This document provides information on various laboratory tests used to diagnose syphilis. It discusses: 1. Dark field microscopy, which examines specimens under oblique illumination to identify the thin, spiral Treponema pallidum bacteria. 2. Serological tests including non-treponemal tests like VDRL that detect anti-lipoidal antibodies, and treponemal tests like FTA-ABS that are more specific and detect antibodies to T. pallidum antigens. 3. Other tests like PCR, animal inoculation and direct fluorescent antibody testing that can also identify T. pallidum from specimens.

1. Lab diagnosis of
syphilis
Dr. Meera Lalu
First Year MD DVL
03.09.2018

2. REFERENCES
• Sexually transmitted diseases and AIDS by V.K.
SHARMA, 2nd edtn.
• Sexually transmitted infections by BHUSHAN
KUMAR & SOMESH GUPTA, 2nd edtn.
• IADVL textbook of dermatology, 4th edtn, Vol III.
2

3. INTRODUCTION
• Spirochaete- Treponema pallidum (subsp.pallidum).
• Not readily cultured or stained by ordinary
reagents.
• Very thin, thus not visualised under normal light
microscope.
3

4. TESTS FOR SYPHILIS
• DIRECT IDENTIFICATION OF T. PALLIDUM
(when lesions are present):
a) Dark ground microscopy
b) Direct antigen detection tests
c) PCR
d) Animal Inoculation 4

5. • SEROLOGICAL TESTS:
a)Non Treponemal tests-screening.
b)Treponemal tests-confirmation.
.
5

6. DARK FIELD MICROSCOPY
• Dark ground illumination (DGI).
• Most specific for diagnosis of syphilis.
• Done immediately after the specimen is obtained: --
-As the viability of the treponemes is necessary to
distinguish T. pallidum from morphologically similar
spirochaetes.
-Delay decreases the motility of treponemes.
6

7. Prevents
transmitted light
from directly
illuminating the
specimen
Blocks centre of
beam of light that
would otherwise
fill the objective
lens
7

8. Only light rays hitting the organism at an oblique
angle enter the microscope objective
luminous appearance against a black background
8

9. • Most productive during
a)primary syphilis
b)secondary syphilis
c)early congenital syphilis
• Can be carried out on lymph node aspirate when
no moist lesions are seen.
• Amniotic fluid (from amniocentesis).
9

10. COLLECTION OF SPECIMEN
• Clean lesion with sterile gauze soaked in saline.
↓
• Gently abrade the lesion with dry gauze.
↓
• Wipe off any blood stained serum, if any.
↓
• Squeeze the lesion to produce clear serous exudate
(If dry, crusted lesion- scrape it).
↓
10

11. • Exudate is transferred on to the glass slide
↓
• If the material is not sufficient - mix it with a drop of
saline to give a homogenous suspension.
↓
• Cover with coverslip.
↓
• Seal the edges of cover slip with petroleum jelly.
↓
• Examine immediately.
11

12. • White,
• Illuminate
• On a dark background.
• Thin
• Spiral
• 6-20µm long
• 6-20 regular spirals
• Corkscrew
movement(rotation
along longitudinal axis).
12

13. RESULT
• T. pallidum in dark field microscopy is
identified by its typical morphology, size and
characteristic movements.
• T. pallidum is differentiated from the other
treponemes by the tightness of spirals and
characteristic cork screw movements.
TREPONEMA PALLIDUM OTHER NON PATHOGENIC TREPONEMES
Has 6-20 regularly wounded coils . Irregularly wounded coils .
And may be longer & thicker .
Shows a slow, deliberate forward & backward
movement, rotating on its long axis, soft
bending and twisting from side to side.
Lack a characteristic motility .
13

14. • Test should be done on three consecutive days.
• Single dark field microscopy, sensitivity: 50%.
• Sensitivity of dark field examination- upto 80%.
• REASONS FOR TESTING NEGATIVE :
-Non syphilitic ulcer.
-Natural resolution.
-Treated patients.
-Prior topical application of antiseptics or antibiotics.
-Insufficient no of organisms present in the specimen.
A NEGATIVE DARK FIELD FINDING DOES NOT EXCLUDE THE
DIAGNOSIS OF SYPHILIS.
14

15. ADVANTAGES
• Easy to do.
• Most specific method to
confirm the diagnosis in
early syphilis.
• High resolution (0.02µ)
allows for easy detection
of thin & extremely fragile
bacteria compared to
ordinary light microscope.
DISADVANTAGES
• Time consuming.
• Needs operator expertise.
• Cannot differentiate T.
pallidum from other
pathogenic treponemes.
• Not recommended for oral
cavity lesions.
15

16. DIRECT FLUORESCENT ANTIBODY TEST FOR
TREPONEMA PALLIDUM
(DFA-TP)
• Detects & differentiates pathogenic treponemes
from non pathogenic treponemes by an Ag-Ab
reaction.
• Cannot distinguish between the pathogenic strains
of Treponema species.
16

17. ADVANTAGES
• More sensitive and specific
than dark field microscopy.
• Samples from oral mucosa
can also be examined.
• Slides need not be
examined immediately.
• Identification of T.
pallidum in tissues.
DISADVANTAGES
• This test cannot
differentiate T. pallidum
subsp pallidum from other
sub species of T. pallidum.
17

18. POLYMERASE CHAIN REACTION
• Is increasingly becoming the investigation of choice
for identifying T. 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.
18

19. PCR CYCLE
• Comprised of 3 steps:
- Denaturation of DNA at 95⁰C
- Primer hybridization (Annealing) at 40-50 ⁰C
- DNA synthesis (Primer extension) at 720C
19

20. ANIMAL INOCULATION
• Animal infectivity testing, done on rabbit testis.
• Oldest method.
• Most sensitive for detecting infectious
treponemes.
• Used as a gold standard for measuring sensitivity
of methods like PCR. 20

21. SEROLOGICAL TESTS
• Useful in the latent stage of the disease, as
treponemes are not readily sustainable in culture
and lesions are usually absent.
• AN IDEAL SEROLOGICAL TEST:
1. high specificity & sensitivity.
2. suitable for treatment monitoring.
3. give a negative result on successful therapy.
4. give a clear cut diagnosis of reinfection. 21

22. • PRINCIPLE:
• T.pallidum infection produces antibodies to more
than 20 different polypeptide antigens.
• 2 types of antibodies:
1. Non specific antibodies (reagins): directed
against lipoidal antigen of T. pallidum as well as
mitochondrial & nuclear membranes of human
cells (autoantibodies).
2. Specific anti-treponemal antibodies: directed
against T. pallidum.
22

23. • Specific anti T.pallidum IgM antibodies develop
during the second week of infection.
• IgG antibody response begins around the fourth
week after infection and usually persists.
• Treatment causes generalized loss of
antibodies. However, IgG antibodies may persist
at a low detectable level.
23

24. 24

25. SEROLOGICAL TESTS
• Non-treponemal tests- for initial
screening.
• Treponemal tests- to confirm diagnosis.
25

26. A) NON TREPONEMAL TESTS
a) VDRL(Venereal Disease Research laboratory)
b) RPR(Rapid plasma reagin) card test
c) RST (Reagin screen test )
d) USR (Unheated Serum reagin test )
e) ART ( Automated Reagin Test)
f)TRUST (Toluidine Red Unheated Serum Test) 26

27. B) TREPONEMAL TESTS
a) Fluorescent Treponemal Antibody Absorption
(FTA-ABS) Test .
b) T. pallidum haemagglutination assay (TPHA).
c) Enzyme immune assay (EIA).
d) Treponema Pallidum Immobilisation test (TPI).
27

28. A) NON TREPONEMAL TESTS
• Based on an antigen composed of alcoholic
solution, containing cardiolipin, cholesterol &
purified lecithin to produce standard reactivity.
• measure anti lipoidal IgM and IgG antibodies.
28

29. • They can be performed as a:
1. Qualitative test ( for initial screening; to check
for presence or absence of antibodies)
2. Quantitative test (to follow treatment; amount
of antibodies present)
• Except for VDRL & RPR tests, most of lipoidal
antigen tests are not used.
29

30. • Antilipoidal antibodies are produced not only
in syphilis & other treponemal diseases, but
also in non-treponemal disease of an acute or
chronic nature in which tissue damage occurs.
30

31. • SPECIMEN OF CHOICE- SERUM.
• But plasma can also be used in RPR card test &
TRUST.
• VDRL- only test used for testing CSF.
• Plasma not used in VDRL, since the samples
must be heated before testing.
31

32. VDRL
• SLIDE FLOCCULATION TEST.
• VDRL antigen: Cardiolipin antigen is an alcoholic
solution composed of 0.03% cardiolipin, 0.9%
cholesterol & 0.21% lecithin.
• Cardiolipin antigen should be freshly constituted each
day of test.
• VDRL slide: glass slide measuring 2 X 3 inches, with 12
concave depressions (each 16mm diameter & 1.75mm
deep). 32

33. VDRL SLIDE 33

34. • Patients serum is inactivated by heating at 56⁰C X
30min in a water bath.
• QUALITATIVE TEST:
• 0.05ml of inactivated serum is taken into 1 well.
↓
• Add 1/60thml (or 1 drop from 18 gauge needle) of
cardiolipin antigen.
↓
• Rotate at 180rpm X 4min.
↓
• View under low power, for flocculation.
• Every test must be accompanied with a known reactive
& nonreactive controls. 34

35. • Medium or large clumps
• Small clumps
• No clumping/ very slight
roughness
• Reactive (R)
• Weakly reactive (W)
• Non reactive (N)
35

36. • Reactive samples are then subjected to quantitative test.
• QUANTITATIVE TEST
• Serum is doubly diluted in saline from 1:2 to 1:256.
↓
• 0.05ml of each dilution is taken in the well.
↓
• 1/60ml of antigen is added to each dilution.
↓
• Rotate → observe under microscope.
The highest dilution showing flocculation is considered as
reactive titre.
VDRL is said to be positive when the titre is >1:8 in dilution. 36

37. VDRL for CSF
• Diagnosis of neurosyphilis.
• Antigen diluted in equal volumes with 10% saline.
• CSF need not be heated.
• Volume of antigen solution taken is 0.01ml (or 1
drop from 21 gauge needle).
• Rotation time- 8min.
37

38. REPORTING OF RESULTS
• REACTIVE:
-past/present infection with a pathogenic T.
pallidum, either treated or not.
-false positive reaction.
• NON REACTIVE:
-no current infection.
-effectively treated infection.
-but does not rule out syphilis in its incubation
period. 38

39. • A four fold increase in titre→infection
reinfection
treatment failure.
• A four fold decrease in titre→effective therapy.
• SERORESISTANT SYPHILIS
(WASSERMANN FASTNESS/ SEROFAST):
When a non treponemal test shows persistent
reactivity with no signs of decline in titre after 6
months of adequate therapy, or fails to show four fold
decrease in titre within a year.
39

40. PROZONE PHENOMENON
• Undiluted serum specimens having high quantity of
reagin antibodies occasionally will give a false
negative reaction, but on further dilutions, it
becomes positive.
• Incidence- low (0.4%).
• It may attain clinical significance:
- patients on continous immunosuppressive
therapy.
- HIV seropositive patients. 40

41. RAPID PLASMA REAGIN TEST (RPR)
• Performed with unheated serum on small plastic
coated cards onto which circles have been imprinted.
• Charcoal particles are added to the VDRL antigen for
easy readability without a microscope.
• Simple; no laboratory equipments required.
• Results available within 5min.
• Costly.
• Lower sensitivity. 41

42. • Card is rotated at 100 rpm for 8 minutes.
• Presence of anticardiolipin antibodies produces
flocculation of charcoal particles→ positive test.
42

43. REAGIN SCREEN TEST (RST)
• Similar to RPR.
• Sudan black dye instead of charcoal.
• Similar results to RPR.
43

44. UNHEATED SERUM REAGIN
(USR) TEST
• Stabilised VDRL antigen.
• Unheated serum.
• Results comparable to VDRL, but less sensitivity &
specificity.
44

45. AUTOMATED REAGIN TEST (ART)
• Reagents of RPR.
• Autoanalyzer.
TOLUIDINE RED UNHEATED SERUM
TEST(TRUST)
• Similar to RPR.
• Antigen remains stable for 6months.
• Toluidine blue instead of charcoal, for better
visualisation.
• Less sensitivity, specificity & reproducibility. 45

46. FALSE POSITIVE REACTIONS
1. Technical false positive reaction.
2. Variation in the normal (BFP reactors): In few
normal individuals, there may be excess production
of reagin.
3. BIOLOGICAL FALSE POSITIVE REACTION:
Polyclonal antiphospholipid autoantibodies
produced against lipoidal antigens present in normal
tissue and in conditions that destroy cell nuclei are
responsible for this reactions.
46

47. TYPES OF FALSE POSITIVE REACTIONS
ACUTE (<6 months)
• Hepatitis
• Infectious Mononucleosis
• Viral pneumonia
• Chicken pox
• Measles
• Malaria
• Pregnancy
• Laboratory accident or
technical error
CHRONIC (>6 months)
• CT disorders- SLE
• Diseases associated with
Ig abnormalities
• Narcotic addiction
• Old age
• Leprosy
• Malignancy
47

48. • Persistently low titre positive reagin tests with
repeatedly negative treponemal tests are the
rule in acute BFP reactions.
• Strongly positive reactions are more common
in chronic BFP reactors.
48

49. TREPONEMAL TESTS
• 1949 – Nelson and Mayer developed the first treponemal
antibody test, the T. pallidum immobilization (TPI) test.
• Uses T. pallidum (Nichol’s strain) grown in rabbit’s testes as
antigen.
• It is based on the ability of patient’s antibody and
complement to immobilise living treponemes , as observed
by dark-field microscopy.
• TPI- less sensitive, less specific, complicated, time consuming.
49

50. TREPONEMAL TESTS
a) Fluorescent Treponemal Antibody Absorption
(FTA-ABS) Test .
b) T. pallidum haemagglutination assay (TPHA).
c) Enzyme immune assay (EIA).
d) Treponema Pallidum Immobilisation test (TPI).
50

51. FTA
• 1957- development of FTA test.
• Used a 1:5 dilution of the patient’s serum in saline
solution, reacting with a suspension of killed
treponemes.
• Fluorescein-labelled anti- human immunoglobulin
was used as conjugate.
• Test was read under microscope with a UV light
source. 51

52. • Later fluorescein isothiocyanate(FITC) was used to
prepare the labeled anti-human globulin conjugate
↓
• Non specific reactions occurred (25%)
(because of shared antigens common to T. pallidum &
non pathogenic treponemes).
↓
• To eliminate these false positive reactions →
FTA-200 TEST
1:200 dilution.
highly specific.
not very sensitive. 52

53. FTA -ABS
• Deacon & Hunter, by preparing a sonicate from
cultures of Reiter’s spirochaete, removed the
common antigens by absorption.
↓
FTA -ABS test
(more specific and sensitive)
• Gold standard for diagnosis.
53

54. FLUORESCENT TREPONEMAL ANTIBODY
ABSORPTION (FTA-Abs) TEST
• Indirect immunofluorescence antibody test.
• The intensity of fluorescence is reported as
nonreactive, borderline or reactive.
• Reactivity begins in the 3rd week of infection.
• Reactivity continues even after succesful therapy.
54

55. • Most sensitive serologic test in the early stages of
syphilis.
• Highly sensitive & specific.
• Can detect recent infection 1-2weeks before other
assays.
• Costlier.
55

56. FTA -ABS
56

57. TREPONEMA PALLIDUM
HEMAGGLUTINATION ASSAY (TPHA)
• 1965 – Rathlev
• Qualitative hemagglutination test, using
formalinised tanned sheep RBCs as the carrier for
T.pallidum antigen.
• Less expensive; less complex.
• Easier to perform.
• Sensitivity is superior to VDRL & FTA-Abs test,
except in primary syphilis.
• If agglutination occurs in a dilution of ≥1:80→
reactive.
57

58. VARIANTS OF TPHA
• Microhemagglutination assay with T. pallidum
antigen (MHA-TP).
• Automated microhemagglutination assay with T.
pallidum antigen (AMHA-TP).
• Hemagglutination treponemal test for syphilis
(HATTS).
• Finger prick MHA-TP.
58

59. TREPONEMA PALLIDUM
IMMOBILISATION (TPI) TEST
• This test detects an antibody, which inhibits the
normal movements of T. pallidum.
• % of treponemes immobilised Result
≥50 % +ve
20-50% doubtful
≤20% -ve
• Nearly 100% positive. 59

60. • Becomes +ve, few days to 1 week later than reagin
test.
• Specificity, thus able to distinquish BFP reactions
from genuine positives.
• With early treatment, becomes –ve.
If it delayed for 5-6months, remains +ve.
• Time consuming
• Expensive.
• Not performed nowadays. 60

61. 61

62. FOLLOW UP
• Non treponemal tests like VDRL/RPR remain the
method of choice for follow up testing, to
demonstrate a decline in titre.
• Patients should be clinically & serologically
examined at 6 & 12 months.
• Decline in titre depends on the factors like initial
titre, stage of infection when treated, treatment
regimen.
62

63. • Tests should be performed at 3 months interval for
atleast 1 year.
• Following adequate therapy for primary and
secondary syphilis, there should be at least 4 fold
decline in titre by 3rd or 4th month and an 8 fold
decline by 6 to 8 months.
• Failure of titres to decline after treatment -
patients treated during late stage of syphilis and in
patients treated for reinfection.
63

64. 64
(VDRL/ RPR)
(FTA-ABS/ TPHA)

65. SYPHILIS & HIV
• Problems in diagnosis of syphilis with HIV are:
1) Confusing signs & symptoms.
2) Lack of serologic response in a patient with
clinically confirmed active syphilis.
3)Failure of nontreponemal test titres to decline
after treatment with standard regimens.
4) Unusually high titres in nontreponemal test.
65

66. 5) Rapid progression to late stages of syphilis and
neurologic involvement even after treatment of
primary or secondary syphilis.
6) Disappearance of treponemal test reactivity over
time.
• BFP reactions for cardiolipin tests (VDRL & RPR) and
PROZONE PHENOMENON can occur.
• Diagnosis is either by observation of T. pallidum or
by appearance of serologic reactivity after
treatment. 66

67. NEUROSYPHILIS
• Non treponemal test is positive- in acute syphilitic
meningitis.
• CSF CHANGES – elevated pressure, mononuclear
pleocytosis of 10 -200 cells/cu.mm, elevated
protein concentration (200 mg/dl), elevated
globulin level & reduction in glucose.
• Presence of positive VDRL/RPR & a raised TPHA
index in CSF indicates neurological involvement.67

68. 68