VAGINAL

SECRETIONS
 examined in the clinical laboratory to diagnose
infections and complications of pregnancy, and for
forensic testing in sexual assault patients.
 Vaginitis is one of the most common conditions
diagnosed by health-care providers for female
patients, particularly women of childbearing age.
 It is characterized by abnormal vaginal discharge
or odor, pruritus, vaginal irritation, dysuria, and
dyspareunia.
 Most often, vaginitis is secondary to bacterial
vaginosis (BV), vulvovaginal candidiasis, or
trichomoniasis; however, vaginitis can also occur with
non-infectious conditions such as vaginal atrophy,
allergies, and chemical irritation.
 Careful microscopic examination of fresh vaginal
secretions is necessary to determine the causative agent
for each syndrome and to provide the appropriate
treatment for the patient and, in some cases, the sexual
partners, to avoid reinfection
 Clinical laboratory personnel performing urine microscopic
examinations should be aware that microscopic constituents
observed in vaginal fluid may also be seen in urine specimens when
the urine specimen is contaminated with vaginal secretions
 tests are performed on vaginal secretions to detect the placental a 1-
microglobulin (PAMG-1) protein to diagnose ruptured fetal
membranes, or fetal fibronectin enzyme to assess the risk of
preterm delivery.
 The fern test is used to identify amniotic fluid that may be present
when the amniotic sac has ruptured.
Specimen Collection and Handling
 The health-care provider collects vaginal secretions
during a pelvic examination.
 A speculum moistened with warm water is used to
visualize the vaginal fornices.
 The specimen is collected by swabbing the vaginal
walls and vaginal pool to collect epithelial cells
along with the vaginal secretions using one or more
sterile, polyester-tipped swabs on a plastic shaft or
swabs specifically designated by the manufacturer.
 Cotton swabs should not be used because cotton is toxic to
Neisseria gonorrhoeae, the wood in a wooden shaft may be
toxic to Chlamydia trachomatis, and calcium alginate can
inactivate herpes simplex virus (HSV) for viral cultures.
 The health-care provider performs a gross examination of the
vaginal secretions and then places the swab in a tube
containing 0.5 to 1.0 mL of sterile physiologic saline
 The swab should be vigorously twirled in the saline to dislodge
particulates from the swab.
 Specimens should be tested with pH paper before being placed
in saline
 The requisition must include:
 patient’s name and unique identifier
 patient medical history that should include
menstrual status
 use of vaginal creams, lubricants, and douches
 recent exposure to sexually transmitted diseases
 Specimens must be kept at room temperature to
preserve the motility of Trichomonas vaginalis
and the recovery of N. gonorrhoeae, whereas
specimens for C. trachomatis and herpes simplex
virus must be refrigerated to prevent overgrowth
of normal flora.
 Specimens for T. vaginalis should be examined
within 2 hours of collection.
Color and Appearance
 Normal vaginal fluid appears white with a flocculent
discharge. Microscopically, normal vaginal flora
includes a predominance of large, rod-shaped, gram-
positive lactobacilli and squamous epithelial cells.
 Abnormal vaginal secretions may appear as an
increased thin, homogeneous white-to-gray
discharge often seen in bacterial vaginosis, or as a
white “cottage cheese”–like discharge particular for
Candida infections
 increased yellow-green, frothy, adherent discharge
associated with T. vaginalis.
 C. trachomatis may present with a yellow, opaque
cervical discharge.
Diagnostic Tests
 pH
 The test should be performed before placing the
swab into saline or KOH solutions.
 Commercial pH test paper with a narrow pH range
is recommended to more accurately evaluate pH
values in the 4.5 range.
 Factors that can interfere with the pH test
contamination of the vaginal secretions with
cervical mucus, semen, and blood.
 pH 4.5 in women with vulvovaginal candidiasis
 > 4.5 in women with bacterial vaginosis,
trichomoniasis, desquamative inflammatory
vaginitis (DIV), and atrophic vaginitis.
 Lactic acid provides an acidic vaginal environment with
a pH value between 3.8 and 4.5.
 This acidity suppresses the overgrowth of infectious
organisms such as Mobiluncus, Prevotella, and
Gardnerella vaginalis, and therefore maintains the
balance of normal vaginal bacteria flora.
 Some lactobacilli subgroups also produce hydrogen
peroxide, which is toxic to pathogens, and helps keep the
vaginal pH acidic to provide protection from urogenital
infections
Microscopic Procedures
 Wet Mount Examination
 For the saline wet mount examination, a cover slip is placed
on the specimen carefully to exclude air bubbles.
 The slide is examined microscopically using the low power
(10×) and high dry power (40×) objective with a bright-
field microscope
 Using the low power objective, the slide is scanned for an
even distribution of cellular components, types and
numbers of epithelial cells, clumping of epithelial cells, and
the presence of budding yeast or pseudohyphae.
 constituents found in vaginal fluid wet mounts:
 squamous epithelial cells
 white blood cells
 red blood cells
 clue cells
 parabasal cells
 basal cells,
 bacteria,
 motile Trichomonas vaginalis,
 Yeast
 hyphae/pseudohyphae.
 Squamous Epithelial Cells
 measure 25 to 70 μm in diameter and exhibit a polygonal
“flagstone” appearance
 contain a prominent centrally located nucleus about the
size of a red blood cell and a large amount of irregular
cytoplasm, lacking granularity, with distinct cell margins
 originate from the linings of the vagina and female
urethra and are present in significant numbers in the
vaginal secretions of a healthy female
 Clue Cells
 abnormal variation of the squamous epithelial cell
and are distinguished by coccobacillus bacteria
attached in clusters on the cell surface
 granular, irregular appearance sometimes
described as “shaggy.”
 diagnostic of bacterial vaginosis caused by G.
vaginalis
 White Blood Cells
 measure 14 to 16 μm in diameter and exhibit a granular
cytoplasm
 often described as polymorphonuclear white blood cells
(PMNs) because of their characteristic multi-lobed
nucleus
 Greater than 3+ WBCs in vaginal secretions suggest
vaginal candidiasis, atrophic vaginitis, or infections with
Trichomonas, Chlamydia, N. gonorrhoeae, or Herpes
simplex.
 Red Blood Cells
 smooth, non-nucleated biconcave disks measuring
approximately 7 to 8 μm in diameter
 not usually seen in vaginal secretions, but they
might be present during menstruation or due to a
desquamative inflammatory process
 Parabasal Cells
 round to oval shaped and measure 16 to 40 μm in diameter.
 NC ratio is 1:1 to 1:2, with marked basophilic granulation or
amorphic basophilic structures (“blue blobs”) in the
surrounding cytoplasm.
 rare to find parabasal cells in vaginal secretions but less
mature cells may be found if the patient is menstruating and
in postmenopausal women
 Increased parabasal cells can indicate desquamative
inflammatory vaginitis.
 Basal Cells
 located deep in the basal layer of the vaginal stratified
epithelium
 round and measure 10 to16 μm in diameter
 nucleus to cytoplasm ratio of 1:2
 round rather than lobed nucleus
 They are not normally seen in vaginal fluid, and if present
and accompanied by large numbers of WBCs and altered
vaginal flora can suggest desquamative inflammatory
vaginitis.
 Bacteria
 Lactobacillus spp. normally comprise the largest
portion of vaginal bacteria.
 They appear as large gram-positive, nonmotile rods
on Gram stain and produce lactic acid, which
maintains the vaginal pH at 3.8 to 4.5
 Other bacteria commonly present include anaerobic
streptococci, diphtheroids, coagulase-negative
staphylococci, and a –hemolytic streptococci.
 The lactobacilli are often replaced by increased numbers
of:
a. Mobiluncus spp - thin, curved, gram-negative, motile rods
b. Prevotella spp., Porphyromonas spp., Bacteroides spp. -
anaerobic gram-negative rods
c. Gardnerella vaginalis - short, gram-variable coccobacilli
d. Peptostreptococcus spp. - gram-positive cocci
e. Enterococcus spp. gram-negative cocci
f. Mycoplasma hominis
g. Ureaplasma urealyticum.
 Trichomonas vaginalis
 atrial flagellated protozoan
 oval shaped, measures 5 to 18 μm in diameter, and has
four anterior flagella and an undulating membrane that
extends half the length of the body
 An axostyle bisects the trophozoite longitudinally and
protrudes from the posterior end, which enables the
organism to attach to the vaginal mucosal and cause tissue
damage
 “jerky” motion
- quickly lose their viability after collection.
- must be examined as soon as possible or, if
necessary, maintained at room temperature for a
maximum of 2 hours before preparing the wet mount
to observe the organism’s motility
 Yeast Cells
 Candida albicans and non–Candida spp. cause most
fungal infections but an occasional yeast in vaginal
secretions
 appear on a wet mount as both budding yeast cells
(blastophores) or as hyphae
 Differentiation with RBC can be made using the
KOH test.
 Yeast cells stain gram positive.
KOH Preparation and Amine Test
KOH Preparation and Amine Test
 The slide is immediately checked for a “fishy”
amine odor. The result is reported as positive
(presence of fishy odor) or negative (absence of
fishy odor).
 The odor results from the volatilization of amines
when the KOH is added.
Other Diagnostic Tests
 Gram Stain
 considered the gold standard in identifying the
causative organisms for bacterial vaginosis
 A scored Gram stain system is a weighted
combination of the following morphotypes:
a. Lactobacillus acidophilus
b. G. Vaginalis & Bacteroides spp.
c. Mobiluncus spp.
 Culture
 gold standard test for detecting yeast and Trichomonas
 more time consuming and requires up to 2 days for a result
 Culture for G. vaginalis is not diagnostic for bacterial
vaginosis because it is part of the normal flora in 50% of
healthy women
 The specimen must be inoculated into the pouch within 30
minutes of collection and then is incubated for 5 days at
37°C in a CO2 atmosphere.
 developed to specifically identify the causative
pathogen for vaginitis
 Affirm VPIII (Becton, Dickinson, Franklin Lakes,
NJ)- for differential diagnosis of G. vaginalis,
Candida spp., and T. vaginalis
- significantly more sensitive than wet mount
microscopy and is less subjective to personnel bias
compared with traditional microscopic tests
 Trichomonas can also be detected by DNA probes
amplified by polymerase chain reaction (PCR).
Enzymes are added to the specimen that amplifies
specific regions of T. vaginalis’ DNA by PCR. The
number of DNA fragments is then calculated.
 This is the most accurate diagnostic method and it
has the advantage of detecting nonviable
organisms.9
Point of Care Tests
 The OSOM Trichomonas Rapid Test (Genzyme
Diagnostics, Cambridge, MA) is an
immunochromatographic strip test that detects T.
vaginalis antigen from vaginal swabs in 10 minutes.
 The test is performed by placing the vaginal swab in
the kit’s sample buffer. The trichomonas proteins
are solubilized into the buffer.
 A visible blue line and a red internal control line
indicate a positive result
 OSOM BVBLUE test (Genzyme Diagnostics,
Cambridge, MA) detects vaginal fluid sialidase, an
enzyme produced by the bacterial pathogens
associated with bacterial vaginosis, such as
Gardnerella, Bacteroides, Prevotella, and
Mobiluncus.
 blue or green is positive, yellow is negative
Vaginal Disorders
 Bacterial Vaginosis
 most common cause of vaginitis, affecting 40% to 50% of
women of childbearing age.
 occurs when there is an imbalance in the ratio of normal
vaginal bacterial flora.
 The predominant organism in the vaginal flora is lactobacilli
 As the vaginal pH becomes alkaline, lactobacilli are replaced
by an overgrowth of G. vaginalis, Mobiluncus spp.,
Prevotella spp., Porphyromonas, Peptostreptococcus,
Mycoplasma hominis, and Ureaplasma spp.
 associated with new or multiple sex partners, frequent
douching, use of intrauterine devices, pregnancy, and a
lack of the protective lactobacilli
 risk factor for the premature rupture of membranes and
preterm labor for pregnant women
 diagnosed by examining the vaginal secretions for
abnormal appearance or quantity, performing the pH and
amine tests, and microscopically observing the wet
mount for the presence of clue cells and the absence of
WBCs and lactobacilli morphotypes
 Three of the following four features must be
present for the diagnosis of BV:
(1) thin, white, homogeneous discharge
(2) vaginal fluid pH greater than 4.5
(3) a positive amine (Whiff) test
(4) presence of clue cells on microscopic
examination
 Therecommended treatments are metronidazole
(Flagyl), metronidazole gel, or clindamycin cream.
 Trichomoniasis
 caused by the parasitic protozoon T. vaginalis
 transmitted by sexual intercourse and causes
vaginitis in women and sometimes urethritis in men
 classified as an STI,frequently occurs with
gonorrhea and Chlamydia infections, and has been
associated with enhanced transmission rates of HIV
 characterized by a green-to-yellow frothy vaginal
discharge, malodor, pruritus, irritation, dysuria,
dyspareunia, and vaginal mucosa erythema, although
some patients are asymptomatic
 Patients may present with a “strawberry cervix” because
of punctuate hemorrhages
 diagnosed with the wet mount examination and
microscopically, visualizing the motile trichomonads in a
fresh specimen; however, this method has a sensitivity of
only 60% to 70%.
 The vaginal pH is greater than 4.5 and the amine
test from the KOH preparation will be positive. If
the wet mount is negative for motile trichomonads,
a culture using Diamond’s medium or the
commercially available pouch system
 Candidiasis
 caused by an infection with the yeast Candida.
 Conditions that can cause a change in the vaginal
environment:
a. the use of broad-spectrum antibiotics, oral
contraceptives, or estrogen replacement therapy
b. hormonal changes that occur with pregnancy
c. Ovulation
d. menopause.
 The infection is predominantly found in women of
childbearing age, who are producing large
amounts of estrogen
 Vulvovaginal candidiasis is not acquired through
sexual intercourse, so treatment of sexual partners
is not indicated
Desquamative Inflammatory
Vaginitis
a syndrome characterized by profuse purulent
vaginal discharge, vaginal erythema, and
dyspareunia.
 The vaginal secretion pH is greater than 4.5 and
the amine test is negative
 treated with 2% clindamycin.
 Hormone replacement therapy is effective for
patients with DIV secondary to atrophic vaginitis.
Atrophic Vaginitis
 syndrome found in postmenopausal women
 caused by thinning of the vaginal mucosa because
of reduced estrogen production and decreased
glycogen production
 Clinical symptoms: vaginal dryness and soreness,
dyspareunia, inflamed vaginal mucosa, and
purulent discharge
Additional Vaginal Secretion
Procedures
 Fetal Fibronectin Test
 adhesive glycoprotein in the extracellular matrix at
the maternal and fetal interface within the uterus.
It is elevated during the first 24 weeks of
pregnancy but then diminishes
 The specimen is obtained by rotating the swab
provided in the specimen collection kit across the
posterior fornix of the vagina for 10 seconds to
absorb the vaginal secretions.
 The methods for detection of the fetal fibronectin
enzyme immunoassay are solid-phase enzyme-
linked immunosorbent assay (ELISA) or lateral
flow, solid-phase immunochromatographic assay
using the Rapid fFN cassette.
 AmniSure Test
 qualitative rapid test that uses an
immunochromatographic device. A sample of vaginal
secretions collected with a swab is placed into a vial
with solvent. The swab is rotated for 1 minute to enable
the solvent to extract the sample from the swab and then
discarded. The AmniSure test strip is placed into the vial.
 A positive test result indicates a membrane rupture and
is indicated by the presence of two lines.
 When only the control line is present, it is reported
as negative for membrane rupture. The test should
be performed immediately after collection, but if
there is a delay in testing, the specimen can be
maintained in a closed sample vial and refrigerated
for 6 hours.
 quickly identifies patients with fetal membrane
rupture, and appropriate intervention can take
place