In the clinical laboratory, Vaginal discharge are examined to diagnose infections and pregnancy complications, as well as for forensic testing (Semen) in sexual assault patients.
Vaginitis is one of the most common conditions diagnosed by doctors in female patients, particularly those of childbearing age. It is distinguished by abnormal vaginal discharge or odor, as well as pruritus, vaginal irritation, dysuria, and dyspareunia. Vaginitis is most commonly caused by bacterial vaginosis (BV), vulvovaginal candidiasis, or trichomoniasis; however, vaginitis can also be caused by non-infectious conditions such as vaginal atrophy, allergies, and chemical irritation.
Although the symptoms of the various vaginitis syndromes are similar, effective treatment for each is dependent on an accurate diagnosis. To avoid reinfection, careful microscopic examination of fresh Vaginal discharge is required to determine the causative agent for each syndrome and to provide the appropriate treatment for the patient and, in some cases, the sexual partners. A saline wet mount examination, potassium hydroxide (KOH) examination, and the Gram stain, which is considered the gold standard, are all microscopic methods. When performing urine microscopic examinations, clinical laboratory personnel should be aware that microscopic constituents seen in vaginal fluid may also be seen in urine specimens if the urine specimen is contaminated with Vaginal discharge. Litmus pH levels, DNA probe testing, culture, and point-of-care test kits are some other tests used for differential diagnosis. The table below summarizes the clinical and microscopic features of the common syndromes.
|Findings||Bacterial Vaginosis||Candidiasis||Trichomoniasis||Desquamative Inflammatory Vaginitis||Atrophic Vaginitis|
|Appearance||Thin, homogeneous, whiteto-gray vaginal discharge||White, curd-like vaginal discharge||Yellow-green frothy adherent vaginal discharge increased in volume||Excessive purulent vaginal discharge, vaginal erythema||Excessive purulent vaginal discharge, vaginal erythema|
|pH||>4.5||3.8 to 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 epithelial cells||Occasional parabasal or basal cells||Occasional parabasal or basal cells|
|>1+ RBCs||>1+ RBCs|
|Other organisms||Increase in small curved bacilli, coccobacilli, and pleomorphic bacilli||Budding yeast cells and pseudohyphae||Trichomonas frequently associated with other organisms||2+ grampositive cocci||Increased grampositive cocci and gramnegative rods; decreased large rods|
|Amine (Whiff) test||Positive||Negative||Positive||Negative||Negative|
|Other tests||Confirmatory tests: DNA probe, proline amino peptidase, OSOM BVBLUE Rapid Test||Confirmatory tests: DNA probe, OSOM BVBLUE Rapid Test||Confirmatory tests: DNA probe or culture, OSOM Trichomonas Rapid Test|
In addition to testing Vaginal discharge for infections, tests are also performed to detect the placental a 1-microglobulin (PAMG-1) protein to diagnose ruptured fetal membranes or the fetal fibronectin enzyme to assess the risk of preterm delivery. The fern test detects amniotic fluid that may be present when the amniotic sac ruptures.
Specimen Collection and Handling
During a pelvic examination, the health-care provider collects Vaginal discharge. Detailed instructions, as well as the specific manufacturer’s collection and transport devices for the organism sought, must be provided. Correct specimen handling and timely transport to the laboratory are critical for detecting the pathogen in question.
- The vaginal fornices are visualized using a speculum moistened with warm water.
- Lubricants containing antibacterial agents should not be used.
- The specimen is collected by swabbing the vaginal walls and vaginal pool with one or more sterile, polyester-tipped swabs on a plastic shaft or swabs specifically designated by the manufacturer to collect epithelial cells as well as Vaginal discharge.
- Cotton swabs should be avoided 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) in viral cultures.
- The health-care provider examines the Vaginal discharge visually before placing the swab in a tube containing 0.5 to 1.0 mL of sterile physiologic saline.
- The tube is sealed before being transported to the laboratory, where the specimen is processed for microscopic examination.
- To dislodge particulates from the swab, vigorously twirl it in the saline. Failure to dislodge particles may result in incorrect results.
- Before immersing specimens in saline, pH paper should be used to test them.
- A sample of vaginal discharge can be diluted in one to two drops of normal saline solution and placed directly on a microscope slide as an alternative method of specimen preparation.
- A second sample is then immersed in a 10% KOH solution in the same manner.
- For microscopic examination, cover slips are placed over both slides.
|Appearance||White, flocculent discharge|
|pH||3.8 to 4.2|
|Amine (Whiff) test||Negative|
|Other cells||Absent (except RBCs during menses)|
|Other organisms||Other lactobacilli subgroups, occasional yeast|
- Specimens that have been properly labeled should be placed in a biohazard bag with the requisition and transported to the laboratory as soon as possible.
- The requisition must include the patient’s name and unique identifier, as well as a medical history that includes menstrual status, the use of vaginal creams, lubricants, and douches, and recent exposure to sexually transmitted diseases.
- Specimens should be analyzed as soon as possible, but if a delay in transport or analysis is required, specimen handling is determined by the suspected pathogen.
- To preserve the motility of Trichomonas vaginalis and the recovery of Neisseria gonorrhoeae, specimens for C. trachomatis and herpes simplex virus must be kept at room temperature, whereas specimens for C. trachomatis and herpes simplex virus must be refrigerated to prevent overgrowth of normal flora.
- T. vaginalis specimens should be examined within 2 hours of collection.
Color and Appearance
- Normal vaginal fluid has a white appearance and a flocculent discharge.
- Normal vaginal flora is dominated by large, rod-shaped, gram-positive lactobacilli and squamous epithelial cells.
- If the patient is menstruating, white blood cells and red blood cells may be present.
- Abnormal Vaginal discharge can manifest as an increased thin, homogeneous white-to-gray discharge, a white “cottage cheese”–like discharge specific to Candida infections, or an increased yellow-green, frothy, adherent discharge associated with T. vaginalis. C. trachomatis can cause yellow, opaque cervical discharge.
When performing a pelvic examination, the health-care provider can perform a vaginal pH test. Before immersing the swab in saline or KOH solutions, the test should be performed. To more accurately evaluate pH values in the 4.5 range, use commercial pH test paper with a narrow pH range. The test paper is immersed in the pooled Vaginal discharge, and the color change is compared to a pH chart. Contamination of Vaginal discharge with cervical mucus, sperm, and blood can all interfere with the pH test.
The pH test aids in determining the cause of vaginitis. In women with vulvovaginal candidiasis, the vaginal pH is usually around 4.5, but it is higher in women with bacterial vaginosis, trichomoniasis, desquamative inflammatory vaginitis (DIV), and atrophic vaginitis.
As previously stated, the bacteria lactobacilli predominate in normal vaginal flora, producing the end product lactic acid from glycogen metabolism. Lactic acid creates an acidic vaginal environment with a pH range of 3.8 to 4.5. This acidity inhibits the growth of infectious organisms such as Mobiluncus, Prevotella, and Gardnerella vaginalis, thereby maintaining the normal vaginal bacteria flora balance. Some lactobacilli subgroups also produce hydrogen peroxide, which is toxic to pathogens and aids in maintaining an acidic vaginal pH to protect against urogenital infections. Bacterial vaginosis has been linked to a lack of lactobacilli that produce hydrogen peroxide. Estrogen production is also required to maintain an acidic vaginal environment.
Microscopic examination is usually used to diagnose vaginal infections. The initial screening tests are saline wet mounts and KOH mounts, and the Gram stain is used as a confirmatory examination for yeast or bacterial vaginosis. Slides are made from the saline specimen solution made immediately after collecting the vaginal swab. If a Gram stain is requested, three clean glass slides are labeled with the patient’s name and a unique identifier. Using a disposable transfer pipette, a drop of specimen is placed on each slide. Another option is to press the swab against the slide and then roll it over the slide. The Gram stain slide is allowed to dry before being heat-fixed for the Gram stain procedure and examination in the laboratory’s microbiology section. Cells and organisms are counted per high power field (hpf) (40) in wet mount examinations; cells and organisms are counted per oil immersion field (100) in Gram stains.
Wet Mount Examination
A cover slip is carefully placed on the specimen to exclude air bubbles before the saline wet mount examination. The slide is examined microscopically with a bright-field microscope’s low power (10) and high dry power (40) objectives. The slide is scanned with a low power objective (100 magnification) for an even distribution of cellular components, epithelial cell types and numbers, epithelial cell clumping, and the presence of budding yeast or pseudohyphae. The slide is then examined with a high power objective (400 magnification), and the organisms and cells are counted and reported per high power field (hpf) using the criteria listed in the table below. Squamous epithelial cells, white blood cells, red blood cells, clue cells, parabasal cells, basal cells, bacteria, motile Trichomonas vaginalis, yeast, and hyphae/pseudohyphae are common constituents found in vaginal fluid wet mounts.
|Rare||Less than 10 organisms or cells/slide|
|1+||Less than 1 organism or cell/hpf|
|2+||1 to 5 organisms or cells/hpf|
|3+||6 to 30 organisms or cells/hpf|
|4+||>30 organisms or cells/hpf|
Intravaginal medications may produce oil droplets, which can obstruct the interpretation of wet mounts. A Gram stain is useful in this case to detect yeast or bacterial vaginosis.
Squamous Epithelial Cells
Squamous epithelial cells measure 25 to 70 µm in diameter and exhibit a polygonal “flagstone” appearance. They 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. These large, flat cells originate from the linings of the vagina and female urethra and are present in significant numbers in the Vaginal discharge of a healthy female. Clumps of epithelial cells are an indication of the presence of increased numbers of yeast.
Clue cells are an abnormal variation of squamous epithelial cells distinguished by coccobacillus bacteria attached in clusters on the cell surface and spreading past the cell’s edges, making the border indistinct or stippled. This gives the cell a granular, irregular appearance that has been dubbed “shaggy.” Clue cells can be used to diagnose bacterial vaginosis caused by G. vaginalis. The presence of clue cells has also been discovered in urine sediment.
White Blood Cells
White blood cells (WBCs) have a granular cytoplasm and are 14 to 16 m in diameter. Because of their multi-lobed nucleus, they are frequently referred to as polymorphonuclear white blood cells (PMNs). WBCs are present in Vaginal discharge in rare to scant numbers in healthy women. Greater than 3+ WBCs in Vaginal discharge suggest vaginal candidiasis, atrophic vaginitis, or infections with Trichomonas, Chlamydia, Neisseria gonorrhoeae, or Herpes simplex.
Red Blood Cells
Red blood cells (RBCs) are smooth, non-nucleated biconcave disks that are 7 to 8 m in diameter. RBCs in vaginal specimens can be slightly distorted. RBCs are not typically found in Vaginal discharge, but they may be present during menstruation or as a result of a desquamative inflammatory process. RBCs can be confused with yeast cells and are distinguished from them by KOH, which lyses the RBCs while leaving the yeast cells intact.
Parabasal cells are round to oval in shape and range in size from 16 to 40 m in diameter. The nucleus-to-cytoplasm ratio is 1:1 to 1:2, with prominent basophilic granulation or amorphous basophilic structures (“blue blobs”) in the surrounding cytoplasm. They are found in the vaginal mucosa’s luminal squamous epithelium. Parabasal cells are uncommon in Vaginal discharge, but less mature cells may be present if the patient is menstruating or in postmenopausal women. If there are a lot of parabasal cells and a lot of WBCs, it could mean you have desquamative inflammatory vaginitis.
Basal cells are located deep in the basal layer of the vaginal stratified epithelium. These cells are round and measure 10 to 16 µm in diameter and have a nucleus to cytoplasm ratio of 1:2. Basal cells are distinguished from WBCs that are similar in size by their 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.2
The vaginal cavity is a nonsterile environment with a complex endogenous bacterial flora that varies with the patient’s age and hormonal status. Lactobacillus spp. make up the vast majority of vaginal bacteria. On Gram stain, they appear as large gram-positive, nonmotile rods that produce lactic acid, which keeps the vaginal pH between 3.8 and 4.5. Lactobacilli subgroups produce hydrogen peroxide, which can help to suppress the growth of other organisms. Anaerobic streptococci, diphtheroids, coagulase-negative staphylococci, and alpha hemolytic streptococci are also common. Vaginitis can occur when conditions exist that cause an imbalance in the normal flora. The absence or decrease in the number of lactobacilli relative to the number of squamous epithelial cells indicates a change in the normal flora. Lactobacilli are frequently replaced by increased numbers of Mobiluncus spp. (thin, curved, gram-negative, motile rods), Prevotella spp., Porphyromonas spp., Bacteroides spp. (anaerobic gram-negative rods), Gardnerella vaginalis (short, gram-variable coccobacilli), Peptostreptococcus spp
Trichomonas vaginalis is an atrial flagellated protozoan that can infect and cause vaginal inflammation in women. The organism is oval in shape, measures 5 to 18 m in diameter, and has four anterior flagella as well as an undulating membrane that runs the length of the body. An axostyle cuts the trophozoite longitudinally and protrudes from the back end, allowing the organism to attach to the vaginal mucosa and cause tissue damage. In a wet mount, the flagella’s “jerky” motion and the undulating membrane of T. vaginalis can be seen. T. vaginalis should not be confused with sperm, which has a single tail, a much smaller head (about one-half the diameter of an RBC), and no axostyle. Furthermore, nonmotile trichomonads can be confused with WBCs.
T. vaginalis organisms lose viability quickly after collection. To observe the organism’s motility, specimens must be examined as soon as possible or, if necessary, kept at room temperature for a maximum of 2 hours before preparing the wet mount. Trichomonas can be found in a urinary microscopic sample, but it cannot be reported unless there is motility, either across the slide or just in the tail. A dead trichomonad is oval in shape and slightly larger than a WBC.
Candida albicans and non–Candida spp. cause most fungal infections but an occasional yeast in Vaginal discharge is considered part of the normal flora. Yeast cells appear on a wet mount as both budding yeast cells (blastophores) or as hyphae, which are long filaments that grow and form a mycelium. Pseudohyphae, multiple buds that do not detach and form chains, can also be seen. It can be difficult to distinguish yeast cells from RBCs on a wet mount because both measure about 7 to 8 µm in diameter; however, differentiation can be made using the KOH test. Yeast cells stain gram positive.
KOH Preparation and Amine Test
The amine (Whiff) test is performed by placing a drop of the saline specimen prepared from the collection swab onto a properly labeled clean slide and adding one drop of 10% KOH solution. The slide is immediately examined for the presence of a “fishy” amine odor. The outcome is either positive (the presence of a fishy odor) or negative (absence of fishy odor). Polyamines are released into the vaginal fluid as the number of anaerobic bacteria in the vagina increases. The odor is caused by the volatilization of amines when KOH is added. Because volatile amines are not present in normal Vaginal discharge, a positive test result indicates bacterial vaginosis caused by an increase in the number of G. vaginalis in combination with Mobiluncus spp. and T. vaginalis.
After the amine test, place a cover slip over the specimen, taking care to avoid air bubbles. Allow the KOH mixture to sit for 5 minutes to dissolve epithelial and blood cells. Heat can be used to accelerate the dissolving process. To prevent specimen deterioration, one drop of 10% glycerin may be added after the KOH. The slide is examined under low magnification (100) for the presence of yeast pseudohyphae and under high magnification (400) to identify smaller blastospores (yeast cells).
Other Diagnostic Tests
Although wet mount and KOH slide examinations, as well as the amine test, are commonly used to diagnose bacterial vaginosis, additional tests may be required for confirmation. Among them are specimen culture, DNA probe testing, Gram stain, proline aminopeptidase assay, and point-of-care test kits.
The Gram stain is considered the gold standard for identifying the organisms that cause bacterial vaginosis. It also keeps a record of the patient’s specimen. A weighted combination of the following morphotypes constitutes a scored Gram stain system: Lactobacillus acidophilus (large gram-positive rods), G. vaginalis and Bacteroides spp. (small gram-variable or gram-negative rods), and Mobiluncus spp (curved gram-variable rods). The various types of bacterial morphophytes are assessed and scored. Lactobacillus morphophytes, for example, are the most common bacteria in normal vaginal flora; thus, if 4+ lactobacillus morphophytes are present on Gram stain and Gardnerella and Bacteroides spp. morphophytes, as well as curved gram-variable rods, the score is 0. A Nugent score of 0 to 3 indicates normal vaginal flora, a score of 4 to 6 indicates intermediate, and a score of 7 or higher indicates bacterial vaginosis.
Culture using various types of media is the gold standard test for detecting yeast and Trichomonas; however, it is more time consuming and can take up to two days to produce a result. G. vaginalis culture is not diagnostic for bacterial vaginosis because it is found in the normal flora of 50% of healthy women.
T. vaginalis requires a special medium known as Diamond’s medium. A commercial transport and culture pouch system for Trichomonas detection is now available (InPouch TV, Biomed Diagnostics, White City, OR). The specimen must be inoculated into the pouch within 30 minutes of collection and then incubated at 37°C in a CO2 atmosphere for 5 days. Every day, the pouch is microscopically examined for motile trichomonads.
To specifically identify the pathogen causing vaginitis, DNA hybridization probe methods have been developed. Affirm VPIII (Becton, Dickinson, Franklin Lakes, NJ) DNA probe testing system is available for the differential diagnosis of G. vaginalis, Candida spp., and T. vaginalis. It is simple to use and produces results in one hour with a sensitivity of 95%. When compared to traditional microscopic tests, this test is significantly more sensitive than wet mount microscopy and less susceptible to personnel bias.
DNA probes amplified by polymerase chain reaction can also detect Trichomonas (PCR). Enzymes are added to the specimen, and PCR is used to amplify specific regions of T. vaginalis’ DNA. The total number of DNA fragments is then determined. This is the most precise diagnostic method, with the added benefit of detecting nonviable organisms.
Point of Care Tests
There are several rapid diagnostic tests available to screen for the causative agents of vaginitis, and they have a higher sensitivity and specificity for the organism sought. Rapid antigen tests, for example, can detect proline aminopeptidase activity in Vaginal discharge to identify G. vaginalis. The OSOM Trichomonas Rapid Test (Genzyme Diagnostics, Cambridge, MA) is a 10-minute immunochromatographic strip test that detects T. vaginalis antigen in vaginal swabs. The test is carried out by immersing the vaginal swab in the sample buffer provided with the kit. Trichomonas proteins are dissolved in the buffer. The anti-trichomonas antibody-coated test stick is inserted into the sample mixture. If trichomonas antigens are present, they will react with the antibodies on the stick as the solution migrates up the stick. A visible blue line and a red internal control line indicate a successful outcome.
The OSOM BVBLUE test (Genzyme Diagnostics, Cambridge, MA) detects vaginal fluid sialidase, an enzyme produced by bacterial pathogens such as Gardnerella, Bacteroides, Prevotella, and Mobiluncus that cause bacterial vaginosis. The test takes one minute to complete and is read by looking at the change in color of the solution: blue or green is positive, yellow is negative.
To identify bacterial vaginosis and trichomonas, commercial tests to measure elevated vaginal pH (VS-Sense Pro Swab) and the presence of amines (FemExam pH and Amines TestCard, Litmus Concepts, Inc., Santa Clara, CA) use pH indicators and an amine test system that is visually read.