The first practical serologic test for syphilis (STS) was the complement fixation (CF) technique invented by Wassermann. He used extract from a syphilitic liver as the antigen that demonstrated the presence of antitreponemal antibodies. Subsequently it was found that the main ingredient of the substance he used actually had nothing to do with syphilitic infection and was present in other tissues besides liver. It is a phospholipid that is now commercially prepared from beef heart and therefore called cardiolipin. The reagent used in current screening tests for syphilis (as a group, sometimes called STS) is a mixture of purified lipoproteins that includes cardiolipin, cholesterol, and lecithin. Apparently, an antibody called “reagin” is produced in syphilis that will react with this cardiolipin-lipoprotein complex. Why reagin is produced is not entirely understood; it is not a specific antibody to T. pallidum. There is a lipoidal substance in spirochetes, and it is possible that this substance is similar enough to the cardiolipin-lipoprotein complex that antibodies produced against spirochetal lipoidal antigen may also fortuitously react with cardiolipin.

The original Wasserman CF test was replaced by a modification called the Kolmer test; but this in turn was replaced by the much faster, easier, and cheaper flocculation tests. In earlier versions of the flocculation reaction the patient’s serum was heated; for unknown reasons, heating seemed to enhance the reaction. Then a suspension of cardiolipin antigen particles is added to the serum and mixed. In a positive (reactive) test result, the reagin antibody in the patient serum will combine with the cardiolopin antigen particles, producing a microscopic clumping or flocculation of the antigen particles. The reaction is graded according to degree of clumping. The current standard procedure for this type of test is the Venereal Disease Research Laboratory (VDRL) test. It was found that the preliminary heating step could be eliminated if certain chemicals were added to the antigen; this modification is called the rapid plasma reagin (RPR) test and gives results very similar to those of the VDRL.

Drawbacks of the cardiolipin serologic tests for syphilis

Variation in test modifications. Not all sera from known syphilitics gave positive reactions in these tests. It was discovered that the number of positives could be increased by altering the ratio of antigen ingredients. However, usually when the percentage of positive results increases significantly, more false positives are reported. One report indicates that about 2% of VDRL or RPR tests in primary and secondary syphilis are falsely negative due to antigen excess (prozone phenomenon).

Effect of antibiotic therapy. A peculiarity of the STS exists when antibiotic treatment is given. If the patient is treated early in the disease, the STS will revert to nonreactive state. In patients with primary syphilis, one study found that the VDRL or RPR returned to nonreactive state in 60% of patients by 4 months and 100% of patients by 12 months. In secondary syphilis, the VDRL became nonreactive in 12-24 months. Another study of patients with primary and secondary syphilis reported that successful treatment usually produced a fourfold decrease in VDRL titer by 3 months and an eightfold decrease by 6 months. The VDRL decline may be slower if the patient had other episodes of syphilis in the past. However, the longer the disease has been present before treatment, the longer the VDRL takes to become nonreactive. In many cases after the secondary stage it will never become nonreactive, even with adequate treatment (this is called “Wassermann fastness”).

Spontaneous loss of test reactivity. In tertiary syphilis, there is a well-documented tendency for a previously reactive VDRL/RPR test result to revert spontaneously to nonreactive, even if the patient is untreated. This is reported to occur in about 20%-30% (literature range, 5%-45%) of patients.

Biologic false positive reactions. Some patients have definitely positive results on RPR but just as definitely do not have syphilis or any exposure to it. These are called biologic false positive (BFP) reactions. The major known causes of BFP reactions can be classified under three headings:

1. Acute BFP reactions, due to many viral or bacterial infections and to many febrile reactions such as hypersensitivity or vaccination. These usually give low-grade or moderate (1 to 2 +) STS reactions and return to normal within a few weeks.
2. Chronic BFP reactions, due to chronic systemic illness such as antiphospholipid antibodies (Chapter 8), rheumatoid-collagen diseases, malaria, or chronic tuberculosis. There is also an increased incidence of BFP reactions in old age. Whereas there is less than 2% incidence of BFP reactions in men before the age of 60 years, some studies report an incidence as high as 9%-10% after age 70.
3. Nonsyphilitic treponemal diseases such as yaws, pinta, Borrelia (Lyme disease), or relapsing fever.

Summary. The cardiolipin STS is cheap, easy to do, and suitable for mass testing. Its sensitivity and specificity are adequate, and positivity develops reasonably early in the disease. Reagents are well standardized and reproducibility is good. Disadvantages are relatively poor sensitivity in primary syphilis, the tendency in late syphilis for spontaneous reversion of a reactive test result to a nonreactive result, and the problem of BPF reactions. To add further to the confusion, some patients with BFP reactions may have syphilis in addition to one of the diseases known to give BFP reactions. Because of this, everyone hoped for a way to use T. pallidum organisms themselves as antigen rather than depend on the nonspecific reagin system.