RDTs for Malaria Diagnosis: Principle, Results, Advantages

Rapid diagnostic tests or RDTs for malaria are a way to test whether a person with malaria-like symptoms has malaria. Malaria parasites produce proteins called antigens. RDTs detect these malarial antigens in a person’s blood. If malaria antigens are present, the test will be positive. If malaria antigens are not present in the person’s blood (RBCs) the test will be negative. Different types of RDTs detect different antigens of malarial parasites.

Some antigens (e.g. pLDH- parasite Lactate dehydrogenase or Aldolase ) are produced by all malarial species but some antigens (HRP2- histidine-rich protein-2 ) are produced by a single species of malarial parasite.

RDTs offer the potential to provide accurate and timely diagnosis, reaching those previously unable to access good quality microscopy services.The RDT works through the lateral flow or Immunochromatographic strip method and notifies the presence of antigens by a color change/formation of bands on an absorbing nitrocellulose strip.

RDTs give results in about 15 minutes, so a patient with malaria can begin treatment right away. There is no need to wait for microscope results.

The RDT test kits come in many formats:

  • Card
  • Dipstick
  • Hybrid cassette-dipsticks
  • Plastic cassette

The three main groups of antigens detected by commercially available RDTs are:

  1. Histidine-rich protein 2 (HRP-2), specific to P. falciparum.
    It is an abundant soluble, heat-stable antigen in the cytoplasm and membrane of infected erytocytes.
  2. Parasite-specific plasmodium lactate dehydrogenase (pLDH), currently available as P. falciparum specific, pan-specific, and P. vivax-specific.
  3. Aldolase (pan-specific). These two antigens are conserved major enzymes in the glycolytic pathway of malaria parasites, they are abundant and are soluble in the parasite.

Note: Pan-specific means that the RDT detects all four types of plasmodia that infect humans.

Target antigens for available RDTs are 

HRP2 PLDH Aldolase
P. falciparum specific  
Pan-specific (all species)  
P. vivax specific    


RDTs for detecting malaria antigens are based on the immunochromatographic test principle. These RDTs capture parasite antigens from peripheral blood using monoclonal antibodies prepared against a target malarial antigen and conjugated to gold particles in a mobile phase.  The test area contains an immobilized monoclonal antibody, which captures the Ag-Ab complex giving a visible line.

If malaria antigen is present in the blood sample, Ag-Ab complex will be formed as it combines with the labeled pan-specific antibody present in the mobile phase. This Ag-Ab complex will migrate along the test strips, which will be captured by the specific antibodies present in the immobile phase (here T1 contains monoclonal antibodies specific to P.falciparum i.e. HRP2 antigen and T2 contains plasmodium pan-specific antibody i.e. pLDH) thus producing a visible colored line.  Control line contains goat anti-mouse antibody and ensures that the system is controlled for migration.

Malaria antigens currently targeted by RDT are HRP-2, pLDH, and Plasmodium aldolase.

HRP-2 : HRP-2 is a water-soluble protein produced by asexual stages and young gametocytes of Plasmodium falciparum. It is expressed on the RBC membrane surface, and because of its abundance in P.falciparum, it is the first antigen to be used to develop an RDT for its detection.

pLDH and aldolase: pLDH, an enzyme found in the glycolytic pathway of the malaria parasite, is produced by sexual and asexual stages of the parasite. Different isomers of pLDH for each of the four Plasmodium spp. infecting humans exist, and their detection constitutes a second approach to RDT development. Several other enzymes of the malaria parasite glycolytic pathway, notably aldolase have been suggested as target antigens for RDT for species other than Plasmodium falciparum.

Materials Required

  • New unopened test packet
  • New unopened alcohol swab
  • New unopened lancet
  • New pair of disposable gloves
  • Buffer
  • Timer
  • Sharps box
  • Pencil or pen.

Test Instructions 

  1. Check the expiry date on the test packet.
  2. Put on gloves. Use new gloves for each patient.
  3. Open the test kit packet and remove, test pad, capillary tube and desiccant sachet.
  4. Write the patient’s name on the test.
  5. Open the alcohol swab. Grasp the 4th finger on the patient’s left hand. (Clonazepam) Clean the finger with the alcohol swab. Allow the finger to dry before pricking.
  6. Open the lancet. Prick patient’s finger to get a drop of blood. Do not allow the tip of the lancet to touch anything before pricking the patient’s finger.
  7. Discard the lancet in the Sharps Box immediately after pricking finger. Do not set the lancet down before discarding it.
  8. Use the capillary tube to collect the drop of blood.
  9. Use the capillary tube to put the drop of blood into the square hole marked “A”.
  10. Discard the capillary tube in the Sharps Box.
  11. Add buffer into the round hole marked “B”.
  12. Wait for 15 minutes after adding buffer.
  13. Read the results (see below)
  14. Dispose of the gloves, alcohol swab, desiccant sachet and packaging in a non-sharps waster container.
  15. Record the test results in your register. Dispose of cassette in non-sharps waste container.

 Interpretation of the test results

RDTs for lab diagnosis of Malaria

In the Kit we are using, ‘T1’ test line is specific for P. falciparum, ‘T2‘ test line is Pan-specific and ‘C‘ is the control line.

Malaria RDT Test One

1. A line in ‘T1‘ and a line in ‘C’ means the patient DOES have falciparum malaria. The test is POSITIVE even if the line in ‘T1‘ is very faint.

2. A line in ‘T2’and a line in ‘C’ means the patient DOES have non-falciparum malaria (P. vivax, P. ovale, P. malariae) or a mixed infection of these.

3. Lines in ‘T1‘ and ‘T2’ and a line in ‘C’ means the patient DOES have falciparum malaria mono-infection or a mixed infection.

4. NO Line in ‘T1’ or ‘T2’ and a line in ‘C’ means the patient DOES NOT have either falciparum malaria or non-falciparum malaria.

5.NO Line in ‘T1‘ or ‘T2’ and NO LINE in ‘C’ means the test is damaged. Results are INVALID.

6. Line in ‘T1’ or ‘T2′ and NO LINE in ‘C’ means the test is damaged. Results are INVALID.

Malaria RDT Test Two

Advantage of Rapid Diagnostic test for Malaria diagnosis

High-quality malaria microscopy is not always available in every clinical setting where patients might seek medical attention. The laboratories associated with these health care settings may now use an RDT to more rapidly determine if their patients are infected with malaria.

  1. RDTs can provide a parasite-based diagnosis in places where microscopy is not possible or practical.
  2. RDTs can be used to distinguish fevers caused by malaria parasites from those caused by other illnesses, such as meningitis and acute respiratory infection (that cause symptom similar to malaria). RDTs can thus help to target anti-malarial treatment (ACTs) to patients who really have malaria. When RDTs show that a febrile patient does not have malaria, that patient is more likely to seek diagnosis and treatment for the illness he/she does have.
  3. It helps to avoid unnecessary use of ACTs on patients who do not have malaria. This will help to prevent or delay drug resistance, making ACTs effective for a longer period.
  4. Relatively easy (with minimal training required) and rapid (giving timely results)
  5. Little or no manipulation of sample required, can be performed in places without laboratories
  6. Most of the RDTs do not require refrigeration, hence tests can be performed where there is no power supply
  7. Uses whole blood (prick or venous blood prick preferred)

Limitation of Rapid Diagnostic test for Malaria diagnosis

  1. RDTs cannot test how many malaria parasites are present in the blood. They can only test whether parasites are present or absent (qualitative result only). Any quantification of parasitemia will require further laboratory tests.
  2. RDTs that detect malaria antigen, Histidine-rich protein 2 or ‘HRP2’ can not be used to check for effectiveness of treatment, because this antigen can remain in the blood for at least two weeks after the parasites are killed by drugs. Other malaria antigens, pLDH, and aldolase disappear from the blood rapidly, so RDTs based on those antigens will usually be negative a few days after effective treatment.
  3. Costs per test may exceed those of microscopy.
  4. Short shelf-life, requiring efficient procurement, transportation, storage, and distribution systems
  5. Intensity of test band varies with the amount of antigen present at low parasite densities-this may lead to
    reader variation in test results.
  6. In many cases, they are less sensitive (and less specific) than laboratory-based tests. The RDT may not be able to detect some infections with a lower number of malaria parasites circulating in the patient’s bloodstream.
  7. The use of the RDT does not eliminate the need for malaria microscopy. All negative RDT must be followed by microscopy to confirm the result. In addition, all positive RDT’s should also be followed by microscopy.

Microscopy is needed to determine the species of malaria detected by the RDT. Microscopy (thick and thin blood smear) is needed to quantify the proportion of infected RBCs, which is an important prognostic indicator.

References and further reading 

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

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