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1 LYNNE S. GARCIA, MS, CLS, FAAM ASCLS-NY SPRING SEMINAR 2015: Diagnostic Medical Parasitology The World Continues to Shrink (Future Challenges in Diagnostic Medical Parasitology) Sponsored by Medical Chemical Corp. 1 PARASITOLOGY TESTS THAT EVERY LAB SHOULD BE ABLE TO PERFORM True STATS: Thick and thin blood films (collection, preparation, examination, reporting) CSF exam for free-living amebae (wet, stain) (Naegleria, Acanthamoeba, Balamuthia, Sappinia); FA/PCR sent to CDC Sendouts: Majority of other specimens; plus specimens for culture and serologies; majority of requests performed in large reference centers, or CDC 2 DIAGNOSTIC PARASITOLOGY: TESTING OPTIONS Microscopy: O&P, Blood Films, Arthropod ID, Fluids/Tissues, Cultures, Fecal Immunoassays Requires visual review, morphological assessment Organism vs artifact, size, morphology Geographic area, collection/processing options Other: Serologies (expertise, reagents, cost, interpretation) FA, EIA, rapids: setups easy, interpretation difficult Diagnostic methods are categorized as high complexity (training, judgment, interpretation) 3 1

2 PARASITOLOGY TESTING: WHAT YOU NEED TO KNOW Minimum: Specimen acceptability, collection, processing, test method, reporting format Relevant Information: Collection/test, specimen acceptability, method, result (make sense?), report formatting and comments, method limitations, clinical disease, disease mimics, geographic endemic areas, optimal methods, correlation of life cycles and diagnostic findings Risk Management: STAT testing (CSF, brain tissue, blood films) 4 STOOL COLLECTION 2 SPECIMENS (O&P) 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 (UNCOMMON) ROUTINE: 2 stools collected in preservative (complete O&P) Data: Cartwright (J. Clin. Microbiol. 37: , 1999) First stool: 75.9% detection Second stool: 92% detection Third stool: May not be cost-effective Data: Hanson and Cartwright (J. Clin. Microbiol. 39:474-8, 1993) Two specimens by either EIA or O&P revealed >90% detection Third Stool: May not be cost-effective 5 STOOL COLLECTION 3 SPECIMENS (O&P) 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 O&P) Data: Nazar (Br. J. Clin. Prac. 47:76-8, 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. 53:36-9, 1995) Yield increased with third stool, 22.7%: Entamoeba histolytica Yield increased with third stool, 11.3%: Giardia lamblia Yield increased with third stool, 31.1%: Dientamoeba fragilis 6 2

3 STOOL PRESERVATIVES and TESTING OPTIONS: O&P O&P Examination (Fresh or Preserved Stool Specimens) Direct Wet Smear (Organism motility): NO if in preservative Concentration: YES, performed for all O&P exams Permanent Stained Smear: Yes, performed for all O&P exams If O&P ordered, BOTH concentration/permanent stained smear must be performed (CAP Checklist, NCCLS/CLSI) Fecal Immunoassays (Fresh, Frozen, Fixatives) 7 EIA: Performed on unspun specimens FA: Concentrated specimen (500 Xg for 10 min) Cartridge Systems: Processing varies Must understand preservative / immunoassay limitations ORGANISMS: O&P EXAM WET MOUNTS (SALINE, IODINE) 8 ORGANISMS: O&P EXAM PERMANENT STAINED SMEARS 9 3

4 STOOL FIXATIVES Formalin: concentration, immunoassays Fixative with PVA: Polyvinyl Alcohol (glue to keep stool on the glass slide) Mercury-based fixatives: phased out due to environmental restrictions Copper or Zinc-based fixatives: zinc-based best morphology being routinely used, including PT Universal Fixatives: (1) Concentration, (2) permanent stain, (3) special stains for coccidia, microsporidia, (4) fecal immunoassays (TOTALFIX) (5) PCR, fecal molecular panels 10 TOTAL-FIX Parasitology site: (Protocols, case histories, exams, etc.) OPTIONS: (1) Concentration, (2) stained smear, (3) special stains for coccidia/microsporidia, (4) fecal IAs, (5) PCR SAF: best with iron-hematoxylin (more picky); albumin used as glue for stool; NO PVA, BUT CONTAINS FORMALIN TOTAL-FIX: NO PVA; NO MERCURY, NO FORMALIN Smears TOTALLY DRY, incubator min STOOL MATERIAL ADHERES TO THE SMEAR 11 WITHOUT PVA OR ALBUMIN TOTAL-FIX Parasitology site: (Protocols, case histories, exams, etc.) (1) Concentration, (2) Routine stained smear, (3) Special stains for coccidia/microsporidia, (4) Fecal IAs, (5) PCR, molecular panels TOTAL-FIX: NO PVA; NO MERCURY, NO FORMALIN 12 4

5 Entamoeba histolytica/e. dispar Entamoeba hartmanni TOTAL-FIX Entamoeba coli TOTAL-FIX Endolimax nana TOTAL-FIX

6 Iodamoeba bütschlii, Blastocystis spp. TOTAL-FIX Giardia lamblia (G. duodenalis, G. intestinalis) TOTAL-FIX Dientamoeba fragilis Chilomastix mesnili TOTAL-FIX

7 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 IV. Cyclospora Autofluorescence 19 Special stains 11 STOOL ORDER RECOMMENDATIONS FECAL IMMUNOASSAYS, ANTIGEN (EIA, FA, Rapid Cartridge) 20 ENZYME IMMUNOASSAY 21 7

8 ENZYME IMMUNOASSAY (EIA) Antigen Detection (Single or batch testing) Limited to certain organisms [Cryptosporidium, Giardia, (Entamoeba histolytica/e. dispar group), E. histolytica] (Dientamoeba, Blastocystis, microsporidia under development) All kits have comparable sensitivity, specificity Color judgment - interpretation if manually read False negatives may result due to low organism 22 numbers (asymptomatic carriers) ENZYME IMMUNOASSAY TIPS Use Unspun Specimen - Fluid If vial is mixed, let settle for 5+ min before testing Thoroughly rinse wells, don t cut any rinse steps Each well MUST receive total number of rinses Squirt buffer directly into wells; squeeze bottle When you slap trays onto paper towels, do so several times; don t be gentle; cups won t fall out Prior to adding last reagents, wells should be empty (not dry, but empty of excess fluid) 23 FLUORESCENCE IMMUNOASSAY USE SEDIMENT FOR TESTING Provides Organism Detection and Differentiation Limited to certain organisms (Cryptosporidium, Giardia) generally 2+ to 4+ All kits have comparable sensitivity, specificity Requires fluorescent microscope (cost issue) Requires color judgment and interpretation False negatives may result due to low organism numbers (asymptomatic carriers) perform centrifugation use sediment 24 8

9 GIARDIA, CRYPTOSPORIDIUM Combination FA Immunoassay Two filters (FITC, background) Giardia lamblia cyst Cryptosporidium spp. oocysts Immunofluorescence (FA scope) One filter (FITC only) 25 FLUORESCENCE TIPS Use Conc Specimen Sediment Looking for cysts & oocysts, not antigen suspension; centrifuged sediment (500 xg 10 min) Prepare thin smears, dry slides (35 C for min); if not dry, stool may fall off; do NOT use heat Gently rinse; allow fluid to flow over wells Organisms may not always fluoresce at 3+ to 4+; may see pale fluorescing bacteria/yeast; may also see very pale Giardia trophs; examine well edges Fluorescence filters; yellow-green = more intense fluorescence; both filters = a bit less intense 26 IMMUNOCHROMATOGRAPHIC ASSAY CARTRIDGE/STRIP Test line will USUALLY be lighter than control line. 27 Too much stool can clog the sample well. If shake vial, allow to stand 5+ min before testing fluid at top. 9

10 3 ORGANISM Cartridge IA CPT Codes: Top three lines = Controls Middle three lines = Tests Bottom line = Negative Control Results: POSITIVE GIARDIA NOTE: EHIST = Entamoeba histolytica/e. dispar group NOT Entamoeba histolytica (true pathogen) 28 CARTRIDGE IMMUNOASSAYS Multiple products antigen detection (membrane flow) possible well clogging with stool debris L All = comparable sensitivity and specificity Excellent; simple to use; clogging L L Single and/or batch testing options Set up for the detection and identification of multiple organisms; note control line color; package insert critical 29 LATERAL FLOW CARTRIDGE TIPS Use Unspun Specimen - Fluid If stool is too thick, reagents will not thin it out enough; if mixture is too thick, fluid will not flow Do NOT mix vial, but use fluid at top of vial; if vial mixed, settle for 5 min+ before testing fluid Control line must be visible all the way across Positive test line is almost always less intense than control; any color should be interpreted as positive; package insert critical per colors Do NOT read after time indicated in directions 30 may get a false positive. 10

11 ANTIBODY DETECTION May or may not be helpful 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 31 6 months to years 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) 32 ENTAMOEBA HISTOLYTICA ENTAMOEBA DISPAR Entamoeba dispar (non-pathogen) Note: Ingested RBCs Entamoeba histolytica (pathogen) 33 11

12 REPORTING Entamoeba histolytica Complex34 If cysts or no ingested RBCs (trophs) are seen or immunoassay is not available: Entamoeba histolytica/e. dispar/e. moshkovskii, E. bangladeshi, Entamoeba histolytica COMPLEX NOTE: Entamoeba moshkovskii (nonpathogen) looks like Entamoeba histolytica/e. dispar; the name is currently not added to the overall report. It is more rare and controversy per pathogenicity (Australia indicates some symptomatic patients) Blastocystis (hominis) spp. Pathogenic Central body (vacuolar) form, large size range 35 Multiple nuclei around central body area Multiple subtypes or species, half pathogenic, Stramenopiles, reclassification, quantitate Rare dissemination, immunocompromised Worldwide: most common stool organism 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: Need 2 stools for NEG; FA trophs 36 12

13 Dientamoeba fragilis: Pathogen Cyst 37 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 Pentatrichomonas hominis (NP) Trichomonas vaginalis (P) P T. vaginalis P. hominis NP No cyst forms Note different position of undulating membrane Nonpathogen (GI tract) and pathogen (urinary/genital tract) (possible urine 38 contamination with stool) Chilomastix mesnili Non Pathogen Nonpathogenic flagellate Trophozoite often looks rounded up (like ameba) Note the curved fibril in the cyst (Shepherd s Crook) Difficult to see without the permanent stained smear

14 Balantidium coli Pathogen 40 Pathogenic ciliate, can penetrate mucosa Trophozoite and cysts are quite large (90+ microns) Covered with cilia, large bean-shaped macronucleus Can penetrate GI tract, diarrhea similar to cholera Uncommon in US (pigs), proficiency testing specimens OPTION SPECIAL STAINS (Coccidia, Microsporidia) Cryptosporidium spp. (C. hominis, C. parvum) Modified AFB, fluorescent stains Cyclospora cayetanensis Modified AFB, autofluorescence Microsporidia Modified trichrome, Calcofluor /DNA DAPI fluorochrome dye Fresh or preserved specimens: concentrated sediment (500 xg for 10 min); smears allowed to air dry 41 SPECIAL STAINS (Less Sensitive than Immunoassays) PROS: Rapid, simple, moderately specific/ sensitive, defined patient situation test orders, patient should fit profiles; low supply costs CONS: Limited to coccidia or microsporidia, Modified Trichrome stains difficult to examine, high labor costs, orders may be inappropriate, requires client education Organism numbers will impact diagnosis; if suspect false negative, retest in days (coccidia) to 1-2 weeks (microsporidia) 42 14

15 SPECIAL STAINS TIPS Use Centrifuged Specimen Sediment Modified acid-fast (coccidia); destain step critical Destain: 1% sulfuric acid recommended; good Cryptosporidium, Cyclospora, Cystoisospora Avoid thick smears; thin preparations best; can use well slides Microsporidia; modified trichrome thin smears helpful; look for horizontal or diagonal line (polar filament) within the microsporidial spores 43 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 cells/mm 3 or higher, good Transplants, water outbreaks 44 CRYPTOSPORIDIUM SPP. C. hominis, C. parvum 45 FA combo reagent for Cryptosporidium and Giardia Modified acid-fast: stool specimen; note sporozoites, 4-6 µm Cyclospora. big Crypto, medium Artifact, small Mod acid-fast 15

16 Cyclospora cayetanensis (Lab confirmed) 1,110 Cases ( )* 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. 46 CYCLOSPORA CAYETANENSIS (Suspected Food Borne Outbreaks) Safranin Stain Modified acid-fast stain Autofluorescence Acid-fast variable Often 1+ to % acid rinse < Crypto FA North American Cyclospora cayetanensis Outbreaks Tap water Fresh Guatemalan raspberries 1996 Fresh Guatemalan raspberries 1996 Basil-Pesto pasta salad 1996 Fresh basil 1997 US, Canada raspberries, mesclun Import voluntarily suspended US, Canada, fruit salad, raspberries 1999 US, chicken pasta, tomato basil salad 2000 Imported raspberries 2001 Canada, Thai basil 2004 Guatemalan snow peas US, fresh basil Cruise ship multiple countries 48 16

17 Cystoisospora (Isospora) belli Clinical - Pathogen Immunocompetent GI tract Self-limiting, profuse watery diarrhea Cramping pain, malabsorption Immunocompromised - Disseminated Severe diarrhea (3-6 liters/day), months Compromised, infants, children, AIDS Extraintestinal dissemination rare, but 49 occurs in immunocompromised Cystoisospora belli Diagnosis Examination for oocysts (15-25 µm) Wet mount good, typical shape Don t confuse with shrunk Giardia cysts Routine stains don t work (O&P stains) Modified acid-fast stains, 1% decolorizer recommended, thin preps, histology Auramine-rhodamine OK, but nonspecific Autofluorescence/shape confirmatory 50 Cystoisospora belli Oocysts 51 17

18 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) 52 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) 53 Fecal immunoassays available in Europe Eye Infections Cytospin MICROSPORIDIA NA aspirate Intestinal Tissue Urine: Calcofluor White Spores, muscle Corneal button Corneal stroma 54 18

19 Microsporidia Genera Clinical Immunocompetent: Ocular 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, 55 rhinitis, disseminated infection Microsporidia Genera Clinical (2) Encephalitozoon hellem Keratoconjunctivitis, sinusitis, rhinitis, pneumonia, bronchiolitis, nephritis, urinary tract infection, prostatic abscess, disseminated infection Trachipleistophora hominis Myositis, keratoconjunctivitis, sinusitis, rhinitis Trachipleistophora antropophthera Encephalitis, myositis, disseminated infection Pleistophora ronneafiei Myositis Vittaforma corneae (Nosema corneum) Corneal stroma, possible kidney (non-hiv) 56 Microsporidia Genera Clinical (3) Anncaliia vesicularum (Brachiola vesicularum) Corneal stroma, skeletal muscle (non-hiv) Anncaliia connori (Brachiola connori) Disseminated infection Anncaliia algerae (Brachiola algerae) Eye, muscle, dissemination, arthropodborne? Singapore Eye Institute 57 19

20 RESULT REPORTING Importance of report comments O&P: O&P: NO ID of Cryptosporidium, Cyclospora, or microsporidia (some exceptions)-iron-hematoxylin stain with carbol fuchsin step; concentration and Cystoisospora belli (wet mount ID) IMMUNOASSAY: 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 58 REPORTING ORGANISMS ORGANISM NAMES: List all names using genus/species/stage (trophozoites, cysts, oocysts, spores, eggs, larvae, etc.) QUANTITATION: Very few parasites are quantitated: Blastocystis spp., some helminth eggs (Trichuris trichiura), viability of helminth eggs (Schistosoma spp.) NON PATHOGENS: These organisms must also be reported (same route as pathogens) 59 REPORTING ARTIFACTS ORGANISM NAMES: Human cells, crystals, yeast (as consult only); PMNs, macrophages, eosinophils, Charcot-Leyden crystals QUANTITATION: General rare, few, moderate, many, packed 60 20

21 MOLECULAR TESTING Most in-house, not FDA approved Blastocystis, Dientamoeba, Microsporidia APTIMA Trichomonas (GenProbe): NAT Affirm VPIII DNA probe Trichomonas (BD) BD MAX Enteric Parasite Panel Giardia, C. hominis, C. parvum, E. histolytica BioFire FilmArray Gastrointestinal panel; Multiplex PCR (biomérieux) Cryptosporidium spp, Cyclospora, Giardia, E. histolytica Luminex NAT 11 viral/bacterial/parasitic Giardia, Cryptosporidium spp. 61 PATHOGENIC FREE-LIVING AMEBAE: Naegleria fowleri Environment Wet environments, domestic water sources, power plant cooling water, well water Isolated in Arctic and sub-antarctic regions (often associated with warmer temperatures) Infected Groups Usually healthy immunocompetent individuals Survival Only 7 out of approximately 300 prior to 2002; death within about a week; no development of immune response 62 Therapy often ineffective; amphotericin B required Can occur northern latitudes climate change/warm Naegleria fowleri - Ameboflagellate Primary Amebic Meningoencephalitis (PAM) 9-year-old male: headache, vomiting, lethargy, neck pain, unable to stand or walk CSF trophozoite and flagellate forms (2 flagella, water) Aggressive treatment with amphotericin B, rifampin Symptoms began 1 week after swimming in man-made lake (Texas); patient expired 63 21

22 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 + 64 Naegleria fowleri Primary Amebic Meningoencephalitis (PAM) 6-month-old male developed PAM High index of suspicion: patients appear to have pyogenic meningitis, but CSF shows no bacteria ( false positive Gram stain) Critical that CSF wet mount be performed for early detection Patient died one other fatal case in infant from India 65 Naegleria fowleri (PAM) 10-year-old male developed PAM (water reservoir); died during 3 rd hospital day 23-year-old male with PAM died on 2 nd hospital day; second case - Venezuela Prevention: nose plugs, no digging in sediment Direct immunofluorescence on brain tissue

23 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 in HIV infected persons Skin lesions present, no CNS involvement Immunocompromised AIDS, lung, kidney, or liver transplants 67 Acanthamoeba keratitis after LASIK 20-year-old woman developed pain, redness, decreased vision, and corneal infiltrate in right eye 15 days after LASIK; NO contact lenses used postoperatively. 3 months later, large corneal infiltrate with multiple satellite lesions in right eye Culture positive for Acanthamoeba cysts; growth 2 months after therapy with topical polyhexamethylene biguanide, chlorhexidine, atropine sulfate, and oral itraconazole infiltrate resolved 68 Disseminated Acanthamoeba Renal Transplant Recipient 36-year-old female renal transplant recipient; on immunosuppressives for 4 years Autopsy showed CNS with chronic granulomatous encephalitis Predominant perivascular infiltrate of amebic cysts, trophozoites, inflammatory cells Both lungs and pancreas also showed infiltration with Acanthamoeba 69 23

24 PATHOGENIC FREE-LIVING AMEBAE (1990) Balamuthia mandrillaris 100 cases Possible history of water exposure (Peru skin plaque) Similar to disease caused by Acanthamoeba (GAE) Headache, nausea, vomiting, confusion, fever, seizures, coma dissemination from cutaneous lesions Granulomatous Amebic Meningoencephalitis more chronic, typically fatal; unknown incubation period 70 Inflammatory response, amebae surrounded by macrophages, lymphocytes, neutrophils CSF wet mounts, bacterial overlay culture NOT effective, IFA, PCR Rare, but almost always fatal some cures; prolonged treatment PATHOGENIC FREE-LIVING AMEBAE Sappinia diploidea, S. pedata 71 Very few cases: Amebic Meningoencephalitis 38-year-old male: visual disturbances, headache, seizure Brain image: solitary mass left temporal lobe (tumor); central necrotic and hemorrhagic inflammation (acute, chronic inflammatory cells without granulomas or eosinophils) Trophozoites have double nuclei; cysts in host unknown Prognosis after surgical excision and medical treatment was favorable PATHOGENIC FREE-LIVING AMEBAE AGAR PLATE CULTURE 72 24

25 Strongyloides stercoralis Short & sexy (infection for years) Pathogenic: Yes, autoinfection, dissemination 73 Acquired: Skin penetration of filariform larvae from soil; ingestion of contaminated food Body site: Intestine, larvae in lungs Symptoms: Pneumonitis, GI complaints, asymptomatic, compromised = severe Clinical specimen: Stool; disseminated = sputum, other tissues (larvae, not eggs) Epidemiology: Worldwide, human to humantail split Control: Improved hygiene, feces disposal, wash fruits, vegetables, ivermectin, albendazole Strongyloides stercoralis Serology: CDC 74 DIROFILARIA SPP. IN U.S. Dog heartworm, mosquitoes Human subcutaneous nodules, lung parenchyma coin lesions x ray Ocular, inflammation, pain, blurring No microfilariae in blood, serologies poor Surgical/autopsy worm ID 75 Often misdiagnosed, harmful interventions Emerging zoonosis in US, many dogs positive 25

26 RARE INFECTIONS in U.S. Baylisascaris procyonis Kinkajous RARE INFECTIONS in U.S. Baylisascaris procyonis Raccoon Serology: CDC Raccoon ascarid, zoonotic disease Human: egg/dirt ingestion Young children, VLM, NLM Lethargy, coma, blindness, death Larval growth (2 mm); very vigorous Diagnosis: (larvae in tissues); raccoon latrines, many extremely resistant eggs 28 ECHINOCOCCUS MULTILOCULARIS Pathogenic: Yes, very hard to treat, poor prognosis Acquired: Ingestion of eggs from worm in intestine of wolf, fox, dog, cat (shrews, voles, mice, squirrels) Body site: Hydatid cysts: Primarily liver Symptoms: Intrahepatic portal hypertension; like metastatic cancer = severe disease Clinical specimen: Imaging, cyst aspirates Epidemiology: Worldwide, into northern U.S. Control: Awareness of contaminated water; washing contaminated berries; surgery, albendazole 78 26

27 ECHINOCOCCUS MULTILOCULARIS (Alveolar Hydatid Disease) 79 LIVER AND LUNG TREMATODES (Flukes) >50 million people infected, >1.1 billion exposed Aquaculture: 48.2% (2012), water/snail exchange; 60 millions tons = 3.6% protein eaten by humans Life cycles: one or more intermediate hosts and definitive hosts (humans) (freshwater snail) Ingestion of metacercariae encysted on plant material or within fish, crabs, crayfish, etc. Clonorchis sinensis, Opisthorchis, Fasciola, and Paragonimus spp. 80 CLONORCHIS SINENSIS (Chinese Liver Fluke) Pathogenic: Yes, cholangiocarcinoma Acquired: Metacercariae in raw or poorly cooked freshwater fish (aquaculture) Body site: Bile ducts and liver; pancreatitis, biliary obstruction; 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, cooking of freshwater fish cultural issues 81 27

28 BLOOD TREMATODES (Flukes) million people in 77 countries Rounded with separate sexes, blood vessels, nonoperculated 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 O O Veins: over bladder S. haematobium Veins: large intestine S. mansoni Veins: small intestine S. japonicum 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. 28

29 MALARIA KEY FACTS u million cases each year u 700, ,000 die each year u 600 US cases 1914, no longer endemic u 2012: 90% deaths/africa, 460,000 before 5 u World s population (~50%), 97 countries u Resistance now seen to artemisinin, core compound in combination therapies u Mosquito, blood/blood products, shared needles, congenital 85 Courtesy of CDC

30 MALARIA PATIENT: LABORATORY ER Evening & night shifts (STAT request) Hematology - microbiology? (late shift?) Finger stick to venipuncture (morphology changes) (parasites continue to grow in EDTA) Automated hematology vs manual examination; low parasitemias missed Thick/thin blood films (read, report); controls Lack of technical expertise (generalist/specialist) Risk management issues (recognition of STAT) 88 Parasitemia Light Microscopy % (5-20/µl) = Positive thick film % (100/µl) = Naïve patients may be symptomatic below this level; remember emergency room patients - travelers 0.2% (10,000/µl) = Level above which immune patients exhibit symptoms; 0.1% (5,000) = BinaxNOW (maximum sensitivity) 2% (100,000/µl) = Maximum parasitemia of P. vivax, P. ovale (young RBCs only) rarely exceeds 2% 2-5% (100, ,000/µl) = Hyperparasitemia, severe malaria, increased mortality 10% (500,000/µl) = Exchange transfusion, high mortality EDTA ANTICOAGULANT Prepare thick and thin films immediately; distortion occurs if >1 to 2 h (Remember when reviewing PT slides) Parasites may be lost if >4 to 6 h delay in smear preparation Adhesion to slide poor if ratio of EDTA/blood incorrect (fill tube to the top) or blood held in EDTA too long P. falciparum: gametocytes round up, confusing morphology P. vivax: ameboid trophs round up, no Schüffner's dots Plasmodium spp: male gametocytes exflagellate resemble Borrelia; related to ph, pco2 (tube room temp 90 with cap off) 30

31 QUALITY CONTROL SLIDES With or Without Plasmodium 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; allow slide to thoroughly dry prior to fixation Fix with methanol, dry, and stain 91 Anticoagulant Artifacts Organism Changes 92 BLOOD PARASITE STAINS Stain Options ALL OK GIEMSA: Historically the blood stain of choice WRIGHT: Commonly used for hematology WRIGHT-GIEMSA: Automated hematology RAPID STAINS: Results in very short time Diff-Quik: (American Scientific Products, McGraw Park, IL) Wright's Dip Stat Stain Set (Medical Chemical Corp., Torrance, CA) OTHER: Field s stain 93 31

32 BLOOD PARASITE STAINS Giemsa, Wright/Giemsa, Rapids, Fields 94 Patient thick/thin blood films are own controls; WBCs look good = parasites look good. NORMOCYTIC VS MACROCYTIC RBCS Plasmodium falciparum Normocytic Growth continues in EDTA ü Plasmodium vivax Macrocytic 95 EXTRACELLULAR FORMS Parasites and Platelets 96 Babesia spp. Extracellular Forms Make sure you know what platelets look like artifact situation. Actual parasites must have both nuclear and cytoplasmic colors. 32

33 Examination Possibilities RBCs Intraerythrocytic protozoa Plasmodium spp. Babesia spp. Extraerythrocytic protozoa Babesia spp. Trypanosoma spp. Extraerythrocytic helminth Microfilariae 97 Examination Possibilities WBCs 98 Within WBCs Leishmania donovani Toxoplasma gondii Outside of WBCs Toxoplasma gondii Trypanosoma spp. Top: Leishmania; Bottom: Toxo, Tryp Toxo Trypanosome Plasmodium vivax 99 33

34 Plasmodium ovale P. ovale curtisi, P. ovale wallikeri 100 Plasmodium malariae 101 Plasmodium falciparum * * Exflagellation can occur in any Plasmodium spp

35 Plasmodium knowlesi Characteristics 24 hr (simian malaria); most rapid cycle Infects any age RBC; heavy infection ; severe No Schüffner s dots (stippling); clumpy dots Delicate rings, two dots of chromatin/ring, appliqué or accolé forms; two rings/rbc Band forms common; round gametocytes Early = P. falciparum; later = P. malariae 103 Plasmodium knowlesi Endemic Areas 104 Sarawak (Malaysian Borneo), Singapore, Thai-Myanmar border; Philippines, Yunnan Province in China; Finland, New York, Australia, Netherlands (travelers-endemic areas) *

36 MIXED MALARIAL INFECTIONS More common than thought Thailand: 30% both P. falciparum, P. vivax Africa: P. falciparum, P. malariae (Malaysia/ P. knowlesi Gambian children: <1% to >60% mixed New Guinea: all four species (PCR) Anopheles : transmit two species at same time 106 Difficult to detect Different parasite levels, low densities, morphologic criteria; PCR ID to species level ANTIGEN DETECTION TESTS External controls now available. TEST PRINCIPLE Detection of HRPII Antigen SPECIES IDENTIFIED P. falciparum, P. vivax BinaxNOW Alere Scarborough P. falciparum Sensitivity/ Specificity 99.7% / 94.2%* P. vivax Sensitivity/Specificity 93.5% / 99.8% Maximum sensitivity - parasitemia >5,000 (parasites/µl) = 0.1% 107 Parasitemia Light Microscopy % (5-20/µl) = Positive thick film % (100/µl) = Naïve patients may be symptomatic below this level; remember ER patients - travelers 0.2% (10,000/µl) = Level above which immune patients symptomatic; 0.1% (5,000) = BinaxNOW (max sens) 2% (100,000/µl) = Maximum parasitemia of P. vivax, P. ovale (young RBCs only) rarely exceeds 2% 2-5% (100, ,000/µl) = Hyperparasitemia, severe malaria, increased mortality 10% (500,000/µl) = Blood replacement, high mortality 36

37 PLASMODIUM Report Comments Plasmodium spp. seen: Unable to rule out Plasmodium falciparum or Plasmodium knowlesi Plasmodium spp. not seen: One negative set of blood films will NOT rule out malaria; submit additional blood specimens every 4-6 hours. Plasmodium spp. seen, possible mixed infection: Unable to rule out Plasmodium falciparum or P. knowlesi NOTE: Mandatory Report parasitemia for every initial and subsequent positive set of malarial films Use the same parasitemia method for all blood film sets on that particular patient. 109 BABESIA SPP. Ixodes scapularis Babesia divergens: Europe Rare, but 42% mortality Babesia microti: US, parts of Europe, Japan Northeast, south to New Jersey; 5% mortality Most cases mild; more severe in immunosuppressed WA1, CA1, MO1 (Babesia divergens-like), states Very serious in splenectomized patients Related to canine pathogen, B. gibsoni WA1, CA5: Now Babesia duncani 110 More pathogenic than B. microti Other blood parasites: Babesia spp. 111 Artifacts Plasmodium falciparum 37

38 CUTANEOUS LEISHMANIASIS : CLINICAL PRESENTATION 112 MUCOCUTANEOUS LEISHMANIASIS L. mexicana Complex u Common throughout Mexico, Belize, Guatemala and southern United States (Arizona, Texas) u Forest rodents (wood rats) are important hosts u Prolonged exposure = chicleros collect chewing gum latex (30% first year), timber cutters, road builders, farm workers u Two culture-positive and four PCR-pos rodents Leishmania-positive. Isolates extend geographic and ecologic range of enzootic leishmaniasis in the U. S. and is new host record. Tucson, Az 113 MUCOCUTANEOUS LEISHMANIASIS

39 LEISHMANIASIS: DIAGNOSIS 115 Organism isolation: smear or culture; histology Lesion sampling: dental broach, slit scrape method, aspiration of lesion edge (multiple samples required), biopsy ( 3 punch spec) Tissue sampling better than other smearing techniques; culture positive in 80% of cases LD bodies seen in about 30% of patients; many lymphocytes/plasma cells in wet ulcerative lesions; dry lesions = granulomas / fewer cells AMERICAN TRYPANOSOMIASIS (No longer South American) Chagas Disease Trypanosoma cruzi Transmission infective organisms in feces of blood-sucking triatomid bugs (kissing bugs, reduviid bugs) Bug s feces contact site of bug bite or mucus membranes; transfusions, congenital infections Mexico, Central, South, and North America (Texas, California); rural areas; dogs, cats, opossums, rodents, armadillos important reservoir hosts 116 CONFIRMED BLOOD DONATIONS: CHAGAS DISEASE Spread of Triatomid Bugs

40 AMERICAN TRYPANOSOMIASIS CLINICAL DISEASE u Initial infection often asymptomatic years later chronic form of disease; acute disease, children u Unilateral periorbital edema (Romaña s sign) u Lesion, fever, lymphadenopathy, myocarditis, hepatosplenomegaly, meningoencephalitis u Chronic disease = cardiomyopathy, congestive heart failure or arrhythmias; dilation of esophagus or colon; MEGASYNDROME 118 xenodiagnosis 119 QUESTIONS