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Microbiology

Culture & Molecular

This section details the laboratory methods used for the detection of several key pathogens in the genital tract: Neisseria gonorrhoeae, Chlamydia trachomatis, Streptococcus agalactiae (Group B Streptococcus, or GBS), and Mycoplasma spp. The focus is on culture and molecular detection methods, which are critical for accurate diagnosis and effective treatment

Neisseria gonorrhoeae

Culture

  • Procedure: Specimens (e.g., endocervical swabs, urethral swabs, urine, etc.) are inoculated onto selective media. Thayer-Martin agar (TMA), Martin-Lewis agar, or New York City (NYC) medium are commonly used
    • Thayer-Martin Agar (TMA): Chocolate agar base with antibiotics (vancomycin, colistin, nystatin) to inhibit the growth of other organisms
    • Martin-Lewis Agar: Similar to TMA but with a different antibiotic combination
    • New York City (NYC) Medium: Contains lysed horse blood and antimicrobial agents
  • Incubation: Cultures are incubated at 35-37°C in a CO2-enriched atmosphere (5-10% CO2)
  • Colony Morphology: N. gonorrhoeae colonies appear as small, translucent, greyish, or tan, convex colonies on selective media. They are oxidase-positive
  • Identification: Presumptive identification is based on colony morphology, Gram stain (Gram-negative diplococci), and oxidase test
    • Confirmation: Confirmatory tests are performed, including carbohydrate utilization tests, rapid tests, or molecular methods
      • Carbohydrate Utilization Test: N. gonorrhoeae ferments glucose but not other carbohydrates like maltose, lactose, or sucrose
      • Rapid Tests: Enzyme immunoassays (EIAs) and immunochromatographic assays are available
  • Sensitivity: Moderate sensitivity, depends on specimen type and culture technique
  • Antibiotic Susceptibility Testing (AST): Performed on isolates to determine antibiotic resistance patterns. Disk diffusion or broth microdilution are used

Molecular Detection

  • Nucleic Acid Amplification Tests (NAATs)
    • Procedure: NAATs amplify the DNA or RNA of N. gonorrhoeae
    • Formats: Polymerase chain reaction (PCR)-based assays, transcription-mediated amplification (TMA), and other amplification methods
    • Specimens: Can be performed on various specimens, including swabs (endocervical, urethral, etc.), urine, and other body fluids
    • Sensitivity: Highly sensitive (95-100%)
    • Specificity: High
    • Advantages: Rapid results, high sensitivity, can be performed on non-invasive specimens (urine)
    • Disadvantages: Requires specialized equipment and trained personnel
  • Probe-Based Tests: Nucleic acid probes detect specific genetic sequences of N. gonorrhoeae. Less common than NAATs

Chlamydia trachomatis

Culture

  • Procedure: Specimens (e.g., endocervical swabs, urethral swabs) are inoculated onto cell cultures. McCoy cells are often used
  • Incubation: Cultures are incubated at 35-37°C
  • Staining: After incubation, the cells are stained with an immunofluorescent antibody (IFA) or Giemsa stain
  • Visualization: C. trachomatis appears as intracytoplasmic inclusions within the cells
  • Sensitivity: Lower sensitivity compared to NAATs
  • Disadvantages: Labor-intensive, time-consuming, and requires specialized cell culture facilities

Molecular Detection

  • Nucleic Acid Amplification Tests (NAATs)
    • Procedure: NAATs amplify the DNA or RNA of C. trachomatis
    • Formats: Polymerase chain reaction (PCR)-based assays, transcription-mediated amplification (TMA), and other amplification methods
    • Specimens: Can be performed on various specimens, including swabs (endocervical, urethral, etc.), urine, and other body fluids
    • Sensitivity: Highly sensitive (95-100%)
    • Specificity: High
    • Advantages: Rapid results, high sensitivity, can be performed on non-invasive specimens (urine)
    • Disadvantages: Requires specialized equipment and trained personnel
  • Advantages: Highly sensitive and specific, fast results, can be performed on urine samples
  • Disadvantages: Requires specialized equipment and trained personnel
  • Other Molecular Methods: Hybridization assays

Other Methods

  • Enzyme Immunoassays (EIAs): Detect C. trachomatis antigens. Less sensitive than NAATs
  • Direct Fluorescent Antibody (DFA) Tests: Use fluorescently labeled antibodies to detect C. trachomatis antigens. Less sensitive than NAATs

Streptococcus agalactiae (Group B Streptococcus, GBS)

Culture

  • Procedure: Vaginal and rectal swabs are collected from pregnant women for prenatal screening. Specimens are inoculated onto blood agar
  • Incubation: Incubated at 35-37°C
  • Colony Morphology: S. agalactiae colonies are small, greyish, translucent, and beta-hemolytic (produce a clear zone around the colonies due to red blood cell lysis) on blood agar
  • Identification
    • Gram Stain: Gram-positive cocci in chains or pairs
    • Catalase Test: Negative
    • CAMP Test: A positive CAMP test is a characteristic feature. S. agalactiae produces a substance that enhances the hemolysis of Staphylococcus aureus when the two organisms are streaked perpendicular to each other on a blood agar plate
    • Serological Tests: Latex agglutination tests can be used to identify S. agalactiae
  • Sensitivity: High if proper culture techniques are used
  • Antimicrobial Susceptibility Testing (AST): Performed on isolates to determine antibiotic resistance patterns if requested

Molecular Detection

  • PCR-based assays: Real-time PCR assays can be used to detect S. agalactiae DNA directly from vaginal and rectal swabs
  • Advantages: Rapid results, high sensitivity
  • Disadvantages: Requires specialized equipment and trained personnel

Mycoplasma spp.

  • Mycoplasmas: are small, cell-wall-deficient bacteria that can colonize the genital tract. Mycoplasma hominis and Ureaplasma urealyticum are the most frequently isolated species. Mycoplasma genitalium is a sexually transmitted pathogen

Culture

  • Procedure: Specimens (e.g., urethral swabs, endocervical swabs) are inoculated onto specialized culture media
    • Media: Requires specialized media containing serum and other growth factors. Mycoplasma hominis requires arginine, while Ureaplasma urealyticum requires urea in the media
  • Incubation: Incubated at 35-37°C
  • Colony Morphology: The colonies are very small and may require microscopic examination to visualize. Mycoplasma hominis may produce “fried-egg” colonies. Ureaplasma urealyticum produces granular colonies
  • Identification
    • Urease Test: Ureaplasma urealyticum produces urease, which can be detected by a color change in the media
    • Biochemical tests
  • Sensitivity: Limited
  • Antibiotic Susceptibility Testing (AST): Can be performed, but not routinely done

Molecular Detection

  • PCR-based assays: PCR assays are the most commonly used method
    • Procedure: PCR assays are available for detection of Mycoplasma genitalium in the genital tract
    • Specimens: Swabs, urine
    • Sensitivity: High
    • Specificity: High
  • Hybridization assays: Less common

Key Considerations

  • Specimen Collection: Proper specimen collection is essential for accurate results. Swabs must be collected from the appropriate sites and transported in appropriate transport media
  • Transport and Storage: Follow laboratory guidelines for transport media, storage temperature, and holding times
  • 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. NAATs are generally more sensitive than culture
  • Antimicrobial Resistance: Antimicrobial resistance is an important consideration for N. gonorrhoeae. AST is performed to guide treatment
  • Reporting: The laboratory report should include the results of all tests performed, along with any relevant interpretive comments
  • Follow-up: In some cases, follow-up testing may be necessary to confirm the diagnosis or to monitor the response to treatment

Key Terms

  • Neisseria gonorrhoeae: The bacterium that causes gonorrhea
  • Chlamydia trachomatis: The bacterium that causes chlamydia
  • Streptococcus agalactiae: Group B Streptococcus (GBS), a bacterium that can cause infections in newborns and pregnant women
  • Mycoplasma spp.: Small, cell-wall-deficient bacteria
  • Culture: A laboratory procedure used to grow microorganisms
  • Molecular Detection: Laboratory tests that detect the presence of genetic material
  • NAAT (Nucleic Acid Amplification Test): A laboratory test that amplifies DNA or RNA to detect microorganisms
  • PCR (Polymerase Chain Reaction): A molecular technique used to amplify DNA
  • TMA (Transcription-Mediated Amplification): A molecular technique used to amplify RNA
  • Selective Media: Culture media that inhibit the growth of certain organisms while promoting the growth of others
  • Antibiotic Susceptibility Testing (AST): Laboratory tests to determine which antibiotics are effective against a specific microorganism
  • CAMP Test: A test used to identify Streptococcus agalactiae
  • Beta-hemolysis: Complete breakdown of red blood cells
  • Intracytoplasmic Inclusions: Structures found inside cells that contain the organism
  • Enzyme Immunoassay (EIA): A laboratory test that uses antibodies to detect antigens
  • Antigen: A substance that triggers an immune response
  • Direct Fluorescent Antibody (DFA) Test: A test that uses fluorescently labeled antibodies to detect antigens
  • Mycoplasma genitalium: A sexually transmitted pathogen
  • Urease: An enzyme that breaks down urea
  • Hybridization assays: A molecular technique used to detect specific DNA sequences