This document discusses diagnostic tests for syphilis caused by the bacterium Treponema pallidum. It describes direct detection methods like darkfield microscopy and fluorescent antibody testing to visualize the bacterium in samples. It also covers non-treponemal tests that detect non-specific reagin antibodies like VDRL and RPR, and treponemal tests that detect antibodies specific to T. pallidum like FTA-ABS. The stages of syphilis and clinical manifestations are also briefly outlined.

1. Dr. Sanjay singh
AIIMS
Diagnostic Evaluation of Syphilis

2. Syphilis
"He who knows syphilis,
knows medicine"
Sir William Osler
2

3.  Caused by Treponema pallidum.
 Motile spiral-shaped gram –ve bacteria
 Characteristic cock-screw motility
 Inability to survive outside in an animal host
 Cannot be cultured in vitro
 Size : approx 10–14 μm in length and 0.1–0.2 μm in diameter, 10 regular
spirals at interval of about 1 μm
 Transmission: sexual; maternal-fetal, and rarely by other means
INTRODUCTION

4. Dr.T.V.Rao MD 4
Ultra Structure
Contains many
strongly antigenic
protein

5. 5

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)

8.  Direct detection of Treponema Pallidum
 Nontreponemal Serological Tests
 Treponemal Serological Tests
Diagnosis of Syphilis

9. TESTS FOR DIRECT DETECTION OF T PALLIDUM
 Animal Inoculation
 Dark Field microscopy
 Direct fluorescent antibody test
 Direct tests for T pallidum in tissue sections
 Nucleic acid amplification methods

10. NONTREPONEMAL SEROLOGICAL TESTS
Microscopic nontreponemal tests
 VDRL (Venereal disease research laboratory test)
 USR (Unheated serum reagin Test)
Macroscopic nontreponemal tests
 RPR (Rapid plasma reagin test)
 TRUST (Toluidine red unheated serum test)

11. TREPONEMAL SEROLOGICAL TESTS
 FTA-ABS (Fluorescent treponemal antibody absorption test)
 FTA-ABS double-staining (Fluorescent treponemal antibody absorption double staining
test)
 TP-PA test (Treponema pallidum particle agglutination test)
 Western blots
 EIAs (Enzyme immunoassays)/Rapid tests

12. Animal inoculation
 Oldest method for detecting infection
 Most sensitive method for detecting infectious treponemes and is used as the gold
standard for measuring the sensitivity of methods such as the PCR
 Rabbit is most commonly used
 Any source of specimen can be used as long as the material is less than 1 h old or was
frozen immediately after collection
 Inoculation of sample : Intratesticular or Intradermal

13.  Incubation period : Inversely proportional to the size of inoculum.
 Sensitivity of RIT approaches 100% if the number of organisms exceeds 23 and patient has
not received antibiotic treatment.

14. Dark Field Microscopy
Ω One of simplest and most reliable for the direct detection of T pallidum
Ω Exudates and fluids from lesions are examined as a wet mount
Ω Examination should be done immediately
Ω Most productive during 1˚, 2˚, early relapsing, and early congenital syphilis when lesions
contains large numbers of treponemes (chancres, condylomata latum, or mucous patches)

15. Comparison of Light Pathways of bright field and dark field Microscopy

16. Procedure
Clean the lesion with a saline soaked gauze and squeeze it between index finger and
thumb to produce a serous exudate (avoid contamination with blood)
Exudate is then transferred onto a glass slide by directly pressing it on the lesion
Normal saline can be added to the exudate to make the material homogenous
Specimen should immediately be examined as delay in examination reduces the
motility of the treponemes

17. Results
 T.pallidum is identified by its typical morphology and characteristic movements
 T.pallidum is differentiated from the other treponemes by the tightness of spirals and
characteristic cork screw movements

18. Organism Location Coils Length (μm) Width (μm) Rotation
T. pallidum subsp.
pallidum
Skin and
mucosal
lesions
Spiral shape,
10–13 coils
Medium, 10
(6–20)
Very thin,
0.13–0.15
Slow to rapid; like a
cork-screw, may rotate
without changing place
T. refringens Normal
genital
flora
Spiral shape,
2–3 coils
Short,5 - 8 Thick,
0.20–0.30
Very rapid; active
serpentine-like, rotates
sometimes so rapidly that it
looks straight
T. phagedenis,
Reiter treponeme
Normal
genital
flora
Spiral shape,
10–12 coils
(10–30)
Medium long,
10–12 (10–30)
Thick, 0.20–0.25
(0.20–0.40)
Slow to rapid; rotates without
changing place
T. denticola Normal
oral
Spiral shape,
6–8 coils (2–8)
Medium, 8
(6–16)
Very thin, 0.15–
0.20
Slow to rapid, often jerky

20. Demonstration of spirochetes

21.  It is a practical alternative to dark field examination
 Specimen collection is same as that of dark field microscopy
 Slide is air dried and fixed with either acetone for 10 min or 100% methanol for 10 sec
 Smear is stained with fluorescein- labeled anti T. pallidum globulin and examined
under fluorescent microscope
Direct fluorescent antibody -T.pallidum (DFA-TP)

23. Advantages
More sensitive and specific than dark field
microscopy
Samples from oral mucosa can also be
examined
Slides need not be examined immediately
Disadvantages
Can’t differentiate T. pallidum subsp from
eachother

24. T pallidum in tissue sections
1˚ Syphilis
• P/V & P/J infiltrate of lymphocytes, plasma cells, and macrophages.
• Capillary endothelial proliferation and subsequent obliteration of small blood vessels
may be appreciable.
• Focal erosion or ulceration is common.

25. 2˚ Syphilis
 Histologically similar to that of the primary chancre but infiltrate is less intense
 “Lichenoid-psoriasiform” configuration with a perijunctional infiltrate of lymphocytes,
histiocytes, and plasma cells
 Sometimes histiocytic component of the infiltrate is prominent, and thus the biopsy
may assume a “lichenoid-granulomatous” configuration

26. Lichenoid infiltrate

29.  Traditionally Warthin starry method has been used for staining of tissue section
 Organisms can also be identified by PCR and a polyclonal antibody against T. pallidum
is available for IHC
 Both PCR and IHC are much more specific than histochemistry in diagnosis of syphilis

30. Secondary syphilis: numerous spirochetes are present (Warthin-Starry stain)

31. Immunoperoxidase staining

32. Immunoperoxidase Conventional silver
stain
Serology
N = 10 9 6 7
Immunoperoxidase technique for detecting spirochetes in tissue
sections : comparison with other methods
Phelps RG, Knispel J, Tu ES et al. Int J Dermatol. 2000 Aug;39(8):609-13.

33. Treponema pallidum distribution patterns in mucocutaneous lesions of primary and
secondary syphilis: an immunohistochemical and ultrastructural study.
Martín-Ezquerra G, Fernandez-Casado A, Barco D et al. Hum Pathol. 2009 ;40(5):624-30.
No. of Patient Warthin-Starry stain IHC p value
Primary Syphilis
(N = 8)
4 8 < 0.05
Secondary Syphilis
(N = 26)
13 21 < 0.05

34. PCR
 Increasingly becoming the investigation of choice for identifyingT.pallidum from the early
lesions of syphilis
 A number of well-preserved DNA sequences have been identified that are specific for
T.pallidum and do not appear to be found in other treponemes
 Assays based on these primers have been shown to be sensitive and specific in the diagnosis
of early syphilis
 Highly sensitive, able to detect as low as 1 to 10 organisms per specimen with high specificity.

35. • Sample size : 12 patients of 2˚ Syphilis
• Polyclonal antibody directed against T. pallidum was positive in 90% of samples
• Bacteria were located in epidermis and upper dermis
• 47-kDa surface protein gene could be amplified by PCR in 75% samples
• When combining both techniques, T. pallidum was detected in 92% of the samples
Diagnosing Treponema pallidum in secondary syphilis by PCR and
immunohistochemistry.
Buffet M, Grange PA, Gerhardt P et al. J Invest Dermatol. 2007;127(10):2345-50.

36. Antitreponemal antibody response
• IgM antibodies are produced ∼2 weeks after exposure, followed by IgG antibodies 2 weeks
after IgM production
• T.pallidum infection produces antibodies to more than 20 different polypeptide antigens.
Antibodies are of two types :
1) Non specific antibodies (reagins) : directed against lipoidal antigen of T. pallidum as well as
mitochondrial & nuclear membranes of human cells
2) Specific anti-treponemal antibodies : directed against T.pallidum
• Early responses are against TpN47 and some of the flagellar proteins, followed by TpN15 and TpN17
• In 2˚ syphilis, there is a disproportionate increase in antitreponemal IgG3-specific responses

37.  Early latent syphilis : Faint – to moderate IgM and strong IgG reactivity are evident
 Late Latent syphilis : Faint IgM & variable IgG
 IgM antibodies decrease rapidly, becoming undetectable within 6–12 months after
treatment
 Several studies suggest that decreasing IgM levels indicate adequacy of treatment.
 In contrast, IgG1 and IgG3 antitreponemal antibodies can persist for years despite therapy

38. NONTREPONEMAL SEROLOGICAL TESTS
Four nontreponemal tests are currently considered standard tests:
• All these non treponemal tests measure anti lipoidal IgM and IgG antibodies
• These tests are used for initial screening and for follow up after treatment
Microscopic tests Macroscopic tests
VDRL RPR
USR TRUST

39. NonTreponemalTests
 They can be performed as a :
1. Qualitative test (to check for presence or absence of antibodies)
2. Quantitative test (to check the amount of antibodies present in the serum)
 Except for VDRL & RPR tests, most lipoidal antigen tests are not used

40. • These tests use basic antigen formula containing standardized amounts of cardiolipin,
cholesterol and lecithin.
• Only tests recommended to monitor the course of disease during and after treatment.
• Nontreponemal tests can also serve to detect reinfection
• Limitations : Reduced sensitivity in primary syphilis and late latent syphilis
False-positive results
False negative results

41. False-positive reactions
Acute False positive reaction
< 6 months
Chronic False positive reaction
> 6 months
 Viral infections
 Malaria
 Immunizations
 Pregnancy
 Laboratory errors
 Connective tissue diseases
 IV drug abusers
 Narcotic addiction
 Ageing
 Leprosy
 Malignancy

42. False Negative Reaction : Prozone Phenomenon
• Occur due to interference by high concentrations of target antibodies in a specimen.
• Such specimens gives a clearly positive reaction when diluted and retested, a process
that brings the antibody-to-antigen ratio within the optimal range.
• Prozone reactions occur in 1 to 2% of patients with secondary syphilis.

43. VDRL
 Venereal Disease Research Laboratory (VDRL) Test is a slide flocculation test employed in
the diagnosis of syphilis
 Since the antigen used in this test is cardiolipin, which is a lipoidal extracted from beef
heart, it is not a specific test.
 Antibodies reacting with cardiolipin antibodies have been traditionally termed “reagin”
 Antigen : lipid component of T pallidum or as a result of tissue injury following infection

44. VDRL-Test Requirements
 Patient’s serum, water bath, freshly prepared cardiolipin antigen, VDRL slide, mechanical rotator,
pipettes, hypodermic syringe with unbeveled needle and microscope.
 Reactive and non-reactive serum controls are also required
 VDRL antigen : 0.03% cardiolipin
0.21% lecithin
0.9% cholesterol
 Cardiolipin antigen must be freshly constituted each day of test. The working antigen is a buffered
saline suspension of cardiolipin.

45. VDRL slide : This is a glass slide measuring 2 X 3 inch with 12
concave depressions, each measuring 16 mm in diameter and
1.75 mm deep.
Patients’ serum is inactivated by heating at 56˚C for 30 minutes
in a water bath to remove non-specific inhibitors (such as
complement).

46.  Qualitative test:
1) 0.05 ml of inactivated serum is taken into one well.
2) 1/60th ml (or 1 drop from 18 gauge needle) of cardiolipin antigen is added with help of
a syringe to the well and rotated at 180 rpm for 4 minutes.
3) Slide is then viewed under low power objective of a microscope for flocculation.
Depending on size, the results are graded as weakly reactive (W) or reactive (R).
4) Reactive samples are then subjected to quantitative test.

47.  QUANTITATIVETEST :
 This is performed to determine the antibody titers
 Serum is doubly diluted in saline from 1 in 2 to 1:256 or more
 Reported as the highest dilution giving a reactive (not weakly reactive) result

48. Reporting of results
 Results of the test are reported as:
1. REACTIVE : Past/ present infection with a pathogenicT.pallidum, which is either
treated or untreated (or) a false positive reaction
2. WEAKLY REACTIVE : Past/present infection, false positive reaction, Serofast
3. NON REACTIVE : No current infection (or) an effectively treated infection , but it does
not rule out syphilis in incubation period

50.  A four fold rise in titer  Infection
Reinfection
Treatment failure
 A four fold decrease in titer  Effective therapy
When a non treponemal test shows a persistent reactivity with no signs of decline in titer
after 6 months of adequate therapy
or
Fails to show a four fold decrease of an initial high titer within 1 year
SERORESISTANCE (SEROFAST)

51. Unheated Serum Reagin test
• USR antigen is VDRL antigen stabilized by addition of EDTA, so need for daily preparation
of an antigen suspension is eliminated
• Choline chloride is added to eliminate the need to heat inactivate the serum.
• Addition of choline chloride also enhances the reactivity of the antigen
• USR test is performed and reported in a manner similar to the VDRL slide test on serum

52. RPR & TRUST
• Both tests are based on USR antigen
• TRUST and RPR card test antigens differ only in the visualization agent added to antigen
• For the RPR card test, sized charcoal particles are added to the antigen
• For the TRUST paint pigment particles are added
• Particles of both tests become entrapped in antigen-antibody lattice formed with a
reactive serum.

53.  Slides are read macroscopically to determine the presence of clumping (flocculation)
 Results of card tests are reported as either reactive, regardless of the size of the
clumps, or nonreactive.
 All serum samples exhibiting any degree of reactivity or roughness should be
quantitated to an endpoint titer

54. Treponemal Tests
 In these tests, entireT.pallidum or its fragments are used as the antigen to detect antibodies
directed against treponemal cellular components
 These tests are used for confirmation of the disease either in past/present
 Treponemal tests become reactive before non treponemal tests but unlike non treponemal tests
they remain positive for many years even after adequate therapy
 Treponemal tests are technically more difficult and costly to perform than nontreponemal tests
and cannot be used to monitor treatment

55. Commonly usedTreponemal tests
 FluorescentTreponemal Antibody Absorption (FTA-Abs) test
 FluorescentTreponemal Antibody Absorption double staining (FTA-Abs-DS) test
 Treponema pallidum Haemagglutination Assay (TPHA)
 Treponemal Enzyme Immunoassay (EIA)

56. Fluorescent Treponemal Antibody Absorption (FTA-Abs) test
Serum for testing is diluted in sorbent (containing extract of Reiter treponemal
culture) to absorb non specific antibodies
Serum is placed on a microscopic slide to which the antigen (a suspension ofT.
pallidum organism) is fixed
Conjugated fluorescein labeled antihuman globulin is added

57. • The intensity of fluorescence is reported as REACTIVE, BORDERLINE, NON REACTIVE.
• False positives and negatives may occur
• It is most sensitive serological test in early stages of syphilis at present
ADVANTAGES
1. High specificity & sensitivity
2. Can detect recent infection 1-2
weeks before other assays
DISADVANTAGES
1. Expensive
2. Time consuming
3. Well trained personnel is required

59. Fluorescent Treponemal Antibody Absorption double staining
(FTA-Abs-DS) test
Serum for testing is diluted in sorbent (containing extract of Reiter treponemal culture) to
absorb non specific antibodies
Serum is placed on a microscopic slide to which the antigen (a suspension ofT. pallidum
organism) is fixed
Class - specific tetramethylrhodamine isothiocyanate – labeled antihuman immunoglobulin G
is added
Counterstain, fluorescein isothiocyanate (FITC)-labeled anti-treponemal globulin, is added to
locate T. pallidum when the slide is examined with the FITC filter.

60. Treponema pallidum Haemagglutination Assay (TPHA)
 A qualitative haemagglutination test using tanned formalinised sheep RBC’s as carrier
forT.pallidum antigen (sensitized cells)
TPPA : Treponema pallidum particle agglutination
 Based on agglutination of coloured particle carriers sensitized withT pallidum antigen
 Uses gelatin particles instead of erythrocytes, thus eliminating nonspecific reactions
with plasma samples

61. Treponemal Enzyme Immunoassay (EIA)
 In this test, serum is added to microwells coated with a treponemal antigen
 An enzyme labeled anti human Ig conjugate & enzyme substrate are added to detect
antigen-antibody reaction after incubation
 It has advantages of higher specificity than FTA-Abs and automated or semi automated
processing and objective reading of results

63. Western Blot

65. Performance of serological tests for syphilis
Percentage of sensitivity by stage of untreated syphilis
Test Primary Secondary Latent Late Specificity
VDRL 78 (74–87) 100 96 (88–100) 71 (34–94) 98 (96–99)
RPR card 86 (77–99) 100 98 (95–100) 73 98 (93–99)
USR 80 (72–88) 100 95 (88–100) 99
TRUST 85 (77–86) 100 98 (95–100) 99(98–99)
FTA-ABS 84 (70–100) 100 100 96 97 (84–100)
FTA-ABS DS 80 (70–100) 100 100 98 (97–100)
TP-PA 88 (86–100) 100 100 96 (95–100)
IgM EIA 93 85 64
Immune capture
EIA
77 100 100 100 99
Chemiluminescence
assay
98 100 100 100 99

66. RPR TPHA IgM EIA
_ _ _ No syphilis or incubating syphilis
_ _ + Early primary syphilis
+ + + Primary or secondary
+ _ + Early infection
+ + _ Late secondary or latent
+ _ _ Biologic false positive, late syphilis
_ + _ Late infection, treated syphilis or false positive
treponemal test
Increasing + Increasing Re- infection, relapse
Interpretation

67. Diagnosis according to stages
1. Early syphilis
 Dark field microscopic examination : - Most specific and sensitive
 Non treponemal tests: - Positive in 80% cases
 Treponemal tests: - Positive in 80 - 90% cases

68. 2. Secondary syphilis
 Dark field microscopic examination: - fluid from moist wet lesions and lymph
node aspirate
 Non treponemal tests: - Always positive, usually at a high dilution
 Treponemal tests: - Always positive

69. 3. Early Latent syphilis
a. Non treponemal tests: - positive in 95-98% cases
b. Treponemal tests: - positive in 97-100% cases
• Diagnosis  based on reactive serological tests- treponemal and non treponemal
in absence of any apparent signs of disease

70. 4. Late Latent syphilis
a. Non treponemal tests: - positive in 34 - 94% cases
b. Treponemal tests: - positive 94 - 96% cases

71. Diagnosis of Neurosyphilis
 CSF examination is done in :
 Patients with neurosyphilis
 In patients with syphilis of more than 2 years duration to exclude asymptomatic
neurosyphilis
 Before retreatment of patients who have had relapses after any form of treatment
 As a follow up procedure for patients who have been treated for neurosyphilis
 In all infants suspected of prenatal syphilis

72.  CSF sample is taken and a cell count is made
 It is further checked for protein abnormalities and subjected toVDRL test
 Diagnosis of neurosyphilis is indicated by
1. Increased cell count (> 10 lymphocytes per mm3 of CSF)
2. Increased proteins (> 40 mg% in the CSF)
3. REACTIVEVDRL test
 SerumVDRL test is reactive in about 2/3rd of the cases

73. Cardiovascular syphilis -
• Serological tests - usually reactive, esp. if extensive involvement
• Negative reaction may accompany a localized lesion
Congenital syphilis :
• Demonstration of T. pallidum by direct examination from nasal discharge or from
early lesions
• Positive treponemal test in a titre, higher than mother or serially rising
• FTA-IgM test is more specific with infection

74. Diagnostic Algorithm

76. Diagnosis of Syphilis in HIV
 Unusual serologic responses have been observed among HIV-infected persons who have syphilis
1. Serologic titers higher than expected
2. False negative serologic test results
3. Delayed appearance of seroreactivity
 Both treponemal and nontreponemal serologic tests should be interpreted in usual manner for
majority of patients who are coinfected with T. pallidum and HIV.

77.  When clinical findings are s/o syphilis but serologic tests are nonreactive, alternative tests (e.g.,
biopsy of a lesion, DGM, or DFA staining of lesion material) might be useful for diagnosis
 Neurosyphilis should be considered in the differential diagnosis of neurologic disease in HIV-
infected persons.