African Trypanosomiasis

- The African sleeping sickness

In 2001 WHO estimated that there were 48,000 deaths caused by sleeping sickness.

Currently It is estimated the number of deaths per year has increased to 50,000 or as high as 100,000.

Most cases are found in Sub-Saharan Africa.

Death rates of sleeping sickness are More than HIV

How can an infectious disease be contained to such a specific region of the world????

Let’s take a closer look at how the disease is spread…

Caused by Trypanosoma brucei

African Sleeping sickness (Humans)

Nagana ( In Animals)

Vector Tsetse fly

Tsetse fly

Phylum - ArthropodaOrder - DiptheraFamily - GlossinidaeGenus - Glossina Species - pallidipes External anatomy

• body Is covered by thick cuticle.

• body is divided into 3 segments head, thorax and abdomen.

• compound eyes are comprised of thousands of small units called ommatidia.

Mouth parts • Mouth parts of the insect are attached to

head by Thecal bulb.• A tube for sucking blood is made up 2

parts thick and darkest labium and thin and transparent labrum.

• Labium has small teeth called labellar teeth used to pierce the host skin.

• At rest mouth parts are point forward and projected by a pair of maxillary palp, while feeding lowered from palp and point downwards.

• A small blood sucking tube called hypopharynx is used to inject saliva which contain anticoagulants.

• It is important to recognize these structures as they are sites for trypanosomes in infected flies.

Thorax• It has 3 pairs of legs and 1 pair of wings attached to it.• Legs being made of four main segments, the coxa,

trochanter, femur and tibia and then five smaller tarsal segments ending with two claws and two small pads called pulvilli.

• it has 2 pairs of spiracles at sides which lead to trachea which helps in respiration.

Abdomen• It has seven abdominal segments.• There are seven pair of pairs of spiracles at one pair of each

segment.• Male fly has a structure at the posterior tip, folded

underneath the last two segments called the hypopygium , forming part of the external genitalia.

• Anus at posterior end.

Internal anatomy• Tse Tse has two salivary glands

stretched through the abdomen, thorax and head to proboscis into which they open into Hypopharynx.

• Glands contain saliva which is anti coagulant prevent the blood of the host from clotting

• Glands are transparent small tubes it is important to recognise as they are sites for development of Trypanosoma brucei-complex trypanosomes.

• When tse tse feeds on the blood it first disengages probocis from maxillary palp into lower skin, labellar teeth penetrates and cuts blood capillaries , blood is sucked through the proboscis by means of muscular pump in pharynx then blood is mixed with saliva from hypopharynx it passes to esophagus from there to muscular proventriculus to crop.

LIFE HISTORY• 30 species and subspecies of tsetse flies exist in the tropical Africa.• Adult measures 6-14mm in length.• Adults of both sexes bite vertebrates and imbibe blood(fly’s only

food).• Males can be distinguished from females by presence of external

genetelia at the end of the abdomen.• Mating between both the sexes is stimulated by the release of

pheromone in the cuticle of female.• Ovulation is induced by female endocrine system only if mating lasts

longer than hour.• Females mate when they are young probably when taking their first

blood meal.

REPRODUCTION IN TSETSE FLY

When mating • spermatophore deposited by males near the end of

spermathecal ducts. • Sperm leaves spermatophore and enters spermathecae.• Egg is ovulated into uterus from right ovary when fly is 9 days

old.• Then it is fertilised by sperm from spermathecae and embryo

develops.• After 4 days the egg hatches into first instar larva which feeds

on milk produced by the uterine glands.• Larva molts twice and occupies most of the mothers abdomen

and then it is deposited on the ground by the female(loose soil shaded by trees).

• Shortly thereafter the female ovulates again and deposits larva about every 7-11 days (depending on ambient temperature).

LARVA• 3 - 8.5 mm length, cream colored

and oval shaped.• Two knobs on posterior end –

respiratory lobes and function for short time following intrauterine life.

• They are ‘–’vely phototactic and ‘+’vely thigmotactic –quickly burrows soil surface.

• After few hours larval integument hardens and becomes black puparium.

• Molting occurs and pupa is formed.• 30 days after formation of puparium

adult flies emerge.• Thoracic flight muscles develop

completely after 9 days.

• Tsetse males survive for about 2-3 weeks.

• Tsetse females survive for about 20 -40 days but may have maximum life span of 3-4 months.

LIFE HISTORY OF TSETSE FLY

Trypanosoma Trypanosoma brucei

African Sleeping sickness ( Humans)

Nagana ( In Animals)

Scientific classification

Kingdom : ExcavataPhylum : EuglenozoaClass : KinetoplasteaOrder : TrypanosomatidaGenus : TrypanosomaSpecies : T. brucei

Cell Structure of Trypanosoma brucei

Basal body anterior of nucleus, with a short, essentially non-functional, flagellum.

Basal body anterior of nucleus, with a long detached flagellum.

Basal body anterior of nucleus, with a long flagellum attached along the cell body.

Basal body posterior of nucleus, with a long flagellum attached along the cell body.

Genome : 11 pairs of chromosomes

Flagellar structure :• 9+2 Microtubule

arrangement

Mainly helps in

Movement and attachment

of parasite to insect gut.

Factors Adverse to Trypanosomes

Variant Surface Glycoproteins(VSG) Coat

Trypanosome is covered with a dense coat of ~107 VSG proteins.

The two main roles of VSG proteins is

1 Shielding : It prevents the identification of parasite surface epitopes, receptors, transporters to the host

immune system.

2 Antigenic variation: VSG undergo gene modification

by ‘Switching’ allowing variant new VSG coat to escape from Immune specific response.

Ligand receptor interactions

 

Host derived molecules: The host derived molecules are present in the body which are necessary for the proliferation of growth of the trypanosome.

These levels may be reduced if patients develop anaemia or cachexia.

Trypanocidal factors in human serum

Trypanosome contains a special factor called trypanosome lytic factor (TLF) which inhibits the growth of T.brucei

Factors favouring trypanosome survival:

-Interferons can markedly proliferate the growth of T.brucei.Trypanosomes release a special factor called trypanosome stimulating factor which stimulates the production of lymphocytes.

-This molecule selectively binds to the CD8 antigen on T-cells which cause them to release IF

Life cycle of Trypanosoma Brucei in tsetse fly

Infection of Vector

Once the tsetse fly bites the infected person the short stumpy parasite T.b enters in to the tsetse fly.

Procyclic trypomastigotes

The short stumpy trypomastigotes transform in to long slender Procyclic trypomastigotes.

Asexual reproduction

The Procyclic trypomastigotes reproduce asexually

By binary fission

Mesocyclic trypomastigotes

The Procyclic trypomastigotes move to the anterior portion of the midgut and elongate to produce mesocyclic trypomastigotes

The mesocyclic trypomastigotes quickly move in to the salivary glands

Ready for Infection

In salivary glands they develop in to epimastigotes , attached by their flagella to the wall of glands

Metacyclic trypomastigotes

They eventually turn in to metacyclic trypomastigotes and now they are ready to inject in to new host

Tsetse fly bites the host

The tsetse fly bites the human being

Inoculation of parasite

The tsetse fly inoculates with the metacyclic trypomastigotes when it takes a blood meal

Metacyclic Trypomastigotes

Transformation

The metacyclic forms quickly in to trypomastigotes .

Asexual reproduction

Reproduce asexually

Parasites spread throughout the body

How it cause disease in Humans ?

Retransformation

After parkistemia they retransform in to smaller, shot, stumpy , non dividing ( they retransform in to smaller that they can survive

Preparing for Vector

Once the tsetse fly bites man they move with blood in to tsetse fly salivary glands ( pharynx)

INTERACTION BETWEEN TRYPANOSOME AND TSETSE FLY

• Progress of trypanosome in tsetse is not stochastic process.• Fly has complex system to create hurdles to throw up the incoming

parasite.• Life cycle divided into two phases :• Establishment• Maturation• Establishment : trypanosome has to establish itself as a dividing

procyclic population in mid-gut.• Trypanosoma brucei –intial establishment occurs in midgut and

maturation in salivary glands.• Transmission cycle starts when susceptible fly bites infected animal.

• Breeding experiments –susceptibility to midgut infection is maternally inherited character.

• Tsetse have midgut lectin activity and these molecules play important role in determining susceptibility.

• Invivo experiments conducted.• Feeding fly with specific lectin inhibitory sugars along with

infective feed.• 100% infection rates achieved.• Feeding with pure procyclin with infective feed showed same

results.• Midgut lectin-kill trypanosomes.• How could this activity be effected by maternal factors?• Symbionts in midgut region showed chitinolytic activity.

Chitin degraded by chitinase enzyme into lectic inhibitory sugars.

Age also significant factor.

Teneral flies showed more susceptibility to infection(low lectin activity with midgut lectin produced in response to blood meal).

PERITROPHIC MATRIX,GUT ENZYMES :

Peritrophic matrix continuously secreted by proventriculus at anterior end of midgut.

• At first peritrophic matrix was thought to act as barrier for infection and also pH of hindgut(5.8) was found to be lethal.

• Electron microscopy studies showed it was wrong.

• Midgut proteases are important in transformation of bloodstream form to procyclic form.

maturation – the trypanosome must leave the midgut and, in the case of the human infective trypanosomes, make its way to the salivary glands.• The second main hurdle of trypahanosoma development in

Tse tse fly is maturation of established procyclic population through epimastigotes to mammalian infective forms (metacyclics) in the salivary glands.

• Only a proportion of established midgut infections mature.• Factor regulating maturation(1) interaction with the midgut lectin(2) fly sex, and (3) trypanosome stockinteraction with the midgut lectinWhile inhibition of the activity of the midgut lectin is important

for trypanosome survival, establishment in the midgut, and essential for successful maturation of the parasites.

• Maturation of midgut infections can be prevented simply by continuous addition of lectin inhibitory sugar to the fly feed.

• Removal of serum from the tsetse diet also prevents maturation as the midgut lectin is secreted in response to serum in the bloodmeal.

Fly sex• Maturation of T. brucei s.l. trypanosomes is greatlyaffected by fly sex• male tsetse produce significantly more mature trypanosome

infections than do females.• Because of these sex differences appears to be the

operation of a product(s) of an X-linked gene that kills or prevents migrating parasites from maturing.

Trypanosome stockThe proportion of midgut infections that eventually mature decreases with increasing maturation time, so that higher rates of maturation are achieved in stocks that are able to mature earlier in tsetse.

Control of disease by Novel Idea

In the tsetse fly studies have revealed that the main function of midgut lectin is to kill the trypanosome

Our Novel Idea

Is to introduce a gene in the insects midgut which over expresses the production of lectin

CLINICAL FEATURES OF AFRICAN TRYPANOSOMIASIS(SLEEPING SICKNESS)

• Disease appears in two stages :

first the haemolymphatic stage

second the meningoencephalitic stage.• Symptoms occur within 1-4 weeks after infection and if left

untreated may lead to coma and death.• Trypanosoma brucei gambiense infection is characterised by

chronic progressive course.• Models based on survival analysis-infection is around 3

years.

• A trypanosomal chancre is rarely seen.(19% patients).

SYMPTOMS OF FIRST STAGE:1. Intermittent fever.(lasts from day to week)

2. Headache.

3. Pruritus.

4. Lymphadenopathy.

5. Hepatosplenomegaly.

SYMPTOMS OF SECOND STAGE:

1.Sleeping disorder is leading symptom.

2.Dysregulation of circadian rhythm of sleep/wake cycle and fragmentation of sleeping pattern.

NEUROLOGICAL SYMPTOMS:

Tremor,fasciculations,general motor weakness, paralysis of limb,hemiparesis,aknesia,abnormal movements such as dyskinesia or chorea-athetosis.

• Parkinson like movements due to muscular hypertension,non-specific movement disorders and speech disorders.

• Abnormal archaic reflexes also arise.

• PSYCHIATRIC SYMPTOMS:• Irritability,psychotic

reactions,aggressive behaviour,inactivity with apathy.

• These disorders increase in frequency with duration of the disease.

DIAGNOSIS• 3 step approach :screening ,diagnostic confirmation and

staging.• Organisms identified by microscopic examination of CSF

sediment.• Increase in WBC and protein, increased IgM or presence of

morula cells.• Increased number of cells(mononuclear):<30/μl(second

month),later 100-400 /μl.• Increased protein:60-100mg/dL with increase in γ-globulin(used

as index to severity of disease and therapeutic response).• Diagnostic methods available are :• CARD AGGLUTINATION TEST FOR

TRYPANOSOMIASIS(CATT)-fast and practical serological test.• 87-98% sensitive and 93-95% specific.

Drop of blood

Mixed with parasites on plastic card

Blue granular deposits = Infection

• Areas with disease prevalence <5%,positive predictive value of positive CATT low for confirmation.

• Results misleading in some areas-absence of LiTat 1.3 antigene.

• Immunofluorescence or ELISA also used.(non-endemic regions).

• Parasitological cconfirmation-examination of lymphnode aspirate and blood.

• Delay b/w sampling and examination kept short.

• Microhaemotocrit centrifugation technique, quantitative buffy coat, miniature anion exchange centrifugation technique used-examination of thin/thick blood films(low sensitivity).

• Serial examination on consecutive days increases sensitivity of blood examination techniques.

• More sensitive approach-detection of parasite nucleic acids by PCR.

TreatmentChemotherapy

Type of drug treatment used depends on the type and stage of African trypanosomiasis.

Type of Trypanosomiasis

Stage 1(Hemolymphatic Stage)

Stage 2(Neurologic [CNS] Stage)

East African trypanosomiasis (caused by T brucei rhodesiense)

Suramin Melarsoprol

West African trypanosomiasis (caused by T brucei gambiense)

Pentamidine or Suramin

Melarsoprol or Eflornithine

Surammin

• Antiparasitic agent used IV in early-stage African trypanosomiasis and onchocerciasis.

• Suramin is given 100-200 mg IV test dose, then 1 g IV on days 1, 3, 7, 14, 21. The recommended dose regimens for suramin last up to 30 days.

• Suramin is more effective and less toxic than pentamidine and excreted in urine in slow rates.

• used for first-stage T b rhodesiense disease, but is generally avoided against T b gambiense disease where Onchocerca spp are also present, its high activity against these parasites can expose patients to the risk of severe allergic reactions

Side affectsHypersensitivity reactions, heamaturia, peripheral neuropathy.

Melarsoprol

used in the late or CNS stage of African trypanosomiasis.• Melarsoprol 2-3.6 mg/kg/d IV for 3 days; after 1 wk, 3.6 mg/kg/d

for 3 days; after 10-21 days, repeat the cycle.• Melarsoprol is practically insoluble in water and must be given

intravenously dissolved in propylene glycol, a solvent that is highly irritant to tissues.

• Therapy is as high as 90-95% successful in clearing the parasitemia.

• However, it can be toxic and even fatal in 4-6% of cases. up to 30% of cases do not respond to the drug

Side effects• encephalopathic syndromes, skin reactions, peripheral motoric

or sensorial neurophathies.

Pentamidine

• Antiprotozoal agent usually used for early stage 1.• Inhibits enzymatic action of parasite.• dosage 4 mg/kg/d IM for 10 d.• Reported to have a >90% cure rate.• Pentamidine does not penetrate the blood-brain barrier M

effectively and, therefore, does not treat CNS infection.Side effects • Hypoglycemia, injection site pain, diarrhea, vomiting etc.

EflornithineGiven 400 mg/kg/d IV in 4 divided doses for 14 days.• Used for patients in whom melarsoprol failsSide effectsConvulsions, diarrhea, vomiting

Combination therapy

May be more effective than monotherapy for the treatment of late-stage T brucei gambiense trypanosomiasis• The trial concluded that a consecutive 10-day low-dose

melarsoprol-nifurtimox combination is more effective than the standard melarsoprol regimen

• Trial that compared the efficacy of eflornithine monotherapy to nifurtimox-eflornithine combination therapy in patients with late-stage T brucei gambiense infection found that cure rates were similar. However, adverse effects were more common in patients who received eflornithine monotherapy .

• Thus, the nifurtimox-eflornithine combination appears to be a promising regimen.

Prevention and control

• No vaccine developed against trypanosome.• chemoprophylaxis is not recommended because of the drugs

adverse effects and low risk of infection.• Prevention is only by reduction of tsetse fly.• Tsetse flies are attracted to dark colored and motion vehicles.• They can bite through the cloth and insect repellant can

provide little protection.• Travelers can take some preventive measures by avoidance

of area where tsetse flies are known to be present or traveling car with closed windows and clothes wrist and ankle length.

• If a person is bitten by tsetse fly he should be monitored.• If a chancre, fever, or other symptom develops, an aspirate of

the chancre, the blood, and possibly a lymph node aspirate should be examined for the presence of trypanosomes.

• In T b gambiense areas the CATT could be used for diagnosis.

• Infected person can remain asymmptomatic for long preiod of time before sighs of sleeping sickness develops, but always act as a reservoir.

• Vector control by use of tsetse fly traps or screens, in combination with odours that attract the flies, or insecticides, helps to reduce the fly density.

SIT• SIT relies on rearing large numbers of male insects in

purposebuilt “fly factories”, sterilizing the males with carefully controlled doses of gamma radiation and finally releasing them by airplane over the target area.

• The radiation induces sterility, but the treated male flies can still fly and mate with wild females.

• Mating between the sterile released males and wild female tsetse flies produces no offspring and the population is eliminated.

• No other insect is affected and there is no adverse impact on the environment.

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