Laboratory diagnosis of Bacterial vaginosis caused by Gardnerella vaginalis

Bacterial vaginosis (BV) is a common condition that affects many women of reproductive age. It is caused by an imbalance of the normal vaginal flora, which leads to an overgrowth of certain bacteria, especially Gardnerella vaginalis. G. vaginalis is a gram-variable rod-shaped bacterium that can adhere to the epithelial cells of the vagina and form a biofilm. This biofilm disrupts the protective function of the vaginal mucosa and increases the risk of infection and inflammation.

BV is associated with various symptoms and complications, such as:

• A thin, white or gray vaginal discharge with a fishy odor
• Vaginal itching, burning or irritation
• Pain during urination or sexual intercourse
• Increased susceptibility to sexually transmitted infections (STIs), such as chlamydia, gonorrhea, herpes and HIV
• Increased risk of pelvic inflammatory disease (PID), which can cause infertility, ectopic pregnancy and chronic pelvic pain
• Increased risk of adverse pregnancy outcomes, such as preterm birth, low birth weight and intrauterine infection

The exact cause of BV is not fully understood, but it is thought to be influenced by various factors, such as:

• Sexual activity: Having multiple or new sexual partners, or having sex with a partner who has BV, can increase the chance of getting BV. However, BV can also occur in women who are not sexually active.
• Hormonal changes: Fluctuations in estrogen and progesterone levels during the menstrual cycle, pregnancy or menopause can affect the vaginal pH and flora.
• Hygiene practices: Douching, using scented soaps or feminine products, or wearing tight or synthetic underwear can alter the vaginal environment and favor the growth of G. vaginalis and other bacteria.
• Antibiotic use: Taking antibiotics for other infections can kill the beneficial lactobacilli that normally keep the vaginal flora in balance and prevent BV.

The diagnosis of BV is based on a combination of clinical and laboratory criteria. The most commonly used methods are:

• The Amsel criteria: These are four clinical signs that indicate BV: a) a thin, white or gray discharge; b) a vaginal pH above 4.5; c) a positive whiff test (a fishy odor when adding potassium hydroxide to the discharge); and d) the presence of clue cells (epithelial cells covered with bacteria) on microscopic examination of the discharge.
• The Nugent score: This is a quantitative method that involves grading a gram stain of the vaginal smear according to the number and type of bacteria present. A score of 0 to 3 indicates normal flora; a score of 4 to 6 indicates intermediate flora; and a score of 7 to 10 indicates BV.

The treatment of BV aims to restore the normal vaginal flora and prevent complications. The most effective antibiotics for BV are metronidazole and clindamycin, which can be taken orally or applied topically. However, recurrence rates are high, and some women may need repeated or prolonged courses of therapy. Alternative treatments, such as probiotics, boric acid or hydrogen peroxide, have been suggested but their efficacy and safety are not well established.

BV is a common and treatable condition that can have serious consequences if left untreated. Therefore, it is important for women to be aware of the signs and symptoms of BV and seek medical attention if they suspect they have it. Moreover, preventive measures, such as practicing safe sex, avoiding douching and using unscented products, can help maintain a healthy vaginal flora and reduce the risk of BV.

The specimen for laboratory diagnosis of BV is usually a vaginal discharge sample collected by the patient or a clinician. The sample should be obtained from the posterior fornix of the vagina, avoiding contamination with cervical or vulvar secretions. The specimen should be transported to the laboratory as soon as possible, preferably within 2 hours of collection. If transport is delayed, the specimen should be refrigerated at 4°C or stored in a transport medium that preserves the viability and morphology of the bacteria.

The vaginal discharge specimen can be used for different types of tests, such as:

• Direct microscopic examination of vaginal fluid or swab: This is the most common and simple test for BV. A drop of vaginal fluid or a swab is placed on a glass slide and stained with Gram stain. The slide is then examined under a microscope for the presence and proportion of different types of bacteria. The diagnosis of BV is based on the Nugent score, which assigns points to different bacterial morphotypes according to their Gram stain characteristics. A score of 0-3 indicates normal flora, 4-6 indicates intermediate flora, and 7-10 indicates BV. Alternatively, the Hay/Ison criteria can be used, which classify the vaginal flora into four grades based on the presence or absence of lactobacilli, Gardnerella/Bacteroides morphotypes, and clue cells (epithelial cells covered with bacteria). A grade of I indicates normal flora, II indicates intermediate flora, III indicates BV, and IV indicates severe BV.
• Culture using enriched media and incubation conditions: This is a more sensitive and specific test for BV, but it is also more expensive and time-consuming. The vaginal discharge specimen is inoculated onto selective and differential media that support the growth of Gardnerella vaginalis and other anaerobic bacteria associated with BV. The media include blood agar, Loeffler`s serum slope, or dextrose starch agar. The plates are incubated at 37°C in an anaerobic atmosphere with 5% carbon dioxide and high humidity for 48 hours. The colonies of Gardnerella vaginalis are small, domed, and surrounded by a zone of beta-hemolysis on blood agar, or a zone of clearing on dextrose starch agar. The colonies can be further identified by biochemical tests, such as catalase, oxidase, hippurate hydrolysis, and indole production.

One of the most common and reliable methods for diagnosing BV is the direct microscopic examination of a vaginal fluid or swab sample. This technique involves staining the sample with a Gram stain, which differentiates bacteria based on their cell wall structure. Gram-negative bacteria appear pink or red, while Gram-positive bacteria appear purple or blue.

In a healthy vaginal flora, the predominant bacteria are Gram-positive lactobacilli, which produce lactic acid and hydrogen peroxide to maintain a low pH and inhibit the growth of other microbes. In BV, the lactobacilli are replaced by a diverse mixture of Gram-negative and Gram-variable bacteria, such as Gardnerella vaginalis, Prevotella spp., Mobiluncus spp., and others. These bacteria produce amines and other substances that increase the pH and cause a foul-smelling discharge.

The microscopic examination of a Gram-stained sample can reveal the presence and relative abundance of these bacteria. The most characteristic finding is the presence of clue cells, which are epithelial cells covered with adherent bacteria. Clue cells are considered diagnostic of BV and can be seen in more than 90% of cases. Other findings include the absence or scarcity of lactobacilli, the presence of many small Gram-negative or Gram-variable rods and cocci, and the presence of curved Gram-negative rods (Mobiluncus spp.).

The direct microscopic examination of a vaginal fluid or swab sample is a simple, inexpensive, and rapid technique that can be performed in any laboratory or clinic. It has a high sensitivity and specificity for diagnosing BV and can also help to rule out other causes of vaginal discharge, such as yeast infections or trichomoniasis. However, it requires trained personnel and quality control to ensure accurate results. It may also miss some cases of BV that have low bacterial counts or mixed infections. Therefore, it is recommended to use this technique in combination with other methods, such as culture or clinical criteria.

Culture is another method to identify the presence of Gardnerella vaginalis in vaginal specimens. Culture can provide more accurate and specific results than direct microscopic examination, but it is also more time-consuming and requires special media and conditions.

To culture Gardnerella vaginalis, the following steps are followed:

• A vaginal swab or fluid sample is collected using a sterile cotton swab or a speculum.
• The sample is inoculated onto an enriched medium, such as human or rabbit blood agar, Loeffler`s serum slope, or dextrose starch agar. These media provide nutrients and indicators for the growth of Gardnerella vaginalis and other bacteria that may be associated with BV.
• The inoculated media are incubated at 37°C for 48 hours in an atmosphere of 5% carbon dioxide and high humidity. This mimics the conditions of the vaginal environment and enhances the growth of Gardnerella vaginalis.
• The cultured media are examined for the presence of characteristic colonies of Gardnerella vaginalis. These colonies are small, domed, and surrounded by a zone of beta-hemolysis (clearing) on blood agar, or a zone of starch hydrolysis (clearing) on dextrose starch agar. The colonies may also have a fishy odor when exposed to air.
• The colonies can be further confirmed by performing Gram stain, biochemical tests, or molecular methods to differentiate them from other bacteria that may grow on the same media.

Culture is a useful technique to diagnose BV caused by Gardnerella vaginalis, but it has some limitations. It may not detect all cases of BV, as some women may have low numbers of Gardnerella vaginalis or mixed infections with other bacteria. It may also yield false-positive results, as some women may have asymptomatic colonization of Gardnerella vaginalis without BV. Therefore, culture results should be interpreted in conjunction with other clinical and laboratory criteria, such as the Amsel criteria.

Besides the Gram stain, BV can be diagnosed by using the Amsel criteria, which are based on four clinical signs:

• Thin, white, yellow, homogeneous discharge. This is the most common symptom of BV and it usually has a foul or fishy odor.
• pH measurement of vaginal fluid >4.5. A normal vaginal pH is between 3.8 and 4.5, which indicates a healthy balance of lactobacilli and other bacteria. A pH higher than 4.5 suggests a shift in the vaginal flora and a possible overgrowth of anaerobic bacteria such as Gardnerella vaginalis.
• Performance of an amine test: Release of a fishy odor on adding alkali—10% potassium hydroxide (KOH) solution (Whiff Test). This test is performed by adding a drop of KOH solution to a sample of vaginal fluid on a slide and sniffing it. A positive result indicates the presence of volatile amines produced by anaerobic bacteria, which react with KOH to produce a characteristic fishy smell.
• Wet mount microscopy of vaginal secretions: observation for clue cells. Clue cells are epithelial cells that are coated with bacteria, giving them a granular or stippled appearance. They are seen under a microscope when a drop of saline is added to a sample of vaginal fluid on a slide. The presence of clue cells is indicative of BV and helps to differentiate it from other causes of vaginitis such as Candida or Trichomonas.

A diagnosis of BV is made when at least three out of the four Amsel criteria are met. However, some studies have suggested that the Amsel criteria may have low sensitivity and specificity, and that other methods such as Nugent scoring (based on Gram stain) or molecular testing (based on PCR) may be more accurate and reliable . Therefore, the Amsel criteria should be used with caution and in conjunction with other clinical and laboratory findings.

Bacterial vaginosis (BV) is a common vaginal infection that can cause symptoms such as discharge, odor, itching, and burning. BV is caused by an imbalance of the normal bacteria in the vagina, which can be triggered by factors such as sexual activity, douching, hormonal changes, or antibiotic use.

BV can sometimes go away on its own, but it can also lead to complications such as pelvic inflammatory disease (PID), preterm labor, low birth weight, and increased risk of sexually transmitted infections (STIs). Therefore, it is important to seek medical attention if you have symptoms of BV or if you are pregnant and have BV.

The main treatment for BV is antibiotics, which can be taken orally or applied topically to the vagina. The most commonly used antibiotics are metronidazole and clindamycin. These antibiotics work by killing the bacteria that cause BV and restoring the normal vaginal flora.

Oral metronidazole is usually taken twice a day for 7 days. It can cause side effects such as nausea, vomiting, metallic taste, headache, and dark urine. It can also interact with alcohol and some other medications, so you should avoid drinking alcohol and inform your doctor about any other drugs you are taking while on metronidazole.

Topical clindamycin is usually applied once a day for 7 days. It can cause side effects such as vaginal irritation, burning, itching, dryness, and yeast infection. It can also weaken latex condoms and diaphragms, so you should use alternative methods of contraception while on clindamycin.

Both oral and topical antibiotics have similar effectiveness in curing BV. However, some studies suggest that topical clindamycin may be more effective in preventing recurrence of BV than oral metronidazole. Therefore, your doctor may recommend one or the other depending on your preference, medical history, and risk factors.

In addition to antibiotics, you can also take some steps to prevent or reduce the recurrence of BV. These include:

• Avoiding douching or using scented products in the vagina
• Wearing cotton underwear and loose-fitting clothes
• Changing tampons or pads frequently
• Washing the vulva with water only or mild soap
• Having regular check-ups and screenings for STIs
• Practicing safe sex by using condoms or dental dams
• Limiting the number of sexual partners or being monogamous
• Taking probiotics or eating yogurt with live cultures

Bacterial vaginosis is a treatable condition that can affect your quality of life and your reproductive health. If you have symptoms of BV or if you are pregnant and have BV, you should consult your doctor for diagnosis and treatment. By following the prescribed antibiotics and taking preventive measures, you can restore your vaginal health and prevent complications.

We are Compiling this Section. Thanks for your understanding.