Nontreponemal Tests: Screening and Monitoring
Nontreponemal tests, such as RPR and VDRL, are cornerstone assays in the Syphilis Diagnosis Algorithm. They serve as initial screening tools and are crucial for diagnosing active infection in symptomatic individuals or known contacts. These tests also play a vital role in assessing treatment efficacy and detecting potential reinfections. While RPR and VDRL remain prevalent in public health labs across the US, other nontreponemal tests exist but are less common. It’s critical to note that results from different nontreponemal tests are not interchangeable due to variations in methodologies and subjective titer interpretations. Consistent monitoring requires using the same test method and specimen type for each patient.
These manual flocculation tests detect antibody-antigen complexes. They rely on microscopic or macroscopic observation of precipitates formed by antibodies binding to a cardiolipin, cholesterol, and phosphatidylcholine antigen mixture. VDRL tests necessitate microscopic reading at 100x magnification. RPR tests utilize charcoal particles for macroscopic reading, and TRUST tests employ toluidine red dye.
Automation is available for certain RPR tests, enhancing throughput and digital result analysis. Regardless of the method, reporting endpoint titers, not just qualitative values, is essential for accurate clinical interpretation. Automated RPR tests may have dilution range limitations, necessitating reflex testing with manual RPR methods to establish endpoint titers beyond these constraints.
Test performance is influenced by specimen type, syphilis stage, autoimmune conditions, and co-infections. Nontreponemal tests might exhibit lower sensitivity in early primary syphilis and tend to decrease over time, even post-treatment. Careful consideration of test and specimen type is crucial before testing, as serum and plasma are not always interchangeable, and some tests require specimen heat treatment.
Subjectivity in manual test interpretation and inter-laboratory variability pose challenges for clinicians, particularly in treatment monitoring and reinfection assessment. Reactive results can be false positives due to recent infections, vaccinations, injection drug use, or underlying chronic conditions. However, when performed by experienced technicians and combined with treponemal tests, clinical history, and physical examination, nontreponemal tests are highly reliable for screening and titer determination.
Serologic Response to Treatment and Endpoint Titers
Nontreponemal antibody titers typically decrease at least fourfold within 12 months post-treatment, especially in early-stage syphilis. Titers may become nonreactive, particularly in pre-secondary stage treatment. However, some patients show less than a fourfold decrease despite effective treatment, with HIV-infected individuals being more prone to inadequate responses in early syphilis. Titers may also persist at low levels (≤1:8), known as the serofast state, especially in late-stage or repeat syphilis cases.
Recommendation for endpoint titers. Endpoint titers are crucial for nontreponemal assays (RPR and VDRL) and must be clearly reported without symbols like “>” or “<“.
Comment and evidence summary. Nontreponemal antibody titers correlate with infection status and are vital for monitoring treatment outcomes. A fourfold titer change is clinically significant. Reporting titers and reflex testing for out-of-range automated RPR tests are necessary for proper clinical management.
Prozone Phenomenon in Syphilis Serology
Agglutination or flocculation assays depend on antigen-antibody complex formation. The prozone effect, or hook effect, is a false-negative phenomenon caused by excess antibodies, typically in undiluted serum, hindering lattice formation. This is more common in early syphilis stages when antibody levels are very high. Diluting the serum sample can resolve the prozone effect.
Studies indicate that prozone effects in RPR tests are rare in general screening populations. However, it’s more frequent in primary and secondary syphilis. While routine dilution of all nonreactive nontreponemal tests is not recommended, laboratories should perform dilution series to rule out prozone when clinically indicated, especially in symptomatic patients with nonreactive undiluted test results or unusual graininess in the test.
Biologic False Positives (BFP)
A biologic false positive (BFP) occurs when a nontreponemal test is reactive due to conditions other than syphilis. BFP reactors have reactive nontreponemal tests but nonreactive confirmatory treponemal tests. BFP reactions are estimated to occur in 0.2%–0.8% of the population and are associated with various conditions, including other infections (malaria, HIV), vaccinations, autoimmune disorders, and injection drug use. Health departments often maintain records of known BFP reactors for future syphilis evaluation.
Treponemal Tests: Confirmation and Beyond
Treponemal tests confirm reactive nontreponemal test results and are valuable in early primary syphilis when nontreponemal tests may be nonreactive. They are also automated for high-throughput screening, particularly in reverse sequence algorithms and blood banks. Treponemal antibodies usually persist for life, even after treatment, except in early primary syphilis where seroreversion can occur in 15%–25% within 2-3 years post-treatment. Seroreversion is less likely after secondary syphilis or longer-duration infections and can also occur in advanced HIV/AIDS.
Unlike nontreponemal tests, treponemal tests are not suitable for monitoring treatment response. For patients with a history of treated syphilis and reactive treponemal tests, repeat treponemal testing is unhelpful for detecting reinfection. Nontreponemal titers, clinical history, physical examination, and risk assessment are crucial in these cases.
Manual treponemal tests include FTA-ABS, TPPA, and EIAs. Automated immunoassays (EIA, CIA, MFIA) are also available. These are often used as initial tests in reverse sequence algorithms. Serum is generally used, but some tests are validated for plasma and dried blood spots (DBS), although DBS is not FDA-cleared.
Test outputs are typically index values (signal-to-cutoff ratio or fluorescence ratio). Equivocal results require retesting per package inserts. The strength of the signal-to-cutoff ratio in immunoassays correlates with reactivity in confirmatory tests. Research suggests that signal-to-cutoff values could potentially reduce the need for additional TPPA confirmation in discordant results.
For discordant nontreponemal and treponemal results, a different treponemal assay (e.g., TPPA) is recommended. Signal-to-cutoff values can be an additional data point in complex situations, like low-risk pregnant individuals. Consultation with the STD Clinical Consultation Network is advised for complex cases.
Blood Bank Screening for Syphilis
Blood donations are mandated to be screened for T. pallidum antibodies. Reactive donations result in donor deferral and notification. FDA guidelines and lists of approved screening tests for blood donors are publicly available.
Syphilis Screening Algorithms: Traditional vs. Reverse
The traditional syphilis diagnosis algorithm starts with a nontreponemal test, with reactive samples confirmed by a treponemal test. This algorithm was historically favored due to the lower cost and complexity of nontreponemal tests.
The reverse sequence algorithm begins with an automated treponemal immunoassay, followed by a quantitative nontreponemal test for reactive samples. Discordant results are resolved using a second, different treponemal assay like TPPA.
Surveys indicate a mix of algorithm usage in US laboratories, with traditional algorithms still more common, particularly in public health labs. However, the reverse algorithm is gaining traction due to automation and efficiency.
Studies comparing both algorithms show that the reverse algorithm may detect more cases but also has a higher false-positive rate, especially in low-prevalence populations. Cost-effectiveness varies depending on syphilis prevalence and local testing costs.
Each algorithm has advantages and disadvantages. The traditional algorithm might miss early or late latent syphilis, while the reverse algorithm might increase false positives in low-prevalence settings. Antibody development for both test types can take up to 2 weeks post-primary infection. Automated treponemal immunoassays offer higher throughput and lower labor costs in high-volume labs. Algorithm selection should consider cost, labor, volume, throughput, and turnaround time. Public health programs and clinicians need both nontreponemal and treponemal results for timely clinical management and reporting. Laboratories must ensure seamless result integration and reporting, especially when using multiple labs for different tests within an algorithm.
Recommendation for syphilis serologic testing algorithm. Combine nontreponemal and treponemal antibody tests for syphilis diagnosis. Avoid sole reliance on a single reactive test. Both traditional and reverse algorithms are acceptable. Algorithm choice should be based on laboratory resources, patient populations, and cost considerations.
Comment and evidence summary. Both algorithms are about 99% concordant. Cost-effectiveness varies by setting. Combining both test types is crucial for accurate syphilis staging and diagnosis.
Specimen Collection and Storage for Syphilis Serology
Serum, plasma, and CSF are suitable specimens for syphilis antibody assays. Specific collection, transport, and storage guidelines vary by test and are detailed in product inserts.
Serum and Plasma Collection and Storage
Serum is the most common specimen. Collect whole blood in tubes without anticoagulants, coagulants, or serum separators (unless specified in product insert). Collect approximately 2.5 times the required serum volume. 1mL is generally sufficient for both nontreponemal and treponemal tests. Allow 15-30 minutes for clotting at room temperature. Avoid refrigeration before clotting. Centrifuge at 1,000–2,000 xg for 10 minutes to separate serum. Transfer serum to polypropylene tubes. Store serum at 2°C–8°C for up to 5 days or ≤−20°C for longer storage. Avoid frost-free freezers. Samples should be hemolysis-free, non-icteric, and non-lipemic. Aliquot serum to prevent repeated freeze-thaw cycles.
Plasma is acceptable for some assays. Collect whole blood in tubes with EDTA, citrate, or heparin anticoagulants. Collect 2.5 times the required plasma volume. Centrifuge at 1,000–2,000 xg for 10 minutes. Transfer plasma to polypropylene tubes. Test within 1-5 days or store frozen according to product inserts. Plasma storage time is generally shorter than serum. Sample quality and aliquotting guidelines are similar to serum.
CSF Collection and Storage
CSF collection requires trained personnel. Collect approximately 1 mL CSF in a polypropylene tube. Store at 2°C–8°C for ≤5 days if testing is delayed or ≤−20°C for longer storage. Avoid blood contamination during collection.
Serologic and CSF Antibody Test Performance: Sensitivity and Specificity
Sensitivity in Primary, Secondary, Latent and Tertiary Syphilis
Nontreponemal tests have variable sensitivity in primary syphilis (48.7%–92.7% for RPR, 50.0%–78.4% for VDRL), potentially lower than treponemal tests in early stages. Sensitivity approaches 100% in secondary syphilis for both RPR and VDRL. Sensitivity decreases in latent syphilis and tertiary syphilis.
Treponemal tests generally show higher sensitivity in primary syphilis compared to nontreponemal tests (FTA-ABS: 78.2%–100%, TPPA: 86.2%–100%). Sensitivity is near 100% in secondary syphilis. Sensitivity remains high in latent and tertiary syphilis, although slightly lower for late latent stages.
Specificity of Serologic Tests
Specificity of RPR and VDRL is generally high and comparable. Treponemal tests like FTA-ABS and TPPA also exhibit high specificity (94%–100%). However, FTA-ABS specificity can be affected by laboratory expertise. TPPA is generally preferred over FTA-ABS due to superior performance. Immunoassays generally show high specificity (94.5%–100%), except for Trep-Sure which has lower specificity.
Recommendation for serologic syphilis testing. Nontreponemal tests are not interchangeable for titer determination. Use the same test for follow-up. TPPA is the preferred manual treponemal test.
Comment and evidence summary. RPR and VDRL have similar sensitivity and specificity. Consistent testing with the same nontreponemal test is crucial for titer monitoring. TPPA demonstrates superior sensitivity compared to FTA-ABS, especially in primary syphilis.
CSF Antibody Tests for Neurosyphilis
Neurosyphilis diagnosis is challenging due to lack of diagnostic consensus and variable reference standards. CSF VDRL is the only FDA-cleared test for neurosyphilis. CSF FTA-ABS is used off-label for its negative predictive value, especially when CSF VDRL is negative but neurosyphilis is still suspected.
Nontreponemal and Treponemal Tests in CSF
CSF VDRL sensitivity ranges from 66.7% to 85.7% and specificity from 78.2% to 86.7% in symptomatic neurosyphilis. CSF RPR shows similar performance. CSF VDRL is the only FDA-cleared test for neurosyphilis.
CSF FTA-ABS sensitivity in definitive neurosyphilis is 90.9%–100%. Specificity varies widely (55%–100%) in patients with syphilis but without neurosyphilis, potentially due to passive antibody diffusion across the blood-brain barrier. A negative CSF FTA-ABS can help rule out neurosyphilis.
Limited data exist for CSF TPPA and automated immunoassays in neurosyphilis. CSF TPPA sensitivity ranges from 75.6%–95.0%. Further research is needed before recommending CSF TPPA for off-label neurosyphilis diagnosis.
CSF Tests for Ocular Syphilis and Otosyphilis
Data are limited on CSF test performance for ocular syphilis and otosyphilis. CSF analysis (cell count, protein, CSF-VDRL) may be helpful in ocular syphilis without neurologic symptoms. CSF testing is generally not recommended for isolated otosyphilis with normal neurologic examination. CSF treponemal tests are not recommended for off-label use in suspected ocular syphilis or otosyphilis without neurosyphilis signs.
Serologic Tests for Congenital Syphilis
Treponemal tests on neonates are not recommended due to passive maternal antibody transfer. Quantitative nontreponemal tests (RPR or VDRL) are recommended for newborns of mothers with reactive syphilis serology. Serum, not umbilical cord blood, should be tested. Use the same nontreponemal test for infant and mother for titer comparison. IgM tests are not recommended for congenital syphilis diagnosis.
Serologic Test Performance in Pregnancy and HIV/AIDS
Serologic tests in pregnancy and HIV/AIDS should be interpreted similarly to non-pregnant and HIV-negative individuals. False positives are rare in pregnancy, similar to the general population. Discordant immunoassay results in pregnant women require TPPA confirmation.
False-positive nontreponemal tests are more frequent in HIV-positive individuals. Treponemal test positivity persists post-treatment similarly in HIV-positive and negative individuals. Seroreversion is not significantly different based on HIV status in treated syphilis.
Recommendation for syphilis serologic testing in pregnant persons and persons living with HIV/AIDS. Interpret nontreponemal and treponemal tests similarly, regardless of pregnancy or HIV status.
Comment and evidence summary. Test interpretation should be consistent across different populations. Discordant results in pregnant individuals require careful adjudication. False positives are more common in HIV-positive persons but overall test performance is comparable.
Direct Detection Tests for T. pallidum
Darkfield Microscopy: Rapid On-Site Diagnosis
Darkfield microscopy is a morphology- and motility-based test for direct T. pallidum detection in lesions. It requires expertise and a darkfield microscope, and must be performed within 20 minutes of specimen collection. It is useful for moist anogenital and secondary syphilis lesions. Oral lesion interpretation is challenging due to commensal treponemes.
Optimal specimens are serous fluid from lesions, collected on a slide using touch preparation or a sterile loop. Maintain or establish darkfield microscopy in high-prevalence STD clinics for rapid POC diagnosis and treatment. Proficiency testing and training are essential.
Sensitivity and specificity for primary lesions range from 75%–100% and 94%–100%, respectively. For secondary lesions, sensitivity is 58%–71% and specificity is 100%. Darkfield microscopy is valuable for definitive diagnosis in primary syphilis, especially when serology is negative. It is less routinely recommended in secondary syphilis except for condylomata lata or when POC serology is unavailable. It might be useful for moist congenital syphilis lesions. Not recommended for oral lesions, CSF, or lymph node aspirates.
Recommendation for darkfield microscopy. Maintain or establish darkfield microscopy in STD clinics for POC diagnosis of primary and secondary syphilis.
Comment and evidence summary. Darkfield microscopy offers rapid detection and treatment, especially in primary syphilis. It is more sensitive than serology in early primary syphilis.
Immunofluorescent Antibody Staining (DFA-TP)
DFA-TP uses fluorescent antibodies to visualize T. pallidum in lesion specimens. Collection is similar to darkfield microscopy, but specimens are fixed and sent to a lab. Sensitivity is comparable to darkfield microscopy, potentially better for older primary lesions. Results take 1-2 days. FDA-cleared DFA-TP is unavailable in the US, requiring lab-validated, non-FDA cleared reagents.
Immunohistochemistry (IHC) and Silver Staining
IHC and silver staining are used on formalin-fixed, paraffin-embedded (FFPE) tissue biopsies (skin, brain, placenta, etc.). Biopsies aid in diagnosing atypical or treatment-resistant lesions. Silver staining is morphology-based, while IHC is immuno-morphology-based. IHC using peroxidase-conjugated avidin-biotin complex (ABC) is commonly used.
IHC ABC shows 100% specificity and 64%–94% sensitivity compared to clinical/serological diagnosis of secondary syphilis. IHC ABC is preferred over silver staining, which has low sensitivity (0%–41%) and interpretation challenges due to background staining. Silver staining is not recommended.
IHC ABC is recommended for atypical lesions and tissue biopsies for suspected syphilis (primary, secondary, congenital, gummatous). For congenital syphilis, test placenta and umbilical cord with IHC ABC or IIF, not silver stain. Collect placenta tissue peripherally near cord attachment and a 3-4 cm umbilical cord section. Fix tissues in 10% buffered formalin immediately.
Recommendation for IHC and silver staining. IHC is preferred over silver staining for FFPE tissue sections.
Comment and evidence summary. IHC is more sensitive and reliable than silver staining for tissue diagnosis of syphilis.
Nucleic Acid Amplification Tests (NAATs)
No FDA-cleared NAATs are available for syphilis, but lab-developed NAATs targeting tp47 or polA genes are used. Sensitivity varies by stage and specimen type (72%–95% on primary lesion exudate, 20%–86% on secondary lesion swabs). Reference standards vary across studies. Highly sensitive reverse transcriptase PCR targeting 16S rRNA and real-time transcription-mediated assay targeting 23S rRNA are also used in research. CLIA-validated PCR tests are used for genital lesions and CSF. Digital droplet PCR is used for saliva research.
NAATs targeting polA gene on secondary maculopapular lesions using a scraping technique showed 84% sensitivity. NAAT sensitivity on secondary lesion biopsies ranges from 26% to 75%. Hologic TMA showed sensitivities of 41.6% and 29.5% for rectal and pharyngeal swabs, respectively, in MSM. NAATs are highly specific (98%–100%).
Laboratory-developed NAATs can be used for primary or possible secondary syphilis lesions in seronegative patients, provided labs establish performance specifications for CLIA compliance. NAATs offer faster primary syphilis diagnosis than serology but have limited benefit over serology in secondary syphilis. NAATs can be adjunct tests for amniotic fluid, neonatal CSF, or neonatal blood in suspected congenital infection, but negative results do not rule out infection due to limited sensitivity. NAATs are not recommended for whole blood or blood fractions due to low sensitivity, and data are insufficient for CSF NAAT in adult neurosyphilis. Insufficient data also exists for ocular fluid or tertiary syphilis lesions.
Point-of-Care (POC) Serologic Testing
POC serologic tests offer rapid syphilis diagnosis and treatment initiation. Only Syphilis Health Check and DPP HIV-Syphilis Assay are FDA-cleared and CLIA-waived in the US. CLIA-waived tests require CLIA certificate of waiver and trained personnel.
Syphilis Health Check Performance
Syphilis Health Check sensitivity and specificity vary across studies. Compared to Trep-Sure EIA, sensitivity ranged from 50.0% to 71.4% and specificity from 91.5% to 95.9%. Compared to RPR and TPPA, sensitivity was 76.9%–90.0% and specificity 98.5%–99.0%. In a CDC retrospective study, sensitivity was 88.7%–95.7% and specificity 93.1%–93.2% when compared to treponemal tests alone or combined with nontreponemal tests. POC testing aims to reach underserved populations.
DPP HIV-Syphilis Assay Performance
DPP HIV-Syphilis Assay showed high sensitivity (95.3%–98.7%) and specificity (100%) compared to TPPA. Further data are needed to guide POC test implementation, cost-effectiveness, and predictive value in different settings.
Syphilis Laboratory Test Reporting
Public Health Reporting
Syphilis cases must be reported to health departments by healthcare providers and/or laboratories, as per state regulations. Report all positive direct detection tests and positive serologic tests, including specimen site. State laws specify reportable tests and timeframes. Report both probable and confirmed cases. Current case definitions are available from the CDC.
Reporting to Health Care Providers
Laboratories should report all tests performed, individual results with interpretations, and the syphilis algorithm used. Communicate any algorithm changes to submitters. Preliminary reports should list pending tests. Final reports should include all tests and results, even if referred to outside labs.
