Regional Medical Laboratory

Syphilis is a disease caused by the spirochete bacterium Treponema pallidum and is most commonly transmitted through sexual contact. It has re-emerged and become an epidemic in Oklahoma. The manifestations of this disease are notorious for having different stages occurring over time in untreated infection. Patients may seek evaluation for symptoms or signs of primary infection (e.g., chancre), secondary infection (e.g., diffuse rash), or tertiary infection (e.g., symptoms of aortic insufficiency). Alternatively, patients may be completely asymptomatic and only identified on routine screening.

The diagnosis of syphilis is most commonly made by serologic testing. There are two types of serologic tests for syphilis: Nontreponemal tests [e.g. Rapid plasma regain (RPR) and Venereal Disease Research Laboratory (VDRL)] and treponemal-specific tests [e.g. Fluorescent treponemal antibody absorption (FTA), T. pallidum particle agglutination assay (TP-PA), T. pallidum enzyme immunoassay (TP-EIA) and T. pallidum Chemiluminescence immunoassay (TP-CIA)]. The use of only one test is insufficient for diagnosis since serologic testing, can be associated with both false positive results and false negative results.

Most commonly, serologic testing for syphilis is associated with false positive results, particularly nontreponemal testing. However, caution must also be taken as false negative results can occur due to the reliance upon a humoral immune response to infection. The use of serologic testing may be limited in patients with advanced immunosuppression and/or early disease. Although the majority of patients have a positive serologic test when they present with a chancre (i.e. 2 to 4 weeks after exposure), approximately 20-30% have a nonreactive nontreponemal test. For such patients, serologic testing generally turns positive within the next 2 to 4 weeks.

The nontreponemal assays are semi-quantitative and the amount of antibody present (both IgM and IgG) generally reflects the activity of the infection. Positive nontreponemal tests are reported as a titer of antibody (e.g. 1:16, which represents the detection of antibody in serum diluted 16-fold). Titers tend to wane over time even without treatment, but successful therapy accelerates the pace of antibody decline. These changes in titer are monitored after treatment to detect a therapeutic response. Treponemal antibody tests are based upon the detection of antibodies directed against specific treponemal antigens and thus are more specific than the nontreponemal tests. Treponemal tests are qualitative only and are reported at “reactive” or “nonreactive”.

Although more specific, treponemal tests have historically been more complex and expensive to perform than nontreponemal tests. Thus, they have traditionally been used as secondary confirmatory tests for syphilis when the nontreponemal tests are reactive. In recent years, these tests have become automated, enhancing their simplicity and facilitating ease of use. As a result, these tests have become increasingly used as an initial screening test for syphilis rather than a confirmatory test. Utilizing treponemal tests for initial screening has been called the “syphilis reverse screening” or “syphilis reverse algorithm”.

In 2014, Regional Medical Laboratory implemented the syphilis reverse screening algorithm for the following reasons:

1. To reduce false positives on the nontreponemal assay
2. False negatives on the nontreponemal assay
3. The RPR was tedious and required repetitive pipette motion by our technologists
4. Expense of time and reagents
5. Significant increase in testing volume
6. The automated treponemal test significantly decreased our turn-around-time, technologist time invested, cost and ease of use
7. It has been well received by the medical community 

After implementation, RML experienced occasional false positive results with the automated treponemal screening test, which was mitigated by determining a window of antibody activity which required further testing with a TP-PA assay. The satisfactory window index was established through the evaluation of over 100 challenging specimens. RML was using a TP-EIA at the time and determined anything having an index of 1 to 10 required further testing with a TP-PA test. RML has since moved to the TP-CIA assay and using the same set of 100 challenging specimens were able to establish a window with a 1 to 5 index that would also require further testing by the TP-PA test. RML is confident that with the further testing in place with TP-PA very few if any false positive are being reported.

Screening with the TP-CIA assay, samples testing negative do not require further testing and the patient is considered negative for syphilis. Samples testing positive by the screening test are reflexed to a nontreponemal test, such as RPR. If the RPR is reactive, the patient is considered positive for untreated or recently treated syphilis. If the RPR is nonreactive and the TP-CIA result is greater than an index of >5 then the patient is considered to have had a previous syphilis infection but if the index is 1 to 5 then a TP-PA test is performed. A positive TP-PA is suggestive of past, successfully treated syphilis, late/latent syphilis, or early syphilis. If the TP-PA is negative, the screening result is typically interpreted as a false positive and a footnote is entered which reads, “The patient’s results are negative (NR) following repeat and further testing by different methodologies for the presence of Treponemal antibody”. These results require a thorough clinical and historical evaluation to assist in the interpretation. See diagram 1.

Many other laboratories across the country have also implemented the reverse sequence screening algorithm using TP-EIA or TP-CIA technology. Their interpretations are identical to RML’s. The published reverse sequence screening algorithm as seen in diagram 2, samples are screened by an automated treponemal test, such as a TP-EIA or TP-CIA. Samples testing negative by the screening assay do not require further testing and the patient is considered negative for syphilis. Samples testing positive by the screening test are reflexed to a nontreponemal test, such as RPR. If the RPR is reactive, the patient is considered positive for untreated or recently treated syphilis. If the RPR is nonreactive, the sample is reflexed to a second treponemal test, the TPPA test. If the TP-PA is negative, the screening result is typically interpreted as a false positive. A positive TPPA is suggestive of past, successfully treated syphilis, late/latent syphilis, or early syphilis. These results require a thorough clinical and historical evaluation to assist in the interpretation of results.

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For more information, please review the following reference article:
“Reverse Sequence Screening for Syphilis: The Advantages and Limitations Compare with the Traditional Algorithms” (link to https://www.aacc.org/publications/cln/articles/2014/november/screening-syphilis).

Syphilis Antibody Testing - Diagram 1
Syphilis Reverse Sequence Screening - Diagram 2