Lecture 2-syphilis serology

INTRODUCTTION TO SYPHILIS
I. Syphilis is a sexually transmitted infection caused
by the bacterium Treponema pallidum subspecies
pallidum. The signs and symptoms of syphilis vary
depending in which of the four stages it presents
(primary, secondary, latent, and tertiary)
II. Syphilis is caused by a spirochete bacterium,
Treponema 2

Morphology and Metabolism of T.
Pallidum
Microscopically T. pallidum appears as fine, spiral (8
to 24 coils) organism, approximately 6-15 μm long.
They are not cultivatable with any consistency in
artificial laboratory media out side the host.
T. pallidum are extremely susceptible to a variety of
physical and chemical agents. However, they may
remain viable for up to 5 days in tissue specimens
removed from diseased animals and from frozen
specimens.
3

Stages of Syphilis
Untreated syphilis is a chronic disease with sub
acute symptomatic periods separated by
asymptomatic intervals, during which the diagnosis
can be made serologically.
The progression of untreated syphilis is generally
divided in to stages. Initially, T.pallidum penetrates
intact mucous membranes or enters the body
through tiny defects in the epithelium.
4

Stages of Syphilis
Primary syphilis
At the end of the incubation period, a patient
develops a characteristics primary inflammatory
lesion called a chancre at the point of initial
inoculation and multiplication of the spirochetes.
Generally it is relatively painless and commonly
located around the genitalia, but in about 10% of
cases lesions may appear almost any where else on
the body e.g. Throat, lip, hands.
5

Stages of Syphilis
Secondary syphilis
Within 2 to 8 weeks after the appearance of the
primary chancre, a patient may develop the sign
and symptoms of secondary syphilis when
organisms gain access to the circulation from the
infected site.
The secondary stage is characterized by a
generalized illness that usually begins with
symptoms suggesting a viral infection headache,
sore throat, low-grade fever and occasionally nasal
6

Stages of Syphilis
Latent syphilis
After resolution of untreated secondary syphilis, the
patient enters a latent non-infectious state in which
diagnosis can be made only by serologic method.
During the first 2-4 years of infection, one fourth of
patients will show relapses of manifestation of
secondary syphilis.
During these relapses, patients are infectious, and
the underlying spirochetemia may be passed
translucently to the fetus. 7

Stages of Syphilis
Late (Tertiary) syphilis
The first manifestations of late syphilis are usually seen
from 3-10 years after primary infection. About 15% of
untreated syphilitic individuals eventually develop late
benign syphilis characterized by the presence of
destructive granulomas.
Untreated patients 10% develop cardiovascular
manifestations.
About 8% of untreated patients, late syphilis involves the
CNS. Initially CNS disease is asymptomatic and can be
detected only by examination of cerebrospinal fluid. 8

Stages of Syphilis
Congenital syphilis
 Congenital syphilis is caused by maternal
spirochetemia and transplacental transmission of
the microorganism usually after 18 weeks of
gestation. Congenital syphilis is diagnosed in three
fourths of the cases in patients over 10 years of
age.
 Early congenital syphilis appears either at birth or
up to two years of age
9

Antibodies in Syphilis
The serologic methods for syphilis measure the
presence of two types of antibodies: treponemal and
non treponemal.
10

Tests for Syphilis
Serologic procedures for syphilis include the
following.
1. Nontreponemal method e.g. Venereal Disease
research laboratory (VDRL) and the rapid plasma
regain (RPR) procedures.
2. Treponemal methods e.g. Fluorescent Treponema
pallidum antibody absorption (FTA-ABS) and
microhemagglutination Treponema pallidum MHA-
TP).
11

Nontreponemal methods
The VDRL procedure is recommended when a patient
suspected of having syphilis has a negative dark field
microscopy result or when atypical lesions are
present. It is further recommended that a
quantitative VDRL assessment be made quarterly for
1 year after treatment for syphilis, or that the
adequacy of treatment in both early and latent
syphilis be monitored
12

Nontreponemal methods
The RPR test can be performed on unheated serum or
plasma using a modified VDRL antigen suspension of
choline chloride with EDTA. The RPR test card test
antigen also contains charcoal for macroscopic
reading. It is about as specific as, and possibly more
sensitive than, the VDRL slide test.
13

Treponemal methods
The FTA – ABS and MHA represent treponemal
methods. Reactive (Positive) regain test can be
confirmed with these two specific treponemal
antigen tests. These procedures, however,
should not be used as primary screening
methods.
14

Sensitivity of commonly useds
erologictests for syphilis
15

Venereal Disease Research
Laboratory(VDRL)
Principle:
heat inactivated serum is mixed with a
buffered saline suspension of cardiolipin–
lecithin–cholesterol antigen. This serum-
antigen mixture is microscopically
examined for flocculation.
16

Laboratory(VDRL)
Specimen collection and preparation:
 The specimen should include all identification, it
must include the patient’s full name, the date the
specimen is collected and the patient’s hospital
identification number.
 Blood should be drawn by an aseptic technique.
The required specimen is a minimum of 2 ml of
clotted blood. The specimen should be promptly
centrifuged and an aliquot of the serum removed.
17

Laboratory(VDRL)
Specimen collection and preparation:
 Severely lipemic or hemolyzed serum is unsuitable
for testing. Before testing, the serum must be heat
in activated at 56C0 for 30 minutes.
 In activated serum should be reheated at 56C0 for
10 minutes, if tested more than 4 hours after the
original in activation.
 Cerebrospinal fluid is also an appropriate fluid for
testing.
18

Laboratory(VDRL)
Equipment required
 VDRL test slide with paraffin of ceramic ring.
 18 gauges hypodermic needle with out bevel (it will
deliver 60 drops) ml of reagents.
 30 ml flat-bottomed glass with stopper, narrow
mouth bottle
 Syringe (1-2ml)
 Rotator
 Serological graduated pipette
 Water bath 56C0
19

Laboratory(VDRL)
Preparation of working antigen suspension
 Dispense 0.4 ml of buffered saline to the bottom of
the 30ml, round, glass-stopper bottle.
 Rapidly add 0.5ml of antigen drop by drop directly
by rotating the bottle in a circular motion on a flat
surface.
 Continue to rotate the bottle for 10 seconds.
 Add 4.1 ml of buffered saline with a 5 ml pipette
 Place the stopper on the bottle and shake up and
down approximately 30 times in 10 seconds.
20

Laboratory(VDRL)
Note: The working antigen suspension can be
stabilized by adding 50 μl of benzoic acid to 5ml of the
diluted working solution instead of discarding within
24 hours. The temperature of the buffer saline and
antigen should be in the range of 230 to 29C0. The
antigen suspension must be used on the day of
preparation.
21

Laboratory(VDRL)
Quality control
Include positive control sera of graded reactivity each
time serologic testing is performed. The antigen
suspension to be used each day is first examined with
these control sera. Store control sera frozen at -20C0
or liquid form for 7 to 10 days.
22

Causes False negative reactions
 Technical error (e.g. unsatisfactory antigen
preparation or techniques.
 The presence of inhibitors in the patient’s serum
 Low antibody titer patients may have syphilis, but
the reagin concentration is too low to produce a
reactive test result.
 Inappropriate temperature
 Prozone reaction
25

LIMITATIONS of VDRL
The VDRL procedure is not specific for syphilis but
may demonstrate positive reactions in other reagin-
producing disorders, infectious disease and
alterations such as pregnancy or aging in normal
physiology
28

Lecture 2-syphilis serology

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