Armenia 2002 diagnostics.2


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Armenia 2002 diagnostics.2

  1. 1. Malaria Diagnostics Gail Stennies, M.D., M.P.H. Medical Officer Malaria Epidemiology Branch DPD/ NCID/ CDC May, 2002
  2. 2. Malaria Diagnosis• Clinical Diagnosis• Malaria Blood Smear• Fluorescent microscopy• Antigen Detection• Serology• Polymerase Chain Reaction
  3. 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. 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. 5. Interpreting Thick and Thin Films• THICK FILM • THIN FILM – lysed RBCs – fixed RBCs, single layer – larger volume – smaller volume – 0.25 μl blood/100 fields – 0.005 μl blood/100 fields – blood elements more – good species concentrated differentiation – good screening test – – requires more time to read positive or negative – parasite density – low density infections can – more difficult to diagnose be missed species
  6. 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. 7. Collection of Blood Smears 1. 4. The second or third Slide must always be finger is usually grasped by its edges. selected and cleaned. 2. 5. Puncture at the side Touch the drop of of the ball of the blood to the slide finger. from below. 3. Gently squeeze toward the puncture site.
  8. 8. Preparing thick and thin films 1. 4. Touch one drop of Carry the drop of blood blood to a clean to the first slide and hold slide. at 45 degree angle. 2. 5. Spread the first Pull the drop of blood drop to make a 1 across the first slide in cm circle. one motion. 3. 6. Touch a fresh drop Wait for both to of blood to the edge dry before fixing of another slide. and staining.
  9. 9. Recognizing Malaria Parasites Blue cytoplasmInside a red blood cell One or more red chromatin dots
  10. 10. Recognizing Erythrocytic Stages: Schematic Morphology Blue Cytoplasm RING Red TROPHOZOITE Chromatin Brown PigmentSCHIZONT GAMETOCYTE
  11. 11. Malaria Parasite Erythrocytic Stages Ring form Trophozoite Schizont Gametocytes
  12. 12. Plasmodium falciparum Infected erythrocytes: normal size M I Gametocytes: mature (M)and immature (I) forms (I is rarelyRings: double chromatin dots; appliqué forms; seen in peripheral blood) multiple infections in same red cell Schizonts: 8-24 merozoites (rarely seen in peripheral blood) Trophozoites: compact (rarely seen in peripheral blood)
  13. 13. Plasmodium vivax Infected erythrocytes: enlarged up to 2X; deformed; (Schüffner’s dots) Rings Trophozoites: ameboid; deforms the erythrocyteSchizonts: 12-24 merozoites Gametocytes: round-oval
  14. 14. Plasmodium ovaleInfected erythrocytes: moderately enlarged (11/4 X); fimbriated; oval; (Schüffner’s dots) “malariae - like parasite in vivax - like erythrocyte” Trophozoites: compact Rings Schizonts: 6-14 merozoites; Gametocytes: round-oval dark pigment; (“rosettes”)
  15. 15. Plasmodium malariae Infected erythrocytes: size normal to decreased (3/4X)Trophozoite: Trophozoite: Schizont: Gametocyte:compact typical 6-12 merozoites; round; coarse, band form coarse, dark pigment dark pigment
  16. 16. Species Differentiation on Thin Films Feature P. falciparum P. vivax P. ovale P. malariae Enlarged infected RBC + + Infected RBC shape round round, oval, round distorted fimbriated Stippling infected RBC Mauer clefts Schuffner Schuffner none spots spots Trophozoite shape small ring, large ring, large ring, small ring, appliqu amoeboid compact compact Chromatin dot often double single large single Mature schizont rare, 12-30 12-24 4-12 6-12 merozoites merozoites merozoites merzoites Gametocyte crescent shape large, large, compact, round round round
  17. 17. Species Differentiation on Thin Films P. falciparum P. vivax P. ovale P. malariaeRingsTrophozoitesSchizontsGametocytes
  18. 18. Species Differentiation on Thick Films Feature P. falciparum P. vivax P. ovale P. malariae Uniform trophozoites + Fragmented trophozoites ++ + Compact trophozoites + + Pigmented trophozoites + Irregular cytoplasm + + Stippling (“RBC ghosts”) + + Schizonts visible very rarely often often often Gametocytes visible occasionally usually usually usually
  19. 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/µl (or count it)parasites/µl = parasites counted X WBC count/µl WBC counted
  20. 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 X 100 total # of RBCs
  21. 21. Calculating Parasite Density• Can interconvert results in % parasitized RBCs and parasites /µl if you know the RBC or WBC counts• If unknown, can assume 4,000,000 RBCs /µl or 8000 WBCs /µl
  22. 22. Parasitemia and clinical correlatesParasitemia Parasites /µl Remarks0.0001-0.0004% 5-20 Sensitivity of thick blood film0.002% 100 Patients may have symptoms below this level, where malaria is seasonal0.2% 10,000 Level above which immunes show symptoms2% 100,000 Maximum parasitemia of P.v. and P.o.
  23. 23. Parasitemia and clinical correlatesParasitemia Parasites/µl Remarks2-5% 100,000- Hyperparasitemia/severe 250,00 malaria*, increased mortality10% 500,000 Exchange transfusion may be considered/ high mortality*WHO criteria for severe malaria are parasitemia > 10,000 /µl andsevere anaemia (haemaglobin < 5 g/l).Prognosis is poor if > 20% parasites are pigment containingtrophozoites and schizonts (more mature forms) and/or if > 5% ofneutrophils contain visible pigment.Hänscheid T. (1999) Diagnosis of malaria: a review of alternatives to conventionalmicroscopy. Clin Lab. Haem. 21, 235-245.
  24. 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. 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. 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. 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. 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. 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. 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. 31. Detection of Plasmodium antigens:pLDH (parasite lactate dehydrogenase)
  32. 32. Detection of Plasmodium antigensA: HRP-2 (histidine-rich protein 2) (ICT)B: pLDH (parasite lactate dehydrogenase)(Flow)C: HRP-2 (histidine-rich protein 2) (PATH)
  33. 33. Malaria Antigen Detection - RDTsFeature PfHRP-2 tests pLDH testsTest Use of monoclonal (Ab) Use of monoclonal andprinciple polyclonal Ab Detects a histidine rich protein Detects a parasite enzyme, of P.f. lactate dehydrogenase Water-soluble protein is pLDH is found in sexual and released from parasitized RBCs asexual forms Not present in mature Differentiation between gametocytes malarial species is based on antigenic differences between pLDH isoforms
  34. 34. Malaria Antigen Detection - RDTsFeature PfHRP-2 tests pLDH testsAdvantages Threshold for parasite Threshold for parasite detection detection as low as 10 ≥ 100 parasites/µl parasites/µl (but sensitivity drops at < 100 parasites /µl) Can detect all species which infect humans Does not cross react with other Can differentiate between P.f. species – P.v., P.o., P.m. and non-falciparum malaria Does not cross react with human LDH Positive only in viable parasites, potentially useful for monitoring success of treatment
  35. 35. Malaria Antigen Detection - RDTsFeature PfHRP-2 tests pLDH testsD Some tests only detect P.f. Cannot differentiate between non-falciparum species Cannot detect mixed infections Cannot detect mixed infections Sensitivity and specificity Sensitivity and specificity decreases < 100 parasites/µl 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
  36. 36. Malaria Antigen Detection - RDTsFeature PfHRP-2 tests pLDH testsSensitivity/ Sensitivity 92-100% Sensitivity P.f. 88-98%Specificity* Specificity 85- 100% P.v. 89-94% Specificity P.f. 93-99% P.v. 99-100%Commercial Approximately US$ 0.60 –1.00 Approximately US$ 2.50cost/test**Commercial 1) PATH falciparum Malaria 1) OptiMAL® - Flow, Inc.products IC Strip test – Program for 2) Binax NOW ®ICT Appropriate Technology in Malaria - Binax, Inc. Health 2) MAKROmed™ 3) Orchid ®* Compared to microscopy, results from multiple studies** Varies by size of order and vendor
  37. 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. 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. 39. Real-Time PCRNew technique based onfluorescencePromising because it haspotential to quantifyparasitemia, decreasescontamination, may detectmultiple wavelengths insame tube identifyingmultiple species in one run,saves hands-on timeNeeds further research andvalidation for malaria
  40. 40. Real-Time PCR
  41. 41. Quantitative Real-Time PCR
  42. 42. Detecting Malaria Parasites/Products: Sensitivity Thresholds HRP-2 pLDHKenya (children) Hospital for Trop. Diseases - LondonParasites/µl n= Sensit.(%) Parasites/µl n= Sensit.(%)1-10 9/23 39 <5 13/22 6011-60 17/21 81 50-500 9/11 8161-100 14/16 88 500-1500 17/18 94101-500 57/57 100 >1500 36/36 100501-1000 12/12 100 Ref: Piper et al. Am J Trop Med Hyg 60: 109-118 (1999)Ref: Beadle et al. Lancet 343: 564-568 (1994) PCR Vietnam (Serial dilution) Detection limits for: - P. falciparum: 0.02-0.08 parasites/µl -P. vivax: 0.8-2.6 parasites/µl Ref: Vu thi Ty Hang et al. Trans R Soc Trop Med Hyg 89: 44-47 (1995)
  43. 43. Preventing Transfusion-Transmitted Malaria (TTM) Detection of Parasites/Parasite Products PCR (0.05 to 0.1 parasites/ l) Microscopy (5 parasites/l) Antigen detection (10 to 100 parasites/ l) 10-5 10-3 10-1 10 103 Parasite densities (parasites/l)
  44. 44. Preventing TTM: Detection of Parasites/Parasite Products 100 parasites/unit PCR (0.05 to 0.1 parasites/l) (25 X 10-5/l) Microscopy (5 parasites/l)10 parasites/unit Antigen detection(2.5 X 10-5/l) (10 to 100 parasites/ l) 10-5 10-3 10-1 10 103 Parasite densities (parasites/l) Detection of 10 parasites/unit requires a sensitivity: -4,000 times better than PCR -200,000 times better than microscopy
  45. 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. 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. 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