TRYPANASOMES

INTRODUCATION;

Trypanosomes are protozoan parasites belonging to the subphylum mastigophora and order
kinetoplastida.

Classification of trypanosomes;

Order: kinetoplastida

Class: zoomastigophorea

Family: trypanosomatidae

Genus: trypanosome

Species: (i) Trypanosoma Brucei complex

 Trypanosome Brucei gambiense

 Trypanosome Brucei rhodesiense
(i) Trypanosoma cruzi

Trypanosomes can also be classified by the type of development in the insect vector and the method of
transmission i.e.

 Salivarian / anterior development

 Stercorarian / posterior type of development.
SALIVARIAN / ANTERIOR STATION TYPE OF DEVELOPMENT

These are a group of trypanosomes in which development takes place in the mid and fore gut of the
vector and transmission is by inoculation when the vector bites.

This means that the infective stages are transmitted through the proboscis as in trypanosomes Brucei
gambiense and rhodesiense.

STERCORARIAN / POSTERIOR STATION TYPE OF DEVELOPMENT

These are a group of trypanosomes in which development takes place in the hind gut of the vector and
transmission is by feacal contamination via the oral route following the biting of the vector as in T.cruzi

MORPHOLOGICAL STAGES

AMASTIGOTE (Leishmania forms)

  This is a stage found in both human and insect host

 It is rounded/ oval in shape, with a kinetoplast but with no flagellum.

 This is a stage in which T.cruzi and leishmania are found intracellular in vertebrate host.

PROMASTIGOTE (Leptomonal)

 This is lanceolate leaf shaped with a kinetoplast anterior to the nucleus from which the
flagellum arises
 It has no undulating membrane.
 It is the infective stage of leishmania parasites found in the mid gut, proboscis
 This stage is lacking in trypanosomes

EPIMASTIGOTE (Crithidial stage)

 This stage is found in arthropods
 It is leaf shaped with kinetoplast anterior and close to the nucleus
 Has under developed undulating membrane
 It is the stage in which T.Gambiense and T.Rhodensiense occurs in the salivary glands of the
tsetse fly vector and in the mid gut of Ruduvid bug in T. cruzi
 It is lacking in leishmania

TRYPOMASTIGOTE STAGE (Trypanosomol stage)

 This is a stage found in the vertebrate host but also found in arthropods( insect vector )as the
infective stage for the human host
  It is spindle shaped with kinetoplast posterior to the nucleus which is usually near to the
posterior end. (Post nuclear kinetoplast). This is the infective stage of trypanosomes.
 This stage is lacking in the leishmania

HUMAN TRYPANOSOMES;
These are mainly 3 species of trypanosomes which include;

1) Trypanosoma Brucei gambiense

2) Typanosoma Brucei Rhodensiense
3) Trypanosome cruzi
 T.gambiense and T.rhodensiense causes African trypanosomiasis in humans.
This disease is commonly known as sleeping sickness. The parasites are closely
related and belong to trypanosome brucei complex.
 Trypanosoma cruzi causes South American trypanasomiasis also known as
chaga’s disease
TRYPANOSOMA BRUCEI GAMBIENSE AND RHODESIENSE

DISTRIBUTION;

 African trypanosomiasis occurs in the tsetse fly infested areas of sub Saharan Africa between 15
degree north and 20 degree south
 tr
 Trypanosoma b. gambiense is found in west Africa and Western central Africa extending from
Ethiopia down to Botswana
 The distribution of T.b.gambiense and t. rhodensiense over-lapse in the region of great lakes of
East Africa.
 In Uganda trypanasomiasis mostly of T.rhodensiense occurs in the busoga region e.g. Jinja,
Iganga and some other parts

TRASMISSION;

 African trypanosomiasis is transmitted by a small number of species of tsetse flies .Both male
and female tsetse flies suck blood and can therefore transmit the disease. The tsetse flies are of
genus Glossina
 The transmission may be cyclical or mechanical

Cyclical transmission;
  This is where the trypanosomes undergo a cycle of development and multiplication in the
digestive tract of the Glossina fly until the infective metacyclic trypomastogote is produced
 Different trypanosome species develop in the different regions of the digestive tract of the flies
and the metacyclic trypomastigote occur either in the biting mouth parts in the salivary glands
 The period from ingestion of infected blood to the appearance of these infective forms varies
from 1-3 weeks
 Once the infective forms are present, the tsetse fly remains infective for the rest of its life
 During the act of feeding, the fly penetrates the skin by the use of its proboscis and the infection
of the host takes place at this stage with the infective metacyclic trypanosomes in the saliva
 T.brucei gambiense is usually transmitted by the lake side and riverine tsetse flies e.g. G.palpalis,
G.fuscipes and G.tachnoides
 T.brucei rhodensiense is transmitted by wood land and savannah tsetse flies e.g. G.morsitans,
G.pallidipes and G.swynnertoni. Both parasites can be transmitted by either group tsetse flies

Mechanical transmission;

a) Biting insects
 The process is purely mechanical where a biting insect passes the blood with
trypomastogotes from an infective animal to another in course of interrupted feeding
period
 The time between the two feeds is crucial for effective transmission because the
trypomastigotes die when the blood dries
 Large biting insects such as tarbanides carry more blood and they are more likely to act
as mechanical vectors than mosquitoes
b) By latrogenic
 This usually occurs when a needle or surgical instrument used on more than one animal
at short interval so that the blood in the needle or instrument does not dry or by
transfusion of fresh blood.
 Transmission can also be by congenital from the mother to the off springs either
through the placenta when the foetus is still in the uterus or when bleeding occurs
during birth
LIFE CYCLE
 This involves two hosts i.e. Human host as a definitive host and tsetse fly as an
intermediate host

IN MAN;

 In the human host, the infective metacyclic trypomastigotes are inoculated

through the skin when an infected tsetse fly takes a blood meal
 The parasite then develop into long cylindrical trypomastigote which multiply at
the site bite causing a painful swelling called a chancre
  Later on they migrate to lymphatic system i.e. lymph nodes, invade the blood
and the tissue fluid, then carried to the heart and various organs of the body
e.g. the spleen and later to the C.N.S where they invade the CSF
 For the life cycle to be continued, the trypomastogotes must be ingested by the
glossina tsetse fly when it takes a blood meal from the human host.

IN TSETSE FLY;

 When the trypomastigote reaches the mid-gut of the fly, they transform into procyclic
trypomastigotes and multiply by binary fission
 Procyclic trypomastigotes leave the mid gut and transform into epimastigotes
 Epimastigotes multiply in the salivary glands and transform into metacyclic trypomastigotes

Pathogenesis & Clinical Features

 There is a painful swelling called a chancre which develops in rhodensiense

trypanosomiasis and less commonly in gambiense infection
 In the early stages of the disease, there is a high irregular fever with shivering, sweating,
increased pulse rate and persistent headache and usually pain in the neck and shoulders
 The lymph glands near to the bite often become swollen. In gambiense infection the
glands at the back of the neck are involved (Winter buttons signs)
 As the disease progresses, the spleen becomes enlarged and there is odema of the eye
lid and often skin rash occurs
 In the male, impotence may occur while in women abortion or amenorrhea may occur
( absence of menestartion)
 In the late stages of infection of the disease, trypanosomes invade the CNS giving
symptoms menigoecephalitis including;
 Trembling
 Inability to speak properly
 Progressive mental dullness
 Excessive sleeping

DIFFERENCES BETWEEN T. BRUCEI GAMBIENSE AND T.BRUCEI RHODENSIENSE

T.GAMBIENSE (West African trypanosome) T.RHODENSIENSE (East African trypanosomes

It causes chronic type of trypanosomiasis with It causes acute type of trypanosomiasis with rapid
early lymphatic evolvement with swelling glands development of encephalitis and symptoms
leading to early death from toxemic or heart
failure
The parasites are usually difficult to find in blood Parasites are easily found in blood and more
but more commonly found in aspirates from commonly in CSF
enlarged lymph glands
Trypanosomes are very few and difficult to detect Trypanosomes can occur in large numbers and can
in direct uncentrifuged preparations be seen easily in un centrifuged preparations
It is not associated with game animals as reservoir It uses game animals as reservoirs
hosts
It is transmitted by lake side and riverine tsetse
It is transmitted by woodland and savannah tsetse
flies i.e. found along lakes and rivers flies found along woodland and savannah areas
Vectored by G.palpalis, G.fuscipes, and Vectored by G.morsitans, G.pallidepes and
G.tachnoides G.swynnertoni
It affects the lymph nodes or glands at the back of
Affects the lymph nodes or glands under jaw,
the neck(winter bottoms sign) armpits base of the elbow and the groin
(kerendel’s sign)
The parasitemia is low The parasitemia is high
LABORATORY DIAGNOSIS;

Specimen;

 Blood
 C.S.F
 Lymph gland aspirates
 Peritoneal aspirate
 Fluid from chancre
 Biopsy

Methods of examination

1) Wet preparation
 This can be performed on blood, C.S.F and other body fluids
 Report the presence of motile trypanosomes
 If no parasite seen, at least 5 preparations should be examined before reporting the
absence of trypanosomes
2) Examination of stained smears
 Make thin and thick smears of the above specimens and stain with field stains , leishman
or giemsa stain
 Examine the smears using x100 objective microscopically and look for detailed
morphological features of trypanosomes and then report as in the wet preparation
method.
 Acridine orange can be used for staining of trypanosomes if facilities of fluorescent
microscopy are available
3) Concentration methods
 Buffy coat
 Triple centrifugation method
 Aminiature anion exchange centrifugation method

AMINIATURE ANION EXCHANGE CENTRIFUGATION METHOD

This is more sensitive for detection of a small number of trypanosomes. This method is very expensive
and only used when parasites cannot be found by other methods
Procedure

 Patients heparinized blood is passed through a buffered anion that negatively charged
exchanged columns of di ethyl-52 cellulose (Buffer)
 As the blood is eluted through the column, the strongly charged blood cells are absorbed onto
the cellulose while the less strongly charged trypanosomes are washed through the column with
buffered saline.
 The elute is then collected, centrifuged and the deposit examined for motile trypanosomes x10,
x40 objectives
4) Animal inoculation
It is used to diagnose trypanosomes in low parasitaemia by first growing them in animals

Procedure

 Inoculate the mice with the specimen suspected to contain trypanosomes e.g. blood
peritoneally.
 Study the mice for about 3 days
Results
If the blood specimen contained trypanosomes, the animal will develop clinical symptoms and
the level of parasitaemia will depend on the strain of the parasite.
Examination
Collect the blood of the mice after 3 days from the tail and examine by wet preparation method,
staining, and concentration method.
5) Serological methods
 These are Antigen-Antibody tests that detect the presence of trypanosomal antibody in
the patient blood
 They include
 Card agglutination test (CATT) for trypanosomes. This is more sensitive to
T.gambiense than T.rhodesiense
 Carry out E.L.I.S.A
 Indirect fluorescent antibody test (I.F.A.T)
 Haem-agglutination test (H.A.T)

Morphology

 There is no morphological difference between T.gambiense and T.rhodensiense

 Both have an “S”-shape
 A Single flagellum arises from the kinetoplast and extends forward along the outer margin of the
undulating membrane and usually beyond it there is a free anterior flagellum.
 A smaller drop like probasal body of the kinetoplast stains darkly
 The nucleus stains dark mauve and is usually centrally placed but posterior nucleus forms can
also occur.
 The cytoplasm stains pale and contains volutin granules
  The parasite is spindle shaped
 They are colorless in wet preparation
 They are found amongst the Rbc therefore they are not intracellular.
 They have a spinning motion
 It shows a variety of forms i.e. long thin forms, intermediate and short stumpy forms.
 They are described as polymorphic trypanosomes

TRYPANOSOMA CRUZI

 This causes South American trypanosomiasis also known as chaga’s disease

 The parasite was first discovered in a vector by Carlos chagas in 1907. He also showed
that the parasite can also cause a disease in humans

Distribution

 Chaga’s disease occurs through south and Central America especially in Brazil, Argentina, and
Mexico. It is estimated that over 24 million people are infected with the species
 It is a zoonotic parasite with over 150 species of wild animals known to habour the parasite e.g.
dogs, pigs, cats and rats.

Transmission

 Chagas disease is mainly transmitted to man through contact with feaces of an infected blood
sucking brightly colored bug belonging to the family Reduvidae and sub family Triatominae
 The bugs are also called kissing bugs
 These also live in he cracks of the wall and roofs of poorly maintained houses and coming out at
night to bit/ feed on exposed parts of the host body mainly around the mouth and eyes
 These faeces containing the infective trypomastigote are deposited on the skin or mucus
membranes as the bugs feeds from its host
 The most important vectors of the disease are those that are adopted to living in human
dwellings and these include;
o Triatoma infestans
o Triatoma braziliensis
 o Rhodnius prolixus
 Transmission can also occur in blood transmission in about 20% of blood donors
 Less commonly also transplacental transmission occurs with the foetus being infected from an
asymptomatic
LIFE CYCLE
 The metacyclic trypomastigotes (infective stage) of the human host are deposited on
the skin as the triatomine bug feeds
 The metacyclic trypomastigote invade the reticulo-endothelial cells near the point of
entries, multiply intracellularly and they become amastigotes.
 The amastigotes then develop into a trypomastigote which are released into the blood
stream when the cell raptures
 Through the blood circulation and lymphatic system, the trypomastigotes reach the
tissue especially those of the heart, nerve, skeletal muscle and the smooth muscles of
the G.I.T where they become masses of psuedocysts.
 Within the psuedocyst a proportion of the amastigotes become elongated and develop
first into epimastigotes and then to trypomastigotes which are released into the blood
when the host cell ruptures.
 Some of the trypomastigotes continue to circulate while the majority of them infect
further tissue cells
 The life cycle is continued when a triatomine bug vector takes a blood meal from an
infected person to ingest circulating trypomastigote
IN THE VECTOR

 The trypomastigotes are transformed and eventually develop into epimastigote which multiply
by binary fission in the hind gut of the bug.
 The short cylindrical and highly motile trypomastigote are formed within 10-15 days and can be
found in the hind gut of the bug ready to be excreated when the vector defecates as it takes a
blood meal.

Pathogenicity & Clinical features

o Many people infected with T.cruzi remain asymptomatic and free from chaga’s disease or
experience an acute infection without progressing to chronic stage. Trypomastigotes and
amastigotes may be seen in the aspirates of chagoma.
o Regional lymph nodules may be infected which frequently involve one side of the face. If the site
of infection is the eye, usually the conjunctiva becomes inflamed and oedema forms, this is
known as Romans signs
o Symptoms may be minor and passed unnoticed however there may be fever, malaise, increased
pulse rate and enlargement of the lymph glands, liver and spleen may occur. Conjunctivitis may
also occur. Muscle pains are persistent and parasites may be seen in blood films
o In the chronic form of the d disease which develops with signs of cardiac muscles damage
including a weak and irregular heartbeat, enlargement of the heart and oedema
 o The parasitic infection of the intestinal muscle may cause damage of the nerves in the intestinal
walls causing loss of the muscular action necessary for the movement of food
o The accumulation and slow movement of food leads to enlargement of the esophagus known as
mega-esophagus and the colon also known mega colon
LABORATORY DIAGNOSIS
 The disease can be diagnosed by the following ways ;
 Examination of blood (thin and thick films)
 Concentration methods
 Xenodiagnoses
 Blood culture
 Serology
Concentration techniques
They include;
 Buffy coat
 Strout method
 Hoff’s method

Strout method;

 In this method 5-10mls of freshly collected venous blood is allowed to clot.

 When the clot has just retracted the supernatant serum is centrifuged at 3000rpm for
15 minutes
 The sediments are examined in wet preparation for motile trypanosomes.

Hoff’s method

 In this method 1ml of fresh EDTA anti-coagulated blood is added to 3mls of ammonium chloride
solution in which the red blood cells are left to lyse in 3 minutes.
 The lysed blood is centrifuged for 15 minutes and the deposit examined as wet preparation for
motile trypanosomes.

Xenodiagnosis

 This is important in detecting trypanosomes in chronic and sub-acute infection in low

parasiteamia
 In this case an un infected healthy susceptible laboratory reared triatomine bug is starved for
2weeks and then fed on the patient’s blood
 Trypanosomes are present, they are ingested by the bug and will multiply and develop into
epimastigotes and trypomastigotes in the hind gut of the bug which can be found in 25-30 days
later in the faeces
 The faeces are got by dissecting the bug and the contents of the hind gut and the rectum
examined microscopically for trypanosomes.
Method of examining
  Collect faeces from the triatomine bug and emulsify with a drop of sterile isotonic saline
 Cover with a cover slip and examine the faeces under a microscope for motile flagellates

Blood culture

 Blood culture is important in diagnosing trypanosomes in the chronic stage of infection when
facilities of Xenodiagnosis are not available
 The parasite is grown on blood broth culture media e.g. NNN medium (Novy-McNeal Nicolle)
 Inoculate aseptically 0.2mls of patient’s blood and incubate at 28c
 After 14-25 days, examine a few drops of broth media for motile trypanosome cruzi
epimatigote.
 Identify the parasite by examining a stained preparation

Serology

 This is important in diagnosis of the chaga’s disease in the chronic stage of the infection when
parasites cannot be found in the blood
 It is used in mass surveys for epidemiological purposes and to screen blood from blood donors
in endemic areas. They include;

 ELISA
 Indirect Florescent Antibody test (IFAT)
 Compliment fixation test
 Indirect Haem-agglutination test

Morphological identification

 It has a single form thus being monomorphic measuring 12-30um in length with a narrow
membrane
 The body is curved always being described as U,V or C shaped depending on the side of view
 The free flagellum is not well developed as in the African trypanosomiasis
 It is medium in length
 The nucleus is centrally placed and stains dark red
 The undulating membrane is not well developed and sometimes it appears bulge at the
posterior end.
PREVENTION AND CONTROL

There is no vaccine or drug for prophylaxis against African trypanosomiasis. Preventive

measures are aimed at minimizing contact with tsetse flies. Local residents are usually aware of
the areas that are heavily infested and they can provide advice about places to avoid. Other
helpful measures include:

 Wear long-sleeved shirts and pants of medium-weight material in neutral colors that
blend with the background environment. Tsetse flies are attracted to bright or dark
colors, and they can bite through lightweight clothing.
 Inspect vehicles before entering. The flies are attracted to the motion and dust from
moving vehicles.
 Avoid bushes. The tsetse fly is less active during the hottest part of the day but will bite
if disturbed.
 Use insect repellent. Permethrin-impregnated clothing and insect repellent have not
been proved to be particularly effective against tsetse flies, but they will prevent other
insect bites that can cause illness.
 Treating the infected person especially in endemic area
 Insecticide spraying of houses and farm building which may harbor tsetse flies
 Health education to the community about the mode of transmission of the disease.