J Lab Med 2024; 48(2): 71–75

Nele Wellinghausen*, Teresa Esthela Rangel Vivar and Dietmar Plonné

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
[Link] as a reference standard. Valid prediction of a TPPA
Received November 27, 2023; accepted February 1, 2024; titer ≥5,120 from TpG result appears not reasonable.
published online February 26, 2024
Keywords: syphilis; serology; Treponema; TPPA; ELISA;
Abstract pregnancy

Objectives: The Treponema pallidum particle agglutination

(TPPA) has been used for decades for serological diagnostics
of syphilis but is no longer available. Therefore, we evalu-
Introduction
ated the Treponema IgG ELISA (TpG) alone and in combi-
Syphilis, caused by the spirochete Treponema pallidum spp.
nation with the Treponema IgM ELISA (TpG+M, both from
pallidum, is a re-emerging sexually transmitted disease in
Euroimmun) as possible substitutes for TPPA as a confir-
several European countries including Germany [1]. Serology
matory test in a two-tier syphilis screening algorithm.
remains the mainstay of laboratory diagnosis of syphilis and is
Furthermore, we investigated whether a TPPA titer of 5,120
usually done by a stepwise approach [2–4]. As screening tests
which is used as cut-off for therapeutic decision in pregnant
polyvalent immunoassays like ELISA or CLIA and CMIA tests
women in Germany can be transferred to an appropriate
which can be run on automated random-access systems are
cut-off value of the TpG.
commonly used. For serological confirmation of syphilis a two-
Methods: All serum samples with reactive syphilis
tier approach is recommended in German guidelines [2, 5]. A
screening test (CLIA, Diasorin) within a 13-months period
reactive result in a syphilis screening test should be confirmed
were included (n=739). In addition to TPPA and rapid plasma
by a second Treponema-specific test, like immunoblot, fluo-
reagin test both ELISA tests were done in all samples.
rescent T. pallidum absorption test (FTA-abs test), T. pallidum
Results: Sensitivity, specificity, positive and negative pre-
particle agglutination (TPPA) test, or another immunoassay
dictive values were 92.2, 100, 100, and 74.5 % for TpG, and
[2, 5, 6]. In addition, a non-treponemal test like the rapid
93.2, 85.4, 96.6, and 74.1 % for TpG+M. By ROC analysis the
plasma reagin (RPR) test is used to determine activity of
cut-off of TpG corresponding to a TPPA titer ≥5,120 was
syphilis and success of antimicrobial therapy. Treponema-
calculated to be 54 RU/mL with a sensitivity of 99.6 % and a
specific IgM antibodies are determined routinely by immu-
resulting specificity of 58.6 %.
noassay or immunoblot for detection of recent infection and,
Conclusions: TpG appears suitable to substitute TPPA as a
in addition to RPR, for evaluation of activity of infection [7]. In
confirmatory test for syphilis diagnostics but TpG-negative
early primary syphilis infection Treponema-specific IgM
samples have to be evaluated by further tests like FTA-Abs or
antibodies appear 2–3 weeks after infection while IgG anti-
immunoblot. Treponema IgM determined in addition to TpG
bodies appear some days after IgM. A positive RPR test is seen
did not improve the test performance compared to the TPPA
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
*Corresponding author: Nele Wellinghausen, MD, MVZ Labor patients the dynamics of IgM and RPR can vary, for instance
Ravensburg, Elisabethenstr. 11, 88212 Ravensburg, Germany,
Treponema-specific IgM antibodies are frequently negative in
E-mail: [Link]@[Link]. [Link]
0002-3185-9526
recurrent infection. A test for detection of Treponema-specific
Teresa Esthela Rangel Vivar and Dietmar Plonné, MVZ Labor IgM and RPR are both done routinely in samples with a pos-
Ravensburg, Ravensburg, Germany itive polyvalent syphilis screening test in most laboratories.
Open Access. © 2024 the author(s), published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 International License.
72 Wellinghausen et al.: Treponema IgG and IgM ELISA for syphilis confirmation

The TPPA test has been commonly used as a syphilis (sample optical density (OD) divided by calibrator OD), with an index <0.8
screening test as well as for confirmation of a reactive regarded negative, 0.8–<1.1 euqivocal, and an index ≥1.1 positive accord-
ing to the instructions of the manufacturer. In our study, index ≥0.8 was
immunoassay screening test since it is very sensitive and
regarded positive. Treponema IgG levels are measured in relative units
specific [5, 6, 8] but it is no longer available on the market. (RU)/ml, with <16 RU/mL interpreted as negative, 16–<22 RU/mL as
Therefore, many laboratories had to revise their algorithm equivocal, and ≥22 RU/mL as positive according to the manufacturer. In
for serological syphilis diagnostics. Enzyme-linked immu- our study, ≥16 RU/mL was regarded positive. Samples with results
nosorbent assays (ELISA) which allow investigation of large 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
amounts of samples with little hands-on time by automated
or positive Treponema IgM ELISA an IgM immunoblot (recomLine
ELISA processors may be an alternative for TPPA [5, 8, 9]. The
Treponema IgM, Mikrogen, Neuried, Germany) was done as a confir-
aim of this study was to evaluate the performance of matory test. If result of Treponema IgG ELISA and TPPA test were
the Treponema IgG ELISA alone and in combination with the discrepant, Treponema IgM and IgG immunoblot (recomline Trepo-
Treponema IgM ELISA (Euroimmun, Germany) as possible nema IgM and IgG, Mikrogen) were done for further evaluation. The
substitutes for the TPPA confirmatory test in a two-tier recomline blots were run on an automated blot processing system
(Dynablot, Mikrogen) and interpretation was done both by the auto-
syphilis screening algorithm. In Germany, the TPPA titer has
mated Recomscan® software (Mikrogen) according to the interpreta-
also been used to guide antibiotic therapy in pregnant tion criteria of the manufacturer. In addition, all blot results were
women: A TPPA titer of 5,120 was the previous suggested cut- confirmed by eye. RPR was done according to the instructions of the
off for the therapeutic decision in pregnant women with manufacturer (Becton Dickinson, Sparks, MD, USA) in dilutions of 1:1
negative Treponema-specific IgM and non-reactive RPR [2]. and 1:4 in order to exclude prozone phenomenon. A titer ≥4 is
considered positive according to German diagnostic guidelines [5]. In
Therefore, it should be clarified whether the TPPA titer of
positive samples the final titer was determined by investigation of
5,120 can be transferred to an appropriate cut-off value of the
further dilutions.
Treponema IgG ELISA. 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.
Materials and methods
Diagnostic tests and algorithm Statistical analysis

All serum samples submitted to our laboratory between December 1st Statistical analyses were performed using R Statistical Software (v4.3.0,
2021 and December 31st 2022 with a request for serological syphilis R Core Team 2023, [Link] RStudio (2023.09.0 Build
diagnostics were eligible for this study. Diagnostic algorithm for sero- 463) and the packages DTComPair [10], cutpointr [11], and epiR [12].
logical syphilis testing included a polyvalent chemiluminescence The results of the TPPA confirmatory test have been used as the true
immunoassay (CLIA) run on the LIAISON® XL system, the LIAISON® state reference for cross-tabulation and performance analysis of the
Treponema Screen assay (Diasorin, Dietzenbach, Germany), as a alternative tests under evaluation. The diagnostic performance (sensi-
screening test. This T. pallidum-specific one-step sandwich CLIA con- tivity, specificity, Cohen‘s kappa) of the index tests (Treponema IgG ELISA,
tains recombinant antigen of T. pallidum (TpN17) which is used to bind combination of Treponema IgG ELISA + IgM ELISA) was compared to the
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 Treponema Screen assay
considered positive. Results between 0.9 and 1.1 including the interval (CLIA)

limits are considered borderline according to the manufacturer. All

samples with an index ≥0.9 in the LIAISON® Treponema Screen assay ReacƟve
Non-reacƟve
(CLIA) were subjected to TPPA test (Fujirebio, Tokyo, Japan), Treponema (n=739)
IgG ELISA, Treponema IgM ELISA (both from Euroimmun, Lübeck,
Germany), and RPR (Becton Dickinson, Sparks, MD, USA). Treponema pallidum parƟcle No laboratory
A serological diagnosis of syphilis using TPPA as confirmatory test aggluaƟnaƟon (TPPA) evidence of syphilis
was established according to the algorithm in Figure 1. TPPA was done
according to the instructions of the manufacturer. All samples were
Non-reacƟve ReacƟve
tested in initial dilutions of 1:80 and 1:2,560 in order to exclude prozone
(n=137) (n=602)
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 No laboratory evidence of
syphilis Treponema pallidum-specific
ELISA (EI 2111-9601 G) and the Treponema IgM ELISA (EI 2111-9601 M) anƟbodies confirmed
False-posiƟve Screening-Test
were performed on the automated EUROLab workstation ELISA (Euro-
immun). Both ELISA tests utilize recombinant p15, p17, p47, and TmpA as
antigens. Results of the Treponema IgM are expressed as index values Figure 1: Diagnostic algorithm for syphilis infection used in this study.
 Wellinghausen et al.: Treponema IgG and IgM ELISA for syphilis confirmation 73

TPPA reference standard with 95 % confidence intervals (CI). The kappa 85.4 % (20 false positive findings out of 137 TPPA negative
statistics were determined to assess the agreement between the TPPA samples, see Table 1). The PPV was 96.6 % and the NPV was
reference standard and the index tests.
comparable to that of the Treponema IgG ELISA alone
Receiver operating characteristic (ROC) analysis was done to
evaluate the areas under the ROC curve (AUC) and to predict the cut-off (74.1 %, see Table 1).
values for treatment decision in pregnant women for Treponema IgG The Treponema IgM ELISA was positive in 242 of the 602
ELISA. The TPPA titer served as the reference standard for treatment TPPA positive samples (40.2 %) and Treponema IgM immu-
decision, with patients having a TPPA titer <5,120 being categorized as noblot confirmed presence of Treponema IgM in 211 of these
not requiring treatment and those with a TPPA titer ≥5,120 as requiring
242 samples (87.2 %). Regarding the 137 TPPA negative sam-
treatment according to German guidelines [2], see also above. To ensure
ples Treponema IgM ELISA was positive in 20 (14.6 %) and
a very high sensitivity of the predicted cut-off for the Treponema IgG
ELISA, the cut-off was calculated at a sensitivity of at least 99 %. IgM was confirmed by immunoblot in only three (15.0 %). In
A p-value <0.05 was considered statistically significant. the 47 TPPA positive but Treponema IgG ELISA negative
samples (see above), Treponema IgM ELISA was positive in 6
(12.8 %) but Treponema IgM was confirmed by immunoblot
Results in only three of them.

Characteristics of the study population

Comparison of the tests.
In total, 739 serum samples were reactive in the Treponema
Screen assay (CLIA) during the study period, including 44 The consistency analysis showed that kappa coefficient
samples with equivocal and 695 samples with positive between Treponema IgG ELISA and TPPA was not signifi-
results. Using the TPPA as confirmatory test, 602 samples cantly different from that between the combination of
(598 positive and four equivocal in the Treponema Screen Treponema IgG ELISA plus IgM ELISA (0.814 (CI 0.763–0.866)
assay) were confirmed as positive for the presence of vs. 0.742 (0.680–0.804), Table 1).
antibodies, while 137 samples (97 positive and 40 equivocal The sensitivity of the combination of Treponema IgG
in Treponema Screen assay) were found to be negative, ELISA plus IgM ELISA was 1 % higher than the sensitivity of
resulting in a false positive rate of 18.5 % for the CLIA when the Treponema IgG ELISA alone (p=0.0143). The specificity
borderline results are considered positive. of the IgG ELISA was significantly higher (p<0.0001) than that
Among the 602 samples confirmed as seropositive, 349 of the combination of the Treponema IgG ELISA plus IgM
samples (58 %) were RPR negative, while 253 samples (42 %) ELISA (proportion difference=14.6 %).
were RPR positive (titer ≥4).

ROC analysis
Performance characteristics of the assays
AUC was calculated with TPPA as the confirmatory test for
Treponema IgG ELISA treatment decision in pregnant women at decision threshold
of titer 5,120. The AUC of the Treponema IgG ELISA was 0.955
All 137 TPPA-negative samples were negative in the Trepo- (0.941–0.967), see Figure 2. For the Treponema IgG ELISA,
nema IgG ELISA, resulting in a specificity of 100 %. Out of the the cut-off for treatment decision in pregnant women was
602 TPPA-positive samples, 47 samples were negative in the calculated to be 54 RU/mL at a sensitivity of 99.6 % and a
IgG ELISA, indicating a sensitivity for Treponema IgG ELISA resulting specificity of 58.8 %, see Figure 2.
of 92.2 % (Table 1). All 47 samples with negative Treponema
IgG ELISA had a TPPA titer ≤640. The PPV of for the Trepo-
nema IgG ELISA was excellent (100 %) but the NPV was only Discussion
74.5 % (Table 1).
The TPPA test was a well-established integral part in diag-
Combination of the Treponema IgG ELISA and the IgM nostic algorithms for serological workup of syphilis infection
ELISA. but is no longer available on the market. The aim of this
study was to evaluate the performance of ELISA tests as
The combination of the two tests Treponema IgG ELISA and possible substitutes for the TPPA confirmatory test in a
IgM ELISA resulted in a sensitivity of 93.2 % (41 false negative two-tier syphilis screening algorithm based on an automated
findings out of 602 TPPA positive samples) and a specificity of CLIA as screening test. Compared to TPPA as a second-line
74 Wellinghausen et al.: Treponema IgG and IgM ELISA for syphilis confirmation

Table : Performance characteristics and comparison of ELISA (Treponema IgG ELISA, combination of Treponema IgG ELISA and IgM ELISA) with TPPA as
reference standard.

TPPAa

Neg Pos Total Sensitivity (%) Specificity (%) PPVb ( % CI) NPVc ( % CI) Kappa value
( % CI) ( % CI) ( % CI)

Treponema IgG . (.–.) . (.–.) . (.–.) . (.–.) . (.–.)
Neg   
Pos   
Total   
Combination of Treponema . (.–.) . (.–.) . (.–.) . (.–.) . (.–.)
IgG and IgM
Neg   
Pos   
Total   
a
TPPA, Treponema pallidum particle agglutination; bPPV, positive predictive value; cNPV, negative predictive value.

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.

confirmatory test Treponema IgG ELISA had a sensitivity of Our study shows that the Treponema IgG ELISA can
92.2 % and a specificity of 100 %. The combination of the substitute the TPPA test as a confirmatory test in serum
separately measured Treponema IgG and IgM was compa- samples with reactive syphilis screening assay (CLIA).
rable to the Treponema IgG ELISA alone in terms of sensi- However, due to its lower sensitivity Treponema IgG nega-
tivity but had a significantly poorer specificity of 85.4 %. tive samples have to be evaluated by further Treponema-
Thus, the Treponema IgM determined in addition to the specific tests like immunoblot or FTA-Abs test. In addition,
Treponema IgG did not improve the test performance Treponema-specific IgM should be tested in order to detect
compared to the TPPA as a reference standard. cases of very early syphilis in these samples. This serological
 Wellinghausen et al.: Treponema IgG and IgM ELISA for syphilis confirmation 75

constellation was observed in 6.4 % of TPPA positive but Informed consent: Not applicable.
Treponema IgG negative samples (data not shown). Author contributions: The author(s) have (has) accepted
The IgG cut-off value for treatment decision in pregnant responsibility for the entire content of this manuscript and
women was calculated by performing ROC analysis using approved its submission.
TPPA titer 5,120 as the reference cut-off value. With a cut-off Competing interests: All other authors state no conflict of
of 54 RU/mL in the Treponema IgG ELISA, 99.6 % of pregnant interest.
women would be treated in accordance with the TPPA titer Research funding: None declared.
recommendation of ≥5,120. But in addition, about 41 % of Data availability: The raw data can be obtained on request
pregnant women would receive therapy that is unnecessary from the corresponding author.
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/ References
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