Malaria

Question 1

Malaria: An Introduction

Answer 1

tropical parasitic disease - distributed in tropical and subtropical zones and imported into the UK
caused by protozoan parasites which infect red blood cells
often transmitted by the bite of an infected Anopheles mosquito
causes much morbidity and mortality around the world

Question 2

Malaria modes of transmission

Answer 2

Mosquito bites (most cases)
Via the placenta
Blood transfusions
Transplantation
Contaminated equipment
Via routes associated with air travel

Question 3

Species of malaria parasites that infect humans

Answer 3

Plasmodium falciparum
Plasmodium vivax
Plasmodium ovale
Plasmodium malariae
Plasmodium knowlesi

Question 4

What makes malaria a global problem

Answer 4

more prevalent in some areas of the world than others due to obvious reasons such as climate and abundance of mosquitoes
Other factors include availability of funding and the degree of cooperation of governments with charities and organisations such as WHO
Education and preventative measures taken (e.g. supply of insecticide treated nets, indoor insecticide sprays, covering of bare skin)
Access and adherence to preventative measures
Accurate diagnosis
Access to treatment

Question 5

Malaria Vaccines

Answer 5

• The first malaria vaccine, RTS,S, was recommended by WHO to prevent malaria in children in October 2021.
• December 2023 the R21/Matrix-MTM malaria vaccine developed by the University of Oxford and the Serum Institute of India, leveraging Novavax’s adjuvant technology, has been awarded prequalification status by the World Health Organization (WHO).

Question 6

Malaria Cycles

Answer 6

The malaria parasite life cycle involves two hosts. During a blood meal, a malaria-infected femaleAnophelesmosquito
1 inoculates sporozoites into the human host
2 Sporozoites infect liver cells
3 mature into schizonts ,
4 which rupture and release merozoites (Of note, inP. vivaxandP. ovalea dormant stage [hypnozoites] can persist in the liver (if untreated) and cause relapses by invading the bloodstream weeks, or even years later.) After this initial replication in the liver (exo-erythrocytic schizogony(A) ), the parasites undergo asexual multiplication in the erythrocytes (erythrocytic schizogony (B)).
5. Merozoites infect red blood cells.
6. The ring stage trophozoites mature into schizonts, which rupture releasing merozoites
7. Some parasites differentiate into sexual erythrocytic stages (gametocytes)
8. Blood stage parasites are responsible for the clinical manifestations of the disease. The gametocytes, male (microgametocytes) and female (macrogametocytes), are ingested by anAnophelesmosquito during a blood meal

Question 7

Schematic model of steps on Pf meozotie invasion

Answer 7

1-Merozoite in bloodstream attaches to receptor on RBC surface.
2-Merozoite re-orientates so that the apical pole is directed towards the RBC surface ( Merozoite attachment to receptor on RBC surface.)
3-Tight junction forms between merozoite and RBC surface accompanied by initial deformation of RBC membrane
4-Entry of merozoite coinciding with formation of parasitophorous vacuole.
5-Closure of RBC and parasitophorous vacuole membranes.
6-Junction between RBC membrane and parasitophorousvacuole severed releasing merozoite into the cell where it transforms into a young trophozoite.

P. falciparum binds to glycophorins A, B, and C.
* P. vivax and P. knowlesi bind to the Duffy antigen.
* The receptors for P. malariae and P. ovale are unknown.

7- Some parasites differentiate into sexual erythrocytic stages (gametocytes)
8- Blood stage parasites are responsible for the clinical manifestations of the disease. The gametocytes, male (microgametocytes) and female (macrogametocytes), are ingested by anAnophelesmosquito during a blood meal

9- The parasites’ multiplication in the mosquito is known as the sporogonic cycle(C). While in the mosquito’s stomach, the microgametes penetrate the macrogametes generating zygotes.

10- The zygotes in turn become motile and elongated (ookinetes)
11- which invade the midgut wall of the mosquito where they develop into oocysts
12- The oocysts grow, rupture, and release sporozoites ,which make their way to the mosquito’s salivary glands. Inoculation of the sporozoites
1- into a new human host perpetuates the malaria life cycle. Source CDC

Question 8

Malaria in the UK - Common groups presenting with malaria

Answer 8

New entrant
Foreign student studying in the UK
Foreign visitor ill whilst in the UK
British citizen who has been working abroad
Armed services
Business travellers
ratio of malaria in UK residents visiting friends and relatives compared with malaria cases acquired in holiday travellers is around 10:1

Question 9

Malaria in the UK - Considerations

Answer 9

Access to medical guidance before travel
Adherence to medical guidance
Awareness of risk
Familiarity with destination
Targeting this group along with their healthcare providers should be considered a priority

Question 10

what determines individual risk?

Answer 10

Being unaware of malaria risk areas
Amount of malaria in the area to be visited
Time of year
Type of parasite(s) present in the area
Preventative measures taken
Immunity or lack of immunity
Appropriate travel advice is very important

Question 11

Immunity in Malaria -

Answer 11

Most pronounced in P.falciparum disease
In areas of high transmission, if a child survives to 5-6 years he or she is likely to have a high degree of immunity
Immunity wanes over a few years without regular exposure
Immunity also wanes during pregnancy
Pregnant women and young children are most at risk

Question 12

Malaria Symptoms

Answer 12

Fever
Chills
Headache
Flu-like symptoms
Muscle aches
Fatigue
Anaemia
Diarrhoea
Vomiting
Cough
Symptoms of cerebral malaria in P. falciparum

Question 13

Lab Diagnosis Tests for Malaria

Answer 13

Full blood count (FBC): non specific and non diagnostic:
Rapid diagnostic tests- Immunochromatography for detection of malarial antigens
Thin and thick blood films
Quantitative buffy coat
Polymerase chain reaction (usually only used in reference laboratories)

Question 14

Malaria: Haematological changes observed from FBC results

Answer 14

• Abnormalities observed from FBC results are non-specific and not diagnostic
• Anaemia seen due to red cell rupture (haemolytic anaemia) and impaired haemopoiesis as well as the removal of parasitized cells via the RE system
• Thrombocytopenia (due to platelet pooling/clearance in the spleen and reduced platelet lifespan due to immune responses)
• WBC count is often normal but may be raised in severe disease: Abnormalities may be observed on the white cell scattergram
• Further tests are needed to confirm that malaria is present.

Question 15

Lateral-flow immunochromatographic techniques

Answer 15

• Variety of formats e.g. dipsticks, strips, cards, wells, and cassettes All the same basic principle.
• Antibodies used that target proteins specific for P. falciparum, such as HRP-2 (PfHRP-2), or Panmalaria proteins present in all species, such as Plasmodium aldolase or Plasmodium lactate dehydrogenase (pLDH), which are enzymes in the glycolytic pathway of Plasmodium species. - P. falciparum LDH (PfLDH)-specific antibodies are also available.
• Blood added to a buffered solution containing a haemolysing agent and one or more antibodies against malaria antigens that are labelled with a marker which can be visualized by the naked eye, such as colloidal gold.
• Antibodies complex with their target antigens if present, then migrate by capillary action along the test strip until they encounter separate immobilized capture antibodies directed against each target antigen in specific sections of the strip.
• Further antibody directed against the labelled antibody, which is the final antibody in the sequence, acting as a control to indicate that the procedure itself has worked. The strip is washed with buffer to remove haemoglobin.

Question 16

NOW malaria kit (BinaxNOW) (rapid diagnostic?

Answer 16

• Rapid immunodiagnostic assay
• Uses two antibodies immobilised on a test strip
• One antibody is specific for histidine-rich protein II of P.falciparum
• Oher is specific for an antigen common to all forms of malaria that infect man (pLDH)
• Colour formation immobilised area after sample and a reagent is added to test strip helps scientist to get an idea of a parasite that may be present
• Control band included
• Only takes about 15 minutes

Question 17

Optimal- IT test ( rapid diagnostic?

Answer 17

• Monoclonal antibodies against metabolic enzyme, parasite lactate dehydrogenase are used to detect presence of malaria
• One antibody specific for P. falciparum
• Other is a pan-specific antibody
• PLDH reacts with specific antibodies against plasmodium falciparum and/or plasmodium spp. inthe presence of plasmodium spp.

Question 18

Rapid malaria diagnostic kits advantages:

Answer 18

Cheap & Quick result
Easy to perform (this helps inexperienced staff make an accurate diagnosis)
Can be used in settings away from the laboratory

Question 19

Rapid malaria diagnostic kits disadvantages:

Answer 19

False positives
False negatives

cross-reacting antibodies such as rheumatoid factor;
pfHRP-2 can cross-react with non-falciparum malaria;
PfHRP-2 can persist after parasites have been cleared from the blood;
the occurance of persistent viable asexual-stage parasitaemia undetectable by light microscopy.

genetic heterogeneity of PfHRP-2 expression;
HRP-2 gene deletions;
antibodies that block immune-complex formation;
prozone effect;
Unknown

prozone effect: Concentration of antibody or antigen is so high that the optimal concentration for maximal reaction with antigen is exceeded and binding is reduced or does not occur.;

Question 20

Quantitative Buffy Coat

Answer 20

Capillary blood is taken into a glass haematocrit tube containing acridine orange (to stain parasite DNA) and potassium oxalate (an anticoaglulant)
A cylindrical float inserted into the tube which is then centrifuged to separate cells according to their densities
Discrete bands are formed which are made larger by the presence of the float
The tube is then placed on a holder and examined under a light microscope with a UV adapter
Parasites can be observed as they fluoresce under the microscope

Question 21

Quantitative Buffy Coat Limitations

Answer 21

almost impossible to differentiate between species, quantification is not possible, false positives can be reported by inexperienced staff due to artefact

Question 22

Polymerase Chain Reaction

Answer 22

• Involves DNA amplification in vitro and is highly sensitive and specific technique

Question 23

PCR advantages

Answer 23

Can detect and differentiate malarial parasites
Technique is tenfold more sensitive than microscopy
More reliable in identifying the species of malaria present

Question 24

PCR disadvantage

Answer 24

Time consuming
Expensive
Requires some degree of experience/expertise

Question 25

Thick and thin films

Answer 25

Should be prepared in all cases where possible
Thin films: conventional ‘wedge’ blood films
Thick films: thicker and should be about 1 cm in diameter
All films should be prepared, stained and examined without delay due to morphological changes that occur over time
Common stains used:Romanowsky, Leishman’s, May Grunwald/Giemsa
Thick films are not fixed
Proper drying is essential prior to examination

Question 26

Thick and thin films ADV and DisAdv

Answer 26

Advantages
‘Gold standard’ in malaria diagnosis
Cheap
Can be used for species identification and quantification

Disadvantages
Parasites can be missed, especially at low levels of infestation
Requires some degree of experience
Can be time consuming
Requires a microscope (not much use in non-lab settings)

Question 27

Identification of Malaria

Answer 27

Knowledge of geographical distribution may give a clue:
P. Vivax: Widest distribution extending through the tropics, subtropics and temperate zones
P. Falciparum: Generally confined to tropics
P. Malariae: Sporadic distribution
P. Ovale: Mainly Central West Africa & some South Pacific Islands

Both the sexual and asexual forms have certain characteristics which enable species identification

Earliest forms of trophozoites in the blood cannot be differentiated but identification of the species becomes possible as they get older

Question 28

Malaria Diagnosis: Maurers cleft & Schuffner dots

Answer 28

Maurer’s clefts appear as irregular red/mauve dots inside red cells infected with P. falciparum.

Schüffner’s dots are multiple, small, brick-red dots inside red cells infected with P. vivax or P. ovale.

Question 29

Plasmodium falciparum - Clinical Presentation/ Pathology

Answer 29

Cycles of development in the blood last 36-48 hours
- liver stage lasts for 8-10 days
- Liver schizont contains 40,000 merozoites
- Mature erythrocytic schizont contains 16-30 merozoites- rarely seen unless patient is gravely ill
- Red blood cells of all ages are invaded
- More than 40% of erythrocytes may be invaded
-Most common and severe form of malaria
- Typical incubation period for P. falciparum is 7–14 days, while P. vivax and P. ovale 12–17 days and P. malariae 18–40 days.

Question 30

Plasmodium falciparum - Microscopic presentation and
Giesma stained thin films

Answer 30

Often only ring forms

No increase in red cell size

Marginal forms (accole)

Fine cytoplasm in young rings

Maurer’s clefts in late ring forms

Dark brown or black pigment

Often higher parasitaemia

Ring forms with 2 or more chromatin dots (not specific)

Often multiple parasites per cell (not specific)

Giemsa-stained thin films of P. falciparum infection (high power).:

Characteristic thin, delicate rings (note the double chromatin dot—headphone appearance—in the infected red cell at the top and the accolé form in the infected red cell at the bottom);
note the multiply infected red cell;
P. falciparum trophozoites are rarely seen in peripheral blood—older, ring forms are referred to as the trophozoites, which tend to have more dense cytoplasm than in younger rings;
mature schizont containing merozoites;
crescent-shaped gametocyte.

Question 31

Plasmodium Vivax - Clinical Presentation/ Pathology

Answer 31

Cycles of development in the blood last 48 hours
All stages are often seen in peripheral blood
Fewer merozoites are seen in liver and erythrocytic stages (e.g. 12-24 merozoites in the erythrocytic stage)
Parasitaemia is much lower than with P. falciparum
Dormant hypnozoites may be formed
Reticulocytes are preferentially invaded

Question 32

Plasmodium Vivax - Microscopic presentation and Giemsa- stained thin films -

Answer 32

Cells may reach twice normal size
Fine cytoplasm in young rings
Amoeboid forms
Schuffner’s dots
Golden brown pigment
Schizonts with average of merozoites (up to 24)
Gametocytes difficult to differentiate

Giemsa-stained thin films of P. vivax infection.
High-power field showing large rings with heavy chromatin dots but faintly stained cytoplasm;
(b) medium-power field showing one ring form and three large, amoeboid trophozoites;
(c) high-power field with mature schizont containing merozoites virtually filling the red cell;
(d) gametocyte filling the entire red cell, which is enlarged.
e) Large trophozoites…

Question 33

P. Ovale- Clinical Presentation / Pathology

Answer 33

Also has a 48 hour erythrocytic stage
8-12 merozoites are seen in erythrocytic schizonts
More restricted in distribution
Hypnozoites occur in this species also
Reticulocytes are preferentially invaded

Question 34

P. ovale - Microscopic presentation and Giemsa stained thin films

Answer 34

Thicker cytoplasm of ring forms
Trophozoites compact and rounded with fine, brown grains of pigment
Cells increase in size but less than P. Vivax
Schuffner’s dots
Schizonts with 6-12 merozoites
Oval red cells with fimbriated edge
Gametocytes difficult to differentiate

Giemsa-stained thin films of P. ovale infection (high power).
a) Ring form with heavy chromatin dot and thick, deep-staining cytoplasm;
(b) ring forms and a developing trophozoite;
(c) Schizont containing merozoites with large nuclei;
(d) gametocyte.

Question 35

Plasmodium malariae - Clinical Presentation/ symptoms

Answer 35

Has an erythrocytic cycle of 72 hours
Has a low prevalence
Liver and erythrocytic schizonts produce fewer merozoites than other species ( 6-12 merozoites in erythrocytic schizonts) so parasitaemias are generally low
P. malariae infects older red blood cells preferentially

Question 36

P. malariae - Microscopic presentation/ giesma thin slides

Answer 36

Cytoplasm noticeably thicker in ring forms
‘Band’ appearance
Dark brown/black pigment
No red cell enlargement
Rarely see granules
Parasites often scanty and difficult to find
Schizonts with 8-10 merozoites arranged in ‘daisyhead’ formation
Mature gametocytes fill the red cell

Giemsa-stained thin films of P. malariae infection (high power).
Ring form with heavy chromatin dot and thick, deep-staining cytoplasm;
(b) band form trophozoite;
(c) mature schizont with fewer merozoites than P. falciparum or P. vivax, central clump of pigment;
(d) gametocyte.

Question 37

Plasmodium knowlesi - Clinical Presentation

Answer 37

Can be found in Southeast Asia
Morphology similar to P. malariae
Higher parasitaemia
Can be fatal

Question 38

P. Knowlesi - Microscopic presentation and giemsa-stained thin films

Answer 38

(a) Ring forms;
(b) ring form and a developing, band form trophozoite;
(c) mature schizont (note the pigment has concentrated into a single mass);
(d) spherical gametocyte.

Giemsa-stained thin films of P. knowlesi infection (high power).
Ring forms;
(b) ring form and a developing, band form trophozoite; (
c) mature schizont (note the pigment has concentrated into a single mass); (
d) spherical gametocyte.

Question 39

What next in that lab?

Answer 39

Laboratory results entered into the hospital computer system so that medical staff can view them

All positive malaria screens are phoned to the requesting doctor and haematology medic on-call

Positive malaria screeb: A patient report form is then filled in and sent to the School of Tropical Medicine. A copy of this also goes to Public Health England.

 (Procedures vary between hospitals)