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A novel ELISA using a recombinant outer membrane protein, rTp0663, as the antigen for serological diagnosis of syphilis

  • Author Footnotes
    1 Man Xu, Yafeng Xie, and Chuanhao Jiang contributed equally to this work.
    Man Xu
    Footnotes
    1 Man Xu, Yafeng Xie, and Chuanhao Jiang contributed equally to this work.
    Affiliations
    Institution of Pathogenic Biology, Medical College, University of South China, Hengyang 421001, China

    Hunan Province Cooperative Innovation Center for Molecular Target New Drug Study, University of South China, Hengyang, China

    Hunan Provincial Key Laboratory for Special Pathogens Prevention and Control, University of South China, Hengyang, China
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  • Author Footnotes
    1 Man Xu, Yafeng Xie, and Chuanhao Jiang contributed equally to this work.
    Yafeng Xie
    Footnotes
    1 Man Xu, Yafeng Xie, and Chuanhao Jiang contributed equally to this work.
    Affiliations
    Institution of Pathogenic Biology, Medical College, University of South China, Hengyang 421001, China

    Hunan Province Cooperative Innovation Center for Molecular Target New Drug Study, University of South China, Hengyang, China

    Hunan Provincial Key Laboratory for Special Pathogens Prevention and Control, University of South China, Hengyang, China
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  • Author Footnotes
    1 Man Xu, Yafeng Xie, and Chuanhao Jiang contributed equally to this work.
    Chuanhao Jiang
    Footnotes
    1 Man Xu, Yafeng Xie, and Chuanhao Jiang contributed equally to this work.
    Affiliations
    Institution of Pathogenic Biology, Medical College, University of South China, Hengyang 421001, China

    Hunan Province Cooperative Innovation Center for Molecular Target New Drug Study, University of South China, Hengyang, China

    Hunan Provincial Key Laboratory for Special Pathogens Prevention and Control, University of South China, Hengyang, China
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  • Yongjian Xiao
    Affiliations
    Institution of Pathogenic Biology, Medical College, University of South China, Hengyang 421001, China

    Hunan Province Cooperative Innovation Center for Molecular Target New Drug Study, University of South China, Hengyang, China

    Hunan Provincial Key Laboratory for Special Pathogens Prevention and Control, University of South China, Hengyang, China

    Department of Clinical Laboratory, The Second Affiliated Hospital of University of South China, Hengyang, China
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  • Xingxing Kuang
    Affiliations
    Institution of Pathogenic Biology, Medical College, University of South China, Hengyang 421001, China

    Hunan Province Cooperative Innovation Center for Molecular Target New Drug Study, University of South China, Hengyang, China

    Hunan Provincial Key Laboratory for Special Pathogens Prevention and Control, University of South China, Hengyang, China
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  • Feijun Zhao
    Affiliations
    Institution of Pathogenic Biology, Medical College, University of South China, Hengyang 421001, China

    Hunan Province Cooperative Innovation Center for Molecular Target New Drug Study, University of South China, Hengyang, China

    Hunan Provincial Key Laboratory for Special Pathogens Prevention and Control, University of South China, Hengyang, China
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  • Tiebing Zeng
    Affiliations
    Institution of Pathogenic Biology, Medical College, University of South China, Hengyang 421001, China

    Hunan Province Cooperative Innovation Center for Molecular Target New Drug Study, University of South China, Hengyang, China

    Hunan Provincial Key Laboratory for Special Pathogens Prevention and Control, University of South China, Hengyang, China
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  • Shuangquan Liu
    Affiliations
    Department of Clinical Laboratory, The First Affiliated Hospital of University of South China, Hengyang, China
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  • Mingxing Liang
    Affiliations
    Institution of Pathogenic Biology, Medical College, University of South China, Hengyang 421001, China

    Hunan Province Cooperative Innovation Center for Molecular Target New Drug Study, University of South China, Hengyang, China

    Hunan Provincial Key Laboratory for Special Pathogens Prevention and Control, University of South China, Hengyang, China
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  • Li Li
    Affiliations
    Institution of Pathogenic Biology, Medical College, University of South China, Hengyang 421001, China

    Hunan Province Cooperative Innovation Center for Molecular Target New Drug Study, University of South China, Hengyang, China

    Hunan Provincial Key Laboratory for Special Pathogens Prevention and Control, University of South China, Hengyang, China
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  • Chuan Wang
    Affiliations
    Institution of Pathogenic Biology, Medical College, University of South China, Hengyang 421001, China

    Hunan Province Cooperative Innovation Center for Molecular Target New Drug Study, University of South China, Hengyang, China

    Hunan Provincial Key Laboratory for Special Pathogens Prevention and Control, University of South China, Hengyang, China
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  • Yimou Wu
    Correspondence
    Corresponding author. Tel.: +86 13707340050; fax: +86 734 8282907.
    Affiliations
    Institution of Pathogenic Biology, Medical College, University of South China, Hengyang 421001, China

    Hunan Province Cooperative Innovation Center for Molecular Target New Drug Study, University of South China, Hengyang, China

    Hunan Provincial Key Laboratory for Special Pathogens Prevention and Control, University of South China, Hengyang, China
    Search for articles by this author
  • Author Footnotes
    1 Man Xu, Yafeng Xie, and Chuanhao Jiang contributed equally to this work.
Open AccessPublished:December 30, 2015DOI:https://doi.org/10.1016/j.ijid.2015.12.013

      Highlights

      • A soluble recombinant version of a new Treponema pallidum outer membrane protein, known as Tp0663, has been produced for the diagnosis of syphilis.
      • The recombinant protein exhibited a superior serodiagnostic performance to the ARCHITECT Syphilis TP chemiluminescent immunoassay (Abbott GmbH & Co. KG) when 3326 characterized serum samples were screened with the two tests.
      • The Tp0663-based ELISA was in perfect agreement with clinical diagnosis (gold standard) with a κ value of 0.99, comparable to that of ARCHITECT Syphilis TP (0.96).
      • Tp0663 may represent a novel candidate with highly accurate diagnosis for the seroscreening of syphilis.

      Summary

      Background

      The lack of Treponema pallidum-specific antigens with highly accurate diagnosis makes the diagnosis of syphilis challenging.

      Methods

      A soluble recombinant version of a new diagnostic protein Tp0663 has been produced. The serodiagnostic potential of this protein was assessed by screening 3326 serum samples simultaneously evaluated by rapid plasma reagin and T. pallidum particle agglutination tests. Kappa (κ) coefficients were used to compare the concordance between clinical diagnosis and the Tp0663-based ELISA or the ARCHITECT Syphilis TP chemiluminescent immunoassay (Abbott GmbH and Co. KG).

      Results

      Using the results of clinical diagnosis as the gold standard, the sensitivity and specificity of Tp0663 were found to be 98.83% (95% confidence interval (CI) 96.61–99.60%) and 100% (95% CI 99.88–100%), respectively. In comparison, the ARCHITECT Syphilis TP assay was found to have a lower sensitivity (97.27%, 95% CI 94.46–98.67%) and specificity (99.61%, 95% CI 99.32–99.78%). In particular, the ARCHITECT Syphilis TP exhibited a false-positive rate of 0.39%. Moreover, the ELISA was in perfect agreement with the gold standard, with a κ value of 0.99, comparable to that of ARCHITECT Syphilis TP (0.96).

      Conclusion

      These results identified Tp0663 as a novel serodiagnostic candidate with great potential for developing novel tests for the diagnosis of syphilis.

      Keywords

      1. Introduction

      Syphilis is a perplexing infection caused by the spirochete Treponema pallidum, which is transmitted primarily through sexual contact. It progresses through multiple clinical stages, with typical clinical features including the hard chancre during primary syphilis, followed by a generalized rash and lymphadenopathy during secondary syphilis. Upon resolution of this stage of syphilis, the infection enters an asymptomatic, latent phase lasting from months to decades. Diverse and chronic symptoms emerge during tertiary syphilis, including neurosyphilis, cardiovascular involvement, and gummas.
      • Singh A.E.
      • Romanowski B.
      Syphilis: review with emphasis on clinical, epidemiologic, and some biologic features.
      The laboratory diagnosis of syphilis is dependent on the use of a multitude of serological tests due to the fact that T. pallidum cannot be cultured in vitro;
      • Norris S.J.
      Polypeptides of Treponema pallidum: progress toward understanding their structural, functional, and immunologic roles. Treponema pallidum Polypeptide Research Group.
      • Wheeler H.L.
      • Agarwal S.
      • Goh B.T.
      Dark ground microscopy and treponemal serological tests in the diagnosis of early syphilis.
      this is in contrast to most bacterial diseases, for which a definite diagnosis can be made by direct detection of the pathogen. The common serological tests for the diagnosis of syphilis are divided into two categories: non-treponemal tests and treponemal tests. The non-treponemal tests, such as the rapid plasma reagin (RPR) test and the venereal disease research laboratory (VDRL) test, are used mainly to determine serological activity and to monitor the therapeutic effect. Despite the advantages of these tests, which are widely available, inexpensive, and simple to perform, the results require further confirmatory screening for the detection of Treponema-specific antibodies. The treponemal tests, such as the T. pallidum particle agglutination (TPPA) assay, the T. pallidum hemagglutination (TPHA) assay, and the fluorescent treponemal antibody absorption (FTA-ABS) test, are used to detect the specific treponemal antibodies. Nevertheless, these tests are labor-intensive and highly operator-dependent. Furthermore, they exhibit poor sensitivities in the detection of early syphilis.
      • Sena A.C.
      • White B.L.
      • Sparling P.F.
      Novel Treponema pallidum serologic tests: a paradigm shift in syphilis screening for the 21st century.
      In recent years, automated enzyme immunoassays (EIAs) and chemiluminescence immunoassays (CIAs) have also been developed for the serodiagnosis of syphilis. These assays, using one or more of the recombinant T. pallidum proteins TpN15 (Tp0171), TpN17 (Tp0435), TpN44.5 (TmpA, Tp0768), and TpN47 (Tp0574),
      • Sun A.H.
      • Mao Y.F.
      • Hu Y.
      • Sun Q.
      • Yan J.
      Sensitive and specific ELISA coated by TpN15–TpN17–TpN47 fusion protein for detection of antibodies to Treponema pallidum.
      • Brito Moreno A.I.
      • Acosta Bas C.
      • Rodriguez M.
      • Baluja Conde I.B.
      • Feal Carballo S.
      • Martinez L.
      Monoclonal antibodies to the recombinant protein TmpA of the Treponema pallidum.
      have gained the interest of researchers in the field of syphilis diagnosis because they are objective, reproducible, automated, and computerized.
      • Hoover K.W.
      • Radolf J.D.
      Serodiagnosis of syphilis in the recombinant era: reversal of fortune.
      More importantly, these tests have led some large laboratories in the USA to screen patients with the reverse algorithm, which begins with a treponemal test; reactive tests are followed by a quantitative non-treponemal test and discordant samples must be rescreened with a second and different treponemal test.
      Centers for Disease Control Prevention
      Discordant results from reverse sequence syphilis screening—five laboratories, United States, 2006–2010.
      • Lee K.
      • Park H.
      • Roh E.Y.
      • Shin S.
      • Park K.U.
      • Park M.H.
      • et al.
      Characterization of sera with discordant results from reverse sequence screening for syphilis.
      Although these proteins have been used in the serodiagnosis of syphilis, there is no general agreement as to which protein antigens are best in terms of serodiagnostic performance, and surface-exposed proteins may have superior sensitivity to the currently used proteins due to their immediate exposure to the immune system. Therefore, it is of great importance to evaluate new recombinant antigens with highly accurate diagnosis for use in serological testing for syphilis.
      The purpose of this study was to analyze the diagnostic potential of recombinant antigen Tp0663, a 28-kDa T. pallidum subsp. pallidum outer membrane protein that has previously been reported in several works as being reactive with sera from syphilis patients.
      • McKevitt M.
      • Brinkman M.B.
      • McLoughlin M.
      • Perez C.
      • Howell J.K.
      • Weinstock G.M.
      • et al.
      Genome scale identification of Treponema pallidum antigens.
      • Brinkman M.B.
      • McKevitt M.
      • McLoughlin M.
      • Perez C.
      • Howell J.
      • Weinstock G.M.
      • et al.
      Reactivity of antibodies from syphilis patients to a protein array representing the Treponema pallidum proteome.
      The sensitivity and specificity of the recombinant antigen were evaluated using sera collected from 3326 individuals, and subsequently compared with the recently launched ARCHITECT Syphilis TP test, a CIA that exhibits excellent ability to automate testing in high-throughput instrumentations. The results showed that the T. pallidum protein Tp0663-based immunoglobulin G (IgG) ELISA exhibited higher overall sensitivity and specificity than the ARCHITECT Syphilis TP. Therefore, Tp0663 represents a promising new candidate that could potentially be incorporated into automated diagnostic tests for syphilis.

      2. Materials and methods

      2.1 Genomic DNA of Treponema pallidum subsp. pallidum Nichols strain

      The T. pallidum subsp. pallidum Nichols strain used in this study was a generous gift from Tianci Yang (Zhongshan Hospital, Medical College of Xiamen University, Xiamen, China) and was propagated in adult New Zealand white rabbits, as described elsewhere.
      • Lukehart S.A.
      • Marra C.M.
      Isolation and laboratory maintenance of Treponema pallidum.
      • Smajs D.
      • McKevitt M.
      • Howell J.K.
      • Norris S.J.
      • Cai W.W.
      • Palzkill T.
      • et al.
      Transcriptome of Treponema pallidum: gene expression profile during experimental rabbit infection.
      All animal experiments were approved by the Institutional Review Committee of the University of South China.

      2.2 Serum samples

      This study was approved by the Human Ethics Committee of the University of South China and was performed in compliance with the Declaration of Helsinki guidelines and national legislation. Informed consent was obtained from all participants. Human serum samples were collected from The First Affiliated Hospital, University of South China (Hengyang, China), The Second Affiliated Hospital, University of South China (Hengyang, China), The First People's Hospital of Changde (Changde, China), and Hunan Provincial People's Hospital (Changsha, China) between February 2015 and May 2015. The serological detection of syphilis in each sample (from 3326 subjects) was performed using the RPR and TPPA tests, which were performed in accordance with the manufacturers’ instructions, after duplicate tests were excluded. All serological tests were performed on the same serum sample, and the results of the two tests were reported simultaneously. The subjects in this study included 948 individuals undergoing routine health examinations, 1358 outpatients, and 1020 inpatients. These subjects underwent syphilis detection for screening (if asymptomatic), for diagnosis (if symptomatic), or to monitor the effects of therapy. In accordance with the staging criteria described in the literature,
      • Larsen S.A.
      • Steiner B.M.
      • Rudolph A.H.
      Laboratory diagnosis and interpretation of tests for syphilis.
      the clinical diagnosis of syphilis was determined by combining serological tests and disease history (including clinical signs and symptoms, and/or the patient's sexual history). Primary syphilis is generally characterized by painless chancre and serous fluids from the lesion, usually coupled with regional lymphadenopathy; there has generally been sexual contact with a person with syphilis, and laboratory confirmation involves dark-field examination and/or positive results of both RPR and TPPA to confirm the diagnosis of syphilis. Secondary syphilis is often characterized by a generalized rash, mucocutaneous lesions, and lymphadenopathy; there is generally a history of sexual exposure to a person with syphilis or primary syphilis, and reactive serological tests (RPR and TPPA) are used to confirm the diagnosis. Latent syphilis is asymptomatic, with a possible history of infection supported by a reactive RPR or TPPA result. An onset of infection within the last 2 years is referred to as early latent syphilis and a duration of latent syphilis >2 years is defined as late latent syphilis. Tertiary syphilis is considered as syphilis presenting with typical clinical symptoms, such as gummatous lesions, neuropsychiatric illness, and cardiovascular involvement, and a history of primary, secondary, or latent syphilis, in addition to clinical confirmation by positive results of both RPR and TPPA. The diagnostic tests used to confirm the serum samples are listed in Table 1.
      Table 1Serological tests used to confirm the characterized serum samples (N = 3326)
      Bacterium/virusSerological testManufacturer
      Treponema pallidumRapid plasma reagin testKeHua Bio-Engineering Inc., Shanghai, China
      Treponema pallidumT. pallidum particle agglutination testFujirebio, Tokyo, Japan
      Hepatitis B virusTotal anti-HBs or anti-HBc testAbbott, Chicago, USA
      Hepatitis C virusHCV testAbbott, Chicago, USA
      Helicobacter pyloriH. pylori IgG testAbbott, Chicago, USA
      CytomegalovirusArchitect cytomegalovirus IgG testAbbott, Chicago, USA
      HBs, hepatitis B surface antigen; HBc, hepatitis B core antigen; HCV, hepatitis C virus.

      2.3 Recombinant protein expression and purification

      The tp0663 gene was amplified by PCR using the forward primer 5′-CCGGAATTCATGAAACAGGGCTGTTTTAT 3′ (EcoRI) and the reverse primer 5′-CCCAAGCTTTCATTTGCCGCTCTCTCCTT 3′ (HindIII) and then cloned into pET-28a expression vector. This construct was transformed into Escherichia coli BL21 (DE3) cells and the positive clone was confirmed by DNA sequencing. Protein expression was induced overnight at 30 °C with 0.1 mM isopropyl-β-d-thiogalactopyranoside (IPTG). Bacteria were harvested and lysed in a buffer containing 50 mM Tris–HCl (pH 7.8), 300 mM NaCl, 10 mM imidazole, 20% glycerol, and 1% Triton X-100. His-tagged protein was purified by affinity chromatography using Ni-NTA beads (Qiagen, Inc., Hilden, Germany). The concentration of the recombinant protein was estimated using a bicinchoninic acid protein assay kit (Pierce, Rockford, USA).

      2.4 Western blotting analysis

      Recombinant protein Tp0663 was electrophoresed in a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and then transferred to a nitrocellulose membrane (Merck Millipore, Darmstadt, Germany). After blocking at room temperature for 2 h with phosphate-buffered saline (PBS) containing 5% nonfat milk and 0.05% Tween 20 (PBSTM), membranes were incubated overnight at 4 °C with human sera (1:500), rabbit sera (1:400), or anti-His-tagged monoclonal antibody (1:1000). Before being incubated separately with corresponding secondary antibodies at 37 °C for 1 h, membranes were washed extensively with PBS containing 0.05% Tween 20 (PBST) (the dilutions of secondary antibodies were as follows: horseradish peroxidase (HRP)-conjugated goat anti-human IgG (Abcam, Cambridgeshire, UK), 1:8000; HRP-conjugated goat anti-rabbit IgG (Abcam), 1:12 000; HRP-conjugated goat anti-mouse IgG (Millipore, Darmstadt, Germany), 1:10 000). Finally, chemiluminescent detection was performed using G:BOX Chemi XXX9 (Syngene, Cambridge, UK) after washing five times with PBST.

      2.5 ELISA

      Recombinant protein Tp0663 was diluted in carbonate buffer (0.1 M, pH 9.6), then 96-well plates (Corning Costar, New York, USA) were coated with 100 μl per well overnight at 4 °C. With intervening washes, the plates were blocked for 2 h at room temperature with 200 μl PBSTM. Serum samples were diluted 1:100 with PBSTM, and 100 μl was added to each well. After incubation for 2 h at 37 °C, the plates were washed extensively with PBST. HRP-conjugated goat anti-human IgG was used at a dilution of 1:10 000, and then 100 μl was added to each well. The mixture was incubated for 1 h at 37 °C. Plates were developed by adding 100 μl tetramethylbenzidine substrate to each well and incubated at room temperature for 30 min. The reaction was stopped by the addition of 100 μl of 1 M sulfuric acid. The absorbance was read at 450 nm. Each sample was assayed in duplicate. The mean optical density value for the uninfected control sera determined by clinical diagnosis plus two times the standard deviation was considered as the cutoff value to determine positivity in each sample. The cutoff value of the ELISA was 0.4121. Absorbance values less than or equal to the cutoff value were determined to be negative, while those greater than the cutoff value were defined as positive.

      2.6 ARCHITECT Syphilis TP

      ARCHITECT Syphilis TP is a two-step immunoassay performed on an fully automated system Architect i2000SR analyzer (Abbott, Chicago, USA), which utilizes recombinant treponemal antigens (TpN15, TpN17, and TpN47) to qualitatively detect the IgG and IgM antibodies to T. pallidum in human serum or plasma. This test has shown perfect performance when compared to the TPPA
      • Wellinghausen N.
      • Dietenberger H.
      Evaluation of two automated chemiluminescence immunoassays, the LIAISON Treponema Screen and the ARCHITECT Syphilis TP, and the Treponema pallidum particle agglutination test for laboratory diagnosis of syphilis.
      and could be used as a reliable test for the detection of syphilis.
      • Dai S.
      • Chi P.
      • Lin Y.
      • Zheng X.
      • Liu W.
      • Zhang J.
      • et al.
      Improved reverse screening algorithm for Treponema pallidum antibody using signal-to-cutoff ratios from chemiluminescence microparticle immunoassay.
      • Jonckheere S.
      • Berth M.
      • Van Esbroeck M.
      • Blomme S.
      • Lagrou K.
      • Padalko E.
      Evaluation of different confirmatory algorithms using seven treponemal tests on Architect Syphilis TP-positive/RPR-negative sera.
      The results of the chemiluminescent reaction are measured in relative light units (RLUs), with an RLU value of <1.00 indicating a negative result and an RLU value of ≥1.00 indicating a positive result. The level of RLUs is directly related to the amount of anti-T. pallidum antibodies in the serum. All serum samples described above, including positives from different stages of infection and nonreactive samples, were screened in duplicate according to the manufacturer's instructions.

      2.7 Statistical analysis

      SPSS software version 18.0 (SPSS Inc., Chicago, IL, USA) was used for the statistical analysis of the data. The diagnostic performances of the two serological tests were evaluated by contrasting them with the gold standard, namely, the combination of the serological tests, imaging diagnosis, and histological examination. The percentages of agreement and kappa (κ) coefficients were also calculated to analyze the agreement between clinical diagnosis and the ELISA or ARCHITECT Syphilis TP. The agreement of the results by κ values was considered near perfect (0.81–1.0), substantial (0.61–0.8), moderate (0.41–0.6), fair (0.21–0.4), slight (0–0.2), or poor (<0).
      • Landis J.R.
      • Koch G.G.
      The measurement of observer agreement for categorical data.

      3. Results

      3.1 Clinical diagnosis of syphilis patients

      In this study, RPR and TPPA tests were performed on samples from 3326 subjects between February 2015 and May 2015. Syphilis was diagnosed clinically by combining serodiagnosis and personal history (including the patient's sexual history and clinical characteristics). Ultimately, 256 subjects were diagnosed with syphilis and 632 subjects were found to be healthy by clinicians. Thus, the prevalence of syphilis was 7.70% (256 of 3326 patients). The clinical data for all of the subjects are given in Table 2.
      Table 2Clinical data for the 3326 subjects evaluated
      CharacteristicNo. (%)

      (N = 3326)
      Sex
       Male1929 (58.00%)
       Female1397 (42.00%)
      Age, years1–91 (mean 47.15)
       Male1–87 (mean 49.28)
       Female2–91 (mean 42.36)
      Ethnicity
       Han3223 (96.90%)
       Other103 (3.10%)
      Married
       Yes2295 (69.00%)
       No1031 (31.00%)
      History of infection
       Yes166 (5.00%)
       No3160 (95.00%)
      Reason for detection
       Screening (asymptomatic subjects)1397 (42.00%)
        Health examination948 (67.86%)
        Subject request155 (11.10%)
        Hospitalization223 (15.96%)
        Other71 (5.08%)
       Diagnosis (symptomatic subjects)1713 (51.50%)
        Sexual exposure to suspected syphilis patient31 (1.81%)
        Chancre, rash, gumma, or neurological involvement138 (8.06%)
        Other1544 (90.13%)
       Monitoring of therapeutic effect216 (6.50%)
      Infection
      Treponema pallidum256 (7.70%)
      Helicobacter pylori17 (0.51%)
       Cytomegalovirus2 (0.06%)
       Hepatitis B virus9 (0.27%)
       Hepatitis C virus3 (0.09%)
       Healthy632 (19.00%)
       Unknown7 (0.21%)
       Other2400 (72.16%)
      Clinical stages of syphilis256 (7.70%)
       Primary11 (4.30%)
       Secondary35 (13.67%)
       Tertiary56 (21.88%)
       Early latent28 (10.94%)
       Late latent126 (49.22%)

      3.2 Recombinant protein expression, purification, and identification

      The tp0663 gene was successfully amplified by PCR (Figure 1). The optimal expression of the His-tagged protein was observed with induction of 0.1 mM IPTG overnight at 30 °C. The soluble recombinant protein was determined through SDS-PAGE analysis to have an estimated purity of >95% (Figure 2). The antigenicity of Tp0663 was confirmed using anti-His monoclonal antibody, rabbit sera infected with T. pallidum Nichols strain (n = 2), and serum samples from syphilis patients (n = 18) (Figure 3). These sera from the syphilis patients, including two samples from patients with primary syphilis, three from patients with secondary syphilis, two from patients with early latent syphilis, seven from patients with late latent syphilis, and four from patients with tertiary syphilis, showed reactivity to recombinant protein Tp0663. However, no reactivity between Tp0663 and normal human sera (n = 5) or serum samples from patients infected with hepatitis B virus (n = 4), hepatitis C virus (n = 1), Helicobacter pylori (n = 1), and cytomegalovirus (n = 1) was observed in this study (Figure 4). These findings showed that the recombinant antigen exhibited 100% sensitivity and 100% specificity in the Western blotting analysis.
      Figure thumbnail gr1
      Figure 1PCR amplification of the tp0663 gene. Lane 1, 100-bp DNA ladder; lane 2, amplified product of the tp0663 gene.
      Figure thumbnail gr2
      Figure 2SDS-PAGE analysis of recombinant protein Tp0663 purification. Lane M, pre-stained protein marker; lane 1, cleared lysate; lane 2, flow through of the lysate; lane 3, eluted fraction of recombinant protein Tp0663.
      Figure thumbnail gr3
      Figure 3Identification of Treponema pallidum protein Tp0663. Western blotting analysis of the immunoreactivity of recombinant protein Tp0663 with sera from uninfected controls (C), anti-His monoclonal antibody (1), rabbit sera infected with T. pallidum Nichols strain (2), and sera from syphilis patients (3).
      Figure thumbnail gr4
      Figure 4Reactivities to recombinant protein Tp0663 of sera from patients at different stages of syphilis, individuals diagnosed with other diseases, and uninfected controls by Western blotting analysis. Western blotting analysis of recombinant protein Tp0663 with serum samples from uninfected controls (C), patients with primary syphilis (1), secondary syphilis (2), early latent syphilis (3), late latent syphilis (4), tertiary syphilis (5), hepatitis B virus (6), hepatitis C virus (7), cytomegalovirus (8), and human immunodeficiency virus (9) using HRP-conjugated goat anti-human IgG.

      3.3 Evaluation of the diagnostic performances of the two syphilis serological tests compared with the gold standard

      With the clinical diagnosis of syphilis as the gold standard, the serodiagnostic potential of the two syphilis serological tests was evaluated in this study. The results indicated that with the ARCHITECT Syphilis TP, 261 of the 3326 subjects would have been diagnosed with syphilis, whereas seven subjects determined to be syphilis patients by clinical diagnosis would not have been diagnosed. Among these syphilis patients, 12 were confirmed to be healthy by clinical diagnosis. The false-positive rate was 0.39% (12 of 3070 subjects) and the missed diagnosis rate was 2.73% (7 of 256 subjects). Furthermore, the sensitivity of the ARCHITECT Syphilis TP was 97.27% (95% confidence interval (CI) 94.46–98.67%) (Table 3). In addition, the seven cases of missed diagnosis were analyzed further. It was found that there was one case of primary syphilis, three of late latent syphilis, and three of tertiary syphilis. Moreover, among these seven cases, three patients were being diagnosed with syphilis for the first time.
      Table 3Comparison of the two syphilis serodiagnosis tests and clinical diagnosis
      Serodiagnosis test and resultClinical diagnosis (gold standard)Sensitivity, % (95% CI)Specificity, % (95% CI)
      PositiveNegative
      ARCHITECT Syphilis TP
       Positive2491297.27 (94.46–98.67)99.61 (99.32–99.78)
       Negative73058
      Tp0663-based ELISA
       Positive253098.83 (96.61–99.60)100 (99.88–100)
       Negative33070
      CI, confidence interval.
      In comparison, using the ARCHITECT Syphilis TP and the Tp0663-based ELISA, 249 and 253 subjects, respectively, had a reactive serodiagnosis. The sensitivity levels of the ARCHITECT Syphilis TP and the ELISA were 97.27% (95% CI 94.46–98.67%) and 98.83% (95% CI 96.61–99.60%), respectively. In addition, the specificities of the ARCHITECT Syphilis TP and the ELISA were 96.61% (95% CI 99.32–99.78%) and 100% (95% CI 99.88–100%), respectively (Table 3). Both the ARCHITECT Syphilis TP and the antigen exhibited strong discriminatory power in the serodiagnosis of syphilis.

      3.4 Comparison of the two serodiagnosis tests at different stages of syphilis

      The 256 cases of syphilis were further classified into different stages of syphilis to evaluate the two serological tests at the different stages (Table 4). It was found that the ARCHITECT Syphilis TP had high sensitivity not only for primary syphilis (90.91%) and secondary syphilis (100%), but also for tertiary syphilis (94.64%). The ELISA exhibited sensitivities of 100%, 97.14%, and 98.21% for primary, secondary, and tertiary syphilis, respectively.
      Table 4Comparison of the two serodiagnosis tests at different stages of syphilis
      DiagnosisNo. of serum samples testedNo. (%) of serodiagnosis positive
      ARCHITECT Syphilis TPTp0663-based ELISA
      Primary1110 (90.91)11 (100)
      Secondary3535 (100)34 (97.14)
      Early latent2828 (100)28 (100)
      Late latent126123 (97.62)125 (99.21)
      Tertiary5653 (94.64)55 (98.21)
      Total256249 (97.27)253 (98.83)

      3.5 Direct comparison of the concordance between clinical diagnosis and the Treponema pallidum recombinant antigen Tp0663-based ELISA or the ARCHITECT Syphilis TP

      For all of the samples, the agreement and κ values of the ELISA and the ARCHITECT Syphilis TP were compared with clinical diagnosis (gold standard). The ELISA showed 99.91% (95% CI 99.88–99.94%) agreement and a κ value of 0.99, and the ARCHITECT Syphilis TP exhibited 99.43% (95% CI 99.36–99.50%) agreement and a κ value of 0.96 (Table 5). The results indicated that the two serological tests had high consistency with clinical diagnosis.
      Table 5Direct comparison of the concordance between clinical diagnosis and the Treponema pallidum recombinant antigen Tp0663-based ELISA or the ARCHITECT Syphilis TP
      Assay and resultClinical diagnosis (gold standard)Agreement % (95% CI)κ value
      PositiveNegativeTotal
      Tp0663-based ELISA
       Positive253025399.91 (99.88–99.94)0.99
       Negative330703073
       Total25630703326
      ARCHITECT Syphilis TP
       Positive2491226199.43 (99.36–99.50)0.96
       Negative730583065
       Total25630703326
      CI, confidence interval.

      4. Discussion

      The identification and characterization of new immunodominant antigens of T. pallidum is central to the improvement of the current laboratory diagnosis of syphilis, a disease that has resurged worldwide in recent years, including in China.
      • Mattei P.L.
      • Beachkofsky T.M.
      • Gilson R.T.
      • Wisco O.J.
      Syphilis: a reemerging infection.
      • Zhu L.
      • Qin M.
      • Du L.
      • Xie R.H.
      • Wong T.
      • Wen S.W.
      Maternal and congenital syphilis in Shanghai, China, 2002 to 2006.
      • Chen Y.
      • Liu Z.
      • Zhang Q.
      • Chen J.
      • Sun W.
      • Yi J.
      • et al.
      Trend in prevalence of syphilis among voluntary blood donors in Xi’an, China from 2006 to 2010.
      A variety of T. pallidum proteins, including TpN44.5, TpN15, TpN17, TpN47, Tp0453, Tp92 (Tp0326), and TpF1 (Tp1038),
      • Sun A.H.
      • Mao Y.F.
      • Hu Y.
      • Sun Q.
      • Yan J.
      Sensitive and specific ELISA coated by TpN15–TpN17–TpN47 fusion protein for detection of antibodies to Treponema pallidum.
      • Brito Moreno A.I.
      • Acosta Bas C.
      • Rodriguez M.
      • Baluja Conde I.B.
      • Feal Carballo S.
      • Martinez L.
      Monoclonal antibodies to the recombinant protein TmpA of the Treponema pallidum.
      • Van Voorhis W.C.
      • Barrett L.K.
      • Lukehart S.A.
      • Schmidt B.
      • Schriefer M.
      • Cameron C.E.
      Serodiagnosis of syphilis: antibodies to recombinant Tp0453, Tp92, and Gpd proteins are sensitive and specific indicators of infection by Treponema pallidum.
      • Smith B.C.
      • Simpson Y.
      • Morshed M.G.
      • Cowen L.L.
      • Hof R.
      • Wetherell C.
      • et al.
      New proteins for a new perspective on syphilis diagnosis.
      • Jiang C.
      • Zhao F.
      • Xiao J.
      • Zeng T.
      • Yu J.
      • Ma X.
      • et al.
      Evaluation of the recombinant protein TpF1 of Treponema pallidum for serodiagnosis of syphilis.
      have been tested in recent decades. Some of these antigens have been used in commercial tests that exhibit high sensitivity and specificity. However, not all patients suffering from syphilis are detected with the use of these antigens, thus more sensitive and specific recombinant T. pallidum antigens would be useful for screening syphilis patients.
      In this study, Tp0663, a 28-kDa T. pallidum subsp. pallidum outer membrane protein that has surface-exposed epitopes,
      • Champion C.I.
      • Blanco D.R.
      • Exner M.M.
      • Erdjument-Bromage H.
      • Hancock R.E.
      • Tempst P.
      • et al.
      Sequence analysis and recombinant expression of a 28-kilodalton Treponema pallidum subsp. pallidum rare outer membrane protein (Tromp2).
      was expressed as a recombinant version in E. coli; this may allow the protein to be produced economically and in large quantities in vitro due to the inherent advantage in the preparation of recombinant antigen compared to the extraction of crude T. pallidum antigen.
      • Van Voorhis W.C.
      • Barrett L.K.
      • Lukehart S.A.
      • Schmidt B.
      • Schriefer M.
      • Cameron C.E.
      Serodiagnosis of syphilis: antibodies to recombinant Tp0453, Tp92, and Gpd proteins are sensitive and specific indicators of infection by Treponema pallidum.
      The molecular weight of the expressed recombinant protein was a little larger than that predicted, possibly due to the His tag at the N terminus. Consistent with the works performed by McKevitt et al.
      • McKevitt M.
      • Brinkman M.B.
      • McLoughlin M.
      • Perez C.
      • Howell J.K.
      • Weinstock G.M.
      • et al.
      Genome scale identification of Treponema pallidum antigens.
      and Brinkman et al.,
      • Brinkman M.B.
      • McKevitt M.
      • McLoughlin M.
      • Perez C.
      • Howell J.
      • Weinstock G.M.
      • et al.
      Reactivity of antibodies from syphilis patients to a protein array representing the Treponema pallidum proteome.
      the recombinant protein identified by SDS-PAGE was found to be reactive with sera from rabbits infected with the T. pallidum Nichols strain and with sera from syphilis patients. However, no reactivity was observed between recombinant protein Tp0663 and uninfected controls and potentially cross-reactive serum samples, showing the recombinant antigen to be a highly sensitive and specific candidate for the serodiagnosis of syphilis in Western blotting analysis.
      The direct comparison of the diagnostic performances of the recombinant T. pallidum antigen Tp0663 and the ARCHITECT Syphilis TP tests is one focus of this study. Using the clinical diagnostic results as the gold standard, the ARCHITECT Syphilis TP exhibited a sensitivity of 97.27% and a specificity of 99.61%, while Tp0663 showed a stronger discriminatory power for the serodiagnosis of syphilis (98.83% sensitivity and 100% specificity). Most surveillance has focused on early syphilis cases, which is the best indicator of the infection and more likely to result in the timely treatment of patients. Thus, the serodiagnosis by the two serological tests was further analyzed at the different stages of syphilis, and especially in early syphilis. It was found that Tp0663 exhibited higher sensitivity and specificity than the ARCHITECT Syphilis TP, although both tests showed high diagnostic significance in early syphilis, as well as in late syphilis. In particular, 12 false-positives would have resulted from the screening of all of the serum samples with the ARCHITECT Syphilis TP. This may be explained by the fact that the ELISA uses outer membrane protein Tp0663 as an antigen, which is thought to be exposed continuously to the immune system and to continually stimulate robust antibody responses, thus having a theoretical advantage in the detection of specific antibodies in individuals with syphilis.
      • Champion C.I.
      • Blanco D.R.
      • Exner M.M.
      • Erdjument-Bromage H.
      • Hancock R.E.
      • Tempst P.
      • et al.
      Sequence analysis and recombinant expression of a 28-kilodalton Treponema pallidum subsp. pallidum rare outer membrane protein (Tromp2).
      In contrast, the ARCHITECT Syphilis TP uses lipoproteins TpN15, TpN17, and TpN47 as the coated antigens; these are thought to be able to induce a strong immune response by activating antigen-presenting cells through toll-like receptor 2.
      • Aliprantis A.O.
      • Yang R.B.
      • Mark M.R.
      • Suggett S.
      • Devaux B.
      • Radolf J.D.
      • et al.
      Cell activation and apoptosis by bacterial lipoproteins through toll-like receptor-2.
      • Brightbill H.D.
      • Libraty D.H.
      • Krutzik S.R.
      • Yang R.B.
      • Belisle J.T.
      • Bleharski J.R.
      • et al.
      Host defense mechanisms triggered by microbial lipoproteins through toll-like receptors.
      Some of these lipoprotein antigens are believed to be concealed in the periplasm of the bacterium,
      • Sellati T.J.
      • Bouis D.A.
      • Caimano M.J.
      • Feulner J.A.
      • Ayers C.
      • Lien E.
      • et al.
      Activation of human monocytic cells by Borrelia burgdorferi and Treponema pallidum is facilitated by CD14 and correlates with surface exposure of spirochetal lipoproteins.
      although they were at first considered to reside on the surface of this pathogen. Moreover, the high overall percentage of agreement and corresponding κ values found in this study indicated that the ELISA was in perfect agreement with clinical diagnosis. Based on these results, recombinant antigen Tp0663 may represent an effective candidate for the seroscreening of syphilis.
      It should be emphasized that Tp0663 may be compatible with commercially available diagnostic tests, such as CIAs and EIAs, since the protein is expressed in a soluble form. Currently, most automated tests used for the serodiagnosis of syphilis utilize multiple recombinant treponemal antigens. This has the potential to confound results because patients may have different serological responses to these antigens regardless of the presence of infection.
      • Martin I.E.
      • Lau A.
      • Sawatzky P.
      • Tsang R.S.
      • Cuff W.
      • Lee C.
      • et al.
      Serological diagnosis of syphilis: enzyme-linked immunosorbent assay to measure antibodies to individual recombinant Treponema pallidum antigens.
      In addition, the development of a test using a single recombinant treponemal protein could cut the costs associated with the preparation of multiple antigens. Furthermore, Tp0663 does show the potential for detecting syphilis patients with high accuracy, which may eliminate the need to perform a variety of tests and thus decrease the medical burden for the patient. In summary, these results suggest that Tp0663 is a promising candidate for incorporation into many test formats for the detection of syphilis.
      A limitation of this study is that it was performed in a distinct geographical location. The subjects tested were all recruited in Hunan, China, which is an area with a relatively high prevalence of syphilis, and samples were not collected from other areas with a lower prevalence. Thus, this study only reflects the characteristics of areas with a high prevalence of syphilis. Similar studies in lower-prevalence regions will be performed in the future. Another limitation is the potential misclassification of patients due to the possible incorrect documentation and inadequate documentation in the medical records. Some patients used false information to protect their privacy (including name, age, history of infection, and inappropriate treatment they had received), which made it difficult to diagnose their syphilis infection and give them timely and effective treatment.
      In conclusion, a new serodiagnostic candidate – Tp0663 – has been identified, which is extremely sensitive (98.83%) and specific (100%) for the detection of all stages of the infection. In a direct comparison, the serodiagnostic performance of the recombinant protein even exceeded that of the commercially available ARCHITECT Syphilis TP. Therefore, recombinant antigen Tp0663 may represent a new candidate for the detection of syphilis, considering the potential for automation, high throughput, and its valuable diagnostic performance.

      Acknowledgements

      This work was supported by the National Natural Science Foundation of China (grant numbers 81471576, 81201331, 30800996, and 81373230), the Hunan Province Cooperative Innovation Center for Molecular Target New Drug Study (2015-351), the Hunan Provincial Key Laboratory for Special Pathogens Prevention and Control Foundation (grant number 2014-5), and the General Project in Hunan Province Science and Technology Program (grant number 2014TT2025).
      We thank Tianci Yang of Zhongshan Hospital, Medical College of Xiamen University, for the gift of the T. pallidum Nichols strain. We are also grateful to Jinhong Xiao of the Hunan Provincial People's Hospital, Changsha, China, to Yanjun Li of The First Affiliated Hospital, University of South China, and to Li Zhang of The First People's Hospital of Changde, Changde, China for providing the serum samples for this study.
      Conflict of interest: None.

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