Use of Treponema pallidum PCR in testing of ulcers for diagnosis of primary syphilis.
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ABSTRACT:
SUBMITTER: Gayet-Ageron A
PROVIDER: S-EPMC4285282 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BACKGROUND: Syphilis is a growing public health problem among men who have sex with men (MSM) globally. Rapid and accurate detection of syphilis is vital to ensure patients and their contacts receive timely treatment and reduce ongoing transmission. METHODS: We evaluated a PCR assay for the diagnosis of Treponema pallidum using swabs of suspected early syphilis lesions in longitudinally assessed MSM. RESULTS: We tested 260 MSM for T pallidum by PCR on 288 occasions: 77 (26.7%) had early syphilis that was serologically confirmed at baseline or within six weeks, and 211 (73.3%) remained seronegative for syphilis. Of 55 men with primary syphilis, 49 were PCR positive, giving a sensitivity of 89.1% (95% CI: 77.8%-95.9%) and a specificity of 99.1% (95% CI: 96.5%-99.9%). Of 22 men with secondary syphilis, 11 were PCR positive, giving a sensitivity of 50% (95% CI: 28.2%-71.8%) and a specificity of 100% (95% CI: 66.4%-71.8%). Of the 77 syphilis cases, 43 (56%) were HIV positive and the sensitivity and specificity of the PCR test did not vary by HIV status. The PCR test was able to detect up to five (10%) primary infections that were initially seronegative, including one HIV positive man with delayed seroconversion to syphilis (72 to 140 days) and one HIV positive man who did not seroconvert to syphilis over 14 months follow-up. Both men had been treated for syphilis within a week of the PCR test. CONCLUSIONS: T pallidum PCR is a potentially powerful tool for the early diagnosis of primary syphilis, particularly where a serological response has yet to develop.
| S-EPMC3541217 | biostudies-literature
Project description:Syphilis serodiagnosis is challenging because distinct clinical forms of the infection may influence serological performance and discordant results between tests make clinical decisions difficult. Several recombinant Treponema pallidum-proteins have already been tested for syphilis diagnosis and they are critical to achieve high accuracy in serological testing. Our aim was to assess the varied from performance of T. pallidum-recombinant proteins TmpA, TpN17 and TpN47 for syphilis serodiagnosis. The proteins were evaluated using sera of 338 T. pallidum-negative, 173 T. pallidum-positive individuals and 209 sera from individuals infected with unrelated diseases. The diagnostic potential was validated by analysis of ROC curves. In the liquid microarray analyses, the ROC curve varied from 99.0% for TmpA and TpN17 to 100% for TpN47. The sensitivity score yielded values of up to 90% for TpN17, 100% for TpN47 and 80.0% for TmpA. The lowest and highest specificity values were presented by TpN47 (91.9%) and TmpA antigens (100%), respectively. TpN47 showed the highest accuracy score (95.5%) among all the recombinant proteins assayed. For the ELISA, the ROC curve was 97.2%, 91.8% and 81.6% for TpN17, TmpA and TpN47, respectively. TpN17 and TmpA yielded a sensitivity of 69.9%, while TpN47 obtained a value of 53.8%. Specificity was almost 100% for all three proteins. No cross-reaction was observed for TpN17 in the serum samples from non-bacterial infections. Regarding leptospirosis-positive samples, cross-reactivity score was varied from 8.6 to 34.6%. This is most probably due to conservation of the epitopes in these proteins across bacteria. The use of recombinant proteins in immunoassays for syphilis diagnosis was showed provide greater reliability to results of the treponemal assays. Despite the low sensitivity, the proteins showed high diagnostic capacity due to the AUC values found. However, an improvement in sensitivity could be achieved when antigenic mixtures are evaluated.
| S-EPMC7302711 | biostudies-literature
Project description:Despite more than a century of research, genetic manipulation of Treponema pallidum subsp. pallidum (T. pallidum), the causative agent of syphilis, has not been successful. The lack of genetic engineering tools has severely limited understanding of the mechanisms behind T. pallidum success as a pathogen. A recently described method for in vitro cultivation of T. pallidum, however, has made it possible to experiment with transformation and selection protocols in this pathogen. Here, we describe an approach that successfully replaced the tprA (tp0009) pseudogene in the SS14 T. pallidum strain with a kanamycin resistance (kanR) cassette. A suicide vector was constructed using the pUC57 plasmid backbone. In the vector, the kanR gene was cloned downstream of the tp0574 gene promoter. The tp0574prom-kanR cassette was then placed between two 1-kbp homology arms identical to the sequences upstream and downstream of the tprA pseudogene. To induce homologous recombination and integration of the kanR cassette into the T. pallidum chromosome, in vitro-cultured SS14 strain spirochetes were exposed to the engineered vector in a CaCl2-based transformation buffer and let recover for 24 hours before adding kanamycin-containing selective media. Integration of the kanR cassette was demonstrated by qualitative PCR, droplet digital PCR (ddPCR), and whole-genome sequencing (WGS) of transformed treponemes propagated in vitro and/or in vivo. ddPCR analysis of RNA and mass spectrometry confirmed expression of the kanR message and protein in treponemes propagated in vitro. Moreover, tprA knockout (tprAko-SS14) treponemes grew in kanamycin concentrations that were 64 times higher than the MIC for the wild-type SS14 (wt-SS14) strain and in infected rabbits treated with kanamycin. We demonstrated that genetic manipulation of T. pallidum is attainable. This discovery will allow the application of functional genetics techniques to study syphilis pathogenesis and improve syphilis vaccine development.
| S-EPMC8284648 | biostudies-literature
Project description:Five immunodominant Treponema pallidum recombinant polypeptides (rTpN47, rTmpA, rTpN37, rTpN17, and rTpN15) were blotted onto strips, and 450 sera (200 from blood donors, 200 from syphilis patients, and 50 potentially cross-reactive) were tested to evaluate the diagnostic performance of recombinant Western blotting (recWB) in comparison with in-house whole-cell lysate antigen-based immunoblotting (wclWB) and T. pallidum hemagglutination (MHA-TP) for the laboratory diagnosis of syphilis. None of the serum specimens from blood donors or from potential cross-reactors gave a positive result when evaluated by recWB, wclWB, or MHA-TP. The evaluation of the immunoglobulin G immune response by recWB in sera from patients with different stages of syphilis showed that rTmpA was the most frequently identified antigen (95%), whereas only 41% of the specimens were reactive to rTpN37. The remaining recombinant polypeptides were recognized as follows: rTpN47, 92.5%; rTpN17, 89.5%; and rTpN15, 67.5%. The agreement between recWB and MHA-TP was 95.0% (100% with sera from patients with latent and late disease), and the concordance between wclWB and MHA-TP was 92.0%. The overall concordance between recWB and wclWB was 97.5% (100% with sera from patients with secondary and late syphilis and 94.6 and 98.6% with sera from patients with primary and latent syphilis, respectively). The overall sensitivity of recWB was 98.8% and the specificity was 97.1% with MHA-TP as the reference method. These values for sensitivity and specificity were slightly superior to those calculated for wclWB (sensitivity, 97.1%, and specificity, 96.1%). With wclWB as the standard test, the sensitivity and specificity of recWB were 98.9 and 99.3%, respectively. These findings suggest that the five recombinant polypeptides used in this study could be used as substitutes for the whole-cell lysate T. pallidum antigens and that this newly developed recWB test is a good, easy-to-use confirmatory method for the detection of syphilis antibodies in serum.
| S-EPMC96096 | biostudies-literature
Project description:Syphilis is a complex multistage sexually transmitted infection (STI) caused by the bacterium Treponema pallidum subspecies pallidum (TP). New diagnostic tools are needed to minimize transmission. This study aimed to assess the additional value of an investigational Transcription Mediated Amplification test for TP (TP-TMA) to routine diagnostics. Between September 2021 and August 2022, visits of all participants of the national PrEP program at the sexual health center (SHC) in Amsterdam were included. Anal, pharyngeal, vaginal and urine samples collected for Chlamydia trachomatis and Neisseria gonorrhoeae screening, were additionally tested with the TP-TMA assay based on detection of 23S rRNA of TP. In total, 9974 SHC visits from 3283 participants were included. There were 191 infectious syphilis cases diagnosed; 26 (14%) primary syphilis, 54 (29%) secondary syphilis and 111 (58%) early latent syphilis. In 79/191 (41%) of these syphilis cases at least one sample was TP-TMA positive. For 16 participants, the positive TP-TMA result was not concordant with routine diagnostics. Of those, two participants were treated for syphilis within a week before the visit. Eight participants were treated for a syphilis notification at the visit or for another STI. Five participants were diagnosed with syphilis in the following visit and one participant was lost to follow-up. Adding the TP-TMA assay to routine diagnostics, we identified 14/191 (7%) additional syphilis infections among participants of the national PrEP program. The TP-TMA assay is a useful diagnostic tool to increase syphilis case finding and thus limit the transmission of syphilis.
| S-EPMC10724458 | biostudies-literature
Project description:Syphilis is a prominent disease in low- and middle-income countries, and a re-emerging public health threat in high-income countries. Syphilis elimination will require development of an effective vaccine that has thus far remained elusive. Here we assess the vaccine potential of Tp0751, a vascular adhesin from the causative agent of syphilis, Treponema pallidum subsp. pallidum. Tp0751-immunized animals exhibit a significantly reduced bacterial organ burden upon T. pallidum challenge compared with unimmunized animals. Introduction of lymph nodes from Tp0751-immunized, T. pallidum-challenged animals to naive animals fails to induce infection, confirming sterile protection. These findings provide evidence that Tp0751 is a promising syphilis vaccine candidate.
| S-EPMC5296639 | biostudies-literature
Project description:BackgroundThe spirochete bacterium Treponema pallidum ssp. pallidum is the etiological agent of syphilis, a chronic multistage disease. Little is known about the global T. pallidum proteome, therefore mass spectrometry studies are needed to bring insights into pathogenicity and protein expression profiles during infection.Methodology/principal findingsTo better understand the T. pallidum proteome profile during infection, we studied T. pallidum ssp. pallidum DAL-1 strain bacteria isolated from rabbits using complementary mass spectrometry techniques, including multidimensional peptide separation and protein identification via matrix-assisted laser desorption ionization-time of flight (MALDI-TOF/TOF) and electrospray ionization (ESI-LTQ-Orbitrap) tandem mass spectrometry. A total of 6033 peptides were detected, corresponding to 557 unique T. pallidum proteins at a high level of confidence, representing 54% of the predicted proteome. A previous gel-based T. pallidum MS proteome study detected 58 of these proteins. One hundred fourteen of the detected proteins were previously annotated as hypothetical or uncharacterized proteins; this is the first account of 106 of these proteins at the protein level. Detected proteins were characterized according to their predicted biological function and localization; half were allocated into a wide range of functional categories. Proteins annotated as potential membrane proteins and proteins with unclear functional annotations were subjected to an additional bioinformatics pipeline analysis to facilitate further characterization. A total of 116 potential membrane proteins were identified, of which 16 have evidence supporting outer membrane localization. We found 8/12 proteins related to the paralogous tpr gene family: TprB, TprC/D, TprE, TprG, TprH, TprI and TprJ. Protein abundance was semi-quantified using label-free spectral counting methods. A low correlation (r = 0.26) was found between previous microarray signal data and protein abundance.ConclusionsThis is the most comprehensive description of the global T. pallidum proteome to date. These data provide valuable insights into in vivo T. pallidum protein expression, paving the way for improved understanding of the pathogenicity of this enigmatic organism.
| S-EPMC5015957 | biostudies-literature
Project description:BackgroundDNA from many pathogens can be detected in saliva. However, the presence and quantity of Treponema pallidum DNA in patients with syphilis in saliva is unknown.Methods234 patients with syphilis with different stages and 30 volunteers were enrolled. Paired saliva and plasma samples were collected from all participants. Consecutive saliva samples from 9 patients were collected every 4 hours following treatment. Treponema pallidum DNA in samples was determined by nested polymerase chain reaction (PCR) and droplet digital PCR targeting polA and Tpp47.ResultsTreponema pallidum DNA detection rates in saliva and plasma were 31.0% (9/29) and 51.7% (15/29) in primary syphilis (P = .11), 87.5% (63/72) and 61.1% (44/72) in secondary syphilis (P < .001), 25.6% (21/82) and 8.5% (7/82) in latent syphilis (P = .004), and 21.6% (11/51) and 5.9% (3/51) in symptomatic neurosyphilis (P = .021), respectively. Median (range) loads of Tpp47 and polA in saliva were 627 (0-101 200) and 726 (0-117 260) copies/mL, respectively, for patients with syphilis. In plasma, however, loads of Tpp47 and polA were low: medians (range) of 0 (0-149.6) and 0 (0-176) copies/mL, respectively. Loads of T. pallidum DNA in saliva during treatment fluctuated downward; the clearance time was positively correlated with the loads of T. pallidum DNA before treatment.ConclusionsCollection of saliva is noninvasive and convenient. The high loads of T. pallidum DNA in saliva and reduction after treatment indicated that saliva can be not only a diagnostic fluid for syphilis but also an indicator of therapeutic effectiveness.
| S-EPMC8563222 | biostudies-literature
Project description:Protein interaction networks shed light on the global organization of proteomes but can also place individual proteins into a functional context. If we know the function of bacterial proteins we will be able to understand how these species have adapted to diverse environments including many extreme habitats. Here we present the protein interaction network for the syphilis spirochete Treponema pallidum which encodes 1,039 proteins, 726 (or 70%) of which interact via 3,649 interactions as revealed by systematic yeast two-hybrid screens. A high-confidence subset of 991 interactions links 576 proteins. To derive further biological insights from our data, we constructed an integrated network of proteins involved in DNA metabolism. Combining our data with additional evidences, we provide improved annotations for at least 18 proteins (including TP0004, TP0050, and TP0183 which are suggested to be involved in DNA metabolism). We estimate that this "minimal" bacterium contains on the order of 3,000 protein interactions. Profiles of functional interconnections indicate that bacterial proteins interact more promiscuously than eukaryotic proteins, reflecting the non-compartmentalized structure of the bacterial cell. Using our high-confidence interactions, we also predict 417,329 homologous interactions ("interologs") for 372 completely sequenced genomes and provide evidence that at least one third of them can be experimentally confirmed.
| S-EPMC2386257 | biostudies-literature
Project description:Extracellular bacteria that spread via the vasculature employ invasive mechanisms that mirror those of metastatic tumor cells, including intravasation into the bloodstream and survival during hematogenous dissemination, arrestation despite blood flow, and extravasation into distant tissue sites. Several invasive bacteria have been shown to exploit normal platelet function during infection. Due to their inherent ability to interact with and influence other cell types, platelets play a critical role in alteration of endothelial barrier permeability, and their role in cancer metastasis has been well established. The highly invasive bacterium and causative agent of syphilis, Treponema pallidum subspecies pallidum, readily crosses the endothelial, blood-brain and placental barriers. However, the mechanisms underlying this unusual and important aspect of T. pallidum pathogenesis are incompletely understood. In this study we use darkfield microscopy in combination with flow cytometry to establish that T. pallidum interacts with platelets. We also investigate the dynamics of this interaction and show T. pallidum is able to activate platelets and preferentially interacts with activated platelets. Platelet-interacting treponemes consistently exhibit altered kinematic (movement) parameters compared to free treponemes, and T. pallidum-platelet interactions are reversible. This study provides insight into host cell interactions at play during T. pallidum infection and suggests that T. pallidum may exploit platelet function to aid in establishment of disseminated infection.
| S-EPMC6338379 | biostudies-literature