TREPONEMA

1. SPIROCHETES-I
Dr. Mohit Bhatia
Assistant Professor
Department of Microbiology
AIIMS, Rishikesh

2. Spirochetes
• Thin, flexible, elongated spirally coiled helical
bacilli
• Speira = coil
• Chaite = hair

3. Classification of Spirochetes
• Class Spirochaetaceae
- Spirocheta
- Cristispira
- Treponema
- Borrelia
• Class Leptospiraceae
- Leptospira
- Leptonema.

4. Ultrastructure of Spirochetes
• Outer membrane
• ™
Periplasmic space containing
flagella
• ™
Peptidoglycan layer
• ™
Inner (cytoplasmic)
membrane
• Motile with endoflagella

5. Morphological differences between Treponema,
Borrelia and Leptospira
Treponema Borrelia Leptospira
Size 6-14μm X
0.2μm
10-30μm X
0.2-0.5μm
6-20 μm X
0.1μm
Spirals 6-12 in no. 3-10 in no. Numerous and
tightly coiled with
hooked ends
Wave length 1μm 3μm 0.5μm
Amplitude of
spiral
1-1.5μm Up to 2μm 0.1μm
Endoflagella
at each pole
3-4 7 - 11 1

6. Spirochetes - Classification
Spirochaetes Disease Transmission
T.pallidum Syphilis Sexual
T.pertenue Yaws
Direct contact
T.endemicum Endemic syphilis
T.carateum Pinta
B.recurrentis Relapsing fever (epidemic) Louse borne
B.duttonii,
B.hermsii
Relapsing fever (endemic) Tick borne
B.burgdorferi Lyme disease Tick borne
L.interrogans Leptospirosis
1.Milder form
2.Severe form (Weil’s disease)
Contact with
rodent urine

7. TREPONEMA

8. TREPONEMA
• Pathogenic species
- T. pallidum subspecies pallidum
- T. pallidum subspecies pertenue
- T. pallidum subspecies endemicum
- T. carateum
• Almost identical in their morphology, antigenic
structure and in genetic composition

9. TREPONEMA PALLIDUM
• ™
Morphology: extremely thin and delicate with tapering
ends
• ™
Size: 6–14 μm × 0.2 μm
• ™
Spirals: 6–12 spirals at intervals of 1 μm
• ™
Motility: flexion extension, translatory and corkscrew
motility
• ™
Endoflagella: About 3–4 flagella - motility & highly
antigenic

10. Microscopy & Culture
• Dark ground or phase contrast microscope
• ™
Staining: Do not take up Gram stain
- Fluorescence staining
- Sliver impregnation methods
• ™
Cultivation: Pathogenic treponemes cannot be grown in artificial
culture media. Maintained in rabbit testes. E.g, „
Nichols strain
• Non-pathogenic Treponemes – grow in Smith Noguchi medium
under strict anaerobic conditions. E.g: Reiter’s strain & Noguchi
strain

11. Antigens
Group-specific antigen: Protein antigen
- Present in all treponemes
- Antibodies detected using antigens of Reiter
treponemes
Species-specific antigen: Polysaccharide
- Antibodies detected by using specific T. pallidum
antigens
Non-specific antigen: Heterophile antigen
- Antibody detected using beef heart antigen

12. PATHOGENESIS OF SYPHILIS
• Mode of transmission:
- Venereal
- Non-venereal - direct contact, blood transfusion or
transplacental
• ™
Spread: T. pallidum penetrates through mucosa or abraded
skin  Enter lymphatics and blood  systemic  primary
lesion
• ™
Incubation period: Variable (9–90 days)
Inversely proportional to the number of organisms
inoculated

13. PRIMARY SYPHILIS
NIFESTATIONS OF SYPHILIS
• Primary (or hard) chancre:
- Single painless papule
ulcerated & indurated
- Covered by thick exudate rich
in spirochetes
- Common sites – penis, cervix
or labia
- Heals within 4–6 weeks

14. CLINICAL MANIFESTATIONS OF
SYPHILIS
• ™
Regional (usually inguinal) lymphadenopathy
- Painless firm, non-suppurative, and often
bilateral
- May persist for months
• ™
™
If acquired by non-venereal mode:
- „
Direct contact → extragenital, usually on the
fingers
- „
Blood transfusion → primary chancre does not
occur

15. SECONDARY SYPHILIS
• Develops 4–8 weeks after
healing of primary lesion
• Skin and mucous
membranes - commonly
affected
• ™
Skin rashes

16. Secondary Syphilis
• ™
Condylomata lata:
Mucocutaneous papules
coalesce to form large pink to
grey lesion in warm moist
intertriginous areas (such as
perianal region, vulva, and
scrotum)
• ™
Mucous patches (superficial
mucosal erosions)

17. Latent Syphilis
• Absence of clinical manifestations of syphilis with
positive serological tests and normal CSF findings.
• ™
Patients are still infectious - bloodstream or in
utero
• ™
Fates:
- Persistent lifelong infection (common)
- Development of late syphilis (rare)
- „
Spontaneous cure

18. Late or Tertiary Syphilis
• Gumma (late benign syphilis): Locally destructive
granulomatous lesions - bone and skin
• ™
Neurosyphilis:
„
- Meningeal syphilis (meningitis)
- Meningovascular syphilis (vasculitis of arteries embolic stroke)
- „
General paresis of insane
„
- Tabes dorsalis
• Cardiovascular syphilis: aneurysm of ascending aorta and aortic
regurgitation

19. Congenital Syphilis
• Transmission - at any stage of pregnancy
• Fetal damage - after fourth month of gestation
Manifestations of congenital syphilis include:
• ‰
Earliest manifestations : (within 2 years of age) & infectious
- Snuffles, mucocutaneous & bone changes, hepatosplenomegaly
• ‰
Late congenital syphilis: (after 2 years) & non-infectious
- Interstitial keratitis, eighth-nerve deafness, bilateral knee effusions
• Residual stigmata - Hutchinson’s teeth (notched central incisors),
Mulberry molars, Saddle nose, and saber shins

20. LABORATORY DIAGNOSIS OF SYPHILIS
• Direct Microscopy
• Dark Ground Microscopy (DGM)
- Slender, flexible, spirally coiled
bacilli with tapering ends
• Motility: Flexion-extension type,
corkscrew motility, Soft bending at
right angle to the midpoint
• Sensitivity of DGM - 80%
• Detection limit of 104 bacilli/mL

21. LABORATORY DIAGNOSIS OF SYPHILIS
• Direct Fluorescent Antibody
Staining
- Distinct, sharply outlined,
apple green fluorescent bacilli
• ™
Sensitivity - 100% when
smear made from fresh
lesions are examined

22. LABORATORY DIAGNOSIS OF SYPHILIS
• Silver Impregnation Staining
• Increase thickness
- Levaditi stain -tissue section
- Fontana stain – exudate smears
• Cultivation- maintained by
subcultures in rabbit testes

23. Serology (Antibody Detection)
• Non-treponemal tests: Detect non-specific reagin
antibody by using cardiolipin antigen derived
from bovine heart
• Treponemal tests: Detect species-specific
antibody by using T. pallidum specific antigen
• Group-specific tests: Detect group or genus-
specific antibody by using Reiter treponemal
strains possessing protein antigen present in all
treponemes.

24. Standard Tests for Syphilis
• Non-treponemal or Non-specific tests or STS (Standard
Tests for Syphilis)
• Detect reagin antibody using cardiolipin antigen extracted
from beef heart
• Cardiolipin antigen - diphosphatidyl glycerol
• Reagin antibodies are IgG or rarely IgM type
- Slide flocculation tests - VDRL, RPR, USR, TRUST
- Wassermann test (e.g. of complement fixation test)
- Kahn test (e.g. of tube flocculation test

25. Venereal Disease Research Laboratory (VDRL)
• ‰
Widely used, simple
and rapid serological
test
• VDRL antigen -
cardiolipin antigen to
which cholesterol and
lecithin are added
• Slide Flocculation test

26. Venereal Disease Research Laboratory
(VDRL)
• Qualitative test: Inactivated serum + a drop of VDRL
antigen rotated at 180 rpm for 4 minutes in a VDRL
rotator  examined under microscope
- Non-reactive: Uniformly distributed fusiform crystals
- Reactive: Medium to large clumps
• ‰
Quantitative test: Test performed with serial dilutions
(1:2, 1:4, 1:8 and so on) of serum done with 0.9%
saline
• ‰
VDRL-CSF: No preheating of CSF is needed

27. VDRL TEST
Positive Negative

28. VDRL v/s RPR
VDRL RPR
Results read microscopically -
clumps are smaller
Results read macroscopically -
Finely divided carbon particles
coated cardiolipin antigens are
used so that larger visible
clumps are formed
Antigen, once reconstituted,
should be used within 24 hours
EDTA is used as stabilizer; hence
RPR antigen can be stored
longer (up to 6 months at 4-
100C)
Preheating of serum is required
to remove non specific
inhibitors
Preheating of serum is not
required as choline chloride is
used to remove inhibitors

29. VDRL v/s RPR
VDRL RPR
Blood, plasma, serum, and CSF
can be tested
Blood, plasma and serum can be
tested but not CSF
Rotation of slide is done for 4
mins
Rotation of card is done for 8
mins
Sensitivity in primary syphilis is
78%
Sensitivity in primary syphilis is
86%
It is cheaper; one vial of VDRL
antigen can be used for 250
tests. It is preferred for field
studies and for antenatal
screening
RPR is expensive than VDRL. It is
preferred when sample load is
less.

30. Other Reagin Antibody Tests
• Unheated Serum Reagin Test (USR) is similar to VDRL
except for:
- EDTA - antigen stabilizer  daily preparation of antigen
is eliminated
- Choline chloride - inhibit the non-specific inhibitors in
serum  pre-heating of serum is not needed
• Toluidine Red Unheated Serum Test (TRUST)
- Modified RPR test where toluidine red pigment
particles
- Does not require microscope for examination

31. Advantages of Non-treponemal Tests
• ™
To monitor the response to treatment
- Reagin tests usually become negative 6–18 months
after the effective treatment
• ™
Neurosyphilis: VDRL detects CSF antibodies
• ™
Detectable 7–10 days after the appearance of primary
chancre (or 3–5 weeks after acquiring the infection)
• Sensitivity: Varies from 78 to 85% in primary stage,
100% in secondary stage and 95–98% in latent stage

32. Disadvantages of Non-treponemal
Tests
• Biological false-positive (BFP) reactions: Positive non-treponemal
tests, with negative treponemal tests, in absence of syphilis and no
technical faults. BFP Antibodies - 1% of normal sera, IgM type
• Conditions - lepromatous leprosy, relapsing fever, malaria, tropical
pulmonary eosinophilia, viral hepatitis, infectious mononucleosis, HIV,
pregnancy and IV drug abusers
• ™
Prozone phenomena
• ™
Sensitivity of non-treponemal tests is low in late stage
• ™
Non-treponemal tests are used as screening tests

33. BIOLOGICAL FALSE POSITIVE(BFP) REACTIONS
As cardiolipin Ag is present both in T.pallidum and in
mammalian tissues, reagin Abs may be induced by
treponemal or host tissue antigens which accounts for
BIOLOGICAL FALSE POSITIVE(BFP)
BFP defined as “positive reactions” obtained in
cardiolipin tests, with negative results in specific
treponemal tests, in the absence of past or present
treponemal infections and not by technical faults
They represent non-treponemal cardiolipin antibody
responses

34. Represent non treponemal cardiolipin Ab responses
BFP reactions-may occur in about 1% of normal sera
Clinically BFP reactions classified as acute & chronic
Acute BFP-last only for weeks or months, associated ē acute infections,
injuries or inflammatory conditions
Chronic BFP-persist longer than 6 months, seen in SLE & other collagen
diseases
Other conditions associated ē BFP reactions are leprosy, malaria, relapsing fever,
infectious mononucleosis, hepatitis , tropical eosinophilia , pregnancy and
menstruation.

35. Syphilis Tests - Sensitivity &
Specificity
Sensitivity (%) Specificity
(%)
Treponemal Primary Secondary Latent Late
FTA-ABS 84 100 100 96 97
TPPA 88 100 100 ? 98
TPHA 76 100 97 94 99
EIA 90 100 100 ? 99
Western
blot
90 100 100 ? 98
TRUST 85 100 98 ? 99

36. Syphilis Tests - Sensitivity &
Specificity
Sensitivity (%) Specificity
(%)
Primary Secondary Latent Late
Non treponemal
VDRL 78 100 95 71 98
RPR 86 100 98 73 98
USR 80 100 95 ? 99
TRUST 85 100 98 ? 99

37. Treponemal or Specific Tests
• T. pallidum Immobilization (TPI) test
- Principle: patient’s antibody and complement to immobilize
the live actively motile T.pallidum (Nichols strain) - observed
under dark ground microscope

38. Treponemal or Specific Tests
Fluorescent Treponemal Antibody-Absorption Test (FTA -ABS)
- Uses killed T.pallidum, indirect fluorescent antibody technique
- Patient’s serum diluted with an extract of non-pathogenic Reiter
treponemes to remove group specific treponemal antibodies
- Layered on a slide previously coated with killed T. pallidum
- Serum antibodies bound to T. pallidum can be detected by addition of
fluorescent labeled anti-human immunoglobulin
- Examined under fluorescent microscope

39. Treponemal or Specific Tests
• ™
Advantages: Highly sensitive and specific in all
stages of syphilis
- First serological test to be positive following
infection
- Detects CSF antibodies
• ™
Disadvantage: False positive results in Lyme
disease

40. Treponemal or Specific Tests
• T. pallidum Hemagglutination Assay (TPHA)
- Tanned sheep RBCs coated with T.pallidum antigens
- Reactive result: Smooth mat of agglutinated cells in microtiter plate
- Nonreactive result: Compact button in the center of the well
• ™
Quantitation - done by serial dilution of patient’s sera
• Advantages: Affordable, easy to perform, available as commercial
kit and no special equipment is needed, detects CSF antibodies
- Sensitivity and specificity of TPHA are excellent

41. TPHA

42. Treponemal or Specific Tests
• Enzyme Immunoassays
- ELISA specific to IgG and IgM, ™
They have excellent sensitivity
and specificity
• Western Blot
- Detects IgG and IgM antibodies separately, highly sensitive
and specific
• Group-specific Test
- Reiter’s protein complement fixation test (RP-CFT)
• Molecular Methods - PCR-based techniques

43. Diagnosis of congenital syphilis
• Definitive diagnosis: Demonstration of T. pallidum by
DGM of umbilical cord, placenta, nasal discharge, or skin
lesion material
• Presumptive diagnosis:
- Infant born to a mother who had syphilis at the time of
delivery regardless of findings in the infant
- Reactive treponemal test in infant

44. Diagnosis of congenital syphilis
• ‰
One of the following additional criteria:
- Clinical signs/symptoms of congenital syphilis
- Abnormal CSF findings without other cause
- Reactive VDRL-CSF test
- Reactive IgM antibody test specific for syphilis (IgM FTA
ABS or IgM ELISA).
• Presence of specific IgM in neonatal serum confirms the
diagnosis

45. DIAGNOSIS OF NEUROSYPHILIS
CSF cell counts of 5cells/cu.mm
Protein values above 40mg/100ml
VDRL(NOT RELIABLE );Non reactive in 30-60%
Negative TPHA of CSF excludes neurosyphilis
Positive TPHA or FTA-ABS of CSF does not necessarily indicate active disease since
reactivity may be caused by transudation of immunoglobulins from serum into CSF.
TPHA index is a reliable parameter
The TPHA index, which relates the CSF-TPHA titre to the albumin quotient and
thus excludes errors from disturbed function of the blood-brain barrier,

46. Syphilis and HIV
• Both syphilis and HIV affect each other’s pathogenesis
• Problems in the diagnosis of syphilis in HIV infected people
are:
- Confusing clinical picture, Lack of serologic response
- Unusually high titers in non-treponemal tests
- Failure of non-treponemal test titers to decline even after
treatment
- Disappearance of treponemal test reactivity over time

47. Treatment Syphilis
• ‰
Penicillin is the drug of choice for all the stages of syphilis
- Primary, secondary, or early latent syphilis: single dose of
Penicillin G
- Late latent CVS or benign tertiary stage: penicillin G is given
single dose weekly for 3 weeks
• ‰
Alternative drug is used in patients with penicillin allergy:
- Primary, secondary, latent, CVS or benign tertiary syphilis—
tetracycline
- Neurosyphilis or pregnancy or associated HIV—
desensitization to penicillin

48. Evaluation after Treatment
• Non-treponemal tests
• ™
For primary and secondary syphilis: at least
fourfold decline in the titer by the third or fourth
month and an eightfold decline in the titer by
sixth to eighth month
• ™
Latent or late syphilis, or patients with multiple
episodes of syphilis: gradual decline in titer, low
titers may persist for years

49. NON-VENEREAL TREPONEMATOSES

50. NON-VENEREAL TREPONEMATOSES
Feature Venereal
Syphilis
Yaws Endemic
Syphilis
Pinta
Agent T. pallidum T. pertenue T. endemicum T.carateum
Mode of
transmission
Sexual,
Transplacental
Blood
Skin-to-skin Household
contacts:
kissing,
sharing
utensils or
insect vector
Skin-to-Skin
Age Adulthood Early
childhood
Early
childhood
Late
childhood

51. NON-VENEREAL TREPONEMATOSES
Feature Venereal
Syphilis
Yaws Endemic
Syphilis
Pinta
Primary lesion Chancre-
painless
non-
indurated
Lymphadenop
athy
Papilloma,
often
ulcerative
Lymphadenop
athy
Rarely seen Non
ulcerating
pruritic
papule
Site of lesion Genital, oral,
anal
Extremities Oral Extremities,
face

52. NON-VENEREAL TREPONEMATOSES
Feature Venereal
Syphilis
Yaws Endemic
Syphilis
Pinta
Secondary
lesions
Skin rashes
Mucosal
patches
Condylomata
lata
Skin lesions-
macular or
papular
Periostitis
Oral mucous
patches
Periostitis,
Lymphadenop
athy
Pintides,
Pigmented &
pruritic
Relapses 25% Common Unknown None

53. NON-VENEREAL TREPONEMATOSES
Feature Venereal
Syphilis
Yaws Endemic
Syphilis
Pinta
Late
complications
Gummas,
CVS and CNS
lesion
Destructive gummas of skin,
bone, cartilage
Destruction of the nose,
maxilla, palate, and pharynx is
termed as gangosa
Non
destructive,
dyschromic
macule

54. YAWS

55. Lesions of Pinta