This is the presentation on Trypanosomiasis that covers classification and diseases caused by Trypanosoma, its life cycle, Geographical distribution, Transmission, diagnosis and treatment and finally its scenario in India. Some flow charts have been taken from published articles, that can be searched directly from net.

1. TRYPANOSOMIASIS
BY: HARPREET KAUR

2. INTRODUCTION
• Definition: any tropical disease caused by trypanosomes
and typically transmitted by biting insects, especially
sleeping sickness and Chagas disease
• Causative agent: Trypanosoma

3. DOMAIN Eukaryota
KINGDOM Protista
PHYLUM Kinetoplasta
CLASS Kinetoplastida
ORDER Trypanosomatida
FAMILY Trypanosomatidae
GENUS Trypanosoma
CLASSIFICATION

4. Sub-genus Species
Schizotrypanum T. cruzi
Duttonella T. vivax
Nannomonas T. congolense
Trypanozoon T. brucei
T. b. brucei
T .b. rhodesiense
T .b. gambiense
T. equipedrum
T. evansi
Pycnomonas T. suis

5. DISEASE CAUSED BY TRYPANOSOMES
Trypanosoma species
Species Distributn. Species
exhibiting
disease
Vector Disease
T. b.
gambiense
West Africa humans Glossina palpalis
(Tsetse fly)
Trypanosomiasis
(Sleeping sickness)
chronic
T. b.
rhodesiense
East Africa humans
Glossina
morsitans
Trypanosomiasis
(Sleeping sickness)
acute
T. b. brucei Africa
cattle,
antelope,
horses, camels
Glossina
pallidipes
Nagana
acute
T.
congolense
Africa
cattle,
antelope,
horses, camels
Glossina
morsitans
Nagana
chronic

6. Species Distributn
Species
exhibiting
disease
Vector Disease
T. vivax Africa
cattle, antelope,
horses, camels
Glossina morsitans
Nagana
acute
T.
equiperdum
Africa horses, donkeys
None (transmitted
during coitus)
Dourine
acute
T. evansi Africa
camels, horses,
deer Tabanid fly
Surra
T. cruzi
South and
Central
America
Reduvid bugs:
Rhodnius prolixus Chagas disease

7. HISTORY
1841: Professor G. Valentin of Berne, Switzerland briefly described a
protozoan found in the trout blood moving by means of its
undulating membrane
1843: David Gruby, Paris discovered a similar organism in frog blood
and because its swimming motion suggested the action of a
corkscrew he called it Trypanosoma sanguinis
1880: Griffith Evans, found trypanosomes horses, mules and camels
blood---- suffering from a fatal wasting disease called surra
1892: Alphonse Laveran, summarized all that was known about
trypanosomes in 11 pages and 18 references

8. 1895: discovered T. brucei as the cause of cattle
trypanosomiasis (cattle nagana)
1901: Robert Michael Forde first observed trypanosomes
in human blood in The Gambia
1902: Joseph Everett Dutton proposed the species name Trypanosoma
gambiense
1902: Aldo Castellani found in CSF of sleeping sickness patients and
suggested that they cause sleeping sickness
1903: Bruce provided conclusive evidence that sleeping sickness is
transmitted by tsetse flies

9. 1904: animal pathogenic trypanosome species T. congolense was
discovered by Alphonse Broden
1905: T. vivax was discovered by Hans Ziemann
1909: Friedrich Karl Kleine showed cyclical transmission of T. brucei in
tsetse flies
1909: Carlos Justiniano Ribeiro Chagas first described Chagas disease
1910: John William Watson Stephens and Harold Benjamin Fantham
recovered human pathogenic trypanosome species, T. rhodesiense
Three major outbreaks have occurred in recent history: one from
1896 to 1906 primarily in Uganda and the Congo Basin and two in
1920 and 1970 in several African countries.

10. Trypanosomiasis
African trypanosomiasis
West African
Trypanosomiasis
East African
Trypanosomiasis
American Trypanosomiasis
Glossina (tsetse fly) Triatoma (winged bug)

11. African Trypanosomiasis
• Also called Sleeping sickness (humans)
• nagana, souma and surra (animals)
• transmitted by infected tsetse flies in rural sub-Saharan Africa
• caused by 2 different parasites:
T. b. gambiense found in 24 countries of Central Africa and
western Africa
Accounts for 98% of the reported cases & cause acute infection
T. b. rhodesiense found in 13 countries of eastern and southern
Africa
Accounts for below 2% of the reported cases & cause chronic
infection

12. -70 million are at risk in36 countries of sub-saharan Africa where
the disease occurs regularly

13. Geographic distribution of both East and West African
strains of T. brucei

14. Tsetse fly
•Include all the species of genus Glossina
•Size of honey bees and grey-brown
•29-31 species have been identified
•Almost all exclusively found on African continent south of Sahara
•6 recognised as vector of sleeping sickness and incriminated in
transmission of two pathogenic human trypanosomiasis
•Both male and female flies feed by cutting through the skin,
rupturing small blood vessels and feeding on the pool of blood
formed, intermittently ejecting saliva, with metacyclic trypanosomes
if infected

15. T.brucei brucei- Glassina morsitans – Wild Games & domestic animals
T.b gambiense- G.palpalis- Man, Reptiles & Birds
G.pallidipes
G.tachinoides- Mammals other than man
- Shade & Moisture loving vector
- Found in Scrubs & Shaddy traces near water
- Not travel far from their breeding place
T. b rhodesiense- G.morsitans
G.swinnertoni Feed on Game animals
G.pallidipes
T. eqipedrum & T. evansi -Horseflies(Tabanus sp.) & Stable flies
(Stomoxys sp.) -So not restricted to African continent

16. MORPHOLOGY
Exist in two interchangable forms:
Trypomastigotes: in blood/ lymph/
tissue spaces of various organs & CNS
is terminal and fatal
Epimastigotes: in salivary gland of
host and culture media.
Trypomastigote (polymorphic trypanosomes)
Spindle shaped----Central nucleus ˗ free flagellum--- undulating
membrane
•Long slender form (30µ): active motile with free flagellum
•Short stumpy form (15µ): sluggish without free flagellum
•Intermediate form (20µ): with a short free flagellum

17. African Trypanosomiasis life cycle
Life cycle of Trypanosoma
b. gambiense & T. b. rhodesiense

19. Mechanism of disease transmission by Glossina

20. Pathogenesis and Clinical Picture
Pathogenesis
Bite of tsetse fly → Trypomastigote enter Subcutaneous pool of blood
Some-Blood stream Majority- Entangle in tissue spaces
Autoimmune Rxn. Damage
Massive release of Kinin
- Large no. of non specific Igs against exoantigens release
- Kinin: Heavy infiltration of Macrophages
Macrophages clear trypomastigotes in tissue spaces
- Oedema: Increased capillary permeability of tissues
Winterbottom
sign
Sleeping
sickness
CNS
- Increase of glial cells throughout the CNS
- Cerebral Perivascular Cuffing with mononuclear cells
- Cerebral softening due to thrombosis of cuffed vessels
- Choroid plexus congestion & infiltration with monocytes &
Parasites
- Heavy infiltration in leftomeninges

21. Diagnosis
Microscopy
Serology
DNA detection
Animal
inoculation

22. Diagnosis
Diagnosis of T. b. gambiense HAT follows a three-step pathway:
screening, diagnostic confirmation, and staging
Diagnosis of Sleeping sickness depends on demonstration of
trypanosomes in: blood, CSF or lymph node aspirates
Primary Screening: includes serology
1) CATT (Card Agglutination Test for Trypanosomiasis)/ T.b.gambensie
2) RDT (Rapid diagnostic test) for T. b. gambiense
3) CLN (Cervical lymph node) palpation and puncture
Antigen detection test
Identification of antibodies that are suitable for antigen detection
assays and antigens for use in developing antibody detection tests are
running concurrently

23. a) Card Agglutination Test for human Trypanosomiasis
• field and laboratory test for serodiagnosis and mass screening of the
• population at risk
• in West and Central Africa
• direct card agglutination test for detection of trypanosome specific
antibodies in blood, serum or plasma
• Freeze dried suspension of purified, fixed and stained trypanosomes
of a cloned predominant Variable Antigen Type (VAT LiTat 1.3)
of T.b.gambiense
• One drop of reagent is mixed with one drop of blood and shaken for
5 min

24. • result visible to the naked eye
• Sensitivity from 87- 98%, specificity- around 95%
• False-negative- Patients infected with strains of trypanosomes
that lack or do not express the LiTat 1.3 gene

25. b) micro-CATT:
 small quantities of antigen (one-fifth of the standard amount)
with blood- impregnated filter paper (FP)
 rapid decrease in sensitivity when FP more than 1 day at
ambient temp.
 reading and interpretation of the agglutination patterns can be
difficult
c) macro-CATT:
 Standard amount of antigen and a higher volume of FP elute
 Sensitivity- 91%
 FP can be stored for up to 2 weeks at ambient temperature (25
to 34°C)

26. 2) Rapid Diagnostic test/ T. b. gambensie
• Alternative to CATT
• Tests are packed individually and are stable at a temperature of
40°C for up to 25 months
• Performed on fresh blood obtained from a finger prick, and no
instrument or electricity is required
• RDT detects host antibodies to infection

27. 3) Cervical lymph node (CLN)
palpation and puncture

28. Other serological tests:
a) LATEX/T. b. gambiense:
- developed- a field alternative to the CATT
- combination of 3 purified VSGs, LiTat 1.3, 1.5, and 1.6, coupled
with suspended latex particles
-higher specificity- 96 - 99% but similar sensitivity- 71 - 100%
b) Immunofluorescence assays:
- highly sensitive and specific
- sensitivity as low as 75% when used with impregnated FP
c) ELISA:
- with serum, FP elutes, and CSF

29. Diagnostic Confirmation by Trypanosome Detection
1. Microscopy: Samples- Blood and Lymph node aspirate (40-80%)
- Should be examined within 20min of collection
- In gambiense- No. of parasites in blood & other body fluids- very
- Multiple sampling & conc. tech required
a) Thick/thin blood films- Good diag. tech
- repeat daily sampling increase probability
- very low sensitivity
Staining Rxn-
• Leishman/Ramnowsky stain
•Cytoplasm & undulating memb. → pale blue
•Nucleus- Reddish purple/Red
• Kinetoplast & flagellum- Dark red

30. Blood smear from a patient with T. b.
gambiense
Blood smear from a patient with T.
b. rhodesiense
Blood films with
T brucei
trypomastigotes

31. 2) Microhematocrit centrifugation technique (Mhct):
• Capillary tube centrifugation technique or as the Woo test
3) Quantitative buffy coat:
• concentrating the parasites by cf & staining nucleus and kinetoplast
of trypanosomes with acridine orange
• 95% sensitivity
• Can detect more patients with low parasitemia than Mhct

32. 4) Mini-anion-exchange centrifugation technique:
• introduced by Lumsden et al., 1974
Stage Determination By CSF Examination
1) White blood cell count:
>20 cells/µl in CSF should be treated for second-stage HAT
2) Trypanosome detection:
Less sensitivity→ double centrifugation method
3) Antibody detection:
- high levels of IgM- marker of second-stage HAT
- latex agglutination test for IgM in CSF (LATEX/IgM)
- sensitivity & specificity- 89 and 93%, respectively

33. Other Diagnostic Approaches
1) Antigen detection tests:
• distinct between active and cured HAT
a) Specific antigen detection by ELISA
b) Card indirect agglutination test for trypanosomiasis
(TrypTect CIATT; Brentec Diagnostics, Nairobi, Kenya)
2) Invitro cultivation of Trypanosomes:
• Not NNN
• Medium of Ringer’s soln. + NaCl +Tvrode’s solution
Citrated human blood
↓
Long-slender forms (Mid-gut forms)
• Not useful for routine diagnosis due to the time consuming method

34. 3) KIVI:
• Kit for Invitro Isolation of trypanosomes in field
• Requires days to culture to get detectable amount
•Imp.- Standard for evaluating Quicker diag. tests
4) Animal Inoculation:
•neonatal, immunosuppressed (e.g., Mastomys natalensis) rodents
5) Proteomic Novel & Accurate Test:
• By identifying distinct serum proteomic signatures characteristic
of HAT by SELDI-TOF
• Sensitivity- 100%
• Specificity- 98.6% (Papadopoulos et al., 2004)

36. 6) Molecular dipstick test:
• HAT-PCR-OC
• For rapid detection of amplified T.brucei DNA
• PCR product visualised on dipstick through hybridization with a
gold-conjugated probe (Oligochromatography)
• 5min
• Controls for PCR & DNA migration, incorporated
• Can detect 5fg of pure T. brucei DNA
• 1 Parasite/180µl of blood
• 100% Sensitivity & Specificity (Deborggraeve et al., 2006)

38. 7) PCR for diag. of T. b. gambiense:
• PCRs targeting repetitive sequences- more sensitive in theory
• None of them have been validated for diagnostic purposes
Sensitivity- 99.4%
Specificity- 98.6%
(Penchenier et al., 2000)

39. Drug Use Drawbacks
Pentamidine
Effective against early-
stage gambiense disease
• Adverse side effect
•Non-oral route
Suramin
Effective against early-
stage
gambiense & rhodesiense
disease
•Adverse side effects
•Non-oral route
Melarsoprol
First line drug for late
stage
gambiense & rhodesiense
disease involving CNS
•Adverse side effects, especially
encephalopathy
•Fatal in 1-5% of cases
•Parasite resistance
•Non-oral route
Eflornithine
Effective against late-
stage gambiense disease
•High cost
•Not effective against T.
rhodesiense
•Non-oral route - has to be
Treatment of African Sleeping Sickness

40. American Trypanosomiasis
• Also called Chagas Disease
• transmitted by infected winged bugs, kissing bugs
• leading cause of heart failure in Latin America
• affects 16 to 18 million people- Central and South America
•killing about 50,000 every year
•100 million people (25%)- Latin America is at risk
•6 million people- Brazil.

41. The human disease occurs in two stages:
Acute stage Chronic Stage
-occurs shortly after initial infection - after a latent period that may
-generally seen in children last many years
-characterised by - asymptomatic
- lesions irreversibly affect
• fever internal organs namely the
•Swelling of lymph nodes heart, oesophagus and colon
•enlargement of the liver and spleen and the peripheral nervous
•local inflammation at the site of system
infection
-Swelling around one eye -25%-cardiac symptoms- death
(Romana’s sign may occur if insect -5% develop digestive damage
faeces are rubbed into it) -3% peripheral nerve damage

42. Triatomine bugs
• Also called reduviid bugs, kissing bugs, assassin bugs, cone-nosed
bugs, and blood suckers
• 138 species of triatomine have potential to transmit T. cruzi
• But 5 are epidemiologically important:
Triatoma infestans
Rhodnius prolixus
Triatoma dimidiata
Triatoma brasiliensis
Panstrongylus megistus R. prolixus
• typically found in the southern United States, Mexico, Central
America, and South America

43. Geographical Distribution of Chagas Disease

44. MORPHOLOGY
Trypomastigote (monomorphic)
Slender shaped (20µ) –Central nucleus
– C or U-shaped–Free flagellum 1/3
Body--Large bulging peripheral
kinetoplast
Amastigote
Obligatory intracellular – mainly in
cardiac & Skeletal muscles – Brain
meninges – Nerve ganglia – cells of GIT
Epimastigote (vector only)
Spindle shape– Kinetoplast anterior to
central nucleus–Undulating membrane
is short – terminal free flagellum

45. American Trypanosomiasis Life cycle

46. Mechanism of Disease Transmission by Triatome

47. Pathogenesis and Clinical Features
Acute Form
Chagoma occurs at the site of bite.
Parasite reaches regional lymph nodes
Blood
Organs and tissues
Symptoms: Fever, enlarged lymph nodes, skin rash, enlarged liver &
spleen.
Romana’s sign (Unilateral conjunctivitis appear suddenly together
with oedema of upper & lower eye lids & cheek)
Meningoencephalitis, heart failure
Death or pass to Chronic form

48. Chronic form
Parasite produces antigens similar to
patient’s self antigens:
The body produces auto-antibodies that cause damage to:
Heart muscle fibres: Exacerbation of infection in
immunosuppressed patients
Oesophageal muscle fibres: megacolon and
constipation
Colon muscle fibres: megaoesophagus and
dysphagia
Destruction of Auerbach’s plexus
CNS or thyroid gland: congestive heart failure
Amastigote form of
T.cruzi

49. Laboratory Diagnosis
Parasitological Diagnosis
Immunological Diagnosis
Molecular Diagnosis
Parasitological Diagnosis
Direct Methods Indirect Methods
- Microscopic Examination - Hemocultures
- Culture - Xenodiagnosis
- Animal Inoculation
Microscopic Examination
 Drop of blood (5 mL)- rapid movements of T .cruzi
trypomastigotes (100 fields)
 Stained thin and thick blood smears
 Less sensitive

50. Microhematocrit
• Collect blood in 1-6 heparinized capillary tubes of 75 mL
• Centrifuged for 5-10 minutes
• Examine the buffy coat in microscopy for trypomastigote
movements
Strout method
• 3 ml of blood incubated for 1 hour at 37°C.
• Serum is collected & centrifuged (at 160g for 3 min)
• The supernatant - second centrifugation (at 400g for 5 min)
• precipitate of last centrifugation examined as fresh blood

52. Culture
 Microscopy fail
 NNN , 22-240 C
 4th day & every week for 6 weeks
 Epimastigotes & Trypomastigotes
Indirect Methods
Multiplication of parasites from the collected samples in the
culture medium
Performed on liver infusion tryptose medium
Maintained at 28°C and observed monthly over 4-6 months
Disadvantages
 Examinations are long and require highly specialized personnel
The results are available only 1-6 months after the test
Less popular
Less sensitive (25-70% )

53. Xenodiagnosis
Insects can be put either directly in contact with the patient's skin or
with anticoagulated blood through a thin latex membrane
intestinal contents of the insects are examined 30-60 days later to
observe for metacyclic trypomastigotes
Animal Inoculation
Specimens are inoculated intraperitoneally into mice
Tail blood collected after 10 days & examined for motile
trypanosomes
Mice killed after 2 months & tissue from heart examined for
amastigotes

54. Immunological Diagnosis
3 commercially available serological tests are used routinely for
investigation of T . cruzi–specific antibodies
 Indirect hemaglutination- Sensitivity- 96-98%
 Immunofluorescence (IFI)
 Enzyme-linked immunosorbent assay (ELISA)
Diagnosis of 98% of patients who are chronically infected IFI
and ELISA are also suitable for the detection of IgM (congenital
infection)
 Complement-mediated lysis- helps to detect antibodies
disappearing soon after treatment
 Direct agglutination test (DAT)- Field Use
 The Western blot- Confirmatory Diagnosis

55. Imaging Studies
Radiography of chest ,colon, esophagus
Electrocardiogram, Holter monitoring & exercise testing ,
Esophageal endoscopy and manometry
Treatment
Benznidazole
5–10 mg/kg per day in two divided doses for 30–60 days
Nifurtimox
8–10 mg/kg in three divided doses after meals for 30–120 days

56. Prevention of African Trypanosomiasis
 No vaccine available against Trypanosomiasis
 Wear long-sleeved shirts and pants of medium-weight material
in neutral colors that blend with the background environment
 Inspect vehicles before entering- flies attracted to the motion
and dust from moving vehicles
 Avoid bushes- 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

57. Prevention of American Trypanosomiasis
Collective prophylaxis
By using sprays & paints containing insecticides
By improving housing & sanitary conditions in rural areas
A mosquito net
Testing of blood donars
Treatment of infected individuals
Personal prophylaxis
Lab personnel should wear gloves & skin and eye protection
Applying insect repellent to exposed skin
Proper washing and cooking

58. Control
Control of African trypanosomiasis rests on two strategies:
• reducing the disease reservoir
• controlling the tsetse fly vector
for T. b. gambiense
• humans are significant disease reservoir
• main control strategy for this subspecies is active case-finding
through population screening
• treatment of the infected persons that are identified
• Tsetse fly traps are sometimes used as an adjunct
For T. b. rhodesiense
• variety of animal hosts- reducing the reservoir of infection is
more difficult
• Vector control- primary strategy- done with traps or screens, in
combination with insecticides and odors that attract the flies

59. Treatment of American Trypanosomiasis
Antitrypanosomal drug treatment is always recommended
For acute, early congenital, and reactivated T. cruzi infection
for chronic T. cruzi infection in children aged <18 years old
In adults, treatment is usually recommended
treatment drugs - benznidazole and nifurtimox
Benznidazole: 5–10 mg/kg per day in two divided doses for 30–
60 days
Nifurtimox: 8–10 mg/kg in three divided doses after meals for
30–120 days

60. Trypanosomiasis in India
•1974- Two cases of self-limiting febrile illness due to Trypanosoma
lewisi reported of an adult couple who lived in a rat-infested village
•symptoms resolved without specific treatment after two to three
days
•2004- the first case of human Trypanosoma evansi -- induced
trypanosomiasis was formally identified in India due to lack of apoL-1
• Unusual case of Trypanosoma evansi reported from rural parts of
Chandrapur district in Maharashtra in an adult male farmhand with
recurrent febrile episodes
•responded to suramin

61. •2007- human T. evansi infection were reported in Maharashtra
State, including a two month old infant in Mumbai
•2007- 57 year old man from district Pune got infection from T.
lewisi
• had chronic intermittent fever, anemia, firm hepatosplenomegaly
and edema on feet
• Treated with suramin
•2010- a 37 day-old infant in Uttar Pradesh
• Trypanosomiasis confined to animals in India
• T. lewisi parasite of rats (Rattus rattus and Rattus norvegicus)
tramsmitted by fleas
• T. evansi infects buffalo, camels, horses, pigs, cattle, dogs, jaguars
and tigers

62. THANK YOU