How malaria RDTs work

Malaria rapid diagnostic tests (RDTs) assist in the diagnosis of malaria by providing evidence of the presence of malaria parasites in human blood. RDTs are an alternative to diagnosis based on clinical grounds or microscopy, particularly where good quality microscopy services cannot be readily provided.
Variations occur between products, such as targets and formats, though the principles of the tests are similar. Malaria RDTs detect specific antigens (proteins) produced by malaria parasites in the blood of infected individuals. Some RDTs can detect only one species (Plasmodium falciparum) while others detect multiple species (P. vivaxP. malariae and P. ovale). Blood for the test is commonly obtained from a finger-prick.
RDTs are lateral flow immuno-chromatographic antigen-detection tests, which rely on the capture of dye-labeled antibodies to produce a visible band on a strip of nitro-cellulose, often encased in plastic housing, referred to as cassettes. With malaria RDTs, the dye-labeled antibody first binds to a parasite antigen, and the resultant complex is captured on the strip by a band of bound antibody, forming a visible line (T - test line) in the results window. A control line (C- control line) gives information on the integrity of the antibody-dye conjugate, but does not confirm the ability to detect parasite antigen.

RDT cassette

A typical RDT cassette

Inside the cassette is a strip made of filter paper and nitrocellulose. Typically, a drop of blood is added to the RDT through one hole (A; sample well), and then a number of drops of buffer usually through another hole (B; buffer well). Buffer carries the blood along the length of the RDT.

Mode of action of common malaria RDT format

 1. The first step of the test procedure involves mixing the patient’s blood with a lysing agent in a test strip or well. This ruptures the red blood cells, releasing more parasite protein.
 2. Dye-labeled antibody, specific for target antigen, is present on the lower end of nitrocellulose strip or in a plastic well provided with the strip. Antibody, also specific for the target antigen, is bound to the strip in a thin (test) line, and either antibody specific for the labeled antibody, or antigen, is bound at the control line.
 3. Blood and buffer, which have been placed on strip or in the well, are mixed with labeled antibody and are drawn up the strip across the lines of bound antibody.
 4. If antigen is present, some labeled antibody-antigen complex will be trapped and accumulate on the test line. Excess-labeled antibody is trapped and accumulates on the control line. A visible control line indicates that labeled antibody has traversed the full length of the strip, past the test line, and that at least some free antibody remains conjugated to the dye and that some of the capturing properties of the antibodies remain intact.
 5. The intensity of the test band will vary with the amount of antigen present, at least at low parasite densities (antigen concentration), as this will determine the amount of dye particles which will accumulate on the line. The control band intensity may decrease at higher parasite densities, as much of the labeled antibody will have been captured by the test band before reaching the control.

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