The document discusses various methods for diagnosing malaria including clinical diagnosis, blood smears, antigen detection, and serology tests. Blood smears, whether thick or thin, remain the gold standard for diagnosis as they allow for species identification and quantification of parasitemia. Rapid diagnostic tests (RDTs) that detect malaria antigens are now widely used and provide results comparable to microscopy. Quantitative Buffy coat analysis and PCR methods can also detect malaria but have some limitations. Clinical diagnosis alone is not very accurate and requires laboratory confirmation.

1. Malaria Diagnostics

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

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

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

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

13. Malaria Parasite Erythrocytic Stages Ring form Trophozoite Schizont Gametocytes

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

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

21. Species Differentiation on Thin Films

22. Parasitemia and clinical correlates Parasitemia Parasites /  l Remarks 0.0001-0.0004% 5-20 Sensitivity of thick blood film 0.002% 100 Patients may have symptoms below this level, where malaria is seasonal 0.2% 10,000 Level above which immunes show symptoms 2% 100,000 Maximum parasitemia of P.v. and P.o.

23. Parasitemia and clinical correlates *WHO criteria for severe malaria are parasitemia > 10,000 /  l and severe anaemia (haemaglobin < 5 g/l). Prognosis is poor if > 20% parasites are pigment containing trophozoites and schizonts (more mature forms) and/or if > 5% of neutrophils contain visible pigment. H änscheid T. (1999) Diagnosis of malaria: a review of alternatives to conventional microscopy. Clin Lab. Haem. 21, 235-245. Parasitemia Parasites/  l Remarks 2-5% 100,000-250,00 Hyperparasitemia/severe malaria*, increased mortality 10% 500,000 Exchange transfusion may be considered/ high mortality

29. The QBC Test

33. RDT Test Format

34. Examples of RDTs: Para Sight F test OptiMal Assay Kit OptiMal assay Result

35. Detection of Plasmodium antigens: pLDH (parasite lactate dehydrogenase)

36. Detection of Plasmodium antigens A: HRP-2 (histidine-rich protein 2) (ICT) B: pLDH (parasite lactate dehydrogenase)(Flow) C: HRP-2 (histidine-rich protein 2) (PATH)

38. THE END T H A N K S