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Microbiology

Vaginitis

This section outlines the laboratory methods used to detect the causative agents of vaginitis, a common condition characterized by inflammation of the vagina. The focus is on the detection of Trichomonas vaginalis, Candida spp., and the diagnosis of bacterial vaginosis (BV)

Trichomonas vaginalis

Microscopic Examination

  • Wet Mount
    • Procedure: A drop of vaginal discharge is mixed with a drop of saline on a microscope slide. A coverslip is applied, and the slide is examined under a microscope at low and high power (400x magnification)
    • Visualization: T. vaginalis is a motile, pear-shaped protozoan with flagella. It may be difficult to visualize if the organism is non-motile or if the sample is old. The organism may be identified by its characteristic jerky movement, which is often described as “twitching”
    • Sensitivity: The sensitivity of wet mount is variable, ranging from 50-80%
  • Gram Stain
    • Procedure: A Gram stain is performed on a smear of vaginal discharge
    • Visualization: T. vaginalis is not readily visible on Gram stain
    • Sensitivity: Not a primary method for detection, but may be seen if present
  • Pap Smear (Papanicolaou Stain)
    • Procedure: A cervical smear is stained using the Pap stain
    • Visualization: T. vaginalis can be visualized on a Pap smear, but the sensitivity is low. The organism appears as a small, oval or pear-shaped organism with a pale cytoplasm and a small, eccentric nucleus
    • Sensitivity: Low sensitivity, not a primary method for detection

Culture

  • Procedure: Vaginal discharge is inoculated onto a specific culture medium designed to grow T. vaginalis. Diamond’s medium or InPouch TV culture system are commonly used
  • Incubation: Cultures are incubated at 37°C for several days
  • Visualization: The culture is examined microscopically for the presence of motile organisms
  • Sensitivity: Higher sensitivity (75-95%) than wet mount, but results take longer
  • Advantage: Can be used for antimicrobial susceptibility testing

Rapid Antigen Tests

  • Procedure: These tests detect T. vaginalis antigens in vaginal discharge. Several commercially available tests are available
  • Formats: Lateral flow immunoassays (similar to home pregnancy tests) and point-of-care tests are available
  • Sensitivity: Sensitivity is variable, but generally higher than wet mount
  • Advantage: Rapid results (10-30 minutes)
  • Limitations: May not be as sensitive as nucleic acid amplification tests (NAATs)

Nucleic Acid Amplification Tests (NAATs)

  • Procedure: These tests amplify the DNA or RNA of T. vaginalis
  • Formats: PCR-based assays, transcription-mediated amplification (TMA)
  • Sensitivity: Very high sensitivity (95-100%) and specificity
  • Advantage: Highly sensitive, can be performed on vaginal swabs or urine samples
  • Disadvantage: Requires specialized equipment and trained personnel

Candida spp.

Microscopic Examination

  • Wet Mount
    • Procedure: Vaginal discharge is mixed with a drop of saline and a drop of 10% potassium hydroxide (KOH) solution on a microscope slide. The KOH lyses epithelial cells and other debris, making the yeast easier to visualize. A coverslip is applied, and the slide is examined under a microscope at low and high power (400x magnification)
    • Visualization: Candida spp. appear as budding yeast cells and/or hyphae (filamentous structures). The presence of hyphae is indicative of a more invasive infection
    • Sensitivity: Moderate sensitivity
  • Gram Stain
    • Procedure: A Gram stain is performed on a smear of vaginal discharge
    • Visualization: Candida spp. appear as Gram-positive, oval-shaped yeast cells. Budding yeast and/or pseudohyphae may be seen
    • Sensitivity: Moderate sensitivity, similar to wet mount
  • Pap Smear
    • Procedure: A cervical smear is stained using the Pap stain
    • Visualization: Candida spp. appear as oval, yeast-like organisms
    • Sensitivity: Lower sensitivity compared to wet mount

Culture

  • Procedure: Vaginal discharge is inoculated onto a culture medium designed to grow yeast, such as Sabouraud’s dextrose agar or CHROMagar Candida
  • Incubation: Cultures are incubated at 30°C for several days
  • Visualization: Candida spp. colonies appear as creamy, white, or buff-colored colonies
  • Identification: Colonies can be identified based on colony morphology, Gram stain appearance, and biochemical tests (e.g., germ tube test, carbohydrate assimilation tests)
  • Sensitivity: Higher sensitivity than microscopic examination
  • Advantage: Allows for species identification and antifungal susceptibility testing (if needed)

Rapid Tests

  • Germ Tube Test
    • Procedure: A small amount of yeast colony is mixed with serum and incubated
    • Visualization: Candida albicans produces germ tubes, which are hyphae-like structures
    • Sensitivity: High for identifying C. albicans
  • Commercial Rapid Tests
    • Procedure: Some commercial tests are available that detect Candida antigens in vaginal discharge
    • Sensitivity: Variable

Bacterial Vaginosis (BV)

BV is characterized by a disruption of the normal vaginal flora, with a decrease in Lactobacillus spp. and an increase in the concentration of other bacteria, including Gardnerella vaginalis, Prevotella spp., and Mobiluncus spp. The diagnosis of BV is primarily based on clinical criteria, but laboratory tests are often used to confirm the diagnosis

Amsel’s Criteria

  • Clinical Diagnosis: BV is diagnosed if at least three of the following four Amsel’s criteria are met:
    1. Homogeneous, thin, white or gray vaginal discharge: The discharge often adheres to the vaginal walls
    2. Vaginal pH > 4.5: Measured using pH paper
    3. Positive “Whiff Test” (amine odor): A drop of 10% KOH is added to a sample of vaginal discharge. A fishy odor (due to the release of amines) is considered a positive result
    4. Presence of “Clue Cells” on Wet Mount
      • Procedure: A wet mount is prepared using vaginal discharge
      • Visualization: Clue cells are vaginal epithelial cells that are coated with bacteria, giving them a characteristic stippled appearance
      • Sensitivity: High specificity

Microscopic Examination

  • Wet Mount
    • Procedure: A wet mount is prepared using vaginal discharge
    • Visualization
      • Clue Cells: The hallmark of BV
      • Reduced or Absent Lactobacilli: Decreased numbers of Lactobacillus spp
      • Increased Numbers of Other Bacteria: Including Gardnerella vaginalis, Mobiluncus spp., and other anaerobic bacteria
      • Motile curved rods: Mobiluncus species may be seen
    • Sensitivity: Moderate to high sensitivity, especially for detecting clue cells
    • Interpretation
      • Nugent Score: Gram stain is used to score bacteria (0-10) based on the presence of Lactobacilli and Gardnerella vaginalis

Gram Stain (Nugent Score)

  • Procedure: A Gram stain is performed on a smear of vaginal discharge
  • Visualization: The bacterial morphotypes are scored based on the presence of Lactobacillus spp., Gardnerella vaginalis, and curved Gram-variable or Gram-negative rods (Mobiluncus spp.)
  • Scoring: The Nugent score is calculated based on the relative abundance of these bacteria. A score of 0-3 is considered normal, 4-6 is intermediate, and 7-10 is diagnostic of BV
  • Sensitivity: High
  • Specificity: High

Culture

  • Procedure: Culture is not routinely used for the diagnosis of BV
  • Disadvantage: Culturing the multiple organisms is not practical

Other Tests

  • Commercial Tests: Some commercial tests are available to detect specific BV-associated organisms (e.g., G. vaginalis)
  • DNA Probe Tests: Tests that detect BV-associated organisms by DNA probe or nucleic acid amplification tests

Key Considerations

  • Specimen Collection: Proper specimen collection is essential for accurate results. Swabs must be collected from the vaginal walls and transported in appropriate transport media
  • Quality Control: Strict quality control measures should be in place to ensure the accuracy and reliability of the laboratory tests
  • Correlation with Clinical Findings: Laboratory results must always be interpreted in conjunction with the patient’s clinical presentation, symptoms, and other relevant information
  • Sensitivity and Specificity: The sensitivity and specificity of each test vary. The choice of test depends on the clinical situation, the availability of resources, and the desired level of accuracy
  • Point-of-Care Testing (POCT): Some rapid tests are available for use at the point of care, allowing for rapid diagnosis and treatment
  • Reporting: The laboratory report should include the results of all tests performed, along with any relevant interpretive comments
  • Antimicrobial Susceptibility Testing: Antimicrobial susceptibility testing is not routinely performed for T. vaginalis or Candida spp
  • Follow-up: In some cases, follow-up testing may be necessary to confirm the diagnosis or to monitor the response to treatment

Key Terms

  • Vaginitis: Inflammation of the vagina
  • Wet Mount: A microscopic preparation of a specimen mixed with saline or KOH
  • KOH (Potassium Hydroxide): A chemical used to lyse cells and debris in wet mounts
  • Clue Cells: Vaginal epithelial cells coated with bacteria, characteristic of bacterial vaginosis
  • Hyphae: Filamentous structures produced by fungi
  • Budding Yeast: A form of yeast cells that are reproducing
  • Germ Tube: A hyphae-like structure produced by Candida albicans
  • Amsel’s Criteria: Clinical criteria used to diagnose bacterial vaginosis
  • Whiff Test: A test for bacterial vaginosis that involves the addition of KOH to vaginal discharge
  • pH: A measure of acidity or alkalinity
  • Nugent Score: A scoring system based on Gram stain findings used to diagnose bacterial vaginosis
  • NAAT (Nucleic Acid Amplification Test): A laboratory test that amplifies DNA or RNA to detect microorganisms
  • Antigen: A substance that triggers an immune response
  • Sensitivity: The ability of a test to correctly identify individuals with a disease
  • Specificity: The ability of a test to correctly identify individuals without a disease
  • Lateral Flow Immunoassay: A rapid test that detects the presence of a substance in a sample
  • Point-of-Care Testing (POCT): Diagnostic testing performed near the patient
  • Antimicrobial Susceptibility Testing: Laboratory tests to determine which antibiotics are effective against a specific microorganism