Incidence and Risk Factors for the Prozone Phenomenon in Serologic Testing for Syphilis in a Large Cohort
Incidence and Risk Factors for the Prozone Phenomenon in Serologic Testing for Syphilis in a Large Cohort
Li-Li Liu 0 1 2
Li-Rong Lin 0 2
Man-Li Tong 0
Hui-Lin Zhang 0
Song-Jie Huang 0
Yu-Yan Chen 0
Xiao-Jing Guo 0
Ya Xi 0
Long Liu 3
Fu-Yi Chen 4
Ya-Feng Zhang 0
Qiao Zhang 0
Tian-Ci Yang 0 2 5
0 Zhongshan Hospital, Medical College of Xiamen University
1 Xiamen Zhongshan Hospital, Fujian Medical University
2 Xiamen Zhongshan Hospital, Fujian University of Traditional Chinese Medicine , Xiamen
3 Department of Chemistry and Biology, College of Science, National University of Defense and Technology , Changsha , China
4 Department of Physiology and Neurobiology, University of Connecticut , Storrs , USA
5 ShenZhen Research Institute of Xiamen University , ShenZhen , China
Background. The prozone phenomenon is known to be associated with high antibody titers; other associations, such as host factors, have not been elucidated. Methods. A retrospective analysis was conducted to evaluate the incidence of the prozone phenomenon of the syphilis rapid plasma reagin (RPR) test among 46 856 clinical samples, between June 2010 and June 2013. Logistic regression was used to analyze the risk factors of the prozone phenomenon. Results. Our results showed that the incidence of the prozone phenomenon was low (0.83%) and could occur during any clinical phase, particularly during primary and secondary syphilis. Pregnancy and neurosyphilis were associated with the prozone phenomenon; sex, age, and whether the patient had been treated were not. The results also revealed that the prozone phenomenon not only occurred in patients with a high titer but also could occur in patients with a moderate/low titer. In fact, almost 31% of the patients with the prozone phenomenon had titers ?1:16. Conclusions. The prozone phenomenon in the RPR test was associated with the phase of syphilis, pregnancy, and neurosyphilis as well as a range of RPR titers between 1:8 and 1:512. This latter finding is in contrast to previous reports that the prozone phenomenon is associated with very high RPR titers.
Syphilis remains a worldwide public health concern [
]. The accuracy of diagnostic testing is critical for
lowering transmission rates and avoiding the complications
observed during late-stage disease. Treponema
pallidum, the causative agent of syphilis, can only be cultured
in vivo and cannot be stained using simple laboratory
methods. Consequently, serological testing (including
nontreponemal and treponemal antibody tests) remains
the mainstay for diagnosing syphilis and monitoring
the success of the subsequent antibiotic treatments
]. However, false-negative results, especially with the
rapid plasma reagin (RPR) test, attributed to the
prozone phenomenon may hinder the prompt diagnosis
and management of syphilis. The prozone
phenomenon generally refers to a false-negative response arising
from cases in which high antibody titers interfere with
the antigen-antibody lattice network formation that is
necessary for visualizing a positive flocculation test.
The prozone phenomenon typically occurs when
undiluted serum is used and can happen during any phase of
]. However, it is still not clear whether the
prozone phenomenon can be associated with other
factors. The few published studies related to this topic
consisted of single case reports [
]. Furthermore, studies
reporting the incidence of the prozone phenomenon are
Liu et al
limited in number and typically utilized small sample sizes [
]. Here, we evaluated the prozone phenomenon of the RPR
test during serological testing for syphilis with a large cohort,
and examined the association between host factors and the
MATERIALS AND METHODS
Study Population and Ethics Statement
We conducted a retrospective analysis of serological tests for
syphilis at Zhongshan Hospital, Medical College of Xiamen
University, between June 2010 and June 2013. During this
period, we included 46 856 subjects who had both RPR and
Treponema pallidum particle agglutination (TPPA) assay at the
same time (after duplicate tests were excluded) from
outpatients, inpatients, and health examination populations. All
serological tests were performed using the same specimen, and the
results of both tests were reported simultaneously. This study
was approved by the Institutional Ethics Committee of Medical
College of Xiamen University and complied with national
legislation and the Declaration of Helsinki guidelines.
Serological Tests and Proof of the Prozone Phenomenon
RPR (InTec, Xiamen, China) and TPPA (Fujirebio, Tokyo,
Japan) tests were performed according to the manufacturer?s
instructions. When the serum was nonreactive toward RPR but
reactive during TPPA, the RPR test was repeated using serum
diluted from 1:1 to 1:32 with a physiological sodium chloride
solution (0.9%) to avoid a false-negative result (the prozone
phenomenon). Results were evaluated immediately with the
naked eye by comparing them to negative and positive controls.
Finally, when diluted serum was reactive and the undiluted
serum was nonreactive, it was considered that the undiluted
sample had exhibited the prozone effect. The serum samples
that reacted with RPR were quantified using 2-fold serial
dilutions. The initial dilution of the serum samples used in the
TPPA reactions was 1:80. TPPA-positive serum samples were
then validated using an automated chemiluminescence
immunoassay (CIA) (Boson Biotechnology, Xiamen, China) to avoid
a false-positive result. Three standard serum samples (400 mIU/
mL, 80 mIU/mL, and 24 mIU/mL) (Beijing Controls &
Standards Biotechnology Co, Ltd, China) were used as positive
controls for the RPR, TPPA, and CIA reactions, respectively. The
serum samples that produced conflicting or inconclusive results
for a particular technique were tested again and the consistent
result was considered the true result. All patients exhibiting the
prozone effect were screened for human immunodeficiency
virus (HIV) type 1/2 antigens/antibodies using enzyme-linked
immunosorbent assay (Beijing Wantai Biological Pharmacy
Enterprise Co, Ltd, China).
Diagnosis of Syphilis
According to the United States Centers for Disease Control and
Prevention and the European Centre for Disease Prevention
and Control [
], syphilis was clinically diagnosed in this
study by combining the serodiagnosis and disease history
(including the clinical characteristics and/or patient?s sexual
history). The diagnosis of primary, secondary, latent, and
tertiary syphilis and neurosyphilis was determined as previously
All statistical analyses were conducted using SPSS version 17.0
for Windows (IBM, Armonk, New York). Logistic regression
was used to analyze the risk factor for the prozone
phenomenon. A 2-sided P value <.05 was considered significant.
Serological Test Results of Syphilis Patients
Among 46 856 subjects included in this study (average age, 49.7
years [range, 1?110 years]), 1573 patients were nonreactive for
RPR but reactive for TPPA. Among these 1573 RPR-negative
subjects, 36 undiluted samples were confirmed to exhibit the prozone
phenomenon. A total of 4298 serum samples were positive for
both RPR and TPPA. Including the 36 samples with the prozone
effect, a total of 4334 subjects had positive RPR results. Therefore,
the incidence of the prozone phenomenon was 0.83% (36/4344)
(Figure 1). Moreover, 121 cases of RPR-positive/TPPA-negative
patients were confirmed by CIA test to have a biological
falsepositive reaction and were excluded from this study.
Incidence of the Prozone Phenomenon in Patients With Different
The clinical characteristics of all 36 syphilis patients exhibiting
the prozone phenomenon are shown in Table 1. All patients
were of Asian race, were not infected with HIV, and had a
mean age of 46.4 years (range, 17?76 years). No significant
difference was found in the incidence of the prozone phenomenon
among syphilis patients with regard to sex, treatment status, or
age (Table 1).
The prozone phenomenon is associated with pregnancy. In
the study, 4 of the 16 female syphilis patients with prozone
phenomenon were pregnant, including 1 in first trimester, 2 in
second trimester, and 1 in third trimester (Table 1). Bivariate
logistic regression showed that compared with nonpregnant
patients (0.71% [12/1682]), there was an increased odds (odds
ratio [OR], 4.123; P = .015) of the prozone phenomenon in
pregnant patients (2.88% [4/139]).
Detailed clinical phase analysis on 36 patients exhibiting
prozone phenomenon is shown in Table 1. There were 2 primary,
10 secondary, 6 tertiary, and 16 latent syphilis. The other 2
Prozone Phenomenon in Syphilis
patients exhibited syphilis at an unknown stage. The incidence
for prozone phenomenon in primary, secondary, tertiary, and
latent syphilis patients were 4.65% (2/43), 1.76% (10/569),
0.63% (6/952), and 0.61% (16/2640), respectively. Among
primary, secondary, and tertiary syphilis, patients with
earlystage syphilis were associated with increased incidence of the
prozone phenomenon. Compared with latent syphilis, primary
syphilis had 8.000-fold higher (P = .007) and secondary syphilis
had a 2.943-fold higher (P = .008) probability of exhibiting
prozone phenomenon. Tertiary syphilis had a slightly increased
odds (OR, 1.04; P = .935) of exhibiting the prozone
phenomenon. We next looked at the association between neurosyphilis
and the prozone phenomenon. Among the 36 syphilis patients,
5 were diagnosed with neurosyphilis (Tables 1 and 2). Patients
with neurosyphilis (3.12% [5/160]) had much higher odds (OR,
4.311; P = .003) of exhibiting the prozone phenomenon,
compared with patients without neurosyphilis (0.74% [31/4174])
The prozone phenomenon is believed to be associated with
higher antibody titer. Surprisingly, the RPR titers of the samples
exhibiting the prozone phenomenon ranged from 1:8 to 1:512
after being serially diluted; nearly 31% of the patients? titers
were ?1:16. In addition, we also found that diluting the
antibody to 1:8 was generally adequate for obtaining the
proper optimal concentration and a readily detectable
reaction. In summary, our results showed that the prozone
phenomenon can also be present in patients with moderate or low
Finally, we analyzed cerebrospinal fluid (CSF) from the 5
cases of neurosyphilis exhibiting the prozone phenomenon
(Table 2). Aside from the second patient listed in Table 2, the
other 4 patients presented with a positive CSF RPR test.
Meanwhile, we found that the 5 patients? CSF nontreponemal tests
did not display the prozone phenomenon. In addition, all 5
patients had a positive CSF TPPA assay. Given that their CSF
white blood cell count of 10 ? 106 cells/L and CSF protein 500
mg/L fell within the normal reference intervals [
], all 5
patients displayed CSF pleocytosis and elevated CSF protein levels,
except for the second patient with a normal CSF protein level.
The prozone phenomenon in syphilitic serologic testing
confounds syphilis diagnosis and generates misleading results
with regard to therapeutic effectiveness. In the current study,
we have conducted a retrospective analysis to evaluate the
Abbreviations: CI, confidence interval; OR, odds ratio; RPR, rapid plasma reagin.
a A total of 4334 had positive RPR, including 36 samples that exhibited the prozone effect, and 4298 serum samples that reacted to both RPR and Treponema
pallidum particle agglutination assay were included our statistical analysis.
b This factor was not included in our statistical analysis.
c RPR titers 1:1?1:4 and RPR titers >1:1024 were not included in the statistical analysis.
incidence of the prozone phenomenon of the RPR test from a
high syphilis prevalence area. Our results indicated that 36
samples exhibited the prozone phenomenon, with an incidence of
0.83%, which is consistent with a previously reported 0.2%?2%
incidence rate in all syphilis cases [
12, 17, 18
]. Our results
indicated that the prozone phenomenon is associated with factors
other than antibody titers, such as pregnancy, the phase of
syphilis, and neurosyphilis.
It is widely accepted that the prozone phenomenon could be
caused by a high antibody titer. However, our results showed
that nearly 31% of the prozone phenomenon occurred in
patients with an RPR titer ?1:16, indicating that moderate/lower
Prozone Phenomenon in Syphilis
Abbreviations: CSF, cerebrospinal fluid; RPR, rapid plasma reagin; TPPA,
Treponema pallidum particle agglutination assay; WBC, white blood cell.
a Five patients? CSF nontreponemal test did not display the prozone
antibody titers can also contribute to the prozone phenomenon.
Similar to a previous report that diluting the antibody to 1:16 is
usually adequate for obtaining the optimal concentration [
we found that diluting the antibody to 1:8 was generally
adequate for obtaining the proper optimal concentration.
Previous studies have indicated that the prozone effect is
usually associated with secondary and early latent syphilis, as well as
early neurosyphilis [
], HIV coinfection [
], and pregnancy
]. Consistently, our results showed that the prozone
phenomenon was highly associated with primary and secondary
syphilis as well as neurosyphilis. It is important to note that in
our study, 2640 patients had latent syphilis, whereas primary
and secondary syphilis only occurred in 43 and 569 patients,
respectively. Such small primary and secondary populations in this
larger cohort may reflect the changes in distribution of syphilitic
stages in recent years due to antibiotic misuse. It has been
reported that late latent syphilis (including subjects with syphilis of
unknown duration) accounted for 59.0%?66.4% of all syphilis cases
]. However, in this study, the small number of primary
syphilis cases may have affected the observed prozone
phenomenon incidence in primary syphilis.
The incidence of congenital syphilis is rising rapidly in China
]. Moreover, false-negative test results hinder efforts to
control its spread, leaving fetuses at risk for congenital syphilis.
Berkowitz et al has recommended that pregnant women who
apparently have negative syphilitic serological results and
signs indicating fetal compromise of unknown etiology,
particularly nonimmune hydrops, should have repeat nontreponemal
testing using diluted serum to prevent a missed syphilis
diagnosis. Serum dilution is recommended as a routine procedure
for all pregnant women in areas with a high prevalence of
syphilis infections [
]. In our study, 4 of the 139 (2.88%)
pregnant syphilis patients exhibited the prozone phenomenon,
supporting the assumption that the prozone phenomenon
appears more commonly in pregnant women [
]. In addition, the
prozone phenomenon has also been reported in the setting of
isolated neurosyphilis [
]. During our study period, 160
neurosyphilis patients with positive serum RPR results were
admitted to the hospital, 5 (3.12%) of whom exhibited the prozone
phenomenon, although their CSF RPR did not display the
prozone phenomenon. Further research is required to understand
the reasons why pregnant women and patients with
neurosyphilis more commonly exhibit the prozone phenomenon.
To detect the prozone effect, samples are often tested both
undiluted and diluted [
]. However, many hospital
laboratories do not routinely test the RPR using diluted serum due to
the labor and reagent costs [
]. In addition, it has been shown
that nontreponemal tests (such as RPR) have low sensitivity,
especially in late syphilis [
], and may show false-negative
results due to the prozone phenomenon . The treponemal
antibody test is proven to be more sensitive and specific for
diagnosing syphilis than the nontreponemal antibody test [
Thus, to avoid the prozone phenomenon and
falsenegative results, we implemented the European Centre for Disease
Prevention and Control?s algorithm for diagnosing syphilis: A
reactive treponemal screening assay ( primary screening test)
is followed by a second and different treponemal assay that is
used as a confirmatory test. The quantitative test (such as
RPR) should only be recommended for assessing the serological
activity of syphilis and monitoring the serological response to
], as it is unnecessary for the diagnosis of syphilis.
However, when a quantitative RPR test is used, sera nonreactive
toward RPR but reactive toward TPPA require a repeated RPR
test using serum diluted from 1:1 to 1:32 with a physiological
solution to avoid the prozone effect and false-negative results.
The limitations of our study should be considered. First, there
was potential misclassification of patients with a prior history of
syphilis due to inadequate medical records or hospital and
public health registries. Second, most of the pregnant subjects at our
hospital were evaluated only by TPPA as the primary screening
test and were excluded from our study. Therefore, our data also
could not reflect the exact number of pregnant subjects with the
prozone phenomenon. Finally, we did not look at the prozone
phenomenon rate in treponemal antibody tests.
Above all, our results indicated that the prozone phenomenon
may be associated with factors other than a high antibody titer. To
reduce false-negative responses, we recommend using the
treponemal antibody test for the serodiagnosis of syphilis, and using
the quantitative tests (such as RPR) to assess serological syphilis
activity and monitor the serological response to treatment.
Acknowledgments. We acknowledge all the colleagues in the clinical
laboratory, Zhongshan Hospital, Medical College of Xiamen University,
for their assistance with the sample collection as well as their support in this
Financial support. This study was supported by the National Natural
Science Foundation Major Research Plan (grant number 91029729), the
National Natural Science Foundation (grant numbers 81171625, 81271335,
81201360, 81101324, 81301501), the Technology Foundation?s Major
Project of Social Development in Fujian Province (grant number 2011Y4009),
the Natural Science Foundation of Fujian Province (grant number
2012D039), the Dedicated Fund for Shenzhen?s Strategic Emerging
Industries Development (grant number JCYJ20130327150714488), the Key
Projects in Fujian Province Science and Technology Program (grant number
2013D025), the projects of Xiamen Science and Technology Program
(grant number 3502Z20139001), and the Key Project of Cultivating
Young Talents in Fujian Province?s Health System (grant number
Potential conflicts of interest. All authors: No reported conflicts.
All authors have submitted the ICMJE Form for Disclosure of Potential
Conflicts of Interest. Conflicts that the editors consider relevant to the
content of the manuscript have been disclosed.
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