This study was approved by the Ethical Review Committee of the Affiliated Brain Hospital, Guangzhou Medical University (#2019-028). Signed informed consent was obtained from all patients or their family members.

Patients diagnosed with neurosyphilis in the Affiliated Brain Hospital of Guangzhou Medical University from January 2019 to December 2021 were retrospectively included. The following three inclusion criteria were applied for patients diagnosed with neurosyphilis: (1) a history of syphilis with signs and/or symptoms of nerve damage; (2) a history of syphilis with unexplained CSF abnormalities (WBC >5*10⁶/L, protein >500 mg/L); (3) a history of syphilis with positive results of a syphilis serological test, a CSF tolulized red unheated serum test (TRUST), and treponema pallidum particle agglutination (TPPA). All patients were negative for HBV, HCV, and HIV. The exclusion criteria were as follows: (1) incomplete clinical and/or relevant laboratory data; (2) severe liver or kidney disease or other infection. All patients were treated with penicillin: aqueous penicillin 18–24 million U per day, once every four hours or continuous intravenous infusion for 10–14 days, followed by intramuscular benzathine penicillin of 2.4 million U per dose once a week for three weeks. Oral prednisone (20 mg) was given once a day for 3 days before penicillin treatment.

A total number of 116 patients met the inclusion criteria. Of them, 81 patients with neurosyphilis had CSF samples and complete clinical data, including clinical examination, brain MRI, CSF analysis, and cognitive assessment (MMSE). All CSF samples were collected during the early active disease stage.

The use of mouse brain sections from C57/BL6J (age: 8–10 weeks) was approved by the Institutional Animal Care and Use Committee, Nanfang Hospital, Southern Medical University, Guangzhou, China. Frozen brain sections 5-μm thick were prepared as previously reported (18). Briefly, ice-cold saline-perfused mouse brain sections were fixed with isopentane-liquid nitrogen and cut to a thickness of 5 μm. For each test, 50 μL of CSF with a dilution of 1:1 was incubated with mouse brain sections at 37°C for 1 h. After washing three times with saline, each section was further incubated with 50 μL of diluted DyLight 488-labeled goat anti-human IgG secondary Ab (1:200 dilution, ab97003, Abcam, Cambridge, MA, USA) at 37°C for another 1 h. Then, the section was washed three times again and photographed under a DM3000 microscope (Leica, Wetzlar, Germany).

CBA is the standard method for the diagnosis of known autoantigens in patients’ samples (serum or CSF), in which HEK293 cells were used and each known autoantigen is overexpressed separately. CSF samples from patients were tested for known autoantibodies, including anti-NMDAR, LGI1, Caspr2, GABAR, AMPAR, mGluR5, GAD65, AQP4, and MOG Abs, using Euroimmun assay kits (Germany) or as previously reported (18).

SPSS 26.0 software was employed for statistical analysis of the data. The measurement data conforming to normal distribution were expressed as x ± s, and the two independent samples t-test was implemented for comparison between groups. Measurement data that did not conform to the normal distribution were represented by the mean [Interquartile range(IQR)], and the Mann–Whitney U-test of two independent samples was applied for comparison between groups. The statistical data were expressed as frequency and percentage, and the chi-squared test or Fisher’s exact test was performed for comparison between groups. Logistic regression and linear regression analysis were used to analyze the univariate variables with statistical differences. For all analyses, p < 0.05 was considered statistically significant.

A total number of 116 cases were diagnosed with neurosyphilis from January 2019 to December 2021, of which 81 were included in this study. Clinical retrospective analysis was performed with all cases that met the inclusion criteria, and CSF samples were subjected to CBA and TBA tests. The study flowchart and results are presented in Figure 1.

The epidemiological and clinical characteristics of the 81 neurosyphilis patients who met the inclusion criteria are displayed in Table 1. Their age range is from 47.75–60 years. The main clinical symptoms were memory decline, in 78 (96.3%) cases, and mental behavior abnormalities, in 76 (93.8%) cases; cases with other symptoms were as follows: dizziness in two cases (2.5%), headache in two cases (2.5%), limb weakness in five cases (6.2%), epilepsy in four cases (4.9%), insomnia in eight cases (9.9%), decreased visual acuity in two cases (2.5%), ataxia in one case (1.2%), and paresthesia in one case (1.2%). Before treatment, the CSF had a WBC count of 8 (2.75–22) 10^6/L and total protein of 0.65 (range 0.51–1.03) g/L. MRI showed parenchymal abnormalities or inflammatory lesions in 28 cases (25.2%), brain atrophy in 56 cases (69.1%), and leukoencephalopathy in 11 cases (9.9%).

The CSF samples of 81 patients were tested with TBA, as can be seen in the flowchart in Figure 2. There were 38 samples (38/81, 46.9%) with positive immunostaining to the mouse brain cells. According to the cellular morphology of the staining, the positive staining was classified as neuronal cells in 21 cases (including cell surface in three cases, nuclei in six cases, and cytosol in 12 cases), glial cells in eleven cases (11/38, 28.9%), and both neuronal cells and gliocytes in six cases, as illustrated in Figure 3.

The known membrane antibodies (anti-NMDAR, LGI1, Caspr2, GABABR, AMPA_A/BR, and mGluR5) and intracellular antibody(GAD65)were analyzed in 21 patients with TBA staining of neuronal cells. Among of them, one case (P14) was positive for the anti-NMDAR antibody and another case (P31) was positive for the anti-GAD65 antibody. These two patients also had serum and CSF tests when they were hospitalized: P14 had a CSF titer of 1:1 anti-NMDAR antibody (Figure 4A) and a serum titer of 1:10; P31 had a CSF titer of 1:1 anti-GAD65 antibody (Figure 4B) and a serum titer of 1:10. Anti-MOG and AQP4 Abs were analyzed in the CSF samples of 11 patients with TBA staining in their glial cells; CBA identified only one case (P6) positive for the anti-AQP4 antibody with a titer of 1:1 (Figure 4C). Serum samples for case P6 are unavailable. Six cases had both neuronal and astrocyte patterns, and no known antibodies were detected.

The detailed clinical manifestations of the three patients are described as follows. P6 (positive anti-AQP4 antibody) was admitted to hospital due to excessive excited talking and decreased sleep for 10 days. The serum and CSF TRUST titers were 1:32 and 1:8, respectively. CSF analysis showed a WBC of 50 × 10⁶/L and a protein level of 0.67 g/L. Brain MRI revealed multiple abnormal signal shadows in the bilateral temporal cortex and subcortex (Figure 5A), MMSE increased from 12 to 18 points post-penicillin treatment. No visual or spinal symptoms were found. The significance of AQP4 Ab positivity was unclear and accompanied AQP4-associated disorder was uncertain.

P14 with the anti-NMDAR antibody was hospitalized for a 10-month memory decline, 5-month lower limb weakness, and 1-month abnormal mental behavior. Upon admission, the serum and CSF TRUST titers were 1:128 and 1:4, respectively. CSF analysis showed increased WBC (3 × 10^6/L) and protein level (0.66 g/L). Brain MRI showed multiple ischemic lesions in the frontotemporal parietal lobe and the lateral ventricle and brain atrophy (Figure 5B). The MMSE score was 7 points. The symptoms continued to progress after the penicillin treatment. Although accompanied anti-NMDAR encephalitis were diagnosed, the patient’s condition meant he was unable to accept glucocorticoid treatment and he refused to receive immunoglobulin treatment due to economic situation. Three weeks later, the patient complicated with severe pneumonia, respiratory failure, and acute coronary syndrome and was transferred to another hospital by his family.

P31 (positive anti-GAD65 antibody) was hospitalized due to mental disorder and abnormal behavior accompanied by memory decline for 3 years and paroxysmal convulsions for 10 days. Serum and CSF TRUST titers were 1:16 and 1:4, respectively. Pleocytosis (WBC of 56 × 10^6/L) and an increased protein concentration (1.64 g/L) were found in the CSF. Brain MRI showed multiple abnormal signals in the bilateral frontal, temporal, and insular lobes, indicating inflammatory lesions and brain atrophy (Figure 5C). There was no obvious improvement in the brain MRI-detected abnormalities after the penicillin treatment. Nevertheless, the cognitive impairment was improved and the MMSE score increased from 12 to 20 points.

The most common symptoms of the 38 TBA-positive patients were cognitive impairment (35 cases, 92.1%) and abnormal mental behavior (37 cases, 97.4%). Other symptoms included dizziness (one case), limb weakness (two cases), convulsions (three cases), insomnia (eight cases), vision loss (one case), ataxia (one case), and paresthesia of the lower extremities (one case). As shown in Table 2, the duration in the TBA-positive group was significantly shorter than that in the TBA-negative group, p = 0.007. There were no significant differences in the pleocytosis and protein levels of CSFs between the TBA-positive and TBA-negative groups. However, both the serum and CSF syphilis titers in the TBA-positive group were higher than those in the TBA-negative group, with significant differences between the groups of p = 0.027 and 0.006, respectively. MRI parenchymal abnormalities in the frontotemporal and hippocampal regions in the TBA-positive group were significantly higher (57.9%) than those in the TBA-negative group (14%) (p < 0.001, Table 2; Figure 6). A significant difference was found in the white matter lesions (p = 0.013) but not diffuse cerebral atrophy between the two groups. The MMSE scores of the patients in the TBA-positive group was lower than that in the TBA-negative group, with a significant difference (p < 0.001) between them (Table 2).

Multivariate logistic regression analysis of factors affecting severe cognitive impairment (MMSE≤10) (Table 3) revealed that a TBA-positive result was an independent risk factor (p = 0.011) for severe cognitive impairment, whereas a positive syphilis TRUST result was not. These results suggest that TBA positivity plays a greater role in the occurrence of severe cognitive impairment in patients with neurosyphilis than that of a positive TRUST result.

In addition, multivariate regression analysis of the parenchymal lesions revealed that a TBA-positive result, but not the serum syphilis titer, was independent risk factor as shown in Tables 2, 4. However, immunostaining in different cell types, neuronal cells or glial cells, had no significant correlation with the site of parenchymal lesions in the brain MRI (Table 5).

In terms of treatment, all 81 patients received penicillin shock therapy. Although prednisolone was administered for three days prior to penicillin use, none received intravenous methylprednisone and/or intravenous immunoglobulin. The pre-disease state of cognition was basically restored in three of the TBA-positive patients, whereas the other 35 patients had only partial cognitive improvement. However, 28 of the TBA-negative patients returned to normal cognition, and 15 patients showed partial improvement. The cognitive outcomes of TBA-negative neurosyphilis patients were better than those of the TBA-positive neurosyphilis patients, with a significant correlation between the two groups (Table 6).