Other Tests

Non-Microscopic Tests

Several attempts have been made to take the malaria diagnosis out of the realm of the microscope and the microscopist. Important advances have been made in diagnostic testing, including fluorescence microscopy of parasite nuclei stained with acridine orange, rapid dipstick immunoassay, and Polymerase Chain Reaction assays.[1] These tests involve identification of the parasitic antigen or the antiplasmodial antibodies or the parasitic metabolic products. Nucleic acid probes and immunofluorescence for the detection of Plasmodia within the erythrocytes; gel diffusion, counter-immunoelectrophoresis, radio immunoassay, and enzyme immunoassay for malaria antigens in the body fluids; and hemagglutination test, indirect immunofluorescence, enzyme immunoassay, immunochromatography, and Western blotting for anti-plasmodial antibodies in the serum have all been developed. These tests have found some limited applications in research, retrograde confirmation of malaria, investigation of cryptic malaria, transfusion blood screening, and investigation of transfusion acquired infections.

Rapid Diagnostic Tests (RDTs) detect species-specific circulating parasite antigens targeting either the histidine-rich protein-2 of P. falciparum or a parasite-specific lactate dehydrogenase. Although the dipstick tests may enhance diagnostic speed, microscopic examination remains mandatory in patients with suspected malaria, because occasionally these dipstick tests are negative in patients with high parasitemia, and their sensitivity below 100 parasites/μl is low.

Tests based on polymerase chain reaction for species-specific Plasmodium genome are more sensitive and specific than are other tests, detecting as few as 10 parasites/μl blood. Antibody detection has no value in the diagnosis of acute malaria. It is mainly used for epidemiologic studies.[6-8]

Polymerase Chain Reaction (PCR): Using the non-isotopically labelled probe following PCR amplification, it is possible to detect malaria parasites. In travelers returning to developed countries, studies based on PCR have been found to be highly sensitive and specific for detecting all 4 species of malaria, particularly in cases of low level parasitemia and mixed infections. The PCR test is reportedly 10-fold more sensitive than microscopy, with one study reporting a sensitivity to detect 1.35 to 0.38 parasites/µL for P. falciparum and 0.12 parasites/µL for P. vivax. The PCR test has also been found useful in unraveling the diagnosis of malaria in cases of undiagnosed fever.

Detection Of Antimalarial Antibodies:

Antibodies to the asexual blood stages appear a few days after malarial infection, increase in titer over the next few weeks, and persist for months or years in semi-immune patients in endemic areas, where re-infection is frequent. In non-immune patients, antibodies fall more rapidly after treatment for a single infection and are undetectable in 3-6 months. Re-infection/relapse induces a secondary response with a rapidly increasing antibody titer.

Malarial antibodies can be detected by immunofluorescence or enzyme immuno assay. It is useful in epidemiological surveys, for screening potential blood donors and occasionally for providing evidence of recent infection in non-immunes. In future, detection of protective antibodies will be important in assessing the response to malaria vaccines.

Intraleucocytic malaria pigment: Intraleucocytic malaria pigment has been suggested as a measure of disease severity in malaria. In a study of 146 children aged 6 months to 14 years in 4 categories – cerebral malaria, mild malaria, asymptomatic malaria and ‘no malaria’- in Ibadan, Nigeria, an area of intense malaria transmission in Africa, the proportion of pigment-containing neutrophils showed a clear rise across the spectrum no malaria–asymptomatic malaria–mild malaria–cerebral malaria (median values 2.0%, 6.5%, 9.0% and 27.0%, respectively; P < 0.0001). The proportion of pigment-containing monocytes did not differ significantly between the mild malaria, asymptomatic malaria and no malaria groups but the cerebral malaria group had a higher median value than the other 3 groups. The ratio of pigment-containing neutrophils to pigment-containing monocytes showed the same trend across the groups of subjects as was observed with the number of pigment-containing neutrophils. The study concluded that the pigment-containing neutrophil count is a simple marker of disease severity in childhood malaria in addition to the parasite count. (Amodu OK, Adeyemo AA, Olumese PE, Gbadegesin RA. Intraleucocytic malaria pigment and clinical severity of malaria in children. Trans R Soc Trop Med Hyg. 1998 (Jan-Feb); 92(1):54-56)

Flowcytometry:

Flowcytometry and automated hematology analyzers have been found to be useful in indicating a diagnosis of malaria during routine blood counts. In cases of malaria, abnormal cell clusters and small particles with DNA fluorescence, probably free malarial parasites, have been seen on automated hematology analyzers and it is suggested that malaria can be suspected based on the scatter plots produced on the analyzer. Automated detection of malaria pigment in white blood cells may also suggest a possibility of malaria with a sensitivity of 95% and specificity of 88%. On flow cytometric depolarized side scatter, the average relative frequency of pigment carrying monocytes was found to differ among semi-immune, non-immune and malaria negative patients.

Mass spectrometry:

A novel method for the in vitro detection of the malarial parasite at a sensitivity of 10 parasites/µL of blood has been recently reported. It comprises a protocol for cleanup of whole blood samples, followed by direct ultraviolet laser desorption time-of-flight mass spectrometry. Intense ion signals are observed from intact ferriprotoporphyrin IX (heme), sequestered by malaria parasites during their growth in human red blood cells. The laser desorption mass spectrum of the heme is structure-specific, and the signal intensities are correlated with the sample parasitemia. Many samples could be prepared in parallel and measurement per sample may not take longer than a second or so. However, the remote rural areas without electricity are not hospitable for existing high-tech mass spectrometers. Future improvements in the equipment and technique can make this method deployable and useful.

Other investigations: Total and differential count, hemoglobin, blood glucose, serum bilirubin, serum creatinine, BUN, AST, ALT, Prothrombin time, urine analysis etc. may be done as needed.

Widal test may be positive, even up to a dilution of 1:320 for ‘O’ and H’ and at lower titres for ‘AH’ and ‘BH’. Any or all the four may be positive, suggesting a non-specific response. A positive Widal test in a patient with confirmed malaria should not therefore be considered as suggestive of typhoid fever.

Further reading:

  1. Hanscheid T, Melo-Cristino J, Pinto BG. Automated detection of malaria pigment in white blood cells for the diagnosis of malaria in Portugal. Am J Trop Med Hyg.2001 May-Jun;64(5-6):290-2
  2. Ben-Ezra J, St. Louis M, Riley RS. Automated malarial detection with the Abbott Cell-Dyn 4000 hematology analyzer. Lab Hematol. 2001;7:61-64
  3. Kramer B, Grobusch MP, Suttorp N et al. Relative frequency of malaria pigment-carrying monocytes of nonimmune and semi-immune patients from flow cytometric depolarized side scatter. Cytometry. 2001 Oct 1;45(2):133-40.
  4. Demirev PA, Feldman AB, Kongkasuriyachai D et al. Detection of malaria parasites in blood by laser desorption mass spectrometry. Anal Chem 2002 Jul 15;74(14):3262-6
  5. Mann M. Mass tool for diagnosis. Nature 2002 Aug 15;418(6899):731-2

 ©malariasite.com ©BS Kakkilaya | Last Updated: Mar 10, 2015

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