syphilis serology ppt, syphilis, laboratory diagnosis of syphilis, vdrl, fta-abs

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PowerPoint Presentation

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 1014 m in length and 0.10.2 m in diameter, 10 regular spirals at interval of about 1 m

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

INTRODUCTION

Dr.T.V.Rao MD4

Ultra StructureContains many strongly antigenic protein

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STAGES OF SYPHILISPrimarySecondaryLatent Early latent Late latentLate or tertiary May involve any organ, but main parts are:NeurosyphilisCardiovascular syphilisLate 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

rabbit is the most practical animal because a local lesion can be produced at the site of inoculation, the tissues remain infective for the life of the animal, infection can be transferred from one animal to another using minced lymph nodes or testes, and serologic tests for syphilis become reactive12

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.

rabbit is the most practical animal because a local lesion can be produced at the site of inoculation, the tissues remain infective for the life of the animal, infection can be transferred from one animal to another using minced lymph nodes or testes, and serologic tests for syphilis become reactive13

Dark Field Microscopy One of simplest and most reliable for the direct detection of T pallidum

Exudates and fluids from lesions are examined as a wet mount

Examination should be done immediately

Most productive during 1, 2, early relapsing, and early congenital syphilis when lesions contains large numbers of treponemes (chancres, condylomata latum, or mucous patches)

Because viability of the treponeme is necessary to distinguish T. Pallidum from morphologically similar saprophytic spirochetes within and near the genitalia, dark-field examination must be accomplished immediately after the specimen is obtained.

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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

OrganismLocationCoilsLength (m)Width (m)RotationT. pallidum subsp. pallidumSkin and mucosallesionsSpiral shape,1013 coilsMedium, 10 (620)Very thin,0.130.15 Slow to rapid; like acork-screw, may rotate without changing placeT. refringensNormal genitalfloraSpiral shape,23 coilsShort,5 - 8Thick, 0.200.30Very rapid; activeserpentine-like, rotates sometimes so rapidly that it looks straightT. phagedenis,Reiter treponemeNormal genitalfloraSpiral shape,1012 coils(1030)Medium long,1012 (1030)Thick, 0.200.25(0.200.40)Slow to rapid; rotates withoutchanging placeT. denticolaNormal oralfloraSpiral shape,68 coils (28)Medium, 8 (616)Very thin, 0.150.20Slow 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

Cant differentiate T. pallidum subsp from eachother

T pallidum in tissue sections1 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

Warthin-Starry time consuming and difficult to interpret29

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

Immunoperoxidase staining

ImmunoperoxidaseConventional silver stainSerologyN = 10967

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.Martn-Ezquerra G, Fernandez-Casado A, Barco D et al. Hum Pathol. 2009 ;40(5):624-30. No. of PatientWarthin-Starry stainIHCp valuePrimary Syphilis(N = 8)48< 0.05Secondary Syphilis(N = 26)1321< 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.

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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 str

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