lOMoARcPSD|16306511

Syphilis - Immunology and Serology Transes

Medical Technology (Universidad de Zamboanga)

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TOPIC: SPIROCHETES
SUBJECT // FINALS
Prof. Nawirah Gaus
SPIROCHETES
Spirochetes are long, slender, helically coiled bacteria
containing axial filaments, or periplasmic flagella, which
wind around the bacterial cell wall and are enclosed by
an outer sheath.
These gram-negative, microaerophilic bacteria exhibit a
characteristic corkscrew flexion or motility.
SYPHILIS
Syphilis is the most commonly acquired spirochete
disease in the United States.
Treponema pallidum- causative agent of syphilis.
- pallidum (subspecies)
-a member of the fam Spirochaetaceae.
Organisms in this family have no natural reservoir in the
environment and must multiply within a living host.
T. PALLIDUM SUBSPECIES
 Pertenue- the agent of yaws. (TROPICS)
 Endemicum- the cause of non venereal endemic
syphilis. (DESERT REGIONS)
 Treponema carateum- the agent of pinta.
(CENTRAL & SOUTH AMERICA)
Trepmonema pallidum varies in length from 6 to 20 mm
and in width from 0.1 to 0.2 mm, with 6 to 14 coils.
The outermembrane of T pallidum is a phospholipid
bilayer with very few exposed proteins.
Treponemal rare outer membrane proteins (TROMPs)-
scarcity of these delay the host immune response.
MODE OF TRANSMISSION
Pathogenic treponemes are rapidly destroyed by heat,
cold, and drying, so they are almost always spread by
direct contact.
Sexual transmission is the primary mode of
dissemination; this occurs through contact of abraded
skin or mucous membranes with an open lesion.
Congenital infections can also occur during pregnancy.
Transmission to the fetus is possible in mothers with
clinically latent disease.
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Transcribed by: QEEN
Other potential means of transmission include
parenteral exposure through contaminated needles or
blood, but this is extremely rare.
STAGES OF DISEASE
1. PRIMARY STAGE
Once contact has been made with a susceptible skin site,
endothelial cell thickening occurs with aggregation of
lymphocytes, plasma cells, and macrophages. This
primary stage lasts from 1 to 6 weeks, during which time
the lesion heals spontaneously
CHANCRE- the initial lesion. Develops between 10 and
90 days after infection, with an average of 21 days.
Chancre is a painless, solitary lesion characterized by
raised and welldefined borders
2. SECONDARY STAGE
Systemic dissemination of the organism occurs. This
stage is usually observed about 1 to 2 months after the
primary chancre disappears.
SYMPTOMS OF THE SECONDARY STAGE
♥ Generalized lymphadenopathy - enlargement of
the lymph nodes
♥ Malaise
♥ Fever
♥ Pharyngitis
♥ Rash on the skin and mucous membranes.The rash
may appear on the palms of the hands and the
soles of the feet
♥ May exhibit neurological signs such as visual
disturbances, hearing loss, tinnitus, and facial
weakness
Lesions persist from a few days up to 8 weeks and
spontaneous healing occurs, as in the primary stage
3. LATENT STAGE
Follows the disappearance of secondary syphilis. This
stage is characterized by a lack of clinical symptoms.
It is arbitrarily divided into early latent (fewer than 1
year’s duration) and late latent, in which the primary
infection has occurred more than 1 year previously.
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TOPIC: SPIROCHETES
SUBJECT // FINALS
Prof. Nawirah Gaus
Patients are noninfectious at this time, with the
exception of pregnant women, who can pass the disease
on to the fetus even if they exhibit no symptoms.
4. TERTIARY STAGE
This stage occurs most often between 10 and 30 years
following the secondary stage.
THREE MAJOR MANIFESTATIONS
A. GUMMATOUS SYPHILIS
Gummas are localized areas of granulomatous
inflammation that are most often found on bones, skin,
or subcutaneous tissue. Such lesions contain
lymphocytes, epithelioid cells, and fibroblastic cells.
They may heal spontaneously with scarring or they may
remain destructive areas of chronic inflammation.
B. CARDIOVASCULAR DISEASE
Usually involve the ascending aorta and symptoms are
caused by destruction of elastic tissue in the aortic arch.
The destruction may result in aortic aneurysm,
thickening of the valve leaflets causing aortic
regurgitation, or narrowing of the ostia, producing
angina pectoris.
C. NEUROSYPHILIS
Neurosyphilis is the complication most often associated
with the tertiary stage, but it actually can occur any time
after the primary stage and can span all stages of the
disease.
During the first 2 years following infection, CNS
involvement often takes the form of acute meningitis.
Late manifestations of neurosyphilis include
degeneration of the lower spinal cord with partial
paralysis and chronic progressive dementia. It usually
takes more than 10 years for these to occur; both are
the result of structural CNS damage that cannot be
reversed.
Fortunately, symptoms of tertiary syphilis are now very
rare because of early detection and effective treatment
with antibiotics such as penicillin.
THE GREAT IMITATOR
Patients with syphilis can be difficult to diagnose
because their clinical presentations can vary widely.
Because the symptoms of syphilis can mimic those of
many other diseases or conditions, the disease has often
been referred to as “The Great Imitator.”
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CONGENITAL SYPHILIS
Congenital syphilis occurs when a woman who has early
syphilis or early latent syphilis transmits treponemes to
the fetus.
The disease can be transmitted at any stage of
pregnancy, typically the fetus is most affected during the
second or third trimester. Fetal or perinatal death occurs
in approximately 10% of the case.
Infants who are liveborn often have no clinical signs of
disease during the first few weeks of life. Some may
remain asymptomatic, but between 60% and 90% of
these infants develop later symptoms if not treated at
birth.
SYMPTOMS
 Clear or hemorrhagic rhinitis, or runny nose.
 Skin eruptions, in the form of a maculopapular rash
that is especially prominent around the mouth, the
palms of the hands, and the soles of the feet, are
also common.
 Generalized lymphadenopathy
 Hepatosplenomegaly
 Jaundice,
 Anemia,
 Painful limbs
 Bone abnormalities
NATURE OF IMMUNE RESPONSE
INTACT SKIN & MUCOUS MEMBRANE- the primary
body defenses against treponemal invasion.
T cells (CD4+ & CD8+) and macrophages play a key role
in the immune response.
Macrophage phagocytosis - heals the primary chancre.
TROMPS- are important in triggering the activation of
complement, which ultimately kills the organism
NOTE! T pallidum is also capable of coating itself with
host proteins, which delays the immune system’s
recognition of the pathogen.
LABORATORY DIAGNOSIS
3 MAIN TYPES
1. DIRECT DETECTION
-requires that the patient have active lesions.
 DARK-FIELD MICROSCOPY
A darkfield condenser is used to keep all incidental light
out of the field except for that captured by the
organisms themselves. It is essential to have a good
specimen in the form of serous fluid from a lesion.
STERILE SALINE- used to clean the lesion when obtaining
serous fluid.
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TOPIC: SPIROCHETES
SUBJECT // FINALS
Prof. Nawirah Gaus
Specimens must be examined as quickly as possible
before they dry out. False-negative results can occur
when there is a delay in evaluating the slides, an
insufficient specimen, or pretreatment of the patient
with antibiotics.
 FLUORESCENT ANTIBODY TESTING
The use of a fluorescent-labeled antibody is a sensitive
and highly specific alternative to darkfield microscopy.
A. DIRECT METHOD
-uses a fluorescent-labeled antibody conjugate to T
pallidum.
B. INDIRECT METHOD
Uses antibody specific for T pallidum and a second
labeled anti-immunoglobulin antibody.
An advantage of these methods is that live specimens
are not required. A specimen can be brought to the
laboratory in a capillary tube and fixed slides can be
prepared for later viewing.
Treponemes can be washed off the slide even after
fixing; therefore, each slide must be handled individually
and rinsing must be carefully done.2 The use of
monoclonal antibodies has made fluorescent antibody
testing very sensitive and specific
If a patient does not have active lesions, as may be the
case in secondary or tertiary syphilis, then serological
testing for antibodies is the key to diagnosis.
2. SEROLOGICAL TESTS (NON-TREPONEMAL AND
TREPONEMAL)
Have traditionally been used to screen for syphilis
because of their high sensitivity and ease of
performance. However, false-positive results are
common because of the nonspecific nature of the
antigen. Therefore, any positive results must be
confirmed by a more specific treponemal test, which
detects antibodies to T pallidum.
August Paul von Wassermann (1906)- a bacteriologist
who developed the first nontreponemal serological test
for syphilis (WASSERMAN TEST based on COMPLEMENT
FIXATION).
WASSERMAN TEST -This test used a crude liver extract
from a fetus that was infected with syphilis as the source
of the lipid antigen. Patient serum was incubated with
cardiolipin antigen in the presence of rabbit serum as
the source of complement; this was followed by a
detection system consisting of antibodycoated sheep
red blood cells (RBCs). If the patient serum contained
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cardiolipin antibody, complexes were formed that
bound the reagent complement and the indicator RBCs
were not lysed.
In contrast, if cardiolipin antibody was not present in the
patient serum, the reagent complement was free to
react with the antibody-sensitized sheep RBCs to cause
hemolysis.
2.1 NON TREPONEMAL SEROLOGICAL TESTS
It determine the presence of an antibody that forms
against cardiolipin.
CARDIOLIPIN-, a lipid material released from damaged cells.
This antibody has sometimes been referred to as reagin. It is
found in the sera of patients with syphilis and several other
disease states.
An antigen complex consisting of cardiolipin, lecithin,
and cholesterol is used in the reaction to detect the
nontreponemal reagin antibodies, which are either of
the IgG or IgM class.
Venereal Disease Research Laboratory (VDRL) test and
the rapid plasma reagin (RPR) test- the most widely
used nontreponemal tests.These tests are based on
flocculation reactions in which patient antibody
complexes with the cardiolipin antigen.
Flocculation is a specific type of precipitation that
occurs over a narrow range of antigen concentrations.
PROZONE PHENOMENON- antibody excess leading to
false negative result. Granular or rough appearance is
typically seen in this case.If a prozone is suspected,serial
twofold dilutions of the patient’s sera should be made to
obtain a titer.
 VDRL test- is both a qualitative and quantitative
slide flocculation test for serum that includes a
modification for use on spinal fluid.Antigen for all
tests must be prepared fresh daily and in a highly
regulated fashion.
ANTIGEN : alcoholic solution of 0.03% cardiolipin, 0.9%
cholesterol, 0.21% lecithin.
-The antigen suspension is prepared by adding the VDRL
antigen with a dropper to a buffered saline solution
while continuously rotating the mixture on a flat surface.
Hamilton syringe is used to deliver one drop of antigen
for the slide test. If the delivery is off by more than 2
drops out of 60, the syringe must be cleaned with
alcohol and recalibrated.
SERUM SPECIMEN- heated at 56°C for 30 minutes to
inactivate complement, after which 0.05 mL is pipetted
into a ceramic ring of a glass slide.
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TOPIC: SPIROCHETES
SUBJECT // FINALS
Prof. Nawirah Gaus Transcribed by: QEEN

-Three control sera are pipetted into separate rings on NOTE! Both have been used to confirm positive
the glass slide in the same manner. Sera and patient nontreponemal test results since these are tests that are
samples are spread out to fill the entire ring. highly specific for syphilis.
-One drop (1/60 mL) of the VDRL antigen is then added
to each ring. The slide is rotated for 4 minutes on a  FTA-ABS test- is one of the earliest confirmatory
rotator at 180 rpm. tests.a dilution of heat-inactivated patient serum is
-It is read microscopically to determine the presence of incubated
flocculation, or small clumps.
-The results are recorded as reactive (medium to large with a sorbent consisting of an extract of nonpathogenic
clumps), weakly reactive (small clumps), or nonreactive treponemes (Reiter strain), which removes antibodies
(no clumps or slight roughness). that crossreact with treponemes other than T pallidum.
-Tests must be performed at room temperature within
the range of 23°C to 29°C (73°F to 85°F). -Diluted patient samples and controls are applied to
individual wells on a test slide fixed with the Nichols
 RPR TEST - is a modified VDRL test involving strain of T pallidum.
macroscopic agglutination. The cardiolipin-
-Following a 30-minute incubation at 37°C, the slides are
containing antigen suspension is bound to charcoal
washed and air-dried and antibody conjugate (anti-
particles; this makes the test easier to read. The
human immunoglobulin conjugated with fluorescein) is
suspension is contained in small glass vials, which
added to each well.
are stable for up to 3 months after opening.
Slides are re-incubated as before and washed to remove
ANTIGEN- is similar to the VDRL antigen with the
excess conjugate. Mounting medium is applied and
addition of the ff, which stabilize the antigen and
coverslips are placed on the slides. They are then
inactivate complement so that serum does not have to
examined under a fluorescence microscope.
be heat-inactivated before use.
-When slides are read under a fluorescence microscope,
 ethylenediaminetetraacetic acid (EDTA)
the intensity of the green color is reported on a scale of
 Thimerosal
0 to 4+. No fluorescence indicates a negative test,
 choline chloride.
whereas a result of 2+ or above is considered reactive.
-Patient serum (approximately 0.05 mL) is placed in an
18-mm circle on a plastic-coated disposable card using a
 PARTICLE AGGLUTINATION (PA) TESTS originally
capillary tube or Dispenstir device.
used sheep RBCs coated with T pallidum antigen
-Antigen is dispensed from a small plastic dispensing
and were referred to as MHA-TP
bottle with a calibrated 20-gauge needle. One free-
(microhemagglutination assay for T pallidum
falling drop is placed onto each test area and the card is
antibody).
mechanically rotated under humid conditions.
a) Serodia T pallidum particle agglutination (TP-PA)
-Cards are read under a high-intensity light source; if
test - one of the current PA tests for T.pallidum. It
flocculation is evident, the test is positive.
uses colored gelatin particles coated with
NOTE! All reactive tests should be confirmed by retesting
treponemal antigens and are more sensitive in
using doubling dilutions in a quantitative procedure. The
detecting primary syphilis.
RPR test appears to be more sensitive than the VDRL in
-Patient serum or plasma is diluted in microtiter plates
primary syphilis
and incubated with either T pallidum-sensitized gel
particles or unsensitized gel particles as a control.
2.2 TREPONEMAL SEROLOGICAL TESTS
-Presence of T pallidum antibodies is indicated by
It detects antibody directed against the T pallidum
agglutination of the sensitized gel particles, which form
organism or against specific treponemal antigens.
a latticelike structure that spreads to produce a smooth
Treponemal tests usually become positive before
mat covering the surface of the well.
nontreponemal tests, although patients with early
-If a sample is negative for the antibody, the gel particles
primary syphilis may be nonreactive
settle to the bottom of the well and form a compact
TWO MAIN TYPES OF MANUAL TREPONEMAL TESTS
button.
 FTA-ABS TEST
 AGGLUTINATION TEST

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TOPIC: SPIROCHETES
SUBJECT // FINALS
Prof. Nawirah Gaus
AUTOMATED IMMUNOASSAYS
( used for the detection of antibodies to T pallidum)
 ENZYME IMMUNOASSAYS (EIAS)
-been manufactured in a variety of formats.
i. two-step sandwich assays
-Antibodies in the patient sample bind to recombinant T
pallidum antigens coated onto microtiter plate wells.
An enzyme-labeled antibody or antigen conjugate and
substrate are added to detect binding. In the immune
capture format, microtiter wells are coated with
antibody to IgM or IgG and are reacted with patient
serum. Antigens that are labeled with an enzyme are
then added.
ii. one-step competitive assays
Competitive EIAs, treponemal antibody in the patient
sample competes with an enzyme-labeled treponemal
antibody conjugate for T pallidum antigens bound to
microtiter plate wells
iii. immune capture assays
Capture EIA tests are especially useful in diagnosing
congenital syphilis in infants because they look for the
presence of IgM, which cannot cross the placenta. They
can also be used in monitoring response to therapy in
the early stages of syphilis because many patients are
negative for IgM treponemal antibodies 6 to 12 months
after treatment.
 CHEMILUMINESCENT IMMUNOASSAYS (CLIA)
-Patient sample is incubated with paramagnetic
microparticles that have been coated with T pallidum
antigens linked to a chemiluminescent derivative.
-After a wash step to remove unbound material, a
catalyst is added and a chemical reaction occurs,
producing emissions of light if the test sample is positive.
-The number of relative light units (RLUs) is proportional
to the amount of treponemal antibody in the sample.
CLIAs have many advantages as compared with EIAs,
including a higher sensitivity in the early stages of
syphilis, faster performance, and more stable reagents
 MULTIPLEX FLOW IMMUNOASSAYS (MFI).
-MFI involves incubation of the patient sample with
microspheres coated with recombinant T pallidum
antigens.
-Microspheres that have bound immune complexes are
detected after addition of a phycoerythrin-labeled
reporter antibody and are analyzed by flow cytometry.
This method can simultaneously detect antibodies to
multiple T pallidum antigens in a small volume of sample
and has a rapid turn-around time.
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CLINICAL APPLICATIONS
♣ Nontreponemal tests are sensitive, inexpensive,
and simple to perform. Their main disadvantage is
that they are subject to false positives. Transient
false positives occur in diseases such as hepatitis,
infectious mononucleosis, varicella, herpes, measles,
malaria, and tuberculosis, as well as during
pregnancy.
Chronic conditions causing sustained false-positive
results include systemic lupus erythematosus (SLE),
leprosy, intravenous drug use, autoimmune arthritis,
advanced age, and advanced malignancy.
♣ Treponemal tests are more difficult to perform, and
have been traditionally used as confirmatory tests
to distinguish false-positive from true-positive
nontreponemal results. They also help establish a
diagnosis in late latent syphilis or late syphilis
because they are more sensitive than
nontreponemal tests in these stages.
TESTING ALGORITHMS
♥ Nontreponemal Test-Screening the sample
♥ Specific Treponemal Test- confirming any positive
results
NOTE! This testing strategy is recommended by the CDC.
A change in testing strategy for syphilis has been
proposed. (inaantok na me, basahin niyo nalang reason
why, sa page 377 ng Stevens book hehe, saranghae)
Here is the newer reverse sequence algorithm method:
♥ Automated treponemal immunoassay- patient
samples are screened by this.
♥ Nontreponemal test- positive results are confirmed
by this
This algorithm has several advantages over the
traditional algorithm:
1. Cost- automated testing can be performed on LIS-
interfaced high-throughput analyzers as opposed to
labor-intensive manual methods, saving time and
reducing errors.
2. Detects more early, late, treated syphilis cases-
higher sensitivity of the specific treponemaltests. In
the traditional algorithm, these may be missed
because testing stops with a negative
nontreponemal test result and the treponemal
specific test is not run.
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TOPIC: SPIROCHETES
SUBJECT // FINALS
Prof. Nawirah Gaus
MOLECULAR TESTING BY PCR
PCR (POLYMERASE CHAIN REACTION)
involves isolating and amplifying a specific sequence of
DNA, has been used to test for the presence of
treponemes in whole blood, spinal fluid, amniotic fluid,
various tissues, and swab samples from syphilis lesions.
Although there are many variations of this procedure,
DNA is basically extracted from the sample and then
replicated using a DNA polymerase enzyme and a primer
pair to start the reaction.
PCR may also be helpful in detecting treponemes in the
blood of neonates with symptoms of congenital syphilis
and in the CSF of patients suspected of having
neurosyphilis.
SPECIAL DIAGNOSTIC AREAS
CONGENITAL SYPHILIS
Cord blood or neonatal serum- where nontreponemal
test for congenital syphilis is performed in order to
detect the IgG class of antibody in addition to IgM.
FTA-ABS test for IgM alone lacks sensitivity
and the test is subject to interference because of the
presence of rheumatoid factor
IgM capture assay is more sensitive and a Western blot
assay using four major treponemal antigens has
demonstrated a high sensitivity and specificity.
The Western blot test is recommended to confirm
congenital syphilis
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CEREBROSPINAL FLUID (CSF)- CSF is typically tested to
determine whether treponemes have invaded the CNS.
Such testing is usually more reliable if CNS symptoms
are present.
The VDRL test and some of the newer ELISA tests- are
the only ones routinely used for the testing of spinal
fluid.
For VDRL spinal fluid testing, the antigen volume used is
less than the serum test and is at a different
concentration. In addition, different slides are used
(Boerner agglutination slides). The test is read
microscopically as in the VDRL serum test. If a test is
reactive, two-fold dilutions are made and retested
following the same protocol.
A positive VDRL test on spinal fluid is diagnostic of
neurosyphilis because false positives are extremely rare.
However, sensitivity is lacking because samples from
fewer than 70% of patients with active neurosyphilis
give positive results.
If a negative test is obtained, other indicators such as
increased lymphocyte count and elevated total protein
(45 mg/dL) are used as signs of active disease. PCR has
been advocated in diagnosing neurosyphilis and may
play an important role in CSF testing in the future.