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

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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

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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 [phone]
• FAX [phone]
• EMAIL Lynnegarcia2_at_verizon.net

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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.

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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)

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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)

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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

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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

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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
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If 1st stool for Giardia NEG, perform IA on one
more stool before reporting NEG! Not required
for Cryptosporidium testing.
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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)

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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!

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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
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MICROSPORIDIA
O
O
O
Ryan Blue Trichrome Weber
Green Trichrome Note horizontal stripes
(polar tubule)
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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.

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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

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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

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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

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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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Plasmodium vivax
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Plasmodium ovale
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Plasmodium malariae
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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

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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.)

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CASE HISTORY 1
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• 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.

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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?

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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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EXAMINATION OF PERMANENT STAINED SMEAR
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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 !!!

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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
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• 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.

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

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CASE HISTORY 2
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• 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.

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CASE HISTORY 2 - COMMENTARY
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• 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.

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CASE HISTORY 2 - SUMMARY
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• 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.).

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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
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• 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.

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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
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• 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.

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AGAR PLATE CULTURE
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CASE HISTORY 3 - SUMMARY
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• 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.

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CASE HISTORY 4
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• 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)
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• 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

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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

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NEGLECTED TROPICAL DISEASES U.S.Toxoplasmosis
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• 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

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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

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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
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• 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
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• 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?
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