Malaria Blood Smear Remains the gold standard for diagnosis Giemsa stain distinguishes between species and life cycle stages parasitemia is quantifiable Threshold of detection thin film: 100 parasites/l thick film: 5 -20 parasites/l Requirements: equipment, training, reagents, supervision Simple, inexpensive yet labor-intensive Accuracy depends on laboratorian skill

Interpreting Thick and Thin Films THICK FILM lysed RBCs larger volume 0.25 μl blood/100 fields blood elements more concentrated good screening test positive or negative parasite density more difficult to diagnose species THIN FILM fixed RBCs, single layer smaller volume 0.005 μl blood/100 fields good species differentiation requires more time to read low density infections can be missed

Malaria Blood Smear Prepare smears as soon as possible after collecting venous blood to avoid Changes in parasite morphology Staining characteristics Take care to avoid fixing the thick smear Risk of fixing thick when thin is fixed with methanol if both smears on same slide Let alcohol on finger dry to avoid fixing thick Be careful if drying with heat

Collection of Blood Smears 1. The second or third finger is usually selected and cleaned. 4. Slide must always be grasped by its edges. 5. Touch the drop of blood to the slide from below. 2. Puncture at the side of the ball of the finger. 3. Gently squeeze toward the puncture site.

Preparing thick and thin films 4. Carry the drop of blood to the first slide and hold at 45degree angle. 1. Touch one drop of blood to a clean slide. 2. Spread the first drop to make a 1 cm circle. 5. Pull the drop of blood across the first slide in one motion. 3. Touch a fresh drop of blood to the edge of another slide. 6. Wait for both to dry before fixing and staining.

Recognizing Malaria Parasites Blue cytoplasm Inside a red blood cell One or more red chromatin dots

Recognizing Erythrocytic Stages: Schematic Morphology RING TROPHOZOITE SCHIZONT GAMETOCYTE Blue Cytoplasm Red Chromatin Brown Pigment

Malaria Parasite Erythrocytic Stages Ring form Trophozoite Schizont Gametocytes

Plasmodium falciparum Infected erythrocytes: normal size M I Gametocytes: mature (M)and immature (I) forms (I is rarely seen in peripheral blood) Rings: double chromatin dots; appliqué forms; multiple infections in same red cell Schizonts: 8-24 merozoites (rarely seen in peripheral blood) Trophozoites: compact (rarely seen in peripheral blood)

Plasmodium vivax Infected erythrocytes: enlarged up to 2X; deformed; (Schüffner’s dots) Rings Trophozoites: ameboid; deforms the erythrocyte Schizonts: 12-24 merozoites Gametocytes: round-oval

“malariae - like parasite in vivax - like erythrocyte” Plasmodium ovale Infected erythrocytes: moderately enlarged (11/4 X); fimbriated; oval; (Schüffner’s dots) “malariae - like parasite in vivax - like erythrocyte” Trophozoites: compact Rings Gametocytes: round-oval Schizonts: 6-14 merozoites; dark pigment; (“rosettes”)

Plasmodium malariae Infected erythrocytes: size normal to decreased (3/4X) Trophozoite: typical band form Trophozoite: compact Schizont: 6-12 merozoites; coarse, dark pigment Gametocyte: round; coarse, dark pigment

Species Differentiation on Thin Films

Species Differentiation on Thin Films

Species Differentiation on Thick Films