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LABORATORY
DIAGNOSIS OF
MALARIA
20 February 2013
Shridhan Anand Patil
• Introduction
• Light microscopy
• Antigen detection - Rapid malaria diagnosis tests
(RDTs)
• Serology
• Other
• QBC - PC...
Introduction
• Accurate diagnosis of malaria
• Chloroquine-resistant malaria
• Ideal test
• Rapid - Easy to use - Reproduc...
Light microscopy
• Gold standard
• 0.0001% parasitemia detection(5-10/microL)
• Species identification
• Response to thera...
Interpreting Thick and Thin Films
• THICK FILM
• lysed RBCs
• larger volume
• 0.25 μl blood/100 fields
• good screening te...
Recognizing Malaria Parasites
Inside a red blood cell
One or more red chromatin dots
Blue cytoplasm
RING TROPHOZOITE
SCHIZONT GAMETOCYTE
Blue
Cytoplasm
Red
Chromatin
Brown
Pigment
Recognizing Erythrocytic Stages:
Schematic...
Malaria Parasite Erythrocytic Stages
Ring form
Trophozoite
Schizont
Gametocytes
Plasmodium falciparum
Rings: double chromatin dots; accole forms;
multiple infections in same red cell
Gametocytes: mature...
Plasmodium vivax
Trophozoites: ameboid; deforms the erythrocyte
Gametocytes: round-ovalSchizonts: 12-24 merozoites
Rings
I...
Plasmodium ovale
Infected erythrocytes: moderately enlarged (11/4 X); fimbriated; oval; (Schüffner’s dots)
“malariae - lik...
Infected erythrocytes: size normal to decreased (3/4X)
Plasmodium malariae
Trophozoite:
compact
Trophozoite:
typical
band ...
Species Differentiation on Thin Films
Feature P. falciparum P. vivax P. ovale P. malariae
Enlarged infected RBC + +
Infect...
Species Differentiation on Thin Films
P. falciparum P. vivax P. ovale P. malariae
Rings
Trophozoites
Schizonts
Gametocytes
Species Differentiation on Thick Films
Feature P. falciparum P. vivax P. ovale P. malariae
Uniform trophozoites +
Fragment...
Calculating Parasite Density - 1
• Using 100X oil immersion lens, select area with 10-
20 WBCs/field
• Count the number of...
Parasitemia and clinical correlates
Parasitemia Parasites / l Remarks
0.0001-0.0004% 5-20 Sensitivity of thick blood
film
...
Parasitemia and clinical correlates
Parasitemia Parasites/ l Remarks
2-5% 100,000-
250,00
Hyperparasitemia/severe
malaria*...
Estimating Parasite Density
Alternate Method
• Count the number of asexual parasites per high-
power field (HPF) on a thic...
Malaria Antigen Detection
• Cannot detect mixed
infections
• Cross reactivity with
rheumatoid factor reportedly
corrected
Malaria Serology – antibody detection
• Not routinely used for diagnosis
• Transfusion blood screening
• Investigation of ...
Polymerase Chain Reaction
• Specific amplification of malaria DNA
• Excellent sensitivity and specificity
• Detects as low...
Quantitative buffy coat
Summary
• Conventional peripheral smear examination is the gold
standard
• RDTs are costly andrequire quality control
• Se...
References
• UpToDate – Malaria diagnosis
• Lab diagnosis of Malaria. J Clin Pathol 1996;49:533-538
• Malaria Diagnosis: A...
Lab diagnosis of malaria
Lab diagnosis of malaria
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Lab diagnosis of malaria

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Includes various description of diagnostic techniques used for Malaria diagnosis.

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Transcript of "Lab diagnosis of malaria"

  1. 1. LABORATORY DIAGNOSIS OF MALARIA 20 February 2013 Shridhan Anand Patil
  2. 2. • Introduction • Light microscopy • Antigen detection - Rapid malaria diagnosis tests (RDTs) • Serology • Other • QBC - PCR • Summary
  3. 3. Introduction • Accurate diagnosis of malaria • Chloroquine-resistant malaria • Ideal test • Rapid - Easy to use - Reproducible • Minimum equipment • Detection of all species • Quantification of infection • Response to therapy • Revert on treatment - Clinical diagnosis
  4. 4. Light microscopy • Gold standard • 0.0001% parasitemia detection(5-10/microL) • Species identification • Response to therapy • Thin and thick smears
  5. 5. Interpreting Thick and Thin Films • THICK FILM • lysed RBCs • larger volume • 0.25 μl blood/100 fields • 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. 6. Recognizing Malaria Parasites Inside a red blood cell One or more red chromatin dots Blue cytoplasm
  7. 7. RING TROPHOZOITE SCHIZONT GAMETOCYTE Blue Cytoplasm Red Chromatin Brown Pigment Recognizing Erythrocytic Stages: Schematic Morphology
  8. 8. Malaria Parasite Erythrocytic Stages Ring form Trophozoite Schizont Gametocytes
  9. 9. Plasmodium falciparum Rings: double chromatin dots; accole 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 M I
  10. 10. Plasmodium vivax Trophozoites: ameboid; deforms the erythrocyte Gametocytes: round-ovalSchizonts: 12-24 merozoites Rings Infected erythrocytes: enlarged up to 2X; deformed; (Schüffner’s dots)
  11. 11. Plasmodium ovale Infected erythrocytes: moderately enlarged (11/4 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
  12. 12. 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
  13. 13. Species Differentiation on Thin Films Feature P. falciparum P. vivax P. ovale P. malariae Enlarged infected RBC + + Infected RBC shape round round, distorted oval, fimbriated round Stippling infected RBC Mauer clefts Schuffner spots Schuffner spots none Trophozoite shape small ring, appliqu large ring, amoeboid large ring, compact small ring, compact Chromatin dot often double single large single Mature schizont rare, 12-30 merozoites 12-24 merozoites 4-12 merozoites 6-12 merzoites Gametocyte crescent shape large, round large, round compact, round
  14. 14. Species Differentiation on Thin Films P. falciparum P. vivax P. ovale P. malariae Rings Trophozoites Schizonts Gametocytes
  15. 15. 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
  16. 16. 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
  17. 17. Parasitemia and clinical correlates Parasitemia Parasites / l Remarks 0.0001-0.0004% 5-20 Sensitivity of thick blood film 0.002% 100 Patients may have symptoms below this level, where malaria is seasonal 0.2% 10,000 Level above which immunes show symptoms 2% 100,000 Maximum parasitemia of P.v. and P.o.
  18. 18. Parasitemia and clinical correlates Parasitemia Parasites/ l Remarks 2-5% 100,000- 250,00 Hyperparasitemia/severe malaria*, increased mortality 10% 500,000 Exchange 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.
  19. 19. 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
  20. 20. Malaria Antigen Detection • Cannot detect mixed infections • Cross reactivity with rheumatoid factor reportedly corrected
  21. 21. Malaria Serology – antibody detection • Not routinely used for diagnosis • Transfusion blood screening • Investigation of cryptic malaria • Epidemiological purposes • Blood stage antigen used
  22. 22. Polymerase Chain Reaction • Specific amplification of malaria DNA • Excellent sensitivity and specificity • Detects as low as ≥ 1 parasite/µL of blood • Useful in identifying drug resistence • Low parasitemia, therapeutic response • Species identification – P. knowlesi
  23. 23. Quantitative buffy coat
  24. 24. Summary • Conventional peripheral smear examination is the gold standard • RDTs are costly andrequire quality control • Serological tests are suitable for epidemiological purpose • Molecular-biological techniques are suitable for research labs, quantification, drug resistence detection and species detection
  25. 25. References • UpToDate – Malaria diagnosis • Lab diagnosis of Malaria. J Clin Pathol 1996;49:533-538 • Malaria Diagnosis: A Brief Review. Korean J Parasitol. Vol. 47, No. 2: 93-102, June 2009 DOI: 10.3347/kjp.2009.47.2.93 • Rapid Diagnostic Tests for Malaria Parasites. CLINICAL MICROBIOLOGY REVIEWS,66–78.2002. Jan. 2002, p. 66–78
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