NOTES ON ANALYSIS OF URINE AND OTHER BODY FLUIDS

Notes Compiled by: Renz Louie Galanto

Strasinger, Seventh Edition

UNIT 17: VAGINAL SECRETIONS

UNIT OUTLINE

• Introduction • Vaginal Disorders

• Specimen Collection and Handling o Bacterial Vaginosis
• Color and Appearance o Trichomoniasis
• Diagnostic Tests o Candidiasis
o pH o Desquamative Inflammatory Vaginitis
o Microscopic Procedures o Atrophic Vaginitis
▪ Wet Mount Examination • Additional Procedures for Vaginal Secretion Associated with
▪ KOH Preparation and Amine Test Pregnancy
o Other Diagnostic Tests o Fetal Fibronectin Test
▪ Gram Stain o AmniSure Test
▪ Culture o Actim PROM
▪ DNA Testing o ROM Plus
▪ Point-of-Care Tests

INTRODUCTION
● Vaginal secretions are examined in the clinical laboratory to diagnose infections and complications of pregnancy, as well as 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
o It is characterized by abnormal vaginal discharge or odor, pruritus, vaginal irritation, dysuria, and dyspareunia
o Most often, vaginitis is secondary to bacterial vaginosis (BV), trichomoniasis, or vulvovaginal candidiasis; however, vaginitis also can occur
with noninfectious conditions, such as vaginal atrophy, allergies, and chemical irritation
o Although the symptoms for the various syndromes of vaginitis are similar, the effective treatment for each depends on an accurate diagnosis
● 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 partner, to avoid reinfection
o Microscopic methods include a saline wet mount examination, potassium hydroxide (KOH) examination, and the Gram stain, which is
considered the gold standard
▪ Other tests used for differential diagnosis include litmus pH levels, DNA probe testing, culture, and point-of-care test kits
● The clinical and microscopic features of the common syndromes are summarized in Table 17-1
● In addition to evaluating vaginal secretions for infections, tests are performed on vaginal secretions to detect the placental α1-microglobulin
(PAMG-1) protein, insulin-like growth factor binding protein-1 (IGFBP-1) 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
● Technical Tip 17-1. Clinical laboratory personnel performing urine microscopic examinations should be aware that microscopic constituents
observed in vaginal fluid also may be seen in urine specimens when the urine specimen is contaminated with vaginal secretions
Table 17-1. Clinical Features and Laboratory Findings in Vaginitis
Findings Bacterial Vaginosis Candidiasis Trichomoniasis Desquamative Atrophic Vaginitis
Inflammatory
Vaginitis
Appearance Thin, homogeneous, White, curd-like Yellow-green frothy Excessive purulent Excessive purulent vaginal
white-to-gray vaginal discharge adherent vaginal discharge vaginal discharge, discharge, vaginal erythema
vaginal discharge increased in volume vaginal erythema
pH >4.5 3.8–4.5 > 4.5 > 4.5 > 4.5
WBCs Rare or absent 3+ to 4+ 2+ to 4+ 3+ to 4+ 3+ to 4+
Lactobacilli Rare or absent Present Absent or present Absent or reduced Decreased
Clue cells >20% Absent Absent or present
Other cells Large clumps of Occasional parabasal Occasional parabasal or
epithelial cells or basal cells basal cells
>1+ RBCs >1+ RBCs
Other Increase in small Budding yeast cells Trichomonas frequently 2+ gram-positive Increased gram-positive
organism curved bacilli, and pseudohyphae associated with other cocci cocci and gram-negative
coccobacilli, and organisms rods; decreased large rods
pleomorphic bacilli
Amine Positive Negative Positive Negative Negative
(whiff) test
UNIT 17: VAGINAL SECRETIONS Strasinger, 7th Edition, 2021
Other tests Confirmatory tests: Confirmatory tests: Confirmatory tests: DNA
DNA probe, proline DNA probe, OSOM probe or culture, OSOM
amino peptidase, BVBLUE Rapid Trichomonas Rapid Test
OSOM BVBLUE Test
Rapid Test

SPECIMEN COLLECTION AND HANDLING

● The health-care provider collects vaginal secretions during a ● An alternative method of specimen preparation is to dilute a
pelvic examination sample of vaginal discharge in one to two drops of normal saline
● Detailed instructions and the specific manufacturer’s collection solution directly on a microscope slide
and transport devices must be provided and are specific to the o Then a second sample is placed in 10% KOH solution in the
organism sought same manner
● Correct specimen handling and timely transport to the laboratory o Cover slips are placed over both slides for microscopic
are important for optimal detection of the responsible pathogen. examination
● A speculum moistened with warm water is used to visualize the ● Properly labeled specimens should be placed in a biohazard bag
vaginal fornices with the requisition form and transported to the laboratory as soon
● Lubricants may contain antibacterial agents and must not be used as possible
● The specimen is collected by swabbing the vaginal walls and o The requisition form must include the patient’s name and
vaginal pool to collect epithelial cells along with the vaginal unique identifier, as well as a patient medical history that
secretions using one or more sterile, polyester-tipped swabs on a should include menstrual status; use of vaginal creams,
plastic shaft or swabs specifically designated by the manufacturer lubricants, and douches; and recent exposure to sexually
● Cotton swabs should not be used because cotton is toxic to transmitted diseases
Neisseria gonorrhoeae, the wood in a wooden shaft may be toxic ● Specimens should be analyzed immediately, but if a delay in
to Chlamydia trachomatis, and calcium alginate can inactivate transport or analysis is necessary, specimen handling is based on
herpes simplex virus (HSV) for viral cultures the suspected pathogen.
● The health-care provider performs a gross examination of the ● Specimens must be kept at room temperature to preserve the
vaginal secretions and then places the swab in a tube containing motility of Trichomonas vaginalis and the recovery of N.
0.5 to 1.0 mL of sterile physiological saline gonorrhoeae, whereas specimens for C. trachomatis and HSV
o The tube is sealed for transport to the laboratory, where the must be refrigerated to prevent overgrowth of normal flora
specimen is processed for microscopic analysis ● Specimens for T. vaginalis should be examined within 2 hours of
o The swab should be twirled in the saline vigorously to collection
dislodge particulates from the swab ● Technical Tip 17-2. Keep specimens for suspected T. vaginalis at
▪ Failure to dislodge particles may lead to erroneous room temperature and examine within 2 hours of collection to
results. visualize movement of the flagella or undulating membrane on a
● Specimens should be tested with pH paper before being placed in wet prep. When not moving, Trichomonas may resemble a white
saline blood cell (WBC), transitional, or renal tubular epithelial (RTE)
cell

COLOR AND APPEARANCE

● Normal vaginal fluid appears white with a flocculent discharge
● Microscopically, normal vaginal flora includes a predominance of Table 17-2. Normal Findings in Vaginal Secretion
large, rod-shaped, gram-positive lactobacilli and squamous Appearance White, flocculent discharge
epithelial cells pH 3.8 – 4.2
● WBCs may be present, and red blood cells (RBCs) will be present Amine (whiff) test Negative
if the patient is menstruating (Table 17-2) WBCs Rare to 2+
● Abnormal vaginal secretions may appear as an increased thin, Lactobacilli Predominant
homogeneous, white-to-gray discharge often seen in BV; as a Clue cells Absent
white “cottage cheese–like” discharge particular for Candida Other cells Absent (except RBCs during menses)
infections; or as an increased yellow-green, frothy, adherent Other organisms Other lactobacilli subgroups, occasional yeast
discharge associated with T. vaginalis
● C. trachomatis may present with a yellow, opaque cervical
discharge

DIAGNOSTIC TESTS

pH__________________________________________________________________________
● The health-care provider can perform a vaginal pH test when ● The test paper is placed in the pooled vaginal secretion, and the
performing a pelvic examination color change is compared with a chart with corresponding pH
● The test should be performed before placing the swab into saline values.
or KOH solutions ● Factors that can interfere with the pH test include contamination
● Commercial pH test paper with a narrow pH range is of the vaginal secretions with cervical mucus, semen, and blood
recommended to evaluate pH values in the 4.5 range more ● The pH test helps to differentiate the causes of vaginitis, as shown
accurately in Table 17-1
UNIT 17: VAGINAL SECRETIONS Strasinger, 7th Edition, 2021
● The vaginal pH is usually about 4.5 in women with vulvovaginal o Then the slide is examined using the high power objective
candidiasis but is above 4.5 in women with BV, trichomoniasis, (400× magnification), and the organisms and cells are
desquamative inflammatory vaginitis (DIV), and atrophic counted and reported per hpf using the criteria in Table 17-3.
vaginitis. ● Typical constituents found in vaginal fluid wet mounts include
● As described previously, normal vaginal flora includes a squamous epithelial cells, WBCs, RBCs, clue cells, parabasal
predominance of the bacteria lactobacilli, which produce the end- cells, basal cells, bacteria, motile T. vaginalis, yeast, and hyphae/
product lactic acid from glycogen metabolism pseudohyphae
o Lactic acid provides an acidic vaginal environment with a ● Intravaginal medications might leave oil droplets that can
pH value between 3.8 and 4.5 interfere with the interpretation of wet mounts
▪ This acidity suppresses the overgrowth of infectious o In this case, a Gram stain is useful to detect yeast or BV
organisms such as Mobiluncus, Prevotella, and ● Squamous Epithelial Cells
Gardnerella vaginalis, and therefore maintains the o Squamous epithelial cells measure 25 to 70 µm in diameter
balance of normal vaginal bacteria flora and exhibit a polygonal “flagstone” appearance
● Some lactobacilli subgroups also produce hydrogen peroxide, o They contain a prominent nucleus that is centrally located
which is toxic to pathogens and helps keep the vaginal pH acidic and about the size of a RBC, as well as a large amount of
to provide protection from urogenital infections irregular cytoplasm, lacking granularity, with distinct cell
● BV has been associated with the absence of hydrogen peroxide– margins (Fig. 17-1)
producing lactobacilli o These large, flat cells originate from the linings of the vagina
● Estrogen production also is necessary to preserve an acidic and female urethra and are present in significant numbers in
vaginal environment the vaginal secretions of a healthy female
● Procedure 17-1. pH Test o Clumps of epithelial cells, as observed in Figure 17-2, are an
o 1. Using a circular motion, gently apply the vaginal secretion indication of the presence of increased numbers of yeast
over the surface of the pH test paper.
o 2. Immediately observe the color reaction on the paper, and
compare it to a color comparison chart to determine the pH
of the sample.
o 3. Record the results

MICROSCOPIC PROCEDURES_______
● Usually, vaginal infections are diagnosed from microscopic
examination
● Saline wet mounts and KOH mounts are the initial screening tests,
and the Gram stain is used as a confirmatory examination for ● Clue Cells
yeast or BV o Clue cells are an abnormal variation of the squamous
● Slides are prepared from the saline specimen solution that was epithelial cell and are distinguished by coccobacillus bacteria
made from the vaginal swab immediately after collection attached in clusters on the cell surface, spreading past the
● Three clean glass slides (if a Gram stain is requested) are labeled edges of the cell and making the border appear indistinct or
with the patient’s name and a unique identifier stippled
o A drop of specimen is placed on each slide using a disposable o Bacteria should cover at least 75% of the epithelial cell
transfer pipette o This gives the cell a granular, irregular appearance
o An alternative method is to press the swab against the slide sometimes described as “shaggy.”
and then roll the swab over the slide o Clue cells are diagnostic of BV caused by G. vaginalis (Fig.
o The slide for Gram stain is allowed to dry and then is heat- 17-3)
fixed for the Gram stain procedure and examination o The presence of clue cells also can be found in urine
performed in the microbiology section of the laboratory. sediment and should be confirmed by the procedures already
● For wet mount examinations, cells and organisms are quantified described
per high power field (hpf) (40×); for Gram stains, cells and
organisms are reported per oil immersion field (100×)
● Technical Tip 17-3. The Seattle STD/HIV Prevention Training
Center has developed the YouTube video titled Examination of
Vaginal Wet Preps, an excellent online overview of performing
the microscopic examination of vaginal secretion. It is available
at https://www.youtube.com/watch?v=8dgeOPGx6YI.
WET MOUNT
WET MOUNT EXAMINATION
● For the saline wet mount examination, a cover slip is placed on
the specimen carefully to exclude air bubbles ● White Blood Cells
o WBCs measure 14 to 16 µm in diameter and exhibit a
o The slide is examined microscopically using the low power
granular cytoplasm
(10×) and high dry power (40×) objective with a bright-field
o Often, they are described as polymorphonuclear (PMN)
microscope
WBCs because of their characteristic multilobed nucleus
o Using the low power objective (100× magnification), the
slide is scanned for an even distribution of cellular (Fig. 17-4)
o Normally, WBCs are present in rare to scanty numbers in
components, types and numbers of epithelial cells, clumping
vaginal secretions
of epithelial cells, and the presence of budding yeast or
o More than 3+ WBCs in vaginal secretions suggest vaginal
pseudohyphae
candidiasis; atrophic vaginitis; or infections with
Trichomonas, Chlamydia, N. gonorrhoeae, or HSV
UNIT 17: VAGINAL SECRETIONS Strasinger, 7th Edition, 2021
o Lactobacillus spp. normally comprise the largest portion of
vaginal bacteria.
o They appear as large, gram-positive, nonmotile rods and
produce lactic acid, which maintains the vaginal pH at 3.8 to
4.5 (Fig. 17-7 A and B)
o Hydrogen peroxide, produced by lactobacilli subgroups, also
can help to suppress the overgrowth of other organisms
o Other bacteria commonly present include anaerobic
streptococci, diphtheroids, coagulase-negative
Figure 17–4 A. White blood cells. Notice the multilobed nucleoli staphylococci, and α-hemolytic streptococci
(×400). B. Stained smear o When conditions are present that cause an imbalance in the
● Red Blood Cells normal flora, vaginitis can occur
o RBCs appear as smooth, nonnucleated, biconcave disks o Absent or decreased numbers of lactobacilli relative to the
measuring approximately 7 to 8 µm in diameter (Fig. 17-5) number of squamous epithelial cells suggests an alteration in
o RBCs can be somewhat distorted in vaginal specimens the normal flora
o Usually, RBCs are not seen in vaginal secretions, but they o Often the lactobacilli are replaced by increased numbers of
might be present during menstruation or due to a any of the following:
desquamative inflammatory process ▪ Mobiluncus spp. (thin, curved, gram-negative, motile
o RBCs can be confused with yeast cells and are distinguished rods)
from yeast cells by KOH, which will lyse the RBCs but allow ▪ Prevotella spp.
the yeast cells to remain intact ▪ Porphyromonas spp.
▪ Bacteroides spp. (anaerobic gram-negative rods)
▪ Gardnerella vaginalis (short, gram-variable
coccobacilli)
▪ Peptostreptococcus spp. (gram-positive cocci)
▪ Enterococcus spp. (gram-negative cocci)
▪ Mycoplasma hominis
▪ Ureaplasma urealyticum

● Parabasal Cells
o Parabasal cells are round to oval-shaped and measure 16 to
40 µm in diameter
o The ratio of nucleus to cytoplasm is 1:1 to 1:2, with marked
basophilic granulation or amorphic basophilic structures
(“blue blobs”) in the surrounding cytoplasm
o They are located in the luminal squamous epithelium of the
vaginal mucosa
o Although it is rare to find parabasal cells in vaginal
Figure 17–7 Bacteria. A. Large rods characteristic of Lactobacilli, the
secretions, less mature cells may be found if the patient is
predominant bacteria in normal vaginal secretions (×400). B. Bacteria
menstruating or if the patient is postmenopausal
with white blood cells (×400)
o Increased numbers of parabasal cells, if present with large
● Trichomonas vaginalis
numbers of WBCs, can indicate DIV (Fig. 17-6)
o T. vaginalis is an atrial flagellated protozoan that can cause
vaginal inflammation and infection in women
o The organism is 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
o 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
(Fig. 17-8)
o The “jerky” motion of the flagella and undulating membrane
characteristic of T. vaginalis can be observed in a wet mount
o Care must be taken not to confuse T. vaginalis with sperm,
● Basal Cells which have only a single tail, a much smaller head
o Basal cells are located deep in the basal layer of the vaginal (approximately one-half the diameter of a RBC), and no
stratified epithelium axostyle
o These cells are round and measure 10 to 16 µm in diameter o In addition, nonmotile trichomonads can be mistaken for
o They have a ratio of nucleus to cytoplasm of 1:2 WBCs (Fig. 17-9)
o Basal cells are distinguished from WBCs, which are similar o T. vaginalis organisms quickly lose their viability after
in size, by their round rather than lobed nucleus collection
o They are not normally seen in vaginal fluid and, if present o Specimens must be examined as soon as possible or, if
and accompanied by large numbers of WBCs and altered necessary, maintained at room temperature for a maximum
vaginal flora, can suggest DIV of 2 hours before preparing the wet mount to observe the
● Bacteria organism’s motility
o The vagina is a nonsterile environment with complex o Trichomonas also can be seen in a urinary microscopic
endogenous bacterial flora that vary with the age and sample but cannot be reported unless motility is observed,
hormonal status of the patient either in movement across the slide or just in the tail
UNIT 17: VAGINAL SECRETIONS Strasinger, 7th Edition, 2021
o A dead trichomonad tends to appear oval and slightly larger 1+ Fewer than 1 organism or cell/hpf
than a WBC. 2+ 1 – 5 organisms or cells/hpf
3+ 6 – 30 organisms or cells/hpf
4+ >30 organisms or cells/hpf
WET MOUNT
KOH PREPARATION AND AMINE TEST
Figure 17–8 Trichomonas
vaginalis. (From Leventhal ● The KOH slide is prepared and the amine (whiff) test is performed
and Cheadle, Medical by placing a drop of the saline specimen prepared from the
Parasitology, 6th edition, collection swab onto a clean slide that is labeled properly and
2012, F.A. Davis Company, adding one drop of 10% KOH solution
Philadelphia, with o The slide is checked immediately for a “fishy” amine odor
permission.) ● The result is reported as positive (presence of fishy odor) or
negative (absence of fishy odor)
● Increased numbers of anaerobic bacteria in the vagina produce
polyamines that are released into the vaginal fluid
● The odor results from trimethylamine, a volatilization product of
amines, when the KOH is added
o Because volatile amines are not present in normal vaginal
secretion, a positive test result suggests BV caused by
increased numbers of G. vaginalis in conjunction with
Mobiluncus spp. and with T. vaginalis
● After the amine test has been performed, place a cover slip over
the specimen, taking care to exclude air bubbles
o Allow the KOH preparation to rest for 5 minutes to dissolve
epithelial and blood cells
o Heat may be applied to speed the dissolving process
o One drop of 10% glycerin may be added after the KOH to
prevent specimen deterioration
o The slide is examined under low power (100× magnification)
for the presence of yeast pseudohyphae and under high dry
● Yeast Cells power (400× magnification) to identify smaller blastospores
o Candida albicans and non-Candida spp. cause most fungal (yeast cells)
infections, but an occasional yeast in vaginal secretions is ● Procedure 17-3. KOH Preparation
considered part of the normal flora o 1. Prepare a clean glass slide labeled with the patient’s name
o Yeast cells appear on a wet mount as both budding yeast cells and unique identifier.
(blastospores) (Fig. 17-10) or as hyphae, which are long o 2. Place one drop of vaginal specimen on the slide.
filaments that grow and form a mycelium (Fig. 17-11) o 3. Add one drop of 10% KOH to the slide.
o Pseudohyphae—multiple buds that do not detach and form o 4. Allow the KOH slide preparation to rest for up to 5
chains—also can be seen minutes to allow cellular tissue and other debris to dissolve.
o It can be difficult to distinguish yeast cells from RBCs on a Gentle heating may speed the dissolving process.
wet mount because both measure about 7 to 8 µm in o 5. Cover the specimen with a cover slip, removing any air
diameter; however, differentiation can be made using the bubbles.
KOH test. Yeast cells stain gram positive. o 6. Examine the slide under the 10× objective for overall
assessment and for yeast pseudohyphae.
o 7. Switch to the 40× objective to examine for budding yeast
cells (smaller blastopore blastospore).
o 8. Record the results
● Procedure 17-4. Amine (Whiff) Test
o 1. Apply one drop of the saline vaginal fluid suspension to
the surface of a clean glass slide.
o 2. Add one drop of 10% KOH directly to the vaginal sample.
o 3. Holding the slide in one hand, gently fan above the surface
of the slide with the other hand and assess for the presence
of a fishy amine odor.
● Procedure 17-2. Saline Wet Mount o 4. Report as positive or negative.
o 1. Prepare a clean glass slide labeled with the patient’s name ▪ Positive: The presence of a fishy odor after adding
and unique identifier. KOH.
o 2. Place one drop of vaginal specimen on the slide. ▪ Negative: The absence of a fishy odor after adding
o 3. Cover the slide with a cover slip, removing any air KOH
bubbles.
o 4. Examine the slide with the 10× objective for epithelial OTHER DIAGNOSTIC TESTS________
cells and any budding yeast cells or pseudohyphae. ● Although the wet mount and KOH slide examinations and the
o 5. Examine the slide with the 40× objective, and quan tify amine test are used commonly to diagnose BV, other tests may be
organisms and cells per hpf. required for a confirmatory diagnosis
o 6. Record the results. o These include Gram stain, specimen culture, DNA probe
Table 17-3. Quantitation Scheme for Microscopic Examinations testing, and point-of-care test kits.
Rare Fewer than 10 organisms or cells/slide
 UNIT 17: VAGINAL SECRETIONS Strasinger, 7th Edition, 2021
WET MOUNT ● A DNA probe testing system, Affirm VPIII (Becton, Dickinson,
GRAM STAIN Franklin Lakes, NJ), is available for differential diagnosis of G.
● The Gram stain is considered the gold standard in identifying the vaginalis, Candida spp., and T. vaginalis. It is easy to perform,
causative organisms for BV and results are available in 1 hour, with a sensitivity of 95%
o It also provides a permanent record of the patient specimen o This test is significantly more sensitive than wet mount
● A scored Gram stain system is a weighted combination of the microscopy and is less subjective to personal bias compared
following morphotypes: Lactobacillus acidophilus (large gram- with traditional microscopic tests
positive rods), G. vaginalis and Bacteroides spp. (small gram- ● New molecular assays are being developed continually for the
variable or gram-negative rods), and Mobiluncus spp. (curved diagnosis of vaginitis
gram-variable rods) ● Trichomonas also can be detected by DNA probes amplified by
● The types of bacterial morphophytes are evaluated and scored polymerase chain reaction (PCR)
o For example, Lactobacillus morphophytes are the o Enzymes are added to the specimen that amplifies specific
predominant bacteria in normal vaginal flora; therefore, if 4+ regions of the DNA of T. vaginalis by PCR
Lactobacillus morphophytes are present on Gram stain, and o Then the number of DNA fragments is calculated
Gardnerella and Bacteroides spp. morphophytes and curved ▪ This is the most accurate diagnostic method, and it has
gram-variable rods are absent, the score is 0 the advantage of detecting nonviable organisms
● As indicated in Table 17-4, a Nugent score of 0 to 3 is considered WET MOUNT
normal vaginal flora, whereas a score of 4 to 6 is reported as POINT-OF-CARE TESTS
intermediate, and a score of 7 or more is diagnostic of BV ● Various rapid diagnostic tests are available to quickly screen for
Table 17-4. Nugent Gram Stain Criteria to Diagnose Bacterial Vaginosis the causative agents of vaginitis, and they provide a higher
Lactobacillus Gardnerella and Bacteroides Curved Gram- Points sensitivity and specificity for the organism sought
spp. Morphophytes Variable Rods o For example, proline aminopeptidase activity in vaginal
4+ 0 0 0 secretions can be detected by rapid antigen tests to identify
3+ 1+ 1+ or 2+ 1 G. vaginalis
2+ 2+ 3+ or 4+ 2 ● The OSOM Trichomonas Rapid Test (Genzyme Diagnostics,
1+ 3+ 3 Cambridge, MA) is an immunochromatographic strip test that
0 4+ 4 detects T. vaginalis antigen from vaginal swabs in 10 minutes
Note: Points are added according to the morphotypes seen. Add the o The test is performed by placing the vaginal swab in the kit’s
points for all three columns for a final sum. A score of 7 or higher sample buffer
indicates bacterial vaginosis o The Trichomonas proteins are solubilized into the buffer
WET MOUNT o The test stick coated with anti-Trichomonas antibodies is
CULTURE placed into the sample mixture
o The solution migrates up the stick and, if Trichomonas
● Culture, using various types of media, is the gold standard test for antigens are present, they will react with the antibodies on
detecting yeast and Trichomonas; however, it is more time the stick
consuming and requires up to 2 days for a result. Culture for G. o A visible blue line and a red internal control line indicate a
vaginalis is not diagnostic for BV because it is part of the normal positive result
flora in 50% of healthy women ● The OSOM BVBLUE test (Genzyme Diagnostics, Cambridge,
● Special media called Diamond medium is required for MA) detects vaginal fluid sialidase, an enzyme produced by the
determining the presence of T. vaginalis bacterial pathogens associated with BV, such as Gardnerella,
● A commercial transport and culture pouch system for the Bacteroides, Prevotella, and Mobiluncus
detection of Trichomonas is now available (InPouch TV, Biomed o The test takes 1 minute to perform and is read by examining
Diagnostics, White City, OR) the change in the color of the solution: blue or green is
o The specimen must be inoculated into the pouch within 30 positive, yellow is negative
minutes of collection and then is incubated for 5 days at 37°C ● Commercial tests to measure an elevated vaginal pH (VS-Sense
in a CO2 atmosphere Pro Swab) and the presence of amines (FemExam pH and Amines
o The pouch is examined microscopically daily for motile TestCard, Litmus Concepts, Santa Clara, CA) use pH indicators
trichomonads and an amine test system that is read visually to identify BV and
MOUNT Trichomonas
DNA TESTING
● DNA hybridization probe methods have been developed to
identify the specific causative pathogen for vaginitis

VAGINAL DISORDERS

BACTERIAL VAGINOSIS______________________________________________________
● BV is the most common cause of vaginitis, affecting 40% to 50% ● As the vaginal pH becomes alkaline, lactobacilli are replaced by
of women of childbearing age an overgrowth of G. vaginalis, Mobiluncus spp., Prevotella spp.,
o It occurs when there is an imbalance in the ratio of normal Porphyromonas, Peptostreptococcus, Mycoplasma hominis, and
vaginal bacterial flora Ureaplasma spp.
● The predominant organism in the vaginal flora is lactobacilli, o The malodor and increased abnormal vaginal discharge
which produce lactic acid that maintains the vaginal pH between result from this mix of organisms and is more apparent after
3.8 and 4.5 intercourse
● Additionally, certain subsets of lactobacilli produce hydrogen
peroxide, which prevents the overgrowth of normal vaginal flora.
UNIT 17: VAGINAL SECRETIONS Strasinger, 7th Edition, 2021
● BV is associated with new or multiple sex partners, frequent ● If the wet mount is negative for motile trichomonads, a culture
douching, use of intrauterine devices, pregnancy, and a lack of the using Diamond medium or the commercially available pouch
protective lactobacilli system (InPouch TV, Biomed Diagnostics, White City, OR) is
o There is evidence that BV is a risk factor for the premature recommended for detection of T. vaginalis
rupture of membranes and preterm labor for pregnant women ● The DNA probe test system, Affirm VPIII (Becton, Dickinson,
● Additional complications include pelvic inflammatory disease Franklin Lakes, NJ), and the point-of-care rapid antigen detection
and endometritis, as well as an increased risk for acquiring some test, OSOM Trichomonas Rapid Test (Genzyme Diagnostics,
sexually transmitted infections (STIs) such as HIV, N. Cambridge, MA), are available with increased sensitivity and
gonorrhoeae, C. trachomatis, and HSV-2 specificity for T. vaginalis
● BV is diagnosed by examining the vaginal secretions for ● The recommended treatment for trichomoniasis is metronidazole
abnormal appearance or quantity, performing the pH and amine o For patients who develop an allergy to metronidazole or for
tests, and microscopically observing the wet mount for the whom treatment is not effective, a newer drug, tinidazole, is
presence of clue cells and the absence of WBCs and lactobacilli available
morphotypes ● All sexual partners of patients, even if asymptomatic, should be
● According to Amsel’s Diagnostic Criteria, three of the following treated to avoid reinfection
four features must be present for the diagnosis of BV:
o 1. Thin, white, homogeneous discharge CANDIDIASIS______________________
o 2. Vaginal fluid pH greater than 4.5
o 3. A positive amine (whiff) test ● Vulvovaginal candidiasis is caused by an infection with the yeast
Candida
o 4. Presence of clue cells on microscopic examination
● It is a common cause of vaginitis, and nearly 75% of adult women
● The Gram stain is the gold standard for determining the ratio of
each bacterial morphotype and offers a definitive diagnosis have at least one yeast infection in their lifetime
● Most yeast infections are caused by C. albicans, but other
● Other tests used to diagnose BV include the DNA hybridization
nonalbican species, such as C. glabrata, C. parapsilosis, C.
probe test; Affirm VPIII (Becton, Dickinson), which detects G.
tropicalis, and C. krusei, have been isolated as the cause.
vaginalis; a proline aminopeptidase test (Pip Activity TestCard,
● Candida is part of the normal vaginal flora, but an infection occurs
Quidel, San Diego, CA); and the OSOM BVBLUE test (Genzyme
when there is a change in the vaginal environment that permits
Diagnostics, Cambridge, MA)
● Treatment is recommended for women to relieve vaginal the overgrowth of Candida and symptoms of the infection to
occur
symptoms and reduce the risk of infection
o Conditions that can cause a change in the vaginal
o In addition, benefits of treatment include reducing the risk of
environment include the use of broad-spectrum antibiotics,
acquiring an STI and reducing the risks associated with
oral contraceptives, or estrogen replacement therapy;
pregnancy
hormonal changes that occur with pregnancy; ovulation; and
o The recommended treatments are metronidazole (Flagyl),
menopause.
metronidazole gel, or clindamycin cream.
● Technical Tip 17-4. The detection of clue cells in the wet mount ● Increased infection rates occur in patients who are
immunocompromised or those with conditions such as diabetes
examination of vaginal discharge is the most useful single
mellitus, iron deficiency, and HIV infection
indicator for the diagnosis of BV.
● The infection is found predominantly in women of childbearing
age, who are producing large amounts of estrogen
TRICHOMONIASIS_________________ o Estrogen causes the vagina to mature and produce glycogen,
● Trichomoniasis is caused by the parasitic protozoon T. vaginalis which facilitates the growth and adherence of C. albicans
o The infection is transmitted by sexual intercourse, and it ● Typical clinical symptoms of vulvovaginal candidiasis include
causes vaginitis in women and, sometimes, urethritis in men genital itching or burning, dyspareunia; dysuria; and the presence
o The infection is classified as an STI, frequently occurs with of an abnormal thick, white, curd-like vaginal discharge
infections of gonorrhea or Chlamydia, and has been ● The pH of the vaginal fluid remains normal (3.8 to 4.5), and the
associated with enhanced transmission rates of HIV. amine test is negative
● In pregnant women, a correlation has been found between a T. ● In vulvovaginal candidiasis, the microscopic examination of the
vaginalis infection and low birth weight, premature rupture of saline and KOH wet prep and Gram stain will reveal budding
membranes, and preterm delivery yeast and pseudohyphae forms, large numbers of WBCs,
● Trichomoniasis is characterized by a green-to-yellow frothy lactobacilli, and large clumps of epithelial cells
vaginal discharge, malodor, pruritus, irritation, dysuria, ● A culture and DNA hybridization probe (Affirm VPIII Microbial
dyspareunia, and vaginal mucosa erythema, although some Identification System; Becton, Dickinson, Franklin Lakes, NJ)
patients are asymptomatic analysis can be performed to confirm the clinical diagnosis and to
● Patients may present with a “strawberry cervix” because of identify the species of Candida
punctuate hemorrhages ● Yeast infections are treated with over-the-counter or pre\scription
o Usually males are asymptomatic or may present with azole antifungal agents
urethritis o They may be intravaginal, suppository, or oral agents, and
● Trichomoniasis can be diagnosed with the wet mount and the regimen depends on the medication
microscopic examinations, visualizing the motile trichomonads in o Over-the-counter intravaginal medications include
a fresh specimen; however, this method has a sensitivity of only butoconazole, clotrimazole, tioconazole, and miconazole
60% to 70% o For patients with recurrent infections (four or more episodes
o The test must be performed within 2 hours of specimen per year), a prescription medication using a longer treatment
collection to preserve the viability of the organism regimen with either oral fluconazole or intravaginal
● WBCs and lactobacilli bacteria also are present with a T. vaginalis butoconazole, nystatin, and terconazole may be more
infection effective
● The vaginal pH is greater than 4.5, and the amine test from the ● Vulvovaginal candidiasis is not acquired through sexual
KOH preparation will be positive intercourse, so treatment of sexual partners is not indicated
UNIT 17: VAGINAL SECRETIONS Strasinger, 7th Edition, 2021
DESQUAMATIVE INFLAMMATORY ATROPHIC VAGINITIS______________
VAGINITIS_________________________ ● Atrophic vaginitis is a syndrome found in postmenopausal
women
● DIV is a syndrome characterized by profuse purulent vaginal
● This syndrome is caused by thinning of the vaginal mucosa
discharge, vaginal erythema, and dyspareunia
because of reduced production of both estrogen and glycogen
● There is a heterogeneous group of causes of DIV; however, β- ● As a result, the vaginal environment changes, and the balance of
hemolytic gram-positive streptococci can be cultured from most
normal flora is altered
patients
● Clinical symptoms include vaginal dryness and soreness,
● The syndrome also can occur secondary to atrophic vaginitis in dyspareunia, inflamed vaginal mucosa, and purulent discharge
postmenopausal women as a result of decreased estrogen ● The vaginal secretion pH is greater than 4.5, and the amine test is
● The vaginal secretion pH is greater than 4.5, and the amine test is
negative
negative
● Microscopic evaluation is similar to that for DIV and includes
● Wet mount and Gram stain microscopic examination of the
large numbers of WBCs and the presence of RBCs, occasional
vaginal secretions reveal large numbers of WBCs, RBCs,
parabasal and basal cells, squamous epithelial cells, and
occasional parabasal and basal cells, squamous epithelial cells,
decreased numbers of lactobacilli that have been replaced by
and reduced or absent lactobacilli that have been replaced by gram-positive cocci and gram-negative rods
gram-positive cocci (refer back to Table 17-1) ● Treatment of atrophic vaginitis is estrogen replacement
● DIV is treated with 2% clindamycin
● Topical vaginal ointments are used initially; however, for
● Hormone replacement therapy is effective for patients with DIV
frequent, recurrent episodes of atrophic vaginitis, oral or
secondary to atrophic vaginitis
transcutaneous (patch) modes are more effective.

ADDITIONAL PROCEDURES FOR VAGINAL SECRETION ASSOCIATED WITH

PREGNANCY
● As noted previously in this chapter, complications from vaginitis o ROM Plus (Clinical Innovations) to detect the amniotic fluid
syndromes can include premature rupture of fetal membranes and proteins alpha-fetoprotein (AFP) and IGFBP-1
a high risk of preterm labor ● The fern test is another test that determines the presence of
● The following tests are used to evaluate these conditions: amniotic fluid in vaginal secretions
o Fetal fibronectin (fFN) enzyme test ● Patient history, amniotic pooling in the fornix of the vagina, a
o AmniSure (AmniSure International) test to detect the vaginal pH greater than 7.0, and a positive fern test are strong
amniotic fluid protein PAMG-1 indicators of amniotic sac rupture
o Actim PROM (Cooper Surgical Medical Devices) test to
detect the amniotic fluid protein IGFBP-1, also referred to as
placental protein 12 (PP12)

FETAL FIBRONECTIN TEST__________________________________________________

● Preterm delivery, defined as delivery before the completion of 37 presence or absence of the fFN is determined
weeks’ gestation, is the leading cause of neonatal mortality and spectrophotometrically at a wavelength of 550 nm
morbidity in the United States. ● The rapid fFN assay is a qualitative test for the detection of fFN
● fFN is an adhesive glycoprotein in the extracellular matrix at the that uses a Rapid fFN cassette kit and a TLiIQ analyzer
maternal and fetal interface within the uterus ● The specimen swab is placed into an extraction buffer and filtered
● It is elevated during the first 24 weeks of pregnancy but then with a plunger filter
diminishes o The filtered sample is dispensed onto the sample application
● The presence of fFN in vaginal secretions between 24- and 34- well of the Rapid fFN cassette
weeks’ gestation is associated with preterm delivery ● The sample flows from an absorbent pad across a nitrocellulose
● The test can be used by health-care providers as a means to better membrane by capillary action through a reaction zone containing
manage patient care, and it can be performed routinely as part of murine monoclonal anti-fFN antibody conjugated to blue
a prenatal visit for asymptomatic women between 22- and 30- microspheres
weeks’ gestation or in symptomatic pregnant women between 24 ● The monoclonal antibody FDC-6 is specific for fFN
and 34 weeks ● The conjugate, embedded in the membrane, is mobilized by the
● Symptoms of preterm delivery include a change in vaginal flow of the sample
secretions, vaginal bleeding, uterine contractions, abdominal or o Then the sample flows through a zone containing goat
back discomfort, pelvic pressure, and cramping polyclonal antihuman fibronectin antibody that captures the
● The specimen is obtained by rotating the swab provided in the fibronectin–conjugate complexes
specimen collection kit across the posterior fornix of the vagina o The remaining sample flows through a zone containing goat
for 10 seconds to absorb the vaginal secretions polyclonal antimouse IgG antibodies, which captures
o The swab must not be contaminated with lubricants, creams, unbound conjugate, resulting in a control line
soaps, or disinfectants that may interfere with the antibody– o After 20 minutes, the intensities of the test line and control
antigen reaction in the test system line are interpreted with the TLiIQ analyzer
● The methods for detection of the fFN enzyme immunoassay are o The results are reported as positive or negative
solid-phase enzyme-linked immunosorbent assay (ELISA) or ● Symptomatic pregnant women with a positive fFN test are at
lateral flow, solid-phase immunochromatographic assay using the increased risk for delivery in less than or equal to 7 to 14 days
Rapid fFN cassette from specimen collection, and asymptomatic pregnant women are
● In the fFN enzyme immunoassay, the vaginal sample is incubated at increased risk for delivery in less than or equal to 34 weeks and
with FDC-6, a monoclonal antibody specific for fFN, and the 6 days of gestation.
UNIT 17: VAGINAL SECRETIONS Strasinger, 7th Edition, 2021
IGFBP-1 accumulates at high concentrations in amniotic fluid
AmniSure TEST_____________________ ●
o When the membranes are ruptured, IGFBP-1 is detectable in
● The risk of premature delivery also may be caused by the
a vaginal swab sample
premature rupture of fetal membranes (PROM)
▪ The swab is held in the vaginal fornix for 10 to 15
● In addition, rupture of fetal membranes can cause infection, fetal
seconds
distress, prolapse of the umbilical cord, postnatal endometritis,
▪ Then the swab is placed in the specimen extraction
and placental abruption
solution and swirled vigorously for 10 to 15 seconds
o A symptom of fetal membrane rupture is leakage of amniotic
o The yellow area of the dipstick is placed into the extraction
fluid
solution and held there until the liquid front reaches the result
● PAMG-1 is present in high levels in amniotic fluid and low levels
area, at which point the dipstick is removed
in blood; therefore, it is a reliable marker of fetal membrane
▪ The test is interpreted in 5 minutes
rupture
▪ A positive result will show two lines on the dipstick,
o A normal level of PAMG-1 in pregnant women ranges from
indicating that the membranes have ruptured
0.05 to 0.22 mg/mL, which might increase to 3 mg/mL when
▪ A negative result will show only one control line,
vaginitis is present
indicating that the membranes are intact
● Fetal membrane rupture causes increased concentrations of
▪ Blood, urine, semen, bath and odor products, common
amniotic fluid in the vaginal secretions and can raise the PAMG-
vaginal infections, and medications will not interfere
1 levels to 2,000 to 25,000 mg/mL
with the testing
● The AmniSure test quickly identifies patients with PROM, and
▪ The test has been designed to minimize interference
appropriate intervention can take place
from bleeding, but in cases of heavy bleeding, the
● The AmniSure ROM test (AmniSure International) is a qualitative
blood present may have a higher concentration of
rapid test that uses an immunochromatographic device
IGFBP-1 protein
● A sample of vaginal secretions collected with a swab is placed
into a vial with solvent
o The swab is rotated for 1 minute to enable the solvent to ROM Plus___________________________
extract the sample from the swab, and then the swab is ● The ROM Plus fetal membranes rupture test is a qualitative
discarded immunochromatographic test for the detection of amniotic fluid
● The AmniSure test strip is placed into the vial in vaginal secretions of pregnant women
o Monoclonal antibodies with colloidal gold particles attached ● This test is unique in that it detects both AFP and IGFBP-1
are located on the pad region of the test strip protein, also known as PP12, from amniotic fluid in vaginal
o The antibodies attach to the PAMG-1 in the sample and secretions.
transport it to the test region ● The ROM Plus test strip is a lateral flow device
o The solution flows from the pad region of the strip to the test ● The sample is collected by placing the swab on the vaginal
region mucosal lining for 15 seconds
▪ The test region of the test strip has antibodies o Then the swab is mixed into a vial containing 400 µL of
immobilized on it. buffer solution, and the diluted sample is applied to the
● If PAMG-1 is present in the patient sample, it will bind with sample pad of the test strip via the sample well on the cassette
antibodies in the test region and produce a line o The liquid moves chromatographically and unidirectionally
o This line is produced by gold dye attached to conjugated toward the absorbent pad
antibodies and indicates a rupture of fetal membranes o During migration, the sample reacts with monoclonal and
o The second control line is designed to indicate that the test is polyclonal antibodies bound to the test strip membrane
functioning well ▪ These antibodies are immunoreactive to a combination
o The test is read immediately or within 10 minutes of AFP and IGFBP-1
o A positive test result indicates a membrane rupture and is ● If the sample contains AFP and IGFBP-1, it
indicated by the presence of two lines binds to the antibody test line, causing the test
o When only the control line is present, it is reported as line to appear and indicate a positive result
negative for membrane rupture ● Two lines will be visible
o The test should be performed immediately after collection, ● If the sample does not contain the amniotic
but if there is a delay in testing, the specimen can be markers, only the control line will be visible,
maintained in a closed sample vial and refrigerated for 6 indicating a negative result
hours ● IGFBP-1 concentration in amniotic fluid is between 10,500 and
o False positives can be caused by the presence of large 350,000 ng/mL
amounts of blood in the vaginal sample o AFP concentration in amniotic fluid is from 2,800 to 26,000
ng/mL
Actim PROM________________________ o Serum IGFBP-1 concentration is from 55 to 242 U/mL
● The Actim PROM (CooperSurgical, Trumbull, CT) is a 5-minute (equivalent to 33 to 290 ng/mL)
o Concentrations of IGFBP-1 in amniotic fluid can be 100 to
test that can be used bedside to diagnose patients with PROM
● It is a rapid immunoassay test that specifically detects IGFBP-1 1,000 times higher than that in maternal serum
in the vaginal fluid

Reference:
Notes from the book by Urinalysis and Body Fluids, Seventh Edition Strasinger (2021)
Disclaimer: All notes in this material are from the following reference above. No additional notes were included for the creation of this material