Test ID: SYPGR
Syphilis IgG Antibody with Reflex, Serum

An aid in the diagnosis of active Treponema pallidum infection

Routine prenatal screening

If syphilis IgG is positive, RRPR/34511 Rapid Plasma Reagin w/ Reflex, S will be performed at an additional charge.

If RRPR is negative, RTPPA/34512 Syphilis Ab, TP-PA, S will be performed at an additional charge.

See Syphilis Serology Algorithm in Special Instructions.

• Syphilis Serology Algorithm

SYPGR: Multiplex Flow Immunoassay

RRPR: Flocculation

RTPPA: Particle Agglutination

Container/Tube:

Preferred: Red top

Acceptable: Serum gel

Specimen Volume: 1 mL

Forms: If not ordering electronically, submit a General Request Form (Supply T239) with the specimen.

Syphilis is a disease caused by infection with the spirochete Treponema pallidum. The infection is systemic and the disease is characterized by periods of latency. These features, together with the fact that Treponema pallidum cannot be isolated in culture, mean that serologic techniques play a major role in the diagnosis and follow-up of treatment for syphilis.

Historically, the serologic testing algorithm for syphilis included an initial nontreponemal screening test, such as the rapid plasma reagin (RPR) or the venereal disease research laboratory (VDRL) tests. Because these tests measure the host's antibody response to nontreponemal antigens, they lack specificity. Therefore, a positive result by RPR or VDRL requires confirmation by a treponemal-specific test, such as the fluorescent treponemal antibody-absorbed (FTA-ABS) or microhemagglutination assay (MHA-TP). Although the FTA-ABS and MHA-TP are technically simple to perform, they are labor intensive and require subjective interpretation by testing personnel.

Recently, enzyme immunoassays (EIA) and multiplex flow immunoassays (MFI) were introduced to assess serologic response to Treponema pallidum. The Bio-Rad BioPlex Syphilis IgG assay is an example of MFI technology, which utilizes specific, treponemal antigens coated on microspheres for the detection of IgG-class antibodies to Treponema pallidum. The BioPlex Syphilis IgG assay is highly sensitive and specific (see Supportive Data), and allows for an objective interpretation of results. Due to several factors including the low prevalence of syphilis in the United States, the increased specificity of treponemal assays, and the objective interpretation of MFI and EIA technology, initial serologic testing by a treponemal-specific assay (eg, EIA or MFI) is now commonly performed in clinical laboratories. Specimens testing positive by the treponemal-specific assay are then tested by RPR to provide supplementary serologic data, as well as to provide an indication of the patient's disease state and history of treatment.

During early primary syphilis, patients may present with nonspecific clinical findings and serology tests may be negative. As the disease progresses into the secondary phase, IgG-class antibodies to Treponema pallidum reach peak titers, and may persist indefinitely regardless of the disease state or prior therapy.

For prenatal syphilis screening, the IgG test is recommended. IgM testing should not be performed during routine pregnancy screening unless clinically indicated.

Treponema pallidum IgG antibodies persist indefinitely, regardless of whether the patient has been treated or not. To determine if a patient has been treated for syphilis, the RPR test is performed. If the RPR is positive, the results may suggest active, untreated syphilis. In contract, a positive syphilis screening test and a negative RPR most likely suggest past, successfully treated syphilis.

Negative

Syphilis screening at Mayo Clinic is performed by using the reverse algorithm, which first tests sera for Treponema pallidum specific IgG antibodies using an automated multiplex flow immunoassay (MFI). A positive IgG treponemal test suggests infection with Treponema pallidum at some point in the past, but does not distinguish between treated and untreated infections. This is because treponemal tests (eg, EIA, MFI, or fluorescent treponemal antibody-absorbed: FTA-ABS) may remain reactive for life, even following adequate therapy. Therefore, the results of a nontreponemal assay, such as rapid plasma reagin (RPR), are needed to provide information on a patient's disease state and history of therapy.

In some patients, the results of the treponemal screening test (syphilis IgG) and RPR may be discordant (eg, syphilis IgG positive and RPR negative). To discriminate between a falsely reactive screening result and past syphilis, CDC recommends performing a second treponemal-specific antibody test using a method that is different from the initial screen test (eg, Treponema pallidum particle agglutination: TP-PA).(2)

In the setting of a positive syphilis IgG screening result and a negative RPR, a positive TP-PA result is consistent with either 1) past, successfully treated syphilis, 2) early syphilis with undetectable RPR titers, or 3) late/latent syphilis in patients who do not have a history of treatment for syphilis. Further historical evaluation is necessary to distinguish between these scenarios (Table 1).

In the setting of a positive syphilis IgG screening result and a negative RPR, a negative TP-PA result is most consistent with a falsely reactive syphilis IgG screen (Table 1). If syphilis remains clinically suspected, a second specimen should be submitted for testing by the SYPHA/34509 Syphilis Antibody Cascade, Serum assay.

Table 1.  Interpretation and follow-up of reverse screening results:

This test is not offered as a screening or confirmatory test for blood donor specimens.

Despite active syphilis, serologic tests may be negative in severely immunosuppressed patients such as those with AIDS.

In very early cases of primary syphilis, serology tests for syphilis may be negative.

In cases of untreated, late/latent syphilis, the result of rapid plasma reagin may be negative. However, the syphilis screening test (MFI) and TP-PA should be positive. A thorough clinical and historical evaluation should be performed to determine if treatment for latent syphilis is required.

Results should be considered in the context of all available clinical and laboratory data.

To evaluate the accuracy of the BioPlex Syphilis IgG assay, 1,008 consecutive, unique serum specimens submitted for routine syphilis IgG testing by EIA were also tested on the BioPlex 2200. Specimens showing discrepant results were repeatedly tested by both methods, and also tested by Treponema pallidum-particle agglutination (TP-PA). The BioPlex 2200 Syphilis IgG assay correctly identified 77 (98.7%) of the 78 specimens that were positive by EIA. The EIA-positive, BioPlex-negative specimen was shown to be truly negative by TP-PA and repeat EIA. In addition, the BioPlex assay correctly identified 916 (98.5%) of the 930 specimens that were negative by EIA. Among the 14 BioPlex-positive, EIA-negative specimens, 8 were shown to be truly positive by TP-PA (see Table 2). The BioPlex Syphilis IgG assay demonstrated an adjusted sensitivity and specificity of 100% and 99.4%, respectively.

Table 2. Comparison of the Bio-Rad Syphilis IgG Assay and the Trep-Chek EIA among Prospective Serum Specimens (n=1,008):

Sensitivity: 77/78 =98.7% (95% Confidence intervals: 92.4%, 99.9%)

Specificity: 916/930 =98.5% (95% Confidence intervals: 97.5%, 99.1%)

To evaluate the accuracy of the TP-PA assay, it was compared to the BioPlex 2200 syphilis IgG MFI assay using 1,200 serum specimens (1,100 prospective and 100 previously characterized sera). The results are summarized in Table 3:

Table 3. Comparison of the BioPlex 2200 syphilis IgG and TP-PA assays using serum samples (n =1,200): 

Sensitivity =90.18%; (95% confidence interval, 83.15%- 94.61%)

Specificity =99.6%; (95% confidence interval, 99.0%- 99.9%)

Overall percent agreement =98.7%; (95% confidence interval, 96.3%- 100%)

All discrepant samples tested negative by RPR.

1. Tramont EC: Treponema pallidum (Syphilis). In Principles and Practice of Infectious Diseases. 5th edition. Edited by GL Mandell, JE Bennet, R Dolin. New York, Churchill Livingstone, 2000, pp 2474-2491

2. CDC. Discordant results from reverse sequence syphilis screening - five laboratories, United States, 2006-2010. MMWR 2011;60(5):133-137

3 Binnicker MJ, Jespersen DJ, Rollins LO: Direct comparison of the traditional and reverse syphilis screening algorithms in a population with a low prevalence of syphilis. J Clin Microbiol 2012 Jan;50(1):148-150

The BioPlex Syphilis IgG is a multiplex flow immunoassay performed on the BioPlex 2200 System (Bio-Rad Laboratories, Hercules, CA). Three types of dyed beads are coated with recombinant proteins associated with Treponema pallidum (15kDa, 17kDa and 47kDa). An aliquot of patient sample, sample diluent, and bead reagent are combined in a reaction vessel, and the mixture is incubated at 37 degrees C. After a wash cycle, antihuman IgG antibody conjugated to phycoerythrin (PE) is added to the dyed beads, and this mixture is incubated at 37 degrees C. Excess conjugate is removed in another wash cycle and beads are resuspended in wash buffer. The bead mixture is then passed through a flow-based detector and identified according to the fluorescence emitted, which is specific to the internal dye composition of the microsphere. The amount of antibody bound to the capture antigen is then determined by measuring the fluorescence of the attached PE. Raw data is calculated in relative fluorescence intensity (RFI).(Package insert: BioPlex 2200 System Syphilis IgG [T.pallidum], Bio-Rad Laboratories Clinical Diagnostic Group, Hercules, CA)

If the IgG result is positive, a rapid plasma regain (RPR) is automatically performed. The RPR test is a macroscopic screening assay done with unheated serum. Reagin reacts with nontreponemal antigen containing colloidal charcoal particles. This reaction results in a visual flocculation of the black particles against the white card background. The test yields a positive or negative result, and all positive samples are titered to determine the highest positive dilution.(Huber TW, Storms S, Young P, et al: Reactivity of microhemagglutination, fluorescent treponemal antibody absorption, Venereal Disease Research Laboratory, and rapid plasma reagin tests in primary syphilis. J Clin Microbiol 1983 Mar;17[3]:405-409)

The Serodia TP-PA test is based on the agglutination of colored gelatin particle carriers sensitized with Treponema pallidum (Nichols Strain) antigen. Serum samples are serially diluted in microplate wells. Sensitized gelatin particles are added to respective wells and the contents of the plate mixed.  The mixture is incubated for 2 hours at room temperature. Serum containing specific antibodies will react with the antigen-sensitized colored gelatin particles to form a smooth mat of agglutinated particles in the microplate well. A compact button formed by the settling of the non-agglutinated particles characterizes negative reactions. The agglutination patterns are read visually to determine interpretation.(Package insert: Serodia -TP PA, Fujirebio Diagnostics, Inc., Tokyo, Japan.)

Monday through Saturday; 9 a.m.

86780-Syphilis antibody

86592-Rapid plasma reagin (if appropriate)

86780-Syphilis Antibody by TP-PA (if appropriate)