VAGINAL

SECRETIONS
AUBF LECTURE FINALS WEEK 4
WHY DO WE NEED TO EXAMINE VAGINAL
SECRETIONS?
To diagnose infections, most commonly vaginitis
To diagnose complications of pregnancy
For forensic testing in sexual assault patients
SPECIMEN COLLECTION AND
HANDLING
 Use speculum moistened
SPECULUM with warm water

Do not use lubricants

Swab the vaginal walls

and vaginal pool to
collect epithelial cells
and vaginal secretions
SWAB USED
Sterile, polyester-tipped on a plastic
shaft
Cotton swabs are toxic to Neisseria
gonorrhoeae
Wooden shafts may be toxic to
Chlamydia trachomatis
Calcium alginate can inactivate herpes
simplex virus for viral cultures
POST-COLLECTION
Perform gross examination, test with pH paper, place swab
in a tube with 0.5 to 1 mL sterile physiologic saline
Alternative method: dilute a sample of vaginal discharge
in 1-2 drops NSS (1 slide) and 10% KOH (1 slide)
COLLECTION GUIDELINES
Place specimens in a biohazard bag
Request form should include:
patient history (includes menstrual status)
Use of vaginal creams, lubricants, and douches
Recent exposure to any STD
Preservation: RT (T. vaginalis, N. gonorrhoeae), refrigeration (C.
trachomatis, HSV)
COLOR AND APPEARANCE
Normal: white, flocculent discharge
Thin, white-to-gray: bacterial vaginosis
White, ‘cottage cheese’-like: Candida infections
Yellow green, frothy, adherent: T. vaginalis
Yellow, opaque: C. trachomatis

Predominant cells: large, Gram-positive lactobacilli (WBC

and RBC may be present)
DIAGNOSTIC TESTS
VAGINAL PH TEST
Commercial pH test paper with a narrow range (4.5)
 4.5 in candidiasis, above 4.5 in other vaginitis causes
Gently apply the vaginal secretion on the surface of the pH paper in a
circular motion
Normally acidic (3.8-4.5) due to the predominance of lactobacilli which
produce lactic acid
Acidic environment suppresses the overgrowth of infectious organisms
Some lactobacilli also produce hydrogen peroxide
MICROSCOPIC PROCEDURES
Saline wet mounts and KOH mounts are the initial screening tests
Gram stain is used as a confirmatory test for yeast and bacterial
vaginosis
Slide preparation:
 Label 2 slides (add 1 if Gram stain is requested)
 Add 1 drop of specimen on each slide

Alternative method: press and roll the swab against the slide
 Rare Less than 10
organisms/cells
WET MOUNT EXAMINATION per slide
1+ Less than 1
organism/cell per
Slide is examined using hpf
LPO: even distribution of cellular 2+ 1-5
components, types and numbers of organisms/cells
epithelial cells, presence of budding per hpf
yeast or pseudohyphae 3+ 6-30
HPO: counting of organisms and cells organisms/cells
per hpf
4+ > 30
organisms/cells
per hpf
SQUAMOUS EPITHELIAL CELLS
25-70 micrometers in diameter
Polygonal flagstone appearance
Originate from vaginal linings and female urethra
Clumps indicate high numbers of yeast
 CLUE CELLS
Abnormal variation of squamous epithelial
cells
With attached coccobacillus bacteria in
clusters on the cell surface
Diagnostic of bacterial vaginosis caused by
G. vaginalis
WHITE BLOOD CELLS
14-16 micrometers in diameter
Granular cytoplasm
> 3 WBCs in vaginal secretions suggest vaginitis or
bacterial/fungal infections
RED BLOOD CELLS
Smooth, non-nucleated biconcave disks
May be present during menstruation or desquamative
inflammatory process
Use KOH to differentiate RBC from yeast
PARABASAL CELLS
Round to oval shaped; 16-40 micrometers in diameter
Marked basophilic granulation/amorphic basophilic structure
(blue blobs) in the cytoplasm
Rare in vaginal secretions
Increased amounts in menstruating and postmenopausal women;
desquamative inflammatory vaginitis
BASAL CELLS
Round; 10-16 micrometers in diameter
Distinguished from WBCs by their round nucleus
Not normally seen in vaginal fluid
Present in desquamative inflammatory vaginitis
 BACTERIA
Normal: vagina contains bacterial
flora (mainly Lactobacillus spp.)
that produce lactic acid and
hydrogen peroxide that suppress
the overgrowth of other organisms
Alteration in normal flora: absent
or decreased numbers of
lactobacilli relative to the number
of squamous epithelial cells
 TRICHOMONAS
VAGINALIS
Atrial flagellated protozoan that can
cause vaginal inflammation and
infection
The axostyle attaches to the vaginal
mucosa and cause tissue damage
‘Jerky’ motion in wet mount
Specimens must be examined ASAP
or maintained at RT for a maximum
for 2 hours
YEAST CELLS
Candida albicans and non-Candida spp. cause most
fungal infections
Occasional yeast is considered part of the normal flora
May appear as both budding cells or as hyphae or
pseudohyphae
KOH PREPARATION AND AMINE TEST
Place 1 drop of saline specimen + 1 drop of 10% KOH
Check for ‘fishy’ amine odor
Increased numbers of anaerobic bacteria produce polyamines that
volatilize when the KOH was added
Positive fishy odor indicates the presence of G. vaginalis, Mobiluncus
spp., and T. vaginalis
Examine slide after 5 minutes and look for pseudohyphae in LPO and
small blastospores in HPO
 Lactobacillu Gardnerella Curved Points
GRAM STAIN s and
Bacteroides
Gram-
variable rods
spp.
Gold standard to identify the 4+ 0 0 0
causative organisms of
3+ 1+ 1+ or 2+ 1
bacterial vaginosis
Use Nugent’s Gram stain 2+ 2+ 3+ or 4+ 2
criteria to diagnose bacterial 1+ 3+ 3
vaginosis
 0-3: normal vaginal flora 0 4+ 4
 4-6: intermediate
 7 or more: bacterial vaginosis
CULTURE
Gold standard for yeast and Trichomonas
Time-consuming and requires up to 2 days for a result
Diamond’s medium is required for Trichomonas
InPouch TV is a commercial transport and culture system for Trichomonas
 Inoculate specimen into the pouch within 30 minutes of collection
 Incubate for 5 days at 37 degrees C in a CO2 atmosphere
 Examine daily for motile trichomonads
DNA TESTING
DNA hybridization probe methods is present to specifically identify
the causative agent of vaginitis
Affirm VPIII is available for the differential diagnosis of G.
vaginalis, Candida spp., and T. vaginalis
DNA probes amplified by PCR can detect T. vaginalis
 Most accurate diagnostic method and can detect nonviable organisms
 POINT OF CARE TESTING
Rapid antigen tests for proline
aminopeptidase is used to
identify G. vaginalis
OSOM Trichomonas rapid test
can detect T. vaginalis from
vaginal swabs in 10 minutes
POINT OF CARE TESTING
OSOM BVBLUE detects vaginal fluid sialidase which is produced by bacterial agents
of vaginosis (Gardnerella, Bacteroides, Prevotella, Mobiluncus)
POINT OF CARE TESTING
VS-Sense Pro Swab: measures elevated vaginal pH
FemExam pH and Amines TestCard: detects
presence of amines to identify bacterial vaginosis
and trichomonas
FETAL FIBRONECTIN TEST
Fetal fibronectin is an adhesive glycoprotein in the extracellular matrix at
the maternal and fetal interface within the uterus
 Elevated during the first 24 weeks of pregnancy and then diminishes
 Presence of fetal fibronectin between 24-34 weeks gestation is associated with
preterm delivery
Obtain specimen by rotating the swab across the posterior fornix of the
vagina for 10 seconds
FETAL FIBRONECTIN TEST METHODS
solid-phase ELISA
Sample is incubated with FDC-6 (an antibody specific for fFN)
Presence or absence of fFN is measured spectrophotometrically
at 550 nm
lateral flow solid-phase immunochromatographic assay
using Rapid fFN cassette
 AMNISURE TEST
Detects placental alpha
macroglobulin-1 (PAMG-1)
Present in high levels in amniotic
fluid
Marker of fetal membrane
rupture which can cause
premature delivery
Normal: 0.05-0.22 mg/mL
Fetal membrane rupture: 2,000-
2,500 mg/mL
VAGINAL DISORDERS
 BACTERIAL VAGINOSIS
caused by an imbalance of the naturally occurring vaginal flora,
 change in the most common type of bacteria present
 increase in the total number of bacteria present
associated with a decline in the overall number of Lactobacilli and with an
increased risk for future STIs
characterized by the presence of clue cells
risk factors for bacterial vaginosis include vaginal douching, multiple sexual
partners, recent antibiotic use, cigarette smoking, and the use of an
intrauterine device
 TRICHOMONIASIS
Trichomonas vaginalis is the causative agent
Complaints: vaginal discharge, pruritus, and irritation
Colpitis macularis (strawberry cervix) is a specific clinical sign
Pathogenesis: adhesion of the parasite and the production of
molecules that are delivered to target cells and mediate cytotoxicity
through damage of the target cell plasma membrane
Tx: metronidazole, tinidazole
 CANDIDAL VULVOVAGINITIS
caused by inflammatory changes in the vaginal and vulvar epithelium
secondary to infection with Candida species, most commonly Candida
albicans
requires both the presence of candida in the vagina/vulva and the
symptoms of irritation, itching, dysuria, or inflammation
risk factors include estrogen use, elevated endogenous estrogens (from
pregnancy or obesity), diabetes mellitus, immunosuppression, and broad-
spectrum antibiotic use
Candida species superficially penetrate the mucosal lining of the vagina
and cause an inflammatory response
Tx: azole antifungals
 DESQUAMATIVE INFLAMMATORY VAGINITIS
inflammatory skin condition of the vagina
may be the result of noninfectious blistering disorders such as pemphigus
vulgaris, lichen planus, and mucous membrane pemphigoid
frequency of DIV in premenopausal, postmenopausal, and postpartum
women and in those receiving antiestrogen therapy for endometriosis
suggests that estrogen deficiency may play a role in the infectious process
most reported symptom is the presence of an intractable, purulent vaginal
discharge, sometimes associated with vulvar soreness, burning, and
dyspareunia
 ATROPHIC VAGINITIS
symptomatic inflammatory process involving the thinned vaginal epithelium
affecting some pre-menopausal and up to 50% of post-menopausal women
Hypoestrogenic state results in the fusion of collagen fibers and
fragmentation of elastin fibers in vulvovaginal tissue and decreased
squamous cells, resulting in decreased mucosal elasticity and decreased
rugae, and narrowing the vagina
estrogen receptor beta is present predominately in the premenopausal state,
leading to the ovarian failure state experienced in menopause