1. Faculty of Medicine and Health Sciences Parasitology Lab Second semester 2013/2014 prepared by: Mohammad Al-Qadi E-mail: m.qadi@najah.edu

2. Outline Topic Weeks Orientation and introduction.W1 Important Parasitological techniques.W2 Amebas.W3 Flagelates.W5 Ciliates and Apicomplexan.W5 Trypanosomes and Leishmania spp.W6 Plasmodium.W7 Revision.W8 Intestinal nematodes.W9 Filaria.W10 Tapeworms.W11 Cestodes.W12 Ectoparasites.W13 Revision.W14 Midterm Exam 40%, Final Exam50%and Quiz, Participation & activity 10 %

3. Safety rules, devices, tools and introduction to Parasitology Lab Lab # 1

4. Safety & Rules Lab coats and gloves. Leave all unnecessary things out side the lab. ( bring only strictly required things as pen and some paper) Smoking, Eating and Drinking are totally banned. Don’t touch face by hands or pens or other things. Nails, Hair and Clothes

5. Safety & Rules Clean your bench area before and after work with proper disinfectant. Deal with all infective material with caution. In case of skin contact with infectious material rinse quickly with antiseptic and wash with soap and water. In case of eye contact with chemicals or biohazardous material wash eye quickly under cool running water. If the infectious material was broken or spilled immediately cover with disinfectant and tell lab technician.

6. Safety & Rules If you use any sharp instruments, dispose of them in a "sharps" container for decontamination. Infected material must be put in autoclavable bags. Put contaminated cotton swabs, slides, pipettes and tips in a container that contains disinfectant. Remove gloves and wash your hands after completing any task involving the handling of fecal material or infectious material Finally, should follow lab instructor commands and guidance, memorizing exactly experiment procedures and don’t hesitate to ask.

7. Devices and Tools Microscope Autoclave Micropipette Laminar flow Tube Glassware Centrifuge Bunsen burner Slide

8. Glassware

9. Centrifuge

10. Bunsen burner and Hot plate

11. Laminar flow

12. Autoclave

13. Microscope

14. Specimen Collection (Stool) Collect the stool in a dry, clean, leakproof (parafilm or other suitable material) container. (Make sure no urine, water, soil or other material gets in the container) Fresh stool should be examined, processed, or preserved immediately. (An exception is specimens kept under refrigeration when preservatives are not available; these specimens are suitable for antigen testing only.) Specimen should be divided and stored in two different preservatives, 10% formalin and PVA (polyvinyl- alcohol), using suitable containers. Add one volume of the stool specimen to three volumes of the preservative. Insure that the specimen is mixed well with the preservative. Formed stool needs to be well broken up. Certain drugs and compounds will render the stool specimens unsatisfactory for examination. (The specimens should be collected before these substances are administered, or collection must be delayed until after the effects have passed. Such substances include: antacids, kaolin, mineral oil and other oily materials, non-absorbable antidiarrheal preparations, barium or bismuth (7-10 days needed for clearance of effects), antimicrobial agents (2-3 weeks), and gallbladder dyes (3 weeks)) Specimen collection may need to be repeated if the first examination is negative. If possible, three specimens passed at intervals of 2-3 days should be examined.

15. Specimen Collection (Stool)

16. Laboratory Diagnosis of Parasitic Infections Purpose – – Confirmation of clinical suspicion – Identification of unsuspected infection Methods same as used in Bacteriology & Virology but significance of different methods varies. Isolation least important, morphological identification very important. Serology relatively less important 16

17. Morphological identification Examination of faeces – – Gross – Microscopy Saline mount Iodine Mount Thick smears – not commonly used Permanent stained smears – Iron hematoxylene – Whearley’s trichrome stain – Concentration methods Floatation techniques Sedimentation techniques 17

18. Examination of Blood – Thin Smear – Thick smear – Wet mount for microfilaria – Stains used 18

19. Entamoeba histolytica and Entamoeba coli Intestinal protozoa

20. Life cycle

21. (1) Infection by Entamoeba histolytica occurs by ingestion of mature cysts in fecally contaminated food, water or hands. (2)Excystation occurs in the small intestine and trophozoites are released. (3)Migrate to the large intestine. Trophozoites multiply by binary fission and produce cysts (4)Cysts are passed in the feces. The cysts can survive days to weeks in the external environment.

22. Biology - structure

23. Structure - trophozoites

24. Structure - cyst

25. Disease Amebiasis – infection of the intestines Symptoms: abdominal cramps, diarrhea (with blood and mucus), weight loss, Entamoeba histolytica invades colon wall, causing colitis, acute dysentery, or long-term diarrhea. Complications: appendicitis, intestinal hemmorhage, perforation Can spread through blood to liver, and rarely, to the lungs, brain or other organs Hepatic amebiasis, acute amoebic hepatitis, liver abscesses, hepatomegaly and cutaneous amebiasis are frequent extra-intestinal complications.

26. Diagnosis Microscopic examination of feces of infected patient  detect trophozoites when in acuted dysentery  detect cysts and trophozoites in chronic and asymptomatic amebosiss

27. Treatment and prevention Treatment depends on severity of infection Usually, metronidazole is given by mouth for 10 days High level of social sanitation, personal hygiene, boiling drinking water, not eating uncooked vegetables or unpeeled fruit. (Tropical countries)

28. Entamoeba coli

29. Non-pathogenic amoeba with world wide distribution Life cycle – similar to E. Histolytica, but does not have invasive stage and does not ingest red blood cells Trophozoite is larger than E.h, from 15-50 micrometers Diagnosis – finding the characteristic cysts or trophozoites microscopically

30. Entamoeba coli

31. Comparison Entamoeba coli can be confused during microscopic examination of stool with entamoeba histolytica This differentiation is usually done by examination of the parasitic cysts with light microscopy

32. Useful Links http://www.cdc.gov/parasites/ http://www.atlas-protozoa.com/ http://www.ncbi.nlm.nih.gov/pubmedhealth/ PMH0001343/ http://www.ncbi.nlm.nih.gov/pubmedhealth/ PMH0001343/ http://www.soton.ac.uk/~ceb/Diagnosis/Vol1. htm#ec http://www.soton.ac.uk/~ceb/Diagnosis/Vol1. htm#ec http://pathmicro.med.sc.edu/parasitology/int est-protozoa.htm http://pathmicro.med.sc.edu/parasitology/int est-protozoa.htm