BMJ Open. Comparing the Performance Characteristics of CSF-TRUST and CSF-VDRL: A Cross-sectional Study

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1 Comparing the Performance Characteristics of CSF-TRUST and CSF-VDRL: A Cross-sectional Study Journal: Manuscript ID: bmjopen Article Type: Research Date Submitted by the Author: 0-Oct-0 Complete List of Authors: Gu, Wei-Ming; Shanghai Skin Disease Hospital, Yang, Yang; Shanghai Skin Disease Hospital, Wu, Lei; Shanghai Skin Disease Hospital, Yang, Sheng; Shanghai Skin Disease Hospital, Ng, Lai-King; National Microbiology Laboratory,Public Health Agency of Canada, <b>primary Subject Heading</b>: Infectious diseases Secondary Subject Heading: Diagnostics, Infectious diseases Keywords: SYPHILIS, LABORATORY METHODS, CHINA : first published as 0./bmjopen on February 0. Downloaded from on March 0 by guest. Protected by copyright.

2 Page of Comparing the Performance Characteristics of CSF-TRUST and CSF-VDRL for Syphilis: A Cross-sectional Study Weiming Gu,*, Yang Yang, Lei Wu, Sheng Yang, Lai-King Ng Shanghai Skin Disease Hospital, Shanghai, China ; and National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Canada. Short title: Laboratory diagnosis of neurosyphilis Word count: total 00, abstract. * Corresponding author Weiming Gu Shanghai Skin Disease Hospital Bao De Road Shanghai 00, China Fax: weiming_gu00@yahoo.com - : first published as 0./bmjopen on February 0. Downloaded from on March 0 by guest. Protected by copyright.

3 Page of ABSTRACT Objective: In this study we aimed to determine the performance characteristics of CSF-TRUST as compared to CSF-VDRL for laboratory diagnosis of neurosyphilis. Design: Cross-sectional study. Setting: STD clinics. Participants and methods: CSF and serum samples were collected from individual STD clinic patients who have syphilis and are suspected to progress to neurosyphilis in a -month period. CSF-VDRL and CSF-TRUST were performed in parallel on the same day when collected. TPPA tests were also performed on the CSF and the serum samples and the Biochemical analysis of the CSF samples was also performed. Results: The overall agreement between CSF-TRUST and CSF-VDRL was.%. All TRUST positive cases were positive in the VDRL; samples of TRUST negative cases tested positive by the VDRL method. Over % of the double positive CSF samples (CSF-VDRL and CSF-TRUST) had an identical titer or a titer within a -fold difference. The agreement of CSF-TPPA and CSF-VDRL was.%. Similarly the agreement of CSF-TPPA and CSF-TRUST was.%. Conclusions: Our results revealed that CSF-TRUST could be used as an option for CSF examination in settings without CSF-VDRL in place. Trial registration: Not available. KEY WORDS Syphilis, Laboratory methods, China - : first published as 0./bmjopen on February 0. Downloaded from on March 0 by guest. Protected by copyright.

4 Page of ARTICLE SUMMARY Article focus VDRL is the method of choice for cerebro-spinal fluid (CSF) examination in patients with suspected neurosyphilis. However, none of commercial VDRL reagents has been approved by China State Food and Drug Administration for use in CSF examination. Serum TRUST performs well and similarly to VDRL for syphilis. The purpose of this study is to evaluate whether CSF-TRUST can be used for neurosyphilis in settings lacking CSF-VDRL. Key messages The overall agreement between CSF-VDRL and CSF-TRUST in CSF samples from individual syphilis patients was.%. Over % of the double positive CSF samples (CSF-VDRL and CSF-TRUST) had an identical titer or a titer within a -fold difference. CSF-TRUST could be used as an alternative for CSF examination of neurosyphilis in settings without CSF-VDRL in place. Strengths and Limitations There are currently no clear criteria for laboratory diagnosis of neurosyphilis. Therefore, this study can only determine the agreement of results for CSF-VDRL and CSF-TRUST. It was not possible to distinguish false positive or false negative for samples with inconsistent results of CSF-VDRL and CSF-TRUST. DATA SHARING STATEMENT Extra data is available by ing the corresponding author. - : first published as 0./bmjopen on February 0. Downloaded from on March 0 by guest. Protected by copyright.

5 Page of Neurosyphilis can occur at any time in the course of syphilis, its symptoms and signs are usually non-specific. Some neurosyphilis cases remain asymptomatic. According to the World Health Organization (WHO) and the Center for Disease Control and Prevention (CDC USA), the Venereal Disease Research Laboratory test on cerebro-spinal fluids (CSF-VDRL) is the method of choice for laboratory diagnosis of neurosyphilis. However, due to the stability issues of VDRL antigens, no manufacturers in China are approved the China State Food and Drug Administration Agency (SFDA) to supply the reagent commercially, which significantly hinders the correct and prompt diagnosis of neurosyphilis in Chinese hospital settings. The toluidine red unheated serum test (TRUST) is a modification of the VDRL procedure by stabilizing the antigen with toluidine red particles. TRUST is mostly used in serum tests for laboratory diagnosis of syphilis. The performance of TRUST for serum examination is similar to that of the rapid plasma regain. The TRUST reagents from commercial sources have been approved by the SFDA for syphilis diagnosis. Therefore, we are interested in determining whether TRUST can be used for CSF examination in suspected neurosyphilis cases. The objective of this study was to determine the performance characteristics of CSF-TRUST when compared to CSF-VDRL in CSF samples collected from syphilis patients. - : first published as 0./bmjopen on February 0. Downloaded from on March 0 by guest. Protected by copyright.

6 Page of MATERIALS AND METHODS CSF and serum samples The criteria for examining CSF of a syphilis patient included one of the follows: () having neurological or psychiatric signs or symptoms, () exhibiting serofast state for more than years or () request from patient because of anxiety of neurosyphilis. CSF samples were collected using lumbar punctures from syphilis patients at the Shanghai Skin Disease Hospital (SSDH) in Shanghai during the period of June 00 February 0. CSF samples containing trace of blood or having hemolysis were excluded from the study. Totally CSF samples from syphilis patients (each sample from one individual patient) were examined in this study. CSF samples were tested in the same day they were collected. CSF white blood cell counts were done within hours after the lumbar puncture, and other tests were conducted within hours. Serum samples were also collected from individuals at the same time and tested for syphilis serology on same day. The study was approved by the Shanghai Skin Disease Hospital Ethical Review Board. All participants provided informed consent. CSF-VDRL, CSF-TRUST and CSF-TPPA CSF samples were centrifuged prior to CSF-VDRL, CSF-TRUST and CSF-TPPA examinations which were performed in parallel. Semi-quantitative tests of CSF-VDRL and CSF-TRUST were performed by a serially two-fold dilution of the CSF samples with 0.% saline and tested with both methods. The highest dilution of - : first published as 0./bmjopen on February 0. Downloaded from on March 0 by guest. Protected by copyright.

7 Page of a sample which gave a positive result was then used to assign a titer value. VDRL antigen with buffered saline was purchased from Becton, Dickinson and Company (Diagnostic Systems, Loveton Circle, Sparks, MD, USA ). CSF-VDRL tests were performed according to the manufacturer s instruction with modifications. A fresh antigen suspension was prepared within h prior to VDRL reactions. CSF-TRUST tests were performed using the TRUST reagent from the Shanghai Rongsheng Biotech Co. (Shanghai, China) according to the routine for serum as manufacturer s instruction. 0 The TRUST antigen suspension was mixed with CSF or diluted CSF sample on a white card, the formation of red clumps indicated a positive result. CSF-TPPA tests were conducted using the Serodia-TPPA reagent from Fujirebio Diagnostics Inc. (Malven, PA, USA). CSF sample and sensitized gelatin particles were added to a microtiter well and incubated at room temperature for two hours. Results were observed according to the manufacturer s instructions. White blood cell (WBC) counts and biochemical analysis of CSF CSF samples were subjected to biochemical tests (Hitachi 00 and Backman-IMMAGE00) to determine levels of chloride, sugar, total protein, and albumin. The normal ranges of these markers and predicted values in neurosyphilis were those previously described. Test results for samples with inconsistent CSF-VDRL and CSF-TRUST testing results were analyzed. - : first published as 0./bmjopen on February 0. Downloaded from on March 0 by guest. Protected by copyright.

8 Page of Serum-TRUST and Serum-TPPA Serum-TRUST and serum-tppa tests were conducted according to the manufacturer s instructions using the Shanghai Rongsheng Biotech Co. TRUST reagent and the Serodia-TPPA reagent from Fujirebio Diagnostics Inc. (Malven, PA, 0 USA), respectively. Statistical analysis The program SPSS.0 from IBM (IBM Corp., New York, USA) was used according to the publisher s instruction. McNemar's test is used to compare paired proportions. A p value of <0.0 was considered significant. - : first published as 0./bmjopen on February 0. Downloaded from on March 0 by guest. Protected by copyright.

9 Page of RESULTS Agreement of results between CSF-VDRL, CSF-TRUST and CSF-TPPA tests The CSF samples were collected from syphilis patients comprising male and female syphilis patients. The average age of the patients was. years old ranging from 0 to years old. For undiluted CSF samples, an overall agreement of.% was observed (% CI:.% -.%) between CSF-VDRL and CSF-TRUST (Table ). Among the CSF samples, positive proportions for VDRL and TRUST were.% and.%, respectively. The positive percents were significantly different between CSF-VDRL and CSF-TRUST (P < 0.00). In addition, the agreement of CSF-TPPA and CSF-VDRL was.%. The positive proportion was significantly higher for CSF-TPPA (.%) than that for CSF-VDRL (.%) (P < 0.00, Table ). Similarly the agreement of CSF-TPPA and CSF-TRUST was.%, and the positive percents for CSF-TPPA and CSF-TRUST were.% and.%, respectively (P < 0.00, Table ). Comparison of titers of CSF-VDRL and CSF-TRUST All 0 CSF samples with negative CSF-TRUST test results were also tested negative by the CSF-VDRL method, and there were CSF samples exhibiting CSF-VDRL positive but CSF-TRUST negative (Table ). There were CSF samples with identical titers, accounting for.% of the CSF samples with both VDRL and TRUST positive (/, shadowed cells of Table ). Moreover, there were CSF samples with only -fold differences in the titers of VDRL and TRUST, accounting - : first published as 0./bmjopen on February 0. Downloaded from on March 0 by guest. Protected by copyright.

10 Page of for.% of the CSF samples with both VDRL and TRUST positive (/, open boxes of Table ). Therefore, the percent of CSF double positive samples (n=) which had identical or a two-fold difference titers was.%. Five CSF samples (/,.%) displayed higher VDRL titers than TRUST ( -fold difference). Analysis of samples with inconsistent results of CSF-VDRL and CSF-TRUST All the CSF-TRUST positive samples in this study were also CSF-VDRL positive (n=). On the other hand, there were CSF-VDRL positive samples (.%) which tested negative by the CSF-TRUST method. All these CSF samples were CSF-TPPA positive. The serum antibody titers of these patients were at the range of : to : for serum-trust and all patients had a titer of higher than :0 for serum-tppa. Physicians clinical diagnosis of the cases included neurosyphilis (n=), primary syphilis (n=), secondary syphilis (n=) and latent syphilis (n=) (Table ). Three cases had a CSF-VDRL titer of : or :, whereas the rest cases exhibited CSF-VDRL positive in undiluted CSF. White blood cell counts (WBC) and biochemical analysis were performed on the CSF samples with inconsistent CSF-VDRL and CSF-TRUST results (Table ). WBC counts ranged from 0 to 0. Seven out of the CSF samples had a remark increase of WBC counts (> x 0 /L, normal ranges 0-x0 /L), which comprised neurosyphilis cases, latent syphilis and secondary syphilis patients. The CSF chloride concentrations ranged from to mmol/l (normal ranges - mmol/l). Eight CSF samples had slightly lower chloride concentration than the - : first published as 0./bmjopen on February 0. Downloaded from on March 0 by guest. Protected by copyright.

11 Page 0 of normal ranges, comprising samples from neurosyphilis and from latent syphilis cases. Sugar concentrations ranged from. to. mmol/l in the CSF samples (normal ranges:.-. mmol/l). The concentrations of total proteins ranged from 0. to.0 mg/l (normal ranges mg/l). Four CSF samples from latent, primary and neurosyphilis cases had slightly higher levels of total proteins than the normal ranges. CSF albumin concentrations ranged from 0. to 0. mg/l (normal ranges g/l). Twelve CSF samples exhibited increased albumin concentrations, comprising latent syphilis, secondary syphilis and neurosyphilis cases : first published as 0./bmjopen on February 0. Downloaded from on March 0 by guest. Protected by copyright.

12 Page of DISCUSSION The combination of the biochemical, WBC examination and VDRL analysis of CSF - sample is the recommended method for the laboratory diagnosis of neurosyphilis. For patients who are neurologically asymptomatic, assessment of the efficacy of neurosyphilis treatment relies solely on normalization of CSF-VDRL measures. However, CSF-VDRL is a complicated process, the antigen suspension needs to be freshly prepared and used within hours. As pointed out by Parham et al, the reproducibility of VDRL method is reagent and operator dependent. In their study, the agreement of CSF-VDRL results between two top CDC laboratories was %. The situation is even more challenging in China as there are no commercial VDRL reagents approved by the SFDA for CSF-VDRL examination. On the other hand the TRUST method is a modified version of the VDRL test, the toluidine red particle labeled antigen is stable at room temperature, can be performed as a simple agglutination test; and there are multiple commercial TRUST reagents approved by SFDA for use in serum examination in the laboratory diagnosis of syphilis. We investigated the potential use of the TRUST method for the laboratory diagnosis of neurophilis with CSF samples collected from syphilis patients. The agreement of CSF-VDRL and CSF-TRUST was.%, and.% of the positive CSF samples had similar titers (with -fold dilutions) determined by the two methods. Moreover, the agreements between CSF-TPPA and CSF-VDRL (.%) and between CSF-TPPA and CSF-TRUST (.%) were similar. This study suggests that - : first published as 0./bmjopen on February 0. Downloaded from on March 0 by guest. Protected by copyright.

13 Page of CSF-TRUST could be an option in CSF-VDRL unavailable settings. Pettit et al evaluated the performance of the TRUST method against the VDRL method on serum samples for the diagnosis of syphilis. Their results indicated a.% agreement between the two methods. Recent publication by Jiang et al revealed the potential use of CSF-TRUST in neurosyphilis diagnosis by examining archived CSF samples from neurosyphilis. The diagnosis of neurosyphilis is based on a combination of clinical presentation and an array of the laboratory tests. Our study has the limitations of defining neurosyphilis status in our study cases because of lack of clinical data. It is difficult to interpret the performance of CSF-TRUST in terms of its specificity, sensitivity and predictive values in STD clinic settings. Nevertheless, our results provide a preliminary indication that CSF-TRUST may be used as an alternative for laboratory diagnosis of neurosyphilis in clinical settings with CSF-VDRL unavailable. Funding This work was supported by the Shanghai Nature Science Fund, Shanghai, China (#0ZB0 and #0DZ00). Competing interests None. Contributorship: WM Gu and Lai-King Ng designed the study. WM Gu wrote the manuscript and data analysis. WM Gu, Y Yang, L Wu and S Yang conduct the study and performed laboratory tests. - : first published as 0./bmjopen on February 0. Downloaded from on March 0 by guest. Protected by copyright.

14 Page of ACKNOWLEDGEMENTS We special thank Raymond S.W. Tsang (National Microbiology Laboratory, Public Health Agency of Canada) and Mingmin Liao (Vaccine and Infectious Disease Organization, University of Saskatchewan, Canada) for their critiques and editing on this manuscript. - : first published as 0./bmjopen on February 0. Downloaded from on March 0 by guest. Protected by copyright.

15 Page of REFERENCE. Christina MM. Update on neurosyphilis. Curr Infect Dis Rep. 00; :-.. Patrick R.M, Ellen JB, James HJ, et al. editors. Chapter: Treponema and Other Human Host-Associated Spirochetes. Manual of clinical microbiology, th edition, Washington D.C. American society for microbiology; 00; -00. Department of Reproductive Health and Research, World Health Organization. The global elimination of congenital syphilis: rationale and strategy for action. Geneva: WHO, 00. ISBN:.. Ronald LM, Christine GC, Teresa FR, et al. Sexually Transmitted Diseases Treatment Guidelines, 00. MMWR; Dec., 00., No. RR-.. Last accessed August, 0.. Pettit DE, Larsen SA, Harbec PS, et al. Toluidine red unheated serum test, a nontreponemal test for syphilis. J Clin Microbiol ;:-.. Pingyu Zhou, Xin Gu, Haikong Lu, et al. Re-evaluation of serological criteria for early syphilis treatment efficacy: progression to neurosyphilis despite therapy. Sex Transm Infect 0;:e. doi:0./sextrans Becton, Dickinson Co., Maryland, USA. Available at: Last accessed August, 0.. Larsen SA, Pope V, Johnson RE, et al. A Manual of Tests for Syphilis. th ed. Washington, DC: American Public Health Association;. 0. Shanghai Rongsheng Biotech Co., Ltd. Shanghai, China. Available at: Last accessed August, 0.. Diagnostics, Inc. Japan. Available at: Last accessed August, 0.. YW Ye edition. National guide to clinical laboratory procedures. Third edition. Medical administration department of ministry of public health, P.R.C. Nov., 00.. Hoffman JI. The incorrect use of Chi-square analysis for paired data. Clin Exp Immunol. Apr; ():-.. French P, Gomberg M, Janier M, et al. 00 European Guidelines on the - : first published as 0./bmjopen on February 0. Downloaded from on March 0 by guest. Protected by copyright.

16 Page of Management of Syphilis. International Journal of STD & AIDS 00; 0: Dijkstra JW. Asymptomatic neurosyphilis. Int J Dermatol, ; :-.. Marra CM, Maxwell CL, Tantalo LC, et al. Normalization of Serum Rapid Plasma Reagin Titer Predicts Normalization of Cerebrospinal Fluid and Clinical Abnormalities after Treatment of Neurosyphilis. CID 00; :.. Parham CE, Pettit DE, Larsen SA, et al. Interlaboratory comparison of the Toluidine Red Unheated Serum Test antigen preparation. JCM, ; 0:-.. Jiang Y, Chen XH, Ma XM, et al. The usefulness of Toluidine Red Unheated Serum test in the diagnosis of HIV-negative neurosyphilis. Sexually Transmitted Diseases 0;:-. The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd to permit this article (if accepted) to be published in Sexually Transmitted Infections and any other BMJPGL products and sublicences such use and exploit all subsidiary rights, as set out in our licence : first published as 0./bmjopen on February 0. Downloaded from on March 0 by guest. Protected by copyright.

17 Page of Table. Agreement of Results between CSF-VDRL and CSF-TRUST CSF-VDRL (n, %) Total Method + - CSF-TRUST + (.%) 0 (0%) (.%) (n, %) - (.%) 0 (.%) (.%) Total 0 (.%) 0 (.%) (00%) - : first published as 0./bmjopen on February 0. Downloaded from on March 0 by guest. Protected by copyright.

18 Page of Table. Agreement of Results between CSF-VDRL and CSF-TPPA or between CSF-TRUST and CSF-TPPA Method CSF-TPPA (n, %) CSF-VDRL (n, %) CSF-TRUST (n, %) + - Subtotal + - Subtotal + 0(.) (.) (.) (.) (0.) (.) - 0(0.0) (.) (.) 0(0.0) (.) (.) Subtotal 0(.) 0(.) (.) (.) P value < 0.00 < : first published as 0./bmjopen on February 0. Downloaded from on March 0 by guest. Protected by copyright.

19 Page of Table. Semi-quantitative Analysis of CSF-VDRL and CSF-TRUST Method CSF-VDRL (N) CSF-TRUST (N) Titer - : : : : : : Subtotal : : 0 : : 0 : 0 0 : Subtotal Note: - negative; Numbers inside the table indicate number of CSF samples with positive reactions for serially : diluted samples. Shadowed boxes indicate samples with identical titers of CSF-VDRL and CSF-TRUST. Open boxes indicate samples having two-fold dilution difference between CSF-VDRL and CSF-TRUST. - : first published as 0./bmjopen on February 0. Downloaded from on March 0 by guest. Protected by copyright.

20 Page of Clinical Diagnosis Table. Biochemical Analysis of CSF Samples with VDRL Reactive but TRUST Non-reactive Number of cases VDRL Titer WBC (0 /ml) Chloride (mmol/l) Sugar (mmol/l) Total Protein (mg/l) CSF- Albumin (mg/l) Neurosyphilis :-: Primary : Secondary : Latent : Total/range :-: : first published as 0./bmjopen on February 0. Downloaded from on March 0 by guest. Protected by copyright.

21 Comparing the Performance Characteristics of CSF-TRUST and CSF-VDRL: A Cross-sectional Study Journal: Manuscript ID: bmjopen r Article Type: Research Date Submitted by the Author: 0-Dec-0 Complete List of Authors: Gu, Wei-Ming; Shanghai Skin Disease Hospital, Yang, Yang; Shanghai Skin Disease Hospital, Wu, Lei; Shanghai Skin Disease Hospital, Yang, Sheng; Shanghai Skin Disease Hospital, Ng, Lai-King; National Microbiology Laboratory,Public Health Agency of Canada, <b>primary Subject Heading</b>: Infectious diseases Secondary Subject Heading: Diagnostics, Infectious diseases Keywords: SYPHILIS, LABORATORY METHODS, CHINA : first published as 0./bmjopen on February 0. Downloaded from on March 0 by guest. Protected by copyright.

22 Page of Comparing the Performance Characteristics of CSF-TRUST and CSF-VDRL for Syphilis: A Cross-sectional Study Weiming Gu,*, Yang Yang, Lei Wu, Sheng Yang, Lai-King Ng Shanghai Skin Disease Hospital, Shanghai, China ; and National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Canada. Short title: Laboratory diagnosis of neurosyphilis Word count: total, abstract. * Corresponding author Weiming Gu Shanghai Skin Disease Hospital Bao De Road Shanghai 00, China Fax: weiming_gu00@yahoo.com - : first published as 0./bmjopen on February 0. Downloaded from on March 0 by guest. Protected by copyright.

23 Page of ABSTRACT Objective: In this study we aimed to determine the performance characteristics of CSF-TRUST as compared to CSF-VDRL for laboratory diagnosis of neurosyphilis. Design: Cross-sectional study. Setting: STD clinics. Participants and methods: CSF and serum samples were collected from individual STD clinic patients who have syphilis and are suspected to progress to neurosyphilis in a -month period. CSF-VDRL and CSF-TRUST were performed in parallel on the same day when collected. TPPA tests were also performed on the CSF and the serum samples and the Biochemical analysis of the CSF samples was also performed. Results: The overall agreement between CSF-TRUST and CSF-VDRL was.%. All TRUST reactive cases were reactive in the VDRL; samples of TRUST negative cases tested reactive by the VDRL method. Over % of the double reactive CSF samples (CSF-VDRL and CSF-TRUST) had an identical titer or a titer within a -fold difference. The agreement of CSF-TPPA and CSF-VDRL was.%. Similarly the agreement of CSF-TPPA and CSF-TRUST was.%. Conclusions: Our results revealed that CSF-TRUST could be used as an option for CSF examination in settings without CSF-VDRL in place. Trial registration: Not available. KEY WORDS Syphilis, Laboratory methods, China - : first published as 0./bmjopen on February 0. Downloaded from on March 0 by guest. Protected by copyright.

24 Page of ARTICLE SUMMARY Article focus VDRL is the method of choice for cerebro-spinal fluid (CSF) examination in patients with suspected neurosyphilis. However, none of commercial VDRL reagents has been approved by China State Food and Drug Administration for use in CSF examination. Serum TRUST performs well and similarly to VDRL for syphilis serological tests. The purpose of this study is to evaluate whether CSF-TRUST can be used for neurosyphilis in settings lacking CSF-VDRL. Key messages The overall agreement between CSF-VDRL and CSF-TRUST in CSF samples from individual syphilis patients was.%. Over % of the double reactive CSF samples (CSF-VDRL and CSF-TRUST) had an identical titer or a titer within a -fold difference. CSF-TRUST could be used as an alternative for CSF examination of neurosyphilis in settings without CSF-VDRL in place. Strengths and Limitations There are currently no clear criteria for laboratory diagnosis of neurosyphilis. Therefore, this study can only determine the agreement of results for CSF-VDRL and CSF-TRUST. It was not possible to distinguish false positive or false negative for samples with inconsistent results of CSF-VDRL and CSF-TRUST. DATA SHARING STATEMENT Extra data is available by ing the corresponding author. - : first published as 0./bmjopen on February 0. Downloaded from on March 0 by guest. Protected by copyright.

25 Page of Neurosyphilis can occur at any time in the course of syphilis, its symptoms and signs are usually non-specific. Some neurosyphilis cases remain asymptomatic., According to the World Health Organization (WHO) and the Center for Disease Control and Prevention (CDC USA), the Venereal Disease Research Laboratory test on cerebro-spinal fluids (CSF-VDRL) is the method of choice for laboratory diagnosis of neurosyphilis. However, due to the stability issues of VDRL antigens, no manufacturers in China are approved by the China State Food and Drug Administration Agency (SFDA) to supply the reagent commercially, which significantly hinders the correct and prompt diagnosis of neurosyphilis in Chinese hospital settings. TRUST is a modification of the VDRL procedure by labeling the antigen with toluidine red particles. TRUST is mostly used in serum tests for laboratory diagnosis of syphilis. The performance of TRUST for serum examination is similar to that of the rapid plasma regain (RPR). The TRUST reagents from commercial sources have been approved by the SFDA for syphilis diagnosis. Therefore, we are interested in determining whether TRUST can be used for CSF examination in suspected neurosyphilis cases. The objective of this study was to determine the performance characteristics of CSF-TRUST when compared to CSF-VDRL in CSF samples collected from syphilis patients. - : first published as 0./bmjopen on February 0. Downloaded from on March 0 by guest. Protected by copyright.

26 Page of MATERIALS AND METHODS CSF and serum samples The criteria for diagnosis of syphilis and syphilis staging were those described by the Ministry of Health China. The criteria for examining CSF of a syphilis patient in this study included one of the follows: () having neurological or psychiatric signs or symptoms, or () exhibiting serofast state for more than years, or () request from patients because of anxiety of neurosyphilis. The diagnosis of neurosyphilis in our hospital followed the criteria set by the National Center for STD Control, China CDC. Briefly, a patient is diagnosed having neurosyphilis when meeting the following criteria: () syphilis history with signs and/or symptoms of neurological damages and serum reactive of nontreponemal-specific serological tests and reactive in treponemal-specific serological tests, and () Syphilis history with abnormal CSF examination results (WBC > 0 /L and protein > 00 mg/l) which cannot explained otherwise, and () syphilis history with CSF-VDRL reactive. In our study analysis, we also categorized patients based on physician s clinical presumptive diagnosis. CSF samples were collected using lumbar punctures from syphilis patients at the Shanghai Skin Disease Hospital (SSDH) in Shanghai during the period of June 00 February 0. CSF samples containing visible particles, exhibiting pink colors or having visible trace of blood were excluded from the study. Totally CSF samples from syphilis patients (each sample from one individual patient) were examined in this study. CSF samples were tested in the same day they were collected. CSF white blood cell counts were done within hours after the lumbar puncture, and other tests were conducted within hours. - : first published as 0./bmjopen on February 0. Downloaded from on March 0 by guest. Protected by copyright.

27 Page of Serum samples were also collected from individuals at the same time and tested for syphilis serology on same day. The study was approved by the Shanghai Skin Disease Hospital Ethical Review Board. All participants provided informed consent in writing. CSF-VDRL, CSF-TRUST and CSF-TPPA The following practice was conducted to minimize potential bias from technicians: ) Two technicians involved in the testing were blinded from patients IDs; ) VDRL and TRUST tests were performed by a separate technician, respectively; ) The laboratory supervisor who was blinded from patients IDs as well combined the two sets of testing results at the end of each day. CSF samples were centrifuged prior to CSF-VDRL, CSF-TRUST and CSF-TPPA examinations which were performed in parallel. Semi-quantitative tests of CSF-VDRL and CSF-TRUST were performed by a serially two-fold dilution of the CSF samples with 0.% saline and tested with both methods. The highest dilution of a sample which gave a reactive result was then used to assign a titer value. VDRL antigen with buffered saline was purchased from Becton, Dickinson and Company (Diagnostic Systems, Loveton Circle, Sparks, MD, USA ). CSF-VDRL tests were performed according to the manufacturer s instruction with modifications as described by Larsen et al. 0 A fresh antigen suspension was prepared within h prior to VDRL reactions. Ten µl of the prepared antigen was incubated with 0 µl CSF samples for min with rotation at 0 rpm on a mechanical rotator. CSF-TRUST tests were performed using the TRUST reagent from the Shanghai - : first published as 0./bmjopen on February 0. Downloaded from on March 0 by guest. Protected by copyright.

28 Page of Rongsheng Biotech Co. (Shanghai, China) according to the routine for serum as manufacturer s instruction. The TRUST antigen suspension was mixed with CSF or diluted CSF sample on a white card, the formation of red clumps indicated a reactive result. CSF-TPPA tests were conducted using the Serodia-TPPA reagent from Fujirebio Diagnostics Inc. (Malven, PA, USA). CSF sample and sensitized gelatin particles were added to a microtiter well and incubated at room temperature for two hours. Results were observed according to the manufacturer s instructions. White blood cell (WBC) counts and biochemical analysis of CSF CSF samples were subjected to WBC counts and biochemical tests (Hitachi 00 and Backman-IMMAGE00) to determine levels of chloride, sugar, total protein, and albumin. The normal ranges of these markers and predicted values in neurosyphilis were those previously described. Test results for samples with inconsistent CSF-VDRL and CSF-TRUST testing results were analyzed. Serum-TRUST and Serum-TPPA Serum-TRUST and serum-tppa tests were conducted according to the manufacturer s instructions using the Shanghai Rongsheng Biotech Co. TRUST reagent and the Serodia-TPPA reagent from Fujirebio Diagnostics Inc. (Malven, PA, USA), respectively., Statistical analysis The program SPSS.0 from IBM (IBM Corp., New York, USA) was used according to the publisher s instruction. McNemar's test is used to compare paired proportions. A p value of <0.0 was considered significant. - : first published as 0./bmjopen on February 0. Downloaded from on March 0 by guest. Protected by copyright.

29 Page of RESULTS Agreement of results between CSF-VDRL, CSF-TRUST and CSF-TPPA tests The CSF samples were collected from syphilis patients comprising male and female syphilis patients. The average age of the patients was. years old, ranging from 0 to years old. For undiluted CSF samples, an overall agreement of.% was observed (% CI:.% -.%) between CSF-VDRL and CSF-TRUST (Table ). Among the CSF samples, reactive proportions for VDRL and TRUST were.% and.%, respectively. The reactive percents were significantly different between CSF-VDRL and CSF-TRUST (P < 0.00). In addition, the agreement of CSF-TPPA and CSF-VDRL was.%. The reactive proportion was significantly higher for CSF-TPPA (.%) than that for CSF-VDRL (.%) (P < 0.00, Table ). Similarly the agreement of CSF-TPPA and CSF-TRUST was.%, and the reactive percents for CSF-TPPA and CSF-TRUST were.% and.%, respectively (P < 0.00, Table ). Comparison of titers of CSF-VDRL and CSF-TRUST All 0 CSF samples with negative CSF-TRUST test results were also tested negative by the CSF-VDRL method, and there were CSF samples exhibiting CSF-VDRL reactive but CSF-TRUST negative (Table ). There were CSF samples with identical titers, accounting for.% of the CSF samples with both VDRL and TRUST reactive (/, shadowed cells of Table ). Moreover, there were CSF samples with only -fold differences in the titers of VDRL and TRUST, accounting for.% of the CSF samples with both VDRL and TRUST reactive (/, open - : first published as 0./bmjopen on February 0. Downloaded from on March 0 by guest. Protected by copyright.

30 Page of boxes of Table ). Therefore, the percent of CSF double reactive samples (n=) which had identical or a two-fold difference titers was.%. Five CSF samples with double reactive (/,.%) displayed significantly higher VDRL titers than TRUST ( -fold difference), whereas no CSF samples had a significantly higher TRUST titers than that of CSF-VDRL. Twenty-two CSF samples exhibited CSF-VDRL reactive but CSF-TRUST negative. Analysis of samples with inconsistent results of CSF-VDRL and CSF-TRUST All the CSF-TRUST reactive samples in this study were also CSF-VDRL reactive (n=). On the other hand, there were CSF-VDRL reactive samples (.%) which tested negative by the CSF-TRUST method. These CSF samples were CSF-TPPA reactive. The serum antibody titers of these patients were at the range of : to : for serum-trust and had a titer of higher than :0 for serum-tppa. The patients were classified based on clinically presumptive diagnosis, pending laboratory testing results, comprising neurosyphilis (n=), primary syphilis (n=), secondary syphilis (n=) and latent syphilis (n=) (Table ). Three cases had a CSF-VDRL titer of : or :, whereas the rest cases exhibited CSF-VDRL reactive in undiluted CSF. The cases with clinically presumptive diagnosis of non-neurosyphilis also exhibited CSF-VDRL reactive. White blood cell counts (WBC) and biochemical analysis were performed on the CSF samples with inconsistent CSF-VDRL and CSF-TRUST results (Table ). Seven out of the CSF samples had a remark increase of WBC counts (> x 0 /L), which comprised neurosyphilis cases, latent syphilis and secondary syphilis - : first published as 0./bmjopen on February 0. Downloaded from on March 0 by guest. Protected by copyright.

31 Page 0 of patients. Eight CSF samples had slightly lower chloride concentration than the normal ranges, comprising samples from neurosyphilis and from latent syphilis cases. Two samples exhibited abnormal CSF sugar concentrations, which were from neurosyphilis patients. Four CSF samples from latent, primary and neurosyphilis cases had slightly higher levels of total proteins than the normal ranges. Twelve CSF samples exhibited increased albumin concentrations, comprising latent syphilis, secondary syphilis and neurosyphilis cases : first published as 0./bmjopen on February 0. Downloaded from on March 0 by guest. Protected by copyright.

32 Page of DISCUSSION The combination of the biochemical, WBC examination and VDRL analysis of CSF sample is the recommended method for the laboratory diagnosis of neurosyphilis. -, For patients who are neurologically asymptomatic, assessment of the efficacy of neurosyphilis treatment relies on normalization of CSF-VDRL measures and other CSF measures such as WBC counts, concentrations of CSF total protein, chloride, sugar and albumin. - However, CSF-VDRL is a complicated process, the antigen suspension needs to be freshly prepared and used within hours. As pointed out by Parham et al, the reproducibility of VDRL method is reagent and operator dependent. In their study, the agreement of CSF-VDRL results between two top CDC laboratories was %. The situation is even more challenging in China as there are no commercial VDRL reagents approved by the SFDA for CSF-VDRL examination. On the other hand, the TRUST method is a modified version of the VDRL test, the toluidine red particle labeled antigen is stable at room temperature (Gu et al, unpublished data), can be performed as a simple agglutination test; and there are multiple commercial TRUST reagents approved by SFDA for use in serum examination in the laboratory diagnosis of syphilis. We investigated the potential use of the TRUST method for the laboratory diagnosis of neurophilis with CSF samples collected from syphilis patients. The agreement of CSF-VDRL and CSF-TRUST was.%, and.% of the reactive CSF samples had similar titers (with -fold dilutions) determined by the two methods. Moreover, the agreements between CSF-TPPA and CSF-VDRL (.%) and between - : first published as 0./bmjopen on February 0. Downloaded from on March 0 by guest. Protected by copyright.

33 Page of CSF-TPPA and CSF-TRUST (.%) were similar. This study suggests that CSF-TRUST could be an option in CSF-VDRL unavailable settings. Pettit et al evaluated the performance of the TRUST method against the VDRL method on serum samples for the diagnosis of syphilis. Their results indicated a.% agreement between the two methods. Recent publication by Jiang et al revealed the potential use of CSF-TRUST in neurosyphilis diagnosis by examining archived CSF samples from neurosyphilis. 0 Discrepancy between CSF-VDRL and CSF-TRUST was observed in this study. Firstly,.% of double reactive samples (/) had significantly higher VDRL titers than TRUST ( -fold difference). Secondly, the CSF-TRUST non-reactive samples had low titers of CSF-VDRL, and a portion of these samples also had abnormal CSF biochemical parameters such as WBC counts, concentrations of sugar, chloride, total protein and albumin. Thirdly, cases were clinically presumptively diagnosed as non-neurosyphilils exhibited CSF-VDRL reactive but CSF-TRUST non-reactive, and these cases also had abnormal values of CSF biochemical markers. This suggests that CSF-TRUST is less sensitive than CSF-VDRL when the antibody titers are low. Therefore, while CSF-TRUST reactive could be diagnostic of neurosyphilis, CSF-TRUST non-reactive patients should be further investigated to rule out neurosyphilis. Earlier studies from others have indicated that a negative CSF treponemal-specific tests may not exclude the diagnosis of neurosyphilis. We also examined the agreement of a CSF treponemal-specific test CSF-TPPA to CSF-VDRL - : first published as 0./bmjopen on February 0. Downloaded from on March 0 by guest. Protected by copyright.

34 Page of and CSF-TRUST. The low percents of agreement (~0%) suggest that CSF-TPPA is not a good method for laboratory diagnosis of neurosyphilis. However, we did not examine the sensitivity and specificity of CSF-TPPA for neurosyphilis in our study. The use of CSF-RPR in neurosyphilis diagnosis have also been evaluated by others.,, Marra et al have recently evaluated CSF-RPR performance on syphilis patients and found that the sensitivities of CSF-RPR for laboratory-diagnosed neurosyphilis was.% (serum RPR method) or % (CSF RPR method) compared to.% of CSF-VDRL. CSF-RPR also had lower titers than CSF-VDRL. Study by Castro et al revealed similar sensitivities for CSF-RPR (%) and CSF-VDRL (0.%), and that both method had specificities of over %. The discrepancy of the results from these two studies may, at least partly, be due to the choice of negative and positive controls. The use of CSF-TRUST for the evaluation of treatment effects of neurosyphilis remains unclear. It was reported that the normalization time (median months) of CSF-VDRL and serum RPR was. months and. months after treatments, respectively. Syphilis stages and HIV-coinfections are some of the factors influencing the normalization time. The diagnosis of neurosyphilis is based on a combination of clinical presentation and an array of the laboratory tests.,,0, Our study has the limitations of defining neurosyphilis status in the study subjects because of lack of clinical data. It is difficult to interpret the performance of CSF-TRUST in terms of its specificity, sensitivity and predictive values in STD clinic settings. It seems that CSF-TRUST and CSF-VDRL - : first published as 0./bmjopen on February 0. Downloaded from on March 0 by guest. Protected by copyright.

35 Page of are congruence in most cases, but CSF-TRUST is less sensitive than CSF-VDRL. Nevertheless, our results provide a preliminary indication that CSF-TRUST may be used as an alternative for laboratory diagnosis of neurosyphilis in clinical settings with CSF-VDRL unavailable. Funding This work was supported by the Shanghai Nature Science Fund, Shanghai, China (#0ZB0 and #0DZ00). Competing interests None. - : first published as 0./bmjopen on February 0. Downloaded from on March 0 by guest. Protected by copyright.

36 Page of ACKNOWLEDGEMENTS We specially thank Raymond S.W. Tsang (National Microbiology Laboratory, Public Health Agency of Canada) and Mingmin Liao (Vaccine and Infectious Disease Organization, University of Saskatchewan, Canada) for their critiques and editing on this manuscript. We thank Yue Chen (Epidemiology and Community Medicine, University of Ottawa) for reviewing statistical analysis. - : first published as 0./bmjopen on February 0. Downloaded from on March 0 by guest. Protected by copyright.

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39 Page of The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd to permit this article (if accepted) to be published in Sexually Transmitted Infections and any other BMJPGL products and sublicences such use and exploit all subsidiary rights, as set out in our licence : first published as 0./bmjopen on February 0. Downloaded from on March 0 by guest. Protected by copyright.