HHS Public Access

Author manuscript
. Author manuscript; available in PMC 2017 August 23.
Author Manuscript

Published in final edited form as:

. 2014 December 15; 36(24): 195–202. doi:10.1016/j.clinmicnews.2014.12.001.

Serologic Testing for Syphilis: Benefits and Challenges of a

Reverse Algorithm
Katherine Soreng, Ph.D.1, Roma Levy, M.S.2, and Yetunde Fakile, Ph.D.3
1Siemens Healthcare Diagnostics Inc., Tarrytown, New York
2Siemens Healthcare Diagnostics Inc., Los Angeles, California
Author Manuscript

3Centers for Disease Control and Prevention, Atlanta, Georgia

Abstract
Syphilis is a human infection of global importance. Its diagnosis can be challenging, requiring
construction of a serologic profile based on the results of at least two types of antibody tests:
treponemal and nontreponemal. The traditional approach to the serodiagnosis of syphilis has been
the use of a nontreponemal screening assay followed by the performance of a treponemal
confirmatory test if the initial nontreponemal screening test was reactive. With the increasing
availability of automated, easier-to-perform, and rapid treponemal assays, an increasing number of
laboratory testing sites are adopting reverse sequence screening for the serodiagnosis of syphilis:
screening with a treponemal assay first, then confirmation with a nontreponemal assay and, when
necessary, discrepant resolution using another treponemal test. In addition to offering automation
Author Manuscript

and increased throughput, a reverse algorithm can increase disease detection, especially in late
latent and early primary stages of infection when the nontreponemal antibody test may be
nonreactive. However, a disadvantage to this approach is that there can be an increase in false-
positive test results. This article reviews the clinical and workflow benefits and limitations of a
reverse testing algorithm and discusses current guidance available from the Centers for Disease
Control and Prevention.

Introduction
Treponema pallidum subsp. pallidum is a fastidious, microaerophilic spirochete that is the
etiologic agent of syphilis. The diagnosis of syphilis, once called the “great imitator”
because of its ability to produce a variety of clinical signs and symptoms of infection that
Author Manuscript

can be easily confused with other diseases, can be a challenge.

Without treatment, syphilis is a chronic and progressive disease that can be associated with
significant morbidity and mortality, especially when transmitted vertically from mother to
child or in patients with advanced tertiary disease. Left untreated, infection is thought to
proceed through a multistage process of primary, secondary, and tertiary stages (Fig. 1) that
is often characterized by episodes of active disease between periods where signs or

Corresponding Author: H. Roma Levy, M.S., 23655 Candlewood Way, West Hills, CA 91307. Tel.: 818-274-5689.
[email protected].
 Soreng et al. Page 2

symptoms are absent (latency); latent disease is typically divided into early latent (less than
Author Manuscript

1 year from primary exposure) and late latent (greater than 1 year) (1). About 30% of
untreated cases are thought to eventually progress to tertiary syphilis within 1 to 20 years of
exposure (2). Primary and secondary disease affects skin, mucosal surfaces, and regional
lymph nodes and can have misleadingly benign manifestations (e.g., painless chancre, rash,
fever, malaise, muscle aches, and lymphadenopathy). In contrast, tertiary disease can occur
in virtually any organ system following a spirochetemia that results in systemic
dissemination of the organism. Tertiary syphilis is associated with serious complications,
which can include cardiac or neurologic damage that can lead to death. Neurosyphilis, a
serious complication, can occur at any stage if the spirochete invades the nervous system.
Symptoms of neurosyphilis include headache, mental disturbance, and a Parkinson’s
disease-like movement disorder (3).

Syphilis is primarily acquired as a sexually transmitted disease (STD), so it is perhaps not

Author Manuscript

surprising that data suggest an increased incidence of syphilis in certain high-risk

populations, such as men who have sex with men and commercial sex workers (3). Beyond
the greater generalized likelihood of contracting an STD through high-risk (e.g.,
unprotected) sexual behavior, having HIV does not appear to place an individual at higher
risk of contracting syphilis (4,5). However, the likelihood of HIV transmission by an HIV-
infected individual to an unprotected sexual partner may be increased if the partner has a
syphilitic chancre that becomes exposed to HIV-bearing secretions (3,5). Thus, syphilis
screening for at-risk and high-risk individuals plays an important role in public health.

Syphilis is usually transmitted sexually; however, it can also be passed vertically from
mother to child either transplacentally (in utero) or perinatally as the newborn comes into
contact with maternal blood and vaginal fluid during birth. Mother-to-child transmission of
Author Manuscript

syphilis can have serious consequences. Approximately 69% of untreated infected pregnant
women will experience an adverse pregnancy outcome, which can include serious birth
defects, low birth weight, and prematurity. The consequences for a child infected with
syphilis in utero can be lifelong. In addition, 25% of in utero infections result in late-term
miscarriage or stillbirth, and 11% culminate in neonatal demise (3,6). Fortunately, routine
screening and treatment of pregnant women have been shown to significantly reduce cases
of congenital syphilis and can help to avoid serious consequences to the fetus or the
newborn. Public health campaigns, such as the Syphilis Elimination Effort, and global
initiatives by the World Health Organization (WHO) and the Pan American Health
Organization in partnership with the United Nations Children’s Fund seek to eliminate
mother-to-child-transmission of syphilis by 2015, in part through targeted prenatal testing
and educational efforts, as well as through improved availability of antenatal care (6).
Author Manuscript

Despite such efforts, syphilis continues to present an important public health challenge in the
United States and many other countries.

Regardless of the mode of transmission and despite the disappearance of symptoms in

latency, T. pallidum infection persists until it has been treated. Antibiotic treatment can
usually eliminate existing infection at any stage; however, there is no immunity associated
with successful therapy, so reinfection can occur with subsequent exposure. The vast
majority of infections are responsive to penicillin, which is still the drug of choice.

. Author manuscript; available in PMC 2017 August 23.

 Soreng et al. Page 3

Fortunately, no penicillin-resistant strains of T. pallidum have been reported; however,

Author Manuscript

penicillin crosses the blood-brain barrier only minimally and so may be of limited use for
the treatment of neurosyphilis. For individuals allergic to penicillin, alternative antibiotics,
such as tetracyclines (tetracycline and doxycycline) and macrolides (azithromycin and
erythromycin), have been used for treatment. Unfortunately, the use of alternative regimens,
in particular the macrolides, has produced antimicrobial resistance in some treponemal
strains (7).

Laboratory Testing for Syphilis

Direct and molecular methods
Reliable testing is essential to establish the correct diagnosis and to institute the appropriate
treatment—especially in latency, when there are no signs or symptoms of disease. In
addition to a careful review of sexual history and physical presentation, several methods can
Author Manuscript

be used to aid in the clinical diagnosis. In primary syphilis, if a patient presents with an open
chancre, direct-visualization methods, such as dark-field or fluorescence microscopy, can be
used to detect motile spirochetes in a fresh specimen collected by swab. Although
microscopic methods can be used earlier in the course of disease than any other test (within
1 week of infection), they also have significant limitations. They are not useful for routine
screening because they are labor-intensive, must be performed at the point of care, must be
examined within minutes of specimen collection, and require the use of specialized and
expensive equipment operated by personnel with appropriate experience and training.
Additionally, a diagnosis can be missed if motile spirochetes are absent in the collection
swab or if the primary chancre is absent.

The use of nucleic acid amplification tests is also currently being explored, and some
Author Manuscript

success has been reported. However, existing methods consist of home brew tests that are
unstandardized, as there are currently no FDA-cleared kits available for commercial use.
Molecular assays, in general, are more expensive than performing standard serologic tests.
Thus, molecular methods cannot be recommended for routine screening and diagnosis at this
time, especially in resource-limited areas of the world (8).

Serologic testing
Serologic testing currently provides the best method for syphilis screening and diagnosis. In
particular, serologic tests may provide the only evidence of infection during the latent
period, and serologic profiles using a combination of test types can help determine the
presence of untreated syphilis versus a patient with a treated past exposure (1). Because
serologic testing for syphilis can be complex and often requires the results of at least two
Author Manuscript

different serologic assays, clinicians need to understand the basis of these tests and the
underlying immunological response to T. pallidum infection to accurately diagnose and
manage disease.

Two types of serologic tests must be used to diagnose and to determine the stage of syphilis:
nontreponemal tests and treponemal tests. Nontreponemal tests detect IgM and IgG
antibodies directed against lipoidal antigens (such as cardiolipin and lecithins) released as a

. Author manuscript; available in PMC 2017 August 23.

 Soreng et al. Page 4

consequence of cell damage from both the host and the bacterium (Fig. 2). Antibodies to
Author Manuscript

these antigens are usually not detected until approximately 6 weeks after infection (Fig. 3)
(9). Examples of nontreponemal tests are the rapid plasma reagin (RPR) test and the
Venereal Disease Research Laboratory (VDRL) method. These tests can be run either
qualitatively or quantitatively and are primarily based on either macroscopic or microscopic
flocculation. Because nontreponemal tests are performed manually, they are labor-intensive
and require a trained operator to conduct the test and interpret the results. While a reactive
test can indicate syphilis infection, specificity is compromised by multiple factors, including
other disease states that result in the production of anti-lipoidal antibodies, generating
biological false-positive results (Table 1) (10).

False-negative results may also occur with nontreponemal tests because of a phenomenon
known as the prozone reaction. This occurs when the nontreponemal antibody concentration
is very high: the test antigen becomes saturated by antibodies, preventing formation of the
Author Manuscript

antigen-antibody matrix required for agglutination (11). The prozone effect may be avoided
by serially diluting the sample until the antibody titer is low enough to yield a positive
response. This should be done if clinical suspicion of syphilis is high and the specimen
yields a serologic result that is weakly reactive or atypical or has a rough, grainy appearance.
Nontreponemal tests are valuable for measuring the efficacy of drug treatment because they
are quantitative. A fourfold drop in titer is indicative of successful antibiotic treatment, and
the nontreponemal antibody titer should be virtually undetectable following effective
antibiotic treatment in most patients (11).

Because nontreponemal tests are not specific for syphilis, reactive sera are typically reflexed
to a treponemal assay for confirmation. Treponemal assays detect the presence of IgM and
IgG antibodies against proteins specific to T. pallidum (Fig. 2). In the native immune
Author Manuscript

response, antibodies are generated against T. pallidum membrane lipoproteins (i.e., TpN15,
TpN17, and TpN47) within ~3 weeks following infection, as shown in Fig. 3. Treponemal
assays are qualitative and generally designed to detect one or more of the antibodies
generated by these membrane antigens. Both laboratory-based and commercial tests have
evolved over the years, and there are several types of formats in use. Manual assays include
the fluorescent treponemal antibody absorption (FTA-ABS) test, which detects the whole
organism, and the Treponema pallidum particle agglutination (TPPA) assay. Manual and
automated versions include line blot assays, microbead immunoassays, enzyme
immunoassays (EIA), and chemiluminescent immunoassays (CIA). Assays may use either
naturally purified or recombinant antigens as the capture ligand. In many countries, rapid
point-of-care testing for treponemal antibody is now also available (12,13). This format is
particularly useful in areas with poor resource settings lacking routine access to laboratory-
Author Manuscript

based methodologies. Some of these tests are being reviewed for WHO pre-qualification.
However, none of the tests are FDA cleared as yet.

Despite the generally high sensitivity and specificity of treponemal assays, there are
limitations inherent to the biology underlying their design. Treponemal assays will detect
other related spirochete subspecies because they are antigenically indistinguishable.
Although treatment is the same, this must be taken into consideration in regions with known
endemic treponematosis that cause other non-venereal diseases, such as yaws (caused by

. Author manuscript; available in PMC 2017 August 23.

 Soreng et al. Page 5

Treponema pallidum subsp. pertenue), pinta (Treponema pallidum subsp. carateum), and
Author Manuscript

bejel (Treponema pallidum subsp. endemicum) (14). As shown in Table 1, treponemal tests
can also generate false-positive results in the presence of a variety of other diseases and
conditions (8).

For establishing the serodiagnosis of patients with syphilis, it is important to understand the
dynamic antibody profiles of both nontreponemal and treponemal assays, as outlined in Fig.
3, and to use the two tests in conjunction. Nontreponemal antibody typically declines in
successfully treated patients as antigenic release from damaged cells is resolved.
Quantitative nontreponemal testing using dilution titers can help identify initial infection; a
reduction in the nontreponemal antibody titer relative to baseline usually signals successful
therapy. Quantitative nontreponemal testing can also be used to determine if a patient who
appears to have failed treatment has been re-infected or is “serofast.” Serofast patients fail to
fully resolve serologically and perpetually exhibit low nontreponemal titers, whereas re-
Author Manuscript

infected patients have persistently higher antibody titers (15). Conversely, treponemal
antibody is generally detected in both infected and resolved cases. Approximately 85% of
patients remain positive for life for treponemal antibody even with successful therapy (7).
Evaluation of patients with reactive treponemal but negative nontreponemal results
(discordant serologies) should be carefully considered for both current and previously
treated infections.

Traditional versus reverse sequence syphilis screening algorithms

For many years, the traditional syphilis testing algorithm employed a nontreponemal assay
for primary evaluation followed by a confirmatory treponemal assay on initially reactive
samples (Fig. 4). Since both assay formats were originally manual, this approach made sense
from both workflow and cost perspectives. However, the commercial availability of an
Author Manuscript

increasing number and range of treponemal assays, including both EIA formats and fully
automated CIA, has suggested an alternative approach to syphilis screening. Unlike
nontreponemal assays, which with few exceptions are currently manual, automated assays
require far less operator interaction, are easier and faster to perform, and are optimized for
high volume and rapid turnaround. Naturally, the workflow advantages offered by automated
treponemal assays suggested that reversing the order of the test types might make syphilis
testing less labor-intensive while retaining diagnostic accuracy. Thus, it is not surprising that
many laboratories have reversed the order in which treponemal and nontreponemal tests are
performed, leading to the development of the reverse sequence syphilis screening (RSSS)
algorithm.

In addition to the potential workflow improvement of RSSS, in the last several years, a
Author Manuscript

growing body of evidence has suggested that the traditional testing approach could be
missing some untreated cases, especially if the patient is in the late latent stage of disease
where seroreactivity to nontreponemal tests declines (16). Use of the traditional screening
approach could miss such cases, because an initial nonreactive nontreponemal test would not
reflex to a treponemal assay. Across different studies and demographic populations
(including HIV patients), up to 40% of untreated late latent cases were found to be

. Author manuscript; available in PMC 2017 August 23.

 Soreng et al. Page 6

nonreactive using nontreponemal assays (17–19). This could create a diagnostic conundrum
Author Manuscript

and could especially impact treatment decision making.

Other advantages offered by RSSS have helped drive the recommendation for its adoption in
countries such as the United Kingdom (20). Treponemal assays detect primary infection at a
slightly earlier stage than a nontreponemal assay (21). Also, published data support the value
of using an RSSS approach for detecting syphilis in both low- and high-risk and low- and
high-prevalence populations (22–24).

In the U.S., an increasing number of laboratories have either changed to or are considering
adopting an RSSS algorithm. Changing paradigms in the order in which treponemal and
nontreponemal tests are conducted is important to patient management, especially in patients
with discordant results, where the current need to treat can be unclear. Laboratory
professionals need to be fully aware of potential discrepancies in the serology to better
Author Manuscript

support practitioner inquiries. Importantly, potential false-positive screening results can arise
with significant frequency when using treponemal assays as the diagnostic starting point
(24). Although the CDC currently continues to recommend a traditional testing approach,
for laboratories or institutions wishing to adopt RSSS, the CDC has provided clear guidance
and a recommended algorithm (Fig. 5) to resolve such discrepancies. In the CDC-
recommended algorithm, initial testing is done with a treponemal assay (EIA or CIA),
followed by a quantitative nontreponemal test for confirmation (quantitative RPR or other
nontreponemal test). Discordant samples are resolved on the basis of a TPPA assay. The
CDC specifically recommends the TPPA assay over the FTA-ABS test, citing concerns over
specificity (24,25). All confirmed results should be reported concurrently to both the
clinician and the appropriate public health agency.
Author Manuscript

However, some studies have suggested that while they have relatively high sensitivity and
specificity, TPPA assays may provide different results in samples that are reactive by other
treponemal assays (representing possible—but unconfirmed—infections) (26,27). In fact,
most of the TPPA assay discrepancies were found in patients who were beyond the primary
stage and who were co-infected with HIV (a population generally at higher risk for syphilis).
Samples from this population have the potential to yield false-positive and false-negative
results using both treponemal and nontreponemal tests (8,28,29). While concerns over use of
the TPPA algorithm have led some laboratories to consider using alternative treponemal
assays on discordant samples that are TPPA negative, the method’s relatively good
performance is generally well accepted, and it is recommended by both the CDC and other
authorities in countries outside the U.S. as an aid in resolving potential screening of false-
positive results. Ongoing studies by the CDC involving a range of commercially available
Author Manuscript

treponemal assays may provide further clarification, once published.

Analytic and clinical considerations with the RSSS

A 2008 study published by the CDC found that using a reverse screening approach (a
treponemal assay followed by confirmation with a nontreponemal method) identified an
additional 3% of patients who would have been missed with the traditional approach (17).
However, the report noted that a relatively high rate of initially reactive treponemal results
(17.2%) were nonreactive according to a second treponemal assay (suggesting the potential

. Author manuscript; available in PMC 2017 August 23.

 Soreng et al. Page 7

for screening false-positive results). This finding was both reinforced and challenged by a
Author Manuscript

2011 study published by the CDC that supported the ability of the RSSS algorithm to
identify samples that would have been missed with traditional screening but also found a
presumptive high rate of treponemal false-positive results. As in the 2008 study, 56.7% of
the specimens were initially treponemal test positive/RPR negative, and 31.6% of these were
nonreactive using an alternate treponemal assay (either TPPA or FTA-Abs, depending on the
laboratory) (24). Regardless of the second treponemal assay used, all laboratories reported a
relatively high rate of false-positive results in both low- and high-prevalence populations
examined. Importantly, out of the 140,176 specimens tested (both low and high risk), the
overall treponemal reactivity was 3.4%, meaning the vast majority of samples were
presumably correctly ruled out following the initial testing step. In the U.S., the probability
that a nonreactive treponemal test represents a true negative is considered to be >98%, as the
overall U.S. prevalence of syphilis is low (30,31).
Author Manuscript

The 2011 study noted that there were discrepancies between the initial and confirmatory
treponemal test methods among the five participating laboratories. To arbitrate the results,
the CDC announced plans for additional studies to compare multiple EIA, CIA, TPPA
assays, and the FTA-ABS test in a head-to-head format using specimens from well-defined
patient populations where clinical histories and risk factors for syphilis were known.
Preliminary, but incomplete, results of this new study suggesting good performance by the
treponemal assays evaluated have recently been published by CDC researchers. Despite
some variability, overall there was good agreement for syphilis seroreactivity among the
seven treponemal assays (three fully automated immunoassays [AIs] and four nonautomated
assays, including the TPPA and FTA-ABS methods). Moreover, a minimum signal-to-cutoff
ratio (which was different for each AI) could be associated with a positive TPPA assay
result, suggesting that a second treponemal test may not be necessary to confirm AI-reactive,
Author Manuscript

RPR-nonreactive sera (32). Additional data from this study are expected to be published and
should further elucidate assay performance, as well as expand our understanding of how
treponemal assays might be used in syphilis testing algorithms.

RSSS in the U.S

In many countries, treponemal screening using a reverse algorithm has become
commonplace, though individual algorithms and discordant-specimen management vary.
Both clinical and labor considerations contribute to the value of reverse screening for
syphilis. In the U.S., the trend toward increased consolidation has meant that sample volume
has often increased in laboratories offering syphilis testing. The current lack of a
commercial, automated option for nontreponemal testing has driven interest in the growing
availability of treponemal assays able to meet the demands of increased throughput. In
Author Manuscript

addition to the nontreponemal assays being more labor-intensive, many clinicians,

laboratorians, and public health agencies have cited concerns over the looming shortage of
trained and experienced laboratory technologists who can accurately perform, interpret, and
report the test results. While nontreponemal tests are clinically important for establishing the
stage of disease and response to therapy, as with any test, both false-positive and false-
negative results can be associated with them, stemming from time and temperature
sensitivity, biologic false-positive results resulting from other diseases and physiological

. Author manuscript; available in PMC 2017 August 23.

 Soreng et al. Page 8

conditions, and the subjective nature of the interpretation. However, the potential for a false-
Author Manuscript

negative result in early primary syphilis or in late latent syphilis may be greater when using
a nontreponemal assay for the initial screen (18,19,24,31,33).

Conclusion
Syphilis immunoassays remain important and commonly requested tests, both in low-risk
populations, such as pregnant women, and in populations at higher risk or with clinical
suspicion of infection. As traditional testing for syphilis (nontreponemal screening followed
by treponemal testing for confirmation) is a long-held practice that is generally well known
to clinicians, it is important for laboratories to be prepared to explain the interpretation of
tests if moving to an RSSS algorithm. Because treponemal testing cannot distinguish
between treated and untreated infections, patients with discordant results should be carefully
considered for therapy, especially if the initial treponemal-test result is confirmed with an
Author Manuscript

alternate treponemal assay. Currently, the CDC recommends the use of the TPPA test in
resolving samples that fail to show reactivity with a nontreponemal test following a reactive
treponemal result (24,25). Clinicians should be informed and educated on both the benefits
and interpretive challenges of a reverse testing algorithm.

References
1. Singh AE, Romanowski B. Syphilis: review with emphasis on clinical, epidemiologic, and some
biologic features. Clin Microbiol Rev. 1999; 12:187–209. [PubMed: 10194456]
2. Centers for Disease Control and Prevention, National Center for HIV/AIDS, Viral Hepatitis, STD,
and TB Prevention, Division of STD Prevention. Self-study STD modules for clinicians— syphilis.
2013. (Accessed January 2014.) http://www2a.cdc.gov/stdtraining/self-study/syphilis/default.htm
3. Centers for Disease Control and Prevention. Syphilis CDC fact sheet. 2012. (Accessed January
Author Manuscript

2014.) http://www.cdc.gov/std/syphilis/STDFact-Syphilis-detailed.htm
4. Skinner JM, et al. Trends in reported syphilis and gonorrhea among HIV-infected people in Arizona:
implications for prevention and control. Public Health Rep. 2014; 129(Suppl. 1):85–94. [PubMed:
24385654]
5. Anonymous. HIV prevention through early detection and treatment of other sexually transmitted
diseases—United States. Recommendations of the Advisory Committee for HIV and STD
prevention. MMWR Recomm Rep. 1998; 47(RR-12):1–24.
6. Hawkes S, et al. Effectiveness of interventions to improve screening for syphilis in pregnancy: a
systematic review and meta-analysis. Lancet Infect Dis. 2011; 11:684–691. [PubMed: 21683653]
7. Lewis DA, Lukehart SA. Antimicrobial resistance in Neisseria gonorrhoeae and Treponema
pallidum: evolution, therapeutic challenges and the need to strengthen global surveillance. Sex
Transm Infect. 2011; 87(Suppl. 2):ii39–43. [PubMed: 22110154]
8. Ratnam S. The laboratory diagnosis of syphilis. Can J Infect Dis Med Microbiol. 2005; 16:45–51.
[PubMed: 18159528]
9. Peeling RW, Ye H. Diagnostic tools for preventing and managing maternal and congenital syphilis:
Author Manuscript

an overview. Bull World Health Organ. 2004; 82:439–446. [PubMed: 15356937]

10. Geusau A, et al. Biological false-positive tests comprise a high proportion of venereal disease
research laboratory reactions in an analysis of 300,000 sera. Int J STD AIDS. 2005; 16:722–726.
[PubMed: 16303064]
11. Larsen, SA., Creighton, ET. Rapid plasma reagin (RPR) 18-mm circle card test. In: Larsen,
SA.Pope, V.Johnson, RE.Kennedy, J., Edward, J., editors. Manual of tests for syphilis. 9th. Centers
for Disease Control and Prevention; Atlanta, GA: 1998. p. 193-207.
12. Benzaken AS, et al. Rapid tests for diagnosing syphilis: validation in an STD clinic in the Amazon
Region, Brazil. Cad Saude Publica. 2007; 23:S456–S464. [PubMed: 17992351]

. Author manuscript; available in PMC 2017 August 23.

 Soreng et al. Page 9

13. Tucker JD, et al. Accelerating worldwide syphilis screening through rapid testing: a systematic
review. Lancet Infect Dis. 2010; 10:381–386. [PubMed: 20510278]
Author Manuscript

14. Lukehart, S. Biology of treponemes. In: Holmes, K.Sparling, P., Stamm, W., editors. Sexually
transmitted diseases. McGraw Hill; New York, NY: 2008. p. 647-659.
15. Workowski KA, Berman S. Sexually transmitted diseases treatment guidelines, 2010. MMWR
Recomm Rep. 2010; 59(RR-12):1–110.
16. Larsen SA, Steiner BM, Rudolph AH. Laboratory diagnosis and interpretation of tests for syphilis.
Clin Microbiol Rev. 1995; 8:1–21. [PubMed: 7704889]
17. Anonymous. Syphilis testing algorithms using treponemal tests for initial screening—four
laboratories, New York City, 2005–2006. MMWR Morb Mortal Wkly Rep. 2008; 57:872–875.
[PubMed: 18701877]
18. Mishra S, et al. The laboratory impact of changing syphilis screening from the rapid-plasma reagin
to a treponemal enzyme immunoassay: a case-study from the Greater Toronto Area. Sex Transm
Dis. 2011; 38:190–196. [PubMed: 20706176]
19. Singh AE, Wong T, De P. Characteristics of primary and late latent syphilis cases which were
initially non-reactive with the rapid plasma reagin as the screening test. Int J STD AIDS. 2008;
Author Manuscript

19:464–468. [PubMed: 18574118]

20. Kingston M, et al. U.K. national guidelines on the management of syphilis. Int J STD AIDS. 2008;
19:729–740. [PubMed: 18931264]
21. Hagedorn, HJ. Laboratory diagnosis of syphilis. In: Gross, G., Tyring, SK., editors. Sexually
transmitted infections and sexually transmitted diseases. Springer-Verlag; Berlin, Germany: 2011.
p. 151-162.
22. Binnicker MJ, Jespersen DJ, Rollins LO. Direct comparison of the traditional and reverse syphilis
screening algorithms in a population with a low prevalence of syphilis. J Clin Microbiol. 2012;
50:148–150. [PubMed: 22090407]
23. Binnicker MJ. Which algorithm should be used to screen for syphilis? Curr Opin Infect Dis. 2012;
25:79–85. [PubMed: 22156894]
24. Anonymous. Discordant results from reverse sequence syphilis screening—five laboratories,
United States, 2006–2010. MMWR Morb Mortal Wkly Rep. 2011; 60:133–137. [PubMed:
21307823]
Author Manuscript

25. Cole MJ, Perry KR, Parry JV. Comparative evaluation of 15 serological assays for the detection of
syphilis infection. Eur J Clin Microbiol Infect Dis. 2007; 26:705–713. [PubMed: 17647033]
26. Binnicker MJ, Jespersen DJ, Rollins LO. Treponema-specific tests for serodiagnosis of syphilis:
comparative evaluation of seven assays. J Clin Microbiol. 2011; 49:1313–1317. [PubMed:
21346050]
27. Maple PA, Ratcliffe D, Smit E. Characterization of Treponema pallidum particle agglutination
assay-negative sera following screening by treponemal total antibody enzyme immunoassays. Clin
Vaccine Immunol. 2010; 17:1718–1722. [PubMed: 20844087]
28. Augenbraun M, et al. Hepatitis C virus infection and biological false-positive syphilis tests. Sex
Transm Infect. 2010; 86:97–98. [PubMed: 20332367]
29. Hicks CB, et al. Seronegative secondary syphilis in a patient infected with the human
immunodeficiency virus (HIV) with Kaposi sarcoma. A diagnostic dilemma. Ann Intern Med.
1987; 107:492–495. [PubMed: 3307583]
30. Gottlieb SL, et al. Prevalence of syphilis seroreactivity in the United States: data from the National
Health and Nutrition Examination Surveys (NHANES) 2001–2004. Sex Transm Dis. 2008;
Author Manuscript

35:507–511. [PubMed: 18356772]

31. Sena AC, White BL, Sparling PF. Novel Treponema pallidum serologic tests: a paradigm shift in
syphilis screening for the 21st century. Clin Infect Dis. 2010; 51:700–708. [PubMed: 20687840]
32. Fakile Y, et al. Head-head comparison of reactivity and signal strength value for reactivity among
seven treponemal assays: a preliminary report. STI and AIDS World Congress Vienna, Austria.
Sex Transm Infect. 2013; 89(Suppl. 1):A363.
33. Sharma M, et al. Syphilis serology in human immunodeficiency virus patients: a need to redefine
the VDRL test cut-off for biological false-positives. J Med Microbiol. 2010; 59:130–131.
[PubMed: 19729459]

. Author manuscript; available in PMC 2017 August 23.

 Soreng et al. Page 10
Author Manuscript
Author Manuscript

Figure 1.
Examples of the three clinical stages of syphilis (2)
a. Primary stage: chancre
b. Secondary stage: palmar rash, full body rash
c. Teritary stage: gummatous lesions in the heart and on the skin
Author Manuscript
Author Manuscript

. Author manuscript; available in PMC 2017 August 23.

 Soreng et al. Page 11
Author Manuscript
Author Manuscript

Figure 2.
Antibodies detected in treponemal and nontreponemal testing
Author Manuscript
Author Manuscript

. Author manuscript; available in PMC 2017 August 23.

 Soreng et al. Page 12
Author Manuscript
Author Manuscript

Figure 3.
Syphilis staging and serology (based on Peeling et al. [9]).
Author Manuscript
Author Manuscript

. Author manuscript; available in PMC 2017 August 23.

 Soreng et al. Page 13
Author Manuscript
Author Manuscript

Figure 4.
The “traditional” syphilis testing algorithm (15,22)
Author Manuscript
Author Manuscript

. Author manuscript; available in PMC 2017 August 23.

 Soreng et al. Page 14
Author Manuscript
Author Manuscript

Figure 5.
CDC algorithm recommended for reverse screening
Author Manuscript
Author Manuscript

. Author manuscript; available in PMC 2017 August 23.

 Soreng et al. Page 15

Table 1

Diseases and conditions that can cause false-positive results using nontreponemal and treponemal tests (8,10)
Author Manuscript

Test type Disease or condition

Nontreponemal Inflammation
Autoimmune diseases (especially lupus erythematosus)
Acute viral infections
Hepatitis C
Pregnancy
Recent immunization
Connective-tissue diseases
HIV infection
Injection drug use
Malignancy
Author Manuscript

Advancing age

Treponemal Thyroiditis
Systemic lupus erythematosus, scleroderma
Infectious mononucleosis, genital herpes
Cirrhosis
Pregnancy
Recent immunization
Hyperglobulinemia
Bacterial infections
Brucellosis
Leptospirosis
Lyme disease
Author Manuscript

Malaria
Hansen’s disease
Injection drug use
Yaws, pinta, bejel
Advancing age
Author Manuscript

. Author manuscript; available in PMC 2017 August 23.