1. 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

2. 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

3. 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

4. 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.

5. 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.

6. Calculating Parasite Density - 1 Using 100X oil immersion lens, select area with 10-20 WBCs/field Count the number of asexual parasites and white blood cells in the same fields on thick smear Count ≥ 200 WBCs Assume WBC is 8000/  l (or count it) parasites/  l = parasites counted WBC counted X WBC count/  l

7. Calculating Parasite Density - 2 Count the number of parasitized and nonparasitized red blood cells (RBCs) in the same fields on thin smear Count asexual stages separately from gametocytes Count 500-2000 RBCs (fewer RBCs if parasitemia is high) % parasitemia = # parasitized RBCs total # of RBCs X 100

8. Calculating Parasite Density Can interconvert results in % parasitized RBCs and parasites /  l if you know the RBC or WBC counts If unknown, can assume 4,000,000 RBCs /  l or 8000 WBCs /  l

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

10. Parasitemia and clinical correlates Parasitemia Parasites/  l Remarks 2-5%100,000- 250,00 Hyperparasitemia/severe malaria*, increased mortality 10%500,000Exchange transfusion may be considered/ high mortality *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. Hänscheid T. (1999) Diagnosis of malaria: a review of alternatives to conventional microscopy. Clin Lab. Haem. 21, 235-245.

11. Estimating Parasite Density Alternate Method Count the number of asexual parasites per high-power field (HPF) on a thick blood film + 1-10 parasites per 100 HPF ++11-100 parasites per 100 HPF +++1-10 parasites per each HPF ++++> 10 parasites per each HPF