Laboratory Diagnosis of Malaria

Once malaria is suspected on clinical grounds, it is mandatory to obtain the laboratory confirmation of the presence of malaria parasites in the patient’s specimen, whenever possible.

Sample: Blood

Blood is taken by pricking a finger or ear lobule before starting treatment with antimalarials. Blood for smear should be collected late in the febrile paroxysm (a few hours after the height of paroxysm) to coincide with presence of highest number of malarial parasites in the peripheral blood.

The diagnosis of malaria may in fact be pursued by the direct demonstration of the parasite whole cell or of parasite’s nucleic acid or products in the blood (direct diagnosis) or by the demonstration of the patient’s immune response to the infection (indirect diagnosis or immunodiagnosis).

The direct demonstration of the whole parasitic cell may be accomplished by several methods, from the old and simple (but still gold standard!) direct microscopic observation of stained blood specimen to the more recent and sophisticated concentration and staining techniques (Buffy coat preparation, Quantitative Buffy Coat (QBC) , acridine orange method).
Finally, the detection of the presence of the genome of the parasite is now possible by the Polymerase Chain Reaction (PCR) technique.The evidence of  specific antibodies is of little, if any, diagnostic value in endemic malaria areas but may provide important information in epidemiological studies and, in some selected setting, in blood donor screening
Rapid and accurate diagnosis of malaria is integral to the appropriate treatment of affected individuals and is preventing the further spread of infection in the community.

a. Microscopy and staining methods 

Microscopic examination remains the “gold standard” for laboratory confirmation of malaria. Microscopy is an established, relatively simple technique that is familiar to most laboratorians. Any laboratory that can perform routine hematology test is equipped to perform a thick and thin blood smear. Within few hours of collecting the blood, the microscopy test can provide valuable information.

Blood specimen collected from the patient is spread as a thick or thin blood smear, stained with a romanovsky stain (most often Giemsa), and examined with 100x oil immersion objective. Visual criteria are used to detect malaria parasites and to differentiate (when possible) the various species.

Various stages of malarial parasites
Once the diagnosis is established, usually by detecting parasites in the thick smear, thin smear can be examined to determine the malaria species and the parasitemia, or the percentage of the patients red cells that are infected with malarial parasites.  Read this post to find out how the species of malarial paraistes are identified and differentiated.

2. Rapid Diagnostic Test for Laboratory Diagnosis of Malaria 

A rapid diagnostic test (RDT) is an alternate way of quickly establishing the diagnosis of malaria infection by detecting specific malaria antigen (e.g. HRP2- Histidine Rich Protein-2, or  PLDH-Parasite Lactate Dehydrogenase or Aldolase) present in patient’s blood.

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 signifies the presence of antigens by a colour change/formation of bands on an absorbing nitrocellulose strip.
The RDTs come in a number of 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 that is present 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 pLDH antibodies.
  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.
Target antigens for commercially available RDTs:
HRP2 PLDH Aldolase
P. falciparum specific
Pan-specific (all species)
P. vivax specific
Note: Pan-specific means that the RDT detects all the four types of plasmodia that infect humans.
Technique: A blood specimen collected from the patient is applied to the sample pad on the test card along with certain reagents. After 15 mins, the presence of specific band in the test card window indicate whether the patient is infected with Plasmodium falciparum or one of the other three species of human malaria.
Malaria lab diagnosis
Advantage of Rapid Diagnostic test for Malaria diagnosis
High quality malaria microscopy is not always available in every clinical settings 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. Relatively easy to use with minimal training required
  2. Relatively rapid, giving timely results
  3. Little or no manipulation of sample required, can be performed in places without laboratories
  4. Most of the RDTs do not require refrigeration, hence tests can be performed where there is no power supply
  5. Uses whole blood (prick or venous blood prick preferred)
Disadvantage of Rapid Diagnostic test (for Malaria diagnosis)
  1. Costs per test may exceed those of microscopy.
  2. Short shelf-life, requiring efficient procurement, transportation, storage and distribution systems
  3. Most tests are qualitative (i.e. gives a yes or no answer). Any quantification of parasitemia will require further
    laboratory-based tests
  4. Intensity of test band varies with amount of antigen present at low parasite densities-this may lead to
    reader variation in test results
  5. 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 lower number of malaria parasites circulating in the patient’s bloodstream.
  6. 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 followed by microscopy.

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

Do you have any queries? Please leave me in the comments section below. I will be happy to read your comments and reply.