UGA Medical Parasitology - Unit 2: Protozoans

Robert Hooke

English scientist that discovered cells in all living thingsand pioneered the use of microscopes to observe microscopic structures.

Anton van Leeuwenhoek

Dutch scientist known as the "father of microbiology" who first observed single-celled organisms and bacteria using a microscope he designed.

Protozoan

a protist with animal like characteristics, also means ‘first animals’

Basic Characteristics of Protozoans

1. Heterotrophic: i.e. has to eat other things.

2. Are unicellular.

3. Most are mobile.

Protozoan Subgroups

SAR

ARCHAEPLASTIDA

EXCAVATA

AMOEBAZOA

OPIS

Protozoan Parasite Focus

SAR: Entamoeba histolytica

EXCAVATA: Giardia/Trichomonas/Trypanosomatids

AMOEBAZOA: Entamoeba histolytica

Entamoeba histolytica

• no defined shape (constantly changing form)

• not free living

• feed via trogocytosis

  • Rather than eat cells whole, E. histolytica

    trophozoites will ingest small bites of host

    cell material.

    • The ‘nibbling’ causes apoptotic death and

    leads to an immune response and much of

    the pathology of infection.

• uses dynamic pseudopodia that extend out to move

• Generally they expand asexually through binary fission but sexual

  recombination is thought to occur

• causes amoebiasis (amoebic dysentery)

• Humans are the only reservoir excreting amoebic cysts. The cysts

  are environmentally stable (~1 month).

  • Transmission occurs through a fecal-oral route (consuming

  contaminated food/water)

How does Entamoeba histolytica move?

actin polymerization at the membrane

*also how your immune cells travel around*

Entamoeba histolytica Life Cycle

Infection begins with cysts being ingested in

contaminated food or water.

• Cysts pass through the stomach and upon reaching

the small intestine the amoeba leaves or “excysts” as

a 4 nuclei containing cell.

• This form, called the ‘trophozoite’, copies its

genomes 1x to give 8 nuclei.

• The parasite then undergoes cytokinesis 3x to make

8 trophozoites from a single cyst.

• The trophozoites migrate to the large intestine, eats

bacteria and begin dividing.

• If they become invasive they can migrate to the

brain, liver and lungs where they cause tissue

destruction and disease.

• Normally, however, they re-encyst and are excreted

by the host to continue the cycle.

Entamoeba histolytica Pathology of Infection

Gradual onset of abdominal pain, watery

stools containing mucus and blood.

• Some patients have only intermittent

diarrhea alternating with constipation.

• Fever is uncommon.

• Parasites begin invading the intestinal

mucosa.

• Infiltration of immune cells and tissue

destruction leads to flask shape ulcers

that eventually allow E. histolytica to

enter into the bloodstream.

• As parasites begin eating and destroying

the colonic epithelium ulcers begin to

form.

• Parasites now have access to

disseminate through the body.

Systemic Phase of Infection:

• Parasites invade the tissues:

• Liver

• Lung

• Brain

• Inflammation and tissue

destruction due to parasite

mediated killing of host cells

can least to systemic

abscesses.

Entamoeba histolytica Treatment and Prevention

Several drugs are available to clear symptomatic

and asymptomatic enteric (luminal) infection:

• Diodohydroxyquin

• Paromomycin

• Diloxanide furoate

• Metronidazole (Flagyl) is the drug of choice for

invasive amoebiasis (90% cure rate).

• Metronidazole should be combined with a luminal

acting drug as it is not fully effective on non-

invasive stages.

• Improved sanitation will help to reduce the

likelihood of transmission.

• Travelers to endemic areas can reduce the risk

of infection by drinking bottled water, not using

ice cubes in drinks, and washing fruits and

vegetables with clean water (or by peeling them

yourself).

Acanthamoeba spp. Life Cycle

Normally free living, these opportunistic pathogens

sometimes infect people through contact with soil,

fresh/sea water, pools, contact lens equipment,

medical devices etc.

• Parasites replicate by mitosis (normal style).

• Acanthamoeba enter the eyes, nose, or broken

skin to gain entry.

• Infection can remain localized at the site of entry or

can spread to other systems of the body and be

deadly.

• Since this is not its normal life cycle of

Acanthamoeba, humans are considered a dead

end host for this organism.

• No known human to human infection.

Acanthamoeba spp. Pathology

If Acanthamoeba enters the eyes it can result in

severe keratitis that can make someone blind.

• This is a serious issue with contact lens users and if

severe will require a corneal graft.

• Systemic infection presents as inflammation of the

lungs or sinuses.

• Skin infections caused by Acanthamoeba can appear

as reddish nodules, skin ulcers, or abscesses in the

skin.

• If the parasite reaches the central nervous system

(CNS) it can cause Granulomatous Amoebic

Encephalitis (GAE).

• GAE starts slowly, with symptoms like headache,

nausea, dizziness, irritability and a low-grade fever.

• GAE is a rare, usually fatal, subacute-to-chronic

central nervous system disease

Acanthamoeba spp. Treatment and Prevention

Miltefosine (Impaido) is a standard prescribed drug

for free-living amoeba infections.

• Several eye medications are available with a

prescription (PHMB-Desomedine, hexamidine and

chlorhexidine)

• To prevent eye infections contact lens users are

advised to carefully follow use and cleaning

directions from their eye care providers.

• It is unknown what can prevent GAE or

disseminated infections as both are extremely rare

anyway.

Balamuthia mandrillaris

Life Cycle/Pathology/Treatment

Can enter the nose or broken skin to gain entry.

• Infection can spread to other systems of the body

and be deadly.

• If the parasite reaches the central nervous system

(CNS) it can cause Granulomatous Amoebic

Encephalitis (GAE).

• Few patients survive Balamuthia infection even

with drug treatment using a combination of

flucytosine, pentamidine, fluconazole, sulfadiazine

and either azithromycin, clarithromycin or even

miltefosine.

• Not known why some people become infected

while others do not. Only ~200 known cases

worldwide.

Naegleria fowleri

“The Brain Eater” Life Cycle

Normally free living these opportunistic pathogens

sometimes come into contact with people who

have fresh water enter deep into the nasal

passages.

• Unlike the other ameobozoons, this is an excavate

and has 3 stages in its life cycle: cyst (1),

trophozoite (2), and flagellate (3). The only

infectious stage of the amoeba is the trophozoite.

• Once penetrating the nasal mucosa the amoeba

migrate to the brain via the olfactory nerves.

• Infection can lead to primary amoebic

meningoencephalitis (PAM).

• No known human to human spread

Naegleria fowleri

Pathology

Enters through nasal cavity, usually when there’s a

sudden rush of water in the cavity while

diving/swimming

• Travels through the cribriform plate and olfactory

bulb where it encounters the brain and begins

feeding.

• Cell death and inflammation causes damage to the

frontal lobe.

• Infection can lead to primary amoebic

meningoencephalitis (PAM).

Stage 1 (2 days)

• Severe frontal headache

• Fever

• Nausea

• Vomiting

• Stage 2 (3 days)

• Stiff neck

• Seizures

• Altered mental status

• Hallucinations

• Coma

Naegleria fowleri

Treatment and Prevention

Only 5 documented cases of people

surviving PAM exist.

• Very few drugs available and only

miltefosine has shown promise as a

potential treatment.

• More research is needed to find

promising therapeutics for these rare

but deadly infections.

• Recommended not to swim in

stagnant or very still lakes and to

avoid aspirating lake water into your

nostrils.

The Kinetoplast

kinet=move plast=organelle

Basic Biology of Trypanosoma cruzi Forms

Normal Eukaryotic Organelles: nucleus, Golgi, ER, mitochondria

Unusual organelles:

• Kinetoplast: Genome of the single giant

mitochondrion containing densly packed DNA

(stains brighter than nuclear DNA).

• Glycosomes: unlike us, trypanosomatids carry out

glycolysis in membrane bound vesicles.

• Reservosomes: pre-lysosomal compartment

present in high amounts in epimastigotes.

• Cytostome/Cytopharynx (SPC): entrance to the

endocytic pathway. Mechanism of large molecule

uptake, i.e. ‘how it eats’.

• Acidocalcisome: calcium storage vesicles.

• Contractile vacuole: similar to the CV in amoebas.

• Flagellum and flagellar pocket: how the parasite

moves and the site of exocytosis.

• Subpellicular microtubules: microtubule skeleton

underlying the entire body of the parasite

Salivarian

transmitted via mouth parts “saliva”

Although salivarians expand in the midgut they then

make their way either out of the intestine and to the

salivary glands or up to the foregut and mouthparts in

order to gain direct access to the host bloodstream

during a bloodmeal

Stercorarian

Transmitted via feces

T. Cruzi Life Cycle

• stercorarian

Infection begins when a reduviid insect host (kissing

bug) takes a blood meal from a warm blooded animal.

• During the bloodmeal the insect vector excretes parasites

in its feces onto the mammalian host.

• These infectious metacyclic trypomastigotes are are

morphologically distinct from epimastigotes and are

primed and ready to enter their new host using the bite

wound or a nearby mucous membrane.

• The parasites then make their way to the bloodstream

and invade any nucleated host cell, thus initiating a new

phase of the infection process.

• Although they enter via a phagocytic-like mechanism,

parasites soon exit from these lysosome-like structures

and begin replicating as flagellar-less amastigotes.

• Once host cell resources are depleted they reconvert to

their flagellated form and lyse the host cell.

• These released parasites are known as trypomastigotes

and are able to perpetuate subsequent rounds of host cell

infection, replication and lysis

Chagas disease Pathology - acute stage

Acute form: 7-10 days incubation period. High

numbers of parasites in the blood or lymphatic

vessels. Parasites may invade almost all nucleated

cell types, preferentially muscle fiber

• Depending on the location and manner of the

initial infection (e.g. mucous membranes or bite

wounds) you have inflammation resulting from

the initial spread of infection.

• During this period, intracellular amastigotes

replicate and begin the process of lysing

infected cells and rapidly spreading.

• This results in initially localized infection and

inflammation resulting in the pathology noted in

Chagoma’s and Romana Sign.

Chagas disease Pathology - indeterminate stage

• During this phase of infection parasites

disappear from the blood.

• Nests of amastigotes are generally found in

very low numbers in muscle tissue.

• Antibodies to T. cruzi antigens can be detected.

• Patients are generally asymptomatic.

• Most patients enter chronic phase within 5 to

15 years.

Chagas disease Pathology - chronic stage

• The chronic stage which appears after a silent

period that may last several years.

• The lesions of the chronic phase irreversibly

affect internal organs namely the heart,

oesophagus and colon and the peripheral

nervous system.

• A slow/steady destruction of smooth and

cardiac muscle and parasympathetic ganglia,

due to the combined effects of cellular

destruction and inflammation, results in a

weakening of the ability to carry out normal

functions.

• Results in Myocardiopathy and Megaviscerae

of the smooth muscle organs of the body

• These autopsy images demonstrate T. cruzi

induced megacolon showing massive dilatation

and elongation of the rectosigmoid and distal

descending colon.

• Colonic involvement leads to prolonged

constipation, abdominal pain and fecaloma.

• Increased risk of volvulus (intestinal knotting)

and consequent bowel ischemia (like a heart

attack for the intestine – cut off of blood flow

• These Barium swallow examinations show

widening of the esophagus (top arrows).

• Dilated tortuous esophagus (top arrowheads).

• Widening of the esophagus with tertiary wave

patterns (bottom left arrowheads) due to

aberrant and non-propulsive peristalsis.

• Typical ‘rat-tail’ sign in the distal portion of the

esophagus (bottom right arrows) connecting to

the stomach (s).

Chagas disease - other facts

• Individuals with HIV and chronic T.

cruzi infection may experience a

reactivation.

• This is most commonly manifested by

meningoencephalitis.

• A reactivation myocarditis is the

second most common manifestation

Chagas Disease Diagnoses

Direct Detection:

o Microscopic detection of parasites in blood.

o Hemoculture (growing parasites) from blood.

o Xenodiagnosis (no longer used) where reduviid

insects feed directly on a person followed by

microscopic evaluation of parasites in the insect

mid/hindgut.

• Serodiagnostics:

o Multiple testing methods exist in a variety of

formats (hemaglutination, immunofluorescence

microscopy, ELISA, Western Blot etc.)

o These tests are based on the presence of

antibodies (made by B-cells) that recognize

antigens (typically parasite proteins).

• Molecular Diagnosis:

o By extracting DNA from the blood and performing

PCR (polymerase chain reaction) you can identify

the presence of extremely rare parasites

(extremely sensitive but expensive)

Chagas Disease treatment - Benznidazole (Radanil)

Orally administered frontline drug in the treatment of

Chagas Disease in use since 1971.

• Is part of the nitroimidazole drug family and works by

the production of free radicals.

• Has a dramatic effect on the intracellular amastigote

stage causing parasite lysis.

• Frequent toxicity (allergic skin reactions, peripheral

neuropathy, leucopaenia, convulsions).

• Partially effective in acute stage (strain variability) but

inactive in chronic stage of the disease.

• Prolonged treatment recommended (up to 60 days)

(5-7 mg/kg per day) but side effects make

compliance difficult.

Chagas Disease treatment - Nifurtimox (Lampit)

Orally administered nitro-anion radical metabolite

that acts through the production of toxic free

radicals. Affects DNA stability.

• Used if Benznidazole fails.

• The parasite is more sensitive due to deficiency in

their antioxidant enzyme repertoire.

• Toxicity is a problem (anorexia, nausea, vomiting,

peripheral neuropathy, skin reactions, CNS effects).

• Only partially effective in acute stage (strain

variability) and inactive in the chronic stage of

disease.

• Requires prolonged treatment (up to 90 days) 98-

10 mg/Kg per day).

• Approved for use in the US in 2020.

• Not available in Canada.

Chagas Disease:

Control and Prevention

• insecticide spraying

• education

• blood supply monitoring

• 
  

Trypanosoma brucei

• hemoflagellate: lives in host circulatory system

• transmitted by blood feeding arthropod

• has many of the same DNA (kDNA)

• Causes African sleeping sickness / human african trypanosomiasis

• salivarian

T. b. gambiense

Reservoir is primarily humans (Human African Trypanosomiasis (HAT)

Causes a chronic disease that

can remain asymptomatic or mild for months or even

years before reaching the central nervous system

T. b. rhodesiense

Zoonotic disease- wild game and cattle reservoir (Nagana)

Causes an acute disease that

rapidly attacks the body, advancing from the blood

stage to the central nervous system within week

Robert Michael Forde (1861–1948)

had the first observation of trypanosomes

T. Brucei Life Cycle

• The female tse tse fly produces larvae, one

  at a time. The egg hatches within the female

  and the larva develops in the female. During

  her life-span a female can only produce a

  maximum of 8-10 offspring.

  • The larva is laid onto the ground

  (larviposition). The female finds a suitable

  place to lay the larva. In damp areas females

  tend not to concentrate their larviposition in

  particular areas. In dry areas larviposition

  takes place mainly in well-shaded spots.

  • The freshly-laid free-living larva, does not

  need to feed and burrows into the soil where

  its skin hardens and blackens into a

  puparium and within the puparium, pupation

  and metamorphosis take place.

T. Brucei Pathology Stage 1

Trypanosomes multiply in the tissue around the

initial bite site.

• This often results in a characteristic local

inflammation known as the trypanosomal “chancre”.

• Usually this is not painful.

• From there they enter the blood and lymphatic

system and begin to disseminate throughout the

circulatory system.

• Enlargement of the lymphatic glands along the

  posterior cervical lymph node chain (especially in

  the posterior triangle of the neck) can be an early

  sign of the disease.

  • This is known as Winterbottom sign, but is not as

  common in T. rhodesiense infection.

  • Aspiration of swollen gland often reveals parasites

T. Brucei Pathology Stage 2

• Symptoms at this stage are rather mild in

gambiense but can be severe in rhodesiense

with an often fatal outcome.

• After 1-2 week period of asymptomatic

incubation, parasites invade blood leading to

fever and headache.

• Once parasites enter blood stream fever sets

in (low and irregular in gambiense and high

and periodic in rhodesiense.

• General toxic symptoms include headache,

facial edema, nausea and vomiting, back and

bone pain.

• The second stage of trypanosomiasis is

characterized by progressive anemia and cachexia.

• Both features are primarily due to extremely high

serum levels of TNFalpha (pro-inflammatory cytokine).

• TNFalpha (tumor necrosis factor also known as

cachexin) was isolated as a factor inducing wasting in

Nagana.

T. Brucei Pathology advanced stages

• In later stages of infection parasites pass the blood

brain barrier and infect the CNS.

• Presence of parasites leads to meningo-

encephalitis with progressive neurological

involvement, which ultimately ends in coma

(sleeping sickness).

• Untreated trypanosomiasis is always fatal

• The progressive encephalitis can cause severe

dementia with sometimes aggressive behavior.

• Disease progression especially CNS invasion is

much faster in rhodesiense.

• Gambiense can take a year or two while

rhodesiense usually passes the blood brain barrier

within a month.

T. Brucei Diagnosis

• At later stages of the disease there might be no

detectable parasites in the blood.

• Lumbar puncture and microscopic identification of

parasites and detection of elevated protein levels

in the CSF are needed for diagnosis.

• Establishment of CNS infection is important for

therapy decisions

OLD T. b. rhodesiense Treatment

Suramin (I.V. administration); only works in the early stage; multiple doses over about 3 weeks

OLD T. b. gambiense treatment

Pentamidine (intramural injection); only works in early stages

OLD T. Brucei Treatment

Melarsoprol(i.v. infusion); works on late stages; works on both t.b. gambiense and t.b. rhodesiense; Severe toxicity (death 5-10%; encephalopathy 10%; pruritus, cardiac failure

T. Brucei Diagnoses

• Trypansosomiasis is best diagnosed by

demonstration of parasites in blood smears,

lymph node exudates or spinal fluid.

• Parasitemia can be very low at times

resulting in false negative results. It is more

likely to find parasites during symptomatic

periods.

• Several techniques can be used to enrich

rare parasites in blood sample

(centrifugation, chromatography).

• The Card Agglutination Test for

Trypanosomiasis (CATT). Sero-diagnostic

test to identify infected individuals

NEW T. Brucei Treatments - Fexinidazole

• The first treatment for sleeping sickness that relies on pills alone

was approved in 2018.

• Simplified treatment: Oral treatment that can be taken at home. The

new drug, fexinidazole, cures all stages of the disease within 10

days.

• Previously, everyone with the parasites found in a blood test also

had to undergo a spinal tap to see if the parasites had reached their

brains.

• The drug was created in the 1980s by Hoechst, a German drug

company, but later abandoned.

NEW T. Brucei Treatments - Acoziborole

• the first single-dose oral treatment for both stages of T.b.

  gambiense sleeping sickness, the most common form of the diseas

• targets CPSF so mRNA processing will not

  occur leading to loss of protein production

T. Brucei Life Cycle

Trypanosoma brucei

• T. brucei is a salivarian kinetoplastid transmitted by the Tsetse fly.

• Two major strains infect humans: T. b. gambiense and T. b. rhodesiense

• If left untreated it is almost always fatal.

• Surveillance and new drug treatments have made this less of an issue for humans but the economic costs for

cattle production due to Nagana is still a major problem.

• The Tsetse fly is viviparous and gives birth to live young and transmits T. brucei via the salivary glands during a

bloodmeal.

• New oral treatment with fexinidazole, cures all stages of the disease within 10 days.

• T. brucei (like all kinetoplastids) have unique mechanisms regulating gene expression.

• They express genes as polycistronic transcripts that are each individually trans-spliced and polyadenylated.

• Stability, and thus likelihood of translation, is dictated by the presence of life-cycle specific RNA binding proteins.

• Parasites exhibit interesting behaviors like social motility the function and mechanistic basis for which remain to be

worked out.

• The social behavior known as “Quorum Sensing” was recently worked out and is the mechanisms parasites uses

to regulate their numbers in a host.

• As part of their immune evasion strategy T. brucei continually changes its surface coat through a mechanism

known as “Antigenic Variation”.

• Antigenic variation involved the selective expression of different coat proteins that change in order to escape

antibody mediated clearance in the host.

What are the replicative stages of T. brucei called?

Long slender bloodstream form, Procyclic form

What are the major strains of T. brucei that infect people?

T. b. gambiense, T. b. rhodesiense

Who discovered T. brucei and demonstrated transmission?

Sir David Bruce

How is T. brucei gene expression distinct?

lacks obvious promoters and can even use RNA polymerase I (Pol I) to express certain protein-coding genes such as VSG (variant surface glycoprotein) and procyclin, which is highly unusual for eukaryotes

What is trans-splicing?

the process by which a Spliced Leader (SL) RNA, a 39-nucleotide mini-exon, is added to the 5′ end of every mRNA in T. brucei. This is necessary because the parasite transcribes genes as long polycistronic RNAs and must convert them into individual mature mRNAs.

Why are RNA binding proteins so important in T. brucei?

RNA-binding proteins are crucial in Trypanosoma brucei because the parasite lacks extensive transcriptional regulation of individual genes and therefore relies primarily on post-transcriptional regulation.