1. Malaria Diagnostics Gail Stennies, M.D., M.P.H. Medical Officer Malaria Epidemiology Branch DPD/ NCID/ CDC May, 2002

2. Malaria Diagnosis Clinical Diagnosis Malaria Blood Smear Fluorescent microscopy Antigen Detection Serology Polymerase Chain Reaction

3. Clinical Diagnosis Hyperendemic and holoendemic areas Laboratory resources not needed Fever or history of fever Sensitivity ranges from poor to high Often has poor specificity and predictive values Overlap with other syndromes

4. 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

5. 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

6. 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

7. 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.

8. 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.

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

10. RINGTROPHOZOITE SCHIZONTGAMETOCYTE Blue Cytoplasm Red Chromatin Brown Pigment Recognizing Erythrocytic Stages: Schematic Morphology

11. Malaria Parasite Erythrocytic Stages Ring form Trophozoite Schizont Gametocytes

12. 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 MI

13. 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)

14. Plasmodium ovale Infected erythrocytes: moderately enlarged (1 1/4 X); fimbriated; oval; (Schüffner’s dots) “malariae - like parasite in vivax - like erythrocyte” Rings Trophozoites: compact Schizonts: 6-14 merozoites; dark pigment; (“rosettes”) Gametocytes: round-oval

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

16. Species Differentiation on Thin Films

18. Species Differentiation on Thick Films

19. 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

20. 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

21. 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

22. 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.

23. 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.

24. 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

25. Fluorescent Microscopy Modification of light microscopy Fluorescent dyes detect RNA and DNA that is contained in parasites Nucleic material not normally in mature RBCs Kawamoto technique –Stain thin film with acridine orange (AO) –Requires special equipment – fluorescent microscope –Staining itself is cheap –Sensitivities around 90%

26. Quantitative Buffy Coat (QBC ®) Fluorescent microscopy after centrifugation AO-coated capillary is filled with 50-100 µl blood Parasites concentrate below the granulocyte layer in tube May be slightly more sensitive than light microscopy but some reports of 55-84%

27. Quantitative Buffy Coat (QBC ®) Useful for screening large numbers of samples Quick, saves time Requires centrifuge, special stains 3 main disadvantages –Species identification and quantification difficult –High cost of capillaries and equipment –Can’t store capillaries for later reference

28. Malaria Serology – antibody detection Immunologic assays to detect host response Antibodies to asexual parasites appear some days after invasion of RBCs and may persist for months Positive test indicates past infection Not useful for treatment decisions

29. Malaria Serology – antibody detection Valuable epidemiologic tool in some settings Useful for –Identifying infective donor in transfusion-transmitted malaria –Investigating congenital malaria, esp. if mom’s smear is negative –Diagnosing, or ruling out, tropical splenomegaly syndrome –Retrospective confirmation of empirically-treated non-immunes

30. Malaria Antigen Detection Immunologic assays to detect specific antigens Commercial kits now available as immunochromatographic rapid diagnostic tests (RDTs), used with blood P. falciparum histidine-rich protein 2 (PfHRP-2) parasite LDH (pLDH) Monoclonal and polyclonal antibodies used in antigen (Ag) capture test Species- and pan-specific Ab Cannot detect mixed infections Cross reactivity with rheumatoid factor reportedly corrected

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

32. 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)

33. Malaria Antigen Detection - RDTs FeaturePfHRP-2 testspLDH tests Test principle Use of monoclonal (Ab) Detects a histidine rich protein of P.f. Water-soluble protein is released from parasitized RBCs Not present in mature gametocytes Use of monoclonal and polyclonal Ab Detects a parasite enzyme, lactate dehydrogenase pLDH is found in sexual and asexual forms Differentiation between malarial species is based on antigenic differences between pLDH isoforms

34. Malaria Antigen Detection - RDTs FeaturePfHRP-2 testspLDH tests AdvantagesThreshold for parasite detection as low as 10 parasites/µl (but sensitivity drops at < 100 parasites /µl) Does not cross react with other species – P.v., P.o., P.m. Threshold for parasite detection ≥ 100 parasites/µl Can detect all species which infect humans Can differentiate between P.f. and non-falciparum malaria Does not cross react with human LDH Positive only in viable parasites, potentially useful for monitoring success of treatment

35. Malaria Antigen Detection - RDTs FeaturePfHRP-2 testspLDH tests Disadvan- tages Some tests only detect P.f. Cannot detect mixed infections Sensitivity and specificity decreases < 100 parasites/µl Can remain positive up to 14 days post treatment, in spite of asexual and sexual parasite clearance, due to circulating antigens Cannot differentiate between non-falciparum species Cannot detect mixed infections Sensitivity and specificity decreases < 100 parasites/µl

36. Malaria Antigen Detection - RDTs FeaturePfHRP-2 testspLDH tests Sensitivity/ Specificity* Sensitivity 92-100% Specificity 85- 100% Sensitivity P.f. 88-98% P.v. 89-94% Specificity P.f. 93-99% P.v. 99-100% Commercial cost/test** Approximately US$ 0.60 –1.00Approximately US$ 2.50 Commercial products 1)PATH falciparum Malaria IC Strip test – Program for Appropriate Technology in Health 2)MAKROmed™ 3)Orchid ® 1)OptiMAL® - Flow, Inc. 2)Binax NOW ®ICT Malaria - Binax, Inc. * Compared to microscopy, results from multiple studies ** Varies by size of order and vendor

37. Polymerase Chain Reaction (PCR) Molecular technique to identify parasite genetic material Uses whole blood collected in anticoagulated tube (200 µl) or directly onto filter paper (5 µl) –100% DNA is extracted –10% blood volume used in PCR reaction

38. Polymerase Chain Reaction (PCR) Threshold of detection at CDC –0.1 parasite/µl if whole blood in tube –2 parasites/µl if using filter paper Definitive species-specific diagnosis now possible Can identify mutations – try to correlate to drug resistance Parasitemia not quantifiable May have use in epidemiologic studies Requires specialized equipment, reagents, and training

39. Real-Time PCR New technique based on fluorescence Promising because it has potential to quantify parasitemia, decreases contamination, may detect multiple wavelengths in same tube identifying multiple species in one run, saves hands-on time Needs further research and validation for malaria

40. Real-Time PCR

41. Quantitative Real-Time PCR

43. Preventing Transfusion-Transmitted Malaria (TTM) Detection of Parasites/Parasite Products Parasite densities (parasites/  l) 10 3 10 10 -1 10 -3 10 -5 PCR (0.05 to 0.1 parasites/  l) Microscopy (5 parasites/  l) Antigen detection (10 to 100 parasites/  l)

44. Preventing TTM: Detection of Parasites/Parasite Products Parasite densities (parasites/  l) 10 3 10 10 -1 10 -3 10 -5 PCR (0.05 to 0.1 parasites/  l) Microscopy (5 parasites/  l) Antigen detection (10 to 100 parasites/  l) 10 parasites/unit (2.5 X 10 -5 /  l) 100 parasites/unit (25 X 10 -5 /  l) Detection of 10 parasites/unit requires a sensitivity: -4,000 times better than PCR -200,000 times better than microscopy

45. Mass Screening for Malaria in Populations for Resettlement Blood smear examinations to detect asymptomatic parasitemia Not useful for predicting individual risks undetectable parasitemias dormant liver phase parasites Can be used to make a decision about the need for mass treatment of the entire group

46. Issues in application of diagnostics Roll Back Malaria objective – At least 60% of those suffering from malaria have prompt access to and are able to use correct, affordable and appropriate treatment within 24 hours of the onset of symptoms Cost should not focus on unit cost alone Must put in context of case management –Amount of drugs being inappropriately dispensed –Increasing drug resistance –Increasingly costly, complex, and toxic alternative drugs –Epidemiology of malaria, populations served –Provider and patient acceptability, esp. of negative results

47. Issues in application of diagnostics Rapid diagnostic tests have potential to complement conventional microscopy or provide a diagnostic modality when none is available Operational research is needed to evaluate best uses and cost effectiveness Potential uses –Epidemics and emergencies –Inadequate or absent lab services, unskilled staff –Mobile clinics –Low transmission areas; areas with high levels of drug resistance –Epidemiologic surveys, seroprevalence data