Microscopic Procedures for Diagnosing Malaria
To establish the diagnosis of malaria, a blood film must be prepared from fresh blood obtained by pricking a patient's finger with a sterile, nonreusable lancet (Figure A-1). Two types of blood films can be used: thin films (as used for hematology) and thick films. Thick and thin films can be made as separate or as combination slides (Figure A-2). Thick blood films are more sensitive in detecting malaria parasites because the blood is concentrated, allowing a greater volume of blood to be examined. However, thick films are more difficult to read.
The thin film should be air-dried, fixed with methanol, and allowed to dry before staining; the thick film also should be thoroughly dried but stained without fixation. For best staining results, blood films should be stained with a 2.5% Giemsa solution (pH of 7.2) for 45 minutes (alternate: 7.5% Giemsa for 15 minutes). A combined Wright-Giemsa stain can also detect malaria parasites but does not demonstrate Schüffner's dots as reliably as Giemsa.
Plasmodium parasites are always intracellular, and they demonstrate, if stained correctly, blue cytoplasm with a red chromatin dot. Common errors in reading malaria films can be caused by platelets overlying a red blood cell, concern regarding missing a positive slide, and misreading of artifacts as parasites. In P. falciparum infections, the parasite density should be estimated by counting the percentage of red blood cells infected (not the number of parasites) under an oil immersion lens on a thin film.
Persons suspected of having malaria, but whose blood films do not indicate the presence of parasites, should have blood films repeated approximately every 12--24 hours for 3 consecutive days. If films remain negative, then the diagnosis of malaria is unlikely. A useful complement to microscopy can be found in polymerase chain reaction (e.g., when microscopy fails to determine parasite species or for confirming negative blood smears). Additional information regarding collection and preparation of blood films is available at CDC's Division of Parasitic Diseases Internet site, DPDx --- Laboratory Identification of Parasites of Public Health Concern (http://www.dpd.cdc.gov/DPDx).
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