Syphilis Serology PDF

Chapter three Syphilis Serology Learning objective  At the end of this chapter, the students should be able to:  Describe the etiology and sign and symptoms of primary, secondary, latent and late (tertiary) syphilis  Discuss the principle and clinical applications of the qualitati...

Chapter three Syphilis Serology Learning objective  At the end of this chapter, the students should be able to:  Describe the etiology and sign and symptoms of primary, secondary, latent and late (tertiary) syphilis  Discuss the principle and clinical applications of the qualitative and quantitative VDRL procedure and RPR card test  Describe specif ic and non specif ic Treponemal antibodies 2 Outline 3.1. Introduction 3.2. The stage of syphilis 3.3. Immune response 3.4. Diagnosis of syphilis 3.5. Serological technique 3.6. Serological technique 3 Introduction Syphilis is a venereal disease caused by spirocheate bacteria called Treponema pallidum. Treponema pallidum Slender, corkscrew-shaped, microaerophilic gram- negative spiral shaped bacteria. Slowly replicating intracellular pathogen Reported in the medical literature as early as 1495. The first diagnostic blood test, the Wassermann test, was developed in 1906. 4 Introduction…  Syphilis is a chronic systemic disease, which leads to lesions on the body.  It is derived from a Greek word "syphilos" meaning crippled, maimed (heart victim).  Is a systematic infection caused by the spirochate Treponema pallidium. 5 Introduction…  Hours to days after penetrates intact mucosa or abraded skin travel via lymphatic's to general circulation and disseminates throughout body (all organs including CNS).  Incubation period directly proportional to size of inoculums.  Host has immune response resulting inf lammation responsible for clinical manifestation. 6 Transmission of Syphilis 1. Sexual transmission, which accounts for the vast majority of cases, and 2. Vertical transmission from mother to fetus in utero. Congenital syphilis can lead to stillbirth, prematurity and to a variety of clinical complications including central nervous system damage. 3. Blood transfusion 7 Stages of syphilis Syphilis has three clinical stages o Primary syphilis o Secondary syphilis o Tertiary syphilis 8 Stages of syphilis … Infection with T. pallidium Primary Syphilis Primary Chancre Secondary Syphilis Secondary lesions 60% 40% Relapse Trans placental Latent Syphilis Transmission Tertiary Syphilis Persistent Asymptomatic Congenital Syphilis Cardiovascular Gumma Tabes Dorsalis Syphilis (20 Yrs) (5 Yrs) (10-15Yrs) 9 Primary syphilis Painless ulcers or chancre at the infection site. o Initially the lesion was painless papule that quickly erodes and becomes indurated. o Appears as a hard erythematus nodule about 1cm in diameter with regional lymph node enlarged. o Persists for some weeks and heals. o In women it commonly develops in the vulva or cervix o In HIV-infected patients, may see multiple or atypical chancres, or no primary lesion. 10  Primary syphilis … Ch an c re of pri m ar y syp hil is Chancres of primary syphilis 11 Secondary syphilis It appears 7 to 10 weeks after primary syphilis. Characterized by  Sore throat, fever, headache, malaise and diffuse lymphadenopathy.  Followed by mucocutaneous rash (on the palms of the hands and soles of the feet).  Condylomata lata (characterized by wart like lesions on the genitals)  During this stage serologic tests are positive. 12 Secondary syphilis … Symptoms last days-weeks In advanced HIV infection, may be more severe or progress more rapidly Distinguish from primary HIV infection 13 Secondary syphilis … Ch syp ancr hil e of is Se co nd ar y Rash of secondary syphilis 14 Secondary syphilis … Rash of secondary syphilis 15 Secondary syphilis… Secondary syphilis Rash and ulcerations of secondary syphilis 16 Latent syphilis Latent infection There are no symptoms, but specific anti-treponemal antibodies are found. The latent period can be divided into early and late stages. In the early latent period, which can last for a year or two after the secondary stage, the symptoms of secondary syphilis can reappear and patients can infect others. In the late latent period, which can last for many years and no symptoms occur, no risk of horizontal sexual transmission, but vertical and blood borne transmission can still occur and there is a balance b/n immunity and pathogen. 17 Tertiary syphilis  Appears irregularly over succeeding years and may cause series and permanent damage by means of chronic inflammation.  If untreated about 25% died directly by late syphilis 18 Tertiary syphilis… 3 basic forms of late syphilis  Gumma- necrotic masses appear in skin, liver, testes and bones  Cardiovascular lesions- lesions on the veins, valves and muscles of the heart.  Neurosyphilis-meningo vascular -general paralysis -tabes dorsalis -degeneration of posterior column of the spinal cord 19 Tertiary syphilis… Neurosyphilis  Neurologic complications or neurosyphilis may occur earlier or progress more rapidly in HIV-positive patients  Meningitis, meningovascular, or parenchymatous disease similar in HIV-uninfected patients  Concomitant uveitis and meningitis more common in HIV- positive patients  Asymptomatic neurosyphilis (CSF with elevated protein, lymphocytosis, or positive serologic test, in absence of symptoms): not a late complication or manifestation 20 Congenital syphilis  Pregnancy does not alter the course of syphilis in adults.  Transmission and adverse outcomes highest with early syphilis.  S y p hi l i s m ay i nc re a se ri sk o f p e ri na t a l H I V transmission to infants = congenital syphilis.  Screening:  At f ir st prenatal v isit in all wom en; in high- prevalence areas or high-risk women, repeat at 28 21 Immune response in syphilis  Infection with T. pallidum involves both CMI and humoral immune response.  Antigens  Wasserman Ag- phospholipid diphosphatidyl glycerol = cardiolipin  Is a normal constituent of host tissue  Antibodies  Wasserman Antibody = Anticardiolipin = Reagin  Is an Ab to Ags of treponemal proteins as carriers and cardiolipin as immunogenic determinant. 22 Immune response in syphilis… Treponemal Antigens From one or more pathogenic species Shared by many/different strains, spps, sub spps or specific to spps or sub spps. Produce anti-treponemal Abs = Abs to components of treponems Treponemal Antibodies could be: Non specif ic directed against proteins common to pathogenic/non-pathogenic treponemes. Specific directed to pathogenic treponems only 23 Diagnosis of syphilis 1. Tests that detect the etiologic agent 2. Serologic tests for syphilis 1.Tests that detect the etiologic agent o Dark field (Dark ground) o Indian ink (Negative stain) o Phase contrast microscopy o Electron microscopy o Silver stain o Fluorescent stain (DF) 24 Diagnosis of syphilis… Morphological characteristics may be studied microscopically. Usual methods: Dark field (Dark ground) Indian ink (Negative stain) Wet preparation Phase contrast microscopy Electron microscopy Silver stain Dry preparation Fluorescent stain (DF) 25 Diagnosis of syphilis… Dark field microscopy  For symptomatic patients with primary syphilis, dark field microscopy is the test choice.  A dark f ie ld examination is also suggested for immediate results in cases of secondary syphilis with a VDRL titer follow-up test. 26 Diagnosis of syphilis… 27 Serologic tests for syphilis  More than 200 tests developed and only few are used currently.  Generally grouped into TWO, based upon the type of Ag used and Ab detected A. Reagin tests for syphilis (Non-treponemal/Non- specific tests) B. Treponemal tests for syphilis (Specific tests) 28 Serologic tests for syphilis… A. Non- Treponemal Tests for Syphilis  A non- treponemal test employs an antigen (E.g., cardiolipin-lecithin),  Are used to detect an antibody like substances or “reagin” antibody,  Are not 100% specific for syphilis antibodies, but are highly sensitive for syphilis 29 Serologic tests for syphilis…  Advantages of being practical, inexpensive and widely available.  Basically of two types: I. Flocculation (tube or slide) and II. Complement fixation tests. 30 Serologic tests for syphilis… I. Flocculation Tests a. Slide flocculation tests,  Needs small amount of clinical specimen and antigen suspension  Are rapidly performed  Results are usually read microscopically  It utilizes cardiolipin, lecithin, cholesterol antigen and heat inactivated serum.  Performed on slide or tube E.g., VDRL 31 Serologic tests for syphilis… b. Tube flocculation tests  Are performed in test tubes  Requires large quantities of specimen and antigen suspension  Are more complicated  Are read with or without magnification. Eg., Kliane flocculation Test Khan flocculation Test VDRL Mazzini test  Hinton (serum) test 32 Serologic tests for syphilis… c. Card flocculation tests (Rapid reagin tests):  RPR (rapid plasma reagin)  PCT- plasma CriT  RPR (Teardrop) card test  RPR (18-mm circle) card tests 33 Serologic tests for syphilis… II. Complement fixation Test (CFT)  Complement components are thermo labile proteins found in normal serum.  It is also found in other animals.  It is destroyed at 56C for 30 minutes.  But up on standing at 7-370C, it may regain part of its activity.  Therefore, previously heated serum must be reheated for 10 min. at 560C before a test can be performed. 34  Complement Fixation Serum with Serum without Abs Antibodies Antigen binds Unbound Antigen Day 1 to antibodies Complement Unbound Complement binds to Ag/Ab complex Hemolysin sensitized Hemolysin sensitized red blood cells RBCs serve as an indicator serve as an indicator Day 2 No lysis Positive Lysis Negative 35 Serologic tests for syphilis… II. Complement fixation Test (CFT)… Antigen suspension inactivated patients serum After period of incubation for Ag-Ab complex Standard complements are added If there is Ag-Ab complex, complements used up RBCs hemolysis inhabited (positive) 36 RPR (Rapid plasma reagin card test for syphilis) Principle  Destructive syphilitic lesions cause tissue damage. Circulating antibodies called reagin are produced against some of the tissue components. The rapid plasma reagin card test uses a modif ie d form of the VDRL (Veneral Disease Research Laboratory) antigen called cardiolipin in suspension with carbon particles. When cardiolipin antigen reacts with reagain antibody in patient’s serum the carbon particles in the suspension clump together. 37 RPR (Rapid reagin card test for syphilis)…. Materials The following are provided in the test kits:  Reagin antigen suspension  Reagin positive control serum  Reagin negative control serum  Reagin test card  Dispensing bottle and needle  Dropper tubes  Mixing sticks 38 RPR (Rapid reagin card test for syphilis)…. Qualitative RPR test method Procedure  Let t h e rea g en t s a n d s p ec i m en s w a rm u p to room temperature.  Dispense one drop of negative control serum on to one circle on the test card using a disposable dropper tube.  Repeat step two with the positive control serum using a clean dropper tube.  Dispense on drop of each sample serum or plasma on to one circle on the card using a clean dropper tube for each 39 RPR (Rapid reagin card test for syphilis)…. Procedure …..  Spread all the drops to cover the whole area of the circles using the mixing sticks.  Mi x w i t h re ag ai n ant i g e n susp e nsi o n i n t he dispensing bottle. Hold the bottle vertically and dispense one drop on to each test sample. Do not mix again.  Place the card on the rotor for 8 minutes at 100rpm. 40 RPR (Rapid reagin card test for syphilis)…. Reading the results  Negative result:  The carbon particles remain in an even suspension = Non reactive  Positive result: The carbon particles clump together = Reactive 41 RPR (Rapid reagin card test for syphilis)…. For the test to be valid the negative control must be non- reactive and the positive control must be reactive 42 RPR (Rapid reagin card test for syphilis)…. Reporting results of a qualitative test  Qualitative results should be reported as reactive or Non-reactive 43 RPR (Rapid reagin card test for syphilis)…. Quantitative RPR test method 1. Dispense 1 drop of 0.85% saline on to circles 1 to 5 on the test card. 2. Dispense 50µl of patient serum or plasma on to the f irst circle and mix by f illing and discharging the pipette at least 6 times (do not make bubbles). You now have a 1 in 2 dilution in the first circle. 3. Transfer 50µl from the f ir st circle to the second circle and repeat the mixing procedure. 4. Continue the dilution procedure to circles 3, 4 and 5 and discard the last 50µl. 44 RPR (Rapid reagin card test for syphilis)…. You know have the following dilutions: Circle 1 2 3 4 5 Dilution ½ ¼ 1/8 1/16 1/32 45 RPR (Rapid reagin card test for syphilis)…. 5. Starting at circler number 5 uses a mixing stick to spread all the drops to cover the whole area of the circles. 6. Mix the regain antigen suspension in the dispensing bottle. Hold the bottle vertically and dispense one drop on to each test sample. Do not mix again. 7. Place the card on the rotor for 8 minutes at 100rpm. 46 RPR (Rapid reagin card test for syphilis)…. Reporting the results of a quantitative test  The titer reported in a quantitative test is the highest sample dilution to show a reactive (positive) result.  In the example below the result would be reported as:  Reactive to 1/8 47 RPR (Rapid reagin card test for syphilis)…. 48 RPR (Rapid reagin card test for syphilis)…. If the highest dilution (1 in 32) is reactive proceed as follows: 8. Prepare a 1in 16 dilution of the sample by adding 0.1ml of serum to 1.5ml of 0.85% saline and mix well. 9. Dispense 1 drop of saline on to circles 6 to 10 on the test card using a dropper tube. 10. Dispense 1 drop of the 1 in 16 dilution on to circle number 6 and proceed as before (step 3 and 4) making doubling dilutions up to well number 10 49 RPR (Rapid reagin card test for syphilis)…. You know have the following dilutions: Circle 6 7 8 9 10 Dilution 1/32 1/64 1/128 1/256 1/512 11. starting at well number 10 spread all the drops as before 12. M i x t h e re a g i n a n t i g e n s u s p e n s i o n i n t h e dispensing bottle. Hold the bottle vertically and dispense one drop on to each circle. Do not mix 50 RPR (Rapid reagin card test for syphilis)…. 13.place the card on the rotor for 8 minute at 100rpm 14.report the titer as the highest dilution to show a positive result 51 RPR (Rapid reagin card test for syphilis)…. Limitation of the test  The rapid regain card test is a non-specif ic test for syphilis.  A positive reaction indicates tissue damage such as that caused by destructive syphilitic lesions.  False negative reactions can occur in the early and later stages of syphilis when there is not a lot of tissue damage. 52 RPR (Rapid reagin card test for syphilis)….  False positive reactions can occur due to other diseases which result in tissue damage such as malaria, leprosy, viral infections, autoimmune diseases and many other conditions including pregnancy.  It is very important that reactive specimens are checked by another test procedure which is specif ic for syphilis.  In the reference laboratories this is done by the 53 VDRL Test Slide Qualitative VDRL Test Principle  During the period of infection with syphilis, reagin, a substance with the properties of an antibody, appears in the serum of affected patients. Reagin has the ability to combine with a colloidal suspension extracted from animal tissue and clump together to form visible masses, a process known as flocculation. 54 VDRL Test… Procedure: 1. Pipette 0.05ml or 1drop of inactivated serum into one ring of the ringed glass slide. 2. Add one-drop (1/60ml) antigen suspension onto each serum. 3. Rotate slide for 4 minutes. (If rotated by hand on a f lat surface, this movement should roughly circumscribed a 2 inch/5mm diameter circle). 4. Tests are read immediately after rotation microscopically with a 10x ocular and a 10x objective. 55 VDRL Test… Reading and reporting of results  Tests are read microscopically with low power objective at 10x magnif ication, which appears short rod forms.  Aggregation of these particles into large or small clumps is interpretedReporting in degrees of reactivity. system No clumping or very slight roughness : Non-reactive (NR) Small clumps : Weakly reactive (WR) Medium and large clumps : Reactive (R) 56 VDRL Test… Advantage of the VDRL test: o It can be performed on serum or cerebrospinal fluid (CSF). The disadvantages of the VDRL test: o The antigen must be prepared fresh daily o The test needs to be read with the aid of a microscope, and o Serum specimens must be heat inactivated before testing. 57 VDRL VS RPR RPR is a more advanced version of VDRL that uses carbon particles RPR is just the VDRL antigen, but it contains carbon (charcoal particles) and stabilized by choline chloride to enhance the reaction and remove inhibitors. VDRL is more sensitive than RPR and can also be used to test cerebrospinal fluid (CSF). RPR is performed on card while VDRL is performed on slide. RPR tests can be done without the use of a microscope. Specimen that will be used for a VDLR requires it to be heated. 58 In an RPR test, the specimen does not need to be heated. Standard Treponemal tests for syphilis 1. T. pallidum immobilization test (TPI) 2. RPCF test ( Reiter protein complement fixation test) 3. Fluorescent Treponnema antibody Absorption test (FTA-ABS-test) 4. The Treponema Pallidum Hemagglutination (TPHA) Test 5. Treponomal pallidum agglutination test. 6. Treponemal pallidum complement fixation test. 59 1. T. pallidum immobilization test (TPI) Principle: Treponema pallidum immobilization test is the most specif ic and extremely valuable test for syphilis. It becomes positive after the second week of infection. The test is however quite complicated and relatively costly to perform. The test, where available, is used for reference and to rule out false- positive sero reactors of other tests. 60 1. T. pallidum immobilization test (TPI)… Method:  Patient’s serum is placed in a test tube with living spirochetes and complement.  After incubation in an atmosphere free of 0 2 , slide preparations are made and examined by dark f ie ld illumination.  The spirochetes will be immobilized by syphilitic serum but will be actively motile in normal serum. The TPI test has its greatest value in conf ir ming syphilis or ruling out biological false positive reaction. 61 1. T. pallidum immobilization test (TPI)… Limitation of the test  It requires live treponemas from infected animals and is difficult to perform.  It does not distinguish the various treponematoses (i.e. yaws, pinta, bejel).  It fails to detect early syphilis.  It cannot be used as an index of therapeutic response.  It is ineffective when the patient is on antibiotics. 62 1. T. pallidum immobilization test (TPI)… Advantage:  On the positive side, the test is the one of choice for spinal f luids, especially for detecting neurosyphilis when reagin tests give non-reactive results. 63 RPCF test ( Reiter protein complement fixation test)  RPCF is a test with treponemal antigen.  Is performed for the diagnosis of syphilis.  Less specific and sensitive than TPI.  The test is simple to perform and quite cheap 64 Fluorescent Treponema antibody Absorption test (FTA-ABS-test)  A modif ied form of f luorescent treponemal antibody test (FTA- Te s t ) w i t h t r e p o n e m a l a n t i g e n e m p l o y i n g i n d i r e c t immunofluorescence.  T h e F TA -A BS u s es a k i l l ed s u s p en s i on of T. pallidum spirochetes as the antigen.  This procedure is performed by over laying whole treponemes f ixed to a slide with serum from patient's suspected of having syphilis.  Fluorescein- conjugated antihuman antibody reagent is then applied as a marker for specif ic anti-treponemal antibodies in 65 the patients' serum. Fluorescent Treponema antibody Absorption test (FTA-ABS-test)… Principle  The FTA-ABS test is a direct method of observation.  Although not recommended for screening, it is the most sensitive serologic procedure in the detection of primary syphilis. Limitation of the test  This test is recommended as a conf ir matory test for syphilis. 66 The Treponema Pallidum Hemagglutination (TPHA) Test Principle In the TPHA test, patient's diluted serum samples are mixed in the wells of a microtiration plate with sheep or avian red cells coated (sensitized) with T. pallidum antigen. Un-sensitized cells added to a second well serve as a control. If antibody is present, the sensitized cells are agglutinated and they settle in a characteristic mat pattern in the bottom of the well. Un-agglutinated cells in a negative test and control well form a button or smooth ring at the bottom of the well. 67 Sensitivity of Syphilitic Serological test Stage Primary Secondary Late Non Treponemal (Reagin tests) VDRL 70% 99% 1% RPR 80% 99% 0% Specific Treponemal test FTA-ABS 85% 100% 95% TPHA-TP 65% 100% 95% TPI 50% 97% - 68 Non standard non treponemal and treponemal test  ELISA  CAPTIA-syphilis G test  CAPTIA-syphilis M test  Syphilis Rapid test device 69 ELISA  The ELISA immuno assays is available for both non- treponemal and treponemal tests.  The ELISA non-treponemal assaya uses the VDRL antigen affixed to a micro titer plate.  On the other hand, at least two different treponemal ELISA assay are commercially available in kit form.  These are the CAPTIA-syphilis G and the CAPTIA- syphilis M tests (trinity Bio tech). 70 CAPTIA-syphilis G test  Used to detect anti-treponemal antibody  Used to measure IgG of Treponemal infection  CAPTIA-syphilis M test  Used to detect anti-treponemal antibodies  This test is particularly useful for diagnosis of congenital syphilis.  Babies whose mothers are infected with syphilis cannot be diagnosed using the tests that measure IgG antibodies.  IgM antibodies do not cross the placenta, the identif ic ation of anti-T. pallidum antibodies in the newborn sera indicates congenital syphilis. 71 Syphilis Rapid test device  I t i s a rap i d q ual i t at i v e c hro m ato g rap hi c immunoassay that uses the af finity of protein A for IgG antibodies to test for treponemal antibodies  Protein A binds to the Fc region of most subclasses of IgG.  One of the advantages of this test is that dilutions are not required and the prozone phenomenon is not an issue as it is for tests whose end points are flocculation or agglutination. 72 Syphilis Rapid test device A drop of serum or plasma is placed on the card and as the sample migrates through the card, it reconstitutes and mixes with selenium-conjugated T. pallidum. The mixture then continues to migrate to the “patient window” site, which contains immobilized T. pallidum antigen. If antibodies to T. pallidum are present in the sample, they bind to the conjugated antigen and to the immobilized antigen, forming a red line at the patient window site. The test can be read after a minimum of 15 minutes up to 24 hours. 73 Summary Lesion Yes No Negative DAF Non treponemal Reactive Non-reactive Positive Titer Repeat Treat Treponemal test Non reactive Reactive False Treat Positive 74  Test for Syphilis Summary… Standard Non Standard Non specific Specific Treponemal and Non treponemal Non treponemal Treponemal TP- complement test Standard Flocculation CF TPI ELISA TPH ABS Kolimer EIA Wassermann RPCF Western Blot TPA agglutination Tube Slide Card test SRTD (syphilis rapid TP. Methylene blue tests test device) VDRL VDRL RPR FTA-ABs Kliane USR Khan PCT-plasma Crit Mazzini Hinton 75 Review question Explain the stages of syphilis Discuss the difference between RPR and VDRL. Write non serological test for syphilis Explain specif ic and non-specif ic serologic test for syphilis 76 Reference 1. Tizard. Immunology an introduction,4 th edition , Saunders publishing,1994 2. Naville J. Bryant Laboratory Immunology and S e ro l o g y 3 r d e d i t i o n. S e ro l o g i c a l s e r v i c e s Ltd.Toronto,Ontario,Canada,1992 3. Ma r y L o ui se.I m m uno l o g y a nd S e ro l o g y i n Laboratory medicine 3rd edition 77