Factors Associated with Serological Cure and the Serofast State of HIV-Negative Patients with Primary, Secondary, Latent, and Tertiary Syphilis
and Tertiary Syphilis. PLoS ONE 8(7): e70102. doi:10.1371/journal.pone.0070102
Factors Associated with Serological Cure and the Serofast State of HIV-Negative Patients with Primary, Secondary, Latent, and Tertiary Syphilis
Tian-Ci Yang 0
Man-Li Tong 0
Li-Rong Lin 0
Gui-Li Liu 0
Hui-Lin Zhang 0
Yan-Li Zeng 0
Wei-Hong Zheng 0
Li-Li Liu 0
Jianqing Xu, Fudan University, China
0 1 Center of Clinical Laboratory, Zhongshan Hospital, Medical College of Xiamen University , Xiamen , China , 2 Xiamen Zhongshan Hospital, Fujian Medical University , Xiamen , China , 3 Department of Neurology, Zhongshan Hospital, Medical College of Xiamen University , Xiamen , China
Background: Some syphilis patients remain in a serologically active state after the recommended therapy. We currently know too little about the characteristics of this serological response. Methods: We conducted a cohort study using the clinical database from Zhongshan Hospital, Medical College of Xiamen. In total, 1,327 HIV-negative patients with primary, secondary, latent, and tertiary syphilis were enrolled. Bivariate and multivariate analyses were utilised to identify factors associated with a serological cure and serofast state in syphilis patients one year after therapy. Chi-square tests were used to determine the differences in the serological cure rate across different therapy time points. Results: One year after the recommended therapy, 870 patients achieved a serological cure, and 457 patients (34.4%) remained in the serofast state. The serological cure rate increased only within the first 6 months. The bivariate analysis indicated that male or younger patients had a higher likelihood of a serological cure than female or older patients. Having a baseline titre #1:2 or $1:64 was associated with an increased likelihood of a serological cure. The serological cure rate decreased for the different disease stages in the order of primary, secondary, latent, and tertiary syphilis. A distinction should be drawn between early and late syphilis. The multivariate analysis indicated that a serological cure was significantly associated with the disease phase, gender, age, and baseline rapid plasma reagin (RPR) titre. Conclusions: The serofast state is common in clinical work. After one year of the recommended therapy, quite a few syphilis patients remained RPR positive. The primary endpoint of the study indicated that disease phase, gender, age and baseline RPR titre were crucial factors associated with a serological cure.
Funding: The current study was supported by the National Natural Science Foundations Major Research Planning (grant 91029729) and the National Natural
Science Foundation (grants 81171625, 81101324 and 81141086) and the Technology Foundations Major Project of Social Development in Fujian Province (grant
2011Y4009). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
The World Health Organization describes syphilis as a
sexually transmitted infection that can be successfully controlled
by public health measures due to the availability of a highly
sensitive diagnostic test and a highly effective and affordable
treatment. Nevertheless, syphilis remains a worldwide public
health problem . The global syphilis statistics show that an
estimated 10 million new infections still occur each year . The
rate of congenital syphilis has been increasing in recent years in
China, with an average annual increase of 71.9% .
Undoubtedly, we are overoptimistic about the prevention of syphilis, and
we still know too little about the disease, especially the serological
response after therapy .
Parenteral penicillin G has been used for more than 50 years,
but no comparative trials have been adequately conducted to
guide the selection of an optimal penicillin regimen (i.e., the dose,
duration, and preparation) . Meanwhile, the basis for evaluating
the therapeutic response remains serological testing . Not all
patients achieve serological reversal after the recommended
treatment, some patients demonstrate a persistent positive
serological reaction that was quite disconcerting for both the
physician and patient . It remains unclear whether the
persistent positive serological reaction indicates persistent foci of
spirochetes or progressive syphilitic lesions or whether it reflects
the persistence of reagin in the circulating blood following
antisyphilitic therapy. For these reasons, a discussion about the
serological response after the recommended therapy is more than
Serological tests are the most widely used laboratory techniques
for diagnosing syphilis and monitoring its post-treatment course
. Serological tests can be divided into two categories:
nontreponemal and treponemal antibody tests. The titre of
nontreponemal antibodies usually correlates with disease activity,
and this titre is the basis for evaluating the therapeutic response
. The nontreponemal titre usually decline after therapy. In
some patients, nontreponemal antibodies can persist for a long
time in a range that differs little from the baseline rapid plasma
reagin (RPR) titre after the recommended therapy. These
antibodies sometimes persist for the lifetime of the patient. In a
previous study, clinical trial data demonstrated that after
undergoing the recommended therapy, approximately 15% of
patients with early syphilis did not exhibit two-dilution declines or
two-dilution increases in the nontreponemal antibody titre. These
individuals were considered to be in a serofast state one year
after treatment . Experience has indicated that, for some
patients, the nontreponemal antibody test results remain in a tight
range one year after the recommended therapy. This response is
called the syphilis serofast state  or sero-resistance .
There is no generally definition of the serofast state (so-called
sero-resistance), but most observers agree that the concept should
be based on a chosen span of time, amount of treatment and the
change of RPR titre [7,14,15]. A fourfold (two dilutions) change in
titre is considered necessary to demonstrate a clinically significant
difference between two nontreponemal test results that were
obtained using the same serologic test . Accordingly, in this
study, after one year of recommended therapy, syphilis patients
were considered to be in a serofast state if their nontreponemal test
remained positive and the titres neither increased nor decreased by
at least four-fold (two dilutions). Syphilis patients with persistent or
recurrent clinical signs of syphilis and whose nontreponemal
antibody titres increased by four-fold or more were considered to
exhibit treatment failure or reinfection. Syphilis patients whose
clinical manifestations disappeared and whose nontreponemal
antibody titres became negative or decreased by four-fold (two
dilutions) were regarded as achieving a serological cure. We still
know little about the serological response, and the factors that
predict the serological response after the recommended therapy
among syphilis patients have not yet been thoroughly studied. In
our previous study [11,12], we found that Tp-IgM could be used
as a serological marker for relapse and syphilis infection. To
further explore the characteristics of the serological response, we
investigated the factors associated with serological cure and the
serofast state in this cohort study.
Study Population and Ethics Statement
All subjects in the present study were collected from the clinical
database of Zhongshan Hospital, Medical College of Xiamen,
between June 2005 and May 2010. This study was approved by
the Institutional Ethics Committee of Zhongshan Hospital,
Medical College of Xiamen University, and was in compliance
with the national legislation and the Declaration of Helsinki
guidelines. Written patient consent was obtained according to the
Laboratory analyses, including RPR (InTec Products, Inc.,
China) and Treponema pallidum particle agglutination (TPPA)
(Fujirebio, Tokyo, Japan), were conducted in accordance with
the instructions of the manufacturers. All serum samples used in
the RPR test were diluted with physiological saline (NaCl 0.9%) at
a ratio of 1:1 to 1:32 (V/V) to avoid prozone effects and
falsenegative results. Readings were taken visually and then compared
with the negative and positive controls. The titers of the serum
samples that were reactive with RPR were quantified using
twofold serial dilutions until the endpoint was determined. Samples
with low titers were randomly and blindly checked by other
technicians for quality control. Three standard serum samples with
low, medium, and high titers from the National Centers for
Clinical Laboratory were used as controls for the RPR reaction.
The initial dilution of the serum samples for the TPPA reactions
Testimony of Diagnosis of Syphilis
In the light of CDC guidelines in USA and European [16,17],
primary syphilis is characterized by an ulcer (chancre), usually with
regional lymphadenopathy, and laboratory confirmation is
darkfield examination/fluorescent antibody method/PCR to detect T.
pallidum in lesion exudate, or/and a reactive nontreponemal test
and a treponemal test to confirm the diagnosis of syphilis.
Secondary syphilis is generally characterised by a maculopapular
rash, and the laboratory test confirmation is a reactive
nontreponemal test and a treponemal test to confirm the diagnosis of
syphilis. For Latent syphilis is asymptomatic with a possible history
of infection supported by reactive treponemal tests and
nontreponemal test and normal cerebrospinal fluid. Tertiary syphilis
was defined as syphilis acquired .1 years previously , clinical
manifestations (i.e., cardiac or gummatous lesions) and a history of
primary, secondary, or latent syphilis, moreover, the laboratory
test confirmation by reactive nontreponemal and treponemal test.
Parenterally administered penicillin G is the preferred drug for
treating all stages of syphilis . The preparation used (i.e.,
benzathine, aqueous procaine, or aqueous crystalline), the dosage,
and the length of treatment depend on the stage and clinical
manifestations of the disease. Generally, nonpenicillin-allergic
participants underwent treatment randomisation to receive
benzathine penicillin (2.4 million U by intramuscular injection
for early syphilis or 7.2 million units total, administered at 3 doses
of 2.4 million units intramuscular injection each at 1-week
intervals for late syphilis). Penicillin allergic participants were
randomised to receive doxycycline (100 mg taken orally twice
daily for 14 days) or azithromycin (2.0 g daily either IM or IV for
1014 days) . The detailed recommended therapy schedules
for different stages of syphilis strictly followed the CDCs
guidelines. Considering that the type of drug used, the dosage,
and the route of administration affected the serological cure.
Therefore, only patients treated with penicillin were included in
our study. After treatment, patients were advised to undergo
serum RPR testing every three months for the first year.
Statistical analysis was conducted using SPSS version 17.0.
Bivariate analysis was utilized to determine the factors associated
with the serological cure. The odds ratios (OR) was estimated with
95% confidence intervals (CIs) from the bivariate analysis, and
factors with P,0.2 were further identified in the multivariate
analysis. Adjusted odds ratios (AOR) with 95% confidence
intervals (CIs) were also estimated from the regression analysis.
Chi-square tests were conducted to identify significant differences
across different therapy time points, and P,0.05 (two sided) was
considered a statistical difference.
Characteristics of the Study Population
A total of 5,460 syphilis patients were found in the Zhongshan
Hospital database from June 2005 to May 2010. After excluding
the records without documentation of HIV testing and records for
HIV-positive or returning patients, 2,707 patients remained. We
further excluded patients who did not receive the recommended
therapy of penicillin, such as irregular treatment, intermittent
treatment, and undertreated therapy. We excluded patients with a
false-positive RPR test to avoid the influence on serological
response by diseases such as leprosy, systemic lupus erythematosus,
HBV, rheumatoid arthritis, thyroid conditions, and respiratory
infections. Individuals who were pregnant, experienced treatment
failure or reinfection during the course of therapy, had no
complement information, or did not agree to participate in our
study were also excluded At last, only 1,327 patients were included
into our study sample (Figure 1). The mean age was 41.02 years,
and 58.9% were men. In this sample, 31.4% patients had latent
syphilis, 22% had primary syphilis, 27.1% had secondary syphilis,
and 19.5% had tertiary syphilis. One year after treatment, 65.6%
exhibited a serological cure, but 34.4% were serofast (Table 1).
Factors Associated with Serological Response
Our study compared the characteristics of 870 patients who
achieved a serological cure with the characteristics of 457 serofast
patients at 12 months after therapy. Male patients were
approximately 2-fold more likely to achieve a serological cure
than female patients. Compared to patients ,40 years of age,
patients between 23 and 29 years of age had a lower probability of
achieving a serological cure, and patients ,23 years old were
comparatively more likely to achieve a serological cure. Patients
with lower or higher baseline RPR titres were easily treated and
achieved serological cure, but patients with a baseline RPR titre of
1:8 or 1:16 had a decreased likelihood of achieving a cure at 12
months. The serological cure rate for the different disease stages
decreased in the order of primary, secondary, latent, and tertiary
syphilis (Table 1).
A multivariate analysis was also conducted using all of the
significant variables identified above. As shown in Table 2, the
results further confirmed that the disease phase, gender, age, and
Serofast (n = 457)
*Abbreviations: OR, odds ratios; CI, confidence interval.
the baseline RPR titre were significantly associated with the
likelihood of a serological cure. An age ,23 years was associated
with a 2-fold greater probability of a serological cure than an age
.40 years, and got the highest AOR. Patients with baseline RPR
titres of 1:1 and 1:2 had high AORs of 2.732 and 2.380,
Serological Response at Different Time Points after
Because the proportion of patients who achieve a serological
cure generally increases with time after treatment, the cure rate
varied over time. In our research, the serological cure rate was
59.8% (794/1,327) at 3 months and improved to 64.1% (850/
1,327) at 6 months. For 612 months after treatment, the
serological cure rate exhibited a slight increase (from 64.1% to
65.6%), but there were no significant differences among different
time points from 6 to 12 months. The serological cure rate did not
change substantially with the time after 6 months (Figure 2).
Serological Responses for Different Baseline RPR Titres
The statistical analysis showed that patients with lower or higher
baseline RPR titres were more likely to achieve serological cure.
Among the 387 patients with baseline RPR titres of 1:1, the
proportion who achieved serological cure was approximately
80.1%, and among the 272 patients with baseline RPR titres of
$1:32, the serological cure rate at 12 months was 60.8%;
however, only 49.1% of patients with baseline RPR titres of 1:8
exhibited a serological cure (Figure 3).
Secondary VS Primary
*Abbreviations: AOR, adjusted odds ratio.
95.0% C.I. for AOR
Difference in Serological Responses between Early
Syphilis and Later Syphilis
It is difficult to distinguish early latent syphilis and late latent
syphilis. We excluded all the latent syphilis patients and further
discussed the difference in the serological cure rate between early
syphilis (including primary and secondary syphilis) and late syphilis
(including tertiary syphilis). The results indicated that patients with
early syphilis had a higher likelihood of a serological cure at 12
months than patients with late syphilis, and the OR was 2.391
Previous research has discussed the serological response to
syphilis treatment . These studies focused primarily on
assessing the effect of HIV infection on the serological response to
the treatment of syphilis [19,20] or on identifying treatment
failures or reinfections . To date, only one other paper has
reported a systematic evaluation to identify predictors of a
serological cure and the serofast state after syphilis treatment
, which focused only on early syphilis patients, and the sample
size was relatively small. In this study, a total of 1,327 cases with
different stages of syphilis met the inclusion criteria and were
included. Our study was an overall evaluation of the factors that
influence the likelihood of achieving a serological cure in patients
with different syphilis stages. The results indicate that among the
1,327 syphilis patients who were treated with the recommended
therapy, 457 patients remained in a serofast state one year after
treatment according to the nontreponemal antibody test, and the
prevalence of the serofast state was 34.4%.
What are the factors that influence the likelihood of achieving a
serological cure? In this study, using bivariate and multivariate
analyses, we found that the disease phase, gender, age, and the
baseline RPR titre were associated with a serological cure. The
serological cure rate is affected by numerous factors, and we
cannot deny the significance of other factors that were not
included in the logistic model with multiple variables. A previous
study indicated that the type of drug used, the dosage, and the
route of administration also affected the serological cure rate .
Meanwhile, the relationship between the serofast reaction and
neurosyphilis has received a great deal of attention . In our
prior study, we analysed the cerebrospinal fluid from 205 patients
in the serofast state, 115 of which (56.1%) were diagnosed with
neurosyphilis . That study demonstrated the close relationship
between the two diseases. Another published study suggested that
the serological response was influenced by the bodys
immunological function .
For many syphilis patients, the serological response varies over
time after the recommended therapy. As stated previously, the
time point used in the analysis is important when assessing the
serological response . Typically, nontreponemal antibody
titres should decrease at least fourfold within 6 months after
treatment of primary or secondary syphilis and within 12 months
after treatment of latent or late infection . In this research, we
compared the serological cure rates at different time points after
treatment. The results indicate that the proportion of serological
cure increased within the first 6 months, but from 6 to 12 months
after treatment the serological cure increased only slightly. These
findings generally support those of other investigations regarding
that the likelihood of a substantial decline in the serofast
proportion over time is low. . Considered in this light, it
would be appropriate to use a 12-month follow-up standard for
syphilis treatment in this research.
In our study, the prevalence of the serofast state in females was
42.8%, which was higher than that in males. This result was
different from the result of a previous study that denied this
association [15,27]. Generally, the immune system differs between
males and females , and the serological response is also
influenced by the immune system , so gender difference could
influence the serological response after treatment. The exact
mechanism of this association was still unclear in present research
and it needs more detailed investigation. Meanwhile, we found
that patients .40 years old had a comparatively lower probability
of achieving a serological cure than patients ,23 years old. A
retrospective analysis found that older age was associated with a
lower probability of a serological cure or reduction in the baseline
RPR titre . This older population usually demonstrates
increasing senescence of the immune system and
immunosuppression that would affect the serological response to syphilis
Our findings also generally support those of other investigations
regarding the relationship between the baseline RPR titre and the
serological response . After therapy, the serological cure rate
first decreased for titres from 1:1 to 1:8 and then gradually
increased as titres increased. A previous study indicated that a high
baseline RPR titre signifies a beneficial inflammatory and immune
Serological cure (n = 604)
Serofast (n = 286)
*The serological cure rates of early and late syphilis patients were compared using bivariate analysis. There was a significant difference in the serological cure rate
between early and late syphilis patients (P ,0.001). The OR was used to access the serological cure.
response to Treponema pallidum (Tp), which facilitated the clearance
of Tp . This observation could be explained by an earlier study
 that demonstrated VDRL-immunised rabbits exhibited
partial protection against reinfection with Tp. It is conformed
that HIV significantly influenced the serological response after
treatment [31,32]. We did not evaluate the effect of HIV in our
study because only 5 HIV-positive patients met our inclusion
criteria, and this was not appropriate for statistical analysis.
The influence of the type of syphilitic infection at the beginning
of treatment on the serological reaction is an infrequently
discussed point . Our results indicated that the serological cure
decreased in the order of primary, secondary, latent, and tertiary
syphilis. There were 29.3% and 26.2% of patients with early
syphilis who did not demonstrate a serological cure at 6 months
and 12 months after therapy, respectively. A previous study
reported that one-fifth of patients treated for early syphilis did not
meet the criteria for serological cure at 6 months after therapy
. This difference could be properly explained here. The
inclusion criteria for our study population differed from the
previous study: returning patients were excluded from our study;
every enrolled individual had differences in immunological
function that would influence the serological response. Another,
we thought the different strains pathogenicity were also a key
factor. Some previous studies found that the Tp subtypes in
different regions were not alike, differing in pathogenicity .
Some scholars have reported that the Tp subtype in China is
different from those in other countries [37,38].
Our study has several limitations. The eligible patients
represented a fraction of the patients diagnosed with syphilis
during the study period, raising a concern for a selection bias.
Nearly 38.1% (2,082/5,460) of the patients were excluded because
they did not have documented HIV test results in our clinical
database. Whether the exclusion of these patients affected the
outcome is unknown. As a retrospective study, collecting
information from medical records previously developed by other
professionals is unavoidable but tends to be less accurate. It was
difficult to entirely exclude from our research patients with
treatment failure or reinfection, which can lead to prolonged
seroactivity . The clinical stages of syphilis often overlap, and
no concrete indications among the different stages of syphilis exist.
Basic research on serological responses is rare. Our report
analyses and discusses the factors associated with a serological cure
and the serofast state in syphilis patients after treatment. We hope
this discussion proves useful to clinicians and public health workers
in identifying patients who may have achieved a serological cure
or become serofast, and aids in determining whether serofast
patients should undergo a repeated clinical treatment. Given that
neurosyphilis is related to the serofast state, cerebral spinal fluid
examination should be considered for persons whose
nontreponemal antibody titres do not respond appropriately within 612
months of therapy or to reinvestigate CSF if the results of a
previous examination were normal.
We thank the patients all for their support in this research and thank the
Zhongshan Hospital colleague for their assistance in collection of research
Conceived and designed the experiments: TCY LLL MLT LRL GLL.
Performed the experiments: MLT GLL HLZ YLZ WHZ. Analyzed the
data: TCY LLL LRL. Contributed reagents/materials/analysis tools:
MLT HLZ GLL YLZ. Wrote the paper: TCY LLL MLT.
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