Post-treatment serological changes in some patients with early syphilis exhibit a parabolic trend

Abstract

Early syphilis accounts for a large proportion of patients with syphilis. Non-Treponema pallidum tests are commonly used to assess treatment effectiveness by analyzing the serological titer before treatment and six months after treatment. However, serological changes during the first three months after completion of treatment have not been completely understood. This prompted us to investigate whether serum titers of patients exhibit a continuous decrease post-treatment and to assess the trend of change in serological titer during this period. One hundred and seventy-three eligible patients with early syphilis were included in the analysis. Pre-treatment serological titers and those at three and six months post-treatment were compared and analyzed. Serological recovery was defined as a 4-fold or greater decrease in titer from pre-treatment level. Forty patients (23.1%) were found to have an increased serum titer at three months after treatment. Among the 40 patients, 13 patients had primary syphilis, 5 patients had secondary syphilis, and 22 patients had early latent syphilis. The proportion of patients with primary syphilis was higher, and their initial titers were significantly lower. No significant differences were observed with respect to age, gender, or initial treatment. The assessment results of 17 patients (9.8% of the total patients) change. Serological changes in some patients exhibit a parabolic pattern that may affect the clinician’s assessment of patient recovery. Therefore, more frequent assessment of serological titer might be required within the first six months post-treatment.

Keywords

Syphilis benzathine penicillin parabolic serological response syphilis patients

Introduction

Syphilis is caused by the spirochete bacterium Treponema pallidum. This disease is transmitted mainly through sexual activity and can also be transmitted from an infected mother to her unborn baby through the placenta. Despite ongoing efforts for prevention and control of syphilis, its incidence continues to increase in both developing and developed countries, such as African countries, the United States, and the United Kingdom.¹ The incidence rates of syphilis in China have greatly increased over the past 30 years;^2,3 the increase has been particularly marked in the last decade (from 6 per 100,000 population in 2000 to 33 per 100,000 population in 2016). In the US and the UK, the incidence rate of syphilis has increased gradually from two per 100,000 population in 2000 to nine per 100,000 population in 2016 (data sources from the US, UK CDC, and the official website of the National Bureau of Statistics of China). Syphilis is now one of the three major infectious diseases in China.^3,4

Currently, benzathine penicillin is recommended as the first-line treatment for the management of syphilis in the United States⁵ and the United Kingdom.⁶ China’s syphilis treatment guidelines regard benzathine penicillin as one of the two first-line treatments.^7,8 However, not all patients achieve serological recovery after treatment, which poses a challenge for both doctors and patients. Non-treponemal tests such as the rapid plasma reagin (RPR) test is commonly used as a standard to determine serological recovery in early syphilis. A 4-fold or greater decrease in titer within 6–12 months post-treatment is considered indicative of serological recovery. In the United States, syphilis treatment guidelines recommend follow-up serological investigation at six months post-treatment in HIV-negative people. However, serological changes within the first three months post-treatment have largely been ignored. Therefore, a retrospective analysis of serological changes was performed in order to determine the necessity of follow-up at three months post-treatment. Our findings may shed some light on the definitive criteria for assessment of serological recovery.

Methods

A retrospective analysis of clinical and laboratory data of syphilitic patients was carried out. These patients were treated as outpatients in the sexually transmitted disease clinic at the Tianjin Third Central Hospital, China between January 2009 and December 2017. In this study, early syphilis includes primary, secondary, and early latent syphilis. According to the diagnostic criteria recommended by the Centers for Disease Control and Prevention, primary syphilis presents with one or more hard chancres and inguinal lymphadenopathy; in addition, the non-treponemal tests (RPR test) and treponemal-specific tests (T. pallidum particle agglutination assay [TPPA] and T. pallidum hemagglutination assay [TPHA]) are positive. For secondary syphilis, in addition to positive laboratory tests for syphilis, there are symptoms including skin and mucous membrane lesions such as rash and maculopapular rash on the trunk and extremities. Some patients may also exhibit superficial lymphadenopathy. In patients with early latent syphilis, the disease course is < 1 year with no clinical signs and symptoms. The patients also required the following criteria in the past one year: (1) history of high-risk sexual behavior (and no high-risk sexual behavior prior to the immediately preceding one year), (2) clinical manifestations of early or secondary syphilis with no history of treatment, (3) sexual partners with history of early syphilis infection, or (4) history of seroconversion based on clinical records of serologic test results. Laboratory tests for RPR and TPPA were both positive with no concomitant abnormalities in cerebrospinal fluid. In the present study, we enquired about the time of infection; accordingly, the disease status was classified as early latent syphilis if the time since infection was less than one year.

Increase or decrease in titer by ≥4-fold (2 dilution) is considered significant, while ≤2-fold (1 dilution) change is not considered significant.

During the follow-up at our hospital, patients were asked by the doctor whether they had any sexual activity or engaged in high-risk sexual behavior since the last visit and this information was recorded in the case history form. Partners of the patients were also requested to come for check-up. High-risk sexual behavior included unprotected sex, multiple sex partners, and sexual intercourse with spouse or partners that have tested positive for syphilis. Patients with early syphilis (primary, secondary, and early latent syphilis) were included in this study. Patients with the following criteria were excluded: tertiary syphilis, HIV-positive, syphilis reinfection (high-risk sexual behavior), abnormal cerebrospinal fluid, other infectious diseases, autoimmune diseases, drug abuse, and those lost to follow-up.

Treatment

The Chinese national guidelines for the treatment of syphilis recommend treatment of early syphilis with three weekly intramuscular injections of 2.4 million units of benzathine penicillin.^7,8 Patients who test positive for penicillin skin test are prescribed alternative therapies. Alternative therapies include 100 mg of doxycycline orally twice a day for a total of 15 days. Serum samples from patients were tested for syphilis at 3, 6, and 12 months after treatment. In addition, a large number of patients underwent serological examination one month after their treatment. This further supports our present study.

Statistical analysis

Age of patients and RPR titers at various time points (before treatment, three and six months post-treatment) were analyzed using comparison of means test, i.e. Mann–Whitney U test. Gender, initial treatment regimen, stages of syphilis, and differences in RPR titer (Tables 3 and 4) were analyzed using geometric means test, i.e. Chi square test. Statistical analysis was performed using SPSS 24.0 software (windows version). P < 0.05 was considered indicative of statistical significance.

Results

A total of 634 sets of patient data were retrieved, of which 173 patients met the inclusion criteria. Out of all patients who enrolled for the initial treatment, 139 were treated with benzathine penicillin while the remaining 34 were given alternative therapies. At three months post-treatment, increased serum titers were observed in 40 patients (23.1%), no changes in serum titers were observed in 14 patients (8.1%), and decreased serum titers were observed in 119 patients (68.8%).

Among patients with increased serum titers, 31 were treated with benzathine penicillin and 9 were treated with alternative therapies (doxycycline). Twenty-two (55%) of these patients were female; the median age of these patients was 42 years (range: 22–71). Thirteen of these patients had primary syphilis, five had secondary syphilis, and 22 had early latent syphilis (Table 1).

Table 1.

Patient characteristics and post-treatment changes in serum titers of patients with early syphilis.

Characteristics	Total (n = 173)	Increased serum titer	No change or decrease
Characteristics	Total (n = 173)	(n = 40)	(n = 133)	P value
Female, n (%)	81 (46.8%)	22 (55.0%)	59 (44.3%)	0.237
Age in years (25th, 50th, and 75th percentiles)	27, 34, 49	27, 42, 50	27, 34, 46	0.460
Syphilis stage, n (%)				<0.01
Primary	21 (12.7%)	13 (32.5%)	8 (6.0%)
Secondary	27 (15.6%)	5 (12.5%)	22 (16.5%)
Early latent	125 (72.3%)	22 (55.0%)	103 (77.5%)
Initial treatment, n (%)				0.605
Benzathine penicillin	139 (80.3%)	31 (77.5%)	108 (81.2%)
Alternatives^a	34 (19.7%)	9 (22.5%)	25 (18.8%)
RPR titers (baseline)				<0.01
≤1:2	21 (12.1%)	9 (22.5%)	12 (9.0%)
1:4	17 (9.8%)	10 (25.0%)	7 (5.3%)
1:8	22 (12.7%)	7 (17.5%)	15 (11.3%)
1:16	29 (16.8%)	7 (17.5%)	22 (16.5%)
1:32	39 (22.5%)	4 (10.0%)	35 (26.3%)
1:64	25 (14.5%)	2 (5.0%)	23 (17.3%)
≥1:128	20 (11.6%)	1 (2.5%)	19 (14.3%)
Geometric mean titer (95% CI)	18 (14,22)	7 (5, 10)	23 (19, 29)	<0.01
25th, 50th, 75th percentiles	1:8,1:16,1:64	1:4, 1:8, 1:16	1:8, 1:32, 1:64	<0.01
RPR titers (3 months)				<0.01
≤1:2	65 (37.6%)	0 (0.0%)	65 (48.9%)
1:4	30 (17.2%)	4 (10.0%)	26 (19.5%)
1:8	28 (16.2%)	8 (20.0%)	20 (15.1%)
1:16	25 (14.5%)	11 (27.5%)	14 (10.5%)
1:32	10 (5.8%)	4 (10.0%)	6 (4.5%)
1:64	9 (5.2%)	7 (17.5%)	2 (1.5%)
≥1:128	6 (3.5%)	6 (15.0%)	0 (0.0%)
Geometric mean titer (95% CI)	6 (4,7)	23 (16, 34)	4 (3, 5)	<0.01
25th, 50th, 75th percentiles	1:2,1:4,1:16	1:8, 1:16, 1:64	1:2, 1:4, 1:8	<0.01
RPR titers (6 months)				<0.01
≤1:2	89 (51.4%)	10 (25.0%)	79 (59.4%)
1:4	27 (15.6%)	4 (10.0%)	23 (17.3%)
1:8	19 (11.1%)	5 (12.5%)	14 (10.5%)
1:16	16 (9.2%)	8 (20.0%)	8 (6.0%)
1:32	13 (7.5%)	8 (20.0%)	5 (3.8%)
1:64	8 (4.6%)	4 (10.0%)	4 (3.0%)
≥1:128	1 (0.6%)	1 (2.5%)	0 (0.0%)
Geometric mean titer (95% CI)	4 (3,5)	10 (6, 15)	3 (2, 4)	<0.01
25th, 50th, 75th percentiles	1:2, 1:2, 1:8	1:4, 1:16, 1:32	1:1, 1:2, 1:4	<0.01

CI: confidence interval; RPR: rapid plasma reagin.

Doxycycline.

In addition, we discovered that some patients have had their serum titers examined at one month post-treatment (Table 2) and the results showed significant increase in serum titers in 21 patients (out of the 40 patients with increased serum titers at three months post-treatment). The increased serum titers at both one month and three months post-treatment demonstrated a coherent trend which peaked at three months. Thereafter, the serum titers decreased to close to baseline at six months, exhibiting a parabolic trend (Figure 1).

Table 2.

Changes in serum titer of some patients with early syphilis one month after benzathine penicillin treatment.

Characteristic	Increased serum titer	No change or decrease
Characteristic	(n = 21)	(n = 132)	P value
RPR titers (baseline)			<0.01
≤1:2	5 (23.8%)	12 (9.1%)
1:4	4 (19.0%)	7 (5.3%)
1:8	5 (23.8%)	15 (11.4%)
1:16	3 (14.3%)	21 (15.9%)
1:32	2 (9.5%）	35 (26.5%)
1:64	1 (4.8%）	23 (17.4%)
≥1:128	1 (4.8%）	19 (14.4%)
Geometric mean titer (95% CI)	7 (4, 12)	23 (19, 29)	<0.01
25th, 50th, 75th percentiles	1:4, 1:8, 1:16	1:8, 1:32, 1:64	<0.01
RPR titers (1 month)			<0.01
≤1:2	2 (9.5%)	25 (18.9%)
1:4	5 (23.8%)	24 (18.2%)
1:8	1 (4.8%)	25 (18.9%)
1:16	6 (28.6%)	34 (25.8%)
1:32	2 (9.5%)	21 (15.9%)
1:64	1 (4.8%)	3 (2.3%)
≥1:128	4 (19.0%)	0 (0.0%)
Geometric mean titer (95% CI)	15 (8, 28)	8 (7, 10)	<0.01
25th, 50th, 75th percentiles	1:4, 1:16, 1:32	1:4, 1:8, 1:16	<0.01

CI: confidence interval; RPR: rapid plasma reagin.

Table 3.

Analysis of the post-treatment titers in patients with early syphilis compared with baseline titers.

Characteristic	Total (n = 173)		Increased serum (n = 40)		P value	No change or decrease (n = 133)		P value
Characteristic	3 months	6 months	3 months	6 months	P value	3 months	6 months	P value
Increased titer	40 (23.1%)	26 (15.0%)	40 (100%)	21 (52.5%)	<0.01	0 (0)	5 (3.8%)	0.065
No change	14 (8.1%)	21 (12.1%)	0 (0)	11 (27.5%)		14 (10.5%)	10 (7.6%)
2-fold decrease in titer	26 (15.0%)	31 (17.9%)	0 (0)	8 (20%)		26 (19.5%)	23 (17.2%)
≥4-fold decrease in titer	93 (53.8%)	92 (53.3%)	0 (0)	0 (0)		93 (70.0%)	92 (69.2%)
Seroreversion	0 (0)	3 (1.7%)	0 (0)	0 (0)		0 (0)	3 (2.2%)

Data presented as n (%).

Table 4.

Analysis of the titer in patients with elevated serum titer at six months post-treatment compared with baseline and three months post-treatment.

Characteristic	Compared with baseline	Compared with three months	P value
Increased titer	21 (52.5%)	4 (10%)	<0.01
No change	11 (27.5%)	10 (25%)
2-fold decrease in titer	8 (20%)	9 (22.5%)
≥4-fold decrease in titer	0 (0)	17 (42.5%)
Seroreversion	0 (0)	0 (0)

Data presented as n (%).

Figure 1.

Changes in the geometric mean titer of the elevated group exhibiting a parabolic trend. CI: confidence interval.

Statistical analysis showed that age, gender, and initial treatment did not affect serum titers of patients. There were, however, significant differences between patients with elevated serum titers and those with no change/decreased serum titers at three months post-treatment. The proportion of patients with primary syphilis in the elevated serum titer group was higher than that in the group with no change/decreased serum titers. Comparing the two groups, the initial mean titer and the geometric mean of the elevated titer group were significantly lower, with 82.5% of the patients having RPR titer ratio of ≤1:16; however, both increased after treatment (Table 1). Patients with elevated serum titers showed significant difference in the serum titers at three and six months after treatment compared with the initial titer; however, no significant difference in this respect was observed among patients with decreased serum titers (Table 3). In addition, the serum titers of patients with elevated titers showed statistically significant difference at six months after treatment when compared with initial titers and titers at three months. The assessment results of 17 patients (9.8% of the total patients) change (Table 4); in addition, when compared with the titers at three months, patients with lower initial titers showed higher rate of recovery (online Supplemental material Table 1).

Discussion

Out of all patients with early syphilis who met the inclusion criteria, 23.1% showed increased serum titer at three months post-treatment with benzathine penicillin or alternative therapies. This result is similar to the study by Holman et al.⁹ in which 20.2% of syphilis patients showed increased RPR titer after treatment. Patients with primary syphilis and early syphilis who had lower initial titers were more likely to exhibit increase in serum titers at three months post-treatment. Subsequent examinations showed a significant decrease in serum titer at six months post-treatment in patients who did not undergo retreatment.

Serological examination is an important basis for the diagnosis of syphilis. Since the treponemal tests (TPPA or TPHA) typically remain positive throughout the life of patients with syphilis, these cannot be used as a standard for these patients. The US syphilis treatment guidelines recommend repeat non-treponemal test and serum titer changes can subsequently be used to understand the current state of infection. Serological examination is usually carried out at 6 and 12 months post-treatment. Treatment is considered effective when there is a 4-fold or greater decrease in titers from the initial levels.^5,6

Despite the advances in laboratory culture techniques for T. pallidum,¹⁰ it is still clinically difficult to determine patient recovery by examining the DNA of T. pallidum. Therefore, serological recovery is used as the main criterion to assess patient recovery. Currently, the most commonly used non-treponemal test is the RPR test which uses an antigen containing cardiolipin, lecithin, and cholesterol.¹¹ Interaction between this antigen and the antibody found in the serum of syphilitic patients results in agglutination and formation of visible macroscopic particles. This allows for detection of active infection via changes in serum titer.^12,13 The reagin is a group of antibodies, including immunoglobulins G and M, which are mainly anti-cardiolipin antibodies. Since the RPR test does not detect specific anti-treponemal antibodies, it lacks specificity. Therefore, any disease that causes the production of anti-cardiolipin antibodies may lead to a positive RPR test, giving rise to biological false positive reactions.^14,15 Apart from syphilis, other conditions such as acute febrile diseases, immunization, pregnancy,¹⁶ chronic diseases (e.g. connective tissue diseases), malignant tumors, leprosy, malaria, tuberculosis, heroin addiction, and hepatitis C^17,18 may lead to a positive RPR test (false positive reaction to syphilis) along with an increase in serum titer. Syphilitic patients who had the above-mentioned diseases were excluded from this study in order to avoid interference with the test results.

The outer membrane of T. pallidum is deficient in cardiolipin, the major lipoidal antigen;¹⁹ hence, it is unable to stimulate the production of reagin in the body. It is believed that the destruction of host tissue cells causes the release of cardiolipin from the mitochondrial membrane, which stimulates the body’s immune response resulting in increased levels of reagin. However, several recent studies have suggested that the lysate of T. pallidum may contain huge amounts of cardiolipin which is released upon the death of the pathogens. This release of cardiolipin antigens induces the production of reagin.^19–21 Therefore, this may be a potential reason for the post-treatment increase in titer (instead of a decrease) observed in some patients with early syphilis. Other reasons may include inter-individual variability with respect to the immune response to T. pallidum. As the duration from the initial response to the peak of infection is unclear, the timing of the treatment is liable to have affected the immune response, leading to difficulty in predicting the results.

Using the RPR titer to determine recovery from syphilis can sometimes be uncertain, especially for serofast and tertiary syphilis patients.^22,23 Our retrospective analysis showed that 17 patients (9.83% of the total patients) in the elevated group did not achieve the criteria for recovery, i.e. the RPR titer at six months post-treatment showed no decrease or a less than 4-fold decrease from the initial titer; however, when compared to RPR titer at three months post-treatment, the six-month titer did show a 4-fold or greater decrease (Table 4). The percentage of patients (9.8%) who did not achieve the criteria for recovery was higher than that in the study by Holman et al. (2.98%). This may be attributable to the different time points used for comparison as Holman et al. had compared the maximal RPR titer (over 14 days) with the initial titer (day 0). These patients with early syphilis did not undergo retreatment within six months and the parabolic change in their RPR titer poses a challenge in determining the state of recovery. Therefore, RPR titer examination may be necessary at three months or even at one month post-treatment in order to avoid variations in determining recovery from syphilis in some patients. This is particularly relevant for patients with early syphilis who have lower initial titer (especially in patients with primary syphilis). Misinterpretation of recovery may affect the subsequent treatment of the patient and potentially increase the financial and psychological burden; this may also lead to complications that may have a negative impact on the patient.²⁴

Penicillin is highly effective in quelling the early manifestations of syphilis; however, its ability to prevent late complications and to completely eradicate T. pallidum in vivo remains questionable. Indeed, studies have documented serological and microbiological failure after conventional penicillin treatment.²⁵ This may be attributable to the following reasons: (1) certain sites of infection such as the central nervous system, bones, joints, eyes, and fibrous encapsulated lesions may not be amenable to complete removal of the pathogen.²⁶ (2) With respect to immunology, patients with early and secondary syphilis exhibit increased secretion of IL-2, IFN, TNF, and MIF by Th1 lymphocytes. These cytokines may stimulate cell-mediated immune responses such as phagocytosis of T. pallidum by macrophages. Other participating cells include CD4 and CD8 lymphocytes; therefore, attenuation of lymphocyte immune response and decrease in the number of CD4 and CD8 lymphocytes may result in treatment failure.²⁷ (3) Animal experiments have shown that small amounts of T. pallidum can evade the host immune response and survive in the host through antigenic variation (concerted switching of antigen expression within a strain, such as T. pallidum repeat gene)²⁸ and phase variation (switching gene expression on and off).²⁹ (4) Bacteria usually respond to harmful environmental stimuli through activation of extracytoplasmic function (ECF) sigma (σ) factors that regulate functions that are important for survival.³⁰ TP0092 is known to be the only gene that encodes syphilis ECF σ factor. Giacani et al.³¹ demonstrated significant elevation of TP0092 in the initial stages of syphilis infection. Therefore, increasing the frequency of serologic tests and combining several drug therapies (enhanced therapy) may be a better strategy to improve the diagnosis and treatment of syphilis.²⁵

This study has certain limitations. First, the enrolled patients denied engaging in any high-risk sexual behavior from the time of initial treatment up to the completion of the follow-up period. Since this information was based on patient’s self-report, its veracity could not be independently determined. Therefore, the possibility of reinfection cannot be completely ruled out. Second, intermittent measurement of RPR titer was performed in this study in contrast to daily dynamic monitoring of the RPR titer, which may have influenced the outcomes. In addition, exclusion of some patients with HIV infection limits the universality of the application. Third, the study does not take into consideration the batch-to-batch variations of the reagent kits used, the environmental conditions, as well as the potential observer bias. Fourth, due to the laboratory constraints at our hospital, we were not able to diagnose early stage syphilis in suspected patients with a single positive result for either TPPA or RPR and in suspected patients with negative results for both TPPA and RPR in the early stage. Therefore, only patients who were compliant and tested positive for both TPPA and RPR were included in this study, which may have resulted in information bias.

Conclusions

The post-treatment parabolic trend observed in the serum titers of some patients with early syphilis may be attributable to the release of antigen after the death of the pathogen or to individual differences, which requires further investigation. Importantly, this may also affect the diagnosis which in turn affects patient recovery. Therefore, increasing the number of patient follow-ups could prevent variation in the results. That said, the uncertainty of this titer change creates an impetus for us to actively search for a method that can accurately assess the effectiveness of treatment and patient recovery.

Supplemental Material

STD871855 Supplementary Material - Supplemental material for Post-treatment serological changes in some patients with early syphilis exhibit a parabolic trend

Supplemental material, STD871855 Supplementary Material for Post-treatment serological changes in some patients with early syphilis exhibit a parabolic trend by Yong Liu, Que-Qiao Bian, Shu-Huan Zhang, Jun Wang, Zhen-Ming Wang and Jun-Yue Li in International Journal of STD & AIDS

Footnotes

Authors’ contribution

YL and QB designed/performed most of the investigation, data analysis, and wrote the manuscript; YL, QB, SZ, JW, ZW, and JL provided data support; YL and QB contributed to interpretation of the data and analyses. All authors have read and approved the manuscript.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declaration of conflicting interests

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

Ethics approval and consent to participate

This study has been approved by the ethics committee at our hospital. Data were extracted from the medical records of the patients; hence, the requirement for informed consent was waived off.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Yong Liu

Supplemental material

Supplemental material for this article is available online.

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