INTRODUCTION

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Syphilis is an infection most often transmitted by sexual contact. The causative agent is the human pathogen Treponema pallidum subsp. pallidum (9, 13). Serodiagnosis can be performed by two types of methods: (i) nontreponemal assays, such as the Venereal Disease Research Laboratory (VDRL) and rapid plasma reagin assays, and (ii) treponemal assays, such as fluorescent treponemal antibody-absorption (FTA-ABS), the microhemagglutination assay for antibody to T. pallidum (MHA-TP) or the T. pallidum hemagglutination assay (TPHA), and various enzyme-linked immunosorbent assays (ELISAs), which are typically more sensitive and yield more objective results than agglutination and fluorescence assays (7). All serological techniques have been shown to be cross-reactive with antibodies directed to other treponema species. False-positive reactivities can arise when conditions such as Lyme borreliosis, autoimmune disease, and human immunodeficiency virus (HIV) infection are present (2, 5, 10, 11, 15). Therefore, the use of only one type of test is insufficient for an accurate diagnosis (16). Moreover, the development of new sensitive assays such as ELISAs points to the questionable empirical threshold values that have been used in agglutination assays (i.e., for TPHA, 1:80) (8, 12, 17).

Although insensitive and of poor specificity, the nontreponemal antibody titers have been correlated with disease activity (1, 11). By contrast, treponema-specific antibody titers correlate poorly with disease activity and cannot be used to assess disease stage. Syphilis disease stage was previously evaluated by the T. pallidum immobilization assay (TPI), but it is rarely used today because it is difficult to perform, time-consuming, and, therefore, very expensive. Anti-T. pallidum titration by FTA-ABS has replaced TPI in many laboratories. However, the interpretation remains subjective and FTA-ABS is not suitable for large-scale testing situations (5, 14). Although Western blotting methods have been suggested as confirmatory assays, they include nonrelevant proteins, thereby inducing indeterminate and false-positive reactivity patterns (6). Furthermore, the reported sensitivity of about 94% (4) has resulted in their limited use.

The present study was designed to validate the new INNO-LIA Syphilis kit as a confirmatory assay for T. pallidum antibodies. We evaluated the assay in a two-phase study. First, we used well-characterized sera to assess sensitivity and specificity. In a second phase, we evaluated the performance with a somewhat less well documented collection of samples and studied any samples with discrepant results in detail. The specificity of the assay was also challenged by testing serum samples from patients with biological conditions, such as autoimmunity and pregnancy, who often test false-positive by assays for T. pallidum. Overall sensitivity and specificity were calculated with reference to consensus diagnostic assay results.

	MATERIALS AND METHODS

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Study design and sera. This study involved a total of 840 human serum specimens tested by the French National Reference Center for Venereal Diseases (Institute Alfred Fournier, Paris, France). The samples had been collected over the past 5 years either for a first screening or for confirmatory testing following a reactivity in a first screening test. All samples were stored at 20°C prior to testing. In the first phase of the study, a unique collection of samples (all tested by TPI) was included (n = 500); of these, 276 were clearly negative (TPHA negative at 1:80 dilution, VDRL negative, and TPI negative) and 224 were clearly positive (TPHA positive at a dilution greater than or equal to 1:80 and TPI positive). The second phase involved the remaining 340 serum samples. In addition to serum samples, 13 cerebrospinal fluid (CSF) samples obtained from patients with clinically documented syphilis at different stages were also evaluated; this collection was tested by classical methods with mixed results, including equivocal serologic results for syphilis. Furthermore, sera from patients with the most common biological conditions that result in false-positive reactivities by serologic assays for syphilis were also investigated by various assays such as assays for anticardiolipin antibodies (n = 8), antinuclear antibodies (n = 10), and rheumatoid factors (n = 11). In addition, Lyme serology-positive sera (n = 12, Epstein-Barr virus capsid antigen immunoglobulin M (IgM)-positive sera (n = 8), sera from pregnant women (n = 10), and isolated sera with VDRL reactivities (n = 31) were tested. The Lyme serology-positive samples were screened by an in-house immunofluorescence technique with Borrelia burgdorferi B31; the results for six samples were further confirmed by Borrelia Western Blot IgG (Gull Laboratories, Bois d'Arcy, France).

Screening assays. The VDRL assay used in this study uses a colloidal suspension of cholesterol-lecithin sensitized with beef heart-extracted cardiolipin (Sypal, Diagast, Loos, France) as a nontreponemal antibody detection system. In addition, treponema-specific antibodies were detected in an initial screening with sensitized sheep erythrocytes (RBCs; MHA-TP; Ames-Bayer, Puteaux, France). Repeat testing, in addition to the previous assay, if needed, was performed by using avian RBCs (LD Serokit TPHA, Labor Diagnostika GmbH, Heiden, Germany). Finally, a screening ELISA was used for further investigations; we used a kit with treponema recombinant antigens (ICE Syphilis; Murex Diagnostics B. V., Aalst, Belgium). All test procedures and interpretation of the results were performed according to the manufacturer's instructions.

Fluorescence methods (FTA-ABS). Samples reactive by a screening method are usually tested by a fluorescent T. pallidum assay with cultured treponema organisms (TrepoSpot IF; BioMérieux, Marcy l'Etoile, France). Specific antibodies are detected with a fluorescein-labeled anti-human immunoglobulin after the absorption of nonspecific antibodies on T. phagedenis extracts (5). If specific fluorescence can be perceived at a sample dilution of 1:100, the sample is considered positive.

IgM detection methods. Two methods have been used to determine IgM in human sera. A commercial assay (FTA-M; Sanofi Diagnostics Pasteur, Marne-la-Coquette, France) and an in-house IgM solid-phase hemadsorption assay (IgM-SPHA) with T. pallidum-sensitized RBCs were used.

TPI. TPI was performed at the Institute Alfred Fournier by an in-house procedure, as follows. A 1/10 dilution of human serum was incubated with a suspension of freshly prepared treponema in the presence of complement proteins for 18 h at 37°C. A control procedure with inactivated complement proteins was also performed for each test. TPI determines the ability of antibody to immobilize live T. pallidum (Nichols strain), as visualized under a dark-field microscope and expressed as a percentage (number of immobilized spirochetes/total count). A sample is considered positive by TPI if at least 70% of Treponema are immobilized compared to the rate of immobilization for the corresponding control. The sample is considered negative if the value by TPI is less than 30% of that for the control, and TPI values between 30 and 70% of that for the control are considered inconclusive. For some TPI-positive samples, titration is obtained at the end dilution that shows immobilization of at least 50% of the T. pallidum spirochetes.

Consensus results by classical tests. For the 340 less well documented samples, we established a consensus serological diagnostic for all samples on the basis of the available results of the classical assays (TPHA, FTA-ABS, and TPI). In some cases, if enough serum was available, repeat testing, in addition to a screening enzyme immunoassay (ICE Syphilis), was performed. In such cases, the global information was considered to define the consensus results. Since VDRL is often negative for patients with true late infections (who are positive by other techniques), VDRL data were not used to determine the consensus results. These consensus results were obtained as follows. A sample was considered positive if all available results were positive; a sample was considered negative if all available results were negative. When discrepant results were shown, the most predominant result was considered the consensus result; if discrepant results were present in equal numbers, no consensus could be reached and the result for the sample was therefore considered to be equivocal.

INNO-LIA Syphilis kit. The INNO-LIA Syphilis kit uses recombinant antigens and synthetic peptides derived from T. pallidum (Nichols strain) membrane proteins. Briefly, the antigens used consisted of three immunodominant proteins (TpN47, TpN17, and TpN15) expressed as full-size proteins in Escherichia coli and one synthetic peptide (TmpA) derived from transmembrane protein A. In brief, the TmpA antigen was synthesized on a solid phase by using Tentagel S resin (Rapp Polymere GmbH, Tübingen, Germany) by standard 9-fluorenylmethoxycarbonyl amino acid chemistry. After completion of peptide synthesis, the peptide was cleaved from the resin and was then purified by reverse-phase high-performance liquid chromatography. The primary amino acid sequence of this antigen was optimized and included an N-terminal biotin residue to allow peptide immobilization on the nylon strips by using streptavidin binding properties.

View larger version (10K): [in this window] [in a new window]   FIG. 1.   Layout of INNO-LIA Syphilis kit strip.

View larger version (40K): [in this window] [in a new window]   FIG. 2.   Representative patterns of reactivity with INNO-LIA Syphilis kit. Control lines are indicated (3+, 1+, and ±); antigen lines are those for the TpN47, TpN17, TpN15, and TmpA antigens, respectively. Sera correspond to the following (from top to bottom): six positive samples with indeterminate (samples 547, 800, and 820) or weakly positive (samples 601, 612, and 668) consensus results, two samples with indeterminate results with one negative (sample 207) and one positive (sample 518) consensus result; and three negative samples (samples 768, 769, and 770) with positive serology for Lyme borreliosis.

Statistics. StatMate software (version 1.01; GraphPad, San Diego, Calif.) was used for the calculation of 95% confidence intervals (CIs) for proportions.

	RESULTS

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INNO-LIA Syphilis kit performance. Among the 500 samples (224 positive and 276 negative samples) studied in the first phase, only 1 sample with a discrepant result was found, yielding a sensitivity of 100% (95% CI, 98.4 to 100%) and a specificity of 99.64% (95% CI, 98.0 to 99.9%) for the INNO-LIA Syphilis kit. The sample with a discrepant result had a negative serology but was scored as indeterminate by the INNO-LIA Syphilis kit. Unfortunately, no follow-up or further testing could be performed with this specimen. In the second phase of the study, among the remaining 340 serum samples with somewhat less well documented serologies, 8 samples had discrepant results, and 5 of these had equivocal serologies by classical testing. None of the Lyme borrelia-positive sera reacted in tests with the INNO-LIA Syphilis kit (the results for three such samples are shown in Fig. 2).

Comparison of INNO-LIA Syphilis kit results with the consensus results. For the determination of the overall assay performance, we combined the samples studied in the two phases and calculated sensitivity and specificity. A summary of the results obtained with the INNO-LIA Syphilis kit testing compared to the consensus data obtained from classical tests is shown in Table 1. Sensitivity was evaluated for a total of 464 serum samples that were considered positive by a consensus of multiple tests (TPHA, FTA-ABS, and TPI) and clinical information. The INNO-LIA Syphilis kit confirmed the results for 462 of 464 samples positive for T. pallidum antibodies, resulting in a sensitivity of 99.6%. Table 2 presents the detailed results available for each sample with discrepant results. Briefly, two samples (samples 518 and 599) considered consensus positive were indeterminate with the INNO-LIA Syphilis kit. Both samples were doubtful by TPHA, positive by FTA-ABS (low titers) and ICE Syphilis EIA, but negative by VDRL. Only one sample was tested by TPI, and it had a negative result. None of the consensus-positive samples was found to be negative with the INNO-LIA Syphilis kit. For evaluating the specificity, 369 of 371 negative serum samples were correctly identified by INNO-LIA Syphilis kit, thereby yielding a specificity of 99.5%. Due to reactivity of a single band, two samples had indeterminate results with the INNO-LIA Syphilis kit. One sample (sample 207) was negative by four techniques but was not tested by the ICE Syphilis EIA; the other sample (sample 666) was negative by VDRL, TPHA, and TPI but positive by FTA-ABS and the ICE Syphilis EIA. None of the samples from this population were false positive. Both overall sensitivity and specificity fit within 95% CIs.

Analysis of discrepant results. Discrepancies between the consensus result and the INNO-LIA Syphilis kit result were again analyzed in the context of their global information. In addition, due to subjective reading of the results obtained by agglutination techniques, if clear-cut positive or clear-cut negative results were not demonstrated at the tested dilution (1:80), a doubtful TPHA result was indicated as D80 (Table 2).

Serum and CSF samples with clinical information. Thirteen CSF specimens obtained from 10 syphilitic patients were also evaluated in this study. Of these, 11 samples were classified as positive by the consensus result as well as with the INNO-LIA Syphilis kit (100% sensitivity). The two remaining CSF samples were considered equivocal by the consensus result (doubtful by TPHA, negative by FTA-ABS, and positive by the ICE Syphilis EIA); with the INNO-LIA Syphilis kit, one sample (sample 699) was positive and one sample (sample 701) was indeterminate (Table 3).

	DISCUSSION

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In the course of the 20th century, syphilis serology testing has significantly contributed to limiting the spread of this venereal disease (15). Furthermore, disease activity has been linked to the results of multiple tests: TPHA-positive and VDRL-negative results are thought to be indicative of latent syphilis, while TPHA-positive and VDRL-positive results are usually indicative of infectious syphilis (1). To establish a conclusive diagnosis, TPI was often used to resolve conflicting results. This complex and expensive methodology has been progressively abandoned and substituted by other strategies for confirmation. For instance, several laboratories perform either TPHA and VDRL titration by serial dilution or TPHA and FTA-ABS titration for confirmation of syphilis (7).

The INNO-LIA Syphilis kit used in this study is a user-friendly methodology that makes use of color-coded reagents and simple interpretation criteria. It shows a high degree of performance as a multiparametric confirmation method. The assay validated in this study has been optimized to confirm the presence of specific antibodies to T. pallidum antigens in human sera and plasma. Nevertheless, testing of a limited series of CSF samples was also possible, and it was suggested that the kit had a higher degree of sensitivity with these samples than agglutination assays do. Since IgM molecules (agglutinins) do not cross the blood-brain barrier, detection of treponemal antibodies was somewhat less sensitive by agglutination techniques (TPHA, VDRL) than by other assays. This was clearly illustrated with the available paired samples from patients (serum and CSF samples) (Table 3). The high degree of sensitivity of the INNO-LIA Syphilis kit allowed the detection of low levels of IgG, like in the CSF of patient 699 (Table 3). Classical testing schemes classified five samples as equivocal due to discrepancies among the different assays (Table 2). Interestingly, these samples were clearly positive with the INNO-LIA Syphilis kit. This can reflect an improved sensitivity on the basis of the use of recombinant antigens versus nontreponemal antigens (in VDRL) (3), extracted treponemal antigens (in TPHA), or whole treponema organisms (in FTA-ABS) (14). The two samples with consensus-negative and INNO-LIA Syphilis kit-indeterminate results may also reflect the high degree of sensitivity of the new assay. On the other hand, since the specificity of the INNO-LIA Syphilis kit has been demonstrated with a large collection of negative samples (data partially shown in the present study) as well as a collection of difficult samples usually prone to false-positive reactivities (Table 4), the two consensus-positive samples that were indeterminate with the INNO-LIA Syphilis kit may illustrate the specificity of the INNO-LIA Syphilis kit. One of these samples (sample 599) had a doubtful result by agglutination techniques, was positive by FTA-ABS and the ICE Syphilis EIA, but was negative by TPI. This can also be due to a differential interference of antibodies to other treponemes that have no clinical significance (5).

In conclusion, the INNO-LIA Syphilis kit can reliably replace various strategies for the confirmation of syphilis infections, which currently requires the performance of several assays. A new prospective study will allow us to determine a possible algorithm for disease staging by this multiparametric assay.