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Title: LYNNE S' GARCIA, MS, FAAM, MTASCP, CLSNCA, BLMAAB


1
LYNNE S. GARCIA, MS, FAAM, MT(ASCP), CLS(NCA),
BLM(AAB)
  • Clinical Laboratory Scientists of Alaska
  • 2009 Conference
  • Diagnostic Medical Parasitology Part I
  • SESSION 17 (Thursday, May 7, 2009)
  • Practical Approach to Patient Testing and Updated
    Information
  • SPONSORED BY
  • MEDICAL CHEMICAL CORPORATION

2
LYNNE S. GARCIA CONTACT NUMBERS
  • Lynne S. Garcia, MS, MT, CLS, BLM, FAAM
  • Director, LSG Associates
  • 512 12th St.
  • Santa Monica, CA 90402-2908
  • PHONE (310) 393-5059
  • FAX (310) 899-9722
  • EMAIL Lynnegarcia2_at_verizon.net

Garcia 2
3
PRESENTATION TOPICS
  • Discuss STAT vs routine methods
  • Describe in-house testing vs outside testing
  • Compare OP, IAs, and special stains (ordering)
  • Update various parasitic pathogens
  • Discuss the risk management aspects of malaria
    testing
  • Discussion of various case histories
  • Discussion of the meaning of Tropical
    Parasitology and how parasitic infections are
    relevant to the entire world, not just warm
    climates.

Garcia 3
4
PARASITOLOGY TESTS THAT EVERY LAB SHOULD BE ABLE
TO PERFORM
  • True STATS
  • Thick and thin blood film examinations (orders,
    collection, processing, examination, reporting)
  • CSF examination for free-living amebae (wet,
    stain)
  • (Naegleria, Acanthamoeba, Balamuthia)
  • Routine
  • (1) OP, (2) fecal immunoassays, (3) special
    stains
  • Sendouts Specimens for culture, serologies,
    arthropod ID Most diagnostic methods are
    categorized as high complexity (training,
    judgment, interpretation)

Garcia 4
5
PARASITOLOGY TESTING WHAT YOU NEED TO KNOW
  • Minimum Specimen acceptability, processing,
    test method, reporting format
  • Relevant Information Collection/test, specimen
    acceptability, method, result (make sense?),
    report formatting, report comments, method
    limitations, clinical disease, disease mimics,
    geographic endemic areas, optimal methods,
    relationship between life cycles and diagnostic
    findings
  • Risk Management STAT testing (CSF, brain
    tissue, blood films)

Garcia 5
6
STOOL PRESERVATIVES and TESTING OPTIONS OP
  • OP Examination (Fresh or Preserved Stool
    Specimens)
  • Direct Wet Smear (Organism motility) NO if in
    preservative
  • Concentration YES, performed for all OP exams
  • Permanent Stained Smear Yes, performed for all
    OP exams
  • If OP ordered, BOTH concentration/permanent
    stained smear must be performed (CAP, CLSI,
    Cumitech)
  • Fecal Immunoassays (Fresh, Frozen, Formalin)
  • EIA Performed on unspun specimens
  • FA Concentrated specimen (500 Xg for 10 min)
  • Cartridge Systems Processing varies
  • Must understand preservative / immunoassay
    limitations

Garcia 6
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STOOL COLLECTION 2 SPECIMENS (OP)
  • 2 Specimens (Fresh or Preserved Stool Specimens)
  • Every other day or every day, but not all in same
    day (within 10 days)
  • If no diarrhea, 1 from normal movement, 1 using
    cathartic
  • ROUTINE 2 stools collected in preservative
    (complete OP)
  • Data Cartwright (J. Clin. Microbiol.
    372408-11, 1999)
  • First stool 75.9 detection
  • Second stool 92 detection
  • Third stool May not be cost-effective
  • Data Hanson and Cartwright (J. Clin. Microbiol.
    39474-8, 1993)
  • Two specimens by either EIA or OP revealed gt90
    detection
  • Third Stool May not be cost-effective

Garcia 7
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STOOL COLLECTION 3 SPECIMENS (OP)
  • 3 Specimens (Fresh or Preserved Stool Specimens)
  • Every other day or every day, but not all in same
    day (within 10 days)
  • If no diarrhea, 2 from normal movements, 1 using
    cathartic
  • ROUTINE 3 stools collected in preservative
    (complete OP)
  • Data Nazar (Br. J. Clin. Prac. 4776-8, 1993)
  • First stool 58.3 of population tested
  • Second stool Added 20.6
  • Third stool Added another 21.1
  • Data Hiatt, et al. (Am. J. Trop. Med. Hyg.
    5336-9, 1995)
  • Third stool Increased 22.7 Entamoeba
    histolytica
  • Third stool Increased 11.3 Giardia lamblia
  • Third stool Increased 31.1 Dientamoeba
    fragilis

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STOOL COLLECTION SUMMARY (OP)
  • Fresh or Preserved Stool Specimens
  • Personal preference
  • Consider ALL testing being ordered (OP, IA,
    special stains)
  • RECOMMENDATION Fixatives eliminate lag time
    problems
  • Number of specimens to Collect
  • Two specimens is acceptable
  • Three is better
  • RECOMMENDATION Three, but two acceptable
  • Testing The three most common options
  • (1) OP, (2) Fecal Immunoassays, (3) Special
    Stains for coccidia and microsporidia
  • Each separate, orderable, billable tests
    (separate CPT codes)

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OPTION FECAL IMMUNOASSAYSINTRODUCTION
Garcia 10
If 1st stool for Giardia NEG, perform IA on one
more stool before reporting NEG! Not required
for Cryptosporidium testing.
11
ENZYME IMMUNOASSAY (EIA)
  • Antigen Detection
  • Limited to certain organisms Cryptosporidium,
    Giardia, (Entamoeba histolytica/E. dispar group),
    E. histolytica
  • All kits have comparable sensitivity, specificity
  • Single or batch testing options
  • Requires color judgment and interpretation
  • Processing performed by Lab Assistants
  • False negatives may result due to low organism
    numbers (asymptomatic carriers)

Garcia 11
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FLUORESCENCE IMMUNOASSAY (FA)
  • Organism Detection and Differentiation
  • Limited to certain organisms (Cryptosporidium,
    Giardia cyst) generally 2 to 4 (faint
    trophs)
  • All kits have comparable sensitivity, specificity
  • Single or batch testing fluorescent microscope
  • Requires color judgment and interpretation
  • Use centrifuged stool sediment (? sensitivity)
  • False negatives may result due to low organism
    numbers (asymptomatic carriers) centrifugation!

Garcia 12
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CARTRIDGE IMMUNOASSAYS
  • Multiple products antigen detection
    (membrane flow)
  • Triage (Biosite)
  • ImmunoCard STAT (Meridian)
  • Para-Tect SIMPLE-READ (Medical Chemical)
  • Xpect (Remel)
  • All comparable sensitivity and specificity
    excellent simple to use single and/or batch
    testing options set up for the detection and
    identification of multiple organisms

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OPTION SPECIAL STAINS(Coccidia, Microsporidia)
  • Cryptosporidium spp. (C. hominis, C. parvum)
  • Modified AFB, fluorescent stains
  • Cyclospora cayetanensis
  • Modified AFB, autofluorescence
  • Microsporidia
  • Modified trichrome, Calcofluor
  • Fresh or preserved specimens concentrated
    sediment (500 xg for 10 min) smears allowed to
    air dry

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CRYPTOSPORIDIUM SPP.CYCLOSPORA CAYETANENSIS
4-6 µm
8-10 µm
Cryptosporidium spp. Modified acid-fast stain,
note sporozoites, infectious when passed
Cyclospora sp Lower power Modified acid-fast
stain, no sporozoites, not infectious when passed
Garcia 15
16
MICROSPORIDIA
O
O
O
Ryan Blue Trichrome Weber
Green Trichrome Note horizontal stripes
(polar tubule)
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17
ORDERING OPTIONSWHATS IMPORTANT AND WHY
  • PATIENT
  • It is very important for the clinician to use
    and understand ordering guidelines this approach
    provides the most clinically relevant information
    for the physician as well as appropriate test
    menu names, CPT codes, and billing.
  • ORDER
  • Specific tests are designed to provide specific
    information OP, fecal immunoassays, special
    stains physician must order tests, not the
    laboratory
  • NOTE
  • If the test ordered is negative AND the patient
    becomes asymptomatic, additional testing may not
    be required.

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ORDERING OPTIONS
  • PATIENT
  • 1. Immunocompromised patient with diarrhea
  • 2. Potential waterborne outbreak (municipal)
  • ORDER
  • Cryptosporidium or Giardia/Crypto immunoassay
  • Negative immunoassay / patient still symptomatic
  • Order OPs, microsporidia, Cyclospora

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ORDERING OPTIONS
  • PATIENT
  • 1. Diarrhea (day care, camper, backpacker)
  • 2. Potential waterborne outbreak (resort)
  • 3. Areas in U.S. where Giardia most common
  • ORDER
  • Giardia or Giardia/Cryptosporidium immunoassay
  • Negative immunoassay / patient still symptomatic
  • Order OPs, microsporidia, Cyclospora

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ORDERING OPTIONS
  • PATIENT
  • 1. Diarrhea, travel history outside of U.S.
  • 2. Past, present resident of developing country
  • 3. Diarrhea, area within U.S. where multiple
    parasites are seen routinely (large metropolitan
    areas NY, LA, DC, etc.)
  • ORDER
  • OP exams
  • Negative OPs / patient still symptomatic
  • Order Cryptosporidium, microsporidia, Cyclospora

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ORDERING OPTIONS
  • PATIENT
  • 1. Diarrhea (may or may not be present)
  • Eosinophilia, unexplained
  • Do not intentionally immunosuppress a patient
    until this issue is resolved!
  • ORDER
  • OP exams, Strongyloides stercoralis (agar
    plate)
  • Negative OPs / patient still symptomatic
  • Order Cryptosporidium, microsporidia, Cyclospora

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ORDERING OPTIONS
  • PATIENT
  • 1. Diarrhea present
  • Suspected food borne outbreak (group activity)
  • Produce (berries, basil, mesclun, snow peas)
  • ORDER
  • Special stain (modified acid-fast) for
    Cyclospora cayetanensis
  • Negative stains/autofluorescence / patient still
    symptomatic
  • Order OPs, immunoassays

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RESULT REPORTING
  • OP On the report, indicate test does NOT allow
    identification of Cryptosporidium, Cyclospora, or
    the microsporidia (there are always some
    exceptions) iron-hematoxylin stain including
    carbol fuchsin step
  • IMMUNOASSAY Indicate method tests for very
    limited and specific organisms only (name each
    organism on the report)

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ENTAMOEBA HISTOLYTICAENTAMOEBA DISPAR
Entamoeba dispar (non-pathogen)
Note Ingested RBCs
Entamoeba histolytica (pathogen)
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REPORTING
  • If cysts or no ingested RBCs (trophs) are seen
    or immunoassay is not available
  • Report as
  • Entamoeba histolytica/E. dispar
  • NOTE Entamoeba moshkovskii (nonpathogen) also
    looks like Entamoeba histolytica/E. dispar
    however, it is not that easy to differentiate, so
    the name is not added to the overall report. It
    also tends to be more rare than the others. Some
    controversy per pathogenicity (Australia studies
    indicate some symptomatic patients), requires
    molecular testing for specificity.

Garcia 25
26
Blastocystis hominis
  • Central body form, large size range
  • Multiple nuclei around central body area
  • Multiple strains (gt12), some pathogenic
  • Undergoing reclassification, quantitate
  • Rare dissemination, immunocompromised
  • More common than Giardia lamblia

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Dientamoeba fragilis
  • ? Very pleomorphic, 1 or 2 nuclei
  • ? Nuclei fragmented chromatin or solid
  • Pathogenic, transmitted via helminth eggs
  • No cyst stage, permanent stained smear
  • Often more common than Giardia lamblia

Garcia 27
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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. (1940s)
  • Worlds population (41), endemic
  • Africa, Asia, Middle East, Central, South
    America, Hispaniola, Oceania
  • Mosquito, blood/blood products, congenital,
    shared needles

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MALARIA PATIENT LABORATORY
  • ER Evening night shifts (always a STAT
    request)
  • Hematology - microbiology? (late shift
    responsibility)
  • Finger stick to venipuncture (organism morphology
    changes)
  • Automated hematology instruments vs manual
    examination (test request format)
  • Thick and thin blood films (prepare, read), buffy
    coat films
  • Lack of technical expertise (generalist/specialist
    )
  • Risk management issues (recognition of STAT!)

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MALARIA PATIENT LABORATORY
  • Low grade fever, malaise, diarrhea, fever not
    periodic
  • History may be inadequate
  • Travel, prophylaxis, self-medication
  • Malaria never considered no STAT coverage
  • Automated exam, no manual review
  • Failure to prepare/read thick thin films
  • Low parasitemia (lt0.1 to 0.0001)
    immunologically naïve symptomatic early in
    primary infection
  • Reported negative, patient released

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QUALITY CONTROL SLIDES
  • PATIENT BLOOD FILMS CAN SERVE AS CONTROLS (IF
    WBCS OK, THEN PARASITES WILL BE OK) positive
    blood films with Plasmodium spp. not required.
  • 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
  • Fix with methanol, dry, and stain

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EDTA ANTICOAGULANTPotential Problems
  • Prepare thick and thin films immediately
    distortion may occur if gt1 to 2 h (Remember this
    when reviewing PT slides)
  • Parasites may be lost if gt4 to 6 h delay in smear
    preparation
  • Adhesion to slide may be problem if ratio of
    EDTA/blood too high or blood held in EDTA too
    long
  • P. falciparum gametocytes may round up, be
    confused with other species temperature
    effects, lag time before processing
  • P. vivax ameboid trophs round up, Schüffner's
    dots may not be visible, lag time before
    processing
  • Plasmodium spp male gametocytes may
    exflagellate may resemble Borrelia related to
    pH, pCO2 (tube room temp with cap off), parasites
    tend to mimic life cycle in the mosquito vector
  • At refrigerator temperature, all organisms round
    up very confusing

Garcia 32
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BLOOD PARASITE STAINSStain Options
  • GIEMSA Historically the blood stain of choice
  • WRIGHT Commonly used for hematology
  • WRIGHT-GIEMSA Automated hematology
  • RAPID STAINS Very rapid results
  • Diff-Quik (American Scientific Products, McGraw
    Park, IL)
  • Wright's Dip Stat Stain Set (Medical Chemical
    Corp., Torrance, CA)
  • OTHER Fields stain
  • RECOMMENDATION Use stain you are familiar with

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BLOOD PARASITE STAINSColor Variations
Color variation is normal parasites will stain
like the PMNs (built in QC)
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THIN BLOOD FILMS
  • Advantages
  • RBC morphology can be seen
  • Compare size of infected RBCs to uninfected RBCs
  • Much easier to identify to species level
  • Easier to calculate parasitemia
  • Mixed infections may still be difficult to detect
  • Disadvantages
  • Much lower sensitivity than thick blood film
  • Infections with low parasitemia may be missed

Garcia 35
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THICK BLOOD FILMS
  • Advantages
  • Examining greater volume of blood
  • May be able to see malaria pigment within
    WBCs
  • May be able to see Schüffner's dots
  • Disadvantages
  • Cant compare sizes of infected and uninfected
    RBCS
  • Organism distortion is difficult to recognize
  • Identification to species level is more difficult
  • PCR may be required to ID to species level
  • (especially in mixed infections New Guinea
    all 4 species)

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Plasmodium spp. Artifacts
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38
Plasmodium vivax
Garcia 38
39
Plasmodium ovale
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40
Plasmodium malariae
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41
Plasmodium falciparum
?
?
Exflagellation can occur in any Plasmodium spp.
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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 now 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
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Parasitemia Light Microscopy
  • 0.0001-0.0004 (5-20/µl) Positive thick film
  • 0.0002 (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 (lowest level of 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

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MIXED MALARIAL INFECTIONS
  • More common than thought
  • Thailand 30 both P. falciparum, P. vivax
  • Africa P. falciparum, P. malariae
  • Gambian children lt1 to gt60 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

Garcia 44
45
PLASMODIUM SPP. Report Comments
  • Plasmodium spp. seen Unable to rule out
    Plasmodium falciparum.
  • 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.
  • 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 (also recommended for Babesia spp.)

Garcia 45
46
CASE HISTORY 1
Garcia 46
  • A patient is diagnosed as having diarrhea and
    specimens are submitted to the laboratory. When
    examining the specimens, the following images are
    seen on the wet mounts.

47
CASE HISTORY 1
  • The test result (direct wet mount and
    concentration sediment only) is reported as
    follows
  • Blastocystis hominis, Endolimax nana cysts,
    Entamoeba histolytica/E. dispar cysts ."
  • Based on the diagnosis of diarrhea and the
    laboratory findings, was the report correct as
    submitted to the physician?
  • Why or why not?

Garcia 47
48
CASE HISTORY 1 - COMMENTARY
  • The images seen were correct HOWEVER, this
    report was not based on the examination of the
    permanent stained smear. Since two of the
    organisms may be nonpathogens, and one may or may
    not be pathogenic, one wonders whether any other
    pathogenic protozoa were present.
  • Without the benefit of the permanent stained
    smear examination, there is always a good chance
    other organisms may have been missed.

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49
EXAMINATION OF PERMANENT STAINED SMEAR
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50
PERMANENT STAINED SMEAR RESULT
  • Two additional pathogens were found (Dientamoeba
    fragilis, Giardia lamblia)
  • Confirmation of prior organisms seen in wet
    mount
  • OP Report was not complete based on wet mount
    only (CAP, CLSI, current literature)
  • Without the permanent stained smear result, the
    report is incorrect and very misleading for the
    physician.
  • The report is incorrect, regardless of the skill
    of the microscopist when examining the wet
    mounts !!!

Garcia 50
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CASE HISTORY 1 - SUMMARY
  • 1. Recognize the limitations of the direct wet
    mount and the concentration wet mount.
  • 2. Understand the importance of the permanent
    stained smear confirm/and detect/identify
    cysts, trophs
  • 3. Understand the definitions of the OP
    examination from the CAP checklist
  • 4. Understand relationship between test menu,
    test options, coding, and reimbursement. Names
    per test menu should match as closely as possible
    CPT codes.

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CASE HISTORY 2
Garcia 52
  • The patient is a 43 year-old male from New
    Hampshire who has just returned from a trip to
    Africa. On his return, he was involved in an
    accident, and required blood transfusions for
    internal injuries. Three weeks post-transfusion,
    he complained of fever, headache, and general
    malaise. Two blood specimens were examined using
    automation and were negative. He continued to
    have fevers and headaches, and several additional
    blood specimens were submitted to the
    microbiology laboratory for examination as thick
    and thin blood films.

53
CASE HISTORY 2
Garcia 53
  • The following images were seen from the third
    blood specimen submitted - please comment on the
    possible diagnosis.

54
CASE HISTORY 2
Garcia 54
  • Why were both thick and thin blood films
    examined?
  • Can the slides be examined using a 60X oil
    objective?
  • How should the slides have been examined prior to
    reporting the results?
  • What are the likely parasites to consider?
  • What needs to be done after seeing the images?
  • How should the case findings be reported?
  • If the organism has been identified to species
    OR
  • If the organism has NOT been identified to
    species.

55
CASE HISTORY 2 - COMMENTARY
Garcia 55
  • Babesiosis was suspected (area, transfusion). The
    accident and blood transfusions were factors.
    However, symptoms were caused by Plasmodium
    falciparum (Africa). He had taken malaria
    prophylaxis, but failed to take it after coming
    home. Because he had never had any contact with
    malaria, he had symptoms with a very low
    parasitemia (undetectable using automation). As
    his symptoms became more severe (about two
    weeks), he finally had gametocytes and numerous
    rings in the peripheral blood. He was very ill
    and almost died.

56
CASE HISTORY 2 - SUMMARY
Garcia 56
  • 1. A complete history is mandatory (where, when,
    symptoms, prophylaxis, prior malaria)?
  • 2. Any set of blood films for parasites is
    considered a STAT procedure 24/7. Both thick
    and thin blood films must be prepared and
    examined prior to report.
  • 4. Dont forget the importance of report
    comments One set of negative blood films does
    not rule out malaria.
  • 5. Know the pros and cons of stain QC and stain
    color differences (patient slide acceptable,
    WBCs parasites).
  • 6. Understand specimen handling prior to
    processing (morphologic changes, distortion,
    etc.).

57
CASE HISTORY 2 OTHER IMPORTANT REPORT COMMENTS
  • The first set of blood films is negative.
  • One set of negative blood films does not rule
    out malaria.
  • Plasmodium spp. seen.
  • Unable to rule out possible Plasmodium
    falciparum infection.
  • Mixed Plasmodium spp. seen.
  • Unable to rule out possible Plasmodium
    falciparum infection.

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CASE HISTORY 3
Garcia 58
  • A 47 year old male was admitted to the hospital
    with complaints of severe mid-epigastric pain
    that had become worse over a period of about a
    week. This patient had received steroids over
    several years and the dose had been increased a
    few weeks before. Previous diagnostic testing
    about 3 weeks ago included an OP examination,
    which was reported as "No Parasites Seen." At
    that time, the patient was also complaining of
    off and on again diarrhea, cough, and fever.

59
CASE HISTORY 3
  • He was treated with supportive care for
    epigastric pain and developing pneumonia, but
    failed to improve. He became comatose and died
    three days later. Autopsy findings included the
    following images (colon). Organisms were also
    found throughout the body.

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CASE HISTORY 3
  • What is significant about his case history? Why?
  • What parasites might be involved?
  • What relevance does pneumonia have? What about
    the fact that the patient became comatose?
  • Why was the first OP examination negative?
  • What would you have recommended in terms of
    additional laboratory testing?
  • What is the patients condition called and why
    might patient history be important?

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CASE HISTORY 3 - COMMENTARY
Garcia 61
  • The image shows Strongyloides stercoralis
    rhabditiform larva in the colon. Eosinophilia may
    or may not be present. In this case, no
    eosinophils were noted (this can occur in the
    hyperinfection syndrome poor prognostic sign).
  • This case represents disseminated
    strongyloidiasis with peritonitis involving
    lungs, liver, peritoneum, small intestine, colon,
    respiratory diaphragm, heart, lymph nodes,
    skeletal muscle and periadrenal and
    peripancreatic fat. Intact rhabditiform and
    filariform larvae of S. stercoralis were seen in
    tissues and stool.

62
AGAR PLATE CULTURE
Garcia 62
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CASE HISTORY 3 - SUMMARY
Garcia 63
  • Understand the relationship between the
    compromised patient and strongyloidiasis.
  • Realize the patient may not have lived in an
    endemic area for years prior to symptomatic
    infection and symptoms may be confusing
    (pneumonia, sepsis, meningitis frequently Gram
    negatives).
  • The routine OP exam may not be positive,
    cultures are recommended (agar plate).
  • Important consideration for any immunosuppressed
    patient, including transplantation, chemotherapy.

64
CASE HISTORY 4
Garcia 64
  • A 14-month old boy was admitted to the hospital
    with complaints of irritability, ataxia, and
    weakness that had developed over a period of
    several weeks. Just prior to admission, he was
    unable to sit up or walk. A complete blood count
    showed 35 eosinophilia lumbar puncture revealed
    an elevated eosinophilia. His neurologic status
    did not improve. Several weeks after admission,
    his MRI revealed severe cortical atrophic changes
    and severe diffuse white matter degeneration. He
    remained in a vegetative state and died several
    months after his initial symptoms.

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CASE HISTORY 4
  • He was not seropositive for Toxocara canis, had
    not lived in or traveled to areas where other
    causes of eosinophilic meningoencephalitis would
    be suspected. At autopsy, the following image
    was seen.

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CASE HISTORY 4
  • Causes of eosinophilic meningoencephalitis?
  • Do you think the age of the child was a factor?
  • What other questions would you ask?
  • Is the eosinophilia important?
  • Can you link eosinophilia and CNS symptoms?
  • Would you have recommended any other parasitic
    procedures? If so, what?

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CASE HISTORY 4 - COMMENTARY
  • The key points include age of child, symptoms,
    CNS symptoms, and eosinophilia. The images also
    convey information allowing the identification of
    the causative agent (general term, not genus).
  • If we suspect a nematode, what might it be? How
    might the images above help with the
    identification?

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CASE HISTORY 4 - SUMMARY
  • 1. This is a case of eosinophilic
    meningoencephalitis caused by Baylisascaris
    procyonis (roundworm).
  • 2. On further questioning, it became clear that
    the child had been exposed to feral raccoons and
    soil contaminated with raccoon feces in his
    backyard. Raccoons were seen in the surrounding
    area, and typical B. procyonis eggs were found in
    the soil.

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Ingestion of infective B. procyonis eggs probably
occurred through ingestion of contaminated soil
or other hand-to-mouth transfer from areas or
articles contaminated with raccoon feces. Common
in the Midwest, Northeast, and on the west coast
of the US. Infection rates can range as high as
82 in raccoons. Infected animals shed an
average of gt25,000 egg/g of feces millions of
eggs are shed every day. Eggs very
environmentally resistant years.
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CLINICAL DISEASE
  • 1. Neural larval migrans by B. procyonis carries
    a poor prognosis.
  • 2. The larvae continue to grow while in the
    brain they are much larger than other larvae
    causing VLM.
  • 3. Treatment must be initiated early to be
    effective often diagnosis is delayed.
  • 4. Also an eosinophil-derived neurotoxin that
    contributes to manifestations of encephalitis.

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PRESENTATION OBJECTIVES
  • Discuss STAT vs routine methods
  • Describe in-house testing vs outside testing
  • Compare OP, IAs, and special stains (ordering)
  • Update various parasitic pathogens
  • Discuss the risk management aspects of malaria
    testing
  • Discussion of various case histories

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TROPICAL PARASITOLOGY ?
  • Tropical diseases are as American as the heart
    attack yellow fever lived happily for centuries
    in Philadelphia malaria liked it fine in
    Washington, not to mention in the Carolinas where
    it took right over. The Ebola virus stopped over
    in Baltimore and Taenia solium settled in
    Brooklyn. Most tropical diseases can be
    considered cosmopolitan.

Dr. Robert S. Desowitz 1926 - 2008
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TROPICAL PARASITOLOGY ?IN THE PAST .
  • Malaria California, North Carolina, Holland,
    marshlands of London
  • Hookworms U.S. southerners, California miners,
    Alpine Swiss tunnel workers
  • Filariasis/elephantiasis Charleston, North
    Carolina
  • American Trypanosomiasis Texas to Detroit and
    Canada triatomid bugs - other states 100,000
    cases suspected serologic evidence of exposure
  • Now Leishmaniasis throughout Texas

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WHAT CAN WE EXPECT IN THE21ST CENTURY ?
  • Cataclysmic hot and cold storms hot summers
    more drought, more floods, more disease
  • World population 9 billion by 2025
  • Political turmoil and terrorism
  • People fleeing to U.S. and other industrialized
    nations
  • CO2 level double by 2100
  • 10-20 of coastal land inundated
  • Wonderful world for insects and diseases they
    carry!

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WHAT CAN WE EXPECT IN THE21ST CENTURY ?
  • Increased temperature by 3-4º C
  • Heat makes insects breed faster
  • Anopheline mosquitoes Maine to Siberia
  • Insects live longer, bite more frequently
  • Parasite cycles within insects also increased
  • Greater parasite infecting dose
  • Greater disease severity
  • INSIDE 37º C
  • OUTSIDE Transmission host to host

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WHAT CAN WE EXPECT IN THE21ST CENTURY ?
  • Of those pathogens now confined to tropical
    countries, most depend on their geographically
    limited hosts.
  • The most tropical pathogens can spread to the
    cooler north and south.
  • The past history of tropical temperate disease
    exchange provides little comfort.
  • Our world has never been compartmentalized.

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THE GLOBAL HEALTH MOVEMENT
  • Millennium Development Goals
  • Eradicate extreme poverty and hunger
  • Achieve universal primary education
  • Promote gender equality and empower women
  • Reduce child mortality
  • Improve maternal health
  • Combat HIV/AIDS, malaria and other diseases
  • Ensure environmental sustainability
  • Develop a global partnership for development

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NEGLECTED TROPICAL DISEASES(NTDs)
  • Forgotten People, Forgotten Diseases ASM
    Press, 2008
  • The neglected tropical diseases and their
    impact on global health and development
  • Dr. Peter J. Hotez
  • The George Washington University and Sabine
    Vaccine Institute, Washington, D.C.

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MAJOR NTDS(Ranked by Prevalence)
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MAJOR NTDS(Ranked by Prevalence)
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KEY POINTS NEGLECTED TROPICAL DISEASES
  • Among most common infections of the poorest
  • Nonemerging, ancient conditions
  • Chronic and disabling, profound stigma
  • High morbidity, low mortality
  • Disability-adjusted life years (DALYs) to those
    of HIV/AIDS, malaria, and tuberculosis
  • Coendemicity with HIV/AIDS and malaria
  • Ranking of the gang of four for death, DALYs
  • HIV/AIDS
  • NTDs
  • Malaria
  • Tuberculosis

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NEGLECTED TROPICAL DISEASES U.S.Soil
Transmitted Helminths (1)
  • Toxocariasis (ingestion of Toxocara eggs dog
    ascarid parasite)
  • Visceral Larva Migrans
  • Ocular Larva Migrans
  • Covert Toxocariasis (resembles asthma)
  • 1970s 4.6-7.3 children (African-Americans
    30)
  • 1990s Connecticut rural urban areas 10
    (serology ) poor Hispanic children (up to 50
    in Bridgeport)
  • New York City 5 children tested for lead were
    positive for previous T. canis infection
  • Possibility of 500,000 inner city American
    Children have/had toxocariasis

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NEGLECTED TROPICAL DISEASES U.S.Soil
Transmitted Helminths (2)
GARCIA 83
  • Strongyloidiasis (enteritis and diarrhea fatal
    disseminated disease in immunocompromised)
  • Endemic rural Appalachian region of U.S. (6.8
    mil) estimated 68,000 cases (Strongyloides
    stercoralis)
  • Eastern Kentucky and Tennessee 1-4 infection
  • Worldwide 30 million in Asia, Africa, Americas

84
NEGLECTED TROPICAL DISEASES U.S.Soil
Transmitted Helminths (3)
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  • Cysticercosis/Neurocysticercosis (Taenia solium
    egg ingestion)
  • Mexico (20 million), Central America (2 million)
    within U.S.
  • Problem in Texas, New Mexico, Arizona,
    California, Chicago, New York
  • 1000-2000 new cases (neuro) yearly one of
    leading causes of epilepsy 2800-3500
    new cases of
    cysticercosis annually
  • Infected may be as high as 41,400
  • Neurocysticercosis 10 (neurology/neurosurgery)
    and 10 of seizures (ER) in Los Angeles

85
NEGLECTED TROPICAL DISEASES U.S.Toxoplasmosis
GARCIA 85
  • Toxoplasma gondii 15 residents antibody
  • Ingestion of oocysts (cat feces) or
    uncooked/poorly cooked meats
  • Often confused with other illnesses
  • Serious infections (congenital) in
    unborn fetus
  • Vision impairment/blindness, hearing loss,
    seizures, mental retardation
  • Of 4 million live births 400-4000
    congenital toxoplasmosis

86
NEGLECTED TROPICAL DISEASES U.S.Giardiasis
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  • CDC estimates 424,120 to 2,120,000 occur
  • Transmission of cysts (fecal-oral route)
    contaminated food or water (diarrhea, chronic or
    acute condition)
  • Considered one of the day care center
    infections
  • Statistically no solid data on number of cases
    among different populations or locations

87
NEGLECTED TROPICAL DISEASES U.S.Leishmaniasis
and Chagas Disease
  • Leishmaniasis (cutaneous) (L. mexicana)
  • Dozens of cases throughout south/central Texas
  • 9 cases now reported in northern Texas
  • Leishmaniasis (visceral) dogs in U.S.
    (foxhounds transmission to humans)
  • American Leishmaniasis (Trypanosoma cruzi)
  • 150,000 Latin American immigrants in U.S. may
    develop chronic Chagas
    Disease
  • Kissing bug vectors found in many areas of U.S.

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NEGLECTED TROPICAL DISEASES Arctic Canada, Alaska
  • Trichinosis (Trichinella spiralis nativa)
  • Inuit, 150,000 indigenous people (rely on

    hunting, fishing)
  • Absence of fruits, vegetables (sea mammals,
    polar
    bears)
  • 60 polar bears infected, walrus often the cause
  • Toxoplasmosis 60 seroprevalence (seal,
    caribou)
  • Echinococcosis
  • Cystic (E. granulosus) (wolves, sled dogs),
    12,000 Inuit
  • Alveolar (E. multilocularis) moving into
    Canada,
    Dakotas, Montana, Wyoming

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MAJOR NTDS U.S., Canada(Ranked by Prevalence)
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NEGLECTED TROPICAL DISEASES Mexico and the
Caribbean
  • Onchocerciasis (Onchocerca volvulus) southern
    states of Chiapas, Oaxaca
  • Use of ivermectin widely used, so no new cases of
    blindness
  • Hookworm, other soil transmitted helminths
  • Cutaneous leishmaniasis cases on the increase
  • Chagas disease gt1,500 new cases (1990-2005)
  • Amebiasis (Entamoeba histolytica)
  • Caribbean Lymphatic filariasis,
    schistosomiasis, hookworm

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MAJOR NTDS(Ranked by DALYs)
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10 LEADING CAUSES OF GLOBAL DALYsDALYs (the
number of healthy life years lost from disability
or premature death) lost annually.
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MAJOR MASS DRUG ADMINISTRATION ORGANIZATIONS
  • Partners for Parasite Control - helminths
  • Mebendazole, albendazole, praziquantel
  • Schistosomiasis Control Initiative
  • Mebendazole, albendazole, praziquantel
  • Global Alliance to Eliminate Filariasis
  • DEC, ivermectin, albendazole
  • African Programme for Onchocerciasis Control
  • Ivermectin
  • Task Force for Child Survival and Development
  • Mectizan, mebendazole, Albendazole
  • Carter Center
  • Dracunculiasis, onchocerciasis, trachoma

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RAPID-IMPACT DRUG PACKAGEPossible Drug Resistance
  • Albendazole or mebendazole (resistance)
  • Diethylcarbamazine or ivermectin
  • Praziquantel
  • Azithromycin
  • NTDS Ascariasis, trichuriasis, hookworm (R),
    schistosomiasis, lymphatic filariasis (R),
    trachoma, onchocerciasis

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ANTIPOVERTY VACCINES
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NEGLECTED TROPICAL DISEASES
  • These are the most common infections of the
    worlds poorest people, in whom they cause
    chronic disability and disfigurement on a massive
    and, at times, almost unimaginable scale.
    Through their poverty-promoting impact on child
    development, pregnancy outcome, and worker
    productivity, the NTDs represent a major reason
    why poor people cannot life themselves out of
    poverty and why the low-income countries where
    they live cannot economically advance. There is
    also a link between the NTDs and long-standing
    conflict (reductions in public health control and
    shifting of resources to military spending).

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SUMMARY
GARCIA 97
  • Tropical Parasitology may be somewhat
    misleading, since these diseases do not seem to
    be confined to particular areas of the world.
  • The world is not as compartmentalized as we may
    think.
  • Potential changes in the 21st century will
    support the spread of these diseases in many
    areas of the world.
  • The Neglected Tropical Diseases (NTDs) and their
    impact on global health will become more widely
    recognized and more important.
  • The expansion of mass drug delivery systems and
    vaccine development will continue to be supported
    by the need to control and eliminate these
    infections.

98
SESSION REVIEW
GARCIA 98
  • The 3 main procedures are (1) OP exam, (2) fecal
    immunoassays, and (3) special stains (coccidia
    and microsporidia).
  • Proper test ordering (physician) is critical for
    good patient care.
  • Certain tests are always considered STATS
    (collection, processing, examination, reporting
    (specimens for free-living amebae, blood films
    for parasites).
  • Tropical Parasitology is misleading, since
    these diseases are not confined to particular
    areas of the world.
  • Potential changes in the 21st century will
    support the spread of these diseases throughout
    the world.
  • Neglected Tropical Diseases (NTDs) will become
    more widely recognized and more important.

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THANKS QUESTIONS?
Garcia 99
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