Syphilis, Lyme, and Tuberculosis Serology

Three infections share one laboratory pattern: the organism is difficult to culture, so the diagnosis rests on detecting the host response. Syphilis testing pairs two antibody classes that answer different questions, Lyme testing chains two reaction steps to control weak specificity, and tuberculosis testing measures a cell-mediated response that the organism itself never enters the bloodstream to trigger. In each, the timing of the specimen against symptom onset decides what a negative result means.

Syphilis

Testing categories

Nontreponemal (lipoidal antigen) tests detect antibody to cardiolipin, cholesterol, and phosphatidylcholine. These lipids appear in damaged host cells and in the T. pallidum membrane, so the antibody response rises with active infection and falls after treatment. The historical name reagin belongs to this response and is unrelated to the IgE term used in allergy. Treponemal tests detect antibody to T. pallidum protein antigens; they appear earlier in primary disease and generally persist for life, so they confirm exposure while nontreponemal titers track 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 100x and remains the only FDA-cleared test for cerebrospinal fluid in neurosyphilis. RPR adsorbs the antigen to charcoal particles, which clumped lattice traps 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 stay on one test method and 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 raises the question of inadequate response, although a minority of appropriately treated patients (14 percent 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 blocks 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 percent, but concentrates in primary and secondary disease, where one study found it in 4.7 percent of primary and 1.8 percent of secondary specimens; resolution required dilutions as high as 1:256 or 1:512 in individual cases. Laboratories rule out prozone by dilution series when a clinician requests it, particularly when signs and symptoms suggest syphilis and an undiluted serum reads nonreactive.¹

False positives. Biologic false-positive reactions occur in roughly 0.2 to 0.8 percent 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

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Treponemal antibody persists in most treated patients, so treponemal tests cannot judge treatment response, and repeat treponemal testing adds nothing once infection is documented. Seroreversion can occur in roughly 15 to 25 percent of patients treated for primary syphilis within 2 to 3 years and in advanced HIV disease.¹ 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 no longer serve in vitro diagnosis in the United States.¹

Testing algorithms

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Both algorithms are acceptable and agree in about 99 percent 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, while 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 percent 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.¹

Neurosyphilis and congenital syphilis

CSF testing. CSF VDRL is highly specific but insensitive; studies place its sensitivity between 13 and 87 percent 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 leaves the evaluation open. Neonatal testing uses serum, with cord blood avoided because maternal blood contamination causes false positives and Wharton jelly causes false negatives, and the same nontreponemal (lipoidal antigen) test, preferably in the same laboratory, is used for infant and mother.¹,²

Direct detection

Darkfield microscopy of lesion exudate identifies motile spirochetes and must be read within 20 minutes of collection; it is useful in primary and secondary disease, carries 75 to 100 percent sensitivity in primary lesions against NAAT reference standards, and is specifically avoided for oral lesions because commensal treponemes resemble T. pallidum. Direct fluorescent antibody staining achieves comparable sensitivity on fixed slides and tolerates delay, though the FDA-cleared product left the United States market. Immunohistochemistry on fixed tissue serves atypical lesions and congenital evaluation, while silver staining, at 0 to 41 percent sensitivity, is no longer recommended. No FDA-cleared nucleic acid amplification test exists for T. pallidum; CLIA-validated laboratory-developed assays target the tp47 or polA genes with 72 to 95 percent sensitivity on primary lesion exudate.¹

Lyme disease

Why serology carries the diagnosis

Borrelia burgdorferi circulates briefly and at low concentration, so culture and PCR on blood or spinal fluid have low yield, and the antibody response carries the diagnosis. An erythema migrans lesion in an endemic area is diagnosed clinically, and serology at that early stage adds little: sensitivity during early localized infection is low, while disseminated manifestations reach above 87 percent sensitivity and 99 percent specificity with two-tier testing.³ Serologic tests may be falsely negative for 4 to 6 weeks after infection.³

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

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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 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. Both steps positive, whether positive or equivocal in either combination, yields an interpretation of antibodies detected and exposure supported.⁴ MTTT runs faster, reads objectively, and requires no reference-laboratory immunoblot expertise, and CDC accepts both algorithms.³

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The 41-kDa flagellin band is the most frequent cross-reactive target in immunoblots, and whole false positives rise in relapsing fever, syphilis, autoimmune disease, and Epstein-Barr virus infection, which is why the band-count criteria matter and immunoblot-only testing without a positive first tier is discouraged.³,⁴ Serologic testing is ordered only with a compatible clinical picture and plausible exposure, because a positive result in a low-prevalence population carries 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

Organism-level biology, specimen selection, and direct identification of Borrelia are covered by the Microbiology subject.

Tuberculosis

Both tuberculosis infection tests measure delayed-type (cell-mediated) hypersensitivity to Mycobacterium tuberculosis antigens. The immune response takes 2 to 8 weeks after infection to become detectable, so both tests carry that window, 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

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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

The reaction is measured as millimeters of induration, transverse to the long axis of the forearm, with erythema ignored. Interpretation uses risk-stratified thresholds:

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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 interferes with both test types, so testing is done on the same day as vaccination or at least 4 weeks 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 laboratories issue the qualitative result together with the numbers, though 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, carries no information about infection; retesting, including a TST, can resolve it.⁶

Tuberculosis disease itself is reported to the health department, and latent infection is reportable in some states.⁶

Interpretation limits shared by all three

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References

1. 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.
2. Centers for Disease Control and Prevention. Congenital syphilis. STI Treatment Guidelines. Accessed August 31, 2026.
3. Centers for Disease Control and Prevention. Clinical testing and diagnosis for Lyme disease. May 15, 2024. Accessed August 31, 2026.
4. Association of Public Health Laboratories. Suggested Reporting Language, Interpretation and Guidance for Lyme Disease Serologic Test Results. April 2024. Accessed August 31, 2026.
5. Centers for Disease Control and Prevention. Clinical testing guidance for tuberculosis: tuberculin skin test. Updated January 31, 2025. Accessed August 31, 2026.
6. Centers for Disease Control and Prevention. Clinical testing guidance for tuberculosis: interferon gamma release assay. Updated May 9, 2024. Accessed August 31, 2026.