Simple presentation of syphilis lab diagnosis

1. Laboratory Diagnosis of
Syphilis
Patricia Antonette E. Pomar, RMT
NRL-SLH/SACCL

2. Objectives
• Short introduction of Syphilis
• Stages of Syphilis Infection
• Laboratory Diagnosis of Syphilis
• Traditional vs. Reverse Algorithm for Syphilis Screening
• Algorithm used in NRL-SLH / SACCL
• Interpretation of result and follow up

3. Genus Treponema
• Etiologic agent: Treponema
pallidum
• helically coiled, cork-screw
shaped
• Cannot be cultured in-vitro
• Transmission
 Sexual
 Trans-placental
 Percutaneous following
contact with infectious
lesions
 Blood Transfusion
Treponema pallidum on Darkfield Microscopy

4. Genus Treponema pallidum
subspecies causes
Treponema pallidum subsp. pallidum Venereal syphilis
Treponema pallidum subsp. pertenue Yaws
Treponema pallidum subsp. endemicum Endemic syphilis / Bejel
Treponema pallidum subsp. carateum Pinta

5. Stages of Syphilis Infection
Primary Secondary Latent Tertiary
Development of primary
lesion or ‘CHANCRE” at the
site of inoculation
Secondary lesions, most
commonly mucocutaneous
Host suppresses infection,
but no lesions are clinically
apparent
Approximately 30% of
untreated patients progress
to the tertiary stage within
1 to 20 years
Painless
May persist from weeks to
months
Only evidence is a positive
serologic test
Rare because of the
widespread availability and
use of antibiotics
Highly infectious
Clinical manifestations:
-Rash (75%–100%)
-Lymphadenopathy (50%–86%)
-Malaise
-Mucous patches (6%–30%)
-Condylomata lata (10%–20%)
-Alopecia (5%)
-Liver and kidney involvement
can occur
-Splenomegaly is occasionally
present
May occur between primary
and secondary stages,
between secondary
relapses, and after
secondary stage
Manifestations
-Gummatous lesions
-Cardiovascular syphilis
Heals within 3-6 weeks
Categories:
Early latent: <1 year
duration
Late latent: 1 year
duration
Serologic test may not be
positive in early primary
syphilis Serologic test are usually
highest in titer

7. Primary Syphilis
Source: CDC/ NCHSTP/ Division of STD Prevention /STD Clinical Slides

8. Secondary Syphilis
Generalized rash Condylomata lata

9. Secondary Syphilis
Nickel / Dime Lesions Alopecia

10. Tertiary Syphilis
Serpiginous Gummata of Forearm Ulcerating Gumma

11. Late Syphilis—Cardiovascular
11
Source: CDC/ NCHSTP/ Division of STD Prevention, STD Clinical Slides

12. Neurosyphilis
• Occurs when T. pallidum invades the central
nervous system (CNS)
• May occur at any stage of syphilis
• Can be asymptomatic
• Early neurosyphilis occurs a few months to a few
years after infection
• Clinical manifestations can include acute syphilitic
meningitis, meningovascular syphilis, and ocular
involvement
• Neurologic involvement can occur decades after
infection and is rarely seen
• Clinical manifestations can include general paresis, tabes
dorsalis, and ocular involvement
• Ocular involvement can occur in early or late
neurosyphilis.
Spirochetes in Neural Tissue

13. Congenital Syphilis
• Occurs when T. pallidum is transmitted from a pregnant
woman to her fetus
• May lead to stillbirth, neonatal death, and infant
disorders such as deafness, neurologic impairment, and
bone deformities
• Transmission can occur during any stage of syphilis; risk is
much higher during primary and secondary syphilis
• Fetal infection can occur during any trimester of
pregnancy
• Wide spectrum of severity exists; only severe cases are
clinically apparent at birth
• Early lesions (most common): Infants <2 years old; usually
inflammatory
• Late lesions: Children >2 years old; tend to be immunologic and
destructive

14. Congenital Syphilis
Mucous Patches Perforation of Palate

15. Laboratory
Diagnosis

16. NON TREPONEMAL TREPONEMAL
• VDRL (Venereal Disease
Research Laboratory)
• RPR (Rapid Plasma Reagin)
• TRUST (Toluidine Red
Unheated Serum Test)
• USR (Unheated Serum Reagin)
• TP-PA (Treponema Pallidum
Particle Agglutination)
• FTA-abs (Fluorescent
Treponemal Antibody -
Absorbed)
• EIA (Enzyme Immunoassay)

17. NON TREPONEMAL TESTS (Screening)
Principles
• T. pallidum infection leads to the production of reagin
Reagin – Antibodies to substances released from cells
damaged by T. pallidum
• Reagin reacts with cardiolipin
Cardiolipin – a phospholipid component of certain
eukaryotic and prokaryotic membranes
• Not specific for T. pallidum
• Titers usually correlate with disease activity and results are
reported quantitatively (can be tittered)
• May be reactive for life, referred to as “serofast”

18. NON TREPONEMAL
Advantages Disadvantages
• Rapid and inexpensive
• Easy to perform and
can be done in clinic
or office
• Quantitative
• Used to follow
response to therapy
• Can be used to
evaluate possible
reinfection
• May be insensitive in
certain stages
• False-positive
reactions may occur
• Prozone effect may
cause a false-negative
reaction (rare)

19. NON TREPONEMAL TESTS
RAPID PLASMA REAGIN (RPR) SERUM
VENEREAL DISEASE RESEARCH LABORATORY (VDRL) CEREBROSPINAL FLUID
Cardiolipin
Charcoal
Reagin
Serum
or
CSF

20. NON TREPONEMAL TESTS

21. NON TREPONEMAL TESTS

22. TREPONEMAL TESTS
• Specific for T. pallidum
• Measure antibody (IgM & IgG) directed against T. pallidum
antigens by particulate agglutination (TP-PA) or
immunofluorescence (FTA-abs)
• May remain positive after treatment
• More sensitive and specific than non-trep. tests
• More expensive and labor intensive
• Can not quantitate…not useful for following response to
treatment

23. TREPONEMAL TESTS
ADVANTAGES DISADVANTAGES
• High Specificity
• Possibly higher sensitivity
during early and late syphilis
stages compared to non-
treponemal tests
• Newer Methods
• Objective result
interpretation
• Automation option
• High throughput
• High reproducibility/
precision
• Remain positive despite
treatment
• Cannot be used to monitor
response to therapy
• Conventional Methods
• Subjective interpretation
requiring technician
expertise to read
• Newer Methods
• Expensive instrumentation
• Higher cost/test

24. Direct Fluorescence Antibody-TP

25. Darkfield Microscopy

26. Enzyme Immunoassay (EIA)

27. TP – Particle Agglutination (TPPA)
TP – Hemagglutination (TPHA)

28. Interpretation of Results
Possible ExplanationTreponemal
Tests
(TP-PA/
FTA-ADS)
Non-
Treponemal
tests
(RPR/ VDRL)
Syphilis - recent or previous
Yaws or pinta
++
No syphilis
False positive
–+
Consistent with previously treated or
untreated Syphilis
Yaws, Pinta, Bejel
+–
No syphilis
Syphilis in incubation period
––

29. Syphilis Screening Algorithms:
Traditional versus Reverse Screening

30. Traditional Algorithm
Non-treponemal test (e.g., RPR)
Treponemal test (e.g., FTA) Negative for syphilis
Non-reactive
Non-reactive
Syphilis Negative for syphilis
Reactive
Reactive

31. Traditional Algorithm
ADVANTAGES DISADVANTAGES
• Results show good
correlation with disease
status
• Rapid, inexpensive
screening method
• Excellent option for
laboratory with small
throughput
• Recommended by the CDC
• Manual (RPR) and subjective
interpretation
• Screening method is non-
specific and may lead to
false-positive results
• Not suitable for high
throughput laboratories
• Potentially lower sensitivity
for detecting early syphilis
and late/latent disease

32. Reverse Algorithm
Treponemal test (eg, EIA)
Non-Treponemal test (eg, RPR) Negative for syphilis
Non-reactive
Non-reactive
Syphilis Second Treponemal Test (e.g., TP-PA)
Reactive
Reactive
Non-reactiveReactive
Evaluation Required* Negative for syphilis

33. Reverse Algorithm
ADVANTAGES DISADVANTAGES
• Objective interpretation of
results
• Results from EIA or MFI
can be interfaced with LIS
• Specific screening test for
anti-T. pallidum
antibodies
• Potentially increased
detection of patients with
early syphilis
• Higher cost/sample
• Higher assay complexity
• Increased detection of
patients with screen (+),
RPR (-) results

34. Follow Up
 Primary or secondary syphilis
 Reexamine at 6 and 12 months.
 Follow-up titers should be compared to the maximum or baseline
nontreponemal titer obtained on day of treatment.
 Latent syphilis
 Reexamine at 6, 12, and 24 months.
 HIV-infected patients
 3, 6, 9, 12 and 24 months for primary or secondary syphilis
 6, 12, 18, and 24 months for latent syphilis
 Neurosyphilis
 Serologic testing as above
 Repeat CSF examination at 6-month intervals until normal