Syphilis, Lyme disease, and tuberculosis infection share one laboratory feature. The organism is difficult to culture, so laboratory diagnosis often depends on detecting the host response. Syphilis testing uses two types of antibody test with different purposes, Lyme testing uses two test tiers to improve specificity, and tuberculosis infection tests measure T-cell memory to selected mycobacterial antigens and do not detect the organism directly. For all three, the timing of the specimen against symptom onset or exposure determines how a negative result is interpreted.
Syphilis
Testing categories
Nontreponemal (lipoidal antigen) tests detect antibody to cardiolipin, cholesterol, and phosphatidylcholine. These lipids appear in damaged host cells and in the Treponema pallidum membrane, so the antibody response rises with active infection and falls after treatment. The historical name reagin refers to this antibody response and is unrelated to the IgE term used in allergy. Treponemal tests detect antibody to T. pallidum protein antigens. Treponemal antibodies appear earlier in primary disease and generally persist for life, so treponemal tests confirm exposure. Nontreponemal titers reflect disease activity.1
Nontreponemal (lipoidal antigen) tests
The Venereal Disease Research Laboratory (VDRL) test and the rapid plasma reagin (RPR) test are flocculation assays using cardiolipin, lecithin (phosphatidylcholine), and cholesterol antigen. VDRL is read microscopically at 100× and remains the only FDA-cleared test for cerebrospinal fluid (CSF) in neurosyphilis. RPR adsorbs the antigen to charcoal particles, which form a clumped lattice for macroscopic reading, and includes choline chloride so unheated serum or plasma can be tested. The toluidine red unheated serum test (TRUST) substitutes toluidine red dye for charcoal.1
Endpoint titer. Every reactive nontreponemal (lipoidal antigen) result is reported as an endpoint titer, the highest dilution that remains reactive, with mathematical symbols such as greater than or less than omitted. Serial titers for one patient must be performed by one test method on one specimen type, because RPR and VDRL titers are not interchangeable.1 Automated RPR platforms with a constrained dilution range require reflex to a manual RPR when the endpoint lies beyond the validated range.1
Titers rise within 1 to 4 weeks of the chancre, peak in secondary disease, and decline after treatment. A fourfold titer change between results from the same test is clinically significant. Failure of a primary or secondary titer to fall fourfold within 12 months of appropriate treatment suggests a possible inadequate response, although a minority of appropriately treated patients (14% in one prospective study) meet that threshold without treatment failure. Persistently low reactive titers after treatment, usually 1:8 or lower, describe the serofast state.1
Prozone. Antibody excess prevents lattice formation, so an undiluted specimen can read weakly reactive or nonreactive with a rough, grainy appearance. Prozone is rare in screened populations, under 0.85%, and occurs mainly in primary and secondary disease, where one study found it in 4.7% of primary and 1.8% of secondary specimens. Resolution required dilutions as high as 1:256 or 1:512 in individual cases. When a clinician requests it, particularly when signs and symptoms suggest syphilis and an undiluted serum reads nonreactive, rule out prozone with a dilution series.1
False positives. Biologic false-positive reactions occur in roughly 0.2 to 0.8% of the population with acute causes (viral infection, recent vaccination, pregnancy) and chronic causes (autoimmune disease, injection drug use, malaria, leprosy, HIV). Every reactive nontreponemal (lipoidal antigen) result therefore requires treponemal confirmation.1
Treponemal tests
Scroll sideways to see all columns
| Test | Format | Laboratory note |
|---|---|---|
| TP-PA (T. pallidum particle agglutination) | T. pallidum antigen-coated gelatin particles; indirect agglutination | Preferred manual treponemal test; highest sensitivity in primary disease (94.5% in one 959-patient study) |
| FTA-ABS (fluorescent treponemal antibody absorption) | Indirect immunofluorescence on fixed T. pallidum (Nichols strain) after serum absorption with Reiter treponeme extract | Read by fluorescence intensity; CSF use is off-label but valued for excluding neurosyphilis when negative |
| EIA (enzyme immunoassay), CIA (chemiluminescence immunoassay), multiplex bead | Automated immunoassays with recombinant antigens | 12 FDA-cleared assays as of 2021; run as reverse-sequence screens in high-volume laboratories |
Treponemal antibody persists in most treated patients, so treponemal tests cannot be used to monitor treatment response. In a patient with documented treated syphilis and a reactive treponemal result, additional treponemal testing is not useful for detecting reinfection. Reinfection is assessed with a quantitative nontreponemal test and the clinical history. Seroreversion can occur in roughly 15 to 25% of patients treated for primary syphilis within 2 to 3 years and in advanced HIV disease. The historical MHA-TP and TPHA agglutination assays, which coated avian or ovine erythrocytes with sonicated T. pallidum antigen, remain available only in some international settings and are no longer used for in vitro diagnosis in the United States.1
Testing algorithms
Scroll sideways to see all columns
| Algorithm | Sequence | Discordance handling |
|---|---|---|
| Traditional | Qualitative RPR or VDRL first; reactive results quantitated, then confirmed with a treponemal test | Treponemal confirmation resolves biologic false positives |
| Reverse sequence | Automated treponemal immunoassay first; reactive results reflex to a quantitative nontreponemal (lipoidal antigen) test | A reactive screen with a nonreactive nontreponemal result is adjudicated with a second, different treponemal test, preferably TP-PA |
Both algorithms are acceptable and agree in about 99% of specimens. The choice depends on cost, volume, staff, and population served. Roughly two thirds of surveyed clinical laboratories used the traditional sequence in 2015. Blood banks and high-throughput laboratories favor the reverse sequence. A reverse-sequence false-positive pattern, treponemal immunoassay reactive with nonreactive RPR and nonreactive TP-PA, occurs in about 0.6% of screened specimens, and interpretation of a discordant treponemal screen includes treated past infection, early primary disease before nontreponemal antibody rises, or a false-positive screen. Low signal-to-cutoff ratios on immunoassay screens correlate with false-positive screens and can support the adjudication decision until test-specific cutoffs are validated. In both sequences, a nonreactive screen is interpreted as syphilis unlikely. A reactive nontreponemal test or a reactive second treponemal test supports previously treated or untreated syphilis. When the second treponemal test is reactive and a previously treated person has possible reexposure, repeat the nontreponemal test in 2 to 4 weeks. When the second treponemal test is nonreactive and clinical and epidemiologic probability is low, the initial treponemal screen is likely a false positive and no further evaluation is needed.1,8
Neurosyphilis and congenital syphilis
CSF testing. CSF VDRL is highly specific but insensitive. Studies place its sensitivity between 13 and 87% depending on the reference standard. A negative CSF VDRL result never excludes neurosyphilis when CSF pleocytosis or elevated protein supports it. CSF FTA-ABS is used off-label, is highly sensitive, and its negative predictive value helps exclude neurosyphilis in a laboratory experienced with the method.1
Congenital syphilis. Maternal IgG crosses the placenta, so neonatal treponemal tests are uninterpretable and no IgM test is recommended. The laboratory comparison is quantitative: a neonatal serum nontreponemal titer fourfold or more above the maternal titer at delivery (two dilutions, such as maternal 1:2 and neonatal 1:8) supports congenital infection, and an equal or lower titer neither confirms nor excludes congenital infection. Test neonatal serum. Avoid cord blood, because maternal blood contamination causes false positives and Wharton jelly causes false negatives. Use the same nontreponemal (lipoidal antigen) test for infant and mother, preferably in the same laboratory, so the two titers can be compared.1,2
Direct detection
Darkfield microscopy of lesion exudate identifies motile spirochetes, so read it within 20 minutes of collection. It is useful in primary and secondary disease and has 75 to 100% sensitivity in primary lesions against reference standards of clinical presentation with serology or PCR. Do not use it for oral lesions, because commensal treponemes resemble T. pallidum. Direct fluorescent antibody staining achieves comparable sensitivity on fixed slides and tolerates delay. The FDA-cleared product is no longer marketed in the United States. Immunohistochemistry on fixed tissue is used for atypical lesions and congenital evaluation. Silver staining, at 0 to 41% sensitivity, is no longer recommended. No FDA-cleared nucleic acid amplification test (NAAT) exists for T. pallidum. CLIA-validated laboratory-developed assays target the tp47 or polA genes with 72 to 95% sensitivity on primary lesion exudate.1
Lyme disease
Serologic diagnosis
Borrelia burgdorferi circulates briefly and at low concentration, so culture and PCR on blood or spinal fluid have low yield, and laboratory diagnosis depends on the antibody response. An erythema migrans lesion in an endemic area is diagnosed clinically. Serology adds little at that early stage because sensitivity during early localized infection is low. For disseminated manifestations, two-tier testing has sensitivity above 87% and specificity of 99%. Serologic tests may be falsely negative for 4 to 6 weeks after infection.3,4
Standard two-tier testing
Standard two-tier testing (STTT) begins with a sensitive first-tier immunoassay, typically an enzyme immunoassay for total antibody or separate IgM and IgG. A negative first tier stops the algorithm. A positive or equivocal first tier reflexes to immunoblots.4
Scroll sideways to see all columns
| Illness duration | Blots interpreted | Positive criteria |
|---|---|---|
| 30 days or less | IgM and IgG | IgM positive with 2 or more of 3 bands (23, 39, 41 kDa), or IgG positive by the 5-of-10 criterion below |
| More than 30 days | IgG only | IgG positive with 5 or more of 10 bands: 18, 23, 28, 30, 39, 41, 45, 58, 66, 93 kDa |
IgM results after 30 days of illness are disregarded because false-positive IgM blots and prolonged IgM seropositivity are common. A positive IgG result supports infection in the recent or remote past and cannot be used to monitor treatment, because antibody persists for months to years after cure. Response to therapy is confirmed clinically.4
Modified two-tier testing
Modified two-tier testing (MTTT) replaces the immunoblot with a second EIA using different B. burgdorferi antigens, in assays FDA-cleared together as a pair. For the FDA-cleared pairs covered by the Association of Public Health Laboratories (APHL) guidance, positive or equivocal results on both steps support an overall positive interpretation. The laboratory follows the cleared pair's package inserts.4 MTTT is faster, is read objectively, and requires no reference-laboratory immunoblot expertise. CDC accepts both algorithms.3
Scroll sideways to see all columns
| Pattern (either algorithm) | Interpretation |
|---|---|
| First tier negative | No laboratory evidence of infection; repeat in 7 to 14 days if recent infection is suspected |
| First tier positive or equivocal, second tier negative | Antibodies not confirmed; no laboratory evidence of infection |
| First tier positive or equivocal, second tier positive | Antibodies detected; consistent with recent or past infection |
The 41-kDa flagellin band is the most frequent cross-reactive target in immunoblots, and false-positive results increase in relapsing fever, syphilis, autoimmune disease, and Epstein-Barr virus infection. For this reason the band-count criteria are required and immunoblot-only testing without a positive first tier is discouraged. Serologic testing is ordered only with a compatible clinical picture and plausible exposure, because a positive result in a low-prevalence population has a low positive predictive value. Testing is specifically discouraged for an erythema migrans rash in a high-incidence area, for patients without tick exposure, and for monitoring therapy.3,4
The Microbiology lessons cover organism biology, specimen selection, and direct identification of Borrelia.
Tuberculosis
Both tuberculosis infection tests measure the cell-mediated immune response to Mycobacterium tuberculosis antigens. The immune response takes 2 to 8 weeks after infection to become detectable, so both tests have that window period, and neither test distinguishes latent infection from active disease. A positive result is followed by evaluation for active disease, with a chest radiograph and bacteriologic examination of sputum.5,6
Scroll sideways to see all columns
| Feature | Tuberculin skin test (TST) | Interferon-gamma release assay (IGRA) |
|---|---|---|
| Method | 0.1 mL of purified protein derivative (PPD) intradermally; induration read at 48 to 72 hours | QuantiFERON-TB Gold Plus: whole blood stimulated with ESAT-6 and CFP-10 peptides, IFN-gamma concentration measured. T-SPOT.TB: a standardized number of isolated peripheral blood mononuclear cells (PBMCs) stimulated, IFN-gamma spot-forming cells counted |
| Formats | Mantoux test with 5 tuberculin units | QuantiFERON-TB Gold Plus, an ELISA measuring IFN-gamma concentration; T-SPOT.TB, an ELISpot counting IFN-gamma-producing cells |
| Visits | Two: placement and reading | One blood draw; collection, transport, and processing deadlines follow the selected assay and collection system |
| Bacille Calmette-Guérin (BCG) and nontuberculous mycobacteria | False positives; reactivity generally wanes with time but repeated testing can boost it | Unaffected by BCG and by most nontuberculous mycobacteria |
| Boosting | A boosted reaction can appear on later tests in previously infected or BCG-vaccinated adults | No booster phenomenon |
CDC guidance encourages IGRAs for most testing, and IGRAs are the preferred test for people who received BCG vaccine, including children, and for people who might not return for a skin-test reading. The TST remains the recommended method for children younger than 5 years. Some experts use IGRAs in that age group, and BCG vaccination in a young child shifts the preference toward an IGRA.5,6
Reading and interpreting the TST
Measure the induration in millimeters, transverse to the long axis of the forearm, and ignore erythema. Interpretation uses risk-stratified thresholds:
Scroll sideways to see all columns
| Induration | Positive for |
|---|---|
| 5 mm or more | HIV infection; recent contacts of infectious TB; organ transplant recipients; immunosuppressed people, including those on prolonged corticosteroid therapy equivalent to 15 mg of prednisone per day or more, or on TNF-alpha antagonists; chest radiograph findings of prior TB |
| 10 mm or more | People born where TB is common; people who misuse drugs or alcohol; residents and employees of high-risk congregate settings; mycobacteriology laboratory workers; conditions that raise TB risk, including silicosis, diabetes, severe kidney disease; low body weight, under 90% of ideal; children younger than 5 years |
| 15 mm or more | People with no known risk factors |
Two-step testing applies when the TST is used for baseline screening of people who will be retested periodically, such as health care personnel: a negative first test is repeated 1 to 3 weeks later, and a positive second result is classified as a boosted reaction that reflects past infection, which makes the baseline result positive. Live-virus vaccination may interfere with both test types, so test on the same day as vaccination or at least 1 month after.5,6
False-negative TST results include anergy, infection within the previous 8 to 10 weeks, very young age, recent live-virus vaccination, recent viral illness, and severe illness including miliary TB and TB meningitis. Recent live-virus vaccination and recent viral illness also depress IGRA responses, and an IGRA within 8 weeks of infection can be negative, so contacts of infectious cases with an initial negative IGRA are retested 8 to 10 weeks after last exposure.5,6
IGRA results
QuantiFERON-TB Gold Plus reports positive, negative, or indeterminate. T-SPOT.TB reports positive, negative, borderline, or invalid. Both report the quantitative antigen, nil, and mitogen values, and the laboratory reports the qualitative result together with the numbers. Specific guidance for interpreting the quantitative values alone is unavailable. An indeterminate or invalid result, usually from a failed mitogen control or a high nil background, provides no information about infection. Retesting, including a TST, can resolve it.6 For QFT-Plus, the nil value must be 8.0 IU/mL or less. The manufacturer subtracts nil from each TB1, TB2, and mitogen value and calls a panel positive when the TB1 minus nil or TB2 minus nil difference is at least 0.35 IU/mL and at least 25% of the nil value in the same tube; negative when both antigen responses fall below that criterion and mitogen minus nil is at least 0.50 IU/mL; and indeterminate when nil exceeds 8.0 IU/mL or mitogen minus nil is below 0.50 IU/mL with no positive antigen response. The magnitude of IFN-gamma cannot be correlated to the stage or degree of infection.7
Tuberculosis disease itself is reported to the health department, and latent infection is reportable in some states.6
Working a QuantiFERON-TB Gold Plus panel. One panel reads Nil 2.00, TB1 2.45, TB2 5.80, and Mitogen 9.20 IU/mL. Subtract nil from each stimulated tube: TB1 − nil = 2.45 − 2.00 = 0.45, TB2 − nil = 5.80 − 2.00 = 3.80, and mitogen − nil = 9.20 − 2.00 = 7.20. The nil value of 2.00 is 8.0 IU/mL or less, so the panel is interpretable, and the mitogen response of 7.20 is adequate. The mitogen value is used in interpretation only when no antigen response meets the positive criterion. TB1 minus nil meets the 0.35 IU/mL cutoff, but 0.45 is below 25% of nil (0.50), so the TB1 tube alone is not positive. TB2 minus nil meets both criteria in the same tube, so the qualitative result is positive and M. tuberculosis infection is likely. Report the qualitative result with the quantitative values. The result does not distinguish latent infection from active disease, so the clinical team follows it with evaluation for active disease by chest radiograph and sputum examination. The IFN-gamma magnitude does not grade the infection.
Interpretation limits shared by all three
Scroll sideways to see all columns
| Limit | Consequence |
|---|---|
| Antibody or cellular response lags infection | Early specimens read negative; repeat on the schedule each algorithm defines: 2 to 4 weeks for a repeat syphilis nontreponemal test when prior treatment and possible reexposure warrant it, 7 to 14 days for Lyme, 8 to 10 weeks for TB contacts |
| Prior infection keeps antibody detectable | Treponemal and Lyme IgG results cannot distinguish current from past infection or monitor treatment |
| Low disease prevalence raises the false-positive share | Each algorithm ties its reflex steps to compatible clinical and exposure findings |
| Method-dependent results | Serial nontreponemal titers use one test; Lyme interpretation depends on FDA-cleared pairs; TST and IGRA results are not interchangeable |
Practice
References
- Papp JR, Park IU, Fakile Y, Pereira L, Pillay A, Bolan GA. CDC laboratory recommendations for syphilis testing, United States, 2024. MMWR Recomm Rep. 2024;73(1):1-32. doi:10.15585/mmwr.rr7301a1 Back to text
- Centers for Disease Control and Prevention. Congenital syphilis. STI Treatment Guidelines. Accessed August 31, 2026. https://www.cdc.gov/std/treatment-guidelines/congenital-syphilis.htm Back to text
- Centers for Disease Control and Prevention. Clinical testing and diagnosis for Lyme disease. May 15, 2024. Accessed August 31, 2026. https://www.cdc.gov/lyme/hcp/diagnosis-testing/index.html Back to text
- Association of Public Health Laboratories. Suggested Reporting Language, Interpretation and Guidance for Lyme Disease Serologic Test Results. April 2024. Accessed August 31, 2026. https://www.cdc.gov/lyme/media/pdfs/2024/05/Suggested-Reporting-Language-Interpretation-and-Guidance-Regarding-Lyme-Disease-Serologic-Test-Results.pdf Back to text
- Centers for Disease Control and Prevention. Clinical testing guidance for tuberculosis: tuberculin skin test. Updated January 31, 2025. Accessed August 31, 2026. https://www.cdc.gov/tb/hcp/testing-diagnosis/tuberculin-skin-test.html Back to text
- Centers for Disease Control and Prevention. Clinical testing guidance for tuberculosis: interferon gamma release assay. Updated May 9, 2024. Accessed August 31, 2026. https://www.cdc.gov/tb/hcp/testing-diagnosis/interferon-gamma-release-assay.html Back to text
- Qiagen. QuantiFERON-TB Gold Plus (QFT-Plus) Instructions for Use. HB-3303-011 R11. August 2025. Accessed September 2, 2026. https://www.qiagen.com/en-US/resources/download/kitHandbookAndProtocol/hb-3303-011-r11-qft-plus-elisa-0825-us Back to text
- Centers for Disease Control and Prevention. Syphilis. STI Treatment Guidelines. Accessed September 24, 2026. https://www.cdc.gov/std/treatment-guidelines/syphilis.htm Back to text