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Case Report

Chronic False Positive Rapid Plasma Reagin (RPR) Tests Induced by COVID-19 Vaccination

Department of Otolaryngology, University of Miami Miller School of Medicine, Miami, FL 33136, USA
Department of Biomedical Engineering, University of Miami, Miami, FL 33136, USA
Department of Neurological Surgery, University of Miami Miller School of Medicine, Miami, FL 33136, USA
Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
Center for Vaccine Research and Pandemic Preparedness (C-VARPP), Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
Department of Pathology, Molecular and Cell-Based Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
Department of Microbiology and Immunology, University of Miami Miller School of Medicine, Miami, FL 33146, USA
Author to whom correspondence should be addressed.
COVID 2023, 3(9), 1304-1309;
Submission received: 1 August 2023 / Revised: 25 August 2023 / Accepted: 28 August 2023 / Published: 30 August 2023


False positive reactive plasmin reagin (RPR) reactivity following a COVID-19 vaccine has been reported, and it is therefore conceivable that individuals who receive frequent coronavirus disease 2019 (COVID-19) vaccinations may exhibit durable RPR responses. Here, we sought to investigate the extent to which repeated mRNA COVID-19 vaccines can elicit chronic false RPR reactivity in a longitudinal cohort. Participants (n = 119) in an IRB-approved (#20201026), longitudinal SARS-CoV-2 cohort study were screened for RPR reactivity via manual RPR card assays. Samples with reactive results underwent additional testing, including follow-on RPR screening at additional timepoints, confirmatory fluorescent treponemal antibody (FTA-ABS) testing and anti-nuclear antibody (ANA) testing. Medical histories were collected. We observed (n = 2) screen-positive RPR results (1.7% [2/119]) following booster vaccination, for which two individuals exhibited chronic, vaccine-induced RPR reactivity for up to 9 months following booster vaccination. Both participants were ANA-negative. It is imperative for clinicians to be mindful of the potential immunologic interference of COVID-19 vaccines with standard infectious disease assays, including RPR testing. Detailed medical histories and clinical contexts, including recent vaccination, should be reviewed prior to proceeding with distressing and invasive workups.
COVID-19; RPR; ID assay

1. Introduction

Syphilis infections caused by the spirochete bacterium Treponema pallidum can result in profound neurological and cardiovascular outcomes, necessitating timely detection and diagnosis. The rapid plasma reagin (RPR) test was first introduced by Portnoy and associates in 1962 [1] and is a simple card assay designed to screen for syphilis, wherein the reagins—antibodies, typically IgE, produced during infection—agglutinate in the presence of beef-derived cardiolipin-lecithin-cholesterol antigen. Many studies of the RPR card test for syphilis support the test and its advantages of rapid performance, simplicity and adequate sensitivity and specificity [2]. However, despite its well-described utility, some studies have reported that non-syphilis diseases and conditions can induce biological false positives. The underlying mechanism by which a biological false positive (or aberrant antibody response to cardiolipin that is not due to T. pallidum infection occurs) is unclear. One study theorized that the globulin responsible for biological false positive reactions differs from the syphilis antibody because it is inhibited by a lecithin associated with the globulin fraction of human serum. Thus, it is posited that the false positive antibody response occurs as the result of auto-immunization by lipids released during exaggerated tissue breakdown [3]. Alternatively, Rusnak et al. suggested that, in the context of HIV-positive individuals, IgM antibodies may be the culprit, as their findings, although not statistically significant, demonstrated that biological false positive tests were correlated with patients with higher IgM levels and did not appear to correlate with anticardiolipin antibody levels or serum IgG or IgA levels [4]. It should be noted, however, that the existing literature suggests that individuals living with HIV may exhibit abnormal serological testing results for syphilis due to the prozone effect. Although this is an interesting phenomenon occurring in the context of false positive serological syphilis testing, the findings of Rusnak et al. involving HIV-positive subjects may not be entirely applicable to the larger population when exploring the underlying mechanism behind false positive testing for syphilis [5]. This population, among others, requires further investigation.
The underlying mechanism leading to false RPR positivity is poorly understood, although its occurrence has been widely demonstrated in the literature. For example, one study tested 63,765 total blood samples obtained at Zhongshan Hospital in the Medical College of Xiamen University from May 2008 to February 2013 and found that 206 (0.32%) individuals had biological false positive reactions in RPR serological testing. This study also characterized relationships between biological false positive tests and any conditions or diseases in these patients, and they found that these biological false positive reactions occurred in association with 17 categories of diseases (e.g., diseases of the respiratory system and diseases of the genitourinary system), including 60 specific diseases. Amongst the 60 diseases associated with false positive RPR reactivity were false labor, megaloblastic anemias, aplastic anemias, redundant prepuce, congenital malformation of the heart and salpingitis [6]. Other causes of non-syphilis-induced RPR-positive tests reported throughout the literature include diseases or conditions such as the hepatitis C virus (particularly those with increased eosinophil counts) [7], leprosy (especially those with the lepromatous form) [2], malaria, respiratory infections, infectious mononucleosis, undulant fever, measles and vaccinia, as well as pregnancy [3]. Most recently, false positive RPR reactivity has been demonstrated when examining baseline non-reactive participant samples that became reactive following COVID-19 vaccination [8]. A recent study by Korentzelos et al. demonstrated that false positivity may depend on the type of RPR test used. In their cohort of 38 participants, 7 (18.4%) were false reactive when tested with the BioPlex RPR as compared to 2 (5.3%) and 1 (2.6%) eliciting false reactivity in the Sure-Vue and Macro-Vue tests, respectively.
Given that COVID-19 vaccination has been demonstrated to generate false positive RPR reactivity, special consideration must be made by clinicians in view of the evolving landscape of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants and subsequent vaccination strategies as recommended by the US Centers for Disease Control and Prevention (CDC) [9]. Current guidelines suggest that individuals may receive “booster” vaccines as early as 8 weeks after the last dose. As chronic false positivity to serological testing extending beyond 6 months has been shown to occur under a number of conditions including but not limited to lymphatic leukemia, pernicious anemia and metastatic cancers [10], and because previous reports demonstrated false RPR reactivity following COVID-19 vaccines [11], it is conceivable that individuals who receive frequent coronavirus disease 2019 (COVID-19) vaccinations could exhibit lasting RPR responses following vaccination. Here, we demonstrate the extent to which repeated mRNA COVID-19 vaccines can elicit chronic false RPR positivity in two distinct participants enrolled in our longitudinal cohort (COVID-19 ImmuniTY “CITY”).

2. Materials and Methods

Participants (n = 119) in our IRB-approved (#20201026), longitudinal SARS-CoV-2 cohort study were screened for RPR positivity at one month following Dose 3 (n = 94) or Dose 4 (n = 25) of a SARS-CoV-2 booster vaccine in order to account for cross-reactivity coinciding with the peak [12] post-vaccination SARS-CoV-2 antibody response. Individuals were excluded if their post-vaccine visits occurred outside of the desired window [20–50 days post boost].
Manual RPR card testing was conducted according to the manufacturer’s (Arlington Scientific, Inc® (ASI), Springville, UT, USA) instructions. Samples with reactive results were diluted in series to achieve endpoint titers at a maximum of 1:16. Endpoint titers were considered the highest dilution in which visible aggregation occurred. All reactive results were confirmed via FTA-ABS testing (Labcorp, Tampa, FL, USA). Screen-positive participants underwent screening at additional timepoints, including pre- and post-vaccines or boosters, to examine the potential relationships between antibody magnitude and the persistence of RPR reactivity. Pre-Dose 2 timepoints were analyzed if available.
SARS-CoV-2 ELISAs were performed using a well-described assay developed by the Icahn School of Medicine at Mount Sinai [13,14]. Briefly, 96-well plates were coated at 4 °C with wild-type Wuhan-Hu-1 SARS-CoV-2 spike protein (2 µg/mL) solution and were incubated overnight. Plates were blocked with 3% non-fat milk prepared in PBS with 0.1% Tween 20 (PBST) and were incubated at room temperature for 1 h. After blocking, serial dilutions of heat-inactivated serum samples were added to the plates and incubated for 2 h at room temperature. Plates were washed three times with 0.1% PBST, followed by the addition of a 1:3000 dilution of goat anti-human IgG–horseradish peroxidase (HRP) conjugated secondary antibody (50 μL) well, and were incubated 1 h. Plates were washed, 100 µL SIGMAFAST OPD (o-phenylenediamine dihydrochloride) solution was added to each well for 10 min, and then the reaction was stopped via the addition of 50 μL per well of 3 M hydrochloric acid. The optical density at 490 nm (OD490) was measured using a Synergy 4 (BioTek [Santa Clara, CA, USA]) plate reader. The background value was set at an OD490 of 0.15; then, discrete titers were reported in values of 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12,800, 1:25,600, 1:51,200, 1:102,400 and 1:204,800. The limit of detection was set at 1:100.
Among our longitudinal cohort (n = 228), we included a total of 119 participants with relevant timepoints in this analysis. Of the participants, 94% received a 3rd mRNA COVID-19 vaccine, with 41% [39/94] receiving Moderna and 59% [55/94] receiving Pfizer. Biological sex distribution was similar (52% female, 48% male) among individuals who received a 3rd dose. The median age was 59 years (range: 21–93). For the 4th dose, we included 25 participants who provided samples at a median of 29 days following booster vaccination. Overall, 56% percent [14/25] received a Moderna booster, and 44% [11/25] received Pfizer. Again, sex was approximately equal with 12 female and 13 male participants. The median age for the participants receiving the 4th dose was 70 years (range: 46–93), and the median day “post-boost” timepoint from receiving the 3rd and 4th dose was 29.5 (SD: 4.39; range: 23 to 41) and 29 (SD: 5.27; range: 20 to 46) days, respectively.

3. Case Description

Following booster (i.e., third or fourth) doses, two screened individuals (1.7% [2/119]) were found to be RPR-positive (Table 1). The first individual, Participant 1, was a 72-year-old heterosexual female and a non-healthcare worker who identified as White and Non-Hispanic. She received two doses of Moderna in early 2021 followed by booster doses of Moderna in July 2021 and April 2022. Thirty-three days had elapsed since her fourth dose, which was the first time point that RPR reactivity was observed. The timepoints demonstrated persistent RPR reactivity, even following the second booster dose, though semi-quantitative titers were very weakly reactive throughout at 1:1. Confirmatory testing (FTA-ABS [LabCorp, Tampa, FL, USA]) was non-reactive for Participant 1. FTA-ABS and ANA testing were non-reactive.
Participant 2 was a 58-year-old heterosexual male healthcare worker who identified as White and Hispanic/Latino. He received two doses of the Pfizer BNT162b2 vaccine in early 2021 with a Pfizer booster dose in October 2021. RPR titers were 1:8 at each timepoint tested following vaccination, with the exception of his visit occurring 131 days following the booster dose, in which the titers decreased to 1:4. His post-fourth-dose RPR titer again escalated to 1:8. Participant 2’s FTA-ABS results were reactive at all post-booster time points available, though none were ANA-positive. Curiously, both RPR and FTA-ABS prior to primary vaccine receipt were non-reactive.
Both individuals were polled for detailed medical and vaccination histories. Neither had a previous history of COVID-19 infection, syphilis, tuberculosis, rickettsial diseases or myocarditis, nor had they received any other vaccines at the time of their SARS-CoV-2 boosters. Participant 1 did report a previous history of breast cancer, though she reported that she had been in remission for many years. The blood type for Participants 1 and 2 were AB+ and O+, respectively. Additionally, the SARS-CoV-2 antibody titer magnitude and kinetics observed in Participants 1 and 2 were consistent with previous work demonstrating a robust peak response followed by a time-dependent, gradual decline [12]. Neither participant demonstrated particularly robust post-vaccination antibody titers in comparison with the rest of the cohort (n = 228) or even among those screened with the RPR assay (n = 119).
Further, there was no clear relationship between SARS-CoV-2 titer magnitude and RPR titer magnitude (Table 1) among either participant found to be RPR reactive. Spearman’s rank correlation demonstrated a weak, non-significant negative correlation between the two assays (r = −0.55, p = 0.12). Surprisingly, SARS-CoV-2 titer magnitude was not predictive of RPR reactivity, as the remaining participants screened at the initial post-booster timepoints (63% [75/119]) had equivalent or higher titers.

4. Discussion

In this study, we investigated the incidence of RPR test false positivity following vaccination with mRNA COVID-19 vaccines in a longitudinal cohort. Approximately 2% of our participants exhibited low, chronic RPR reactivity, sustained for as long as 9 months following each dose of the vaccine. This finding builds on previous work examining false positive RPR results following COVID-19 vaccination [8] and supports persistent reactivity for up to a year and half, which is rare for smaller immune challenges such as vaccination [15]. Although we cannot rule out the distinct possibility that Participant 2 acquired an active syphilis infection in the time subsequent to Dose 2, the nature of the RPR titers reported (results < 1:8 may persist over the course of a lifetime even in treated syphilis cases and are considered “low” [16]) and proximity to COVID-19 vaccination are more suggestive of a chronic false positive result.
Further, Participant 2 had no past medical history or sociodemographic risk factors that could predispose them to a greater risk of syphilis infection or any history of known autoimmune disorders or infectious diseases. Thus, although the chronic false positive result elicited in Participant 2 may have transpired due to a number of different mechanisms, we theorize that the interesting findings of Participant 2’s non-reactive pre-vaccine RPR and FTA-ABS test and subsequent RPR-positive test may be a result of COVID-19 vaccine-induced cross-reactivity. Theoretical COVID-19 vaccine-specific explanations include the generation of and cross-reactivity with anti-PEG antibodies, as SARS-CoV-2 mRNA vaccines have been demonstrated to induce or boost PEG-specific antibodies [9], which may function similarly to syphilis-specific antibodies. Responsible mechanisms may also be due to underlying genetic susceptibility, by which aberrant molecular mimicry [10] (the production of particular autoimmune autoantibodies) or bystander activation [11] (the activation of T-cells without antigen recognition) may explain Participant 2′s false RPR test positivity.
The interesting findings detailed in this case report provide useful information to clinicians and the greater scientific community. In addition to the inherent strengths of following a longitudinal cohort (n = 228), the authors emphasize the non-zero likelihood of chronic RPR false positivity following the administration of the SARS-CoV-2 vaccine and boosters outside of this cohort. This should be considered in the context of routine RPR screening, particularly in individuals with known co-morbidities (i.e., autoimmune diseases). The limitations of this work include the small percentage of false RPR reactivity represented (~2%), though it stands to reason that, if applied to the total population, these serological findings would result in a rather substantial number of affected individuals. Indeed, when applied to all SARS-CoV-2-vaccinated individuals within the global population, this would equate to approximately 94,000,000 people affected worldwide.
In summation, given that vaccination remains the preeminent strategy in mitigating SARS-CoV-2 transmission and disease severity, it is critically important for the clinical community to be aware of possible vaccine cross-reactivity with RPR assays. RPR assays, as well as other serological tests that do not align with clinical presentations following SARS-CoV-2 vaccination, may need to be repeated or supplemented with additional diagnostic tools. Consequences for patients include time-consuming and invasive workups and interventions, including lumbar puncture [12], necessitating detailed screening questions for recent vaccinations, including any COVID-19 boosters, at the time of RPR screening. Future work employing large sample populations is needed to better understand the incidence and duration of false reactive RPR tests, particularly in view of CDC recommendations regarding COVID-19 vaccination strategies.

5. Conclusions

A greater investigation is needed to look into the underlying mechanisms leading to false RPR assays. Understanding possible mechanisms, including autoimmunization following exaggerated breakdown, high levels of IgM antibodies, anti-PEG antibodies and underlying genetic susceptibility, may aid in avoiding a misdiagnosis of syphilis in COVID-19 vaccination recipients as well as in individuals with various diseases and conditions that have been shown to generate false RPR test results.

Author Contributions

Conceptualization: E.W., S.P. (Suresh Pallikkuth) and S.P. (Savita Pahwa); Investigation: E.W. and J.M.C.; Resources: S.P. (Suresh Pallikkuth), S.P. (Savita Pahwa), M.H. and F.K.; Writing—review and editing: E.W., D.J.K., M.H., J.M.C., F.K., S.P. (Savita Pahwa) and S.P. (Suresh Pallikkuth); Funding: S.P. (Savita Pahwa) and F.K. All authors have read and agreed to the published version of the manuscript.


This work was partly funded by the NIAID Collaborative Influenza Vaccine Innovation Centers (CIVIC) contract 75N93019C00051 as part of the PARIS/SPARTA studies.

Institutional Review Board Statement

This case report was the result of a sub-set analysis conducted under the COVID-19 Immunity Study (“CITY”), which was IRB approved at the University of Miami (#20201026). All participants provided written informed consent.

Informed Consent Statement

The study participants described herein provided written informed consent for their results to be submitted as a case report.

Data Availability Statement

The dataset generated is not publicly available given the sensitive nature of the information contained within the dataset, including personal and medical details.

Conflicts of Interest

Florian Krammer has consulted for Curevac, Seqirus and Merck and is currently consulting for Pfizer, Third Rock Ventures, Avimex and GSK. He is named on several patents regarding influenza virus and SARS-CoV-2 virus vaccines, influenza virus therapeutics and SARS-CoV-2 serological tests. Some of these technologies have been licensed to commercial entities, and Dr. Krammer is receiving royalties from these entities. Dr. Krammer is also an advisory board member of Castlevax, a spin-off company formed by the Icahn School of Medicine at Mount Sinai to develop SARS-CoV-2 vaccines. The Krammer laboratory has received funding for research projects from Pfizer, GSK and Dynavax, and three of Dr. Krammer’s mentees have recently joined Moderna. All other authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper.


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Table 1. RPR positive participant characteristics.
Table 1. RPR positive participant characteristics.
IDAgeSexSARS-CoV-2 Vaccine ManufacturerTimepointRPR TiterFTA-ABS ResultANA Result eDays ElapsedSARS-CoV-2 Titer
172FemaleModerna bPre 2nd Dose---- --a
Post 2nd Dose1:1-- 51 d1600
Pre 4th dose1:1-- 252 d51,200
Post 4th dose1:1Non-ReactiveNegative33 d25,600
Post 4th dose1:1-- 129 d51,200
258MalePfizer bPre-VaccineNon-ReactiveNon-ReactiveNegative----
Post 2nd Dose1:8ReactiveNegative266 c3200
Pre 3rd Dose1:8----273 d200
Post 3rd dose1:8ReactiveNegative31 d25,600
Post 3rd Dose1:4ReactiveNegative131 d6400
Post 4th Dose1:8ReactiveNegative211 d1600
a Timepoint not available as participant was not enrolled in the study prior to vaccine receipt. b Primary/booster vaccines were homologous in each participant described. c Days elapsed between pre-vaccine timepoint and the post 2nd dose timepoint. d Days elapsed between receipt of a COVID-19 vaccine/booster. e ANA results were reported as negative (<1:80); borderline (1:80); positive (>1:80).
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MDPI and ACS Style

Williams, E.; Kennedy, D.J.; Hoffer, M.; Carreño, J.M.; Krammer, F.; Pallikkuth, S.; Pahwa, S. Chronic False Positive Rapid Plasma Reagin (RPR) Tests Induced by COVID-19 Vaccination. COVID 2023, 3, 1304-1309.

AMA Style

Williams E, Kennedy DJ, Hoffer M, Carreño JM, Krammer F, Pallikkuth S, Pahwa S. Chronic False Positive Rapid Plasma Reagin (RPR) Tests Induced by COVID-19 Vaccination. COVID. 2023; 3(9):1304-1309.

Chicago/Turabian Style

Williams, Erin, Devin J. Kennedy, Michael Hoffer, Juan Manuel Carreño, Florian Krammer, Suresh Pallikkuth, and Savita Pahwa. 2023. "Chronic False Positive Rapid Plasma Reagin (RPR) Tests Induced by COVID-19 Vaccination" COVID 3, no. 9: 1304-1309.

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