F. When the specimen is examined microscopically, always confirm that no fecal contamination (artifacts, vegetable debris, etc.) is present. This type of contam-ination is rare and would probably be limited to a urine specimen. However, if a urine or other urogenital specimen was contaminated with fecal material, it is possible that Pentatrichomonas hominis (nonpathogen found in the intestinal tract) could be misidentified as T. vaginalis, an identification that implies sexual transmission.
IX. LIMITATIONS OF THE
PROCEDURE A. If the specimen is left at room temperature or held at refrigerator temperature for a prolonged period (usually1 h), the organisms will round up, lose their motility, and eventually die. Motility may occasionally be enhanced by warming the specimen to 37C, but this will not revive dying organisms.
B. Wet mounts have been reported to detect T. vaginalis in 75 to 85% of infected patients. Alternative diagnostic methods may include culture, use of monoclonal antigen detection kits, use of permanent stained slides, and collection of a sec-ond sample for examination.
C. If the patient has a P. hominis intestinal infection and the urogenital specimen becomes contaminated with fecal material, a false-positive T. vaginalis result may be reported, because P. hominis and T. vaginalis are similar in shape.
REFERENCE 1. Garcia, L. S. 2001. Diagnostic Medical Par-asitology, 4th ed., p. 806. ASM Press, Wash-ington, D.C.
SUPPLEMENTAL READING Eschenbach, D., H. M. Pollock, and J. Schach-ter. 1983. Cumitech 17, Laboratory Diagnosis of Female Genital Tract Infections. Coordinating ed., S. J. Rubin. American Society for Microbi-ology, Washington, D.C.
Holmes, K. K., P. Mardh, P. F. Sparling, and P. J. Wiesner. 1990. Sexually Transmitted Dis-eases. McGraw-Hill, New York, N.Y.
䊓 Indicate the expiration date on the label and in the work record or on the manufacturer’s label.
A. 0.85% NaCl
1. Dissolve in distilled water in an appropriate glass flask by using a magnetic stirrer.
sodium chloride (NaCl) ... 850 mg distilled water ... 100 ml 2. Store in a glass bottle.
3. Label as 0.85% NaCl with a preparation date and an expiration date of 6 months.
Store at room temperature.
VIII. PROCEDURE NOTES (continued)
Include QC information on reagent container and in QC records.
APPENDIX 9.6.6–1 Reagents
B. Modified D’Antoni’s stock iodine
1. Dissolve in distilled water with an appropriate glass flask by using a magnetic stirrer.
potassium iodide (KI) ...1.0 g powdered iodine crystals ...1.5 g distilled water ... 100 ml
2. The D’Antoni’s solution should be saturated with iodine, with some excess crystals left in the bottle. Store in a brown bottle at room temperature. The stock solution remains good as long as an excess of iodine crystals remains on the bottom of the bottle.
3. Label as D’Antoni’s stock iodine with the preparation date and an expiration date of 1 year.
4. Small amounts of stock iodine solution can be aliquoted into brown dropper bottles for routine daily use. The expiration date will be from 30 to 60 days, depending on the amount of fading of the solution from the normal strong-tea color (small dropper bottles and the use of clear glass will result in a shorter expiration time). The use of a brown bottle will lengthen the expiration time.
APPENDIX 9.6.6–1 (continued)
9.6.7.1
9.6.7 Urogenital Specimens: Permanent Stained Smear (Giemsa)
[Updated March 2007]
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 Trichomonas vaginalis infections are pri-marily diagnosed from direct saline (wet) mounts by detecting live motile flagel-lates. Permanently stained smears can be made from patient specimens for specific
II. SPECIMEN A. Vaginal discharge B. Urethral discharge C. Penile discharge
D. Urethral-mucosa scrapings
E. First-voided urine with or without prostatic massage
Collect specimens with a platinum loop, cotton-Dacron swab, or speculum.
Place these specimens in a small amount (1.0 ml) of 0.85% NaCl in a test tube, or smear them directly onto a microscope slide. Use a drop of 0.85% NaCl to dilute the direct smear when it is placed on the slide. Air dry slides prepared in this manner before transporting them to the laboratory. Place specimens col-lected with a cotton-Dacron swab in Amies transport medium if the specimen cannot be processed immediately. Organisms will remain viable for approxi-mately 24 h in Amies transport medium. Collect urine specimens in a clean-catch urine collection container. Centrifuge the urine at 500⳯ g for 5 min, and examine the sediment for T. vaginalis. Hold all specimens at room temperature, because refrigerator temperatures have a deleterious effect on the organisms.
Returning the specimen to room temperature will not reverse these deleterious morphological changes. Reject any specimens more than 24 h old.
III. MATERIALS A. Reagents (see Appendix 9.6.7–1) 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
5. 2 Coplin jars 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)
identification of the organism. Although a number of stains can be used, Giemsa and Papanicolaou stains are the ones most fre-quently used to diagnose T. vaginalis in-fections.
Observe standard precautions.
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. Check the direct-mount reagents each time they are used.
1. The saline should be clear, with no visible contamination.
2. Giemsa stain
A peripheral blood film may be used to quality control the Giemsa stain. For staining characteristics, see procedure 9.8.5.
3. Phosphate buffer
Check the buffer each time you use it. The buffer should be clear, with no signs of visible contamination or precipitates. The pH should be between 6.8 and 7.2.
4. Review the Giemsa-stained control slide before searching the patient’s spec-imen for the organism. If there was potential fecal contamination of the specimen, you may have to differentiate T. vaginalis from Pentatrichomonas hominis.
B. The microscope should have been calibrated within the last 12 months, and the