Overview IMPORTANCE CLASSIFICATION SPECIMEN COLLECTION PROTOZOA WORMS BLOOD PARASITES ARTHROPODS DELUSIONAL PARASITOSIS QUIZZES GROSS

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2 IMPORTANCE Overview CLASSIFICATION SPECIMEN COLLECTION PROTOZOA WORMS BLOOD PARASITES ARTHROPODS DELUSIONAL PARASITOSIS QUIZZES GROSS

3 - Of 6.6 billion people on Earth, 2.7 billion live on <$2/day - 1 in 4 suffer from ascariasis, trichuriasis, or hookworm infection - 1 in 10 suffers from schistosomiasis

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5 Transmission of Intestinal Parasites

6 Classification Protozoa: Microsporidia Encephalitozoon; Enterocytozoon; Microsporidium Protozoa Trichomonas; Naegleria; Leishmania; Trypanosoma; Acanthamoeba; Balamuthia; Entamoeba; Cryptosporidium; Toxoplasma; Babesia; Plasmodium Nematodes (roundworms; microfilaria) Trichinella; Loa loa; Onchocerca; Wuchereria; Brugia; Mansonella; Strongyloides; Dracunculus Trematodes (flukes) Schistosoma; Fasciola; Paragonimus Cestodes (tapeworms) Taenia; Echinococcus; Diphyllobothrium

7 Protozoa Protozoa Versus Helminths Helminths Microscopic (2-100 μm) Macroscopic (<1 mm -10 m) Single-celled Multicellular Simple (like yeast) Ascaris Differentiated lumbricoides tissues Entamoeba histolytica 10 μm

8 Parasitology Specimen Collection

9 Specimen Collection Stool Blood Other types of specimens

10 Why Collect Stool? Amoeba E. histolytica E. coli (non path) G. lamblia Round worm Ascaris Hookworm Enterobius vermicularis Strongyloides Trematodes Clonorchis sinensis Paragonimus westermani Schistosomas(not Sh) mansoni Cestodes D. latum T. saginata T. solium

11 Relationship of stool consistency to protozoan stage Active trophozoites can be found in fresh or liquid stool Cysts are found in formed stool

12 More formed = more cysts Less formed = more trophs

13 Specimen Collection Clean / dry / leak-proof container Unpreserved, store 1 hr at RT / 4 hr refrigerated Preserved: store at RT (examine within hours) Collect 3 specimens over 2-3 days if first is negative Preserve specimen ASAP usually using 2 preservatives 10% formalin PVA Combine 1 part specimen : 3 parts pres.

14 Collection containers hardydiagnostics.com

15 Interfering Substances Delay collection for the indicated period of time if the patient consumes the following: 7-10 days Antacids Kaolin Mineral oil Non-absorbed anti-diarrheal preparations Barium or bismuth 2-3 weeks Antimicrobial agents 3 weeks Gall bladder dyes

16 10% Formalin Advantages Preserves the morphology of helminth eggs, larvae, protozoan cysts, coccidia Can be concentrated Can be used to prep specimens for acid fast, chromotrope (eg. For Microsporidia) stains, some DFA Compatible with epifluorescence microscopy Disadvantages Does not work well with trichrome stain Does not preserve troph morphology well Can interfere with PCR / some EIA

17 Polyvinyl Alcohol (PVA) (non mercuric) Advantages Good preservation of morphology of trophs and cysts Easy to prep permanent slides Stabilizes specimens for months Disadvantages Does not preserve helminth eggs / larvae / coccidia / microsporidia Can t be concentrated No acid fast, safranin or chromotrope stains

18 Concentration Floatation (Zinc sulfate / Sheather s sugar) Solution has increased specific gravity so organisms rise while debris sink Advantages clean material Disadvantages walls of eggs and cysts can collapse, some eggs do not float

19 Concentration Sedimentation (formalin ethyl acetate) Solution has decreased specific gravity so organisms sink Advantages less prone to technical errors Disadvantages less clean

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21 Microscopic Examination Calibrate ocular Wet mount Stain with iodine Seal edges with 1:1 wax / petroleum jelly Scan at 10X and move to higher magnification if suspicious things are seen Direct mount / UV exam (cyclospora) Under UV, fluoresces blue Under intense blue, fluoresces green

22 Saline and Iodine Wet mounts EC troph, cyst atlas-protozoa.com commons.wikimedia.org Eh troph dpd.cdc.gov

23 Trichrome Stain Eh troph atlas-protozoa.com Blastocystis hominis badbugs.org

24 Modified Kinyoun Stain

25 Microsporidia

26 Other Preps from Stool Scotch tape prep E. vermicularis Stick scotch tape on the anus several hours after sleeping or just after waking before visiting the bathroom or taking a shower Must have 4-5 negatives for definitive negative diagnosis

27 Pinworm Paddle mtherald.com practicalscience.com

28 Detection of Parasite Antigens EIA E. histolytica (req. freash unpreserved stool) Cryptosporidia (10% formalin is OK) Giardia (10% formalin is OK) Direct fluorescence assay ID oocysts in concentrates and unconcentrated specimens with a FITC labeled monoclonal Cryptosporidium Giardia Rapid immunochromogenic tests available (rapid tests) E. histolytica Giardia Cryptosporidium Must be confirmed by other assays

29 Giardia and Cryptosporidia IFA dpd.cdc.gov

30 Molecular Methods PCR can be used to look for pathogens Need specimen with no preservative or preserved in absolute ethanol CDC will test for Giardia, cryptosporidium, cyclospora, microsporidium, E. histolytica

31 Blood Specimens Collected for to look for Malaria Filarial worms Trypanosomes Babesiosis Collect before treatment with anti-parasite medication Thin and thick smears (advantages and disadvantages) Stains Wrights stain (suboptimal) Giemsa stain

32 Other Specimen Types Urine Schistosoma haematobium (seen in iodine stain of concentrate Urogenital Trichomonas vaginalis Tissues toxoplasma, leishmania, filarial worms, Taenia, Echinococcus, Clonorchis, Paragonimus CSF T. brucei, Naeglaria fowleri, Ancanthamoeba

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