Evaluation of an Immunochromatographic Point-of-Care Test for the Simultaneous Detection of Nontreponemal and Treponemal Antibodies in Patients With Syphilis

Abstract

We described the evaluation of the Syphilis Screening & Confirm Assay for the simultaneous detection of nontreponemal and treponemal antibodies. A total of 248 samples were evaluated. The sensitivity of the tests was 98.8%, 99.5% and 98.9%, while specificity was 94.7%, 88.9% and 93.2%, respectively, as compared with the rapid plasma reagin, Treponema pallidum hemagglutination assay, and fluorescent treponemal antibody absorption tests.

In Brief

Syphilis Screening & Confirm Assay is a POC test that detects treponemal and nontreponemal antibodies and shows a sensitivity and specificity similar to the syphilis standard laboratory tests.

Syphilis is a systemic sexually transmitted infection caused by Treponema pallidum that affects 12 million people each year.¹ In Portugal, syphilis seroreactivity rates are high, especially in some population groups, such as sex workers (8.7%) and in men who have sex with men (MSM; 4.2%; unpublished results. Early diagnosis and treatment of syphilis are essential to prevent transmission to sexual partners, especially pregnant women and their neonates. Because direct detection of T. pallidum requires the use of a dark-field microscope, the usefulness of this technique is limited because it demands considerable expertise in distinguishing T. pallidum from other subspecies.² On the other hand, the use of polymerase chain reaction for the identification of T. pallidum DNA is not sensitive enough, especially in the latent stages of the infection.³ This technique is also not performed routinely at primary health care settings. Nontreponemal tests are inexpensive, are simple to perform, and may be used to evaluate treatment response. However, reactive samples need to be confirmed with a treponemal test. If treponemal tests are used for screening, then a reactive result cannot distinguish between active and treated syphilis cases and needs to be confirmed with a nontreponemal test. Meanwhile, a number of treponemal rapid point-of-care (POC) tests have been developed, in accordance with the ASSURED (Affordable, Simple, Sensitive, Specific, User friendly, Rapid and robust, Equipment free and Deliverable to end user) criteria,⁴ but they still have to be confirmed by a nontreponemal test. The DPP Syphilis Screening & Confirm Assay (SSCA) used in the present study is an immunochromatographic POC diagnostic test manufactured by Chembio Diagnostics Systems Inc, Medford, NY. It is stable at room temperature for 1 year. A sample of whole blood, serum, or plasma is placed into well No. 1, and a running buffer to hydrate the colloidal gold conjugate is placed into well No. 2 of the device (Fig. 1). Nontreponemal and treponemal antibodies are simultaneously detected within 10 to 15 minutes.⁵

Figure 1: Structure of the dual POC test for syphilis indicating the location of the antigen lines. A, Dissected view after testing of a reactive serum. B, Complete cassette after testing of a reactive serum.

We evaluated the sensitivity and specificity of the DPP SSCA in detecting nontreponemal and treponemal antibodies in individuals to be screened for syphilis.

Two hundred forty eight serum samples collected from individuals with known stages of syphilis, including some who have been treated, were tested. This was an exempted study. Sera samples were collected as excess samples from clinics. However, the information related to the stage of syphilis was made available by health officials of the various clinics. No participants were enrolled from and during a period, and information regarding age or HIV status was unknown.

Thirty-six were in the primary stage, 39 had secondary syphilis, and 99 had latent syphilis. Fifteen individuals were diagnosed as having had past syphilis and been successfully treated, and 60 presented with no signs and symptoms of syphilis and were nonreactive in both rapid plasma reagin (RPR) and T. pallidum hemagglutination assay (TPHA) tests.

Samples were evaluated by a quantitative RPR test (Macro-Vue^R RPR Card Tests; Benton, Dickinson and Company, Sparks, MD), qualitative TPHA (Lab21 Healthcare Ltd, Kentford, UK), fluorescent treponemal antibody absorption test (FTA-Abs; Fluorescent Treponema Antibody Absorbed; EUROIMMUN, Lübeck, Germany), and the DPP SSCA (Chemio Diagnostic Systems, Medford, NY). Each assay was performed as per manufacturers’ instructions.

Sensitivity and specificity results of the SSCA nontreponemal test (SSCA-NT) in comparison with the RPR test are described in Table 1. From the 248 samples, 171 and 71 were reactive and nonreactive in both tests, respectively, with a concordance rate of 97.6% (242/248). The sensitivity and specificity of the evaluated test were 98.8% (171/173) and 94.7% (71/75), respectively.

TABLE 1: Comparison of the SSCA-NT Test With the RPR Test

The comparison of the treponemal SSCA test (SSCA-TT) with the TPHA is described in Table 2. One hundred eighty four serum samples from the total number of samples (248) were reactive in both SSCA-TT and the TPHA tests, and 56 were nonreactive in any of these tests, with the concordance rate being 96.8% (240/248). The sensitivity was 99.5% (184/185) and the specificity was 88.9% (56/63).

TABLE 2: Comparison of the SSCA-TT Test With the TPHA Test and With FTA-Abs Tests

In comparing the SSCA-TT and the FTA-Abs (Table 2), the sensitivity was 98.9% (187/189), the specificity was 93.2% (55/59), and the concordance rate was 97.6% (242/248). One hundred eighty seven samples were reactive, and 55 were nonreactive.

The treponemal and nontreponemal sensitivity of the SSCA, the TPHA, and the FTA-Abs tests, in accordance with the various stages of syphilis, is referred in Table 3. The sensitivity in primary syphilis was 100% (36/36) for all tests, with the exception of the TPHA, which showed a sensitivity of 97.2% (35/36). For secondary syphilis, the sensitivity was 100% (39/39) for both test and comparators. For patients with latent syphilis, the sensitivity was 99% (97/98) for both the RPR and the SSCA-NT tests, 98% (96/98) for the SSCA-TT, and 99% (97/98) and 100% (98/98), respectively, for the comparators TPHA and FTA-Abs.

TABLE 3: Sensitivity of Tests Used in This Study by Clinical Stage of Syphilis

The specificity of the SSCA-NT and SSCA-TT was evaluated in relation to the FTA-Abs test in patients presenting with no signs and symptoms of syphilis, whose samples were nonreactive in the RPR and TPHA tests. The specificity of the DPP SSCA was 93.2% (55/59) for the treponemal line and 98.3% (58/59) for the nontreponemal line (Table 4).

TABLE 4: Specificity of the SSCA-NT and SSCA-TT Compared With the FTA-Abs in Patients With No Clinical Evidence of Syphilis

The diagnosis of sexually transmitted infections, including syphilis, is based on clinical manifestations and serological testing.

The early serological diagnosis of syphilis is essential to initiate immediate treatment. When results are not available, patients may not return for counseling and treatment, and this may result in further spreading the disease to sexual partners and newborns in cases of pregnant women.

Serological determination for syphilis is normally performed in laboratories, requiring appropriate equipment and trained personnel to interpret the results that may not be available for several days or weeks. In developing countries with resource-poor settings, laboratory testing is significantly more challenging because of the lack of adequate energy source, transportation, and infrastructure conditions. Available POC tests, which have been developed to overcome these problems, detect only treponemal antibodies,⁶ and they are unable to distinguish between active and past infections. Although a POC test that detects both treponemal and nontreponemal antibodies has been developed, the detector conjugate needed refrigeration.⁷ The test used in the present study can be performed with whole blood; it was developed to simultaneously detect both treponemal and nontreponemal antibodies; the device is stable at room temperature and does not require refrigeration. This study demonstrated that the sensitivity and specificity of the test are as good as the standard diagnostic tests for syphilis. The test can be performed at a clinical site, and results are available within 10 to 15 minutes. The comparison between the SSCA and the comparator (RPR, TPHA, and FTA-Abs) tests showed similar sensitivities for both the nontreponemal (98.8%) and treponemal lines (99.5%) for the comparison with the TPHA and 98.9% for the FTA-Abs. The specificity results were also comparable between the SSCA-TT and the FTA-Abs (93.2%) and for the SSCA-NT when compared with the RPR (94.7%). The specificity of the SSCA-TT with the TPHA was 89%, with 7 samples shown to be reactive in the first test and nonreactive in the last test. It should be noticed that 3 of these samples were also reactive in the FTA-ABS test. One of them was a case of primary syphilis with a reactive RPR and SSCA-NT. The other 3 samples could be from patients with past infection that had been treated and cured.

Sensitivity was 100% in the primary and secondary stages for all tests, with the exception of a nonreactive TPHA (97.2%) obtained in serum from a patient with primary syphilis. This sample was further submitted to a polymerase chain reaction technique, and T. pallidum DNA was amplified. In latent syphilis, results were similar, varying from a sensitivity of 98% of the nontreponemal line for both SSCA and RPR to 100% for the FTA-Abs test. Although we feel that biological false-positive samples are important to assess the use of the test, especially of the SSCA-NT, we did not have available samples with other diseases than syphilis reactive against cardiolipin antigen.

Specificity was established by criteria such as a patient with any signs or symptoms of syphilis, whose samples were nonreactive in the TPHA and RPR tests and was 98.3% when compared with the comparator test (FTA-Abs). These results showed that both treponemal and nontreponemal lines of the DPP SSCA had a sensitivity and a specificity similar to the syphilis standard laboratory tests. It also allows for screening of both treponemal and nontreponemal antibodies. Therefore, the DPP Syphilis Screening & Confirm POC test can be considered a useful POC test for the serological diagnosis of syphilis, including in resource-poor settings, where there is a need to provide counseling and treatment on site and thus prevent the further spread of the disease.