The Rapid Plasma Reagin Test
and the Venereal Disease
Research Laboratory Assay
(VDRL)
Serological Diagnostic Screening
Tests for Syphilis
Syphilis: Causative Agent and
Disease Description



Treponema pallidum - the causative agent for the
disease known as syphilis.
Order: Spirochaetales Family: Treponemataceae.
Genus: Treponema, a slender, tightly coiled,
pointed-ended, 6-10 axial filamentous gram
negative rod.
Causative Agent

T. pallidum- a microaerophilic, obligate parasite
that requires mammalian cells for survival.
Covered in a sheath, aids this organism in it’s
pathogenicity by reducing or limiting antigenic
stimulation of the host’s immune system.

Members of this genus contain of a number of
species that can exist as either commensal flora or
human pathogens.
Treponema Speciation

Nonpathogenic-exists as commensal flora of the
oral cavity, gastrointestinal and urogenital tract.
Are also considered to be strict anaerobes.

Pathogenic-transmitted by person-to-person
contact. The microaerophilic subspecies of
pallidum are associated with various TreponemaRelated Diseases in humans
Treponema pallidum sub speciation

T. pallidum subspecies
pallidum - syphilis

T. pallidum subspecies
pertenue - yaws

T. pallidum subspecies
endemicum - non-venereal
(endemic) syphilis/bejel

T. carateum - pinta
Species- indistinguishable
serologically and
morphologically.
Differentiation- based on
clinical & epidemiological
characteristics.
Disease State


Untreated, syphilis is a chronic infectious disease
with many clinical manifestations that occur in
distinct stages consisting of subacute symptomatic
periods separated by asymptomatic intervals.
Initially, T. pallidum penetrates intact mucous
membranes or enter the body through tiny defects
in the epithelium. Upon entrance, the
microorganism is carried by the circulatory system
to every organ of the body.
Clinical Manifestation


The progression of untreated syphilis is generally
divided into into four stages: Primary, Secondary,
Latent and Tertiary (Late) Syphilis, with Tertiary
Syphilis being further divided into Neurosyphilis
and Congenital Syphilis.
Spirochetemia occurs very early in infections
before the appearance of the first lesion or reactive
blood test described as clinical or serological
detection. In this state, syphilis appears to be in an
incubating state that can range from 10 to 90 days.
Primary Syphilis

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Small, solitary nodule at the point of initial
inoculation.
Primary inflammatory lesion enlarges and
ulcerates developing into a characteristic painless
ulcer or chancre that heals spontaneously.
Chancre are located on vaginal mucosa or cervix
of the female and external genitalia of the male.
Secondary Syphilis


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Systemic: within 2-8 weeks of visible chancre.
Generalized illness being with viral-like
symptoms: headache, sore throat, low-grade fever
and occasionally a nasal discharge.
Generalized rash of skin and mucous membranes.
Secondary Syphilis
Development of lymphadenopathy and
lesions of the skin and mucous membranes.
 Lesions contain a large number of
spirochetes and are highly contagious when
exposed on the skin surface.
 Macular lesion and/or condylomata lata-flat
lesions resembling warts in moist areas of
the body (around the anus or vagina).

Latent Syphilis
Latency- exhibit no clinical symptoms, a
noninfectious state which diagnosis can be
made only by serologic methods.
 During the first 2-4 years, patient may
exhibit one or more mucocutaneous relapses
of secondary syphilis manifestation.
 During these relapses, patient is highly
infectious and may transmit the disease.

Tertiary Syphilis


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Characterized by the presence of destructive
granulomas that produce lesions resembling
segments of circles that heal with superficial
scarring.
Exhibited 3-10 years after primary infection.
Skeletal system involvement
Cardiovascular system-involves the aortic
endothelium, weakening the blood vessels of the
aortic arch known as syphilitic aneurysm.
Other Clinical Manifestations
of Tertiary Syphilis

Neurosyphilis: Asymptomatic involvement of the
CNS and detected only by examination of
cerebrospinal fluid (CSF).

Congenital Syphilis: Caused by maternal
spirochetemia and transplacental transmission of
microorganism.
Neurosyphilis

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Acute syphilitic meningitis
Meningovascular syphilis manifested as a seizure.
Cerebrovascular accident - stroke
General paresis, personality changes, dementia
and delusional states.
Tabes dorsalis- involvement of the posterior
column and dorsal root, characterized by broadbased gait with impotence and bladder
dysfunction.
Congenital Syphilis

Hutchinsonian triad: Hutchinson’s teeth,
interstitial keratitis, and nerve deafness.

Other characteristics- fissuring around the
mouth and anus, skeletal lesions,
perforation of the plate and collapse of nasal
bones to produce a saddle-nose deformity.
Immunological Manifestations
Specific antibodies- produced against T. pallidum
are IgM and IgG antibodies.
 Detected by Treponemal serological procedures
Nonspecific antibodies-produced against the protein
antigen group common to pathogenic spirochetes
formed and are known as nontreponemal
antibodies.
 Detected by nontreponemal serological procedures
Diagnostic Evaluation




The diagnosis of syphilis is based on the clinical picture in
conjunction with demonstration of microorganisms in a
lesion and serologic testing.
Darkfield Microscopy
Nontreponemal Methods- Venereal Disease Research
Laboratory (VDRL) and the Rapid Plasma Reagin (RPR)
procedures are used to screen patient’s serum.
Treponemal Methods-Fluorescent Treponema pallidumabsorbed (FTA-ABS) and the microhemagglutination
Treponema pallidum [MHA-TP]are used for confirmation.
Clinical Limitations Associated with
Serological Testing


Infectious disease - measles, chicken pox,
hepatitis, infectious mononucleosis, leptospirosis,
malaria, leprosy, rickettsial disease,
trypanosomiasis, lymphogranuloma venereum
(LGV)
Noninfectious conditions - autoimmune
disorders, drug addiction, old age, pregnancy and
recent immunization
RPR test
The RPR test is a nontreponemal
testing procedure for the serologic
detection of syphilis.
Principle of RPR



The RPR Card antigen suspension is a carbon
particle cardiolipin antigen that detects reagin.
Reagin is an antibody like substance present in
serum or plasma from individuals with syphilis.
The reagin binds to the test antigen which consists
of cardiolipin-lecithin coated particles that cause
macroscopic flocculation.
Principle of RPR
When a specimen such as serum or plasma
contains antibody, flocculation occurs with
the resulting aggregation of the carbon
particles.
 The flocculation appears as black clumps
against the white background of the plastic
coated card.

Principle of RPR

Antibodies associated with syphilis begin to
appear in the blood 4 to 6 weeks after
infection. Nontreponemal tests determine
the presence of reagin. Reagin is a
nontreponemal autoantibody directed
against cardiolipin antigens.
Materials for RPR
RPR Test Cards
 RPR Control Cards
 RPR Antigen
 Distilled Water
 Dispenstirs
 Rotator

RPR Test Background
The RPR test uses a white plastic coated
card that consist of several circles that are
18 mm in diameter.
 The controls which are strongly reactive,
moderately reactive, and non-reactive are
contained on the control card in a dried
form.

Procedure for Controls

A. Use Dispenstir to draw up distilled water
B. Drop 1 drop on the card test circle for each patient
sample.
C. Invert Dipenstir and spread the water in the circle until
the dried control is completely reconstituted.
D. Add antigen as described for the patients

E. Rotate for 8 minutes at 100 rpm

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Reactions for Controls
The following reactions should be observed to
compare against the test results:
 Reactive control - characteristic strong clumping.
 Reactive moderate control - moderate clumping.
 Non-reactive control - smooth, grayish
appearance of unclumped particles
Specimen Collection
Specimen Collection

*The addition of
choline chloride,
which inactivates
complement enables
the serum to be
tested without prior
heating.

Unheated serumcentrifuge for
sedimentation of cellular
elements, serum may be
frozen until time of
testing.
Heated Serum- transfer
serum to clean tube and
place in 56°C water bath
for 30 minutes
Specimen Collection
Specimen Collection

Unheated Plasma specimen should be
collected with an
anticoagulant such as
EDTA or heparin,
plasma must be stored
at 2°C to 8°C. Plasma
must be tested within
in 24 hrs of collection.
Procedure for Test
Label rings on test card with numbers of
samples to be tested
 Use Dispenstir to draw up serum sample.
 Hold Dispenstir in a perpendicular position
directly over the test circle to which the
specimen is to be delivered.
 Squeeze Dispenstir to allow 1 drop to fall
on to each circle

Procedure for Test
Invert Dispenstir,and using the sealed end
spread the specimen in the confines of the
circle.
 Reconstitute the antigen bottle, by shaking.
Holding the bottle in a straight vertical
position drop one or two drops in the upper
corner of each test circle, then place one
“free falling” drop on each test area.

Procedure for Test
Rotate card for 8 minutes on a mechanical
rotator at 100 rpm. The test card she also be
covered with a humidifier cover.
 After rotating mechanically, the test card
should be rotated manually by hand 3 to
four rotations and then read immediately
macroscopically in the “wet” state under a
high intensity lamp.

Results of Test
The test results should be reported as
reactive (even if minimally reactive) or nonreactive.
 All test results that are nonreactive should
be quantitated.

Explanation of Results
A negative RPR test may indicate one of the
following:
1. The patient does not have syphilis.
2. The infection is too recent for antibodies to be
produced. (Repeated tests should be administered
at 1 week, 1 month, and 3 month intervals to
establish presence or absence of disease).
3. The syphilis is latent or inactive
4. Faulty immunodefense mechanism
5. Faulty lab techniques

Explanation of Results
A positive reaction is not conclusive for
syphilis. Several conditions produce
biologic false positive results for syphilis.
(False positive means that the test revealed
a positive reaction when it was actually
negative).
 False positives may reveal the presence of
other serious diseases.

Non-syphilitic Conditions Giving
Biologic False-Positive Results
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Malaria
Leprosy
Relapsing fever
Infectious
Mononucleosis
Atypical pneumonia

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Viral pneumonia
Lupus erythematosus
Measles
pregnancy
drug abuse
Resolving False Positive RPR
Tests

False positive RPR tests may be resolved by
testing the patient’s serum with a specific
treponemal antigen test.
Explanation of Results
Diagnosis of syphilis requires correlation of
geographical area or country, patient
history, physical findings, and results of
syphilis antibody tests.
 T. pallidum is diagnosed when both the
screening and confirmatory test are reactive.

Explanation of Results

Treponemal tests are more specific than
non-treponemal tests

Treponemal tests confirm syphilis when a
reactive or positive non-treponemal result is
obtained.
Confirmatory Tests for Syphilis
I. FTA-ABS
flourescent
treponemal antibody test
 II. TP-PA
particle aggluntination T. pallidum test
 III. MHA-TP
Microhemagglutination assay - T.
pallidum

Confirmatory Tests for Syphilis

Treponemal tests are used to confirm
reactive non-treponemal procedures.

A positive FTA-ABS test almost always
remains positive and therefore is not
recommended for monitoring therapy.
Theory of Antigen-Antibody
Reaction
It is theorized that reagin is an antibody
against tissue lipids.
 Lipids are presumed to be liberated or free
from body tissue in the normal course of
activity.
 As a result of being free in the body, these
lipids may induce antibody formation.

Interfering Factors
Alcohol decreases reaction intensity in tests
and therefore should be avoided for a least
24 hrs before blood is drawn.
 Avoid drawing the blood sample
immediately after the patient has eaten.

Clinical Information
Sexual partners of patients with syphilis
should be evaluated for the disease.
 After treatment, patients with early stage
syphilis should be tested at 3 month
intervals for one year to monitor declining
reactivity of the syphilis.

The VDRL Procedure
Background

The Venereal Disease Research Laboratory
(VDRL) test is one of two variations of
flocculation procedures used for serological
testing of syphilis, the other being the Rapid
Plasma Reagin (RPR). Flocculation testing is
based on antibody detection with the interaction of
soluble antigen with an antibody that results in a
precipitate formation of fine particles.
Background

The VDRL is a confirmatory serological
microflocculation slide test used for the detection
of syphilis antibodies. In a VDRL procedure, the
patient’s serum is heat-inactivated and mixed with
a buffered saline suspension of VDRL Antigen
containing cardiolipin, lecithin and cholesterol that
binds with Reagin, an antibody-like protein. A
combination of Reagin and VDRL Antigen form
microscopic clumping called flocculation.
Background


The VDRL can be used for qualitative and
quantitative measurements and is recommended
when a patient suspected of having syphilis has a
negative dark field microscopy result or when
atypical lesions are present.
Syphilis is a venereally transmitted disease that
has a serious and potentially life threatening
sequelae if untreated.
VDRL Serological Procedure
Principles

VDRL Antigen is a nontreponemal antigen
composed of cardiolipin cholesterol and lecithin.
The nontreponemal tests measures anti-lipid
antibodies, which are formed by the host in
response to lipids released from damaged host
cells early in infection with T. pallidum, and lipidlike material form the treponemal cell surface.
During syphilis infection, an antibody-like
substance called reagin can be detected in the
patient’s serum or CSF.
Procedural Principles

Reactive nontreponemal tests confirm the
diagnosis in the presence of early or late lesion
syphilis and offer a clue in latent subclinical
syphilis. It is also an effective tool for detecting
cases in epidemiologic investigations and are
superior to the treponemal test for following the
response to therapy.
Procedural Principles

Nontreponemal antigen tests are not entirely
specific for syphilis and do not have satisfactory
sensitivity in all stages of syphilis. Whenever the
results of a nontreponemal antigen test disagree
with the clinical impression, a treponemal antigen
test such as the FTA-ABS should be performed.
Quality Assurance

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

Prepare a fresh antigen suspension each testing day. Once
prepared, it should be used within 8 hours.
Store prepared suspension at 23-29)C.
Test antigen suspension reactivity with control sera
(Reactive, Weakly reactive and Nonreactive). Test serum
dilutions within 1 hour after heat inactivation.
Use antigen suspension only if it produces the expected
reactivity with the control sera comparable to results
obtained with the reference antigen.
Required Materials


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VDRL Antigen with buffered saline solution containing
1% sodium chloride, pH 6.0+/-0.1 with 0.05%
formaldehyde preservative
Reactive, weakly reactive and nonreactive serum
0.9% saline, non-disposable 1cc glass syringe and
calibrated needles without bevel-18 gauge(serum) or 21-22
gauge(CSF), slide cards(serum) or concavity slides(CSF)
Stirrers
Rotator
Specimen Collection and
Preparation for Serum


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Collect 5-8 ml of blood by aseptic venipuncture in a red
top tube.
Allow blood to clot at room temperature then centrifuge to
obtain serum.
Heat the test sera at 560C for 30 minutes.
Specimen must be at 23-290C when tested.
Specimen must be clear of hemolysis and show no visible
evidence of bacteria contamination.
Store at room temperature for 4 hours, after which store at
2-80C, maybe refrigerated up to 5 days, then frozen at <200C.
Specimen Collection and
Preparation for CSF
Centrifuge and decant the specimen
 Specimens do not require heat inactivation
before testing.
 Spinal fluids that are visibly contaminated
or that contain gross blood are
unsatisfactory

Antigen Suspension Preparation


Pipette 0.4ml of VDRL buffered saline to the bottom of a
round 30 ml glass stoppered bottle with a flat inner-bottom
surface. Gently tilt bottle so that VDRL buffered saline
will cover the entire inner-bottom surface of the bottle.
Add 0.5 ml of VDRL Antigen directly into the saline while
continuously but gently rotating the bottle on a flat surface
from the lower half of a 1.0 ml pipette graduated cylinder
to the tip. Add antigen drop by drop at a rate that allows
about 6 sec for 0.5 ml of antigen. Keep pipette tip in the
upper third of the bottle and do not splash saline unto the
pipette.
Antigen Suspension Preparation




Expel the last drop of antigen without touching pipette to
the saline and continue rotation of the bottle for 10 sec.
Add 4.1 ml of buffered saline from a 5 ml pipette. Do not
drop saline directly on antigen; allow it to flow down the
side of the bottle.
Cap the bottle and mix by gentle inversion. Allow to stand
for 5 minutes but no more than 2 hours. The suspension is
ready for use.
Remix suspension by swirling only
Antigen Suspension
Mix by gentle
inversion. Allow
to stand at least 5
minutes but no
more than 2 hours.
Procedure: Step 1
Wells should be
labeled R, WR,
NR, and PS for
reactive, weakly
reactive, and
nonreactive,
respectively.
Procedure: Step 2
Pipette .05ml of specimen into one concavity
of an agglutination slide.
Procedure: Step 3
Add one drop
(.01 ml) of
sensitized
antigen
suspension to
each specimen
with a 21 or
22 gauge
needle.
Procedure: Step 4

Rotate slides for 8
minutes on a
mechanical rotator
at 180 rpm. Note:
when the tests are
performed in a dry
climate, the slides
may be covered
with a box lid to
prevent
evaporation.
Step 5a for Positive CSF Samples
Quantitative tests are run
on all spinal fluids
found to be reactive in
the qualitative test.
Prepare fluid as
follows:
A. Pipette 0.2 ml of
0.9% saline into each
of 5 or more tubes.
Step 5b for Positive CSF
Samples
Add 0.2ml
of unheated
spinal fluid to
tube 1, mix
well and
transfer 0.2
ml to tube 2 .
Step 5c for Positive CSF Samples
Continue mixing
and transferring 0.2
ml from one tube to
the next until the
last tube is reached.
The respective
dilutions are 1:2,
1:4, 1:8, 1:16. Etc.,
Step 5: for Positive CSF Samples
2. Test each spinal fluid dilution and undiluted
spinal fluid as described under “VDRL slide
qualitative on spinal fluid.”
3. Report results in terms of the greatest spinal
fluid dilution (dils) that produces a reactive
result.
Reading Slide Results
Results for Serum Specimen



Qualitative Testing - Medium to large clumps
(Reactive); Small clumps (Weakly Reactive); No
clumping or very slight roughness (Nonreactive).
Verify control sera results for expectation. If reactions are
not as expected, the test is invalid and results can not be
reported.
Perform a quantitative test to endpoint on all serum
samples that produce reactive, weakly reactive or “rough”
nonreactive results in the qualitative slide test.
Results for Serum Specimen

Quantitative Testing - Report the titer as the
highest dilution that produces a Reactive (not
weakly reactive) results
Results for CSF Specimen

Qualitative Testing - No clumping or very slight
roughness (Nonreactive); Definite clumps (Reactive)
A quantitative test should be performed on any reactive
specimen.

Quantitative Testing - Report the titer in terms of the
highest dilution that produces a reactive (not weakly
reactive) result.
Interpretation

Results of the serum VDRL Test must be confirmed by a
treponemal test.

Diagnosis depends upon results of VDRL, treponemal
confirmatory test, clinical signs and symptoms and risk
factor.

Reactive VDRL - may indicate past or present infection
with microorganism or false positive. A false positive is
determined if the confirmatory treponemal test is negative
Interpretation



Nonreactive VDRL - with clinical evidence may indicate
early primary syphilis, a prozone reaction in secondary or
late syphilis.
Nonreactive VDRL - with no clinical evidence may
indicate no current infection or an effectively treated
infection.
Quantitative VDRL - detects changes in reagin titer.
Serum samples displaying a fourfold increase in titer on a
repeated sample may indicate an infection, reinfection or
treatment failure. A fourfold decrease during treatment
indicates adequate therapy.
Sources of Error



False positive reactions - occur in 10% to 30% of
positive serological tests for syphilis and consist
of nonsyphilitic positive VDRL. reactions with
cardiolipin type antigens.
False negative reactions - consist of conditions
and a variety of situations.
Weakly reactive - caused by very early infection,
lessening of the activity of the disease after
treatment and improper technique or questionable
reagents.
False Positive Reactions

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Lupus erythematosus
Rheumatic fever
Vaccinia and virus
pneumonia
Pneumococcal
pneumonia
Infectious
mononucleosis

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Infectious hepatitis
Leprosy
Malaria
Rheumatoid arthritis
Pregnancy
Aging individuals
False Negative Reactions
Technical error - unsatisfactory antigen or
technique.
 Low antibody titers
 Presence of inhibitors in the patient’s serum
 Reduced ambient temperature (below 230 to
290)
 Prozone reaction

References
Wasley G.D. (1988). Syphilis serology.
Oxford press, New York.
 Abbot laboratories, Abbott Park, IL 60064.
 Center for disease control (1999).
Guidelines for evaluation and acceptance of
new syphilis serology tests for routine use.
US department of health, education and
welfare publication, Atlanta.

Photos
Thomas B. Wiggers, Associate Professor
Clinical Laboratory Sciences, UMMC
 Additional photos:www.Kumc.EDU
