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2013 PARASITOLOGY WORKSHOP Lynnegarcia2@verizon.net

2013 PARASITOLOGY WORKSHOP Lynnegarcia2@verizon.net. LYNNE S. GARCIA, MS, FAAM, CLS, BLM(AAB) Diagnostic Medical Parasitology Workshop 2013 UPDATE – PART 4 MALARIA SPONSORED BY MEDICAL CHEMICAL CORPORATION www.med-chem.com. PRESENTATION TOPICS. Discuss STAT vs routine methods

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2013 PARASITOLOGY WORKSHOP Lynnegarcia2@verizon.net

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  1. 2013 PARASITOLOGY WORKSHOPLynnegarcia2@verizon.net LYNNE S. GARCIA, MS, FAAM, CLS, BLM(AAB) Diagnostic Medical Parasitology Workshop 2013 UPDATE – PART 4 MALARIA SPONSORED BY MEDICAL CHEMICAL CORPORATION www.med-chem.com

  2. PRESENTATION TOPICS • Discuss STAT vs routine methods • Describe in-house testing vs outside testing • Discuss potential problems with EDTA anticoagulant • Review blood staining and stain/QC options • Discuss risk management of blood parasite testing • Discussion of various case histories (blood parasites)

  3. MALARIA – KEY FACTS • 300-500 million cases each year • 700,000 – 2.7 million die each year • 600 cases in U.S. in 1914 • Malaria not endemic in U.S. (1940’s) • World’s population (41%), endemic • Africa, Asia, Middle East, Central, South America, Hispaniola, Oceania • Mosquito, blood/blood products, congenital, shared needles

  4. MALARIA PATIENT: LABORATORY • ER – Evening & night shifts (STAT request) • Hematology or microbiology? (late shift ??) • Finger stick to venipuncture (organism morphology changes) (parasites grow in EDTA!) • Automated hematology vs manual examination Thick and/or thin blood films (prepare, read) • Lack of technical expertise (generalist/specialist) • Risk management issues (recognition of STAT!)

  5. MALARIA PATIENT: LABORATORY • No periodic fever, malaise, diarrhea; poor history • Traveler, prophylaxis, self-medication • Malaria never considered; no STAT coverage • Automated exam, no manual review • Failure to prepare/read thick & thin films • Low parasitemia (<0.01% to 0.0001%) – traveler immunologically naïve = symptomatic early • Reported negative, patient released (false negative)

  6. QUALITY CONTROL SLIDESWith or without Plasmodium PATIENT BLOOD FILMS CAN SERVE AS CONTROLS (IF WBCS OK, THEN PARASITES WILL BE OK); positive blood films with Plasmodium spp. NOTREQUIRED • Make thin films; do not fix; mark side film is on • Once dry, individually wrap in foil • Box slides, wrap box in foil (will keep 5 years) • Store in freezer at –70C or –20C • Allow wrapped slide to come to room temperature prior to unwrapping; allow slide to thoroughly dry prior to fixation • Fix with methanol, dry, and stain

  7. EDTA ANTICOAGULANT • Prepare thick and thin films immediately; distortion occurs if >1 to 2 h (Remember when reviewing PT slides) • Parasites may be lost if >4 to 6 h delay in making smears • Adhesion to slide poor if ratio of EDTA/blood too high or blood held in EDTA too long • P. falciparum: gametocytes round up, confusing • P. vivax: ameboid trophs round up, no Schüffner's dots • Plasmodium spp: male gametocytes exflagellate –resemble Borrelia; pH, pCO2 (room temp with cap off); false negative diagnosis of spirochetes

  8. Anticoagulant ArtifactsOrganism Changes

  9. TEMPERATURE EFFECTSOrganism Changes • P. falciparum: at warm temp, rings will continue to grow; mimic P. malariae • P. falciparum: gametocytes will round up; mimic P. malariae • Plasmodium spp.: all rings will continue development at more rapid rate; no longer small • Plasmodium spp.: at refrigerator temp, all organisms tend to round up

  10. BLOOD PARASITE STAINSStain Options • GIEMSA:Historically the blood stain of choice • WRIGHT: Commonly used for hematology • WRIGHT-GIEMSA: Automated hematology • RAPID STAINS: Results in very short time • Diff-Quik: (American Scientific Products, McGraw Park, IL) • Wright's Dip Stat Stain Set (Medical Chemical Corp., Torrance, CA) • OTHER: Field’s stain

  11. BLOOD PARASITE STAINSColor Variations

  12. BLOOD STAIN ISSUESTechnique Considerations • It is more appropriate to use a stain with which you are familiar, rather than Giemsa • PMNs will serve as the QC “organism” for any of the blood stains; color differences not critical • Any parasites present will stain like the PMNs, regardless of the stain used • Rapid stains are acceptable • The CAP Checklist DOES NOT mandate the use of Giemsa Stain.

  13. THIN BLOOD FILMS • Advantages • RBC morphology seen; RBCs preserved • Compare size of infected to uninfected RBCs • Much easier to identify to species level • Easier to calculate parasitemia (%/100 RBCs) • Disadvantages • Much lower sensitivity than thick blood film • Infections with low parasitemia may be missed

  14. THICK BLOOD FILMS • Advantages • Examining greater volume of blood • May see malaria pigment within WBCs • May be able to see Schüffner's dots • Disadvantages • Can’t compare infected, uninfected RBCS • Organism distortion is difficult to recognize • Identification to species level is more difficult

  15. THIN BLOOD THICK BLOOD FILMS FILMS

  16. THICK BLOOD FILMSTips for Improvements • After films are dry, fix the smear with acetone for few seconds (dip twice); then dry. • This approach will improve the durability of the thick film, and will NOT interfere with the lysis of the RBCs. • Films tend to have a clean background, making the parasites easier to see. • Overall adherence to the slide is improved.

  17. Plasmodium spp. Artifacts

  18. Parasitemia Light Microscopy • 0.0001-0.0004% (5-20/µl) = Positive thick film • 0.002 % (100/µl) = Naïve patients may be symptomatic below this level; remember emergency room patients - travelers • 0.2% (10,000/µl) = Level above which immune patients exhibit symptoms; 0.1% (5,000) = BinaxNOW (maximum sensitivity) • 2% (100,000/µl) = Maximum parasitemia of P. vivax, P. ovale (young RBCs only) – rarely exceeds 2% • 2-5% (100,000-250,000/µl) = Hyperparasitemia, severe malaria, increased mortality • 10% (500,000/µl) = Exchange transfusion, high mortality

  19. Plasmodium vivaxCharacteristics • 48 hr cycle (tertian malaria) • Tends to infect young RBCs (2 rings/cell) • Enlarged RBCs as ring grows • Schüffner’s dots (stippling) after 8-10 hrs • Delicate ring (EDTA will continue to grow) • Very ameboid trophozoite • Mature schizont contains 12-24 merozoites

  20. Plasmodium vivaxClinical Disease (Young RBCs) • Primary attack 7-10 days; 2 weeks – 2 months+ • Symptoms: headache, photophobia, muscle aches, anorexia, nausea, vomiting • Early infection: steady low-grade fever • True relapses (liver): after weeks – 5 years+ • Severe complications “always possible” (cerebral, pulmonary); resistance (chloroquine, primaquine) • Spleen: Splenomegaly, leukopenia, leukocytosis (fever), nephrotic syndrome possible • Paroxysms: moderate to severe (48 hr periodicity) GARCIA 20

  21. Plasmodium vivax

  22. Plasmodium ovaleCharacteristics • 48 hr cycle (tertian cycle like P. vivax) • Tends to infect young RBCs • Enlarged RBCs with fimbriated edges • Schüffner’s dots (stippling) beginning of cycle • Smaller ring than P. vivax (EDTA changes) • Trophozoites less ameboid than P. vivax • Mature schizont contains ave. 8 merozoites

  23. Plasmodium ovaleClinical Disease (Young RBCs) • Primary attack 7-10 days; recovery 2-3 wks • Symptoms: headache, photophobia, muscle aches, anorexia, nausea, vomiting (less severe than P. vivax) • Early infection: steady low-grade fever • True relapses (liver): after weeks – 1 year • Severe complications: possible but rare • Spleen: no particular problems, not like P. vivax • Paroxysms: mild, no rigors (48 hr periodicity) Garcia 23

  24. Plasmodium ovale

  25. Plasmodium malariaeCharacteristics • 72 hr cycle (quartan malaria) • Tends to infect old RBCs • Normal to small size RBCs as ring grows • No true stippling present • Thick ring, large nucleus • Trophozoite forms “bands” across RBC • Mature schizont contains 6-12merozoites

  26. Plasmodium malariaeClinical Disease (Old RBCs) • Primary attack 27- 40 days, can survive in body 20-40 yrs+ • Symptoms: headache, photophobia, muscle aches, anorexia, nausea, vomiting (similar to P. vivax) • Early infection: regular periodicity (72 hr), old RBCs only • True relapses (liver): relapse NO, recrudescence YES • Severe complications: CNS rare, nephrotic syndrome very common (proteinuria) • Kidney: problems from deposition antigen-antibody complexes; glomerulonephritis; chronic kidney disease not reversible • Paroxysms: severe, longer cold stage, severe during hot phase, collapse during sweating phase 26

  27. Plasmodium malariae

  28. Plasmodium falciparumCharacteristics • 36-48 hr (malignant tertian malaria) • Tends to any RBC regardless of age; very heavy infection • All sizes of RBCs • No Schüffner’s dots (stippling); Maurer’s dots (bluish, single dots) • Delicate rings, may have two dots of chromatin/ring, appliqué or accolé forms; may have two rings/RBC • Very ameboid trophozoite • Crescent-shaped gametocytes (may take 10 days+)

  29. Plasmodium falciparumClinical Disease (All RBCs) • Primary attack 8-12 days; preceded by 3-4 days vague • Symptoms: headache, photophobia, muscle aches, anorexia, nausea, vomiting; onset symptoms more severe • Early infection: steady low-grade fever, no periodicity AND no crescent-shaped gametocytes (ER patients) • True relapses: relapse NO, recrudescences up to a year • Severe complications: can occur any time during infection; related to plugging of capillaries in various internal organs; symptoms may be quite severe even with LOW PARASITEMIA (cerebral malaria very dangerous) • Late symptoms: Extreme fevers, different organs impacted • Paroxysms: severe, anemia severe (all age RBCs)

  30. Plasmodium falciparum   Exflagellation can occur in any Plasmodium spp.

  31. Plasmodium knowlesiCharacteristics • 24 hr (simian malaria); rapid cycle • Tends to infect any RBC; heavy infection • All sizes of RBCs; Duffy factor like P. vivax • No Schüffner’s dots (stippling); clumpy dots later on • Delicate rings, may have two dots of chromatin/ring, appliqué or accolé forms; may have two rings/RBC • Band form trophozoites commonly seen • Gametocytes round, tend to fill the cell • Early stages = P. falciparum; later stages = P. malariae

  32. Plasmodium knowlesiClinical Disease (All RBCs) • Primary attack: similar to P. falciparum • Symptoms: headache, photophobia, muscle aches, anorexia, nausea, vomiting; onset symptoms severe • Early infection: steady low-grade fever, no periodicity; parasitemia increases quickly – cycle every 24 h • True relapses: relapse NO • Severe complications: similar to that seen with P. falciparum • Late symptoms: Extreme fevers, different organs • Paroxysms: severe, anemia severe (all age RBCs)

  33. Plasmodium knowlesi

  34. Plasmodium knowlesi Cox-Singh J, B Singh, Trends Parasitol 24:406-10, 2008 Errors in identification: Comparison of P. knowlesi, P. falciparum, and P. malariae

  35. MIXED MALARIAL INFECTIONS • More common than thought • Thailand: 30% both P. falciparum, P. vivax • Africa: P. falciparum, P. malariae (Malaysia/ P. knowlesi • Gambian children: <1% to >60% mixed infections • New Guinea: all four species (confirmed by PCR) • Anopheles mosquitoes: can transmit two species at the same time • Difficult to detect • Different parasite levels, low organism densities, confusion among morphologic criteria • PCR becoming test of choice for ID to species level

  36. OTHER DIAGNOSTIC TESTS • Buffy Coat Smears • Centrifugation, removal of plasma, leaving small amount above buffy coat • Aspirate remaining plasma, buffy coat, RBCs immediately below buffy coat • Prepare thick and thin films, process and stain as usual • Can be difficult to perform well

  37. ANTIGEN DETECTION TESTS(Plasmodium falciparum) • Rapid Detection Test Systems (RDTs) • Histidine-rich protein-2 (HRP-2) • ParaSight-F (BD), ICT Malaria (Amrad) (Binax) • HRP-2 = long half-life (followup problems) • Plasmodial lactate dehydrogenase (pLDH) • OptiMAL, OptiMAL IT (2nd Generation) • Flow • pLDH = correlation with viable parasites

  38. ANTIGEN DETECTION TESTS The BinaxNOW Malaria Test is a rapid immuno-diagnostic assay for differentiation and detection of circulating Plasmodium falciparum (P.f.) antigen and the antigen common to all to Pan malarial species: Plasmodium vivax (P.v.), Plasmodium ovale (P.o.), and Plasmodium malariae (P.m.) in whole blood. It is FDA approved (June, 2007). Test line 1 Positive = P. falciparum Test line 2 Positive = P. vivax, P. malariae, or P. ovale Test lines 1,2 Positive = P. falciparum and possible mixed infection

  39. ANTIGEN DETECTION TESTS External controls now available. TEST PRINCIPLE Detection of HRPII Antigen SPECIES IDENTIFIED P. falciparum, P. vivax BinaxNOW Alere P. falciparum Sensitivity/Specificity– 99.7% / 94.2%*  P. vivax Sensitivity/Specificity– 93.5% / 99.8% Maximum sensitivity - parasitemia >5,000 (parasites/µl) = 0.1%

  40. Parasitemia Light Microscopy • 0.0001-0.0004% (5-20/µl) = Positive thick film • 0.002 % (100/µl) = Naïve patients may be symptomatic below this level; remember emergency room patients - travelers • 0.2% (10,000/µl) = Level above which immune patients exhibit symptoms; 0.1% (5,000) = BinaxNOW (maximum sensitivity) • 2% (100,000/µl) = Maximum parasitemia of P. vivax, P. ovale (young RBCs only) – rarely exceeds 2% • 2-5% (100,000-250,000/µl) = Hyperparasitemia, severe malaria, increased mortality • 10% (500,000/µl) = Exchange transfusion, high mortality

  41. PLASMODIUM SPP. Report Comments • Plasmodium spp. seen: Unable to “rule out” Plasmodium falciparum or P. knowlesi • Plasmodium spp. not seen:One negative set of blood films will NOT “rule out” malaria; submit additional blood specimens every 4-6 hours. • Plasmodium spp. seen, possible mixed infection: Unable to “rule out” Plasmodium falciparum or P. knowlesi • NOTE: It is mandatory that the parasitemia be calculated and reported for every initial and subsequent positive set of malarial films – the same method for determining parasitemia must be used for all blood film sets on that particular patient.

  42. RISK MANAGEMENT ISSUES • Blood film request = ALWAYS a STAT • Inadequate history, use of routine automation = “false negative” results • Failure to identify malaria = patient death • Incorrect collection, processing, exam of blood films = “false negative” results • Inexperience, lack of parasitology knowledge = great risk for patient

  43. WHAT CAUSES PROBLEMS? • Use of unclean/new slides (remove oil) • Failure to use Standard Precautions • Failure to collect full tube of blood (ratio) • Allow blood to stand too long prior to film preparation; morphology changes • Send blood out without blood film preparation and examination!

  44. COMBINATION THICK/THIN BLOOD FILM • Advantages Blood does not tend to fall off like thick film Can be stained as either thick or thin film

  45. CASE STUDY 1 PATIENT HISTORY July, ’06: 47 year old male traveler returned from Sub-Saharan Africa; low grade fever, malaise, some diarrhea; had taken incorrect prophylaxis Testing: Thick and thin blood films prepared and read that evening; both blood films reported as negative; blood films examined by two different individuals following morning Final Report: Patient diagnosed with Plasmodium species; “Unable to rule out Plasmodium falciparum”

  46. PARASITIC INFECTION: Sub-Saharan Africa Case Study 1

  47. CASE STUDY 1 - QUESTIONS • Why do you think the blood films were originally reported as negative? • What history might be missing? • What did you see on the thin blood film? • What was seen on the thick blood film the following morning? • What key points does this case emphasize?

  48. CASE STUDY 1 - REVIEW • Patient from Africa; traveler with no prior exposure to malaria (immunologically naïve) • Patient became symptomatic very early in the infection; 0.0002% (100/µl) naïve patients may be symptomatic below this level! • Inappropriate prophylaxis did not prevent malaria • Thick and thin blood films: 300 oil immersion fields (1,000x) or more based on history • Recognition of malaria as possibility from history

  49. CASE STUDY 2 – PATIENT HISTORY May, ’03: 21 year old male returned from Iraq and Afghanistan June, ’03: Cyclic fevers, chills, rigors, severe malaise; diagnosed with malaria, patient remained in-house June, ’03: Appropriate therapy – BUT patient intubated, respiratory failure Sept. ’03: Patient discharged after very difficult course

  50. CASE STUDY: Parasite Images

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