CM and PARA - Fecalysis and Perianal swab (Day 8 and 9) PDF

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I. Clinical Microscopy and Parasitology Day 8-9: July 16-17, 2020Topics: Fecalysis Peri-anal swabReferences: Video AUBF 6thGen by StrasingerFecal AnalysisBasic Guidelines in Stool Analysis Do not eat, drink, smoke, apply cosmetics or manipulate contact lenses in work area.  Adopt universal precaut...

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CLINICAL INTERNSHIP 1 Day 8-9: July 16-17, 2020

I. Clinical Microscopy and Parasitology Topics: Fecalysis Peri-anal swab

References: Video AUBF 6th Gen by Strasinger

Fecal Analysis Basic Guidelines in Stool Analysis 

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Do not eat, drink, smoke, apply cosmetics or manipulate contact lenses in work area. Adopt universal precautions as well as standard microbiological laboratory practices (Biosafety Level 2). Wear protective safety glasses, gloves and laboratory coat when processing specimens. Use biological safety cabinets as needed. If you have cuts or abrasions on the skin of your hands, cover them with adhesive dressing. If you use any sharp instruments, dispose of them in a “sharps” container for decontamination. Remove gloves and wash your hands after completing any task involving the handling of fecal material. Decontaminate work surface at least once a day and after any spill of potentially infectious material.

Specimen Collection, Transport, and Preservation Procedures

Amoebiasis / Intestinal giardiasis - recommend at least 3 stool (every other day)  Direct Wet Mount  Fecal Concentration Technique  Permanent Stained Smear Specimens must be collected before the start of treatment regimens or collection must be delayed until after the effects have passed because it may result to suppression of parasites. → 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)  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.*** 4.

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Collect the stool in a dry, clean, leakproof container. Make sure no urine, water, soil or other material gets in the container. → →

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Stool sample should be collected on a clean cardboard or a pan. *transfer to a clean, dry, sterile, wide-mouthed container with a tight-fitting lid with sufficient moisture before submitting to the lab. NEVER CONTAMINATE WITH URINE AND WATER.

Possible stool contaminants:  Urine - known to destroy some parasites.  Toilet bowl water - free-living protozoa and nematodes may be confused with human parasites.  Water - destroy select parasites, such as schistosome eggs and amebic trophozoites.  Toilet paper - mask parasites or make examination of the sample difficult. 2.

Containers should be properly labeled at the side (body) of the container (not on the lid) with necessary information (Complete name, date and time of collection).

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Stool sample can be passed and collected on a sufficient amounts so repetition of examination can be done if necessary. → 2-5 grams → Pea / walnut / thumb size

The image on the right demonstrates the distribution of protozoa in relation to stool consistency and should be taken into consideration when specimens are received.

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. → Fresh stool samples should be delivered and processed into the laboratory  Liquid stool: within 30 minutes  Semi-solid stool: within 1 hour  Formed stool: within 1 hour → Preserved with fixative solution if there will be a delay in processing.*** REMEMBER!  Trophozoite: Watery stool  Cyst: Formed stool (easier to identify)

CLINICAL INTERNSHIP 1 Day 8-9: July 16-17, 2020

I. Clinical Microscopy and Parasitology Topics: Fecalysis Peri-anal swab

References: Video AUBF 6th Gen by Strasinger

Specimen Transport 1. Unpreserved Specimens → Place stool sample in a clean container as quickly as possible and kept under refrigeration until necessary arrangements are made for pick-up and delivery. → Ensure that the specimen remains cold during transport 2. Preserved Specimens → Same as for unpreserved specimens except that they do not need refrigeration. Specimen Preservation  If using a commercial collection kit, follow the kit’s instructions. If kits are not available, the specimen should be divided and stored in two different preservatives, 10% formalin and PVA  Add one volume of the stool specimen to three volumes of the preservative.  Insure that the specimen is mixed well with the preservative.

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Two-vial system - a formalin vial for the concentration techique and a PVA vial for the stained slide. 3.

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Schaudinn’s Fixative Advantage Good preservation of trophozoites and cysts Easy preparation of permanent stained smear

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General Preservatives for Stool Specimen 1.

Formalin Advantage

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All purpose fixative Easy to prepare Long shelf life Good preservation of morphology of helminth eggs, larvae, protozoan cysts, and coccidia Suitable for concentration procedures and UV fluorescence microscopy Suitable for acid-fast, safranin, and chromotrope stains Compatible with immunoassay kits and UV fluorescence microscopy

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Disadvantage 

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Not suitable for some permanent smears stained with trichrome Trophozoites usually cannot be recovered and morphologic details of cysts and eggs may fade with time Can interfere with PCR (extended fixation time)

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Advantage Components both fix and stain Easy to prepare • Long shelf life Useful for field survey Suitable for concentration procedures

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Low Viscosity Polyvinyl Alcohol Advantage Good preservation of morphology of protozoan trophozoites and cysts Easy preparation of a permanent stained smear Long shelf life when stored at room temperature (several months)

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Disadvantage Inadequate preservation of morphology of helminth eggs, larvae, coccidia and microsporidia Contains mercuric chloride Difficult and expensive to dispose Difficult to prepare in

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Disadvantage Not suitable for some permanent smears stained with trichrome Inadequate preservation of morphology of protozoan trophozoites Iodine interferes with other stains and fluorescence Iodine may cause distortion of protozoa

Modified PVA Advantage

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Disadvantage Less suitable for concentration procedures Contains mercuric chloride Inadequate preservation of morphology of helminth eggs and larvae, coccidia and microsporidia Poor adhesion of liquid or mucoid specimens to slides

Merthiolate-Iodine-Formaldehyde (MIF)

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the lab Not suitable for concentration procedures Not for immunoassay kits Not suitable for acidfast, safranin, and chromotrope stains

Permanent smears can be made and stained with trichrome Zinc is preferred over copper No mercuric chloride

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Staining is not consistent Organism morphology of cysts and trophozoites is poor Copper-morphology of cysts and trophozoites Zinc- better morph but not comparable to LV-PVA

CLINICAL INTERNSHIP 1 Day 8-9: July 16-17, 2020

I. Clinical Microscopy and Parasitology Topics: Fecalysis Peri-anal swab

References: Video AUBF 6th Gen by Strasinger

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One-Vial Fixatives (Single) →

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Advantage Free of formalin and mercury Can be used for concentration techniques and permanent stained smears Can be used for performing fecal immunoassays

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Disadvantage Do not provide the same quality of preservation as mercury-based fixatives Organism identification will be more difficult from permanent stained slides Sometimes more expensive than formalin and LV-PVA

Sodium Acetate-Acetic Acid- Formalin (SAF) Advantage Easy to prepare Long shelf life Suitable for concentration procedures and preparation of permanent stained smears Suitable for acid-fast, safranin, and chromotrope stains Compatible with immunoassay kits

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Acid Fast Stain (Cryptosporidium, Cyclospora, and Isospora) *Direct Immunofluorescent Assay (Giardia and Cryptosporidium) *Safranin Stain (Cyclospora)

3. Specimens in PVA Fixative →Trichrome Stain (Protozoa) Macroscopic Examination A. Consistency  Check for the consistency of the specimen.  Manner of reporting the consistency:  Formed  Soft  Loose  Watery

Disadvantage Requires additive for adhesion of specimens to slides Permanent stains not as good as PVA or Scaudinn’s fixative

Testing of Fecal Specimens Preserved in Formalin and PVA (*indicates special test)

Type 1: Separate hard lumps (hard to pass) Type 2: Sausage shaped, but lumpy Type 3: Like a sausage, but with cracks on its surface Type 4: Like a sausage or snake, smooth and soft Type 5: Soft blob with clear cut edges (passed easily) Type 6: Fluffy pieces with ragged edges, mushy stool Type 7: Watery, no solid pieces-entirely liquid B. Color 1. Specimens in 10% Formalin → Wet Mount (helminths and protozoa) → *ELISA (Giardia and Cryptosporidium) → *Chromotrope Stain (Microsporidia) 2. Formalin-Ethyl Acetate Concentration → Wet Mount (helminths and protozoa) → *Direct Mount (epifluorescence for Cyclospora and Isospora)

Table 14-3

Macroscopic Stool Characteristic

Color / Appearance Black

Possible Cause

Red

Upper GI bleeding Iron therapy Charcoal Bismuth (antacids) Lower GI bleeding Rifampin

CLINICAL INTERNSHIP 1 Day 8-9: July 16-17, 2020

I. Clinical Microscopy and Parasitology Topics: Fecalysis Peri-anal swab

Pale yellow, white, gray Green Bulky/frothy Ribbon-like Mucusor blood-streaked mucus

References: Video AUBF 6th Gen by Strasinger

Bile-duct obstruction Barium Sulfate Biliverdin/ oral antibiotics Green vegetables Bile-duct obstruction Pancreatic disorders Intestinal constriction Colitis Dysentery Malignancy Constripation

Direct Saline and Iodine Wet Mounts 1. Write the patient’s name or accession number and the date at the left-hand end of the slide.

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Place a drop of saline in the center of the left half of the slide and place a drop of iodine solution in the center of the right half of the slide.

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Pick up a small portion of the specimen (size of a match head) using an applicator stick and mix with the drop of saline.

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Pick up a small portion of the stool and mix it with the drop of iodine, to prepare the iodine wet mount.

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Cover the drop of saline and the drop of iodine with a coverslip. Hold the coverslip at an angle, touch the edge of the drop, and lower gently on to the slide.

TAKE NOTE!  If there are several samples received at the same time, those containing blood and mucus should be examined first, followed by the liquid specimens. Question Why do we need to prioritize stool samples with blood and mucus? → The presence of mucus-coated stools indicates intestinal inflammation or irritation. → Mucus-coated stools may be caused by pathologic colitis, Crohn disease, colon tumors, or excessive straining during elimination. → Blood-streaked mucus suggests damage to the intestinal walls, possibly caused by bacterial or amebic dysentery or malignancy. → The presence of mucus should be reported. Microscopic Examination Categories of stool and appropriate techniques to be used Technique to use Consistency Protozoan Saline Iodine Buffered stage most methylene likely to be blue (if found trophozoites are seen Formed Cysts + + Soft Cysts + + + (occasionally trophozoites) Trophozoites Loose + + Trophozoites Watery + + *worm eggs and larvae may be found in stools of any consistency

A. Wet Mount Materials and reagents  Coverslips and microscopic slides  Dropping bottles containing saline solution, Lugol’s iodine, and buffered methylene blue  Microscope  Pens or markers for labelling  Applicator sticks

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It is recommended to use direct saline and direct iodine wet preparations on each sample. Systematically scan the entire coverslip area using the 10× objective as illustrated in Figure C on the right. If something suspicious is seen, a higher magnification may be necessary.

CLINICAL INTERNSHIP 1 Day 8-9: July 16-17, 2020

I. Clinical Microscopy and Parasitology Topics: Fecalysis Peri-anal swab

References: Video AUBF 6th Gen by Strasinger

Enterobius vermicularis adult worm

B. Direct Saline Wet Preparation → Detects motile troph

C. Stained Slide Preparation: → Permanent stained slides are used for identification of protozoan trophozoites and cysts and for confirmation of species. → The microscope should be calibrated before examination begins. → Normally 3 × 1 slides are used to prepare permanent stained slides. If the specimen is unpreserved, prepare a thin even smear of the material by streaking the material back and forth on the slide with an applicator stick. If necessary dilute feces with saline. → For PVA fixed specimens, apply two or three drops of the specimen to the slide and with a rolling motion or an up and down dabbing motion spread the specimen evenly to cover an area roughly the size of a 22 by 22 mm coverslip. For other fixatives, check manufacturers instructions. Systematically examine the smear microscopically utilizing the 100× oil objective. Examine at least 200 to 300 oil immersion fields

Area and Time of Specimen Collection  Area: perianal skin  Time: early-morning sample (before the patient has bathed, or used the toilet)  *Up to six successive day morning samples should be collected before a negative result is issued. Materials  PPE  Tongue depressor  Scotch tape  Glass Slide  Microscope Procedure: A. Scotch Tape Test Procedure 1. Fold the end of a 10 cm piece of transparent tape, adhesive out, over the end of a tongue depressor.

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Press the tape firmly several times against the right and left perianal folds.

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Smooth the tape back on a clean glass microscope slide, adhesive side down.

Perianal Swab  Principle: The clear-cellulose tape preparation is the most widely used procedure for the detection of human pinworm infections. Enterobius vermicularis egg

CLINICAL INTERNSHIP 1 Day 8-9: July 16-17, 2020

I. Clinical Microscopy and Parasitology Topics: Fecalysis Peri-anal swab

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References: Video AUBF 6th Gen by Strasinger

Label the glass slide with patient name, birthday, initial of collector and date and time of collection.

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Examine the slide under a microscope using the low power (10x) objective. → E. vermicularis eggs at LPO

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Cellophane tape Method for Pinworm Detection The following test should be performed in the morning before the patient (children 1-10 years old) have washed or defecated because the eggs generally deposited in the perianal region at night. Pinworm infection should not be ruled out until at least five consecutive daily negative preparations have been examined.

Prepare tools and materials  Pencil/marker  Adhesive label  Clean microscope slide  Cellotape (2x 1.5cm)  Wooden applicator stick →

E. vermicularis eggs at HPO

Procedures:  Note: Before starting the examination, greet and inform your patient first about the purpose and the procedure of the examination. There may be some discomfort when his/her anus is pressed with the tape, but convince the patient that the examination will be done as soon as possible. 1. 2.

Comparison of E. vermicularis in different microscopic identification technique

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Eggs of E. vermicularis in a cellulose-tape preparation. (A) Eggs of E. vermicularis in a wet mount. (B) Egg of E. vermicularis in an iodine-stained wet mount from a formalin concentrate. ( C)

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Reporting of Results:  Report the organism and stage.  Example: Enterobius vermicularis eggs present  Example: Enterobius vermicularis adult worm present.

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Wash hands, wear mask and wear hand gloves because pinworm eggs are usually infective. Write down the patient’s identity (name, age, and gender) and sample’s collection date on an adhesive label, and put a label of the left side of a clean slide. Ask the patient to bent on his/her knees or lie down on the left side of his/her chest with knee and right thigh drawn up. Spread the buttocks and gently apply the cellotape on the anal and perianal and press with your fingertip or with wooden applicator. Slowly release the cellotape from the anal and perianal. Place the tape on the slide and press gently to make sure there are no bubbles. Put the slide under the microscope. Take off the right hand glove, so the macro and micrometer of the microscope can be operated easily. Keep the left hand glove in case we need to contact with the slides or other things that might be infectious. Examine systematically with low power objective (10x) or dry objective (40x) if needed. Determine the species of eggs seen under a microscope. If eggs (+), count and categorize the number of eggs to be one of the categories below:  1-5 eggs = +  6-10 eggs = ++  11-20 eggs = +++  > 20 eggs = ++++ Permanent slide can be made from positive slide, otherwise soak all the gloves, the slides and other unused material in chlorinated solution to avoid infection. After doing the examination, wash your hand and write down the report for the patient....