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. 2023 Sep 28;18(9):e0292044. doi: 10.1371/journal.pone.0292044

Changes in rapid plasma reagin titers in patients with syphilis before and after treatment: A retrospective cohort study in an HIV/AIDS referral hospital in Tokyo

Kazuhiko Ikeuchi 1,2, Kazuaki Fukushima 1,*, Masaru Tanaka 1, Keishiro Yajima 1, Makoto Saito 2, Akifumi Imamura 1
Editor: Antonella Marangoni3
PMCID: PMC10538775  PMID: 37768989

Abstract

Introduction

Although the rapid plasma reagin (RPR) test is used to determine treatment efficacy for syphilis, animal studies show that it decreases gradually after an initial increase even without treatment. Pre-treatment changes in RPR titer in humans and its relationship with post-treatment changes in RPR titer are not well known.

Methods

We retrospectively analyzed the clinical records of syphilitic patients who underwent automated RPR (Mediace) testing twice before treatment (i.e., at diagnosis and treatment initiation) within 1–3 months at an HIV/AIDS referral hospital in Japan between 2006 and 2018. The RPR values were expressed as the ratio to the value at treatment initiation. The mean monthly relative change in the RPR after treatment was calculated on the log2 scale for each patient and analyzed by multivariable linear regression.

Results

Sixty-eight patients were identified. The median age was 45 (interquartile range [IQR], 38–50), 98.5% (67/68) were men, and 97.1% (66/68) had HIV. The median RPR titer ratio at treatment initiation/diagnosis was 0.87 (IQR, 0.48–1.30). The RPR titer decreased more than twofold in 26.5% (18/68) and more than fourfold in 10.3% (7/68) before treatment. In the multivariable analysis, higher age (predicted monthly RPR relative change on the log2 scale 0.23/10 years [95% confidence interval [CI], 0.090–0.37]), history of syphilis (0.36 [95% CI, 0.07–0.65]), and a lower ratio of RPR at treatment initiation/diagnosis (−0.52/every 10-fold increase [95% CI, −0.81 to −0.22]) were associated with a slower RPR decrease after treatment.

Conclusions

In a mostly HIV patient population, RPR titer can show more than four-fold spontaneous increase or decrease within 1–3 months. Pre-treatment spontaneous decrease of RPR titer was associated with a slower decrease in post-treatment RPR titer.

Introduction

Syphilis is a sexually transmitted infection that is caused by the spirochete Treponema pallidum subsp. pallidum. Typically, T. pallidum causes primary syphilis at the invasion site and disseminates throughout the body, causing secondary syphilis and its associated symptoms, such as skin rashes [1]. T. pallidum invades the central nervous system (CNS) in 25%–60% of early syphilis, but 95% are asymptomatic [1]. Although T. pallidum that invades the CNS is eliminated in most patients with intramuscular or oral antibiotics [2,3], patients who fail to eliminate T. pallidum from CNS are at risk of progressing to symptomatic neurosyphilis [1], which requires intravenous treatment. Therefore, it is important to treat promptly with effective antibiotics and monitor the treatment efficacy.

Nontreponemal tests (e.g., rapid plasma reagin [RPR] test) detect antibodies against the cardiolipin-cholesterol-lecithin antigen. Nontreponemal tests are highly sensitive, cheap, and decrease in response to treatment and are used as a marker of treatment efficacy [4–6]. The current guidelines recommend that patients with primary and secondary syphilis should undergo nontreponemal tests at 6 and 12 months and, if the titer does not decrease fourfold, clinicians should consider cerebrospinal fluid (CSF) tests [7].

However, some researchers and clinicians have reported that the nontreponemal titer could change without treatment. In a rabbit model of syphilis infection, the natural course of the nontreponemal titer shows a bell-shaped curve with a longer right tail; the titer increases shortly after inoculation, then decreases gradually, and can become negative even without treatment [8]. We hypothesized that if nontreponemal titer increases and decreases spontaneously in humans, then pre-treatment changes in nontreponemal titer might affect post-treatment response.

Since even early latent syphilis is transmittable to other persons and can progress to symptomatic syphilis [9], immediate treatment should be started after diagnosis. Therefore, there are only a few data about the natural course of the nontreponemal titer before treatment, and it is unethical to conduct such prospective clinical studies. However, in the real world, patients may sometimes be unable to receive treatment on the day of diagnosis for some reasons [10]. For example, people with human immunodeficiency virus (HIV) (PWH) could be incidentally diagnosed with syphilis by routine screening for sexually transmitted infections, and unfortunately, treatment may be delayed to the next visit to the hospital particularly when the patients were asymptomatic. Using the routinely collected clinical data of syphilitic patients who had a gap between diagnosis and treatment, we conducted a retrospective cohort study to elucidate the pre-treatment changes in nontreponemal titer and of its influence on the post-treatment nontreponemal response.

Methods

Setting and patients

We retrospectively reviewed the clinical records of patients diagnosed with syphilis between January 2006 and December 2018 at Tokyo Metropolitan Cancer and Infectious Disease Center Komagome Hospital, which is an HIV/acquired immune deficiency syndrome (AIDS) referral hospital in Tokyo, Japan. The records of the patients with syphilis whose RPR titer was measured twice before treatment (i.e., at diagnosis and at treatment initiation) were extracted for this study. Some of these patients were included in our earlier study [11]. An episode of syphilis was defined as an elevation of the RPR titer (≥8.0 R.U. [RPR unit]) and a positive T. pallidum latex agglutination test result (Sekisui Medical, Mediace RPR, and TPLA). If the baseline RPR was positive (i.e., having a history of syphilis), a more than fourfold increase in RPR titer was defined as a new infection.

Patients with confirmed neurosyphilis by CNS tests and/or ocular syphilis were excluded. The Japanese medical insurance system allows a maximum of 3 months to prescribe medication and, therefore, the interval between the routine outpatient visits is usually 1–3 months. Patients were excluded if their interval between the time of diagnosis and treatment initiation were outside this stipulated period (i.e., <15 days and >105 days). For patients with multiple episodes, only the most recent episode was included in the analysis.

Data collection

Information on the following variables were extracted from the clinical records: age, sex, history of syphilis, clinical stage of syphilis, RPR titer at diagnosis, RPR titer at treatment initiation, TPLA titer at treatment initiation, antibiotics, serological cure, HIV infection, CD4 counts, and HIV viral load. The RPR titer was assessed until it decreased by at least fourfold from the value recorded at treatment initiation. The RPR titer was followed until December 2019. Serological cure was defined as a fourfold decrease in the RPR titer within 12 and 24 months in early symptomatic syphilis and in latent syphilis, respectively.

Primary and secondary syphilis were classified as early symptomatic syphilis. Early latent syphilis was defined as asymptomatic syphilis acquired within 12 months. Early symptomatic syphilis and early latent syphilis were classified as early syphilis, and late latent syphilis and latent syphilis of unknown duration were classified as late syphilis.

The automated RPR test, which is commonly used in Japan and Korea [12,13], is a quantitative method for evaluating the agglutination reaction between anti-lipid antibodies and latex. The coefficient of variation (CV) of the conventional manual RPR tests is reported to be 25%–40%, whereas the CV of the automated RPR titer is reported to be 10%–13% around the cut-off RPR titer (1.0 R.U), and only <5% at higher levels [14]. Considering the CV of the automated RPR test, a ratio of RPR at treatment initiation/diagnosis of <80%, 80–120%, and >120% was defined as indicating a decreased-, stable-, and increased-RPR group, respectively.

This study was approved by the Institutional Committee on Research Ethics of our hospital (approval number: 2990). This study was conducted in an opt-out method on our hospital website to obtain informed consent.

Statistical analysis

First, the pre-treatment change in the RPR titer was illustrated using the ratio of RPR titer at treatment initiation/diagnosis. Second, we assessed whether the pre-treatment change in the RPR titer (i.e., the ratio of RPR measurements at treatment initiation/diagnosis) could affect the slope of the post-treatment relative change in the RPR titer. The ratio (i.e., relative change) of the RPR titer at post-treatment/treatment initiation was used for the analysis. The averaged monthly change in the RPR ratio (i.e., speed of the decrease in RPR) after treatment was calculated on the log2 scale for each patient by using the slope of linear approximation (i.e., log-linear was assumed). The mean of the monthly change in the RPR ratio was compared between the groups on the log2 scale using the Student’s t-test and antilog was used for describing the mean and 95% Confidence Intervals (CI). Univariable and multivariable linear regression analyses were conducted to assess the variables that affect the monthly change in RPR titer ratio. Variable selection was conducted by backward elimination using the P-values obtained with the Wald test. Statistical significance was defined as a 2-sided P-value <0.05. For highly correlated variables, one value was chosen based on clinical importance. Data analysis was performed using R version 4.0.2 and Stata MP 16.1 (StataCorp, TX, USA).

Results

Between January 2006 and December 2018, 395 patients experienced 522 syphilis infection (Fig 1). After excluding 25 neurosyphilis and/or ocular syphilis, 371 patients (93.9%, 371/395) had 497 syphilis infections (95.2%, 497/522). Among them, 94 patients (25.3%, 94/371) experienced 111 syphilis infections (22.3%, 111/497) in which RPR titer was tested twice before treatment: at diagnosis and at treatment initiation. Twenty-six patients were excluded because the gap between diagnosis and treatment initiation was outside the defined time period (<15 days, n = 1; >105 days, n = 25). A total of 68 patients were tested for RPR titer twice before treatment in 1–3 months. They experienced 79 such infection episodes and their last episodes were analyzed.

Fig 1. Flow chart of the study procedures.

Fig 1

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Abbreviations: RPR, Rapid plasma regain.

The interval between diagnosis and treatment initiation was 1, 2, and 3 months in 9 (13.2%), 39 (57.4%), and 20 patients (29.4%), respectively. The median age was 45 years (interquartile range [IQR], 38–50 years), and 98.5% (67/68) were men. Among them, 66 men were PWH. Thirty-eight patients (55.9%, 38/68) had a history of syphilis. Most patients were treated with oral or intravenous regimen (amoxicillin, n = 45; oral benzylpenicillin, n = 22; intravenous penicillin G, n = 1), because intramuscular benzathine penicillin G had not been authorized in the study period in Japan. The median RPR titer was 163 R.U. (IQR, 56–328 R.U.) at diagnosis and 132 R.U. (IQR, 62–252 R.U.) at treatment initiation. The median RPR titer ratio at treatment initiation/diagnosis was 0.87 (range, 0.11–44.25; IQR, 0.48–1.30). The RPR titer increased more than twofold in 17.6% (12/68) and fourfold in 11.8% (8/68), whereas it decreased more than twofold in 26.5% (18/68) and fourfold in 10.3% (7/68) of patients before treatment. After treatment, serological cure was achieved in all patients who were followed RPR titer for defined duration (58/58). RPR titers were not followed until serological cure in the rest.

Based on the change in the RPR titer before treatment, the patients were categorized into three groups: increased-RPR group (n = 20), stable-RPR group (n = 18), and decreased-RPR group (n = 30). The temporal changes in the RPR titer from before and after treatment for each patient are shown in Fig 2, and the characteristics of each group are shown in Table 1. The mean monthly change in the ratio of the RPR titer after treatment was 0.47-fold/month (95% confidence interval [CI], 0.35–0.63/month) on linear scale in the increased-RPR group (the reference group), 0.59-fold/month (95% CI, 0.46–0.76/month) in the stable-RPR group (P = 0.23), and 0.71-fold/month (95% CI, 0.65–0.78/month) in the decreased-RPR group (P = 0.002).

Fig 2. Temporal changes in the RPR titer from before to after treatment.

Fig 2

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The time course of the RPR titer from before to after treatment in each group is shown (2A, increased-RPR group; 2B, stable-RPR group; 2C, decreased-RPR group). Each line represents the time course of the RPR titer for each patient. The black line represents the average post-treatment RPR change in each group. Abbreviations: RPR, rapid plasma regain.

Table 1. Clinical characteristics of patients in the increased-, stable-, and decreased-RPR groups.

Increased-RPR group Stable-RPR group Decreased-RPR group
Characteristics n = 20 n = 18 n = 30
Age (years) 46 (41–50) 44 (40–51) 45 (37–51)
Male 20 (100%) 17 (94.4%) 30 (100%)
HIV infection 20 (100%) 16 (88.9%) 30 (100%)
CD4 counts (/μL) 508 (349–675) 496 (316–609) a 482 (370–605)
HIV-RNA <50 copies/mL 17 (85.0%) 14 (87.5%) a 26 (86.7%)
Stage of syphilis
    Early symptomatic 5 (25.0%) 4 (22.2%) 5 (16.7%)
    Early latent 14 (70.0%) 9 (50.0%) 20 (66.7%)
    Late latent 1 (5.0%) 1 (5.6%) 0 (0%)
    Late with unknown duration 0 (0%) 4 (22.2%) 5 (16.7%)
History of syphilis 12 (60.0%) 6 (33.3%) 20 (66.7%)
RPR titer at diagnosis (R.U.) 64 (19–261) 110 (76–262) 229 (92–359)
RPR titer at treatment initiation (R.U.) 212 (126–377) 118 (73–248) 103 (30–183)
TPLA at treatment initiation (T.U.) 12,045 (4,187–24,935) 7,985 (4,756–16,615) 7,619 (3,231–16,470)
Treatment
    Oral amoxicillin 14 (70.0%) 11 (61.1%) 20 (66.7%)
    Oral benzylpenicillin 5 (25.0%) 7 (38.9%) 10 (33.3%)
    Intravenous benzylpenicillin G 1 (5.0%) 0 (0%) 0 (0%)
Serological cure 20 (100%) 15 (100%) b 23 (100%)b
Mean monthly change in the ratio of the RPR titer after treatment c 0.47 (0.35–0.63) 0.59 (0.46–0.76) 0.71 (0.65–0.78)
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Data are presented as median (interquartile range) or number (percentage) unless otherwise specified.

a Only people living with HIV (n = 66) were analyzed.

b RPR titers were not followed until serological cure in 10 patients (RPR-stable group, n = 3; RPR-decreased group, n = 7).

c Mean (95% confidence interval) were calculated on log2 scale and are expressed in antilog.

Abbreviations: RPR, rapid plasma reagin; R.U., RPR unit; TPLA, Treponema pallidum latex-agglutination; T.U., titer unit.

The results of the linear regression are shown in Table 2. In the univariable analysis, higher age, HIV-RNA >50 copies/mL, late-stage syphilis, history of syphilis, and a lower ratio of RPR titer at treatment initiation/diagnosis were associated with larger slopes of post-treatment RPR, which means a slower post-treatment decrease (i.e., the downslope becoming closer to zero). In the multivariable analysis, higher age (predicted mean monthly change in the RPR ratio on the log2 scale 0.23/10 years [95% CI, 0.090–0.37], P = 0.002), history of syphilis (0.36 [95% CI, 0.07–0.65], P = 0.02) and a lower ratio of RPR at treatment initiation/diagnosis (−0.52/every 10-fold increase [95% CI, −0.81 to −0. 22], P = 0.001) were associated with a slower post-treatment decrease in RPR. Other variables were not associated with the post-treatment relative changes in RPR titer.

Table 2. Association between the slope of the ratio of the post-treatment RPR titer compared to the baseline titer and other characteristics using linear regression analysis.

Univariable Multivariable
Coefficient P-value Coefficient P-value
Age (per 10 years) 0.23 (0.067–0.39) 0.006 0.23 (0.090–0.37) 0.002
CD4 counts (per 100μL) 0.066 (−0.017 to 0.15) 0.12
HIV-RNA <50 copies/mL 0.67 (0.19–1.16) 0.008
Late syphilis* 0.37 (−0.09 to 0.82) 0.12
History of syphilis 0.45 (0.12–0.78) 0.008 0.36 (0.07–0.65) 0.02
RPR titer at diagnosis (per 10R.U.) 0.0060 (−0.00036 to 0.012) 0.06
RPR titer at treatment initiation (per 10R.U.) 0.0020 (−0.0078 to 0.012) 0.69
Ratio of RPR titer at treatment initiation/diagnosis (per every 10-fold increase) −0.57 (−0.90 to −0.25) 0.001 −0.52 (−0.81 to −0.22) 0.001
TPLA titer (per 100T.U.) 0.00054 (−0.00072 to 0.0018) 0.40
Amoxicillin therapy −0.27 (−0.63 to 0.08) 0.13
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Coefficients are shown in log2 scale. Abbreviations: RPR, rapid plasma reagin; R.U., RPR unit; TPLA, Treponema pallidum latex-agglutination; T.U., titer unit.

*compared with early syphilis.

Discussion

We have shown that the RPR titer could change dramatically within 1–3 months even without treatment. Surprisingly, the RPR titer decreased spontaneously by fourfold in 10% of patients in our cohort. Furthermore, the pre-treatment decreases in the RPR titer, in addition to the higher age and a history of syphilis were significantly associated with a slower post-treatment decline in the RPR titer.

Our data showed that the RPR titer could decrease more than fourfold before treatment, which means that the treatment success criteria could be achieved without treatment in some patients. In a rabbit model, non-treponemal titers increased after infection and then spontaneously decreased more than fourfold within 6 months even without treatment [8]. Even after the RPR titer became negative, however, viable T. pallidum was detected in the lymph nodes of rabbits. Therefore, even patients with low RPR titers need to receive antibiotic treatment if they have not received appropriate treatment.

Recently, Pandey K, et al. reported that RPR changed more than fourfold in 14.8% of syphilis patients within 14 days before treatment [10], despite the difference that most patients were non-PWH and the RPR card test (Becton Dickinson) was used in their study. In their study, fewer patients showed a fourfold decrease in pre-treatment RPR titer than those with an increase (increase, 12.3%; decrease, 2.5%), while they were similar in our study (increase, 11.8%; decrease, 10.3%). In the rabbit model, the spontaneous RPR decrease is much slower than the initial increase. The longer duration of the pre-treatment period in our study can be one reason for the higher proportion of patients with decreased titer observed in our study.

The strength of our study is that we evaluated the association between pre- and post-treatment RPR change, in addition to the description of pre-treatment changes in RPR titer. Of note, the lower ratio of the RPR titer at treatment initiation/diagnosis, rather than a lower RPR titer at treatment initiation, was associated with a slower post-treatment RPR decrease. This result is highly understandable if the natural course of RPR titer in humans is similar to that of rabbits (i.e., a bell-shaped curve): when the treatment is initiated during the increasing or apex of the bell-shaped curve (i.e., increased-RPR group and some patients in the stable-RPR group), the rate of post-treatment RPR decrease is expected to be faster, because the RPR titer decreases spontaneously even without treatment; however, when the treatment is initiated during the decreasing or right-tail of the curve (i.e., stable-RPR group and decreased-RPR group), the rate of the post-treatment RPR decrease is expected to be slower. However, it is impossible to ascertain the pre-treatment RPR change in most cases who are treated promptly. Thus, even if the RPR titer, stage, and age at treatment initiation were the same, the speed of the post-treatment RPR decrease could be completely different depending on the timing from the infection. Generally, a slow post-treatment RPR decrease is used as an indicator of treatment failure, mainly due to neurosyphilis, but there is little evidence [9]. Future studies are needed to determine whether clinicians should evaluate neurosyphilis in all patients with a slow decrease in post-treatment RPR.

It has been reported that the slow RPR decrease or serological cure after treatment is associated with various factors, such as higher age, a more advanced stage of syphilis, prior history of syphilis, and lower baseline RPR titers [15]. Among them, higher age was associated with a slower RPR decrease in our multivariable model, too. Based on our hypothesis, it is reasonable that the advanced stage of syphilis, in which RPR titers have already decreased spontaneously, is associated with slow RPR decrease after treatment, but we did not find a statistically significant association between the advanced stage and slow RPR decrease in the present study. It could be attributed to the limited number of late syphilis, comprising only two patients with definite late-stage latent syphilis, while nine patients had syphilis with unknown duration. Similar to the present study, prior history of syphilis was associated with a slower RPR response in some studies, indicating potential differences in post-treatment RPR change between initial infection and reinfection [15–17]. A lower baseline RPR titer is a well-known risk factor for slower RPR decrease after treatment [15,17], but pre-treatment RPR decrease was more significant than baseline RPR titer itself in the multivariable analysis in our study. A lower baseline RPR titer might be a result of a pre-treatment RPR decrease.

HIV infection also known to affect the baseline RPR titer and its response after treatment. For example, HIV is a major risk factor for serofast (i.e., more than four-fold decrease in RPR titer but still testing positive), resulting in higher baseline RPR titers in repeated infection in PWH [17–19]. Biological false-positives are also common in PWH due to the production of antiphospholipid antibodies caused by dysregulated B cell activation [19]. Most patients were PWH in our study and more than half had a history of syphilis, so these backgrounds might affect the baseline RPR titer and the speed of RPR decrease. Immunocompromised status, such as CD4 <350/μL and untreated HIV also reported as risk of serofast [20,21]. Although HIV-RNA <50 copies/mL was associated with a slow RPR decrease in our univariable analysis, it is counterintuitive that HIV-uncontrolled patients had a better treatment course for syphilis [21]. This apparent association between HIV-RNA and RPR change was probably confounded by age and the history of syphilis in our cohort.

It is not ideal that many patients were not treated on the date of diagnosis in our hospital (15.1% of total infections, 79/522). This percentage was not so different from the previous study (15.6%, 766/4903) [10]. When PWH are diagnosed with latent syphilis by screening blood tests, they usually leave the hospital before the results become available. Although PWH do not miss their syphilis treatment as they are scheduled for their next visit, to prevent progression of the disease and the potential spread of infection [9], clinicians should contact them for prompt treatment even if they are asymptomatic. In addition, we should reevaluate the RPR titer, because it might be different from diagnosis.

There are several limitations of our study. First, we used an automated RPR test, which is not a global standard. The automated RPR test "Mediace" used in this study correlated strongly with the conventional manual RPR card test [13]. Mediace automated RPR has been reported to be less sensitive when titers are low [14,22], but the titer in this study was high enough in most cases. Compared to the conventional manual RPR tests, the seroconversion rate after 12 months of treatment in Mediace RPR tests was almost the same, but the Mediace RPR titer was reported to decrease often faster [13,22]. Future studies are needed to determine whether our results are reproducible when the conventional manual method is used. Second, because most patients were PWH in the present study, serological response could have been different from non-PWH as discussed above. While the guidelines state that there is no need to change the criteria for treatment success for PWH [9], they point out that nontreponemal titer may decrease slower. Third, this was a retrospective study that followed the RPR titer for only 1–3 months. To reveal the natural course of RPR, it is required to follow patients with syphilis without treatment for a long time prospectively, but this is ethically unacceptable. Fourth, the treatment of non-neurosyphilis in Japan is different from that in many other countries. Intramuscular benzathine penicillin G, the gold standard for the treatment of syphilis, was not authorized in Japan until 2021. Therefore, the patients treated by intramuscular benzathine penicillin G were not included in this study. In Japan, oral benzylpenicillin was previously used as an alternative to intramuscular benzathine penicillin G in the 2000s, and was gradually replaced by oral amoxicillin in Japan because of the low bioavailability of oral benzylpenicillin. In previous studies, oral amoxicillin showed approximately 95% efficacy for non-neurosyphilis, and approximately 90% efficacy for even late-stage latent syphilis [11,23]. The slope of the post-treatment RPR titer in our study might be different from that after treatment with intramuscular penicillin G. Fifth, we did not perform CSF tests for patients with slower post-treatment RPR response to rule out the asymptomatic neurosyphilis. However, in general, a higher RPR titer is a risk factor for neurosyphilis [24,25], so it is unreasonable to hypothesize that a patient with a decreasing RPR titer is more likely to have neurosyphilis. Whether the slow RPR decrease is useful in predicting neurosyphilis requires further study. Sixth, there is a possibility that patients received antibiotics before our treatment. However, because antibiotics are not available over the counter without a prescription in Japan, self-treatment is almost impossible. Furthermore, approximately 80% of participants were asymptomatic syphilis patients; therefore, there was no reason for them to take antibiotics for syphilis by themselves. Although there is a possibility that they received antibiotics for other reasons in other hospitals, it is difficult to explain the high percentage of patients with decreased pre-treatment RPR titers.

Conclusions

Approximately 10% of patients with syphilis achieved “serological cure”, based on the RPR titer, before treatment. When we cannot start treatment on the day of diagnosis for some reasons, we should reevaluate the RPR titer and, even if RPR titer decreases, should treat syphilis when patients have not been treated before. Higher age, a history of syphilis, and a lower rate of RPR titer at treatment initiation and diagnosis were associated with a slower post-treatment RPR decrease. Therefore, even if patients have the same characteristics and the same RPR titer at treatment initiation, the rate of RPR decrease after treatment could be quite different. Because our results depend on the automated RPR tests from PWH treated with oral regimen, further studies are desired for the generalizability of our findings.

Supporting information

S1 File. * months from treatment initiation.

RPR ratio compared with RPR at treatment initiation is shown.

(XLSX)

Click here for additional data file. (26.2KB, xlsx)

Acknowledgments

We would like to thank Editage (www.editage.com) for English language editing.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

The authors received no specific funding for this work.

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Decision Letter 0

Antonella Marangoni

12 Apr 2023

PONE-D-22-35264The natural course of nontreponemal antibody titers in patients with syphilis before treatment: A retrospective cohort studyPLOS ONE

Dear Dr. Fukushima,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Dear Authors, Your study was found interesting but I agree with the first referee that it has important and severe limitations. In particular, all the statements included in the text are based on the use of Mediace aRPR and Sekure RPR. Both methods require improvement before they can be used to diagnose syphilis or evaluate treatment efficacy in clinical practice. Second, some statements should be mitigated as they sound at least hazardous (in some cases, […], the elevation of nontreponemal titer was identified […] and treatment may be initiated at the next visit to the hospital after a few months). Third, the point that has raised the greatest concerns: all the post treatment statements are based on the routinary use of other antibiotics than benzathine penicillin G which is the first line therapy and gold standard for treatment of early syphilis according to current guidelines. This should be discussed and data about the use of standard benzathine penicillin G should be added Finally, the title and abstract should be modified.

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PLOS ONE

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At this time, please address the following queries:

a)        Please clarify the sources of funding (financial or material support) for your study. List the grants or organizations that supported your study, including funding received from your institution.

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Additional Editor Comments:

Dear Authors,

Your study was found interesting but I agree with the first referee that it has important and severe limitations.

In particular, all the statements included in the text are based on the use of Mediace aRPR and Sekure RPR. Both methods require improvement before they can be used to diagnose syphilis or evaluate treatment efficacy in clinical practice.

Second, some statements should be mitigated as they sound at least hazardous (in some cases, […], the elevation of nontreponemal titer was identified […] and treatment may be initiated at the next visit to the hospital after a few months).

Third, the point that has raised the greatest concerns: all the post treatment statements are based on the routinary use of other antibiotics than benzathine penicillin G which is the first line therapy and gold standard for treatment of early syphilis according to current guidelines.

This should be discussed and data about the use of standard benzathine penicillin G should be added

Finally, the title and abstract should be modified

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Title of the article:

The natural course of nontreponemal antibody titers in patients with syphilis before treatment: A retrospective cohort study

Reference number:

PONE-D-22-35264

Comments:

Thank you for the opportunity to review your interesting manuscript. I enjoyed reading it as I consider the topic of this manuscript of great relevance. I have to say that the title might be misleading, and some statement should be mitigated throughout the text as serology in syphilis is a tricky issue (see Major Compulsory Revisions). The abstract should be carefully reorganized as the aims are not explicit and the results are not fully coherent with the title of the text (see Major Compulsory Revisions). Moreover, the conclusion paragraph of the manuscript is missing (see Major Compulsory Revisions). The main issue here is the use of Mediace automated RPR (aRPR) and Sekure rapid plasma reagin (RPR-S) (Sekisui Diagnostics) which accuracy and sensitivity require improvement before they can be used to diagnose syphilis and evaluate treatment efficacy in clinical practice (see Major Compulsory Revisions). A thorough focus in regards of the limitations of the tests used should be included to make this article suitable for publication.

- Major Compulsory Revisions

Line 2-3: Please, consider modifying the title of your manuscript. The title of the text is “The natural course of nontreponemal antibody titers in patients with syphilis before treatment: A retrospective cohort study”. However, 98.5% (67/68) are men and, moreover, 97.1% (66/68) are PLWH (people living with HIV). Additionally, a consistent part of the manuscript regards the serological response after treatment. It is known that PLWH are considered a special population in regards of their serological response [Ren M. AIDS. 2020].

Line 40-41: Please, consider explicating your aims and purposes in the abstract introduction. Moreover, a consistent part of your manuscript regards the serological response after treatment in a special population. Please, include this aspect in your aims part here.

Line 56-58: The abstract conclusion part might be misleading as the presented results might not be applicable to the general population. In your manuscript there is no evidence that more reliable markers of treatment efficacy are required in patients with syphilis. Please, try to mitigate your statement as 97.1% (66/68) of the sample considered are PLWH.

Line 74: Please, try to mitigate this sentence. Currently, various treponemal-specific immunoassays are increasingly being used for syphilis screening (reverse approach) including EIAs, CIAs, MBIAs, among others. These assays can be automated and are relatively nonspecific. For this reason, a nontreponemal test (eg, RPR) is used as a confirmatory test on initially reactive specimens [Park IU, Clin Infect Dis. 2020]

Line 89-93: Writing this sentence might be hazardous. As international guidelines suggest at least an annual screening for syphilis in PLWH, it is undoubtedly true that a non-treponemal titer elevation might occur unexpectedly. However, starting syphilis treatment “few months” after the prove of an infection might be considered at least hazardous as the patient is contagious and a danger for himself and the population. I would rather re-write this sentence pointing out your efforts in treating as soon as possible new cases of syphilis enlightening that, occasionally (due to the difficulty in contacting patients to schedule a treatment visit) few months might elapse between the diagnosis and treatment.

Line 104-106: The main issue here is the use of Mediace automated RPR (aRPR) and Sekure rapid plasma reagin (RPR-S) (Sekisui Diagnostics). These tests present the advantage of being automated, but Mediace aRPR has the disadvantage of a poor sensitivity in low titers [Leroy AG, Diagn Microbiol Infect Dis. 2022] and the sensitivity and accuracy of the RPR-S test requires improvement before it can be used to diagnose syphilis and evaluate treatment efficacy in clinical practice [Osbak K, J Med Microbiol. 2017]. In the discussion (line 195-200) the authors point out the pros of using aRPR. However, a thorough focus in regards of the limitations of the tests used must be included to make this article suitable for publication.

Line 180: Please, it is crucial to report the descriptive analysis of the treatment part. In other words, it is essential to explicit that none of the patient received intramuscular benzathine penicillin G (which is, as you stated in the discussion part, the gold standard for treatment of early syphilis).

Line 272-278: The conclusion part is missing. Please, insert this paragraph in your manuscript. Please, mitigate the statements in this paragraph in light of the limitation of the study (non-treponemal assays used, antimicrobial therapy administered, etc).

- Minor Essential Revisions

Line 70-72: It has been recently proven that polymerase chain reaction (PCR) tests might detect a T. pallidum infection even in individuals with a negative diagnostic serological test for syphilis [Junejo MH, Sex Transm Infect. 2022]. Perhaps it is not correct to state that nontreponemal tests are used for monitoring treatment efficacy “as a result” of a PCR low sensitivity especially thinking about early or primary syphilis. Please, try to re-organize these sentences pointing out the several pros of using non treponemal tests in clinical practice, according to the current guidelines.

Line 80-82: The popular study cited [Zhou P, Sex Transm Infect. 2012] has several limitations as the authors stated in their manuscript. I would rather enlighten in the text that the analysis cited is a case series in which multiple different antimicrobial regimens have been used to treat secondary syphilis.

Line 99: Please, clarify the methods used to rule out neurosyphilis. Please, explain in this paragraph how a diagnosis of “non-neurosyphilis” has been made.

Line 105: Please, make explicit the abbreviation and acronyms never used before in the manuscript.

Line 161-163: Among the 68 included, please, enlighten in the text how many patients had an history of syphilis (as you have already done in Table 1). This is crucial as serological trend in case of reinfection, especially among PLWH, might be anomalous [Marchese V, J Clin Med. 2022].

Line 169-171: Please, clarify this sentence.

Level of interest:

This is an interesting work as serological trend in syphilis infection is a tricky issue which requires further studies to better understand both the serological natural course and the serological response to treatment.

Reviewer #2: This interesting paper retrospectively evaluated the natural course of RPR titers in patients with syphilis before treatment, and also identified the association between changes in RPR titers before treatment and that after treatment. The data on natural course of RPR titers are limited since performing a prospective study is unethical. Through analyzing RPR testing results at diagnosis and at treatment initiation, the study found that RPR titers can spontaneously decrease before treatment and post-treatment decrease was slower in patients whose titers decreased before treatment. I have some minot comments as follows.

Minor comments:

Results:

1. Page 9, Line 164: The abbreviation "R.U." should be defined upon first appearance.

2. A recently published study (Pandey K et al. Clin Infect Dis. 2023;76(5):795-799.) showed 2.5% patients had ≥4-Fold decrease and 12.3% patients had ≥4-Fold increase in RPR titers before treatment. The differences between the 2 studies could be discussed in the Discussion section.

3. Page 10, Line 170: The authors claimed that "RPR titers were not followed until serological cure in 10 patients; however, based on the predicted change of the posttreatment RPR titer, it was expected that all patients would achieve serological cure." How do the authors perform the prediction?

Discussion:

4. Typo: page 11, line 194: "were" significantly associated with...

5. Page 11, Line 203: The included patients might still receive antibiotics with activity against T. pallidum from other hospitals/clinics for other reasons (e.g. pharyngitis, pneumonia, cellulitis...). If information on medication use from other hospitals/clinics are not available, the bias should be stated in the limitation.

6. Page 13: The association between posttreatment RPR decrease and history of syphilis was not discussed.

7. Page 14, Line 257: The reason of unavailability of IM BPG in Japan could be provided in short or with a cited reference.

8. Page 14, Line 264: The treatment efficacy of BPG enhanced with oral amoxicillin vs BPG alone was compared in a RCT conducted by Rolfs RT et al, which showed similar efficacy between the 2 treatment groups (83% vs 82%).

Tables:

9. Table 2: The coefficient and p values in multivariable analysis were different from that in the manuscript text.

10. Table 2: The reason that the variable "HIV-RNA <50 copies/mL" was not selected in the multivariable analysis should be stated.

**********

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

**********

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PLoS One. 2023 Sep 28;18(9):e0292044. doi: 10.1371/journal.pone.0292044.r002

Author response to Decision Letter 0

20 May 2023

Editor comments to the author

1. In particular, all the statements included in the text are based on the use of Mediace aRPR and Sekure RPR. Both methods require improvement before they can be used to diagnose syphilis or evaluate treatment efficacy in clinical practice.

Response: Thank you for your important advice. As you mentioned, automated RPR is not a global standard method for diagnosis and evaluation of treatment efficacy. We used Mediace RPR method in this study. According to the reviewers’ comments, we revised the title, abstract and discussions to clear the limitations of aRPR. Please see our reply to the seventh comment from reviewer 1.

2) Second, some statements should be mitigated as they sound at least hazardous (in some cases, […], the elevation of nontreponemal titer was identified […] and treatment may be initiated at the next visit to the hospital after a few months).

Response: Thank you for your comment. We deleted potentially hazardous description and mitigated this expression. Please see our reply to the sixth comment from reviewer 1.

3) Third, the point that has raised the greatest concerns: all the post treatment statements are based on the routinary use of other antibiotics than benzathine penicillin G which is the first line therapy and gold standard for treatment of early syphilis according to current guidelines. This should be discussed and data about the use of standard benzathine penicillin G should be added.

Response: Thank you for pointing out an important problem. In Japan, intramuscular penicillin G became authorized only in November 2021. Amoxicillin or doxycycline had been the first-line treatment for early syphilis recommended by Japanese guidelines. We don’t have much data of post-treatment RPR change after intramuscular penicillin G use in Japan. Please also see our reply to the eighth and nineth comments from reviewer 1 and the seventh and eighth comments from reviewer 2.

5) Finally, the title and abstract should be modified

Response: Thank you for your advice. We revised the title and abstract as follows: “Changes in rapid plasma reagin titers in patients with syphilis before and after treatment: a retrospective cohort study in an HIV/AIDS referral hospital in Tokyo.”

Reviewer 1

1. I have to say that the title might be misleading, and some statement should be mitigated throughout the text as serology in syphilis is a tricky issue (see Major Compulsory Revisions). The abstract should be carefully reorganized as the aims are not explicit and the results are not fully coherent with the title of the text (see Major Compulsory Revisions). Moreover, the conclusion paragraph of the manuscript is missing (see Major Compulsory Revisions). The main issue here is the use of Mediace automated RPR (aRPR) and Sekure rapid plasma reagin (RPR-S) (Sekisui Diagnostics) which accuracy and sensitivity require improvement before they can be used to diagnose syphilis and evaluate treatment efficacy in clinical practice (see Major Compulsory Revisions). A thorough focus in regards of the limitations of the tests used should be included to make this article suitable for publication.

Response: Thank you for your important and helpful comments. According to your comments, we carefully revised title, abstract and manuscripts as below.

- Major Compulsory Revisions

2. Line 2-3: Please, consider modifying the title of your manuscript. The title of the text is “The natural course of nontreponemal antibody titers in patients with syphilis before treatment: A retrospective cohort study”. However, 98.5% (67/68) are men and, moreover, 97.1% (66/68) are PLWH (people living with HIV). Additionally, a consistent part of the manuscript regards the serological response after treatment. It is known that PLWH are considered a special population in regards of their serological response [Ren M. AIDS. 2020].

Response: Thank you very much for your advice. We agree with your comment and changed the title to “Changes in rapid plasma reagin titers in patients with syphilis before and after treatment: a retrospective cohort study in an HIV/AIDS referral hospital in Tokyo.”

3. Line 40-41: Please, consider explicating your aims and purposes in the abstract introduction. Moreover, a consistent part of your manuscript regards the serological response after treatment in a special population. Please, include this aspect in your aims part here.

Response: Thank you for your advice. We clarified our objective of this study as the association between pre- and post-treatment changes in automated RPR (title, Lines 38-41, 56-58, 80-81, 89-92 in the clear version).

4. Line 56-58: The abstract conclusion part might be misleading as the presented results might not be applicable to the general population. In your manuscript there is no evidence that more reliable markers of treatment efficacy are required in patients with syphilis. Please, try to mitigate your statement as 97.1% (66/68) of the sample considered are PLWH.

Response: Thank you for your important comment. We revised title and abstract accordingly to clarify that this study used the data collected at an HIV/AIDS referral hospital (title, Lines 42-44, 56-58 in the clear version). We also added this point to the Limitations (lines 254-264 in the clear version).

5. Line 74: Please, try to mitigate this sentence. Currently, various treponemal-specific immunoassays are increasingly being used for syphilis screening (reverse approach) including EIAs, CIAs, MBIAs, among others. These assays can be automated and are relatively nonspecific. For this reason, a nontreponemal test (eg, RPR) is used as a confirmatory test on initially reactive specimens [Park IU, Clin Infect Dis. 2020]

Response: Thank you for your comment. The sentence is misleading and the specificity of non-treponemal and treponemal tests is not necessarily important in this context, so we removed this sentence (Line 70 in the clear version).

6. Line 89-93: Writing this sentence might be hazardous. As international guidelines suggest at least an annual screening for syphilis in PLWH, it is undoubtedly true that a non-treponemal titer elevation might occur unexpectedly. However, starting syphilis treatment “few months” after the prove of an infection might be considered at least hazardous as the patient is contagious and a danger for himself and the population. I would rather re-write this sentence pointing out your efforts in treating as soon as possible new cases of syphilis enlightening that, occasionally (due to the difficulty in contacting patients to schedule a treatment visit) few months might elapse between the diagnosis and treatment.

Response: Thank you for your helpful advice. We emphasized that clinicians should ideally start treatment as soon as the diagnosis was made, in the introduction section (Lines 82-89 in the clear version). In addition, according to your advice, we added a paragraph that enlightens that treatment often delays in the real world and that treatment should be initiated as soon as possible (Lines 246-253 in the clear version).

7. Line 104-106: The main issue here is the use of Mediace automated RPR (aRPR) and Sekure rapid plasma reagin (RPR-S) (Sekisui Diagnostics). These tests present the advantage of being automated, but Mediace aRPR has the disadvantage of a poor sensitivity in low titers [Leroy AG, Diagn Microbiol Infect Dis. 2022] and the sensitivity and accuracy of the RPR-S test requires improvement before it can be used to diagnose syphilis and evaluate treatment efficacy in clinical practice [Osbak K, J Med Microbiol. 2017]. In the discussion (line 195-200) the authors point out the pros of using aRPR. However, a thorough focus in regards of the limitations of the tests used must be included to make this article suitable for publication.

Response: Thank you for your important comment. We modified the title and abstract to indicate that we used an automated RPR test. We agree that these automated RPR tests are controversial and not necessarily accepted globally. Therefore, we have moved the discussion of automated RPR tests to the limitation section from the section on the strength of this study (Title, Lines 43, 254-261 in the clear version).

8. Line 180: Please, it is crucial to report the descriptive analysis of the treatment part. In other words, it is essential to explicit that none of the patient received intramuscular benzathine penicillin G (which is, as you stated in the discussion part, the gold standard for treatment of early syphilis).

Response: Thank you for your advice. In Japan, unfortunately, intramuscular benzathine penicillin G became available only in November 2021, and oral amoxicillin or doxycycline was used for syphilis except neurosyphilis. This was the reason why none of the patients in our study were treated with intramuscular benzathine penicillin G. Although the lack of use of this global first-line treatment is a limitation of our study, we believe that this will not affect our findings on pre-treatment changes in RPR (Lines 267-276 in the clear version). We added this point to the results section (Lines 164-167 in the clear version).

9. Line 272-278: The conclusion part is missing. Please, insert this paragraph in your manuscript. Please, mitigate the statements in this paragraph in light of the limitation of the study (non-treponemal assays used, antimicrobial therapy administered, etc).

Response: Thank you for your important comment. We made the conclusion section, and mitigated the expression. We removed the sentence that the RPR test is unreliable for determining treatment efficacy. We also added the limitations to the conclusion section (Lines 289-298 in the clear version).

- Minor Essential Revisions

10. Line 70-72: It has been recently proven that polymerase chain reaction (PCR) tests might detect a T. pallidum infection even in individuals with a negative diagnostic serological test for syphilis [Junejo MH, Sex Transm Infect. 2022]. Perhaps it is not correct to state that nontreponemal tests are used for monitoring treatment efficacy “as a result” of a PCR low sensitivity especially thinking about early or primary syphilis. Please, try to re-organize these sentences pointing out the several pros of using non treponemal tests in clinical practice, according to the current guidelines.

Response: Thank you for your advice. We revised the explanation of nontreponemal tests. We removed the misleading description of PCR tests. We emphasized the strength of nontreponemal tests in this paragraph (Lines 70-72 in the clear version).

11. Line 80-82: The popular study cited [Zhou P, Sex Transm Infect. 2012] has several limitations as the authors stated in their manuscript. I would rather enlighten in the text that the analysis cited is a case series in which multiple different antimicrobial regimens have been used to treat secondary syphilis.

Response: Thank you for your comment. According to your advice, we chose not to emphasize the inaccuracy of RPR as a method of determining treatment efficacy. Therefore, we removed this reference (Line 80).

12. Line 99: Please, clarify the methods used to rule out neurosyphilis. Please, explain in this paragraph how a diagnosis of “non-neurosyphilis” has been made.

Response: Thank you for your important comment. As you pointed out, “non-neurosyphilis” is not a widely used term. We added the confirmed neurosyphilis and/or ocular syphilis as the exclusion criteria. We also revised the study flow chart (Lines 106, 154-155 in the clear version, Figure 1).

13. Line 105: Please, make explicit the abbreviation and acronyms never used before in the manuscript.

Response: Thank you for pointing it out. We added the abbreviation (Line 102 in the clear version, Table 1, Table 2)

14. Line 161-163: Among the 68 included, please, enlighten in the text how many patients had an history of syphilis (as you have already done in Table 1). This is crucial as serological trend in case of reinfection, especially among PLWH, might be anomalous [Marchese V, J Clin Med. 2022].

Response: Thank you for your comments. We added this point to the manuscript (Lines 163-164 in the clear version).

15. Line 169-171: Please, clarify this sentence.

Response: Thank you for your comment. The problems in this sentence were also pointed out by reviewer 2. We expected it from the log2 slope of RPR decrease, but it is not a usual way to predict the treatment success. Because it is misleading, we removed this sentence (Line 173 in the clear version).

Reviewer 2

Minor comments:

1. Page 9, Line 164: The abbreviation "R.U." should be defined upon first appearance.

Response: Thank you for your comment. “R.U.” is RPR unit, and “T.U.” is titer unit. We added the abbreviation (Line 102 in the clear version, Table 1, Table 2).

2. A recently published study (Pandey K et al. Clin Infect Dis. 2023;76(5):795-799.) showed 2.5% patients had ≥4-Fold decrease and 12.3% patients had ≥4-Fold increase in RPR titers before treatment. The differences between the 2 studies could be discussed in the Discussion section.

Response: Thank you for your advice. We have already read this recently published article after the initial submission of our article. Study design of this study is similar to ours, but we analyzed longer pre-treatment periods (1-3 months vs < 14 days). Another difference and the novelty of our study is that we analyzed the association with pre- and pos-treatment RPR change. Patient characteristics (low proportion of PWH) and RPR method (conventional manual method) were different, too. We added a new paragraph to explain these differences (Lines 206-213).

3. Page 10, Line 170: The authors claimed that "RPR titers were not followed until serological cure in 10 patients; however, based on the predicted change of the posttreatment RPR titer, it was expected that all patients would achieve serological cure." How do the authors perform the prediction?

Response: Thank you for your pointing out. We have removed this sentence as we have received the same comment from reviewer 1. Please see also our reply to the 15th comment from reviewer 1.

4. Typo: page 11, line 194: "were" significantly associated with...

Response: Thank you for your comment. We revised this sentence (Line 198 in the clear version).

5. Page 11, Line 203: The included patients might still receive antibiotics with activity against T. pallidum from other hospitals/clinics for other reasons (e.g. pharyngitis, pneumonia, cellulitis...). If information on medication use from other hospitals/clinics are not available, the bias should be stated in the limitation.

Response: Thank you for your comment. The lack of information on the use of antibiotic use outside of our hospital is an important limitation, even though antibiotics are not available over the counter in Japan and it is not very likely to receive oral antibiotics for such a long time for minor infections. We added this point to the limitation (Lines 281-287 in the clear version).

6. Page 13: The association between posttreatment RPR decrease and history of syphilis was not discussed.

Response: Thank you for your comments. We added the discussion (Lines 240-242) as follows: “Prior history of syphilis, associated with a slower RPR response in the present study, was associated in some studies but not in others.”

7. Page 14, Line 257: The reason of unavailability of IM BPG in Japan could be provided in short or with a cited reference.

Response: Thank you for your comment. We unfortunately could not find out the exact reason why IM BPG was withdrawn from the market in 1980s and had not been available until late 2021 in Japan. Some believe it is because a well-known person died of anaphylactic shock from IM BPG, but there are no detailed references.

8. Page 14, Line 264: The treatment efficacy of BPG enhanced with oral amoxicillin vs BPG alone was compared in a RCT conducted by Rolfs RT et al, which showed similar efficacy between the 2 treatment groups (83% vs 82%).

Response: Thank you for your comment. This sentence is not relevant, so we have corrected this sentence in order to avoid making unnecessary confusions (Line 275 in the clear version).

Tables:

9. Table 2: The coefficient and p values in multivariable analysis were different from that in the manuscript text.

Response: Thank you for your careful review. We corrected the manuscript (Lines 187-189 in the clear version).

10. Table 2: The reason that the variable "HIV-RNA <50 copies/mL" was not selected in the multivariable analysis should be stated.

Response: Thank you for your important point. In the univariable analysis, HIV-RNA <50 copies/ml was significantly associated with slower RPR decline after treatment. However, as clearly mentioned in the Methods (Lines 147-148 in the clear version), variable selection for the multivariable model was conducted by backward elimination using the P-values obtained with the Wald test. HIV-RNA <50 copies/mL became not significant in the multivariable model, therefore, it was dropped from the final multivariable model. In the previous studies, RPR responses were slower in HIV-untreated patients (who were supposedly with higher HIV-RNA). In our study, HIV-untreated patients were younger and had less history of syphilis, which were likely to confound the univariable results for HIV-RNA <50 copies/mL. We added a brief explanation to the results section (Lines 190-191 in the clear version).

The authors have no conflicting financial interests, all authors concur with the submission and have contributed to this work, and the material submitted has not been previously reported nor is under consideration for publication elsewhere.

Sincerely,

Kazuaki Fukushima, M.D., Ph.D.

Attachment

Submitted filename: Response to reviewers_RPR_revised manuscript.doc

Click here for additional data file. (73KB, doc)

Decision Letter 1

Antonella Marangoni

21 Jun 2023

PONE-D-22-35264R1Changes in rapid plasma reagin titers in patients with syphilis before and after treatment: a retrospective cohort study in an HIV/AIDS referral hospital in TokyoPLOS ONE

Dear Dr. Fukushima,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

The revised version has been appreciated but I strongly sugggest to update the references' list and to improve the discussion

==============================

Please submit your revised manuscript by Aug 05 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Antonella Marangoni, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments:

The referee appreciated the revised version of the manuscript and I agree with the few comments.

In particular, you should update the bibliography as suggested and expand the discussion by including also other clinical conditions that might influence the serological response.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

**********

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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Reviewer's report

Title of the article

Changes in rapid plasma reagin titers in patients with syphilis before and after treatment: a retrospective cohort study in an HIV/AIDS referral hospital in Tokyo

Reference number

PONE-D-22-35264R1

Comments:

Thank you for the opportunity to review again your interesting manuscript. I enjoyed reading it as I feel the manuscript improved and suitable for publication. I recommend updating the bibliography (see Major Compulsory Revisions), and expanding the discussion in certain points (see Major Compulsory Revisions).

- Major Compulsory Revisions

Line 64-66: Please, try to update the bibliography as, for instance, reference [2] goes back to 1968. There are several most recent manuscripts that can be cited here [Tiecco G, Pathogens. 2021 or Ramchandani MS, Infect Dis Clin North Am. 2023].

Line 154-160: Please, add percentage in the main text in order to make data more easily comparable.

Line 232-236: Please, expand the discussion by including also other clinical conditions that might influence the serological response, and, if it is possible, compare your data to the available literature. Post-treatment RPR might be different in case of first infection treatment or syphilis reinfection management [Marchese V, J Clin Med. 2022]. Moreover, several atypical conditions might exist especially in PLWH such as serological-non-responder or serofast status [Seña AC, BMC Infect Dis. 2015 or Ghanem KG, Sex Transm Dis. 2021].

- Minor Essential Revisions

None

Level of interest

This is an interesting work as serological trend in syphilis infection is a tricky issue which requires further studies to better understand both the serological natural course and the serological response to treatment.

Quality of written English

English syntax is optimal.

Statistical review

This is a methodologically rigorous work.

**********

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Reviewer #1: No

**********

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PLoS One. 2023 Sep 28;18(9):e0292044. doi: 10.1371/journal.pone.0292044.r004

Author response to Decision Letter 1

3 Jul 2023

Reviewer 1

1. Line 64-66: Please, try to update the bibliography as, for instance, reference [2] goes back to 1968. There are several most recent manuscripts that can be cited here [Tiecco G, Pathogens. 2021 or Ramchandani MS, Infect Dis Clin North Am. 2023].

Response: Thank you for your advice. We have added the reference you suggested (Line 66).

2. Line 154-160: Please, add percentage in the main text in order to make data more easily comparable.

Response: Thank you for your advice. We have added the percentage of included patients (Line 155-158).

3. Line 232-236: Please, expand the discussion by including also other clinical conditions that might influence the serological response, and, if it is possible, compare your data to the available literature. Post-treatment RPR might be different in case of first infection treatment or syphilis reinfection management [Marchese V, J Clin Med. 2022]. Moreover, several atypical conditions might exist especially in PLWH such as serological-non-responder or serofast status [Seña AC, BMC Infect Dis. 2015 or Ghanem KG, Sex Transm Dis. 2021].

Response: Thank you for your valuable comments. We have added an explanation addressing the potential impact of prior syphilis infection on post-treatment RPR change and the potential influence of serofast and serological non-responder, which are commonly observed in HIV patients. We have also added the references you suggested (Lines 242-246).

Attachment

Submitted filename: Response_230703.doc

Click here for additional data file. (33KB, doc)

Decision Letter 2

Antonella Marangoni

18 Jul 2023

PONE-D-22-35264R2Changes in rapid plasma reagin titers in patients with syphilis before and after treatment: a retrospective cohort study in an HIV/AIDS referral hospital in TokyoPLOS ONE

Dear Dr. Fukushima,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The manuscript has definitely improved, but some further modifications are still necessary.

In particular, I think that the discussion should be improved. 

Please submit your revised manuscript by Sep 01 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Antonella Marangoni, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments:

Dear authors

The manuscript has definitely improved, but some further modifications are still necessary.

In particular, I think that the discussion should be improved. Moreover, please fulfill all the requests of the referee

[Note: HTML markup is below. Please do not edit.]

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2023 Sep 28;18(9):e0292044. doi: 10.1371/journal.pone.0292044.r006

Author response to Decision Letter 2

1 Aug 2023

Reviewer 1

Comment 3. Line 232-236: Please, expand the discussion by including also other clinical conditions that might influence the serological response, and, if it is possible, compare your data to the available literature. Post-treatment RPR might be different in case of first infection treatment or syphilis reinfection management [Marchese V, J Clin Med. 2022]. Moreover, several atypical conditions might exist especially in PLWH such as serological-non-responder or serofast status [Seña AC, BMC Infect Dis. 2015 or Ghanem KG, Sex Transm Dis. 2021].

Based on this comment, we revised the discussion section. In this latest revision, we have focused on the factors that influence the speed of RPR decrease after treatment and conducted a thorough comparison of our study with relevant references (Lines 234-255). Additionally, we have added a new paragraph about the complex effect of HIV on RPR dynamics based on provided references (Lines 256-265).

Thank you once again for your careful consideration of our work. We look forward to receiving further guidance from you regarding the next steps in the publication process.

The authors have no conflicting financial interests, all authors concur with the submission and have contributed to this work, and the material submitted has not been previously reported nor is under consideration for publication elsewhere.

Sincerely,

Kazuaki Fukushima, M.D.

Attachment

Submitted filename: Response to reviewers_230801.doc

Click here for additional data file. (32KB, doc)

Decision Letter 3

Antonella Marangoni

12 Sep 2023

Changes in rapid plasma reagin titers in patients with syphilis before and after treatment: a retrospective cohort study in an HIV/AIDS referral hospital in Tokyo

PONE-D-22-35264R3

Dear Dr. Fukushima,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Antonella Marangoni, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Thank you for the opportunity to review again your interesting manuscript. I feel the manuscript improved and suitable for publication.

Reviewer #2: (No Response)

**********

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

**********

Acceptance letter

Antonella Marangoni

20 Sep 2023

PONE-D-22-35264R3

Changes in rapid plasma reagin titers in patients with syphilis before and after treatment: a retrospective cohort study in an HIV/AIDS referral hospital in Tokyo

Dear Dr. Fukushima:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

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on behalf of

PhD Antonella Marangoni

Academic Editor

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Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 File. * months from treatment initiation.

    RPR ratio compared with RPR at treatment initiation is shown.

    (XLSX)

    Click here for additional data file. (26.2KB, xlsx)
    Attachment

    Submitted filename: Response to reviewers_RPR_revised manuscript.doc

    Click here for additional data file. (73KB, doc)
    Attachment

    Submitted filename: Response_230703.doc

    Click here for additional data file. (33KB, doc)
    Attachment

    Submitted filename: Response to reviewers_230801.doc

    Click here for additional data file. (32KB, doc)

    Data Availability Statement

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