SYPHILIS SEROLOGY-2.pptx

OUTLINE
• CASE PRESENTATION
• INTRODUCTION TO SYPHILIS
• MORPHOLOGY AND METABOLISM OF SYPHILIS
• STAGES OF SYPHILIS
• ANTIBODIES IN SYPHILIS
• SEROLOGICAL TESTS FOR SYPHILIS
• TREATMENT OF SYPHILIS
• REFERENCES
2

CASE PRESENTATION
• This is a case of a 27-year-old male from Makole who
presented to the outpatient department with a chief
complaint of an allergic reaction.
• The patient states that he was in his usual state of health
until three days ago when he began to develop a rash on
his stomach that is now on the palms of his hands and
soles of his feet. The rash is not painful or itchy.
• The patient states that he had a strange ‘’ulcer-like’’ thing
on his penis a couple of weeks ago but it went away and
never really hurt.
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• The patient is single and sexually active with two to three
concurrent female partners. He has had unprotected sex with
"at least one of his partners in the past couple of weeks. He
doesn't know the sexual histories of his current or past sexual
partners and admits to over 15-lifetime partners.
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PMHx
-Ibuprofen 200 mg PO PRN pain(has taken 4 doses in the last
day)
-The patient got the Hepatitis B vaccine about 6 months ago.
-He reports to have never tested for HIV.
Surgical Hx
-None
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Family-Social Hx
-Father had HTN and passed away from a stroke 4 years ago;
mother is still living and has type 2 DM
-Client has multiple sexual partners; (+) drinking alcohol, (-)
Tobacco,(+) Marijuana (occasional), (-) Illicit drugs
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On Examination
• awake, alert, slightly underweight man, Diffuse
mucocutaneous rash noted on the abdomen, back, upper
extremities (including palms of hands), and soles of feet.
VITALS SIGNS.
Temp 38.3 0C,
PR 72 bpm,
RR 16 breaths per minute,
BP 141/85 mm Hg,
SPO2 94% on room air
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Review of other systems.
Respiratory system.
-Clear auscultation with no wheezing or rhonchi
Genitourinary system
-Abdominal rash extends to the genital region and base of the
penis;
-noted a healing wound/ulcer on the dorsal aspect of the penis
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Cont..
Musculoskeletal system.
-No joint or muscle pain
-Extremities are well-perfused and warm to touch; noted rash as
described above
CNS
-Normal
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Gastrointestinal System
-No tenderness on palpation of the abdomen, Normal bowel
sounds were heard; noted rash as described above
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Impression: Secondary Syphilis
PLAN
-RPR
-TP-PA assay
-HIV test.(-ve)
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LABORATORY WORK UP
EQUIPMENT
-Sterile syringe
-Sterile gloves
-Sanitizer
-specimen collecting bottle without anticoagulant
SPECIMEN.
-Blood
-Blood was drawn from the patient aseptically and put into the specimen
bottle.
TRANSPORTATION
-The blood sample was immediately taken to the laboratory in a sample
transporting container for processing.
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Cont..
PROCESSING.
-The sample bottle was taken to the serology section for
processing.
-The sample bottle was put into the centrifuge machine and
centrifugation was done for about 5 minutes.
-After centrifugation, serum was obtained.
-A Rapid syphilis strip was dipped into the test tube with patient
serum and read after 15 minutes.
-There was appearance of a Test and Control line on the strip
showing the presence of syphilis.
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Cont..
• Another test like TP-PA or FTA-ABS should have been done to
confirm the presence of syphilis but due to lack of the test, it
was not done.
FEEDBACK
The patient was advised to go to a facility where both tests are
performed before getting medication.
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INTRODUCTION TO SYPHILIS
• Syphilis is a sexually transmitted infection caused by the bacterium
Treponema pallidum subspecies pallidum. The signs and symptoms of
syphilis vary depending on which of the four stages it presents
(primary, secondary, latent, and tertiary)
• Syphilis is caused by a spirochete bacterium, Treponema pallidum.
15

MORPHOLOGY AND METABOLISM OF
T. PALLIDUM
16
Treponema pallidum is a spirochete bacterium responsible for causing syphilis, a
sexually transmitted infection. Here are key aspects of the morphology and
metabolism of T. pallidum:
Morphology
1.Shape
• T. pallidum is a helically shaped, spiral, or corkscrew-like bacterium. It has a
distinctive morphology among bacteria.
2.Size:
• It is a relatively thin and long bacterium, with a diameter of about 0.2
micrometers and a length ranging from 6 to 20 micrometers.
3.Motility:
• T. pallidum is highly motile, and its spiral shape contributes to its corkscrew-
like movement. This motility is facilitated by axial filaments or endoflagella,
which run within the periplasmic space and rotate, allowing the bacterium to
move in a spiral manner.

Cont..
4. Cell Wall
– T. pallidum has a cell wall, but it lacks the typical
peptidoglycan layer found in many other bacteria. The
outer membrane is less complex than that of other Gram-
negative bacteria.
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Metabolism
1. Energy Metabolism
• T. pallidum is an obligate human pathogen, meaning it
cannot survive outside the host. It has limited metabolic
capabilities and relies on host-derived nutrients.
• The exact metabolic pathways of T. pallidum are poorly
understood due to the inability to culture the bacterium in
vitro. It lacks many conventional metabolic pathways
found in free-living bacteria.
2. Anaerobic Conditions
• T. pallidum is generally considered anaerobic, thriving in
environments with low oxygen levels. However, it can
survive in microaerophilic conditions as well.
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3. Nutrient Uptake
• T. pallidum obtains nutrients from the host,
particularly during the various stages of syphilis
infection. It is known to extract nutrients such as
glucose and other essential components from the host
tissue.
4. Sensitivity to Environmental Conditions
• T. pallidum is sensitive to changes in environmental
conditions, and its survival is optimized within the
host's specific temperature range and pH.
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Cont..
• Understanding the unique morphology and metabolic
characteristics of T. pallidum is crucial for developing effective
diagnostic methods and targeted treatments for syphilis.
• The inability to culture T. pallidum in the laboratory has posed
challenges in studying its metabolism and physiology directly,
and much of the information is inferred from genetic studies
and observations during infection.
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STAGES OF SYPHILIS
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• Syphilis is a sexually transmitted infection caused by the bacterium
Treponema pallidum.
• The infection progresses through several stages, each characterized
by distinct clinical manifestations.
• The stages of syphilis are traditionally categorized into :
i. Primary
ii. Secondary
iii. Latent
iv. Tertiary stages.

Cont..
i. Primary Syphilis
Symptoms: The initial stage begins with the appearance of a
painless sore or ulcer known as a chancre at the site of
infection, typically genital, anal, or oral. The chancre is highly
infectious.
Timing: The chancre usually appears 10 to 90 days (average
21 days) after exposure to the bacterium.
Healing: The chancre heals spontaneously within a few
weeks, even without treatment.
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ii. Secondary Syphilis
Symptoms: This stage is marked by a rash that may
appear on the palms of the hands and soles of the feet,
along with flu-like symptoms, swollen lymph nodes, and
mucous membrane lesions. The rash is highly
contagious.
Timing: Secondary syphilis typically occurs a few weeks
to several months after the appearance of the chancre.
Healing: Symptoms may resolve on their own, but
without treatment, the infection progresses to the
latent stage.
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iii. Latent Syphilis
Symptoms: In this stage, there are no apparent
symptoms, but the bacterium remains in the body.
Latent syphilis is further divided into early latent (within
the first year of infection) and late latent (after the first
year).
Timing: Early latent syphilis occurs within the first year
of infection, while late latent syphilis occurs after the
first year.
Healing: Without treatment, latent syphilis can progress
to tertiary syphilis.
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iv. Tertiary Syphilis
Symptoms: Tertiary syphilis can occur years after
the initial infection and is characterized by severe
complications affecting various organs, including
the heart, brain, and nerves. Gummas (soft, non-
cancerous growths) may form.
Complications: Neurosyphilis, cardiovascular
syphilis, and gummatous syphilis are examples of
complications that can occur in the tertiary stage.
Healing: Tertiary syphilis can be life-threatening
and requires prompt medical intervention.
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Cont..
• It's important to note that not everyone with syphilis
progresses through all stages, and the course of the infection
can vary among individuals.
• Early detection and treatment with antibiotics, typically
penicillin, are crucial to preventing the progression of syphilis
to later stages and reducing complications.
• Regular testing and safe sexual practices are essential for
preventing and managing syphilis infections.
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Cont..
• Congenital syphilis is a condition that occurs when a pregnant
woman with syphilis passes the infection to her baby during
pregnancy or at birth.
• Treponema pallidum, the bacterium causing syphilis, can cross the
placenta and infect the developing fetus.
• Congenital syphilis can result in serious health complications for the
newborn.
• The most common mode of transmission is through the
bloodstream from an infected mother to the fetus during
pregnancy.
• Transmission can also occur during childbirth as the baby passes
through the birth canal if the mother has active syphilis lesions.
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Cont..
• Congenital syphilis is divided into Early and late congenital
syphilis.
• Early Congenital Syphilis may be present at birth or within the
first few weeks of life.
• Manifestations can include rash, snuffles (nasal discharge),
hepatosplenomegaly (enlarged liver and spleen), and other
systemic signs.
• Late Congenital Syphilis If left untreated, can progress to later
stages, leading to dental abnormalities (Hutchinson's teeth),
bone abnormalities, interstitial keratitis (eye inflammation),
and neurological complications.
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Cont..
• Early detection of syphilis in pregnant women is crucial.
Routine prenatal care includes syphilis screening during the
first trimester and, if necessary, repeat testing during the third
trimester.
• Testing the newborn for syphilis is essential, especially if the
mother has a positive syphilis test or has not received
adequate treatment.
• Congenital syphilis is a preventable condition through timely
and effective prenatal care. It underscores the importance of
routine testing, early detection, and proper treatment of
syphilis in pregnant women to protect the health of both the
mother and the unborn child.
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ANTIBODIES IN SYPHILIS
• In syphilis, the immune system produces antibodies in
response to the presence of the bacterium Treponema
pallidum.
• Two main types of antibodies are commonly measured in
syphilis testing:
i. Non-treponemal antibodies
ii. Treponemal antibodies.
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Cont..
i. Non-Treponemal Antibodies
• Non-treponemal antibodies are antibodies produced in response to
lipoidal material released from damaged host cells during syphilis
infection.
• These antibodies are not specific to Treponema pallidum but are
used in screening tests for syphilis. These are cardiolipin.Cholesterol
and lecithin.
ii. Treponemal Antibodies
• Treponemal antibodies are antibodies that specifically target
components of Treponema pallidum.
• These antibodies are more specific to syphilis and are used in
confirmatory tests.These are anti-treponeme IgG, IgM and to a
lesser degree IgA.
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SEROLOGICAL TESTS FOR SYPHILIS
• Serological tests for syphilis are crucial for the diagnosis and
management of the infection.
• These tests detect the presence of antibodies produced by
the immune system in response to the bacterium Treponema
pallidum, which causes syphilis.
• The two main categories of serological tests for syphilis are
non-treponemal tests and treponemal tests.
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Cont..
i. Non-treponemal tests
• These are serological tests used for the screening and
monitoring of syphilis.
• Unlike treponemal tests, which detect antibodies specific to
the bacterium Treponema pallidum, non-treponemal tests
detect antibodies that react with components released from
damaged host cells during syphilis infection.
• These tests are considered nonspecific but are valuable for
initial screening and monitoring treatment response.
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Cont..
Common non-treponemal tests are:
1. VDRL (Venereal Disease Research Laboratory)
• VDRL is a blood test that detects antibodies produced in
response to syphilis.
• It does not directly detect antibodies specific to the
bacterium Treponema pallidum but instead detects
antibodies that react with cardiolipin, a substance released
from damaged host cells during syphilis infection.
• It measures the degree of flocculation (clumping) when
patient serum is mixed with an antigen containing
cardiolipin, cholesterol, and lecithin.
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Cont..
PRINCIPLE OF VDRL
• The test is based on the principle of detecting antibodies
produced by the immune system in response to infection
with the bacterium Treponema pallidum, the causative
agent of syphilis.
i. Antigen Used
• The VDRL test uses an antigen composed of a mixture
containing cardiolipin, cholesterol, and lecithin.
• Cardiolipin is a substance found in the cell membranes
of bacteria, including Treponema pallidum.
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ii. Formation of Complexes
• When patient serum is mixed with the VDRL antigen,
antibodies present in the patient's blood that react to
cardiolipin and other components will form complexes or
aggregates.
• These complexes cause flocculation, which is the clumping
of particles in the mixture.
iii. Observation of Flocculation
• The degree of flocculation is observed visually or through a
microscope.
• The presence and extent of clumping are indicative of the
presence of syphilis antibodies in the patient's serum.
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Cont..
iv. Quantitative Assessment:
• The VDRL test provides a quantitative result, expressed as
a titer, which indicates the dilution of the patient's serum
at which flocculation is still observed.
• The titer provides information about the concentration of
syphilis antibodies in the patient's blood.
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Cont..
PROCESS OF THE VDRL TEST.
1. Sample Collection
• A blood sample is collected from the patient through venipuncture.
2. Serum Separation
• The blood sample is centrifuged to separate the serum from other
blood components.
3. Antigen-Antibody Interaction
• The VDRL test uses an antigen suspension containing cardiolipin,
cholesterol, and lecithin. The patient's serum is mixed with this
antigen.
• If the patient has syphilis, antibodies (non-treponemal antibodies)
present in the serum will react with the cardiolipin and other
components in the antigen.
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Cont..
4. Formation of Complexes
• The interaction between the antibodies and the VDRL
antigen leads to the formation of complexes or
aggregates in the mixture.
5. Flocculation
• The complexes cause the particles in the mixture to
clump together, a phenomenon known as flocculation.
The degree of flocculation is observed visually or under a
microscope
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Cont..
6. Reading and Titer Determination
• The test results are read based on the degree of
flocculation. A positive result indicates the presence of
syphilis antibodies.
• The test provides a quantitative titer, which indicates the
highest dilution of the patient's serum that still shows
visible flocculation. The titer provides information about
the concentration of antibodies in the patient's blood.
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Interpretation
• A positive VDRL test result suggests exposure to syphilis or an
active infection. However, the test is nonspecific and can yield
false positives in certain conditions.
• Confirmatory testing using treponemal tests (e.g., FTA-ABS,
TP-PA) is necessary to confirm the diagnosis of syphilis.
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Cont..
Limitations
• Nonspecificity
• The VDRL test is nonspecific and can yield false-positive
results due to other conditions such as autoimmune
diseases such as lupus, certain infections such HIV, and
pregnancy.
• Stages of Infection
• The sensitivity of the VDRL test may vary in different stages
of syphilis. It is generally more sensitive in primary and
secondary syphilis.
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Cont..
• The VDRL test is an initial screening tool, and a positive result
should be followed by confirmatory testing using treponemal
tests (e.g., FTA-ABS, TP-PA) for a definitive diagnosis of
syphilis.
• It's crucial to interpret test results in the context of the
patient's clinical history and conduct further testing as
needed. Consultation with a healthcare professional is
essential for accurate diagnosis and appropriate management
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Cont..
2. RAPID PLASMA REAGIN (RPR)
• The Rapid Plasma Reagin (RPR) test is a non-treponemal
serological test used for the screening and diagnosis of
syphilis.
• Like the VDRL (Venereal Disease Research Laboratory)
test, the RPR test is a non-specific test that detects
antibodies produced in response to syphilis infection.
• It is a rapid and convenient screening tool that is
commonly used in clinical settings.
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PRINCIPLE OF RPR
i. Antigen-Antibody Interaction
• The RPR test uses an antigen suspension that contains
cardiolipin, cholesterol, and lecithin. These antigens are
similar to those used in the VDRL test.
ii. Formation of Complexes
• When patient's serum is mixed with the RPR antigen,
antibodies (non-treponemal antibodies) present in the
patient's blood react with the cardiolipin and other
components in the antigen.
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Cont..
iii. Agglutination reaction
• The interaction between antibodies and the RPR antigen leads to
the formation of visible clumps or aggregates, a process known as
agglutination. The degree of agglutination is macroscopically
observed.
iv. Quantitative Titer
• The RPR test provides a quantitative result known as a titer.
• The titer represents the highest dilution of the patient's serum
at which agglutination is still observed.
• It is expressed as a ratio (e.g., 1:8) and provides information
about the concentration of syphilis antibodies in the patient's
blood.
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Cont..
PROCESS OF THE RPR TEST
1. Sample Collection
• A blood sample is collected from the patient through
venipuncture.
2. Serum Separation
• The blood sample is centrifuged to separate the
serum (liquid portion of blood) from other blood
components.
3. Antigen-Antibody Interaction
• The RPR test uses an antigen suspension containing
cardiolipin, cholesterol, and lecithin. The patient's
serum is mixed with this antigen.
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Cont..
4. Agglutination Reaction
• If the patient has syphilis, antibodies (non-treponemal
antibodies) in their serum will react with the RPR antigen.
• This interaction leads to the formation of visible clumps or
aggregates, known as agglutination.
5. Macroscopic Observation
• The degree of agglutination is macroscopically observed.
• The test is often performed on a glass slide, and the
appearance of clumps is indicative of a positive reaction.
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Cont..
6. Quantitative Titer
• The test provides a quantitative result known as a titer.
The titer indicates the dilution of the patient's serum at
which agglutination is still observed.
• It is expressed as a ratio (e.g., 1:8), providing information
about the concentration of syphilis antibodies.
7. Result Interpretation
• A positive result indicates the presence of syphilis
antibodies in the patient's serum. The titer provides
additional information about the antibody concentration
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Clinical Use
1.Screening for Syphilis
• The RPR test is commonly used as an initial
screening tool for syphilis. A positive result
prompts further testing and evaluation.
2.Monitoring Treatment Response
• Serial RPR tests are performed during
syphilis treatment to monitor the effectiveness
of the treatment. A decreasing titer indicates a
positive response to treatment.
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Limitations
• Nonspecificity: The RPR test is not specific to syphilis and can
yield false-positive results in conditions other than syphilis.
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Cont..
ii. Treponemal Tests
• Treponemal tests are serological assays designed to detect
antibodies specifically directed against the bacterium
Treponema pallidum, the causative agent of syphilis.
• Unlike non-treponemal tests (such as VDRL and RPR), which
detect antibodies that react with cardiolipin and other
components, treponemal tests provide a more specific
confirmation of syphilis infection.
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Cont..
The common types of treponemal tests are:
i. Fluorescent Treponemal Antibody Absorption Test (FTA-
ABS)
ii. Treponema pallidum Particle Agglutination (TP-PA) Assay
iii. Treponemal Enzyme Immunoassay (EIA) or
Chemiluminescent Immunoassay (CIA)
iv. Line Immunoassay (LIA)
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I. FLUORESCENT TREPONEMAL ANTIBODY ABSORPTION TEST (FTA-ABS)
PRINCIPLE
• FTA-ABS is a microscopic test that uses a
microscope to observe the reaction of patient serum
with a preparation of T. pallidum that has been treated
with fluorescent dye. If antibodies against T. pallidum
are present in the patient's serum, they will bind to the
spirochetes, causing visible fluorescence.
Clinical Use
• FTA-ABS is used as a confirmatory test for syphilis. A
positive FTA-ABS result indicates the presence of
specific antibodies against T. pallidum.
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PROCESS OF FTA-ABS
1. Antigen Preparation
• Treponema pallidum antigens are prepared and fixed to
glass slides. The antigens can be obtained from cultured T.
pallidum organisms.
2. Patient Serum Incubation
• Patient serum, which may contain antibodies against T.
pallidum, is mixed with the prepared antigen-coated slides.
• The mixture is incubated to allow any specific antibodies
present in the patient's serum to bind to the T. pallidum
antigens on the slides.
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Cont..
3. Washing
• Unbound antibodies and other components are washed
away to remove non-specific reactions.
4. Secondary Antibody Incubation
• Fluorescent-labeled anti-human antibodies (secondary
antibodies) are added to the slide.
• These antibodies specifically bind to any antibodies that
have attached to the T. pallidum antigens on the slide.
5. Washing
• Excess secondary antibodies are washed away to reduce
background fluorescence.
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6. Fluorescence Observation
• The slide is examined under a fluorescent microscope. If
specific antibodies against T. pallidum are present in the
patient's serum, they will be bound to the T. pallidum
antigens on the slide, and fluorescence will be observed.
7. Interpretation
• The presence or absence of specific fluorescence is
interpreted. A positive result indicates the presence of
antibodies against T. pallidum.
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Clinical Considerations
• Confirmation of Syphilis
• The FTA-ABS test is used as a confirmatory test
for syphilis. It provides evidence of exposure to T.
pallidum.
• Persistent Positivity
• Treponemal antibodies, including those detected
by FTA-ABS, may persist for a long time even
after successful treatment.
• Therefore, FTA-ABS is not suitable for monitoring
treatment response.
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• Specificity
• FTA-ABS is specific for T. pallidum antibodies
and has higher specificity compared to non-
treponemal tests.
• Clinical Correlation
• Interpretation of FTA-ABS results should
consider the patient's clinical history,
symptoms, and risk factors
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Limitations
• False Positives
• While FTA-ABS is highly specific, false-
positive results can occur in certain
conditions, such as other treponemal
infections or autoimmune diseases.
• Use in Early Primary Syphilis
• FTA-ABS may not always be sensitive in early
primary syphilis, and other tests like darkfield
microscopy or PCR may be more appropriate.
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Cont..
• The FTA-ABS test is part of the algorithm for syphilis testing
and is often used as a confirmatory test following a positive
result in a non-treponemal test (e.g., VDRL or RPR).
• Interpretation of results and clinical decision-making should
be done by healthcare professionals based on the specific
context of the patient's presentation.
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Cont..
II. TREPONEMA PALLIDUM PARTICLE AGGLUTINATION (TP-
PA) ASSAY
Principle
• TP-PA is an agglutination test where particles coated
with T. pallidum antigens are mixed with patient
serum. If antibodies specific to T. pallidum are
present, they will cause agglutination of the particles.
Clinical Use
• TP-PA is used as a confirmatory test for syphilis. A
positive TP-PA result suggests the presence of
treponemal antibodies.
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Cont..
PROCESS OF TP-PA ASSAY
1. Antigen Preparation
• Treponema pallidum antigens are prepared and coated
onto particles, such as gelatin particles or red blood cells.
pallidum, is mixed with the prepared antigen-coated
particles.
• The mixture is incubated to allow any specific antibodies
present in the patient's serum to bind to the T. pallidum
antigens on the particles.
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Cont..
3. Agglutination Reaction
• If antibodies specific to T. pallidum are present in the
patient's serum, they will cause agglutination or clumping
of the antigen-coated particles.
4. Macroscopic or Microscopic Observation
• The degree of agglutination is observed either
macroscopically or microscopically. Positive agglutination is
indicative of the presence of antibodies against T.
pallidum.
5. Interpretation
• The presence or absence of agglutination is interpreted. A
positive result indicates the presence of antibodies against
T. pallidum.
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Cont..
• Confirmation of Syphilis
• The TP-PA assay is used as a confirmatory test for syphilis.
It provides evidence of exposure to T. pallidum.
• Specificity
• TP-PA is specific for T. pallidum antibodies and has higher
specificity compared to non-treponemal tests.
• Use in Confirmatory Testing
• TP-PA is often used in conjunction with other treponemal
tests, such as FTA-ABS or EIA, to confirm the presence of T.
pallidum antibodies.
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Cont..
Limitations
• False Positives
• While TP-PA is highly specific, false-positive results can
occur in certain conditions, such as other treponemal
• TP-PA may not always be sensitive in early primary syphilis,
and other tests like darkfield microscopy or PCR may be
more appropriate.
• Confirmation Algorithm
• TP-PA is typically used as part of the algorithm for syphilis
testing, and results should be interpreted in conjunction
with the patient's clinical history and other test results.
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Cont..
• The TP-PA assay is valuable for confirming the presence of
antibodies against T. pallidum, and its specificity makes it
useful in the diagnosis of syphilis.
• Healthcare professionals interpret the results based on the
specific context of the patient's presentation, and
confirmatory testing is an important component of the
syphilis diagnostic algorithm.
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Cont..
iii. Treponemal Enzyme Immunoassay (EIA) or
Chemiluminescent Immunoassay (CIA)
Principle
• These tests use enzyme or chemiluminescent labels to
detect antibodies against T. pallidum. The patient's serum
is incubated with a solid phase coated with T. pallidum
antigens. If specific antibodies are present, a reaction
occurs, and the labeled antibodies generate a measurable
signal.
Clinical Use
• Treponemal EIAs or CIAs are commonly used for syphilis
screening and confirmation. They are automated and
provide quantitative results
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Cont..
PROCESS OF TREPONEMAL EIA/CIA
1. Coating Solid Phase
• A solid phase, such as a microplate or membrane, is coated
with Treponema pallidum antigens. These antigens may
include various proteins from the T. pallidum bacterium.
pallidum, is added to the coated solid phase.
• The serum is allowed to interact with the T. pallidum
antigens, facilitating the binding of specific antibodies to
the antigens.
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3. Washing
• Unbound components, including non-specific antibodies,
are washed away to reduce background interference.
• Enzyme-labeled or chemiluminescent-labeled secondary
antibodies are added to the solid phase.
• These secondary antibodies specifically bind to antibodies
present in the patient's serum that have attached to the T.
pallidum antigens.
5. Washing
background signals and enhance specificity.
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6. Enzyme Reaction or Chemiluminescent Reaction
• For EIA, an enzyme substrate is added. The enzyme-
catalyzed reaction produces a color change, and the
intensity of the color is proportional to the amount of
bound antibodies. For CIA, the chemiluminescent-
labeled antibodies emit light during a reaction.
7. Detection
• The developed color or emitted light is measured
using a spectrophotometer (for EIA) or a luminometer
(for CIA). The results are quantified and compared to a
calibration curve.
71

Cont..
8. Interpretation
• The presence or absence of antibodies against T. pallidum
is determined based on the measured signal. A positive
result indicates the presence of specific antibodies.
72

Cont..
• Screening and Confirmation
• Treponemal EIA or CIA is commonly used for both
screening and confirmation of syphilis.
• These tests are part of the two-step testing algorithm,
often following a non-treponemal test (e.g., VDRL, RPR).
• Quantitative Results
• EIA or CIA provides quantitative results, allowing for the
assessment of the antibody concentration in the patient's
serum.
• Specificity
• These tests are specific for T. pallidum antibodies and have
higher specificity compared to non-treponemal tests.
73

Cont..
Limitations
• False Positives
• While Treponemal EIA or CIA is highly specific, false-
positive results can occur in certain conditions, such as
other treponemal infections or autoimmune diseases.
• These tests may not always be sensitive in early primary
syphilis, and other tests like darkfield microscopy or PCR
may be more appropriate.
• Results should be interpreted as part of the algorithm for
syphilis testing, and clinical correlation is essential.
74

Cont..
• Treponemal EIA or CIA is a valuable tool in the diagnosis of
syphilis, providing sensitive and specific results.
• Interpretation of results should be done by healthcare
professionals based on the patient's clinical context, history,
and other test results. Confirmatory testing is an important
component of the syphilis diagnostic algorithm.
75

Cont..
iv. Line Immunoassay (LIA)
Principle
• Similar to Western blot, LIA uses a strip containing
separated T. pallidum proteins. Patient serum is
applied, and if specific antibodies are present, they
bind to the corresponding proteins on the strip.
Clinical Use
• Line immunoassays are used for confirmation of
syphilis, providing information about the specific
proteins targeted by the antibodies.
76

Cont..
PROCESS OF LINE IMMUNOASSAY (LIA)
1. Antigen Separation
• Treponema pallidum antigens, including various proteins from
the bacterium, are separated using techniques such as
electrophoresis or chromatography.
2. Coating the Strip
• The separated T. pallidum antigens are coated onto a solid
support, typically a nitrocellulose or nylon membrane, as lines
or bands.
pallidum, is applied to the strip. The serum is allowed to interact
with the separated T. pallidum antigens on the membrane.
77

Cont..
4. Binding of Antibodies
• If antibodies specific to T. pallidum are present in the
patient's serum, they will bind to the corresponding
antigens on the membrane.
5. Washing
• Unbound components, including non-specific antibodies,
are washed away to reduce background interference.
• Enzyme-labeled or chemiluminescent-labeled secondary
antibodies are applied to the strip.
• These secondary antibodies specifically bind to antibodies
present in the patient's serum that have attached to the T.
pallidum antigens.
78

Cont..
7. Washing
background signals and enhance specificity.
8. Enzyme Reaction or Chemiluminescent Reaction
• For LIA, an enzyme substrate is applied to the strip. The
enzyme-catalyzed reaction produces a visible color change
at the bands corresponding to the T. pallidum antigens. For
chemiluminescent versions, emitted light is detected.
9. Detection
• The developed color or emitted light is visually observed or
measured using a suitable instrument.
• The presence or absence of bands indicates the presence
of specific antibodies against T. pallidum
79

Cont..
10. Interpretation
• The pattern of bands and their intensity is interpreted. A
positive result indicates the presence of specific antibodies
against T. pallidum
80

Cont..
• Screening and Confirmation
• Line Immunoassay (LIA) is commonly used for both
screening and confirmation of syphilis. It is often part
of the two-step testing algorithm following a non-
treponemal test (e.g., VDRL, RPR).
• Visual Interpretation
• LIA results are often interpreted visually, with the
presence or absence of bands indicating the
presence of specific antibodies.
• Specificity
• LIA is specific for T. pallidum antibodies and has
higher specificity compared to non-treponemal tests.
81

Cont..
Limitations
• False Positives
• While LIA is highly specific, false-positive results can
occur in certain conditions, such as other treponemal
• LIA may not always be sensitive in early primary
syphilis, and other tests like darkfield microscopy or
PCR may be more appropriate.
• Results should be interpreted as part of the algorithm
for syphilis testing, and clinical correlation is essential.
82

Cont..
• Line Immunoassay is a valuable tool in the diagnosis of
syphilis, providing sensitive and specific results.
• Interpretation of results should be done by healthcare
professionals based on the patient's clinical context, history,
and other test results. Confirmatory testing is an important
component of the syphilis diagnostic algorithm.
83

TREATMENT OF SYPHILIS
• The treatment of syphilis typically involves the use of antibiotics.
• The choice of antibiotic and duration of treatment depend on the stage of
syphilis and individual factors such as allergies or other medical
conditions.
The most common antibiotics used to treat syphilis include:
1. Penicillin G: This is the preferred and most effective treatment for syphilis.
The type and dosage of penicillin may vary based on the stage of syphilis.
Intramuscular injection of long-acting penicillin is often used.
2. Doxycycline or Tetracycline: In cases where penicillin is not suitable due to
allergies or other reasons, doxycycline or tetracycline can be used.
However, these antibiotics are generally not recommended for pregnant
women or children.
84

Cont..
• It's crucial to follow the prescribed treatment plan and
complete the full course of antibiotics as directed by a
healthcare professional. Regular follow-up testing is also
necessary to ensure the infection has been adequately
treated.
The stages of syphilis and their corresponding treatments
are as follows:
i. Primary and Secondary Syphilis: A single dose of
penicillin G is typically sufficient. In some cases, multiple
doses may be required, especially if the infection has been
present for an extended period.
85

Cont..
ii. Early Latent Syphilis: Penicillin G is administered in multiple
doses over a span of one to three weeks.
iii. Late Latent and Tertiary Syphilis: Penicillin G is given in
weekly doses for three weeks.
iv. Neurosyphilis: Intravenous penicillin G is usually required,
and the duration of treatment may be longer.
86

Cont..
• It's important to note that prevention, regular testing, and
early detection of syphilis are essential in managing the
infection.
• Safe sexual practices, including the use of condoms, can help
prevent the spread of syphilis and other sexually transmitted
infections.
• If you suspect you have syphilis or have been exposed to it, it
is crucial to seek prompt medical attention for diagnosis and
appropriate treatment.
87

REFERRENCES
1. Bailey & Scott’s Diagnostic Microbiology (14 edition) by Tille P
(2017).
2. Geo F.B, Janet S.B., and Stephen A.M. (2019). Jawetz, Melnick and
Adelberg’s Review of Medical Microbiology.28TH EDITION. Publ. Lange
Medical Publications, New York.
3. Subhash Chandra Parija.(2012). Textbook of Microbiology and
Immunology. 2ND Edition.
4. Connie R. Mahon, Donald C. Lehman, George Manuselis (2015)
Textbook of diagnostic microbiology.—Fifth edition
5. Sherris and Ryan’s Medical Microbiology, 8TH Edition (2022) by
Kenneth J Ryan.
6. Brock Biology of Microorganisms, 14Th Edition (2015) by Michael T
Madigan, John M Martinko, Kelly S Bender, Daniel H Buckley, and David
A Stahl
88

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