Clinical Education Center
Vaginitis — Molecular Testing To Address Diagnostic Challenges
Vaginitis is the most common gynecologic diagnosis in the primary care setting, affecting one in three women in their lifetime, but diagnosing its cause can be challenging.1
Dr. Dale Schwab, Senior Scientific Director, Infectious Disease, Quest Diagnostics Nichols Institute and Focus Diagnostics Reference Laboratory, discusses the etiology of vaginitis and reviews how advances in test technology are assisting physicians in their differential diagnosis.
“A complex microbiome exists in the vagina,” says Dr. Schwab. “This biome consists of a wide range of organisms but usually at low numbers with the exception of Lactobacillus spp.” These organisms include Gram-positive cocci,such as Streptococcus spp., Gram variable rods such as Gardnerella vaginalis, Gram-negative organisms such as E. coli, anaerobes such as Bacteriodes spp, and the cell-wall deficient organisms, Mycoplasma spp. and Ureaplasma spp. as well as low numbers of yeast such as Candida albicans.2, 3
“The levels of these various organisms vary in each woman, and the concentrations will vary during a woman’s menstrual cycle,” continues Dr. Schwab. “However, in a healthy vagina, the Lactobacilli are always predominant. The metabolism of these organisms maintains the optimal vaginal pH between 3.8 and 4.2, and some species produce hydrogen peroxide, which is toxic to other organisms. Vaginitis - inflammation of the vagina, often accompanied by discharge - develops when the vaginal flora has been altered by introduction of a pathogen or by changes in the vaginal environment that allow organisms generally present at a low number to proliferate.”
The three diseases most frequently associated with vaginitis are bacterial vaginosis (BV), caused by an overgrowth of bacteria including Gardnerella vaginalis and anaerobes, vaginal trichomoniasis (TV), caused by Trichomonas vaginalis, and vulvovaginal candidiasis, usually caused by Candida albicans. Cervicitis can also sometimes cause a vaginal discharge but because the diagnostic tests and treatments for cervicitis are different from those for vaginitis, it is important to differentiate these conditions. 4 - 6
“Any inflammation in the genital tract increases susceptibility to other diseases,” notes Dr. Schwab. “So, if a woman has vaginitis she is more prone to an infection with Neisseria gonorrhoeae (NG), Chlamydia trachomatis or even HIV. BV and TV are both associated with adverse pregnancy outcomes, including premature rupture of the membranes, preterm labor and delivery, and low birth weight. The Centers for Disease Control and Prevention (CDC) therefore recommends treatment of BV and TV for symptomatic pregnant women.” 4
Obtaining an accurate diagnosis can have a significant impact on therapy. “Some physicians may judge that Candida isn’t the cause, so they prescribe Flagyl, as that treats Trichomonas and BV,” says Dr. Schwab. “But there’s a big problem with that assumption since Trichomonas is sexually transmitted, while BV is not. So, if you’re not diagnosing adequately, you’re not treating the partner and the woman is going to get re-infected. It is also possible to have dual-infections.”
Traditional Diagnostic Approaches
If the findings of the patient’s history and/or physical examination suggest that the patient has vaginitis, a sample of the vaginal discharge is obtained for examination. A standard office examination assesses the discharge according to the Amsel criteria. This involves using a microscopic wet-mount preparation to look for clue cells, testing the pH of the vaginal fluid and performing a “whiff” test to detect amines. The drawback of this approach is that most physicians don’t have the time to conduct the test or they may not be sufficiently proficient with the microscope.
The traditional laboratory-based test for bacterial vaginosis is the Nugent Gram stain test. This is a quantitative Gram stain that looks at the relationship between Lactobacillus morphotypes and organisms that are morphologically similar to Gardnerella vaginalis, Bacteroidesor Mobiluncus, and provides a score based on that relationship. A high score, reflecting a low number of Lactobacilli and a high number of the other morphotypes, is consistent with a diagnosis of bacterial vaginosis.
“A complicating factor with the traditional tests,” says Dr. Schwab, “is that there’s not always agreement between the Amsel and Nugent criteria. Furthermore, interpreting the Nugent test can be somewhat subjective: if a woman is clearly normal or abnormal it’s easy to read, but in the middle section of the scoring it’s very subjective.”
If testing did not indicate BV, a physician would typically perform a Gram stain or wet mount for Candida species. “With a very severe infection you’ll see yeast and hyphae, but some Candida, like Candida glabrata, doesn’t produce hyphae - it just appears as small round cells, which are often mistaken for epithelial cells or white cells. These account for between 10% and 20% of infections, and can easily be missed. For Trichomonas no sensitive test has been available - wet mounts and Pap smears are only around 50% sensitive.” 7
Molecular Testing – Accurate and Convenient
Since traditional testing could result in misdiagnosis or under-diagnosis, there have been efforts over recent years to develop molecular tests, which can provide greater sensitivity and specificity. The first test, BD Affirm TM VP III, to address this was a direct molecular test to detect the DNA of Candida species, Trichomonas vaginalis, and Gardnerella vaginalis. To account for the number of Gardnerella vaginalis present in normal flora the test detects 2 x 105 CFU or more. For Candida spp. it detects 1,0000 CFU and for Trichonomas vaginalis, 5,000 CFU. While the test was a significant advance on traditional testing, and less subjective, it became evident that it was over-sensitive for G. vaginalis and not sufficiently sensitive for T. vaginalis.
Molecular nucleic acid amplification tests (NAAT) are now available, which provide high levels of sensitivity and specificity, and can be ordered individually or as a panel.“The Quest Diagnostics test for BV quantifies the level of Lactobacillus species compared with that of indicator organisms, recognizing that BV is not caused by any single organism but by an imbalance,” explains Dr. Schwab. “We’re looking at situations where you get a decrease in the Lactobacilli and an increase in the indicator organisms,” explains Dr. Schwab. “The levels referenced by the test are based on some important studies performed on the vaginal microbiome to determine relative levels in normal women and in women with vaginitis.8-10 We feel it is important to provide a quantitative result, not just a qualitative one. Our BV test provides a clear indication as to whether or not an imbalance exists versus conventional qualitative approaches that test for a broad array of organisms.The Candidiasis NAAT test detects and differentiates four species of Candida: albicans, glabrata, tropicalis, parapsilosis. The non-C.albicans species tend to be more resistant to standard treatment, so they are important to detect. Trichomonas vaginalis is detected by an FDA cleared NAAT and can detect < 1 organism /mL as the assay targets ribosomal RNA which is present in greater than a million copies/organism.”11
Convenience and Accuracy
The panels now available, based on advanced technology, offer several benefits to physicians. “In the current environment where physicians have a larger case load and are seeing increasingly more patients per hour, it’s very difficult to spend enough time with the patient to apply the Amsel criteria and perform some of the testing. The advanced tests now available allow physicians to get an accurate diagnosis with one vaginal swab and multiple tests, consistent with current guidelines, enabling them to provide adequate treatment and follow-up. Our test also allows physicians to test for chlamydia and gonorrhea at the same time, so when appropriate they can take that opportunity to test for those organisms with the same sample.”
Addressing the Complexities of Cervicitis
Diagnosing cervicitis also presents particular challenges, since the exact etiology is often unknown. The organisms that are known to cause disease and can be diagnosed are C. trachomatis and N. gonorrhoeae. Recently a third organism has been shown to play a significant role and that is Mycoplasma genitalium. “Culturing this organism requires special medium and about a month to grow in the lab, so the only way you can effectively diagnose it is molecular,” notes Dr. Schwab. ”So, the molecular approach is also very useful for diagnosing cervicitis, as it allows testing for CT, NG and Mycoplasma genitalium with just one endocervical swab.”
- Egan M and Lipsky M. Diagnosis of Vaginitis. Am Fam Physician. 2000 Sep 1;62(5):1095-1104. http://www.aafp.org/afp/2000/0901/p1095.html Accessed June 17, 2014
- Larsen B and Monif G. Understanding the Bacterial Flora of the Female Genital Tract Clin Infect Dis. (2001) 32 (4): e69-e77.doi: 10.1086/318710
- Priestley C, Jones B, Dhar J, Goodwin L. Genitourin Med 1997;73:23-28 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1195755/pdf/genitmed00001-0030.pdfAccessed June 17, 2014
- Diseases Characterized by Vaginal Discharge. Centers for Disease Control and Prevention. http://www.cdc.gov/std/treatment/2010/vaginal-discharge.htm Accessed June 17, 2014
- Gynecologic Problems. Frequently Asked Questions. American College of Obstetricians and Gynecologists. http://www.acog.org/~/media/For%20Patients/faq028.pdf?dmc=1&ts=20140617T1524236447 Accessed June 17, 2014
- Changes in the 2010 STD Treatment Guidelines What Adolescent Health Care Providers Should Know. American College of Obstetricians and Gynecologists. https://www.acog.org/About_ACOG/ACOG_Departments/Adolescent_Health_Care/Changes_in_the_2010_STD_Treatment_Guidelines__What_Adolescent_Health_Care_Providers_Should_Know
- Wiese W, Patel SR, Patel SC, Ohl CA, Estrada CA. A meta-analysis of the Papanicolaou smear and wet mount for the diagnosis of vaginal trichomoniasis. Am J Med. 2000 Mar;108(4):301-8.
- Fredricks DN, Fiedler T, Marrazzo JM. Molecular identification of bacteria associated with bacterial vaginosis. N Engl J Med. 2005; 353(18): 1899-1911.
- Srinivasan S, Hoffman N, Morgan M, Matsen F, Fiedler T et al Bacterial Communities in Women with Bacterial Vaginosis: High Resolution Phylogenetic Analyses Reveal Relationships of Microbiota to Clinical Criteria http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0037818 Accessed June 17, 2014
- Ravela J, Gajera P Abdob Z, Schneiderc GM, Koeniga S.Vaginal microbiome of reproductive-age women http://www.pnas.org/content/early/2010/06/02/1002611107.full.pdf Accessed June 17, 2014