section epub:type=”chapter” id=”c0013″ role=”doc-chapter”> After studying this chapter, the student should be able to: The most common gynecologic complaints encountered by health care providers are vaginal discharge, vaginal discomfort, and vaginal odor. The three major causes for these symptoms are bacterial vaginosis, candidiasis, and trichomoniasis. Whereas the causative agent for each of these conditions is distinctly different, the clinical presentations can be nonspecific and similar (Table 13.1). Because treatment can differ significantly, determining the causative agent before initiating therapy is important, and in some cases, treating sexual partners is also necessary to avoid reinfection. Table 13.1 KOH, 10% potassium hydroxide; RBC, red blood cell; WBC, white blood cell. aValues from healthy nonmenstruating women. bExternal dysuria is pain experienced during urination owing to the passage of urine over inflamed tissue. cThese large (Gram-positive) rods are lactobacilli, the predominant microbe in vaginal secretions of healthy individuals. These conditions are usually differentiated using a sample of vaginal secretions and a few direct microscopy tests: wet mount examination, amine or “whiff” test, KOH examination, and Gram stain. Even though these microscopy tests are simple and easy to perform, the accuracy of the results obtained depends directly on the skill and expertise of the microscopist. This fact should not be minimized. If personnel with the necessary technical skills and expertise are unavailable, testing should be referred to a laboratory with qualified personnel. The Clinical Laboratory Improvement Act has classified the wet mount examination and the KOH examination of vaginal secretions as provider-performed microscopy tests. The act also states that when nonlaboratory personnel (i.e., clinical practitioners such as physicians, physician assistants, nurse practitioners, and nurses) perform these tests, the designated laboratory director is responsible for ensuring the accuracy and reliability of the testing performed. Timely and accurate testing of vaginal fluid specimens can identify offending organisms that cause vaginal discharge and discomfort, enabling health care providers to immediately diagnose and treat common causes of vaginitis/vaginosis. Appropriate collection and handling of vaginal secretion specimens optimizes the recovery and detection of microorganisms and other cellular elements. Based on the presence, absence, or combination of elements observed microscopically, the cause of vaginitis or vulvovaginitis can be determined. A health care provider collects vaginal secretions during a pelvic examination. A nonlubricated speculum, moistened only with warm water, is used to provide access to the vaginal fornices. Speculum lubricants are avoided because they often contain antimicrobial agents. The specimen collection device is usually a sterile, polyester-tipped (e.g., Dacron) swab on a plastic shaft or, alternatively, a sterile wire loop. Selection of the sampling device is important because cotton has been toxic to Neisseria gonorrheae, whereas wooden shafts have been toxic to Chlamydia trachomatis.1 The health care provider uses one or more swabs or a collection loop to obtain vaginal secretions from the posterior vaginal fornix and the vaginal pool. After collection, the labeled specimen should be transported to the laboratory as soon as possible in a biohazard bag accompanied by a requisition slip. In addition to the standard patient identification information on the request slip, an appropriate medical history should be provided, such as the patient’s menstrual status, exposure to sexually transmitted diseases, and use of vaginal lubricants, creams, and douches. Tests on vaginal secretion specimens should be performed as soon as possible. If a delay in transport or analysis is unavoidable, these specimens should be kept at room temperature. Refrigeration adversely affects the recovery of N. gonorrheae and the detection of the trophozoite Trichomonas vaginalis, whose identification depends on observing its characteristic “flitting” or jerky motility. However, for detection of C. trachomatis or viruses (e.g., herpes simplex virus), refrigeration is preferred to prevent overgrowth of the normal bacterial flora. When a health care provider examines the vaginal secretions immediately after collection (i.e., provider-performed microscopy testing), the swab is often placed directly into 0.5 mL to 1.0 mL of sterile physiologic saline (0.9% NaCl) and the microscopic examinations are performed. An alternate approach is to place a small drop of sterile physiologic saline onto a microscope slide into which the swab of vaginal secretions is directly rolled for microscopic viewing. The pH of vaginal secretions must be determined at the time of specimen collection by the healthcare provider. Commercial pH paper can be placed directly on the lateral wall of the vagina for about 1 minute to absorb the secretions. Subsequently, the color change of the pH paper is compared with a color chart to determine the pH value. Alternatively, a swab is used to collect vaginal secretions and the liquid sample is immediately expressed onto pH paper. Regardless of which technique is used, it is important to avoid contact with any cervical discharge because of its high alkaline pH. Note that once a sampling swab has been placed into saline (pH ~7), the pH value has been altered by the saline and can no longer be determined using that swab. Vaginal secretions should have a pH in the range of 3.8 to 4.5. The predominant bacteria in a healthy vagina are lactobacilli, and it is lactic acid, their major metabolic end product, that maintains this normally acidic pH. Studies have demonstrated that some lactobacilli also produce hydrogen peroxide, which further enhances the healthy acidic environment of the vagina. In part, the bactericidal qualities of hydrogen peroxide prevent overgrowth of some indigenous microbes, such as Gardnerella vaginalis. In fact, the reduction or absence of hydrogen peroxide–producing lactobacilli is associated with bacterial vaginosis.2,3 The determination of the vaginal pH is a useful diagnostic tool because it assists in the differential diagnosis of vaginitis (see Table 13.1). A pH greater than 4.5 is associated with bacterial vaginosis, trichomoniasis, and atrophic vaginitis. A pH less than 4.5 is normal, but can also be present with candidiasis. Microscopic examinations should be performed as soon as possible on vaginal secretion specimens, particularly for detection of T. vaginalis, for which identification depends solely on observing actively motile organisms. The vaginal swab is placed into a tube containing approximately 1.0 mL of sterile physiologic saline (0.9% NaCl) and is agitated or twirled for a few seconds to release the secretions from the swab. Two microscope slides are typically prepared using the vaginal swab. One slide is used for a direct wet mount examination and the second slide for a 10% KOH preparation and the amine or “whiff” test. Depending on the laboratory protocol, a third slide may also be prepared for Gram stain. Each slide is prepared from the saline suspension made using the vaginal swab. A drop of this suspension is placed onto a clean, labeled microscope slide by using a disposable transfer pipette or, alternatively, by pressing and rolling the moistened swab on the microscope slide to express liquid onto the slide. The wet mount and KOH slides, as well as the slide for Gram stain (if requested), are usually prepared at the same time. The YouTube video titled Examination of Vaginal Wet Preps created by the Seattle STD/HIV Prevention Training Center is available online and provides an excellent detailed overview of performing the microscopic examination.4 When preparing the direct wet mount slide, a coverslip is placed on the drop of saline-suspended specimen, taking care not to trap air bubbles. This slide is examined using brightfield or phase-contrast microscopy at low-power (×100) and high-power (×400) magnifications. Low-power magnification is used to assess the overall distribution of the specimen components and to evaluate epithelial cells, such as the number and type, and whether clumping is present. Subsequently, high-power magnification is used to quantify the elements using criteria similar to those listed in Table 13.2.5,6 The following elements are typically identified and reported when observed in wet mount preparations of vaginal secretions: red blood cells, white blood cells, yeast, hyphae/pseudohyphae, trichomonads, clue cells, and squamous epithelial cells. In some institutions, wet preps are performed in the microbiology department, in which case predominant bacterial morphotypes may also be reported. Table 13.2 In health, white blood cells are present in vaginal secretions, and their numbers range from a few observed in an entire preparation to several cells observed in every high-power field of view. This variation is often associated with a woman’s menstrual cycle, with increased white blood cells present during ovulation and menses.7 In contrast, red blood cells usually are not present unless the specimen was collected around or during menstruation. This highlights the need for a current patient history to accompany the results of each vaginal fluid specimen. The vagina has a complex bacterial flora with the characteristically large rods of lactobacilli accounting for 50% to 90% of the microbes in a healthy vagina.8 These morphologically distinct, large, nonmotile, Gram-positive rods produce lactic acid as their major metabolic waste product, which is principally responsible for the acidic (pH 3.8–4.5) environment of a healthy vagina (Fig. 13.1). In addition, a subset of these lactobacilli produce hydrogen peroxide, which helps maintain balance in the vaginal flora by preventing the proliferation of other bacteria, in particular, G. vaginalis and Prevotella bivia. Any decrease in the number of lactobacilli relative to the number of squamous epithelial cells present in the preparation is an indication of an imbalance in the microbial flora. Whereas small numbers of other bacterial morphotypes may be observed in normal vaginal secretions, the presence of increased numbers or a preponderance of them is considered abnormal. These morphotypes include small, nonmotile, Gram-variable coccobacilli (e.g., G. vaginalis); thin, curved, Gram-variable, motile rods (e.g., Mobiluncus spp.); Gram-positive cocci (e.g., Peptostreptococcus spp., staphylococci, and streptococci); Gram-negative cocci (e.g., Enterococcus spp.); and Gram-negative rods (e.g., Prevotella spp., Porphyromonas spp., Bacteroides spp., coliforms). An occasional yeast or blastoconidium can be present in normal vaginal secretions. Because of the visual similarity of yeast and red blood cells, the KOH preparation, which lyses red blood cells, is useful in distinguishing these two entities. Yeast cells are typically 10 μm to 12 μm in diameter and are Gram positive. Increased numbers (1 + or greater) of yeast or the presence of hyphae or pseudohyphae is considered abnormal and indicates candidiasis (i.e., a fungal or yeast infection) (Fig. 13.2). The vagina is a thick-walled fibromuscular tube lined with stratified squamous epithelium. Therefore when the vaginal mucosa is swabbed during the collection of vaginal secretions, a significant number of squamous epithelial cells are recovered. These cells predominate in wet mounts from a healthy vagina and are identified easily by their large (30–60 mm), thin, flat, flagstone-shaped appearance. They have a small, centrally located nucleus and a large amount of cytoplasm, which becomes finely granulated as the cell ages (Fig. 13.3). This intracellular keratohyalin granulation caused by cell degeneration is distinctly different from and must not be confused with the shaggy appearance of clue cells, which are formed when numerous small bacteria adhere to the membranes of epithelial cells (Fig. 13.4; also see Fig. 7.68). Clue cells, a diagnostic indicator of bacterial vaginosis, appear soft and finely stippled with indistinct cellular borders because of the numerous bacteria adhering to them. In these bacteria-laden cells with shaggy-appearing edges, the nuclei may not be visible. To be considered a clue cell, the bacteria do not need to cover the entire cell; however, bacteria should cover at least 75% of the cell surface and the bacterial organisms must extend beyond the cell’s cytoplasmic borders.4,6 Sometimes these cells are described as “bearded.” The skill and expertise of the microscopist prevent the intracellular keratohyalin granulation of normal degenerating squamous cells from being misidentified as adherent bacteria of clue cells. Two microscopic characteristics facilitate this differentiation: (1) keratohyalin granules vary in size, and (2) they are usually larger and more refractile than bacteria. Note that epithelial cells covered with numerous large lactobacilli rods are not clue cells. Parabasal cells reside below the surface or luminal squamous epithelium of the vaginal mucosa. Therefore no or at most a few parabasal cells are present in normal vaginal secretions. However, increased numbers may be found during menstruation or in specimens from postmenopausal women. These cells are 15 to 40 μm in diameter and are oval to round with distinct cytoplasmic borders (Fig. 13.5). In size and shape, these cells closely resemble transitional epithelial cells of the urinary system; however, their nucleus-to-cytoplasm ratio is smaller—that is, 1:1 to 1:2. Increased numbers of parabasal cells are usually observed in vaginal secretions from women with atrophic vaginitis and desquamative inflammatory vaginitis.
Analysis of Vaginal Secretions
Key Terms1
Healthy/Normala
Candidiasis
Bacterial Vaginosis
Trichomoniasis
Atrophic Vaginitis
Patient complaints
—
Vulvovaginal itching and soreness, discharge, “external” dysuria,b dyspareunia
Malodorous discharge
Vulvovaginal soreness, malodorous discharge, “external” dysuria,b dyspareunia
Vaginal dryness, dyspareunia
Discharge characteristics
—
White, curd-like
Foul-smelling, thin, gray, homogeneous; adherent to mucosa
Copious, yellow-green, frothy; may be foul-smelling, adherent to mucosa
—
pH
3.8–4.5
3.8–4.5
>4.5
5.0–6.0
5.0–7.0
Direct Wet Mount Microscopy findings
Bacteria
Large rodsc predominate
Large rodsc predominate
Increase in Gram-variable coccobacilli; rare to absent large rodsc
Mixed bacterial flora
Decreased large rods,c increase in Gram-positive cocci and Gram-negative rods
WBCs
Rare to 2 +
3 + to 4 +
Rare
2 + to 4 +
3 + to 4 +
Other
Budding yeast, pseudohyphae
Clue cells
Motile trichomonads (60%)
RBCs: 1 + or greater; parabasal cells present
KOH microscopic examination
Negative
Budding yeast, pseudohyphae
Negative
Negative
Negative
Amine or “whiff” test
Negative
Negative
Positive
Positive, often
Negative
Miscellaneous
—
If microscopy negative, use culture or DNA probe analysis
If results inconclusive, use DNA probe analysis; culture of no value
If microscopy negative, use culture or DNA probe analysis
Specimen Collection and Handling
pH
Microscopic Examinations
Wet Mount Examinations
Results
Number of Cells or Organisms
Viewing Area
Rare
<10
Per slide
Occasional
<1
Per 10 hpfs
1+ (Few)
<1
Per hpf
2+ (Few)
1–5
Per hpf
3+ (Mod)
6–30
Per hpf
4+ (Many)
>30
Per hpf
Blood Cells
Bacterial Flora
Yeast
Epithelial Cells
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Analysis of Vaginal Secretions
Learning Objectives
