Diagnosis Of Malaria Flashcards
Global Impact of malaria - WHO World Malaria Report
- 3.3 billion people live in areas at risk of malaria transmission in 106 countries and territories
- 2015 WHO - 219 million clinical episodes and 435000 deaths
- African Region (92%), followed by the South-East Asia region (6%) and the Eastern Mediterranean Region (2%)
- In 2017, malaria killed an estimate 266,000 under 5s globally
- 61% of malaria deaths are of under five years old
Phylum - Apicomplexa
Theileria - animal parasite (East coast Fever in cows). Babesia (certain species can infect humans)
Toxoplasma - human pathogen
Cats definite host - problematic for pregnant women and immune compromised
6 species of parasites that cause malaria in humans
- Plasmodium falciparum - most widespread - causes majority of fatalities
- Plasmodium vivax - widespread - few fatalities - dormant liver stage
- Plasmodium Knowlesi - Zoonotic reservoir Asia
- Plasmodium ovale Curtis I and allikeri) -few fatalities (Africa, Asia, Oceania) - dormant liver stage
- Plasmodium malariae - Africa, Asia, S.America
Diagnosis of Malaria:
- Giemsa stained blood smear - considered gold standard
- requires trained and experiences user
- Can diagnose different parasitic species
Diagnosis
- Presumptive diagnosis: no longer recommended in most regions
- leads to misuse of drugs and in-necessarily high drug pressure in population - implications for spread of drug resistance
- Current aim - diagnose malaria before treatment
The HRP2 rapid diagnostic test
- HRP2 is synthesised in the parasite and exported into the RBC
- Released into the blood during parasite egress (going in or coming out)
- Reaches very high levels in infected blood (approx. 100 micrograms/ml - this is very high) - levels likely correlate with disease severity
- function of the protein is unclear
- This protein is used in many RApid diagnosis tests (tests detect the parasite protein not he antibodies against the protein)
Exported parasite proteins are released into the blood each time parasites egress
Step 1: add drop of blood and a drop of buffer
- Nitrocellulose strip (inside RDT)
- Blood and buffer will mix
- buffer contains detergent that will break open blood cells and parasites and release more protein (including HRP2)
- The strip also contains a dye labelled antibody that recognises HRP2 (usually gold labelled) - this becomes mixed with lyses blood and will bind to HRP2
- Lysed blood + anti-HRP2:Hrp2 start to soak into the membrane and move in this direction (towards the results window)
key point: strip of membrane has another antibody that also recognises HFRP2 - this antibody is stuck to the membrane and does not move
- A malaria positive sample will result in a pink band on the RDT in the ‘test’ window. This is due to specific trapping of antiHRP2: gold particles on the strip
- key point* - strip of membrane also has another antibody immobilised (control). This antibody wil recognise the anti-HRP2 antibody. This is in the control window.
if the test is functional this should also give a pink band in the control window
Check notes for diagram
Test result
- positive test - pink band present in test and control window
- Negative test - pink band in control window only - HRP2 was undetectable
- NO BAND - neither test nor control window has a band - this means that the test did not work and the diagnosis is unknown (test is expired or damaged in some way)
Rapid diagnostic tests
- Important diagnostic tool
- cheap and fast
- prevent unnecessary use of anti malarial drugs
- HRP2 RDT use widespread to detect P.falciparum - generally very successful. Only detects P.falciparum (not Knowlesi, vivax, ovale, malariae)
- significant numbers of HRP2 negative parasites in S.America and now in Africa
Current status: The RTS,S vaccine
• First malaria vaccine to go beyond phase III clinical
trials (results published 2015).
• Currently undergoing large trials in Africa.
• Based on the CS protein (Circumsporozoite protein
CS protein is on the surface of sporozites
RTS,S in phase III trials
Protection is partial and wanes over time. 5-17 month children – vaccinated 0-1-2 months
-51% reduction in clinical malaria over the first year.
-26% reduction in clinical malaria over 48 months.
48 months - 39% (four dose vaccination).
Currently undergoing large pilot studies in Kenya,
Malawi, and Ghana.
Some indications that sequence variation in CS
protein may be problematic
R21 vaccine
- RTS,S – only approx. 20% of the Hepatitis B
surface antigen is coupled to CS.
• R21 – closer to 100%
Other vaccine approaches?
• Attenuated sporozoites are very promising
vaccine candidates
• Attenuation by irradiation – this has worked in
trials performed in 1970s.
• Genetic attenuation – very promising in
mouse models of malaria
Aim
• Develop a malaria parasite that does not
cause disease but can illicit an immune
response without causing malaria.
• i.e. a live attenuated liver stage vaccine
Making an attenuated liver stage malaria
vaccine using the mouse model system
(Plasmodium berghei)
- Find a gene that is essential for parasite growth in the liver - UIS3
- Knockout the gene
- Check that the parasite can ‘make it to the liver’ but no further -i.e does not actually cause malaria
- Test whether mice that have been infected with attenuated uis3 - parasites are protected against subsequent infections with wildtype parasites
Can UIS3 knockout parasites get
through the mosquito stage?
-Allow mosquitos to feed on mice infected with the uis3- parasites.
-Check to see if parasites are able to get to the
mosquito salivary gland – they can
Allow mosquitos to bite uninfected
mice
Parasites can invade hepatocytes
In vivo (green spots are parasites in the liver – visualised by immunofluorescence).
Parasites can invade hepatocytes but cannot mature
UIS3- parasites do not cause malaria
UIS3- parasites do not cause malaria
Key point: even though the mice were infected with uis3- parasites, they do not get malaria.
Summary
• 1. The gene for UIS3 is deleted in Plasmodium
berghei.
• 2. UIS3- parasites are taken up by the mosquito + form sporozoites in the salivary gland.
• 3. They invade liver cells but never mature and do not cause malaria.
• Can these parasites be used as a malaria vaccine -
YES??
Conclusion
- Genetically attenuated parasites that fail to
mature in the liver may be used as a vaccine in
mice.
• Will this work in people – currently testing
genetically modified parasites with three different genes deleted.
• Is this practical – can we make enough sporozoites and can the attenuated sporozoites be preserved
Transmission blocking vaccines
Antibodies target proteins on the surface of gametes
Key points relating to transmission
blocking vaccines
• Parasites replicate asexually in the blood
• Some of these parasites will differentiate to
form sexual stages (gametocytes).
• There are male and female gametocytes.
• In the mosquito midgut these differentiate
into gametes that egress from red blood cells
and must fuse to form a zygote in order to
continue the lifecycle.
