Value of CXCL13 in diagnosing asymptomatic neurosyphilis in HIV-infected patients

Abstract

Diagnosing asymptomatic neurosyphilis (ANS) in HIV-infected patients is difficult. A recent report suggested that CXCL13 is a promising diagnostic marker for neurosyphilis in HIV-positive patients. However, whether CXCL13 can be a diagnostic marker for ANS in HIV-infected patients remains unknown. The purpose of our study was to determine the role of CXCL13 in diagnosing ANS in HIV-infected patients. This study comprised two study and three control groups. Two study groups included 12 HIV-infected patients with ANS and 25 patients with syphilis and HIV co-infection (without ANS). Three control groups included 9 patients with ANS without HIV infection, 25 HIV-infected patients without syphilis and 10 healthy volunteers. Concentrations of CSF CXCL13 were measured before and after neurosyphilis therapy. Our results showed that CSF CXCL13 concentrations were significantly increased in all of the HIV-infected patients with ANS, the 25 HIV patients with syphilis and the 9 ANS patients without HIV, but not in the patients of the other two control groups. CSF CXCL13 concentrations declined in the two study groups of patients following neurosyphilis therapy. Therefore, CSF CXCL13 concentrations could improve the diagnosis of ANS in HIV-infected patients.

Keywords

Syphilis HIV AIDS cerebrospinal fluid CXCL13 asymptomatic neurosyphilis human immunodeficiency virus diagnosis

Introduction

Human Immunodeficiency Virus (HIV) infection and syphilis affect similar high-risk populations, share modes of transmission and syphilitic lesions can increase the likelihood of HIV transmission; thus co-infections with HIV and Treponema pallidum (TP), the spirochete that causes syphilis, are very common.^1,2 HIV can infect human CD4+ T lymphocytes, dendritic cells and monocytes-macrophages and induce immunosuppression, which can lead to the inability to clear TP.³ At the same time, HIV can infect neuroglial cells and destroy the meninges, thus enabling TP to more easily penetrate the blood–brain barrier.^3,4 Therefore, patients with this co-infection tend to have a higher incidence and more rapid progression of neurosyphilis.^5–8 In China, approximately 780,000 patients are infected with HIV, more than 46.5% of whom were infected through sexual contact (the most common mode of transmission).⁹ The rise in syphilis rates has been accompanied by an increased incidence and prevalence of neurosyphilis, particularly among patients who are also infected with HIV.^10,11 Neurosyphilis can be symptomatic or asymptomatic. Symptomatic neurosyphilis is characterised by various clinical manifestations, including symptomatic meningitis, brain infarction, tabes dorsalis, syphilitic optic atrophy and general paresis. Asymptomatic neurosyphilis (ANS) is defined as an infection of the central nervous system (CNS) with cerebrospinal fluid (CSF) abnormalities but no clinical symptoms.¹² Patients with ANS are likely to damage the CNS before more severe symptoms occur.

At present, the diagnosis of ANS in HIV patients requires the detection of anti-cardiolipin antibodies in CSF by Venereal Disease Research Laboratory (VDRL) and CSF white blood cells (WBCs). If the CSF VDRL test is positive, the diagnosis of neurosyphilis is confirmed. When the CSF VDRL is negative, CSF WBC levels higher than 20/µl is recommended to improve the specificity of neurosyphilis diagnosis.^2,13 However, in patients with HIV and syphilis co-infection, the presence of WBCs in the CSF can be associated with syphilis, HIV or both. Therefore, it is very difficult to confirm a diagnosis of ANS in patients with HIV and syphilis co-infection using the level of WBCs in the CSF.^14,15

A recent report indicated that the levels of the B lymphocyte chemoattractant chemokine (C-X-C motif) ligand 13 (CXCL13) are increased in patients with Lyme neuroborreliosis (LNB) (i.e. infection with Borrelia burgdorferi).¹⁶ Spirochetal lipoproteins are critical for the induction of CXCL13 in monocytes; CXCL13 levels can therefore be elevated in the early phases of infection.^16,17 A study showed that CSF concentrations of CXCL13 are elevated in HIV-positive patients with neurosyphilis, suggesting that CXCL13 could be a useful marker of neurosyphilis in HIV-positive patients.¹⁸ However, all of the patients in this study were diagnosed with neurosyphilis (either symptomatic or asymptomatic) based on clinical symptoms, a positive VDRL test or CSF WBC levels greater than 20/µl. Patients with CSF WBC levels ≤ 20 but ≥ 10/µl were not classified in this study. The initial phases of neurosyphilis usually result in asymptomatic meningeal reactions or a mildly abnormal CSF.⁸ It remains unclear whether CXCL13 levels can be used as a diagnostic marker of ANS in HIV-positive patients. Therefore, the purpose of this study was to investigate the possible utility of CSF CXCL13 concentrations in diagnosing ANS in patients with HIV and syphilis co-infection and a negative CSF VDRL test.

We focused on patients who were co-infected with HIV and ANS and analysed their CSF CXCL13 levels before and after receiving therapy for neurosyphilis. We conducted this study to systematically assess the ability of CXCL13 to diagnose ANS in patients co-infected with HIV and syphilis with a negative CSF VDRL test. Our ultimate aim was to establish CSF CXCL13 as a diagnostic marker of ANS in HIV-infected syphilis patients.

Methods

Study participants

We collected CSF samples from patients of the Department of Dermatology and Sexually Transmitted Infections of the Guangzhou Eighth people’s Hospital. Two groups of the study participants had confirmed HIV infection and had received confirmatory syphilis antibody tests. Physical examination was performed on patients by clinical doctors. All of the study patients had no neurological abnormalities on examination, no hearing or vision loss, no headache or dizziness. For all the patients with high CSF WBCs and negative CSF VDRL, all other infective causes of CSF pleocytosis were excluded.

The study participants were divided into two groups: (1) HIV infected with ANS (HIV+/ANS, serum RPR positive, CSF VDRL positive and/or CSF WBCs > 20/µl, n = 12); (2) HIV infected with syphilis, but without ANS (HIV+/Syph+, serum RPR positive, CSF VDRL negative, and 10/µl ≤ CSF WBCs ≤ 20/µl, n = 31).

The control participants were divided into three groups: (1) HIV uninfected with ANS (HIV−/ANS, serum RPR positive, CSF VDRL positive and/or WBCs >10 /µl, n = 9); (2) HIV infected without syphilis or other infections (HIV+/Syph−, serum RPR negative, TPHA negative, n = 25); 3) healthy volunteers (Norm, n = 10).

Ethics statement

Written informed consent was obtained from all patients and volunteers, and the study was approved by the ethics committee of the Guangzhou Eighth People’s Hospital.

Laboratory methods

Serum rapid plasma reagin (RPR) was measured using RPR test kit (KHR, Shanghai, China) and CSF VDRL titre was measured using VDRL Antigen Kit (BD Company, USA). HIV viral loads were measured using COBAS AmpliPrep/COBAS TaqMan 48 HIV-1 test Kit (Roche, USA). CD4+ T lymphocyte was measured using CD45/CD3/CD4/CD8 Antibody Kit (BD Company, USA). Routine serum and CSF analyses were performed by the laboratory of the Guangzhou Eighth Hospital.

ELISA

CSF CXCL13 concentrations were measured using a human CXCL13 development kit (Quantikine; R&D Systems, Minneapolis, MN, USA). The analysis was conducted according to the manufacturer’s instructions.

Statistical methods

Data were analysed using SPSS version 12 (SPSS Inc, Chicago, IL, USA). The data from different patient groups were compared with non-parametric Kruskal–Wallis rank tests. Correlation analyses were conducted using Spearman’s rank coefficients (Rs).

Results

Patient characteristics

A total of 87 participants were enrolled in this study from June 2011 to March 2013. Participant demographics, medical history and laboratory values were shown in Table 1. Four patients in HIV+/ANS group and nine patients in HIV−/ANS group had received benzathine penicillin G injections before but had experienced treatment failure. There was a single patient in the HIV+/ANS group and four patients in the HIV+/Syph+ group diagnosed with secondary syphilis. The other syphilis patients were all latent syphilis. Patients in the two study groups all had normal nerve reflexes and had no hearing or vision loss, headache or dizziness.

Table 1.

Participant demographics, medical history and laboratory values.

	HIV+/ANS	HIV+/Syph+	HIV−/ANS	HIV+/Syph−	Norm
Number	12	25	9	25	10
M/F	11/1	21/4	2/7	13/12	4/6
Age (median, range)	40 (29–60)	35 (23–76)	36 (28–65)	37 (25–67)	48 (42–69)
MSM/MSW/WSM	10/1/1	16/5/4	0/2/7	6/7/12	0/4/6
PG treatment	4	0	9	/	/
Skin rash	1	4	0	/	/
Receiving HAART	3	4	/	6	/
1/serum RPR (median, range)	8 (4–32)	8 (4–32)	4 (4–32)	/	/
Plasma HIV-RNA	11500	4100	/	5600	/
(copies/ml) (median, range)	(<20–340,000)	(<20–420,000)		(<20–240,000)
Blood CD4+ T	375	199	/	233	/
(cells/µl) (median, range)	(98–515)	(2–380)		(1–455)
CSF WBC (cells/µl) (median, range)	24 (14–30)	13 (10–18)	13 (8–16)	2 (0–4)	2 (0–4)
Positive CSF VDRL	17% (2/12)	0 (0/25)	44% (4/9)	/	/

Note: Data are presented as median (range).

M/F: male/female; MSM: men who have sex with men; MSW: men who have sex with women; WSM: women who have sex with men; PG treatment: received benzathine penicillin G injection before; HAART: Highly Active Antiretroviral Therapy; CSF: cerebrospinal fluid; VDRL: Venereal Disease Research Laboratory.

Serum and CSF samples from the four groups of patients were routinely analysed (Table 1). The parameters evaluated in routine laboratory tests of the healthy volunteers were within normal limits.

CSF CXCL13 concentrations by patient group

The CXCL13 concentrations in the CSF of the patients of the five groups are shown in Table 2. The CSF CXCL13 concentrations of the volunteers in the Norm group were statistically significantly different from those of the patients in the other four groups (p ≤ 0.001; Figure 1). CSF CXCL13 concentrations were also significantly higher in the HIV+/ANS, HIV+/Syph+ and HIV−/ANS patients than in the HIV+/Syph− patients (p < 0.001; Figure 1).

Table 2.

CSF CXCL13 concentrations in the five groups.

CXCL13 median concentrations (IQR)
Group	Before therapy	After therapy (pg/ml)
HIV+/ANS	186.2 (50.5–779.4)	9.5 (7.4–10.3)
HIV+/Syph+^a	102.2 (19.1–204.5)
HIV+/Syph+^b	109.1 (76.2–215.2)	10.1 (7.8–13.2)
HIV−/ANS	229.4 (136.9–283.4)
HIV+/Syph−	3.5 (2.5–5.5)
Norm	0 (0–2.9)

Note: CSF CXCL13 concentrations in the Norm group were statistically significantly different from the other four groups (p ≤ 0.001); CSF CXCL13 concentrations were higher in the HIV+/ANS, HIV+/Syph+* and HIV−/ANS groups than in the HIV+/Syph− group (p < 0.001); there was no significant difference in the HIV+/ANS, HIV+/Syph+* and HIV−/ANS groups (p > 0.001).

HIV+/Syph+: n = 31.

HIV+/Syph+: n = 25.

Figure 1.

Box plots of CSF CXCL13 concentration. The concentrations in the norm group were statistically different from the other four groups (p ≤ 0.001). The concentrations in each syphilis group were higher than those of the HIV+/Syph− patients (p < 0.001). No statistical difference in the CSF CXCL13 concentrations of HIV+/ANS, HIV+/Syph+ and HIV−/ANS patients was observed. The outlying value was 452 pg/ml in Box 2, and the patient had high RPR titre (1/32) and low CD4 count (112 cells/µl) without PG treatment before; the outlying values in Box 3 were 75.7 pg/ml and 62.57 pg/ml, and these two patients had received PG treatment before and CSF VDRL tests were negative.

The CSF CXCL13 concentrations of the 12 patients in the HIV+/ANS group were highly elevated. The CSF CXCL13 concentrations of the 25 patients in the HIV+/Syph+ group were higher than 10 pg/ml. These patients were presumed to have ANS and received the appropriate treatment. The median CSF CXCL13 concentration of the patients in the HIV-/ANS group was 229.4 pg/ml, which was higher than that of the patients in the HIV+/ANS group (186.2 pg/ml). However, the levels in the patients of these two groups were not statistically significantly different (p = 0.943).

Decline in CSF CXCL13 concentrations following neurosyphilis therapy

The CSF CXCL13 concentrations of the 25 patients in the HIV+/Syph+ group were higher than 10 pg/ml. These patients and the 12 HIV+/ANS patients received 24 million units of crystalline penicillin G per day administered in intravenous doses of four million units every 4 hours or in the form of a continuous infusion for 14 days; the patients then received 2.4 million units of benzathine penicillin G administered intramuscularly every week for three weeks. Three months after the treatment, the two groups of patients returned to the hospital and provided a second sample of CSF. The levels of WBCs in the CSF returned to normal levels in all patients following therapy, and CSF CXCL13 concentrations decreased significantly following treatment in the two study groups (Figure 2).

Figure 2.

Comparisons of CSF CXCL13 before and after treatment of neurosyphilis. Visit 1 is the study entry visit and visit 2 is the visit three months after treatment. Decline in CSF CXCL13 concentrations in HIV+/ANS patients (p = 0.002) and in HIV+/Syph+ patients (p < 0.001). The outliers in Figure 2(b) were 452 pg/ml and 404.69 pg/ml. These two patients had high RPR titre (1/32, 1/16) and low CD4 count (112 cells/µl, 50 cells/µl), without PG treatment before.

Correlation analysis

In both study groups, CSF CXCL13 concentrations before therapy were positively correlated with serum RPR titres (Rs = 0.871, p = 0.001 in the HIV+/ANS group; Rs = 0.563, p = 0.003 in the HIV+/Syph+ group).

In the HIV+/Syph+ group, the CSF CXCL13 concentrations and blood CD4+ T lymphocytes cell counts were not significantly correlated (p = 0.245). In contrast, the CSF CXCL13 levels and the blood CD4+ T lymphocytes cell counts were negatively correlated in the HIV+/ANS group (Rs = −0.72, p = 0.008).

CSF CXCL13 concentrations were not correlated with plasma HIV-RNA viral loads in HIV+/ANS or HIV+/Syph+ patients. In addition, no correlation between CSF CXCL13 concentrations and CSF WBCs counts was observed in either group of patients.

Discussion

CXCL13 was first described in 1998.¹⁹ This chemokine was believed to be mainly responsible for establishing and maintaining lymphatic tissue. Many recent retrospective and prospective studies have shown that spirochetes can invade the CSF, thereby inducing the production of CXCL13 in human monocytes/macrophages and dendritic cells. CXCL13 was confirmed to be a useful marker for the diagnosis of LNB.^20–22 A recent study showed that CSF concentrations of CXCL13 are elevated in HIV-positive patients with neurosyphilis.¹⁸ However, the diagnostic value of CXCL13 in HIV-infected patients with ANS remains unknown.

This study found a significant increase in CSF CXCL13 concentrations in patients co-infected with HIV and syphilis with 10/µl ≤ CSF WBCs ≤ 20/µl. Diagnosing neurosyphilis in a timely manner in patients co-infected with HIV and syphilis with abnormal levels of CSF WBCs but no neurological symptoms is very important.^23,24

To assess the diagnostic performance of CSF CXCL13 in patients with ANS, we systematically compared the study groups with the control groups. Our results showed that CSF CXCL13 concentrations were higher in patients with ANS than in HIV-positive patients without syphilis. A decrease in CSF CXCL13 levels following treatment was also evident. Therefore, we showed that CSF CXCL13 concentrations are elevated in patients with ANS and HIV infection. We also showed that CSF CXCL13 concentrations declined significantly following treatment with standard crystalline penicillin G, indicating that CSF CXCL13 may be used to evaluate the therapy effectiveness through a long-term follow-up.

We found that CSF CXCL13 concentrations and serum RPR titres were positively correlated in patients co-infected with HIV and syphilis. It is well accepted that serum RPR titres are indicative of the severity of syphilis; however, some studies have shown that serum RPR titres may be affected by the immune status of HIV-infected patients.^24,25 Nevertheless, it is likely that RPR titres can be used for diagnostic purposes.²⁶ In summary, our findings provided evidence that CXCL13 levels could be used to diagnose ANS in HIV-positive patients. Because of the limited number of participants in our study, further studies on the relationship between CSF CXCL13 concentrations and serum RPR titres are necessary.

In the study by Marra et al.,²⁷ neurosyphilis was significantly more common in patients with CD4+ T cell count ≤ 350 cells/µl. However, in our study, the median CD4 count in the HIV+/ANS group was 375 cells/µl and in the HIV+/Syph+ group was 199 cells/µl. The CD4 count >350 in the HIV+/ANS group might be due to 3 of 12 patients having received HAART; 4 of 12 patients had experienced treatment failure and this was a small sample. The impact on immune status and neurosyphilis was therefore uncertain. In future studies, we recommend increasing the sample size and dividing the participants into more groups based on CD4+ T lymphocyte cell counts. If CSF CXCL13 concentrations are higher than 10 pg/ml, further assessment and treatment might be necessary. If CSF CXCL13 levels are to be used in clinical practice, their sensitivity and specificity must be estimated using relevant control groups, and recommended cut-off levels must be established. The diagnostic value of CSF CXCL13 levels in HIV-positive patients with ANS should be further studied.

Footnotes

Conclusions

We confirmed a previous report that found highly elevated CSF CXCL13 concentrations in HIV-positive patients with ANS. CSF CXCL13 concentrations declined significantly following treatment with crystalline penicillin G, which confirms the diagnosis efficacy of this regimen. CSF CXCL13 concentrations could improve the diagnosis of ANS in HIV-infected patients and might represent a potential tool to further study with which to monitor the efficacy of treatment.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by Science and Technology Planning Project of Guangdong Province China Grant 2012B031800332, Guangzhou City medical science and technology project 20121A011079.

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