Microbiology, Inflammation, and Viral Infections

Inflammation and Cellular Obscuring

Overgrowth of microbes in the vaginal milieu may result in obscuring of morphologic details in the smear ( Fig. 7-1 ). In such smears, numerous polymorphonuclear leukocytes occur, often interspersed with a large number of histiocytes. Excessive bacterial growth may also contribute to cellular obscuring. It must be realized that no meaningful evaluation of cellular change may be possible on such smears. In all such cases, it is almost mandatory that appropriate therapy be initiated and a repeat smear examined before an opinion is rendered. Atrophic epithelium of the vagina is particularly prone to inflammatory changes ( Fig. 7-2 ) that may mimic atypia. In such cases local hormonal application generally helps in proper interpretation of cells. In specimens with overwhelming inflammatory exudates, it may not be possible to differentiate vaginitis from cervicitis and endocervicitis. Inflammatory exudates and vaginal microbial flora are considerably altered in liquid-based gynecologic slides (LBGSs).

Background, inflammatory exudate and microbiologic flora may not be obvious in LBGSs.

Bleeding

In infectious processes, both fresh and old blood may be observed. Postmenopausal women with atrophic, thin vaginal mucosa may bleed more easily. Similar changes may occur in Trichomonas vaginalis infection, which produces the typical “strawberry” cervical lesion. Whereas the fresh bleeding is recognizable without much difficulty, old bleeding, observed as fibrin, should be differentiated from mucus. In direct smear preparations, fibrin threads are uniformly thick and reveal nodal formations at the points of intersections of interlacing threads. Sometimes, hemosiderin pigment may be observed within the macrophages and extracellularly. Sometimes, old hemorrhage may contain hematoidin crystals that appear as “cockleburs,” as described by Hollander and Gupta ( Fig. 7-3 ).

Cytolysis, the process of cellular degeneration due to bacterial overgrowth, commonly affects the intermediate squamous epithelial cells. The process is believed to be glycogen dependent. Late menstrual cycle and pregnancy as well as hormonal contraceptives often cause lactobacilli overgrowth. Pale staining, vesicular nuclei with little or no cytoplasm of the intermediate cells predominate in such smears. Numerous lactobacilli may occur interspersed with the remaining cellular remnants ( Fig. 7-4 ).

Degeneration

In inflammatory states cytoplasm of the squamous and columnar cells may be completely or partially disintegrated. However, the major changes are observed within the nuclei. These have been detailed by Frost. Briefly: the nuclei may become compact, dense, and pyknotic with loss of all chromatin details ( Fig. 7-5 ). Such nuclei may have a distinct circumferential cytoplasmic clearing or hollow, causing a perinuclear “halo” ( Fig. 7-6 ), often seen in association with Trichomonas and other infections. Nuclei undergoing degenerative changes frequently lose the sharp details of their nuclear envelope, the chromatin, and the interphase. The nuclear chromatin may clump irregularly or appear beaded along the nuclear margins. They may become blurry and opaque. Other changes include nuclear swelling, with partial or total disintegration of the nuclear envelope, karyorrhexis, and karyopyknosis ( Fig. 7-7 ).

Degenerative, Regenerative (Repair), and Metaplastic Changes in Inflammation

The epithelial cells of the lower genital tract (ectocervix, endocervix, and transformation zone), under the influence of persistent irritation (infectious and non-infectious) and repair, undergo morphologic changes commonly referred to as hyperplasia and metaplasia. These essentially reflect a benign process of tissue repair. An overestimation of reparative changes is a most common error in interpretive cytology. Geirsson and co­workers referred to these as atypical reparative changes (ARCs). The majority of these changes are believed to be derived from the columnar and squamous epithelia, but reserve or pluripotential cells may be involved in the genesis of the metaplasia. It must be appreciated that there is a continuum of changes observed in the healing phase. Cytomorphologic changes, for convenience, are grouped under the term metaplasia. Some of these morphologic changes can be indistinguishable from “early” intraepithelial dysplastic alterations, observed within both the squamous and the glandular epithelia. These cellular features are grouped as “atypical squamous or glandular cells of undetermined significance” (ASC, AGC).

The earliest discernible changes are referred to as presquamous metaplasia: columnar differentiation phase, or type I ARCs of the cervical columnar epithelium. Under the effect of chronic irritation and repair processes, the surface cells of the columnar epithelium continue to mature, and there is a proliferation of the basal or reserve cells. These small, undifferentiated cells commonly occur in small tissue fragments. They have high nucleus-to-cytoplasmic ratios and prominent nucleoli ( Fig. 7-8 ). The nuclear chromatin is fine and uniformly distributed, and the nuclear membrane is well delineated and thin. These may have an inflammatory background. The cells of subluminal origin (progenitor cells) can often be mistaken for undifferentiated neoplasia.

As the changes evolve, the subluminal cells differentiate from the germinal layers upward. These changes reflect the immature squamous metaplastic epithelium and have been referred to as ARC II. The cells may appear to be columnar and have excessive goblet cell proliferation and mucus production. Signet-ring forms may be recognized. These cells can have numerous macronucleoli, coarse chromatin, and modest but pale cytoplasm. If not carefully examined, the changes can be mistaken for a neoplastic lesion of the endocervix ( Fig. 7-9 ). Presence of heavy acute inflammation generally helps in the correct interpretation of these changes. In the LBGS, inflammation is less obvious and these cells often occur as small tissue fragments and may be reported as AGC.

As the changes progress from the subluminal, via the pre­squamous, to the keratinizing stratified squamous phase , the cells become oval or polygonal with sharp borders and dense cytoplasm. They lie in sheets and reveal no obvious cilia and mucus. Intercellular bridges may be seen at times. These cells with their metaplastic changes have been called ARC type III. The nuclear changes may be reactive or degenerative with pyknosis. These cells may be mistaken for squamous cell carcinoma ( Fig. 7-10 ).

Squamous Epithelium

When stressed, as by a chronic irritation or an infective injury, squamous epithelium responds in a number of ways. These changes essentially represent alteration of functional differentiation of the affected cells and are mostly cytoplasmic in nature. Proper identification of these cytoplasmic features is necessary, as they may mask a more serious underlying disease process. These changes–hyperkeratosis, parakeratosis, basal cell hyperplasia, pseudoparakeratosis, and dyskeratosis–have been discussed elsewhere in this book. They reflect abnormalities of maturation with normal keratin formation in cells that normally do not reveal these changes.

Endocervical Columnar Epithelium

In addition to the squamous metaplasia discussed earlier, endocervical cells may undergo other morphologic changes including columnar cell hyperplasia and hyperplastic polyp formation.

Endometrium

Heavy acute inflammation, with pronounced reactive, degenerative, and metaplastic changes may be observed in these cells during the latter half of menstrual bleeding, following instrumentation, in the postpartum period, and in association with the usage of an IUD. Retained gestational products and foreign bodies may result in extensive squamous metaplasia, multinucleated giant cell reaction, and calcification. Some of these changes are discussed later in this chapter.

Micrococcus Vaginitis (Toxic Shock)

This entity is now rarely observed in current practice. This group of microbes includes a large number of Gram-positive coccoid organisms commonly observed in female genital tract smears, and Gram-negative diplococci. Staphylococcus aureus may be recovered from the vagina in about 5% of normal women. These organisms frequently cause vaginitis and vaginal discharge and may produce toxic shock syndrome. This association was documented by Shands and coworkers in 1980. These organisms characteristically occur singly and can be seen within the polymorphonuclear leukocytes or other infected epithelial cells. In vaginal smears, occasionally fragments of tampon fibers may be observed ( Fig. 7-16 ). However, the finding of coccoid organisms or tampon fibers in the vaginal smear does not have any correlation with the clinical occurrence of toxic shock syndrome.

Lactobacillus Vaginitis (Cytolytic Vaginosis)

Lactobacilli are a heterogeneous group of organisms normally present in the vaginal flora. They occur in abundance in the late luteal phase and in pregnancy, prefer an acid environment, and are common among women using hormonal preparations (contraceptives and replacements) and in the premenarchal and menopausal age groups. They are Gram-positive, immobile, non-spore-forming anaerobes or facultative anaerobes. Certain species may be aerobic in their growth characteristics. In the presence of lactobacilli, glycogen-rich intermediate cells are often lysed. Smears in such cases show cellular crowding, cytolysis with cytoplasmic debris, and numerous bare nuclei occurring in a predominantly bacillary background. False clue cells can be reported in these cases as the lactobacilli adhere to the edges of squamous cells. Lactobacilli may be observed in up to 50% of healthy women depending on the day of the menstrual cycle. In the symptomatic population, the observed figure may be lower, about 20%. It is debatable whether pure lactobacilli (an unlikely occurrence) produce vaginitis, although vaginal discharge and leukorrhea may occur as a result of excessive cytolysis.

Gonococcus Vaginitis

These Gram-negative diplococci cause abundant, purulent vaginal exudates. The infection affects the urethra and the perivaginal glands. On the surface of squamous cells, these organisms occur as bean-shaped diplococci. The gonococci are better observed in the air-dried areas of the smears, such as the edges of the smear. This is an uncommon occurrence in properly prepared LBGSs. Within the air-dried distended polymorphonuclear leukocytes, diplococci may be present in large numbers ( Fig. 7-17 ). Gonococcus vaginitis is a venereal infection with important social and medical implications. Although it is detectable cytologically, we do not advise rendition of such a diagnosis on cytologic examination of Papanicolaou-stained smears alone; they may be indistinguishable from other cocci organisms, phagocytosed debris, or Chlamydia organisms.

Curved Anaerobic Bacterial Vaginitis

These motile, anaerobic, rod-shaped organisms resemble Wolinella and have been recognized as a cause of nonspecific vaginitis by Hjelm and colleagues. In the Papanicolaou-stained smears, these bacteria cannot be easily diagnosed but are better detected in wet-mount preparations. Clinically, the presentation is of nonspecific vaginitis.

Vaginal Lactobacillosis

A recently recognized clinical picture has been reported among women who have used antifungal local medications for genital Candida infection for a prolonged period of time, generally more than 20 months. These result in the proliferation of giant lactobacilli often accompanying the yeast forms of Candida organisms. A correct morphologic recognition of this condition is important for specific treatment with appropriate antibiotics ( Fig. 7-18 ).

Foreign-body Vaginitis

A forgotten tampon is the most common cause of this type of vaginitis, in which there is a secondary overgrowth of anaerobic organisms. The tampons may irritate and ulcerate the vaginal wall and ectocervix. Occasionally, fragments of tampons can be observed in vaginal smears. Their presence is not diagnostic of vaginitis. Heavy acute inflammation, mucus, and foreign-body giant cells may be observed.