Blood and tissue flagellates

notes 2014

1. BLOOD AND TISSUE FLAGELLATES

Trypanosoma cruzi
DISEASE: Chagas’ Disease or American Trypanosomiasis
 the ONLY trypanosome that has an intracellular
amastigote stage
VERTEBRATE HOST: humans, domesticated and wild
animals
VECTOR/INTERMEDIATE HOST: Reduviid bug/ Kissing
bug/Triatomid bug/Assasin bug
 Triatoma infestans
 Triatoma sordida
 Panstrongylus megistus
 Rhodnius prolixus
GEOGRAPHIC DISTRIBUTION
 Southern part of United States, Mexico, Brazil
Central America, South America
4 STAGES OF DEVELOPMENT
1. Amastigote or leishmania form - found
intracellularly in a human host. No free
flagellum
2. Promastigote or leptomonas form – elongated
spindle-shaped with pointed ends and a short
free flagellum that arises from the kinetoplast
at the anterior end
3. Epimastigote or Crithidia –elongate spindle-
shaped with the free flagellum from the
anterior continuing backwards along the margin
of the undulating membrane and ends at the
kinetoplast which is situated anterior to the
nucleus
4. Trypomastigote or Trypanosoma form – found
in the human host bloodstream as well as
infective stage metacyclic trypomastigote in the
fly vector. In trypomastigotes the kinetoplast is
near the posterior end of the body, and the
flagellum lies attached to the cell body for most
of its length by an undulating membrane.



MODE OF TRANSMISSION
 Bite of an infected Reduviid bug
(Infective metacyclic trypomastigotes found in
the feces of reduviid bug penetrates the skin as
the bite wound is rubbed/scratched)
 Blood transfusion
 Transplacental route
 Accidental ingestion of infected insect
STAGE: TRYPOMASTIGOTE
 Found in the peripheral blood
 Length: 16 -20 um
 Pointed posterior end with large kinetoplast
 Centrally located nucleus
 Undulating membrane runs the entire length of
the parasite and extends as a free flagellum
 In stained specimens, they are C-shaped/U-
shaped/ S-shaped
 Cork-screw-like motility


STAGE: AMASTIGOTE
 Develops in cardiac muscles, brain tissue,
visceral tissue (INTRACELLULAR)
 Round or ovoid in shape
 1.4 um – 4 um in diameter
 Large red nucleus
 Has a dark-staining rod-shaped kinetoplast


LIFE CYCLE
All hemoflagellates are dimorphic.
2 phases of the Life Cycle:
1. within humans
2. Within insect vector or the intermediate host

2. LIFE CYCLE IN MAN
Infected reduviid bug partakes a mealdefecates
passing out metacyclic trypomastigotepenetrates the
skin via mucus membranestrypomastigotes engulfed
by histiocytesdevelops into amastigote
intracellularly multiply by binary fissionmay
transitionally pass through a promastigote
stageepimastigotetrypomastigoteinfected
histiocyte ruptures in 4-5 days releasing
trypomastigotes in the bloodstreaminfects new
histiocytes and replicate again as
amastigotestrypomastigote in bloodstream is
ingested by reduviid bug as it takes a blood meal
LIFE CYCLE IN THE INSECT VECTOR
Trypanosomes pass through the posterior portion of the
midgut of reduviid bug develop into
epimastigotemultiplies by longitudinal binary
fissiondevelop into infective metacyclic
trypomastigoteappears in the insect’s rectum 8-10
days after infectionexcreted in the bug’s
fecesenters human host via scratch on skin or
through mucous membranes that are rubbed with
fingers contaminated with bug’s feces
INFECTED CELLS
Frequently infects:
1. Reticuloendothelial cells of spleen, liver, cardiac
muscles, smooth and skeletal muscles
2. Skin
3. Gonads
4. Intestinal mucosa
5. Placenta
PATHOGENESIS AND CLINICAL MANIFESTION
 Chagoma – localized inflammation at the site of
infection (usually in the face)
 Small, painful, reddish nodule that takes
2-3 months to resolve
 Trypomastigotes and amastigotes may
be aspirated from Chagoma

ACUTE FORM
 4 days – 2 wks after the insect bite
 High fever, lymphadenopathy, swelling of the
entire body
 Severe symptoms are seen in young children
with CNS involvement early in the infection.
Amastigotes are seen in the meningeal tissues.
Leads to death within a few days or weeks
 Meningoencephalitis in neonates
 Romaña’s Sign – conjunctivitis and unilateral
edema of the eyes

 Some patients will experience complete
recovery after the acute stage but most will
progress to Chronic Chagas’ Disease
CHRONIC FORM
 More common in adults
 More common than the acute form
 Cardiac damage– most common serious form of
the disease
 CHF (Congestive Heart Failure)
 Mega syndrome
 Megaesophagus
 Megacolon
 Cardiomegaly
 CNS involvement - signs of agitation,
disorentiation, aphasia, comadeath if left
untreated
DIAGNOSIS
 Patient history – being in an endemic area
 Clinical Presentation: Febrile episodes,
enlargement of the lymph nodes, Chagoma,
Romaña’s sign, myocarditis, CNS and digestive
problems
 Demonstration of parasites in blood, CSF, and
tissue  Giemsa staining
 Centrifugation – examination of the buffy coat
layer
 Blood cultures – if parasites are scanty
 Animal inoculation
 Xenodiagnosis – successful even in the later
part of the disease when blood films are
negative

3. Xenodiagnosis

SEROLOGIC TESTS
 Complement fixation
 Direct and Indirect Hemagglutination
 Indirect Immunofluorescent Antibody Test
(IFAT)
 PCR
 ELISA – directs T. cruzi antigen in urine
TREATMENT
 Nifurtimox (aka Lampit or Bayer 2502) – DOC
 Allupurinol
 Benznidazole
 Megaesophagus and Megacolon –surgical
intervention
PREVENTION
 Education of the endemic population to raise
awareness about the disease
 Insect control – application of insecticide on the
roof and walls
 Housing improvement to eliminate cracks and
crevices where the insect resides
 Screening of blood for transfusion
 Routine addition of gentian violet dye
to blood bottles in final concentration
of 0.025% to kill T. cruzi
______________________________________________
Trypanosoma brucei complex
 Both sp. are morphologically indistinguishable
 Same life cycle
 Both are pathogenic for humans in Africa
 “brucei-gambiense-rhodesiense complex”
 Disease: African Sleeping Sickness
STAGES OF DEVELOPMENT
 Polymorphic trypanosomes – seen in blood and
CSF
 Epimastigotes – seen in vector
Trypanosoma brucei gambiense
 Disease: West African Sleeping Sickness/
Gambian Trypanosomiasis
 Has a chronic course which ends with
CNS involvement leading to death after
several years
 Parasite can be found in the wet lowlands and
rainforest of West and Central Africa
MODE OF TRANSMISSION
 Bite of an infected Tsetse fly (Intermediate host
and vector)
(Riverine tsetse flies of the palpalis group)
 Glossina palpalis
 Glossina tachinoides (tsetse fly)
 Glossina fuscipes
 Day biters
 Blood transfusion
 Organ transplant
 Transplacental route

LIFE CYCLE


Trypomastigotes are ingested by tsetse fly when it takes
a blood meal on infected humans  develop into
epimastigotemultiplies within the fly in the gut and
salivary glands. As tsetse fly takes another blood meal,
saliva containing metacyclic trypomastigotes is
transmitted to the human hosttrypomastigotes
multiply in the bloodstream (may be ingested by
fly)goes to CNS causing sleeping sickness
CLINICAL MANIFESTATIONS AND PATHOGENESIS
1
st
Stage
 Asymptomatic incubation period
 Trypanosomal chancre at the insect bite
2
nd
Stage
 Trypomastigotes found in the bloodstream and
lymphatic system
 1
st
distinct symptom: fever followed by afebrile
periods
 Headache, malaise, anorexia, night sweats,
weakness, joint and muscle pain, tachycardia
 Lymphadenopathy and glandular enlargement
 Winterbottom’s sign – enlargement of the post-
cervical chain of lymph nodes

4.  Erythematous (red )rash, pruritus, edema
 Kerandel’s sign – delayed sensation to pain
3
RD
Stage
 Takes 6 mos. – 1 yr after the onset of first
symptoms
 Meningoencephalitic stage
 Increased fatigue, mental dullness, apathy,
diminished motor control
 Somnolence (excessive sleepiness)
 Demonstration of trypomastigotes in px’s CSF
 Sleepiness progresses to coma death
LABORATORY DIAGNOSIS
 Demonstration of trypomastigotes in:
 blood, lymph node aspirates, bone
marrow – early stage
 CSF – late stage
 Direct wet mounts examined for motile
trypanosomes
 Concentration techniques - Trypanosomes can
be found in the buffy coat layer of blood after
centrifugation
 Serologic Tests: Card Agglutination
Trypanosomiasis Test (CATT)
TREATMENT
 Pentamidine –2
nd
stage
 Suramin – for 1
st
and 2
nd
stage
 more toxic than pentamidine
 May be prescribed during pregnancy
 Melarsoprol – 3
rd
stage; CNS involvement
 Eflornithine - the resurrection drug
PREVENTION
 Control, management, and avoidance of insect
vector
 Wearing protective clothing against tsetse fly
 Application of repellents
 Clearing of vegetation where tsetse fly breeds
 When traveling to endemic areas, wear khaki or
olive drab clothing
Trypanosoma brucei rhodesiense
 East African Sleeping Sickness/Rhodesian
Trypanosomiasis
 Geographical distribution: Central and Eastern
Africa
 Pathology is similar to Gambian form but more
severe and fatal (terminating within 1 yr)
 More common in males than in females
 Trypomastigotes are found in the peripheral
blood during febrile periods
 Glomerulonephritis
 Myocarditis
 Somnolence – indicates CNS involvement
 Entire course of the disease takes 9-12 months
MODE OF TRANSMISSION
 Bite of an infected tsetse fly
 Glossina pallidipes
 Glossina morsitans
 Glossina swynnertoni
Reservoir host: antelopes, game animals, domesticated
cattle, waterbuck, impala, and warhog
LABORATORY DX
 Same as gambian trypanosomiasis
______________________________________________
Leishmania spp.
Basis of differentiation of Leishmania sp:
 Geographic distribution
 Pathogenesis
Stages of Development:
Promastigoteamastigote
Serologic tests to differentiate the species:
 Kinetoplast DNA (kDNA)
 DNA hydridization
______________________________________________
Leishmania tropica complex
 L. tropica
 L. aethiopica
 L. major
Vector
 Sand fly (Stomoxys calcitrans)
Infective Stage
 Promastigote
Diagnostic Stage
 Amastigote (intracellular in mononuclear
phagocytic cells)
MODE OF INFECTION
 Bite of infected sand fly
 Blood transfusion
DISEASE
 Old World Cutaneous Leishmaniasis
 Recidivans
 Chronic Relapsing Cutaneous Leishmaniasis
 Oriental Sore
 Aleppo or Baghdad or Delhi boil
 Dry or Urban Cutaneous Leishmaniasis
EPIDEMIOLOGY
Areas bordering Mediterranean, Middle East, Republic
of Georgia, and India
LIFE CYCLE IN THE VECTOR
sand fly ingests amastigote from infected human after
taking a blood meal within the insect, amastigote
transforms into promastigotemultiply
promastigotes migrate to the pharynx of sand
flyTransmits the promastigote to humans
LIFE CYCLE IN HUMAN HOSTS
Promastigotes are engulfed by phagocytestransform
into amastigotesmultiply inside cellcell ruptures
releasing amastigotes which invade other macrophages

5. then multiply intracellularly leading to tissue
destruction
CLINICAL MANIFESTATIONS AND PATHOGENESIS
 Incubation period: 2-24 months
 First Sign/Early Lesion: Oriental sore
 Late Lesion: multiple on exposed surface of the
body
 Healing occurs over 1-2 yrs without treatment
leaving a depigmented scar
 Diffuse Cutaneous Leishmaniasis occurs in
patients with impaired immunity
 Lesions are loaded with parasites
COMPLICATIONS
 Secondary bacterial infections
 Leishmania recidiva – due to an exaggerated
delayed hypersensitivity response to parasite
antigen
 Facial lesions with scanty parasites
LABORATORY DX
 Characteristic feature of lesion: elevated and
indurated margin of the ulcer
 Demonstration of amastigotes from lesions or
biopsy
 Giemsa or Wright’s stain
 Presence of promastigotes from specimen
cultured on NNN media
 Montenegro (Leishmanin) Skin Test – delayed
hypersensitivity reaction
 Promastigotes are administered
intradermally
 (+) result: induration and erythema of
4-5 mm or more in diameter
SEROLOGIC TESTS
 Indirect fluorescent Antibody Assay (IFAT)
 Complement Fixation
 Immunoperoxidase test
 Direct/Indirect Hemagglutination
TREATMENT
 Sodium stibogluconate (Pentostam) – DOC
 Meglumine antimonate (Glucantime)
 Amphoterecin B and ketoconazole
 Prompt TX for individuals with active lesions to
prevent autoinfection
PREVENTION
 Vector and reservoir control
______________________________________________
Leishmania mexicana complex
Leishmania brasiliensis, L. panamensis, L. peruviana, L.
guyanensis
STAGES OF DEVELOPMENT
 Amastigote
 Promastigote
DISEASE
 New World Cutaneous Leishmaniasis
 American Leishmaniasis
 Espundia
 Uta – form of the disease with mucosal features
 Mucocutaneous Leishmaniasis
 Pain bois (forest yaws)
VECTOR: Lutzomyia sandfly
MODE OF TRANSMISSION
 Contamination of the bite wound
 By contact
GEOGRAPHIC DISTRIBUTION
 From Mexico to Argentina
CLINICAL MANIFESTATIONS
 Mucocutaneous lesions - Growth of polyp-like
appendages in the nasal cavity

 Development of ulcers on or around the oral
and nasal mucosa
 Needs to be differentiated with lepromatous
leprosy
 Tapir nose – destruction of oropharyngeal
mucosa
 Chiclero ulcer erosion of the pinna of ear of
forest workers

LABORATORY DX
 Same for Leishmania sp.
TREATMENT
 Pentostam – DOC
 Camolar and Amphoterecin B
______________________________________________
Leishmania donovani complex
 L. donovani, L. infantum, L. chagasi
MORPHOLOGY
 In Mammalian tissues
- Amastigotes (leishmania stage)
 In the gut of sandflies or in cultures
- promastigote form (leptomonas stage)
DISEASE (fatal if not treated)
 Visceral Leishmaniasis
 Kala azar or Black Disease
 Dum-dum fever
 Death Fever
 Ponos
 Tropical splenomegaly

6. GEOGRAPHIC DISTRIBUTION
 India, Pakistan, Thailand, parts of Africa, China
VECTOR
 Phlebotomus sandflies
 Lutzomyia
RESERVOIR HOST
 Humans, dog, wild animals
CLINICAL MANIFESTATIONS
 More common in males
 Affects the spleen, liver, bone marrow,
peripheral blood, lymphatics
 Most prominent symptoms:
 Fever
 Splenomegaly
 Cachexia
 Anemia
 Recovery from kala-azar leads to lasting
immunity
 Butterfly rash
LABORATORY DIAGNOSIS
 History and PE
 Demonstration of the parasites from the blood
and tissues
 Culture using Novy-MacNeal-Nicolle Medium –
promastigotes
 Bone marrow aspirate and splenic puncture
SEROLOGIC TESTS
 Indirect hemagglutination test
 ELISA
 Formol-gel aldehyde test of Napier
 Direct Agglutination Test
TREATMENT
 Pentostam and Lomidine – DOC
 Amphoterecin B
 Allopurinol or gamma intervention + Pentostam
PREVENTION
 Avoid and destroy infected dogs
 Destroy breeding places
 Use repellents
 Vector control
 Health education