Serologic Testing for Syphilis

Expires June 2021

Influencing Test Utilization: The Clinical Laboratory's Expanding Role

This Hot Topic will focus on improving test utilization: both decreasing unnecessary test orders and increasing appropriate test orders. The knowledge gained can be used by the laboratory and shared with physicians to ensure that the correct test is ordered for the correct patient, that the best test methodologies are utilized, and that accuracy and quality remain at the excellent level, ensuring the best medical care.

Syphilis is a disease caused by infection with the spirochete Treponema pallidum and serologic techniques play a major role in the diagnosis and follow-up of this disease. Dr. Matthew Binnicker explains the serological testing algorithms and interpretation for syphilis.

Presenter: Matt Binnicker, Ph.D.



Welcome to Mayo Medical Laboratories' Hot Topics. These presentations provide short discussions of current topics and may be helpful to you in your practice.

Our presenter for this program is Dr. Matthew Binnicker from the Division of Clinical Microbiology at Mayo Clinic in Rochester, Minnesota. Dr. Binnicker will discuss serologic testing for the diagnosis of syphilis. He explains the syphilis testing algorithms and interpretation of results.

Thanks Sharon, and thanks to all of the viewers who have joined me today for this video hot topic. In this presentation, we’ll discuss the laboratory methods used to assist in the diagnosis of syphilis, with an emphasis on how serology should be used and interpreted in cases of suspected syphilis infection. I should preface our discussion by highlighting that the diagnosis of syphilis can be extremely challenging, and unfortunately, each of the current laboratory methods and testing algorithms have significant limitations. So it’s very important to have a solid understanding of the advantages and disadvantages of the various tests and algorithms so that an accurate interpretation can be made.

Presentation Outline

In this presentation, we'll first review some background information on syphilis, including the causative agent and the clinical manifestations of the disease. Second, we'll discuss the various laboratory methods that can be used to assist in the diagnosis of syphilis, with an emphasis on serologic testing. Third, I'll review the traditional algorithm for syphilis serologic testing, and discuss the advantages and limitations of that approach. I'll then discuss how changes in the incidence of syphilis over the past several decades has contributed, in part, to many clinical laboratories adopting a modified, contemporary algorithm for serologic testing. And finally, we'll review key points to consider to ensure the accurate interpretation and proper follow-up of patients with suspected syphilis infection. So let's get started.

Syphilis — The "Great Imitator"

Syphilis is a sexually transmitted disease caused by the spirochete bacterium, Treponema pallidum. You can see the cork-screw shaped, brown-staining organisms in the image on the bottom-right-hand side of your screen. Syphilis is often-times referred to as the Great Imitator, due to the fact that the clinical signs and symptoms are often indistinguishable from other diseases. During the primary stage of the disease, patients may present with a sore, or chancre, typically located at the site of infection. Patients may then show symptoms of rash, fever and general malaise during the secondary stage of disease, and if left untreated, the infection may result in organ damage, paralysis, or mental status changes in the late or latent phase of the illness.

Diagnosis: Laboratory Testing

There are a number of different laboratory methods that have been developed to assist in the diagnosis of syphilis, but many of these tests are not routinely used in the clinical laboratory. The rabbit infectivity test, or RIT assay, demonstrates high sensitivity and specificity for the detection of Treponema pallidum. However, this method is time-consuming and laborious and is limited to research settings. A second method is dark- field microscopy, which allows for the visualization of the spirochete in clinical samples as you can see in the image on this slide. Dark-field microscopy possesses relatively high specificity, but this method is not routinely performed in many clinical labs due to the low prevalence of positive samples. Similarly, microscopy following immunostaining, such as direct fluorescent antibody or silver staining, can be applied to tissue samples. But this method is also uncommon in clinical labs. Polymerase chain reaction, or PCR, is a technology that offers a lot of promise for improving the laboratory diagnosis of syphilis. Several studies have shown that PCR may allow for a rapid and sensitive detection of Treponema pallidum in clinical samples; however, there are currently no commercially-available PCR assays, and this technology is not widely available in clinical laboratories.

Diagnosis: Nontreponemal Serology Tests

That brings us to serology, which has been the mainstay of syphilis diagnosis for decades. There are 2 classes of syphilis serology tests, with the first being the nontreponemal assays. These tests are designed to detect antibodies to nonspecific antigens, such as cardiolipin, that are produced in most patients with syphilis. Examples of nontreponemal tests include the rapid plasma reagin, or RPR, and the venereal disease research laboratory, or VDRL, assays. These tests have demonstrated good performance over the years, but can show low sensitivity in very early or late disease. Importantly, these tests usually revert to negative after successful treatment, and so they can be used to monitor response to therapy.

Diagnosis: Treponemal Serology Tests

The second class of syphilis serology tests is the treponemal assays, which are designed to detect antibodies against specific antigens from Treponema pallidum. Examples include fluorescent treponemal antibody, or FTA, Treponema pallidum particle agglutination, or TP-PA, and newer generation assays such as enzyme immunoassays. Although these tests are typically more specific than nontreponemal assays, they will remain positive for years despite treatment, and this has significant implications that we’ll discuss later in the presentation.

Diagnosis: Traditional Algorithm

Now that we’ve reviewed the laboratory methods that can be used to assist in the diagnosis of syphilis, I wanted to spend a few minutes focusing on how serology is used and results interpreted. For decades, clinical laboratories have used a traditional algorithm for syphilis serology that is outlined on this slide. In this approach, serum samples are first tested using a non-treponemal screening test, such as RPR. If the result of the RPR is negative, the interpretation is that there is no serologic evidence of syphilis and no further testing is required. However, if the RPR is positive, it is recommended that a treponemal test, such as TP-PA or FTA be performed to confirm the results of the RPR. If the treponemal test is negative, the results would not be consistent with syphilis and the RPR would be considered a false-positive result. However, if the treponemal test is positive, this would be consistent with syphilis and treatment would be considered.

As I mentioned, this traditional algorithm has been in place for decades and has several advantages, including the fact that it is a reliable method, especially in high-prevalence settings. In addition, this algorithm utilizes a rapid, inexpensive screening method that is economical and easily implemented in most hospitals and small clinics. However, this approach has limitations, including a low throughput and subjective interpretation of the screening RPR results. This subjectivity, along with the fact that RPR detects mainly non-specific antibodies can translate into higher rates of false-positive results, especially in low-incidence settings.

Syphilis — Incidence

The incidence of disease is of particular importance when selecting an assay or testing algorithm, as the positive predictive value of a laboratory method is impacted by how prevalent the disease is in the population. As you can see on this slide, the incidence of syphilis has declined significantly over the past 60 years.

With the highest rates occurring in large cities along the east and west coasts and in the southern portion of the United States. Because of the limitations of the traditional testing algorithm, and the fact that the overall rate of syphilis nationwide is low, many clinical labs have adopted a reverse algorithm for syphilis serologic testing.

Diagnosis: Contemporary Algorithm

In this approach, serum samples are first tested by a treponemal-specific screening test, such as one based on enzyme immunoassay, or EIA, technology. Negative results by the screening EIA are interpreted as no serologic evidence of syphilis, and no further testing is required unless early syphilis is suspected. If, however, the treponemal screening test is positive, the sample should be tested by a nontreponemal test to assess the patient’s disease and treatment status. A positive result by the nontreponemal test generally is suggestive of syphilis, which is either untreated or recently treated. In contrast, a negative result by the nontreponemal test can yield several different interpretations based on the patient’s clinical and treatment history. We’ll go over these scenarios in more detail in just a few minutes.

The contemporary algorithm for syphilis serology has several advantages, including a specific screening test that yields an objective result. In addition, this approach lends itself to high throughput, automated screening by methods such as EIA, chemiluminescence immunoassay or multiplex flow immunoassay technologies, so this is algorithm is often appealing to high-volume reference laboratories.

However, this reverse testing algorithm comes with several significant limitations that we should discuss. First, most treponemal assays, especially those based on newer technologies such as EIA or multiplex platforms, are more expensive compared to RPR. Second, treponemal tests remain positive following therapy, so the implementation of the reverse algorithm has led to many health care providers and laboratorians asking, “How do we interpret results from patients that are treponemal screen positive, but negative by nontreponemal tests?”

Diagnosis: Proposed Algorithm with Treponemal Screening Test

This important question prompted members of the CDC and the Association of Public Health Laboratories to meet to discuss syphilis diagnostic testing, and subsequently propose that the following algorithm be followed when a treponemal-specific test is used for screening. In this algorithm, samples that are positive by a treponemal test but are negative by RPR should be tested by a second treponemal assay, such as the Treponema pallidum particle agglutination assay. The implementation of a second treponemal test will likely assist in the interpretation of results.

So at this point, you may still be asking yourself, “How do I interpret the results of syphilis serology and what follow-up action is required when I’m confronted with different patterns of results?”

To address these questions, let’s walk through several of the common scenarios and discuss which testing is recommended, and whether or not follow-up is required.

Interpretation and Follow-up

In the first scenario, you’re seeing a patient with no history, or an unknown history, of syphilis. The result of the screening treponemal test is negative, so no additional testing is required. We interpret this result as “no serologic evidence of syphilis” and in most situations no further testing is required, unless clinically indicated, such as a suspected early syphilis infection.

In the second scenario, a patient presents with an unknown history of syphilis, and the results of both the screening treponemal test and the RPR are positive. In this situation, a second treponemal test is not required, and we would interpret these results as either untreated syphilis, or recently treated syphilis. For follow-up, I would refer you to the CDC website for up-to-date treatment guidelines and also recommendations on further testing, which generally includes following RPR titers to demonstrate a response to therapy.

In the third scenario, a patient with no history, or an unknown history, of syphilis tests positive by the screening treponemal assay, but the reflex testing by RPR is reported as negative. In this situation, a second treponemal test is performed on the same serum sample and is reported as negative. These results would be consistent with a false-positive screening test, and no further testing is required unless clinically indicated. I should highlight that the second treponemal test should be a different method than the one used for screening, and the CDC has recently recommended that TP-PA be used to resolve discordant screening treponemal and RPR results.

In the fourth scenario, a patient with unknown history tests positive by the treponemal screening assay, negative by RPR, but is also positive by the second-tier treponemal test. These results could be consistent with several different interpretations, depending on the historical and clinical evaluation. If the patient has been treated in the past with an antimicrobial with anti-Treponemal activity, these results could be suggestive of past, successfully treated disease. However, if the patient lacks any treatment history, these results may be consistent with latent syphilis, which may require treatment. These varying interpretations stress the importance of a thorough historical and clinical evaluation in patients with these results.

Finally, you see a patient with a known history of syphilis who tests positive by the treponemal screening test but is negative by RPR. If the patient was treated for syphilis in the past, then these are the expected results and would indicate past, successfully treated disease. In this situation, no further testing or follow-up would be required.

So as you can see from these different scenarios, interpretation of syphilis serology results is complex and relies on careful consideration of the laboratory and clinical findings.


In summary, the traditional serology algorithm, which includes a nontreponemal screening test followed by a treponemal confirmatory test, is still recommended in high-incidence settings. However, the reverse algorithm is widely used due to advantages in specificity and laboratory workflow. When a treponemal test is used for screening, a nontreponemal should be performed on screen-positive samples to assess the patient’s disease and treatment status. And a second treponemal test, such as the TP-PA assay, should be used on samples with discordant screen-positive, but RPR-negative results.

Before we wrap up, I just wanted to make a few additional concluding remarks. First, routine screening for syphilis in healthy, pregnant women should be performed by RPR or an IgG-based treponemal assay. Including syphilis IgM testing in healthy, asymptomatic patients increases the potential for false-positive results. Second, the pretest probability is critical when ordering and interpreting syphilis serology. In other words, you should always question and investigate positive results in patients without symptoms and in patients with low risk and low pretest probability of disease. And finally, it is imperative that health care providers perform a thorough historical and clinical evaluation following positive syphilis serology results so that an accurate interpretation can be made.


MCL Education

MCL Education

This post was developed by our Education and Technical Publications Team.