Considerations for Endpoint Titer Determination in Syphilis Testing Using Newly Marketed, Automated Rapid Plasma Reagin Instruments

Syphilis is a sexually transmitted disease (STD) caused by the bacterium Treponema pallidum, subspecies pallidum (T. pallidum). ¹ The resurgence of syphilis in the last 2 decades is a major public health concern in the United States. In 2020, a total of 133 945 cases of all stages of syphilis were reported in the United States, an increase of more than 70% since 2015. The national rate of congenital syphilis has increased 254% since 2016. In 2020, a total of 2148 cases of congenital syphilis were reported, including 149 congenital syphilis–related stillbirths and infant deaths. ² However, syphilis diagnosis is still challenging, partially because of overlapping and ambiguous clinical presentation, particularly during early infection. ³ For laboratory testing, darkfield microscopy examinations and direct molecular detection of T. pallidum from clinical specimens are definitive methods for diagnosing early syphilis, but both methods are currently not widely performed at local, reference, or public health laboratories and have challenges in identifying asymptomatic or early-stage infection without the presence of visible lesions for suitable specimen collection. ⁴ Darkfield microscopy requires special equipment and experienced operators for reliable results. ⁵ US Food and Drug Administration (FDA)–cleared molecular tests for syphilis are still not available, and laboratory-developed molecular tests are limited in availability. Therefore, serological tests that detect treponemal and nontreponemal antibodies remain the mainstay for routine laboratory diagnosis of active syphilis infection. ⁶

Treponemal tests are specific for detection of T. pallidum antibodies, which can become reactive as early as 2 weeks after infection and remain detectable for life, even after successful treatment. ⁷ Therefore, reactivity to a treponemal test alone, while implying infection, does not determine whether the infection is recent or past or whether it has been treated. Syphilis infection leads to the production of nonspecific antibodies directed against lipoidal material released from damaged host cells as well as lipoprotein-like material and possibly cardiolipin released from the treponemes.^6,8 Nontreponemal tests detect these nonspecific antibodies, which appear later than treponemal antibodies and may not be reactive until about 6 weeks after infection. ⁷ These nontreponemal antibodies can also be induced by various diseases or medical conditions unrelated to syphilis (biological false-positive), such as autoimmune disease, acute viral infection, vaccinations, connective-tissue diseases, malignancy, injection drug use, pregnancy, and aging.^9-14 False-negative nontreponemal test results can occur due to the prozone effect, which occurs when high antibody titers prevent antibody/antigen lattice formation.^6,15 The amount of antibody produced in response to infection is measured and reported as a titer. Two nontreponemal tests have been widely used: the Venereal Disease Research Laboratory (VDRL) test and the rapid plasma reagin (RPR) test. The VDRL test is a microscopic flocculation assay that uses an antigen containing cardiolipin, lecithin, and cholesterol. Flocculates will form when the VDRL antigen combines with lipoidal antibodies in sera or cerebrospinal fluid from syphilis patients. The RPR card test is a macroscopic nontreponemal flocculation test that uses a modified VDRL antigen suspension containing choline chloride to eliminate the need to heat inactivate serum. It also contains finely divided charcoal particles as a visualizing agent, which makes RPR tests relatively easy to perform. ⁶ Thus, RPR is the most common first-line nontreponemal test used to screen for syphilis infection. Up to 72.8% of US public health laboratories were conducting RPR tests in 2016.^6,16 In a manual RPR test, antigen is mixed with unheated serum or plasma on plastic-coated cards; if antibodies are present, flocculation reactions take place. Results are interpreted by visually reading the cards in good light. Recently, automated RPR tests have been introduced for high-throughput testing and more objectively interpreted nontreponemal screening using automated methods. This commentary provides insights on the newly evolved automated RPR test platforms and outlines considerations for using these new tests for syphilis testing.

Automated RPR Test

Recently, 3 automated nontreponemal RPR platforms have been cleared by the FDA: AIX 1000 RPR Automated Test System (Gold Standard Diagnostics, Inc; hereinafter referred to as AIX 1000), ASI Evolution automated RPR syphilis test (Arlington Scientific, Inc; hereinafter referred to as ASI Evolution), and BioPlex 2200 Syphilis Total & RPR Assay (Bio-Rad Laboratories, Inc; hereinafter referred to as BioPlex RPR) (Table). The AIX 1000 is the world’s first fully automated system for nontreponemal RPR and received FDA 510(k) clearance in November 2015. It is a flocculation test consisting of the AIX 1000 Analyzer and RPR test reagents that determines the presence of reagin antibodies in human serum. The AIX 1000 Analyzer delivers serum from collection tubes into test wells. After the antigen suspension is added and mixed with sera, if antibodies are present, antigen–antibody will form black flocculants due to the presence of carbon particles. A high-resolution image is captured by an onboard camera and analyzed by proprietary software. ¹⁷ The AIX 1000 performs the RPR test procedure for qualitative screens of up to 192 samples in 90 minutes.

Table.

Summary of features of automated rapid plasma reagin (RPR) instruments for the testing of syphilis, United States, 2022 ^a

Instrument (manufacturer)	FDA decision date	Assay technology	Sample matrix	Sample capacity	Default range of titers detected	510(k) reference	Product URL
AIX 1000 Rapid Plasma Reagin Automated Test System (Gold Standard Diagnostics)	November 2015	Flocculation test	Serum	192 tests/90 min	1:2-1:256	https://www.accessdata.fda.gov/cdrh_docs/reviews/K150358.pdf	https://www.gsdx.us/aix-1000
ASI Evolution Automated RPR Analyzer (Arlington Scientific)	June 2018	Flocculation test	Serum or plasma	190 tests/60 min	1:1-1:2048	https://www.accessdata.fda.gov/cdrh_docs/reviews/K173376.pdf	https://www.arlingtonscientific.com/asi-evolution
BioPlex 2200 Syphilis Total and RPR Assay (Bio-Rad Laboratories, Inc)	May 2017	Multiplex flow immunoassay	Serum or plasma	200 tests/60 min	1:4-1:2048 b	https://www.accessdata.fda.gov/cdrh_docs/reviews/K170413.pdf	https://info.bio-rad.com/bioplex-syphilis

^aInformation is current as of October 2022. See US Food and Drug Administration (FDA) websites for updates on these and future tests.

^bSome dilutions may require a 1-step offline dilution.

The ASI Evolution was cleared by the FDA in June 2018 for diagnostic screening, blood donor screening, and cadaveric (non–heart-beating) tissue screening. Similar to AIX 1000, it is a flocculation test for the detection of reagin antibodies in human serum and plasma. The ASI Evolution automates the dispensing of serum or plasma samples and the dispensing of carbon antigen reagent. The instrument also automates the RPR agglutination measurement and image-processing algorithm using an internal charge-coupled device camera. The ASI Evolution can process and analyze up to 190 samples per 60 minutes for qualitative screens. ¹⁸

The FDA cleared BioPlex RPR in May 2017. It is a multiplex flow immunoassay and the only dual treponemal/nontreponemal test. It is intended for the qualitative detection of total (immunoglobulin G/immunoglobulin M) antibodies to T. pallidum and the qualitative detection and/or titer determination of nontreponemal reagin antibodies in human serum or plasma. The BioPlex RPR uses T. pallidum fusion protein and cardiolipin antigen-coated fluoromagnetic beads with unique fluorescent signatures to identify the presence of antibodies to T. pallidum and reagin in a 2-step assay format. BioPlex RPR can run up to 200 samples in 60 minutes. In December 2021, the FDA issued a letter about possible false RPR reactivity with BioPlex RPR following a COVID-19 vaccine. ¹³ Therefore, the BioPlex RPR was taken off the US market until further notice from the manufacturer. This false-positive issue is not further discussed here.

Nontreponemal (RPR) Titer

The Centers for Disease Control and Prevention (CDC) recommends that all samples with a reactive qualitative nontreponemal test be tested quantitatively to determine the actual titer. ³ The titer is a measure of the amount of antibodies produced in response to infection. Titers of nontreponemal antibodies usually decline or become nonreactive with effective treatment. Titer change of nontreponemal antibodies can be used to monitor treatment responses. A 4-fold change in titer, equivalent to a change of 2 dilutions (eg, from 1:16 to 1:4 or from 1:8 to 1:32), is considered necessary for demonstrating a clinically significant difference when the same serologic test is used. ³ Serological cure is defined as a ≥4-fold decline in nontreponemal titers or as seroreversion to nonreactive results after treatment. ³ However, in certain cases, nontreponemal antibodies might decrease <4-fold (serological nonresponse) or might persist with low-level titers without seroreversion after initial ≥4-fold decline (serofast status). ¹⁹ In cases of reinfection, nontreponemal titers could increase ≥4-fold from the individual’s prior posttreatment test. In addition, nontreponemal titer of a neonate, as compared with the mother’s titer at delivery, is critical for congenital syphilis diagnosis. Neonates with reactive nontreponemal tests should also receive serologic testing (ie, RPR every 2 to 3 months until the test becomes nonreactive). ²⁰ Therefore, nontreponemal test antibody titers are critical for syphilis diagnosis and monitoring treatment responses.

In manual RPR card tests, each reactive sample is initially quantitated to 1:16 dilutions. If the highest dilution tested (1:16) is reactive, samples are further diluted down to establish actual endpoint titers. However, the 3 FDA-cleared automated RPR tests were approved only for a limited RPR titer range, and after that, manual titration must be performed. The AIX 1000 performs the semiquantitative titers from 1:2 to 1:256 (Table). ²¹ The quantitative titer range begins at 1:2. For specimens that are reactive by qualitative testing and nonreactive at 1:2 when performing the quantitative test, the titer is considered 1:1 (undiluted). The ASI Evolution quantitative titer range begins at 1:1 (undiluted) and serially dilutes samples up to 1:2048 to determine endpoint titers. ¹⁸ Qualitative RPR results using BioPlex RPR are classified as nonreactive (<1.0 Antibody Index [AI]) and reactive (≥1.0 AI). RPR reactive samples are titered using the onboard dilution procedure, and available ranges are from 1:4 to 1:64 and could be extended further from 1:128 to 1:2048 following the manufacturer’s instructions (Table). ²²

CDC laboratory staff received reports that some diagnostic laboratories, while using automated RPR tests, did not perform the necessary manual titrations when RPR titers reached the end of the approved spectrum. Instead of reporting an actual endpoint titer, the highest or lowest titers from the automated instruments were reported or less than or greater than the lower and upper limits titers were reported (eg, <1:4 or >1:256). All entities that received the laboratory results for an automated test may not be aware of the test limitations and/or reporting language and interpretation when the new tests were introduced. Without accurate RPR titers reported, these patients might undergo unnecessary or missed evaluations, treatment, or follow-up visits for serological monitoring.

Lessons Learned for Diagnostic Laboratories

CDC guidance states that serum should be diluted to identify the endpoint titer by any method, including automated methods. ³ It is necessary to perform serial dilutions on all RPR reactive samples until an endpoint titer has been identified. Manufacturer instructions should be followed for automated RPR instruments, which may require off-instrument manual dilutions or, alternatively, separate endpoint titer determination using manual RPR. For consistency, if performing additional manual testing after automated RPR, manual RPR but not VDRL should be used. VDRL and RPR are equally valid assays, but results from these 2 tests cannot be compared directly. ³ Sequential serologic RPR tests for a patient should be performed using the same method, preferably by the same laboratory. ³

The best laboratory practice is to take the following steps when using automated RPR tests:

1. The report should clearly state the actual endpoint titer of the final laboratory RPR test result. Reports should not contain “greater than” or “less than” symbols.

2. If an immediate RPR result is requested, laboratories can consider reporting an initial qualitative RPR result or a preliminary titer result (defined as RPR titers from automated instruments at the lower or upper limit of the default titer range that has not been serially diluted to determine an endpoint titer). At a minimum, the report should clearly (a) state the dilution range tested on preliminary results, (b) indicate that endpoint titer determination is pending, and (c) specify a defined turnaround time that will not delay treatment, counseling, and partner services. A final endpoint titer should be completed and reported.

3. The laboratory should ensure that electronic clinical laboratory data entry and reporting systems are set up for correct data input and transmission of the preliminary laboratory result, including the name of assay used for testing, titer range, and turnaround time for pending endpoint titers. Automated RPR results that have a “greater than” or “less than” symbol should not be imported as a simple quantitative value (eg, if the result is “>32,” then “>32” should be imported, not “32”) to allow accurate transmission of results for specimens that have not yet been subjected to endpoint titration.

It is also a good practice for federal agencies and organizations and other public health professionals (eg, in public health departments) to assist in this effort with action steps that include the following:

• Communicating with testing laboratories that use automated RPR systems to ensure that endpoint titration is done by laboratories.

• Alerting health care providers about the increasing use of automated RPR testing systems and the need to ensure that endpoint titration is done by laboratories.

• Working together with laboratories to support best practices for patient clinical management by ensuring determination and reporting of endpoint titers if they are using any automated RPR instrument.

• Reviewing and conducting quality control of electronic laboratory reports, case reports, and/or data feeds of RPR results to determine whether any RPR results with a “greater than” or “less than” symbol are received as originally reported, and ensuring that RPR results are not entered as a simple value (eg, if the result is “>32,” then “>32” should be imported, not “32”). Doing so will allow for accurate reporting of results for specimens that have not yet been subjected to endpoint titration.

Conclusion

The advent of FDA-cleared automated RPR systems as part of laboratories’ testing algorithm for clinical syphilis diagnostic purposes can facilitate high-volume testing and expedited turnaround. Automated RPR systems are set up to perform both qualitative and quantitative RPR, but the quantitative range may be limited. Laboratories may be reporting results only within the range of the instrument and indicating results for specimens with titers that exceed the range as less than or greater than the lower and upper limits rather than a traditional endpoint titer. All reactive RPR results should have an actual endpoint titer. Individual patients’ titers are used for temporal comparison, and quantitative titers are critical for health care providers to determine response to treatment or possible reinfection, as stated in the CDC STI Treatment Guidelines. ³