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1 New methods for the diagnosis of malaria Tom van Gool Academic Medical Centre, Amsterdam Rogier van Doorn

2 The importance of malaria diagnosis Without vaccines being available the major strategy to combat malaria is currently prompt diagnosis, treatment and prevention.

3 Fast diagnosis: low parasitaemia, low risk for complications!! Number parasites per ul blood (%) (10) (9) (8) (7) (6) (5) (4) (3) (2) (1) risk of death Increasing level of parasitaemia P. falciparum 2%: maximum parasitaemie P. vivax, P. ovale en P. malariae Infection Start complaints days

4 Differences in morphology with microscopy make the species!

5 New(er) tests for malaria diagnosis Quantitative Buffy Coat (QBC) Automatic analysers (CD4000) Polymerase Chain Reaction (PCR) Antigen detection (RDT)

6 QBC Quantitative Buffy Coat

7 QBC = Quantitative Buffy Coat capillary tube internally coated with acridine orange nucleus and cytoplasm of malaria parasites are stained with acridine orange Buffy coat and erythrocytes are compressed by float in small space beneath wall of tube

8 Sampling blood for QBC

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14 QBC-tube in fluorescence microscopy Buffy coat Concentration of parasites.

15 Parasites are easy and clearly recognizable!

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17 Summary QBC in routine practice: Excellent screening method for malaria: - fast results (5 min) - eexcellent sensitivity (1 parasite/ul) - high specificity (100%) Disadvantage: no recognition of different species. Investment in apparatus

18 Sensitivity of different microscopic techniques, in routine clinical practice Thick smear, thin smear and QBC

19 Techniques studied (I): Thick smear: Giemsa and Fields stained Thick smear study-time 100 fields (1000x): 0.3 ul blood/ 3 min. 200 fields (1000x): 0.6 ul blood/ 6 min. 400 fields (1000x): 1.2 ul blood/ 12 min. 800 fields (1000x): 2.4 ul blood/24 min. (= Gold standard)

20 Techniques studied II Thin smear (Diff Quick) 200 fields (1000 x), 5 min.

21 Techniques studied III QBC (Netherlands) 2 lanes: 5 min. search 2x

22 Study site Total no. cases studied Cases without malaria* Cases with asexual stages of malaria* P. falciparum P. vivax P. ovale P. malariae Study 1 Netherlands Study 2 Surinam *: according to Giemsa thick smear 800 fields (Gold Standard) # not further included in study analysis Patients included in the study: Netherlands and Surinam Gold standard: 800 fields 1000 x Giemsa

23 Sensitivity different microscopic techniques in Netherlands Results GTS800 fields "Gold standard" 100 fields (3 min) Field's thick smear 200 fields (6 min) 400 fields (12 min) 100 fields (3 min) Results with different techniques Giemsa thick smear 200 fields (6 min) 400 fields (12 min) 5 min Giemsa thick smear 100 fields Giemsa thick smear 200 fields Giemsa thick smear 400 fields P. falciparum P. vivax P. ovale P. malariae Sp. Unknown All species (total) Sensitivity 100% 98,2% 98,2% 99,1% 98,2% 100% 100% 93,6% 98,2% 100% 100% 100% Thick smear Fields 200 fields: 98% Thin smear Thin smear combined with: Thick smear Giemsa 200 fields: 100% QBC 100% QBC

24 Conclusions Thick smear Giemsa 200 fields (1000 x, = 6 min) good standard (100% sensitivity in Netherlands) for excluding malaria (WHO advise.: 100 fields) Less experience of technicians?: 400 fields (=12 min) Giemsa performs slightly better as Fields stain Fields faster and better conservation of parasite morphology Thin smear 5 min standalone surprisingly good sensitivity (93-94 %)!

25 New(er) tests for malaria diagnosis Quantitative Buffy Coat (QBC) Automatic analysers (CD4000) Polymerase Chain Reaction (PCR) Antigen detection (RDT)

26 The Cell-Dyn 4000 haematology analyser RBC measurements reticulocyte measurements absolute WBC count WBC differential counts WBC viability measurements platelet analysis many more complex things detection of malaria?? Abbott

27 Results microscopy Microscopic diagnosis in 112 patients: 46 x P. falciparum malaria 25 x parasitemia 0.5% 21 x parasitemia < 0.5% 11 x P. vivax or P.ovale 55 x no malaria

28 Results Cell-Dyn 4000 Specificity 96% Overall sensitivity (all species): 63% Sensitivity P. falciparum > 0.5%: 96%!

29 Molecular detection of Plasmodium spp. - Correct identification of species, also quantative - Possibility of drug resistance testing - Increased sensitivity (equal as QBC)

30 Serology Not for diagnosis in a case suspected of malaria! Useful for epidemiological studies and screening blood donors Good test: (Newmarket EIA): 3 recombinant antigens of P. falciparum and one for P. vivax Sensitivity acute phase: P. falciparum (83%), P. vivax (85%) and P. ovale (70%) Vox Sanguinis (2004), 87, Kitchen et al.

31 New(er) tests for malaria diagnosis Quantitative Buffy Coat (QBC) Automatic analysers (CD4000) Polymerase Chain Reaction (PCR) Antigen detection (RDT)

32 Antigen-detection for malaria Fast method (5-15 min) Easy to use, no specific expertise needed No specific apparatus needed

33 Antigens used in current tests: 1) HRPII (Histidine Rich Protein II, P. falciparum) 2) Aldolase (present in all species) 3) Parasitic Lactate Dehydrogenase (pldh) - a) pldh specific for P. falciparum - b) pldh specific for P. vivax - c) pan-pldh: present in all species

34 ICT NOW Malaria and ICT Combo Cassette Combo cassette Positive control P.falciparum HRP II Aldolase

35 OptiMal-IT Positive control Pan malaria LDH P.falciparum LDH

36 Palutop +4 and Core Malaria Positive control Pan malaria LDH P.vivax LDH P.falciparum HRP II

37 Experience with antigen tests (RDT) from literature Rapid, fast and reliable Senstivity from % Specificity from % So.

38 Is with the new tests classical microscopy outdated? Remote areas in tropics? Places in tropics with some basic infrastructure? Laboratories in tropics with good infrastructure..? Laboratories in western countries at daytime.? Laboratories in western countries at night shifts..?

39 Study towards use of dipsticks in routine clinical practice in the Netherlands and Surinam

40 Microscopic methods used studied (I): Thick smear (Giemsa and Fields stained,1000 x) Thin smear (Diff Quick) 200 fields (1000 x), 5 min. QBC

41 ICT-NOW Netherlands HRPII and aldolase Optimal 48/IT Netherlands pldh Pf pldh Ps Combo Surinam HRPII and aldolase Core Surinam HRPII pldh Pf pldh Pv pldh Pan

42 Study site Total no. cases studied Cases without malaria* Cases with asexual stages of malaria* P. falciparum P. vivax P. ovale P. malariae Study 1 Netherlands Study 2 Surinam *: according to Giemsa thick smear 800 fields (Gold Standard) # not further included in study analysis Patients included in the study Gold standard: 800 fields 1000x Giemsa

43 Sensitivity and specificity antigen tests

44 Results of GTS 800 fields "Gold standard" Study 1 N L Sensitivity of antigen tests Defense NL Study 2 Sur Study 1 Netherlands Study 2 Surinam ICT Optimal Combo Core P. falciparum (98,8%) 72 (87,8%) 74 (98,7%) 66 (88,0%) P. vivax (75,0%) 20 (100%) 47 (95,9%) 45 (91,8%) P. ovale (20,0%) 2 (40,0%) 0 0 P. malariae (100%) 1 (100%) 6 (75,0%) 6 (75,0%) All species (total) (90,8%) 95 (88,0%) 127 (96,2%) 117 (88,6%) ICT sensitivity over all 91%. P. falciparum: 99% P. vivax: 75% Surinam

45 Specificity of antigen tests A ntigen test C o untry P f P v, P o o r P m ICT NOW Surinam e /122 (98,4) 2 0 ICT NOW Netherlands /272 (97,8) 5 1 Com bo Surinam e /122 (99,1) 1 0 Com bo Netherlands /272 (98,5) 3 1 Optim al Surinam e 12 12/12 (100) 0 0 Optim al Netherlands /272 (98,9) 2 1 * according to gold standard: GTS 800 N o.cases micro sco pic negative fo r asexual stages o f malaria N o. cases negative in antigen tests (specificity %) N o. patients with false po sitive band

46 Combination of antigen test and microscopy: the best of two worlds?

47 Conclusions antigen tests Antigen tests easy to use, fast results Good sensitivity (less than QBC and Thick Smear) HRPII better for P.falciparum as specific pldh Aldolase and pldh good results with other species In general good specificity Can occasionally be false negative with high(er) parasitaemia Can occasionally be false positive

48 Conclusions antigen test and thin smear Combination of antigen tests and thin smear highly efficient: sensitivities: 97%- 99% With this combination no major mistakes can be made and parasitemia can reliable be calculated. Combination easy to perform, also in less experienced hands!

49 Can with an antigen test the parasitemia of P. falciparum be established?

50 Relation with aldolase band and parasitemia? HRPII and aldolase Study among 123 patients with P. falciparum with known parasitemia

51 (%) positive aldolase-band Positive Aldolase-band related to parasitemia in patients (n=123) with P. falciparum infection <500 (<0,1%) n= ,000 (0,01-0,02%) n=12 1,000-5,000 (0,02-0,1%) n=25 5,000-25,000 (0,1-0,5%) n=22 25,000-50,000 (0,5-1,0%) n=20 >50,000 (>1,0%) n=26 P. falciparum trofozoites µl (%)

52 Use of aldolase band for estimation parasitaemia P. falciparum infection Use of aldolase for calculation of parasitemia of P. fal ICT NOW Malaria Only HRPII (T1)? HRPII and aldolase positive? P. falciparum infection, parasitemia always lower as1%. Parasitaemia P. falciparum most likely higher as 1% but lower parasitaemia also possible

53 Persistence of positive bands for P. falciparum after treatment for malaria

54 Duration of positivity of the QBC/Thick Smear and Parasight-F after start of treatment with Halfan (H) or Riamet (C) (n:17) parasitaemia (parasites/100 leuco s) at start of therapy R H H H H H H H R H R H days 28 Parasight-F QBC/ Thick Smear Recrudescence No additional samples for Parasight F availabe R R R R R

55 Is with the new tests classical microscopy outdated? Remote areas in tropics yes. Places in tropics with some basic infrastructure? Laboratories in tropics with good infrastructure.. no Laboratories in western countries at daytime Laboratories in western countries at night shifts: antigen test and thin smear! no

56 End