ORIGINAL STUDY
Toll-Like Receptor Polymorphisms Are Associated With Increased Neurosyphilis Risk Christina M. Marra, MD,* Sharon K. Sahi, BS,* Lauren C. Tantalo, BS,* Emily L. Ho, MD, PhD,* Shelia B. Dunaway, MD,Þ Trudy Jones, MN, ARNP,* and Thomas R. Hawn, MD, PhDÞ Background: Single-nucleotide polymorphisms (SNPs) in toll-like receptors (TLR) 1, 2, and 6 impair cell signaling in response to spirochetal lipoproteins. We investigated whether common SNPs in TLR1, TLR2, or TLR6 were associated with laboratory- or clinically-defined neurosyphilis. Methods: Polymorphisms in the genes for TLR1 (a TYG mutation at position 1805), TLR2 (a GYA mutation at position 2258), and TLR6 (a CYT mutation at position 745) were sought in 456 white patients with syphilis. Laboratory-defined neurosyphilis included a reactive cerebrospinal fluid (CSF)YVenereal Disease Research Laboratory (VDRL) test. Clinically-defined neurosyphilis included new vision or hearing loss. Controls had CSF white blood cells of 5/KL or less, nonreactive CSFYVenereal Disease Research Laboratory, and no vision or hearing loss. Results: Overall, 26.2% of patients had laboratory-defined and 36.2% had clinically-defined neurosyphilis. Compared with controls, patients with any of the 3 SNPs were more likely to have laboratory-defined neurosyphilis. Those with TLR2 or TLR6 SNPs were more likely to have clinically-defined neurosyphilis. These associations were independent of serum rapid plasma reagin titer. Conclusions: A common TLR1 polymorphism is associated with an increased risk of laboratory-defined neurosyphilis, and common TLR2 and TLR6 polymorphisms are associated with an increased risk of both laboratory- and clinically-defined neurosyphilis. These data suggest that host factors impact the natural history of syphilis.
T
reponema pallidum spp. pallidum (hereafter termed T. pallidum), the bacterium that causes syphilis, is highly neuroinvasive. Studies from the early 20th century demonstrated that isolation of the organism from cerebrospinal fluid (CSF) in early syphilis was common.1 However, even before the advent of antibiotics, not every patient with syphilis developed neurosyphilis. Host and pathogen-related factors may influence susceptibility. For example, patients with high serum rapid plasma reagin (RPR) titers2Y4 and those infected with HIV, particularly if they have low peripheral blood CD4+ T cells or are not taking antiretroviral agents, may be at higher risk for asymptomatic and symptomatic neurosyphilis.2,3,5 In addition,
From the *Departments of Neurology and †Medicine, University of Washington, Seattle, WA
Portions of this work were presented at the 20th Conference on Retroviruses and Opportunistic Infections; Atlanta, GA; March 3Y6, 2013. This work was supported by the National Institute of Neurological Disorders and Stroke of the National Institutes of Health (Grant No. NS34235). The authors have no conflicts of interest relevant to this work. Correspondence: Christina M. Marra, MD, Harborview Medical Center, Box 359775, 325 9th Ave, Seattle, WA 98104. E-mail: [email protected]. Received for publication February 5, 2014, and accepted May 5, 2014. DOI: 10.1097/OLQ.0000000000000149 Copyright * 2014 American Sexually Transmitted Diseases Association All rights reserved.
440
particular T. pallidum strain types may be more likely to cause neurosyphilis.6 Clinicians in the early 20th century posited that race influenced susceptibility to neurosyphilis, citing a decreased risk in African Americans compared with whites.7 Subsequent work suggested a genetic basis for such differences, with an increased risk of syphilitic dementia, but not other forms of neurosyphilis, in patients with certain HLA types8 that differed in African Americans compared with whites.9 Although more recent reports suggest that there may be genetic contributions to syphilis susceptibility,10Y13 to the best of our knowledge, there have been no recent investigations of genetic susceptibility to neurosyphilis. Human toll-like receptors (TLRs) are a family of 10 proteins that differentially recognize pathogen-associated molecular patterns. Ligation of cellular TLRs activate signaling cascades that lead to initiation of the innate immune response and cytokine production, ultimately culminating in antimicrobial host defenses.14 T. pallidum expresses many lipoproteins15 that stimulate innate immune cells via TLR2 as a heterodimer with TLR1 or TLR6.16,17 In a microarray analysis of skin from patients with secondary syphilis, transcripts for both TLR1 and TLR2 were up-regulated, whereas TLR6 transcripts were not.18 Single-nucleotide polymorphisms (SNPs) in TLR1, TLR2, and TLR6 impair the innate immune response to spirochetal lipopeptides and lipoproteins. A GYA mutation in the TLR2 gene at position 2258 (TLR2_G2258A) occurs in 3% to 9% of whites and is associated with decreased cell signaling in response to stimulation with the T. pallidum lipoprotein T47L.19,20 Similarly, a TYG mutation in the TLR1 gene at position 1805 (TLR1_T1805G) is common, with 52% of Seattle European whites being GG, and is associated with decreased TLR1 surface expression21 and decreased cell signaling in response to Borrelia burgdorferi, a spirochetal organism closely related to T. pallidum.22 Finally, a CYT mutation in the TLR6 gene at position 745 (TLR6_C745T) is also common (seen in 46% of European Americans23) and is associated with decreased cell signaling in response to B. burgdorferi.22 Although the TLR1 and TLR2 SNPs have been associated with altered susceptibility and course of Borrelia infections,24,25 it is not known if SNPs in TLR1, TLR2, or TLR6 are associated with the clinical course of syphilis. In this study, we investigated whether common and functionally defined TLR1/2/6 SNPs were associated with neurosyphilis in HIVinfected and HIV-uninfected patients with syphilis.
METHODS Study Participants Participants were enrolled in a study of CSF abnormalities in syphilis conducted in Seattle, WA, from March 1997 through August 2013.2 Eligibility criteria included clinical or serological evidence of syphilis and assessment by the referring provider that the patient was at risk for neurosyphilis. Reasons
Sexually Transmitted Diseases
&
Volume 41, Number 7, July 2014
Copyright © 2014 by the American Sexually Transmitted Diseases Association. Unauthorized reproduction of this article is prohibited.
TLR Polymorphisms Increase Neurosyphilis Risk
for referral to the study included the following: (1) neurological findings, particularly vision or hearing loss; (2) serum RPR titer Q1:32; and (3) in HIV-infected individuals, peripheral blood CD4+ T cell count of 350/KL or less. The latter criteria are based on published data.2Y4 Participants underwent a structured history and neurological examination that included assessment of vision and hearing, lumbar puncture, and venipuncture. The study protocol was reviewed and approved by the University of Washington Institutional Review Board, and human experimentation guidelines were followed in the conduct of this research. Written informed consent was obtained from all participants.
to calculate R2 and D¶ as measurements of linkage disequilibrium (LD) between the TLR1 and TLR6 polymorphisms. Comparison of median values between groups was performed using the Mann-Whitney U test, and comparison of proportions was performed using W2 or Fisher exact tests. Logistic regression was used for multivariate analysis, with results reported as odds ratios (ORs) with 95% confidence intervals (95% CI); variables with P values greater than 0.20 in univariate analysis were not included in multivariate analysis. P values less than 0.05 were considered statistically significant. Adjustments were not routinely made for multiple comparisons.
RESULTS
Laboratory Methods DNA was extracted from blood, or in 3 instances from serum, using the QIAamp DNA Blood Midi Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. The TLR2_G2258A SNP (reference SNP identification number [rs] 5743708 in the Single Nucleotide Polymorphism Database) was detected by restriction fragment length polymorphism analysis.20 The TLR1_T1805G (rs5743618) and TLR6_C745T (rs5743810) SNPs were detected using TaqMan SNP genotyping assays (Applied Biosystems, Carlsbad, CA) with iTaq Supermix with ROX (Bio-Rad, Hercules, CA) on an ABI 7900HT or Viia7 instrument, according to the manufacturer’s instructions. Genotyping failed for the TLR2 SNP in 2 patients and for the TLR6 SNP in 1 patient. Serum RPR and CSFY Venereal Disease Research Laboratory (VDRL) were performed using standard methods.26
Statistical Methods All patients in this study had syphilis based on clinical and serological criteria.27 Patients with laboratory-defined neurosyphilis had a reactive CSF-VDRL test. Patients with clinically-defined neurosyphilis had new vision or hearing loss as assessed by history and physical examination; patients with previously documented vision or hearing abnormalities were excluded from analysis. Controls were defined as patients with normal CSF white blood cell counts (e5 cells/KL), nonreactive CSF-VDRL, and no vision or hearing loss. We restricted the analysis to self-reported whites. Stata program PWLD was used TABLE 1.
Participant Characteristics To examine the association of TLR polymorphisms with susceptibility to neurosyphilis, we used a case-control study design in 456 syphilis patients who underwent lumbar puncture for evaluation of neurosyphilis and were categorized as laboratory- or clinically-defined neurosyphilis or as controls. Overall, most study participants were men and 77.2% were HIV infected, consistent with the demographics of syphilis in Seattle. The proportion of patients who were treated for uncomplicated (nonneurological) syphilis before study entry and serum RPR titers differed significantly in the controls compared with the neurosyphilis cases (Table 1). Cerebrospinal fluidYVDRL was available for 453 patients and was reactive in 91 (20.1%). One hundred forty-five (32.2%) of 450 assessable patients had symptoms or signs of vision or hearing loss. Of 447 patients with both assessments, 49 (11.0%) only had a reactive CSFVDRL, 106 (23.7%) only had vision or hearing loss, and 36 (8.1%) had both.
Frequencies of TLR1, TLR2, and TLR6 Polymorphism Genotypes in Patients With Laboratory- and Clinically-Defined Neurosyphilis and Controls We compared genotype frequencies of the 3 SNPs between those with the 2 neurosyphilis phenotypes and controls. None of the controls violated Hardy-Weinberg equilibrium (Table 2). There was a significant association between the
Patient Characteristics
Characteristic
P
Clinical Neurosyphilis (n = 145)
P
251 (98.0%) 38 (31Y44) 200 (78.1%)
90 (98.9%) 41 (33Y47) 70 (76.9%)
1.000 0.063 0.813
141 (97.2%) 40 (32Y46) 111 (76.6%)
0.728 0.142 0.717
195 (77.4%) 57 (22.6%) 32 (8Y128)
62 (68.1%) 29 (31.9%) 256 (128Y512)
0.081
0.885
G0.001
110 (78.0%) 31 (22.0%) 128 (64Y256)
G0.001
130 (50.8%)
31 (34.1%)
0.006
58 (40.0%)
0.038
59 (23.0%)
17 (18.7%)
0.387
30 (20.7%)
0.585
Controls (n = 256) Laboratory Neurosyphilis (n = 91)
Sex (men) Age, y HIV-infected Syphilis stage* Early† Late‡ Reciprocal serum RPR titer§ Treated for uncomplicated syphilis before entry Previous syphilis
Controls had no vision or hearing loss and CSF white blood cells e5/KL and a nonreactive CSF-VDRL. Patients with laboratory-defined neurosyphilis had a reactive CSF-VDRL. Patients with clinically-defined neurosyphilis had new vision or hearing loss. Values are n (%) or median (interquartile range). Comparison of median values between neurosyphilis groups and controls was performed using the Mann-Whitney U test, and comparison of proportions between neurosyphilis groups and controls was performed using W2 or Fisher exact tests. *Stage was available for 252 controls and 141 patients with clinically-defined neurosyphilis. †Early syphilis includes primary, secondary, and early latent stages. ‡Late syphilis includes late latent syphilis and syphilis of unknown duration. § RPR titer was available for 255 controls and 143 patients with clinically-defined neurosyphilis.
Sexually Transmitted Diseases
&
Volume 41, Number 7, July 2014
441
Copyright © 2014 by the American Sexually Transmitted Diseases Association. Unauthorized reproduction of this article is prohibited.
Marra et al.
TABLE 2.
TLR1, TLR2, and TLR6 Polymorphism Gene Frequencies in Controls and in Patients With Neurosyphilis
Genotype Frequencies*
TLR1 Control Laboratory-defined neurosyphilis Clinically-defined neurosyphilis TLR2 Control Laboratory-defined neurosyphilis Clinically-defined neurosyphilis TLR6 Control Laboratory-defined neurosyphilis Clinically-defined neurosyphilis
n (%)
n (%)
n (%)
TT 45 (17.6) 10 (11.0) 17 (11.7) GG 254 (99.2) 86 (94.5) 133 (93.0) CC 125 (48.8) 33 (36.3) 41 (28.5)
TG 110 (43.0) 32 (35.2) 67 (46.2) GA 2 (0.8) 5 (5.5) 10 (7.0) CT 104 (40.6) 42 (46.2) 82 (56.9)
GG 101 (39.5) 49 (53.8) 61 (42.1) AA 0 (0) 0 (0) 0 (0) TT 27 (10.5) 16 (17.6) 21 (14.6)
P
HWE P value 0.119
0.048 0.297 0.950 0.015 0.001 0.443 0.064 G0.001
Controls had no vision or hearing loss and CSF white blood cells e5/KL and a nonreactive CSF-VDRL; patients with laboratory-defined neurosyphilis had a reactive CSF-VDRL, and patients with clinically-defined neurosyphilis had new vision or hearing loss. P value calculated from a genotypic model with a 2 3 W2 or Fisher exact test. *Genotypes designate common homozygote (AA), heterozygote (Aa), and uncommon homozygote (aa). HWE indicates Hardy-Weinberg equilibrium.
TLR1 SNP and laboratory-defined neurosyphilis, but no significant association with clinically-defined neurosyphilis (Table 2). The TLR2 SNP was significantly associated with both laboratory- and clinically-defined neurosyphilis, and the TLR6 SNP was marginally associated with laboratory-defined and significantly associated with clinically-defined neurosyphilis (Table 2). Based on the experimentally determined genetic models of the 3 well-characterized SNPs, we then examined the association of the TLR1 SNP using a recessive model, and the TLR2 and TLR6 SNPs using dominant models21,24,28,29 (Table 3). The associations between the 3 SNPs and laboratorydefined neurosyphilis were significant for all 3 SNPs, with ORs ranging from 1.68 to 7.38 for the ‘‘deficient’’ genotype (TLR1_1805GG, TLR2_2258GA, and TLR6_745CT/TT) (Table 3). The associations between the TLR2 and TLR6 deficient genotypes and clinically-defined neurosyphilis had a higher magnitude (OR, 9.55 and 2.40) and remained
TABLE 3.
significant even after taking into account multiple comparisons (Table 3).
Haplotype and Diplotype Analysis of the TLR1 and TLR6 Polymorphisms Because of close genomic proximity of TLR1 and TLR6 on chromosome 4, we next ascertained whether these associations were independent by measuring LD among the loci using pairwise LD analysis. R2 was 0.31, suggesting a low to moderate level of LD between these polymorphisms. To determine whether there were additive effects from deficiency in both TLR1 and TLR6, we examined diplotypes that were double deficient (TLR1_1805GG and TLR6_745CT/TT, n = 154) versus those with at least 1 sufficient variant (TLR1_1805GT/ TT and/or TLR6_745CC, n = 301). Results were consistent with the findings in patients who were TLR1 deficient. Specifically, the odds of laboratory-defined neurosyphilis were significantly higher in double-deficient individuals compared with those with
TLR1, TLR2, and TLR6 Polymorphisms in Controls and Patients With Neurosyphilis
TLR1 Control Laboratory-defined neurosyphilis
TT/TG 155 42
TLR2 Control Laboratory-defined neurosyphilis Clinically-defined neurosyphilis
GG 254 86 133
TLR6 Control Laboratory-defined neurosyphilis Clinically-defined neurosyphilis
CC 125 33 41
Recessive model GG 101 49 Dominant model GA/AA 2 5 10 Dominant model CT/TT 131 58 103
OR (95% CI)
P
1.79 (1.11Y2.90)
0.018
7.38 (1.41Y38.75) 9.55 (2.06Y44.21)
0.018 0.004
1.68 (1.03Y2.75) 2.40 (1.55Y3.71)
0.040 G0.001
Controls had no vision or hearing loss and CSF white blood cells e5/KL and a nonreactive CSF-VDRL; patients with laboratory-defined neurosyphilis had a reactive CSF-VDRL, and patients with clinically-defined neurosyphilis had new vision or hearing loss. Comparison of proportions between the neurosyphilis groups and controls was performed using W2 or Fisher exact tests. Univariate logistic regression was used to derive ORs with 95% CIs.
442
Sexually Transmitted Diseases
&
Volume 41, Number 7, July 2014
Copyright © 2014 by the American Sexually Transmitted Diseases Association. Unauthorized reproduction of this article is prohibited.
TLR Polymorphisms Increase Neurosyphilis Risk
marginally increased the odds of laboratory-defined neurosyphilis (Table 4). In multivariate analysis, the odds of laboratory-defined neurosyphilis remained significantly higher in those with TLR1 and TLR6 deficiency, even after taking the above measures into account. Similarly, in univariate analysis, the odds of clinicallydefined neurosyphilis were significantly higher in patients with higher serum RPR titers, and those treated for uncomplicated syphilis before study enrollment, and older age marginally increased the odds of clinically-defined neurosyphilis (Table 4). In multivariate analysis, the odds of clinically-defined neurosyphilis remained significantly higher in those with TLR6 deficiency, even after taking the above measures into account (Table 4).
at least 1 sufficient variant (OR, 1.82 [1.11Y2.98], P = 0.017), but the odds of clinically-defined neurosyphilis were not (OR, 1.44 [0.94Y2.21], P = 0.098).
Genetic and Nongenetic Risk Factors for Neurosyphilis Higher serum RPR titers increase neurosyphilis risk,2Y4 as we saw in our study participants (Table 1). Infecting strain type6 also can increase the risk of neurosyphilis. We thus sought to include these factors in our analysis. Although serum RPR titer was available for 453 patients, we were only able to determine infecting strain type for 83 (18.2%) patients. We have previously shown that circulating strain types in Seattle underwent a significant change over time, particularly when comparing before and after 2005.6 We thus used year of sample collection (1997 through 2004 vs. 2005Y2013) as a surrogate for strain type in our analyses. We also included age, syphilis stage (early [primary, secondary or early latent stages] vs. late [late latent stage or unknown duration]), treatment of uncomplicated syphilis before study enrollment, and any previous episode of uncomplicated syphilis in our analysis of risk for neurosyphilis (Table 1). Table 4 shows the results of this analysis for the deficient TLR1 and TLR6 SNPs and laboratorydefined neurosyphilis and the deficient TLR6 SNP for clinicallydefined neurosyphilis; there were too few outcomes to allow for multivariate analysis of the TLR2 SNP and neurosyphilis phenotypes. In univariate analysis, the odds of laboratory-defined neurosyphilis were significantly higher in patients with higher serum RPR titers, those infected before 2005, and those who were not treated for uncomplicated syphilis before study enrollment (Table 4). In addition, older age and late-stage syphilis
TABLE 4.
Genetic and Nongenetic Risk Factors for Neurosyphilis in HIV-Infected Individuals The TLR6 deficient SNP was more common in HIVinfected compared with HIV-uninfected individuals (214 [61.0%] of 351 vs. 52 [50.0%] of 104, P = 0.046), but the other 2 deficient TLR SNPs were equally common in HIV-infected and HIVuninfected individuals. In addition to serum RPR titer and infecting strain type, lower peripheral blood CD4 and nonuse of antiretroviral medications may increase neurosyphilis risk in HIV-infected individuals.2,3 We repeated the above analysis among HIV-infected participants, including HIV-related factors that influence neurosyphilis risk (Table 5). Although the number of outcomes was too small to perform multivariate analyses for the TLR2 polymorphism, the associations between laboratoryand clinically-defined neurosyphilis and the deficient genotype that were seen in the group as a whole persisted in the HIVinfected subset (for laboratory-defined neurosyphilis: OR, 7.62 [1.44Y40.20, P = 0.017]; for clinically-defined neurosyphilis: OR, 8.91 [1.89Y42.02, P = 0.006]).
Associations Between Neurosyphilis and TLR1 and TLR6 SNPs and Clinical Variables
Univariate Analysis OR (95% CI) Laboratory-defined neurosyphilis TLR1 (GG vs. TT/TG) RPR titer (Q1:32 vs. G1:32) Year (G2005 vs. Q2005) Stage (late vs. early) Age (per 10-y increase) Treatment of uncomplicated syphilis before enrollment Previous syphilis TLR6 (CT/TT vs. CC) RPR titer (Q1:32 vs. G1:32) Year (G2005 vs. Q2005) Stage (late vs. early) Age (per 10-y increase) Treatment of uncomplicated syphilis before enrollment Previous syphilis Clinically-defined neurosyphilis TLR6 (CT/TT vs. CC) RPR titer (Q1:32 vs. G1:32) Year (G2005 vs. Q2005) Stage (late vs. early) Age (per 10-y increase) Treatment of uncomplicated syphilis before enrollment Previous syphilis
Multivariate Analysis P
1.79 14.27 1.78 1.60 1.24 0.50 0.77 1.68 14.27 1.78 1.60 1.24 0.50 0.77
(1.11Y2.90) (5.08Y40.08) (1.03Y3.08) (0.94Y2.72) (0.99Y1.55) (0.30Y0.82) (0.42Y1.40) (1.03Y2.75) (5.08Y40.08) (1.03Y3.08) (0.94Y2.72) (0.99Y1.55) (0.30Y0.82) (0.42Y1.40)
0.018 G0.001 0.038 0.082 0.060 0.006 0.388 0.040 G0.001 0.038 0.082 0.060 0.006 0.388
2.40 2.82 1.34 0.96 1.18 0.65 0.87
(1.55Y3.71) (1.73Y4.59) (0.82Y2.20) (0.59Y1.58) (0.98Y1.42) (0.43Y0.98) (0.53Y1.43)
G0.001 G0.001 0.238 0.885 0.081 0.038 0.585
OR (95% CI) 2.10 22.69 2.00 2.87 1.27 0.51 2.57 27.09 2.10 3.17 1.36 0.52
(1.22Y3.62) (7.48Y68.83) (1.06Y3.77) (1.50Y5.49) (0.98Y1.65) (0.29Y0.89) V (1.45Y4.55) (8.74Y83.95) (1.11Y3.96) (1.64Y6.13) (1.04Y1.76) (0.30Y0.92) V
2.71 (1.71Y4.30) 3.51 (2.09Y5.89) V V 1.22 (1.01Y1.49) 0.71 (0.46Y1.09) V
P 0.008 G0.001 0.031 0.001 0.072 0.018 V 0.001 G0.001 0.023 0.001 0.022 0.024 V G0.001 G0.001 V V 0.043 0.119 V
Controls had no vision or hearing loss and CSF white blood cells e5/KL and a nonreactive CSF-VDRL; patients with laboratory-defined neurosyphilis had a reactive CSF-VDRL, and patients with clinically-defined neurosyphilis had new vision or hearing loss. Logistic regression was used for univariate and multivariate analysis with results reported as ORs with 95% CIs; variables with P values greater than 0.20 in univariate analysis were not included in multivariate analysis.
Sexually Transmitted Diseases
&
Volume 41, Number 7, July 2014
443
Copyright © 2014 by the American Sexually Transmitted Diseases Association. Unauthorized reproduction of this article is prohibited.
Marra et al.
TABLE 5.
Associations Between Neurosyphilis and TLR1 and TLR6 SNPs and Clinical Variables in HIV-Infected Patients
Univariate Analysis
Laboratory-defined neurosyphilis TLR1 (GG vs. TT/TG) RPR titer (Q1:32 vs. G1:32) Year (G2005 vs. Q2005) ARVs (current use vs. none) CD4 (e350 vs. 9350 cells/KL) Stage (late vs. early) Age (per 10-y increase) Treatment of uncomplicated syphilis before enrollment Previous syphilis TLR6 (CT/TT vs. CC) RPR titer (Q1:32 vs. G1:32) Year (G2005 vs. Q2005) ARVs (current use vs. none) CD4 (e350 vs. 9350 cells/KL) Stage (late vs. early) Age (per 10-y increase) Treatment of uncomplicated syphilis before enrollment Previous syphilis Clinically-defined neurosyphilis TLR6 (CT/TT vs. CC) RPR titer (Q1:32 vs. G1:32) Year (G2005 vs. Q2005) ARVs (current use vs. none) CD4 (e350 vs. 9350 cells/KL) Stage (late vs. early) Age (per 10-y increase) Treatment of uncomplicated syphilis before enrollment Previous syphilis
Multivariate Analysis
OR (95% CI)
P
OR (95% CI)
P
1.43 (0.83Y2.47) 25.13 (5.99Y105.41) 1.71 (0.91Y3.19) 0.21 (0.12Y0.40) 1.45 (0.79Y2.64) 1.49 (0.78Y2.83) 1.15 (0.88Y1.50) 0.47 (0.26Y0.85) 0.81 (0.42Y1.59) 1.57 (0.89Y2.76) 25.13 (5.99Y105.41) 1.71 (0.91Y3.19) 0.21 (0.12Y0.40) 1.45 (0.79Y2.64) 1.49 (0.78Y2.83) 1.15 (0.88Y1.50) 0.47 (0.26Y0.85) 0.81 (0.42Y1.59)
0.197 G0.001 0.095 G0.001 0.232 0.227 0.303 0.012 0.547 0.120 G0.001 0.095 G0.001 0.232 0.227 0.303 0.012 0.547
1.80 (0.90Y3.59) 23.36 (5.28Y103.40) 2.20 (0.76Y6.39) 0.24 (0.12Y0.48) V V V 0.55 (0.27Y1.12) V 2.40 (1.18Y4.89) 23.44 (5.35Y102.70) 2.18 (0.73Y6.51) 0.25 (0.12Y0.50) V V V 0.57 (0.28Y1.17) V
0.097 G0.001 0.146 G0.001 V V V 0.099 V 0.016 G0.001 0.163 G0.001 V V V 0.126 V
2.09 (1.26Y3.44) 4.00 (2.22Y7.21) 1.37 (0.78Y2.40) 0.36 (0.21Y0.61) 1.10 (0.64Y1.90) 1.07 (0.60Y1.94) 1.12 (0.90Y1.40) 0.69 (0.43Y1.11) 0.90 (0.51Y1.57)
0.004 G0.001 0.270 G0.001 0.719 0.811 0.325 0.122 0.705
2.79 (1.54Y5.05) 6.12 (2.95Y12.69) V 0.36 (0.20Y0.64) V V V 0.90 (0.51Y1.59) V
0.001 G0.001 V 0.001 V V V 0.706 V
Controls had no vision or hearing loss and CSF white blood cells e5/KL and a nonreactive CSF-VDRL; patients with laboratory-defined neurosyphilis had a reactive CSF-VDRL, and patients with clinically-defined neurosyphilis had new vision or hearing loss. Comparison of proportions was performed using W2 or Fisher exact tests. Logistic regression was used for univariate and multivariate analysis with results reported as ORs with 95% CIs; variables with P values greater than 0.20 in univariate analysis were not included in multivariate analysis. ARVs indicate antiretrovirals or antiretroviral drugs.
In univariate analysis, the odds of laboratory-defined neurosyphilis were significantly higher in those with higher serum RPR titers, in those not taking antiretroviral agents, and in those who were not treated for uncomplicated syphilis before study enrollment (Table 5). Although the TLR1- and TLR6 deficient SNPs were not significantly associated with laboratory-defined neurosyphilis in HIV-infected patients in univariate analysis, the odds of laboratory-defined neurosyphilis were significantly higher in those with the deficient TLR6 SNP in multivariate analysis, taking into account the above factors. Similarly, in univariate analysis, the odds of clinically-defined neurosyphilis were significantly higher in those with higher serum RPR titers and in those not taking antiretroviral agents; treatment of uncomplicated syphilis before study entry was weakly associated with clinically-defined neurosyphilis (Table 5). The odds of clinically-defined neurosyphilis were significantly higher in those with the deficient TLR6 SNP in univariate analysis and remained so in multivariate analysis after taking into account the above factors.
DISCUSSION We found that laboratory-defined neurosyphilis was more common in patients who harbored the TLR1_1805GG, TLR2_2258GA, and TLR6_745CT/TT genotypes than those
444
who did not, and clinically-defined neurosyphilis was more common in patients who harbored the TLR2_2258GA and TLR6_745CT/TT genotypes than in those who did not. In addition, in the subset of HIV-infected patients, the higher odds of laboratory-defined neurosyphilis in those with the TLR1_1805GG genotype was less evident, perhaps because of smaller numbers or because the heightened risk of neurosyphilis due to HIV-induced immunodeficiency outweighs the modest risk conferred by the TLR1 SNP. This hypothesis is supported by the strongly protective effect of antiretroviral use that we saw in our analyses, with a 60% to 88% reduction in the odds of laboratory-defined disease in HIV-infected patients on antiretroviral therapy. The first step in neurosyphilis pathogenesis is invasion of the CSF by T. pallidum via the bloodstream, presumably via the blood-CSF barrier. Cerebrospinal fluid inflammation may ensue, but the patient remains asymptomatic. This stage is termed asymptomatic neurosyphilis. Asymptomatic neurosyphilis can progress to subsequent tissue injury and symptomatic disease. A study using a human brain microvascular endothelial cell line and rat primary brain endothelial cells showed that cerebral microvascular endothelium expresses TLR2 and TLR6, but not TLR1,30 and that ligation of these receptors increased vascular permeability in vitro. To the extent that in vitro studies of brain microvascular endothelium reflect the blood-CSF barrier, these results may offer insight into why we did not see an association between the TLR1 SNP and clinically-defined neurosyphilis:
Sexually Transmitted Diseases
&
Volume 41, Number 7, July 2014
Copyright © 2014 by the American Sexually Transmitted Diseases Association. Unauthorized reproduction of this article is prohibited.
TLR Polymorphisms Increase Neurosyphilis Risk
TLR1 signaling may not be operative at the blood-CSF barrier and thus may not influence T. pallidum neuroinvasion at the local site. However, TLR1 signaling could still affect neuroinvasion if it is involved in the initial immune response to peripheral T. pallidum infection. For example, defective signaling in mononuclear cells or macrophages could result in poorer control of peripheral infection, resulting in greater levels of bacteremia and greater likelihood of CSF invasion on the basis of a higher organism burden. Limitations of this work should be considered in interpreting our data. Our sample size, although large for a syphilis study, is modest for a candidate gene study. Nonetheless, our sample was sufficient to allow us to identify statistical significance. We tested 3 SNPs whose choice was based on biological plausibility, limiting false-positive associations. Most of our patients were infected with HIV, consistent with the demography of syphilis in our region. It is possible that there is an interaction between TLR SNPs and HIV-associated immunodeficiency. In fact, the TLR6 deficient SNP was more common in our HIV-infected participants. However, the associations that we identified between the TLR2 and TLR6 SNPs and laboratory or clinically defined neurosyphilis persisted when HIV was taken into account. Our laboratory definition of neurosyphilis, reactive CSF-VDRL, is quite stringent. Although we excluded from analysis patients who had preexisting neurological abnormalities, it is possible that the clinical abnormalities that we recorded were not due to neurosyphilis and that our clinical definition of neurosyphilis was thus less stringent than our laboratory definition. However, the associations that we identified with both neurosyphilis diagnoses were concordant for the TLR2 and TLR6 SNPS, adding to the credibility of our findings. Moreover, if misclassification of clinical neurosyphilis occurred, there is no reason to expect that it would have been more likely in those with the SNPs of interest. Rather, it would have weakened, not strengthened, the associations that we observed. Similarly, although all our patients self-identified as ‘‘Caucasian,’’ genetic heterogeneity cannot be excluded. However, this would also have been more likely to weaken, rather than strengthen the associations that we found. We were not able to validate our findings in an independent cohort of patients, and we hope that this work will stimulate others to replicate it. It should be noted that our patients were referred because of a concern regarding possible neurosyphilis. This bias could result in our ‘‘controls’’ being less representative of patients at low risk for neurosyphilis than the general population of patients with syphilis. As such, the ORs that we provide may be underestimates of the true effect of the SNPs that we studied. Our data support the contention made in the preantibiotic era that host factors impact the natural course of syphilis. Future work will be required to replicate these results as well as to determine the underlying mechanisms.
REFERENCES 1. Chesney AM, Kemp JE. Incidence of Spirochaeta pallida in cerebrospinal fluid during early stage of syphilis. JAMA 1924; 83: 1725Y1728. 2. 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; 189:369Y376.
Sexually Transmitted Diseases
&
3. Ghanem KG, Moore RD, Rompalo AM, et al. Neurosyphilis in a clinical cohort of HIV-1Yinfected patients. AIDS 2008; 22: 1145Y1151. 4. Libois A, De Wit S, Poll B, et al. HIV and syphilis: When to perform a lumbar puncture. Sex Transm Dis 2007; 34:141Y144. 5. Taylor MM, Aynalem G, Olea LM, et al. A consequence of the syphilis epidemic among men who have sex with men (MSM): Neurosyphilis in Los Angeles, 2001Y2004. Sex Transm Dis 2008; 35:430Y434. 6. Marra CM, Sahi SK, Tantalo LC, et al. Enhanced molecular typing of Treponema pallidum: Geographical distribution of strain types and association with neurosyphilis. J Infect Dis 2010; 202: 1380Y1388. 7. Stokes JH, Beerman H, Ingraham NR. Modern Clinical Syphilology. Diagnosis, Treatment, Case Study. 3rd ed. Philadelphia: WB Saunders, 1944. 8. Strandberg Pedersen N, Axelsen NH, Svejgaard A. General paralysis of the insane associated with HLA-Aw32. Acta Pathol Microbiol Scand C Immunol 1981; 89:181Y184. 9. Whitsett C, Turner WJ, Lee TD, et al. HLA antigen frequencies in paralytic dementia. J Neurol Sci 1976; 30:417Y420. 10. Jiang HW, Tian HQ, Liu H, et al. Association of the HLA-DRB1 locus with syphilis in a Chinese population. Int J Infect Dis 2011; 15:e342Y5. 11. Zhuang YL, Ren GJ, Tian KL, et al. Human leukocyte antigen-C and killer cell immunoglobulin-like receptor gene polymorphisms among patients with syphilis in a Chinese Han population. Acta Pathol Microbiol Immunol Scand 2012; 120:828Y835. 12. Zhuang YL, Song Y, Zhu C, et al. Association of KIR genotypes and haplotypes with syphilis in a Chinese Han population. Scand J Immunol 2012; 75:361Y367. 13. Zhuang YL, Zhu CF, Zhang Y, et al. Association of KIR2DS4 and its variant KIR1D with syphilis in a Chinese Han population. Int J Immunogenet 2012; 39:114Y118. 14. Palm NW, Medzhitov R. Pattern recognition receptors and control of adaptive immunity. Immunol Rev 2009; 227:221Y233. 15. McGill MA, Edmondson DG, Carroll JA, et al. Characterization and serologic analysis of the Treponema pallidum proteome. Infect Immun 2010; 78:2631Y2643. 16. Lien E, Sellati TJ, Yoshimura A, et al. Toll-like receptor 2 functions as a pattern recognition receptor for diverse bacterial products. J Biol Chem 1999; 274:33419Y33425. 17. Takeuchi O, Akira S. Pattern recognition receptors and inflammation. Cell 2010; 140:805Y820. 18. Cruz AR, Ramirez LG, Zuluaga AV, et al. Immune evasion and recognition of the syphilis spirochete in blood and skin of secondary syphilis patients: Two immunologically distinct compartments. PLoS Negl Trop Dis 2012; 6:e1717. 19. Lorenz E, Mira JP, Cornish KL, et al. A novel polymorphism in the Toll-like receptor 2 gene and its potential association with staphylococcal infection. Infect Immun 2000; 68:6398Y6401. 20. Schroder NW, Hermann C, Hamann L, et al. High frequency of polymorphism Arg753Gln of the Toll-like receptor-2 gene detected by a novel allele-specific PCR. J Mol Med (Berl) 2003; 81:368Y372. 21. Johnson CM, Lyle EA, Omueti KO, et al. Cutting edge: A common polymorphism impairs cell surface trafficking and functional responses of TLR1 but protects against leprosy. J Immunol 2007; 178:7520Y7524. 22. Oosting M, Ter Hofstede H, Sturm P, et al. TLR1/TLR2 heterodimers play an important role in the recognition of Borrelia spirochetes. PloS one 2011; 6:e25998. 23. Tantisira K, Klimecki WT, Lazarus R, et al. Toll-like receptor 6 gene (TLR6): Single-nucleotide polymorphism frequencies and preliminary association with the diagnosis of asthma. Genes Immun 2004; 5:343Y346. 24. Schroder NW, Diterich I, Zinke A, et al. Heterozygous Arg753Gln polymorphism of human TLR-2 impairs immune activation by Borrelia burgdorferi and protects from late stage Lyme disease. J Immunol 2005; 175:2534Y2540.
Volume 41, Number 7, July 2014
445
Copyright © 2014 by the American Sexually Transmitted Diseases Association. Unauthorized reproduction of this article is prohibited.
Marra et al.
25. Strle K, Shin JJ, Glickstein LJ, et al. Association of a toll-like receptor 1 polymorphism with heightened Th1 inflammatory responses and antibiotic-refractory Lyme arthritis. Arthritis Rheum 2012; 64:1497Y1507. 26. Larsen SA, Pope V, Johnson RE, et al. A Manual of Tests for Syphilis. 9th ed. Washington, DC: American Public Health Association; 1998:361. 27. Sexually transmitted diseases treatment guidelines. MMWR Recomm Rep 2010; 59:1Y110.
446
28. Hawn TR, Misch EA, Dunstan SJ, et al. A common human TLR1 polymorphism regulates the innate immune response to lipopeptides. Eur J Immunol 2007; 37:2280Y2289. 29. Shey MS, Randhawa AK, Bowmaker M, et al. Single nucleotide polymorphisms in toll-like receptor 6 are associated with altered lipopeptide- and mycobacteria-induced interleukin-6 secretion. Genes Immun2010; 11:561Y572. 30. Nagyoszi P, Wilhelm I, Farkas AE, et al. Expression and regulation of toll-like receptors in cerebral endothelial cells. Neurochem Int 2010; 57:556Y564.
Sexually Transmitted Diseases
&
Volume 41, Number 7, July 2014
Copyright © 2014 by the American Sexually Transmitted Diseases Association. Unauthorized reproduction of this article is prohibited.
Toll-Like Receptor Polymorphisms Are Associated With Increased Neurosyphilis Risk Christina M. Marra, MD,* Sharon K. Sahi, BS,* Lauren C. Tantalo, BS,* Emily L. Ho, MD, PhD,* Shelia B. Dunaway, MD,Þ Trudy Jones, MN, ARNP,* and Thomas R. Hawn, MD, PhDÞ Background: Single-nucleotide polymorphisms (SNPs) in toll-like receptors (TLR) 1, 2, and 6 impair cell signaling in response to spirochetal lipoproteins. We investigated whether common SNPs in TLR1, TLR2, or TLR6 were associated with laboratory- or clinically-defined neurosyphilis. Methods: Polymorphisms in the genes for TLR1 (a TYG mutation at position 1805), TLR2 (a GYA mutation at position 2258), and TLR6 (a CYT mutation at position 745) were sought in 456 white patients with syphilis. Laboratory-defined neurosyphilis included a reactive cerebrospinal fluid (CSF)YVenereal Disease Research Laboratory (VDRL) test. Clinically-defined neurosyphilis included new vision or hearing loss. Controls had CSF white blood cells of 5/KL or less, nonreactive CSFYVenereal Disease Research Laboratory, and no vision or hearing loss. Results: Overall, 26.2% of patients had laboratory-defined and 36.2% had clinically-defined neurosyphilis. Compared with controls, patients with any of the 3 SNPs were more likely to have laboratory-defined neurosyphilis. Those with TLR2 or TLR6 SNPs were more likely to have clinically-defined neurosyphilis. These associations were independent of serum rapid plasma reagin titer. Conclusions: A common TLR1 polymorphism is associated with an increased risk of laboratory-defined neurosyphilis, and common TLR2 and TLR6 polymorphisms are associated with an increased risk of both laboratory- and clinically-defined neurosyphilis. These data suggest that host factors impact the natural history of syphilis.
T
reponema pallidum spp. pallidum (hereafter termed T. pallidum), the bacterium that causes syphilis, is highly neuroinvasive. Studies from the early 20th century demonstrated that isolation of the organism from cerebrospinal fluid (CSF) in early syphilis was common.1 However, even before the advent of antibiotics, not every patient with syphilis developed neurosyphilis. Host and pathogen-related factors may influence susceptibility. For example, patients with high serum rapid plasma reagin (RPR) titers2Y4 and those infected with HIV, particularly if they have low peripheral blood CD4+ T cells or are not taking antiretroviral agents, may be at higher risk for asymptomatic and symptomatic neurosyphilis.2,3,5 In addition,
From the *Departments of Neurology and †Medicine, University of Washington, Seattle, WA
Portions of this work were presented at the 20th Conference on Retroviruses and Opportunistic Infections; Atlanta, GA; March 3Y6, 2013. This work was supported by the National Institute of Neurological Disorders and Stroke of the National Institutes of Health (Grant No. NS34235). The authors have no conflicts of interest relevant to this work. Correspondence: Christina M. Marra, MD, Harborview Medical Center, Box 359775, 325 9th Ave, Seattle, WA 98104. E-mail: [email protected]. Received for publication February 5, 2014, and accepted May 5, 2014. DOI: 10.1097/OLQ.0000000000000149 Copyright * 2014 American Sexually Transmitted Diseases Association All rights reserved.
440
particular T. pallidum strain types may be more likely to cause neurosyphilis.6 Clinicians in the early 20th century posited that race influenced susceptibility to neurosyphilis, citing a decreased risk in African Americans compared with whites.7 Subsequent work suggested a genetic basis for such differences, with an increased risk of syphilitic dementia, but not other forms of neurosyphilis, in patients with certain HLA types8 that differed in African Americans compared with whites.9 Although more recent reports suggest that there may be genetic contributions to syphilis susceptibility,10Y13 to the best of our knowledge, there have been no recent investigations of genetic susceptibility to neurosyphilis. Human toll-like receptors (TLRs) are a family of 10 proteins that differentially recognize pathogen-associated molecular patterns. Ligation of cellular TLRs activate signaling cascades that lead to initiation of the innate immune response and cytokine production, ultimately culminating in antimicrobial host defenses.14 T. pallidum expresses many lipoproteins15 that stimulate innate immune cells via TLR2 as a heterodimer with TLR1 or TLR6.16,17 In a microarray analysis of skin from patients with secondary syphilis, transcripts for both TLR1 and TLR2 were up-regulated, whereas TLR6 transcripts were not.18 Single-nucleotide polymorphisms (SNPs) in TLR1, TLR2, and TLR6 impair the innate immune response to spirochetal lipopeptides and lipoproteins. A GYA mutation in the TLR2 gene at position 2258 (TLR2_G2258A) occurs in 3% to 9% of whites and is associated with decreased cell signaling in response to stimulation with the T. pallidum lipoprotein T47L.19,20 Similarly, a TYG mutation in the TLR1 gene at position 1805 (TLR1_T1805G) is common, with 52% of Seattle European whites being GG, and is associated with decreased TLR1 surface expression21 and decreased cell signaling in response to Borrelia burgdorferi, a spirochetal organism closely related to T. pallidum.22 Finally, a CYT mutation in the TLR6 gene at position 745 (TLR6_C745T) is also common (seen in 46% of European Americans23) and is associated with decreased cell signaling in response to B. burgdorferi.22 Although the TLR1 and TLR2 SNPs have been associated with altered susceptibility and course of Borrelia infections,24,25 it is not known if SNPs in TLR1, TLR2, or TLR6 are associated with the clinical course of syphilis. In this study, we investigated whether common and functionally defined TLR1/2/6 SNPs were associated with neurosyphilis in HIVinfected and HIV-uninfected patients with syphilis.
METHODS Study Participants Participants were enrolled in a study of CSF abnormalities in syphilis conducted in Seattle, WA, from March 1997 through August 2013.2 Eligibility criteria included clinical or serological evidence of syphilis and assessment by the referring provider that the patient was at risk for neurosyphilis. Reasons
Sexually Transmitted Diseases
&
Volume 41, Number 7, July 2014
Copyright © 2014 by the American Sexually Transmitted Diseases Association. Unauthorized reproduction of this article is prohibited.
TLR Polymorphisms Increase Neurosyphilis Risk
for referral to the study included the following: (1) neurological findings, particularly vision or hearing loss; (2) serum RPR titer Q1:32; and (3) in HIV-infected individuals, peripheral blood CD4+ T cell count of 350/KL or less. The latter criteria are based on published data.2Y4 Participants underwent a structured history and neurological examination that included assessment of vision and hearing, lumbar puncture, and venipuncture. The study protocol was reviewed and approved by the University of Washington Institutional Review Board, and human experimentation guidelines were followed in the conduct of this research. Written informed consent was obtained from all participants.
to calculate R2 and D¶ as measurements of linkage disequilibrium (LD) between the TLR1 and TLR6 polymorphisms. Comparison of median values between groups was performed using the Mann-Whitney U test, and comparison of proportions was performed using W2 or Fisher exact tests. Logistic regression was used for multivariate analysis, with results reported as odds ratios (ORs) with 95% confidence intervals (95% CI); variables with P values greater than 0.20 in univariate analysis were not included in multivariate analysis. P values less than 0.05 were considered statistically significant. Adjustments were not routinely made for multiple comparisons.
RESULTS
Laboratory Methods DNA was extracted from blood, or in 3 instances from serum, using the QIAamp DNA Blood Midi Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. The TLR2_G2258A SNP (reference SNP identification number [rs] 5743708 in the Single Nucleotide Polymorphism Database) was detected by restriction fragment length polymorphism analysis.20 The TLR1_T1805G (rs5743618) and TLR6_C745T (rs5743810) SNPs were detected using TaqMan SNP genotyping assays (Applied Biosystems, Carlsbad, CA) with iTaq Supermix with ROX (Bio-Rad, Hercules, CA) on an ABI 7900HT or Viia7 instrument, according to the manufacturer’s instructions. Genotyping failed for the TLR2 SNP in 2 patients and for the TLR6 SNP in 1 patient. Serum RPR and CSFY Venereal Disease Research Laboratory (VDRL) were performed using standard methods.26
Statistical Methods All patients in this study had syphilis based on clinical and serological criteria.27 Patients with laboratory-defined neurosyphilis had a reactive CSF-VDRL test. Patients with clinically-defined neurosyphilis had new vision or hearing loss as assessed by history and physical examination; patients with previously documented vision or hearing abnormalities were excluded from analysis. Controls were defined as patients with normal CSF white blood cell counts (e5 cells/KL), nonreactive CSF-VDRL, and no vision or hearing loss. We restricted the analysis to self-reported whites. Stata program PWLD was used TABLE 1.
Participant Characteristics To examine the association of TLR polymorphisms with susceptibility to neurosyphilis, we used a case-control study design in 456 syphilis patients who underwent lumbar puncture for evaluation of neurosyphilis and were categorized as laboratory- or clinically-defined neurosyphilis or as controls. Overall, most study participants were men and 77.2% were HIV infected, consistent with the demographics of syphilis in Seattle. The proportion of patients who were treated for uncomplicated (nonneurological) syphilis before study entry and serum RPR titers differed significantly in the controls compared with the neurosyphilis cases (Table 1). Cerebrospinal fluidYVDRL was available for 453 patients and was reactive in 91 (20.1%). One hundred forty-five (32.2%) of 450 assessable patients had symptoms or signs of vision or hearing loss. Of 447 patients with both assessments, 49 (11.0%) only had a reactive CSFVDRL, 106 (23.7%) only had vision or hearing loss, and 36 (8.1%) had both.
Frequencies of TLR1, TLR2, and TLR6 Polymorphism Genotypes in Patients With Laboratory- and Clinically-Defined Neurosyphilis and Controls We compared genotype frequencies of the 3 SNPs between those with the 2 neurosyphilis phenotypes and controls. None of the controls violated Hardy-Weinberg equilibrium (Table 2). There was a significant association between the
Patient Characteristics
Characteristic
P
Clinical Neurosyphilis (n = 145)
P
251 (98.0%) 38 (31Y44) 200 (78.1%)
90 (98.9%) 41 (33Y47) 70 (76.9%)
1.000 0.063 0.813
141 (97.2%) 40 (32Y46) 111 (76.6%)
0.728 0.142 0.717
195 (77.4%) 57 (22.6%) 32 (8Y128)
62 (68.1%) 29 (31.9%) 256 (128Y512)
0.081
0.885
G0.001
110 (78.0%) 31 (22.0%) 128 (64Y256)
G0.001
130 (50.8%)
31 (34.1%)
0.006
58 (40.0%)
0.038
59 (23.0%)
17 (18.7%)
0.387
30 (20.7%)
0.585
Controls (n = 256) Laboratory Neurosyphilis (n = 91)
Sex (men) Age, y HIV-infected Syphilis stage* Early† Late‡ Reciprocal serum RPR titer§ Treated for uncomplicated syphilis before entry Previous syphilis
Controls had no vision or hearing loss and CSF white blood cells e5/KL and a nonreactive CSF-VDRL. Patients with laboratory-defined neurosyphilis had a reactive CSF-VDRL. Patients with clinically-defined neurosyphilis had new vision or hearing loss. Values are n (%) or median (interquartile range). Comparison of median values between neurosyphilis groups and controls was performed using the Mann-Whitney U test, and comparison of proportions between neurosyphilis groups and controls was performed using W2 or Fisher exact tests. *Stage was available for 252 controls and 141 patients with clinically-defined neurosyphilis. †Early syphilis includes primary, secondary, and early latent stages. ‡Late syphilis includes late latent syphilis and syphilis of unknown duration. § RPR titer was available for 255 controls and 143 patients with clinically-defined neurosyphilis.
Sexually Transmitted Diseases
&
Volume 41, Number 7, July 2014
441
Copyright © 2014 by the American Sexually Transmitted Diseases Association. Unauthorized reproduction of this article is prohibited.
Marra et al.
TABLE 2.
TLR1, TLR2, and TLR6 Polymorphism Gene Frequencies in Controls and in Patients With Neurosyphilis
Genotype Frequencies*
TLR1 Control Laboratory-defined neurosyphilis Clinically-defined neurosyphilis TLR2 Control Laboratory-defined neurosyphilis Clinically-defined neurosyphilis TLR6 Control Laboratory-defined neurosyphilis Clinically-defined neurosyphilis
n (%)
n (%)
n (%)
TT 45 (17.6) 10 (11.0) 17 (11.7) GG 254 (99.2) 86 (94.5) 133 (93.0) CC 125 (48.8) 33 (36.3) 41 (28.5)
TG 110 (43.0) 32 (35.2) 67 (46.2) GA 2 (0.8) 5 (5.5) 10 (7.0) CT 104 (40.6) 42 (46.2) 82 (56.9)
GG 101 (39.5) 49 (53.8) 61 (42.1) AA 0 (0) 0 (0) 0 (0) TT 27 (10.5) 16 (17.6) 21 (14.6)
P
HWE P value 0.119
0.048 0.297 0.950 0.015 0.001 0.443 0.064 G0.001
Controls had no vision or hearing loss and CSF white blood cells e5/KL and a nonreactive CSF-VDRL; patients with laboratory-defined neurosyphilis had a reactive CSF-VDRL, and patients with clinically-defined neurosyphilis had new vision or hearing loss. P value calculated from a genotypic model with a 2 3 W2 or Fisher exact test. *Genotypes designate common homozygote (AA), heterozygote (Aa), and uncommon homozygote (aa). HWE indicates Hardy-Weinberg equilibrium.
TLR1 SNP and laboratory-defined neurosyphilis, but no significant association with clinically-defined neurosyphilis (Table 2). The TLR2 SNP was significantly associated with both laboratory- and clinically-defined neurosyphilis, and the TLR6 SNP was marginally associated with laboratory-defined and significantly associated with clinically-defined neurosyphilis (Table 2). Based on the experimentally determined genetic models of the 3 well-characterized SNPs, we then examined the association of the TLR1 SNP using a recessive model, and the TLR2 and TLR6 SNPs using dominant models21,24,28,29 (Table 3). The associations between the 3 SNPs and laboratorydefined neurosyphilis were significant for all 3 SNPs, with ORs ranging from 1.68 to 7.38 for the ‘‘deficient’’ genotype (TLR1_1805GG, TLR2_2258GA, and TLR6_745CT/TT) (Table 3). The associations between the TLR2 and TLR6 deficient genotypes and clinically-defined neurosyphilis had a higher magnitude (OR, 9.55 and 2.40) and remained
TABLE 3.
significant even after taking into account multiple comparisons (Table 3).
Haplotype and Diplotype Analysis of the TLR1 and TLR6 Polymorphisms Because of close genomic proximity of TLR1 and TLR6 on chromosome 4, we next ascertained whether these associations were independent by measuring LD among the loci using pairwise LD analysis. R2 was 0.31, suggesting a low to moderate level of LD between these polymorphisms. To determine whether there were additive effects from deficiency in both TLR1 and TLR6, we examined diplotypes that were double deficient (TLR1_1805GG and TLR6_745CT/TT, n = 154) versus those with at least 1 sufficient variant (TLR1_1805GT/ TT and/or TLR6_745CC, n = 301). Results were consistent with the findings in patients who were TLR1 deficient. Specifically, the odds of laboratory-defined neurosyphilis were significantly higher in double-deficient individuals compared with those with
TLR1, TLR2, and TLR6 Polymorphisms in Controls and Patients With Neurosyphilis
TLR1 Control Laboratory-defined neurosyphilis
TT/TG 155 42
TLR2 Control Laboratory-defined neurosyphilis Clinically-defined neurosyphilis
GG 254 86 133
TLR6 Control Laboratory-defined neurosyphilis Clinically-defined neurosyphilis
CC 125 33 41
Recessive model GG 101 49 Dominant model GA/AA 2 5 10 Dominant model CT/TT 131 58 103
OR (95% CI)
P
1.79 (1.11Y2.90)
0.018
7.38 (1.41Y38.75) 9.55 (2.06Y44.21)
0.018 0.004
1.68 (1.03Y2.75) 2.40 (1.55Y3.71)
0.040 G0.001
Controls had no vision or hearing loss and CSF white blood cells e5/KL and a nonreactive CSF-VDRL; patients with laboratory-defined neurosyphilis had a reactive CSF-VDRL, and patients with clinically-defined neurosyphilis had new vision or hearing loss. Comparison of proportions between the neurosyphilis groups and controls was performed using W2 or Fisher exact tests. Univariate logistic regression was used to derive ORs with 95% CIs.
442
Sexually Transmitted Diseases
&
Volume 41, Number 7, July 2014
Copyright © 2014 by the American Sexually Transmitted Diseases Association. Unauthorized reproduction of this article is prohibited.
TLR Polymorphisms Increase Neurosyphilis Risk
marginally increased the odds of laboratory-defined neurosyphilis (Table 4). In multivariate analysis, the odds of laboratory-defined neurosyphilis remained significantly higher in those with TLR1 and TLR6 deficiency, even after taking the above measures into account. Similarly, in univariate analysis, the odds of clinicallydefined neurosyphilis were significantly higher in patients with higher serum RPR titers, and those treated for uncomplicated syphilis before study enrollment, and older age marginally increased the odds of clinically-defined neurosyphilis (Table 4). In multivariate analysis, the odds of clinically-defined neurosyphilis remained significantly higher in those with TLR6 deficiency, even after taking the above measures into account (Table 4).
at least 1 sufficient variant (OR, 1.82 [1.11Y2.98], P = 0.017), but the odds of clinically-defined neurosyphilis were not (OR, 1.44 [0.94Y2.21], P = 0.098).
Genetic and Nongenetic Risk Factors for Neurosyphilis Higher serum RPR titers increase neurosyphilis risk,2Y4 as we saw in our study participants (Table 1). Infecting strain type6 also can increase the risk of neurosyphilis. We thus sought to include these factors in our analysis. Although serum RPR titer was available for 453 patients, we were only able to determine infecting strain type for 83 (18.2%) patients. We have previously shown that circulating strain types in Seattle underwent a significant change over time, particularly when comparing before and after 2005.6 We thus used year of sample collection (1997 through 2004 vs. 2005Y2013) as a surrogate for strain type in our analyses. We also included age, syphilis stage (early [primary, secondary or early latent stages] vs. late [late latent stage or unknown duration]), treatment of uncomplicated syphilis before study enrollment, and any previous episode of uncomplicated syphilis in our analysis of risk for neurosyphilis (Table 1). Table 4 shows the results of this analysis for the deficient TLR1 and TLR6 SNPs and laboratorydefined neurosyphilis and the deficient TLR6 SNP for clinicallydefined neurosyphilis; there were too few outcomes to allow for multivariate analysis of the TLR2 SNP and neurosyphilis phenotypes. In univariate analysis, the odds of laboratory-defined neurosyphilis were significantly higher in patients with higher serum RPR titers, those infected before 2005, and those who were not treated for uncomplicated syphilis before study enrollment (Table 4). In addition, older age and late-stage syphilis
TABLE 4.
Genetic and Nongenetic Risk Factors for Neurosyphilis in HIV-Infected Individuals The TLR6 deficient SNP was more common in HIVinfected compared with HIV-uninfected individuals (214 [61.0%] of 351 vs. 52 [50.0%] of 104, P = 0.046), but the other 2 deficient TLR SNPs were equally common in HIV-infected and HIVuninfected individuals. In addition to serum RPR titer and infecting strain type, lower peripheral blood CD4 and nonuse of antiretroviral medications may increase neurosyphilis risk in HIV-infected individuals.2,3 We repeated the above analysis among HIV-infected participants, including HIV-related factors that influence neurosyphilis risk (Table 5). Although the number of outcomes was too small to perform multivariate analyses for the TLR2 polymorphism, the associations between laboratoryand clinically-defined neurosyphilis and the deficient genotype that were seen in the group as a whole persisted in the HIVinfected subset (for laboratory-defined neurosyphilis: OR, 7.62 [1.44Y40.20, P = 0.017]; for clinically-defined neurosyphilis: OR, 8.91 [1.89Y42.02, P = 0.006]).
Associations Between Neurosyphilis and TLR1 and TLR6 SNPs and Clinical Variables
Univariate Analysis OR (95% CI) Laboratory-defined neurosyphilis TLR1 (GG vs. TT/TG) RPR titer (Q1:32 vs. G1:32) Year (G2005 vs. Q2005) Stage (late vs. early) Age (per 10-y increase) Treatment of uncomplicated syphilis before enrollment Previous syphilis TLR6 (CT/TT vs. CC) RPR titer (Q1:32 vs. G1:32) Year (G2005 vs. Q2005) Stage (late vs. early) Age (per 10-y increase) Treatment of uncomplicated syphilis before enrollment Previous syphilis Clinically-defined neurosyphilis TLR6 (CT/TT vs. CC) RPR titer (Q1:32 vs. G1:32) Year (G2005 vs. Q2005) Stage (late vs. early) Age (per 10-y increase) Treatment of uncomplicated syphilis before enrollment Previous syphilis
Multivariate Analysis P
1.79 14.27 1.78 1.60 1.24 0.50 0.77 1.68 14.27 1.78 1.60 1.24 0.50 0.77
(1.11Y2.90) (5.08Y40.08) (1.03Y3.08) (0.94Y2.72) (0.99Y1.55) (0.30Y0.82) (0.42Y1.40) (1.03Y2.75) (5.08Y40.08) (1.03Y3.08) (0.94Y2.72) (0.99Y1.55) (0.30Y0.82) (0.42Y1.40)
0.018 G0.001 0.038 0.082 0.060 0.006 0.388 0.040 G0.001 0.038 0.082 0.060 0.006 0.388
2.40 2.82 1.34 0.96 1.18 0.65 0.87
(1.55Y3.71) (1.73Y4.59) (0.82Y2.20) (0.59Y1.58) (0.98Y1.42) (0.43Y0.98) (0.53Y1.43)
G0.001 G0.001 0.238 0.885 0.081 0.038 0.585
OR (95% CI) 2.10 22.69 2.00 2.87 1.27 0.51 2.57 27.09 2.10 3.17 1.36 0.52
(1.22Y3.62) (7.48Y68.83) (1.06Y3.77) (1.50Y5.49) (0.98Y1.65) (0.29Y0.89) V (1.45Y4.55) (8.74Y83.95) (1.11Y3.96) (1.64Y6.13) (1.04Y1.76) (0.30Y0.92) V
2.71 (1.71Y4.30) 3.51 (2.09Y5.89) V V 1.22 (1.01Y1.49) 0.71 (0.46Y1.09) V
P 0.008 G0.001 0.031 0.001 0.072 0.018 V 0.001 G0.001 0.023 0.001 0.022 0.024 V G0.001 G0.001 V V 0.043 0.119 V
Controls had no vision or hearing loss and CSF white blood cells e5/KL and a nonreactive CSF-VDRL; patients with laboratory-defined neurosyphilis had a reactive CSF-VDRL, and patients with clinically-defined neurosyphilis had new vision or hearing loss. Logistic regression was used for univariate and multivariate analysis with results reported as ORs with 95% CIs; variables with P values greater than 0.20 in univariate analysis were not included in multivariate analysis.
Sexually Transmitted Diseases
&
Volume 41, Number 7, July 2014
443
Copyright © 2014 by the American Sexually Transmitted Diseases Association. Unauthorized reproduction of this article is prohibited.
Marra et al.
TABLE 5.
Associations Between Neurosyphilis and TLR1 and TLR6 SNPs and Clinical Variables in HIV-Infected Patients
Univariate Analysis
Laboratory-defined neurosyphilis TLR1 (GG vs. TT/TG) RPR titer (Q1:32 vs. G1:32) Year (G2005 vs. Q2005) ARVs (current use vs. none) CD4 (e350 vs. 9350 cells/KL) Stage (late vs. early) Age (per 10-y increase) Treatment of uncomplicated syphilis before enrollment Previous syphilis TLR6 (CT/TT vs. CC) RPR titer (Q1:32 vs. G1:32) Year (G2005 vs. Q2005) ARVs (current use vs. none) CD4 (e350 vs. 9350 cells/KL) Stage (late vs. early) Age (per 10-y increase) Treatment of uncomplicated syphilis before enrollment Previous syphilis Clinically-defined neurosyphilis TLR6 (CT/TT vs. CC) RPR titer (Q1:32 vs. G1:32) Year (G2005 vs. Q2005) ARVs (current use vs. none) CD4 (e350 vs. 9350 cells/KL) Stage (late vs. early) Age (per 10-y increase) Treatment of uncomplicated syphilis before enrollment Previous syphilis
Multivariate Analysis
OR (95% CI)
P
OR (95% CI)
P
1.43 (0.83Y2.47) 25.13 (5.99Y105.41) 1.71 (0.91Y3.19) 0.21 (0.12Y0.40) 1.45 (0.79Y2.64) 1.49 (0.78Y2.83) 1.15 (0.88Y1.50) 0.47 (0.26Y0.85) 0.81 (0.42Y1.59) 1.57 (0.89Y2.76) 25.13 (5.99Y105.41) 1.71 (0.91Y3.19) 0.21 (0.12Y0.40) 1.45 (0.79Y2.64) 1.49 (0.78Y2.83) 1.15 (0.88Y1.50) 0.47 (0.26Y0.85) 0.81 (0.42Y1.59)
0.197 G0.001 0.095 G0.001 0.232 0.227 0.303 0.012 0.547 0.120 G0.001 0.095 G0.001 0.232 0.227 0.303 0.012 0.547
1.80 (0.90Y3.59) 23.36 (5.28Y103.40) 2.20 (0.76Y6.39) 0.24 (0.12Y0.48) V V V 0.55 (0.27Y1.12) V 2.40 (1.18Y4.89) 23.44 (5.35Y102.70) 2.18 (0.73Y6.51) 0.25 (0.12Y0.50) V V V 0.57 (0.28Y1.17) V
0.097 G0.001 0.146 G0.001 V V V 0.099 V 0.016 G0.001 0.163 G0.001 V V V 0.126 V
2.09 (1.26Y3.44) 4.00 (2.22Y7.21) 1.37 (0.78Y2.40) 0.36 (0.21Y0.61) 1.10 (0.64Y1.90) 1.07 (0.60Y1.94) 1.12 (0.90Y1.40) 0.69 (0.43Y1.11) 0.90 (0.51Y1.57)
0.004 G0.001 0.270 G0.001 0.719 0.811 0.325 0.122 0.705
2.79 (1.54Y5.05) 6.12 (2.95Y12.69) V 0.36 (0.20Y0.64) V V V 0.90 (0.51Y1.59) V
0.001 G0.001 V 0.001 V V V 0.706 V
Controls had no vision or hearing loss and CSF white blood cells e5/KL and a nonreactive CSF-VDRL; patients with laboratory-defined neurosyphilis had a reactive CSF-VDRL, and patients with clinically-defined neurosyphilis had new vision or hearing loss. Comparison of proportions was performed using W2 or Fisher exact tests. Logistic regression was used for univariate and multivariate analysis with results reported as ORs with 95% CIs; variables with P values greater than 0.20 in univariate analysis were not included in multivariate analysis. ARVs indicate antiretrovirals or antiretroviral drugs.
In univariate analysis, the odds of laboratory-defined neurosyphilis were significantly higher in those with higher serum RPR titers, in those not taking antiretroviral agents, and in those who were not treated for uncomplicated syphilis before study enrollment (Table 5). Although the TLR1- and TLR6 deficient SNPs were not significantly associated with laboratory-defined neurosyphilis in HIV-infected patients in univariate analysis, the odds of laboratory-defined neurosyphilis were significantly higher in those with the deficient TLR6 SNP in multivariate analysis, taking into account the above factors. Similarly, in univariate analysis, the odds of clinically-defined neurosyphilis were significantly higher in those with higher serum RPR titers and in those not taking antiretroviral agents; treatment of uncomplicated syphilis before study entry was weakly associated with clinically-defined neurosyphilis (Table 5). The odds of clinically-defined neurosyphilis were significantly higher in those with the deficient TLR6 SNP in univariate analysis and remained so in multivariate analysis after taking into account the above factors.
DISCUSSION We found that laboratory-defined neurosyphilis was more common in patients who harbored the TLR1_1805GG, TLR2_2258GA, and TLR6_745CT/TT genotypes than those
444
who did not, and clinically-defined neurosyphilis was more common in patients who harbored the TLR2_2258GA and TLR6_745CT/TT genotypes than in those who did not. In addition, in the subset of HIV-infected patients, the higher odds of laboratory-defined neurosyphilis in those with the TLR1_1805GG genotype was less evident, perhaps because of smaller numbers or because the heightened risk of neurosyphilis due to HIV-induced immunodeficiency outweighs the modest risk conferred by the TLR1 SNP. This hypothesis is supported by the strongly protective effect of antiretroviral use that we saw in our analyses, with a 60% to 88% reduction in the odds of laboratory-defined disease in HIV-infected patients on antiretroviral therapy. The first step in neurosyphilis pathogenesis is invasion of the CSF by T. pallidum via the bloodstream, presumably via the blood-CSF barrier. Cerebrospinal fluid inflammation may ensue, but the patient remains asymptomatic. This stage is termed asymptomatic neurosyphilis. Asymptomatic neurosyphilis can progress to subsequent tissue injury and symptomatic disease. A study using a human brain microvascular endothelial cell line and rat primary brain endothelial cells showed that cerebral microvascular endothelium expresses TLR2 and TLR6, but not TLR1,30 and that ligation of these receptors increased vascular permeability in vitro. To the extent that in vitro studies of brain microvascular endothelium reflect the blood-CSF barrier, these results may offer insight into why we did not see an association between the TLR1 SNP and clinically-defined neurosyphilis:
Sexually Transmitted Diseases
&
Volume 41, Number 7, July 2014
Copyright © 2014 by the American Sexually Transmitted Diseases Association. Unauthorized reproduction of this article is prohibited.
TLR Polymorphisms Increase Neurosyphilis Risk
TLR1 signaling may not be operative at the blood-CSF barrier and thus may not influence T. pallidum neuroinvasion at the local site. However, TLR1 signaling could still affect neuroinvasion if it is involved in the initial immune response to peripheral T. pallidum infection. For example, defective signaling in mononuclear cells or macrophages could result in poorer control of peripheral infection, resulting in greater levels of bacteremia and greater likelihood of CSF invasion on the basis of a higher organism burden. Limitations of this work should be considered in interpreting our data. Our sample size, although large for a syphilis study, is modest for a candidate gene study. Nonetheless, our sample was sufficient to allow us to identify statistical significance. We tested 3 SNPs whose choice was based on biological plausibility, limiting false-positive associations. Most of our patients were infected with HIV, consistent with the demography of syphilis in our region. It is possible that there is an interaction between TLR SNPs and HIV-associated immunodeficiency. In fact, the TLR6 deficient SNP was more common in our HIV-infected participants. However, the associations that we identified between the TLR2 and TLR6 SNPs and laboratory or clinically defined neurosyphilis persisted when HIV was taken into account. Our laboratory definition of neurosyphilis, reactive CSF-VDRL, is quite stringent. Although we excluded from analysis patients who had preexisting neurological abnormalities, it is possible that the clinical abnormalities that we recorded were not due to neurosyphilis and that our clinical definition of neurosyphilis was thus less stringent than our laboratory definition. However, the associations that we identified with both neurosyphilis diagnoses were concordant for the TLR2 and TLR6 SNPS, adding to the credibility of our findings. Moreover, if misclassification of clinical neurosyphilis occurred, there is no reason to expect that it would have been more likely in those with the SNPs of interest. Rather, it would have weakened, not strengthened, the associations that we observed. Similarly, although all our patients self-identified as ‘‘Caucasian,’’ genetic heterogeneity cannot be excluded. However, this would also have been more likely to weaken, rather than strengthen the associations that we found. We were not able to validate our findings in an independent cohort of patients, and we hope that this work will stimulate others to replicate it. It should be noted that our patients were referred because of a concern regarding possible neurosyphilis. This bias could result in our ‘‘controls’’ being less representative of patients at low risk for neurosyphilis than the general population of patients with syphilis. As such, the ORs that we provide may be underestimates of the true effect of the SNPs that we studied. Our data support the contention made in the preantibiotic era that host factors impact the natural course of syphilis. Future work will be required to replicate these results as well as to determine the underlying mechanisms.
REFERENCES 1. Chesney AM, Kemp JE. Incidence of Spirochaeta pallida in cerebrospinal fluid during early stage of syphilis. JAMA 1924; 83: 1725Y1728. 2. 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; 189:369Y376.
Sexually Transmitted Diseases
&
3. Ghanem KG, Moore RD, Rompalo AM, et al. Neurosyphilis in a clinical cohort of HIV-1Yinfected patients. AIDS 2008; 22: 1145Y1151. 4. Libois A, De Wit S, Poll B, et al. HIV and syphilis: When to perform a lumbar puncture. Sex Transm Dis 2007; 34:141Y144. 5. Taylor MM, Aynalem G, Olea LM, et al. A consequence of the syphilis epidemic among men who have sex with men (MSM): Neurosyphilis in Los Angeles, 2001Y2004. Sex Transm Dis 2008; 35:430Y434. 6. Marra CM, Sahi SK, Tantalo LC, et al. Enhanced molecular typing of Treponema pallidum: Geographical distribution of strain types and association with neurosyphilis. J Infect Dis 2010; 202: 1380Y1388. 7. Stokes JH, Beerman H, Ingraham NR. Modern Clinical Syphilology. Diagnosis, Treatment, Case Study. 3rd ed. Philadelphia: WB Saunders, 1944. 8. Strandberg Pedersen N, Axelsen NH, Svejgaard A. General paralysis of the insane associated with HLA-Aw32. Acta Pathol Microbiol Scand C Immunol 1981; 89:181Y184. 9. Whitsett C, Turner WJ, Lee TD, et al. HLA antigen frequencies in paralytic dementia. J Neurol Sci 1976; 30:417Y420. 10. Jiang HW, Tian HQ, Liu H, et al. Association of the HLA-DRB1 locus with syphilis in a Chinese population. Int J Infect Dis 2011; 15:e342Y5. 11. Zhuang YL, Ren GJ, Tian KL, et al. Human leukocyte antigen-C and killer cell immunoglobulin-like receptor gene polymorphisms among patients with syphilis in a Chinese Han population. Acta Pathol Microbiol Immunol Scand 2012; 120:828Y835. 12. Zhuang YL, Song Y, Zhu C, et al. Association of KIR genotypes and haplotypes with syphilis in a Chinese Han population. Scand J Immunol 2012; 75:361Y367. 13. Zhuang YL, Zhu CF, Zhang Y, et al. Association of KIR2DS4 and its variant KIR1D with syphilis in a Chinese Han population. Int J Immunogenet 2012; 39:114Y118. 14. Palm NW, Medzhitov R. Pattern recognition receptors and control of adaptive immunity. Immunol Rev 2009; 227:221Y233. 15. McGill MA, Edmondson DG, Carroll JA, et al. Characterization and serologic analysis of the Treponema pallidum proteome. Infect Immun 2010; 78:2631Y2643. 16. Lien E, Sellati TJ, Yoshimura A, et al. Toll-like receptor 2 functions as a pattern recognition receptor for diverse bacterial products. J Biol Chem 1999; 274:33419Y33425. 17. Takeuchi O, Akira S. Pattern recognition receptors and inflammation. Cell 2010; 140:805Y820. 18. Cruz AR, Ramirez LG, Zuluaga AV, et al. Immune evasion and recognition of the syphilis spirochete in blood and skin of secondary syphilis patients: Two immunologically distinct compartments. PLoS Negl Trop Dis 2012; 6:e1717. 19. Lorenz E, Mira JP, Cornish KL, et al. A novel polymorphism in the Toll-like receptor 2 gene and its potential association with staphylococcal infection. Infect Immun 2000; 68:6398Y6401. 20. Schroder NW, Hermann C, Hamann L, et al. High frequency of polymorphism Arg753Gln of the Toll-like receptor-2 gene detected by a novel allele-specific PCR. J Mol Med (Berl) 2003; 81:368Y372. 21. Johnson CM, Lyle EA, Omueti KO, et al. Cutting edge: A common polymorphism impairs cell surface trafficking and functional responses of TLR1 but protects against leprosy. J Immunol 2007; 178:7520Y7524. 22. Oosting M, Ter Hofstede H, Sturm P, et al. TLR1/TLR2 heterodimers play an important role in the recognition of Borrelia spirochetes. PloS one 2011; 6:e25998. 23. Tantisira K, Klimecki WT, Lazarus R, et al. Toll-like receptor 6 gene (TLR6): Single-nucleotide polymorphism frequencies and preliminary association with the diagnosis of asthma. Genes Immun 2004; 5:343Y346. 24. Schroder NW, Diterich I, Zinke A, et al. Heterozygous Arg753Gln polymorphism of human TLR-2 impairs immune activation by Borrelia burgdorferi and protects from late stage Lyme disease. J Immunol 2005; 175:2534Y2540.
Volume 41, Number 7, July 2014
445
Copyright © 2014 by the American Sexually Transmitted Diseases Association. Unauthorized reproduction of this article is prohibited.
Marra et al.
25. Strle K, Shin JJ, Glickstein LJ, et al. Association of a toll-like receptor 1 polymorphism with heightened Th1 inflammatory responses and antibiotic-refractory Lyme arthritis. Arthritis Rheum 2012; 64:1497Y1507. 26. Larsen SA, Pope V, Johnson RE, et al. A Manual of Tests for Syphilis. 9th ed. Washington, DC: American Public Health Association; 1998:361. 27. Sexually transmitted diseases treatment guidelines. MMWR Recomm Rep 2010; 59:1Y110.
446
28. Hawn TR, Misch EA, Dunstan SJ, et al. A common human TLR1 polymorphism regulates the innate immune response to lipopeptides. Eur J Immunol 2007; 37:2280Y2289. 29. Shey MS, Randhawa AK, Bowmaker M, et al. Single nucleotide polymorphisms in toll-like receptor 6 are associated with altered lipopeptide- and mycobacteria-induced interleukin-6 secretion. Genes Immun2010; 11:561Y572. 30. Nagyoszi P, Wilhelm I, Farkas AE, et al. Expression and regulation of toll-like receptors in cerebral endothelial cells. Neurochem Int 2010; 57:556Y564.
Sexually Transmitted Diseases
&
Volume 41, Number 7, July 2014
Copyright © 2014 by the American Sexually Transmitted Diseases Association. Unauthorized reproduction of this article is prohibited.
