Treponema denticola infection is not a cause of false positive Treponema pallidum serology

THE NEW MICROBIOLOGICA, 28, 215-221, 2005

Treponema denticola infection is not a cause of false positive Treponema pallidum serology Antonella Marangoni1, Vittorio Sambri1, Francesca Cavrini1,2, Alessia Frabetti1, Elisa Storni1,2, Silvia Accardo1, Dora Servidio3, Federico Foschi3, Lucio Montebugnoli3, Carlo Prati3, Roberto Cevenini1 Section of Microbiology, DMCSS, University of Bologna, St. Orsola Hospital, Bologna, Italy; 2 Centro Regionale di Riferimento per le Emergenze Microbiologiche, Bologna, Italy; 3 Department of Oral Sciences, University of Bologna, Bologna, Italy

1

SUMMARY It has long been assumed that parodontal disease can be a cause of false positive results in syphilis serology, but so far there are no definitive data supporting this hypothesis. In this study we tested 250 serum samples obtained from blood donors all of which were negative when routinely screened for antibodies against Treponema pallidum. Then, all these samples were tested by immunoenzymatic (ELISA) and Western Blot (WB) assays to investigate reactivities against T. denticola. Thirteen samples showed a strong positivity when tested by both methods. When tested by WB against T. pallidum no sample met the positivity criteria. Nevertheless, bands with molecolar weights of about 30-35 KDa (endoflagellar core antigens) were recognized. All the 13 subjects serologically T. denticola positive underwent oral clinical and radiological observation: all showed a very poor parodontal status (CPSS>103). Eleven crevicular fluid samples out of the total of 13 patients were T. denticola positive by Real Time PCR carried out using a LightCycler system. In this study we demonstrated that the presence of T. denticola in the crevicular fluid samples obtained from patients with a severe periodontal status and/or a positive serology against T. denticola is not a cause of false positive results in syphilis serology. KEY WORDS: Treponema denticola, syphilis, serology Received April 14, 2005

INTRODUCTION The laboratory diagnosis of syphilis is still a crucial point in the epidemiological and diagnostic evaluation of the disease (Larsen et al., 1995). Available serologic tests for syphilis are divided into nontreponemal and treponemal assays. Nontreponemal tests include the Venereal Disease Research Laboratory (VDRL) and the

Corresponding author Vittorio Sambri, MD, PhD Section of Microbiology, DMCSS St. Orsola Hospital, University of Bologna Via Massarenti 9, 40138 Bologna, Italy e-mail: [email protected]

Accepted June 10, 2005

Rapid Plasma Reagin (RPR) card test. Treponemal tests include the serum fluorescent treponemal antibody absorption test (FTA-ABS), the T. pallidum hemagglutination test (TPHA), the immunoenzymatic assay (ELISA) and the Western Blot (WB) assay; both ELISA and WB tests can be based on either whole-cell lysate (Farshi et al., 1985; Halling et al., 1999) or recombinant (Castro et al., 2003; Sambri et al., 2001a; Sambri et al., 2001b) treponemal antigens. A small percentage of false positive results (about 1%) still exits when treponemal tests are used as screening procedures (Norris et al., 2003). Oral spirochetes are certainly involved in the etiology of periodontitis (Riviere et al., 1997; Loesche and Grossman, 2001). It has been previously reported that patients with necrotizing ulcera-

216 A. Marangoni, V. Sambri, F. Cavrini, A. Frabetti, E. Storni, S. Accardo, D. Servidio, F. Foschi, L. Montebugnoli, C. Prati, R. Cevenini

tive gingivitis or periodontitis have detectable antibodies to T. pallidum antigens, in particular TpN47, TpN15 and TpN37 (Tzagaroulaki and Riviere, 1999). It has long been assumed that parodontal disease can be a cause of false positive results in syphilis serology, but this hypothesis has not yet been fully investigated. The purpose of this study was to investigate if the presence of T. denticola in the crevicular fluid obtained from patients with poor periodontal status and the presence of high levels of circulating antibodies against T. denticola could be a cause of false positive syphilis serology.

METHODS Study group Two hundred and fifty serum samples were obtained from the blood bank of the St. Orsola Hospital, Bologna, Italy. All the blood donors gave their informed consent to this study. Commercial syphilis serologic tests The following tests were used: recomWell Treponema (Mikrogen, Martinsried, Germany), RPR (Radim, Pomezia, Italy) and TPHA (AlfaWasserman, Milan, Italy). Testing was performed following the manufacturer’s instructions. recomWell Treponema, used as screening test, is a quantitative immunoenzymatic assay for the detection of IgG or IgM antibodies against T. pallidum in human serum or plasma samples (Sambri et al., 2001b). This test, prepared with a recombinant form of the following T. pallidum antigens TpN47, TpN17 and TpN15, is based on the principle of an indirect “sandwich” enzyme immunoassay. Bacterial strains T. denticola (ATCC 35405) was grown anaerobically in modified NOS Medium-ATCC 1494 (Riviere et al., 1999) at 37°C for 72 h. The spirochetes were harvested and the antigen preparations were made and stored as previously reported (Cevenini et al., 1992). T. pallidum subsp. pallidum (Nichols strain) was originally obtained from the Statens Serum Institute (Copenhagen, Denmark) and maintained by passage in testicles of adult male New Zealand White rabbits every ten to fourteen days. The animals were

given antibiotic-free food and water ad libitum. Treponemes were extracted from infected testicles and prepared to be used as antigen as described elsewhere (Marangoni et al., 2000) after the animal had been killed with sodium pentothal. T. denticola enzyme-linked immunosorbent assay (ELISA) Serum samples were assayed for the presence of anti-T. denticola antibodies using an ELISA technique, as already reported (Gemmel et al., 2002; Pussinen et al., 2002). Briefly, 100 µl of a sonicated T. denticola suspension were coated onto 96-well high-binding plates (Maxisorb Immunoplates, Nunc, Roskilde, Denmark) at a protein concentration of 5 µg/ml. After blocking nonspecific sites with 3% bovine serum albumin (BioSPA, Milan, Italy) in phosphate-buffered saline (PBS)-Tween 20 (0.05%), 1:100 diluted serum samples were added. Antigen-antibody complexes were detected using a peroxidaselabeled rabbit antiserum to human IgG (DAKO, Copenhagen, Denmark) diluted 1:2000 in PBSTween. 100 µl of tetramethylbenzedine substrate solution were pipetted in each well; after 30 minutes of incubation at room temperature the enzymatic reaction was stopped by adding 100 µl of stop solution (H2SO4 0.5 N) to each well. The results were read at 450 nm after subtraction of the OD values obtained at the reference wavelength of 650 nm. Unspecific binding was monitored by blank wells which contained no sample, whereas negative controls (n=10) consisted of pooled serum samples obtained from healthy subjects, with no clinical signs of periodontitis. Subjects were considered seropositive for T. denticola when the corresponding antibody value was ≥0.8 OD, which represented the mean antibody level plus 2xSD of the negative controls. SDS-PAGE and Western Blotting (WB) Separation of polypeptides was performed with a Laemmli buffer system (Laemmli, 1970) using a 12% acrylamide gel (Marangoni et al., 1999). The Western Blot procedure was performed according to Towbin (Towbin et al., 1979), as previously described (Sambri et al., 2001a). A T. denticola WB test was considered positive when at least 6 different antigens with molecular weights ranging between 100 KDa and 12 KDa were clear-

Treponema denticola infection and syphilis serology

ly and strongly recognized. The interpretation criteria of T. pallidum WB were the following, as previously described (Sambri et al., 2001a): a test was considered positive when at least three bands out of TpN47 (47 KDa), TmpA (44.5 KDa), TpN17 (17 KDa) and TpN15 (15.5 KDa) were clearly recognized. The apparent molecular weight of each band was determined by plotting the positions of Rainbow low molecular weight standards (Amersham Biosciences, Little Chalfont, UK). Oral clinical observation A detailed description of the oral status was given in each patient using the total number of teeth and the probing pocket depth (PPD). PPD was taken at 6 aspects per tooth and measured at a force of 0.3 N with a manual pressure-sensitive probe(PCP-UNC 15 probe tip, Hu Friedy, Chicago, IL, equipped with a Brodontic spring device (Dentramar, Waalwijk, Holland) from the gingival margin to the bottom of the gingival sulcus. The clinical periodontal sum score (CPSS: the sum of the number of sites with probing pockets depths ?4 mm, number of gingival sites with bleeding after probing, visible suppuration on probing, number of furcation lesions exceeding grade 1) was used to evaluate the degree of periodontal status (Mattila et al., 2000; Cavrini et al., 2005). Crevicular fluid samples Crevicular fluid specimens were obtained from patients undergoing clinical periodontal observation by sampling at multiple sites using paper cones inserted into the periodontal pockets. All the specimens from each individual patient were pooled and stored at -80 °C until processed for DNA extraction. DNA extraction The extraction of bacterial and plaque samples DNA was performed by using the QIAamp DNA Blood Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. The concentration of plaque DNA was calculated with a DU Series 500 Spectrophotometer (Beckman Instruments, Fullerton, CA, USA). Real-time PCR For the detection of T. denticola in crevicular fluid samples a quantitative Real-time PCR was car-

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ried out using a LightCycler system (Roche Diagnostics GmbH, Penzberg, Germany) with SYBR Green I dye. A specific pair of primers (5’TAATACCGAATGTGCTCATTTACAT-3’, 5’-TCAAAGAAGCATTCCCTCTTCTTCTTA-3’) targeted to T. denticola 16S rDNA gene was used (Siqueira et al., 2000) and was custom synthesized by PRIMM, Milan, Italy. The predictive specificity was studied using the BLAST software available on-line at http://www.ncbi.nlm.nih.gov/blast. Each PCR was performed in a total volume of 20 µl containing 4 µl of a ready-to-use reaction mix (LightCycler Fast Start DNA Master PLUS SYBR Green I, Roche), 1 µl of each primer (final concentration, 0.5 µM each), 5 µl of template DNA extracted from crevicular fluid samples or from scalar dilutions of T. denticola culture and 9 µl of sterilized DNase-RNase-free water. The amplification reactions were performed in glass LightCycler Capillaries with a pre-incubation at 95°C for 10 min for Fast StartTaq DNA Polymerase activation and 55 PCR cycles including denaturation at 95°C for 10 seconds, annealing at 60°C for 10 seconds and extension at 72°C for 13 seconds. The melting curve analysis was performed by heating at 20°C second-1 to 95°C, cooling at 20°C-1 to 65°C for an incubation step of 15 seconds, and slowly heating (0.1°C second1) to 95°C. The melting curve analysis was followed by a final cooling at 40°C for 30 seconds. The target amplification profile demonstrated a specific main peak with a melting temperature (Tm) at 88°C. A negative reference sample, made with DNA extracted from T. pallidum subsp. pallidum (Nichols strain) (Sambri et al., 2001a), was tested in each run. Standards for T. denticola quantification were assessed by 10-fold scalar dilution of T. denticola cell suspension. The stock solution contained 2.5x109 treponemes/ml. After DNA extraction, as reported above, the concentration of T. denticola DNA was calculated with a DU Series 500 Spectrophotometer (Beckman Instruments, Fullerton, CA, USA) and the stock solution of standard acid nucleic was adjusted to a concentration of 1x107 copies/µl. The fluorescence values of serial diluted standards were used to generate a standard curve by plotting the Ct values versus the logarithms of the concentration of known quantities of DNA (1x107 to 1x102 copies/µl). The LightCycler soft-

218 A. Marangoni, V. Sambri, F. Cavrini, A. Frabetti, E. Storni, S. Accardo, D. Servidio, F. Foschi, L. Montebugnoli, C. Prati, R. Cevenini

ware (3.5) calculated a linear regression line through the data points, allowing the interpolation of any unknown sample (via its Ct). The limit of detection of the T. denticola genome was 50 copies for reaction mixture. Samples from each patient were run in duplicate and the amount of T. denticola cells in each subgingival plaque sample was represented as the average value of copies number per ng of plaque DNA.

RESULTS All the blood donors sera were first evaluated for the presence of antibodies against T. pallidum

using the commercial ELISA recomWell Treponema IgG and IgM; no sample was identified as positive or doubtful. Afterwards, all the specimens were investigated for the presence of an immune reaction to T. denticola by a “home made” T. denticola ELISA. 25 sera proved reactive, with OD values ranging between 1.05 and 3.0. When tested by T. denticola WB assay, 13 sera were confirmed positive, since they showed a strong reaction against at least 6 antigens, with molecular weights ranging between 100 and 12 KDa. No serum among the 25 T. denticola ELISA positive was reactive when tested by TPHA, RPR and T. pallidum WB. In particular, when tested by T. pallidum WB the 13 sera confirmed T. denticola positive showed the following reactions against diagnostic bands: TpN47 was recognized by 8 sera, and TmpA was recognized by two sera; furthermore, bands with molecular weights of about 30-35 KDa were recognized. Figure 1 shows an example of WB reactivity against T. pallidum and T. denticola, respectively. The sensitivity of Real-time PCR assay was measured with DNA extracted from a 10-fold dilution series of T. denticola and this method detected from 102 to 107 cells of T. denticola with a correlation coefficient of -1.00. Melting curve analysis determined the product identification showing a specific amplification product with a Tm of 88°C in each positive sample, whereas no aspecific product was detected (Figure 2a). T. pallidum DNA gave negative results by Real-time PCR assay in all the runs. The 13 subjects identified as immune reactive to T. denticola underwent oral clinical and radiological examinations. All of them had a very poor periodontal status resulting from a high value of CPSS (>103). Eleven patients had the sample from dental pockets positive for T. denticola DNA by Real-time PCR (Figure 2b). The number of spirochetes ranged from 35 to 109,090 cells/ng of plaque DNA (Table 1).

FIGURE 1 - Western Blot analysis for antibodies to T. pallidum (lane A) and T. denticola (lane B). The molecular weight (MW) standards are shown on the right (protein dimensions are expressed in KDa).

Treponema denticola infection and syphilis serology

219

a

FIGURE 2 - T. denticola detection in crevicular fluid samples by using Real-time PCR. a Melting curve analysis. DNA extracted from each crevicular fluid sample gave a specific amplification product with a Tm of 88°C, whereas no aspecific product was detected when T. pallidum DNA was tested as negative control. b Results obtained by testing crevicular fluid samples (curves 1-6 and 8); negative control (curve 9); T. denticola standard (5x105 copies/µl, curve 7).

b

TABLE 1 - Summary of results obtained when the 13 crevicular fluid samples were tested by Real-time PCR. The samples were obtained from the blood donors serologically reactive to T. denticola. All these 13 subjects had a poor periodontal status Patients

T. denticola WB results

T. pallidum WB results

T. denticola Real-time PCR results

T. denticola cells identified in crevicular samples

1

+

-

+

89 cells/ng DNA plaque

2

+

-

+

35 cells/ng DNA plaque

3

+

-

+

100 cells/ng DNA plaque

4

+

-

+

139 cells/ng DNA plaque

5

+

-

+

3023 cells/ng DNA plaque

6

+

-

+

416 cells/ng DNA plaque

7

+

-

+

3541 cells/ng DNA plaque

8

+

-

+

123 cells/ng DNA plaque

9

+

-

+

1559 cells/ng DNA plaque

10

+

-

+

109,090 cells/ng DNA plaque

11

+

-

+

244 cells/ng DNA plaque

12

+

-

-

-

13

+

-

-

-

220 A. Marangoni, V. Sambri, F. Cavrini, A. Frabetti, E. Storni, S. Accardo, D. Servidio, F. Foschi, L. Montebugnoli, C. Prati, R. Cevenini

DISCUSSION The strong immune response to T. denticola demonstrated by both ELISA and WB tests in 13 cases of subjects suffering from acute periodontal disease suggested that these patients had been infected by this oral spirochete. When analyzed by T. denticola WB, the sera reacted against many antigens with molecular weights ranging between 100 KDa and 12 KDa. In particular, a 55 KDa-polypeptide (probably T. denticola Msp) and several bands with molecular weights of 3038 KDa (flagellar antigens) were clearly recognized. In addition, the presence of T. denticola DNA in crevicular fluid samples in 11 cases was also demonstrated by Real time PCR, thus corroborating the hypothesis that all 11 subjects were still actively infected by T. denticola. Conventional PCR assays are one of the most sensitive and rapid methods for determining the prevalence of periodontal bacteria in subgingival plaque. However conventional PCR methods are unable to quantify the number of bacteria so that quantitative Real time PCR assays have recently been developed to detect the number of periodontal pathogens colonizing the subgingival pockets, to investigate the correlation between the presence of bacteria and the severity of periodontal disease (Yasuyuki et al., 2002; Kuboniwa et al., 2004; Yoshida et al., 2004). In this study an SYBR Green-based Real-time quantitative PCR assay was developed to detect the number of treponemes in oral specimens. In this report, spirochetes found in crevicular samples ranged from 35 to 109,090 cells/ng of plaque DNA. None of these patients showed a positive serology for T. pallidum when investigated by three conventional serologic tests (a recombinant-antigens based ELISA, TPHA and RPR). When tested by WB against T. pallidum no sample met the positivity criteria, even if some bands were recognized, in particular bands with molecular weights of about 30-35 KDa (endoflagellar core antigens). These findings clearly demonstrated that the past or present gingival infection by T. denticola is not immunologically detectable using highly specific (Castro et al., 2003; Sambri et al., 2001a; Sambri et al., 2001b) serologic methods for syphilis. These results showed that T. denticola periodontal infection is not among the caus-

es of the small percentage of false positive syphilis serology results still present when treponemal methods are used (Norris et al., 2003). ACKNOWLEDGEMENTS This study was supported in part by grant “Centro di Riferimento Regionale per le Emergenze Microbiologiche - CRREM” from Regione Emilia Romagna - 2003.

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