Significance of a Diagnosis of
Microorganisms on Pap Smear
Valerie A. Fitzhugh, MD and Debra S. Heller, MD
Department of Pathology and Laboratory Medicine, New Jersey Medical School,
University of Medicine and Dentistry of New Jersey, Newark, NJ
h Abstract: The Pap smear has been in use for more than
half a century as the primary screening test for preinvasive
and invasive lesions of the uterine cervix. Although not
the primary use and an imperfect test, it can be extremely
useful in the diagnosis of some microorganisms. This
review focuses on the use of the Pap smear in the
diagnosis of several microorganisms including Actino-
myces, Chlamydia trachomatis, Candida, Trichomonas
vaginalis, Leptothrix vaginalis, Herpes Simplex Virus, the
causative agents of bacterial vaginosis, and other rarer
organisms. The accuracy of diagnosis using the smear
varies among the different organisms in question. h
Key Words: vaginal smears, vaginitis, cytology
T he Pap smear has been in use for more than half a
century to screen women for the precursors of
cancer of the uterine cervix. Although not the primary
goal and not mandatory in current Bethesda System
terminology, it is common practice to make the
diagnosis of organisms seen on the Pap smear. Is the
Pap smear truly an accurate enough test for the diagnosis
of various microorganisms? The purpose of this article is
to review the literature in regards to microorganisms
seen on the Pap smear and see if the test is sensitive and
specific in the diagnosis of microorganisms.
Actinomyces
Actinomyces species are gram-positive, nonYacid-fast,
obligate anaerobic bacteria that demonstrate branching,
filamentous growth. They are normal inhabitants of the
Reprint requests to: Debra S. Heller, MD, Department of Pathology and
Laboratory Medicine, New Jersey Medical School, University of Medicine
and Dentistry of New Jersey, UH/E158, 185 South Orange Avenue, Newark,
NJ 07101. E-mail: hellerds@umdnj.edu
Ó 2007, American Society for Colposcopy and Cervical Pathology
Journal of Lower Genital Tract Disease, Volume 12, Number 1, 2008, 40Y51
Copyright @ 2007 American Society for Colposcopy and Cervical Pathology. Unauthorized reproduction of this article is prohibited.
oropharynx as well as the bowel. Actinomyces israelii is
the most common pathogen in human infections [1].
Other members of the genus, such as Actinomyces
naeslundii, Actinomyces viscosus, Actinomyces neuii,
and Actinomyces eriksonii, cause similar infections in
the clinical setting [1].
If patients do not have mucosal injury, Actinomyces
cannot cross mucosal barriers. In a low-oxygen environ-
ment where mucosal injury has occurred, actinomycosis
may result [1]. This genus grows grossly as colonies that
breach anatomical boundaries, forming abscesses sur-
rounded by dense fibrous tissue and containing many
neutrophils. The exudate frequently contains yellow/
brown sulfur granules that contain Actinomyces colo-
nies, calcium phosphate, and tissue debris [1].
Actinomyces has been recognized for many years in
the cervicovaginal canal of women who used intrauter-
ine devices (IUDs) for periods longer than 1 year [1,
2Y13]. Infection with Actinomyces has also been seen in
women who have no history of IUD use, enhancing the
need for familiarity with this organism on cervicovaginal
smears [7, 14, 15]. Forgotten tampons and pessaries are
also a fertile ground for Actinomyces growth [3, 4].
Ascending Actinomyces infection is thought to derive
from the perineum or from orogenital or anogenital
contact [1, 8].
One of the earliest reports of Actinomyces in the
cervicovaginal canal associated with IUD usage was in
1976 by Gupta et al. [2]. This group investigated the
appearance of these organisms in 3 of 13 patients who
were IUD users. In the Pap-prepared slides, the
Actinomyces organisms were most abundant in the cer-
vical portion of the smear. Under low power, the
organisms formed small islands of amorphous material
[2]. The organisms stained grayish blue to black,
depending on the hematoxylin stain used. According to

Figure 1. Actinomyces: A mass of tangled organisms is the
characteristic finding on Pap smear.
Gupta et al. [2], sulfur granules are infrequent, and other
bacterial infections are associated with Actinomyces
infection. At high power, the filaments appear dark blue,
black, or golden brown. In the center of the masses, the
filaments lie in a haphazard fashion, branching at acute
angles. The tips of most filaments are blunted slightly
and clubbed [2] (Figure 1).
This early work by Gupta et al. [2] spurred a series of
studies on the presence of Actinomyces in cervicovaginal
smears, but the question remains, is the Pap smear
sensitive and specific for finding these organisms? Many
of the articles that followed Gupta et al. [2] claimed that
Actinomyces-like organisms could be easily identified on
Pap-prepared smears [1, 3Y13], with high sensitivity and
specificity [1, 3, 4, 8, 11Y13]. Although no mathematical
calculations were demonstrated in any of these articles,
it appeared to the authors that Actinomyces did not cause
a diagnostic dilemma. Many of the published studies
confirmed their findings via use of culture techniques
or immunofluorescent antibodies [1, 3, 5, 6, 8, 12] but
maintained not only that the diagnosis could be made on
Pap smear but also that treatment decisions could be
made based on the presented evidence.
It appears based on the literature that Actinomyces
can be identified and diagnosed confidently on Pap-
prepared slides with high sensitivity and specificity.
Chlamydia trachomatis
Chlamydia trachomatis is an obligate intracellular
bacterium that infects and multiplies within epithelial
Copyright @ 2007 American Society for Colposcopy and Cervical Pathology. Unauthorized reproduction of this article is prohibited.
Microorganisms on Pap Smear & 41
cells and macrophages. Chlamydiae cause prolonged
infections in their host. Neutrophils are involved in the
early host response to infection, but lymphocytes, mono-
cytes, and macrophages eventually predominate [16].
Chlamydiae have 2 morphological forms, known as
elementary and reticulate bodies. These forms represent
the 2 stages of the chlamydial life cycle. Elementary
bodies are the immature form, which mature into
reticulate bodies. Once maturation is complete, the
reticulate bodies again differentiate into elementary
bodies, which are released from the host cell, and the
cycle begins anew [16].
One of the earliest articles written on the subject of
diagnosing C. trachomatis on Pap-prepared slides was
authored by Gupta et al. [17], stating that Chlamydia-
infected cells were most commonly seen in the cervical
component of a cervicovaginal smear, although cells
were occasionally seen in the vaginal component. They
defined 3 stages of infection. In the first stage, these
‘‘atypical’’ cells occurred either singly or in groups of 2 to
6 cells with distinct crisp outlines. The cytoplasm was
cyanophilic and rarely acidophilic. The changes were
usually focal and localized to the perinuclear region.
Within these areas were finely granular, uniformly sized
objects termed coccoid bodies.
In the second stage of infection, the cytoplasm was
textured and finely vacuolated. The organisms had
assumed a perinuclear location and were eosinophilic or
basophilic. Some inclusion bodies could be seen among
the coccoid bodies [17].
In the third stage of infection, multiple inclusion
bodies occurred randomly or in the perinuclear areas of
multinucleated cells. The intracytoplasmic inclusions
were uniform, frequently molding and overlapping, and
had a zone of clearing around them. Columnar cells
were most commonly affected, with occasional involve-
ment of metaplastic cells. The findings in this article
were confirmed with immunofluorescence, and the
authors concluded that Pap-prepared smears were
indeed useful to make a preliminary diagnosis of
Chlamydia infection [18].
Shortly after this article was published, other authors
attempted to, but could not, replicate the findings of
Gupta et al. Some authors stated that they could not
confidently use these criteria in the diagnosis of
Chlamydia within cervical smears [18, 19]. In 1985,
Kiviat et al. [20] attempted to expand on diagnostic
criteria put forth by Gupta et al. In addition to looking
for the vacuoles previously described (Figure 2), they
attempted to tie those findings to the amount of
 42 & FITZHUGH AND HELLER
Figure 2. Changes suggestive of Chlamydia: Intracytoplasmic
vacuoles with inclusions are seen. This should be followed with
confirmatory testing.
inflammation in the smear. They also attached signifi-
cance to the transformed lymphocytes [20]. They
admitted that light microscopic diagnosis of Chlamydia
infection was insensitive and nonspecific and only with
the addition of culture and immunofluorescence were
sensitivity and specificity increased [20].
Shiina [21] concluded that ancillary studies were
needed to make an accurate diagnosis of C. trachomatis
infection. Shiina’s important contribution was the
description of the nebular body, or nebular inclusion,
which was classified as types II to V according to the size
of the inclusions, the presence of a membranous
structure around the inclusions, and the release of
particles into the extracellular space [21]. Some authors
have argued that the finding of these nebular inclusions
is the most reliable indicator of Chlamydia infection on
Pap smear [22].
Many authors have attempted to determine if the Pap
smear is sensitive and specific enough to be used for
daily use in diagnosing Chlamydia infection. Measures
of sensitivity ranged from 0% to 63%, with most studies
falling in the range of 10% to 20% [21, 22], an abysmal
value for a screening test. Measures of specificity over a
series of studies range from 20% to 93% [22] and
averaged 79% over a series of studies according to
Bernal et al. [23]. Findings such as these have lead many
authors to conclude that the Pap smear is not a reliable
method of diagnosing C. trachomatis infection and that
ancillary studies should be used in these cases [18Y20,
22Y28].
Some authors have attempted to modify the Pap
procedure to increase the sensitivity and specificity of
Copyright @ 2007 American Society for Colposcopy and Cervical Pathology. Unauthorized reproduction of this article is prohibited.
the smear in the diagnosis of Chlamydia infection. Vela
et al. [29] described a modification to the Pap stain
with a buffered Wright solution. They claimed that they
were able to make more diagnoses of infection on Pap
smear with the modified stain than with the regular
Pap stain. No further work appears to have been done
on this area, however.
The idea of the Pap smear as a diagnostic modality for
Chlamydia infection has fallen by the wayside, in no
small part due to the lack of sensitivity and specificity of
the method. For many years, culture was, and still
remains, the criterion standard for diagnosis, with a
sensitivity as low as 50% and specificity reported to be
100% [30]. However, culture is time-consuming, and
molecular diagnostics, more specifically, polymerase
chain reaction (PCR), have become the leading means
of diagnosing infection by C. trachomatis.
Herpes Simplex Virus
Herpes simplex virus (HSV) is a member of the
herpesvirus family and consists of 2 types, HSV-1
and HSV-2. These viruses are similar but differ by
their biologic properties [16]. Herpes simplex virus
organisms multiply in parabasal and intermediate
epithelial cells at their site of entry into the body. As
the viruses multiply, infected cells fuse with adjacent
cells to form syncytia. However, viral replication
causes cell lysis [16]. Immunocompetent patients are
able to clear primary infection with a vigorous
cytotoxic T-lymphocyte response. Immunocompro-
mised patients, on the other hand, may suffer from
disseminated disease.
Clinically, these viruses infect only humans, and
patients are infected by direct contact with infected
secretions. Herpes simplex virus 1 is most commonly the
cause of gingivostomatitis, which results from contact
with oral secretions [16]. Herpes simplex virus 2 is most
commonly spread via sexual contact, so most infections
occur after puberty. This is problematic in women
because women with genital HSV infection can transmit
the virus to their offspring. The infant’s chance of
becoming infected is 3% to 4% if the mother’s disease
is recurrent and increases to 30% to 40% if the mother
is undergoing primary infection. Vaginal delivery is
contraindicated in these patients if the infection is
active [16].
The principal site of HSV infection in female patients
is the cervix. Primary infection involves the cervix in
more than 80% of cases. Both the endocervix and
ectocervix may be involved [31].

In 1966, Naib et al. [32] described the morphology of
HSV infection on cytology. The characteristic changes
include homogenization of the nuclear contents, mar-
gination of chromatin leading to a ground-glass appear-
ance, multinucleation leading to the formation of giant
cells, presence of intranuclear eosinophilic inclusions,
and molding of the nuclei (Figure 3).
Few studies have attempted to answer the question of
whether the Pap smear is sensitive and specific enough to
be used reliably as a diagnostic tool in HSV infection, as
diagnosis of HSV has moved away from glass slides and
into the molecular era. A study performed by Brown
et al. [33] in 1979 tested the sensitivity and specificity of
the Pap smear against immunofluorescence and crystal
violet staining. The Pap smear was highly specific, but
the sensitivity was less than 40% [33], again poor when
considering a screening test. Kapur et al. [34] compared
the results of direct immunofluorescence with Pap-
prepared smears. Thirty smears were examined, and 17
of those smears were positive for HSV antibodies on
immunofluorescence. Of the 17 positives, 9 had minimal
to no cytological atypia and 8 others were diagnostic.
Again, the sensitivity was low in the conventional
cytology smears [34].
Another study, performed by Puthavathana et al. [35]
in 1998 moved into a more modern era, evaluating the
Pap stain, immunoperoxidase staining, and PCR for
Figure 3. Herpes: Multinucleation with nuclear molding and
ground-glass nuclei are seen.
Copyright @ 2007 American Society for Colposcopy and Cervical Pathology. Unauthorized reproduction of this article is prohibited.
Microorganisms on Pap Smear & 43
sensitivity and specificity. Ninety-six cases of suspected
HSV were assessed. The Pap stain was the most poorly
performing, with a sensitivity of 62.7% and a specificity
of 81.0% [35]. The concern in this study was that if the
Pap smear is used as the primary screening study, then
cases would be missed secondary to the low sensitivity.
Furthermore, there are mimics of herpesvirus that can
cause confusion on cytological specimens. Stowell et al.
[36] published a study in 1994 where they found that
endocervical cell artifacts secondary to endocervical
brushing can result in a false-positive diagnosis of HSV
infection. They also studied the diagnostic criteria of
HSV infection in cases that were correctly diagnosed. Of
the criteria studied, only ground-glass chromatin had
high sensitivity (95%) and high specificity (95%).
Intranuclear inclusions, a finding supposedly pathogno-
monic for HSV infection, had low sensitivity (42%) [36],
showing that a combination of findings is important
when attempting to diagnose HSV infection on cytology.
In summary, cytology has some value in diagnosing HSV
infection, but ancillary studies should be considered.
Candida
Candida albicans is the most common member of the
genus seen in cervicovaginal smears and causes most of
vaginal infections [37]. The organism is an opaque white
to tan fungus that, in the tissues, may assume a mixture
of pseudohyphae, true hyphae, and spores. It is usually
found in small numbers on mucosal surfaces and has the
ability to invade the mucosa and multiply when the
opportunity presents itself [16].
There are multiple conditions that favor the devel-
opment of candidiasis, including diabetes, pregnancy,
and immunocompromise [16]. The organisms can
infect the vulva, vagina, or cervix. Patients may be
asymptomatic or they may present with genital burn-
ing, itching, and a thick white discharge. It is believed
that the profound immune response that accompanies
candidiasis is largely responsible for the clinical
manifestations [16].
On the Pap smear, the organisms tend to be pink.
Long pseudohyphae are commonly seen, and at times,
the pseudohyphae appear to be skewering the surround-
ing squamous cells. These structures have been described
as ‘‘shish kebabs’’ [37] (Figure 4A). Spores may also be
seen, and clusters of small spores are consistent with
Torulopsis glabrata (Figure 4B).
Very few authors have examined the sensitivity and
specificity of the Pap smear in regards to infection by
Candida. In 2001, Audisio et al. [38] attempted to
 44 & FITZHUGH AND HELLER

Figure 4. A, Candida: Pseudohyphae and yeast forms are present, and the squamous cells are clustered with the organisms, giving a
‘‘shish-kebab’’ appearance. B, Torulopsis glabrata: Clusters of small yeast forms are seen overlying squamous cells.

validate the Pap smear in the diagnosis of Candida transmission of HIV in the presence of trichomoniasis,
species. They screened 824 smears and compared the which is thought to be secondary to local inflammation
results against Gram stain of the vaginal exudate. Of the and disturbance of the cervical epithelium [40]. Tricho-
824 cases, 22% were positive for organisms consistent moniasis has also been linked to premature rupture of
with Candida species. They found the sensitivity of the the membranes in pregnant women, preterm birth,
Pap smear to be 31% and the specificity to be 98.5%. cervical intraepithelial neoplasia, pelvic inflammatory
They concluded that the low sensitivity of the Pap smear disease, and infertility [40].
renders it inadequate as a screening test. The organism is seen on the Pap smear as a pear-
Another recent study by Takei et al. [39] compared shaped organism with a pale, eccentrically placed
the effectiveness of liquid-based preparations to con- nucleus (Figure 5). Well-preserved trichomonads often
ventional pap smears. Candida was one of the organisms
examined. They found that the liquid-based preparation
was more sensitive and specific for the identification of
these fungal organisms, which they attributed to the
large size of the organism in comparison to other
organisms such as bacteria and Trichomonas vaginalis.
They felt that the large organism may not be eliminated
in the liquid preparation processing, leading to higher
numbers of the organisms on the smear.

Trichomonas vaginalis
Trichomonas vaginalis is the largest of the trichomonads
and is the only one known to infect humans [16]. The
trophozoite is the form seen in the tissues. They range
from 5 to 29 Km in length. Each has 4 flagella that
project anteriorly from the organism, which contains a
single nucleus. A rod called the axostyle transects the
organism and extends outward posteriorly [16].
Clinically, trichomoniasis is one of the most common
sexually transmitted diseases in the United States, with 8
million new cases diagnosed yearly. The most common Figure 5. Trichomonas vaginalis: The organisms are small and
pear shaped, with an elongated nucleus. The flagellum is not
manifestation in infected women is symptomatic vagi-
seen as in a wet smear. Two organisms are seen here near a
nitis [40]. The disease does not appear to cause disease in squamous cell. Note the small perinuclear halo in a neighboring
men. There is a known 2-fold increase in the risk of cell (pseudokoilocyte).

Copyright @ 2007 American Society for Colposcopy and Cervical Pathology. Unauthorized reproduction of this article is prohibited.

contain red cytoplasmic granules. An associated finding
is the presence of squamous cells with a small, narrow,
perinuclear halo that is distinct from, and should not be
confused with, the large halo seen in human papilloma-
virus infection [37].
There are multiple studies regarding the sensitivity
and specificity of the Pap smear in diagnosing
T. vaginalis infection. An early study was performed
comparing the conventional smear to other methods in
1988 by Thomason et al. [41]. The Pap smear was the
least sensitive of all the methods tested. Trichomonas
is much more difficult to recognize when high numbers
of leukocytes are present, a problem more common to
conventional smears than in liquid-based preparations.
This can lead to false-positive diagnoses, especially in
places where Trichomonas is prevalent [41]. A more
recent study by Aslan et al. [42] showed that not only
was the conventional smear an inaccurate test for the
diagnosis of trichomonads but that liquid-based pre-
parations are far more sensitive and specific. Lara-
Torre and Pinkerton [43] also assessed the adequacy of
the liquid-based Pap stain. They found that the liquid-
based Pap smear had a sensitivity of 61.4% and a
specificity of 99.4%. They concluded that the presence
of T. vaginalis on the liquid-based preparation is
accurate and that treatment may commence without
further testing.
A meta-analysis of the Pap smear in the diagnosis of
vaginal trichomoniasis was performed by Wiese et al.
[40] in 2000. The purpose of their study was to
obtain a reliable estimate of the sensitivity and
specificity of the Pap smear as well as the wet mount.
They pooled a series of articles written between 1976
and 1998 that examined the test characteristics of the
Pap smear. The sensitivity of the articles included was
57%, whereas the specificity was 97%. They con-
cluded that a Pap smear containing trichomonads is
diagnostic among patients from high prevalence set-
tings but is indeterminate in medium- to low-prevalence
settings [40].
The conventional smear does not appear to be a
useful tool in the diagnosis of T. vaginalis. However,
liquid-based cytology appears to be more reliable.
Investigation on the use of PCR to confirm the diagnosis
made on Pap smear is ongoing and may become more
useful as tests continue to be developed.
Bacterial Vaginosis
Bacterial vaginosis is caused by a shift in vaginal flora
leading to overgrowth of a mixed flora that includes
Copyright @ 2007 American Society for Colposcopy and Cervical Pathology. Unauthorized reproduction of this article is prohibited.
Microorganisms on Pap Smear & 45
Gardnerella vaginalis. The vaginal flora shifts from the
dominant aerobic Lactobacillus to predominantly anae-
robic organisms including G. vaginalis, Bacteroides
species, Mobiluncus species, and Mycoplasma hominis
[44, 45]. Under normal circumstances, lactobacilli
produce an acidic medium in the vagina by the
production of hydrogen peroxide, which transforms
glycogen to lactic acid. This lactic acid suppresses the
growth of other organisms. However, if the balance is
tipped away from the lactobacilli, they are replaced by
anaerobes [44].
Clinically, patients often present with a clinical
picture of itching, pain, and vaginal discharge or odor.
The criterion standard for the clinical diagnosis was first
described by Amsel et al. [46] and has been repeated in
numerous works. Three of the 4 objective signs must be
present: (1) a milky homogenous vaginal discharge, (2) a
vaginal pH higher than 4.5, (3) a fishy odor with the
addition of KOH (whiff test), and (4) clue cells on a
saline wet mount [44, 47, 48]. Bacterial vaginosis has
been implicated in preterm labor, premature rupture of
the membranes, chorioamnionitis, puerperal endome-
tritis, pelvic inflammatory disease, postoperative vaginal
cuff cellulitis, and urinary tract infection [48]. The
classic finding for bacterial vaginosis on the Pap stain is
the presence of clue cells (Figure 6). Clue cells are
squamous cells that are completely covered by cocco-
bacilli. Interestingly, neutrophils are not abundant in
these specimens.
Figure 6. Bacterial vaginosis: A clue cell, a squamous cell coated
with bacteria, is seen.
 46 & FITZHUGH AND HELLER
The Pap smear has been compared with various
microbiological methods of diagnosing bacterial vagi-
nosis. Multiple studies compared the Gram stain to the
Pap stain for diagnostic accuracy. The Pap smear was
shown to be a sensitive test, with values ranging from
55% to 93%. The Pap smear was also specific, with a
range of 94% to 98%. Each of the studies concluded
that the Pap smear would give accurate diagnostic
results [44, 45, 48]. A large study was undertaken by
Giacomini et al. [47] in 1997. They examined 1,896
smears, of which 166 were positive for bacterial
vaginosis. In this study, the sensitivity of the Pap smear
was found to be 88.7%. Furthermore, the specificity was
98.8%. They were able to conclude that the Pap smear is
a valid test for the diagnosis of bacterial vaginosis and
suggest that the smear be used to screen for the disease in
appropriate patients.
The Bethesda system recommends that these cases be
signed out in cytology reports as a shift in vaginal flora
suggestive of bacterial vaginosis [49]. This shift in and of
itself is not sufficient for the clinical diagnosis of
bacterial vaginosis because specimens obtained from
any single site are not necessarily representative of the
entire flora of the cervix and vagina. However, in the
proper clinical setting, the Gram stain can be used as an
adjuvant to assist in the diagnosis [49]. It appears then
that the Pap smear can be used to support a diagnosis of
bacterial vaginosis based on the literature at this time.
Microbiological testing can be used in conjunction with
the Pap test to fulfill clinical criteria.
Leptothrix vaginalis
Leptothrix vaginalis is a Gram-positive anaerobic rod
that ranges from 45 to 70 Km in length [50]. The
organisms are filamentous and segmented but non-
branching. These organisms are in the same family as
lactobacilli but are morphologically different. In some
women, these organisms cause the symptoms of bacte-
rial vaginosis, specifically itching, pain, and vaginal
discharge or odor [50].
On the Pap smear, these organisms are segmented,
long, and filamentous (Figure 7). They tend to take on
a blue hue. Of diagnostic importance, the appearance
of Leptothrix is associated in many cases with
T. vaginalis [37].
To date, very few articles have been published
regarding this organism. Furthermore, of those pub-
lished, none have performed an assessment of the
sensitivity or specificity of the Pap smear. The evaluation
of these parameters remains to be seen.
Copyright @ 2007 American Society for Colposcopy and Cervical Pathology. Unauthorized reproduction of this article is prohibited.
RARE ORGANISMS
Cytomegalovirus
Cytomegalovirus (CMV) is also a member of the
herpesvirus family. This virus is known to cause a number
of clinical syndromes in both children and adults.
Exposure to and infection by CMV is a rather frequent
event, although actual clinical manifestations of the
disease are far less frequent [2]. Because there is so much
genomic and phenotypic variability, numerous strains of
CMV exist. Antigenic variations have also been observed
but are not clinically important [16]. The incidence of
serum antibodies to CMV has been reported to be at least
40% in many populations [16]. There are 2 age peaks in
which infection is most common. The first peak occurs in
infancy, and the second is in early adulthood, coinciding
with the increase of infectious mononucleosis in this
population [16]. The disease is now seen more frequently
in immunocompromised populations [16]. Clinically,
some infants acquire the disease in utero, resulting in
congenital CMV syndrome. Defects include hepatosple-
nomegaly, jaundice, anemia, thrombocytopenia, low birth
weight, microcephaly, intracerebral calcifications, and
chorioretinitis [16]. Perinatal infections can occur more
rarely if the virus is in the cervix, because the child
acquires the virus while traveling through the birth canal
[16]. Approximately one third of primary maternal
infections are transmitted to the fetus, and most are
within the first trimester [16]. In healthy young adults, on
the other hand, CMV infection can cause an infectious
mononucleosis-like syndrome. In immunocompromised
patients, the infection can become disseminated, leading
to death in some cases [16].
Figure 7. Leptothrix: These thin filamentous organisms are
usually abundant when seen and thinner than candidal hyphae.

Most of the literature of cervical infection by CMV is
in the form of case reports. The virus is described
microscopically as producing nuclear inclusions, also
known as owl’s-eye cells, which are large, dark cells
surrounded by clear haloes and chromatin marginated
peripherally around the nuclear envelope [51] (Figure 8).
The cells also tend to have ample cytoplasm with
multiple granular inclusions [51].
An early study that evaluated the presence of CMV on
Pap-stained smears was that of Morse and Gardner [52]
in 1974. In this study, only one slide showed a single
inclusion that was diagnostic of CMV. Clearly, there is
not enough power there to determine sensitivity or
specificity of the Pap smear in this diagnosis. In the
various case reports that followed [51, 53, 54], CMV
was detected on Pap smear and confirmed by immuno-
cytochemistry [53, 54] or real-time PCR [54]. Because
the number of cases in the literature is so small and
because the finding of CMV in the cervix is so rare, it is
impossible at this time to determine if the Pap smear is
sensitive or specific enough to routinely diagnose CMV
infection.
Enterobius vermicularis
Enterobius vermicularis is commonly known as pin-
worm. The adult female is more commonly seen and is
approximately 10 mm long [16]. It is cream colored and
has a sharply pointed tail, hence the name (Figure 9).
Figure 8. Cytomegalovirus: Characteristic large intranuclear
inclusions are seen in this specimen from a bronchoalveolar
lavage.
Copyright @ 2007 American Society for Colposcopy and Cervical Pathology. Unauthorized reproduction of this article is prohibited.
Microorganisms on Pap Smear & 47
Figure 9. Enterobius vermicularis: Cross-section of 2 adult pin-
worms in an appendix. Note the numerous ova in these gravid
parasites, oval on one side and flat on the other. The worms are
rarely seen on Pap smear. Image courtesy of Dr. Chitra
Pushpanathan, Janeway Children’s Health & Rehabilitation
Centre, St. John’s, Newfoundland and Labrador, Canada.
The worm has small ridges that run down its sides that
widen to alae. Adult worms lie attached to the cecum.
When the female worm is near the time when she needs
to lay her eggs, she travels down the colon, out the anal
canal, and lays her eggs on the perineum. She can lay up
to 20,000 eggs at one time [16]. The eggs become
infectious shortly after deposition and can cause
infection to others by handling infected bed sheets,
transfer from the hands (via scratching the pruritic area)
to the oral cavity, or inhalation of the eggs and
swallowing. This cycle takes approximately 2 weeks to
complete [16].
Clinically, the most common presenting symptom is
anal pruritus, secondary to the eggs that have been laid
there [16]. In female patients, the female worms can
travel up the vaginal canal, causing infection of the
vagina and cervix, and can even enter the uterus,
resulting in endometritis, and the fallopian tubes,
resulting in salpingitis. The female worms can also
enter the urethra, causing cystitis [16].
Pinworm infection of the vagina and cervix is a
thing of case reports. One of the first reports presented
was in 1969 by Langlinais [55], in which a smear
taken from an 18-year-old woman was submitted for
cytological examination. Numerous parasite eggs were
distributed throughout. Most of the eggs were
orangeophilic and contained mature larva. The refrac-
tile shell was composed of an outer triple layer and an
 48 & FITZHUGH AND HELLER
inner monolayer. A female worm was also identified
carrying 88 ova [55].
A second case report observed in 1970 by Rad and
Jeannot [56] was that of a 31-year-old woman who had
a cervical smear. The findings of this smear were nearly
identical to that of Langlinais [55]. Wong and Becker
[57] examined a series of 4 cases that occurred between
1971 and 1980. In addition to the finding of pinworm in
the 4 smears, 3 of the 4 cases showed extreme
inflammation, with mostly neutrophils and increased
mucus [57]. Of course, such a small number of cases are
not nearly enough to try to determine sensitivity and
specificity of the Pap smear when trying to diagnose
pinworm infection. However, it is prudent for the
cytologist to be wary of this diagnosis in that the
cervical examination may be the first clue to pinworm
infestation.
Schistosoma
Schistosoma is a genus of trematodes (flukes). Five
species infect humans, and only 3, Schistosoma man-
soni, Schistosoma hematobium, and Schistosoma japo-
nicum, are clinically important [16]. These organisms
are seen most commonly in Africa, the Middle East,
Southeast Asia, the Caribbean, and South America.
These organisms are responsible for approximately
200,000 deaths per annum [16]. The organisms have
Figure 10. Schistosoma hematobium: Note the terminal spine in
this organism found in a urine specimen.
Copyright @ 2007 American Society for Colposcopy and Cervical Pathology. Unauthorized reproduction of this article is prohibited.
a ventral sucker and, once in their blood vessel of
choice, lay eggs daily for a span of 4 to 35 years. The
organisms are differentiated from each other by their
shape and the presence and location of a spine [16]
(Figure 10).
Clinically, S. hematobium causes bladder disease and
can be isolated from the bladder by cytology or biopsy
[16]. S. mansoni and S. japonicum are more commonly
found in the bowel and are, therefore, diagnosed by
biopsy or stool samples [16]. In cases where the female
genital tract is involved, the vagina and cervix are most
frequently involved. The eggs can also be found in the
uterus, ovary, and fallopian tubes [58].
Very few reports of Schistosoma infection of the
cervix identified on Pap smear exist. The earliest case
report was produced by Youssef et al. [59] in 1962,
where they described the finding of Schistosoma
organisms in vaginal smears. The organisms are most
commonly S. hematobium. In a later case report,
produced by DeMille et al. [60] in 1995, a Liberian
woman presented with infertility. On cervical smear
stained by the Pap method, S. hematobium ova were
scattered among the numerous inflammatory cells. The
ova were in various stages of development. The bulk of
the ova identified contained miracidium showing an
eosinophilic cytoplasm, hematoxylin-stained granules,
and, most importantly, the pathognomonic terminal
spine. Occasionally empty egg shells were also present
[60], but one must be mindful of the fact that, to
document active infection, living organisms must be
present inside the eggs if only eggs are present. A unique
case was reported in 1998 by Mainguene et al. [61] in
which a European woman who had visited Senegal
9 years previously presented with S. hematobium on Pap
stain. Another unusual case was offered in 2005 by Jain
et al. [62]. A 69-year-old psychiatric patient had
pinworm and S. hematobium on the same smear.
Again, sensitivity and specificity of detection of
schistosomes on Pap smear has yet to be determined.
There simply are not enough cases in the literature or in
any one institution to make such a determination
possible. However, special care should be made when
examining the smears of women from endemic areas,
and caution should be taken in women who have
traveled to these endemic areas.
CONCLUSION
The Pap smear has been heralded as one of the greatest
screening tests of our time. However, no screening test is

completely accurate. When it comes to the assessment of
microorganisms, the sensitivity and specificity of the test
are widely variable. Although organisms should be
reported when identified, the less sensitive and specific
tests can be combined with a confirmatory test, such
as PCR, if necessary, to provide the best result for
the patient.
REFERENCES
1. Fiorino AS. Intrauterine contraceptive device-associated
actinomycotic abscess and Actinomyces detection on cervical
smear. Obstet Gynecol 1996;87:142Y9.
2. Gupta PK, Hollander DH, Frost JK. Actinomycetes in
cervico-vaginal smears: an association with IUD usage. Acta
Cytol 1976;20:295Y7.
3. Spence MR, Gupta PK, Frost JK, King TM. Cytologic
detection and clinical significance of Actinomyces israelii in
women using intrauterine contraceptive devices. Am J Obstet
Gynecol 1978;131:295Y8.
4. Jones MC, Buschmann BO, Dowling EA, Pollock HM.
The prevalence of Actinomycetes-like organisms found in
cervicovaginal smears of 300 IUD wearers. Acta Cytol
1979;23:282Y6.
5. Fry R, Linder AM, Bull MM. Actinomycete-like
organisms in cervicovaginal smears. S Afr Med J 1980;57:
1041Y3.
6. Traynor RM, Parratt D, Duguid HLD, Duncan I.
Isolation of actinomycetes from cervical specimens. J Clin
Pathol 1981;34:914Y6.
7. Petitti DB, Yamamoto D, Morgenstern N. Factors
associated with Actinomyces-like organisms on Papanicolaou
smear in users of intrauterine contraceptive devices. Am J
Obstet Gynecol 1983;145:338Y41.
8. Nayar M, Chandra M, Chitraratha K, Kumari Das S,
Rai Chowdhary G. Incidence of actinomyces infection in
women using intrauterine contraceptive devices. Acta Cytol
1985;29:111Y6.
9. Merki-Feld GS, Lebeda E, Hogg B, Keller PJ. The
incidence of Actinomycetes-like organisms in Papanicolaou
stained smears of copper- and levonorgestrel-releasing intrau-
terine devices. Contraception 2000;61:365Y8.
10. Demirezen S, Kaya D, Beksac S. Cytologic findings in
pap smears with Actinomyces-like organisms. Acta Cytol
2005;49:257Y61.
11. Luff RD, Gupta PK, Spence MR, Frost JK. Pelvic
actinomycosis and the intrauterine contraceptive device:
a cyto-histomorphologic study. Am J Clin Pathol 1978;69:
581Y6.
12. Bhagavan BS, Gupta PK. Genital actinomycosis and
intrauterine contraceptive devices: cytopathologic diagnosis
and clinical significance. Hum Pathol 1978;9:567Y78.
13. Hagar WD, Douglas B, Majmudar B, Naib ZM,
Williams OJ, Ramsey C, et al. Pelvic colonization with
Copyright @ 2007 American Society for Colposcopy and Cervical Pathology. Unauthorized reproduction of this article is prohibited.
Microorganisms on Pap Smear & 49
Actinomyces in women using intrauterine contraceptive
devices. Am J Obstet Gynecol 1979;135:680Y4.
14. Zakut H, Achiron R, Treschan O, Kutin E. Actino-
myces invasion of placenta as a possible cause of preterm
delivery. Clin Exp Obstet Gynecol 1987;14:89Y91.
15. Abadi MA, Abadi J. Actinomyces chorioamnionitis
and preterm labor in a twin pregnancy: a case report. Am J
Obstet Gynecol 1996;175:1391Y2.
16. Walker TS. Microbiology. Philadelphia: WB. Saunders,
1998: 198, 216Y22, 315Y7, 356Y61, 363Y4, 436Y7, 463Y7, 478Y9.
17. Gupta PK, Lee EF, Erozan YS, Frost JK, Geddes ST,
Donovan PA. Cytologic investigations in Chlamydia infection.
Acta Cytol 1979;23:315Y20.
18. Purola E, Paavonen J. Routine cytology as a diagnostic
aid in chlamydial cervicitis. Scand J Infect Dis Suppl 1982;32:
55Y8.
19. Geerling S, Nettum JA, Lindner LE, Miller SL, Dutton L,
Wechter S. Sensitivity and specificity of the Papanicolaou
stained cervical smear in the diagnosis of Chlamydia trachoma-
tis infection. Acta Cytol 1985;29:671Y5.
20. Kiviat NB, Peterson M, Kinney-Thomas E, Tam M,
Stamm WE, Holmes KK. Cytologic manifestations of cervical
and vaginal infections. II. Confirmation of Chlamydia
trachomatis infection by direct immunofluorescence using
monoclonal antibodies. JAMA 1985;253:997Y1000.
21. Shiina Y. Cytomorphologic and immunocytochemical
studies of chlamydial infections in cervical smears. Acta Cytol
1985;29:683Y91.
22. Arroyo G, Linnemann C, Wesseler T. Role of the
Papanicolaou smear in diagnosis of chlamydial infections. Sex
Transm Dis 1989;16:11Y4.
23. Sekhri A, LeFaou AE, Tardieu JC, Antz M, Fabre M.
What can be expected from the cytologic examination of
cervicovaginal smears for the diagnosis of Chlamydia tracho-
matis infections? Acta Cytol 1988;32:805Y10.
24. Bernal JN, Martinez MA, Dabancens A. Evaluation of
proposed cytomorphologic criteria for the diagnosis of
Chlamydia trachomatis in Papanicolaou smears. Acta Cytol
1989;33:309Y13.
25. Clark RB, Schneider V, Gentile FG, Pechan B, Dalton
HP. Cervical chlamydial infections: diagnostic accuracy of the
Papanicolaou smear. South Med J 1985;78:1301Y3.
26. Lindner LE, Geerling S, Nettum JA, Miller SL,
Altman KH. The cytologic features of chlamydial cervicitis.
Acta Cytol 1985;29:676Y82.
27. Forster GE, Cookey I, Munday PE, Richman PI, Jha R,
Coleman D, et al. Investigation into the value of Papanicolaou
stained cervical smears for the diagnosis of chlamydial cervical
infection. J Clin Pathol 1985;38:299Y402.
28. Pandit AA, Kihilnani PH, Powar HS, Mali BN, Joshi JV,
Krishna U. Value of Papanicolaou smear in detection of
Chlamydia trachomatis infection. Diagn Cytopathol 1993;9:
164Y7.
29. Vela C, Mendoza N, Otiniano L. Cytologic diagnosis
 50 & FITZHUGH AND HELLER
of Chlamydia in cervicovaginal secretions: use of a Papanico-
laou stain modification with buffered Wright solution. Acta
Cytol 1998;42:954Y8.
30. Watson EJ, Templeton A, Russell I, Paavonen J, Mardh PA,
Stary A, et al. The accuracy and efficacy of screening tests for
Chlamydia trachomatis: a systematic review. J Med Microbiol
2000;51:1021Y31.
31. Elnashar AM, Aboelea A, Tantawy TA. Cytologic,
colposcopic, and virologic detection of cervical herpes simplex
virus. Int J Gynecol Obstet 2003;81:69Y70.
32. Naib ZM, Nahimas AJ, Josey WE. Cytology and
histopathology of cervical herpes simplex infection. Cancer
1966;19:1026Y31.
33. Brown ST, Jaffe HW, Zaidi A, Filker R, Herrmann KL,
Lyleria HC, et al. Sensitivity and specificity of diagnostic tests
for genital infection with herpesvirus hominis. Sex Transm Dis
1979;6:10Y3.
34. Kapur S, Patterson K, Chandra R. Detection of herpes
simplex virus in cytologic smears. Arch Pathol Lab Med
1985;109:464Y5.
35. Puthavathana P, Horthongkham N, Roongpisuthipong A,
Matyhangkul P, Kanyok R, Boonsirikhamchai P, et al. Compar-
ison between virus isolation method, Papanicolaou stain,
immunoperoxidase stain and polymerase chain reaction in the
diagnosis of genital herpes. Asian Pac J Allergy Immunol 1998;
16:177Y83.
36. Stowell SB, Wiley CM, Powers CN. Herpesvirus
mimics. A potential pitfall in endocervical brush specimens.
Acta Cytol 1994;38:43Y50.
37. Cibas ES, Ducatman BS. Cytology: Diagnostic Princi-
ples and Clinical Correlates, 2nd ed. New York: Saunders,
2003:20Y2.
38. Audisio TA, Pigini T, de Riutort SV, Schindler L, Ozan M,
Tocalli C, et al. Validity of the Papanicolaou smear in the
diagnosis of Candida spp., Trichomonas vaginalis, and bacterial
vaginosis. J Lower Gen Tract Dis 2001;5:223Y5.
39. Takei H, Ruiz B, Hicks J. Cervicovaginal flora:
Comparison of conventional Pap smears and a liquid-based
thin-layer preparation. Am J Clin Pathol 2006;125:855Y9.
40. Wiese W, Patel SR, Patel SC, Ohl CA, Estrada CA. A
meta-analysis of the Papanicolaou smear and wet mount for
the diagnosis of vaginal trichomoniasis. Am J Med 2000;108:
301Y8.
41. Thomason JL, Gelbart SM, Sobun JF, Schulien MB,
Hamilton PR. Comparison of four methods to detect
Trichomonas vaginalis. J Clin Microbiol 1988;26:1869Y70.
42. Aslan DL, Gulbahce HE, Stelow EB, Setty S, Brown CA,
McGlennen RC, et al. The diagnosis of Trichomonas vaginalis in
liquid-based Pap tests: correlation with PCR. Diagn Cytopathol
2005;32:341Y4.
43. Lara-Torre E, Pinkerton JS. Accuracy of detection of
Trichomonas vaginalis organisms on a liquid-based Papanico-
laou smear. Am J Obstet Gynecol 2003;188:354Y6.
44. Vardar E, Maral I, Inal M, Ozguder O, Tasli F, Postaci H.
Copyright @ 2007 American Society for Colposcopy and Cervical Pathology. Unauthorized reproduction of this article is prohibited.
Comparison of Gram stain and Pap smear procedures in the
diagnosis of bacterial vaginosis. Infect Dis Obstet Gynecol
2002;10:203Y7.
45. Tokyol C, Aktepe OC, Cevrioglu AS, Altindis M, Dilek FU.
Bacterial vaginosis: comparison of Pap smear and microbiological
test results. Mod Pathol 2004;17:857Y60.
46. Amsel R, Totten PA, Spiegel CA, Chen KC, Eschenbach
D, Holmes KK. Nonspecific vaginitis. Diagnostic criteria and
microbial and epidemiologic associations. Am J Med 1983;74:
14Y22.
47. Giacomini G, Calcinai A, Moretti D, Cristofani R.
Accuracy of cervical/vaginal cytology in the diagnosis of
bacterial vaginosis. Sex Transm Dis 1998;25:24Y7.
48. Davis JD, Connor EE, Clark P, Wilkinson EJ, Duff P.
Correlation between cervical cytologic results and Gram stain
as diagnostic tests for bacterial vaginosis. Am J Obstet Gynecol
1997;177:532Y5.
49. Young NA, Bibbo M, Buckner SB, Colgan TJ, Prey
MU. Non-neoplastic findings. In: Solomon D, Nayar R, eds.
The Bethesda System for Reporting Cervical Cytology (2nd
ed.). New York: Springer; 2004:30, 32.
50. Korenek P, Britt R, Hawkins C. Differentiation of the
vaginoses-bacterial vaginosis, lactobacillosis, and cytolytic
vaginosis. Internet J Adv Nurs Pract 2003;6. Available at:
http://www.ispub.com/ostia/index.php?xmlFilePath=journals/
ijanp/vol6n1/vaginosis.xml.
51. Gideon K, Zaharopoulos P. Cytomegalovirus endocer-
vicitis diagnosed by cervical smear. Diagn Cytopathol 1991;7:
625Y7.
52. Morse AR, Coleman DV, Gardner SD. An evaluation
of cytology in the diagnosis of herpes simplex virus and
cytomegalovirus infection of the cervix uteri. J Obstet
Gynaecol Br Commonw 1974;81:393Y8.
53. Hunt JL, Baloch Z, Judkins A, LiVolsi VA, Montone
KT, Gupta PK. Unique cytomegalovirus inclusions in ectocer-
vical cells on a cervical/endocervical smear. Diagn Cytopathol
1998;18:110Y2.
54. Sekhon HS, Press RD, Schmidt WA, Hawley M, Rader A.
Identification of cytomegalovirus in a liquid-based gyneco-
logic sample using morphology immunohistochemistry, and
DNA real time PCR detection. Diagn Cytopathol 2004;30:
411Y7.
55. Langlinais PC. Enterobius vermicularis in a vaginal
smear. Acta Cytol 1969;13:40Y1.
56. Rad MJ, Jeannot A. Enterobius vermicularis in a
cervical smear. Acta Cytol 1970;14:466Y7.
57. Wong JY, Becker SN. Enterobius vermicularis ova in
routine cervicovaginal smears: light and scanning electron
microscopic observations. Acta Cytol 1982;26:484Y7.
58. Kjetland EF, Ndhlovu PD, Mduluza T, Gomo E,
Gwanzura L, Mason PR, et al. Simple clinical manifestations
of genital Schistosoma hematobium infection in rural Zim-
babwean women. Am J Trop Med Hyg 2005;72:311Y9.
59. Youssef AF, Fayad MM, Shafeek MA. The diagnosis of
 Microorganisms on Pap Smear & 51

genital bilharziasis by vaginal cytology. Am J Obstet Gynecol
1962;83:710Y4.
60. DeMille PA, Bourquin P, Sun CC, Kauffman L.
Cytologic diagnosis of Schistosoma haematobium in routine
cervicovaginal smear from an infertile woman. Diagn Cyto-
pathol 1995;13:181Y2.
61. Mainguene C, Clement N, Gabriel S, Loubiere R,
Hofman P. Urogenital schistosomiasis: an unusual discovery on
cervical smears from a Caucasian female. Acta Cytol
1998;42:1045Y6.
62. Jain S, Sharma P, Gupta R. Dual cervical parasitosis in
a psychiatric patient. Cytopathology 2005;16:53Y4.

Copyright @ 2007 American Society for Colposcopy and Cervical Pathology. Unauthorized reproduction of this article is prohibited.