The microscopic evaluation of scale and vaginal fluid is crucial to the diagnosis of some types of vaginitis and for the diagnosis of tinea in a setting that could alternatively represent psoriasis, contact dermatitis, lichen simplex chronicus, erythrasma, etc. In the case of the evaluation of vaginal infections, even a self-collected swab can be far, far more useful than a telephone diagnosis on the basis
We have found that women can be given a culturette tube to capture vaginal fluid and mail to the office with excellent concordance to testing in the office for candidiasis, bacterial vaginosis, and estrogen maturation index. Most culturette brands yield obvious sheets of buds after 48 hours, but some can take as long as a week to become positive (Fig. 4-4
Sadly, more and more gynecology offices have no microscope, severely limiting the evaluation of vaginitis.
Fungal preparations are essential to the diagnosis of some anogenital diseases. Men generally interpret all anogenital itching as produced by “jock itch” or tinea infection, whereas women assume that all vulvovaginal itching represents a yeast infection. The confirmation or elimination of these conditions is vital.
KOH 10%-20% solution is a basic agent that dissolves the keratin of epithelial (squamous) cells, allowing spores as well as fungal hyphae and pseudohyphae to be seen more clearly. The reliability of this test depends on the choice of the lesion to be sampled, adequate dissolution of cells so fungal elements are best visualized, and the experience of the examiner in distinguishing fungal elements from artifacts such as hair, fabric fibers, cell membranes, and fractures in crusts.
A fungal preparation of vaginal fluid is much easier than that of scale from skin, because there are fewer artifacts, and the fragile mucosal squamous epithelial cells dissolve quickly. However, some infections may produce a small number of organisms that are missed on microscopic examination. Vaginal secretions are collected with a cotton-tipped applicator from secretions remaining on the blade of the withdrawn speculum, from a pool of secretions within the vagina (avoiding the cervical os) or by gently rolling the cotton tip along the vaginal walls when there is no pool of vaginal secretions. Then, a dot of vaginal fluid is touched to the slide, rather than smeared on, which can distort the preparation. Care should be taken to avoid a too-thick preparation that prevents a careful examination. If the vagina is dry and there are no obvious secretions, then the cotton tip should be rolled onto the slide.
The best areas of skin to sample for tinea cruris (dermatophytosis) or cutaneous candidiasis include the peripheral scale from plaques of tinea, and pustule roofs, or white, cheesy material produced by suspected yeast. These elements are removed by gently scraping with the rounded surface of a number 15 scalpel blade. In the damp areas of the genitalia, the sample usually adheres to the scalpel blade and can be wiped onto the glass slide. Dry, hairbearing skin should be moistened with water so that the scale sticks to the blade until it is smeared onto the slide.
Once the vaginal secretions or scale is applied to the glass slide, a drop of KOH is placed on the material to dissolve the keratin from cells and to enhance the visibility of fungal elements. A coverslip is applied, and firm pressure with slight agitation of the coverslip with the back of a fingernail or a pencil eraser (to avoid distracting fingerprints on the coverslip) flattens and augments the dissolution of keratin. Although the nonkeratinized epithelial (squamous) cells in vaginal smears deteriorate quickly after exposure to KOH, the scale of keratinized skin requires further attention to dissolve so that fungi and yeasts can be easily detected. The examiner can use KOH mixed with dimethyl sulfoxide or DMSO (available at medical supply facilities) to enhance dissolution. Or the examiner can simply allow 10-15 minutes for the KOH to disintegrate the cells. In addition, scale dissolves more quickly after addition of KOH if the slide is gently warmed over an alcohol flame.
Fungal elements are best visualized by lowering the condenser to increase the contrast between fungi elements and epithelial cells. Spores, buds, and hyphal elements appear refractile and sometimes very slightly green, and these are much smaller than common artifacts, such as hair and fibers. Dermatophytes appear as branching, septate hyphae that cross over cell membranes (Fig. 4-5
). Candida albicans
and Candida tropicalis
appear as budding yeasts with hyphae or nonseptate, branching pseudohyphae (Fig. 4-6
). At times, cell membranes in the process of dissolving resemble hyphae or pseudohyphae (Fig. 4-7
). When this is suspected, pressure and slight agitation of the coverslip disrupts the cell membranes more efficiently and differentiates between true hyphae/pseudohyphae and this artifact. Non-albicans/tropicalis
species of yeast are characterized by buds without mycelia (Fig. 4-8
). The specific dermatophyte species of tinea cruris cannot be recognized from the smear. A culture is required to speciate a dermatophyte on the rare occasion that this is desired. Oil droplets and air bubbles can be
confused with yeast buds at times, but the variability in size and very round shape distinguish these from yeast buds (Fig. 4-9
). “Tinea” versicolor (which occurs uncommonly in the genital area) is a misnomer because it is a yeast rather than a dermatophyte or true “tinea” infection. It exhibits short, curved hyphae and spores/buds microscopically (Fig. 4-10
Fig. 4-5. A fungal preparation of skin cells scraped from the surface of tinea shows long dark hyphae (arrows) that are easily visible when the squamous cells dissolve from the KOH.
Saline “Wet Mount” Preparation (See Also Chapter 14)
Information from an examination of vaginal secretions is sometimes extremely important in the evaluation of the vulva since irritating or infected vaginal secretions touch the vulva. A microscopic evaluation of vaginal secretions using normal saline under a coverslip permits an evaluation of the morphology of cells, a screen for inflammation and estrogen effect, and a crude survey of colonizing and infecting organisms. The diagnosis of bacterial vaginosis can be made only with a wet mount that shows clue cells and the absence of lactobacilli in conjunction with a positive whiff test and a pH >4.5.
Fig. 4-6. Candidiasis shows not only branching mycelia but also budding yeast on higher power.
Fig. 4-7. When cell membranes are only partially dissolved, they can mimic the branching hyphae of tinea and candidiasis.
A common mistake in the interpretation of a wet mount is a quick directed scan for yeast and clue cells, although even more common is submission of secretions for expensive molecular testing for infection with the incorrect assumption that this covers all causes of vaginitis. Secretions are collected as described above for evaluation for Candida
spp. However, the secretions are transferred from the cotton-tipped applicator by touching (if abundant) or gently rolling (if scant) onto the glass slide. Care should be taken to avoid the thick application of secretions, which interferes with visualization.
Some clinicians prefer inserting the cotton-tipped applicator into a test tube and adding a few drops of saline to dilute the secretions and separate the cells to better evaluate the morphology. This author has tried both methods but has found that the evaluation of white blood cells is problematic when the vaginal secretions are first diluted. First, inflammatory cells stick to the sides of a test tube (and the sides of a culturette tube if secretions on the cotton-tipped applicator are inserted into a culturette tube for later evaluation). Second, the sample is diluted so that detection of lactobacilli can be difficult.
Fig. 4-8. Non-albicans Candida shows only buds, without mycelia, on wet mount. These elongated almost caplet-shaped buds represent Candida krusei.
Fig. 4-9. Air bubbles or oil droplets can mimic buds of yeast. However, these do not exhibit budding, and they show marked variability in size compared to the buds of non-albicans Candida.
A knowledge of the normal components of vaginal secretions and common artefacts is needed to evaluate vaginal secretions beyond the absence of yeast, clue cells, and trichomonads (Fig. 4-11
). Mature epithelial cells shed from a well-estrogenized vaginal epithelium appear as large, often folded, polygonal cells with abundant cytoplasm and a small, condensed nucleus. A crude maturation index can be performed, in which the degree of maturation of epithelial cells is estimated (and this is all that is needed for the evaluation of vulvar discomfort). Immature epithelial cells, or parabasal cells, are much smaller and rounder than mature cells, with proportionately larger nuclei (Fig. 4-12
). These less mature cells are seen in several settings and serve as a marker for atrophic, estrogen-deficient vaginal epithelium, erosions of the epithelium, and rapidly proliferative inflamed vaginal mucosa. Clue cells are a distinct abnormality of epithelial cells that are pathognomonic for bacterial vaginosis. These clue cells occur when bacteria adhere to epithelial cells and obscure the sharp borders of the cells so that the
edge appears ragged and the cytoplasm appears granular (Fig. 4-13
Fig. 4-10. Uncommon on anogenital skin is pityriasis (“tinea”) versicolor; this fungal preparation shows clusters of buds (black arrow) and short hyphe (blue arrows) that generally do not branch.
Fig. 4-11. A wet mount is crucial in the diagnosis of chronic vulvovaginal symptoms; squamous cells should be large, flat, folded, and mature, there should be only one white blood cell (black arrows) per squamous epithelial cell, and bacteria should be predominately the small rods of lactobacilli (blue arrow).
Fig. 4-12. These round cells represent parabasal cells (blue arrow), immature epithelial cells shed from thin estrogen-deficient vaginal walls, and normal flat, folded mature squamous epithelial cells are scant (black arrow).
Fig. 4-13. The presence of clue cells, epithelial cells coated with bacteria so that the borders are ragged (arrow), is pathognomonic for bacterial vaginosis, and a more important sign than the presence of bacteria that suggests this diagnosis on molecular testing.
Although many different organisms are normal vaginal inhabitants, lactobacilli are the most common bacteria seen on a saline preparation of normal vaginal secretions of a well-estrogenized woman. These appear as rods of variable lengths. Occasionally, lactobacilli attach to each other from end to end, to form very long filaments, formerly believed to represent Leptothrix
). These strands are sometimes confused with the hyphae of C albicans or C tropicalis.
However, the filaments of lactobacilli are smaller in caliber than yeast, and these are nonbranching, when compared to the hyphae and pseudohyphae of C albicans.
Other parameters evaluable from a saline preparation are the numbers and types of white blood cells. Leukocytes are regularly present, normally at a ratio of 1:1 or less with epithelial cells. Inflammation, which can be produced by Trichomonas
infection, an irritated or superinfected atrophic vagina, erosive vaginal dermatoses, or desquamative inflammatory vaginitis, is characterized by an increase in white blood cells (Fig. 4-15
). The inflammatory cells can be either lymphocytes or neutrophils, but there is no published information on the implications of this difference. Some clinicians believe that bacterial infections (excluding bacterial vaginosis) are more likely to be characterized by neutrophilic inflammation, whereas noninfectious inflamed dermatoses, such as lichen planus, are more likely to result in an influx of lymphocytes. This author has found this distinction to be unreliable.
Fig. 4-14. Sometimes, lactobacilli line up end to end to form long filaments. Although some believe this occurs as a result of treatment for candidiasis and causes symptoms of itching and irritation, these authors find this to represent a normal variant.
Fig. 4-15. Inflammation is characterized by increased numbers of white blood cells (arrow); this wet mount shows sheets of lymphocytes. Vaginal inflammation is often characterized by the presence of round parabasal cells as well.
Microscopic Examination for Infestations
The three infestations that produce marked anogenital symptoms are scabies, pinworms, and pubic lice. Lice and their nits can be seen by the very careful naked eye or the simple magnifiers mentioned earlier, although a lowpower microscopic examination of a hair with a nit is more conclusive.
The most important aspects of an evaluable scabies preparation are the correction selection of a burrow and the aggressive rather than gentle removal of the affected epidermis. The microscopic confirmation of scabies is often difficult and requires practice. The average patient with scabies has few mites despite widespread rash, most of which results from the immunologic response to the mite and the secondary eczema produced by itching and rubbing. Although one would expect a burrow to present as a linear papule, the burrow of these microscopic parasites actually appear as burrow an edematous, oval papule, with the best burrows for sampling being unscratched. Usually, even those patients with genital
scabies have more accessible and appropriate burrows between fingers or on the ventral wrists. The classic nodules of scabies most common on the penis and scrotum are not appropriate for scraping, as the mite is usually deeper in the skin. A thin layer of epidermis is shaved with a number 15 scalpel blade, and the skin specimen is laid flat on a glass slide. A drop of normal saline or immersion oil is applied to the material, and a coverslip is affixed. The presence of ova, feces (scybala), or the mite itself is definitive evidence of infestation (Fig. 4-16
). The mite, if present, is not subtle and is rarely missed. Eggs are regular, smooth, and fairly large, so they also are usually identified without difficulty. Scybala, however, are regular, clustered, small, golden brown globules that require some experience to identify with confidence. A negative scraping does not eliminate scabies as a diagnosis. A biopsy adds information in the absence of a positive scraping, by either identification of the mite or by the presence of eosinophils, suggests a parasite of some type. A biopsy can also be useful in the patient with genital scabietic nodules but no burrows since that biopsy specimen will be interpreted as an arthropod bite consistent with but not diagnostic of scabies, although the mite usually is not identified.
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