Treponema pallidum immobilization (TPI) test. Syphilitic spirochetes can grow in rabbits. Nelson devised a Treponema pallidum immobilization (TPI) test in which syphilitic spirochetes are incubated with the patient’s serum. If specific antisyphilitic antibody is present, it will attack the spirochetes and immobilize them, causing them to stop moving when viewed under the microscope. This involves an antibody that is different from reagin and is specific against pathogenic Treponema spirochetes. Besides syphilis, other Treponema spirochetal diseases such as yaws may give positive reactions. The main disadvantages of this test are that it requires working with live spirochetes and use of an animal colony, is difficult to perform accurately, and is expensive. The TPI test has been replaced by the FTA-ABS and the microhemagglutination (MHA) tests (to be discussed later), and it is now very difficult, almost impossible, to obtain a TPI study in the United States.

Reiter protein complement fixation (RPCF) test. The TPI test is done using the Nichol strain of pathogenic spirochetes. It was discovered that a certain nonpathogenic Treponema spirochete called the “Reiter strain” could be cultured more easily and cheaply on artificial media. Antigen prepared from this organism was adapted to a CF technique, and the result was the Reiter protein complement fixation (RPCF) test.

The Reiter antibody is different from the Treponema-immobilizing antibody of the TPI test. Apparently, the nonpathologic Reiter and the pathologic Nichol spirochete share a common protein antigen, and it is this protein that is used in the RPCF test. In addition, the Nichol organism has a specific antigen that results in the immobilizing antibody response of the TPI assay. Several investigators have found the RPCF test to be almost as sensitive and specific as the TPI test, although others are less enthusiastic about its sensitivity in late syphilis. The Reiter antibody also appears at a different time than does the TPI antibody. The disadvantages of the RPCF are those inherent in all CF tests. The RPCF test is practically never used in the United States.

Fluorescent treponemal antibody (FTA) test. In the FTA procedure, dead Nichol strain spirochetes are fixed to slides; these spirochete-containing slides can be prepared in the laboratory or obtained ready for use from commercial sources. The patient’s serum is placed on the slide with the spirochetes and allowed to incubate with the organisms. Antispirochete antibody in the patient serum coats the surface of the spirochetes. The serum is then washed off and replaced by antibodies against human gamma globulin that are tagged with a fluorescent dye. Since human antibodies against syphilis produced after the first month of infection are gamma globulins, the antibodies against human gamma globulin, with the fluorescein dye attached, will attack and adhere to any patient antitreponemal antibody that coats the spirochetes on the slide. The spirochetes will then appear fluorescent when viewed with an ultraviolet microscope.

Unfortunately, fluorescent work is not as simple as this description or the recent literature would imply. Many technical problems remain. These tests at present are not suitable for mass screening, although they are less time consuming than the RPCF test and easier than the TPI test. Many substances give varying degrees of natural fluorescence, and it is sometimes difficult to decide whether a preparation is actually positive or not. There may be nonspecific antigen-antibody binding of the cross-reacting type, as well as specific reaction. When the animal anti-human globulin antibody is conjugated with fluorescein, not all of the fluorescein binds to it. Any remaining free fluorescein may nonspecifically stain various proteins, including the spirochetes, when the tagged mixture is added to the patient’s serum.

Because of the problem of nonspecific fluorescence, the FTA underwent modification to become the FTA test with absorption (FTA-ABS). Reiter Treponema antigen is used to absorb nonspecific cross-reacting antibodies out of the patient’s serum. Antibody to T. pallidum is not absorbed out by this technique. The absorbed patient serum replaces nonabsorbed patient serum in the standard FTA procedure.

The FTA-ABS is a well-established test. It has relatively good sensitivity in primary syphilis (except in very early disease) and is reported to be even more sensitive than the TPI in tertiary syphilis. If the patient is treated adequately in the primary or secondary stage, the FTA-ABS response will usually return to nonreactive state, but after the secondary stage it usually will not become nonreactive in spite of therapy. It is said to be at least as specific as the TPI, possibly even more so.

Drawbacks. Weak reactions may cause interpretation problems. Official recommendations are that equivocal or 1+ reactive specimens should be repeated and the 1+ reclassified as borderline if the repeat test result is nonreactive and called 1+ reactive if reactivity remains 1+. This is important because some false positive and false negative results may occur in the FTA-ABS due to laboratory technical factors. Some studies have revealed a 5%-10% variance between laboratories. This is much more likely to happen in weakly (1+) reactive specimens than with specimens having reactivity graded 2+ to 4+.

No laboratory test is free from the possibility of nonhuman error, and the FTA-ABS is no exception. Occasional false positive FTA-ABS results have been reported in persons with hyperglobulinemia due to macroglobulins and in patients with antinuclear antibodies. In addition, atypical fluorescent patterns (“beaded”) that could be misinterpreted as reactive have occurred in some patients with systemic lupus erythematosis. Cross-reaction may occur in other spirochetal diseases such as Borrelia (Lyme disease), leptospirosis, and relapsing fever. Problems of nonspecific fluorescence mentioned earlier have been reduced but not entirely eliminated. Occasional false negatives may occur even with the FTA-ABS test. Several reports suggest that concurrent infection by the human immunodeficiency virus type 1 (HIV-1) can sometimes delay the development of a positive VDRL or FTA-ABS reaction.

Microhemagglutination (MHA-TP) test. An MHA test is available using formalin-treated RBCs coated with Nichol strain T. pallidum material. Patient serum is preabsorbed with Reiter Treponema reagent in the same manner as the FTA-ABS technique. Antibody to T. pallidum prevents normal RBC agglutination when the test is performed. About 1%-2% of sera contain nonspecific Forssman-type antibodies, so that reactive sera must be retested with nonsensitized control RBCs.

The MHA test is not as sensitive in primary syphilis as the FTA-ABS test, although it is reactive in more than 50% of patients. It seems equally as sensitive as the FTA-ABS test in secondary and possibly in late syphilis. Compared to FTA-ABS results, various studies have shown 90%-98% overall correlation. Our laboratory has performed a comparison of this type and found that nearly 85% of the disagreements represented either nonreactive or 1+ reactive MHA results and nonreactive or 1+ FTA-ABS results. Therefore, most disagreements seem to occur at low reactivity levels in which the accuracy of either test result is open to some question. This being the case, there is reason to conclude that the MHA could be substituted for the FTA-ABS, except possibly in primary syphilis (in which case an FTA-ABS test could be done if the MHA test results were nonreactive). The MHA test is much easier to perform than the FTA-ABS test and is less expensive.

More recently, several enzyme-linked immunosorbent assays (ELISA) have been reported, with preliminary results similar to those of the FTA-ABS. However, more independent evaluations are needed. Several research centers have also developed Western blot methods. Several investigators have published nucleic acid probe tests amplified by polymerase chain reaction (PCR). Again, initial very good results must be verified by others.