Molecular Diagnosis of Syphilis in Brazilian Ambulatory Patients: Detection of Treponema pallidum subsp. pallidum in Serum Using Ancient DNA Protocols

TL;DR

This study shows that ancient DNA methods can improve syphilis detection in blood samples, offering a better alternative to traditional tests.

Contribution

The study introduces the use of ancient DNA protocols for molecular diagnosis of syphilis in serum samples.

Findings

aDNA protocols successfully amplified T. pallidum DNA in 82.35% of serum samples.

T. pallidum sequences were confirmed in 70.59% of the tested samples.

Molecular approaches using aDNA methods show potential for improving syphilis detection and surveillance.

Abstract

The rising incidence of syphilis in recent decades underscores the need to improve diagnostic and control strategies. The infection caused by Treponema pallidum subsp. pallidum is commonly diagnosed using serological tests. However, these methods exhibit limitations in the early or late stages of disease, when antibody responses and/or bacterial loads are low. Molecular biology detection using serum samples is also hampered by low circulating bacterial loads during asymptomatic periods. Ancient DNA (aDNA) studies apply methods adapted to recovering low concentrations and degraded DNA. In this study, we evaluated the effectiveness of aDNA protocols applied to the molecular diagnosis of T. p. subsp. pallidum in serum samples from ambulatory patients from Rio de Janeiro, Brazil. A PRISMA-based systematic review was also performed to identify studies using molecular biology diagnosis from serum. Twenty serums screened by TPHA (Treponema pallidum Hemagglutination assay) and with different VDRL titers (Venereal Disease Research Laboratory test) were analyzed. Amplification of tpp15 gene was observed in 14/17 (82.35%) samples; T. pallidum sequence was confirmed in 12/17 (70.59%). The findings demonstrate the potential of molecular approaches based on aDNA-adapted protocols as alternatives to conventional serological diagnosis, contributing to improved detection of infection and strengthening epidemiological surveillance of syphilis.

Full text

1. Introduction

Syphilis remains a sexually transmitted and congenital infection of global relevance. Despite advances in diagnostic and therapeutic methods, the disease continues to have high incidence and prevalence in several regions worldwide, reflecting the need for epidemiological surveillance and prevention strategies [1,2,3]. Treponema pallidum subspecies pallidum belongs to the Spirochaetaceae family, characterized by its helical shape and high motility, which favor its dissemination in host tissues [4]. This subspecies is the etiological agent of syphilis, a primary sexually transmitted and congenital infection that represents a major concern for global public health. Syphilis is distinguished by its remarkable ability to mimic a variety of dermatological, neurological, and systemic manifestations, a characteristic that has earned it the classic title of “the great imitator” (magna simulans) [5].

The genus Treponema also includes other species and subspecies with different clinical presentations: T. pallidum subspecies endemicum, which causes bejel; T. pallidum subspecies pertenue, the etiological agent of yaws; and T. carateum, responsible for pinta. Despite sharing similar morphology, these bacteria differ in their modes of transmission and geographic distribution [6]. Syphilis progresses through well-defined clinical stages, shaped by the complex interaction between T. pallidum and the host immune response [7]. The primary stage typically presents with painless ulcers at the inoculation site, appearing two to three weeks after exposure [8]. Most untreated individuals progress to secondary syphilis, characterized by systemic symptoms and heterogeneous cutaneous lesions, frequently affecting palms and soles, an indicative feature of infection [9,10]. After resolution of these lesions, the agent may persist in a latent state and, without treatment, evolve into more severe forms with cardiovascular, neurological, or osseous complications [10].

Diagnostic strategies for syphilis include direct and indirect detection of T. pallidum. Direct methods include dark-field microscopy, silver staining, direct fluorescent antibody testing, and rabbit infectivity testing. Although classical, these techniques present important limitations, including variable sensitivity that depends on bacterial structure, the clinical stage of infection, and specimen quality [11]. Due to these challenges, serological tests, both treponemal and non-treponemal, remain the primary diagnostic tools. The VDRL (Venereal Disease Research Laboratory) and the RPR (Rapid Plasma Reagin) are widely used for screening and therapeutic follow-up. On the other hand, treponemal tests, such as TPHA (Treponema pallidum Hemagglutination Assay), FTA-ABS (Fluorescent Treponemal Antibody Absorption), and immune assays (EIA/ELISA), have higher specificity and are used for diagnostic confirmation [12,13].

Serological tests for syphilis are subject to false-positive and false-negative results, which may be related to the clinical stage of infection, the presence of co-infections, or the prozone effect [14,15,16]. Consequently, molecular biology techniques, particularly Polymerase Chain Reaction (PCR), have increasingly been used to directly detect T. pallidum DNA in various biological samples. However, the application of PCR in serum still presents significant challenges. The primary limitation is the low and variable bacterial load, due to transient spirochetemia [17,18]. Blood and serum contain natural inhibitors of PCR, such as proteins and lipids, which can interfere with the enzymatic amplification and reduce the sensitivity of the analysis [18]. On the other hand, samples from lesions or tissue exudates are considered more suitable for molecular detection of T. pallidum, as they have higher bacterial loads. A systematic review by Simpore and coauthors (2022) found a significant increase in the adoption of molecular biology methods between 2009 and 2019, with a predominance of conventional PCR and real-time PCR. Both performed satisfactorily, with consistently high specificity, although sensitivity varies with the clinical stage of the infection [19].

Ancient DNA (aDNA) studies have demonstrated the potential to detect Treponema spp. in archaeological materials, despite the challenges inherent to recovering fragmented DNA resulting from taphonomic processes [20,21,22,23]. To overcome these limitations, methodologies adapted to the analysis of highly degraded DNA or at low concentrations have been successfully applied [24]. Kolman and co-authors (1999) were the first to report the identification of T. p. ssp. pallidum in human remains from Easter Island, dated to approximately 240 ± 50 years BP, confirming infection in archaeological material [20]. Guedes and coauthors (2018) investigated the presence of T. pallidum in young individuals without signs of pathology, dated to the seventeenth and nineteenth centuries in Rio de Janeiro [21]. These results are part of a historical period marked by outbreaks of syphilis and yaws in the city, when it is estimated that about one-fifth of the population was infected [25]. These advances showed that, with optimized, sensitive protocols, it is possible to detect T. pallidum DNA even in highly degraded samples. In this context, the present study proposes applying a methodological protocol adapted from paleogenetic studies to detect T. pallidum in serological samples with low bacterial concentrations. Additionally, a PRISMA-guided systematic review was conducted to identify studies of molecular biology methods for syphilis diagnosis in serum samples, providing context for the interpretation of our findings. We hypothesize that the molecular detection of T. p. subsp. pallidum in serum samples can be achieved using protocols originally developed for archaeological DNA recovery.

2. Materials and Methods

2.1. Samples

Twenty serum samples from patients were analyzed, including sixteen with positive serology for syphilis, and as negative controls, two weakly reactive, and two non-reactive samples, as determined by VDRL and TPHA testing. Serological titers ranged from 1:2 to 1:8, and none of the individuals had a prior history of treatment. The samples were provided by the Laboratório de Diagnóstico, Ensino e Pesquisa (LADEP) of the Sergio Arouca National School of Public Health (ENSP/FIOCRUZ). Their use had been previously approved by the ENSP Research Ethics Committee under opinion No. 12/2013. Authorization to receive sera for molecular biology analyses was granted by the institution’s Scientific Committee, enabling the development of Guedes’ master’s thesis in 2014 [26]. The samples were subsequently stored in the biorepository of the Laboratório de Parasitologia Integrativa e Paleoparasitologia (LPIP/IOC/FIOCRUZ) for future investigations.

2.2. Molecular Biological Diagnostic Using aDNA Protocol

DNA extraction was carried out using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), with protocol modifications designed to enhance aDNA recovery. For each sample, 200 μL of serum was processed, and a negative extraction control was included. In addition to the standard chemical digestion step, a physical lysis procedure was incorporated by exposing the samples to liquid nitrogen or dry ice, followed by vigorous vortex agitation at maximum speed to enhance cellular disruption. Centrifugation was performed at 14,000 rpm for 5 min, replacing the original protocol step of 8000 rpm for 2 min. Elution was conducted in a final volume of 40 μL to increase DNA concentration. DNA quantification was subsequently performed using the Quantus™ fluorometer (Promega, Madison, WI, USA).

A reconstructive polymerization pretreatment was applied to all extracted samples to facilitate the reconstruction and amplification of degraded DNA fragments, following the procedure described by Iñiguez in 2021 [24], a widely used technique in paleogenetic analyses. For the detection of T. pallidum, PCR amplification targeting the tpp15 gene was performed using the primers TL243F (5′-GAGCAGGATGTCTCTATGAGTTATAAAAGA-3′) and TH123R (5′-GAAGCCACTACCGATGTGCG-3′) [20], PCRs were set up in a final volume of 25 μL, containing 1× buffer, 1.75 mM MgCl_2_, 0.2 mM dNTPs, 10 μM of each primer, 3 U Platinum Taq DNA Polymerase (Promega), and 5 μL of extracted DNA (1–5 ng). The amplification protocol consisted of an initial denaturation at 95 °C for 5 min, followed by 50 cycles of denaturation at 95 °C for 40 s, annealing at 60 °C for 40 s, and extension at 72 °C for 40 s, with a final extension at 72 °C for 7 min. Reactions were carried out in a SimpliAmp™ thermocycler (Thermo Fisher Scientific, Waltham, MA, USA), and PCR negative controls were included to monitor for possible contamination.

The PCR products (pPCR) were analyzed by electrophoresis on a 2% agarose gel, stained with GelRed™ (Biotium, Fremont, CA, USA), and visualized under ultraviolet light using the Bio-Rad Transilluminator 2000 system (Hercules, CA, USA). Samples that did not yield visible amplicons were subjected to a second round of amplification under the same conditions used for the tpp15 marker. Positive amplicons were purified using the ExoSAP-IT™ reagent (Thermo Fisher Scientific, Waltham, MA, USA). DNA sequencing was performed with the BigDye™ Terminator v3.1 Cycle Sequencing Kit on the RPT01A DNA Sequencing Platform at FIOCRUZ, using the Applied Biosystems ABI 3730 sequencer (Foster City, CA, USA). The resulting sequences were processed and edited using BioEdit v5.0.9 and SeqMan v7.00 (DNASTAR Lasergene, Madison, WI, USA) and subsequently compared with reference sequences in the GenBank database via the NCBI/BLAST tool available at https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 12 July 2024.

In order to investigate the possible presence of inhibitors in the amplification reaction, the remaining negative samples were subjected to PCR using the 12S rDNA marker, widely used for the identification of vertebrates, as described by Kitano and coauthors (2008) [27]. The PCR with a final volume of 25 μL was performed with 1X buffer, 2 mM MgCl_2_, 0.2 mM dNTP, 10 μM each primer, 2U Platinum Taq DNA Polymerase (Promega, USA), and 5 µL or 1–10 ng/μL of DNA. The cycling included an initial cycle of 95 °C for 3 min, followed by 35 cycles of 30 s at 95 °C, 30 s at 57 °C, and 30 s at 72 °C with an extension of 10 min at 72 °C. The pPCR were analyzed as described above.

2.3. Comparative Evaluation of Ancient DNA and Conventional Molecular Protocols

To compare the ancient DNA protocol with a conventional molecular approach, PCR assays were performed with and without a reconstructive polymerization pre-treatment. The reconstructive polymerization pre-treatment is commonly employed in aDNA analyses prior to amplification. In parallel, PCR assays were conducted without this pre-treatment, following a conventional molecular workflow described above (Section 2.2).

2.4. Systematic Review

A systematic review framework was employed to identify studies that addressed the molecular biology diagnosis of syphilis in serum samples (Supplementary Figure S1).

3. Results

3.1. Molecular Biology Diagnosis of Syphilis in Serological Samples

The results revealed that 14 of 17 of the TPHA-positive or weakly reactive serum samples (82.35%) exhibited amplification of the T. pallidum marker. The presence of bacterial DNA was shown despite the low concentration of DNA obtained after the extraction step (Table 1). The TPHA-negative serum samples used as negative controls showed no tpp15 amplifications. Similarly, no amplification was detected in weakly reactive samples.

A total of 12/14 (85.71%) PCR-positive samples generated sequences that met quality criteria for comparison with the GenBank/NCBI database. The presence of T. p. subsp. pallidum was confirmed in 10/12 (83.33%) of the sequenced samples, based on the characteristic polymorphism (T191943C) of the subspecies (Supplementary Figure S2). Among the samples confirmed as T. p. subsp. pallidum, 10 presented serological titers of 1:2, 1:4, or 1:8 in the VDRL tests, showing concordance between serological and molecular data. However, in two samples (SR02 and SR13) with titers of 1:2 and 1:4, although amplification was successful and the sequences were of good quality, the region obtained did not include the segment corresponding to the differential polymorphism, limiting identification to the T. pallidum species level (Supplementary Figure S2). Two other samples (SR08 and SR12) with titers of 1:4 and 1:8 presented amplified products with the expected size, but not quality sequences. The sequences obtained were compromised by base noise, low signal intensity, and overlapping peaks in the electropherograms, making the comparative analysis with GenBank unfeasible. All negative samples showed 12S rDNA amplification with the molecular marker applied as an endogenous DNA control, indicating the integrity of the extracted DNA and the absence of inhibitory compounds that compromised the PCR activity.

3.2. Comparative Methodological Analysis

The comparative methodological analysis demonstrated differences in amplification efficiency between the conventional molecular protocol and the aDNA approach (Supplementary Figure S3). PCR assays performed without reconstructive polymerization pre-treatment yielded amplification in 7/18 samples (38.9%), indicating a lower detection rate when compared to assays preceded by reconstructive polymerization, which yielded amplifications in 14/18 samples (77.8%). In addition, PCR products obtained without reconstructive polymerization consistently exhibited weak low-intensity bands on agarose gels, which impairs the following sequencing procedures and consequently the confirmation of syphilis diagnosis. Electrophoretic profiles are provided in Supplementary Figure S3.

3.3. Systematic Review

Results of the systematic review showed a limited number of eligible studies addressing the molecular diagnosis of syphilis in serum samples, including four scientific articles and two master’s dissertations (Supplementary Table S1). The studies predominantly employed conventional PCR and real-time PCR targeting single-copy genes (Supplementary Table S1).

4. Discussion

In the present study, the T. pallidum detection rate was 82.35% TPHA seropositive individuals. Among the PCR-positive samples, 85.71% were identified as T. p. subsp. pallidum sequence. The serum samples were collected in 2013 and originally intended for the VDRL and TPHA serological tests of LADEP/FIOCRUZ. It is important to highlight that, even though these samples were stored for about 12 years, the application of aDNA recovery protocols enhanced the sensitivity of the PCR applied. The analysis not only enabled the detection of treponemal DNA in the serum samples but also provided insights into the limitations and potential of aDNA-based protocols within a contemporary clinical framework, particularly in samples with low bacterial loads.

The application of molecular methods in serological samples for the diagnosis of syphilis has been investigated since the end of the twentieth century [17,28]. Although the serum does not present the best performance for the direct detection of T. pallidum, due to the low concentration of circulating treponemas that hinder its lysis and DNA recovery [4,17], this material remains of great interest as it is widely used in clinical practice and often is the first sample collected during diagnostic investigation [29,30,31]. This availability underscores the importance of evaluating its potential in molecular approaches, although PCR analyses on serum samples are still infrequent. Nevertheless, conventional PCR-based molecular approaches are associated with higher costs and longer processing times, which may limit their routine application [31]. Molecular approaches enable more detailed epidemiological analyses, allowing the identification of circulating T. pallidum lineages (Nichols or SS14 clade) and the detection of genetic markers associated with antimicrobial resistance. Molecular surveillance is further justified by the increasing documentation of antimicrobial macrolide resistance in T. pallidum, associated with the 23S rRNA gene [32,33,34]. Evidence of reduced susceptibility or resistance to penicillin has also been documented [35,36,37]. The disease management relies primarily on penicillin G, while macrolides are commonly used in cases of penicillin allergy and have demonstrated efficacy as alternative therapeutic options [35,36,37,38].

Previous studies have reported detection rates in cases of primary and secondary syphilis in serological samples, ranging from 28.57% to 75% [28,39]. Martin and coauthors (2009) [31] reported that, in cases of primary syphilis, the molecular diagnosis performed on ulcer swabs or scrapings showed sensitivity close to 75%. On the other hand, in blood and serum samples from patients with secondary syphilis, positivity was approximately 50%. According to the authors, PCR performance is superior in primary lesions, where the bacterial load is higher and precedes complete seroconversion [31]. Blood and/or serum may be adequate when the bacteria is systemically disseminated. Durán Rodríguez and coauthors (2019) developed a qPCR targeting the T. pallidum tpp47 gene for the detection in serum from patients with suspected gestational and congenital syphilis, achieving 75% positivity and good agreement with serological tests [40]. Oliveira (2016) [41] conducted a molecular biology investigation involving patients with and without a history of HIV infection who were evaluated at the Souza Araújo Outpatient Clinic—FIOCRUZ. A number of 16/26 serum samples yielded T. p. subsp. pallidum sequences, and the A2058G mutation was detected in two of them, indicating the circulation of macrolide-resistant variants [41]. The detection of T. p. subsp. pallidum across multiple clinical specimen types, including saliva, serum, and CSF, was assessed in patients with primary, secondary, latent, and suspected neurosyphilis in China, totaling 1023 analyzed samples [42]. In serum, T. p. subsp. pallidum DNA was identified in 17 cases, with positivity concentrated in the secondary (2/59) and latent (10/412) stages, and no amplification was obtained from individuals with primary syphilis. Among patients with suspected neurosyphilis, 9.1% (5/55) of serum samples were positive. Overall, serum detection remained low (3.1%, 17/543), likely reflecting the limited circulating bacterial burden, which restricts the release of detectable nucleic acids into the bloodstream. The detection of treponemal DNA in individuals with latent syphilis suggests that this clinical stage may function as a persistent reservoir capable of sustaining transmission [42]. In the present study, the detection rates were higher than those reported in the literature identified through the PRISMA-based methodology.

The World Health Organization has established global strategic guidelines for the control and elimination of major public health challenges, including HIV, viral hepatitis, and other sexually transmitted infections (STIs), with particular emphasis on syphilis [43]. In the national context, Brazil reflects this epidemiological pattern: between 2010 and 2025, a total of 1,902,301 cases of acquired syphilis were reported [44]. In parallel, Brazilian public health strategies agenda for the elimination of congenital syphilis, establishing 2030 as a target year for its elimination through strengthened surveillance, expanded access to diagnosis and treatment [45,46,47].

The global response to syphilis involves stopping mother-to-child transmission, strengthening surveillance systems, timely diagnosis, and scaling up more accurate diagnostic tools capable of differentiating between active infection and specific manifestations, such as neurosyphilis and congenital syphilis [48]. In this context, molecular diagnostic testing is particularly relevant, as it allows confirmation of infection and monitoring of the therapeutic response. Despite the limited number of samples analyzed in the present study, we demonstrated the applicability of the molecular approach for serum diagnosis. Future investigations will include a larger number of samples to confirm the robustness of the approach presented here.

The present study has some limitations that should be considered when interpreting the results. First, clinical information regarding the disease status of patients, including the clinical stage of syphilis, was not available. The serum samples analyzed were archived samples originally collected at a single center (LADEP, ENSP/FIOCRUZ) and provided to our laboratory exclusively for use as control material in a paleogenetic study conducted in 2014 [25], under conditions that restricted access to associated clinical or epidemiological data. Consequently, limiting the evaluation of molecular detection rates in relation to disease stage. Second, the small number of samples analyzed may restrict the strength of the conclusions of the findings and preclude more detailed analyses across clinical or epidemiological subgroups. Third, the specificity of the assay was not systematically evaluated against a comprehensive panel of potential biological confounders or interfering agents. Although no false-positive results were observed in the samples analyzed. Nevertheless, samples that failed to yield T. p. subsp. pallidum amplifications were further evaluated for potential PCR inhibition. Finally, the use of long-term archived serum samples may have introduced uncontrolled pre-analytical variables affecting DNA integrity and detection efficiency.

Improving the supply of diagnostic methods, access to treatment, and the incorporation of prevention strategies in health services focused on acquired and congenital syphilis are essential. Strengthening molecular diagnosis by standardizing procedures, rigorously implementing quality control, and continuously training teams is important to ensure reliable results and support clinical decisions. In this scenario, the increasing application and dissemination of molecular biology techniques for STI diagnosis represent a major strength, as they promote technical consolidation, facilitate assay execution, and progressively reduce per-sample costs, thereby enhancing their feasibility for diagnostic and surveillance purposes.

In summary, the present study demonstrated an aDNA protocol as a reliable and accurate alternative for the diagnosis of active syphilis, showing the specific detection of T. p. subsp. pallidum polymorphism, so excluding T. p. subspecies endemicum (bejel), T. p. subspecies pertenue (yaws), and T. carateum (pinta); absence of false-positive results, which frequently occur in serological assays; and a level of detection in patients with serological titers 1:2–1:8, low values indicative, mainly, of early infections, but also recent latent syphilis (asymptomatic phase) suggesting possible active infection, which could allow a precocious treatment and follow-up until cure.