Rapid Malaria Diagnosis
By Mary-Louise Scully, MD
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A previously healthy 6-year-old boy who recently immigrated to the United States from Honduras presented with a history of fever, abdominal pain, and headache. Although initially afebrile, while in the emergency department his axillary temperature reached 41.6°C and he vomited 3 times. His physical exam was remarkable for a palpable spleen tip and a liver that extended 1 cm below the right costal margin. A routine white blood cell (WBC) count was performed by use of the Cell-Dyn 4000 automated blood-cell analyzer (Abbott) giving the scattergram shown in Figure 1.
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In the process of obtaining a routine WBC count, red blood cells (RBCs) are lysed and free malarial parasites were then detected as a distinct population on the basis of light-scattering traits, such as cytoplasmic and nuclear optical characteristics (ie, size, granularity, lobularity, and complexity). The malaria organisms in Figure 1 appear as a population that is slightly smaller than lymphocytes with a low angle of scatter (1'- 3') but greater granularity and surface complexity as shown by the pattern of orthogonal scatter (90'). P vivax malaria was confirmed in this patient by examination of Wright-Giemsa-stained slides.
Comment by Mary-Louise Scully, MD
Automated blood cell analyzers can contribute to the detection of malaria, especially in cases when there is no clinical suspicion. The Abbott, CD 3500 blood-cell analyzer has been reported to detect levels of parasitemia as low as 1500 parasites/µl. However, an experienced pathologist can detect as few as 5-20 parasites/µl of blood using light microscopy of Giemsa-stained blood films. Therefore, it should be emphasized that the automated blood cell analyzer is not an appropriate screening test for malaria, but may play a role in situations where malaria is not suspected.
The careful examination by a trained microscopist of a well-prepared stained blood film remains the "gold standard" of malaria diagnosis having excellent sensitivity, the ability to characterize all 4 Plasmodium species as well as being used to quantify parasitemia. The disadvantages of the Giemsa-stained thick blood films (G-TBF) are that it is labor-intensive, and its success depends on having well trained microscopists. PCR has even greater sensitivity than the G-TBF as shown by several authors who reviewed PCR positive/G-TBF negatives and confirmed that "false"positives were in fact true positives.1 Some experts now question if indeed the G-TBF is still the proper yardstick by which to measure all other tests.
In a recent review, Moody defines a rapid diagnostic test for malaria as a method that requires 1 hour or less.2 Fluorescent dyes such as acridine orange (AO) and benzothiocarboxypurine (BCP) have affinity for the nucleic acid in the parasites’ nucleus. Which will exhibit an apple green or yellow fluorescence when excited by UV light at a wavelength of 490 nm. The centrifugal quantitative buffy coat or QBC II (QBC, Becton Dickinson) combines an AO-coated capillary tube and an internal float to separate layers of granulocytes and platelets using centrifugation. Parasites usually concentrate in the granulocyte-erythrocyte interface and can be viewed using a long-focal-length objective (paralens) with a fluorescent microscope. Results of experimental and field studies to assess the sensitivity of QBC have varied, but the majority show sensitivities of more than 90%. The largest study of 18,845 blood samples showed a positivity rate of 25% for QBC (100% sensitivity) and 18% for G-TBF.3
The major advantage of the QBC is its speed and relative ease of interpretation. The disadvantages would be the need for electricity, the cost of capillary tubes and equipment, and the difficulty in species identification and quantification. An added benefit of the QBC may lie in its ability to diagnose other diseases in the febrile patient such as relapsing fever, African trypanosomiasis, and filariasis.4-6 The QBC also gives the clinician in the field a simultaneous hematocrit and platelet count. These are extremely useful data since anemia and thrombocytopenia are important indicators of severe illness.
Another fluorescent microscopy technique is the Kawamoto technique in which a fluorescent microscope is fitted with an interference filter and AO is used to stain thin blood films. Expertise is needed to distinguish parasites from the Howell-Jolly bodies since they will stain with AO as well. Another fluorochrome technique using a solution of benzothiocarboxypurine (BCP) applied to an unfixed, dry, thick blood film has a reported sensitivity and specificity of greater than 95% for P falciparum. This method overcomes the necessity for rapid examination that is often needed in other fluorescence techniques.
PCR, although strictly not a rapid method, is certainly going to play an important future role in malaria diagnosis, perhaps even modifying our "gold standard." The value of PCR lies in its excellent sensitivity (able to detect £ 5 parasites/µl of blood) and its ability to detect all species of Plasmodium in nested or multiplex assays. PCR-based methods are useful for malaria studies on strain variation, mutations, and the study of parasite genes involved in drug resistance. As PCR technology improves, this technique may be able to be performed fast enough to be more useful to the clinician.
A recent congress of the World Health Organization produced a document entitled New Perspectives in Malaria Diagnosis. In this document, the term rapid diagnostic tests (RDTs) was restricted to immunochromatographic methods to detect Plasmodium-specific antigens in a fingerprick blood sample.7 These tests can be performed in about 15 minutes by persons with minimal training and require no electricity or special equipment. These tests often have a test strip or dipstick bearing monoclonal antibodies directed against the target parasite antigen (see Table).
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One antigen targeted is the histidine-rich protein II (HRP-II), a water-soluble protein produced by trophozoites and young (not mature) gametocytes of P falciparum. Three commercially available HRP-II antigen dipstick tests with significant published data are the Parasite F, ICT Pf, and the Path Falciparum Malaria IC tests. Sensitivities of these kits are generally more than 90% at parasite densities greater than 100 parasites/µl of blood. Below this level, sensitivities fall. False negatives may be due to a gene deletion for the production of HRP-II. Therefore, a strong positive with a test such as ICT Pf is highly predictive of P falciparum parasitemia, but a negative test should not exclude the diagnosis of malaria.8
These 3 RDTs using HRP-II will not detect nonfalciparum malaria. False positives with rheumatoid factor may occur, though less frequently with the monoclonal IgM antibodies used in the ICT Pf and Path Falciparum Malaria tests. A major disadvantage of RDTs using HRP-II antigens is that in many patients the tests remain positive for 7-14 days following treatment—results that may be confused with drug resistance or treatment failure.
Parasite lactate dehydrogenase (pLDH) is a soluble glycolytic enzyme produced by asexual and sexual stages (gametocytes) of all 4 species of malaria parasites. The OptiMAL tests for P vivax and P falciparum uses 3 monoclonal antibodies that can bind to active pLDH. Two of the antibodies are panspecific, recognizing all 4 malaria species while the other detects only for P falciparum. These monoclonal antibodies do not seem to cross-react with LDH from other organisms such as pathogenic bacteria, fungi, Leishmania, or Babesia spp. In one series, the sensitivity of the OptiMAL for P falciparum was 88% and for P vivax 94%.9 Lower sensitivities are found at levels of parasitemia less than 100 parasites/ml or for P ovale and P malariae. In the future, a more sensitive monoclonal antibody may help improve detection of P ovale and P malariae.
Clearance of parasites from the blood during therapy for malaria correlates with a fall in pLDH levels that can be detected with the OptiMAL test. Therefore, tests to detect or measure pLDH might play a future role in monitoring response to therapy, especially in areas where blood films are not easily available.
Aldolase is another enzyme in the glycolytic cycle of the malaria parasite. In the ICT Pf/Pv test, panspecific monoclonal antibodies against Plasmodium aldolase are combined in a test with HRP-II to detect P vivax and P falciparum. Results for P vivax have been disappointing at lower levels of parasitemia. The ICT Pf/Pv had 96% sensitivity for P vivax if there were more than 500 parasites/µl of blood but had only 29% sensitivity for parasite levels less than 500 parasites/ml.10
According to the WHO document, an ideal RDT for malaria should 1) detect all 4 species of malaria at least as accurately as microscopy; 2) have a sensitivity of 100% for levels of 100 parasites/µl (0.002% parasitemia); 3) have a specificity of at least 90% for all species; and 4) provide quantitative information on parasite density. Also these test kits should not require refrigeration, be reliable in extreme heat, and have a shelf life of 1-2 years. Further testing in the field and technical improvements are still needed, but someday these test kits may even play a role in the self-diagnosis of malaria in travelers to remote areas.
Dr. Scully is a member of the Group Health Cooperative of Puget Sound, Seattle, Wash.
References
1. Seesod N, et al. An integrated system using immunomagnetic separation, polymerase-chain reaction and colorimetric detection for the diagnosis of P falciparum. Am J Trop Med Hyg. 1997;56:322-328.
2. Moody A. Rapid diagnostic tests for malaria parasites. Clin Microbiol Rev. 2002;15(1):66-78.
3. Damodar SU. Evaluation of acridine-orange staining of centrifuged parasites in malarial infection. Indian J Med Sci. 1996;50(7):228-230.
4. Cobey FC, et al. Short report: Detection of Borrelia (relapsing fever) in rural Ethiopia by means of the quantitative buffy coat technique. Am J Trop Med Hyg. 2001;65(2):164-165.
5. Baily JW, et al. The use of acridine orange QBC technique in the diagnosis of African trypanosomiasis. Trans R Soc Trop Med Hyg. 1992;86(6):630.
6. Freedman DO, et al. Rapid diagnosis of Bancroftian filariasis by acridine orange staining of centrifuged parasites. Am J Trop Med Hyg. 1992;47(6):787-793.
7. World Health Organization. 2000. WHO/MAL/2000.1091. New Perspectives in Malaria Diagnosis. World Health Organization, Geneva, Switzerland.
8. Wongsrichanalai C, et al. Comparison of a rapid field immunochromatographic test to expert microscopy for the detection of Plasmodium falciparum asexual parasitemia in Thailand. Acta Trop. 1999;73:263-273.
9. Palmer CJ, et al. Evaluation of the OptiMAL test for rapid diagnosis of Plasmodium vivax and Plasmodium falciparum malaria. J Clin Microbiol. 1998;36: 203-206.
10. Tjitra E, et al. Field evaluation of the ICT malaria Pf/Pv immunochromatographic test for detection of Plasmodium falciparum and Plasmodium vivax in patients with presumptive clinical diagnosis of malaria in eastern Indonesia. J Clin Microbiol. 1999;37: 2412-2417.
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