Blood & Tissue Dwelling Protozoa

(Hemoflagellates)

Morphologic Characteristics of Hemoflagellates

minute actively motile
fusiform body with the nucleus central in position with a large, oval central karyosome
flattened side to side, tapering anterior and blunt posterior end
single anterior flagellum which travels in a wavy spiral motion; arising from a kinetoplast near the anterior end
reproduce by binary fission
nourishment is attained from blood, plasma, lymph, CSF and product of cellular disintegration

4 stages of development
Amastigote ( Leishmania )
spherical/ovoid in shape
has no free flagellum
non-flagellated, non-motile
a basal body at the base of the flagellum
the kinetoplast is usually detectable as a darkly staining body near the nucleus

Promastigote ( Leptomonas )
kinetoplast is towards the anterior end with a
free flagellum with no undulating membrane
spindle shaped body, shorter
single nucleus with karyosome at the center

Epimastigote ( Crithidia )
elongate, spindle-shaped body, longer
single nucleus with central karyosome
the kinetoplast is more centrally located, usually just anterior to nucleus
The single free flagellum emerges from the middle of the parasite and forms a shorter
undulating membrane

Trypomastigote ( Trypanosoma )
morphologically similar to epimastigote
kinetoplast is located on the posterior end
the attachment of the flagellum to the body forms an undulating membrane that spans
the entire length of the parasite and the free flagellum emerges from the anterior end
Genus Leishmania
obligate intracellular parasite capable of causing human disease
all species are morphologically identical
differentiation among species causing disease in human is based on clinical grounds
Various species are transmitted by sandflies
reproduce by binary fission

2 morphologic stages of development of Leishmania :

1) Amastigote
- present in the vertebrate host (human)
- found always intracellularly in the cells of the reticuloendothelial system, at times are
present in the blood stream in large mononuclear cells

2) Promastigote
- present in the invertebrate host
- infectious form found in the midgut & proboscis of intermediate host ( sandfly )
- spindle shaped body
- measuring 15 20m L x 1.5 3.5 m W

Life Cycle of Leishmania

Leishmaniasis
caused by 3 large species complexes, which differ in clinical manifestations, geographical distribution and sandfly vectors
Clinical diseases:
Cutaneous Leishmaniasis involves the skin
Mucocutaneous Leishmaniasis - involves the skin & mucous membranes
Visceral Leishmaniasis - involves the visceral organs
Mode of transmission:
bite of a female sandfly
Rarely: Congenital transmission, blood transfusion or contamination of bite wounds and by direct contact
Cutaneous Leishmaniasis
Synonyms: Oriental sore/ Tropical ulcer
Delhi ulcer/ Baghdad boil
Site of involvement: skin
not found peripheral blood
rarely disseminate, mainly cutaneous
Most common causative agent: Leishmania tropica
2 types of Cutaneous Leishmaniasis :
Old world cutaneous leishmaniasis
New world cutaneous leishmaniasis

Old world cutaneous leishmaniasis

Cause the Oriental sore
Produce Leishmaniasis belonging to Leishmania tropica Complex
vector: sandfly of genus Phlebotomus
L. major - lesions occuring in the lower limbs, assoc. with moist lesions which tend to ulcerate very early
( Wet/ Rural cutaneous leishmaniasis)
L. tropica - characterized by dry lesions that ulcerate only after several months
(Dry / urban cutaneous leishmaniasis)

New world cutaneous leishmaniasis

Caused by species of the Leishmania mexicana Complex:
L. mexicana - assoc. with Chiclero ulcer/Bay ulcer
- lesions appear single, usually in the ear where they can be quite destructive to the cartilage

Caused by L. Braziliensis complex:

L. braziliensis - cause a disease known as Uta
- skin lesions that are self-healing and very similar to L. tropica

Lab. Diag:
1. demonstration of parasite from skin biopsy, exudate from ulcer edge stained with Wright/Giemsa stain
-amastigote stage inside mononyclear cells
2. culture NNN medium (Novy-MacNeal-Nicolle)
3. serology Indirect flourecent antibody test
4. Dermal test (Montenegro Test) intradermal injection of killed promastigote suspension

Treatment:
1. Antimonial compounds:
Sodium stibogluconate (Pentostam)
- is the drug of choice for all types of leishmaniasis
- 20mg/kg for 20 days of intravenous therapy
Meglumine antimonate (Glucantime)
- 50mg/kg daily for 10-12 days
- used in areas where Pentosam is not available
nd
2. 2 line:
Amphotericin B - unresponsive to pentavalent antimonials
Pentamidine
Metronidazole
Itraconazole used in India to treat cutaneous leishmaniasis
Mucocutaneous Leishmaniasis
Synonyms: American leishmaniasis
Most common causative agent: Leishmania braziliensis
Geographical Dist. : Central & South America, Brazil, Eastern Peru, Bolivia, Parugay, Ecuador, Colombia, Venezuela
In Brazil known as Espundia
Site of involvement: skin with development of ulcers in the mouth or nasal mucosa and disfigurement
Insect vector: genus Lutzomyia

Clinical Manifestation:
Lesions appears 1-4 weeks after the bite
outstanding feature:
- ulcers on the oronasal mucosa or pharyngeal mucosa causing highly disfiguring tissue destruction swelling
the soft and hard palates are also affected; (+) deformity of the cheek and lips
symptoms include: fever, weight loss, anemia, weakness, and hepatosplenomegaly
Death is due to secondary bacterial infxn

Lab dx & treatment: same with cutaneous leishmaniasis

Visceral Leishmaniasis
Synonyms: Kala-azar / Death fever / Black disease / Dumdum Fever / Tropical splenomegaly
Most common causative agent: Leishmania donovani

L. donovani - India, Burma, East Pakistan, Sumatra, Thailand, Southern Russia, East Africa, Ethiopia, Sudan, and China
L. chagasi Cental and South America
L. infantum Europe and Africa, Siberia
Insect vector: sandfly
L. donovani - genus Phlebotomus
L. chagasi - genus Lutzomyia
Site of involvement: Endothelial cells of visceral organ ( spleen & liver, intestinal mucosa, mesenteric glands)
most often associated with massive splenomegaly
Most severe form of leishmaniasis

Lab diagnosis:
demonstration of the parasite ( blood & tissue smear, bone marrow, lymph nodes, liver, and spleen)
Splenic puncture effective method for securing reticuloendothelial cells for study
Bone marrow aspiration diagnostic procedure of choice
Culture - NNN medium
Serological Fluorescent antibody test , ELISA

Treatment
Antimony Sodium stibogluconate (Pentostam)
the drug of choice
2 line drugs:
nd

amphotericin-B - effective in treating Pentosam resistant visceral leishmaniasis

Pentamidine
Miltefosine used in India since 2002 as 1st line drug
Interferon gamma enhance the killing of Leishmania amastigote
Genus Trypanosoma
Trypanosomes are hemoflagellates (those that occur in the blood and tissue fluids of the majority of vertebrate animals)
Many species of trypanosomes can live in harmony with their hosts producing no pathogenic effect, but the best known
species are those that are pathogenic to their definitive hosts
The disease in caused by the pathogenic types is called trypanosomiasis (Sleeping sickness)
Reservoir host are domestic cattle and wild animals

represented by trypomastigote stage
spindle shaped, elongated body (pleomorphic - from long slender to short and blunt)
nucleus with centrally located karyosome
kinetoplast found at the posterior end of the body
undulating membrane originating from the blepharoplast
Anterior flagellum runs along the edge of the undulating membrane
Salivarian Trypanosomes
Trypanosoma brucei rhodesiense
Trypanosoma brucei gambiense
The metacyclic trypanosomes are found in the proboscis of the insect vector --- infection is inoculative
causative agent of African Trypanosomiasis
These are known as salivarian trypanosomes as they complete their development in the salivary system
Transmission takes place by inoculation of the metacyclic trypomastigote stage

Stercorarian Trypanosomes
Trypanosoma cruzi
The metacyclic trypanosomes occupy a posterior position in the gut of the insect vector and are passed out in the feces ---
infection is contaminative
causative agent of American Trypanosomiasis
These trypanosomes are known as stercocarian as they complete their development in the posterior region of the vector,
so that the infective forms appear in the insects feces
African Sleeping Sickness
exists in 2 morphologically identical subspecies:
Trypanosoma brucei gambiense
Trypanosoma brucei rhodesiense
Transmission from one vertebrate to another is carried out by blood-sucking invertebrates, usually an insect
The vector: Tsetse fly (Glossina spp.)
human host: trypomastigotes live in the blood, reticular tissue of the lymph and spleen, and CSF
In chronic cases: trypomastigotes invade the CNS

2 stages of development in trypanosoma brucei infection MOT:

1. Trypomastigote 1. bite of tstse fly
- seen among vertebrate host (human, others like hog, goat and cattle) 2. congenital
- can be seen in the blood stream, CSF and lymph 3. human-fly-human
2. Epimastigote
- seen among invertebrate host
- developed from procyclic trypomastigote in the tse-tse flys midgut

Symptomatology:
1.winterbuttom sign enlargement of cervical lymph nodes
2. kerandel sign pain in nerves CNS involvement

Lab:
1. microscopic demonstration of trypanosome in the blood, LN, BM acute stage; CSF- chronic stage
2. serological detect anti-typanosome AB

Rx:
Acute stage: Pentamidine , Suramin
Late stage w/ CNS involvement: Metarsoprol

American Sleeping Sickness

Synonyms : South American Trypanosomiasis / Chagas disease
Geographical Distribution : Central & South America
Causative agent: Trypanosoma cruzi
Insect Vector: Reduviid bug/ Triatomid bug/ Assassin bug Kissing bug/ Cone-nosed bug

Morphology
represented by Trypomastigotes
spindle shaped body w/c assumes a C , U or S - shaped in stained blood smear
has free flagellum about 1/3 of body length
nucleus centrally located and a large kinetoplast
the undulating membrane is best demonstrated by electron microscopy

Clinical Manifestation
primary lesion
-Multiplication of T. cruzi at the site of infection can produce an inflamed swelling (chagoma) which persists for weeks
Romanas sign
- Unilateral edema of the upper and lower eyelid may occur along with conjunctivitis
Diagnosis:
Microscopic demonstration of parasite from stained blood smear or lymph node aspirate
Xenodiagnosis
best choice for chronic cases
placing uninfected bug on the patient suspected of having the disease and allow to feed. Later crushed the bug
and examined for any stage of the parasite
Blood culture using NNN medium
Serological methods: Indirect Hemagglutination
ELISA
Immunofluorescent antibody test

Treatment:
Nifurtimox
drug of choice
Inhibits intracellular development of T. cruzi
Premaquine
partially effective in acute cases