Evaluation of a Treponema IgG ELISA alone and in combination with an IgM ELISA as substitutes for Treponema pallidum particle agglutination (TPPA) as confirmatory tests in a two-tier diagnostic algorithm for diagnosis of syphilis infection

Introduction

Syphilis, caused by the spirochete Treponema pallidum spp. pallidum, is a re-emerging sexually transmitted disease in several European countries including Germany [1]. Serology remains the mainstay of laboratory diagnosis of syphilis and is usually done by a stepwise approach [2], [3], [4]. As screening tests polyvalent immunoassays like ELISA or CLIA and CMIA tests which can be run on automated random-access systems are commonly used. For serological confirmation of syphilis a two-tier approach is recommended in German guidelines [2, 5]. A reactive result in a syphilis screening test should be confirmed by a second Treponema-specific test, like immunoblot, fluorescent T. pallidum absorption test (FTA-abs test), T. pallidum particle agglutination (TPPA) test, or another immunoassay [2, 5, 6]. In addition, a non-treponemal test like the rapid plasma reagin (RPR) test is used to determine activity of syphilis and success of antimicrobial therapy. Treponema-specific IgM antibodies are determined routinely by immunoassay or immunoblot for detection of recent infection and, in addition to RPR, for evaluation of activity of infection [7]. In early primary syphilis infection Treponema-specific IgM antibodies appear 2–3 weeks after infection while IgG antibodies appear some days after IgM. A positive RPR test is seen 4–6 weeks after infection, i.e. 2–3 weeks after the appearance of Treponema-specific IgM antibodies [5]. In recurrent syphilis infection as well as in special patient groups like HIV-positive patients the dynamics of IgM and RPR can vary, for instance Treponema-specific IgM antibodies are frequently negative in recurrent infection. A test for detection of Treponema-specific IgM and RPR are both done routinely in samples with a positive polyvalent syphilis screening test in most laboratories.

The TPPA test has been commonly used as a syphilis screening test as well as for confirmation of a reactive immunoassay screening test since it is very sensitive and specific [5, 6, 8] but it is no longer available on the market. Therefore, many laboratories had to revise their algorithm for serological syphilis diagnostics. Enzyme-linked immunosorbent assays (ELISA) which allow investigation of large amounts of samples with little hands-on time by automated ELISA processors may be an alternative for TPPA [5, 8, 9]. The aim of this study was to evaluate the performance of the Treponema IgG ELISA alone and in combination with the Treponema IgM ELISA (Euroimmun, Germany) as possible substitutes for the TPPA confirmatory test in a two-tier syphilis screening algorithm. In Germany, the TPPA titer has also been used to guide antibiotic therapy in pregnant women: A TPPA titer of 5,120 was the previous suggested cut-off for the therapeutic decision in pregnant women with negative Treponema-specific IgM and non-reactive RPR [2]. Therefore, it should be clarified whether the TPPA titer of 5,120 can be transferred to an appropriate cut-off value of the Treponema IgG ELISA.

Materials and methods

Diagnostic tests and algorithm

All serum samples submitted to our laboratory between December 1st 2021 and December 31st 2022 with a request for serological syphilis diagnostics were eligible for this study. Diagnostic algorithm for serological syphilis testing included a polyvalent chemiluminescence immunoassay (CLIA) run on the LIAISON^® XL system, the LIAISON^® Treponema Screen assay (Diasorin, Dietzenbach, Germany), as a screening test. This T. pallidum-specific one-step sandwich CLIA contains recombinant antigen of T. pallidum (TpN17) which is used to bind T. pallidum-specific IgM and IgG antibodies. The results are reported qualitatively but an index value is reported by the analytical system. An index value of <0.9 is interpreted as negative, while an index value >1.1 is considered positive. Results between 0.9 and 1.1 including the interval limits are considered borderline according to the manufacturer. All samples with an index ≥0.9 in the LIAISON^® Treponema Screen assay (CLIA) were subjected to TPPA test (Fujirebio, Tokyo, Japan), Treponema IgG ELISA, Treponema IgM ELISA (both from Euroimmun, Lübeck, Germany), and RPR (Becton Dickinson, Sparks, MD, USA).

A serological diagnosis of syphilis using TPPA as confirmatory test was established according to the algorithm in Figure 1. TPPA was done according to the instructions of the manufacturer. All samples were tested in initial dilutions of 1:80 and 1:2,560 in order to exclude prozone phenomenon. All samples with a TPPA titer <80 were defined as being seronegative and all with a TPPA titer ≥80 as being seropositive. In positive samples the final titer was determined. The Treponema IgG ELISA (EI 2111-9601 G) and the Treponema IgM ELISA (EI 2111-9601 M) were performed on the automated EUROLab workstation ELISA (Euroimmun). Both ELISA tests utilize recombinant p15, p17, p47, and TmpA as antigens. Results of the Treponema IgM are expressed as index values (sample optical density (OD) divided by calibrator OD), with an index <0.8 regarded negative, 0.8–<1.1 euqivocal, and an index ≥1.1 positive according to the instructions of the manufacturer. In our study, index ≥0.8 was regarded positive. Treponema IgG levels are measured in relative units (RU)/ml, with <16 RU/mL interpreted as negative, 16–<22 RU/mL as equivocal, and ≥22 RU/mL as positive according to the manufacturer. In our study, ≥16 RU/mL was regarded positive. Samples with results exceeding the linear range of the IgG ELISA test (>166 RU/mL) were measured repeatedly in a dilution of 1:10. In samples with an equivocal or positive Treponema IgM ELISA an IgM immunoblot (recomLine Treponema IgM, Mikrogen, Neuried, Germany) was done as a confirmatory test. If result of Treponema IgG ELISA and TPPA test were discrepant, Treponema IgM and IgG immunoblot (recomline Treponema IgM and IgG, Mikrogen) were done for further evaluation. The recomline blots were run on an automated blot processing system (Dynablot, Mikrogen) and interpretation was done both by the automated Recomscan^® software (Mikrogen) according to the interpretation criteria of the manufacturer. In addition, all blot results were confirmed by eye. RPR was done according to the instructions of the manufacturer (Becton Dickinson, Sparks, MD, USA) in dilutions of 1:1 and 1:4 in order to exclude prozone phenomenon. A titer ≥4 is considered positive according to German diagnostic guidelines [5]. In positive samples the final titer was determined by investigation of further dilutions.

Figure 1:

Diagnostic algorithm for syphilis infection used in this study.

In addition to Treponema IgG alone, the results of the combination of Treponema IgG ELISA and Treponema IgM ELISA were analysed. The outcome of this combination was considered negative if both tests were negative and positive if at least one of the two tests was positive.

Results

ROC analysis

AUC was calculated with TPPA as the confirmatory test for treatment decision in pregnant women at decision threshold of titer 5,120. The AUC of the Treponema IgG ELISA was 0.955 (0.941–0.967), see Figure 2. For the Treponema IgG ELISA, the cut-off for treatment decision in pregnant women was calculated to be 54 RU/mL at a sensitivity of 99.6 % and a resulting specificity of 58.8 %, see Figure 2.

Figure 2:

ROC analysis of Treponema IgG ELISA with TPPA at decision threshold of titer 5,120 as the confirmatory test for treatment decision in pregnant women.

Discussion

The TPPA test was a well-established integral part in diagnostic algorithms for serological workup of syphilis infection but is no longer available on the market. The aim of this study was to evaluate the performance of ELISA tests as possible substitutes for the TPPA confirmatory test in a two-tier syphilis screening algorithm based on an automated CLIA as screening test. Compared to TPPA as a second-line confirmatory test Treponema IgG ELISA had a sensitivity of 92.2 % and a specificity of 100 %. The combination of the separately measured Treponema IgG and IgM was comparable to the Treponema IgG ELISA alone in terms of sensitivity but had a significantly poorer specificity of 85.4 %. Thus, the Treponema IgM determined in addition to the Treponema IgG did not improve the test performance compared to the TPPA as a reference standard.

Our study shows that the Treponema IgG ELISA can substitute the TPPA test as a confirmatory test in serum samples with reactive syphilis screening assay (CLIA). However, due to its lower sensitivity Treponema IgG negative samples have to be evaluated by further Treponema-specific tests like immunoblot or FTA-Abs test. In addition, Treponema-specific IgM should be tested in order to detect cases of very early syphilis in these samples. This serological constellation was observed in 6.4 % of TPPA positive but Treponema IgG negative samples (data not shown).

The IgG cut-off value for treatment decision in pregnant women was calculated by performing ROC analysis using TPPA titer 5,120 as the reference cut-off value. With a cut-off of 54 RU/mL in the Treponema IgG ELISA, 99.6 % of pregnant women would be treated in accordance with the TPPA titer recommendation of ≥5,120. But in addition, about 41 % of pregnant women would receive therapy that is unnecessary according to former TPPA criteria. On the other hand, using such a cut-off, about 19 % of TPPA positive pregnant women could be spared from unnecessary therapy (IgG 16 to <54 RU/mL). Guidance of antibiotic therapy in pregnant women by the ELISA result appears in our opinion not reasonable since (1) only a minority of pregnant women would benefit from the determined cut-off and (2) the TPPA cut-off of 5,120 is not evidence-based but was established by expert consensus [13], [14], [15]. Recommendation of antibiotic therapy to all pregnant women with serologically confirmed syphilis infection without past antibiotic therapy would be a possible approach until current German guidelines regarding syphilis treatment in pregnant women have been revised. Alternatively, indications for antibiotic treatment of syphilis infection in pregnancy could be adapted to the recommendations of non-pregnant patients following the European guideline [3]. For this procedure, recommendation of therapy could be limited to women with a clear positive IgG result ≥22 RU/mL, since this cut-off is still below the determined cut-off of 54 RU/mL.

Although we included a large amount of serum samples our study has some limitations. Anamnestic data of the patients concerning possible previous laboratory results and antibiotic therapies were not available. Therefore, we analyzed the data on an individual sample basis. This approach best reflects the analytical work-up in a diagnostic laboratory where clinical and anamnestic data are not routinely available. In addition, Treponema IgM immunoblot was not done in all samples and, thus, sensitivity and specificity of the Treponema IgM ELISA could not be calculated. In a recent study, sensitivity of the Treponema IgM ELISA (Euroimmun) was 89.4 % in initial diagnosis of syphilis in a study population of mainly HIV-positive patients [7]. Finally, the transfer of the therapeutic TPPA cut-off for pregnant women to the Treponema IgG ELISA result was done on a non-selected population of samples from syphilis patients since a sufficient number of samples from pregnant women with syphilis was not available.