• • Serologic tests for syphilis are the most common means for diagnosis of syphilis and the best currently available tool for evaluating response to therapy.
  
• • Serologic diagnosis of past or present syphilis infection is based on the results of two tests: a reactive nontreponemal serologic test for syphilis, confirmed by a test for syphilis based on unrelated treponemal antigens.
  
• • Testing may be complicated by atypical immunologic responses by cross-reactive antibodies leading to false-positive test results, and by difficulties in distinguishing current from past infection.

Syphilis is a chronic sexually transmitted disease (STD) with worldwide prevalence. From the time of acquisition of infection until diagnosis, the course of untreated infection may span decades. At any point in its natural history, a diagnosis of untreated syphilis warrants treatment. Although the causative organism, Treponema pallidum, may be demonstrated using darkfield microscopy during its earliest (primary and secondary) stages, this test is not useful for diagnosis except in the early stages of disease. Equally important, darkfield microscopy is often not performed even when lesions are present because a microscope equipped with a darkfield condenser is rarely available in the settings where patients with syphilis are seen. As a result, most syphilis diagnosis, as well as follow-up to ascertain response to therapy, is carried out using serologic tests for syphilis. The practical utility of serologic testing may be complicated by atypical immunologic responses by cross-reactive antibodies leading to false-positive test results, and by difficulties in distinguishing current from past infection.

Serologic tests for syphilis become reactive early in the course of untreated infection as infected individuals begin to produce antibodies to T pallidum. It is estimated that up to 10% of patients with syphilitic chancres of primary syphilis will have nonreactive serologic tests for syphilis, most of which occur in the first few days following appearance of the lesion. After primary lesions have been present for several days, the likelihood of nonreactive serologic tests declines markedly. Over the course of untreated infection, serologic titers tend to rise and then may fall, reflecting disease activity and duration of infection. Nearly all patients with untreated secondary or early latent syphilis will have reactive serologic tests. With continuing infection and production of anti—T pallidum antibodies, serologic titers tend to increase. Thus, patients with primary syphilis tend to have somewhat lower serologic titers than those with secondary or early latent infection.

After rising over the first few years of untreated infection (ie, from primary through secondary and into the latent stage), serologic titers peak and then may decline gradually over time. Although serologic titers may roughly reflect duration of infection, there is far too much person-to-person variation in antitreponemal antibody production to allow currently available serologic test titers to be considered as precise reflections of duration of infection. Studies done more than 50 years ago suggested that a small (but poorly quantified) proportion of individuals with untreated late latent or late syphilis will have nonreactive serologic tests for syphilis, although the proportion of cases in which this occurs is unclear.

At present, most syphilis diagnosis is based on a two-stage testing process. Patients are first tested using a “nontreponemal” serologic test that uses synthesized cardiolipin-lecithin-cholesterol antigens to detect cross-reacting antibodies to T pallidum. For purposes of confirmation, specimens that produce reactive nontreponemal results are subsequently tested with a second, “treponemal” test that uses T pallidum antigens. By utilizing this two-step algorithm to detect reactivity to two relevant, unrelated antigens, the proportion of false-positive test results is markedly reduced (see Table 26–1). Reactive nontreponemal tests that are not confirmed by reactive treponemal tests are typically considered to be false-positive serologic tests for syphilis.

Nontreponemal Tests

As mentioned, virtually all currently available nontreponemal tests currently utilized for syphilis testing are based on the reactivity of human immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies to T pallidum with a cardiolipin-lecithin-cholesterol antigen, typically performed as a flocculation test in which the antigen-antibody lattice formed by mixing patient plasma or serum with an antigen suspension is detected. The prototypic nontreponemal test for syphilis is the Venereal Disease Research Laboratory (VDRL) test. Other widely used nontreponemal tests include the rapid plasma reagin (RPR), automated reagin test (ART), and toluidine red unheated serum test (TRUST). An important characteristic of most nontreponemal tests is that they can be used both as qualitative tests, in which results are reported as either reactive or nonreactive, and as quantitative tests, in which the patient specimen is tested in a series of dilutions. The results are reported either as the minimum dilution of reactivity (ie, 1:1, 1:2, 1:4, 1:8, etc) or as the reciprocal of that dilution (ie, 1, 2, 4, 8, etc). The ability to provide a semiquantitative measure of antitreponemal serologic tests allows them to be used to evaluate response to therapy following treatment.

Treponemal Tests

This broad category of serologic tests for syphilis comprises those tests that use treponemal antigens as reagents. In the past these antigens were most often generated through purification of T pallidum that had been repeatedly passed in rabbits and then utilized to make tests in a number of different formats (immunofluorescence, hemagglutination, enzyme-linked immunosorbent assay [ELISA], etc). More recently, as information on the sequence of the T pallidum genome has become available, cloned treponemal antigens are increasingly being used for diagnostic syphilis testing. Among the advantages of these cloned antigens are that they can be produced in large quantities and that the antigens are not contaminated by potentially cross-reactive antigens, as is the case for antigen preparations generated from animal-passaged T pallidum.

Although investigators have periodically evaluated quantitative treponemal serologic tests for syphilis diagnosis, staging, or evaluation of response to therapy, the utility of these tests for these purposes is controversial. In most settings, treponemal tests are used qualitatively as confirmatory tests for patients with reactive nontreponemal serologic tests. In general, and unlike the nontreponemal tests, treponemal serologic test reactivity is life-long in the majority of persons with syphilis and is of little use for evaluating response to therapy. As for the nontreponemal tests, a small proportion of persons who are not infected with T pallidum or other pathogenic treponemes (see later discussion) will have reactive test results (ie, false positive). As a result, screening efforts that rely solely on treponemal tests—without confirmatory nontreponemal testing—are not recommended.

A recent development relating to treponemal serologic tests is the advent of ELISA screening assays, which can be used to efficiently test large numbers of specimens for T pallidum antibodies (ie, in blood banks or by some large commercial laboratories). Although ELISA test results are often presented in quantitative fashion, there is little experience in using these tests to evaluate response to therapy. Rather, persons identified as having syphilis using ELISA tests should then be tested using a quantitative nontreponemal assay for the purpose of evaluating response to therapy.