Syphilis Serology

Aka: Syphilis Serology, Syphilis Antibody, Syphilis Testing, Treponema Pallidum Antibody Measurement, Syphilis Screening, Syphilis Diagnosis, Syphilis False Positive

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II. Indication

  1. Syphilis (Treponema pallidum) detection
  2. Syphilis Screening at least annually for all Men who have Sex with Men and others at high risk of Syphilis
  3. Skin lesions or other clinical findings suggestive of Syphilis
  4. Confirmation of positive Screening Test
  5. Genital Ulcer

III. Labs: Non-Treponemal Tests

  1. Background
    1. Non-Treponemal Test (Syphilis Serology)
      1. Syphilis infection and its associated cellular injury releases multiple Antigens
      2. Released Antigens (e.g. cardiolipin, lecithin) trigger Antibody development in Syphilis infected patients
    2. Antigen is an extract from beef heart (Non-Treponemal Derived Substance)
      1. Combination of Cholesterol, cardiolipin and lecithin (extracts of beef heart)
      2. Antigen mix is a similar for RPR and VDRL
    3. Antigen mix precipitates Antibody
      1. Non-specific Syphilis antibodies bind Antigens, and result in observed Agglutination (RPR, VDRL)
  2. Tests
    1. Venereal Disease Research Laboratory (VDRL)
      1. VDRL mixes serum and Antigen on a glass slide, and uses light microscopy to visualize Agglutination
    2. Rapid Plasma Reagin (RPR)
      1. RPR uses a a disposable card with impregnated carbon particles (allows for reading with the naked eye)
      2. Agglutination (clumping) indicates serum contains IgG or IgM Antibody to RPR Antigen
      3. Similar to VDRL Test, but faster and more simple to perform (RPR does not require a microscope)
    3. Serum Toluidine Red Unheated Serum Test (TRUST)
      1. Similar, to RPR, but impregnates paint particles into disposable cards
      2. TRUST is typically associated with a higher Test Sensitivity than RPR
    4. Other tests
      1. Automated Reagin Test (ART)
      2. Standard Test for Syphilis (STS)
  3. Efficacy
    1. Serum Test Specificity: 85-99%
      1. See False Positive causes below
    2. Serum Test Sensitivity in untreated Syphilis
      1. Primary Stage (Chancre stage)
        1. False Negatives are most common in the first 4 weeks
        2. After one week: 30%
        3. After three weeks: 90%
      2. Secondary Stage: 100%
      3. Tertiary Stage: 90%
      4. Latent Stage: may be unreactive
    3. CSF Test Sensitivity
      1. VDRL has a higher Test Sensitivity in CSF samples (Neurosyphilis) than RPR
      2. Marra (2012) Sex Transm Dis 39(6):453-7 +PMID: 22592831 [PubMed]
  4. False Negative Causes
    1. Testing too early after infection (esp. first 4 weeks)
    2. Prozone Phenomenon
      1. High Antibody levels result in a paradoxically negative sample
  5. False Positives
    1. See Non-Treponemal Test
    2. Recent infections (e.g. Malaria, HIV, Tuberculosis)
    3. Other Treponemal organisms (Yaws, Pinta, Bejel)
    4. Autoimmune disorders (Systemic Lupus, Rheumatoid Arthritis)
    5. Pregnancy
    6. Recent Immunizations (e.g. COVID19 Vaccine)
    7. Intravenous Drug Abuse

IV. Labs: Treponemal Specific Tests

  1. Background
    1. Treponemal Antigen precipitates Antibody (False Negatives in the first 2 weeks)
    2. Treponemal Specific Tests are identifying Treponemal antibodies
      1. Contrast with Non-Treponemal Tests which identify cardiolipin and lecithin antibodies
    3. Treponemal Tests do NOT differentiate Treponemal species
      1. Other Treponemal infections (Yaws, Pinta, Bejel) will cause false positive Treponema pallidum
  2. Rapid Immunoassays (used as first step in reverse sequence Syphilis Screening below)
    1. Methadologies (dozens of available immunoassays)
      1. Enzyme immunoassay (EIA)
      2. Chemiluminescence immunoassays (CIA)
      3. Microbead Immunoassays (MBIA)
    2. Composite Immunoassay efficacy
      1. Test Specificity >94% (except 83% for TrepSure)
      2. Test Sensitivity: 94 to 100%
        1. Primary Syphilis: >94% (most assays)
        2. Secondary Syphilis: 100%
        3. Early Latent Syphilis: 95% to 100%
        4. Late Latent Syphilis: 92% to 100%
  3. Manual Assays (highest Test Specificity, used as definitive confirmatory test)
    1. Treponema Pallidum Particle Agglutination Assay (TP-PA)
      1. Highest efficacy of the Manual Treponemal Assays
      2. Test Specificity: 99%
      3. Test Sensitivity
        1. Primary Syphilis: 86 to 100%
        2. Secondary Syphilis: 100%
        3. Early Latent Syphilis: 94 to 100%
        4. Late Latent Syphilis: 87 to 100%
    2. Fluorescent Treponemal Antibody (FTA-ABS)
      1. Test Specificity: 87-100%
      2. Test Sensitivity
        1. Primary Syphilis: 78 to 100%
        2. Secondary Syphilis: 93 to 100%
        3. Early Latent Syphilis: 94 to 100%
        4. Late Latent Syphilis: 84 to 93%
    3. Microhemagglutination - Treponema pallidum (MHA-TP)
      1. Test Specificity: 99%
      2. Test Sensitivity
        1. Primary Syphilis: 46 to 89%
        2. Secondary Syphilis: 90 to 100%
        3. Early Latent Syphilis: 94 to 100%
        4. Late Latent Syphilis: 97%
  4. References
    1. Park (2020) Clin Infect Dis 71(Suppl 1):S13-20 +PMID: 32578866 [PubMed]

V. Labs: Dark-field Microscopy

  1. Most specific test if primary or Secondary Syphilis with active lesions available to sample
    1. Most specific if Chancre or Condyloma Latum is present
    2. May detect Syphilis at any stage if an active lesion may be sampled
  2. Can result in immediate diagnosis in the first week without the 3 week delay witing for IgM to develop
  3. Accuracy varies with experience of technician and a negative test does not exclude Syphilis

VI. Protocol: Reverse Sequence Syphilis Screening

  1. Protocol employed by many U.S. labs as of 2022
  2. Step 1: Automated Treponemal Antibody Test
    1. Testing by enzyme immunoassay (EIA, CLIA) or similar allows for rapid, high volume sample screening
    2. Risk of False Positives in low risk populations
    3. Treponemal Antibody tests are positive for life after initial infection (regardless of treatment)
  3. Step 2: Nontreponemal Tests (e.g. RPR or VDRL)
    1. Confirmation testing requires human read cards/slides for Agglutination
    2. Levels fall with treatment and over time since infection
    3. Four fold increase in titers suggests reinfection
  4. Step 3: Manual manual Treponema pallidum–specific assay (e.g. TP-PA Agglutination Assay)
    1. Indicated if there is a discrepancy, with a positive step 1, but a negative step 2
    2. Not needed if Step 1 and Step 2 are both positive

VII. Protocol: Testing

  1. Screening
    1. Nontreponemal tests (RPR or VDRL)
      1. Syphilis Screening (positive within 3 weeks of developing primary Chancre)
      2. Syphilis RPR positive test will be returned with titer (e.g. 1:16)
      3. After treatment, by 6 months, RPR should fall by a factor of 4 (e.g. 1:4)
      4. On subsequent infection, expect the RPR titer to once again rise
    2. HIV Screening (test all patients who are positive for Syphilis)
      1. HIV coinfection with Syphilis is common
      2. HIV patients are at higher risk of Neurosyphilis
  2. Negative test with lesions present or other strong clinical indicators
    1. Repeat screening in 2-3 weeks
  3. Confirmation of positive Screening Test
    1. Fluorescent Treponemal Antibody (FTA-ABS)
    2. Microhemagglutination - Treponema pallidum (MHA-TP)
  4. Neurosyphilis CSF Evaluation
    1. See Neurosyphilis
    2. Indications for Lumbar Puncture with CSF Exam
      1. All patients with Syphilis and neurologic symptoms
      2. All patients with serologic or exam findings consistent with treatment failure
      3. HIV patient specific criteria
        1. CD4 Count <350 cells/mm3 or
        2. Rapid plasmin reagin (RPR) >1:32
  5. Monitoring response to treatment
    1. Non-Treponemal Antibody test (e.g. RPR) will normalize after treament
      1. Levels decline overtime, and then increase >4 fold with reinfection
    2. Treponemal Tests (Antibody) will remain positive for life despite treatment

VIII. Efficacy

  1. Diagnostic Test Sensitivity in Primary Syphilis
    1. Dark-field Exam of Chancre: 80%
    2. Non-Treponemal Tests (e.g. RPR): 78-86%
    3. Treponemal Tests (e.g. FTA-ABS): 76-84%
  2. Diagnostic Test Sensitivity in Secondary Syphilis
    1. Dark-field Exam of Chancre: 80%
    2. Non-Treponemal Tests (e.g. RPR): 100%
    3. Treponemal Tests (e.g. FTA-ABS): 100%
  3. Diagnostic Test Sensitivity in Latent Syphilis
    1. Non-Treponemal Tests (e.g. RPR): 95-100%
    2. Treponemal Tests (e.g. FTA-ABS): 97-100%
  4. Diagnostic Test Sensitivity in Tertiary Syphilis
    1. CSF evaluation required (see below)
    2. Non-Treponemal Tests (e.g. RPR): 71-73%
    3. Treponemal Tests (e.g. FTA-ABS): 94-96%

IX. Resources

  1. Diagnostic Tests for Syphilis
    1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4999316/

X. Reference

  1. Kirk, McHugh and Parnell (2023) Crit Dec Emerg Med 37(8): 23-9
  2. Mason and von Reinhart (2018) EM:Rap 18(6): 19-20
  3. Bakerman (1984) ABCs of Interpretive Lab Data, p. 392
  4. Larsen (1995) Clin Microbiol Rev 8:1-21 [PubMed]

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