Microscopy

The diagnosis of malaria is usually made by the examination of Giemsa-stained thick and thin blood smears for intraerythrocytic ring stage parasites using an oil immersion lens (magnification x 1000) (Figure 3.2A,B). Thick films made from a drop of blood dried on a microscope slide, then stained with water-based Giemsa stain allow concentration of parasites (with lysis of the red

Fig. 3.7 Mature parasite form in a patient with severe P. falciparum malaria cells). They are 20-40 times more sensitive than thin films in samples with low parasitaemia (depending on the expertise of the personnel staining and examining the films). Reasonable sensitivity can be achieved if fields containing 500-1000 leukocytes are examined for parasites or half an hour of examination is completed before deciding that parasites are undetectable. Thin smears are fixed with anhydrous methanol to preserve parasite and erythrocyte morphology, and are used to differentiate parasite species as well as to quantify the percentage of infected erythrocytes (Figure 3.3). The yield from blood films may be highest at or near the peak of fever but blood should be collected when the diagnosis is first considered rather than waiting for the next febrile episode, since patients may be afebrile at presentation. A direct smear from intradermal blood is sensitive in skilled hands but if anticoagulants are used, smears should be prepared within 3 hours, as parasite and red cell morphology may deteriorate with prolonged exposure to the anticoagulant (Ree and Sargeaunt, 1976).

The method of estimation of parasitaemia is shown in Table 3.5. An accurate assessment of parasitaemia is an important prognostic indicator in P. falciparum infections and is required to monitor response to therapy. Immature and mature asexual stages and sexual stages (gam-etocytes) are observed in the peripheral blood in P. vivax, P. ovale and P. malariae infections. In P. falciparum infections, immature asexual parasites are the usual finding (Figure 3.2). The presence of late trophozoites or schizonts in the peripheral blood is a predictor of mortality, with more than 10 000 mature trophozoites or schi-zonts/^ having a sensitivity of 90% and a specificity of 72% for mortality (Silamut and White, 1993; Warhurst and Williams, 1996) (Figure 3.7). Occasionally malaria parasites are detected in a bone marrow smear or, at autopsy, in a brain smear.

Identification of malaria parasites in blood smears may be difficult, and depends on the experience of the microscopist and the parasitae-mia in the peripheral blood at the time of blood collection. With synchronous replication, there may be very few or even no parasites present, despite severe complications from sequestered parasites. Semi-immunity, chemoprophylaxis with antimalarial drugs, and treatment with some antibiotics (tetracycline, azithromycin, clindamycin, trimethoprim-sulphamethoxazole, erythromycin and fluoroquinolones) may also have an effect on parasitaemia. Therefore, if malaria is suspected, thick and thin smears

Table 3.5 Estimation of parasitaemia in thick and thin blood films

Thick films1 Count the number of parasites per 200 leucocytes (WBC)2'3

„ . , , , , , Parasite count x WBC count4 Parasites/^l blood =-

Approximate % parasitaemia =

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