RAPID PLASMA REAGIN (RPR)
.Prepare a 1:50 dilution of
nonreactive serum in 0.9% saline to be used for making 1:32 and higher
dilutions of the specimen to be tested.
RAPID PLASMA REAGIN (RPR) 18-MM
CIRCLE CARD TEST
Sandra A. Larsen, Ph.D. and Ernest
T. Creighton, M.P.H.*
CONTENTS
Test Principles
Specimen Collection and Handling
Specimen
Collection
Handling
Materials
Reagents
Equipment
Calibration
Pipettors and Tips
Needles
Rotator
Quality Control
RPR Test Antigen
Procedure for Testing
Daily Controls
Procedures
Qualitative Test
Quantitative Test
Calculations and Ranges
Interpretation of Results
Acceptable Variations
Sources of Error
Test Limitations
References
*Deceased 1996
RAPID PLASMA REAGIN (RPR) 18-mm
CIRCLE CARD TEST
TEST PRINCIPLES
The rapid plasma reagin (RPR) 18-mm
circle card test is a macroscopic, nontreponemal flocculation card test used to
screen for syphilis.1-4 The antigen is prepared from a modified Venereal
Disease Research Laboratory (VDRL) antigen suspension containing choline
chloride to eliminate the need to heat inactivate serum,
ethylenediaminetetraacetic acid (EDTA) to enhance the stability of the
suspension, and finely divided charcoal particles as a visualizing agent. In
the test, the RPR antigen is mixed with unheated or heated serum or with
unheated plasma on a plastic-coated card. The RPR test measures IgM and IgG
antibodies to lipoidal material released from damaged host cells as well as to
lipoprotein-like material, and possibly cardiolipin released from the
treponemes.5,6 The antilipoidal antibodies are antibodies that are produced not
only as a consequence of syphilis and other treponemal diseases, but also in
response to nontreponemal diseases of an acute and chronic nature in which
tissue damage occurs.7 If antibodies are present, they combine with the lipid
particles of the antigen, causing them to agglutinate. The charcoal particles
coagglutinate with the antibodies and show up as black clumps against the white
card. If antibodies are not present, the test mixture is uniformly gray. The
test can be purchased in kit form or in component parts from many commercial
sources. Without some other evidence for the diagnosis of syphilis, a reactive
nontreponemal test does not confirm T. pallidum infection.
SPECIMEN COLLECTION AND HANDLING
Specimen
1.Avoid accidental infection when
collecting and processing samples by observing universal precautions (Chapter
2).
2.Serum and plasma are both suitable
specimens for the qualitative test; however serum is the preferred sample for
the quantitative test.8 Test plasma samples within 48 hrs after collection.8
3.An acceptable specimen should not
contain particulate matter that would interfere with reading test results.
Specimens that are excessively hemolyzed, grossly contaminated with bacteria,
chylous or otherwise extremely turbid are unsatisfactory. A specimen is too
hemolyzed for testing when printed matter cannot be read through it.
Note: Hemolysis may be caused by
transporting blood in freezing or extremely hot weather without proper
insulation.
4Not all unsuitable specimens should
be discarded or not analyzed. When an unsatisfactory sample is received in the
laboratory, notify the requesting physician and discuss whether that specimen
should be tested. If the ordering physician still desires a
3
test result, then the condition of
the sample must be stated on the report, and a notation made of any limitation
on interpretation of the test result.9
Collection
The procedures for the collection
and processing of venous blood is given in detail in Chapter 3.
1.Serum- Collect whole blood into a
clean, dry tube without an anticoagulant.
2.Plasma- Collect blood in a tube
containing EDTA as an anticoagulant. Completely fill the tube or collect blood
until the vacuum in the collection tube has been exhausted.
3.Label each specimen with patient
identifier, and date.
Handling
A. Serum
1.Allow sufficient time
(approximately 20 minutes) at room temperature for the specimen to clot.
2.Centrifuge the specimen at room
temperature at 1000 to 1200 x g for at least 5 minutes to sediment cellular
elements (see Chapter 3).
3.Keep serum specimens in the
original collection tube if testing will be performed within a few hours.
Remove serum from clot and store at refrigerator temperature (2° - 8°C) if
testing is to be delayed. If a delay of more than 5 days is anticipated before
testing, freeze the specimen at -20°C or lower. Avoid repeated freeze-thawing
of specimens. Although unheated serum specimens may be used, serum may be
heated at 56°C for 30 minutes without affecting test outcome. Specimens must be
at room temperature (23° - 29°C; 73° - 85°F) at the time of testing.
4.If serum samples are to be shipped
to a testing site, specimen containers must be
leakproof and placed within a
leakproof plastic bag. Paperwork should be submitted in a separate plastic bag,
if included with the sample.10
B. Plasma
1.Centrifuge the specimen at room
temperature at 1000 to 1200 x g for at least 5 minutes to sediment cellular
elements. Plasma may be retained in the original collection tube if the test is
to be performed immediately. If not, plasma should be removed from cellular elements.
4
2.Store plasma specimens at
refrigerator temperature (2° - 8°C) and test within 48 hours. Plasma samples
must be at 23° - 29°C (73° - 85°F) at the time of testing. Do not heat plasma.
3.Do not use plasma specimens for
confirmatory treponemal tests.
MATERIALS
Reagents
Purchased
1.RPR antigen suspension. RPR
antigen suspension is a stabilized combination of 0.003% cardiolipin,
0.020-0.022% lecithin, 0.09% cholesterol, 10% choline chloride,
0.0125M EDTA, 0.01875% charcoal,
0.01M Na2HP04, 0.01M KH2P04, 0.1% thimerosal in distilled water.1 The antigen
suspension is packaged in ampules. Store unopened ampules at 2° to 8°C; do not
store the antigen in bright sunlight or in temperatures above 29°C; do not
freeze. An unopened ampule of antigen is stable up to the expiration date.
2.Control serum samples. Control
serum samples are lyophilized reactive (R), minimally reactive (Rm), and
nonreactive (N) control serum specimens on a card, or liquid or lyophilized
serum samples of graded reactivity. If quantitative tests are to be performed,
a control serum that can be titered to at least a 1:4 dilution should be used.
Store control cards or serum samples according to the manufacturer=s
directions.
(Reagents may be purchased from
Ampcor, Bridgeport, NJ; ASI, Arlington, TX;
Baxter Healthcare Corp., Miami, FL;
Becton-Dickinson Microbiology Systems, Cockeysville, MD; and Remel, Augusta,
GA)
Prepared
1.0.9% Saline. Add 0.9 g of dry
sodium chloride (ACS) to 100 ml of distilled water.
2.Diluent. Prepare a 2% solution of
human serum in 0.9% saline, by diluting a human serum nonreactive for syphilis
1:50 in 0.9% saline.
Provided in kit
1.Disposable, calibrated 20-gauge
needle without bevel, silicone treated
2.Plastic antigen dispensing bottle,
1 dram
3.Plastic-coated RPR cards, with 10
circles, each approximately 18 mm in diameter. Store cards at room temperature.
5
4. Dispenstirs, a disposable
(plastic) dispensing-stirring device that delivers 50 µl
Not provided in kit
1.Mechanical rotator, fixed-speed or
adjustable to 100 2 rpm, circumscribing a circle 3/4-inch in diameter on a
horizontal plane
2.Humidifying cover
3.High-intensity incandescent lamp
4.Safety pipetting device with
disposable tip that delivers 50 µl
5.Calibrated dropper that delivers
50 µl in a single drop (optional)
6.Discard containers and
disinfectants
7.Disposable latex gloves, safety
glasses, and protective clothing
CALIBRATION
Pipettors and Tips
With the pipettors currently
available, the measurement of small serum volumes is routine. Most
manufacturers include in the specifications of their pipettors the accuracy for
frequently used microliter volumes. Daily use may affect pipettors, making them
lose their initial accuracy. The differences in disposable tips from sources
other than the manufacturer of the pipettor, is probably the most common error.
For budgetary reasons, a less expensive brand of pipette tips may be
substituted for those of the manufacturer. Although the less expensive brand
may be satisfactory, the laboratory should verify the accuracy and precision of
the substitute pipet tips in their test system. Commercial kits to check
pipettor accuracy are available. Also, manufacturers provide procedures for
checking the accuracy of their equipment. Historically, the gravimetric or
spectrophotometric procedures, which use the weight of water or the absorbance
of a substance at a given wavelength, have been the most accepted methods used
to calibrate pipettors. These procedures should not be used instead of those
specified by the manufacturer's.
Needles
6
1.Check the calibrated needle each
time a new needle is used, when needle has been dropped or wiped, or when the
control pattern is not met to ensure the delivery of the correct volume of
antigen suspension (60 drops 2 drops per ml; 17 µl per drop).
2.Place the needle on a 1-ml syringe
or on a 2-ml pipette. Fill the syringe or pipette with RPR antigen suspension.
Holding the syringe or pipette in a vertical position, count the number of
drops delivered in 0.5 ml. The needle is correctly calibrated if 30 drops + 1
drop is delivered in 0.5 ml.
3.Replace the needle if it does not
meet this specification. Be sure to test the calibration of the replacement
needle.
Rotator
1.Speed - For rotators without a
digital readout, the speed can be estimated by counting the number of rotations
made per minute. To count the rotations place your finger next to the rotator
and count the number of times the rotator touches your finger in 15 seconds. If
the rotator is properly adjusted, the count should be 25. The rotator=s speed
should be calibrated each day it is used.
2.Time - The rotator=s timer should
be checked against another laboratory timer or stop watch. The rotator=s timer
should be within 15 seconds of the set time.
QUALITY CONTROL
It is the responsibility of the
laboratorian to ensure that reagents are of good quality and standard
reactivity. Chemicals and distilled water should be of high quality, and
solutions should be prepared according to the directions specified for each
technique.
Test each new lot of RPR antigen for
the RPR 18-mm circle card test in parallel with a reference reagent to verify
that the two antigens are comparable before placing the new antigen into routine
use.
Parallel testing should be performed
on at least two testing days, by using different specimens of graded reactivity
for each test period. Tests should be performed following the techniques
described below. Record the results of all check testing.
Individual specimens of graded
reactivity for check testing may be obtained by selecting specimens from the
daily test runs and storing them in the freezer. Reactive serum diluted with
non-reactive serum to produce various degrees of reactivity may also be used.
If possible, fresh serum specimens from routine test runs should be used for
the nonreactive specimens.
RPR Test Antigen
Criteria of acceptability
7
1.Test results on reference control
serum specimens of graded reactivity and individual serum specimens in the
qualitative and the quantitative tests must be the same as those obtained with
the reference antigen suspension.
2.The antigen suspension should show
in tests with non-reactive serum specimens the complete dispersion of antigen
particles and no more roughness than is seen with the reference antigen
suspension.
Procedure for Testing
1.Using controls of graded
reactivity, the reactivity of the new antigen suspension and the reference
antigen suspension is tested by using the appropriate test technique.
2.If the new antigen suspension
shows obvious deviations from the established reactivity pattern of the
controls in comparison with the reference antigen suspension, the product is
considered unsatisfactory and no further testing is performed.
3.If the new antigen suspension
gives the established reactivity pattern of the controls of graded reactivity
or shows only a slight deviation in comparison with the reference antigen
suspension, specimens may be tested with this reagent.
4.Compare the new antigen suspension
and the reference antigen suspension by qualitative testing of individual serum
specimens of graded reactivity. Test at least 3 reactive, 10 intermediate
(minimally reactive to 1:2 dilution), and 7 nonreactive serum samples. Test
serum specimens side by side with the new and the reference antigen
suspensions.
5.Select three reactive serum
specimens for quantitative testing. Prepare serial dilutions of each serum in
0.9% saline (1:2, 1:4, 1:8, 1:16) in test tubes. Dilutions of 1:32 or greater
should be prepared in diluent (2% nonreactive human serum in 0.9% saline). Test
each serum dilution side by side with the new and the reference antigen
suspensions.
Note: It is important to make these
master serial dilutions in test tubes so that the exact same serum dilutions
are being compared between antigens.
6.Record results of all testing.
7.Review test results and determine
whether the new antigen suspension meets the criteria of acceptability.
Daily Controls
8
1.Check room temperature. For reliable
and reproducible test results, the RPR Card antigen suspension, controls, and
test specimens must be at room temperature (23° - 29°C; 73° - 85°F), when tests
are performed.
2.At each routine test run, check
the expiration date on the ampule.
3.Determine antigen suspension
reactivity with control cards or control serum specimens of graded reactivity
(reactive, minimally reactive, and nonreactive). If quantitative tests are to
be performed, then a control serum that can be titered to at least a 1:4 dilution
should be used.
4.Use only RPR antigens that
reproduce the established reactivity pattern of the controls.
PROCEDURES
Qualitative Test
1.To prepare antigen for testing,
attach the hub of the dispensing needle to the fitting on the plastic
dispensing bottle. Shake the antigen ampule to resuspend the particles. Open
the ampule. Squeeze the dispensing bottle to collapse it. Insert the needle
into the ampule and withdraw all the antigen suspension into the dispensing
bottle.
2.Place 50 µl of serum or plasma
onto a 18-mm circle of the RPR test card, using a disposable Dispenstir or a
safety pipetting device.
3.Using the inverted Dispenstir
(closed end) or flat toothpicks, spread the serum or plasma to fill the entire
circle. Do not spread the specimen beyond the confines of the circle.
4.Gently shake the antigen
dispensing bottle to resuspend the particles.
5.Holding the dispensing bottle and
needle in a vertical position, dispense several drops to
clear the needle of air. Then add
exactly 1 free-falling drop (17 µl) of antigen suspension to each circle
containing serum or plasma. Do not mix.2, 3
6.Place the card on the mechanical
rotator under a humidifying cover. Rotate the card for 8 minutes at 100 2 rpm.
7.Immediately remove the card from
the rotator; briefly rotate and tilt the card by hand (three or four to-and-fro
motions) to aid in differentiating nonreactive from minimally reactive results.
8.Perform the quantitative test on
serum specimens showing any degree of reactivity (clumping) or Aroughness.@
9
Reading and Reporting Qualitative
Results
1.Read the test reactions in the
Awet@ state under a high-intensity incandescent lamp. Read the test without
magnification.
2.Report the results as follows.
Reading
|
Report
|
Characteristic clumping ranging
from
|
|
marked and intense (reactive) to
|
Reactive (R)
|
slight but definite (minimally to
|
|
moderately) reactive
|
|
Slight roughness or no clumping
|
Nonreactive (N)
|
Note: Only two reports with the RPR
card test are possible: reactive, no matter how much clumping, or nonreactive.
Quantitative Test3
1.Dilute to an endpoint titer all
serum specimens with rough nonreactive results in the qualitative test. Test
each specimen undiluted (1:1), and in 1:2, 1:4, 1:8, and 1:16 dilutions (see
Fig 10:1).
2.Place 50 µl of 0.9% saline in
circles numbered 2 through 5. Do not spread the saline.
3.Using a safety pipette device,
place 50 µl of serum in circle 1 and 50 µl of serum in circle 2 (Fig 10:1).
4.Mix the saline and the serum in
circle 2 by drawing the mixture up and down in a safety pipette eight times.
Avoid forming bubbles.
5.Transfer 50 µl from circle 2 (1:2)
to circle 3, and mix.
6.Transfer 50 µl from circle 3 (1:4)
to circle 4, and mix.
7.Transfer 50 µl from circle 4 (1:8)
to circle 5 (1:16), mix, and then discard the last 50 µl.
10
*Begin dilutions in 2% normal
human serum
|
|
Figure 10:1. Diagram of card for
quantitative test
|
|
.
|
Using the broad end of a clean
Dispenstir, spread the serum dilution to fill the entire
|
surface of circle 5, the highest
dilution (1:16). Using the same Dispenstir, repeat for
|
|
circle 4(1:8), 3(1:4), 2(1:2), and
1 (undiluted).
|
9.Gently shake the dispensing bottle
to resuspend the antigen particles.
10.Holding the antigen dispensing
bottle in a vertical position, dispense 1 or 2 drops to clear the needle of
air. Then add exactly 1 free-falling drop (17 µl) of antigen suspension in each
circle. DO NOT MIX.
11.Place the card on the rotator
under the humidifying cover and rotate the card for 8 minutes at 100 2 rpm.
12.Immediately remove the card from
the rotator; briefly rotate and tilt the card by hand (three or four to-and-fro
motions) to aid in differentiating nonreactive from minimally reactive results.
13.If the highest dilution tested
(1:16) is reactive, continue as follows:
b.Prepare a 1:16 dilution of the
test specimen by adding 0.1ml of serum to 1.5ml of 0.9% saline. Mix thoroughly.
11
c.Place 50 µl of the 1:50
nonreactive serum diluent in circles 2 through 5 of an RPR card.
d.Using a safety pipetting device
with disposable tip, place 50 µl of the 1:16 dilution of the test specimen in
circle 1 and 50 µl in circle 2.
e.Using the same pipette and tip,
make serial twofold dilutions. Complete test as described in steps 4 through 13
(see AQuantitative Test@). Use a clean tip for each specimen tested. Prepare
higher dilutions if necessary in 1:50 nonreactive serum diluent.
14.After completing the day=s tests,
remove the needle from the antigen dispensing bottle. Rinse needle in distilled
water, and air dry. Do not wipe needle (wiping removes the silicone coating). A
satisfactory needle may be retained as a spare for replacement of an
unsatisfactory needle.
15.Recap the plastic dispensing
bottle containing the antigen suspension and refrigerate at 2° to 8°C. Do not
freeze the antigen. Antigen stored in the dispensing bottle will retain its
reactivity for 3 months or until the expiration date, whichever is sooner.
Reading and Reporting quantitative
results
1.Read the test reaction in a Awet@
state under a high-intensity incandescent lamp as for the qualitative test.
2.Report the results in terms of the
highest dilution that has given a reactive result, including a minimally
reactive result,
TP = True Positive, the number of
persons who test reactive that actually have syphilis FN = False Negative, the
number of persons who test nonreactive that have syphilis
TN = True Negative, the number of
persons who test nonreactive that do not have syphilis FP = False Positive, the
number of persons who test reactive that do not have syphilis
INTERPRETATION OF RESULTS
1.The RPR card test is an aid in the
diagnosis of syphilis. Clinicians combine the RPR card test with results of
other serologic tests, darkfield examinations, clinical signs and symptoms, and
risk factors in arriving at a syphilis diagnosis. Without some other support
for the diagnosis of syphilis, a reactive RPR card test is commonly unrelated
to T. pallidum infection. The predictive value of a reactive RPR card test in a
serologic diagnosis of syphilis is increased when combined with a reactive
treponemal test, such as the fluorescent treponemal antibody absorption
(FTA-ABS) test or the microhemagglutination assay for antibodies to T. pallidum
(MHA-TP).
2.A reactive RPR card test may
suggest past or present infection with a pathogenic treponeme; however, it may
also be a false-positive reaction. False-positive reactions can result from
laboratory error as well as serum antibodies unrelated to syphilis infection.
Technical errors are detected by a nonreactive RPR card test with a second
serum specimen. False-positive RPR card tests from infections with
nontreponemal diseases or other disease conditions are identified by an
accompanying nonreactive treponemal test.
3.A nonreactive RPR card test
without clinical evidence of syphilis may suggest no current infection or an
effectively treated infection. A nonreactive RPR card test with clinical
evidence of syphilis can be seen in early primary syphilis; in secondary
syphilis, as a result of the prozone reaction; and in some cases of late
syphilis. A nonreactive RPR card test result does not rule out an incubating
syphilis infection.
13
4.When the quantitative RPR card
test is performed on patients with syphilis, a fourfold rise in titer in a
repeat specimen may suggest an infection, a reinfection, or a treatment failure;
a fourfold decrease, e.g. 1:16 to 1:4, in titer following treatment for early
syphilis usually indicates that therapy was adequate.
5.All reactive qualitative RPR card
tests should be diluted to an endpoint and the endpoint titer reported.
Unusually high RPR card test titers can be seen with concurrent human
immunodeficiency virus type 1 (HIV-1) infection. Unusually high false-positive
titers may also be seen in patients with lymphomas.
ACCEPTABLE VARIATIONS
1.Prepare a 1:16 dilution for
further quantitation, using 50 µl of specimen to 750 µl of 0.9% saline.
2.If, after screening, the serum to
be quantitated seems likely to exceed 1:16, prepare all dilutions directly on
the card, using a 1:50 nonreactive serum as the diluent beginning with circle 6
(1:32) continuing for the remainder of the card.
SOURCES OF ERROR
1.If the temperatures of the sera,
reagents, or testing area are less than 23°C (73°F), test reactivity decreases;
if temperatures are greater than 29°C (85°F), test reactivity increases.
2.If the speed of the mechanical
rotator is too fast or too slow, improper antigen-antibody interaction will
cause unpredictable test results.
3.If the time of rotation is too
long test reactivity may be increased, or if too short test reactivity may be
decreased.
4.If the card is excessively rotated
and tilted (to-and-fro motions) by hand after removal from the rotator, a
false-reactive result may occur.
5.If lighting produces a glare on
the card, the reactions may be obscured.
6.If the antigen is outdated or not
adequately tested for standard reactivity, the results may be inaccurate.
7.If the serum is unevenly spread in
the circle, the antigen and antibody may not mix properly.
14
8.If hemolyzed, contaminated, or
improperly collected serum or plasma specimens are tested, the reaction may be
masked.
9.If the moistened humidifying cover
is not used to cover tests as they are being rotated, proper humidity will not
be maintained, and test components may dry on card giving rise to false
reactive results.
TEST LIMITATIONS
1.The RPR card test cannot be used
to test spinal fluids.12
2.A prozone reaction may be
encountered occasionally. In a prozone reaction, complete or partial inhibition
of reactivity occurs with undiluted serum (maximum reactivity is obtained only
with diluted serum). The prozone phenomenon may be so pronounced that only a
rough reading is produced in the qualitative test by a serum that will be
strongly reactive when diluted. All test specimens producing any degree of
roughness or reactivity with the RPR card test antigen in the qualitative test
should be retested by using the quantitative procedure. In addition, a specimen
should be tested for the prozone phenomenon when the clinician suspects
syphilis, but the qualitative RPR is nonreactive.
3.The RPR card test may be reactive
in persons from areas where yaws, pinta or
nonvenereal syphilis is endemic.
Generally, residual titers from these infections will be <1:8.13, 14
4.Biological false-positive (BFP)
reactions occur occasionally with cardiolipin antigens, mainly in specimens
from persons who abuse drugs; who have diseases such as lupus erythematosus,
mononucleosis, malaria, leprosy, or viral pneumonia; or who have recently been
vaccinated.
5.Nontreponemal test titers of
persons who have been treated in latent or late stages of syphilis or who have
become reinfected do not decrease as rapidly as do those of the persons in the
early stages of their first infection. In fact, these persons may remain
Aserofast,@ retaining a low-level reactive titer for life.15
REFERENCES
1.Portnoy J, Brewer JH, Harris A.
Rapid plasma reagin card test for syphilis and other treponematoses. Public
Health Rep 1962;77:645-52.
2.Portnoy J. Modifications of the
rapid plasma reagin (RPR) card test for syphilis, for use in large scale
testing. Am J Clin Pathol 1963;40:473-9.
3.Portnoy J. A note on the
performance of modifications of the rapid plasma reagin (RPR) card test for
syphilis, for use in large scale testing. Public Hlth Lab 1965;23:43.
4.RPR Macro-Vue Card Test - Procedures
Manual. Hynson Westcott and Dunning: Baltimore, MD 1977.
15
5.Matthews HM, Yang TK, Jenkin HM.
Unique lipid composition of Treponema pallidum (Nichols virulent strain).
Infect Immun 1979;24:713-9.
6.Belisle JT, Brandt ME et al. Fatty
acids of Treponema pallidum and Borrelia burgdorferi lipoproteins. J Bacteriol
1994;176:2151-7.
7.Catterall, RD. Presidential
address to the M.S.S.V.D.: Systemic disease and the biological false-positive
reaction. Br J Vener Dis 1972;48:1-12.
8.Larsen SA, Pettit DE, Perryman MW,
Hambie EA, Mullally R, Whittington W. EDTA- treated plasma in the rapid plasma
reagin card test and the toluidine red unheated serum test for serodiagnosis of
syphilis. J Clin Microbiol 1983;17:341-5.
9.Department of Health and Human
Services, Health Care Financing Administration. Clinical laboratory improvement
amendments of 1988; Final Rule. Federal Register 1992 (Feb. 28):7183 [42CFR
493.1703 (c)].
10.National Committee for Clinical
Laboratory Standards. Tentative Standard M29-T2. Protection of laboratory
workers from infectious disease transmitted by blood, body fluid and tissue,
National Committee for Clinical Laboratory Standards, Villanova, PA
1991;11(14):31-2.
11.National Committee for Clinical
Laboratory Standards. Document I/LA18-A. Specifications for immunological
testing for infectious diseases; Approved guideline, 1994;14(18):38.
12.Larsen SA, Hambie EA, Wobig CH,
Kennedy EJ. Cerebrospinal fluid serologic tests for syphilis: Treponemal and
nontreponemal tests. In: Morisset R, Kurstak E, eds. Advances in sexually
transmitted diseases. VNW Science Press, Utrecht, The Netherlands 1986:157-62.
13.Gershman KA, Rolfs RT, Larsen SA,
Zaidi A, Palafox NA. Seroepidemiological characterization of a syphilis
epidemic in the Republic of the Marshall Islands, formerly a Yaws endemic area.
Int J Epidemiol 1992;21-599-606.
14.McDermott J, Steketee R, Larsen
S, Wirima J. Syphilis-associated perinatal and infant mortality in rural
Africa. Bulletin WHO 1993;6:773-80.
15.Fiumara NJ. Serologic responses
to treatment of 128 patients with late latent syphilis. Sex Transm Dis
1979;6:243-6.