Part II Leishmania& Trypanosome

Debre Berhan University College of medicine Dep MLS Medical parasitology 2012/2020
3.3 BLOOD AND TISSUE FLAGLLATES

 Belonging to the family Trypomastidae
 Six genera but only two of them are responsible to cause disease to man
 GenusLeishmania
 GenusTrypanosome
3.3.1 General Characteristics blood & tissue flagellates

 Reproduces bysimple longitudinal binary fission
 Transmission occurs through biological insect vectors as intermediate hosts
& human as definitive host
The following are the main developmental forms
1 | To save your mind read at home, To save your life stays at home. Compiled by Moges L.
Fig1. Leishmania morphology
1.Amastigote (Leishmanial form)

 Rounded body, central nucleus, no free flagellum.
 No undulating membrane, The only intracellular forms of all Leishmania species and
Trypanosome cruzi.
2. Promastigote (Leptomonad form)
 Elongated body, central nucleus, anterior kinetoplast,Single anterior flagellum
arises found in the invertebrate host, and in culture media (of allLeishmania
species) and in man forTryponosoma cruzi
3. Epimastigote /crithidial/ forms
 Elongated body, single free flagellum, single nucleus, Has undulating membrane,
found in the invertebrate host and in culture media (ofTrypanosomespecies)
4. Trypomastigote (* see in diagnosis of trypanosome )
 Pleomorphic, it can be as “U” or “C” shape
single flagellum arises posteriorly , Has undulating membrane ,found in the
peripheral blood of vertebrates and is the diagnostic stage of
Trypanosomespecies.
5. Metacyclic Trypomastigote /Trypanosomal/ Forms.
 Morphologically similar to trypomastigote stage but it is short and stumpy, final
developmental stage in the gut of the insect vectors, infective stageTrypanosomes
of
species.
3.3.2 Leishmania
Sir William Leishman (1900)discovered L. donovani in spleen smears of a soldierwho died of

fever at Dum-Dum, India. The disease wasknown locally asDum-Dum fever or kala-azar.
 In the human host, Leishmania are intracellular parasites that infect the
mononuclear phagocytes.
 Human infection is caused by about 21 of 30 species that infect mammals. These
include.
 Leishmaniasis caneasily classified based onclinically . It is Neglected tropical
diseases (NTDs)
3.3.2.1 Cutaneous leishmaniasis (CL)
 L. tropica ,L. major and L. aethiopica

2. Visceral leishmaniasis (VL)
 L. donovani, L. infantumand L. Chagasi
3. Mucocutaneous leishmaniasis (MCL)
 L. panamensis,L. guyanensis and L. Brazilliensis
4 .Diffuse cutaneous leishmaniasis (DCL)
 L. amzonensis,L. aethiopica
Geographical distribution
Cutaneous and visceral Leishmaniasis are endemic in Ethiopia. Cutaneous Leishmaniasis
occurs in most areas of the Ethiopian highland plateau (elevation 1500m / 4921ft to
2700m / 8858ft), including Addis Ababa. Areas of risk for visceral Leishmaniasis include
the northwestern, southwestern, and southern lowlands, and the northeastern low -lying
arid areas along the Red Sea coast
.
 Geographical distribution of leishmaniasis is limited by:
 Sand flytendency to take blood from humans or animals only, and
 Its capacity to support the internal development of specific species of
leishmania
 In Ethiopia
 Four species ofLeishmania is found, namely,
 L. aethiopica,L.major ,L. tropica and L. donovani
Common mode of transmission. Bite of sandfly
 GeneraPhlebotomus inOld world and Lutzomyia in New world
 It is transmitted through the bite of infected female sandflies belonging to the Phlebotomus,
Lutzomyia, and Psychodopygus species.
 Uncommon modes of transmission:
 Congenital transmission, Blood transfusion, and rarely inoculation of

cultures
Life cycle:
1. Leishmaniasis is transmitted by the bite of female phlebotomine sandflies.The

sandflies inject the infective stage, promastigotes, during blood meals.
2. Promastigotes that reach the puncture wound are phagocytized by macrophages
and transform into amastigotes.
3. Amastigotes multiply in infected cells and affect different tissues, depending in part
on the Leishmaniaspecies.
4. This originates the clinical manifestations of leishmaniasis. Sandflies become
infected during blood meals on an infected host when they ingest
macrophages
infected with amastigotes.
5. In the sandfly's midgut, the parasites differentiate into promastigotes, which
multiply and migrate to the proboscis.
Clinical features and pathology
1. Cutaneous Leishmaniasis
WHO case definition:
A person showing clinical signs (skin lesions). A papule

appears, which may enlarge to become an indolent ulcerated
nodule or plaque. The sore remains in this stage for a
variable time before self healing and typically leaves a
depressed scar. Other atypic
al forms may occur.
Incubation period
 L. major: At least one week. Usually less than 4 months.

 L. tropica: At least one week. Usually –
28 months.
L. tropica (SW Asia, N.Africa )

 Anthroponotic or dog reservoir
 L. tropica is transmitted by P. sergenti from person to person.
 Very rarely, L. tropica through transfusion.
 Dry urban oriental sore -70mm diameter
'dry painless lesio
 Are self-healing, 1-2yrs,Often leave disfiguring scars and Immune to re
-infection.
L. major
 L. major is transmitted by Phlebotomuspapatasi from the animal reservoir to
humans.
 (central Asia, middle East, Africa)wet oreintal sore,Wet lesion
 Early papules is inflamed(5-10mm).Develop to large uneven ulcer
 Self-healing (3-6mths)
 Protect against reinfection & also withL.tropica
L. aethiopica
• highlands of Kenya and Ethiopia
• Zoonotic and common reservoir hyrax
• Similar to oreintal sore,Self-heal 1-3 yrs
• Can cause DCL
3.3.2.2 Diffuse Cutaneous Leishmaniasis
• Lesion develop over large areas of the body, Scaly, not ulcerated, nodules
• Chronic and painless and Numerous parasites in lesions.
• L. aethiopica,scaly, not ulcerated, noduleschronic and painless, numerous parasites
in lesions
3.3.2. 3 Mucocutaneous Leishmaniasis
Skin changes similar to oriental sore. Some forms tend to spread to mucosa and cause
severe tissue destruction.leads to partial or total destruction of mucous membranes of the
nose, mouth and throat. Over 90% of mucocutaneous leishmaniasis cases occur in Bolivia
(the Plurinational State of), Brazil, Ethiopia and Peru.
3.3.3 Visceral Leishmaniasis
(VL), also known as kala

-azar is fatal if left untreated in over 95% of cases. It is
characterized by irregular bouts of fever, weight loss, enlargement of the spleen and liver,
and anaemia. It remains one of the top parasitic diseases with outbreak and mortality
potential. In 2018, more than 95% of new cases reported to WHO occurred in 10 countries:
Brazil, China, Ethiopia, India, Iraq, Kenya, Nepal, Somalia, South Sudan and Sudan.
 Caused by theLeishmania donovani, L. infantum and L. chagasi.
Clinical Presentation
 Incubation period
• generally 2-6 months ,can range 10 days to years
 Depressed hematopoiesis
• severe anemia,leucopenia
• thrombopenia  petechial hemorrhages in mucosa
3.3.3.1 Post-kala-azar dermal leishmaniasis (PKDL)

Post-kala-azar dermal leishmaniasis (PKDL) is a complication of visceral leishmaniasis
(VL) in areas where Leishmania donovani is endemic; it is characterized by a
hypopigmented macular, maculopapular, and nodular rash usually in patients who have
recovered from VL . It usually appears 6 months to 1 or more years after apparent cure
of the disease but may occur earlier or even concurrently with visceral leishmaniasis
especially in the Sudan.PKDL heals spontaneously in the majority of casesin Africa but
rarely in patients in India. It is considered to have an important role in maintaining and
contributing to transmission of the diseaseparticularly in interepidemic periods of VL,
acting as a reservoir for parasites.
Diagnosis of CL, MCL, DCL
Suspected because of:
 Geographical presence of parasite history of sandfly bite

 Skin lesion ; nasopharyngeal lesions ,nodular lesions
 Demonstration of parasite amastigotes (scrapings, biopsy, aspirates)
1. Collection and examination of slit skin smears for amastigotes

 The quality of the sampling procedure is essential. Local anaesthesia
will reduce pain during the procedure, making sampling easier and
of higher quality.
 Should be taken from the inflamed raised swollen edge of an ulcer or
nodule not from its base or centre which usually contains only
necrotic tissue.
 If bacterial infection is present, examination for Leishmania
amastigotes is best delayed until antimicrobial treatment has been
completed and the bacterial infection has cleared.
2. Culture of ulcer material
 When cutaneous leishmaniasis is suspected and parasites cannot be found in

smears.
 Material for culture is best obtained by injecting and then aspirating a small
quantity of sterile physiological saline in and out of the hardened margin of
the
ulcer.
 A few drops of the final aspirate is used to inoculate the culture medium.

Laboratory diagnosis of Visceral leishmaniasis
Sample can be
 Bone marrow aspirate

 Splenic aspirate
 Lymph node
 Tissue biopsy
Common method Microscopy and Culture in NNN Medium
1. Microscopy
Leishmania (Leishman-
Donovan or LD bodies). Lying
in macrophage cells from liver.
Giemsa.
Cultures are required for:
 Obtaining a sufficient number of organisms to use an antigen for immunologic

diagnosis and speciation,
 Obtaining parasites to be used in inoculating susceptible experimental animals,
 In vitro screening of drugs, and
 As a supplement to other methods orto provide a diagnosis when routine methods
have failed
 Leishmania strains can be maintained as promastigotes in artificial culture
medium
 Some of the culture media used are :Novy
–Nicolle-MacNeal(NNN), M199, or
Grace’s and Tobies medium
Animal inoculation
 Inoculation of laboratory animals (such as hamsters, mice or guinea pigs) with

infected specimen
 Not usually employed as a diagnostic test, since several months may be required to
obtain a positive result.
 Can be infected via many routes,including across mucous membranes,
intraperitoneal and intrasplenic routes
 Both amastigotes and promastigotes can infect the animal
the animal is examined
weekly for signs of infection
 In the absence of signs of obvious infection, the animal is generally sacrificed after 4
months, at which point liver and spleen samples are examined for the presence of
the parasite
Prevent and control
1. Early detection by serological diagnosis

(VL) and treatment of infected persons
2. Personal protection from sandfly bites by:
 Using insect replants
 Avoiding endemic areas especially at times when sandifies are most active
 Use of pyrethroid impregnated bed nets and curtains
3. Vector control bythe use of, sticky paper traps, or residual insecticide spraying of houses
. Destruction of stray dogs and infected domestic dogs
5. Elimination and control of rodents
3.3.4 Trypanosomes
Classified in to two groups bas

ed on the type of developmentin the insect vector and mode
of transmission.
• Two distinct forms occur in humans

– African Trypanosomiasis
• Trypanosoma brucei gambiense
• Trypanosoma brucei rhodesiense
• Transmited by the Tsetse fly
– American Trypanosomiasis
• Trypanosoma cruzi
• Transmited by the tritomine bug
African Trypanosomiasis
Human African sleeping sickness
• It is caused by the flagellate protozoan, Trypanosoma brucei
• exists in 2 morphologically identical subspecies:
– Trypanosoma brucei gambiense
• Disease: West African or Gambian Afr ican trypanosomiasis
– Trypanosoma brucei rhodesiense
• Disease: East African or Rhodesian African trypanosomiasis
• Mode of Transmission:
1. Bite of infected Tse
-Tse fly –Glossina spp.
2. Congenital
3. Sexual contact
4. Human-fly-human transmissio
Current WHO report
 The latest data released by the World Health Organization confirms the sustained
decrease in the number of new cases of human African trypanosomiasis
 977 cases of HAT were reported in 2018, down from 2,164 in 2016.
 The area at moderate or high risk of HAT has shrunk
to less than 200,000 square
kilometres. More than half of this area is in the Democratic Republic of the Congo.
Life cycle
1. During a blood meal on the mammalian host, an infected tsetse fly (genus Glossina)
injects metacyclic trypomastigotes into skin tissue.
2. The parasites enter the lymphatic system and pass into the bloodstream. Inside the
host, they transform into bloodstream trypomastigotes
3. Are carried to other sites throughout the body, reach other blood fluid
s (e.g., lymph,
spinal fluid), and continue the replication by binary fission.
4. The entire life cycle of African Trypanosomes is represented by extracellular
stages. The tsetse fly becomes infected with bloodstream trypomastigotes when
taking a blood meal on an infected mammalian host .
5. In the fly’s midgut, the parasites transfo
by binary fission.
6. Leave the midgut, and transform into epimastigotes.
7. The epimastigotes reach the nue multiplication
fly’s by salivar
binary fission.
8. The cycle in the fly takes approximately 3 weeks.Humans are the main reservoir
for Trypanosoma brucei gambiense , but this species can also be found in
animals. Wild game animals are the main reservoir ofT. b. rhodesiense
The lifecycle of Trypanosoma cruziand Trypanosoma brucei.The life cycle of T. cruzi is

shown on the left, that of T. brucei on the right.T. brucei escapes from the immune
response by expressing a new VSG. These surface antigens vary after a period of time
allowing the parasite to continually evade the host immune system.Variant surface
glycoprotiens (VSG) and variant mucins. However, in the arthropod host the parasites
express invariant mucins and procyclic acidic repertoire protein (PARP).
Clinical features:
Infection occurs in 3 stages.
1. Bite reaction A trypanosomal chancre can develop on the site of inoculation.

2. Parasitemia by a hemolymphatic stage with symptoms that include fever,
lymphadenopathy, and pruritus.
3. CNSstage, invasion of the central nervous system can cause headaches, somnolence,
abnormal behavior, and lead to loss of consciousness and coma (continus sleep). The
course of infection is much more acute withT. b. rhodesiense
than T. b. gambiense
.
Laboratory diagnosis
• A definitive diagnosis requires detection of trypanosomes
– In blood, Lymph nodes, CSF, Skin chancre aspirates, or Bone marrow.
• Blood smear
– Giemsa-stained thick smear (more sensitive)
– wright and leishman stains are inadequate.
• Chancreaspirate
• Serologic antibody detection- Field diagnosis
American trypanosomiasis (Chagas disease)
Causedby Trypanosoma cruzi,occurs in Central and South America. Unlike African forms of
the disease, infection is not transmitted by insect bite, butrather by insect feces
contaminating the conjunctiva or a break in the skin.
Control
Tsetse fly populations have been reduced successfully by the use of insecticides or traps
with an attractant bait plus insecticide. No reliable vaccine is available, and the variability
in antigenic composition of the blood populations makes vaccination a difficult goal.
 Drugs such as pentamidine and the arsenical suramin, are successful in treatme
nt
 Sterile insect technique

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Debre Berhan University College of medicine Dep MLS Medical parasitology 2012/2020

3.3 BLOOD AND TISSUE FLAGLLATES

3.3.1 General Characteristics blood & tissue flagellates

The following are the main developmental forms

Fig1. Leishmania morphology

1.Amastigote (Leishmanial form)

Sir William Leishman (1900)discovered L. donovani in spleen smears of a soldierwho died of

3.3.2.1 Cutaneous leishmaniasis (CL)

 L. tropica ,L. major and L. aethiopica

 Congenital transmission, Blood transfusion, and rarely inoculation of

1. Leishmaniasis is transmitted by the bite of female phlebotomine sandflies.The

Clinical features and pathology

WHO case definition:

A person showing clinical signs (skin lesions). A papule

 L. major: At least one week. Usually less than 4 months.

L. tropica (SW Asia, N.Africa )

3.3.2. 3 Mucocutaneous Leishmaniasis

3.3.3 Visceral Leishmaniasis

(VL), also known as kala

 Caused by theLeishmania donovani, L. infantum and L. chagasi.

• generally 2-6 months ,can range 10 days to years

3.3.3.1 Post-kala-azar dermal leishmaniasis (PKDL)

Suspected because of:

 Geographical presence of parasite history of sandfly bite

1. Collection and examination of slit skin smears for amastigotes

2. Culture of ulcer material

 When cutaneous leishmaniasis is suspected and parasites cannot be found in

Laboratory diagnosis of Visceral leishmaniasis

 Bone marrow aspirate

Cultures are required for:

 Obtaining a sufficient number of organisms to use an antigen for immunologic

 Inoculation of laboratory animals (such as hamsters, mice or guinea pigs) with

Prevent and control

1. Early detection by serological diagnosis

Classified in to two groups bas

• Two distinct forms occur in humans

The lifecycle of Trypanosoma cruziand Trypanosoma brucei.The life cycle of T. cruzi is

1. Bite reaction A trypanosomal chancre can develop on the site of inoculation.

American trypanosomiasis (Chagas disease)

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