LYNNE S. GARCIA, MS, CLS, FAAM Lynnegarcia2@verizon.net

CAPHLD 65th Institute

Future Challenges in Diagnostic Medical Parasitology

SPONSOR: MEDICAL CHEMICAL

CORPORATION

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UNIVERSAL FIXATIVES ♦ OPTIONS: (1) Concentration, (2) permanent stained

smear, (3) special stains for coccidia/microsporidia, (4) fecal immunoassays, (5) PCR

♦ SAF: works best with iron-hematoxylin stain (a bit more difficult/picky); often used with albumin as glue for stool; no PVA, BUT CONTAINS FORMALIN

♦ TOTAL-FIX: NO PVA; NO MERCURY, NO FORMALIN – Critical to make sure stool smears are TOTALLY DRY – Drying in the 37ºC incubator highly recommended (on a tray);

minimum of 30 min to 1 h or more – IF THE SMEARS ARE TOTALLY DRY, THE STOOL MATERIAL

WILL ADHERE TO THE SMEAR WITHOUT USING PVA OR ALBUMIN 2

♦ Fresh or Preserved Stool Specimens – Personal preference – Consider ALL testing being ordered (O&P, 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 – O&P, Immunoassays, Special Testing

11 STOOL ORDER

RECOMMENDATIONS

IV. Cyclospora Autofluorescence Special stains

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FECAL IMMUNOASSAYS - ANTIGEN 4

RESULT REPORTING

♦O&P: Indicate test does NOT allow ID of Cryptosporidium, Cyclospora, or microsporidia (there are always some exceptions)-iron-hematoxylin stain with carbol fuchsin step; concentration and Cystoisospora belli

♦IMMUNOASSAY: Indicate method tests for very limited and specific organisms only (name each organism on the report)

♦SPECIAL STAINS: Remember to name organisms on the report – both pos/neg 5

ANTIBODY DETECTION

♦ Recent travel to endemic area – Positive = recent infection

♦ Resident of endemic area – Positive = infection unrelated to current clinical status

♦ Protozoa specific; Helminths = cross reactivity Amebiasis, babesiosis, malaria, Chagas’, Toxoplasma, trypanosomiasis, Angiostrongylus, Ascaris, cysticercosis, echinococcosis, paragonimiasis, fascioliasis, filariasis, toxocariasis, trichinosis, strongyloidiasis, schistosomiasis, Baylisascaris; PCR (blood parasites)

♦ Antibodies may/may not decline with time/therapy – 6 months to years

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Entamoeba histolytica

♦ Clinical Symptoms Intestinal: diarrhea, dysentery Extraintestinal: right upper quadrant pain, fever

♦ Clinical specimens Intestinal: stool, sigmoidoscopy Extraintestinal: liver aspirate, biopsy, serology

♦Therapy Intestinal: Iodoquinol, Diloxanide furoate (cysts) Symptomatic: Metronidazole (trophozoites)

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ENTAMOEBA HISTOLYTICA ENTAMOEBA DISPAR

Note: Ingested RBCs

Entamoeba histolytica (pathogen)

Entamoeba dispar (non-pathogen)

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REPORTING

♦ If cysts or no ingested RBCs (trophs) are seen or immunoassay is not available: Entamoeba histolytica/E. dispar/E. moshkovskii, E. bangladeshi

NOTE: Entamoeba moshkovskii (nonpathogen) looks like Entamoeba histolytica/E. dispar; it is not easy to differentiate, so the name is currently not added to the overall report. It is more rare than the others. Controversy per pathogenicity (Australia indicates some symptomatic patients)

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Blastocystis (hominis) spp. Pathogenic

• Central body (vacuolar) form, large size range • Multiple nuclei around central body area • Multiple subtypes, some pathogenic, common #1 • Stramenopiles, reclassification, quantitate • Rare dissemination, immunocompromised • Group of strains or species (some pathogenic)

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Giardia lamblia (duodenalis, intestinalis) Pathogen

• Teardrop shape, spoon • Two nuclei, stain pale • Curved median bodies • Linear axonemes • Pathogen, 19,733 in 2005 • Water, food borne • Typical motility, but caught up in mucus •Fecal immunoassays may require 2 stools for POS

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Dientamoeba fragilis: Pathogen

• Very pleomorphic, 1 or 2 nuclei • Nuclei fragmented chromatin or solid • Pathogenic, transmitted via helminth eggs • Cyst: animal reservoir, permanent stain • As common or more common than Giardia

Cyst

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Cryptosporidium spp. – Clinical 10,500 Cases Reported in 2010

♦ Immunocompetent – GI tract Self-limiting, profuse watery diarrhea Cramping pain, nausea, anorexia

♦ Immunocompromised - Disseminated Severe diarrhea (3-6 liters/day), weeks HIV patients, CD4 cell count marker 180-200 cells/mm3 or higher, good Transplants, water outbreaks

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CRYPTOSPORIDIUM SPP. C. hominis, C. parvum

Modified acid-fast: stool specimen; note sporozoites, 4-6 µm

FA combo reagent for Cryptosporidium and Giardia

Cyclospora. big Crypto, medium Artifact, small Mod acid-fast

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Cyclospora cayetanensis (Lab confirmed) 1,110 Cases (1997-2008)*

♦ Immunocompetent – GI tract Malaise, fever, watery diarrhea Fatigue, anorexia, vomiting, weight loss

♦ Immunocompromised – May disseminate Relapses for many weeks – in sputum Up to 12 weeks, biliary disease – AIDS TMP-SMX effective *Does not include year of big outbreaks, 1996 – U.S. 15

CYCLOSPORA CAYETANENSIS (Suspected Food Borne Outbreaks)

Modified acid-fast stain Autofluorescence Acid-fast variable Often 1+ to 3+ 1% acid rinse < Crypto FA

Safranin Stain

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MICROSPORIDIA Pathogen (now Fungi)

♦ Group of obligate intracellular, spores protozoa/fungi: 10 cases up to 1985 ♦ Term for phylum Microspora, 100 genera ♦ Genera (7), 14 species = human pathogens ♦ Possibilities include person-to-person and animal-to-person – Insects??? (water & foodborne; widespread antibodies) ♦Questions remain (reservoir hosts, congenital infections)

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Microsporidia Diagnosis Order: Stool & Urine

♦ Modified trichrome stains (chromotrope) ♦ 10X amount of chromotrope 2R, dye in routine Wheatley’s trichrome (O&P) ♦ Tissue Gram stains recommended ♦ PAS, silver stains acceptable, H&E NO ♦ Calcofluor, but non specific (stool) ♦ Fecal immunoassays under development; available in Europe 18

Microsporidia – Polar Tubule

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MICROSPORIDIA

Intestinal Tissue Urine: Calcofluor White

Spores in NA aspirate

Spores, muscle Corneal button Corneal stroma 20

Eye Infections Cytospin

Microsporidia Genera - Clinical

♦ Enterocytozoon bieneusi - IMPORTANT • Enteritis, cholangitis, cholecystitis,

pneumonia, bronchitis, sinusitis, rhinitis ♦ Encephalitozoon intestinalis - IMPORTANT

• Enteritis, cholangitis, cholecystitis, nephritis, urinary tract infection, sinusitis, rhinitis, bronchitis, keratoconjunctivitis, disseminated

♦ Encephalitozoon cuniculi • Hepatitis, peritonitis, encephalitis, urinary

tract, intestinal, keratoconjunctivitis, sinusitis, rhinitis, disseminated infection

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MOLECULAR TESTING Most in-house, not FDA approved

♦ APTIMA Trichomonas (GenProbe): NAT

– High sensitivity/specificity; No monitor therapy ♦ Affirm™VPIII DNA probe Trichomonas (BD) ♦ BioFire FilmArray Gastrointestinal panel; Multiplex

PCR – Cryptosporidium, Cyclospora, E. histolytica, Giardia

♦ Luminex NAT – 11 viral/bacterial/parasitic – Giardia, Cryptosporidium

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Naegleria fowleri Primary Amebic Meningoencephalitis

(PAM) – Neti pot sinus irrigation

♦ 28-year-old male developed PAM after a history of irrigating sinuses daily with tap water and neti pot ♦ Admitted with severe headache, vomiting, fever, neck and back pain; CSF = bacterial meningitis; antibiotics ♦ Wet mount of CSF = amebae; patient expired ♦ 51-year-old female PAM after 3 days of altered mental status, nausea, vomiting, high fever ♦ Died 4 days later; neti pot use; faucets PCR +

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PATHOGENIC FREE-LIVING AMEBAE: Acanthamoeba

♦ Environment Soil, air, fresh water, salt water, sewage Washing the face in pond water, sand/dust in eye, inhalation, traumatic injection, entry through existing wounds or lesions

♦ Disseminated Infections Skin, brain, bones Rhinosinusitis, keratitis, otitis, vasculitis, endophthalmitis

reported in HIV infected persons Skin lesions present in absence of CNS involvement

♦ Immunocompromised AIDS, lung, kidney, or liver transplants 24

PATHOGENIC FREE-LIVING AMEBAE AGAR PLATE CULTURE

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Strongyloides stercoralis

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DIROFILARIA SPP. IN U.S.

♦ Dog heartworm, mosquitoes ♦ In humans, subcutaneous nodules, lung

parenchyma “coin lesions” – routine x ray ♦ Ocular disease, inflammation, pain, blurring ♦ No microfilariae in blood, serologies poor ♦ Surgical/autopsy worm ID ♦ Often misdiagnosed, harmful interventions ♦ Emerging zoonosis in US, many dogs positive

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RARE INFECTIONS in U.S. Baylisascaris procyonis - Raccoon

♦Raccoon ascarid, serious zoonotic disease ♦Human infections, egg ingestion, dirt

–Young children, VLM, NLM, death common –Lethargy, loss of muscle coordination, coma –Blindness, delayed development IF survive –Larval growth (2 mm); very vigorous migrations

♦Diagnosis: process of elimination (larvae in tissues); raccoon latrines, many extremely resistant eggs 28

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ECHINOCOCCUS MULTILOCULARIS

(Alveolar Hydatid Disease)

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LIVER AND LUNG TREMATODES (Flukes)

♦ >50 million people infected, >1.1 billion exposed ♦ Aquaculture: 48.2% (2012), water/snail exchange ♦ Life cycles tend to be complex with one or more

intermediate hosts as well as definitive hosts (require freshwater snail in life cycle)

♦ Humans serve as the definitive host ♦ Ingestion of metacercariae encysted on plant

material or within fish, crabs, crayfish, etc. ♦ Most well known infections: Clonorchis sinensis,

Opisthorchis, Fasciola, and Paragonimus spp.

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CLONORCHIS SINENSIS (Chinese Liver Fluke)

♦ Pathogenic: Yes, worm burden, cholangiocarcinoma ♦ Acquired: Ingestion of infective metacercariae encysted in

raw or poorly cooked freshwater fish (aquaculture) ♦ Body site: Bile ducts and liver ♦ Symptoms: None to acute pancreatitis, biliary tract

obstruction ♦ Clinical specimen: Stool ♦ Epidemiology: China, Japan, Korea, Malaysia, Singapore,

Taiwan, Vietnam, human to human; animal to human (dogs, cats, fish-eating mammals)

♦ Control: Improved hygiene, fecal waste disposal, adequate cooking of freshwater fish 31

BLOOD TREMATODES (Flukes)

♦ 200 – 300 million people in 77 countries ♦ Rounded with separate sexes, blood vessels, non-

operculated eggs, no encysted metacercariae ♦ Life cycles tend to be complex, requiring

freshwater snail in life cycle ♦ Humans serve as the definitive host ♦ Skin penetration by cercariae released from the

freshwater snail. ♦ Most well known infections: Schistosoma

mansoni, S. haematobium, S. japonicum

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SCHISTOSOMA SPP. (Blood Flukes) ♦ Potential diagnostic problem: premature hatching ♦ Concentration: use saline, not water (stool, urine)

Note expansion of miracidium larva once released from shell.

PLEASE GO TO PART 2

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