Recognizing Tricomonads in Gram-Stained Smears

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1 MICROBIOLOGY Brian John Harrington, PhD, MPH Debra Lynn Williams, MT(ASCP) Recognizing Tricomonads in Gram-Stained Smears ABSTRACT Using duplicate smears of 600 genital specimens, we compared the Gram stain with low-ph acridine orange (AO) stain for detection of Trichomonas in women. Trichomonads were found in 70 specimens (11.7%) with theao stain and in 34 (5.7%) with the Gram stain. Initially, only 10% of specimens (1 of the first 10) positive for trichomonads with the AO stain were identified with the Gram stain, but this improved to >75% (15 of the last 20) by the end of the study. With increased awareness, experience, and confidence, the routine Gram stain can be used successfully for detection of trichomonads in many cases that might otherwise go unsuspected. were experienced in recognizing trichomonads in smears of genital tract specimens. Materials and Methods Specimens were collected from 600 female patients using Culturette II swabs (Becton Dickinson Microbiology Systems, Cockeysville, Md) or double-swabbed Starswabs (Starplex Scientific, Etobicoke, Ontario, Canada), both of which use a liquid, modified Stuart medium for transport. Two smears were made from one of the swabs by rolling on microscope slides and allowing to air dry (the second swab was used for routine bacterial culture). Randomly, one smear was used for the Gram stain and the other for the AO stain. Smears need not be fixed or stained immediately, and unfixed smears of known positive specimens can be kept for use as positive controls for both stains. From the Pathology Department, St Vincent Mercy Medical Center, Toledo, Ohio. Reprint requests to Dr Harrington, Department of Public Health, Medical College of Ohio, 3015 Arlington Ave, Toledo, OH Dr Harrington is currently with the Department of Public Health, Medical College of Ohio, Toledo. Gram Stain Air-dried smears were fixed with Gram decolorizer (70:30 mixture of 95% ethanol-acetone) for 2 minutes prior to staining. Similar in effect to methanol fixation, this provides better preservation of cellular material and a cleaner background VOLUME 30. NUMBER 12 ana Of the common causes of vaginitis in women negative for HIV infection, bacterial vaginosis accounts for 40% to 50% of cases, Candida species for 20% to >25%, infection with Trichomonas for 15% to >20%, and mixed infections for as many as 20%.',2 Infection with Trichomonas may be a risk factor for HIV,3,4 and trichomoniasis in women with HIV is thought to be common.2 It has been recommended that women be tested for Trichomonas infection before any reproductive tract surgery, after changing sexual partners, and during pregnancy, because of the association with postsurgical complications,5 preterm delivery, premature rupture of membranes, and infection in the neonate.6 Symptoms of trichomoniasis include cervicitis, urethritis, and prostatitis. Many infections in both female and male patients are asymptomatic, and infection is often difficult or impossible to diagnose clinically.6-9 Prevalence values range from <5% to >75%, depending on the population studied, but typically are 10% to 20%. 1,3 ' 9-11 While culture for Trichomonas is usually accepted as the most effective diagnostic method, any one medium alone may not enable detection of all cases.9'11-13 Laboratory diagnosis is usually based on microscopic examination of clinical material, most commonly as a wet mount or more rarely as a stained smear. The sensitivity of wet mount method ranges from 30% to >80%, depending on the population studied,7,14 the experience of the microscopist,10'14 and delay in examining the specimen.15,16 The buffered, low-ph acridine orange (AO) stain is twice as sensitive as the wet mount 17 and has been the standard method for diagnosis of Trichomonas infection in our laboratory since We compared the detection rates with Gram stain and AO stain. Also reported is our laboratory detection rate of trichomonads in Gram-stained smears for a 12month period after the AO stain-gram stain study, when the technologists reading the Gram stains

2 than heat fixation. To enhance the staining of trichomonads, the safranin step in the routine Gram stain procedure was extended to 2 minutes. The Gram-stained smears were read by technologists as part of their routine work on those specimens. Acridine Orange Stain Fig 1. Vaginal smear exhibits four trichomonads (arrows). Nuclei (N) are visible in two, and flagella (F) in one (Gram stain, original magnification x50 oil immersion). 1. Fix air-dried smears with Gram decolorizer for 2 minutes, then drain off. 2. Stain with AO solution for 2 minutes. Wash off with water. 3. Flood slide with 0.3 mol/l calcium chloride solution for 30 seconds. Wash off with water. 4. Coverslip the smear in the water remaining after the excess has drained off. The calcium chloride solution step is not always needed, but in many cases enhances the clarity of the fluorescence, and was always performed in our study. The AO-stained smears were read using epi-illumination from a 100-W mercury vapor lamp with blue excitation as used for fluorescein isothiocyanate (Olympus Corporation of America, New Hyde Park, NY). Fig 2. Vaginal smear exhibits seven trichomonads (arrows). Nuclei (N), and flagella (F) are visible in some (Gram stain, original magnification x50 oil immersion)..»» *, * /»' **» Fig 3. Vaginal smear exhibits five trichomonads (arrows). Nuclei are visible in some (Gram stain, original magnification X50 oil immersion). m i VOLUME 30. NUMBER 12 Results Typical appearances of trichomonads and human cellular material in clinical specimens stained with Gram stain are shown in Figures 1 through 5, and with AO stain in Figure 6. Trichomonads are usually oval, but their shape may be distorted. With Gram stain, the flagella are often visible, the nucleus and axostyle may be seen, and the cytoplasm may appear finely granular or vacuolated. Criteria used to identify Gram-stained trichomonads were visualization of the flagella or the nucleus and axostyle. Of the 600 specimens in the Gram-stain vs AOstain blind study, trichomonads were seen in 70 (11.7%) with the AO stain and in 34 (5.7%) with the Gram stain. This produced an overall detection rate for the Gram stain of 48.5% of the AOpositive specimens. However, of the first 10 AO-positive specimens only 1 (10%) was detected with Gram stain, in contrast to 15 (75%) of the last 20 AO-positive specimens. No specimens were positive with Gram stain and negative with AO stain. Of the 600 specimens, 180 (30%) had moderate or large numbers of neutrophils, in contrast Buffered, low-ph AO stain (Becton Dickinson Microbiology Systems, Sparks, Md) was used as previously described for trichomonads in genital tract specimens17:

3 Discussion Despite numerous studies that show its lack of sensitivity, the wet mount probably is still the most widely used microscopic procedure for detection of Trichomonas. With only slight modification, the low-ph AO stain can be used, and is twice as sensitive as the wet mount. 17 Fluorochrome and enzyme-labeled immunologic stains have been described,12,18,19 and these and other immunoassays and gene probe methods have the potential to be highly specific and sensitive, but are relatively expensive. Although it has been reported that trichomonads can often be recognized on Gram-stained smears,23 others 2,24-27 report negative results of the Gram stain and other stains for their detection, typically citing difficulty in recognizing trichomonads among other cellular materials. The criteria used in our study, visualization of the flagella or of the nucleus and axostyle, led to high specificity but low sensitivity. With experience, individual readers may relax these criteria, in which case sensitivity would increase. It is important to note that the detection rate improved markedly with experience with the Gram stain, to >75% by the end of our study. Not only is it possible to detect trichomonads in Gram-stained smears, but the Gram stain can be more sensitive than the wet mount method. Technologists experienced in reading wet mounts for Trichomonas had a detection rate of only 50% compared with the detection rate with the AO stain,17 whereas by the end of our study the detection rate with the Gram stain was >75% compared to that with the AO stain. Unlike wet mounts, Gram-stained smears have the advantage that they can be saved and reviewed later. The presence of neutrophils in vaginal discharge may not be due solely to trichomonads,28 but several reports7,8,9,25 have noted an association with Fig 4. Vaginal smear exhibits squamous epithelial cell and two trichomonads (arrows) with vacuolated cytoplasm. Flagella (F) are visible on trichomonad at right (Gram stain, original magnification x50 oil immersion). Fig 5. Vaginal smear exhibits two trichomonads (arrows) with vacuolated cytoplasm (Gram stain, original magnification x50 oil immersion). Fig 6. Vaginal smear exhibits three trichomonads with squamous epithelial cells, mucus, and bacteria. Flagella are never seen with acridine orange (AO) stain, but fluorescence of the nucleus (yellow) and cytoplasm (orange) is characteristic (Low ph AO stain, original magnification x50 oil immersion). VOLUME 30, NUMBER to 58 (83%) of the 70 specimens in which trichomonads were seen. After completion of the Gram and AO stain comparison study, the technologists' routine Gram stain reports of genital tract specimens received for routine culture for a 12-month period were reviewed. Trichomonads were seen in 125 (7.2%) of 1,738 smears reported, and of these, 97 (77.6%) were found to have one or more trichomonads per field (X 100 oil immersion objective). Sensitivity of the Gram stain was directly related to the number of trichomonads present in the specimen.

4 Trichomonas infection. It was noted in our study that moderate or large numbers of neutrophils, the presence of which on Gram-stained smear should be an indication for the reader to examine it closely for trichomonads, accompanied many of the identified Trichomonas infections. It is now recognized that the Gram stain is important as a diagnostic aid in bacterial vaginosis, 29,30 and if only one microscopic examination is done in the microbiology laboratory on genital tract specimens, it is most likely to be a Gram stain. The review of routine Gram stain results for 1,738 specimens showed that trichomonads were seen in 125 (7.2%), and only 24 (19.2%) of those specimens had been ordered for "Trichomonas examination." Of the 600 specimens in the Gram stain-ao stain study, only 8 (11 %) of the 70 specimens in which trichomonads were seen had been so ordered. If a specific test for Trichomonas is performed only on physician order, it is important that any unsuspected Trichomonas infections be detected if possible, and their recognition on Gram-stained smears assumes increased importance, not only in revealing unsuspected cases of trichomoniasis but also in suggesting the possibility of other sexually transmitted diseases being present as coinfections. 6 ' 9,25 Conclusions With the current emphasis on the need for cost-effective and timely, relevant results from the microbiology laboratory, few will routinely use culture techniques for Trichomonas, even for those specimens negative by microscopy. The sensitivity of culture depends on both the medium used and the duration of incubation. Immunologic methods, while potentially sensitive and specific, are certainly more costly than the wet mount method, or AO or Gram stain. For those laboratories with a fluorescence microscope, we recommend the AO stain for microscopy when a test for Trichomonas is ordered. Although the Gram stain should not replace more sensitive procedures when trichomoniasis is specifically suspected, the realization that, in addition to its value in alerting to other infections, trichomonads may be seen on the routine Gram stain will markedly improve the clinical relevance of Gram stain reports when a test for Trichomonas has not been specifically requested. 6. Pastorek JG, Cotch MF, Martin DH, et al. Clinical and microbiological correlates of vaginal trichomoniasis during pregnacy. Clin Infect Dis. 1996;23: Fouts AC, Kraus SJ. Trichomonas vaginalis: reevaluation of its clinical presentation and clinical diagnosis. / Infect Dis. 1980;141: McLellan R, Spence MR, Brockman M, et al. The clinical diagnosis of trichomoniasis. Obstet Gynecol. 1982:60: Wolner-Hansenn P, Kreiger JN, Stevens CE, et al. Clinical manifestations of vaginal trichomoniasis. JAMA. 1989;261: Draper D, Parker R, Patteron E, et al. Detection of Trichomonas vaginalis in pregnant women with the InPouch TV culture system. / Clin Microbiol. 1993;31: Poch F, Levin D, Levin S, et al. Modified thioglycollate medium: a simple and reliable means for detection of Trichomonas vaginalis. ] Clin Microbiol. 1996;34: Kreiger IN, Tarn MR, Stevens CE, et al. Diagnosis of trichomoniasis. JAMA. 1988;259: Thomason JL, Gelbart SM, Sobun JF, et al. Comparison of four methods to detect Trichomonas vaginalis. J Clin Microbiol. 1988;26: Lossick JG. The diagnosis of vaginal trichomoniasis. JAMA. 1988;259: Greenwood JR, Kirk-Hillaire K. Evaluation of acridine orange stain for detection of Trichomonas vaginalis in vaginal specimens. / Clin Microbiol. 1981;14: Healy GR, Garcia LS. Intestinal and urogenital protozoa. In: Murray PR, Baron EJ, Pfaller MA, et al, eds. Manual of Clinical Microbiology. 6th ed. Washington, DC: American Society for Microbiology; 1995: Harrington BJ, Gaydos JM. Low ph acridine orange stain for trichomonads. Lab Med. 1984;15: O'Hara CM, Gardner WA, Bennett BD. Immunoperoxidase staining of Trichomonas vaginalis in cytologic material. Acta Cytol. 1980;24: Smith RF. Detection of Trichomonas vaginalis in vaginal specimens by direct fluorescence assay. / Clin Microbiol. 1986;24: Yule A, Gellan MCA, Oriel JD, et al. Detection of Trichomonas vaginalis antigen in women by immunoassay. / Clin Pathol. 1987;40: Carney IA, Unadkat P, Yule A, et al. New rapid latex agglutination test for diagnosing Trichomonas vaginalis infection. / Clin Pathol. 1988;41: Briselden AM, Hillier SL. Evalution of Affirm VP microbial identification test for Gardnerella vaginalis and Trichomonas vaginalis. J Clin Microbiol. 1994;32: Sobrepena RL. Identification of Trichomonas vaginalis in Gram-stained smears. Lab Med. 1980;11: Thomason JL, Wilcoski LM, McLaughlin CA. Trichomoniasis. Clin Micro Newslett. 1986;8: Rein MF, Muller M. Trichomonas vaginalis and trichomoniasis. In: Holmes KK, Mardh PA, Sparling PF, et al, eds. Sexually Transmitted Diseases. 2nd ed. New York, NY: McGraw-Hill Information Services; 1990: Garcia LS. Laboratory methods for diagnosis of parasitic infections. In: Baron EJ, Finegold SM, eds. Diagnostic Microbiology. 8th ed. St Louis, Mo: Mosby; 1990: Melvin DM, Healy GR. Intestinal and urogenital protozoa. In: Lenette EH, Balows A, Hausler WJ Jr, et al, eds. Manual of Clinical Microbiology. 4th ed. Washington, DC: American Society for Microbiology; 1985: Peipert JF, Boardman L, Hogan JW, et al. A prospective study of labo References ratory tests for detecting acute upper genital tract infection in women. 1. Sweet RL, Gibbs RS. Infectious Diseases of the Female Genital Tract. 2nd Obstet Gynecol. 1996;87: ed. Baltimore, Md: Williams & Wilkins; Nugent RP, Krohn MA, Hillier SL. ReliaDility of diagnosing bacterial 2. Sobel JD, Schuman P. Vaginitis in HIV-infected women. In: Mayer KH, vaginosis is improved by a standardized method of Gram stain interpretation. / Clin Microbiol. 1991;29: ed. Opportunistic Complications of HIV. Vol 2. Philadelphia, Pa: Meniscus Health Care Communications; Spiegel CA. Bacterial vaginosis. Clin Microbiol Rev. 1991;4: Laga M, Manoka A, Kivuvu M, et al. Nonulcerative sexually transmitted diseases as risk factors for HIV-1 transmission in women: results from a cohort study. AIDS. 1993;7: Sewankambo N, Gray RH, Wawer MI, et al. HIV-1 infection associated with abnormal vaginal flora morphology and bacterial vaginosis. Lancet. 1997:350: Heine P, McGregor JA. Trichomonas vaginalis: a reemerging pathogen. Clin Obstet Gynecol. 1993;36: VOLUME 30, NUMBER 12

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