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Lab diagnosis of malaria

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Shridhan Patil

This document discusses various methods for laboratory diagnosis of malaria, including light microscopy, antigen detection tests, serology, and molecular techniques. Light microscopy examination of thick and thin blood films is the gold standard for diagnosis and can identify parasite species, but requires trained technicians. Rapid diagnostic tests provide rapid results but cannot detect mixed infections. Serology is useful for epidemiological purposes rather than direct diagnosis. Molecular techniques like PCR provide high sensitivity but are best for research and special cases due to cost and complexity. No single method is ideal so a diagnostic approach depends on the clinical situation and available resources.

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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 - PCR Summary
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
Light microscopy Gold standard 0.0001% parasitemia detection(5-10/microL) Species identification Response to therapy Thin and thick smears
Interpreting Thick and Thin Films THICK FILM lysed RBCs larger volume 0.25 亮l 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 亮l blood/100 fields good species differentiation requires more time to read low density infections can be missed
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 Morphology
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 (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
Plasmodium vivax Trophozoites: ameboid; deforms the erythrocyte Gametocytes: round-ovalSchizonts: 12-24 merozoites Rings Infected erythrocytes: enlarged up to 2X; deformed; (Sch端ffners dots)
Plasmodium ovale Infected erythrocytes: moderately enlarged (11/4 X); fimbriated; oval; (Sch端ffners dots) malariae - like parasite in vivax - like erythrocyte Rings Trophozoites: compact Schizonts: 6-14 merozoites; dark pigment; (rosettes) Gametocytes: round-oval
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
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
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 + Fragmented trophozoites ++ + Compact trophozoites + + Pigmented trophozoites + Irregular cytoplasm + + Stippling (RBC ghosts) + + Schizonts visible very rarely often often often Gametocytes visible occasionally usually usually usually
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
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.
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. H辰nscheid T. (1999) Diagnosis of malaria: a review of alternatives to conventional microscopy. Clin Lab. Haem. 21, 235-245.
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
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 cryptic malaria Epidemiological purposes Blood stage antigen used
Polymerase Chain Reaction Specific amplification of malaria DNA Excellent sensitivity and specificity Detects as low as 1 parasite/袖L of blood Useful in identifying drug resistence Low parasitemia, therapeutic response Species identification P. knowlesi
Quantitative buffy coat
Lab diagnosis of malaria
Lab diagnosis of malaria
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
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,6678.2002. Jan. 2002, p. 6678

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Lab diagnosis of malaria

  • 2. Introduction Light microscopy Antigen detection - Rapid malaria diagnosis tests (RDTs) Serology Other QBC - PCR Summary
  • 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. Light microscopy Gold standard 0.0001% parasitemia detection(5-10/microL) Species identification Response to therapy Thin and thick smears
  • 5. Interpreting Thick and Thin Films THICK FILM lysed RBCs larger volume 0.25 亮l 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 亮l blood/100 fields good species differentiation requires more time to read low density infections can be missed
  • 6. Recognizing Malaria Parasites Inside a red blood cell One or more red chromatin dots Blue cytoplasm
  • 8. Malaria Parasite Erythrocytic Stages Ring form Trophozoite Schizont Gametocytes
  • 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. Plasmodium vivax Trophozoites: ameboid; deforms the erythrocyte Gametocytes: round-ovalSchizonts: 12-24 merozoites Rings Infected erythrocytes: enlarged up to 2X; deformed; (Sch端ffners dots)
  • 11. Plasmodium ovale Infected erythrocytes: moderately enlarged (11/4 X); fimbriated; oval; (Sch端ffners dots) malariae - like parasite in vivax - like erythrocyte Rings Trophozoites: compact Schizonts: 6-14 merozoites; dark pigment; (rosettes) Gametocytes: round-oval
  • 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. 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. Species Differentiation on Thin Films P. falciparum P. vivax P. ovale P. malariae Rings Trophozoites Schizonts Gametocytes
  • 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. 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. 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. 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. H辰nscheid T. (1999) Diagnosis of malaria: a review of alternatives to conventional microscopy. Clin Lab. Haem. 21, 235-245.
  • 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. Malaria Antigen Detection Cannot detect mixed infections Cross reactivity with rheumatoid factor reportedly corrected
  • 21. Malaria Serology antibody detection Not routinely used for diagnosis Transfusion blood screening Investigation of cryptic malaria Epidemiological purposes Blood stage antigen used
  • 22. Polymerase Chain Reaction Specific amplification of malaria DNA Excellent sensitivity and specificity Detects as low as 1 parasite/袖L of blood Useful in identifying drug resistence Low parasitemia, therapeutic response Species identification P. knowlesi
  • 26. 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
  • 27. 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,6678.2002. Jan. 2002, p. 6678