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A negative nontreponemal and/or specific antitreponemal IgM test does not

exclude active infectious syphilis: evidence from a rabbit infectivity test

Article in Medicine · August 2016

DOI: 10.1097/MD.0000000000004520

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MD-D-16-01681

Clinical Case Report Medicine ®

OPEN

A negative nontreponemal and/or specific

antitreponemal IgM test does not exclude active
infectious syphilis: evidence from a rabbit
infectivity test
A case report
Li-Rong Lin, MDa, Man-Li Tong, MSa, Kun Gao, BSa, Xiao-Zhen Zhu, BSa, Jin-Yi Fan, BSa,
a b b a a,∗
AQ1 Wei-Hong Zheng, MS , Shu-Lian Li, BS , Hui-Ling Lin, BS , Li-Li Liu, MD , Tian-Ci Yang, PhD

Abstract
Background: The diagnostic criteria for active infectious syphilis in the clinic are important matter of controversy and debate. So
far, clinicians habitually do use the negative results of the nontreponemal and/or the specific antitreponemal IgM as the evidences of
disease-free or active infection-free status.
Method: We present a case study involving a patient who was admitted to Zhongshan Hospital because of cerebral infarct. Clinical
examination indicated he had a history of latent syphilis with negative nontreponemal and specific antitreponemal IgM tests. The
cerebrospinal fluid sample from the patient was inoculated into seronegative New Zealand rabbit.
Results: Motile Treponema pallidum was detected by a rabbit infectivity test in the patient’s cerebrospinal fluid. This syphilis strain
was confirmed by DNA subtyping form of “CDC subtype/tp0548 sequence type”, and the strain type was 14d/f. Treatment with
benzathine penicillin provided no apparent benefit, but treatment with aqueous crystalline penicillin G, especially recommended for
neurosyphilis, led to disease regression. No evidence of cerebral infarct was observed during a 2-year follow-up period.
Conclusion: The definitive differential diagnosis of active infectious syphilis should be reconsidered. Moreover, selecting the
appropriate penicillin preparation is important because T pallidum can reside in sequestered sites. It is necessary to treat a patient
with known invasion of the central nervous system with aqueous crystalline penicillin G, if previous treatment for syphilis failed and
patients had some clinical neurological presentation that is otherwise unexplained, but that could represent neurosyphilis. Additional
studies are needed to confirm the results in other syphilis patients.
Abbreviations: CNS = central nervous system, CSF = cerebrospinal fluid, FTA-Abs TP-IgM = fluorescent treponemal antibody-
absorption Treponema pallidum-IgM, RIT = rabbit infectivity test, RPR = rapid plasma regain, TPPA = Treponema pallidum particle
agglutination.
Keywords: active infectious syphilis, antitreponemal IgM test, nontreponemal test, rabbit infectivity test

1. Introduction
clinical setting.[1] Clinicians generally rely on serological markers
Currently, there are still controversies existing on which criteria to diagnose active syphilis. For example, syphilis-specific IgM can
to be used to diagnose cases of active infectious syphilis in a be detected as early as 2 weeks after infection,[2] nontreponemal

Editor: Ismael Maatouk.

L-RL, M-LT, and KG contributed equally to this work.
Funding: This work was supported by the National Natural Science Foundation [grant numbers 81271335, 81201360, 81471967, 81471231, 81401749, 81301501,
81271895], the Key Projects in Fujian Province Science and Technology Program [grant numbers 2013D025, 2014D021], the Projects of Xiamen Science and
Technology Program [grant numbers 3502Z20144044, 3502Z20154015, WSK2010–01], the Key Project of Cultivating Young Talent in Fujian Province’s Health System
[grant number 2014-ZQN-ZD-34], the National Science Foundation for Distinguished Young Scholars of Fujian [grant number 2014D001], the Youth Foundation Project
of Fujian Provincial Health Department [grant numbers 2014-2-68, 2013-2-90], the Medical Innovation Project of Fujian Health Development Planning Commission
[grant number 2014-CXB-40], the Major Special Projects of Serious Illness in Xiamen [grant number 3502Z20159016], the Natural Science Foundation of Fujian
Province [grant number 2016J01628, 2013J01358], and the 863 Program [grant numbers 2014AA021401, 2014AA02230].
The authors declare that they have no competing interests.
a b
AQ2 Zhongshan Hospital, Medical College of Xiamen University, Xiamen Huli District Maternity and Child Care Hospital, Xiamen, China.
∗
AQ3 Correspondence: Tian-Ci Yang, Zhongshan Hospital, Medical College of Xiamen University, Xiamen 361004, China (e-mail: yangtianci@xmu.edu.cn).
Copyright © 2016 the Author(s). Published by Wolters Kluwer Health, Inc. All rights reserved.
This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
Medicine (2016) Vol:No
Received: 10 March 2016 / Received in final form: 22 June 2016 / Accepted: 13 July 2016
http://dx.doi.org/10.1097/MD.0000000000004520

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antibodies can be detected 5 to 7 weeks after initial infection, and sex. The patient refused a lumbar puncture examination. The
both of these are generally correlated with disease activity.[3] The syphilis and neurosyphilis were diagnosed or excluded as
rabbit infectivity test (RIT) is another method used to establish described previously.[7,8] The initial diagnoses included: multiple
infection with viable Treponema pallidum, and it has historically infarction; diabetes; hypertension; and latent syphilis of
been considered as the gold standard for detecting active syphilis. unknown duration (suggestion: having a lumbar puncture if he
This method has a test sensitivity high enough to detect as few as a experienced continued neurological discomfort to rule out or
single organism when repeated passages are performed in confirm neurosyphilis). Supporting treatment for cerebral
rabbits.[4] However, to perform this test, access to an animal infarction (including management for diabetes and hypertension)
facility is required, and it is extremely time-consuming and was initiated based on the 2008 European Stroke Organization
expensive.[5] For this reason, it is most commonly used in a guidelines,[9] and treatment with benzathine penicillin for syphilis
research setting, but is impractical and expensive as a routine (7.2 million units total, administered in 3 doses of 2.4 million
diagnostic procedure. units intramuscular injection per dose at 1-week intervals) was
Without treatment, an active syphilis infection can develop into according to the US CDC guidelines.[10] The patient was AQ4
symptomatic late syphilis. The most dreaded complications of discharged with improved function after 30 days.
this disease are neurosyphilis and the involvement of the aortic The patient was re-admitted to the same hospital on April 20,
valve and root.[1] Here, we report a patient with cerebral infarct 2011 due to sudden weakness in the right extremities and
and presumed latent syphilis with negative serum rapid plasma language disorders. His clinical signs and symptoms were
reagin (RPR) and syphilis-specific IgM tests, even though motile identical as his last visit. A brain MRI and DWI indicated
T pallidum was detected in the patient’s cerebrospinal fluid (CSF) multiple cerebral infarctions observed in the pons varolii (Fig. 1K
by RIT. This study was approved by the Institutional Ethics and L) and the left side of the mesencephalon (Fig. 1M and N),
Committee of Zhongshan Hospital, and it was performed in and the right side of the thalamus (Fig. 1O and P). Blood tests
compliance with national legislation and the guidelines of the revealed negative RPR (ruling out the prozone phenomenon),
Declaration of Helsinki. Written patient consent was obtained negative specific antitreponemal IgM, but positive TPPA (titer:
according to institutional guidelines. All rabbit experiments were 1:640). The patient denied having extramarital sex after the first
performed using protocols approved by the animal experimental admission. Two days after admission, lumbar puncture exami-
ethics committee of the Medical College of Xiamen University. nation indicated that the following: 4.0  106/L CSF-WBCs,
The CSF sample (undiluted) was inoculated into seronegative 413.10 mg/L CSF-protein, a negative CSF-RPR test, and a
New Zealand white male mature rabbits as previously negative CSF-TPPA test. All tests for microorganisms were
reported.[6] negative (Table 1). Clinical laboratory indices for neurosyphilis
were all negative. The clinical diagnosis on April 20, 2011 was:
multiple cerebral infarct; diabetes; hypertension; and latent
2. Case report
syphilis of unknown duration. Supporting treatment for cerebral
A 60-year-old married man, complaining of weakness in his right infarction was only provided according to the 2008 European
limbs and difficulty with verbal expression, was admitted to guidelines,[9] but therapy for syphilis was not provided at this
Zhongshan Hospital Xiamen University for the first time on time of re-admission. After 20 days of treatment, the patient was
September 4, 2010. He had a history of diabetes mellitus discharged because of increased strength in the right extremities
(controlled in normal blood sugar level) for more than 5 years and improved language functions.
and tobacco use for 30 years. General physical examination was However, 1.0 mL of CSF (undiluted) from the patient at his
normal except for high blood pressure of 178/115 mm Hg and second admission was inoculated into a seronegative New
low-density lipoprotein (LDL) of 3.92 mmol/L (CHOL: 5.55 mm Zealand White male rabbit on April 22, 2011. After 3 months,
mmol/l, TG:1.355 mm mmol/l). A clinical neurological examina- the rabbit showed neither seroconversion nor orchitis. Then the
tion showed that he was suffering from mild expressive aphasia, rabbit was euthanized, and samples from the testes were
right central facial palsy, and mild right hemiparesis. Muscle transferred to a naive rabbit in a “blinded" manner. Twenty-
strength in the patient’s right limbs was grade 4. In addition, the eight days later, the rabbit developed orchitis, and RPR (1:16)
right Babinski sign was positive. However, sensation was normal, and TPPA (1:1280) were positive, indicating seropositivity for T
and Kernig sign was negative with a supple neck. The pallidum. Furthermore, motile T pallidum were observed
echocardiogram showed no abnormalities with the heart. A in isolated testicular fluids under dark-field microscopy.
brain computed tomography (CT) scan was normal (Fig. 1A), but The RIT was therefore positive for T pallidum. This syphilis
brain magnetic resonance imaging (MRI) and DWI showed new strain was confirmed by DNA subtyping using the form “CDC
onset of lacunar infarction involving the left basal ganglia subtype/tp0548 sequence type,” and the strain type was identified
(Fig. 1B–D), the left vermis (Fig. 1E); and other lacunar infarction as 14d/f.
in the left-side mesencephalon (Fig. 1F and G), and also right-side On August 25, 2011, the patient was admitted for a third time
pons (Fig. 1H and I). MRA indicated no intracranial artery to the same hospital for re-treatment of syphilis. Before
stenosis, but had stiff blood vessels (Fig. 1J). A blood specimen treatment, blood and CSF data were collected (Table 1).
taken at the time of admission revealed the patient had negative Although clinical data did not support a diagnosis of neuro-
RPR and specific antitreponemal IgM, and was positive for T syphilis, the positive RIT by patient’s CSF provided definitive
pallidum particle agglutination (TPPA) (titer, 1:320). Diluted evidence that T pallidum had invaded the central nervous system
serum was tested to exclude a false-negative test due to the (CNS). Treatment with penicillin specifically for neurosyphilis
prozone phenomenon of the syphilis RPR. The patient claimed no was administered according to US CDC guidelines, as follows[10]:
history of sexually transmitted disease or suspicious clinical aqueous crystalline penicillin G was administered intravenously
symptoms before and around the time of admission, but he at 24 million units per day (4 million units every 4 hours) for 14
admitted to having had extramarital sex 1 time several years ago. days. At discharge, the patient was advised to undergo serological
Meanwhile, his wife had no history of syphilis or extramarital testing and CSF examination (including a RIT test for scientific

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Figure 1. Brain imaging data. At the first admission, a brain CT scan was normal (A), but brain MRI and DWI showed new onset of lacunar infarction involving the left
basal ganglia (B: T1 WI, C: T2 WI, D: DWI), the left vermis (E: DWI); and other lacunar infarction in the left side of the mesencephalon (F: T1 WI, G: T2 WI), and also the
right side of the pons (H: T1 WI, I: T2 WI). MRA indicated no intracranial artery stenosis, but had stiff blood vessels (J). At the second admission, images revealed
multiple lacunar infarcts observed in the pons varolii (K: T1 WI, L: T2 WI), the left side of the mesencephalon (M: T1 WI, N: T2 WI), and the right side of the thalamus (O:
T1 WI, P: T2 WI). CT = computed tomography, MRI = magnetic resonance imaging.

research) every 6 months. No evidence of new cerebral infarct or active treponemal disease in such a patient would be unusual.
neuro-invasion was observed during a 2-year follow-up period Although a positive antitreponemal IgM test indicates
(Table 1). active infection, a negative antitreponemal IgM test does not
exclude active infection, especially if the test is performed during
the late stages of infection.[11] Nevertheless, clinicians habitually
3. Discussion
use negative antitreponemal IgM or Venereal Disease Research
European CDC guidelines state that titers of nontreponemal and Laboratory test/RPR results as the evidence of disease-free and
specific antitreponemal IgM are correlated with disease activity in active infection-free status.[11,12] This is the first study showing
carriers of active infectious syphilis.[11] However, the European the successful recovery of motile T pallidum using RIT with the
CDC also states that a titer of <1:32 or a negative Venereal CSF derived from a cerebral infarct patient with latent syphilis
Disease Research Laboratory test/RPR test is not sufficient to whose nontreponemal and specific antitreponemal IgM tests were
exclude an active syphilis infection, although the presence of an both negative. These results indicate that negative results in

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Table 1
Clinical test data.
Test results
First admission on Second admission on Third admission on First follow-up on Second follow-up on Third follow-up on
Variable September 4, 2010 April 20, 2011 August 25, 2011 March 23, 2012 September 20, 2012 October 8, 2013
HIV antibody Neg — — — — —
Serum RPR Neg Neg Neg Neg Neg Neg
Serum TPPA 1:320 1:640 1:640 1:320 1:320 1:160
Serum FTA-Abs TP-IgM Neg Neg Neg Neg Neg Neg
CSF RPR — Neg Neg Neg Neg Neg
CSF TPPA — Neg Neg Neg Neg Neg
CSF WBC, per/L — 4.0  106 5.0  106 2.0  106 3.0  106 5.0  106
CSF protein, mg/L — 413.10 438.20 328.10 269.4 313.2
CSF microorganism — Neg Neg Neg Neg Neg
∗
examination
RIT test — Pos Pos Neg Neg Neg
CSF = cerebrospinal fluid, FTA-Abs TP-IgM = fluorescent treponemal antibody-absorption Treponema pallidum-IgM, neg = negative, pos = positive, RIT = rabbit infectivity test, RPR = rapid plasma reagin,
TPPA = Treponema pallidum particle agglutination, WBC = white blood count.
∗
Including Gram stain, India ink method, acid fast bacteria stain, and routine cultures of the second CSF sample (including fungal testing) detection.

nontreponemal and/or specific antitreponemal IgM tests do not is that these findings are based on a single case. Therefore,
exclude the presence of active infectious syphilis. additional studies are needed to confirm the results in other
It is important to select the correct form of penicillin to treat syphilis patients.
syphilis. This fact reflects the ability of T pallidum to sequester in
the CNS and/or the aqueous humor, which are sites that are
relatively inaccessible to some forms of penicillin.[13] In the References
present case study, the patient was treated for latent syphilis using [1] Brown DL, Frank JE. Diagnosis and management of syphilis. Am Fam
benzathine penicillin as a result of his first hospital admission. At Physician 2003;68:283–90.
[2] Herremans M, Notermans DW, Mommers M, et al. Comparison of a
his second hospitalization, for cerebral infarct, the clinical
Treponema pallidum IgM immunoblot with a 19S fluorescent trepone-
symptoms were identical, but a RIT performed using the patient’s mal antibody absorption test for the diagnosis of congenital syphilis.
CSF was positive for motile T pallidum, which indicated that the Diagn Micr Infec Dis 2007;59:61–6.
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[4] Unemo M, Ballard R, Ison C, et al. Laboratory diagnosis of sexually
neurosyphilis. The patient was therefore hospitalized and treated transmitted infections, including human immunodeficiency virus. World AQ6
with aqueous crystalline penicillin G, which is the commonly Health Organisation Bull 2013.
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study was diagnosed with cerebral infarct, but the clinician did characteristics in patients suffering from general paresis in the modern
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not diagnose him with neurosyphilis. In hindsight, it is not [8] Tong ML, Lin LR, Liu LL, et al. Analysis of 3 algorithms for syphilis
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identified in the patient’s CNS by the tests performed on April 20, [9] Hacke W, Ringleb PA, Bousser MG, et al. Guidelines for management of
ischaemic stroke and transient ischaemic attack 2008: The European
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and better able to evade immune responses in the CNS.[14] [10] Workowski KA, Berman S. Sexually transmitted diseases treatment
In conclusion, this individual case study shows that negative guidelines, 2006. MMWR Recomm Rep 2006;55:1–94.
results in nontreponemal and/or specific antitreponemal IgM [11] French P, Gomberg M, Janier M, et al. IUSTI: 2008 European guidelines
on the management of syphilis. Int J Std Aids 2009;20:300–9.
tests do not exclude active infectious syphilis. Moreover, selecting [12] Donkers A, Levy HR, Letens-van Vliet A. Syphilis detection using the
the appropriate penicillin preparation is important because T Siemens ADVIA Centaur Syphilis treponemal assay. Clin Chim Acta
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indicates the necessity of treating a patient with known invasion [13] Workowski KA, Berman S. Sexually transmitted diseases treatment
guidelines, 2010. MMWR Recomm Rep 2010;59:1–10.
of the CNS with aqueous crystalline penicillin G, if previous
[14] Marra C, Sahi S, Tantalo L, et al. Enhanced molecular typing
treatment for syphilis failed and patients had some clinical of treponema pallidum: geographical distribution of strain types
neurological presentation that is otherwise unexplained, but that and association with neurosyphilis. J Infect Dis 2010;202:
could represent neurosyphilis. One limitation of the current study 1380–8.

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