CDC updates on syphilis testing for the clinical laboratory

The recent release of the 2022 Sexually Transmitted Infection (STI) Surveillance Report by the Centers for Disease Control (CDC) has confirmed the continuing increase in reported syphilis cases, which have reached numbers not seen since 1950. These statistics correlate with the ongoing opioid epidemic and the CDC calls the two “intersecting epidemics.” The data also confirm that syphilis continues to disproportionately affect minoritized racial and ethnic populations.

The rise in syphilis among women of childbearing age is particularly alarming, with congenital syphilis rates increasing by 937.3% since 2013. The clinical laboratory has a significant opportunity to help reduce the spread of syphilis by providing timely, accurate, and appropriate screening and treatment monitoring.

With the increase in syphilis and the recognized importance of clinical laboratory testing, the CDC in February 2024 released the first ever recommendations for syphilis testing in the clinical laboratory (MMWR Recomm Rep 2024; doi: 10.15585/mmwr.rr7301a1). This extensive document provides a review of current research on syphilis biology and clinical laboratory tests, as well as specific recommendations for clinical laboratories. Here we’ll review a few highlights in updates of the science and terminology, as well as the recommendations from the CDC for performing testing.

Using the right tests for syphilis

Syphilis diagnosis relies on two types of serologic tests that detect the host’s antibody response to the infection. The first type, known as treponemal tests, identifies antibodies (usually IgM and IgG) specific to proteins found on Treponema pallidum. These antibodies typically remain detectable for life, even after successful treatment.

The second type, non-treponemal tests, has been thought to detect antibodies generated in response to tissue damage caused by infection. More recent work, highlighted in the CDC guidelines, indicates that the lipoidal antigens used in non-treponemal tests (cardiolipin, cholesterol, phosphatidylcholine) are found in the membranes of T. pallidum and hosts alike. These tests therefore are more properly termed “lipoidal antigen” tests.

Unlike treponemal antibodies, non-treponemal (lipoidal antigen) antibodies generally decline after treatment, making these tests useful for monitoring treatment effectiveness and distinguishing between new, active infections and those that have been previously treated. Non-treponemal tests include the rapid plasma regain (RPR) and Venereal Disease Research Laboratory (VDRL) tests. Positive tests are titrated to provide a semi-quantitative titer result.

Importantly, these lipoidal antigens are also found in host cell membranes. As a result, reactive tests may not be associated with syphilis, resulting in biological false positive tests. Biological false positives can account for as much as 11% of positives tests in some populations, thus a positive non-treponemal test alone is insufficient to diagnose syphilis.

Following the CDC-recommended algorithm

With these two types of tests available, CDC recommends that laboratories perform screening through algorithmic testing (Figure 1). The original CDC-recommended screening algorithm is the forward or traditional algorithm, which starts with a non-treponemal test and, when positive, is assessed by treponemal testing to ensure the reactive test is specific for syphilis.

As treponemal testing became more automated, the reverse algorithm was introduced. In this algorithm, a reactive treponemal test is followed by a non-treponemal test to determine whether the infection is active. If the non-treponemal test is negative, the laboratory performs a second treponemal test to ensure that the first treponemal test was not falsely positive. Each algorithm has limitations; however, the reverse algorithm may be more likely to catch early and late syphilis infections.

Key CDC recommendations

The laboratory recommendations for syphilis testing from the CDC that pertain to serologic testing are listed below in italics.

Recommendation for endpoint titers. This is a reporting recommendation that all non-treponemal (lipoidal antigen) titers be reported down to a 1:1 titer and up to an end point titer (i.e. – no “>” or “<” in result reporting). For laboratories using automated RPR testing that may not report down to a 1:1 titer, these specimens are recommended to be reflexed to a manual RPR method. Many laboratories do not currently perform endpoint titers, but rather dilute to a specific titer and report as “>”.

Recommendation for syphilis serologic testing algorithm. Full forward or reverse syphilis serologic algorithms should be used for diagnosis (Figure 1). This recommendation highlights the need for clinical laboratories to incorporate reflex testing algorithms that facilitate compliance with either a forward or reverse algorithm without requiring the ordering provider to select the subsequent test. This approach also reduces the need for patients to return to their healthcare provider or a blood draw site for a final diagnosis.

Recommendation for serologic syphilis testing. The same non-treponemal (lipoidal antigen) test should be used when monitoring patients for treatment response or reinfection; RPR and VDRL titers are not interchangeable. The second treponemal test in the reverse algorithm — and the treponemal test in the forward algorithm — is often a manual test with high specificity, and the Treponema pallidum particle agglutination (TPPA) test is the preferred manual treponemal test based on performance characteristics compared to other manual treponemal tests.

It is notable that there is little published data on the performance of automated treponemal tests stratified by syphilis stage. Available literature seems to indicate comparable performance overall with TPPA; however, additional data are needed.

Recommendation for syphilis serologic testing in pregnant persons and recommendation for syphilis serologic testing in persons living with HIV/AIDS. Historically there has been a lack of clarity on the best testing and interpretation in these populations. Current evidence indicates that these populations should receive the same testing and interpretation regardless of pregnancy or HIV status.

The CDC Laboratory Recommendations for Syphilis Testing is a comprehensive document with additional information on test choice, test performance, recommendations for sample processing, result reporting, and current research. Amid this ongoing epidemic, the CDC emphasizes that laboratories are critical in the public health response. Implementing the CDC recommendations is an important first step for clinical laboratories in reducing the public health burden.

Sarah Wheeler, PhD, FADLM, CC(NRCC) is an associate professor in the department of pathology and the associate medical director of clinical immunopathology at the University of Pittsburgh Medical Center. She is also the medical director of the automated laboratory at UPMC Mercy Hospital and medical director of clinical chemistry at UPMC Children’s Hospital of Pittsburgh. Email: [email protected]