New methods for the diagnosis of malaria Tom van Gool Academic Medical Centre, Amsterdam Rogier van Doorn
The importance of malaria diagnosis Without vaccines being available the major strategy to combat malaria is currently prompt diagnosis, treatment and prevention.
Fast diagnosis: low parasitaemia, low risk for complications!! Number parasites per ul blood (%) 500.000 (10) 450.000 (9) 400.000 (8) 350.000 (7) 300.000 (6) 250.000 (5) 200.000 (4) 150.000 (3) 100.000 (2) 50.000 (1) risk of death Increasing level of parasitaemia P. falciparum 2%: maximum parasitaemie P. vivax, P. ovale en P. malariae Infection Start complaints days
Differences in morphology with microscopy make the species!
New(er) tests for malaria diagnosis Quantitative Buffy Coat (QBC) Automatic analysers (CD4000) Polymerase Chain Reaction (PCR) Antigen detection (RDT)
QBC Quantitative Buffy Coat
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
Sampling blood for QBC
QBC-tube in fluorescence microscopy............. Buffy coat Concentration of parasites.
Parasites are easy and clearly recognizable!
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
Sensitivity of different microscopic techniques, in routine clinical practice Thick smear, thin smear and QBC
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)
Techniques studied II Thin smear (Diff Quick) 200 fields (1000 x), 5 min.
Techniques studied III QBC (Netherlands) 2 lanes: 5 min. search 2x
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 383 272 109 83 20 5 1 Study 2 Surinam 331 195 132 75 49 0 8 *: 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
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 83 72 77 78 77 79 80 74 77 79 80 0 P. vivax 20 20 20 20 20 20 20 20 20 20 20 0 P. ovale 5 3 3 4 3 4 5 5 5 5 5 0 P. malariae 1 1 1 1 1 1 1 1 1 1 1 0 Sp. Unknown 0 11 6 5 6 5 3 2 4 4 3 109 All species (total) 109 107 107 108 107 109 109 102 107 109 109 109 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
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 %)!
New(er) tests for malaria diagnosis Quantitative Buffy Coat (QBC) Automatic analysers (CD4000) Polymerase Chain Reaction (PCR) Antigen detection (RDT)
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
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
Results Cell-Dyn 4000 Specificity 96% Overall sensitivity (all species): 63% Sensitivity P. falciparum > 0.5%: 96%!
Molecular detection of Plasmodium spp. - Correct identification of species, also quantative - Possibility of drug resistance testing - Increased sensitivity (equal as QBC)
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), 87,150-155. Kitchen et al.
New(er) tests for malaria diagnosis Quantitative Buffy Coat (QBC) Automatic analysers (CD4000) Polymerase Chain Reaction (PCR) Antigen detection (RDT)
Antigen-detection for malaria Fast method (5-15 min) Easy to use, no specific expertise needed No specific apparatus needed
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
ICT NOW Malaria and ICT Combo Cassette Combo cassette Positive control P.falciparum HRP II Aldolase
OptiMal-IT Positive control Pan malaria LDH P.falciparum LDH
Palutop +4 and Core Malaria Positive control Pan malaria LDH P.vivax LDH P.falciparum HRP II
Experience with antigen tests (RDT) from literature Rapid, fast and reliable Senstivity from 80-100% Specificity from 90-100% So.
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..?
Study towards use of dipsticks in routine clinical practice in the Netherlands and Surinam
Microscopic methods used studied (I): Thick smear (Giemsa and Fields stained,1000 x) Thin smear (Diff Quick) 200 fields (1000 x), 5 min. QBC
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
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 383 272 109 83 20 5 1 Study 2 Surinam 331 195 132 75 49 0 8 *: 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
Sensitivity and specificity antigen tests
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 83 75 82 (98,8%) 72 (87,8%) 74 (98,7%) 66 (88,0%) P. vivax 20 49 15 (75,0%) 20 (100%) 47 (95,9%) 45 (91,8%) P. ovale 5 0 1 (20,0%) 2 (40,0%) 0 0 P. malariae 1 8 1 (100%) 1 (100%) 6 (75,0%) 6 (75,0%) All species (total) 109 132 99 (90,8%) 95 (88,0%) 127 (96,2%) 117 (88,6%) ICT sensitivity over all 91%. P. falciparum: 99% P. vivax: 75% Surinam
Specificity of antigen tests A ntigen test C o untry P f P v, P o o r P m ICT NOW Surinam e 122 120/122 (98,4) 2 0 ICT NOW Netherlands 272 266/272 (97,8) 5 1 Com bo Surinam e 122 121/122 (99,1) 1 0 Com bo Netherlands 272 268/272 (98,5) 3 1 Optim al Surinam e 12 12/12 (100) 0 0 Optim al Netherlands 272 269/272 (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
Combination of antigen test and microscopy: the best of two worlds?
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
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!
Can with an antigen test the parasitemia of P. falciparum be established?
Relation with aldolase band and parasitemia? HRPII and aldolase Study among 123 patients with P. falciparum with known parasitemia
(%) positive aldolase-band 100 75 50 25 0 Positive Aldolase-band related to parasitemia in patients (n=123) with P. falciparum infection <500 (<0,1%) n=18 500-1,000 (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 (%)
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
Persistence of positive bands for P. falciparum after treatment for malaria
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 3700 2700 2000 1200 900 500 277 163 136 129 111 86 80 60 41 35 24 R H H H H H H H R H R H 2 7 14 21 days 28 Parasight-F QBC/ Thick Smear Recrudescence No additional samples for Parasight F availabe R R R R R
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
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