microbiology by man bahadur rana

1. GENITAL DISEASE
MAN BAHADUR RANA
BPH
ACAS,NEPAL

2. Treponema pallidum
(SYPHILIS)

3. CONTD....

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6. Treponema pallidum
T. Pallidum is a
spirochaete bacterium
with subspecies that cause
treponemal disease such
as syphilis , bejel, pinta and
yaws.

7. Contd...
Four known subspecies of
Treponema.
1. Treponema pallidum pallidum .
2. Treponema pallidum
endemicum.
3. Treponema pallidum carateum
4. Treponema pallidum pertenue

8. - These are long, slender,
helically tightly coiled
bacteria.
- Gram negative.
- Can be free living or
parasitic.
- They have tapering
ends.

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- cork screw motility.
- Because of thinness of the
spirals T. pallidum cannot
be seen in wet film by light
microscope.
- Its morphology and
motility can be seen by
dark field illumination.

11. Contd...

12. PATHOGENESIS
- T. pallidum is a strict parasite and
its life outside the animal body is
short. Infection is usually acquired
by sexual contact with infected
individual and is commenest in
the most sexually active age
group of 15-30 yrs old.
- The treponemes are present in
the superficial genital lesions and
enters the body from one partner
to other via skin and mucous
membrane through abrasions
during sexual contact.

13. Contd...
- The disease may also be
transmitted transplacentally .
- Medical personnel are
occasionally infected by an
accidental finger prick with an
infected needle.
- T. pallidum travels via the
lymphatic system to regional
lymph nodes and then
throughout the body via the
blood stream.

14. Contd.....
- Invasion of the CNS can occur
during any stage of syphilis.
- The bacteria rapidly enter the
lymphatics , are widely
disseminated via the blood
stream and may lodge in any
organ. The exact infectious
dose for man is not known,
but in experimental animals
fewer than 10 organisms are
sufficient to initiate infection.

15. Contd....
- The bacteria multiply at the site
of entry forming a chancre, and
after an incubation period of
about a month(range 10-90 days)
the clinical disease set in.
- The clinical manifestation falls
into four stages:
- Primary
- Secondary
- Latent
- tertiary

16. Contd....
Primary Syphilis:
- The primary lesions of
Syphilis is the chancre . The chancre
is painless and most frequently on
the external genitalia , but it may
occur on the cervix, perianal area, in
the mouth or anal canal.
- In some cases it may be on
lips , cheeks and nipples(when it is
acquired through kissing).
- Chancres usually occurs
singly but in immunocompromised
individuals, multiple or persistent
chancres may develop.

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- The chancre usually heals
spontaneously with in 3-6 weeks
even without treatment leading a
thin scar.

23. Secondary syphilis
- This usually starts to manifest itself
3-8 weeks after healing of primary
chancre.
- The secondary lesions are due to
wide spread multiplication of the
treponemes and their dessimination
through the blood.
- Clinical signs include:
. Low grade fever
. Malaise
. Lymphadenopathy
. Alopecia areata (irregular
loss)

24. Contd...
- On skin , the rash is most
often maculopapular and is
found on the palms and
soles.
- On mucous membranes, the
rash appears in the form of
discoloured patches.
. Condylomalata (i.e
raised sessile lesions) are
characteristics and found on
the anal mucosa.

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- The manifestation of
secondary syphilis are transient
(8-12) weeks because most
patient with secondary syphilis
build a sufficient immune
response and the disease
caused spontaneously.
- About 13-15% untreated cases
will progress to a latent stage
and finally to tertiary stage.

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Latent stage:
In latent stage,
the infection remain
dormant (latent) for
variable periods (2 years
is the usual benchmark)
without any clinical
symptoms but with
positive serology.

31. Contd....
Tertiary Syphilis:
- Decades after the
primary infection , patient
may develop late or
tertiary syphilis.
- It is a slowly progressive
, destructive inflammatory
disease that may affect
any organ.

32. Contd....
It may lead to:
. Gummata (rubery
tumors)
. Bone deformities
. Blindness
. Loss of
coordination
. Paralysis
. Insanity

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37. CULTIVATION
- Pathogenic treponemes cannot
be cultivated in artificial media
and are maintained by
subculture in susceptible
animals.
- The virulent strains e .g (Nichols
strain) of T. pallidum has been
maintained by serial passages in
rabbit testis for several decades
since it was isolated in 1913 from
CSF of a patient with
neurosyphilis.

38. Contd....
- Cultivable treponemes
such as T .phagedenis
(Reiter treponeme) are
non pathogenic. They
can be grown under
strictly anaerobic
condition in Smith
Noguchi medium.

39. Laboratory testing:
Evaluation based on three factor:
- Clinical findings.
- Demonstration of
spirochetes in clinical
specimen.
- Present of antibodies
in blood or cerebrospinal fluid.

40. Contd....
- Direct examination of clinical
specimen by Dark – Field
microscopy or Fluorescent
antibody testing of sample.
- Non specific or non- treponemal
serological test to detect reagnin,
utilized as screening test only.
- Specific Treponemal antibody test
are used as a confirmatory test
for a positive reagin test.

41. Contd...
Note:
Every patient with positive
syphilis serology should be
tested for HIV with the patients
informed consent and vice-versa.
HIV infected patient who
acquire syphilis run a more
severe course and may fail to
produce anti treponemal
antibodies.

42. Contd...
Direct demonstration of
Treponema.
Treponema cam be
demonstrated in fluid or
scraping from chancre
(primary stage),mucous
patches and condyloma of
secondary syphilis and
congenital syphilis by dark
ground microscopy.

43. Contd...
Dark ground microscopy
(DGI):
- Clean the chancre with saline.
- A drop of exudate is collected on
a slide by applying gentle pressure
at the base of the lesion.
- The wet film is then covered by a
coverslip
- Examine under dark ground
illumination.
- T. pallidum appears as a slender,
spiral organism exhibiting flexion
and extension movement.

44. Contd...
- For positive microscopic
findings , the number of
treponemes must be 104 or
more per ml of exudate.
- This technique requires
considerable skills and is often
unavailable in out patient
clinics.

45. Direct fluorescent Antibody
Treponemal test (DFA-Tp
test)
- Smear made from blood films or
from clinical material is fixed in
acetone or 95% ethanol for 5 min
and dried.
- The fluorescent labelled
antibodies are brought into
contact with antigens fixed on the
slide (eg blood film).
- Incubate at 370 C for half an hour.
- Allowing them to react and
washed.

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- Examine under
fluorescent microscope.
- In positive cases ,
treponemes fluoresce
displaying typical
morphology.

47. Serological test
1. Non treponemal (reagin) tests:
- VDRL/ RPR
2. Treponemal test (TPHA)
VDRL(Veneral disease Research
Laboratory Test) :
It is the most widely used
simple and rapid test.
The VDRL antigen must
be prepared daily.

48. Contd...
- In the standard test for syphilis ,
reagin antibodies in patients serum
is detected by cardiolipin antigen.
- Cardiolipin antigen is an alcoholic
extract of bovine heart muscle to
which lecithin and cholesterol are
added.
- Cardiolipin reacts with non-specific
reagin (a mixture of IgG and Ig M
antibodies).
- Before performing the test, the
patient serum is inactivated by
heating in a water bath at 560 C for
30min to destroy the complement.

49. Contd...
Method: Preparation of antigen:
1. Pipette 0.4 ml buffered saline
and transfer into a round
bottomed screw cap bottle.
2. Transfer 0.5 ml VDRL Antigen
with the help of a tuberculin
syringe, drop by drop while gently
rotating the bottle.
3. Shake the bottle vigorously.
4. Transfer the remaining 4.1 ml of
buffered saline into the bottle.

50. Contd..
5. Shake the bottle vigorously
and keep it aside for 15-30
min for maturation.
6. The working antigen must be
used on the same day itself.
Inactivate the serum sample
in a water bath at 560 C for 30
minutes.

51. Contd...
Procedure:
- Transfer 50 micro litre of serum
into the VDRL slide.
- Transfer 50ul of positive and
negative control.
- One drop of working antigen is
placed on each well.
- The VDRL slide is placed on a
shaker and rotate for 4 min.

52. Contd...
- The test is read using a compound
microscope with low power
objective.
Results:
Positive control: Medium
to large clumps.
Negative control: No
clumps

53. RPR (Rapid Plasma
Reagin) test
In this test VDRL antigen is
adsorbed on finely divided carbon
particles and suspended in
choline chloride which blocks
inhibitory factor in the serum
thus eliminating the need to heat
the serum before testing.
Procedure:
- One drop of patients serum ,
positive and negative controls are
placed with in circles.

54. Contd...
- 15-20ul of antigen is dropped
over the serum samples using
the antigen delivery dropper.
- Serum and antigen is mixed well
using the stick and the card is
rotated at 1800rpm for 4
minutes.
- The card is then rocked gently
and observe under strong source
of light.

55. Contd...
- In a positive case the
flocculation of carbon
particles (black aggregates) is
visible with naked eye.
- In a negative test , there is a
complete absence of black
aggregates.

56. TPHA (Treponema pallidum
haemagglutination test)

57. Prophylaxis
- At present there is no effective
vaccine against syphilis.
- As transmission of the infection is
by direct contact, it is possible to
protect against syphilis by
avoidance of sexual contact with
an infected individual.
- The use of mechanical barrier
such as condom in which
prevention of direct contact
between infected mucous
membrane is achieved.

58. Treatment
Penicillin is the drug of choice for
Syphilis.
Early syphilis :
Benzathine penicillin , 24lacs unit
IM route once.
Procaine penicillin 600000 units.
IM daily for 10 days.
If the patient is unable to take penicillin
then give tetracycline or erythromycin
500mg 4 times a day by mouth.
OR
Doxycycline 100mgx2 for 15 days