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PARASITOLOGY July 8, 2011 Lecture Accurate Diagnosis of parasitic infections is important to decrease the prevalence and incidence of parasitism.

Confirm a clinical impression that the condition is parasitic in nature Rule out a diagnosis Aid the physician in the choice of proper medication Help in monitoring effect of a treatment regimen Reliability of Results is dependent on the following: Proper collection of specimen Proper handling and processing of specimens Skill of the laboratory analyst Quality of equipment used in the examination

Diagnosis of Parasitic infections can be done by: 1. Demonstration of parasites provide a definitive diagnosis Possible only during the patent stage of the infection - Adult - cysts - Eggs - oocysts - Larvae - trophozoites 2. Detection of host immune response to the parasites. - Provides presumptive evidence of infection Specimen use for Parasitic Exam Stool Urine Blood Tissue aspirate orifice swabs sputum CSF tissue biopsies

Fresh specimens in sufficient amounts are valuable Methods of Parasite Identification / Examination A. Microscopic Examination of Stools - Most important and common method of intestinal parasite diagnosis through the demonstration of the following: Eggs Cysts Trophozoites Adults Larva Oocytes Stool specimens for examination should have the following information: - Patients name - age / sex / gender - Date / time of collection - requested procedure - Requesting physician - presumptive diagnosis - Travel history - prior infections

Consideration for a useful parasitic diagnosis: 1. Drugs which could interfere with the identification of parasites. - These drugs contain crystalline residues. Stool samples should be collected a week after the last intake of these drugs. 2. Intake of antibiotics - Usually decreases the number of protozoan. 3. Amount of stool to be collected. - For routine exam; thumb sized specimen of formed stool. - About 5-6 tbsp. of waters stool. 4. Contamination with toilet water, urine and soil must be avoided - This can destroy protozoan trophozoites - Soil and water may contain free-living organisms that would complicate diagnosis of infection 5. Age of the sample (it is advisable that we would not accept specimens that would exceed 30 mins to 1 hour of age) 6. Delay in the examination may require preservation to ensure that parasites are present in the identifiable stage. If not to refrigerate, add chemicals 7. Refrigerate (1-6 C; 1-5C; 3-5C in a 12-24 hour period) - Never freeze stool samples - Never keep them in incubators 8. Avoid drying of the specimens Elements which may be found in stool specimens aside from parasites: WBC PMN / eosinophils (polymorphonuclear leukocytes) - May indicate inflammation RBC - Ulcerations / bleeding Macrophages Bacterial and parasitic infections Charcot-Leyden Crystal - Hypersensitivity Epithelial cells from the intestinal tract Eggs of arthropods/plant nematodes may be mistaken for human parasites. Fungal spores (yeast and yeast-like fungi) Elements of plant origin which may resemble a parasite - Plant cells/fibers - vegetable spirals - Pollen grains - starch granules Plant element and animal hairs that may look like helminth larvae Techniques of Microscopic Stool Examination 1. DFS (Direct Fecal Smear) - Detection of motile protozoan trophozoites/cysts 2. Kato thick smear - Can be used in mass stool examination - Good in detecting eggs with thick shells - Diarrheic/watery stools are not recommended. Does not detect protozoan cysts/trophozoites 3. Concentration - Recommended in light infections or if there is a need to recover more parasites Sedimentation Floatation (another ex. Brine Floatation)

4. Stool Culture methods - Larval differentiation Coproculture Harada mori / test tube method culture 5. Egg counting procedures - To identify the severity/intensity of infection or Katokatz method / cellophane covered thick smear Short egg count 6. Perianal Swab - Enterobius and Taenia species Cellulose tape / scotch tape method B. Macroscopic Examination - Stool specimens submitted to the lab can be on a fresh state or preserved. In fresh stools, the laboratory should classify the following: Consistency - Formed - soft - Semi-formed - loose / watery Color - Presence of blood should always be reported Presence of Mucus Common Stool Preservatives Appropriate fixation of parasites in stool will presume the morphological features of the parasites and destruction of eggs and larvae. Ratio: stool - preservative (1:3) 1. Formalin buffered with sodium phosphate - 5% for protozoan cysts - 10% for helminth egg and larvae 2. Schaudinns Solution - For fresh stools for staining 3. PVA - Plastic resin serves to adhere a stool sample onto a slide - For protozoan cysts and trophozoites for permanent staining 4. MIF (Merthiolate-Iodine-Formaldehyde) - For fixation of protozoans, helminth eggs and larvae 5. SAF (sodium acetate acetic acid formalin) - Does not contain mercuric chloride Examination of Blood For filarial and protozoan parasites Methods: 1. Finger-prick a. Wet/fresh preparation - Microfilariae and tryposmastigote b. Stained smear - Thick films (rapid identification) - Thin films (proper identification) Blood stains usually used for parasite identification: a. Giemsa b. Wrights stain c. Delafield hematoxylin stain

c. Capillary tube method - Using heparinized capillary tube Buffy coat films - Trypanosomes and leishamania Quantitative buffy coat - Capillary tube which is precooled with acridine orange and potassium oxalate - Babesia 2. Venous Blood Detect microfilaria a. Knotts concentration - 1 ml of blood is concentrated with 10 ml of 2% formalin b. Membrane Filtration - Using syringe with a swinney filter holder attached - Draw 1 ml of fresh / anticoagulated blood and lysed by adding distilled water - Microfilaria will be recovered Sputum Examination Parasites that can be recovered from sputum: 1. Migrating larvae off ascaris, strongyloides, and hookworms 2. Paragonimus ova 3. Echinococcus granulosus hooklets 4. Protozoa - Entamoeba histolytica - Cryptosporidium parvum oocytes - Entamoeba gingivalis Considerations: 1. Gross and Macroscopic Examination a. Consistency - Serous - Mucoid - Purulent - Bloody - Or combination b. Color Yellow may indicate pus Greenish tint pseudomonas Bright red bleeding Rust-colored hemoglobin breakdown 2. Microscopic Examination a. Wet mount using saline or iodine - Useful for protozoan trophozoites b. Sputum concentration (3% NaOH) - Sediments are studied 3. Serological Methods To detect antibodies / antigens Immunodiagnosis and DNA diagnosis - IHA - immunoblot - IF - RIA - ELISA - immunodiffusion

- PCR