Comparison of "Lumipulse anti-Treponema pallidum" and "Architect Syphilis TP" and further examination.

Abstract Background Syphilis is a sexually transmitted disease caused by Treponema pallidum (TP) infection. In recent years, diagnostic reagents with fully automated immunoassay instruments have become mainstream. However, these positive screening tests with high sensitivity, which are performed with full automation, need confirmation by Treponema pallidum particle agglutination (TPPA) to be judged as positive. Methods We evaluated the diagnostic performance of Lumipulse G TP‐N assay (Lumi‐TP, Fujirebio Inc) in 223 preselected TP‐positive samples and 1041 TP‐negative samples, compared it with that of the TP gold standard test (TPPA) and Architect Syphilis TP test (Archi‐TP, Abbott). Results The concordance rates for the results for the positive and negative samples between Lumi‐TP and TPPA were 100%. On the other hand, the rates for the results between Archi‐TP and TPPA were 100% for positive samples and 99.14% (1032/1041) for negative samples. Correlation tendency and rate between Archi‐TP and Lumi‐TP were 2.549 and 0.841 in positive specimens up to 160 detected values in Lumi‐TP. However, the detection value of Archi‐TP reached a plateau when it exceeded about 40. Furthermore, according to the comparison of each value obtained from Archi‐TP and Lumi‐TP with the strength of the staining of each line in the immune‐chromatography assay kit, ESPLINE TP (Fujirebio Inc) for TP major antigens, Tp15‐17 and TpN47, it was found that Lumi‐TP obtained higher values than Archi‐TP, particularly for TpN 47. Conclusions Lumi‐TP has high specificity and is useful not only for screening but also for determining the amount of anti‐TP antibodies.


| INTRODUC TI ON
Recently, the epidemiological re-emergence of syphilis has occurred in many countries, including China. 1 If the diagnosis and subsequent medical treatment of the early stage of syphilis are delayed, this leads to serious complications. Therefore, a high sensitivity and specificity syphilis screening examination that can be administered rapidly with decreased labor costs is required. For the syphilis examination in China several years ago, manual kits, such as point-of-care testing (POCT) and ELISA kits, were used in addition to the gold-standard Treponema pallidum particle agglutination (TPPA) test. Furthermore, fully automated chemiluminescent immunoassay reagents have been used as routine assays in hospitals.
Wellinghausen and colleagues reported that the concordance rates for positive and negative serum were calculated for TPPA and chemiluminescent immunoassay kits made by LIAISON (DiaSorin) and Architect Syphilis TP (Archi-TP), and the positive concordance rates were 100% (18/18), 100% (17/17), and 100% (18/18), respectively. Furthermore, the negative concordance rates were 100%, 99.8%, and 99.6%, respectively. Archi-TP showed less specificity than the TPPA. 2 On the other hand, it was reported that the LIAISON kit has higher sensitivity and that TPPA has higher specificity among nine serological TP screening assays, including the LIAISON, Archi-TP, and TPPA. 3 Similarly, 149 were judged positive by the Archi-TP. Thirty-seven out of 149 samples showed different results according to TPPA and Archi-TP. Eight were judged (21.6%) as positive, 11 (29.7%) as indeterminate, and 18 (48.6%) as negative by other dot-blot methods. In this article, further analysis by TPPA after Archi-TP screening examination is recommended. 4 It may be necessary to clarify the sensitivity and specificity of commercial anti-TP reagents.
Among the Treponema pallidum polypeptides, at least five (TpN15, TpN17, TpN37, TmpA, and TpN47) have proved to be of diagnostic relevance. 5,6 The LIAISON Treponema pallidum-specific and Immulite syphilis screens, however, use only TpN17, suggesting that TpN17 could be the main TP antigen. 7 Furthermore, the components that are used by the HISCL anti-TP, Elecsys Syphilis, Archi-TP, and Chemclin CLIA reagents are TpN15, TpN17, and TpN47. 7 Therefore, the performance differences of these TP kits might be due to differences in the amount of antigen used.
Lefevre and colleagues showed that for the detection of anti-TP antibody for each stage of syphilis, anti-TP IgM was positive for 16/17 cases of primary syphilis, 11/13 of secondary syphilis, 9/14 of early latent syphilis, and 0/33 of late latent syphilis. On the other hand, anti-TP IgG was positive for 14/17, 13/13, 14/14, and 33/33 of samples at each stage. 8 Western blotting of IgM against Treponema pallidum antigen was performed for 39 pairs of maternal/infant serum. Fetal IgM antibodies in each case were detected specifically. The combined data suggested that fetal serum IgM reactivity with the 47-KDa antigen of TP could be used as an important molecular marker for the diagnosis of congenital syphilis. It was found that the anti-TP IgM antibody was positive in the early stage of infection. 9,10 This study aims to evaluate the diagnostic performance of a new anti-TP screening kit, Lumipulse G TP-N assay (Lumi-TP), comparing with the Archi-TP and to confirm the judgment by the TP gold standard test, TPPA. Furthermore, the secondary purpose is to investigate the reactivity of the Lumi-TP and Archi-TP assays against the TP major antigens TpN15, TpN17, and TpN47 by using an immune-chromatography kit, ESPLINE TP, to detect two lines for Tp15-17 and TpN47.

| Materials used for each syphilis testing kit
The principle and the materials used for the syphilis testing kits, in-

| Lumipulse G TP-N assay
The Lumipulse G TP-N (Lumi-TP) assay is an anti-TP IgG and IgM qualitative kit used with human serum and plasma that is based on a two-step sandwich chemiluminescent immunoassay. The Tp15-17 and TpN47 antigens are bound to ferrite particles (generated inhouse) as a solid phase and coupled with alkaline phosphatase (ALP, Oriental Yeast Corp) as a conjugate. Lumi-TP assays were performed using the automated immunoassay tool Lumipulse G1200 (Fujirebio Inc). If the assay signal in the specimen was higher than or equal to the cutoff value, the signal was judged as positive.
As Lumi-TP compares the correlation between the measured value and the distribution of negative specimens with Archi-TP, the values for Lumi-TP were recalculated as three digits as was done for Archi-TP.

| Architect Syphilis TP assay
The Architect Syphilis TP (Archi-TP) assay with microparticles coated with recombinant TP antigens (TpN15, TpN17 and TpN47) and acridinium-labeled anti-human IgG and IgM conjugate is a two-step immunoassay for the qualitative detection of antibody to TP in human serum or plasma using a fully automated immunoassay instrument, Architect (Abbott). The specimen is considered reactive for anti-TP if the signal is greater than or equal to the cutoff value.

| Serodia TP-PA
The Serodia TP-PA (TPPA) assay (Fujirebio Inc) uses sensitized colored gelatin particles as carriers of the native TP antigen, which was purified from cultured Nichols strain. After each specimen was added, the gelatin particles bound with native TP antigen, and the degree of purified protein agglutination was observed. This quantitative analysis was carried out by measuring the titer required to change the agglutination degree by using an initial titration scheme for each serum. TPPA was used as a gold standard to measure the performance of all other assays.

| Western blotting
Western blotting is a manual test used to compare the reactivity of anti-TP IgG and IgM using membrane-spotted Tp15-17 or TpN47.

| Performance evaluation study
Each measured value of Lumi-TP and Archi-TP for the 1041 negative

| Reactivity for Tp15-17 and TpN47
The Lumi-TP and Archi-TP values at each level from (−) to (3+) according to the ESPLINE Tp15-17 and TpN47 lines were compared.
Then, 223 positive specimens with ESPLINE data were classified into four groups as shown below: Group 1 contained specimens with stronger colored lines for Tp15-17 than for TpN47.
Group 2 contained specimens with stronger colored lines for TpN47 than for Tp15-17.

Group 3 contained specimens with weakly colored lines for both
Tp15-17 and TpN47.

Group 4 contained specimens showing discrepancies between the Archi-TP and Lumi-TP values.
Group 5 contained other 208 positive specimens.  Table 2. The number of false-positive samples identified by Lumi-TP and Archi-TP was 0 (0%) and 9 (0.86%), respectively, and the false-positive values for

F I G U R E 2
The flowchart of the tests used for the anti-TP assay kits  blotting results, and it was also shown to be positive at up to a 640-fold dilution according to TPPA (Table 3).

| D ISCUSS I ON
Lumi-TP appears to show better diagnostic performance in terms of specificity than Archi-TP, as shown by the narrow distribution of the values for the negative samples and the correlation of the TP testing results with those of the gold standard assay, TPPA. Lumi-TP has a higher margin for the cutoff value as a result.
Therefore, for Lumi-TP, the certainty of the test results for anti-TP positive samples may be increased. Then, Lumi-TP might lead to a reduced need for retesting.
There was a report that the use of anti-human antibodies tended to cause the deterioration of specificity and an increase in false positives due to the influence of nonspecific components, such as  In the eight specimens in group 1 of Table 3  showed higher Lumi-TP values. In three cases with only Tp15-17 staining, Archi-TP showed a higher value than Lumi-TP. Therefore, it is considered that although the reactivity of Lumi-TP and Archi-TP   against anti-TP antibodies are similar, Archi-TP mainly detects antibodies against TpN15 and TpN17, and Lumi-TP mainly detects antibodies against Tp15-17 and TpN47. On the other hand, in one specimen in group 2 shown in Table 3 that showed stronger ESPLINE staining for the TpN47 band, Lumi-TP showed higher values. This also indicates that Lumi-TP is highly reactive for the TpN47 antigen. In the early stage in syphilis, there were more specimens with detection of anti-TP IgM than those with detection of anti-TP IgG. 9 On the other hand, it has been reported that TpN47 is highly reactive in the early stage of syphilis or in patients with a previous infection. 6,12 It is possible that Lumi-TP might detect anti-TP antibodies in the infectious early stage.
According to Western blotting, eleven cases in groups 1-4, as shown in Table 3 ies, while other methods use a double-antigen method that uses TP antigen as the solid phase and a labeled conjugate. The discrepancies might be due to differences in the assay formats, although further study will be required to confirm this.