1. Parasitology Lab methods

2. Parasitology: Laboratory methods (1 hour) 1.1.Lists the laboratory methods in parasitology (microscopy, culture, serology, DFA and PCR). 1.2.Defines the sample taking. 1.3.Lists the stains in microscopy.

3. Parasitic Evaluation of Stool Specimen: Collection and Processing Techniques If a faecal sample is not properly collected and taken care of before examination, they will be of little or no value for accurate diagnosis. If a faecal sample is not properly collected and taken care of before examination, they will be of little or no value for accurate diagnosis. Amoebic trophozoites begin to degenerate 1-2 hours after passage, as do flagellate trophozoites. Amoebic trophozoites begin to degenerate 1-2 hours after passage, as do flagellate trophozoites. Cysts will deteriorate if the faecal specimens are left standing for many hours or overnight, especially at high temperatures. Cysts will deteriorate if the faecal specimens are left standing for many hours or overnight, especially at high temperatures. Helminth eggs and larvae are less affected by the age of the specimen than are protozoa. Helminth eggs and larvae are less affected by the age of the specimen than are protozoa. changes may occur that could affect their identification changes may occur that could affect their identification

4. A clean dry container must be used for the collection of faecal samples. Urine and water will destroy trophozoites, if present, and the presence of dirt also causes identification problems. A clean dry container must be used for the collection of faecal samples. Urine and water will destroy trophozoites, if present, and the presence of dirt also causes identification problems. Ideally the specimen should be brought to the lab as soon as it is passed, to avoid deterioration of protozoa and alterations of the morphology of protozoa and helminths. Ideally the specimen should be brought to the lab as soon as it is passed, to avoid deterioration of protozoa and alterations of the morphology of protozoa and helminths. The specimen container should be clearly labelled with the patients name, date, and time of passage of the specimen. The specimen container should be clearly labelled with the patients name, date, and time of passage of the specimen. An amount of stool adequate for parasite examination should be collected and a repeat sample requested if too little is supplied. An amount of stool adequate for parasite examination should be collected and a repeat sample requested if too little is supplied. Diarrhaeal specimens, or those containing blood and mucus, should be examined promptly on arrival in the laboratory. Diarrhaeal specimens, or those containing blood and mucus, should be examined promptly on arrival in the laboratory.. if specimens cannot be examined as soon as they arrive, they should be put in the refrigerator.. if specimens cannot be examined as soon as they arrive, they should be put in the refrigerator. Specimens from patients with HIV should be left in 10% formalin for hour before proceeding with parasite examination Specimens from patients with HIV should be left in 10% formalin for hour before proceeding with parasite examination

5. Three grams of sample is required for most parasite analysis. Three grams of sample is required for most parasite analysis. Stool should be free of urine, because urea inhibits some parasites. Stool should be free of urine, because urea inhibits some parasites. Fresh stools are needed for amebae and flagellate trophic forms. Fresh stools are needed for amebae and flagellate trophic forms. Liquid stools are best to detect tropic forms, while formed stools are best to detect ova and cyst forms. Liquid stools are best to detect tropic forms, while formed stools are best to detect ova and cyst forms. Stools should not be frozen or allowed to stand at room temperature. Stools should not be frozen or allowed to stand at room temperature. Polyvinyl alcohol (PVA) and Sodium Acetate formalin (SAF) can be used to preserve stool samples. Polyvinyl alcohol (PVA) and Sodium Acetate formalin (SAF) can be used to preserve stool samples. Gross examination of stool may detect adult forms Visual observation of the faecal sample Gross examination of stool may detect adult forms Visual observation of the faecal sample

6. It is important to observe the macroscopic appearance of the stool as this can give a clue to the type of organisms present. It is important to observe the macroscopic appearance of the stool as this can give a clue to the type of organisms present. the consistency; formed, unformed or liquid; the consistency; formed, unformed or liquid; the colour the colour the presence or absence of an exudate the presence or absence of an exudate the presence of adult worms can also be seen in a freshly passed stool eg adult stages of Ascaris lumbricoides and Enterobius vermicularis. Proglottids of Taenia species can also be seen. the presence of adult worms can also be seen in a freshly passed stool eg adult stages of Ascaris lumbricoides and Enterobius vermicularis. Proglottids of Taenia species can also be seen.

7. Types of Microscopic evaluation techniques: Simple method: evaluation with physiological saline evaluation with lugol solution Concentration method: Sheathersugarflotation Formalin-ethyl acetate concentration Zinc sulfate flotation Blood concentration methods: The knott method uses low speed centrifugation to concentrate blood samples suspected of containing minimal parasite amounts. Buffy coat slides are used for Leishmania or Trypanosoma detection

8. Permanent stained samples :Permanent stained smears is used to enhance parasite morphology, and to allow for future study. Iron hematoxylin stain Trichome stain (Wheatley or Gomori) is the most commonly used stain for parasite study. Modified acid-fast stain is used to detect Cryptosporidium Methylene-Blue stain and Giemsa stain is used to evaluate morphologies of eggs and trophozoite form.

9. Collection methods of Stool Samples: The Cellophane tape method is used to collect Enterobius vermicularis eggs. The Enterotest (string test) is used to obtain duodenal contents for parasitic examination. The sigmoidoscope is used to collect colon material.

10. Sample types and associated parasites (genus) Blood: Leishmania, Trypanosoma and onchocerca Skin: Onchocerca. Vaginal or urethral: Trichomonas. Oral (mouth or nasal): Entamoeba, Trichomonas, or Naegleria. Eye scrapings: Acanthamoeba. Tissue: Naegleria, Acanthamoeba, and Leishmania. Urine: Schistosoma and Trichomonas. Sputum: Ascaris, Strongyloides, and Entamoeba.

11. Diagnostic Test Direct Fluorescent Antibody (DFA): used to identify Trichomonas vaginalis. Direct Agglutination Test (DAT): used to diagnose leishmaniasis and Chagas‘ disease. ELISA: used to identify Toxoplasma gondii. Complement fixation (CF): used to diagnose leishmaniasis, Chagas' disease, and pneumocystosis. Gel-Diffusion Precipitin (GDP) test: used to detect amebic infections. Indirect Immunofluorescent Antibody (IFA): used to diagnose amebic, malarial, toxoplasmosis, and schistosome infections. DNA probes, flow cytometry, and polymerase chain reactions are being developed for parasite diagnostic use.