Laboratory diagnosis of malaria

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NARMADA PRASAD TIWARI

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Laboratory diagnosis of malaria

  1. 1. Laboratory diagnosis of MalariaMODERATOR - DR. A. PANCHONIA
  2. 2. Malaria and Man
  3. 3. Ancient History of Malaria• Malaria parasites have been with us since the dawn of time. They probably originated in Africa (along with mankind), and fossils of mosquitoes up to 30 million years old, show that the malaria vector, the malaria mosquito, was present well before the earliest history.
  4. 4. Hippocrates and Malaria• Hippocrates, a physician born in ancient Greece, today regarded as the "Father of Medicine", was the first to describe the manifestations of the disease, and relate them to the time of year and to where the patients lived.
  5. 5. History – Events on Malaria• 1753- name of malaria was given.• 1847-Meckel observed presence of pigment in organs.• 1880 - Charles Louis Alphose Lavern discovered malarial parasite in wet mount. wins Nobel Prize in 1907• 1883 - Methylene blue stain - Marchafava• 1891 - Polychrome stain- Romanowsky• 1898 - Roland Ross - Life cycle of parasite transmission, wins Nobel Prize in 1902• 1948 - Site of Exoerythrocytic development in Liver by Shortt and Garnham
  6. 6. Major Developments in 20th Century• 1955 - WHO starts world wide malaria eradication programme using DDT• 1970 – Mosquitos develop resistance to DDT Programme fails• 1976 – Trager and Jensen in vitro cultivation of parasite
  7. 7. Lavern and Ronald RossPioneered the Events on Malaria
  8. 8. Introduction• Malaria is probably one of the oldest diseases known to mankind that has had profound impact on our history.• It is a huge social, economical and health problem, particularly in the tropical countries.• Malaria is a vector-borne infectious disease caused by single- celled protozoan parasites of the genus Plasmodium.• Malaria is transmitted from person to person by the bite of female mosquitoes.
  9. 9. Global problem
  10. 10. What causes Malaria• Malaria is caused by a parasite called Plasmodium, which is transmitted via the bites of infected mosquitoes. In the human body, the parasites multiply in the liver, and then infect red blood cells.• Transmission of Malaria do not occur <160c and >330c• Do not occur > 2000 meters altitude.
  11. 11. Etiology of Malaria• Five Species known to infect Man1 Plasmodium vivax – Benign Tertian, Tertian Malaria(Grassi and Feletti)2 P.ovale - Ovale tertian Malaria(Stephens)3 P.malariae – Quartan malaria (Laveran)4 P.falciparum – Falciparum malaria or Malignant Tertian malaria(Welch)5 P. knowlesi (Sinton and Muller)
  12. 12. LIFE CYCLE OF MALARIA Oocyst Sporozoites Zygote Stomach Wall Salivary GlandGametocytes Pre-erythrocytic (hepatic) cycle Exo-erythrocytic (hepatic) cycle Erythrocytic Hypnozoites Cycle Adapted from:
  13. 13. LIFE CYCLE OF MALARISA PARASITEIN MAN IN MOSQUITO• PRE ERYTHROCYTIC • GAMETOCYTE SCHIZOGONY- • ZYGOTE CRYPTOZOITES. • OOKINETE• ERYTHROCYTIC CYCLE- • OOCYST MEROZOITES. • SPOROZOITE• GAMETOGONY- GAMETOCYTES• EXOERYTHROCYTIC CYCLE- PHANEROZOITES.
  14. 14. Period of Pre erythrocytic cycle• 1 P.vivax 8 days• 2 P.falciparum – 6 days• 3 P.malariae - 13 – 16 days,• 4 P.ovale 9 days On maturation Liver cells ruputure Liberate Merozoites into blood stream
  15. 15. Affinity of Parasite to Erythrocytes• P.vivax Young red blood cells• P.malariae Old red blood cells• P.ovale Young red blood cells• P.falciparum Infects all age groups Also adhere to the endothelial lining of Blood vessesl Causes the obstruction, Thrombosis and Local Ischemias
  16. 16. Erythrocytic Schizogony• Liberated Merozoites penetrate RBC• Three stages occur 1 Trophozoites 2 Schizont 3 Merozoite
  17. 17. Trophozoites• After invasion grow and feed on hemoglobin• Blue cytoplasm and red nucleus, Called as Signet ring appearance• Hence called ring form
  18. 18. Schizont• When the Trophozoite is fully developed becomes compact.• Malarial pigments are scattered through the cytoplasm• The Nucleus is large and lies at the periphery starts dividing.• Becomes Schizont
  19. 19. Exo Erythrocytic Schizogony• Some Sprozoites do not undergo sporogony in the first instance• But go into resting stage called as Hypnozoites,( hibernation )• Within 2 years reactivate to form Schizonts release Merozoites and attack red cell and produce relapses• Absent in P falciparum
  20. 20. Gametogony• Merozoites differentiate into Male and female gametocytes• Macrogametocytes also called female gametocytes• Microgametocyte also called as male gametocytes• They develop in the red cells• Found in the peripheral blood smears• Microgametocyte of all species are similar in size• Macro gametocytes are larger in size.
  21. 21. Mosquito cycleA definitive Host – Mosquito
  22. 22. Clinical Features of Malaria
  23. 23. Malaria DiagnosisClinical DiagnosisMalaria Blood SmearFluorescent microscopyAntigen DetectionSerologyPolymerase Chain Reaction
  24. 24. 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
  25. 25. 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.
  26. 26. 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.
  27. 27. Recognizing Malaria Parasites Blue cytoplasmInside a red blood cell One or more red chromatin dots
  28. 28. Recognizing Erythrocytic Stages: Schematic Morphology Blue Cytoplasm RING Red TROPHOZOITE Chromatin Brown Pigment SCHIZONT GAMETOCYTE
  29. 29. Malaria Parasite Erythrocytic Stages Ring form Trophozoite Schizont Gametocytes
  30. 30. Plasmodium falciparum Infected erythrocytes: normal size M I Gametocytes: mature (M)and immature (I) forms (I is rarelyRings: double chromatin dots; accole forms; seen in peripheral blood) multiple infections in same red cell Schizonts: 18-32 merozoites (rarely seen in peripheral blood) Trophozoites: compact (rarely seen in peripheral blood)
  31. 31. Plasmodium vivax Infected erythrocytes: enlarged up to 2X; deformed; (Schüffner’s dots) Rings Trophozoites: ameboid; deforms the erythrocyteSchizonts: 12-24 merozoites Gametocytes: round-oval
  32. 32. Plasmodium ovaleInfected erythrocytes: moderately enlarged (11/4 X); fimbriated; oval; (Schüffner’s dots) Trophozoites: compact Rings Schizonts: 6-14 merozoites; Gametocytes: round-oval dark pigment;
  33. 33. Plasmodium malariae Infected erythrocytes: size normal to decreased (3/4X)Trophozoite: Trophozoite: Schizont:compact typical 6-12 merozoites(rosette like); band form coarse, dark pigment
  34. 34. Species Differentiation on Thin FilmsFeature P. falciparum P. vivax P. ovale P. malariaeEnlarged infected RBC + +Infected RBC shape round round, oval, round distorted fimbriatedStippling infected RBC Maurer Schuffner Schuffner Ziemann dots dots dots dotsTrophozoite shape Small ring, large ring, large ring, small ring, accoleform amoeboid compact compactChromatin dot often double single large singleMature schizont rare, 18-32 12-24 8-12 6-12 merozoites merozoites merozoites merzoitesGametocyte crescent shape large, large, compact, round round round
  35. 35. Species Differentiation on Thin Films P. falciparum P. vivax P. ovale P. malariaeRingsTrophozoitesSchizontsGametocytes
  36. 36. Species Differentiation on Thick FilmsFeature P. falciparum P. vivax P. ovale P. malariaeUniform trophozoites +Fragmented trophozoites ++ +Compact trophozoites + +Pigmented trophozoites +Irregular cytoplasm + +Stippling (“RBC ghosts”) + +Schizonts visible very rarely often often oftenGametocytes visible occasionally usually usually usually
  37. 37. Calculating Parasite Density - 1 • Using 100X oil immersion lens, select area with 10-20 WBCs/field on Thick smear • 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
  38. 38. Calculating Parasite Density - 2 • Count the number of parasitized and nonparasitized red blood cells (RBCs) in the same fields on thin smear • Count 1000 RBCs (fewer RBCs if parasitemia is high)Number of parasite in 1 µlOf blood = RBC IN million/cmm X Parasite %
  39. 39. 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
  40. 40. Differentiating Babesia from Malaria
  41. 41. High Power• Ring shaped trophozites • White eccentric “food• The intraerythrocytic vacuole” in a ring form. trophozoites multiply by binary • Very transient stage in Malaria. fission or schizogony, forming Very rarely seen. two to four separate merozoites. .
  42. 42. the famous Maltese Cross• Presence of 4 daughter merozoites in a tetrad ispathomnemonic.• However, rarely seen.• Never seen in malaria.
  43. 43. 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 – Nuclei of malaria parasites floresce bright green and cytoplasm red. – Staining itself is cheap – Sensitivities around 90%
  44. 44. Quantitative Buffy Coat (QBC ®)• Useful for screening large numbers of samples• Quick, saves time• Requires centrifuge, special stains• Malaria parasite floresce green yellow against dark red –black background.• 3 main disadvantages – Species identification and quantification difficult – High cost of capillaries and equipment – Can’t store capillaries for later reference
  45. 45. Principle of QBC System
  46. 46. 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
  47. 47. 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
  48. 48. Target antigens for malaria RDT pLDH HRP2 Pf-onlyPf and pan-specific bands Persists after parasite deathClosely reflects parasite viability Aldolase Pan-specificAsexual and sexual stages Closely reflects parasite viability? Potential for monitoring treatment efficacyPv, Po, Pm-specific Mabs developed
  49. 49. RDTs : test formats
  50. 50. Current RDT formats• Card / cassette / dipstick• HRP2• HRP2 & aldolase• pLDH Pf & pan• pLDH Pf & Pv "COMBO" tests• HRP2, pLDH pan• HRP2, pLDH pan & pLDH Pv• aldolase
  51. 51. RDT procedure
  52. 52. Plastic cassette format ofRDT
  53. 53. Card format of RDT
  54. 54. Dipstick format of RDT
  55. 55. Potential applications for RDTs. Diagnosis in Confirmation of remote areas dubious microscopy diagnosisRapid outbreak investigationand surveillance Laboratory-based screening / diagnosis
  56. 56. Polymerase Chain Reaction (PCR)• Molecular technique to identify parasite genetic material• Uses whole blood collected in anticoagulated tube or directly onto filter paper
  57. 57. Polymerase Chain Reaction (PCR)• Threshold of detection – 5 parasites/µl• 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
  58. 58. PCR: identification of malaria species Lane S: Molecular base pair standard (50-bp ladder). Black arrows :size of standard bands. Lane 1: P. vivax (size: 120 bp). Lane 2: P. malariae (size: 144 bp). Lane 3: P. falciparum (size: 205 bp). Lane 4: P. ovale (size: 800 bp).
  59. 59. Comparison of methods for diagnosing Plasmodium infection in blood PARAMETER MICROSCOPY PCR FLUORESCENCE Dipstick HRP-2 Dipstick pLDH, ICT-Pf/PvSensitivity 50 5 50 >100 >100(parasites/micol) P.f good, others P. falciparum and P.vivax good P.oSpecificity All species All species P. falciparum difficult and P.m only Pldhprarasite density crude Yes No No crude estimationor parasitemia estimationtime for result 30-60 min 24 hr 30-60 min 20 min 20 minskill level High High Moderate Low Low QBC apparatus PCR or directequipment Microcsope Kit only Kit only appratus fluorescence microscopecost /test Low High moderate/low Moderate Moderate
  60. 60. Hame jindgi apni kamiyo ko door karne ke bajay.Bhagwan ne jo hame khubiya di hai unka upyog karne me gujarni chahiye. SPEAKER-DR. NARMADA PRASAD TIWARI
  61. 61. a consequence was natural selection forsickle-cell disease, thalassaemias, glucose-6-phosphatedehydrogenasedeficiency, ovalocytosis, elliptocytosis andloss of the Gerbich antigen (glycophorin C)and the Duffy antigen onthe erythrocytes because such blooddisorders confer a selective advantageagainst malaria infection (balancingselection).[7] The three major types ofinherited genetic resistance (sickle-celldisease, thalassaemias, and glucose-6-phosphate dehydrogenase deficiency)were present in the Mediterranean world bythe time of the Roman Empire, about 2000years ago.[8]
  62. 62. The term miasma was coinedby Hippocrates of Kos who used it todescribe dangerous fumes from the groundthat are transported by winds and cancause serious illnesses.[13] The namemalaria, derived from ‘mal’aria’ (bad airin Medieval Italian). This idea came fromthe Ancient Romans who thought that thisdisease came from the horrible fumes fromthe swamps

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