Appendix -- Microscopic Procedures for Diagnosing Malaria
To establish the diagnosis of malaria, a blood smear must be
prepared
from fresh finger-prick blood ((Figure_A1) and
(Figure_A2)). * The
thin smear is fixed in methanol before staining; the thick smear is
stained
unfixed. Many hospitals have a Wright-Giemsa stain available, which
is
acceptable; however, Wright stain alone will not reliably stain
Plasmodium
parasites. For best results, the smear should be stained with a 3%
Giemsa
solution (pH of 7.2) for 30-45 minutes. 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 on a thin film.
Thick blood smears are more sensitive in detecting malaria parasites because the blood is concentrated, allowing a greater volume of blood to be examined. However, thick smears are more difficult to read, and thin smears may be preferred by laboratories that have limited experience. Plasmodium parasites are always intracellular, and they demonstrate, if stained correctly, blue cytoplasm with a red chromatin dot. Common errors in reading malaria smears are caused by platelets overlying a red blood cell, concerin about missing a positive slide, and misreading artifacts as parasites. Persons suspected of having malaria but whose blood smears do not demonstrate the presence of parasites should have blood smears repeated approximately every 12-24 hours for 3 consecutive days. If smears remain negative, then the diagnosis of malaria is unlikely.
For rapid diagnosis, make the thick and thin films on separate slides. Air dry the thin film, fix it with methyl alcohol, and immediately stain it. If no parasites are found on the thin film, wait until the thick film is dry and examine it for organisms that may not have been detected on the thin preparation.
In Figures A1 and A2, the hands are shown ungloved to better illustrate their placement during the procedures. However, wearing gloves while processing blood specimens is recommended to prevent transmission of Figure_A1
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