syphilis serology ppt, syphilis, laboratory diagnosis of syphilis, VDRL, FTA-ABS
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Transcript of syphilis serology ppt, syphilis, laboratory diagnosis of syphilis, VDRL, FTA-ABS
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.