The vaginal epithelium is a hormone responsive non-keratinized stratified squamous epithelium. With estrogen production, you see the maturation of the cells with a thickening of the mucosa. The normal vaginal flora of reproductive-age women includes multiple aerobic, facultative anaerobic and obligate anaerobic species. Anaerobes dominate aerobes 10:1. The bacteria are symbiotic with the host and are alterable depending on the vaginal microenvironment.
Normal Vaginal Flora
- Staphylococcus aureus
- Staphylococcus epidermidis
- Group B Streptococci
- Enterococcus faecalis
- Staphylococcus spp
- Escherichia coli
- Klebsiella species
- Proteus species
- Enterobacter species
- Acinetobacter species
- Citrobacter species
- Pseudomonas species
Anaerobic gram-positive cocci
- Peptostreptococcus species
- Clostridium species
Anaerobic gGram-positive bacilli
- Lactobacillus species
- Propionibacterium species
- Eubacterium species
- Bifidobacterium species
- Actinomyces israelii
- Prevotella species
- Bacteroides species
- Bacteriodes fragillis species
- Fusobacterium species
- Veillonella sspeciesp
The function of vaginal bacterial colonization is not clear. The flora produces lactic acid and hydrogen peroxide that inhibit pathogenic bacteria.
The composition of the vaginal flora is responsible for the pH of the vagina. With estrogen comes the production of glycogen from the vaginal mucosa. The glycogen is the nutrient necessary for many vaginal ecosystem species seen in reproductive age women, including Lactobacilli. The glycogen is metabolized to lactic acid contributing to the normal vaginal pH of 3.8-4.2. This acidity suppresses the overgrowth of infectious organisms such as Mobiluncus, Prevotella, and Gardnerella vaginalis.
In pre-pubertal girls and post-menopausal women, the lack of estrogen leads to a deficiency of glycogen and thus a paucity of lactic acid producing flora. The normal pre-pubertal and post-menopausal vaginal pH is 6 to 7.5. Females can be more prone to infections at these times when the only commensal flora is mainly of skin origin, but infections are still more common in reproductive years.
Changing any element of the vaginal ecology can alter the population characteristics of the vaginal bacteria. Changes in hormonal status, as previously mentioned with estrogen, can greatly shift the makeup of the flora. Menses can act as a nutrient base for some bacterial species leading to their overgrowth, but there is no clear evidence that this is associated with pathogens or infection. Broad-spectrum antibiotic use can lead to alteration of the vaginal bacterial flora leading to Candida species overgrowth. Douching and unprotected vaginal intercourse can increase pH as well.
History and Physical
Normal Vaginal Secretions
Few vaginal secretions are expected in reproductive age women. The secretions are white and flocculent and consist of the desquamated vaginal mucosa, vaginal epithelium transudate, mucous secretions from the endocervix, endometrial gland secretions, lactic acid, Bartholin gland secretions and sebaceous gland secretions from the vulva. There will be a wide spectrum of what is normal from patient-to-patient.
Symptoms prompting evaluation include patient reports of abnormal vaginal discharge, malodorous discharge, vaginal irritation, dysuria, and dyspareunia. The differential diagnosis includes bacterial vaginosis, vulvovaginal candidiasis, trichomoniasis, desquamative inflammatory vaginitis, atrophic vaginitis, allergies, chemical irritation, cervicitis, and foreign bodies.
The evaluation should begin with a patient history noting the character of symptoms (discharge, pruritus, pain, bleeding, etc.) and their timing. The patient should be questioned about previous episodes of similar symptoms. Inquire about hygienic practices including the use of lubricants and douches. Obtain a sexual history and note previous sexually transmitted infections. Search for possible new allergen exposures. Finally, review the medical history looking for immune suppression and recent steroid or antibiotic use.
The gross examination is completed to characterize any discharge present. pH testing should be completed with the testing strip placed directly into the vaginal pool or along the side wall. Cervical mucus, semen, and blood will all falsely elevate the pH.
A speculum exam should be performed with warm water. Lubricants can contain antibacterial agents that will affect the specimens. Make use of polyester-tipped swabs on plastic shafts, or specifically designated swabs from diagnostic test’s manufacturers. Cotton is toxic to Neisseria gonorrhea, and the wood in the shafts of swabs can be toxic to Chlamydia trachomatis.
Diagnosis of Vaginitis
The Wet Prep
After specimen collection is completed during the exam, the swab is transferred to a tube containing 0.5 mL to 1 mL of physiologic saline. The swab should be vigorously swirled to dislodge particulates. Three slides are prepared by placing a droplet utilizing a disposable transfer pipette. The three slides are a wet mount, potassium hydroxide mount and a slide for gram stain.
Exams should proceed immediately after specimen collection. If this is not possible, the specimen should be kept at room temperature and examined within 2 hours. Cooling will limit the trichomonas motility.
The wet mount is examined immediately. The 10X objective is used to assess the types of the epithelial cells present (mature, parabasal, basal or clue cells) and to establish the presence of budding yeast or pseudohyphae. The 40X objective is used to count organisms and cells per the high-power field (HPF).
- Squamous epithelial cells are polygonal cells. The central nucleus is roughly the size of a red blood cell (RBC). There is a large amount of irregular cytoplasm. The cells’ margins are distinct. They are present in large numbers in the vaginal secretions of healthy women.
- Clue cells are an abnormal variation of the squamous epithelial cell. The cell is distinguished by coccobacillus bacteria which is attached in clusters to the cell surface. This makes the cells edges stippled and the borders indistinct. The cell is granular and irregular. Clue cells, when present in abundance (greater than 20% of squamous epithelial cells), indicate Gardnerella vaginalis overgrowth.
- White Blood Cells will appear one-half to one-third the size of squamous epithelial cells. They exhibit a granular cytoplasm. The multi-lobed nucleus lends to the name polymorphonuclear white blood cells (PMN’s). In normal secretions, they are present in small numbers. Greater than 3% white blood cells (WBC) suggest vaginal candidiasis, atrophic vaginitis or infections with trichomonas, chlamydia, gonorrhea or HSV.
- RBCs are one-half the size of WBCs and are smooth, non-nucleated biconcave disks. RBCs can be confused with yeast, but KOH will lyse them while yeast cells remain on the KOH mount.
- Parabasal Cells are larger than a WBC but smaller than squamous epithelial cells. They are round to oval shaped with a nucleus to cytoplasm level ratio of 1:1 or 1:2. The cytoplasm contains basophilic granulation or amorphic basophilic structures. These are rarely seen in normal vaginal secretions unless women are menstruating or postmenopausal. Parabasal cells present with many WBCs indicates desquamative inflammatory vaginitis.
- Basal Cells are roughly the same size as WBCs but with round nuclei. The nucleus-to-cytoplasm ratio is 1:2. These are not normally found in vaginal secretions. Their presence can indicate vaginal atrophy or in the presence of excessive WBCs, desquamative inflammatory vaginitis.
- Lactobacillus species should predominate in the healthy reproductive age vagina. These will appear as large, nonmotile rods.
- Trichomonas vaginalis is a flagellated protozoan slightly larger on average than a WBC. There are four anterior flagella and an undulating membrane that extends half the body length. An axostyle bisects the trophozoite longitudinally and protrudes from the posterior end. This enables the organism to attach to the vaginal mucosa.
- Yeast cells (blastospores) are of similar size to RBC’s. Pseudohyphae are multiple buds that instead of detaching, form chains. Yeast is best observed on the 10-fold objective.
A separate slide is used for the KOH prep. The saline-diluted specimen is added to the slide along with a drop of 10% KOH. Increased numbers of anaerobic bacteria (G. vaginalis, Mobiluncus, Trichomonas as opposed to a predominance of Lactobacillus) will lead to the production of amines. KOH will lead to volatilization of these amines leading to a “fishy” amine odor, thus the “Whiff test.” After performing the “Whiff test” place the coverslip and leave the slide to rest for 5 minutes. Letting the slide rest will allow for the dissolution of the epithelial cells and the RBCs. The microscopic exam can then be performed to search for yeast pseudohyphae and blastospores without interference from other cell types.
The gram stain remains the gold standard for identifying the causative agent for bacterial vaginosis but is only routinely used in the research setting. The gram stain slide must be heat-fixed. It is evaluated using the Nugent score. The Nugent score is calculated based on the observed quantities of Lactobacillus acidophilus, Gardnerella vaginalis, Bacteroides species and Mobiluncus species.
Cell culture has a limited role in the evaluation of vaginitis. It remains the gold standard for the detection of yeast. Unfortunately, the results are not timely. The culture of Gardnerella vaginalis is not useful as it is part of the normal flora in 50% of women. Trichomonas vaginalis can be cultured, but it requires a specific medium, Diamond’s medium, and is time-consuming and labor-intensive. Beyond recurrent yeast, culture is not clinically important to the evaluation of vaginitis.
DNA hybridization probes are available for G. vaginalis, Candida species, Trichomonas vaginalis, chlamydia, and gonorrhea and are being used increasingly in the evaluation of abnormal vaginal discharge. The sensitivities are very high and turn around is quick.
Various point-of-care tests exist for Trichomonas vaginalis, and Gardnerella vaginalis are commercially available. Most require in-house equipment (exception, OSOM for trichomonas which has the lowest sensitivity), but the turn-around time is 15 to 60 minutes. Their sensitivities are 90% or better.
Treatment / Management
BV is an imbalance of the normal vaginal flora. Loss of vaginal acidity can lead to a loss of Lactobacilli dominance, further alkalization of the vagina and overgrowth of pathogens such as G. vaginalis, Mobiluncus, Prevotella, Prophyromonas, Peptostreptococcus, Mycoplasma hominis, and Ureaplasma. Alternatively, loss of vaginal estrogen can lead to loss of glycogen. Without glycogen, Lactobacilli will not have the substrate for lactic acid production, and the pH will rise.
Risk factors for BV include new or multiple sex partners, frequent douching, intrauterine contraceptive devices and pregnancy. PROM, PPROM, PTL, PID, endometritis, sexually transmitted infections, and post-hysterectomy cuff cellulitis are all associated with BV.
The signs and symptoms of BV include a nonirritating, malodorous vaginal discharge. The vaginal mucosa and cervical epithelium will appear normal. There will be no cervical motion tenderness or pelvic pain on palpation.
First line diagnostics for bacterial vaginosis should include a physical exam, pH testing, and a wet prep. Amsel’s Diagnostic Criteria for BV has a 95% PPV. You must have three of the four criteria for the diagnosis of BV. The criteria are a thin, white, homogeneous vaginal discharge, a pH greater than 4.5, a positive amine whiff test and the presence of clue cells on microscopic examination. Again, the Gram stain is mainly for research purposes.
DNA hybridization probe for concentrations of Gardnerella, point-of-care vaginal sialidase testing (associated with BV pathogens) and bacterial morphology testing available from local labs are acceptable compared to Amsel and Nugent.
Recommended Regimens per the Centers for Disease Control and Prevention (CDC):
Metronidazole 500 mg orally twice a day for 7 days
Metronidazole gel 0.75%, one full applicator (5 g) intravaginally, once a day for 5 days
Clindamycin cream 2%, one full applicator (5 g) intravaginally at bedtime for 7 days
Tinidazole 2 g orally once daily for 2 days
Tinidazole 1 g orally once daily for 5 days
Clindamycin 300 mg orally twice daily for 7 days
Clindamycin ovules 100 mg intravaginally once at bedtime for 3 days
Patients should be advised to abstain from alcohol for 24 hours after completion of the metronidazole course and 72 hours after the tinidazole course to avoid the disulfiram-like reaction. There is no evidence to support the use of Lactobacillus probiotics in BV treatment or the recurrence of symptoms.
Eighty percent to 90% cure rates are expected 1 week from treatment. No routine follow-up is necessary for the asymptomatic patient after treatment, though 30% will recur at 3 months or later. With recurrent disease, the first step is to confirm the diagnosis of bacterial vaginosis again. For recurrence, women can be treated with the same regimen again or one of the alternative regimens. Multiple recurrences after completion of a recommended regimen can be treated with 0.75% metronidazole gel twice weekly for 4 to 6 months. While this has been shown to reduce recurrences, the benefit does not persist when suppressive therapy is discontinued. Another option for the persistent or recurrent disease is an oral nitroimidazole followed by intravaginal boric acid 600 mg daily for 21 days and then suppressive 0.75% metronidazole gel twice weekly for 4 to 6 months.
Trichomonas vaginalis is a parasitic protozoan, transmitted by sexual intercourse. It causes vaginitis in women and occasionally urethritis in men, but most males are asymptomatic. Trichomoniasis is associated with other STIs such as gonorrhea and chlamydia and can enhance transmission of HIV. As with all other STI’s, the diagnosis of trichomoniasis should prompt screening for other STIs. Trichomonas vaginalis infection in pregnancy is associated with low birth weight, PROM, and preterm delivery.
The signs and symptoms of Trichomoniasis include malodorous, green-to-yellow, frothy vaginal discharge. They may have vaginal pruritic and irritation, dysuria and dyspareunia. A physical exam will reveal vaginal mucosa erythema and punctate hemorrhages of the cervix.
The wet prep 60% to 70% sensitivity for trichomoniasis. The pH will be greater than 4.5. An amine test can be positive. Culture with Diamond’s medium has a 75% to 95% sensitivity but is rarely used as the available PCR test is 95% sensitive and produces quicker results. Overall, start with the wet prep and move on to the DNA probes if you do not identify trichomonas on the slide, but you are still suspicious.
Recommended Regimen per the CDC:
Metronidazole 2 gm by mouth once or tinidazole 2 gm by mouth once.
Metronidazole 500 mg by mouth two times per day for 7 days.
The nitroimidazoles are the only class of antimicrobial medications known to be effective against T. vaginalis infections. Only metronidazole and tinidazole have been cleared by FDA for the oral or parenteral treatment of trichomoniasis. Metronidazole gel does not reach therapeutic levels in the urethra and paravaginal glands and is not recommended for the treatment of trichomoniasis. Tinidazole is avoided in pregnancy and contraindicated in the first trimester.
The recommended metronidazole regimens have cure rates of approximately 84% to 98%, and the recommended tinidazole regimen have cure rates of approximately 92% to 100%.
Because of the high rate of reinfection among women treated for trichomonas (17% within three months in one study), retesting for T. vaginalis is recommended for all sexually active women within three months. Testing by nucleic acid amplification can be conducted as soon as two weeks after treatment. Metronidazole resistance occurs in 4% to 10% of cases of vaginal trichomoniasis, and tinidazole resistance in 1%, but most cases of “persistent” infection will be reinfection. When reinfection can be reliably ruled out a multi-day course such as metronidazole 500 mg orally twice daily for seven days can be ordered. The next regimen would be metronidazole or tinidazole at 2 g by mouth daily for seven days. If these regimens fail, consider referral to an infectious disease specialist and susceptibility testing.
Yeast vulvovaginitis is most often secondary to Candida albicans, but less common pathogens can include Candida glabrata, Candida parapsilosis, Candida tropicalis and Candida krusei. While Candida is a normal constituent of the vagina, risk factors for pathogenic overgrowth include antibiotic uses, combination oral contraceptives, estrogen therapy, pregnancy, diabetes, corticosteroid use and all forms of immune compromise.
The infection is most common during the childbearing years when estrogen is plentiful. Glycogen is key to facilitating Candida growth and adherence. Signs and symptoms of yeast vulvovaginitis include genital burning, pruritis, dyspareunia, dysuria and a thick, white, curd-like discharge.
The wet prep has a 60% to 70% sensitivity to yeast vaginitis. Budding yeasts, pseudohyphae, large numbers of WBCs, lactobacilli, and clumps of epithelial cells will be seen on the wet mount. The pH will be less than 4.5, and the amine “whiff” test will be negative.
A yeast culture is rarely needed. It can be ordered if nonalbicans species are suspected. Candida glabrata will not for pseudohyphae and is difficult to recognize on microscopy. DNA testing is also available to identify species.
Uncomplicated Vulvovaginal Candidiasis
Complicated Vulvovaginal Candidiasis
Recurrent (four or more episodes in a year), or
Non-Albicans species, or
Women with diabetes mellitus, HIV, debilitation, immunosuppressive therapy (corticosteroids) or other immunocompromised.
Uncomplicated: A short course (single dose; 1-day or 3-day course) of over-the-counter topical antifungals will result in cure rates of 80% to 90% for uncomplicated vulvovaginal candidiasis (clotrimazole, miconazole, tioconazole, butoconazole, itraconazole). A single dose by mouth of fluconazole 150 mg by mouth is also effective. No follow-up is needed if the symptoms resolve.
Recurrent candidiasis: 7 to 14 days of topical therapy or a 100-mg, 150-mg, or 200-mg oral dose of fluconazole every third day for a total of 3 doses [day 1, 4, and 7) can be used. Oral fluconazole (i.e., 100-mg, 150-mg, or 200-mg dose) weekly for 6 months is the first line maintenance regimen. 30% to 50% of women will have recurrent disease after maintenance therapy is discontinued.
Severe candidiasis: 7 to 14 days of topical azole or 150 mg of fluconazole in two sequential oral doses 72 hours apart.
Nonalbicans candidiasis: 7 to 14 days of a non-fluconazole azole regimen (oral or topical) as first-line therapy. If recurrence occurs, 600 mg of boric acid in a gelatin capsule is recommended, administered vaginally once daily for 2 weeks.
Desquamative Inflammatory Vaginitis
Signs and Symptoms of desquamative inflammatory vaginitis include profuse, purulent vaginal discharge, vaginal erythema, and dyspareunia. Causes are heterogeneous. Most will grow beta-hemolytic gram-positive Streptococcus in vaginal cultures. It is often found with vaginal atrophy.
The wet mount and gram stain will reveal large numbers of WBCs, RBCs, occasional parabasal and basal cells, squamous epithelial cells, reduced or absent lactobacilli and increased gram-positive cocci. The vaginal pH will be greater than 4.5, and the amine test will be negative. It is treated with vaginal 2% clindamycin.
Signs and Symptoms of atrophic vaginitis include vaginal dryness, dyspareunia, vaginal inflammation, thinned mucosa, loss of rugae and occasionally purulent discharge. A wet mount will demonstrate large numbers of WBCs, occasional parabasal and basal cells, decreased lactobacilli and increased gram-positive cocci and gram-negative rods. This is best treated with vaginal estrogen therapy.