Syphilis Serology serological test for medical laboratory.ppt

Serology test

1. Chapter three
SyphilisSerology

2. Learning objective
Attheendofthischapter,thestudents
shouldbeableto:
Describetheetiologyandsignandsymptomsof
primary,secondary,latentandlate(tertiary)
syphilis
Discusstheprincipleandclinicalapplicationsof
thequalitativeandquantitativeVDRLprocedure
andRPRcardtest
DescribespecificandnonspecificTreponemal
antibodies

3. Outline
3.1. Introduction
3.2. The stage of syphilis
3.3. Immune response
3.4. Diagnosis of syphilis
3.5. Serological technique

4. Definition
Syphilis a systematic infection caused by the
spirochate Treponema pallidium.
It 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).
3.1. Introduction

5. Reportedinthemedicalliteratureasearlyas
1495.
In1905itwasdiscoveredthatsyphiluiswas
causedbyaspirochetetypeofbacteria,
T.pallidum(originallycalledspirochaetapallida).
Thefirstdiagnosticbloodtest,theWassermann
test,wasdevelopedin1906.
3.1. Introduction

6. Transmittedby:
MainlySexualcontact(Venerealsyphilis)
Lesscommonlyviatheplacenta(congenital
syphilis)OR
Byaccidentalinoculationfrominfectious
material
e.g.freshbloodtransfusion
3.1. Introduction

7. Hourstodaysafterpenetratesintactmucosaor
abradedskintravelvialymphatic'stogeneral
circulationanddisseminatesthroughoutbody(all
organsincludingCNS)
Incubationperioddirectlyproportionaltosizeof
inoculums
Hosthasimmuneresponseresultinginflammation
responsibleforclinicalmanifestation
3.1. Introduction

8. 3.2. Stages of syphilis
Primary Syphilis
Secondary Syphilis
Latent Syphilis
Tertiary SyphilisPersistent Asymptomatic
Relapse Trans placental
Transmission
Congenital Syphilis
40%
60%
Primary Chancre
Secondary lesions
Infection with T. pallidium
Gumma
(5 Yrs)
Cardiovascular
Syphilis
(10-15Yrs)
Tabes Dorsalis
(20 Yrs)

9. Primarychancre
Appearsatthesiteofinoculation,initiallypainless
papulethatquicklyerodesandbecomesindurated
Appearsasaharderythematusnoduleabout1cmin
diameterwithregionallymphnodeenlarged.
Persistsforsomeweeksandheals
Inwomenitcommonlydevelopsinthevulvaor
cervix
InHIV-infectedpatients,mayseemultipleor
unusualchancres,ornoprimarylesion
3.2. Stages of syphilis

10. Chancres of primary syphilis
3.2. Stages of syphilis

11. Secondarysyphilis
Alwayswidespreaderythematusskinabout1cmin
diameterwithregionallymphnodeenlarged.
Ulcerateswithaclearrim
Theulcerispainless,persistsforsomeweeksand
heals
Inwomenitcommonlydevelopsinthevulvaor
cervix
3.2. Stages of syphilis

12. Secondarysyphilis(2-8weeksafterprimary
inoculation)
Inconsistentsymptoms,mayinclude:
Rash
Generalizedlymphadenopathy
Constitutionalsymptoms (fever,malaise,
anorexia,headache)
CNSsymptoms
3.2. Stages of syphilis

13. Symptoms last days-weeks
In advanced HIV infection, may be more
severe or progress more rapidly
3.2. Stages of syphilis

14. Secondary syphilis
Rash of secondary syphilis
3.2. Stages of syphilis

15. Secondary syphilis
Rash of secondary syphilis
3.2. Stages of syphilis

16. Secondary syphilis
Rash and ulcerations
of secondary syphilis
3.2. Stages of syphilis

17. Latentsyphilis
no overt signs/symptoms, though relapse of
manifestations of secondary syphilis may occur
Tertiary syphilis(Late 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
neurosyphilis, cardiovascular syphilis, gummatoussyphilis; or
slowly progressive disease in any organ system
3.2. Stages of syphilis

18. 3basicformsoflatesyphilis
Gumma-necroticmassesappearinskin,liver,
testesandbones
Cardiovascularlesions-lesionsontheveins,
valvesandmusclesoftheheart.
Neurosyphilis-meningovascular
-generalparalysis
-tabesdorsalis
–degenerationofposterior
columnofthespinalcord
3.2. Stages of syphilis

19. Congenitalsyphilis
Pregnancydoesnotalterthecourseofsyphilisin
adults
Transmissionandadverseoutcomeshighestwith
earlysyphilis
SyphilismayincreaseriskofperinatalHIV
transmissiontoinfants=congenitalsyphilis
Screening:
Atfirstprenatalvisitinallwomen;inhigh-
prevalenceareasorhigh-riskwomen,repeatat
28weeksandatdelivery
3.2. Stages of syphilis

20. 3.3. Immune response
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.

21. TreponemalAntigens
Fromoneormorepathogenicspecies
Sharedbymany/differentstrains,spps,sub
sppsorspecifictosppsorsubspps
Produceanti-treponemal Abs=Absto
componentsoftreponems
TreponemalAntibodiescouldbe:
-Nonspecificdirectedagainstproteinscommon
topathogenic/non-pathgenictreponemsor
-Specificdirectedtopathogenictreponemsonly
3.3. Immune response

22. 3.4. Diagnosis of syphilis
1.Tests that detect the etiologic agent(directly)
2.Serologictestsforsyphilis
1.Teststhatdetecttheetiologicagent
Darkfield(Darkground)
Indianink(Negativestain)
Phasecontrastmicroscopy
Electronmicroscopy
Silverstain
Fluorescentstain(DF)

23. Morphological x-ics may be studied microscopically.
Usual methods:
Dark field (Dark ground)
Indian ink (Negative stain)
Phase contrast microscopy
Electron microscopy
Silver stain
Fluorescent stain (DF)
Wet preparation
Dry preparation
3.4. Diagnosis of syphilis

24. Darkfieldmicroscopy
Forsymptomaticpatientswithprimarysyphilis,
darkfieldmicroscopyisthetestchoice.
Adarkfieldexaminationisalsosuggestedfor
immediateresultsincasesofsecondarysyphilis
withaVDRLtiterfollow-uptest.
3.4. Diagnosis of syphilis

25. 3.4. Diagnosis of syphilis

26. 3.4. Diagnosis of syphilis

27. 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)(indirect
method)
B. Treponemal tests for syphilis (Specific
tests)(direct method)
3.5. Serologic tests for syphilis

28. 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,
They detect biomarkers that are released during
cellular damage that occurs from the syphilis
spirochete.
Are not 100% specific for syphilis antibodies, but are
highly sensitive for syphilis
3.5. Serologic tests for syphilis

29. Nontreponemal tests are screening tests, very rapid
and relatively simple, but need to be confirmed by
treponemal tests.
Syphilitic infection leads to the production of
nonspecific antibodies that react to cardiolipin.
This reaction is the foundation of “nontreponemal”
assays
All nontreponemal tests measure immunoglobulins G
(IgG) and M (IgM) anti-lipid antibodies formed by the
host in response both to lipoidal material released
from damaged host cells early in infection and to lipid
from the cell surfaces of the treponeme itself.

30. 3.5. Serologic tests for syphilis
These nontreponemal tests are widely used for
qualitative syphilis screening. However, their
usefulness is limited by decreased sensitivity in
early primary syphilis and during late syphilis, when
a large number of untreated patients will be
negative by these methods.
With nontreponemal tests, false-positive reactions
can occur for a large number of reasons, the most
common of which is other infections, both viral and
bacterial.

31. Advantages of being practical, inexpensive and
widely available.
Basically of two types:
I. Flocculation(tube or slide) and
II. Complement fixation tests.
3.5. Serologic tests for syphilis

32. 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
3.5. Serologic tests for syphilis

33. b.Tubeflocculationtests
areperformedintesttubes
requireslargequantitiesofspecimenand
antigensuspension
aremorecomplicated
arereadwithorwithoutmagnification.
Eg.,
Klianeflocculation Test
Khan flocculation Test
VDRL
Mazzini test
Hinton (serum) test
3.5. Serologic tests for syphilis

34. C. Card flocculation tests (Rapid reagintests):
RPR (rapid plasma reagin)
RPR (Teardrop) card test
RPR (18-mm circle) card tests
3.5. Serologic tests for syphilis

35. II.Complement fixationTest(CFT)
Complement components arethermolabile
proteinsfoundinnormalserum.
Itisalsofoundinotheranimals.
Itisdestroyedat56Cfor30minutes.
Butuponstandingat7-37
0
C,itmayregainpart
ofitsactivity.
Therefore,previouslyheatedserummustbe
reheatedfor10min.at56
0
Cbeforeatestcanbe
performed.
3.5. Serologic tests for syphilis

36. Complement Fixation
Serum without AbsSerum with
Antibodies
Antigen binds
to antibodies
Unbound Antigen
Complement
binds to Ag/Ab
complex
Unbound Complement
No lysis Positive Lysis Negative
Hemolysin sensitized
RBCs serve as an
indicator
Hemolysin sensitized
red blood cells
serve as an indicator
Day 1
Day 2

37. 3.6. Serological technique
RPR(Rapidreagaincardtestforsyphilis)
Principle
Destructivesyphiliticlesionscausetissuedamage.
Circulatingantibodiescalledreagainareproduced
againstsomeofthetissuecomponents.Therapid
regaincardtestusesamodifiedformoftheVDRL
(VernalDiseaseResearchLaboratory)antigen
calledcardiolipininsuspensionwithcarbon
particles.Whencardiolipinantigenreactswith
reagainantibodyinpatient’sserumthecarbon
particlesinthesuspensionclumptogether.

38. 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
3.6. Serological technique

39. QualitativeRPRtestmethod
Procedure
Letthereagentsandspecimenswarmuptoroom
temperature.
Dispenseonedropofnegativecontrolserumonto
onecircleonthetestcardusingadisposable
droppertube.
Repeatsteptwowiththepositivecontrolserum
usingacleandroppertube.
Dispenseondropofeachsampleserumorplasma
ontoonecircleonthecardusingacleandropper
tubeforeachspecimen.
3.6. Serological technique

40. Procedure
Spreadallthedropstocoverthewholeareaofthe
circlesusingthemixingsticks.
Mixwithreagainantigensuspensioninthe
dispensingbottle.Holdthebottleverticallyand
dispenseonedropontoeachtestsample.Donot
mixagain.
Placethecardontherotorfor8minutesat
100rpm.
3.6. Serological technique

41. Reading the results
Negative result:
The carbon particles remain in an even
suspension = Non reactive
Positive result:
The carbon particles clump together
= Reactive
3.6. Serological technique

42. Forthetesttobevalidthenegativecontrolmustbenon-
reactiveandthepositivecontrolmustbereactive
3.6. Serological technique

43. Reportingresultsofaqualitativetest
Qualitativeresultsshouldbereportedasreactive
orNon-reactive
3.6. Serological technique

44. QuantitativeRPRtestmethod
1.Dispense1dropof0.85%salineontocircles1to
5onthetestcard
2.Dispense50µlofpatientserumorplasmaonto
thefirstcircleandmixbyfillinganddischarging
thepipetteatleast6times(donotmake
bubbles).Younowhavea1in2dilutioninthe
firstcircle.
3.Transfer50µlfromthefirstcircletothesecond
circleandrepeatthemixingprocedure.
4.Continuethedilutionproceduretocircles3,4
and5anddiscardthelast50µl.
3.6. Serological technique

45. You know have the following dilutions:
Circle 1 2 3 4 5
Dilution ½ ¼ 1/8 1/16 1/32
3.6. Serological technique

46. 5.Startingatcirclernumber5usesamixingstickto
spreadallthedropstocoverthewholeareaofthe
circles.
6.Mixtheregainantigensuspensioninthe
dispensingbottle.Holdthebottleverticallyand
dispenseonedropontoeachtestsample.Donot
mixagain.
7.Placethecardontherotorfor8minutesat
100rpm.
3.6. Serological technique

47. Reportingtheresultsofaquantitativetest
Thetiterreportedinaquantitativetestisthe
highestsampledilutiontoshowareactive
(positive)result.
Intheexamplebelowtheresultwouldbe
reportedas:
Reactiveto1/8
3.6. Serological technique

48. 3.6. Serological technique

49. Limitationofthetest
Therapidregaincardtestisanon-specifictest
forsyphilis.Apositivereactionindicatestissue
damagesuchasthatcausedbydestructive
syphiliticlesions.
Falsenegativereactionscanoccurintheearly
andlaterstagesofsyphiliswhenthereisnoalot
oftissuedamage.
3.6. Serological technique

50. Falsepositivereactionscanoccurduetoother
diseaseswhichresultintissuedamagesuchas
malaria,leprosy,viralinfections,autoimmune
diseasesandmanyotherconditionsincluding
pregnancy.
Itisveryimportantthatreactivespecimensare
checkedbyanothertestprocedurewhichis
specificforsyphilis.
3.6. Serological technique

51. VDRLTEST
SlideQualitativeVDRLTest
Principle
Duringtheperiodofinfectionwithsyphilis,
reagin,asubstancewiththepropertiesofan
antibody,appearsintheserumaffectedpatients.
Reaginhastheabilitytocombinewithacolloidal
suspensionextractedfromanimaltissueand
clumptogethertoformvisiblemasses,aprocess
knownasflocculation.
3.6. Serological technique

52. Procedure:
1.Pipette0.05mlor1dropofinactivatedseruminto
oneringoftheringedglassslide.
2.Addone-drop(1/60ml)antigensuspensiononto
eachserum.
3.Rotateslidefor4minutes.(Ifrotatedbyhandon
aflatsurface,thismovement shouldroughly
circumscribeda2inch/5mmdiametercircle).
4.Testsarereadimmediatelyafterrotation
microscopicallywitha10xocularanda10x
objective.
3.6. Serological technique

53. Readingandreportingofresults
Testsarereadmicroscopicallywithlowpower
objectiveat10xmagnification,whichappearsshort
rodforms.Aggregationoftheseparticlesintolarge
orsmallclumpsisinterpretedindegreesof
reactivity.
Reporting system
No clumping or very slight roughness :Non-reactive (NR)
Small clumps : Weakly reactive(WR)
Medium and large clumps : Reactive (R)
3.6. Serological technique

54. B.Treponemal testsforsyphilis
StandardTreponemaltestsforsyphilis
1. T. pallidumimmobilization test (TPI)
2. RPCF test ( Reiter protein complement fixation
test)
3. Fluorescent Treponnemaantibody Absorption test
(FTA-ABS-test)
4. TreponomapallidumMethylene Blue tests.
5. Treponomalpallidumagglutination test.
6. Treponemalpallidumcomplement fixation test.
3.6. Serological technique

55. 1.T.pallidumimmobilizationtest(TPI)
Principle:
Treponema pallidumimmobilizationtestis
themostspecificandextremelyvaluabletest
forsyphilis.Itbecomespositiveafterthe
secondweekofinfection.Thetestishowever
quitecomplicatedandrelativelycostyto
perform.Thetest,whereavailable,isused
forreferenceandtoruleoutfalse-positive
seroreactorsofothertests.
3.8. Serological technique

56. Method:
Patient’sserumisplacedinatesttubewithliving
spirochetesandcomplement.
Afterincubationinanatmospherefreeof0
2,slide
preparationsaremadeandexaminedbydarkfield
illumination.
Thespirocheteswillbeimmobilizedbysyphilitic
serumbutwillbeactivelymotileinnormalserum.
TheTPItesthasitsgreatestvalueinconfirming
syphilisorrulingoutbiologicalfalsepositive
reaction.
3.6. Serological technique

57. 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.
3.6. Serological technique

58. Advantage:
Onthepositiveside,thetestistheoneofchoice
forspinalfluids,especiallyfordetecting
neurosyphiliswhenreagintestsgivenon-reactive
results.
3.6. Serological technique

59. Fluorescent Treponnema antibody Absorption
test (FTA-ABS-test)
Amodifiedformoffluorescenttreponemal
antibodytest(FTA-Test)withtreponemalantigen
employingindirectimmunofluorescence
FTA-usedfordiagnosisofsyphilis
Itisaspecificandsensitivetest
3.6. Serological technique

60. Principle
TheFTA-ABStestisadirectmethodof
observation.Althoughnotrecommended for
screening,itisthemostsensitiveserologic
procedureinthedetectionofprimarysyphilis.
Limitationofthetest
Thistestisrecommended asaconfirmatorytest
forsyphilis.
Itisrecommended forscreeningtest.
AreagintestsuchastheVDRLorRPRisnot
recommended forscreening.
3.6. Serological technique

61. Stage
Primary Secondary Late
Non Treponemal (Reagintests)
VDRL 70% 99% 1%
RPR 80% 99% 0%
Specific Treponemal test
FTA-ABS 85% 100% 95%
TPHA-TP 65% 100% 95%
TPI 50% 97% -
3.6. Serological technique

62. Non standard non treponemaland treponemal
test
ELISA
CAPTIA-syphilis G test
CAPTIA-syphilis M test
Syphilis Rapid test device
3.6. Serological technique

63. ELISA
TheELISAimmunoassaysisavailableforboth
non-treponemalandtreponemaltests.
TheELISAnon-treponemalassayausestheVDRL
antigenaffixedtoamicrotiterplate.
AtleasttwodifferenttreponemalELISAassayare
commerciallyavailableinkitform.
ThesearetheCAPTIA-syphilisGandtheCAPTIA-
syphilisMtests(trinityBiotech).
3.6. Serological technique

64. CAPTIA-syphilisGtest
usedtodetectanti-treponemalantibody
usedtomeasureIgGofTreponemalinfection
CAPTIA-syphilisMtest
usedtodetectanti-treponemalantibodies
Thistestisparticularlyusefulfordiagnosisof
congenitalsyphilis.
Babieswhosemothersareinfectedwithsyphilis
cannotbediagnosedusingtheteststhat
measureIgGantibodies.
IgMantibodiesdonotcrosstheplacenta,the
identificationofanti-T.pallidumantibodiesin
thenewbornseraindicatescongenitalsyphilis
3.6. Serological technique

65. SyphilisRapidtestdevice
it is a rapid qualitative chromatographic
immunoassay that uses the affinity of protein A
for IgGantibodies to test for treponemal
antibodies
Protein A binds to the Fcregion 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.
3.6. Serological technique

66. Test for Syphilis
Non StandardStandard
Treponemal and
Non treponemal
Standard
ELISA
EIA
Western Blot
SRTD (syphilis rapid
test device)
Non specific Specific
Non treponemal
TP-complement test
TPI
TPH ABS
RPCF
TPA agglutination
TP. Methylene blue tests
FTA-ABs
Flocculation
Kolimer
Wassermann
Tube
VDRL
Kliane
Khan
Mazzini
Hinton
Cardtest
RPR
USR
PCT-plasma Crit
Slide
VDRL
CF
Treponemal
Summary

67. Reviewquestion
Explainthestagesofsyphilis
DiscussthedifferencebetweenRPRandVDRL.
Writenonserologicaltestforsyphilis
Explainspecificandnon-specificserologictestfor
syphilis

68. Reference
1.Tizard.Immunology anintroduction,4
th
edition
,Saunderspublishing,1994
2.NavilleJ.BryantLaboratoryImmunology and
Serology3
rd
edition.Serologicalservices
Ltd.Toronto,Ontario,Canada,1992
3.MaryLouise.Immunology andSerologyin
Laboratorymedicine3
rd
edition