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medical help for various personal and social reasons.
and infectious inflammatory reactions. Although most of these 
infections remain confined locally; they can progress and the 
organisms may ascend to the fallopian tubes and the ovaries. 
Occasionally, microbial infections from the lower genital tract 
can disseminate via the peritoneal space or the hematogenous 
or lymphatic routes.
It must be appreciated that while a large number of women har-
boring genital infections may remain asymptomatic, vaginal infec-
tion can produce a number of clinical symptoms. Increased vaginal 
secretions, along with sloughed vaginal epithelial cells, other infec-
tive organisms, bacteria, and inflammatory cells, constitute acute 
vulvovaginitis. Inappropriate use of over-the-counter medications, 
personal hygiene, tight clothing, impermeable panty hose (panty 
hose vulvitis), reaction to various laundry detergents, and washed 
clothes may contribute to the symptoms of vulvovaginitis.1 Often 
personal and social reasons delay medical intervention.
Key features of vulvovaginitis
 • Increased vaginal secretions;
 • Sloughed vaginal epithelial cells;
 • Bacteria;
 • Infectious organisms; and
 • Inflammatory cells.
Vaginitis accounts for nearly 6 million visits to the healthcare 
Vaginal Microbiology
Among healthy women, the vaginal milieu is polymicrobial and 
contains a large number and variety of aerobic as well as obli-
gate and facultative anaerobic organisms.2 The most frequently 
recovered bacteria include lactobacilli, Streptococcus viridans, 
and Staphylococcus epidermidis; none of which cause symptoms. 
Bacteroides and Gardnerella vaginalis may be culturable from 20 
and 30%, and 50% of the asymptomatic women, respectively. 
Staphylococcus occurs infrequently in the healthy vaginal flora. 
Table 7.1 lists the microorganisms that can be commonly 
 recovered from vaginal specimens.
Pregnancy, besides causing a growth of lactobacillary flora, 
does not appear to affect the microbial composition of the 
vagina significantly. Estrogenic hormones and similar substances 
help in the epithelial maturation of the vagina and support the 
growth of an extraordinary number of microbes. Transplacental 
hormonal exchanges also influence the vaginal epithelium of 
the newborn infant. Bacterial composition and an adult type of 
microenvironment may occur in a newborn female infant.
Menarche and menopausal changes also affect the bacterial 
makeup of the vagina. Hormone or hormone-like medications, 
contraceptives, intrauterine contraceptive devices (IUDs), bar-
rier diaphragms, pessaries, and other similar substances and 
S e c t i o n A
Female Genital Tract 
Micro
The lower female genital tract includes the vulva, the vagina, the 
cervix, and the uterine cavity. It is in direct communication with 
the external environment, and prone to various noninfectious 
Introduction
Contents
Introduction
Vaginal Microbiology
General Features
Background Changes
Cellular Changes
Infections of the Female Genital Tract
Bacterial Infections
Viral Infections
providers per year with an annual cost of over a billion dollars 
to society.1 Vulvovaginal irritation, itching, pain, ulceration 
with bleeding, dyspareunia, and warty growths are some of the 
 common presenting features of vaginal infections. Most often, 
biology, Inflammation, 
and Viral Infections
Prabodh K Gupta and Cindy McGrath
Chlamydial Infection
Fungal Infections
Parasitic Infections
Concluding Remarks
C H A P T E R 7
a woman may remain minimally symptomatic and may not seek 
91
contraception may directly or indirectly influence the micro-
bial balance of the lower genital tract.3 Common factors influ-
encing the vaginal microbial flora are presented in Table 7.2. 
It must be appreciated that the vaginal flora is in a dynamic 
92
Diagnostic CytologyPART TWO
state physiologically and in health. It contains a large number 
of organisms that, under poorly understood conditions, may 
become pathogenic and cause disease.
General Features
A number of general cytological features represent the various 
effects of infective processes. These include the changes listed 
in Table 7.3. Specific cytological changes frequently are associ-
ated with certain infections and are described in their respective 
areas. Only some of these responses may occur under specific 
inflammatory conditions.
Background changes
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 bacte-
rial 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 man-
datory 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 appli-
cation generally helps in proper interpretation of cells. In speci-
mens with overwhelming inflammatory exudates, it may not be 
possible to differentiate vaginitis from cervicitis and endocervici-
tis. Inflammatory exudates and vaginal microbial flora is consid-
erably altered in the liquid-based gynecological slides (LBGS).4
Physiologic Diseases and drugs Local factors
Parturition Hepatic disorders Infections
Pregnancy Hormonal imbalance IUD
Menstruation Metabolic diseases Pessary
Menopause Erosion and infections Diaphragm
Oral contraceptives Vaginal douche
Hormonal mimic drugs Surgery
Antibodies Trauma
Abortion
Sexual exposure
Table 7.2 Common factors influencing vaginal microbial flora
Table 7.1 Common microbial organisms in the vaginal flora
Lactobacilli Bacteroides species
Diphtheroids Peptococcus species
Staphylococcus species Peptostreptococcus species
Streptococcus species Fusobacterium species
Enterobacter (not group A) Clostridium species
Gardnerella vaginalis Bifidobacterium
Postmenopausal atrophy and atypia:
 • Important to treat with local estrogens;
 • Repeat cytologic examination after 6 weeks; and
 • Immediate colposcopy not recommended.
Cellular specimens from specific areas, e.g. the vaginal, cervi-
cal, and endocervical smear,5 or vulvar or lateral vaginal wall 
scrapings, may reveal inflammatory response in one or more 
preparations that can help localize the infective process within 
the lower genital tract. A specific cervical inflammation with 
predominant lymphohistiocytic reaction may occur in follicular 
cervicitis (discussed later). Granulomatous reaction may occur 
in the presence of foreign bodies (e.g. suture material, surgical 
clips, IUD) or specific infections such as tuberculosis.
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 Gupta6 (Fig. 7.3).
Cytolysis, the process of cellular degeneration due to bacte-
rial overgrowth, commonlyaffects the intermediate squamous 
epithelial cells. The process is believed to be glycogen depend-
ent. 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 intermedi-
ate cells predominate in such smears. Numerous lactobacilli 
may occur interspersed with the remaining cellular remnants 
(Fig. 7.4).
Table 7.3 Cytologic features of vaginopancervical smears in infective 
processes
 
 
General
Cellular 
 degenerative 
changes
 
 
Cellular reactive
Background 
changes
Nuclear Hyperplasia and 
repair
Acute and chronic 
inflammation and 
cellular obscuring
Cytoplasmic 
 
Degenerative 
 
Fresh and old blood Metaplasia
Cytolysis Parakeratosis
Cell distribution 
changes
Hyperkeratosis 
Pseudoparakeratosis
Multinucleation
Histiocytic 
 proliferation
Dysplasia
Microbiology, inflammation, and Viral infections
7
BA
Fig. 7.1 Heavy, acute inflammatory exudate. (A) Vaginopancervical smear (Papanicolaou × LP). (B) Note the reduced background inflammation in LBGS 
(Papanicolaou × LP)
BA
Fig. 7.2 Atrophic smear with inflammatory background and some “atypical” cells (arrows). (A) Vaginopancervical smear. (B) Same patient after topical 
estrogen application. Note the cellular maturation and obvious “atypical” cells (arrow). LBGS (Papanicolaou × MP).
BA
Fig. 7.3 Hematoidin crystals, also known as cockleburs, are seen associated with macrophage response. (A) Vaginopancervical smear (Papanicolaou × MP). 
(B) Hematoidin crystals LBGS. Note the clean background and lack of obvious macrophages (Papanicolaou × MP).
93
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Diagnostic CytologyPART TWO
cellular changes
In the cervicovaginal smears, intermediate and superficial cells 
occur predominantly among healthy and normally menstruating 
women. Heavy inflammation frequently causes an exfoliation 
of parabasal cells in the vaginal smear. Although often present 
in postmenopausal women and in the immediate postpartum 
period, occurrence of these three types of cells in the premeno-
pausal age group should be carefully evaluated. Caution need to 
be exercised in rendering hormonal evaluation in smears with 
excessive inflammation. The parabasal cells may exfoliate from 
ulceration of the squamous epithelium of the vagina and the 
ectocervix.
Under the persistent effect of various microbial infections 
and inflammatory reactions, both squamous and columnar 
 epithelial cells may undergo degenerative changes. Almost all 
these changes are nonspecific, but their identification helps in 
the proper interpretation of more serious cellular alterations. 
This is critical because most degenerative changes may be 
accompanied by concurrent regenerative, reactive, and meta-
plastic changes. An extremely heterogenous group of cellular 
changes that includes reactive, degenerative, metaplastic, and 
neoplastic features is termed atypical squamous cells (ASC). 
Morphological features of these cells overlap and generally are 
nonspecific, meaning that they cannot be precisely separated 
between neoplastic and non-neoplastic changes. Additional 
studies are often necessary for further characterization of these 
changes (discussed elsewhere).
Degeneration
In inflammatory states cytoplasm of the squamous and colum-
nar cells may be completely or partially disintegrated. However, 
the major changes are observed within the nuclei. These have 
been detailed by Frost.7 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 inter-
phase. 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).
Fig. 7.5 Nuclear degeneration. Vaginopancervical smear 
(Papanicolaou × HP).
Fig. 7.7 Nuclear degeneration among endocervical cells infected with 
adenovirus infection. Vaginopancervical smear (Papanicolaou × HP).
Fig. 7.4 Lactobacilli. Vaginopancervical smear (Papanicolaou × HP).
 Fig. 7.6 Perinuclear clearing or halos. Vaginopancervical smear 
(Papanicolaou × MP).
Microbiology, inflammation, and Viral infections
7
Degenerative, Regenerative (Repair), and Metaplastic 
Changes in Inflammation
The epithelial cells of the lower genital tract (ectocervix, endocer-
vix, and transformation zone), under the influence of persistent 
irritation (infectious and non-infectious) and repair, undergo 
morphologic changes commonly referred to as 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 co-workers8 referred to 
these as atypical reparative changes (ARC). 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 glan-
dular epithelia. These cellular features are grouped as “atypi-
cal squamous or glandular cells of undetermined significance” 
(ASC, AGC) (discussed elsewhere).
The earliest discernible changes are referred to as presquamous 
metaplasia: columnar differentiation phase, or type I ARC 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 
(N/C) ratios and prominent nucleoli (Fig. 7.8). The nuclear chro-
matin is fine and uniformly distributed, and the nuclear mem-
brane 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 exces-
sive goblet cell proliferation and mucus production. Signet-
ring forms may be recognized. These cells can have numerous 
macronucleoli, coarse chromatin, and modest but pale cyto-
plasm. If not carefully examined, the changes can be mistaken 
for a neoplastic lesion of the endocervix (Fig. 7.9). Presence of 
Fig. 7.8 Squamous metaplasia (atypical reactive cells, ARC I). LBGS 
(Papanicolaou × MP).
heavy acute inflammation generally helps in the correct inter-
pretation 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 pre-
squamous, to the keratinizing stratified squamous phase, the cells 
become oval or polygonal with sharp borders and dense cyto-
plasm. They lie in sheets and reveal no obvious cilia and mucus. 
Intercellular bridges may be seen at times. These cellswith 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 differen-
tiation of the affected cells and are mostly cytoplasmic in nature. 
Proper identification of these cytoplasmic features is necessary, 
Fig. 7.9 Squamous metaplastic changes with columnar cell 
hyperplasia (ARC II). LBGS (Papanicolaou × MP).
Fig. 7.10 Metaplastic changes revealing keratinizing stratified 
squamous metaplasia (ARC III). LBGS (Papanicolaou × MP).
95
96
Diagnostic CytologyPART TWO
as they may mask a more serious underlying disease process. 
These changes—hyperkeratosis, parakeratosis, basal cell hyper-
plasia, 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.
Squamous epithelial cell changes
 • Hyperkeratosis;
 • Parakeratosis;
 • Basal cell hyperplasia;
 • Pseudoparakeratosis; and
 • Dyskeratosis.
Dyskeratosis is mentioned here because of its relationship 
with viral infections and developing cancer. This represents an 
abnormality of the squamous cells in which the cytoplasmic 
maturation is altered. The affected cells reveal premature, hyper-
mature, or atypical keratinization. It is a common occurrence 
in the presence of chronic infections, such as those caused by 
human papillomavirus (HPV). The cytomorphologic features 
are further detailed in the appropriate sections.
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.
Columnar Cell Hyperplasia
Endocervical cells frequently enlarge and produce excessive 
mucus. Such changes occur with chronic irritation of the endo-
cervical canal, such as among women using IUDs8 or hormonal 
contraceptives, and who are exposed to certain infections; these 
are discussed separately.
Hyperplastic Polyp
Hyperplastic endocervical columnar cells may proliferate to 
produce finger-like epithelial processes—polyps. As described 
by Ramzy9 and Frost,10 these polyps are three-dimensional struc-
tures with three distinct planes—a floor or base composed of a 
sheet of polygonal cells, a middle plane that makes up the sides 
of the polyp, and a top or surface layer that, like the base, is also 
a sheet of polygonal cells. In the center of the polyp, a connec-
tive tissue core that contains fibroblasts, collagen, and capillary 
vessels, may be recognizable.
Tubal Metaplasia
Generally, endocervical epithelium may contain ciliated colum-
nar cells only in small numbers (5–10%). Under conditions 
of chronic irritation, sheets of ciliated cells representing tubal 
metaplasia may occur in the specimens collected from the trans-
formation zone and the adjacent endocervix. These cells may 
show pseudostratification and atypia and can be a common 
source of misinterpretation as neoplastic cells.11 Tubal metaplas-
tic cells may stain positively with p16INK 4A antibodies,12 which 
may lead to confusion with neoplastic processes.
Columnar epithelial cell changes
 • Hyperplasia
 • Polyp formation
 • Squamous metaplasia
 • Tubal metaplasia
Endometrium
Heavy acute inflammation, with pronounced reactive, degen-
erative, and metaplastic changes may be observed in these 
cells during the later 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.
Infections of the Female Genital Tract
Bacterial infections
Bacteria most commonly infect the female genital tract. Bibbo 
and Wied13 reported nonspecific organisms including mixed 
bacteria and coccobacilli in nearly 20% of patients. Among chil-
dren these infections occur commonly and may be hormonally 
dependent. Vaginal or vaginopancervical smears often reveal a 
number of bacilli and coccid organisms (mixed infections) as 
detailed by Wied and Bibbo.14 These organisms, although dif-
fusely scattered, may occur in clumps and as microcolonies. 
Appropriate microbiologic isolation techniques are necessary 
for specific species identification but is generally not considered 
necessary for clinical management of the disease.
Bacterial vaginitis (nonspecific vaginitis) was first described 
by Gardner and Dukes.15 They stated, “Any woman whose ovar-
ian activity is normal and who has a gray, homogenous, mal-
odorous vaginal discharge with a pH of 5.0 to 5.5 that yields no 
Trichomonads is likely to have Haemophilus vaginalis vaginitis.” 
It is also known as nonspecific vaginosis/vaginitis so named by 
Blackwell and Barlow,16 or bacterial vaginosis (BV), a term used 
by the International Agency for Research on Cancer.17 This is the 
most common cause for the clinical entity of bacterial vaginosis. 
Instead of making a specific diagnosis of G. vaginalis infection, 
reporting of a “shift in the bacterial flora” is the current term 
used to describe the organism variously named as Haemophilus 
vaginalis and Corynebacterium vaginalis. Gardner and Dukes18 
first described these organisms. Regarding the etiology of BV, the 
statement by Fredricks and Marrazzo that “BV probably results 
from infection with complex communities of bacteria that 
consist of metabolically interdependent (syntrophic) species” 
appears true.19
Morphologically, the organisms are Gram-negative or Gram-
variable, are 0.1–0.8 nm in diameter, and appear bacillary or coc-
cobacillary. The microbe, although it shares many characteristics 
with Corynebacterium, is catalase-negative and is now classified 
separately. Petersdorf and colleagues20 and Ledger and associ-
ates21 found that as many as 40–50% of women may have vaginal 
infection with G. vaginalis and be asymptomatic. Among symp-
tomatic women, leucorrhoea and pruritus with inflamed vaginal 
mucosa and occasional punctate hemorrhages are commonly 
observed. Increased growth and concentration of these organ-
isms may not denote pathogenicity.17 It is believed that patients 
with pure G. vaginalis infection are asymptomatic when the 
vaginal pH is less than 4.5. Secondary organisms interplay with 
G. vaginalis and alter this synergistic relationship. A raised pH 
over 4.5 (5.0 to 6.5) and an interaction with various bacteroides 
and peptococci may produce clinical disease. Recently, consider-
able interest has been exhibited in the study of BV. Molecular 
identification of the associated bacteria has revealed the presence 
Microbiology, inflammation, and Viral infections
7
of three bacteria in the Clostridiales order. These are named as 
bacterial vaginosis-associated bacterium (BVAB1, BVAB2, and 
BVAB3). BVAB are considered highly specific for BV infection.22
Patients with a high pH of the vagina have a vaginal discharge 
with a distinct fishy odor. When the pH is further raised by 
potassium hydroxide (KOH), this odor is manifest in the “whiff 
test.”23 Such preparations of vaginal reactions and KOH, when 
examined microscopically, have the diagnostic “clue cells” (Fig. 
7.11). These refer to normal polygonal squamous cells having 
thin, transparent cytoplasm covered by tiny coccobacillary forms 
of G. vaginalis. Edges of the “infected” cells reveal the BV changes. 
The cell borders may be indistinctand on a different plane of 
focus. Similar clue cells are observed in the fixed and stained 
Papanicolaou preparations (Fig. 7.12). A variable amount of 
acute inflammation may be present in the background. Mere 
complete or partial covering of the squamous epithelial cells by 
the organisms (Fig. 7.13) or their sticking to the cellular mar-
gins (Fig. 7.14) per se should not be considered diagnostic for 
G. vaginalis. To be diagnostic, clue cells should have bacterial 
organisms not only covering the surfaces of the affected cells 
but also spreading beyond the margins of the squamous cells. 
In LBGS preparations, organisms appear in a clean background 
(Fig. 7.15). Detection of BV is reported to be considerably less in 
Fig. 7.11 “Clue cells” phase contrast. Vaginopancervical smear 
(unstained × MP). 
Fig. 7.12 Gardnerella vaginalis (BV) infection. Vaginopancervical smear 
(Papanicolaou × LP).
LBGS-based preparations than in conventional slides.4 This may 
not be an entirely true observation. A high degree of diagnostic 
accuracy exists in cytologic detection of clue cells and culture 
confirmation for G. vaginalis. Schnadig and co-workers24 cul-
tured G. vaginalis in nearly 90% of the cases that contained clue 
cells. This infection is believed to be sexually transmissible, and 
an accurate diagnosis is necessary.
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 docu-
mented by Shands and co-workers in 1980.25 These organisms 
characteristically occur singly and can be seen within the poly-
morphonuclear 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 correla-
tion with the clinical occurrence of toxic shock syndrome.
Fig. 7.13 Partial obliteration of the squamous cell by the coccobacillary 
organisms. Vaginopancervical smear (Papanicolaou × MP).
Fig. 7.14 Organisms sticking to squamous cell. Vaginopancervical 
smear (Papanicolaou × MP).
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Diagnostic CytologyPART TWO
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, immo-
bile, 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 men-
strual cycle. In the symptomatic population, the observed figure 
may be lower, about 20%. It is debatable whether pure lactoba-
cilli (an unlikely occurrence) produce vaginitis, although vagi-
nal discharge and leucorrhoea may occur as a result of excessive 
cytolysis.
Fig. 7.16 Tampon fibers. Occasionally, these may have a core center that 
may contain red blood cells. Vaginopancervical smear (Papanicolaou × LP).
Fig. 7.15 “Clue cells” in LBGS (Papanicolaou × LP).
Gonococcus Vaginitis
These Gram-negative diplococci cause abundant, purulent vagi-
nal 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 LBGS. 
Within the air-dried distended polymorphonuclear leukocytes, 
diplococci may be present in large numbers (Fig. 7.17). Gono-
coccus 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 exami-
nation of Papanicolaou stained smears alone; they may be indis-
tinguishable 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.2,26,27 In the Papanicolaou stained smears, 
these bacteria cannot be easily diagnosed but are better detected 
in wet mount preparations. Clinically, the presentation is of non-
specific 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 lacto-
bacilli accompanying often the yeast forms of Candida organisms. 
A correct morphologic recognition of this condition is important 
for specific treatment with appropriate antibiotics28,29 (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.
Fig. 7.17 Gonococcal organisms. These reveal diplococci within the 
polymorphonuclear leukocytes, and on the surface. Vaginopancervical smear 
(Papanicolaou × OI).
Microbiology, inflammation, and Viral infections
7
Allergic and Acute Vaginitis
Numerous eosinophils may occur in the cervical samples obtained 
from women with vaginal discharge. Generally, the causes are 
noninfectious and associated with an allergic reaction to vaginal 
douche, contraceptives, or various items of clothing30 (Fig. 7.19).
Desquamative Inflammatory Vaginitis (DIV)
This clinical condition is noninfectious in nature and may result 
from a number of blister-forming disorders including pemphi-
gus vulgaris, lichen planus, and pemphigoid.31
Pemphigus vulgaris may exfoliate parabasal size cells that 
have extremely prominent single or multiple nucleoli, pale chro-
matin, and features of reactive cells. Mitosis may be observed. 
Nuclear and cytoplasmic changes can simulate squamous cell 
carcinoma or atypical endocervical or metaplastic cells (Figs. 
7.20, 7.21).32
Granuloma Inguinale
Gram-negative, encapsulated coccobacillary organisms called 
Calymmatobacterium granulomatis cause this venereally transmit-
ted infection. The infection produces large, ulcerated lesions 
that histologically reveal inflammatory granulation tissue and 
Fig. 7.18 Vaginal lactobacillosis. This picture reveals numerous “giant” 
lactobacilli along with some yeast forms of Candida (Papanicolaou × HP).
Fig. 7.19 Allergic vaginitis. Note the numerous eosinophils in this 
preparation. Cervicovaginal smear (Papanicolaou × MP).
numerous macrophages. These macrophages are easily identi-
fiable in ethanol-fixed Papanicolaou stainedsmears. They are 
plump and swollen and have a lobulated cytoplasm. Within the 
cytoplasm, a large number of coccobacillary (1–2 μm) struc-
tures (Donovan bodies) are seen. These are safety-pin shaped 
with terminal or polar thickening of the cell walls (Fig. 7.22). 
The organisms stain faintly with hematoxylin and eosin (H + E) 
dyes. They can be stained with Romanowsky or silver stains. 
Varying degrees of acute inflammation are commonly observed 
in the smears. The infection is more common in the tropics, 
and the incidence is high in India and New Guinea. Although 
reported, an association of granuloma inguinale and squamous 
cell carcinoma is controversial. It is true that the infection may 
cause extremely bizarre pseudoepitheliomatous hyperplasia of 
the squamous epithelium that can mimic neoplasm.
Tuberculosis (Granulomatous Cervicitis)
This is a disease of the tropics and is usually secondary to 
extragenital, most often pulmonary, tubercular infection. 
Involvement is more common in the fallopian tubes and the 
endometrium and is thus difficult to detect cytologically. Angrish 
and Verma33a reported a number of cases of cervical tuberculosis 
that were detected cytologically. The cervical smears reveal large 
B
A
Fig. 7.20 Pemphigus vulgaris. In (B) cells show metaplastic changes. 
These features can be confused with neoplastic as well as viral changes. 
Vaginopancervical smear (Papanicolaou × MP).
99
100
Diagnostic CytologyPART TWO
 aggregates of epithelioid cells. These appear as pale, cyanophilic 
cells in a syncytial formation with indistinct and arborizing bor-
ders and vesicular, oval nuclei (Fig. 7.23). Intermixed with these 
one may occasionally observe Langhans-type multinucleated 
giant cells (Fig. 7.24). These cells may contain as many as 20 
to 30 peripherally arranged vesicular nuclei. A variable number 
of lymphocytes may be present in the background. Secondary 
infection is common in these ulcerated lesions, and heavy, acute 
inflammatory exudates may be present. A cytologic diagnosis of 
granulomatous disease, probably tuberculosis, can be suggested 
under appropriate clinical and cytologic settings.
Malacoplakia
Malacoplakia is a rare disorder that may affect the cervix. We 
have observed two cases occurring in postmenopausal women 
with atrophic smears and persistent vaginal discharge.33 Numer-
ous macrophages with the characteristic intracytoplasmic, lami-
nated inclusions (Michaelis-Gutmann bodies) may be observed 
(Fig. 7.25). They can be stained for calcium salts including cal-
cium phosphates and carbonates by histochemical techniques 
such as Von Kossa’s method.34
Fig. 7.22 Donovan bodies. A single macrophage reveals the cytoplasmic 
lobules containing numerous safety-pin-shaped bacillary structures. 
Vaginopancervical smear (Papanicolaou × OI).
Fig. 7.21 Pemphigus vulgaris, cervix. Tissue biopsy reveals cellular changes 
similar to those seen in the smear in Fig. 7.20. Cervical biopsy (H&E × MP).
Langerhans Cell Histiocytosis
This rare disease of unknown etiology may involve the lower 
female genital tract and the endometrium. It has been included 
here because the lesions can both clinically and cytologically 
may be indistinguishable from inflammatory or neoplastic 
processes. It can occur as “pure” genital tract or part of the gen-
eralized systemic disease. Cervical cytology may contain atypi-
cal histiocytic cells (Fig. 7.26), numerous macrophages with 
intranuclear grooves, eosinophils, and an occasional multinu-
cleated giant cell (Fig. 7.27). This diagnosis may be considered 
in the presence of intranuclear grooves in the macrophages, and 
eosinophils in the smear. The exact morphologic features vary 
from the stage of the disease. Immunohistochemical stains S100 
and CD1a as well as ultrastructural demonstration of Birbeck 
 granules are helpful diagnostically.35,36
Actinomyces
These organisms belong to the order of higher bacteria that also 
include Mycobacteriaceae and Streptomycetaceae. There are three 
common species of Actinomyces—A. israelii, A. bovis, and A. naes­
lundii. These bacteria are nonmotile, non-spore-forming, and 
Fig. 7.24 Cervical tuberculosis (H&E × LP).
Fig. 7.23 Epithelioid cells. Notice the syncytial formation of cells with ill-
defined margins. Vaginopancervical smear (Papanicolaou × MP). 
Microbiology, inflammation, and Viral infections
7
anaerobic or facultative anaerobes. They are Gram-positive and 
occur in filamentous and diphtheroid forms. With the recent 
increase in IUD usage, genital Actinomyces infection appears to 
also be increasing in prevalence. It is, however, true that the new 
device designs and the judicious usage make the clinical disease 
less likely.37
Actinomyces occur commonly within the tonsillar crypts, tar-
tar of teeth, and the alimentary tracts. Actinomyces do not occur 
as commensals in the vaginal flora. In the female genital tract, 
ascending infection is the most common mode of occurrence of 
clinical disease; however, rarely, hematogenous and lymphatic 
spread, or dissemination of infection from the alimentary tract or 
other distant sources, may occur. Ascending infection occurs in 
the presence of intrauterine or intravaginal contraceptives, IUDs 
of various types being the most common. Vaginal pessaries, surgi-
cal clamps, and foreign bodies, including forgotten tampons, all 
have been associated with vaginal Actinomyces. Among untreated 
women, clinical disease may be manifest for as much as 12 
months after the removal of the Actinomyces-associated IUD.
Fig. 7.25 Michaelis-Gutmann bodies. Intracytoplasmic laminated 
structures from a case of malacoplakia of the cervix. Vaginopancervical smear 
(Papanicolaou × HP). 
Fig. 7.26 Langerhans cell histiocytosis. Note numerous histiocytic cells 
with intranuclear grooves (arrowhead) and an occasional eosinophil (arrow). 
Vaginopancervical smear (Papanicolaou × HP).
Gupta has reviewed the subject and the relationship of Actin­
omyces with clinical female genital tract disease.38 It is appropri-
ate to say that nearly 10% of women using an IUD may develop 
vaginal Actinomyces infection at some stage. If such users have 
symptoms of lower genital tract infection such as pelvic pain, 
vaginal discharge, bleeding, fever, or lower abdominal tender-
ness, approximately one-quarter of these women may have geni-
tal Actinomyces infection. Of the women using an IUD and being 
admitted to the hospital for clinically suspected pelvic inflam-
matory disease, about 40% may harbor the organism in the 
lower genital tract.39,40 Dissemination of the infection to distant 
sites has been documented by de la Monte and co-workers,41 
and by Hager and Majmudar.42
Cytomorphology of Actinomyces
In close proximity to the calcified and mineralized fragments of 
a disintegrating IUD, the Actinomyces organism can be detected 
in Papanicolaou stained vaginal smears. Typically, the organisms 
appear as spidery, amorphous clumps that are darker in the center 
(Fig. 7.28A). Morphologic features of the Actinomyces colonies are 
more distinct in LBGS-based slides (Fig. 7.28B). These aggregates 
of Actinomyces in the cervicovaginal smears have been referred 
to as “Gupta bodies” by Hager and Majmudar.42 Upon careful 
examination, numerous filamentous organisms with acute angle 
branching patterns are recognizable in these clumps (Fig. 7.29). 
They can be uniformly thick and beaded. The filaments generally 
extend to the outer limits of the dark clumps. Only a few delicate, 
branching filamentous forms may occur scattered randomly in 
the smear. In Papanicolaou stained smears, calcified filamentous 
forms that may not be stainable by antigen antibody techniques, 
club forms, or the Splendore Hoeppli phenomenon may be iden-
tified. Typical sulfur granules may be observed in smears obtained 
from symptomaticpatients (Fig. 7.30). These per se are not diag-
nostic of Actinomyces, and proper morphologic identification of 
the filamentous forms is necessary in all cases. Gupta and co-
workers have detailed various other morphologic forms.38,43
Actinomyces organisms can be stained with modified Gram, peri-
odic acid-Schiff (PAS), and silver stains. A definitive species diagnosis 
requires specific antigen antibody reaction using an immunoenzy-
matic or immunofluorescence or bacterial culture procedures.44
Fig. 7.27 Langerhans cell histiocytosis. Note atypical histiocytic cells and 
numerous eosinophils. This smear was obtained 14 months after that in Fig. 7.26. 
Vaginopancervical smear (Papanicolaou × HP).
101
102
Diagnostic CytologyPART TWO
A number of organisms, including Candida, dermatophytes, 
and Nocardia, along with bacterial aggregates, and foreign 
 substances such as sulfa drug crystals and contraceptive creams, 
may resemble Actinomyces organisms. Hematoidin crystals 
described by Hollander and Gupta6 have a resemblance to sulfur 
granules. The differential diagnosis of Actinomyces as seen in the 
vaginopancervical smear is presented in Table 7.4.
We believe that genital Actinomyces is an exogenous infection. 
Orogenital contact may be an important mode of acquiring the 
genital Actinomyces infection. The “tail” of the IUD most likely 
acts as a carrier for the ascent of the organisms. The tissue dam-
age produced by the body and edges of the IUD causes a change 
in the oxygen reduction potential and alteration in the micro-
bial milieu of the lower genital tract. The changed environment 
is conducive to the growth of these organisms. Actinomyces 
has been observed with all types of IUDs, including currently 
 marketed models. Infection is more common with devices 
with polyfilamentous thread and with angular forms.
Key features of genital Actinomyces
 • Always associated with an IUD or a foreign body;
 • May cause no symptoms;
 • Occur as dark, woolly clumps (Gupta bodies);
B
A
Fig. 7.28 Actinomyces. (A) Vaginopancervical smear (Papanicolaou × LP). 
(B) LBGS (Papanicolaou × LP).
 • Parallel filaments, branching at acute angle;
 • Difficult to culture; and
 • May be confirmed by special stains.
Occasionally, Actinomyces may occur in association with 
“black yeast,” a fungus Aureobasidium pullulans, commonly found 
in areas with poor hygienic conditions. It has large, dark-fruiting 
bodies (Fig. 7.31). As reported by de Moraes-Ruehsen and asso-
ciates, Entamoeba gingivalis, a protozoan of the oral cavity, may 
be found in association with Actinomyces in vaginal specimens45 
(Fig. 7.32). An orogenital route of this Actinomyces infection is 
a distinct possibility. These nonpathogenic protozoa should be 
distinguished from Entamoeba histolytica that occur in the alimen-
tary tract and which may also cause lower genital tract infection.
IUD-Associated Cellular Changes
In addition to the alterations in the microbial environment and 
Actinomyces infection, usage of the IUD is associated with cellular 
changes occurring in the various genital tract epithelia, as early 
as 10–12 weeks after an IUD insertion. These result from chronic 
irritation by the IUD tail and the body affecting the adjacent 
 tissues within the endocervix and the uterine cavity. It is impor-
tant to recognize these morphologic features as they can mimic 
B
A
Fig. 7.29 Colonies of Actinomyces. (A) Note the numerous filamentous 
structures radiating from the center. Vaginopancervical smear (Papanicolaou 
× HP). (B) Higher magnification of the colonies of Actinomyces 
(Papanicolaou × HP).
Microbiology, inflammation, and Viral infections
7
and be confused with dysplastic and neoplastic cellular changes 
of the squamous, metaplastic, endocervical, and endometrial 
epithelia. These changes appear more pronounced in LBGS and 
interpretation can be problematic especially in the paucity of 
an inflammatory background. There is no definite evidence for 
the association of squamous dysplastic changes and IUD usage. 
Squamous cell changes are essentially reactive and reparative in 
nature. These occur in about 40% of women using IUDs. DNA 
analysis of IUD-associated cellular changes does not reveal any 
aneuploidy.
The morphological picture is further complicated by inter-
play among the reactive-proplastic and degenerative-retroplastic 
changes occurring over a prolonged period and affected by 
 polymicrobial and physiologic factors.
Endocervical columnar cells may become hyperplastic with 
large papillary tissue fragment formations. Bibbo and co- 
workers46 and Gupta and colleagues47 have systematically 
reviewed these changes. Columnar cell hyperplastic changes 
should be distinguished from adenocarcinoma (Fig. 7.33). They 
may mimic papillary tumors of ovarian or endometrial origin. 
Single cells can be extremely bizarre and resemble neoplasia. 
Cells may show large cytoplasmic vacuoles referred to as “bub-
ble gum” cells. The presence of heavy inflammation and degen-
erative changes helps diagnostically. The salient features of these 
cellular changes are summarized in Table 7.5. The presence of 
psammoma or calcified bodies among IUD users is not an indi-
cation of neoplasm.
Another cell type, best described as indeterminate cell changes 
or “IUD cells,” probably arises from the endometrial surface. 
Fig. 7.30 Sulfur granule. In the center are radiating filamentous structures 
of Actinomyces organisms. Vaginopancervical smear (Papanicolaou × MP).
Table 7.4 Differential diagnosis of Actinomyces in vaginal smears
Other organisms Candida, Aspergillus, Nocardia, Penicillium, 
Trichophyton, Leptotrichia, lactobacilli
Miscellaneous structures Filamentous structures: Fibrin, mucus, 
sulfa crystals, cotton and synthetic fibers
Nonfilamentous structures: Contraceptive 
cream, bacterial clumps, hematoxylin 
pigment, spermatozoa, hematoidin, 
foreign material (spores, pollen, douche 
ingredients)
Fig. 7.31 Aureobasidium pullulans. These black yeast organisms can 
vary in color from light yellow, gold-brown, to black. Vaginopancervical 
smear (Papanicolaou × HP). Reproduced with permission from Gupta PK: 
Intrauterine contraceptive device: Vaginal cytology, Pathologic changes, and 
their clinical implications. Acta Cytol 1982;26:571-613.
Such conclusions are supported by the work of Gupta and co-
workers.47 These cells with a high nucleus-to-cytoplasmic ratio 
should be distinguished from the third type of cell described 
by Graham48 and from in situ carcinoma (HSIL, CIN III) cells. 
Nuclear degeneration, the presence of nucleoli, and a hiatus 
between normal and abnormal cells help differentiate these 
cells from true neoplastic cells (Fig. 7.34). Table 7.6 summarizes 
the salient features of these cells. Occasionally, the endometrial-
type reactive cells and the IUD cells may occur together.
Binucleated and multinucleated giant forms and psammoma 
body formation are other findings that may be observed in 
the presence of the IUD and Actinomyces. These develop from 
endometrial surface changes. Extensive squamous metaplasia of 
the endometrial surface may occur in some cases as the result of 
prolonged endometritis accompanying the IUD.
Key features of IUD-associated cellular changes
 • Bubble gum cells;
 • IUD cells;
 • Metaplastic cells;
 • Mesenchymal proliferation;
 • Multinucleation; and
 • Psammoma body formation.
Leptotrichia buccalis
These microbes, also known as just Leptotrichia or Leptothrix, are 
Gram-negative, non-spore-forming anaerobic organisms. They 
occur in the oral and vaginal cavities as very thin, segmented, 
large, filamentous structures. Occasionally, branching may be 
observed (Fig. 7.35). Morphologically they may be indistin-
guishable from certain forms of Doederlein’s bacillus. Most 
frequently(75–80%), cases of Leptotrichia have concomitant 
T. vaginalis infection. Numerous other infective organisms, 
including Candida and G. vaginalis, may occur in the presence of 
L. buccalis infection.
Bibbo and Wied13 made an investigative study on the preva-
lence of Leptotrichia in cervicovaginal smears. They observed 
 Leptotrichia organisms in 75% cases with trichomonads, 1.5% 
with Doederlein’s bacillus, and about 1% among patients with 
103
104
Diagnostic CytologyPART TWO
fungal or BV infection. Nearly half (47%) of the 1,000 patients 
studied were oral contraceptive users. Pregnancy and menopause 
were other physiologic features, followed by the postpartum 
state, that were often associated with the presence of L. buccalis 
in cervical smears. Sometimes acute inflammatory changes may 
be observed in the presence of Leptotrichia.
Mycoplasma
These are the smallest known organisms capable of growing in cell-
free media. Jones and Davson documented a correlation between 
the occurrence of a “dirty” smear and mycoplasma,61 and Mardh 
and co-workers confirmed these findings and reported the occur-
rence of coccoid organisms both on the surface and in between the 
squamous epithelial cells in dirty smears in cases of Mycoplasma.49 
Such features appear to have limited practical value.48a,49
Follicular Cervicitis
Also referred to as lymphocytic cervicitis, this is a specific type 
of cervical, and sometimes vaginal, lesion in which the pre-
dominant feature is the occurrence of lymphoid follicles in the 
subepithelial areas. When examined cytologically, numerous 
mature and reactive lymphoid cells and germinal macrophages 
(tingible bodies) are seen (Fig. 7.36A).50 Cellular changes are 
uncommonly recognized in LBGS (Fig. 7.36B) and are often 
more problematic to interpret. Small lymphoid cells appear in 
aggregates especially in LBGS and do not clearly reveal tingi-
ble bodies.51 Lack of mucus and small size of the lymphocytes 
appear to contribute to these morphologic changes. At times, 
a capillary from the germinal center of the lymphoid follicle 
may be scraped and observed in the smear (Fig. 7.37). There is 
evidence that nearly 50% of the cases of follicular cervicitis are 
BA
Fig. 7.33 “Bubble-gum” cells occurring in a patient with an IUD. (A) Vaginopancervical smear (Papanicolaou × HP). (B) IUD-associated glandular cells LBGS 
(Papanicolaou × MP).
Table 7.5 Comparison of IUD-associated columnar-type cells 
and adenocarcinoma cells
Feature IUD columnar cells Tumor cells
Tumor diathesis Absent Present
Distribution Endocervical Random
Inflammation Present Variable
Cellular 
degeneration
Present Absent 
“Bubble gum” cyto-
plasm
Present Absent 
Bare nuclei Absent Present
Cellular preservation Poor Good
Atypical histiocytic 
cells
Absent Present 
A
Fig. 7.32 Entamoeba gingivalis. (A) Vaginopancervical smear (Papanicolaou ×
B
 HP). (B) LBGS (Papanicolaou × HP).
Microbiology, inflammation, and Viral infections
7
 • Must identify tingible-body macrophage;
 • Difficult to diagnose in LBGS due to lymphocytic 
the nature of the affected tissues, the virus, general and local 
immune responses, and particular enzymatic derangements are 
 dispersion; and
 • Cells should be distinguished from:
 – Lymphoma;
 – Metastatic tumor cells;
 – Endometrial cells; and
 – Histiocytes.
Viral infections
Diseases caused by these intracellular organisms are among the 
most common in the human body and include a most hetero-
geneous group of clinical conditions. Although some of the viral 
infections have been affecting humanity for thousands of years, 
changes in society, social habits, medical practice, and advances 
in diagnostic capabilities have resulted in a great many new 
important in determining the cytomorphologic changes and the 
nature of the tissue injury or injuries. In some common viral 
infections, these cellular changes may be quite typical and con-
sidered of diagnostic significance.
General Features of Viral Infection
Inclusion Formation
Inclusions are discrete, dense, homogeneous, round, or oval 
intracellular structures consisting of viral particles in a matrix, 
and generally represent a stage in the replication of the virus. 
They do not occur in all viral infections, their formation 
depending upon a particular agent and on the affected tissue. 
Certain inclusions are typical and diagnostic. Inclusions may be 
observed within the nuclei, the cytoplasm, or both.
associated with Chlamydia infection.52,53 Follicular cervicitis is 
not uncommonly seen in postmenopausal atrophic smears. The 
precise pathogenesis of this condition in this age group is not 
well understood. These cells should be distinguished for malig-
nant lymphomas, histocytes, endometrial cells, and metastatic 
tumor cells.
Key features of follicular cervicitis
BA
Fig. 7.34 “IUD cells.” These high nucleocytoplasmic (N/C) ratio cells appear to
degeneration, and nucleoli. If not carefully examined, these can be easily mistake
(Papanicolaou × HP). (B) IUD cells in LBGS (Papanicolaou × OI).
Table 7.6 Comparison of IUD cells and cervical intraepithelial neoplasia 
(CIN/HSIL) cells
Feature IUD cell CIN/HSIL cell
Distribution Endocervical Endocervical
Tissue fragments Rare Common (LBGS)
Inflammation Present Absent
Cellular 
 degeneration
Present Absent 
Preservation Poor Good
Cellular hiatus Present Absent
Nucleoli Present Absent
Multinucleation Present Absent
IUD columnar cells Present Absent
viral diseases. Even though smallpox has been eradicated from 
the world, with antibiotics and immunosuppressive therapies, 
numerous dormant viral infections have become manifest.
Being intracellular by nature, viruses co-opt cellular meta-
bolic processes in their replication cycles. In addition to 
 be of endometrial origin. They frequently show multinucleation, nuclear 
n for cervical intraepithelial neoplasm. (A) Vaginopancervical smear 
Fig. 7.35 Leptotrichia buccalis. Vaginopancervical smear 
(Papanicolaou × HP).
105
106
Diagnostic CytologyPART TWO
Hydropic or Ballooning Degeneration
This particular cellular change is often an effect of organelle 
membrane damage caused by the virus. Certain degenerative 
changes precede or accompany the development of inclusion 
bodies and are often used in the diagnostic evaluation of 
cellular changes (Fig. 7.38).
Necrosis
Viruses may cause coagulative necrosis and characteristic cyto-
plasmic changes. Most often, the cytoplasm becomes opaque 
and thickened and loses its transparency and crispness. Nuclear 
degenerative changes with karyolysis and karyorrhexis may 
occur (Fig. 7.39). Only ghost forms of the infected cells may 
remain.
Giant Cell Formation
Alterations in the membrane composition of the infected cells 
contribute to the fusion of cells to produce syncytial and giant 
forms. Sometimes nuclear inclusions may occur within the 
multinucleated forms. Fig. 7.39 Nuclear degeneration. LBGS (Papanicolaou × HP).
A
Fig. 7.36 (A) Follicular cervicitis. Vaginopancervical smear (Papanicolaou × OI
macrophages (arrow) and lymphocytes may be observed (Papanicolaou × HP).
Fig. 7.37 Follicular cervicitis. Note the occurrence of a germinal follicle 
and a capillary in this picture. Vaginopancervical smear (Papanicolaou × LP).
B
). (B) Follicular cervicitis, LBGS. Diagnosis is difficult to make. Tingible 
Fig. 7.38 Hydropic degeneration. Vaginopancervical smear 
(Papanicolaou × HP).
Microbiology, inflammation, and Viral infections
7
Cellular Proliferation
Transient cellular proliferation is commonly seen in viral infec-
tions. These changes may be extreme and mimic dyskaryosis 
and neoplastic forms (Fig. 7.40).
Cellular Cohesion
In certain viral infections, the initial step is attachment to thehost cell; viral proteins (antireceptors) adsorb to the cell surface 
furnished with appropriate receptors. The interaction may alter 
not only the surface of the infected cell but also the structure 
of the virion. Although not fully understood, viral detachment 
and readsorption perhaps contribute to cell clumps or plaque 
formation.
Cytoskeleton Changes
Cytoplasmic and nuclear changes frequently occur not as a 
result of the damage caused by the virus, but rather as a result 
of specific reorganization of the cellular or skeletal elements 
necessary for its growth. Alteration in intermediate keratin fila-
ments and microtubules, and cellular metabolism contribute to 
the formation of ciliocytophthoria (CCP) seen in certain viral 
infections. It should be distinguished from detached ciliary tufts 
A
B
Fig. 7.40 (A) Atypical cellular proliferation, LBGS (Papanicolaou × MP). 
(B) “Atypical” cellular changes in cervical herpes, LBGS (Papanicolaou × HP).
(DCT) described by Hollander and Gupta that may be observed 
in lower genital tract smears in the absence of a viral infection.6,54 
In vivo hemadsorption observed occasionally may be a related 
phenomenon (Fig. 7.41).
Oncogenesis
Both in vitro and in vivo neoplastic transformation of viral-
infected cells may occur. Numerous DNA viruses and a group of 
retroviruses are capable of neoplastic transformation. These com-
monly manifest as dyskaryosis and atypical nuclear alterations.
Quite often, in the presence of florid viral infection, no dis-
cernible morphologic changes may occur in the infected cells 
and tissues.
The previously mentioned cellular manifestations may or 
may not be reflected in all cytologic preparations and in the 
presence of all viral infections.
Specific Infections
Specific viral infections commonly observed in the female geni-
tal tract include herpes infection: Herpes is a Greek word mean-
ing “to creep.” It is believed that this word was used in relation 
to certain clinical features of an infection that eventually was 
found to be related to the particular DNA virus. There are at least 
six different viruses in this group causing disease in humans. 
These are:
 • Herpes simplex virus, type 1 and type 2 (HSV 1, HSV 2);
 • Cytomegalovirus (CMV);
 • Varicella-zoster virus;
 • Epstein-Barr (EB) virus; and
 • Lymphoma-associated viruses.
In the cytologic preparations from the female genital tract 
herpes, CMV and varicella may be detected.
Herpes Simplex Virus
Distinction between HSV 1 and HSV 2 was made based on sero-
logic studies by Schneweis.55 Most people acquire antibodies to 
HSV 1 during the first 2 years of their life. Herpetic vulvovagin-
itis or stomatitis due to HSV 1 may occur at the time of initial 
infection, generally in infancy or early adolescence. Infection 
is mostly asymptomatic, or it may be accompanied by upper 
Fig. 7.41 Hemadsorption. This patient had herpes infection at the time 
when these and many other similar cells were seen. Vaginopancervical smear 
(Papanicolaou × HP).
107
108
Diagnostic CytologyPART TWO
 respiratory tract or ocular symptoms. Morphologically, HSV 1 
and HSV 2 cellular changes appear identical.
Although congenital or neonatal transmission may occur, 
HSV 2 generally occurs after puberty and the onset of sexual 
 activity. Cutaneous lesions, commonly vesicles, tend to occur 
in the same area repeatedly; the interval between successive 
eruptions varies considerably even in the same individual. 
Stress, menses, and other unrelated ailments may precipitate an 
eruption in an otherwise healthy person. Following the initial 
infection, the virus remains dormant in the sacral (S2 through 
S4) dorsal root ganglia in the spinal cord. McDougall56 has 
 documented its presence in the spinal cord.
Recently, there has been an increase in the occurrence of HSV 
2 cases; it is generally attributed to changed sexual and social 
habits. Using seroepidemiologic data, an association of HSV 2 
and cervical cancer has been reported by Kessler and others.55,57–63 
The precise role of HSV 2 in the development of human cervical 
cancer is far from resolved; evidence, however, accepts HPV as 
the most important causative infection.
Herpes Simplex Genitalis Virus, Type 2 For nearly 2500 years, 
people have used the word herpes in medical literature. Corey 
has briefly discussed the history of genital herpes.64 John Astruc 
first described genital herpes in 1736 in the French literature. 
More than 100 cases of “herpes progenitalis” were reported in 
the late nineteenth century. Lipschutz established experimental 
transmission of herpes in human beings in 1921.65 He concluded 
that there were differences between oral (HSV 1) and genital 
herpes (HSV 2) infections.
HSV 2 infection is one of the most common sexually transmit-
ted genital infections; more than 300,000 new cases are recorded 
in the United States annually. The prevalence of infection varies 
depending on the group studied. Although in general popula-
tions the incidence of infection is not well established, genital 
HSV infection was diagnosed among 4.2% of those attending 
the Sexually Transmitted Disease Clinic in Seattle, Washington, 
in 1980. Women presenting at student health services have been 
found to have HSV 2 infection about seven to ten times more 
commonly than gonorrhea. Data from the Centers for Disease 
Control and Prevention (CDC) suggest that the prevalence of 
HSV 2 is increasing and that the infection is occurring in social 
groups that previously did not have the disease.
Primary infection may be asymptomatic or accompanied by 
severe constitutional symptoms. Commonly, fever, headache, 
and myalgia occur before the appearance of mucocutaneous 
lesions. Visible lesions appear between 2 and 7 days following 
exposure to the virus. Local pain and itching, dysuria, vaginal 
discharge, and inguinal lymphadenopathy may be present. The 
lesions are painful and often multiple. Large ulcerations that 
start as papules or vesicles spread rapidly. They form pustules 
that coalesce and break down. Unless complicated by second-
ary infection, these ulcers heal in 5 to 10 days with reepitheli-
alization. Residual scarring is uncommon. Systemic symptoms 
and inguinal lymphadenopathy occur mainly in primary HSV 2 
infection.
The cytologic diagnosis of HSV infection is important and 
must be made on well-preserved cells that have typical diagnos-
tic features and have not been altered by air-drying, fixation, or 
inflammation. Such a diagnosis may determine proper man-
agement of patients, especially pregnant women with geni-
tal ulcerations. An HSV diagnosis, with its social and medical 
 implications, should be rendered only when unequivocal 
 evidence is present.
In addition to diagnosis in the standard Pap test, direct sam-
pling of visible lesions can be performed. Such smears should be 
prepared from the edge and bed of the ulceration, not from the 
contents of the vesiculae. The latter generally contain serosan-
guineous material with acute inflammatory cells, eosinophils, 
and some macrophages. Although use of air-dried smears and 
their examination after Romanowsky stain (Tzanck prepara-
tion)66 have been advocated, we do not recommend this for 
genital lesion diagnoses. Heavy inflammation, cellular obscur-
ing, and degeneration often make interpretation difficult and 
may severely compromise the diagnostic value of air-dried 
smears. Cellular samples obtained from the cleared ulcer beds 
should be immediately fixed in 95% ethanol and examined after 
Papanicolaou staining.
The virus may infect the immature squamous, metaplastic, 
and endocervical columnar cells. Initially, the changes are pro-
plastic and somewhat nonspecific. The infected cells can occur 
singly, in groups, and in tissue fragments. There is cytomegaly 
and karyomegaly,and the nucleocytoplasmic ratio is not much 
altered. These cells demonstrate a combination of reactive (pro-
plastic) and degenerative (retroplastic) changes. The nuclei of 
the infected cells show changes in the chromatin structure con-
sisting of hydropic or ballooning degeneration. The chromatin 
material becomes extremely finely divided and is uniformly 
dispersed in the nuclear sap. The chromatin–parachromatin 
interphase is obliterated, and nuclei assume a faintly hematoxy-
linophilic, homogenized appearance. Some chromatin material 
may be matted against the inner leaf of the nuclear envelope, 
which may appear uniformly thick and conspicuous. The altered 
nuclear morphology is commonly referred to as ground glass, 
bland, gelatinous, glassy, or opaque. In some cases the redistri-
bution of chromatin may result in a beaded appearance of the 
nuclear margins. Nucleoli may be present and conspicuous, may 
have associated chromatin, and may not appear typically bright 
acidophilic. Although the nucleoli generally remain round or 
oval, sometimes irregular shapes may be observed.
In the later stages of HSV infection, the cells undergo the 
effects of viral replication and DNA integration. The cells may 
assume multinucleation, which is observed in nearly 80% of the 
smears from cases of genital HSV infection. The infected nuclei 
may have the same homogeneous chromatin pattern described 
previously. The nuclei appear tightly packed within the cells 
and reveal distinct internuclear molding (Fig. 7.42). At times 
they may be overlapping and not molding. Large and single 
intranuclear inclusions appear within these nuclei. The nuclear 
inclusions are generally round or oval. They can be angulated 
and sharp (Fig. 7.43). They lack structure and are densely eosi-
nophilic. Depending on the staining procedure employed, they 
may appear cherry red. A clear zone, or halo, which separates it 
from the nuclear membrane, surrounds the intranuclear inclu-
sion. Most often the halo is as clear as the background of the 
slide. Sometimes it may retain delicate, homogeneous, diffuse 
hematoxylinophilia. Small, inconspicuous chromatin granules 
can occur in the peri-inclusion halo. Inclusions may occur in 
infected single cells observed in nearly one-third of the cases of 
HSV 2 infection. Intranuclear inclusions may not be present in 
all of the nuclei within the multinucleated giant cells.
The cytoplasm in the infected cells at this early stage of HSV 
infection is dense. It may lose its transparent appearance and 
become opaque. Often it stains bright cyanophilic.
HSV-infected cells can become atypical; the enlarged cells may 
assume bizarre shapes (Fig. 7.44). They may be hyperchromatic 
Microbiology, inflammation, and Viral infections
7
or degenerated and may be misinterpreted as tumor cells. The 
cytoplasm may show changes of the cytoskeletal structure and 
become dense or opaque uniformly or focally. The latter may 
represent keratohyaline material. Degenerative vacuoles, the 
ectoplasmic–endoplasmic differentiation with spiral fibrils of 
Erbeth, as described by Patten,67 may be present between the 
two zones. The fibrillary apparatus of Herxheimer appears as 
delicate, uniformly thin spirals that originate at the nucleus, 
travel down the cytoplasm, and may be observed in cells with 
squamous differentiation features.
Key features of herpes genitalis infection
 • Multinucleated giant cells not diagnostic;
 • Nuclear changes critical for diagnosis; and
 • Primary and secondary infection cannot be distin-
guished morphologically.
Multinucleated giant cells per se do not establish the diag-
nosis of HSV infection. Proper nuclear features and inclusions 
must be identified for such diagnosis. Virus-infected cells or 
virocytes should be distinguished from other giant cells such 
as trophoblasts, foreign-body giant cells following extraneous 
intervention with foreign bodies or surgery, nonspecific giant 
Fig. 7.42 “Early” herpes genitalis infection. Vaginopancervical smear 
(Papanicolaou × HP).
Fig. 7.43 Herpes genitalis infection. Vaginopancervical smear 
(Papanicolaou × HP).
cells seen in postmenopausal smears, and reactive multinucle-
ated cells found in cases of cervicitis.
Some workers have described morphologic differences that 
may distinguish between primary and recurrent herpes. Paucity 
of intranuclear inclusions and occurrence of chromatin homo-
geneity and “ground-glass” changes were often reported in 
primary herpes, whereas inclusions were predominant among 
cases with post primary infection. The studies of Vesterinen and 
associates63 and other workers have not confirmed this obser-
vation, although the World Health Organization has adapted 
these observations differentiating primary and recurrent herpes. 
Morphologically, HSV 2 and HSV 1 cannot be differentiated. 
Either such diagnosis can be rendered only by appropriate sero-
logic reactions or viral culture studies (Fig. 7.45).
Cytomegalovirus
These large (1800–2000 Å) DNA viruses belong to the herpes 
group that includes HSV 1, HSV 2, varicella-zoster, and EB virus. 
CMV is ubiquitous and circulates commonly in the general 
population. As with herpes viruses, CMV establishes itself in the 
host and causes persistent infection and recurrent disease. In the 
genital tract, reinfection may occasionally occur. Unlike other 
Fig. 7.44 Bizarre columnar cells in a smear with herpes genitalis infection. 
LBGS (Papanicolaou × HP).
Fig. 7.45 Herpes genitalis. Note both intranuclear inclusions and “ground-
glass” nuclei in the same group of cells. LBGS (Papanicolaou × MP).
109
110
Diagnostic CytologyPART TWO
herpes viruses, however, clinically overt manifestations of viral 
replication are seen only rarely. The infection spreads by inti-
mate contact through body secretions, including saliva, tears, 
urine, endocervical mucus, semen, and transplanted organs.
Nearly 50–60% of adult women have circulating antibod-
ies to CMV. Serologic evidence of infection is more common in 
low socioeconomic groups. Cervical shedding of CMV occurs 
in nearly 10% of the female population.21 These figures vary in 
different populations; e.g. the recovery rate for CMV in cervical 
specimens has been reported to be 28% in Japan. In a number 
of studies summarized by Kumar and co-workers,68 the inci-
dence of primary genital CMV infection has been reported as 
between 0.2 and 2.2%. Most cases of primary genital CMV infec-
tion are clinically asymptomatic. Stagno and colleagues reported 
symptomatic episodes in only 1 of 21 cases,68a and Griffiths and 
associates69 in only 1 of 14 pregnant women with primary CMV 
infection. Almost always these symptoms are infectious mono-
nucleosis-like and include lethargy, malaise, and fever.
Characteristically, epithelial tissues, including salivary gland, 
alimentary tract, bronchial and alveolar lining cells, hepatocytes, 
renal tubule cells, hematopoietic cells, and endocervical and 
endothelial cells, are targeted by CMV infection. Cytologically, 
the infected cells, endocervical columnar, perhaps, occur more 
commonly in the cervical smears than is recognized. According 
to Naib,70 the CMV-bearing cells occur within the endocervical 
glands, and not many cells may be observed in the epithelium 
of the endocervical canal. Proper cellular specimens, as can be 
obtainable with a Cytobrush or similar technique, may be more 
rewarding. In addition, because almost all women are asymp-
tomatic, very little effort may be made to screen these smears 
critically for CMV-associated changes. In a certain number of 
patients, concomitant CMV and HSV infections may occur. It is 
not infrequent to overlook the not-so-obvious CMV-infected cells 
in such cases. At times, the morphologic identification of CMV 
and its differentiation from HSV may be extremely difficult.The CMV-infected cells may be multinucleated and some-
what enlarged. The nuclear degenerative changes may be simi-
lar to those in HSV. We have observed that the internuclear 
 molding tends to be less obvious in CMV-infected cells than in 
those infected with HSV. Infected endocervical cells are aniso-
cytotic. They contain round intranuclear inclusions, generally 
A
Fig. 7.46 (A) Cytomegalovirus cervix. Note the chromatin threats. (B) Intracyt
acidophilic, which are disproportionately large when compared 
with the total size of the nuclei. These inclusions have a clear 
zone of halo around them, and frequently threads of chromatin 
material may stretch between the inclusion and the inner leaf of 
the nuclear membrane, which may be considerably thickened 
with the chromatin material in apposition against it, giving a 
wheel spoke appearance. The infected cells may have an intra-
cytoplasmic, irregular inclusion (Figs 7.46 and 7.47).
Infected endocervical cells may be buried among inflamma-
tory or other epithelial cells and may be hard to screen for in 
a routine fashion. In selected cases, CMV-specific monoclonal 
antibodies and in situ techniques can be used to establish the 
presence of CMV-infected cells.
Herpes zoster
Varicella-zoster is related to varicella (chickenpox) that is so 
common in childhood and infancy. Herpes zoster (shingles) 
infection may involve the vulva and vagina. Lesions often appear 
in patients who were exposed to the varicella virus in childhood. 
They occur in older individuals along the distribution of sensory 
nerves as extremely painful vesiculae or blisters, and tend to be 
unilateral.
B
oplasmic inclusions, LBGS (Papanicolaou × OI).
Fig. 7.47 Cytomegalovirus cervicitis (H&E × MP).
Microbiology, inflammation, and Viral infections
7
In humans, using molecular hybridization restriction enzyme 
analyses and polymerase chain reaction (PCR), greater than 100 
distinct HPV types have been identified. These different HPV 
types tend to be site-specific. Approximately 40 different HPV 
types affect the anogenital tract. HPV types are generally sepa-
rated into two major groups, low and high risk, depending on 
their risk for the development of cervical cancer. Low-risk types 
of HPV (most commonly types 6 and 11) have essentially no risk 
association for invasive cervical carcinoma, but tend to cause 
condyloma acuminatum, flat condyloma, and some low-grade 
squamous dysplasia (cervical intraepithelial neoplasia [CIN] 
I).71 The high-risk or “oncogenic” HPV types (16, 18, 31, 33, 35, 
39, 45, 51, 52, 56, 58, 59, 66, 68, 73, and 82) are often found in 
high-grade cervical dysplasia (CIN II and III), cervical carcinoma 
in situ, and invasive squamous cell carcinoma and adenocar-
cinoma and its precursors. High-risk HPV types are also com-
monly seen in CIN I lesions72a, 72b (Table 7.7).
HPV replicates in the nuclei of squamous epithelial cells. In 
the superficial squamous epithelium, the virus reaches full matu-
rity as a permissive infection and undergoes koilocytic changes 
Papillomavirus and Cancer The virus is believed to enter the 
body through small, inconspicuous cuts or abrasions on the skin 
or mucous membrane. It stimulates the growth of the prickle 
cell layer. The growth is by clonal expansion and, as suggested 
by Broker and Butcher,74 it pushes aside the normal epithelium 
to form benign warts. In these low-grade processes virus replica-
tion occurs episomally in the nucleus.
In addition to the numerous benign, self-limiting warty 
growths, papillomaviruses are associated with a number of 
 neoplasms occurring in animals, such as rabbits and cattle. The 
most convincing evidence for the association of HPV with cer-
vical cancer comes from the studies by Bosch et al.75 and Wal-
boomers et al.,76 who elegantly demonstrated the presence of 
HPV DNA in 99.7% of cervical cancers (932 cervical cancer cases 
from 32 hospitals in 22 countries). HPV DNA and RNA has also 
been demonstrated in at least 80% of all cervical, vulvar, and 
penile squamous cell carcinomas, in a similar proportion of pre-
malignant CIN lesions, and in 95% of genital condylomas.77,78 
HPV types 6 and 11 predominate in benign warty lesions, 
whereas types 16 and 18 occur in 60–70% of all cervical tumors 
A smear from the base of the lesion often reveals numer-
ous multinucleated giant cells with little intercellular mold-
ing. Numerous infected single cells may occur. Intranuclear 
inclusions may be basophilic, large, and inseparable from the 
markedly thickened inner nuclear membrane. Infected paraba-
sal-type cells may show some cytoplasmic degenerative changes. 
Intracytoplasmic vacuolation and hyalinization may be present 
(Fig. 7.48).
Human Papillomavirus (HPV)
HPVs belong to the family Papovaviridae, which includes dou-
ble-stranded DNA members, papillomaviruses, and polyoma-
viruses. The papillomavirus genome is approximately 8000 base 
pairs in length. It has three functioning areas, including genes 
for early viral function, the late region containing genes for viral 
structural proteins, and a noncoding regulatory region. The viral 
capsid has two proteins and polypeptides. Papillomavirus has 
been isolated from more than 60 animal species, including 
mammals, reptiles, and amphibians. The vast majorities of these 
viruses infect epithelial surfaces of either the skin or mucosa 
and cause self-limiting warty growth. The papillomaviruses are 
 species-specific and generally do not cross-infect.
A
Fig. 7.48 Herpes zoster. (A) Note the basophilic intranuclear inclusion. (B) High
smear (Papanicolaou (A) × MP, (B) × OI).
with HPV virions filling the nucleus and upon cell death, capa-
ble of reinfecting other cells. In the basal layers of squamous 
and glandular epithelium, the virus is latent and its DNA can 
only be detected by molecular techniques.73 In many low-grade 
precancerous lesions, HPV DNA is usually retained intact in an 
episomal form. In contrast, in invasive cancers and some high-
grade precancers, viral DNA is integrated into the host DNA, 
and retains only genes associated with oncogenesis.73
B
er power showing the texture and details of the inclusions. Vaginopancervical 
Table 7.7 Common human papillomavirus lesions (over 100 types)
HPV type Lesion
1, 2, 3, 4, 10, 28 Common warts
6, 11, 31, 42 Anogenital condyloma, low-risk 
(CIN grade I) lesions
16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 
58, 59, 66, 68, 73, 82 
 
High-risk (CIN grade II and III) 
lesions—anogenital, laryngeal, 
esophageal, lung cancer 
(possibly)
26, 27 Warts, immune deficiency, renal 
transplant
111
112
Diagnostic CytologyPART TWO
with cervical cancer has resulted in the development of vaccines 
directed against the most common oncogenic HPV types 16 and 
18.108 These vaccines have been shown to prevent infection and 
the development of precancerous lesions due to these subtypes, 
and one has been FDA approved for use in girls aged 9–26.
Historical Perspective The typical cytomorphologic changes 
now associated with HPV infection were first documented by 
Ayre in 1949.85 Papanicolaou, in his Atlas of Exfoliative Cytology 
published in 1954, presented magnificent illustrations depict-
ing cellular changes currently recognized as HPV-associated.86 
Koss and Durfee in 1956 used the term koilocytic atypia to 
describe the surface epithelial changes of the cervix and its rela-
tionship with cancer.87 Naib and Masukawa in 1961 published a 
paper entitled “Identification of Condyloma Acuminata Cells in 
Routine Vaginal Smears.”88 Additional papers on the same topic 
were published by Ayre,89 Sagiroglu,90 and De Girolami.91 Ayre 
for the first time suggested a possible viral etiology of cervical 
dysplastic lesions.89 In the mid-1970s, Meisels and Fortin, and 
Purola and Savia independently defined a set of