Because the infectious agent of syphilis cannot be cultured in vitro, diagnostic measures must rely mainly on serologic testing, microscopic detection of T pallidum in lesions, and other examinations (biopsies, lumbar puncture, x-rays) for evidence of tissue damage.

A. Serologic Tests for Syphilis
There are two general categories of serologic tests for syphilis: (1) Nontreponemal tests detect antibodies to lipoidal antigens present in either the host or in T pallidum. The original antigens employed to measure these nonspecific antibodies (reagin) were crude extracts of beef heart or liver and resulted in significant numbers of false-positive reactions. The cardiolipin-cholesterol-lecithin preparation presently used is much purer and gives fewer false-positive reactions. (2) Treponemal tests employ live or killed T pallidum as antigen to detect antibodies specific for pathogenic treponemes.

1. Nontreponemal Antigen Tests - The most commonly used nontreponemal antigen tests are the Venereal Disease Research Laboratory (VDRL) and rapid plasma reagin (RPR), which measure the ability of heated serum to flocculate a suspension of cardiolipin-cholesterol-lecithin. The flocculation tests are inexpensive, rapid, and easy to perform and are therefore used primarily for routine screening. Quantitative expression of the reactivity of the serum, based upon titration of dilutions of serum, is valuable in establishing the diagnosis and in evaluating the efficacy of treatment, since titers usually correlate with disease activity.

Nontreponemal tests generally become positive 4-6 weeks after infection, or 1-3 weeks after the appearance of a primary lesion; they are almost invariably positive in the secondary stage, with titers ? 1:32. In the late stages, titers tend to be lower (< 1:4). These serologic tests are not highly specific and must be closely correlated with other clinical and laboratory findings. The tests are positive in patients with non-sexually transmitted treponematoses (see below). More importantly, “false-positive” serologic reactions are frequently encountered in a wide variety of nontreponemal states, including connective tissue diseases, infectious mononucleosis, malaria, febrile diseases, leprosy, intravenous drug use, infective endocarditis, old age, hepatitis C viral infection, and pregnancy. False-positive tests also occur more commonly in HIV-seropositive patients (4%) than in HIV-seronegative patients (0.8%). False-positive reactions are usually of low titer and transient and may be distinguished from true positives by specific treponemal antibody tests. False-negative results can be seen when very high antibody titers are present (the prozone phenomenon). If syphilis is strongly suspected and the nontreponemal test is negative, the laboratory should be instructed to dilute the specimen to detect a positive reaction. The RPR and VDRL tests are equally reliable, but titers of RPR tend to be higher than the VDRL. Thus, when these tests are used to follow disease activity, the same testing method should be used and preferably should be performed at the same laboratory.

Nontreponemal antibody titers are used to assess adequacy of therapy. The time required for the VDRL or RPR to become negative depends on the stage of the disease, the height of the initial titer, and whether the infection is an initial or repeat episode. In general, individuals with repeat infections, higher initial titers, and more advanced stages of disease at the time of treatment have a slower seroconversion rate and are more likely to remain serofast (ie, titers do not become negative). Older data derived from more intensive treatment regimens than are presently used indicate that in primary and secondary syphilis, the VDRL usually decreases fourfold by 3 months and eightfold by 6 months. Furthermore, seronegativity was seen in 97% of those with primary syphilis and 76% of those with secondary syphilis at 2 years. More recent data based on currently recommended treatment regimens (see below) suggest that decreases in titer may be slower - ie, in primary and secondary syphilis it may take 6 months to see a fourfold decrease in titer and 12 months to see an eightfold drop. In patients with early latent syphilis, response is even slower, with a fourfold drop in titer taking 12-24 months. Seronegativity was seen in 72% of patients with primary syphilis and only 56% of those with secondary syphilis after 3 years. Additional studies support a slower decline in titers with currently recommended treatment regimens.

2. Treponemal Antibody Tests - The fluorescent treponemal antibody absorption (FTA-ABS) test measures antibodies capable of reacting with killed T pallidum after absorption of the patient’s serum with extracts of nonpathogenic treponemes. The FTA-ABS test is of value principally in determining whether a positive nontreponemal antigen test is “false-positive” or is indicative of syphilis. Because of its great sensitivity, particularly in the late stages of the disease, the FTA-ABS test is also of value when there is clinical evidence of syphilis but the nontreponemal serologic test for syphilis is negative. The test is positive in most patients with primary syphilis and in almost all patients with secondary syphilis. Like nontreponemal antigen tests, the specific treponemal antibody test may revert to negative with adequate therapy. This is seen almost exclusively in initial infections in individuals with primary syphilis. In one study, 11% of individuals with a first episode of primary syphilis were seronegative by the FTA-ABS test at 1 year posttreatment, and 24% were negative by 3 years. Immunologic status may also affect antibody titers. Seven percent of asymptomatic HIV-infected patients became seronegative after treatment, as opposed to 38% of symptomatic HIV-infected individuals. The long-held belief that a positive FTA-ABS persists indefinitely is clearly not valid, and this test therefore cannot be used as a reliable marker of previous infection. False-positive FTA-ABS tests occur rarely in systemic lupus erythematosus and in other disorders associated with increased levels of ?-globulins, malaria, leprosy, and other spirochetal infections. It is noteworthy that Lyme disease may cause a false-positive FTA-ABS test but rarely causes a false-positive reaginic test. The T pallidum hemagglutination (TPHA) test and the T pallidum particle agglutination (TPPA) test are comparable in specificity and sensitivity to the FTA-ABS. The TPPA test, because of ease of performance, has supplanted the FTA-ABS test as the means of confirming the diagnosis of syphilis.

Final decisions about the significance of the results of serologic tests for syphilis must be based upon a total clinical appraisal.

B. Microscopic Examination

In infectious syphilis, T pallidum may be shown by darkfield microscopic examination of fresh exudate from lesions or material aspirated from regional lymph nodes. The darkfield examination requires considerable experience and care in the proper collection of specimens and in the identification of pathogenic spirochetes by observing characteristic features of morphology and motility. Repeated examinations may be necessary. Spirochetes usually are not found in late syphilitic lesions by this technique.

An immunofluorescent staining technique for demonstrating T pallidum in dried smears of fluid taken from early syphilitic lesions is available. Slides are fixed and treated with fluorescein-labeled antitreponemal antibody that has been preabsorbed with nonpathogenic treponemes. The slides are then examined for fluorescing spirochetes in an ultraviolet microscope. Because of its simplicity and convenience to clinicians (slides can be mailed), this technique has replaced darkfield microscopy in most health departments and medical center laboratories.

C. Spinal Fluid Examination

Cerebrospinal fluid findings in neurosyphilis are variable. In “classic” cases there is an elevation of total protein, lymphocytic pleocytosis, and a positive cerebrospinal fluid reagin test (VDRL). However, cerebrospinal fluid may be completely normal in neurosyphilis, and the VDRL may be negative. In one study, 25% of patients with primary or secondary syphilis in whom T pallidum was isolated from cerebrospinal fluid had a normal cerebrospinal fluid examination. In later stages of syphilis, normal cerebrospinal fluid analysis in the presence of infection can occur, but it is unusual. Because false-positive reagin tests rarely occur in the cerebrospinal fluid, a positive test confirms the presence of neurosyphilis. Because the cerebrospinal fluid VDRL may be negative in 30-70% of cases of neurosyphilis, a negative test does not exclude neurosyphilis. The use of cerebrospinal fluid FTA-ABS in the diagnosis of neurosyphilis is controversial. It is a highly sensitive test but lacks specificity, and a high serum titer of FTA-ABS may result in a positive cerebrospinal fluid titer in the absence of neurosyphilis. However, because the test is so sensitive a negative cerebrospinal fluid FTA-ABS is strong evidence against the diagnosis of neurosyphilis.

Cerebrospinal fluid examination is recommended depending on the clinical manifestations and the stage of disease, as discussed below. Asymptomatic neurosyphilis (ie, positive cerebrospinal fluid findings without symptoms) requires prolonged penicillin treatment as given for symptomatic neurosyphilis. Adequate treatment is indicated by gradual decrease in cerebrospinal fluid cell count, protein concentration, and VDRL titer. Rarely, serologic tests of cerebrospinal fluid may remain positive for years after adequate treatment of neurosyphilis even though all other parameters have returned to normal.