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Abstract
Background Considering the unknown prevalence of neurosyphilis in West China, and the confusing diagnosis of neurosyphilis, the role of CSF_CXCL13 and syphilis serology was studied to provide a more accurate reference for the clinical detection and diagnosis of neurosyphilis.
Methods A retrospective data set I was used to investigate the prevalence of neurosyphilis, as well as the laboratory characteristics of 244 patients. Besides, to explore the diagnostic value of CSF_CXCL13 and syphilis serology for neurosyphilis, another 116 CSF_serum paired samples (data set II) were collected from 44 neurosyphilis and 72 non-neurosyphilis/syphilis patients.
Results About 6.25% (156 out of 2494) syphilis was neurosyphilis. When Treponema pallidum infection occurs, syphilis serology (sero_TRUST ≥1:16 and sero_TPPA titre ≥1:10240) can be good predictors of neurosyphilis, as well as syphilis CSF serology (CSF_TPPA ≥1:320, CSF_TRUST and venereal disease research laboratory). The sensitivity of serology in neurosyphilis can be complemented by CSF_CXCL13, which could be the therapy monitor of neurosyphilis.
Conclusion Due to the lack of ideal biomarkers for neurosyphilis, the importance of syphilis serology cannot be ignored, and their combination with CSF_CXCL13 or other biomarkers should be further investigated.
- syphilis
- serology
- diagnosis
Data availability statement
All data relevant to the study are included in the article or uploaded as supplementary information. No data or models were generated or used during the study.
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Introduction
Syphilis, a re-emerging STI that was caused by the spirochete Treponema pallidum (TP), is a health problem of increasing incidence in recent years. Neurosyphilis (NS) is the neurological manifestations of syphilis, including asymptomatic, parenchymatous, meningovascular, ocular NS and otitis, which may occur at any stage during the whole infection phase. In recent years, related data showed that the antibody to TP prevalence rate is 2.38% in our hospital,1 where the high prevalence of HIV/syphilis coinfection was observed.2 It is generally accepted that HIV coinfection persons are more likely to develop NS. Usually, NS is confirmed by reactive results in non-treponemal (NTTs) and treponemal (TTs) serological tests, together with neurological or ophthalmological symptoms and cerebrospinal fluid (CSF) abnormalities, such as high white cell count (WCCs), high protein concentrations or positive venereal disease research laboratory (VDRL) test or fluorescent treponemal antibody absorption (FTA-ABS) test results. While in practice, it is difficult to make a clear diagnosis of NS due to the lack of VDRL and FTA-ABS tests in China. Although the rapid plasma regain (RPR) and unheated serum regain test (USR) were identified as excellent alternatives of VDRL for NS diagnosis, the sensitivity of CSF NTTs is still questionable. Negative TTs on CSF makes a non-NS diagnosis but cannot exclude the diagnosis of NS.3 The false positive of CSF serology examination is less uncommon due to blood contamination and/or disturbed function of the blood–brain barrier (BBB).4 Quantitation of serum TPPA or RPR titres is worthwhile to screen out NS and predict the normalisation of CSF abnormalities after treatment.5 6 Although the definitive diagnosis of NS is challenging, more and more potential biomarkers or algorithms were proposed or proved. Algorithms for individuals with suspected NS are suggested by Canadian Public Health Laboratory Network.7 Excepted for the traditional and widely used syphilis serology detection, molecular biology techniques have also been used extensively. With recent advances in molecular diagnostics, PCR is considered to be the most reliable and practical method for laboratories to implement for syphilis diagnosis.8 However, available data indicate that the sensitivity of CSF TP PCR (ranged from 40% to 70%) was low compared with CSF serological assays.9 However, when a CSF sample tests positive for TP in a well-validated PCR assay, this result is definitive for the presence of the organism. In recent years, the CSF CXCL13, CXCL8, CXCL10 and macrophage migration inhibitory factor were also proved to be valuable biomarkers for differentiating NS from non-NS/syphilis in HIV-negative patients.10–14 Yu et al 15 showed that CXCL13/CXCR5 play a pivotal role for B cell recruitment to the CSF, involving in aberrant humoral immune responses participated in neurological damage in patients with NS, although elevated CSF_CXCL13 is unspecific and is seen in other neurological disorders, such as Lyme neuroborreliosis. Up to date, the combination role of CXCL13 and syphilis serology is unknown. Meanwhile, the prevalence of NS and its laboratory characteristics are not yet known. We conducted a retrospectively investigation to learn the prevalence and laboratory characteristics of NS patients, and then we assessed the diagnostic value of CSF CXCL13 and syphilis serology testing in patients with NS.
Materials and methods
Definitions
According to Diagnosis for Syphilis of China (WS 273–2018, published in March 2018), NS is defined as syphilis patients with both of the following: (1) elevated CSF protein (>500 mg/L) and/or leucocyte count (≥10×106/L) in the absence of other known causes of these abnormalities and (2) clinical symptoms or signs consistent with NS without other known causes for these clinical abnormalities. Confirmed NS is defined as suspected NS with reactive CSF_VDRL and/or CSF_TRUST. NS group included NS with or without reactive CSF_VDRL and/or CSF_TRUST. Non-neurosyphilis (N-NS) group was defined as syphilis cases without evidence of NS.
Population enrolled and study design
We retrospectively collected data without personal identifiers from West China Hospital from January 2013 to June 2018 (data set I). A total of 2691 syphilis patients were enrolled, including 296 patients with CSF examination results. Patients (52 cases) with HIV infection, intracranial infection or other intracranial diseases were then excluded. Lastly, a total of 244 patients were included in data set I, including 156 NS cases and 88 N-NS cases. From July 2018 to January 2019, 142 syphilis patients with CSF examinations were enrolled (data set II). Their CSF–serum specimen pairs were collected for subsequent analysis. CSF samples with macroscopic blood contamination or from duplicate patients were excluded. A total of 116 specimen pairs were included finally, which were 44 NS and 72 N-NS. More detailed process of patient enrolled was illustrated in figure 1. The different NS prevalence rates between two data sets are caused by the population and selection bias.
Flow chart process of data enrolled and excluded. Retrospective data set I of syphilis cases was developed based on hospital information system (HIS) from January 2013 to June 2018. Data set II was built on purposeful collection of data and specimens of syphilis patients from July 2018 to January 2019. CSF, cerebrospinal fluid.
Tests
The syphilitic serological tests for each sample were performed using TRUST (Rongsheng, China), TPPA (Fujirebio, Japan) tests, Lumipulse G TP-N Syphilis (Fujirebio, Japan) and VDRL (BD, America) according to the manufacturer’s instructions. CSF_CXCL13 was measured by enzyme immunoassay (BOSTER, China) as the instruction said. Qalb [Qalb=(albumin CSF/albumin serum)×103] were calculated. Other related laboratory parameters, including CSF results and sera results, were collected from hospital information system.
Statistical analysis
The Mann-Whitney U test or ANOVA was used for continuous variables with skewed distribution, and a χ2 test or Fisher’s exact test was used for categorical variables. Spearman’s rank correlation was used to analyse the correlation between variables. The above-mentioned statistical analyses were performed using SPSS V.26.0 for Windows. Receiver operating characteristic (ROC) analysis was performed to determine the performance of serum RPR and TPPA titre, and the optimal cut-off were determined corresponding to the maximal Youden’s index (sensitivity+specificity−100%). Comparison of ROC curves was carried out using Medcal V.12.3.0 (Broekstraat, Belgium). P<0.05 was considered statistically significant.
Ethic statements
These samples were analysed anonymously. For researchers, all personal or private information was blind. Written or oral informed consents can be exempted according to rules of Ethics Committee of West China Hospital of Sichuan University
Results
Characteristics of the study participants
In total, 344 HIV-negative patients (190 NS and 154 N-NS) were enrolled. We found 151 of 190 patients with NS were diagnosed or suspected as NS by checking medical record, and 23 of 154 N-NS patients were misdiagnosed or suspected as NS. The rate of escape diagnosis in NS group and the rate of misdiagnosis in N-NS group were 20.5% and 14.94%, respectively. The prevalence of NS was about 6.26% (156 out of 2492) in patients with syphilis (figure 1). NS can be found in any stage of syphilis, and latent syphilis and stage III syphilis were more common in NS group (table 1). There was no significant difference in age distribution between N-NS and NS patients (p>0.05). Totally, 74.0% (174/235) of these participants were males, accounting for 55.8% in the N-NS group and 84.6% in the NS (p<0.05). Obviously, CSF abnormalities, increased protein levels, lower glucose level and elevated WBCs were significantly different between two groups (p<0.05). As shown in table 1, the NS group had significantly higher sero_TRUST titres (1:16, 1:4~1:32) and serum TP_CLIA S/co values than N-NS patients (1:2, 1:1~1:8）(p<0.001). The IgG rate, the IgG index and QALB in the NS group were also significantly higher than those in N-NS group (p<0.05).
Characteristic of the study population in data set I
Serology roles in NS in data set II
While sero_TPPA titre can discriminate NS (1:20480, 1:10240~1:20480) from N-NS (1:1280, 1:40~1:20480) with an area under curve (AUC) (0.868, 0.787~0.927), providing a sensitivity of 66.67% and a specificity of 89.06%. The sero_TRUST titre in NS group (1:32, 1:8~1:128) was higher than that in N-NS group (1:1, 0~1:4), and the AUC for sero_TRUST titre was 0.863 (95% CI 0.786 to 0.919). The optimal cut-off points of serological tests for the identification of NS was sero_TRUST titre ≥1:16 and sero_ TPPA titre ≥1:10 240. We collected 116 serum-CSF sample pairs to obtain CSF_TPPA titre and CSF_VDRL result. We found an excellent association between CSF_VDRL (1:2,0~1:8) and CSF_TRUST (1:2, 0~1:8) (r=0.951, p<0.05). Differences in qualitative results and more than one-fold titre were deemed as inconsistency. In this prerequisite, 112 (96.55%) samples between two assays were consistent. Three qualitative results were different, and one semiquantitative result was different. The AUC of CSF_TRUST was 0.818 (0.736, 0.884), providing a sensitivity of 63.64% and specificity of 100%, which was equal to the AUC of CSF_VDRL (0.830, 0.749~0.893) (p>0.05). CSF_TPPA titre can discriminate NS (1:5120, 1:640~1:20480) from N-NS. The AUC of CSF_TPPA was 0.911 (0.844, 0.956), with its optimal cut-off points for the identification of NS was titre ≥1:320, providing a sensitivity of 84.09% and specificity of 88.89%. More detailed information about data set II was list in the supplementary material (online supplemental table S1).
Supplemental material
CXCL13 roles in NS
The NS group had significantly higher CSF_CXCL13 (469.13 pg/mL, 20.27 pg/mL~2178.70 pg/mL), especially NS patients reactive with CSF_VDRL or CSF_TRUST (2001.2 pg/mL, 17 pg/mL~10 654 pg/mL), especially higher than N-NS group (63.83 pg/mL, 1.5 pg/mL~126.16 pg/mL) (p<0.001). The CXCL13 can provide a ROC curve with AUC 0.874 (0.803, 0.944). The optimal cut-off point of serological tests for the identification of NS was CXCL13 >13.37 pg/mL, providing a sensitivity of 84.9% and specificity of 78.87%. As illustrated in figure 2, CSF_CXCL13 shows the same diagnostic performance as CSF_TPPA. Elevated CXCL13 levels were also found in patients with intracranial infection (tuberculous meningitis, cryptococcal meningitis and purulent meningitis), intracranial space-occupying lesion and multiple cranial nerve damage. Four follow-up cases showed that the CXCL13 clearly descended with the decreasing of CSF_TPPA (table 2).
Comparison ROCs of five methods used in neurosyphilis. The AUCs of CSF_TPPA, CSF_CXCL13 and sero_TRUST is significantly bigger than those of CSF_VDRL and CSF_TRUST (p<0.05). The AUC of sero_TRUST was 0.863 (0.786, 0.919), with its optimal cut-off points for the identification of NS was titre ≥1:16, providing a sensitivity of 68.2% and a specificity of 90.3%; the AUC of CSF_TRUST was 0.818 (0.736, 0.884), providing a sensitivity of 63.64% and a specificity of 100%; the AUC of CSF_VDRL was 0.830 (0.749, 0.893), providing a sensitivity of 65.9% and a specificity of 100%; the AUC of CSF_TPPA was 0.911 (0.844, 0.956), with its optimal cut-off points for the identification of NS was titre ≥1:320, providing a sensitivity of 84.09% and a specificity of 88.89%; the AUC of CSF_CXCL13 was 0.875 (0.801, 0.929), with its optimal cut-off points for the identification of NS was titre >13.37 pg/mL, providing a sensitivity of 84.09% and a specificity of 88.89% and a specificity of 79.17%. CSF, cerebrospinal fluid; ROC, receiver operating characteristic.
Four follow-up results of patients with neurosyphilis
Discussion
In this study, we found 6.25% (156 out of 2494) syphilis were NS in data set I (figure 1), which can develop NS at any stage (table 1), and the distribution stages of syphilis were significantly different among two groups. Early stages (I and II) of syphilis are difficult to be diagnosed or identified as NS. Here, NS is easy to escape diagnosis (20.5%) or be misdiagnosed (14.9%). Merritt et al 16 reported that almost 30% of all patients with syphilis (including early stage syphilis) had NS. Therefore, the concept of NS should not be ignored when patients with syphilis are identified, especially asymptomatic syphilis. However, the estimate of NS prevalence is unreliable due to the lack of gold standard tests.17
Once NS is suspected, we must use syphilis algorithms composed of TTs and NTTs first.18 TTs using CSF was useful in identifying two patients with non-reactive CSF-VDRL.19 Besides, Xiao et al 6 also showed the serum TPPA titres performed better in screening symptomatic NS. In other word, TPPA titre is worthwhile in NS. Sero_TPPA can discriminate NS from N-NS with an AUC (0.868, 0.787~0.927), providing a sensitivity of 66.67% and a specificity of 89.06%. The NS group had significantly higher serum and CSF TPPA titres and sero TP_CLIA S/co values than N-NS group (p<0.05). Xiao et al 5 showed the serological activity of TPPA might reflect CSF abnormalities less sensitive than serological RPR for predicting normalisation of CSF abnormalities. A positive CSF TT (TPHA/TPPA) does not confirm the diagnosis of NS but a negative CSF TPPA result is highly unlikely in NS.20 Reactive CSF_TPPA results also can be seen in N-NS group. The TPPA index (QCSF_TPPA) relates the TPPA titre to the Qalb and attempts to exclude errors from disturbed function of the BBB.21 Luger21 showed that TPPA index in patients with active NS were above 500.
The CSF_VDRL test remains the standard for the diagnosis of NS today, although it has very high (99.8%) specificity, its sensitivity is only 50% (range 30%–70%).17 22 Many studies showed that CSF_RPR titre is an alternative of CSF_VDRL or a good predictor for treatment efficacy,23 24 as well as the USR test25 and TRUST,24 particularly when the CSF_VDRL is not available. Although the nice consistency between TRUST and VDRL was found, two cases did not match in between the CSF_VDRL and with CSF_TRUST. So, a negative TTs on CSF makes a diagnosis of NS unlikely but cannot exclude the diagnosis.3 It is still a debate which CSF NTTs, VDRL, USR, TRUST or PRR is better. However, at least, VDRL is not the only indicator any longer. In addition, biological false positive of VDRLs or TRUST should be avoided by establishing a serological diagnosis of syphilis.4 Correspondingly, false negative results can occur in NTTs in the prozone phenomenon, when antibody levels are so high. Here, we can use CSF_TRUST to support diagnosis NS where VDRL is not available.
This study indicated that CXCL13 can be a potential marker for NS in HIV-infected individuals and the added value of CSF_CXCL13 to conventional CSF biomarkers, such as the RPR, in diagnosing NS. CSF_CXCL13 levels were significantly higher in patients with NS when NS was diagnosed by CSF-VDRL/TRUST than in the syphilis control group. The sensitivity (84.09%) and specificity (78.87%) of CSF_CXCL13 (cut-off >13.37 pg/mL) to diagnosis NS, which were different from the observation (cut-off ≥4.871 pg/mL) conducted by Zeng et al 10 and were different from the study (cut-off ≥76.3 pg/mL) conducted by Mothapo et al.26 Marra27 showed that the lower cut-off (10 pg/mL) of CSF_CXCL13 has higher sensitivity and the higher cut-off (250 pg/mL) has higher specificity in HIV infected patients with syphilis.27 Zen et al investigated the QCXCL13 to estimate the intrathecal synthesis of CXCL13, excluding the influence of blood CXCL13 contamination. We also found elevated CXCL13 levels in patients with intracranial infection (tuberculous meningitis, Cryptococcal meningitis and purulent meningitis), intracranial space-occupying lesion and multiple cranial nerve damage. Taken this into consideration, the use of CXCL13 must be in combination with syphilis serology, which also can show good potential diagnostic roles in NS. Four follow-up cases (table 2) may suggest CSF concentrations of CXCL13 declined after treatment for NS.27
In conclusion, when infection of T. pallidum occurs, syphilis serology assays (sero_TRUST ≥1:16 and/or sero_TPPA titre ≥1:10240) can be good predictors of NS, as well as syphilis CSF_serology (CSF_TPPA≥1:320, reactive CSF_TRUST and VDRL) as demonstrated. The role of serology in NS can be complemented by CSF_CXCL13, which may monitor the therapy of NS. Several limitations of our study should be acknowledged, such as the potential misclassification of different groups of patients with NS. Additionally, the bias may be caused the selection of lumbar puncture. FTA-ABS test was not taken in this study as well.
Key messages
The role of CSF_CXCL13 and syphilis serology was evaluated for the clinical detection and diagnosis of neurosyphilis.
Syphilis sero_serology (sero_TRUST ≥1:16 and sero_TPPA titre ≥1:10240) can be good predictors of neurosyphilis.
Syphilis CSF_serology (CSF_TPPA≥320, CSF_TRUST and VDRL) also showed good predictive ability for neurosyphilis.
Abstract translation
This web only file has been produced by the BMJ Publishing Group from an electronic file supplied by the author(s) and has not been edited for content.Data availability statement
All data relevant to the study are included in the article or uploaded as supplementary information. No data or models were generated or used during the study.
Ethics statements
Patient consent for publication
Ethics approval
The Ethics Committee of West China Hospital of Sichuan University approved this study [2020(920)].
Acknowledgments
We would like to thank Mrs Mengna Zou and Dr Zhonghao Wang for reviewing the English style and the grammar in the manuscript.
References
Supplementary materials
Supplementary Data
This web only file has been produced by the BMJ Publishing Group from an electronic file supplied by the author(s) and has not been edited for content.
Footnotes
Handling editor Anna Maria Geretti
Contributors DL: conceptualisation, funding acquisition, resources, visualisation and writing – original draft. XH: data curation and resources. MS: investigation, methodology and validation. LL: methodology and resources. CT: conceptualisation, project administration and supervision.
Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.
Competing interests None declared.
Provenance and peer review Not commissioned; externally peer reviewed.