SYPHILIS

By:-Dr. Amit kr. Patel

JRA
Microbiology Department
RIMS, Ranchi
 Treponemas :-
• The genus Treponema belongs to:-
Order- spirochaetales
Family- Spirochaetaceae
• The genus Treponema includes-
-invasive human pathogens
-commensals in mouth (T.denticole),
intestine and genitalia
• The clinical manifestation produced by
Treponema is called Treponematoses.
 Introduction :-
• Discovered by Schaudinn and Hoffmann (1905)

• Thin, delicate, spiral with tapering ends.

• 10-15 µm x 0.1-0.2µm, about 6-14 regular spirals.

• Corkscrew motility, flexion and extension, rotation

and translatory movement.
• Dark-ground or phase contrast microscopy .
• Stained by silver impregnation method, Giemsa stain.
 Morphology:-
• Have axial filaments.
• T.pallidum cytoplasm is surrounded by trilaminar
cytoplasmic membrane.
• Peptidoglycan provides rigidity and shape.
• It has lipid rich membrane layer.
• Three to four endoflagella originate.
 Resistance :-
• Organism can not survive outside host.
• T.pallidum is very delicate and readily inactivated by
drying or by heat ( 41 - 42°c in 1 hr.)
• Killed at 0-4°c in 1-3 days.
• Stored frozen at -70°c in 10% glycerol or in liquid
nitrogen (-130°c) for 10-15 years.
 Cont..

• Inactivation by contact with oxygen, distilled

water ,arsenic bismuth, common antiseptic agents
and antibiotics.
 Cultivation:-

Pathogenic strain:- virulent T.pallidum strain

(Nichole’s strain) can not be cultivated in artificial
medium.

• have been maintained for many decades by serial

testicular passage in rabbits.
 Conti..

Non-pathogenic strain:-
• Cultivated in artificial medium .
• The Reiter treponem (T. phagedenis, T.refringens)
grow well in thioglycollate medium containing serum
or Smith-Niguchi medium.
 Antigenic structure:-

1) Diphosphatidyl glycerol produces reagin

antibody:-
• It is similar with cardiolipin produced by OX heart.
• React in the standard or non-specific test for syphilis
such as Wassermann , Kahn and VDRL.
2) Group antigen:- found in T.pallidum as well as
non-pathogenic cultivable treponemes like Reiter
treponeme.
• Protein in nature.
3) Species specific antigen:-
• Polysaccharide in nature and species specific.
• Antibody to this antigen is demonstrated by specific
T.pallidum tests and is positive only with pathogenic
treponemes.
 Virulence factors:-

Virulence factors Biological functions

Outer membrane protein adherence to the surface of the host cell.

Enzyme hyaluronidase Facilitate perivascular infiltration

Fibronectin Prevents phagocytosis by macrophages

Epidemiology:-
 Epidemiology:-
• VDRL reactivity showed a declining trend with
1.56% prevalence in 2007 to 1.05% in 2016
with significant fall in reactivity for females.

Declining trends of syphilis seroprevalence in India

NCBI Pubmed 2021
 Pathogenesis:-

1) Venereal syphilis:- sexual contact

2) Non-venereal :- occupational- in doctor, nurses

usually in fingers, no primary chancre.
-rarely by blood transfusion.
 SYPHILIS

• Syphilis can be acquired by the –

(1) Venereal
(2)non-venereal route
(3)congenital or acquired
 Venereal syphilis:-

• Infective dose:-60 treponemes

• Generation time:-30-33 hours
• I.P. :- 10 – 90 days
• Acquired by sexual contact.
• Infectivity of a patient to the sexual partner is
maximum during the first two years of the disease
 3 stages of primary syphilis :-

I. Primary
II. Secondary
III. Tertiary
 Primary syphilis:-
• Chancre at the site of entry.
• Painless, avascular, circumscribed, indurated,
superficially ulcerated lesion.
• It is known as “hard chancre”.
• The regional lymph nodes are swollen , discrete,
rubbery and non-tender.
 Secondary syphilis:-
• Spirochetes multiply and spread through blood.
• Most infectious stage.
• Non-itchy roseolar or papular skin rash often affecting
the palms and soles, mucocutaneous lesions,
generalized lymphadenopathy.
• In moist areas of the body the rash becomes flat
broad whitish lesions called condylomata lata.
• Secondary lesions are usually undergo spontaneous
healing, sometimes even take 4-5 years.
 Latent syphilis:-

• After the secondary lesions disappear ,there is a

period of quiescence known as latent syphilis.
• In this stage positive serological test for syphilis but
no clinical evidence of treponemal infection.
• Patient is infective in this stage.
-Materno-fetal transmission can occurs(10%).
Early latent is known for relapses.
- Mucocutaneous relapses are most common.
Late latent phase is known for resistance to
re-infection and relapses.
- Transmission of infection is possible.
 Tertiary syphilis:-
• Occurs in one-third of the patients.
• 3 different forms:-
1) Late benign syphilis (15%) :- granulomatous
lesion in skin called gummata( tumor like lesion
destroys surrounding tissue due to active cellular
immune response.
2) Neurosyphilis :-meningo-vascular syphilis.
(3)Cardiovascular syphilis(1%):-
Syphilitic endarteritis( ascending aorta),aortic
aneurism, dilatation of aortic ring.
Late tertiary/quaternary syphilis:-
-after several decades of initial infections may
develops neurological manifestation like general
paralysis.
- Tabes dorsalis.
 Congenital syphilis:-

• Infection is transmitted from mother to fetus

transplacentally.
• Transmission can take place at any stage of
pregnancy.
• Women with early syphilis can infect her fetus much
more commonly 75-95%.
• Lesions of congenital syphilis usually develop only
after the fourth month of gestation, when fetal
immune competence starts appearing.
• 40% untreated maternal infections cause late
abortion, stillbirth.
• Death of neonates due to secondary bacterial
infection.
 Newborn:-

• Premature or low weight baby.

• Hepatomegaly and splenomegaly with skin rash.
• Early rash:- small vesicles on the palm and sole.
• Late rash:-copper colored flat or maculopapular rash
on the face, palms, soles.
• Rash at the junction of the skin and mucus
membrane of the mouth, genitalia.
• saddle nose.
• Severe congenital pneumonia, referred on x-rays as
“whiteout “ or pneumonia alba.
 Older children:-
• Bone pain.
• Clutton’s joint- joint swelling.
• Notched and peg shaped teeth called Hutchinson
teeth.
• Gray, mucous-like patches on the anus and outer
vagina (condyloma lata).
 Lab.diagnosis:-
A. Specimen:-
• Specimen should be collected with carefully because
lesions are highly infectious.
• The lesion is cleaned with gauze soaked in warm
saline and the margins gently scraped so that the
superficial epithelium is abraded.
• Gentle pressure is applied to the base of the lesion
and the serum that exudes is collected and
preventing admixture with blood.
 Direct microscopy:-
• Demonstration of Treponemes from superficial lesion
of primary, secondary and congenital syphilis.
1) Direct ground microscopy( DGM) :-
• T.pallidum appears as slender, flexible, spirally coiled
bacilli with tapering ends, measuring 6-20µm in
length and contains 6-20 spirals.
Motility:-
i. Slow to rapid flexion-extension type of movement.
ii. Rotation around its longitudinal axis (cork screw
motility).
iii. Rotation may be accompanied by a soft bending at
right angle to the mid point.
 Direct fluorescent antibody staining for
T.pallidum (DFA-TP)
• Smear stained with fluorescent labelled monoclonal
antibody targeted against T.pallidum surface antigen.
• Appears as distinct, sharply outline apple green
fluorescent colored bacilli.
• Sensitivity is 100% when smear made from fresh
lesions.
 Silver impregnation staining:-
• Treponemes reduce silver nitrate to metallic silver
that is deposited on the surface making them thicker.
• Fontana stain is used for staining smears made from
exudate.
 Serology (antibody detection):-
• Depending upon the types of antigen used, three
types of tests are available to detect antibody in
patient’s serum.
1) Non-treponemal test/non-specific test/STS
2) Treponemal test/specific treponemal test
3) Group specific test.
 Non-treponemal test :-
• Non-specific test.
• STS( standard test for syphilis)
• Detect non-specific reagin antibody by using
cardiolipin antigen derived from ox heart.
• Cardiolipin antigen is chemically diphosphatidyl
glycerol. Similar lipid haptens have been detected on
the surface of T.pallidum.
 Non-treponemal test or standard test for
syphilis :-
Older method:-
• Wassermann test (CFT)
• Kahn test (Tube flocculation test)
Newer method :- (Slide flocculation test)
• VDRL
• RPR
• TRUST
• USR
 VDRL test:-
Principle:-
• Slide flocculation test.
• Reagin antibody reacts with VDRL antigen
(cardiolipin-lecithin cholesterol antigen) and form
floccules.
Method :-
• VDRL kit contains (a) VDRL antigen
(b) Buffered saline
(Titration of samples)
 Rapid plasma reagin (RPR) test:-

• Slide flocculation test using disposable plastic cards.

• Similar to VDRL test but some differences are:-

 VDRL RPR
-Result read microscopically as clumps are -Result read microscopically ; carbon
smaller in size. particles coated cardiolipins are used so
that large visible clump are formed

-Antigen, once reconstituted should be -EDTA is used as stabilizer; so that RPR

used within 24 hrs. antigen can stored longer (upto 6 months
at 4-10°c)

-Pre-heating of serum is required to -Pre-heating of serum not required as

remove non-specific inhibitors. choline chloride is used to remove
inhibitors.

-Blood, plasma, serum and CSF can be -Blood, plasma, serum can be tested but
tested. not CSF..

-Rotation of slide is done for 4 minutes. -Rotation of slide is done for 8 minutes.

-Sensitivity in primary syphilis is 78% -Sensitivity in primary syphilis is 86%

Toludine red unheated serum test(TRUST):-
• Modified RPR test.

• Toludine red pigment particles are used instead of

carbon particles.

• It does not require microscope for examination.

 USR(unheated serum reagin) test:-
Similar to VDRL except:-

• EDTA is used as antigen stabilizer.

• Choline chloride is used in serum to inhibit the non-

specific inhibitors; hence pre-heating of serum is not
needed.
 advantage of non-treponemal test disadvantage of non-treponemal test

Most recommended test to monitor Biological false positive reaction.

treatment response .

VDRL can be used to detect CSF Sensitivity is low in late stages.

antibodies ( neurosyphilis).

Reagin antibodies become detectable 7- -Screening test:-

10 days after appearance of primary Non-treponemal test need to confirmed
syphilis. by treponemal test.
 Interpretation:-
Non-treponemal test(RPR/VDRL)

Reactive Non-reactive

Treponemal test No syphilis or

(EIA,TPHA,FTA-ABS) very reacent
infection

Reactive Non-reactive

Syphilis(new case or Biological false- positive

previously treated
case
 Specific treponemal test:-
1) Fluorescent treponemal antibody-absorption (FTA-
ABS) test.
2) T.pallidum haemagglutination assay(TPHA)
3) T.pallidum immobilization(TPI) test
4) Enzyme immuno assay (EIA)
5) Western blot
 Fluorescent treponemal antibody-absorption
test (FTA-ABS)
• Used killed T.pallidum.
• Patient’s serum is first diluted with extract of non-
pathogenic Reiter treponemes to remove group
specific treponemal antibodies.
• Patient serum is layered on a slide previously coated
with killed T.pallidum.
• Serum antibodies bound to T.pallidum can be
detected by addition of fluorescent labelled anti-
human immunoglobulin and then slide is examined
under fluorescent microscope.
 Treponema pallidum haemagglutination
assay (TPHA)
A qualitative haemagglutination test using tanned
formalinised sheep RBC’s as the carrier for
T.pallidum antigen (sensitized cells)

Serum is tested with both sensitised and non

sensitised RBC’s
 T.pallidum immobilization test (TPI):-
Principle:-
• Ability of patient’s antibody and complement to
immobilize the live actively motile T.pallidum (Nichols
strain) ,to observe under dark ground microscope.

• One of the widely used test for syphilis in the past ,

now not in use.
 Enzyme immunoassay (EIA):-
• Serum is added to micro-wells coated with a
treponemal antigen.
• An enzyme labeled antihuman
immunoglobulin conjugate and enzyme
substrate are added to detect Ag-Ab reaction.
• IgM ELISA is more sensitive than IgM FTA-ABS
for diagnosis of congenital syphilis.
 Western blot:-
• Detect IgG and IgM antibodies seperately.
• It is highly sensitive and specific.
• Detect surface antigen of Nichols strain of
T.pallidum( TpN15,TpN17, TpN47).
 Polymerase chain reaction (PCR):-

• Used for detection of early lesions of syphilis.

• PCR amplify T.pallidum specific genes such as gene
coding for 47-kDa surface antigen (lipoprotein) and
39-kDa basic membrane protein.
• Important in diagnosis of congenital and
neurosyphilis.
 RAPID TEST:-
• TP Syphilis Test card
 Recombinant antigens are dry immobilised at the
end of nitrocellulose membrane strip.
 Visible red line is seen if TP antibodies are captured
by TP antigens.
 Result shown in less than 30 minutes
 Interpretation:-
Non-treponemal test treponemal test likely interpretation

Positive Positive syphilis

Positive Negative no syphilis (false positive)

Negative Positive primary or latent syphilis,

previously treated or
untreated syphilis.

Negative Negative no syphilis , incubating

syphilis
 Diagnosis of congenital syphilis:-
(A) Definitive diagnosis.
Demonstration of T.pallidum by DGM of umbilical
cord , placenta, nasal discharge or skin lesion material.
(B) Presumptive diagnosis :-
• Infant born to mother having syphilis at time of
delivery regardless of findings in the infant.
• Reactive treponemal tests in infants.
(C) Additional criteria :-
• Sign/symptoms of congenital syphilis.
• Reactive VDRL-CSF test.
• Reactive IgM antibody test specific for syphilis ( IgM
FTA-ABS or IgM ELISA).
 Treatment :-
(1) penicillin:- drug of choice for primary, secondary,
early latent syphilis, single dose of penicillin-G is
given.
• Late latent CVS or benign tertiary stage:-
penicillin-G is given single dose weekly for 3 weeks.
• Neurosyphilis or associated HIV:- aqueous
crystalline or procaine penicillin-G is given for 10-14
days.
(2) Tetracycline :- used in case of penicillin allergy.

Prevention :-
• Treatment of cases and contacts (sexual partner).
• Education about safe sex practices.
• Prophylactic use of barrier contraceptive method.