathetic system

Quiescent infections may be exacerbated by immunosuppression

T
...
LEISHMANIA

Animal reservoir

Transmission via: Sand flies; acquired in horses, wild animals as
reservoirs
o Old World: genera Phlebotomus
o New World: genera Lutzomyia

Amastigote form in mammalian host, promastigote in insect
vector

T
...
g
...
cruzi infection requires
reactivity on two tests using two different methodologies
and/or two different T
...
Tropica

L
...
Major
May become viscerotopic



“rural or wet ulcer”



More aggressive cutaneous infection, may
also form diffuse cutaneous leishmaniasis
Multiple skin nodules resembling
lepromatous leprosy

L
...
Donovani
L
...
Amazonensis



May produce diffuse cutaneous lesion



“uta” - Benign cutaneous lesion
primarily occurring in children
Domestic dogs – main reservoir



miltefosine

Visceral Leishmaniasis

New World
L
...
Peruviana





L
...
Infantum
L
...
g
...
venezeuelensis
L
...
pifanoi
L
...
hyanensis

Treatment of cutaneous leishmaniasis

endemic settings outside of the Americas
o no risk of mucosal dissemination
o cosmetically unimportant lesions may be treated topically
(e
...
, cryotherapy) or allowed to self-heal

For individuals at risk of aggressive, disfiguring, or disseminated
disease

systemic therapies with sodium stibogluconate

meglumine antimoniate

miltefosine

paromomycin – both oral and topical forms may also be used
Mucocutaneous Leishmaniasis

Caused by: (1) L
...
g nasal, oral, and pharyngeal mucosa



Treated with
o sodium stibogluconate

o

amphotericin

Leishmania amastigotes




Smears: 2 to 3 mm incision at the border of lesion
o Recover small amount of tissue from cut surface
o Smear and imprint must be treated with Giemsa stain
Biopsies should be submitted to histopathology and/or submitted
for culture
Visceral Leishmaniasis:
o (1) buffy coat preparations;
o (2) LN and BM aspirates;
o (3) spleen and liver biopsies

Tissue biopsy

Culture

More sensitive

Allows for identification of species/subspecies

Culture medium:
o Novy-MacNeal-Nicolle Medium

o



Schneiders Drosophila Medium supplemented with calf
serum
Detection of (+) promastigote in 2 to 5 days
Culture should be held for 4 weeks

Promastigotes in culture


Amastigotes can be found in imprints, smears, and tissue sections
o Morphology: Small size (2–4 μm); delicate cytoplasm;
nucleus; kinetoplast
o may appear smaller in In tissue sections due to shrinkage
during fixation
o must be differentiated from other intracellular organisms e
...

yeast cells of H
...
gondii trophozoites

Leishamania Amastigotes; kinetoplast (arrow)





Differential diagnosis: H
...
and T
Title: Laboratory DIangosis of Trypanosomiasis and Leishmaniasis
Description: This is an overview of the biology and laboratory diagnosis of Trypanosomiasis and Leishmaniasis, which are the most clinically important human hemoflagellates.