diagnosis of intestinal protozoa


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diagnosis of intestinal protozoa

  2. 2. INTRODUCTIONIntestinal protozoa can be divided into pathogenic and non-pathogenicprotozoans. Laboratory diagnosis is important to differentiate betweenthe harmless and the medically important parasites. This is most oftenbased upon the morphology of respective organisms. There are somesteps that need to be followed in order to successfully diagnoseintestinal protozoan. The steps include:  Collecting specimens  Preparation of specimens  Evaluation under microscopePreparation of stool specimen includes direct smear (wet mount),concentration method, fixative and preservation, staining methods andcultivation. 1
  3. 3. SPECIMENS AND COLLECTION OF SPECIMENS The specimen is stool as the intestine is the habitat of the protozoa. Duodenal aspirate and material from ulcer or abscess can also be examined. The stool is collected in a dry, clean and leakproof screw-capped container. Loose and watery stool are examined for trophozoites while formed stool is examined for cysts. (Diagram 1.1) Diagram 1.1 Stool is best examined fresh as motile trophozoites may be seen and increases the chances to discover protozoa. The stool specimen must be free of urine and contaminants. If specimens need to be stored, they must be preserved immediately. They are then sealed well and stored properly. If preservative is unavailable, the specimen is kept in a refrigerator. These specimens are not suitable to examine motile form of protozoa. Screw-capped container to collect and store stool 2
  4. 4. PREPARATION OF STOOL SPECIMENSa. DIRECT SMEAR (WET MOUNT)Direct fecal smears can be used as a quick screening test to check for anyintestinal parasite, but the small size of the sample limits its usefulness. Wetmounts are useful for detecting motile organisms. The disadvantages are dirtybackground and less detection of parasites. Protozoa are often detected via adirect fecal smear, and are best acquired from the surface of fresh feces. Theprocedure of direct smear is as follow: First of all, a drop of saline or iodine is placed on a clean glass slide using a dropper. A pea-sized amount of fresh stool is taken using an applicator stick and mixed together with the saline/iodine drop. The smear is mounted with a coverslip and examined under microscope at 40x objective. Trophozoites, cysts, ova can be seen. Feces is mixed with saline using an The fecal material is mixed until it is well applicator stick dispersed to produce a smear 3
  5. 5. b.CONCENTRATION METHODSA concentration procedure is performed mainly to separate the parasites from fecaldebris. The concentration procedure not only increases the numbers of parasites inthe sediment but it also unmasks them, making them more visible by removingdebris.Formalin ether sedimentation techniqueFormalin acts both a fixative and preservative of protozoan eggs, larvae and cysts. The specificgravity of protozoan cysts and helminthes eggs is greater than that of water. Fecal debris isextracted into the ether phase so that the parasitic forms can be separated and thenregimented by centrifugation. The procedure is as follow:  A small amount of stool is mixed with 5 ml of 10% formal saline in a centrifuge tube using an applicator stick.  The mixture is then strained into a paper cup containing cotton gauze as a filter.  The filtered suspension is poured back into centrifuge tube.  10% formal saline is added until it reaches 8 ml.  2 ml of ether is added.  The centrifuge tube is plugged with a rubber stopper and shaked vigorously for few minutes.  It is then centrifuged at 2500 rpm for 2 minutes.  The debris plug is loosened with an applicator stick and the supernatant is discarded in a single hand motion.  The sediment is allowed to mix with the remaining fluid and placed on a clean glass slide.  The sediment is mounted with a coverslip and viewed under microscope. 4
  6. 6. BRINE FLOATATION TECHNIQUEThis technique is best used in diagnosis of helminthes but can also beused to recover protozoa which are light weighted. The protozoa whichare lighter than the solution will float and can be lifted with glass slideto be examined. The procedure is as follow:  Saturated solution of sodium chloride is prepared (brine solution).  A small amount of feces is mixed with 2ml of brine solution in a bijou bottle.  More brine solution is added till the brim of bijou bottle while stirring.  Drops of brine solution are added up to the surface of the bottle without overspilling.  A clean glass slide is placed over the solution surface and left for 30 minutes exactly.  The slide is lifted in a single hand motion and examined under microscope. Bijou bottle used in brine floatation technique. 5
  7. 7. FIXATIVE AND PRESERVATIONPreservation of specimens is necessary when stool specimens cannot be examined within the prescribedtime interval. Various preservatives are available with the two most commonly used being Merthiolateiodine formaldehyde (MIF) and Polyvinyl alcohol (PVA).FIXATIVE ADVANTAGE DISADVANTAGEPolyvinyl alcohol (PVA)  Permanent smears can  Staining not consistent be made and stained  Organism morphology with trichrome may be poor  Zinc is preferred over  Copper-morphology of copper cysts and trophozoites is  No mercuric chloride poorMerthiolate iodine  Components both fix  Not suitable for someformaldehyde (MIF) and stain organisms permanent smears  Easy to prepare stained with trichrome  Long shelf life  Inadequate preservation  Useful for field surveys of morphology of  Suitable for protozoan trophozoites concentration  Iodine interferes with procedures other stains and fluorescence  Iodine may cause distortion of protozoa10% Formalin  All purpose fixative  Not suitable for some  Easy to prepare permanent smears  Long shelf life stained with trichrome  Good preservation of  Inadequate preservation morphology of helminth of morphology of eggs, larvae, protozoan protozoan trophozoites cysts, and coccidia  Can interfere with PCR,  Suitable for especially after extended concentration fixation time procedures and UV  fluorescence microscopy  Suitable for acid-fast, safranin, and chromotrope stains  Compatible with immunoassay kits and UV fluorescence microscopy  6
  8. 8.  Polyvinyl alcohol (PVA) This is a mixture of fixative and water-soluble resin that is specifically used to fix and preservetrophozoites of intestinal amoebic organisms. These trophozoites are very fragile and willbecome distorted or disintegrate completely within a few hours after stool passage. Thisfixative will preserve trophozoites for long periods of time and will make it easier to identifythem. PVA is primarily used for preserving fresh specimens to be shipped to centrallaboratories. Permanently stained films can be prepared from the preserved material. Thesolution serves as an adhesive as well as a preservative and prevents the loss of organismsduring staining. This is advantageous when preparing smears from liquid specimens.Preparation of reagents:Polyvinyl Alcohol Fixative (PVAEthyl alcohol (95 %) 50 mlSaturated mercuric chloride 100 mlGlacial acetic acid10 gGlycerol3 mlPolyvinyl alcohol powder (PVA) 9 gProcedure:STEP 1: Mix a portion of feces with three times the amount of formalin.STEP 2: Allow settling for at least one hour and store in screw-cap bottles.STEP 3: If shipment or prolonged storing is required, dip the top portion of the tightly fastenedcontainer, to include the cap, two or three times into a hot paraffin bath. This will preventspillage and reduce the rate of evaporation 7
  9. 9. PERMANENT STAININGIt is a routine diagnosis. Two methods are used in permanent staining of intestinal protozoa, trichromeand iron-hematoxylin. TRICHROME STAINING METHOD The Trichrome stain is a rapid staining procedure which provides excellent differentiation of internal structures of intestinal parasites as well as facilitating the separation of these organs from background material and artifacts. The Trichrome stain (Wheatley Modification) is to be used for staining of intestinal protozoan cysts and trophozoites in PVA fixed specimens. Sediments from the Formalin-ethyl acetate concentration technique cannot be stained by this method. All liquid stool specimens should receive a trichrome stain as trophozoites will occur in liquid specimens and not formed specimens. Soft stool specimens may be stained if requested or the concentrates are suspicious.Preparation of staining solution 1) Tincture of iodine:  Iodine crystals 7g  Potassium iodine 5g  70 % alcohol 100ml Procedure: 1. The iodine and potassium iodide is grinded in a mortar 2. The mixture is then dissolved and rinsed in alcohol 3. Tincture is stored in a brown bottle 2) Modified trichrome stain  Chromotrope 2R 0.6g  Light green SF 0.3g  Phosphotungstic acid 0.7g  Acetic acid 1.0ml  Distilled water 100 ml 8
  10. 10. Trichrome staining procedure 1. A thin smear is made of fresh stool on a slide 2. After the smear is dried, it is placed in PVA fixative 3. It is then let to stand overnight 4. The smear is then stained as follow : Solution Duration Tincture of iodine 1 minute 70 % alcohol 1 minute Trichrome stain 8 minute Acid alcohol 10 seconds Absolute alcohol 1 minute xylene 1 minute 5. It is then mounted withdeepex or Canada balsamThe outcome of trichrome staining(100x oil immersion) Protozoa-Entamoeba histolytica cyst Cytoplasm stain blue-green Nucleus stain red Glycogen vacuoles are clear( dissolved by fixatives Background is usually various shades of green 9
  11. 11. IRON HEMATOXYLIN STAINThis staining method gives maximum detail and more reliable outcome but is more time consuming thanthe trichrome staining. It is often used with fresh stoolProcedure: 1. Direct smear of fresh stool 2. Slides are immersed in schaudinn’s solution for overnight 3. Dip slides in iodine tincture for 5 min 4. Place slides in 70% ethanol for 5 min. 5. Place slides in 50% ethanol for 2 min. 6. Wash well with distilled water. 7. Place slides in hematoxylin working solution for 10 min. 8. Place slides under running tap water (best if tepid) for 10 min. 9. Differentiate slides (one by one) in destaining solution or for 30 s. 10. Place slides under running tap water for 10 min. 11. Place slides in 95% ethanol for 5 min. 12. Place slides in 100% ethanol for 5 min. 13. Place slides in 100% ethanol for 3 min. 14. Place slides in xylene for 5-10 min. 15. Mount slides with DPX Entamoeba histolytica 10
  12. 12. RESULTS (MICROSCOPIC EVALUATION) Entamoeba histolytica cyst- wet mount (high power) Entamoeba histolytica trophozoite- iron hematoxylin oil immersion 11
  13. 13. Entamoeba coli cyst-iodine stain oil immersion Entamoeba coli cyst and trophozoite- trichrome stain oil immersion Iodamoeba buetschlii cyst- iodine stain high power12
  14. 14. Iodamoeba buetschlii trophozoite and cyst- trichrome stain oil immersion Giardia lamblia trophozoite- trichrome stain oil immersion Giardia lamblia cyst- iron hematoxylin stain oil immersion13
  15. 15. Balantidium coli trophozoite- trichrome stain oil immersionBalantidium coli cyst in formalin preserved stool- unstainedhigh power Cryptosporidium parvum oocyst- acid fast stain oil immersion 14
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