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Detection of the 47-kilodalton membrane immunogen gene of Treponema pallidum in various tissue sources of patients with syphilis
Diagnostic Microbiology and Infectious Disease 51 (2005) 143 – 145 www.elsevier.com/locate/diagmicrobio
Detection of the 47-kilodalton membrane immunogen gene of Treponema pallidum in various tissue sources of patients with syphilis Alexander Vasilevich Kouznetsov, Peter Weisenseel, Paul Trommler, Susanne Multhaup, Joerg Christoph Prinz* Laboratory of Immune Pathogenesis and Serology, Department of Dermatology and Allergology, Ludwig-Maximilians-University of Munich, 80337 Munich, Germany Received 17 June 2004; accepted 4 October 2004
Abstract Polymerase chain reaction (PCR) was used to detect the 47-kDa immunogen gene of Treponema pallidum in peripheral blood mononuclear cells (PBMCs), skin lesions, and serum, but less consistently in purified granulocytes or ejaculates of patients with manifest and latent syphilis. Therefore, skin lesions and PBMCs may serve as the most reliable sources for a PCR-based diagnosis of syphilis. D 2005 Elsevier Inc. All rights reserved. Keywords: Polymerase chain reaction; Treponema pallidum; PBMCs; Syphilis
1. Note In certain clinical situations such as false serological tests, the diagnosis of syphilis may require additional diagnostic measures. The polymerase chain reaction (PCR) has been used to detect Treponema pallidum infection. T. pallidum has been found in the blood (Pietravalle et al., 1999; Marfin et al., 2001), lymph nodes (Kouznetsov and Prinz, 2002), rashes (Zoechling et al., 1997; Sutton et al., 2001), stomach (Inagaki et al., 1996), aortal wall (O’Regan et al., 2002), or cerebrospinal fluid (Noordhoek et al., 1991). Nevertheless, there are no data available about which of the blood compartments could be most reliable for the molecular detection of T. pallidum. Furthermore, PCR has not yet been used to identify T. pallidum in ejaculate samples, which is a major source of infection during sexual intercourse. By a PCR-based approach, we studied the presence of 47-kDa immunogen gene (Hsu et al., 1989) in cellular and noncellular blood fractions [peripheral blood mononuclear cells (PBMCs), granulocytes, serum, and plasma] of 15
This work was presented in part at the 29th Annual Meeting of German Society of Dermatological Research (ADF), February 28 to March 2, 2002, Berlin, Germany (Abstract No P 129). * Corresponding author. Tel.: +49-89-5160-6010; fax: +49-89-51606064. E-mail address: [email protected] (J.C. Prinz). 0732-8893/$ – see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2004.10.003
syphilis patients before antibiotic treatment. Eight of them had clinically manifest (2 primary, 6 secondary) and 7 latent syphilis. Diagnosis was confirmed by treponema pallidum haemagglutination assay (TPHA), Venereal Diseases Research Laboratory (VDRL), and 19S-IgM-fluorescent treponemal antibody absorption (19S-IgM-FTA-ABS) tests. Two smears from chancres, four ejaculates from patients strictly lacking syphilis lesions on hands and genitals, and four 3-mm biopsies from secondary syphilis were also collected for investigation. Blood samples from 10 patients that had been treated for syphilis within the last 24 months were included into the study. According to the criteria of the Centers for Disease Control and Prevention (1997), these patients originally had had primary (1/10 patients), secondary (2/10), early latent (4/10), or late latent syphilis (3/10). All of them still had a positive 19S-IgM-FTA-ABS test. Serum or plasma was obtained from peripheral blood by centrifugation. PBMCs containing lymphocytes and monocytes or granulocytes were isolated from 50 mL of heparinized peripheral blood by standard methods of density centrifugation using Ficoll (density 1.077 g/mL, Biochrom, Berlin, Germany) or Polymorphprep (density 1.113 g/mL, Axis-Shield, Oslo, Norway), respectively. Swab smears from chancres were vigorously agitated in 1 mL of sterile saline. DNA isolation from these samples or from 1.5 mL of serum, plasma, or ejaculate was performed using DNAzolRBD reagent kit (Gibco BRL; Invitrogen;
144
A.V. Kouznetsov et al. / Diagnostic Microbiology and Infectious Disease 51 (2005) 143 –145
Karlsruhe, Germany). Each 2 107 PBMCs or 5 107 granulocytes (one fifth of the total PBMCs or granulocytes preparation) were used for DNA isolation. Skin biopsies were homogenized in DNAzolRBD. Isolated DNA was dissolved in 20 AL of water. Nested bhot startQ PCR (35 cycles) for amplification of a DNA sequence of the 47-kDa immunogen gene was performed in an automated thermocycler (GeneAmp PCR System 9700, Perkin-Elmer, Norwalk, CT) under stringent conditions using an external oligonucleotide primer pair, Trep-336 (5V-CAGCAGGGGAAGAAAAAAGTGGG-3V) (MWG-Biotech, Ebersberg, Germany) and Trep-714 (5VAAGGTCGTGCGGGCTCTCCAT-3V) for a 379-bp fragment, and an internal primer pair, Trep-370 (5VGACCCAAGCGTTACTAAGATGG-3V) and Trep-563 (5VACCGCAACTGGGACAAACTTCAT-3V), for a 194-bp fragment. Both PCRs comprised a final volume of 25 AL. Four microliters of template and 14.7 AL of amplicon were taken for first and second PCR, respectively. All reactions were performed with positive (heat-killed T. pallidum Nichols, Biologische Analysensystem, Lich, Germany), human h-actin gene, and negative controls (water, corresponding samples from healthy individuals). T. pallidum amplicons were fractionated by gel electrophoresis, blotted to nylon membranes, and hybridized with a [g32 P]ATP-labeled 19-bp oligonucleotide (5V-GCAGTAGCGTTGGCGGATC-3V) corresponding to nucleotides 481–499. PCR sensitivity was determined by serial dilution of heat-killed T. pallidum Nichols strain (Biologische Analysensystem). The absolute PCR threshold was 10 trepo-
nemes per PCR sample. For each sample, DNA preparation and PCR were performed in independent triplicates. The 47-kDa immunogen gene of T. pallidum was detected in 12 of 15 patients (80%) with syphilis (Table 1). Treponemal DNA was not found in the blood of patient 1 who has primary syphilis probably because extensive hematogenous dissemination had not yet occurred. Instead, T. pallidum DNA was detected in the chancre secretion of this patient. In patients 14 and 15 who had latent syphilis but tested negative by PCR, the bacterial load in blood may have decreased with disease duration below the threshold of PCR. Thus, in this stage, PCR analysis of blood fractions may not be sufficient to contribute to the diagnosis of syphilis (Marfin et al, 2001). Of the different blood samples, PBMCs were positive in all patients with secondary syphilis, in 1 of the 2 patients with primary syphilis and in 5 of 7 with latent syphilis. All other blood fractions (serum, plasma, and granulocytes) gave less consistent results (Table 1). Thus, PBMC appears as a more reliable source for the detection of T. pallidum than serum or other blood fractions. In this respect, our results correspond to those of Pietravalle et al. (1999) who detected T. pallidum in serum of 5 (83.3%) of 6 persons with manifest syphilis and in 6 (67%) of 9 persons with latent syphilis. Even lower detection rates were reported by Marfin et al. (2001) who detected T. pallidum DNA in whole blood in 2 (25%) of 8 patients with manifest syphilis and in 8 (61.5%) of 13 patients with latent syphilis. All 6 skin lesions (chancres and biopsies) were positive by PCR. The lesions of primary and secondary syphilis
Table 1 Clinical characteristics, syphilis serology, and PCR results of various tissue sources of 15 patients with syphilis Patient no., stage of infection
Syphilis serology TPHA (titer)
1. Primary 1:320 2. Primary 1:40 3. Secondary 1:640 4. Secondary 1:2560 5. Secondary 1:10 240 6. Secondary 1:5120 7. Secondary 1:2560 8. Secondary 1:81 920 PCR positive samples/total samples 9. Early latent 1:640 10. Early latent 1:81 920 11. Early latent 12. Early latent 1:320 13. Late latent 1:1280 14. Late latent 1:10 240 15. Late latent 1:10 240 PCR positive samples/total samples
T. pallidum PCR samples
VDRL (titer)
19S-IgM
1:2 1:16 1:32 1:64 1:32 1:64 1:32 1:32
(+) (+) + + + + +
1:16 1:16
+
1:16 1:8 1:64 1:4
(+) + F + F
PBMC + + + + + + + 7/8 + + + + +
5/7
Serum + + + + + + 6/8
Granulocyte
+ +
+ + 4/8 +
Plasma
+
1/8
+ +
2/7
1/7
0/7
Skin lesion
Ejaculate
+a +a,c +b +b ND +b +b ND 6/6 ND ND ND ND ND ND ND ND
ND + ND ND ND + ND 2/3 ND ND ND ND ND ND 0/1
19S-IgM was determined by 19S-IgM-FTA-ABS test; +, positive; (+), weakly positive; F, borderline positive; , negative; ND, not done. bEarly latentQ and blate latentQ are defined as syphilis of less and more than 1-year duration, respectively, according to the criteria of the Centers for Disease Control and Prevention (1997). a Swab. b Biopsy. c Perianal erosion.
A.V. Kouznetsov et al. / Diagnostic Microbiology and Infectious Disease 51 (2005) 143 –145
usually have sufficient bacterial DNA for molecular detection (Zoechling et al., 1997; Sutton et al., 2001). Furthermore, for the first time, we could confirm by PCR the infectivity of ejaculates from syphilis patients. Further studies need to determine if the bacteria were located intracellularly in monocytes, attached to the cell surface, or just enriched between the plasma and Ficoll fractions in the PBMC fraction. Density-gradient centrifugation of blood at a density of 1.051 g/mL can enrich treponemes (Hanff et al., 1984). Ficoll density gradient centrifugation may therefore have contributed to the sensitivity of PCR in the PBMC samples. All blood specimens from the 10 treated patients were negative by PCR. Because these patients had not been tested before antibiotic treatment, it is difficult to speculate about a successful eradication of T. pallidum. The negative test results, however, may support that sufficient treatment eliminates T. pallidum from the circulation. Wicher et al. (1998) had proven by PCR in an experimental animal system that dead T. pallidum are cleared from soft tissue within 30 days. Yet, there have been no time-related studies about the treatment-induced clearance of T. pallidum from the blood of syphilis patients. Hybridization was used to corroborate the specificity of the experimental approach, but it did not increase its sensitivity. Ethidium bromide staining may therefore be sufficient for the detection of 47-kDa gene amplicons and thus facilitate a broader use. In summary, PCR may detect T. pallidum in ejaculates, skin lesions, and in cellular and noncellular fraction of peripheral blood. PBMC fraction may be the best source for molecular detection of manifested and latent syphilis when serology is inconclusive and syphilitic skin lesions are lacking.
145
Acknowledgment A.V. Kouznetsov, MD, was supported by research scholarship from the president of the Russian Federation and the German Academic Exchange Service (DAAD). References Centers for Disease Control and Prevention (1997) Case definitions for infectious conditions under public health surveillance. MMWR Morb Mortal Wkly Rep 46:34 – 37. Hanff PA, et al (1984) Purification of Treponema pallidum, Nichols strain, by Percoll density gradient centrifugation. Sex Transm Dis 11:275 – 286. Hsu P-L, et al (1989) Sequence analysis of the 47-kilodalton major integral membrane immunogen of Treponema pallidum. Infect Immun 57: 196 – 203. Inagaki H, et al (1996) Gastric syphilis: polymerase chain reaction detection of treponemal DNA in pseudolymphamatous lesions. Hum Pathol 27:761 – 765. Kouznetsov AV, Prinz JC (2002) Molecular diagnosis of syphilis: The Schaudinn-Hoffmann lymph-node biopsy. Lancet 360:388 – 938. Marfin AA, et al (2001). Amplification of the DNA polymerase I gene of Treponema pallidum from whole blood of persons with syphilis. Diagn Microbiol Infect Dis 40:163 – 166. Noordhoek GT, et al (1991) Detection by polymerase chain reaction of Treponema pallidum DNA in cerebrospinal fluid from neurosyphilis patients before and after antibiotic treatment. J Clin Microbiol 29: 1976 – 1984. O’Regan AW, et al (2002) Barking up the wrong tree? Use of polymerase chain reaction to diagnose syphilitic aortitis. Thorax 57:917 – 918. Pietravalle M, et al (1999) Diagnostic relevance of polymerase chain reaction technology for T. pallidum in subjects with syphilis in different phase of infection. Microbiologica 22:99 – 104. Sutton MY, et al (2001) Molecular subtyping of Treponema pallidum in an Arizona County with increasing syphilis morbidity: Use of specimens from ulcer and blood. J Infect Dis 183:1601 – 1606. Wicher K, et al (1998) Identification of persistent infection in experimental syphilis by PCR. Infect Immun 66:2509 – 2513. Zoechling N, et al (1997) Molecular detection of Treponema pallidum in secondary and tertiary syphilis. Br J Dermatol 136:683 – 686.
Detection of the 47-kilodalton membrane immunogen gene of Treponema pallidum in various tissue sources of patients with syphilis Alexander Vasilevich Kouznetsov, Peter Weisenseel, Paul Trommler, Susanne Multhaup, Joerg Christoph Prinz* Laboratory of Immune Pathogenesis and Serology, Department of Dermatology and Allergology, Ludwig-Maximilians-University of Munich, 80337 Munich, Germany Received 17 June 2004; accepted 4 October 2004
Abstract Polymerase chain reaction (PCR) was used to detect the 47-kDa immunogen gene of Treponema pallidum in peripheral blood mononuclear cells (PBMCs), skin lesions, and serum, but less consistently in purified granulocytes or ejaculates of patients with manifest and latent syphilis. Therefore, skin lesions and PBMCs may serve as the most reliable sources for a PCR-based diagnosis of syphilis. D 2005 Elsevier Inc. All rights reserved. Keywords: Polymerase chain reaction; Treponema pallidum; PBMCs; Syphilis
1. Note In certain clinical situations such as false serological tests, the diagnosis of syphilis may require additional diagnostic measures. The polymerase chain reaction (PCR) has been used to detect Treponema pallidum infection. T. pallidum has been found in the blood (Pietravalle et al., 1999; Marfin et al., 2001), lymph nodes (Kouznetsov and Prinz, 2002), rashes (Zoechling et al., 1997; Sutton et al., 2001), stomach (Inagaki et al., 1996), aortal wall (O’Regan et al., 2002), or cerebrospinal fluid (Noordhoek et al., 1991). Nevertheless, there are no data available about which of the blood compartments could be most reliable for the molecular detection of T. pallidum. Furthermore, PCR has not yet been used to identify T. pallidum in ejaculate samples, which is a major source of infection during sexual intercourse. By a PCR-based approach, we studied the presence of 47-kDa immunogen gene (Hsu et al., 1989) in cellular and noncellular blood fractions [peripheral blood mononuclear cells (PBMCs), granulocytes, serum, and plasma] of 15
This work was presented in part at the 29th Annual Meeting of German Society of Dermatological Research (ADF), February 28 to March 2, 2002, Berlin, Germany (Abstract No P 129). * Corresponding author. Tel.: +49-89-5160-6010; fax: +49-89-51606064. E-mail address: [email protected] (J.C. Prinz). 0732-8893/$ – see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2004.10.003
syphilis patients before antibiotic treatment. Eight of them had clinically manifest (2 primary, 6 secondary) and 7 latent syphilis. Diagnosis was confirmed by treponema pallidum haemagglutination assay (TPHA), Venereal Diseases Research Laboratory (VDRL), and 19S-IgM-fluorescent treponemal antibody absorption (19S-IgM-FTA-ABS) tests. Two smears from chancres, four ejaculates from patients strictly lacking syphilis lesions on hands and genitals, and four 3-mm biopsies from secondary syphilis were also collected for investigation. Blood samples from 10 patients that had been treated for syphilis within the last 24 months were included into the study. According to the criteria of the Centers for Disease Control and Prevention (1997), these patients originally had had primary (1/10 patients), secondary (2/10), early latent (4/10), or late latent syphilis (3/10). All of them still had a positive 19S-IgM-FTA-ABS test. Serum or plasma was obtained from peripheral blood by centrifugation. PBMCs containing lymphocytes and monocytes or granulocytes were isolated from 50 mL of heparinized peripheral blood by standard methods of density centrifugation using Ficoll (density 1.077 g/mL, Biochrom, Berlin, Germany) or Polymorphprep (density 1.113 g/mL, Axis-Shield, Oslo, Norway), respectively. Swab smears from chancres were vigorously agitated in 1 mL of sterile saline. DNA isolation from these samples or from 1.5 mL of serum, plasma, or ejaculate was performed using DNAzolRBD reagent kit (Gibco BRL; Invitrogen;
144
A.V. Kouznetsov et al. / Diagnostic Microbiology and Infectious Disease 51 (2005) 143 –145
Karlsruhe, Germany). Each 2 107 PBMCs or 5 107 granulocytes (one fifth of the total PBMCs or granulocytes preparation) were used for DNA isolation. Skin biopsies were homogenized in DNAzolRBD. Isolated DNA was dissolved in 20 AL of water. Nested bhot startQ PCR (35 cycles) for amplification of a DNA sequence of the 47-kDa immunogen gene was performed in an automated thermocycler (GeneAmp PCR System 9700, Perkin-Elmer, Norwalk, CT) under stringent conditions using an external oligonucleotide primer pair, Trep-336 (5V-CAGCAGGGGAAGAAAAAAGTGGG-3V) (MWG-Biotech, Ebersberg, Germany) and Trep-714 (5VAAGGTCGTGCGGGCTCTCCAT-3V) for a 379-bp fragment, and an internal primer pair, Trep-370 (5VGACCCAAGCGTTACTAAGATGG-3V) and Trep-563 (5VACCGCAACTGGGACAAACTTCAT-3V), for a 194-bp fragment. Both PCRs comprised a final volume of 25 AL. Four microliters of template and 14.7 AL of amplicon were taken for first and second PCR, respectively. All reactions were performed with positive (heat-killed T. pallidum Nichols, Biologische Analysensystem, Lich, Germany), human h-actin gene, and negative controls (water, corresponding samples from healthy individuals). T. pallidum amplicons were fractionated by gel electrophoresis, blotted to nylon membranes, and hybridized with a [g32 P]ATP-labeled 19-bp oligonucleotide (5V-GCAGTAGCGTTGGCGGATC-3V) corresponding to nucleotides 481–499. PCR sensitivity was determined by serial dilution of heat-killed T. pallidum Nichols strain (Biologische Analysensystem). The absolute PCR threshold was 10 trepo-
nemes per PCR sample. For each sample, DNA preparation and PCR were performed in independent triplicates. The 47-kDa immunogen gene of T. pallidum was detected in 12 of 15 patients (80%) with syphilis (Table 1). Treponemal DNA was not found in the blood of patient 1 who has primary syphilis probably because extensive hematogenous dissemination had not yet occurred. Instead, T. pallidum DNA was detected in the chancre secretion of this patient. In patients 14 and 15 who had latent syphilis but tested negative by PCR, the bacterial load in blood may have decreased with disease duration below the threshold of PCR. Thus, in this stage, PCR analysis of blood fractions may not be sufficient to contribute to the diagnosis of syphilis (Marfin et al, 2001). Of the different blood samples, PBMCs were positive in all patients with secondary syphilis, in 1 of the 2 patients with primary syphilis and in 5 of 7 with latent syphilis. All other blood fractions (serum, plasma, and granulocytes) gave less consistent results (Table 1). Thus, PBMC appears as a more reliable source for the detection of T. pallidum than serum or other blood fractions. In this respect, our results correspond to those of Pietravalle et al. (1999) who detected T. pallidum in serum of 5 (83.3%) of 6 persons with manifest syphilis and in 6 (67%) of 9 persons with latent syphilis. Even lower detection rates were reported by Marfin et al. (2001) who detected T. pallidum DNA in whole blood in 2 (25%) of 8 patients with manifest syphilis and in 8 (61.5%) of 13 patients with latent syphilis. All 6 skin lesions (chancres and biopsies) were positive by PCR. The lesions of primary and secondary syphilis
Table 1 Clinical characteristics, syphilis serology, and PCR results of various tissue sources of 15 patients with syphilis Patient no., stage of infection
Syphilis serology TPHA (titer)
1. Primary 1:320 2. Primary 1:40 3. Secondary 1:640 4. Secondary 1:2560 5. Secondary 1:10 240 6. Secondary 1:5120 7. Secondary 1:2560 8. Secondary 1:81 920 PCR positive samples/total samples 9. Early latent 1:640 10. Early latent 1:81 920 11. Early latent 12. Early latent 1:320 13. Late latent 1:1280 14. Late latent 1:10 240 15. Late latent 1:10 240 PCR positive samples/total samples
T. pallidum PCR samples
VDRL (titer)
19S-IgM
1:2 1:16 1:32 1:64 1:32 1:64 1:32 1:32
(+) (+) + + + + +
1:16 1:16
+
1:16 1:8 1:64 1:4
(+) + F + F
PBMC + + + + + + + 7/8 + + + + +
5/7
Serum + + + + + + 6/8
Granulocyte
+ +
+ + 4/8 +
Plasma
+
1/8
+ +
2/7
1/7
0/7
Skin lesion
Ejaculate
+a +a,c +b +b ND +b +b ND 6/6 ND ND ND ND ND ND ND ND
ND + ND ND ND + ND 2/3 ND ND ND ND ND ND 0/1
19S-IgM was determined by 19S-IgM-FTA-ABS test; +, positive; (+), weakly positive; F, borderline positive; , negative; ND, not done. bEarly latentQ and blate latentQ are defined as syphilis of less and more than 1-year duration, respectively, according to the criteria of the Centers for Disease Control and Prevention (1997). a Swab. b Biopsy. c Perianal erosion.
A.V. Kouznetsov et al. / Diagnostic Microbiology and Infectious Disease 51 (2005) 143 –145
usually have sufficient bacterial DNA for molecular detection (Zoechling et al., 1997; Sutton et al., 2001). Furthermore, for the first time, we could confirm by PCR the infectivity of ejaculates from syphilis patients. Further studies need to determine if the bacteria were located intracellularly in monocytes, attached to the cell surface, or just enriched between the plasma and Ficoll fractions in the PBMC fraction. Density-gradient centrifugation of blood at a density of 1.051 g/mL can enrich treponemes (Hanff et al., 1984). Ficoll density gradient centrifugation may therefore have contributed to the sensitivity of PCR in the PBMC samples. All blood specimens from the 10 treated patients were negative by PCR. Because these patients had not been tested before antibiotic treatment, it is difficult to speculate about a successful eradication of T. pallidum. The negative test results, however, may support that sufficient treatment eliminates T. pallidum from the circulation. Wicher et al. (1998) had proven by PCR in an experimental animal system that dead T. pallidum are cleared from soft tissue within 30 days. Yet, there have been no time-related studies about the treatment-induced clearance of T. pallidum from the blood of syphilis patients. Hybridization was used to corroborate the specificity of the experimental approach, but it did not increase its sensitivity. Ethidium bromide staining may therefore be sufficient for the detection of 47-kDa gene amplicons and thus facilitate a broader use. In summary, PCR may detect T. pallidum in ejaculates, skin lesions, and in cellular and noncellular fraction of peripheral blood. PBMC fraction may be the best source for molecular detection of manifested and latent syphilis when serology is inconclusive and syphilitic skin lesions are lacking.
145
Acknowledgment A.V. Kouznetsov, MD, was supported by research scholarship from the president of the Russian Federation and the German Academic Exchange Service (DAAD). References Centers for Disease Control and Prevention (1997) Case definitions for infectious conditions under public health surveillance. MMWR Morb Mortal Wkly Rep 46:34 – 37. Hanff PA, et al (1984) Purification of Treponema pallidum, Nichols strain, by Percoll density gradient centrifugation. Sex Transm Dis 11:275 – 286. Hsu P-L, et al (1989) Sequence analysis of the 47-kilodalton major integral membrane immunogen of Treponema pallidum. Infect Immun 57: 196 – 203. Inagaki H, et al (1996) Gastric syphilis: polymerase chain reaction detection of treponemal DNA in pseudolymphamatous lesions. Hum Pathol 27:761 – 765. Kouznetsov AV, Prinz JC (2002) Molecular diagnosis of syphilis: The Schaudinn-Hoffmann lymph-node biopsy. Lancet 360:388 – 938. Marfin AA, et al (2001). Amplification of the DNA polymerase I gene of Treponema pallidum from whole blood of persons with syphilis. Diagn Microbiol Infect Dis 40:163 – 166. Noordhoek GT, et al (1991) Detection by polymerase chain reaction of Treponema pallidum DNA in cerebrospinal fluid from neurosyphilis patients before and after antibiotic treatment. J Clin Microbiol 29: 1976 – 1984. O’Regan AW, et al (2002) Barking up the wrong tree? Use of polymerase chain reaction to diagnose syphilitic aortitis. Thorax 57:917 – 918. Pietravalle M, et al (1999) Diagnostic relevance of polymerase chain reaction technology for T. pallidum in subjects with syphilis in different phase of infection. Microbiologica 22:99 – 104. Sutton MY, et al (2001) Molecular subtyping of Treponema pallidum in an Arizona County with increasing syphilis morbidity: Use of specimens from ulcer and blood. J Infect Dis 183:1601 – 1606. Wicher K, et al (1998) Identification of persistent infection in experimental syphilis by PCR. Infect Immun 66:2509 – 2513. Zoechling N, et al (1997) Molecular detection of Treponema pallidum in secondary and tertiary syphilis. Br J Dermatol 136:683 – 686.