Laboratory Diagnosis of Malaria

Malaria is one of the most common infectious diseases and one of the leading causes of death. It is caused primarily by four plasmodia: Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium falciparum. A fifth species, Plasmodium knowlesi is found in Southeast Asia. Cases of P. falciparum and P. vivax are more common compared to P. malariae and P. ovale. P. vivax is the most widely distributed, and P. falciparum causes the most serious disease.

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

Once malaria is suspected on clinical grounds, it is mandatory to obtain laboratory confirmation of the presence of malaria parasites in the patient’s specimen whenever possible. Rapid and accurate diagnosis of malaria is integral to appropriately treating affected individuals and preventing the further spread of infection in the community. 

Sample Collection

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

Microscopy and Staining Methods 

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 specimens to the more recent and sophisticated concentration and staining techniques (buffy coat preparationQuantitative Buffy Coat (QBC), acridine orange method). 

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

Examination of Thick and Thin Blood Smear

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. A thick smear is used to screen for the presence of organisms, and a thin smear is used for species identification. Identification of species is required as the treatment protocol may vary based on the species involved.

Visual criteria such as the number of infected red blood cells, presence of crescent-shaped gametocytes, presence of Schuffne’rs dots, etc., are used to detect and differentiate (when possible) species of malarial parasites.

Various stages of malarial parasites

Read more about thick and thin smears for the diagnosis of malaria.

Quantitative buffy coat

Quantitative Buffy Coat
Falciparum malaria in QBC Test

The quantitative buffy coat (QBC) method uses acridine orange staining, a fluorescent nucleic acid stain, to identify organisms in infected RBCs. This method provides a simple but rapid screening tool for malaria diagnosis. The test uses a 50 μL tube containing all the reagents (anticoagulant) and stain (acridine orange) needed for completion of the assay.

Read more about quantitative buffy coat (QBC) here

Antigen-Based Tests

Several rapid malaria tests are commercially available to detect malarial antigens such as histidine-rich protein 2 (HRP2), Plasmodium aldolase, or species-specific parasite lactate dehydrogenase (pLDH) using monoclonal antibodies.

RDT for Malaria Diagnosis

These tests are based on an antigen capture approach in dipstick or cartridge formats. BinaxNOW rapid malaria test is a US FDA-approved commercial malaria test kit.

Read more: RDTs for Malaria Diagnosis: Principle, Procedure, and Results.

Serologic Tests

The evidence of specific antibodies against malarial antigens is of little diagnostic value in endemic areas. Antibodies are produced late in infection, so they are not useful for patient management. It is also extremely difficult to differentiate a previous disease from an acute malaria infection.   

Still, serology may provide important information in epidemiological studies and some selected settings, such as blood donor screening. 

Molecular Diagnosis

Detecting the presence of the parasite’s genome is possible using the polymerase chain reaction (PCR) technique. PCR amplification of target DNA sequence followed by direct detection of the five malarial parasites using specific DNA sequences is a new diagnostic practice in some laboratories. The high sensitivity (detection is possible for as few as 5-10 parasites per microliter of blood), rapidity, and simplicity of molecular methods make them popular.

Laboratory-based molecular methods are not FDA-approved, so it is still recommended to use them and think blood smears to quantitate the level of parasitemia.

Finished reading? Now, it is quiz time: Test your understanding of the Malaria life cycle, pathogenesis, and diagnosis 

References and further readings

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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