Malaria Diagnostic Tests

Malaria is diagnosed by finding Plasmodium parasites or their products in blood. Prompt diagnosis separates malaria from other causes of fever, directs treatment, and detects severe disease, which kills within hours. Any fever after travel to or residence in a malaria endemic area is malaria until excluded.

Microscopy of Giemsa stained thick and thin blood films is the reference method. The thick film concentrates parasites for detection and counting, and the thin film identifies species and stages.

Rapid diagnostic tests (RDTs) detect parasite antigens (histidine rich protein 2, parasite lactate dehydrogenase, aldolase) on a lateral flow strip in 15 to 20 minutes. Molecular tests (PCR, loop mediated isothermal amplification) detect low density infection and confirm species. Serology shows past exposure only and is not used for diagnosis.

There were an estimated 249 million malaria cases and 608 000 deaths in 2022, 95% in sub-Saharan Africa and most in children under 5 years. Plasmodium falciparum causes the most severe disease and most deaths. Plasmodium vivax is the most widespread, Plasmodium ovale and malariae cause milder illness, and Plasmodium knowlesi (a zoonosis in South-East Asia) can be severe.

Indications.

• Fever (or history of fever in the last 48 hours) in a patient who lives in or has travelled to a malaria endemic area within the last year (and up to 3 months for most falciparum cases, longer for vivax and ovale).
• Unexplained anaemia, thrombocytopenia, jaundice, splenomegaly, impaired consciousness, seizures, respiratory distress, hypoglycaemia, kidney injury or shock in such a person.
• Screening of blood donors and pregnant women in endemic areas.
• Assessment of treatment response.

Specimen. Capillary (finger prick) or venous blood in EDTA. Make thick and thin films immediately, or within 3 hours of collection from EDTA (parasite morphology changes with time). Use fresh Giemsa at pH 7.2 for stain quality. Send a repeat sample every 12 to 24 hours for 3 days if the first result is negative and suspicion remains. For RDTs, use fresh blood and read at the time stated by the manufacturer.

Microscopy.

• Thick film: sensitivity detects 5 to 20 parasites/microL in experienced hands. Parasitaemia is counted against 200 to 500 white cells, assuming 8000 white cells/microL, or against red cells.
• Thin film: percentage of infected red cells (parasitaemia) and species identification. Detection limit about 100 to 200 parasites/microL.
• Species identification: P. falciparum shows ring forms (often multiple per cell), banana shaped gametocytes, and rarely schizonts in peripheral blood. P. vivax and ovale show enlarged red cells with Schüffner dots and all stages. P. malariae shows band forms and basket schizonts. P. knowlesi resembles P. malariae or falciparum.
• Negative film: three films 12 to 24 hours apart exclude malaria with high confidence.

Parasitaemia and severity. For falciparum malaria, parasitaemia over 2 to 5% (non-immune patients) or over 10% (in any patient) defines hyperparasitaemia, a feature of severe disease. In high transmission areas, patients have partial immunity and may tolerate high counts. Peripheral schizonts in falciparum malaria indicate severe disease and a poor prognosis.

Rapid diagnostic tests.

• Histidine rich protein 2 (HRP2) detects P. falciparum only, with sensitivity over 95% at 100 or more parasites/microL, and stays positive for 2 to 4 weeks after cure (so it cannot show treatment failure).
• Plasmodium lactate dehydrogenase (pLDH) detects all species and falls quickly with treatment. Aldolase is a pan species antigen.
• Combination RDTs (HRP2 plus pLDH) identify P. falciparum and non-falciparum infection.
• Deletion of the HRP2 and HRP3 genes in P. falciparum (reported in the Horn of Africa and South America) causes false negative HRP2 tests. Laboratories are replacing them with pLDH-based tests where this occurs.
• RDTs have a prozone effect with very high parasitaemia, causing false negatives, and false positives with rheumatoid factor and persistent antigen.

PCR. Detects 0.02 to 5 parasites/microL, identifies species including mixed infections, and detects drug resistance markers. It is used for confirmation, for low density infections in returning travellers, and for relapsing malaria.

Associated laboratory findings.

• Thrombocytopenia (platelets under 150 x 10^9/L in 60 to 80%), anaemia (haemolysis), raised bilirubin and LDH, raised CRP, lymphopenia, and a normal or low white cell count.
• Hypoglycaemia (in children, pregnant women and quinine treated patients), lactic acidosis, raised creatinine, and elevated transaminases.
• Disseminated intravascular coagulation in severe disease.

Features of severe falciparum malaria.

• Impaired consciousness (Glasgow Coma Score under 11) or seizures, prostration, respiratory distress and pulmonary oedema.
• Shock (systolic blood pressure under 80 mmHg).
• Acute kidney injury (creatinine over 265 micromol/L).
• Jaundice (bilirubin over 50 micromol/L) with other organ dysfunction.
• Hypoglycaemia (under 2.2 mmol/L).
• Severe anaemia (haemoglobin under 70 g/L).
• Acidosis (bicarbonate under 15 mmol/L or lactate over 5 mmol/L).
• Abnormal bleeding, haemoglobinuria, and parasitaemia over 10%.

Critical values and findings needing immediate action.

• Any positive result for P. falciparum, or a mixed or unspecified infection: treatment starts immediately, and the laboratory telephones the result.
• Severe malaria features: intravenous artesunate without delay, with glucose, fluids, and care in a high dependency unit.
• Parasitaemia over 10%, or over 2 to 5% with clinical signs: this is severe malaria.
• Hypoglycaemia or seizures in a child with fever: glucose and anticonvulsant, and test for malaria.
• Negative test with ongoing suspicion: repeat films at 12 to 24 hours (a total of three).
• P. vivax or ovale: radical cure with primaquine or tafenoquine after G6PD testing.

Interferences and pitfalls.

• A single negative film or RDT does not exclude malaria. Low parasitaemia early in the illness, sequestration of mature falciparum parasites, prior partial treatment or prophylaxis, and poor technique cause false negatives.
• Microscopy depends heavily on training, and species errors (mistaking P. knowlesi for P. malariae, ovale for vivax) occur.
• Mixed infections are easily missed, and PCR helps.
• Pitfalls on films: platelets, stain precipitate and bacteria overlying red cells mimic parasites. Babesia in the thin film resembles falciparum rings.
• HRP2-based tests remain positive after treatment, so cannot be used to judge cure. After treatment, microscopy shows response.
• HRP2 deletion strains give false negative RDTs.
• Do not wait for laboratory confirmation to treat severe malaria if testing is delayed.
• Gametocytes may persist for days to weeks after cure of falciparum malaria and do not mean failure.
• Always test for G6PD before primaquine or tafenoquine.
• Test other causes of fever in parallel, such as bacterial sepsis, typhoid, dengue and viral haemorrhagic fever, since co-infection is common.

Special groups.

• Children: severe anaemia, cerebral malaria and hypoglycaemia are the main severe presentations.
• Pregnancy: placental malaria with low parasitaemia in peripheral blood, miscarriage, low birth weight and maternal anaemia. Intermittent preventive treatment is given in endemic areas.
• Travellers and migrants: non-immune, develop severe disease with low parasitaemia.
• Splenectomised and immunosuppressed patients: severe disease, including with P. knowlesi.
• Regions: in high transmission areas, asymptomatic parasitaemia is common, so a positive test with another obvious cause of fever may not explain illness. RDT-based management is common where microscopy is not available.

Follow up tests.

• Repeat thick and thin films at 12 to 24 hours, then daily until negative.
• Full blood count, glucose, urea, creatinine and electrolytes, liver function tests, lactate and blood gas.
• G6PD assay, blood cultures, HIV test, urine analysis and coagulation tests.
• Chest radiograph in respiratory distress.
• Lumbar puncture when meningitis cannot be excluded.
• PCR for species confirmation and drug resistance markers.