Clinical and Biological Characteristics of 40 Patients With Neurosyphilis and Evaluation of Treponema pallidum Nested Polymerase Chain Reaction in Cerebrospinal Fluid Samples

Clinical Infectious Diseases MAJOR ARTICLE Clinical and Biological Characteristics of 40 Patients With Neurosyphilis and Evaluation of Treponema pallidum Nested Polymerase Chain Reaction in Cerebrospinal Fluid Samples Clélia Vanhaecke,1 Philippe Grange,1 Nadjet Benhaddou,2 Philippe Blanche,3 Dominique Salmon,3 Perrine Parize,4 Olivier Lortholary,4 Eric Caumes,5 Isabelle Pelloux,6 Olivier Epaulard,7 Jérôme Guinard,8 and Nicolas Dupin1; on behalf of the Neurosyphilis Networka 1 Laboratoire de Recherche en Dermatologie, Institut Cochin INSERM U1016, Centre National de Référence Syphilis, Faculté de Médecine, Université Paris Descartes, 2Service de Bactériologie and Service de Médecine Interne, Hôpital Cochin, 4Service de Maladies Infectieuses et Tropicales, Hôpital Necker-Enfants Malades, Centre d’Infectiologie Necker Pasteur, IHU Imagine, 5Service de Maladies Infectieuses et Tropicales, Hôpital Pitié Salpêtrière, AP-HP, Paris, 6Service de Bactériologie, and 7Service de Maladies Infectieuses, Centre Hospitalier Universitaire de Grenoble, and 8Service de Microbiologie, Centre Hospitalier Régional d’Orléans, France 3 Background. Syphilis remains a significant public health problem. We conducted a prospective study to define more precisely the clinical and biological characteristics of patients with neurosyphilis (NS), and we assessed the diagnostic value of nested polymerase chain reaction (PCR) testing for Treponema pallidum in cerebrospinal fluid (CSF) samples. Methods. From 2001 to 2013, we included 40 patients (90% men; 45% infected with human immunodeficiency virus) with NS, defined as syphilis with neurological and/or ophthalmological symptoms and CSF abnormalities. Results. Thirty patients (75%) had early, 5 (12.5%) had late, and 5 had meningovascular NS. Twenty-four patients (80%) with early NS had ophthalmological symptoms, 14 (47%) had neurological symptoms, and 8 (26%) had both. All patients with meningovascular NS had only neurological symptoms. All patients with late NS had neurological symptoms, and 2 (40%) also had ocular symptoms. Ophthalmological symptoms were present in 65% of all patients with NS, and neurological symptoms in 60%. Seventeen patients (42.5%) had CSF white blood cell counts >20/μL (mean, 57/μL), and 27 (67.5%) had high CSF protein levels (>0.5 g/L; mean value, 1 g/L). CSF PCR results were positive in 42%, and CSF VDRL results in 30%. The nested PCR assay had an overall sensitivity of 42.5%, a specificity of 97%, a positive predictive value of 77%, and a negative predictive value of 86%. Conclusions. Early NS is the most frequent presentation, with an overrepresentation of polymorphous ophthalmological symptoms. PCR is highly specific and of potential value when used with other biological parameters. Keywords. neurosyphilis; PCR; CSF; T. pallidum. Neurosyphilis (NS) has multiple clinical presentations, and there is no consensus concerning its definition. Early NS is defined as symptoms of meningitis or ocular symptoms with positive serological results within 12 months of infection. Meningovascular NS is defined as endarteritis of central nervous system (CNS) vessels, and parenchymatous late NS as general paresis and tabes dorsalis. The diagnosis of NS is challenging. Early invasion of the CNS by Treponema pallidum has been detected with the rabbit inoculation test in cerebrospinal fluid (CSF) samples from patients with early syphilis, with or without neurological symptoms or CSF abnormalities. However, the isolation of live spirochetes from the CSF with the neurosyphilis seems too insensitive Received 17 April 2016; accepted 6 July 2016; published online 1 September 2016. a Neurosyphilis Network members are listed in the Notes. Correspondence: N. Dupin, Dermatology, Hôpital Cochin, 89 rue d’Assas, Paris, France (nicolas.dupin@cch.aphp.fr). Clinical Infectious Diseases® 2016;63(9):1180–6 © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com. DOI: 10.1093/cid/ciw499 1180 • CID 2016:63 (1 November) • Vanhaecke et al (sensitivity, 15%–40%) and impractical for routine use [1–3]. A reactive nontreponemal test (usually the Venereal Disease Research Laboratory [VDRL] test) performed in CSF samples (CSF VDRL) is the current reference standard for NS diagnosis [4], despite its poor sensitivity, with negative results obtained for up to 70% of individuals with NS [5]. A positive VDRL result for serum is required for the diagnosis of NS [6]. Tests for detecting Treponema-specific antibodies in CSF samples, such as fluorescent Treponema antibody absorption (FTA-Abs), have been evaluated, with inconsistent results. The likelihood of NS is low if negative results are obtained, although this diagnosis cannot be excluded in cases of strong clinical suspicion [7, 8]. Other criteria are used, such as a high CSF white blood cell (WBC) count or a high CSF protein level, despite their lack of specificity. In practice, NS is defined as positive results in nontreponemal and treponemal serological tests, together with neurological or ophthalmological symptoms and CSF abnormalities, such as high WBC, high protein concentrations, or positive VDRL or FTA-Abs test results [9, 10]. The usefulness of polymerase chain reaction (PCR) in this setting is unclear. The detection of T. pallidum DNA with Downloaded from https://academic.oup.com/cid/article/63/9/1180/2402965 by guest on 14 June 2022 (See the Editorial Commentary by Musher on pages 1187–8.) Fifteen of the remaining 55 patients with suspected NS were excluded because key data were missing or because they did not meet the criteria for NS criteria. Serological Tests The VDRL test (Latex Pasteur; Bio-Rad), the T. pallidum hemagglutination assay (TPHA; Newmarket), and the FTA-Abs test (Trepo Spot IF; BioMérieux) were carried out in accordance with the manufacturers’ instructions. We tested for human immunodeficiency virus (HIV) types 1 and 2 antigens and antibodies in serum with an automated enzyme-linked immunosorbent assay (Genscreen ULTRA HIV Ag-Ab; Bio-Rad). Positive results for antibody detection were confirmed by immunoblotting for HIV-1 and HIV-2 (New Lav-Blot I and New Lav-Blot II; Bio-Rad). Blood CD4 T-cell count, CSF WBC count, and CSF protein level were determined with standard methods at each of the participating study sites. We used ≥2 dilutions when testing samples to confirm positive FTA-Abs results in CSF samples. DNA Extraction MATERIALS AND METHODS DNA was extracted from CSF samples with the NucleoSpin Blood kit (Macherey-Nagel EURL), according to the manufacturer’s instructions. Diagnosis of NS Nested PCR Method The patients were adults with NS, defined as follows: (1) positive treponemal and nontreponemal serum test results; (2) positive CSF VDRL or positive CSF FTA-Abs test result and 1 CSF abnormality in laboratory tests, such as pleocytosis (cell count, >20/μL) or high protein levels (>0.5 g/L); and (3) clinical symptoms. Symptoms were classified into 3 groups: early NS included meningitis (headache, photophobia, nausea, vomiting, cranial nerve palsies, and subjective hearing loss) and/or ocular disease (visual impairment, ocular inflammation, uveitis, and retinitis); meningovascular NS included endarteritis of vessels anywhere in the CNS, confirmed by magnetic resonance imaging, with the exclusion of other cerebrovascular causes; and late NS included general paresis, dementia, motor or sensory deficit, and confusion, with the exclusion of other causes confirmed by the improvement of symptoms after treatment. Asymptomatic NS was excluded because this condition has not been clearly defined and the criteria for CSF examination are unclear. The nested PCR (nPCR) assay for the detection of T. pallidum DNA was based on amplification of the target gene tpp47 and was performed as described elsewhere [14] (Supplement). The limit of detection of our nPCR was 20 bacteria per milliliter. Samples CSF samples were collected retrospectively and prospectively at the Syphilis Reference Center of Cochin Hospital in Paris, France. These samples were stored at the Bacteriology Laboratory of Cochin Hospital between 2001 and 2013. Clinical data and laboratory test results were collected retrospectively. CSF samples giving negative results for VDRL and FTA-Abs tests from patients with negative serum treponemal and nontreponemal test results were used as negative controls. We analyzed 202 samples, including 147 from patients who did not have NS. Statistical Analysis We assessed the concordance between the clinical and biological diagnosis of NS and T. pallidum nPCR results, by determining sensitivity, specificity, positive predictive value, negative predictive value, Youden index, and likelihood ratios. We used Fisher exact test to assess associations between discrete variables, with differences considered significant at P ≤ .05. RESULTS Clinical Characteristics of Patients With NS Thirty patients (75%) had early NS, 5 (12.5%) had meningovascular NS, and 5 (12.5%) had late NS. Men accounted for 90% of the 40 patients (mean age, 46 years). Eighteen patients (45%) were HIV positive, and the median CD4 T-cell count for these patients was 385/μL. Six of the HIV-positive patients (35%) were receiving highly active antiretroviral therapy. Ophthalmological symptoms were present in 26 patients (65%), neurological symptoms in 24 (60%), and both in 10 (25%); 16 patients (40%) had only ophthalmological and 14 (35%) had only neurological symptoms. The ophthalmological symptoms observed were uveitis in 14 patients, retinitis in 6 (including 3 cases of necrotizing retinitis), isolated hyalitis in 1, optical neuritis in 3, and papillary edema Neurosyphilis and PCR • CID 2016:63 (1 November) • 1181 Downloaded from https://academic.oup.com/cid/article/63/9/1180/2402965 by guest on 14 June 2022 PCR in CSF samples from patients at any stage of syphilis can confirm that the CNS has been invaded by the spirochete [9]. However, only a few studies have evaluated the use of PCR for the diagnosis of NS, and these studies have yielded conflicting results. Some authors found no correlation between the presence of T. pallidum in CSF samples shown by a PCR assay amplifying the tpp47 gene and the criteria for NS diagnosis [10], and one study reported a sensitivity of about 25% for a PCR assay amplifying the bmp gene [10]. In parallel, other authors have reported different sensitivity results for PCR tests: 60% for the detection of T. pallidum by a PCR assay amplifying the tpp47 gene from the CSF from neonates born to syphilitic mothers [11], 57% for the diagnosis of NS in 40 patients tested with a PCR assay amplifying the tpp47 gene [12], and 61% in 65 patients tested with an reverse-transcription (RT) PCR assay amplifying ARN16S [5]. Only a few large studies on patients with NS [5, 10, 12, 13] have been published. The aim of the current study was to describe the clinical and biological characteristics of 40 patients with well-documented NS and to assess the diagnostic value of a PCR test for use in CSF samples. Table 1. Clinical and Biological Characteristics of Patients With Neurosyphilis, by Clinical Presentation, and Negative Control Patients Patients With NS by Clinical Presentation, No.a Characteristic Early (n = 30) Meningovascular (n = 5) Late (n = 5) Total (n = 40) Negative Controls, No.a (n = 147) CSF nPCR results Positive 12 3 2 17 5 Negative 18 2 3 23 142 Male 27 5 4 36 93 Female 3 0 1 4 54 41 (26–60) 53.4 (41–69) 64 (39–80) 46 (26–80) 51.7 (17–92) 0 Sex Age, mean (range), y HIV infection 17 1 CD4 T-cell count, mean, cells/μL 391 254 18 26 384 230 Positive 10 1 1 12 0 Negative 20 4 4 28 147 Positive 30 5 5 40 0 Negative 0 0 0 0 147 Ophthalmological 24 0 2 26 28 Neurological 14 5 5 24 100 Syphilis serological results CSF VDRL Clinical symptoms Neurological and ophtalmological symptoms CSF WBC count, mean (range), cells/μL CSF WBC count >20/μL CSF protein level, mean (range), g/L CSF protein level >0.5 g/L 8 0 2 10 57 (0–304) 23 (8–60) 7 (0–13) 47 (0–304) 17 2 0 19 97 0.92 (0.36–2.63) 1.08 (0.45–2.40) 2.17 (0.50–6.96) 1.08 (0.36–6.96) 0.92 (0.27–8.63) 27 4 4 35 113 126 (0–2750) Abbreviations: CSF, cerebrospinal fluid; HIV, human immunodeficiency virus; nPCR, nested polymerase chain reaction; NS, neurosyphilis; TPHA, Treponema pallidum hemagglutination assay; VDRL, Venereal Disease Research Laboratory; WBC, white blood cell. a Unless otherwise specified, data represent No. of patients or controls. in 2. Three patients had a combination of symptoms: uveitis and hyalitis, uveitis and retinitis, and hyalitis and retinitis in 1 patient each. The neurological symptoms observed were headache in 6 patients (isolated in 2 and associated with meningitis in 4), tinnitus in 3, hearing loss in 3, motor disorder in 4, behavioral problems in 3, and language problems in 2. Among the 30 patients with early NS, 24 (80%) had ophthalmological symptoms, 14 (46%) had neurological symptoms and 8 (27%) had both. All 5 patients with late NS had neurological symptoms, and 2 (40%) also had ophthalmological symptoms. All 5 patients with meningovascular NS had only neurological symptoms. The patients with late NS were older than the other patients (Table 1). Clinical and Biological Results for Controls with Negative Serum and CSF Serological Results Biological Features of Patients With NS The nPCR results were positive for 17 CSF samples. The nPCR assay had an overall sensitivity of 42.5% and a specificity of 97%. The Youden index was 0.395, the positive predictive value was 77%, and the negative predictive value was 86%, indicating moderate agreement between the nPCR results and the physician’s diagnosis of NS. The difference in detection rates between patients and controls was significant (P < .001), but the likelihood positive ratio was 14.17 and the likelihood negative ratio 0.59, indicating that the confirmation of NS diagnosis by the analysis of CSF samples was moderately likely. Nineteen patients (47.5%) had CSF WBC counts >20/μL (mean, 47/μL), and 35 (87.5%) had high CSF protein levels (>0.5 g/L; mean, 1.1 g/L). The CSF WBC count was higher in patients with early NS than in the other patients (Table 1). No significant difference was observed between clinical presentations (Table 2). Twelve of 34 patients (35%) had positive results for the VDRL-CSF test, including 1 with meningovascular and 1 with late NS. All patients without positive CSF VDRL results had a positive CSF FTA-Abs result. 1182 • CID 2016:63 (1 November) • Vanhaecke et al We tested 147 negative control patients. All had negative results for serum and CSF VDRL and TPHA tests, and 24% were HIV positive. These patients presented without CNS involvement (35%; cancer, sarcoidosis, Behçet disease, uveitis), with neurological disease (30%; epilepsy, meningitis, lupus, Behçet disease, cerebral vasculitis, lymphoma), with HIV infection and HIVrelated encephalopathy (10%), or with HIV infection and other opportunist infections (25%; cytomegalovirus infection, lymphoma, Castleman disease). nPCR Testing of CSF Samples Downloaded from https://academic.oup.com/cid/article/63/9/1180/2402965 by guest on 14 June 2022 Serum VDRL and TPHA Table 2. Clinical and Biological Characteristics of Patients With Neurosyphilis, by Symptom Type, and Negative Control Patients Patients With NS by Type of Symptoms, No.a Ophthalmological Only (n = 16) Neurological Only (n = 14) Both Types (n = 10) Total (n = 40) Positive 7 7 3 17 5 Negative 9 7 7 23 142 Male 14 12 10 36 93 Female 2 2 0 4 54 51.7 (17–92) Characteristic Negative Controls, No.a (n = 147) CSF nPCR results Sex 41 (26–58) 50 (26–67) 47 (27–80) 46 (26–80) HIV infection Age, mean (range), y 10 4 4 18 26 CD4 T-cell count, mean, cells/μL 354 366 460 384 230 Syphilis serological results Positive 6 2 4 12 0 Negative 10 12 6 28 147 Positive 16 14 10 40 0 Negative 0 0 0 0 147 Early 16 6 8 30 28 Late 0 3 2 5 100 Serum VDRL and TPHA Clinical presentation of NS Meningovascular CSF WBC count, mean (range), cells/μL CSF WBC count >20/μL CSF protein level, mean (range), g/L CSF protein level >0.5 g/L 0 5 0 5 45 (0–167) 17 (0–60) 90 (0–304) 47 (0–304) 10 4 5 19 97 0.86 (0.4–2.63) 1.35 (0.36–6.96) 1 (0.54–1.7) 1.08 (0.36–6.96) 0.92 (0.27–8.63) 14 12 9 35 113 126 (0–2750) Abbreviations: CSF, cerebrospinal fluid; HIV, human immunodeficiency virus; nPCR, nested polymerase chain reaction; NS, neurosyphilis; TPHA, Treponema pallidum hemagglutination assay; VDRL, Venereal Disease Research Laboratory; WBC, white blood cell. a Unless otherwise specified, data represent No. of patients or controls. Positive nPCR results in CSF samples were obtained for 12 patients (40%) with early, 3 (60%) with meningovascular, and 2 (40%) with late NS, whereas the CSF VDRL result was positive for 10 patients (33%) with early, 1 (20%) with meningovascular, and 1 (20%) with late NS. A positive result for nPCR was not significantly associated with any particular clinical presentation or biological characteristics and tended to be associated with a CSF protein level of up to 0.5 g/L and a time between sampling and the PCR assay of <1 month (Table 3). HIV-Infected Patients All 18 HIV-infected patients were men. These patients had a median age of 39 years; 17 (95%) had early, 1 had meningovascular, and none had late NS. Fourteen patients (77%) had ophthalmological symptoms, 8 (44%) had neurological symptoms, and 4 (22%) had both. The mean WBC count was 53/μL, and the mean protein level was 1.08 g/L. Eight patients (44%) had positive nPCR results in CSF samples. DISCUSSION Our study described the clinical characteristics of 40 patients with NS and showed that early NS is the most frequent presentation, with a high prevalence of ophthalmological symptoms. To avoid including patients without NS, we included only patients with symptoms. There was a high proportion of HIVinfected patients, but there were no clinical and/or biological differences between them and HIV-noninfected patients. The CSF VDRL result was positive in thirty percent of patients, whereas PCR detection of treponemal DNA had a positive result in 42.5%, with a high specificity. One key finding of this study was the high diversity of both ophthalmological and neurological symptoms. NS seems to be as heterogeneous as secondary syphilis, with symptoms extending from isolated headache to cerebrovascular accidents, and from tinnitus to necrotizing retinitis. This suggests that clinicians may be underprescribing tests for NS owing to a lack of awareness of the broad range of symptoms observed in this disease. We did not include patients with asymptomatic NS because of the lack of consensus in France about whether and under what conditions lumbar puncture should be performed in patients without symptoms. Another objective of our study was to evaluate the place of PCR in the diagnosis of NS from CSF samples. Previous studies have clearly shown that the performance of CSF treponemal Neurosyphilis and PCR • CID 2016:63 (1 November) • 1183 Downloaded from https://academic.oup.com/cid/article/63/9/1180/2402965 by guest on 14 June 2022 CSF VDRL Table 3. Clinical and Biological Characteristics of Patients With Neurosyphilis, by Cerebrospinal Fluid Nested Polymerase Chain Reaction Results CSF nPCR Results Characteristic Total Positive Negative 95% CI P Value Patients with NS Total 40 17 23 6–77 <.001 Early 30 12 18 .12–3.6 .70 Meningovascular 5 3 2 .22–29.5 Late 5 2 3 .075–10.28 >.99 .60 Type of symptoms Ophthalmological only 16 7 9 .25–4.66 >.99 Neurological only 14 7 7 .35–7.2 .52 Both 10 3 7 .07–2.73 .47 CSF WBC count >20 19 8 11 .23–4.05 >.99 CSF protein level >0.5 g/L 35 17 18 .73 to infinity HIV positive 18 8 10 .27–4.85 23 13 10 .890866–22.66 Negative control patients 147 5 142 . . . . . . .054 All patients 187 22 165 . . . . . . Abbreviations: CI, confidence interval; CSF, cerebrospinal fluid; HIV, human immunodeficiency virus; nPCR, nested polymerase chain reaction; NS, neurosyphilis; WBC, white blood cell. antibody tests depends on the criteria used for diagnosing NS. Studies that used a positive CSF VDRL result as the sole criterion to diagnose NS captured only subjects with definitive syphilis. Therefore, the first difficulty is confirming the diagnosis of NS, because the reference standard (ie, positive CSF VDRL result) is not sensitive enough. Indeed, only 35% of our patients had positive CSF VDRL results. The nPCR test had a sensitivity of 44% and a specificity of 97%. These results are similar to those of previous studies reporting sensitivities of about 25% in a PCR assay amplifying the bmp gene in a study of 27 cases [10], 57% in a PCR assay amplifying the tpp47 gene in a study of 40 cases [12], and 61% in an RT-PCR assay amplifying 16S RNA in a study of 65 cases [5]. Another study reported a sensitivity of 47% and a specificity of 93% for a total of 49 samples, but there were only 2 cases of confirmed NS, both of which yielded positive results for CSF PCR, and 2 positive cases of NS, with positive PCR results obtained for only 1. The other cases corresponded to primary, secondary or late syphilis not meeting the criteria for NS [9]. However, the detection sensitivity in our study was too low for this test to be considered suitable for use as a diagnostic test. Positive PCR results were not associated with other CSF abnormalities, consistent with previously reported findings [13]. The low sensitivity observed may be due, in part, to the effects of the various handling and storage conditions on the stability of T. pallidum DNA in CSF. Our results suggest that some of the treponemal DNA originally present in the clinical samples was degraded (Table 3). This is consistent with a previous study reporting that quantitative PCR amplification of the tpp47 gene was more sensitive with fresh blood samples than with frozen blood samples [15]. Nevertheless, a study testing a 1184 • CID 2016:63 (1 November) • Vanhaecke et al classic PCR amplifying the tpp47 gene reported a similar detection sensitivity for CFS samples stored at room temperature or at 4°C and after freeze-thaw cycles [16]. We obtained discordant results for 5 patients with positive nPCR results but no diagnosis of NS. Three of these patients had HIV infection and severe immunodeficiency, and 2 had neurological involvement, due to cytomegalovirus invasion of the CNS in 1 and HIV infection in 1. These patients also had other opportunistic infections and were treated with multiple antibiotics. We cannot exclude the possibility that they had very early syphilis and had not yet produced antibodies [9]. Moreover, we were unable to determine whether syphilis would have developed later, owing to the multiple antibiotic treatments they received for opportunistic diseases linked to their immunodeficiency. TPHA and VDRL test results were not checked, because the PCR assay was performed many years after the lumbar puncture. None of our negative controls yielded positive results at any time, so contamination cannot account for these false-positive results. Our results for PCR contrast with those obtained for directly sampled skin or mucosal lesions from patients with primary and secondary syphilis, for which sensitivities of 57%–97% have been reported [15, 17–19]. This may be because the skin is the target of Treponema in primary or secondary syphilis, whereas the CSF may not be the only target of the spirochete in patients with NS. The various expressions of NS depend on the site at which Treponema is found: the meninges and eyeball for acute NS, the vessels for meningovascular NS, and the brain for parenchymatous late NS. Thus, the detection of Treponema in CSF samples may not be the most efficient method possible, but it is the only method currently available. Moreover, only small numbers of spirochetes may be required to cause Downloaded from https://academic.oup.com/cid/article/63/9/1180/2402965 by guest on 14 June 2022 <1 mo between sampling and nPCR assay .06 >.99 Supplementary Data Supplementary materials are available at http://cid.oxfordjournals.org. Consisting of data provided by the author to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the author, so questions or comments should be addressed to the author. Notes Acknowledgements. Clelia Vanhaecke was a recipient of a personnal grant from the Société Française de Dermatologie. Neurosyphilis Network members. Sophie Galimard, Service de Médecine Interne, Hôpital Cochin, AP-HP; Timothée Boyer Chammard, Service de Maladies Infectieuses et Tropicales, Hôpital Necker, AP-HP; Benoit Henry and Loïc Epelboin, Service de Maladies Infectieuses et Tropicales, Hôpital Pitié Salpêtrière; Neila Sedira and Emmanuel Heron, Service de Médecine Interne, Centre Hospitalier National d’Ophtalmologie des QuinzeVingts, Paris; Isabelle Alcaraz, Service de Maladies Infectieuses et du Voyageur, Hôpital de Tourcoing; Nathalie Franck, Service de Dermatologie, Hôpital Cochin; Hélène Chaussade and Adrien Lemaignen, Service de Médecine Interne et Maladies Infectieuses, Centre Hospitalier Régional Universitaire de Tours; Claire Demangeot and Antoine Petit, Service de Dermatologie, Hôpital Saint Louis, AP-HP; Magdalena Gerin, Service de Médecine Interne, Hôpital Jean Verdier; Christophe Guier, Laboratoire de Biologie Médicale, Hôpital Drôme Nord; Nadia Idri, Service de Bactériologie, Centre de Soins et d’Accueil Hospitalier de Nanterre, Bertrand Issartel, Centre de Vaccinations Internationales et de Médecine des Voyages du Tonkin, Lyon; Etienne Lagier, Service de Bactériologie, Centre Hospitalier d’Aix; Anne Léger, Unité de Neurovasculaire, Hôpital Pitié Salpêtrière, AP-HP; Isabelle Mahé and Emmanuel Mortier, Service de Médecine Interne, Hôpital Louis Mourier, Paris; Eric Monlun, Service de Neurologie, Centre Hospitalier de Pau; Christophe Rapp, Service de Maladies Infectieuses et Tropicales, Hôpital d’Instruction des Armées Begin, Saint Mandé; Claude Remy, Service de Neurologie, Hôpital Drôme Nord; Laurent Renier, Service de Neurologie, Centre Hospitalier d’Aix; Olivier Saladini, Service de Psychiatrie, Hôpital Drome Nord; François Sellal, Service de Neurologie, Hôpital de Colmar; Valérie Serry, Service de Biologie, Centre Hospitalier Intercommunal de Fréjus; Laurence Weiss, Service d’Immunologie Clinique, Hôpital Européen Georges Pompidou, AP-HP. Author contributions. All authors had full access to all of the data for this study and take responsibility for the integrity of the data and the accuracy of the data analysis. Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. References 1. Ghanem KG. Neurosyphilis: a historical perspective and review. CNS Neurosci Ther 2010; 16:e157–68. 2. Lukehart SA, Hook EW III, Baker-Zander SA, Collier AC, Critchlow CW, Handsfield HH. Invasion of the central nervous system by Treponema pallidum: implications for diagnosis and treatment. Ann Intern Med 1988; 109:855–62. 3. Rolfs RT, Joesoef MR, Hendershot EF, et al. A randomized trial of enhanced therapy for early syphilis in patients with and without human immunodeficiency virus infection. N Engl J Med 1997; 337:307–14. 4. Marra CM, Tantalo LC, Maxwell CL, Ho EL, Sahi SK, Jones T. The rapid plasma reagin test cannot replace the Venereal Disease Research Laboratory test for neurosyphilis diagnosis. Sex Transm Dis 2012; 39:453–7. 5. Marra CM, Maxwell CL, Smith SL, et al. Cerebrospinal fluid abnormalities in patients with syphilis: association with clinical and laboratory features. J Infect Dis 2004; 189:369–76. 6. Wöhrl S, Geusau A. Neurosyphilis is unlikely in patients with late latent syphilis and a negative blood VDRL-test. Acta Derm Venereol 2006; 86:335–9. 7. Workowski KA, Berman SM. Centers for Disease Control and Prevention sexually transmitted disease treatment guidelines. Clin Infect Dis 2011; 53(suppl 3): S59–63. 8. Harding AS, Ghanem KG. The performance of cerebrospinal fluid treponemalspecific antibody tests in neurosyphilis: a systematic review. Sex Transm Dis 2012; 39:291–7. Neurosyphilis and PCR • CID 2016:63 (1 November) • 1185 Downloaded from https://academic.oup.com/cid/article/63/9/1180/2402965 by guest on 14 June 2022 neurological disease, and the techniques used to detect them may have been insufficiently sensitive [9]. The lower sensitivity of nPCR in CSF than in skin samples may be due to the fact that T. pallidum is only transiently present in CSF, at a time point not coinciding with lumbar puncture. A previous study in a primate model showed that T. pallidum was detected with the rabbit inoculation test in CSF samples from all primates at 2 weeks, but not thereafter [20]. Moreover, the first nPCR assay was negative for 1 of our patients with late NS. This patient’s symptoms had improved 3 months later, after intravenous ceftriaxone treatment. Lumbar puncture was repeated and nPCR yielded positive results. The detection of T. pallidum by PCR after antibiotic treatment has already been reported for 29 CSF samples, suggesting that T. pallidum DNA can remain present in CSF for long periods after the effective killing of the bacteria by appropriate treatment and that the detection of this DNA is not predictive of relapse [10]. We cannot exclude the possibility that the nPCR assay detects DNA from killed bacteria. We were unable to determine whether our patients presented any subsequent relapses. Moreover, a previous study reported the detection of T. pallidum by RT-PCR in CSF samples from rabbits infected with the Nichols strain, 2 and 4 weeks after inoculation, whereas CSF VDRL results were positive at 4 and 6 weeks, and CSF pleocytosis was never observed [21]. However, nervous system invasion and the immune reaction to Treponema may differ between rabbits and humans, because CSF pleocytosis is observed in humans infected with the Nichols strain. These results confirm the difficulties involved in studying the invasion of the CNS by T. pallidum. The nPCR results obtained for patients with meningovascular and late NS were unexpected. We hypothesized that T. pallidum would be absent from the CSF in these patients but that it would persist in the vessels and/or parenchyma. The nPCR results were positive for 2 of the 5 cases of late NS (sensitivity, 40%) and for 3 of the 5 cases of meningovascular NS (sensitivity, 60%). Moreover, 1 patient with neurological symptoms (tetrapyramidal syndrome and ischemic cerebral abnormalities seen with magnetic resonance imaging), a positive VDRL and TPHA result, and HIV infection was excluded, because he did not meet the inclusion criteria for CSF WBC counts and protein levels. He had a negative CSF VDRL result and it was impossible to perform an FTA-Abs test, but nPCR gave a positive result. A diagnosis of NS was retained. This case highlights the potential value of nPCR in cases of meningovascular features, for which the CSF VDRL test is inefficient. The diagnosis of NS remains challenging for clinicians and biologists. Despite its lack of sensitivity for use alone as a diagnostic test, nPCR amplifying tpp47 seemed more sensitive to T. pallidum in CSF samples from patients with NS than the currently recommended CSF VDRL test. This nPCR test should therefore be preferred for the diagnosis of NS. Studies in a larger population are required to confirm these preliminary results. 9. Hay PE, Clarke JR, Taylor-Robinson D, Goldmeier D. Detection of treponemal DNA in the CSF of patients with syphilis and HIV infection using the polymerase chain reaction. Genitourin Med 1990; 66:428–32. 10. Noordhoek GT, Wolters EC, de Jonge ME, van Embden JD. Detection by polymerase chain reaction of Treponema pallidum DNA in cerebrospinal fluid from neurosyphilis patients before and after antibiotic treatment. J Clin Microbiol 1991; 29:1976–84. 11. Grimprel E, Sanchez PJ, Wendel GD, et al. Use of polymerase chain reaction and rabbit infectivity testing to detect Treponema pallidum in amniotic fluid, fetal and neonatal sera, and cerebrospinal fluid. J Clin Microbiol 1991; 29:1711–8. 12. Molepo J, Pillay A, Weber B, Morse SA, Hoosen AA. Molecular typing of Treponema pallidum strains from patients with neurosyphilis in Pretoria, South Africa. Sex Transm Infect 2007; 83:189–92. 13. Chung KY, Lee MG, Lee JB. Detection of Treponema pallidum by polymerase chain reaction in the cerebrospinal fluid of syphilis patients. Yonsei Med J 1994; 35:190–7. 14. Grange PA, Gressier L, Dion PL, et al. Evaluation of a PCR test for detection of Treponema pallidum in swabs and blood. J Clin Microbiol 2012; 50:546–52. 15. Cruz AR, Pillay A, Zuluaga AV, et al. Secondary syphilis in Cali, Colombia: new concepts in disease pathogenesis. PLoS Negl Trop Dis 2010; 4:e690. 16. Villanueva AV, Podzorski RP, Reyes MP. Effects of various handling and storage conditions on stability of Treponema pallidum DNA in cerebrospinal fluid. J Clin Microbiol 1998; 36:2117–9. 17. Orle KA, Gates CA, Martin DH, Body BA, Weiss JB. Simultaneous PCR detection of Haemophilus ducreyi, Treponema pallidum, and herpes simplex virus types 1 and 2 from genital ulcers. J Clin Microbiol 1996; 34:49–54. 18. Palmer H, Higgins S, Herring A, Kingston M. Use of PCR in the diagnosis of early syphilis in the United Kingdom. Sex Transm Infect 2003; 79:479–83. 19. Bruisten SM, Cairo I, Fennema H, et al. Diagnosing genital ulcer disease in a clinic for sexually transmitted diseases in Amsterdam, The Netherlands. J Clin Microbiol 2001; 39:601–5. 20. Marra CM, Castro CD, Kuller L, et al. Mechanisms of clearance of Treponema pallidum from the CSF in a nonhuman primate model. Neurology 1998; 51:957–61. 21. Tantalo LC, Lukehart SA, Marra CM. Treponema pallidum strain-specific differences in neuroinvasion and clinical phenotype in a rabbit model. J Infect Dis 2005; 191:75–80. Downloaded from https://academic.oup.com/cid/article/63/9/1180/2402965 by guest on 14 June 2022 1186 • CID 2016:63 (1 November) • Vanhaecke et al Please excuse the presence of this and the following test pages, which have been added to a small number of article PDFs for a limited time as part of our process of continual development and improvement. academic.oup.com/cid academic.oup.com/cid 1 of 4 Lorem ipsum dolor sit amet, consectetur adipiscing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum. Lorem ipsum dolor sit amet, consectetur adipiscing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum. Lorem ipsum dolor sit amet, consectetur adipiscing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum. Lorem ipsum dolor sit amet, consectetur adipiscing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum. Lorem ipsum dolor sit amet, consectetur adipiscing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum. Lorem ipsum dolor sit amet, consectetur adipiscing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum. Lorem ipsum dolor sit amet, consectetur adipiscing elit, sed do academic.oup.com/cid 2 of 4 eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum. Lorem ipsum dolor sit amet, consectetur adipiscing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum. Lorem ipsum dolor sit amet, consectetur adipiscing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum. Lorem ipsum dolor sit amet, consectetur adipiscing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum. Lorem ipsum dolor sit amet, consectetur adipiscing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum. Lorem ipsum dolor sit amet, consectetur adipiscing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum. Lorem ipsum dolor sit amet, consectetur adipiscing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. academic.oup.com/cid 3 of 4 Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum. Lorem ipsum dolor sit amet, consectetur adipiscing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum. Lorem ipsum dolor sit amet, consectetur adipiscing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum. Lorem ipsum dolor sit amet, consectetur adipiscing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum. Lorem ipsum dolor sit amet, consectetur adipiscing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum. Lorem ipsum dolor sit amet, consectetur adipiscing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum. academic.oup.com/cid 4 of 4