Labarotory diagnosis of syphilis

1. Laboratory diagnosis in
syphilis
Presented by- Dr Avono Dominica Kulnu, JR 3
Moderated by- Dr Aurobindo Bhoi, Asst Prof
SCB Medical College, Cuttack

2. Syphilis
• Syphilis is an infectious disease caused by the spirochaetal bacterium
Treponema pallidum usually acquired through sexual contact

4. Direct diagnostic methods for syphilis
• Identify the organism
• Direct Microscopy
• Dark-field microscopic examination
• Direct fluorescent antibody staining of T. pallidum (DFA-TP)

5. • Materials required:
1. Dark field microscope
2. Thin glass slides (1mm)
3. Thin cover slips (.1mm)
4. Glass marking pencil / marker
5. Normal Saline
Dark field microscopy

6. • Collection from genital ulcers
1. Remove any scab or crust covering the lesion.
2. Secondary infection exudate, removed with a gauze sponge.
3. Compress the base of the lesion, promote the accumulation of tissue
fluid on the ulcer surface.
4. Apply a glass slide to the oozing lesion or use a sterile
bacteriological loop to transfer the fluid from the lesion to the glass
slide.
5. Place a cover glass to remove air bubbles
6. Examine the slide immediately (within 5-20mins) because the
motility of the treponemes may be reduced or completely lost.

7. • Dry papulo squamous lesions of the skin
• Cervical/vaginal lesions
• Mucous patches
• Lymph node
• Reading:
• Morphology: In a black background, silvery-white and bright, thin, spiral
organism, 6-14µm long, with 8-14 spirals, pronounced and regular.
• Motility: moves by rotation, bending and twisting and increasing and
decreasing in length.
• After examining a slide, discard it into a container of 70% alcohol or 1%
sodium hypochlorite.

8. Disadvantages:
• Requires a trained, experienced personnel
• Not for oral, rectal and non penile genital lesions- commensals
• Not recommended for dry sores– dilution with saline is disadvantageous
• Examination should be done immediately after specimen collection.
• Limited sensitivity
• Failure to detect T. pallidum by this test does not rule out syphilis
• False negative- if treatment with antibiotics

9. Direct Fluorescent Antibody staining of T. pallidum(DFA-TP)
Method :
• Material collected from lesion and stained with
fluorescein labelled anti T. pallidum globulin.
• Distinct, sharply outlined, apple green colored
spirochaetes under fluorescent microscope

10. • Advantages:
• Easier to perform than dark-field microscopy
• More sensitive and specific than dark field microscopy
• Living, motile treponemes are not required.
• Slides need not be examined immediately
• Specimen : lesion exudates, (including rectal, intestinal and oral
lesions), tissues, body fluids can be examined

11. Natural Course of Infection and Serological
Response
• Specific anti-TP IgM- end of the 2nd week of infection;
• Anti-TP IgG, development of symptoms- about 4 weeks
• Following treatment of early syphilis,
• Titers of non-specific antibody and specific IgM decline rapidly
• Specific IgG antibodies generally persist.
• HIV infection
• May reduce or delay the antibody response in primary syphilis
• In most cases the response is exaggerated.

13. • Non-treponemal tests: Detect non-specific reagin antibody (IgG,
rarely IgM) by using cardiolipin antigen derived from bovine heart
• Treponemal tests: Detect species-specific antibody by using T.
pallidum specific polysaccharide antigen
Serology (Indirect diagnostic method)

14. Serological tests
Treponemal tests:
For screening
• Slide flocculation tests
• VDRL test
Variants of VDRL test:
• RPR test
• TRUST
• USR test
• RST
• ART
• Wassermann test (complement
fixation test)
• Kahn test (tube flocculation test)
Non treponemal tests:
For confirmation
• TPI test
• FTA-Abs test
• TPHA
• RPCF
• EIA/ELISA - Enzyme Immunoassay
• CLIA/CIA - Chemiluminescense
Immunoassay
• Microbead - Immune Assays (Bioplex)
• Western blot technique

15. NON TREPONEMAL
TESTS

16. Wassermann reaction/ test
• First blood test for syphilis, complement fixation test
• Developed by Wassermann, Julius Citron, and Albert Neisser in 1906
• Sample of blood or CSF taken  introduced to the cardiolipin
antigen extracted from bovine muscle or heart.
• Treponemal nonspecific antibodies react with the lipid – the
Wassermann reaction (WR) of antiphospholipid antibodies (APAs)
• Newer tests, such as RPR and VDRL tests, have mostly replaced it.

17. Venereal Disease Research Laboratory test
• Slide Flocculation test
• Widely used, simple and rapid serological test
• Mechanism: detects IgG, IgM
• formed by host in response to lipoidal material released by
damaged host cells early in infection and
• lipid from the cell surfaces of the treponeme
• VDRL antigen – 0.03%cardiolipin, 0.9%cholesterol,
0.21%lecithin

18. • Reagents:
1) VDRL antigen
2) VDRL buffered saline
3) Normal saline (0.9%)
• Process:
1. Preparation of antigen emulsion
2. Standardization of antigen suspension
3. Preparation of Serum: In a serological waterbath with a temperature of 56
C, heat the specimen serum for 30minutes.
4. Inactivated serum + a drop of VDRL antigen and rotated at 180 rpm for 4
minutes in a VDRL rotator
5. Examine under microscope (10x)

19. Reading:
- Non-reactive: No clumping or slight
roughness
- Reactive: Medium to large clumps
- Weakly reactive : small clumps
• Quantitative test: Test performed with
serial dilutions (1:2, 1:4, 1:8 and so on)
of serum with 0.9% saline
• VDRL-CSF: No preheating of CSF is
needed

21. Variants of Venereal Disease Research Laboratory
test
RPR Rapid Plasma Reagin test-
• Results within 5 min
• Conventional RPR card test
• Uses cardiolipin Ag
• Carbon particles to detect reagin Ab
• Automated RPR test
• Immunoassay technique using latex
• Particles coated with lecithin and cardiolipin

22. RPR Test
• Reading:
• Small to large clumps – Reactive
• No clumps or slight roughness-
Non- Reactive
• If clumps occur, perform the
tests in serial dilutions starting
from 1in2,4,8,16 and so on. Find
out the last dilution in which
clumping occurs

23. VDRL RPR
Results read microscopically - clumps are
smaller
Results read macroscopically - Finely
divided carbon particles coated cardiolipin
antigens are used so that larger visible
clumps are formed
Antigen, once reconstituted, should be used
within 24 hours
EDTA is used as stabilizer; hence RPR
antigen can be stored longer (up to 6
months at 4-100C)
Preheating of serum is required to remove
non specific inhibitors
CSF-no heating
Preheating of serum is not required as
choline chloride is used to remove
inhibitors
Blood, plasma, serum, and CSF can be
tested
Blood, plasma and serum can be tested but
not CSF
Rotation of slide is done for 180 rpm for 4
mins
(CSF-8 min )
Rotation of card is done for 8 mins
Sensitivity in primary syphilis is 78% Sensitivity in primary syphilis is 86%
It is cheaper; one vial of VDRL antigen can
be used for 250 tests. It is preferred for field
studies and for antenatal screening
RPR is expensive than VDRL. It is
preferred when sample load is less.

24. Variants of Venereal Disease Research Laboratory
test
• Unheated serum regain (USR ) test: similar to VDRL except for:
- EDTA- antigen stabilizer
- pre-heating of serum is not needed
• Toluidine red unheated serum test (TRUST)
- Modified RPR test- toluidine red pigment particles used
- Does not require microscope for examination
• Reagin screen test (RST)
- Sudan black diazo dye used
• Automated reagin test (ART)

25. Interpretation
• Qualitative test- nonreactive, reactive, and weakly reactive
• Reactive- past/ present/ treated/ untreated infection, false positive
• Non reactive- no current infection, effectively treated infection, does not
rule out infection in incubation period
• In reactive test:
• A fourfold increase in titer – infection, reinfection, or treatment failure
• A fourfold decrease in titer - effective therapy
• Examination of serial dilutions of serum
• follow the course of illness, response to therapy

26. 1 : 1024
1 : 512
1 : 256
1 : 128
1 : 64
1 : 32
1 : 16
1 : 8
1 : 4
1 : 2
1 : 1
Dilutions of Non-specific Tests (RPR/VDRL)
2 dilution or
“4 fold”
decline
1 dilution or
“2 fold”
decline

27. Seroresistance or serofast state
• When a nontreponemal serologic test
• shows persistent reactivity with no signs of decline of titer at 6 months
after adequate therapy or
• fails to show a fourfold decrease of initial high titer within 1 year
• Causes-
• Persistent low level T. pallidum infection
• Variability of host antibody response to infection
• Tissue injury due to nonsyphilitic inflammatory conditions

28. Advantages
1. Inexpensive and simple
2. Mass screening
3. Baseline titer can be used to follow-up
the treatment response.
4.Detectable 7–10 days after the
appearance of primary chancre (or 3–5
weeks after acquiring the infection)
5. Neurosyphilis: VDRL detects CSF
antibodies
Disadvantages
1. Reduced sensitivity in primary
syphilis and late latent syphilis
2. False-positive results
3. False-negative results

29. False-negative reaction
• PROZONE phenomenon
• Undiluted serum specimens reagin antibody excess
• On serial dilutions, the test becomes positive.
• Clinically significant in certain populations at risk :
• Those who are on continuous immunosuppressive drugs
• Those who are HIV seropositive
• POSTZONE phenomenon
• Antigen excess

30. In VDRL test
Zone of equivalence : Optimal ratio of the antigen antibody  yields insoluble
visible precipitate  test positive

31. False positive reactions
• Technical error
• Variation in normal- excess production of regain
• Biologically false positive reactions (BFP)-
• Polyclonal antiphospholipid autoantibodies against lipoidal
antigens in normal tissues and conditions that destroy cell nuclei
• Acute- less than 6 months, low titre positive test with repeated
negative treponemal tests
• Chronic- longer than 6 months, strongly positive reactions

32. Acute BFP Chronic BFP
Infections- Malaria Autoimmune diseases- SLE
Leprosy RA
Typhus PAN
Viral pneumonia Dysgammaglobulinemias
Infectious mononucleosis
Filariasis
Trypanosomiasis
Lyme disease
Pregnancy

33. TREPONEMAL TESTS

34. Treponema pallidum immobilization test
• Principle: ability of patient’s antibody and complement to
immobilize the live actively motile T.pallidum (virulent Nichols
strain from rabbits) observed under dark ground microscope
• Interpretation-
• positive- 50% or more of the treponemes are immobilized
• 20% or less- negative
• 20-50%- doubtful
• Becomes positive few days to a week later than the VDRL test
• Specificity - 100%
• Time consuming, expensive, and hazardous- not performed
nowadays.

35. Fluorescent treponemal antibody absorption test
• Indirect Immunofluorescence antibody test
1. Patient’s serum diluted with an extract of nonpathogenic Reiter
treponemes to remove group specific treponemal antibodies
2. Layered on a slide previously coated with killed T. Pallidum
3. Serum antibodies bound to T. Pallidum can be detected by addition of
fluorescent labeled anti-human immunoglobulin
4. Examined under fluorescent microscope : Presence of antibody in
patient’s serum is indicated by fluorescence.
• IgM-FTA-ABS test-for congenital syphilis

38. • Advantages:
1. Highly sensitive and specific in all stages of syphilis
2. Reactivity begins in the 3rd week of infection
3. Most sensitive serological test in the early stage of syphilis.
4. Detects CSF antibodies- highly sensitive
• Disadvantage:
1. False positive results- autoantibodies like RF, Borrelia
infections
2. Time consuming
3. Well trained personnel

39. Fluorescent treponemal antibody absorption
double-staining test
• A fluorochrome-labelled counterstain for T. pallidum and antihuman IgG
conjugate labeled with tetramethylrhodamine isothiocyanate to detect
antibody in patient’s serum  visible test reaction
• False positivity- 1%

40. Treponema pallidum haemagglutination assay (TPHA)
• A qualitative haemagglutination test using tanned formalinised sheep RBC’s
coated with T. pallidum antigens (sensitized cells)
• Patients serum is diluted in absorbing diluent which contains:
• Sonicated sheep & bovine red cell stroma
• Normal rabbit testicular extract
• Reiter treponemal sonicate
• Normal rabbit serum
• Stabilizers
• Serum is tested with both sensitised and non sensitised RBC’s

43. Reporting
• Preliminary results are available in 3-4 hrs, final results in 18 hrs
• REACTIVE : if agglutination occur in a dilution of 1:80 or more
• Reactivity is positive around 4th-5th week of infection
• False positive may be due to heteroagglutination, non specific group
antibodies

44. Variations of TPHA
• Microhemagglutination assay with T.pallidum antigen (MHA- TP)
• Performed on microtiter plates
• Less duration- 4hrs
• Cost reduced
• Automated microhemagglutination assay with T.pallidum antigen
(AMHA-TP)
• Haemagglutination treponemal test for syphilis (HATTS)
• Finger prick MHA-TP

45. Treponemal EIA/ ELISA
• Recombinant treponemal antigen is fixed to wells in microtiter plates
• Serum is added and rinsed off after 30–60 min
• Antibody to T. Pallidum binds to antigen and reacts in the 2nd incubation
with an enzyme labeled antihuman globulin & enzyme substrate
• Enzyme- horseradish peroxidase, alkaline phosphatase
• Substrate- hydrogen peroxide, p nitrophenyl phosphate
• Result- 3–4 h.
• Appropriate alternative to the combined VDRL/RPR and TPHA test
• They can be used to detect IgM, IgG or combined

47. Advantages:
1. Automated (or semi-automated) processing
2. Objective reading of results
3. IgM-specific assays can assist in distinguishing between early and late
infections.

48. Western blot technique
• Based on the whole T. pallidum lysate antigen
• The presence of antibodies to the immunodeterminants with molecular
weights 15.5, 17, 44.5, and 47 kDa - diagnostic for acquired syphilis
• IgM-specific conjugate - in the diagnosis of congenital syphilis.
• Detects IgG and IgM antibodies separately
• Highly sensitive and specific

49. Limitations of treponemal tests
• Remain reactive for years with or without treatment
• Treponemal test antibody titers correlate poorly with disease activity
• Should not be used to evaluate response to therapy, relapse, or
reinfection in previously treated patients
• Does not differentiate venereal syphilis from endemic syphilis (yaws and
pinta).

50. Sensitivities of serological tests
Stage of disease (% positive)
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
TPPA 76 (69-90) 100 97 (97-100) 94
EIA 93 100 100

51. Other tests- Oral fluid test for syphilis
• Time-resolved fluorescence immunoassay
• To detect antibodies to T. pallidum recombinant antigens in the oral fluid
specimens collected using “Oracol”swab
• In early syphilis –
• Sensitivity: 100%
• Specificity: 97.9%
• Patients with positive syphilis serology –
• Sensitivity: 76.5%
• Specificity: 96.9%
• Potentially useful when collection of blood is not practicable.

52. Point of care tests for syphilis
• Aim: To integrate rapid, simple, and technologically appropriate
syphilis testing at venues with limited resources and areas with high
syphilis prevalence
• Two varieties of tests used:
• Immunochromatography strip tests
• Particle agglutination tests
• Also detects antibodies to other diseases like HIV, Hepatitis

53. Cerebrospinal fluid examination in syphilis
Indications
1. Neurological, ophthalmic, or auditory symptoms and signs
2. Other clinical evidence of active infection – aortitis, gumma or iritis
3. Treatment failure
4. HIV infection
5. A nontreponemal serum titer of >32 if the duration of syphilis is
over 1 year
6. A non penicillin-based treatment regimen is planned
7. All infants suspected of prenatal syphilis.

54. Typical CSF findings of neurosyphilis
1. Moderate mononuclear pleiocytosis (10–400 cells/mL)
2. Elevated total protein (0.46–2.0 g/L)
3. Positive CSF VDRL criteria.
CSF VDRL Reading:
Visible clumping - Reactive (R)
No clumping or very slight roughness - Non-reactive (NR)

55. • CSF VDRL test- highly specific
• False-positive results- rare in the absence of blood contamination
• Early syphilis- examine CSF after 1–2 years of post treatment
follow-up
• In untreated asymptomatic late syphilis, a CSF examination should
always be done
• A normal CSF is by definition an essential prerequisite for a diagnosis
of latent syphilis.

56. Nucleic acid testing
• PCR-based methods- real-time, in-house and multiplex PCRs
developed for the detection of T.pallidum in clinical specimens
• Highly sensitive, able to detect as low as one to 10 organisms per
specimen with high specificity
• Promise as a test of choice for congenital syphilis, neurosyphilis and
early primary syphilis when traditional tests have limited sensitivity

57. Histopathology
• Perivascular infiltration of
lymphocytes and plasma cells
with intimal proliferation of both
arteries and veins (endarteritis
obliterans)
• Organisms in walls of capillaries
and lymphatic vessels can be
demonstrated by: Levaditi’s
silver stain, fluorescent antibody
technique and
immunohistochemistry

58. • An important principle of syphilis serology- detection of TP antibody by
• screening test, followed by
• confirmatory test- equivalent sensitivity, greater specificity, independent
methodologically
• A second specimen should be tested
• to confirm the results obtained from the first specimen and
• A quantitative non-TP test
• to assess the stage of infection and
• to monitor the effect of treatment.

61. Guidelines for screening of syphilis in pregnancy
• The WHO STI guideline- screening of all pregnant women for syphilis
• during the first antenatal care (ANC) visit
• repeat early in the 3rd trimester
• at the time of delivery

62. Guidelines for screening of syphilis in newborns
• Newborns born to syphilis-positive women detected during ANC should be
tested by
• RPR using infant serum and
• not cord blood as it can yield false-positive result
• All newborns showing fourfold rise in titer compared to that of their
mothers’ titer
• Hospitalize- to initiate penicillin treatment for 10 days.
• Newborns born to high risk mothers should be tested
• Prenatally, at delivery and at 4-8wks

63. Serological tests in HIV
Unusual serologic responses in co-infected patients
• Increased biological false positivity
• Seronegative cases of syphilis.
• Delayed titre responses after treatment
• Return of titres to nonreactivity as immunosuppression advances

64. Serological tests in HIV
• Periodic screening
• At least annually and
• 2–4 times yearly among high-risk groups, such as men who have sex with
men (MSM)
• Irrespective of signs and symptoms, all HIV-positive patients should have
• Baseline VDRL screening and
• Follow-up at 3 months to rule out the possibility of false-negative results,
as seroconversion generally takes about 4–6 weeks after infection
• HIV testing for all new syphilis patients

65. What’s new in syphilis

67. • The new app offers quick and
easy access to streamlined STI
prevention, diagnostic, and
treatment recommendations.
• September 19, 2022

70. References
• Ravindra K, Gulati S. Syphilis. In: S Sacchidanand, editor. IADVL
Textbook of Dermatology, 5th ed. Mumbai: Bhalani Publishing House;
2022. p. 3415-69.
• Kinghorn GR, Omer R. In: Griffiths C, editor. Rook’s Textbook of
Dermatology, 9th ed. West Sussex: John Wiley & Sons, Ltd; 2016. p. 29.3-
.27.
• Naco.gov.in [internet]. New Delhi: Laboratory manual for diagnosis of
sexually transmitted and reproductive tract infections. [updated 2014
January 9]. Available from
https://naco.gov.in/sites/default/files/STI_Lab%20manual_
• Cdc.gov [internet]. Atlanta: STI guidelines. [updated 2021 July 23].
Available from https://www.cdc.gov/std/treatment-guidelines/STI-
Guidelines-2021

71. Thankyou