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TRANSCRIPT
10/19/2012
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Serologic Testing for SyphilisComparison of the Traditional and Reverse Screening Algorithms
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Elli S. Theel, Ph.D.Director, Infectious Diseases Serology LaboratoryAssistant Professor of Laboratory Medicine and PathologyDivision of Clinical MicrobiologyMayo Clinic, Rochester, Minnesota
October 24, 2012
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Disclosures
None
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Objectives
• Describe the treponemal and non-treponemal assays for syphilis screening
• Discuss the advantages and limitations of both the traditional and reverse syphilis screening algorithms
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algorithms
• Result interpretation from the reverse syphilis screening algorithm
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Outline
• Syphilis Infection• Causative Agent• Clinical Manifestations
• Laboratory Tests for Diagnosis of Syphilis• Non-treponemal Tests
T l T
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• Treponemal Tests
• Traditional Algorithm for Syphilis Screening
• Reverse Algorithm for Syphilis Screening
• Interpretation and Follow-up
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Treponema pallidum – The Agent of Syphilis
• Spirochete
• Obligate human parasite
• Transmission• Sexual
wadsworth.org
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• Trans-placental• Percutaneous following contact with infectious
lesions• Blood Transfusion
• No reported cases of transmission since 1964
g
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Syphilis – The “Great Imitator”
• Infectious Dose: ~57 organisms1
• Incubation Period – 21 days (median)
• 3 clinical stages of syphilis • Primary:
• Painless sore (chancre) at inoculation site
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• Secondary:• Rash, Fever, Lymphadenopathy, Malaise
• Tertiary/Latent:• CNS invasion, organ damage
• “The physician that knows syphilis knows medicine.” – Sir William Osler
1Magnuson HJ, et al. Inoculation of syphilis in human volunteers. Medicine 1956;35:33-82http://www.cdc.gov/std/syphilis/stdfact-syphilis.htm
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Laboratory Diagnosis of SyphilisThe Uncommon Methods
• Rabbit Infectivity Test (RIT)• High Sensitivity and Specificity• Long turn-around-time• Limited to research settings
• Dark Field Microscopy
http://www.els.net
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• Useful only during primary infection• Technician expertise required
• Immunostaining• Direct fluorescent antibody or silver stain
• Polymerase Chain Reaction (PCR)• Not commercial available
textbookofbacteriology.net
CDC/NCHSTP/Dividion of STD Prevention
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Laboratory Diagnosis of SyphilisThe Common Methods
• Serology• Mainstay for syphilis testing• Two classes of serologic tests
• Non-treponemal
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p• Treponemal
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• Principle:• T. pallidum infection leads to the production of
reagin• Reagin – Antibodies to substances released from
cells damaged by T. pallidum
Serologic Tests for Syphilis:Non-Treponemal Assays
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cells damaged by T. pallidum• Reagin reacts with cardiolipin
• Cardiolipin – a phospholipid component of certain eukaryotic and prokaryotic membranes
• Examples of non-treponemal tests:• Rapid Plasma Reagin (RPR)• Venereal Disease Research Laboratory (VDRL)
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• RPR and VDRL are agglutination assays
Serologic Tests for Syphilis:Non-Treponemal Assays
Cardiolipin
Charcoal
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• RPR and VDRL are agglutination assays
Reagin
Serologic Tests for Syphilis:Non-Treponemal Assays
Cardiolipin
Charcoal
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Serumor
CSF
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• Rapid turnaround time – Minutes
• Inexpensive
• No specialized instrumentation required
• Usually revert to negative following therapy
Non-Treponemal Tests:Advantages
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• Usually revert to negative following therapy • Can be used to monitor response to therapy
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• Results are subjective• Intra- and Inter-laboratory variability
• Non-specific• False positive results can result from other
i f ti i f ti diti
Non-Treponemal Tests:Limitations
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infectious or non-infectious conditions• EBV, Lupus, etc.
• Limited sensitivity in early/primary syphilis and in late/latent syphilis
• Low throughput • Problematic for high volume laboratories
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• Possibility for prozone effect• High levels of antibody may inhibit the
agglutination reaction• To identify prozone, labs must serially dilute
samples
Non-Treponemal Tests:Limitations, continued
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p
Undilute 1:2 1:4 1:8 1:16
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• Principle:• Infection leads to production of specific antibodies
directed against T. pallidum
• Treponemal tests detect IgG or total IgM/IgG antibodies directed against T pallidum
Serologic Tests for Syphilis:Treponemal Assays
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antibodies directed against T. pallidum
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• Microhemagglutination assay (MHA)
• Fluorescent treponemal antibody (FTA-ABS)
• Treponema pallidum particle agglutination (TP-PA)
• Enzyme Immunoassay (EIA)
M l i l Fl I (MFI)
Serologic Tests for Syphilis:Treponemal Assays
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• Multiplex Flow Immunoassay (MFI)
FTA-ABS
www.mastgrp.bizYellow wells = positive
Conventional EIATP-PA
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Syphilis IgM Syphilis IgG
Patient Serum Added
Treponemal Assays:Multiplex Flow Immunoassays
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Added
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Syphilis IgM Syphilis IgG
Patient Serum Added
Labeled anti-IgM andanti-IgG reporterantibody added
Treponemal Assays:Multiplex Flow Immunoassays
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Added
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Syphilis IgM Syphilis IgG
Patient Serum Added
Labeled anti-IgM andanti-IgG reporterantibody added
Treponemal Assays:Multiplex Flow Immunoassays
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Added
Bound beads are passedthrough the laser detector
Laser 1 identifies the bead(IgM vs. IgG)
Laser 2 determines if the target antibody is present(presence or absence of fluor)
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• High Specificity
• Possibly higher sensitivity during early and late syphilis stages compared to non-treponemal tests
• Newer Methods
Treponemal Assays:Advantages
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• Objective result interpretation• Automation option• High throughput• High reproducibility/precision
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• Remain positive despite treatment• Cannot be used to monitor response to therapy
• Conventional Methods• Subjective interpretation requiring technician
ti t d
Treponemal Assays:Limitations
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expertise to read
• Newer Methods• Expensive instrumentation• Higher cost/test
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Syphilis Screening Algorithms:Traditional versus Reverse Screening
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Traditional Algorithm
Non-treponemal test (e.g., RPR)
Treponemal test (e.g., FTA) Negative for syphilis
Non-reactive
Non-reactive
Reactive
Reactive
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Syphilis Negative for syphilis
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Traditional Algorithm
Non-treponemal test (e.g., RPR)
Treponemal test (e.g., FTA) Negative for syphilis
Non-reactive
Non-reactive
Reactive
Reactive
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Advantages:
• Results show good correlation with disease status• Rapid, inexpensive screening method• Excellent option for laboratory with small throughput• Recommended by the CDC
Syphilis Negative for syphilis
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Traditional Algorithm
Non-treponemal test (e.g., RPR)
Treponemal test (e.g., FTA) Negative for syphilis
Non-reactive
Non-reactive
Reactive
Reactive
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Disadvantages:
• Manual (RPR) and subjective interpretation• Screening method is non-specific and may lead to false-
positive results • Not suitable for high throughput laboratories• Potentially lower sensitivity for detecting early syphilis and
late/latent disease
Syphilis Negative for syphilis
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• Incidence of disease impacts the positive predictive value of the assay
(in th
ousa
nds) Primary and secondary
Early latentTotal syphilis
The Traditional Syphilis Algorithm:If it works, why change it?
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http://www.cdc.gov/std/stats09/figures/33.htm
Rate per 100,000 population
0.2 0.21-2.2 >2.2
Cas
es (
Year
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Reverse Algorithm
Treponemal test (eg, EIA)
Non-Treponemal test (eg, RPR) Negative for syphilis
Non-reactive
Non-reactive
Reactive
Reactive
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Syphilis Second Treponemal Test (e.g., TP-PA)
Non-reactiveReactive
Evaluation Required* Negative for syphilis
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• Automated treponemal screening assays are available (i.e., EIA, MFI)2
• > 500 sera/9 hr shift by MFI vs. ~ 200 sera/9 hr shift by manual methods
• Objective interpretation of results
• Results from EIA or MFI can be interfaced with LIS
Reverse Algorithm:Advantages
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• Specific screening test for anti-T. pallidum antibodies
• Potentially increased detection of patients with early syphilis3:
• Among 560 patients with lesions, 18 (3.2%) were EIA (+), DFA (+) and RPR (-)
• Among 9,137 patients with EIA (+), RPR (-) results, 54 became RPR (+) on follow-up testing
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• Higher cost/sample
• Higher assay complexity
• Increased detection of patients with screen (+), RPR (-) results4,5:
• CDC - ~56% of EIA reactive samples are non-reactive by RPR
Reverse Algorithm:Limitations
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• How do we interpret these results?
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Case #1• 37-year-old with HIV
• Presents to primary care physician with a 2-week history of fatigue, intermittent fever and new rash on palms and soles
P i l l d it l l i
Reverse Syphilis Screening Algorithm:Result Interpretation
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• Previously resolved genital lesion
• Syphilis serology ordered• Syphilis IgG by EIA: positive• RPR: positive, titer of 1:64
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Case #1 Conclusion
• No further testing needed on this sample
• Interpretation: “Untreated or recently treated syphilis.” Follow CDC treatment guidelines4
• For treatment follow-up:
Reverse Syphilis Screening Algorithm:Result Interpretation
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• Samples can be tested directly by RPR.
• A 4-fold decrease in RPR titers (eg, 1:64 to 1:16) is interpreted as response to therapy
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Case #2
• 23-year-old female
• Evaluated during first-trimester, routine pregnancy visit
• Previously healthy
Reverse Syphilis Screening Algorithm:Result Interpretation
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• Syphilis serology ordered
• Syphilis IgG by EIA: positive
• RPR: negative
• Second treponemal test, TP-PA: negative
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Case #2 Conclusion
• Interpretation: “Probable false-positive screening test. Negative for syphilis.”
• False-positive serologic tests are not uncommon during pregnancy and confirmatory testing is often required
Reverse Syphilis Screening Algorithm:Result Interpretation
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required
• Syphilis IgM testing not recommended for routine pregnancy screening
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Case #3
• 50-year-old immigrant from Somalia
• Pre-kidney transplant evaluation
• Syphilis serology ordered• Syphilis IgG by EIA: positive
Reverse Syphilis Screening Algorithm:Result Interpretation
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Syphilis IgG by EIA: positive• RPR: negative• TP-PA: positive
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Case #3 Conclusion
• Interpretation: “Historical and clinical evaluation required.”
• During evaluation with provider, patient indicates no known history of treatment for syphilis.
Reverse Syphilis Screening Algorithm:Result Interpretation
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• Patient treated for possible latent syphilis
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Case #4
• 30-year-old inmate
• Past history of syphilis (10 years prior)
• Syphilis serology ordered• Syphilis IgG by EIA: positive
Reverse Syphilis Screening Algorithm:Result Interpretation
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• Syphilis IgG by EIA: positive• RPR: negative
• Interpretation: “Past, successfully treated syphilis. No further testing for syphilis required.”
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Patient historyTreponemal
screen RPR2nd
treponemala Interpretation Follow-up
Unknown history of syphilis NEG NA NA
No serologic evidence of syphilis
None, unless clinically indicated (eg, early syphilis)
Unknown history of syphilis POS POS NA
Untreated or recently treated syphilis
See CDC treatment guidelines; follow RPR titers
Reverse Syphilis Screening Algorithm:Summary
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Unknown history of syphilis POS NEG NEG
Probable false-positive screening test
No follow-up testing, unless clinically indicated
Unknown history of syphilis POS NEG POS
Possible syphilis (eg, latent) or previously treated syphilis
History and clinical evaluation required
Known history of syphilis POS NEG POS or NA
Past, successfully treated syphilis
None
aSecond treponemal test should be TP-PA or a different method than screening testFor CDC treatment guidelines see http://www.cdc.gov/std/treatment/default.htm
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Patient historyTreponemal
screen RPR2nd
treponemala Interpretation Follow-up
Unknown history of syphilis NEG NA NA
No serologic evidence of syphilis
None, unless clinically indicated (eg, early syphilis)
Unknown history of syphilis POS POS NA
Untreated or recently treated syphilis
See CDC treatment guidelines; follow RPR titers
Reverse Syphilis Screening Algorithm:Summary
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Unknown history of syphilis POS NEG NEG
Probable false-positive screening test
No follow-up testing, unless clinically indicated
Unknown history of syphilis POS NEG POS
Possible syphilis (eg, latent) or previously treated syphilis
History and clinical evaluation required
Known history of syphilis POS NEG POS or NA
Past, successfully treated syphilis
None
aSecond treponemal test should be TP-PA or a different method than screening testFor CDC treatment guidelines see http://www.cdc.gov/std/treatment/default.htm
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Patient historyTreponemal
screen RPR2nd
treponemala Interpretation Follow-up
Unknown history of syphilis NEG NA NA
No serologic evidence of syphilis
None, unless clinically indicated (eg, early syphilis)
Unknown history of syphilis POS POS NA
Untreated or recently treated syphilis
See CDC treatment guidelines; follow RPR titers
Reverse Syphilis Screening Algorithm:Summary
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Unknown history of syphilis POS NEG NEG
Probable false-positive screening test
No follow-up testing, unless clinically indicated
Unknown history of syphilis POS NEG POS
Possible syphilis (eg, latent) or previously treated syphilis
History and clinical evaluation required
Known history of syphilis POS NEG POS or NA
Past, successfully treated syphilis
None
aSecond treponemal test should be TP-PA or a different method than screening testFor CDC treatment guidelines see http://www.cdc.gov/std/treatment/default.htm
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Patient historyTreponemal
screen RPR2nd
treponemala Interpretation Follow-up
Unknown history of syphilis NEG NA NA
No serologic evidence of syphilis
None, unless clinically indicated (eg, early syphilis)
Unknown history of syphilis POS POS NA
Untreated or recently treated syphilis
See CDC treatment guidelines; follow RPR titers
Reverse Syphilis Screening Algorithm:Summary
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Unknown history of syphilis POS NEG NEG
Probable false-positive screening test
No follow-up testing, unless clinically indicated
Unknown history of syphilis POS NEG POS
Possible syphilis (eg, latent) or previously treated syphilis
History and clinical evaluation required
Known history of syphilis POS NEG POS or NA
Past, successfully treated syphilis
None
aSecond treponemal test should be TP-PA or a different method than screening testFor CDC treatment guidelines see http://www.cdc.gov/std/treatment/default.htm
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Patient historyTreponemal
screen RPR2nd
treponemala Interpretation Follow-up
Unknown history of syphilis NEG NA NA
No serologic evidence of syphilis
None, unless clinically indicated (eg, early syphilis)
Unknown history of syphilis POS POS NA
Untreated or recently treated syphilis
See CDC treatment guidelines; follow RPR titers
Reverse Syphilis Screening Algorithm:Summary
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Unknown history of syphilis POS NEG NEG
Probable false-positive screening test
No follow-up testing, unless clinically indicated
Unknown history of syphilis POS NEG POS
Possible syphilis (eg, latent) or previously treated syphilis
History and clinical evaluation required
Known history of syphilis POS NEG POS or NA
Past, successfully treated syphilis
None
aSecond treponemal test should be TP-PA or a different method than screening testFor CDC treatment guidelines see http://www.cdc.gov/std/treatment/default.htm
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Conclusions• Syphilis is typically diagnosed by serologic means
• Two main classes of syphilis serologic tests:• Non-treponemal (e.g., RPR, VDRL)• Treponemal (e.g., FTA, TP-PA, EIA, MFI)
• Traditional Algorithm• Non-treponemal test first
b
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• Screen by RPR• If RPR positive use treponemal test to confirm
• Advantages• Recommended by CDC• Cost-effective• Suitable for most lower throughput labs
• Limitations• May miss very early or late/latent infection
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Conclusions
• Reverse Algorithm• Treponemal test first
• Screen by EIA or MFI• Screen positive samples tested by non-
treponemal test: RPR• EIA/MFI and RPR discordant samples should be
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tested by a second treponemal test: TP-PA• Advantages
• Allows for automation and increased sample throughput
• Limitations• Result interpretation can be challenging
• Good communication with providers is critical
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References1 Magnuson HJ, et al. Inoculation of syphilis in human volunteers.
Medicine (Baltimore) 1956;35:33-82.
2 Binnicker MJ, et al. Treponema-specific test for serodiagnosis of syphilis: comparative evaluation of seven assays. J Clin Microbiol 2012;49:1313-1317
3 Mishra S, et al. The laboratory impact of changing syphilis screening from the rapid-plasma reagin to a treponemal enzyme i t d f th t T t S
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immunoassay: a case study from the greater Toronto area. Sex Transm Dis 2011; 38:190-196
4 CDC. Discordant results from reverse sequence syphils screening: five laboratories, United States, 2006-2010. Morb Mortal Wkly Rep 2011;60:133-137
5 Binnicker MJ, et al. Direct comparison of the traditional and reverse syphilis screening alorithms in a population with a low prevalence for syphilis. J Clin Microbiol 2012; 50:148-150
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Questions & Discussion
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Questions & Discussion
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