Future Challenges in Diagnostic Medical Parasitology

Transcription

1 LYNNE S. GARCIA, MS, CLS, FAAM CAPHLD 65 th Institute Future Challenges in Diagnostic Medical Parasitology SPONSOR: MEDICAL CHEMICAL CORPORATION 1

2 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

3 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 STOOL ORDER O&P, Immunoassays, RECOMMENDATIONS Special Testing IV. Cyclospora Autofluorescence 3 Special stains 11

4 FECAL IMMUNOASSAYS - ANTIGEN 4

5 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

6 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 6 months to years

7 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) 7

8 ENTAMOEBA HISTOLYTICA ENTAMOEBA DISPAR Entamoeba dispar (non-pathogen) Note: Ingested RBCs Entamoeba histolytica (pathogen) 8

9 REPORTING 9 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)

10 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) 10

11 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 11

12 Dientamoeba fragilis: Pathogen Cyst 12 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

13 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 13

14 CRYPTOSPORIDIUM SPP. C. hominis, C. parvum 14 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 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. 15

16 CYCLOSPORA CAYETANENSIS (Suspected Food Borne Outbreaks) Safranin Stain Modified acid-fast stain Autofluorescence Acid-fast variable Often 1+ to % acid rinse < Crypto FA

17 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) 17

18 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

19 Microsporidia Polar Tubule 19

20 Eye Infections Cytospin MICROSPORIDIA Spores in NA aspirate Intestinal Tissue Urine: Calcofluor White Spores, muscle Corneal button Corneal stroma 20

21 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, 21 rhinitis, disseminated infection

22 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 22

23 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 + 23

24 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

25 PATHOGENIC FREE-LIVING AMEBAE AGAR PLATE CULTURE 25

26 Strongyloides stercoralis 26

27 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 27

28 RARE INFECTIONS in U.S. Baylisascaris procyonis - Raccoon Raccoon ascarid, serious zoonotic disease 28 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

29 ECHINOCOCCUS MULTILOCULARIS 29 (Alveolar Hydatid Disease)

30 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. 30

31 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

32 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 32

33 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.

34 PLEASE GO TO PART 2 34