Serologic Tests for Syphilis by mikeholy

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									Laboratory Tests for Syphilis

        Diana Nurutdinova MD
            Instructor in Medicine
   Washington University School of Medicine
                St. Louis, MO
Laboratory tests for syphilis
    Confirm clinical suspicion of disease
    Screen populations at risk
    Monitor response to therapy
    Determine treatment failure and need
    for lumbar puncture
Electron micrograph of Treponema pallidum on
cultures of cotton-tail rabbit epithelium cells
Types of laboratory tests
   Direct examination of lesion material
    – darkfield microscopy
   Serologic testing of blood samples
    – non-treponemal tests (screening)
    – treponemal tests (confirmatory)
    – direct fluorescent antibody (DFA)
    – histologic staining (biopsy)
Silver Stain
Darkfield microscopy

   Extremely specific for T. pallidum
   Test of choice for moist genital ulcers
   Offers immediate diagnosis
   Opportunity for immediate treatment
Darkfield Microscopy
   Confirms diagnosis of syphilis by
   directly demonstrating T. pallidum in
   material from suspected lesions or
   regional lymph nodes
   May be possible to diagnose primary
   syphilis before serologic tests become
Darkfield Microscopy
Principle of Darkfield
    Special microscope
     – Light rays strike
       objects in field
     – No direct rays enter
       microscope objective
     – Object appears self-
       luminous against a
       dark background
How Darkfield Works
Darkfield Microscopy
   All genital lesions should be considered
   syphilitic until a darkfield exam proves
   Other lesions (skin, mucous membrane)
   should also be examined if syphilis is
   However       darkfield exams require trained
   personnel, proper equipment, and
Primary syphilis - chancre
Primary syphilis - chancre
Moist lesions
   Remove scab, crust, exudate
   Squeeze base of lesion to produce
      tissue fluid on lesion surface
   Apply glass slide directly to exudate, or
      collect fluid with sterile loop
   Alternative method: “wash” lesion with
      sterile saline, transfer fluid to slide
Dry skin lesions
   Gently remove superficial skin with
     scalpel, needle tip, or mechanical
   Squeeze lesion base to collect tissue
   Avoid contamination of specimen with
Cervical/vaginal mucosal lesions
    Use speculum to better visualize lesion
    Remove cervical or vaginal discharge
        or exudate
    Compress lesion to produce serous
        fluid accumulation at base of lesion
    If necessary, use Kelly clamp to
Specimen Collection / Preparation

   Always observe universal precautions!
   Rapid transport from patient to
   Patience in viewing and interpreting
Examination of lesion material
   Place slide on darkfield microscope
   Apply oil to light source AND the slide
   Dim external room lighting
   Scan slide slowly for characteristic
      “corkscrew”-appearing organisms
   Minimum 10-minute review of slide
Remember. . .
  Don’t perform darkfield exam on ORAL
   lesions !
    – these can be colonized with non-
      pathogenic oral treponemes
    – presence of spirochetes in oral lesions
      does not necessarily rule in T. pallidum
Confounders of darkfield exam
   Use of topical antibiotics by patient may
      kill some organisms
   Lack of sufficient serous exduate on
      lesion surface
   Insufficient time spent scanning slide
   All of these factors can lead to a
      false-negative interpretation
Confirmatory testing for syphilis
    Stat serologic test at time of clinical
    Careful evaluation of patient sexual
       exposure history and lesion
When in doubt. . .

                     Treat patient
                     empirically for
                     syphilis, even in
                     the face of
                     negative test
Serologic tests for syphilis
    “A blood test”
    Detects antibody in serum
    Requires blood sample centrifugation
    Requires laboratory processing
    Follow universal precautions at every
Syphilis - Non-treponemal tests
    Used for screening large samples
    Used for clinical follow-up
    Cardiolipin-cholesterol-lecithin antigen
    Sensitive but not 100% specific
    Two tests commonly available
    – Rapid Plasma Reagin test (RPR)
    – Venereal Disease Research Laboratory
       test (VDRL)
Non-treponemal test sensitivity

  Test     1o    2o    EL     LL

  VDRL    78%   100%   95%   71%

  RPR     86%   100%   98%   73%
Technique of the RPR test
   Add 1 drop of antigen per circle
   Rotate under cover: 8 min. at 100 rpm
   Read tests immediately under high
   light, rotating and tilting card manually
    – intense clumping of antigen: reactive
    – slight definite clumping: minimally reactive
    – no clumping: nonreactive
Reading the RPR
Reactive control VDRL Slide Test
RPR: Serial Dilutions
Comments on RPR test
   Store serum in refrigerator for up to 5 d.
   Freeze at -20o C for indefinite storage
   Test accuracy of antigen needle
   Run control card daily
   Prozone effect: false negative test in
      undiluted samples of high titer
      (1-2% of secondary syphilis pts.)
Reporting results: examples
  1:1 1:2 1:4 1:8 1:16       Report

  Min.   N   N      N   N   Reactive 1:1
   R     R   N      N   N   Reactive 1:2
   R     R   Min.   N   N   Reactive 1:4
   R     R   R      R   N   Reactive 1:8
False-positive RPR / VDRL
   General population: 1-2%
   Injection drug users: 10%
   Transient false-positive
   – pregnancy
   – febrile illnesses
   Chronic false-positive
   – autoimmune disorders
False Positive RPR / VDRL
   Infectious                  Noninfectious
    – Lyme disease              – Drug addiction
                                – Any connective
    – Leptospirosis               tissue disease
    – Leprosy                   – Rheumatoid heart
    – Tuberculosis                disease
    – SBE                       – Transfusions
    – Rickettsial disease       – Pregnancy
    – Malaria                   – “Old age”
    – Hepatitis                 – Chronic liver disease
    – Vaccinia (vaccination)
    – Many others
Following RPR / VDRL titers
   Expect 4-fold decrease in 6 months
   Expect 8-fold decrease in 12-18 months
   Most patients with early disease are
      seronegative within 1 year of Rx
   Low titer may persist for life in
      adequately treated latent disease
   4-fold increase needs re-treatment
Syphilis - Treponemal tests
   Used for confirmation of infection
   Detects antibodies against T. pallidum
      cellular components
   More expensive, more specific
   Commonly available tests include:
    – Fluorescent Treponemal Antibody
       Absorption (FTA-ABS)
    – T. pallidum particle agglutination (TP-PA)
    – (MHA-TP no longer commercially available)
Sensitivities of Serologic Tests
 Test     Primary      Secondary     Latent       Late

VDRL     78 (74–87)       100      95 (88–100) 71 (37–94)

RPR      86 (77–100)      100      98 (95–100)     73

FTA-     84 (70–100)      100         100          96
MHA-TP   76 (69–90)       100      97 (97–100)     94
Interpreting Treponemal tests
    86% of syphilis cases remain reactive
    for life
    Not used to monitor efficacy of
    treatment or reinfection
    1% false-positive rate in general
   Screening test in specific settings
   – Blood bank screening
   Provides high volume testing
EIA Testing for Syphilis
Other serologic tests
  Other tests should be ordered at the
   time of syphilis serology:
      HIV serology
      Hepatitis B serology
      Herpes simplex virus - type
      specific serology
      Chlamydia/GC urine testing
Syphilis serology

  How do you ascertain who is infectious?

  How do you ascertain who should be
  tracked for partner notification?
Syphilis serology interpretation
   All darkfield + patients are infectious
   Many seropositive patients are
   infectious or have been infectious in the
   past several weeks
   Partner notification reduces disease
       rate in the exposed population
Relation of serostatus to
stage of disease
   20% with primary syphilis have negative
   RPR or VDRL
   98-99% with secondary syphilis have
   positive RPR or VDRL
   98-99% of positive RPR or VDRL are
   true positives (actually have syphilis)
   Difficult to distinguish between re-
   infection vs. treatment failure
Special Considerations
   HIV and syphilis
    – Seronegative primary and secondary
    – Increased incidence of prozone
    – Loss of reactivity to specific treponemal
      tests in previously treated disease
    – Altered therapeutic responses
Special Considerations: CSF testing
     Darkfield microscopy
      – 100% sensitive 96%-97% specific
      – sensitivity 50%-60%; highly specific
     RPR is unsuitable for CSF
      – higher false-positive rate than VDRL
     Typical CSF findings of neurosyphilis:
      – moderate mononuclear pleocytosis
      – elevated total protein (0.46–2.0g/l)
Congenital Syphilis Testing
   Maternal history of syphilis diagnosis
   and treatment
   Maternal antibody transfer to fetus
   Non-treponemal testing at birth
   Evaluate infant CSF for VDRL
  Laboratory tests are important
  component of syphilis clinical care
  Darkfield microscopy and serologic
  tests for syphilis are essential for
  accurate diagnosis and treatment
  Non-treponemal test titers are useful for
  monitoring response to syphilis therapy

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