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P R E A N A L Y T I C A L C O N S I D E R A T I O N S

I. PRINCIPLE

The clear-cellulose-tape preparation is the most widely used procedure for the detec-tion of human pinworm infecdetec-tions (2, 4).

Adult Enterobius vermicularis worms in-habit the large intestine and rectum; how-ever, the eggs are not normally found in

fecal material. The adult female migrates out the anal opening and deposits the eggs on the perianal skin, usually during the night. The eggs, and occasionally the adult female worms, stick to the sticky surface of the cellulose tape. These cellulose tape

preparations are submitted to the labora-tory, where they are examined under the microscope. Commercial collection sys-tems are also available (pinworm collec-tion kit; Evergreen Scientific, Los Ange-les, Calif.) (1).

II. SPECIMEN The specimen is collected from the skin of the perianal area first thing in the morn-ing, before the patient has bathed or used the toilet. Preparations should be taken for at least 4 to 6 consecutive days with negative results before a patient is consid-ered free of the infection.

III. MATERIALS A. Reagent

䊓 Indicate the expiration date on the label and in the work record or on the manufacturer’s label.

Xylene (or xylene substitute [ethyl acetate]) or toluene

B. Supplies

1. Clear cellulose tape (not Magic mend tape)

2. Tongue depressors

3. Glass slides (1 by 3 in., or larger if you prefer)

C. Equipment

1. Binocular microscope with 10⳯, 40⳯, and 100⳯ objectives (or the approximate magnifications for low-power, high dry power, and oil immersion examination)

2. Oculars should be 10⳯. Some workers prefer 5⳯; however, over-all smover-aller magnification may make final organism identifications more difficult.

A N A L Y T I C A L C O N S I D E R A T I O N S

A. The microscope should be calibrated, and the objectives and oculars used for the calibration procedure should be used for all measurements on the micro-scope. Post the calibration factors for all objectives on the microscope for easy access (multiplication factors can be pasted right on the body of the microscope) (see procedures 9.1 and 9.3.2). Although there is not universal agreement, the microscope should probably be recalibrated once each year. This recommen-dation should be considered with heavy use or if the microscope has been bumped or moved multiple times. If the microscope does not receive heavy use, then recalibration is not required on a yearly basis.

Observe standard precautions.

Include QC information on reagent container and in QC records.

IV. QUALITY CONTROL

B. Pictures of Enterobius eggs (with measurements) should be available for com-parison with the clinical specimen.

C. Record all QC results.

V. PROCEDURE A. Place a strip of clear cellulose tape (adhesive side down) on a microscope slide as follows: starting ca. 1.5 cm from one end, run the tape toward the same end, and wrap the tape around the slide to the opposite end. Tear the tape even with the end of the slide. Attach a label to the tape at the end torn flush with the slide.

B. To obtain a sample from the perianal area, peel back the tape by gripping the labeled end, and, with the tape looped (adhesive side outward) over a wooden tongue depressor that is held firmly against the slide and extended about 2.5 cm beyond it, press the tape firmly several times against the right and left perianal folds (see Fig. 9.6.1–1).

C. Smooth the tape back on the slide, adhesive side down.

D. Label with patient name and date.

E. Submit the tapes and slides to the laboratory in a plastic bag.

F. Examine the slide directly under the low-power objective (10⳯) of the micro-scope. To make the eggs more visible, lift the tape from the slide and add a drop of xylene or toluene to the slide. Press the tape back down on the slide.

Examine with low light intensity on low power.

VI. RESULTS A. Typical pinworm eggs are thick shelled and football shaped, with one flattened side, and may contain a partially or fully developed larva.

B. Adult female worms are occasionally seen on the tape preparation. The worms are approximately 1 cm in length, are white or cream colored, and have a pointed tail.

Figure 9.6.1–1 Collection of E. vermicularis eggs by the cellulose tape method (from reference 1).

P O S T A N A L Y T I C A L C O N S I D E R A T I O N S

A. Report the organism and stage. Do not use abbreviations.

Example: Enterobius vermicularis eggs present.

B. Report adult worms.

Example: Enterobius vermicularis adult worm present.

IV. QUALITY CONTROL (continued)

VII. REPORTING RESULTS

Examination for Pinworm: Cellulose Tape Preparation 9.6.1.3

VIII. PROCEDURE NOTES A. Pinworm eggs are usually infectious. The use of glass slides and tapes may expose laboratory personnel to these eggs.

B. Some recommend the use of the Swube (paddle with sticky adhesive coat; Bec-ton Dickinson) as a safer alternative. The petrolatum swab is another alternative (3). Another option is the collection system available from Evergreen Scientific (Fig. 9.6.1–2).

C. If opaque tape is submitted by mistake, a drop of immersion oil on the top of the tape will clear it enough to proceed with the microscopic examination.

IX. LIMITATIONS OF THE

PROCEDURE A. The female pinworm deposits eggs on the perianal skin only sporadically.

B. Without multiple tapes (taken consecutively, one each morning), it is not pos-sible to determine if the patient is positive or negative for the infection.

C. Occasionally, a parent will bring in an adult worm collected from the perianal skin or from the surface of the stool. The identification of the adult worm (almost always the female) confirms the infection.

Figure 9.6.1–2 Diagram of a commercial kit (Evergreen Scientific) for use in sampling the perianal area for the presence of pinworm (E. vermicularis) eggs. On the left is the vial containing the sampler, which has sticky tape around the end. Once this is applied to the perianal area and eggs are picked up on the tape, the label area is placed at one end of the slide. The sticky tape is rolled down the slide and attaches to the glass. This device is easy to use and provides an area sufficient for adequate sampling. A minimum of four to six consecutive negative tapes are required to rule out a pinworm infection; most laboratories are accepting four rather than requesting the full six. (From reference 1.)

C. Report negatives.

Example: No Enterobius vermicularis eggs or adults seen.

REFERENCES 1. Garcia, L. S. 2001. Diagnostic Medical Par-asitology, 4th ed., p. 802–804. ASM Press, Washington, D.C.

2. Graham, C. F. 1941. A device for the diag-nosis of Enterobius vermicularis. Am. J. Trop.

Med. 21:159–161.

3. Markey, R. L. 1950. A Vaseline swab for the diagnosis of Enterobius eggs. Am. J. Clin.

Pathol. 20:493.

4. Melvin, D. M., and M. M. Brooke. 1982.

Laboratory Procedures for the Diagnosis of Intestinal Parasites, 3rd ed. U.S. Department of Health, Education and Welfare publication no. (CDC) 82-8282. U.S. Government Printing Office, Washington, D.C.

VII. REPORTING RESULTS (continued)

9.6.2.1

The direct smear (1, 2) is primarily used to detect motile parasites that are found in the colon (the organism in question is usu-ally Entamoeba histolytica or Entamoeba histolytica/E. dispar). Specific ulcerated areas should always be sampled; in the ab-sence of specific lesions, the mucosa is

randomly sampled. On low-power (⳯100) examination of the smear, motil-ity of trophozoites and/or human cells might be detected. At high dry power (⳯430), organisms might be tentatively identified on the basis of size, nucleus/cy-toplasm ratio, appearance of the

cyto-plasm, and motility (saline only). The di-rect smear can be prepared either with 0.85% NaCl or with iodine (Lugol’s or D’Antoni’s). Presumptive findings using this procedure must be confirmed by some type of permanent stained smear.

II. SPECIMEN A. The specimen may consist of mucosal lining, mucus, stool, and/or a combination of the three. The specimen will be taken by the physician and either prepared at bedside for immediate review or submitted to the laboratory for subsequent examination.

B. Prepare direct wet mounts on clean, new glass slides (1 by 3 in.). Depending on the specimen type, the following amounts should be used.

1. For mounts of mucus or similar material, place approximately 1 or 2 drops on the slide.

2. For mounts of stool, place approximately 1 or 2 drops on the slide.

3. If the material is very wet (watery), place 1 or 2 drops on the slide.

C. If the specimen must be transported to the laboratory, the material can be placed in a small amount of 0.85% NaCl (0.5 to 1.0 ml) to keep the specimen from drying out. These specimens should be transported to the laboratory within no more than 30 min from collection time and should be examined immediately.

If this is not possible, then the material can be preserved and processed as a permanent stained smear (see procedure 9.6.3).

III. MATERIALS A. Reagents (see Appendix 9.6.2–1) B. Supplies

1. Disposable glass or plastic pipettes 2. Glass slides (1 by 3 in., or larger if

you prefer)

3. Coverslips (22 by 22 mm; no. 1, or larger if you prefer)

4. Small tubes containing 0.5 to 1.0 ml of 0.85% NaCl

C. Equipment

1. Binocular microscope with 10⳯, 40⳯, and 100⳯ objectives (or the

approximate magnifications for low-power, high dry power, and oil immersion examination)

2. Oculars should be 10⳯. Some workers prefer 5⳯; however, over-all smover-aller magnification may make final organism identifications more difficult.

3. Tabletop centrifuge (for tubes con-taining specimen and 0.85% NaCl if the specimen is submitted in sa-line)

Observe standard precautions.