Haematology and Transfusion Practice BIOL33451 Malaria and blood parasites I

Malaria: An Introduction

• Malaria is a tropical parasitic disease caused by protozoan parasites that infect red blood cells.

• It is typically transmitted through the bite of infected Anopheles mosquitoes.

• Malaria causes significant morbidity and mortality worldwide and is prevalent in tropical and subtropical zones.

• Malaria can be transmitted via:

  • Mosquito bites (most common).

  • The placenta.

  • Blood transfusions.

  • Transplantation.

  • Contaminated equipment.

  • Air travel.

Species of Malaria Parasites

• The species of malaria parasites known to infect humans include:

  • Plasmodium falciparum

  • Plasmodium vivax

  • Plasmodium ovale

  • Plasmodium malariae

  • Plasmodium knowlesi

World Malaria Report 2023 Key Messages

• Malaria disproportionately affects pregnant women and children in Africa.

• Significant progress in reducing malaria burden occurred between 2000-2015, demonstrating that progress is possible.

• Stepped-up action including new tools, strategies, and financing is needed in all endemic countries to get back on track.

• Universal health coverage is crucial for success.

Malaria Transmission Areas

• Malaria prevalence varies globally due to climate, mosquito abundance, funding, government cooperation with organizations like WHO, education, preventative measures, accurate diagnosis, and access to treatment.

Malaria Vaccine

• The first malaria vaccine, RTS,S, was recommended by WHO in October 2021.

• The vaccine has reached nearly 2 million children in Ghana, Kenya, and Malawi since 2019 through the Malaria Vaccine Implementation Programme (MVIP).

• In December 2023, the R21/Matrix-MTM malaria vaccine, developed by the University of Oxford and the Serum Institute of India, was prequalified by the World Health Organization (WHO).

Malaria Cycles

• The malaria cycle involves stages in both humans and mosquitoes. In humans, the parasite infects liver cells (exo-erythrocytic cycle) and red blood cells (erythrocytic cycle).

Pf Merozoite Invasion

• Steps involved in Pf merozoite invasion:

  1. Merozoite attachment to a receptor on the RBC surface.

  2. Merozoite re-orientation towards the RBC surface.

  3. Formation of a tight junction between merozoite and RBC, causing initial deformation.

  4. Entry of merozoite, forming a parasitophorous vacuole.

  5. Closure of RBC and parasitophorous vacuole membranes.

  6. Severing the junction, releasing the merozoite into the cell where it transforms into a young trophozoite.

• Plasmodium falciparum binds to glycophorins A, B, and C.

• Plasmodium vivax and Plasmodium knowlesi bind to the Duffy antigen.

• The receptors for Plasmodium malariae and Plasmodium ovale are unknown.

Malaria in the UK

• The ratio of malaria cases in UK residents visiting friends and relatives compared with those acquired by holiday travelers is approximately 10:1.

• Important considerations include access to and adherence to medical guidance before travel, awareness of risk, and familiarity with the destination. Targeting this high-risk group and their healthcare providers is a priority.

• Other groups presenting with malaria include new entrants, foreign students, foreign visitors, British citizens working abroad, armed services, and business travelers.

Individual Risk Factors

• Factors determining individual risk include:

  • Lack of awareness of malaria risk areas.

  • Amount of malaria in the visited area.

  • Time of year.

  • Types of parasites present.

  • Preventative measures taken.

  • Immunity or lack thereof.

  • Appropriate travel advice.

Immunity in Malaria

• Immunity is most pronounced in Plasmodium falciparum disease.

• In high transmission areas, children surviving to age 5-6 often have a high degree of immunity.

• This immunity wanes without regular exposure and during pregnancy.

• Pregnant women and young children are most at risk.

Malaria Symptoms

• Common symptoms include:

  • Fever

  • Chills

  • Headache

  • Flu-like symptoms

  • Muscle aches

  • Fatigue

  • Anaemia

  • Diarrhoea

  • Vomiting

  • Cough

• Symptoms of cerebral malaria can occur in Plasmodium falciparum infections.

Laboratory Diagnosis

• Diagnostic tests include:

  • Full blood count (FBC)

  • Rapid diagnostic tests (immunochromatography for malarial antigens)

  • Thin and thick blood films

  • Quantitative buffy coat

  • Polymerase chain reaction (PCR, usually in reference laboratories).

Haematological Changes

• Abnormalities observed in FBC results are non-specific and not diagnostic.

• Anaemia can occur due to red cell rupture (haemolytic anaemia), impaired haemopoiesis, and removal of parasitized cells.

• Thrombocytopenia is common due to platelet pooling/clearance in the spleen and reduced platelet lifespan.

• WBC count is often normal but may be raised in severe disease.

• Abnormalities may be observed on the white cell scattergram.

Rapid Diagnostic Tests (RDTs)

• RDTs use lateral-flow immunochromatographic techniques.

• Antibodies target proteins specific for Plasmodium falciparum, such as HRP-2 (PfHRP-2), or panmalaria proteins like Plasmodium aldolase or Plasmodium lactate dehydrogenase (pLDH).

• P. falciparum LDH (PfLDH)-specific antibodies are also available.

NOW Malaria Kit (BinaxNOW)

• A rapid immunodiagnostic assay using two antibodies immobilized on a test strip.

• One antibody is specific for histidine-rich protein II of Plasmodium falciparum, and the other for an antigen common to all malaria forms infecting humans (pLDH).

• Color formation indicates the presence of specific parasites.

• Includes a control band and takes about 15 minutes.

Optimal-IT Test

• Uses monoclonal antibodies against parasite lactate dehydrogenase to detect malaria.

• One antibody is specific for P. falciparum, and the other is a pan-specific antibody.

• PLDH reacts with specific antibodies in the presence of plasmodium species.

Advantages of Rapid Diagnostic Kits

• Cheap and quick results.

• Easy to perform, aiding accurate diagnosis by inexperienced staff.

• Can be used in settings away from the laboratory.

Disadvantages of Rapid Diagnostic Kits

• False positives: cross-reacting antibodies.

• False negatives:

  • PfHRP-2 can cross-react with non-falciparum malaria.

  • PfHRP-2 can persist after parasites have been cleared.

  • 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.

Quantitative Buffy Coat (QBC)

• Capillary blood is taken into a glass haematocrit tube containing acridine orange and potassium oxalate.

• A cylindrical float is inserted, and the tube is centrifuged to separate cells by density.

• The tube is examined under a light microscope with a UV adapter, where parasites fluoresce.

• Limitations include difficulty in species differentiation, inability of quantification, and false positives due to artifacts.

Polymerase Chain Reaction (PCR)

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

• Advantages include detection and differentiation of malarial parasites in addition to being tenfold more sensitive than microscopy.

• Disadvantages include high cost, time consumption, and the requirement of expertise.

Thick and Thin Films

• Should be prepared in all cases where possible.

• Thin films are conventional ‘wedge’ blood films, while thick films are thicker (about 1 cm in diameter).

• Films should be prepared, stained, and examined without delay due to morphological changes.

• Common stains used include Romanowsky, Leishman’s, and May Grunwald/Giemsa stains.

• Thick films are not fixed before staining.

Advantages of Thick and Thin Films

• ‘Gold standard’ in malaria diagnosis.

• Cheap.

• Can be used for species identification and quantification.

Disadvantages of Thick and Thin Films

• Parasites can be missed at low levels of infestation.

• Requires expertise and can be time-consuming.

• Requires a microscope.

Identification of Malaria

• 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

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

Maurer’s Clefts and Schuffner’s Dots

• 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.

Morphological Differentiation of Human Malarial Parasites

• Refer to species differentiation table in Haematology (Moore Knight and Blann) for revision.

Plasmodium falciparum

• Cycles of development in the blood last 36-48 hours.

• The liver stage lasts for 8-10 days.

• Liver schizont contains 40,000 merozoites.

• Mature erythrocytic schizont contains 16-30 merozoites.

• Red blood cells of all ages are invaded.

• More than 40% of erythrocytes may be invaded

• Typical incubation period is 7–14 days.

P. falciparum Characteristics

• Often only ring forms are seen.

• No increase in red cell size.

• Marginal forms (accolé) are common.

• 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.

• Often multiple parasites per cell (not specific).

Plasmodium vivax

• 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.

P. vivax Characteristics

• Cells may reach twice normal size.

• Fine cytoplasm in young rings.

• Amoeboid forms.

• Schuffner’s dots are a key feature.

• Golden brown pigment.

• Schizonts with average of merozoites (up to 24).

• Gametocytes difficult to differentiate.

Plasmodium ovale

• 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.

P. ovale Characteristics

• 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.

Plasmodium malariae

• 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.

P. malariae Characteristics

• 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.

Plasmodium knowlesi

• Can be found in Southeast Asia.

• Morphology similar to P. malariae.

• Higher parasitaemia.

• Can be fatal.

P. knowlesi Morphology

• (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.

Post-Laboratory Procedures

• Laboratory results are entered into the hospital computer system for medical staff to view.

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

• 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)

Further Reading

• Moore, G. Knight, G. and Blann, A. (2021) Haematology Oxford University

• WHO malaria report 2023: https://www.who.int/teams/global-malaria-programme/reports/world-malaria-report-2023

• Government website: https://www.gov.uk/government/publications/malaria-in-the-uk-annual-report