Dr. Sanjay singhAIIMS

Diagnostic Evaluation of Syphilis

Syphilis

"He who knows syphilis, knows medicine"

Sir William Osler

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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 m in length and 0.1–0.2 m in diameter, 10 regular μ μ spirals at interval of about 1 mμ

Transmission: sexual; maternal-fetal, and rarely by other means

INTRODUCTION

Dr.T.V.Rao MD 4

Ultra Structure

Contains many strongly antigenic

protein

5

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)

Direct detection of Treponema Pallidum

Nontreponemal Serological Tests

Treponemal Serological Tests

Diagnosis of Syphilis

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

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)

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

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

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.

Dark Field MicroscopyΩ 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)

Comparison of Light Pathways of bright field and dark field Microscopy

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

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

Organism Location Coils Length (μm) Width (μm) Rotation

T. pallidum subsp. pallidum

Skin and mucosallesions

Spiral shape,10–13 coils

Medium, 10 (6–20)

Very thin,0.13–0.15

Slow to rapid; like acork-screw, may rotate without changing place

T. refringens Normal genitalflora

Spiral shape,2–3 coils

Short,5 - 8 Thick, 0.20–0.30

Very rapid; activeserpentine-like, rotates sometimes so rapidly that it looks straight

T. phagedenis,Reiter treponeme

Normal genitalflora

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 withoutchanging place

T. denticola Normal oralflora

Spiral shape,6–8 coils (2–8)

Medium, 8 (6–16)

Very thin, 0.15–0.20

Slow to rapid, often jerky

Demonstration of spirochetes

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)

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

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.

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

Lichenoid infiltrate

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

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

Immunoperoxidase staining

Immunoperoxidase Conventional silver stain

Serology

N = 10 9 6 7

Immunoperoxidase technique for detecting spirochetes in tissue sections : comparison with other

methodsPhelps RG, Knispel J, Tu ES et al. Int J Dermatol. 2000 Aug;39(8):609-13.

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

PCR

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

Highly sensitive, able to detect as low as 1 to 10 organisms per specimen with high specificity.

• 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.

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

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

NONTREPONEMAL SEROLOGICAL TESTSFour 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

Non Treponemal Tests 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

• 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

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

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.

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

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.

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).

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.

QUANTITATIVE TEST :

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

Reporting of results

Results of the test are reported as:1. REACTIVE : Past/ present infection with a pathogenic T.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

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)

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

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.

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

Treponemal Tests

In these tests, entire T.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

Commonly used Treponemal tests

Fluorescent Treponemal Antibody Absorption (FTA-Abs) test

Fluorescent Treponemal Antibody Absorption double staining (FTA-Abs-DS) test

Treponema pallidum Haemagglutination Assay (TPHA)

Treponemal Enzyme Immunoassay (EIA)

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 of T. pallidum organism) is fixed

Conjugated fluorescein labeled antihuman globulin is added

• 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

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 of T. 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.

Treponema pallidum Haemagglutination Assay (TPHA)

A qualitative haemagglutination test using tanned formalinised sheep RBC’s as carrier

for T.pallidum antigen (sensitized cells)

TPPA : Treponema pallidum particle agglutination

Based on agglutination of coloured particle carriers sensitized with T pallidum antigen

Uses gelatin particles instead of erythrocytes, thus eliminating nonspecific reactions

with plasma samples

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

Western Blot

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

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

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

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

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

4. Late Latent syphilis

a. Non treponemal tests: - positive in 34 - 94% cases

b. Treponemal tests: - positive 94 - 96% cases

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

CSF sample is taken and a cell count is made

It is further checked for protein abnormalities and subjected to VDRL 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. REACTIVE VDRL test

Serum VDRL test is reactive in about 2/3rd of the cases

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

Diagnostic Algorithm

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.

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.