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Serological Diagnosis of Syphilis (PowerPoint) by TummalapalliRao

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Serological Diagnosis of Syphilis

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									Dr.T.V.Rao MD




   Dr.T.V.Rao MD   1
            Syphilis
                    

"He who knows
 syphilis, knows
   medicine"

Sir William Osler
                Dr.T.V.Rao MD   2
Syphilis was a Taboo
         
            Poster for testing
             of syphilis,
             showing a man
             and a woman
             bowing their
             heads in shame
             (ca. 1936).
        Dr.T.V.Rao MD             3
                   SYPHILIS
         INTRODUCTION
                    
Caused by Treponema pallidum.
Transmission: sexual; maternal-fetal, and rarely
 by other means.
Primary and secondary syphilis in the US dropped
 by ~ 90 %t from 1990 to 2000, the number of
 cases have gone up since then.
A dramatic increase in cases in men from 2000 to
 2002 reflected syphilis in MSM.
Syphilis increases the risk of both transmitting
 and getting infected with HIV.
                        all patients with syphilis.
 Perform HIV testing inDr.T.V.Rao MD                4
  Introduction to Syphilis
Syphilis is one of a
                      
 group of diseases
 caused by spirochete
 organisms of the
 genus Treponema.
 Sexually acquired
 syphilis occurs
 worldwide and is
 caused by T. pallidum
 subspecies pallidum.
                    Dr.T.V.Rao MD   5
Other Related to
  Treponemes
       Related Treponemes
                        cause the non-
                        venereal
                        treponematoses
                        bejel, or endemic
                        syphilis (T. pallidum
                        endemicum), yaws (T.
                        pallidum pertenue),
                        and pinta (T.
                        carateum).
        Dr.T.V.Rao MD                           6
         STAGES OF SYPHILIS
                             
1. Primary
2. Secondary
3. Latent
       Early latent
       Late latent
4. Late or tertiary
       May involve any organ, but main parts are:
           Neurosyphilis
           Cardiovascular syphilis
           Late benign (gumma)


                            Dr.T.V.Rao MD            7
     Diagnosis of Syphilis
                                
 The nontreponemal tests, VDRL and rapid plasma reagent (RPR),
  are antilipoidal antibodies seen in other disease states, pregnancy,
  and occasionally after vaccination. They are nonspecific and
  cannot rule in disease. These tests have sensitivities approaching
  80% in patients with symptomatic primary syphilis and virtually
  100% in patients with secondary syphilis.
 – A positive VDRL/RPR should be quantified and titers followed at
  regular intervals after treatment. As such, its value is in response
  to treatment. However, it does not correlate with symptom
  resolution.
 – Most patients have nonreactive nontreponemal tests within
  several years after successful treatment for syphilis, but a
  significant number have persistently positive tests, the so-called
  serofast reaction.           Dr.T.V.Rao MD                             8
                                           Diagnosis



  Laboratory Diagnosis
                    
Identification of Treponema pallidum in
  lesions
  Darkfield microscopy
  Direct fluorescent antibody - T. pallidum
   (DFA-TP)
Serologic tests
  Nontreponemal tests
  Treponemal tests

                                                       9
                                               Diagnosis


Nontreponemal Serologic Tests
                      (continued)
                           
    Advantages:                   Disadvantages:
 Rapid and inexpensive         May be insensitive
 Easy to perform and can        in certain stages
  be done in clinic or office   False-positive
                                 reactions may
 Quantitative                   occur
 Used to follow response       Prozone effect may
  to therapy                     cause a false-
 Can be used to evaluate        negative reaction
  possible reinfection           (rare)
                                                       10
                Diagnosis
                         
 Patients with a reactive VDRL or RPR should have the
  result confirmed by specific treponemal testing. FTA-
  ABS and or EIA.
 • Tertiary syphilis Serology is used in the diagnosis.
  Evaluation of neurosyphilis requires a lumbar puncture
  (LP) and evaluation of the CSF.
 – The CDC currently recommends LP only if the patient
  is seroreactive and HIV positive, has symptoms of
  neurosyphilis

                        Dr.T.V.Rao MD                      11
          Tests to Confirm
                             
 Syphilis may be confirmed either via blood tests or direct
  visualization using microscopy. Typical diagnosis is with
  blood tests using nontreponemal and/or treponemal tests.
  Nontreponemal test are used initially and include venereal
  disease research laboratory (VDRL) and rapid plasma
  regain however as these test occasionally are falsely positive
  confirmation is required with a treponemal test such as
   treponemal pallidum particle agglutination
   (TPHA) or fluorescent treponemal antibody
   absorption test (FTA-Abs)
                            Dr.T.V.Rao MD                          12
Dr.T.V.Rao MD   13
     VDRL - Background
                     Laboratory
The Venereal Disease Research
 (VDRL) test is one of two variations of
 flocculation procedures used for serological
 testing of syphilis, the other being the Rapid
 Plasma Reagin (RPR). Flocculation testing is
 based on antibody detection with the
 interaction of soluble antigen with an antibody
 that results in a precipitate formation of fine
 particles.
                    Dr.T.V.Rao MD                  14
       VDRL Test Basics
                     
The VDRL is a confirmatory serological micro
  flocculation slide test used for the detection of
  syphilis antibodies. In a VDRL procedure, the
  patient’s serum is heat-inactivated and mixed
  with a buffered saline suspension of VDRL
  Antigen containing cardiolipin, lecithin and
  cholesterol that binds with Reagin, an
  antibody-like protein. A combination of Reagin
  and VDRL Antigen form microscopic clumping
  called flocculation.
                      Dr.T.V.Rao MD                   15
  VDRL – A Standard Test for
           Syphilis
 The VDRL can be used
                      
  for qualitative and
  quantitative
  measurements and is
  recommended when a
  patient suspected of
  having syphilis has a
  negative dark field
  microscopy result or
  when atypical lesions
  are present.
                      Dr.T.V.Rao MD   16
     VDRL Serological Procedure
             Principles
                      
VDRL Antigen is a nontreponemal antigen
 composed of cardiolipin cholesterol and
 lecithin. The nontreponemal tests measures
 anti-lipid antibodies, which are formed by the
 host in response to lipids released from
 damaged host cells early in infection with T.
 pallidum, and lipid-like material form the
 treponemal cell surface. During syphilis
 infection, an antibody-like substance called
 reagin can be detected in the patient’s serum or
 CSF.
                     Dr.T.V.Rao MD                  17
 Preparation of Antigen
                           each testing day.
 Prepare a fresh antigen suspension
  Once prepared, it should be used within 8 hours.
 Store prepared suspension at 23-29)C.
 Test antigen suspension reactivity with control sera
  (Reactive, Weakly reactive and Nonreactive). Test
  serum dilutions within 1 hour after heat inactivation.
 Use antigen suspension only if it produces the expected
  reactivity with the control sera comparable to results
  obtained with the reference antigen.

                         Dr.T.V.Rao MD                      18
    Required Materials
                      
VDRL Antigen with buffered saline solution
 containing 1% sodium chloride, pH 6.0+/-0.1
 with 0.05% formaldehyde preservative
Reactive, weakly reactive and nonreactive
 serum
0.9% saline, non-disposable 1cc glass syringe
 and calibrated needles without bevel-18
 gauge(serum) or 21-22 gauge(CSF), slide
 cards(serum) or concavity slides(CSF)
Stirrers           Dr.T.V.Rao MD                19

Rotator
       Specimen Collection and
        Preparation for Serum
                          
 Collect 5-8 ml of blood by aseptic venipuncture in a red
  top tube.
 Allow blood to clot at room temperature then
  centrifuge to obtain serum.
 Heat the test sera at 560C for 30 minutes.
 Specimen must be at 23-290C when tested.
 Specimen must be clear of hemolysis and show no
  visible evidence of bacteria contamination.
 Store at room temperature for 4 hours, after which
  store at 2-80C, maybe refrigerated up to 5 days, then
  frozen at <-200C.
                         Dr.T.V.Rao MD                       20
       Specimen Collection and
         Preparation for CSF
Centrifuge and
                            
 decant the specimen
Specimens do not
 require heat
 inactivation before
 testing.
Spinal fluids that are
 visibly contaminated
 or that contain gross
 blood are            Dr.T.V.Rao MD   21

 unsatisfactory
Antigen Suspension Preparation
                          saline to the bottom of
 Pipette 0.4ml of VDRL buffered
  a round 30 ml glass stoppered bottle with a flat inner-
  bottom surface. Gently tilt bottle so that VDRL
  buffered saline will cover the entire inner-bottom
  surface of the bottle.
 Add 0.5 ml of VDRL Antigen directly into the saline
  while continuously but gently rotating the bottle on a
  flat surface from the lower half of a 1.0 ml pipette
  graduated cylinder to the tip. Add antigen drop by
  drop at a rate that allows about 6 sec for 0.5 ml of
  antigen. Keep pipette tip in the upper third of the
  bottle and do not splash saline unto the pipette.
                         Dr.T.V.Rao MD                      22
Antigen Suspension Preparation
                            without touching pipette
 Expel the last drop of antigen
  to the saline and continue rotation of the bottle for 10
  sec.
 Add 4.1 ml of buffered saline from a 5 ml pipette. Do
  not drop saline directly on antigen; allow it to flow
  down the side of the bottle.
 Cap the bottle and mix by gentle inversion. Allow to
  stand for 5 minutes but no more than 2 hours. The
  suspension is ready for use.
 Remix suspension by swirling only
                          Dr.T.V.Rao MD                      23
    Antigen Suspension
                            
 Cap the bottle and mix
  by gentle inversion.
  Allow to stand for 5
  minutes but no more
  than 2 hours. The
  suspension is ready for
  use.
 Remix suspension by
  swirling only

                        Dr.T.V.Rao MD   24
Procedure: Step 1
                       Wells should
                       be labeled as
                       reactive ®,
                       weakly
                       reactive (WR),
                       and
                       nonreactive
                       (NR),
       Dr.T.V.Rao MD               25
Procedure: Step 3
                       Add one drop
                        (.01 ml) of
                        sensitized
                        antigen
                        suspension to
                        each specimen
                        with a 21 or 22
       Dr.T.V.Rao MD
                        gauge needle.26
     Procedure: Step 4
                        
 Rotate slides for 8
  minutes on a
  mechanical rotator at
  180 rpm. Note: when
  the tests are
  performed in a dry
  climate, the slides may
  be covered with a box
  lid to prevent
  evaporation.
                       Dr.T.V.Rao MD   27
        Results for Serum
                Specimen
 Qualitative Testing -
                        
  Medium to large clumps
  (Reactive); Small clumps
  (Weakly Reactive); No
  clumping or very slight
  roughness (Nonreactive).
 Verify control sera
  results for expectation.
  If reactions are not as
  expected, the test is
  invalid and results can
  not be reported.
                             Dr.T.V.Rao MD   29
         Reporting the Results
                            
 Perform a quantitative test
  to endpoint on all serum
  samples that produce
  reactive, weakly reactive or
  “rough” nonreactive results
  in the qualitative slide test.
 Quantitative Testing -
  Report the titer as the
  highest dilution that
  produces a Reactive (not
  weakly reactive) results

                             Dr.T.V.Rao MD   30
                                                                Diagnosis

 Diagnosis of CNS Infection with
             Syphilis
                                  
No test can be used alone to diagnose neurosyphilis.
 VDRL-CSF: highly specific but insensitive
   Diagnosis usually depends on the following factors:
       Reactive serologic test results,
       Abnormalities of CSF cell count or protein, or
       A reactive VDRL-CSF with or without clinical manifestations.
   CSF leukocyte count usually is elevated (>5 WBCs/mm3) in
    patients with Neurosyphilis.
   The VDRL-CSF is the standard serologic test for CSF,
    and when reactive in the absence of contamination of
    the CSF with blood, it is considered diagnostic of
    Neurosyphilis.
                                                                        31
      Specimen Collection and
        Preparation for CSF
Centrifuge and
                              
 decant the specimen
Specimens do not
 require heat
 inactivation before
 testing.
 Spinal fluids that are
  visibly contaminated
  or that contain gross
  blood are
  unsatisfactory         Dr.T.V.Rao MD   32
       Testing CSF Samples
Quantitative tests are
 run on all spinal fluids
 found to be reactive in
 the qualitative test.
 Prepare fluid as
 follows:
A. Pipette 0.2 ml of 0.9%
 saline into each of 5 or
 more tubes.

                       Dr.T.V.Rao MD   33
   Testing of CSF Samples
Add 0.2ml of
unheated
spinal fluid to
tube 1, mix
well and
transfer 0.2 ml
to tube 2 .
                  Dr.T.V.Rao MD   34
    Testing of CSF Samples
Continue mixing and
transferring 0.2 ml
from one tube to the
next until the last tube
is reached. The
respective dilutions
are 1:2, 1:4, 1:8, 1:16.
Etc.,
                     Dr.T.V.Rao MD   35
Reporting CSF Samples
                           
2. Test each spinal fluid
   dilution and undiluted
   spinal fluid as described
   under “VDRL slide
   qualitative on spinal
   fluid.”
3. Report results in terms of
   the greatest spinal fluid
   dilution (dils) that
   produces a reactive
   result.
                          Dr.T.V.Rao MD   36
 All Positive Samples tested
  by Quantitative Method
In Quantitative
                        
 Testing - Report
  the titer in terms
  of the highest
  dilution that
  produces a
  reactive (not
  weakly reactive)
  result.
                       Dr.T.V.Rao MD   37
Quantitative Testing and
       Reporting
           
                  In Quantitative
                   Testing - Report
                          the titer in terms
                          of the highest
                          dilution that
                          produces a
                          reactive (not
          Dr.T.V.Rao MD
                          weakly reactive)     38
                          result.
            Interpretation
                    clinical evidence may
Nonreactive VDRL - with
  indicate early primary syphilis, a prozone reaction in
  secondary or late syphilis.
 Nonreactive VDRL - with no clinical evidence may
  indicate no current infection or an effectively treated
  infection.
 Quantitative VDRL - detects changes in reagin titer.
  Serum samples displaying a fourfold increase in titer
  on a repeated sample may indicate an infection,
  reinfection or treatment failure. A fourfold decrease
  during treatment indicates adequate therapy.

                          Dr.T.V.Rao MD                     39
        Sources of Error
                      - occur in 10% to
False positive reactions
 30% of positive serological tests for syphilis
 and consist of nonsyphilitic positive VDRL.
 reactions with cardiolipin type antigens.
False negative reactions - consist of
 conditions and a variety of situations.
Weakly reactive - caused by very early
 infection, lessening of the activity of the disease
 after treatment and improper technique or
 questionable reagents.

                      Dr.T.V.Rao MD                    40
False Positive Reactions
 Lupus
                                 Infectious
  erythematosus
                                   hepatitis
 Rheumatic fever
 Vaccinia and virus              Leprosy
  pneumonia                       Malaria
 Pneumococcal                    Rheumatoid
  pneumonia
                                   arthritis
 Infectious
  mononucleosis                   Pregnancy
                  Dr.T.V.Rao MD
                                  Aging individuals
                                                   41
    False Negative Reactions
 Technical error -
                    
  unsatisfactory antigen or
  technique.
 Low antibody titers
 Presence of inhibitors in
  the patient’s serum
 Reduced ambient
  temperature (below 230
  to 290)
 Prozone reaction
                        Dr.T.V.Rao MD   42
The RPR test is a nontreponemal testing
procedure for the serologic detection of
                syphilis.




                Dr.T.V.Rao MD              43
       Principle of RPR Test
 The RPR Card antigen
                                
  suspension is a carbon particle
  cardiolipin antigen that detects
  reagin.
 Reagin is an antibody like
  substance present in serum or
  plasma from individuals with
  syphilis.
 The reagin binds to the test
  antigen which consists of
  cardiolipin-lecithin coated
  particles that cause macroscopic
  flocculation.

                               Dr.T.V.Rao MD   44
Principle of RPR
        When a specimen such
        
                as serum or plasma
                contains antibody,
                flocculation occurs with
                the resulting aggregation
                of the carbon particles.
               The flocculation appears
                as black clumps against
                the white background of
                the plastic coated card.

      Dr.T.V.Rao MD                         45
         Principle of RPR
 Antibodies associated      
  with syphilis begin to
  appear in the blood 4 to
  6 weeks after infection.
  Nontreponemal tests
  determine the presence
  of reagin. Reagin is a
  nontreponemal
  autoantibody directed
  against cardiolipin
  antigens.
                         Dr.T.V.Rao MD   46
    Materials for RPR
                   
RPR Test Cards
RPR Control
 Cards
RPR Antigen
Distilled Water
Dispenstirs
Rotator       Dr.T.V.Rao MD   47
RPR Test Background
          The RPR test uses a
          
                        white plastic coated
                        card that consist of
                        several circles that are
                        18 mm in diameter.
                 The controls which are
                  strongly reactive,
                  moderately reactive,
                  and non-reactive are
                  contained on the
                  control card in a dried
                  form.

        Dr.T.V.Rao MD                              48
      Specimen Collection
                            
Unheated Plasma -
 specimen should be
 collected with an
 anticoagulant such as
 EDTA or heparin,
 plasma must be stored
 at 2°C to 8°C. Plasma
 must be tested within
 in 24 hrs. of collection
                        Dr.T.V.Rao MD   49
   Specimen Processing
                      Unheated serum-
                                 centrifuge for
*The addition of choline         sedimentation of cellular
  chloride, which                elements, serum may be
  inactivates                    frozen until time of
  complement enables             testing.
  the serum to be               Heated Serum- transfer
                                 serum to clean tube and
  tested without prior           place in 56°C water bath
  heating.                       for 30 minutes

                   Dr.T.V.Rao MD                       50
Prepare the Card
        Labelnumbers of samples
        
          with
               rings on test card

                to be tested
               Use Dispenstir to draw up
                serum sample.
               Hold Dispenstir in a
                perpendicular position
                directly over the test circle
                to which the specimen is to
                be delivered.
               Squeeze Dispenstir to
                allow 1 drop to fall on to
                each circle
      Dr.T.V.Rao MD                             51
      Performing the Test
 Invert Dispenstir,and         
  using the sealed end
  spread the specimen in
  the confines of the circle.
 Reconstitute the antigen
  bottle, by shaking.
  Holding the bottle in a
  straight vertical position
  drop one or two drops in
  the upper corner of each
  test circle, then place
  one “free falling” drop on
                           Dr.T.V.Rao MD   52
  each test area.
 Rotate card at Regulated Speed
 Rotate card for 8 minutes
  on a mechanical rotator at
                            
  100 rpm. The test card she
  also be covered with a
  humidifier cover.
 After rotating
  mechanically, the test card
  should be rotated
  manually by hand 3 to four
  rotations and then read
  immediately
  macroscopically in the
  “wet” state under a high
  intensity lamp.


                           Dr.T.V.Rao MD   53
 Procedure for Controls
 A. Use Dispensator to draw
                               
  up distilled water
 B. Drop 1 drop on the card
  test circle for each patient
  sample.
 C. Invert Dispensator and
  spread the water in the circle
  until the dried control is
  completely reconstituted.
 D. Add antigen as described
  for the patients
 E. Rotate for 8 minutes at
  100 rpm

                              Dr.T.V.Rao MD   54
Reactions of Controls
           should be observed to
           The following reactions
                   compare against the test
                   results:
                  Reactive control -
                   characteristic strong
                   clumping.
                  Reactive moderate
                   control - moderate
                   clumping.
                  Non-reactive control -
                   smooth, grayish
                   appearance of unclumped
                   particles

         Dr.T.V.Rao MD                        55
Observe for Reactivity
          




         Dr.T.V.Rao MD   56
A non reactive RPR sample
           




          Dr.T.V.Rao MD     57
Repeat all Positive Samples after
            Dilutions
               




              Dr.T.V.Rao MD         58
      Explanation of Results
                            one of the following:
A negative RPR test may indicate
1. The patient does not have syphilis.
2. The infection is too recent for antibodies to be produced.
   (Repeated tests should be administered at 1 week, 1
   month, and 3 month intervals to establish presence or
   absence of disease).
3. The syphilis is latent or inactive
4. Faulty immunodefense mechanism
5. Faulty lab techniques
                           Dr.T.V.Rao MD                 59
  Explanation of Results
                   not conclusive for
A positive reaction is
 syphilis. Several conditions produce
 biologic false positive results for syphilis.
 (False positive means that the test
 revealed a positive reaction when it was
 actually negative).
False positives may reveal the presence
 of other serious diseases.
                    Dr.T.V.Rao MD                60
Nontreponemal positive Tests
    Need Confirmation
 Nontreponemal antigen     
  tests are not entirely
  specific for syphilis and
  do not have satisfactory
  sensitivity in all stages of
  syphilis. Whenever the
  results of a
  nontreponemal antigen
  test disagree with the
  clinical impression, a
  treponemal antigen test
  such as the FTA-ABS
  should be performed.

                           Dr.T.V.Rao MD   61
Non-syphilitic Conditions Giving
 Biologic False-Positive Results
                        
  Malaria                           Viral pneumonia
  Leprosy                           Lupus erythematous
  Relapsing fever                   Measles
  Infectious                        pregnancy
   Mononucleosis                     drug abuse
  Atypical pneumonia




                    Dr.T.V.Rao MD                          62
 Resolving False Positive RPR Tests

False positive RPR
                   
 tests may be
 resolved by
 testing the
 patient’s serum
 with a specific
 treponemal
 antigen tests.
                   Dr.T.V.Rao MD      63
Dr.T.V.Rao MD   64
 Confirmatory Tests for Syphilis
                   
Treponemal tests are used to confirm
 reactive non –treponemal procedures.
 TPHA testing is now routinely done

A positive FTA-ABS test almost always
 remains positive and therefore is not
 recommended for monitoring therapy.
                  Dr.T.V.Rao MD          65
              TREPONEMAL TESTS
FTA-ABS
 Used as a confirmatory tests.
                                      
 Sensitivity and specificity high.
     85% of patients with primary syphilis are reactive
     99% with secondary syphilis
     > 95% with late syphilis (It may be the only test with a positive result for
      patients with cardiovascular or neurologic syphilis).
 Remains reactive for life in most, despite adequate therapy. Only 15-25
   % of those treated for primary syphilis may turn negative by 2-3 yrs.
 False positive in other treponemal diseases (pinta, yaws..) and other
  spirochete diseases (Lyme, leptospirosis…)

MHA-TP test (microhemagglutination assay for T. pallidum; agglutination of
   RBCs to which T. pallidum antigens have been fixed is the basis).
  TPHA and FTA-ABS Testing are
commonly used Confirmatory Tests
               




              Dr.T.V.Rao MD        67
 For Routine Testing a Combination of VDRL
     or RPR and TPHA is highly preferred

 TPHA Test is a sensitive
                               
  passive haemagglutination
  test, that detects specific
  Treponema pallidum
  antibodies in serum within
  one hour. Used in
  combination, the VDRL or
  RPR and TPHA Tests provide
  accurate and reliable
  confirmation of active syphilis
  infection. No specialized
  equipment is required and
  results are clearly visible and
  easily interpreted.

                              Dr.T.V.Rao MD   68
Microhemagglutination assay (MHA-TP)and
TPHA (T. pallidum hemagglutination assay)
                          
 The MHA-TP was the earlier iteration of the TPHA.
 Occasionally, these tests are simply referred to as an
 indirect hemagglutination assay (IHA). The
 hemagglutination tests generally are simpler to
 perform than the fluorescent antibody tests and lend
 themselves to automation. The MHA-TP and TPHA
 tests are very rarely used currently. Both tests are
 quickly being replaced by newer and easier TP-PA and
 EIA-based tests (see below), including lateral flow strip
 test
                         Dr.T.V.Rao MD                       69
Microhemagglutination assay
                          
 The MHA-TP and TPHA are used to confirm a syphilis
  infection after another method tests positive for the
  syphilis bacteria. The MHA-TP and TPHA tests detect
  antibodies to the bacteria that cause syphilis and can
  be used to detect syphilis in all stages, except during
  the first 3 to 4 weeks when antibody levels are too low.
  These tests are also suitable for use as a screening
  procedure. Neither of these tests is suitable for use on
  cerebrospinal fluid (CSF).

                         Dr.T.V.Rao MD                       70
Principles of TPHA Test
                  
TPHA (Treponema Pallidum
 Hemagglutination) is an indirect
 hemagglutination assay carried out on micro
 plates for the qualitative and semi-qualitative
 detection of anti- Treponema pallidum specific
 antibodies in human serum. Avian blood cells
 stabilized and sensitized with a solution of T.
 pallidum antigen agglutinate in the presence of
 anti-T Pallidum antibodies, exhibiting a typical
 agglutination pattern.
                     Dr.T.V.Rao MD                  71
       Reading TPHA Results
                
 The upper, left-hand well contains
  a positive control test. The red cells
  have had treponemal antigens
  attached and antibodies in the
  serum have caused these cells to
  agglutinate and form a mat across
  the bottom of the well. These
  antibodies can be presumed to be
  specific for Treponemes, since
  otherwise identical red cells that
  have not had the treponemal
  antigens attached do not cause
  haemagglutination, as seen in the
  bottom, left-hand well. A negative
  serum test is shown in the center
  and a patient's test is on the right.
  This result supports a diagnosis of
  syphilis.
                                     Dr.T.V.Rao MD   72
      EIA tests for Syphilis
                        
 Enzyme immunoassay (EIA), also known as an enzyme
  linked immunosorbent assay (ELISA), for syphilis is a
  relatively new invention first appearing on the market in the
  mid-1990s. There are numerous benefits to the EIA
  platform over earlier technologies. Firstly, the majority of
  diseases that are considered to be of clinical and public
  health importance already exist in an EIA format, which is
  highly standardized even across international boundaries.
  This familiarity allows new EIAs to be readily accepted by
  clinicians and technicians with minimal difficulty. It also
  limits the need to purchase new capital equipment since
  most labs will already be equipped to handle EIAs.
                           Dr.T.V.Rao MD                          73
     EIA tests Syphilis Gaining
            Importance
There have been
                           
 several
 developments in
 particularly the
 advent of enzyme
 immunoassays
 (EIAs) and, lately, the
 commercial
 availability of
 recombinant
 antigen-based tests Dr.T.V.Rao MD   74
    Fluorescent Treponemal
 Antibody Absorption (FTA-abs)
                       
The FTA-abs test detects antibodies to T.
 pallidum and can be used to detect syphilis
 infection at any stage except during the first 3
 to 4 weeks after exposure (which is about the
 same time frame that the VDRL/RPR tests
 become effective) and in tertiary stages of the
 disease. In the secondary stage of syphilis, the
 FTA-abs test is most reliable and is reportedly
 positive in 100 percent of cases. It can be
                      Dr.T.V.Rao IgG
 adapted to detect either MD or IgM antibody.       75
Gold Standard Confirmatory test
                              
 The FTA-abs is still
  generally regarded as the
  ‘gold standard’, but it has a
  number of limitations. It is
  a subjective test and
  difficult to standardize. It
  is sensitive, but the TPHA
  is more sensitive, except in
  the third and fourth weeks
  of infection; the TPHA is
  also more specific2.
                             Dr.T.V.Rao MD   76
             Sensitivity of Serological Tests in
                             Untreated Syphilis
                               Stage of Disease (Percent Positive [Range])

 Test                         Primary          Secondary             Latent            Tertiary

 VDRL                       78 (74-87)              100           95 (88-100)         71 (37-94)
 RPR                        86 (77-99)              100           98 (95-100)              73
 FTA-ABS*                  84 (70-100)              100                100                 96
 Treponemal
                            76 (69-90)              100           97 (97-100)              94
 Agglutination*
 EIA                              93                100                100

*FTA-ABS and TP-PA are generally considered equally sensitive in the primary stage of disease.
                                                                                                   77
                                                                                           Diagnosis

       Causes of False-Positive Reactions in
           Serologic Tests for Syphilis
     Disease                                       RPR/VDRL             FTA-ABS           TP-PA
     Age
     Autoimmune Diseases
                                                      Yes
                                                                            Yes
                                                                            Yes
     Cardiovascular Disease                                                 Yes            Yes
     Dermatologic Diseases                             Yes                  Yes            --
     Drug Abuse                                        Yes                  Yes
     Febrile Illness                                   Yes
     Glucosamine/chondroitin sulfate                                     Possibly
     Leprosy                                           Yes                  No             --
     Lyme disease                                                           Yes
     Malaria                                           Yes                  No
     Pinta, Yaws                                       Yes                  Yes            Yes
     Pregnancy                                        Yes*
     Recent Immunizations                              Yes                   --            --
     STD other than Syphilis                                                Yes
     *May cause increase in titer in women previously successfully treated for syphilis
                                                                                                   78

Source: Syphilis Reference Guide, CDC/National Center for Infectious Diseases, 2002
        AIDS and Syphilis
                            
The Serological Tests
 in AIDS and HIV
 related infections
 should be
 interpreted with
 caution and
 expertise, need a
 better understanding
 of the progress of the
 Disease.             Dr.T.V.Rao MD   79
 Nontreponemal Serologic Tests
Principles                
   Measure antibody directed against a cardiolipin-lecithin-
    cholesterol antigen
   Not specific for T. pallidum
   Titers usually correlate with disease activity and results
    are reported quantitatively
   May be reactive for life

Nontreponemal tests include VDRL, RPR,
  TRUST,                                                     80
               References
                          
 Thomas B. Wiggers, Associate Professor Clinical
  Laboratory Sciences, UMMC
 Additional photos:www.Kumc.EDU
 Center for disease control (1999). Guidelines for
  evaluation and acceptance of new syphilis serology tests
  for routine use. US department of health, education and
  welfare publication, Atlanta.
 Wasley G.D. (1988). Syphilis serology. Oxford press,
  New York.
 Abbot laboratories, Abbott Park, IL 60064.
                         Dr.T.V.Rao MD                       81
                 
Created / Designed by Dr.T.V.Rao
 MD for ‘e’ Learning Resources for
     Medical and Paramedical
 Students in the Developing World
                 Email
         doctortvrao@gmail.com

                Dr.T.V.Rao MD        82

								
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