Parasites Flashcards
Entamoeba Histolytica
- Motile amoeba that uses pseudopodia to propel
- Fecal-oral route : common in tropics where sanitation is poor and readily spread in freshwater
- Disease in around 10% (90% asymptomatic?)
- Invasive in large intestine Dysentery (bloody diarrhoea)
- Spreads through blood to other organs : liver, lung , brain injury
- Mortality: 50,000 - 100,000 annually
What illness does E. histolytica cause
Intestinal amoebiasis by E. histolytica:
* Clinical outcomes:
90% asymptomatic
Mild symptomatic (non-invasive) infection
Amoebic colitis (inflamed colon lining)
Acute invasive amoebic dysentery (perforated colon)
Abscesses of the liver (amoebic liver abscess), lungs,
heart, brain, kidney etc.
* Diagnosis
o Stool examination (trophozoites and cysts)
o Colonoscopy
* Treatment: metronidazole+tinidazole (effective against many anaerobic parasites) why still so deadly?
Why is E. histolytica still claiming 100k lives per year?
- Delayed diagnosis and treatment: many are asymptomatic or have generic
clinical symptoms (diarrhea) parasite has more time to cause extensive
damage! - Lack of access to healthcare/medication: only seek medical care when
symptoms are severe, may not have enough money for medications - Malnutrition and co-infection: patients more susceptible to other
infections and increased risk of complications - Drug resistance: particularly in areas where the disease is endemic
How to Diagnose E. Histolytica
NOTE: Cysts of Entamoeba dispar (non pathogenic) are INDISTINGUISHABLE!!!
E. Histolytica II (Techlab): monoclonal antibody-based ELISA for the rapid detection of E. histolytica adhesin in stool
Balantidium coli
- Only known pathogenic ciliate (propelled by rows of cilia that beat with synchronized wavelike motion)
- Large size (up to 0.2mm) and forms cysts
- Diagnosis: microscopy
- Pigs are natural reservoirs
- Only affects immunocompromised (vomitting,
diarrhoea, dysentery) - Treatment: metronidazole or tetracycline
- Trophs cause disease
- Cysts are infective
Trichomonas
Trichomonas vaginalis
- Resides in genitourinary tract, not associated with mortality
- STD: Trichomoniasis
- No cysts
- Disease:
- 50% are asymptomatic
- Mainly in females vaginitis (tender with greenish yellow
discharge)
- Treatment of both partners: metronidazole or tinidazole
- Diagnosis: InPouchTV, staining, wet-mount
T. vaginalis
* No Cysts!
* Obligate
endoparasite
Giardia
- Most common flagellate in clinical specimens
- Heart/face-like shape
- Forms cysts
- Ventral sucking disc, non-invasive
- Disease: malabsorption syndrome, diarrhoea (fatty stools)
- Mortality: 4600/year
- Entero-Test capsule (String test): to sample duodenal contents
- RDTs (ImmunoCardSTAT! Cryptosporidium/Giardia)
G. lamblia
* Trophs cause disease
* Cysts are infective
T. Cruzi
- T. cruzi (American)
Chagas disease
Vector: Triatomine (kissing bug) faeces+scratching
South America, mortality: 10k/year
Acute infection >90% treatment success, but may go unnoticed
(Romana’s sign : self-resolving localized swelling)
Chronic infection (30yrs++) internal organs affected (smooth muscles of
heart, oesophagus & colon, also peripheral nervous sys), leads to organ failure (heart enlargement/rupture) and death, treatment <10%
T. Brucei
- T. brucei (African)
African sleeping disease, mortality: 7-9k/yr
Vector: tsetse fly
T.b. rhodesiense (S+E Africa): fast onset (animal reservoir)
T.b.gambiense (Cen+W Africa): slow onset (human reservoir)
Crosses blood-brain barrier
Evades host immune system
T. Brucei Diagnosis
Diagnosis of T. brucei (African)
* African trypanosomiasis has two stages:
– 1st stage (acute): parasite found in blood
– 2nd stage: crosses BBB and invades CNS
* T. b. rhodesiense (fast onset: weeks to mths) parasites easily be found in blood, lymph node fluid or in biopsy of the painful sores (chancre)
* T. b. gambiense (slow onset: mths to yrs) difficult to detect in blood and requires microscopic examination of lymph node aspirate
* Untreated infection with either subspecies leads to coma and death
Apicomplexa:
- Possess specialized invasive stages with apical complexes
- No specific organelles for movement yet has “gliding motility”
(reliant on adhesions and small static myosin motors) - Flagella only in specific sexual stages
- Has non-motile stages (merozoite or intracellular stages)
- Obligate endoparasites
- Some are intracellular: Plasmodium (mortality >400k/yr),
Toxoplasma (1.5k/yr) - Some extracellular: Cryptosporidium (48k/yr)
Plasmodium spp.
- Causes malaria, the most devastating parasitic disease
- At risk: 92 countries and > 3.4 billion ppl
- Malaria cases/deaths lower by 18%/28% since 2010
- Annually: 241M new infections & 627k deaths (2021 WHO estimates, c.f. dengue: 390M infected & 21k deaths)
- 95% cases and 96% deaths in Sub-Saharan Africa, mostly children <5yr (80% of all deaths)
- Increasing burden due to: parasite drug resistance (practical), insecticide resistance, civil unrest/travel/population growth, global warming
- Six clinical species: P. falciparum, P. vivax, P. ovale, P. malariae and zoonotic P. knowlesi and P. cyanomolgi
- Vector: Anopheles mosquito
- Symptoms: bouts of chills and fever, vomitting, anemia, renal damage, ketoacidosis, respiratory distress, cerebral malaria, placental malaria…
Malaria, microscopy
- Microscopy pros: simple, cheap, sensitive, quantitative
- Microscopy cons: slow, tedious, requires training & microscope, risk of blood borne pathogens (HepB & HIV)
- Thick smear: thick/dense blood film UNFIXED RBC lysis/ parasite staining with Giemsa water rinse dry observe thick “cover” a much larger quantity of blood per field RBC lysis get rid of opaque background RBCs but ugly!
- Thin smear: thin/sparse blood film FIXED staining with Giemsa water rinse dry observe fixed much better morphology and can differentiate species
Malaria, fluorescent stains
Acridine orange (nucleic acid stain)
- only infected RBCs will fluoresce
- QBC (centrifugation + fluorescence) sensitive, fast, cannot speciate/quantify, expensive, cannot store, fragile capillaries
Malaria, antigen-capture
Antigen Capture
- Rapid Diagnostic Test:
* Requires blood drawing and liquid reagents
* Easy to perform and interpret (10-20min)
* Expensive (USD3-5 per test)
- Component #1: lactate dehydrogenase (all spp)
- Component #2: histidine-rich protein-2 (only Pf)
RDT test
- Mix patient’s blood with a lysing agent to rupture RBCs & release more parasite protein.
- Dye-labelled antibody, specific for target antigen, is present on the lower end of strip. Antibody to same antigen also bound to the Test band. Antibody specific for the free antibody is bound at the control line.
- Blood and buffer are mixed with labelled antibody and are drawn up the strip across the lines of bound antibody.
- If antigen is present, some labelled antibody-antigen complex will be trapped and accumulate on the test line. Excess-labelled antibody is trapped and accumulates on the control line. A visible control line indicates that labelled antibody has traversed the full length of the strip, past the test line, and that at least some free antibody remains conjugated to the dye and that some of the capturing properties of the antibodies remain intact.
- The intensity of the Test band will vary with the amount of antigen present (at least at low parasite densities), as this will determine the amount of dye particles which will accumulate on the line. The control band intensity may decrease at higher parasite densities, as much of the labelled antibody will have been captured by the test band before reaching the control.
Malaria, Molecular Approaches
Molecular
- Essential for eradication agenda: pre-elimination challenges include asymptomatic patients, very low parasitemias & gametocytes
- PCR: Primers to parasite DNA, DNA extraction, PCR amplification, run gel to visualize
- Variants: quantitative PCR, loop mediated isothermal amplification(LAMP) etc.
- Field-adapted methods w/o thermocycler or gel!
- Also, 18S rRNA seq to distinguish 5 human-infecting species
- Pros: Semi-quantitative, species-specific, sexual vs asexual stages
- Cons: Equipment/electricity, slow, trained technicians, costs
- Excellent sensitivity… But is that good enough for eradication?
Singapore and Malaria Today
- Malaria-free since 1982: fogging, draining swamps, surveillance/treatment
- Recent outbreaks: 2003, 2009 (Pf, Pv and Pk)
- Anopheles vector still present but have active surveillance (reportable disease) and active tackling of disease (reduce possibility of transmission)
- 80.5% of wild long-tailed macaques are natural reservoir for P. knowlesi (47.5%) and P. cynomolgi (71.5%)
- Army boys stopped taking antimalarial pills
Malaria and past Singapore
- Malaria became prevalent in Singapore from 1880
- 1907-1910 about 2,000 deaths annually
- In 1911 peaked at 2930 deaths, with 127 people dead within a week in June
- anti-malaria committee was set up by Sir Malcolm Watson antimalarial drainage system & oiling program huge success!
- Japanese occupation (1942-1945) drains not maintained (2,771 deaths in 1945)
- Control measures intensified post-war no malaria from 1955 to 1963
- Fuyong estate outbreak in 1964: 33 cases, P. vivax malaria as a notifiable disease
- 1969 outbreak at P. Tekong with 25 cases, CQR arrival
- 1975 Whampoa-Kallang outbreak: 82 cases, P. vivax (some Pf also) imported malaria + vectors + no immunity = high local transmission
- New control strategy in 1975 efficiently identifying focus of transmission (active & passive case detection, mass blood surveys) for aggressive epidemic control (weekly oiling, spraying of workers quarters, vector virtually eradicated)
- Sustained improvements between 1975-1982
- No sustained local transmission 22 Nov 1982 declared malaria free!
Why is malaria detection so important in the big picture?
Progression of the malaria eradication war:
* Much resources invested thus far but reducing ROI
* No second-chances due to financial/political/cultural mistrust
* Rebound malaria – waning immunity, worse outcome if re-infected
* Outclassed by growing parasite/vector resistance
Eradication requires detection of hidden reservoirs (ultra low
parasitemias) and low level transmission (asymptomatic)!
Singapore situated near the “hotspot” for malaria drug- resistance development but with only 100-300 imported cases a year, should we care?
* Anopheles vector may adapt to our “city in garden”
* We have no immunity any infection is serious
* Diabetes & obesity are risk factors for severe malaria
* Limited alternatives to ACTs
Switching
Antigenic variation: periodic switching of surface molecules
- New Ag expressed on parasites (switch rate of 0.1% per division)
- Not recognized by current wave of antibodies
- Allows persistent and eventually lethal infection
12-15 nm thick variant surface glycoprotein (VSG) surface coat
Conserved anchor sequence buried in plasma membrane
VSG is immunogenic and Abs clear parasites with same VSG
10 7 copies of VSG genes per cell (10% of genome)
But mostly frame-shift or truncated pseudogenes
>1000 distinct VSG genes
3 distinct pools of VSG genes: >1000 at sub-telomeric ends, 200 in mini-chromosomes and 30-40 in telomeric expression sites
Allelic exclusion: Only ONE VSG gene expressed at any time
Switch mechanism: (1) in situ switch, (2) telomere exchange, (3) gene conversion
Tackling Switching
Exploiting this knowledge…
1. Inhibit switching mechanism: current batch of parasites cleared with no “switched” parasites remaining.
2. Dysregulate switching: results in multiple VSG being expressed simultaneously leading to increased likelihood of parasite clearance.
But this remains very challenging to do scientifically & therapeutically!
Why Malaria resistance rising
Patients from the Pailin (Western Cambodia) require longer treatment with either artesunate(plan F) (shown in red) or artesunate– mefloquine (shown in purple) therapy than
the parasites from the Wang Pha (Western Thailand, shown in
yellow and blue) to be cleared.
- foreign workers on the borders seek cheaper alternatives/don’t follow up to treatment
Basis of resistance
Microarray transcriptomics to ID resistance mechanism
Rings and trophozoites stages have reduced expression of many basic metabolic and cellular pathways suggesting slower growth/maturation -> quiescent stage!
In schizonts stage there is increased expression of all genes associated with protein metabolism -> increased protein synthesis in later stage to compensate!
- K13 mutation observed via sequencing(fwd genetics) and using genetic modification, “survival rates decreased from 13 to 49% to 0.3 to 2.4% after the removal of K13 mutations. Conversely, survival rates in wild-type parasites increased from ≤0.6% to 2 to 29% after the insertion of K13 mutations”(reverse genetics)
