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Journal of Clinical Microbiology, June 2004, p. 2833-2835, Vol. 42, No. 6
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.6.2833-2835.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Evaluation of a New Particle Gel Immunoassay for Determination of Antibodies against Treponema pallidum

Bruno L. Schmidt*

Department of Dermatology, Hospital of the City of Vienna—Lainz, A-1130 Vienna, Austria

Received 10 October 2003/ Returned for modification 24 November 2003/ Accepted 22 February 2004


   ABSTRACT
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A new particle gel immunoassay (DiaMed AG, Cressier sur Morat, Switzerland) with three recombinant Treponema pallidum antigens was evaluated with serum samples from patients with syphilis (n = 124) and patients without syphilis (n = 490). It proved to be a simple, rapid (20 min), and useful test with sensitivity, specificity, and positive and negative predictive values of 91.9, 99.8, 99.2, and 98%, respectively.


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Treponema pallidum-specific tests use either whole organisms as the antigen, as in the fluorescence T. pallidum absorption (FTA-ABS) test, or sonicates of the pathogen. Preparing a reproducible antigen is a difficult task because the bacterium cannot be cultured continuously in vitro (1) and must be maintained in rabbits. The use of recombinant T. pallidum antigens in place of a poorly defined mixture of antigens from wild-type T. pallidum has the potential for improving the specificity of serologic tests. Recombinant antigens can be produced in large quantities to ensure reproducibility and cost-effectiveness.

Of the about 40 proteins of T. pallidum that can be seen by immunoelectrophoresis, only a few have been shown to be suited for diagnostic purposes (6). While proteins responsible for transport of nutrients or motility (flagellum complex) or involved in biochemical pathways have epitopes in common with other bacteria, high specificity has been demonstrated for three proteins, TpN15, TpN17, and TpN47 (5). Not surprisingly, commercially available kits based on recombinant antigens of these proteins have been introduced (2, 3, 8, 10-14).

Particle gel immunoassays (PaGIAs) represent established methods in blood group serology and have been introduced for detection of infectious diseases (7). The PaGIA for syphilis antibody (DiaMed, Cressier sur Morat, Switzerland) is a new particle immunoassay that contains recombinant antigens for detection of T. pallidum antibodies. We compared data obtained by the PaGIA method to those obtained by the Venereal Disease Research Laboratory (VDRL) test, the T. pallidum particle agglutination (TP-PA) test, and the ICE Syphilis immunoassay (11). The PaGIA consists of a microtube containing a gel matrix and red polymer particles sensitized with recombinant antigens TpN15, TpN17, and TpN47 in a ready-to-use suspension.

The PaGIA was performed as recommended by the manufacturer. Briefly, 10 µl of serum or plasma was pipetted into the funnel of the appropriate microtube, a 50-µl volume of vortexed particles was added, and the mixture was incubated at room temperature for 5 min. After centrifugation for 10 min in a special identification (ID) centrifuge, results were read visually. If antibodies to T. pallidum are present in the sample, particles are agglutinated. Positive results are typically indicated by the presence of a tight red line on the top of the gel surface. In rare cases, when only small amounts of specific antibodies (and/or low avidity) are present in the serum, agglutinated beads can be seen distributed within the gel matrix. Negative results are indicated by the presence of a pellet of colored particles at the bottom of the microtube following centrifugation with no agglutinated particles present within or on top of the gel (Fig. 1).



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  		FIG. 1. Close-up of two gel tubes (original length, 1 in.) of an ID cardholder with six tubes. After centrifugation, red beads coated with recombinant antigens TpN15, TpN17, and TpN47 remain on top of the gel if specific antibodies are present in the serum (left tube) or get concentrated at the bottom with serum samples negative for T. pallidum antibodies.

 
The other Treponema tests included the Serodia TP-PA (Fujirebio) test, a passive particle agglutination test using gelatin particle carriers sensitized with purified T. pallidum (Nichols strain); the ICE-Syphilis enzyme-linked immunosorbent assay (ELISA; Abbott-Murex); the FTA-ABS test (Biomerieux); and the VDRL test (Dade Behring). Immunoglobulin M (IgM)-specific antibody was detected with the Mercia Syphilis M (Microgen Bioproducts, Camberley, United Kingdom) and 19S IgM FTA-ABS (9) tests.

A panel of serum samples from untreated patients with primary syphilis (n = 60), secondary syphilis (n = 27), early latent syphilis (n = 32), and neurosyphilis (n = 5) were selected to determine the sensitivity of the assay. Clinical diagnoses were confirmed by dermatologists and/or neurologists. Serum samples from patients with early latent syphilis (duration of less than 1 year) were all reactive in the FTA-ABS test and both IgM-specific tests.

Specificity was determined with 432 fresh serum or plasma samples (all nonreactive in the TP-PA and ICE Syphilis tests) from patients without a history of syphilis and 20 possibly cross-reacting serum samples taken from patients with confirmed Lyme borreliosis (all reactive in IgG- and IgM-specific ELISAs [Dade Behring or DAKO, Glostrup, Denmark] and by Western blotting [Viramed] and negative in the TP-PA and the FTA-ABS test). In addition, we tested 38 serum samples that demonstrated some degree of reactivity in the FTA-ABS test (n = 7), the VDRL test (n = 25; median titer, 1:4; range, 1:1 to 1:32), or the Mercia Syphilis M ELISA (n = 6; mean antibody index, 5.1; range, 2.4 to 7.2). For the latter 38 serum samples, infection with T. pallidum could be excluded by anamnesis. This study was approved by the ethics commission of the city of Vienna, Austria.

The PaGIA was reactive with 114 (91.9%) of 124 serum samples from patients with syphilis. The sensitivities of the PaGIA and the other tests performed are presented in Table 1. Patients with secondary syphilis or neurosyphilis were reactive in all tests. No significant difference in sensitivity (P < 0.05, chi-square statistics) could be found between the PaGIA and the treponemal tests compared, even with samples from patients with primary or early latent syphilis. Ten serum samples out of 92 from patients with primary or early latent syphilis were missed by the PaGIA, compared to 15 serum samples missed by the VDRL test.


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  		TABLE 1. Sensitivities of serologic tests with serum samples from untreated patients with syphilis

 
Details of the 15 VDRL-negative serum samples and 8 of the 10 PaGIA-negative serum samples are given in Table 2 (patients with negative TP-PA and/or VDRL test results); the remaining 2 were weakly reactive in the TP-PA, the VDRL test, the FTA-ABS test, and both IgM-specific tests. The reactivity of the PaGIA could be demonstrated with TP-PA test-negative serum samples. Out of four serum samples negative in the TP-PA and VDRL tests, two were reactive in the PaGIA. One serum sample, negative in the TP-PA test but reactive in the VDRL test, was positive in the PaGIA (Table 2).


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  		TABLE 2. Details of 16 serum samples from patients with primary or early latent syphilis that were negative in the VDRL and/or the TP-PA testa

 
Only one of the fresh serum samples was reactive in the PaGIA. None of the 38 serum samples with false-positive results in other syphilis tests or the 20 possible cross-reacting serum samples from patients with confirmed Lyme borreliosis were reactive in the PaGIA. It is known that cross-reacting antibodies can cause false-positive results in the FTA-ABS test (4). However, all 20 serum samples from patients with confirmed Lyme borreliosis were nonreactive in the PaGIA. My own studies using Western blotting to analyze cross-reacting proteins have shown that mainly proteins of the flagellum complex are responsible for false-positive syphilis serology outcomes (unpublished results). The fact that recombinant proteins of the flagellum complex are not included in the PaGIA may be responsible for its high specificity. However, because of the small sample size, cross-reaction with Borrelia burgdorferi antibodies cannot be excluded completely. My specificity data demonstrate that the PaGIA yielded a specificity of 99.8%. The positive and negative predictive values were 99.1 and 98%.

In summary, the PaGIA offered excellent specificity and a sensitivity similar to that of other treponemal tests. In comparison to agglutination or ELISAs, the new assay has the advantages of simplicity of operation (no washers and readers are necessary; as equipment, only a centrifuge holding the ID cards is needed) and of a reaction time of only 20 min, which make the PaGIA an attractive choice for detection of T. pallidum antibodies.


   FOOTNOTES
 
* Mailing address: Department of Dermatology, Hospital of the City of Vienna—Lainz, Wolkersbergenstr. 1, A-1130 Vienna, Austria. Phone: 431 80110 2536. Fax: 431 80110 2828. E-mail: bruno.schmidt{at}kavwien.at. Back


   REFERENCES
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Journal of Clinical Microbiology, June 2004, p. 2833-2835, Vol. 42, No. 6
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.6.2833-2835.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.




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