OBJECTIVES: Treponema pallidum subsp. pallidum (T. pallidum) is the etiological agent of syphilis, a sexually transmitted disease of global public health importance. The objective of this study was to introduce a novel in vitro protocol for isolation of T. pallidum directly from patients' clinical samples, eliminating the need for rabbit propagation. METHODS: Four oral and five genital swabs were collected from nine epidemiologically unrelated patients at two hospitals in Brno, Czech Republic. Swabs were submerged in TpCM-2 medium for transport. Samples were then placed on a 0.4 μm filters and incubated for 2.5 hours. During this period, spiral T. pallidum cells passed through the filter pores to the well containing TpCM-2 medium and rabbit feeder cells (Sf1Ep). Stable T. pallidum cultures (containing >1 × 107 treponemes) were achieved by subculturing every 7 days into fresh well. RESULTS: A successful protocol for in vitro isolation of T. pallidum was established. From the nine clinical specimens processed, six T. pallidum cultures (MU1-MU6) were derived after 14 to 112 days of cultivation. Five of these strains (MU1-MU5) belonged to SS14-like cluster and shared the same allelic profile 1.3.1. The remaining strain (MU6) was identified as a Nichols-like strain with an allelic profile 9.16.3. DISCUSSION: The introduced in vitro protocol enables isolation of T. pallidum from clinical material, including frozen samples, without the need for experimental rabbits. This method facilitates the isolation of contemporary, clinically relevant treponemal strains.
- MeSH
- bakteriologické techniky * metody MeSH
- králíci MeSH
- lidé MeSH
- pohlavní orgány mikrobiologie MeSH
- syfilis * mikrobiologie diagnóza MeSH
- Treponema pallidum * izolace a purifikace genetika klasifikace MeSH
- ústa mikrobiologie MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
Syphilis is a multistage sexually transmitted disease caused by Treponema pallidum ssp. pallidum (TPA). This study analyzed clinical samples collected from patients with a diagnosed syphilis infection from 2004-2022, isolated in the Czech Republic. Mucocutaneous swab samples (n = 543) from 543 patients were analyzed, and from these samples, 80.11 % (n = 435) were PCR positive, and 19.89 % (n = 108) were PCR negative for TPA DNA. Swabs were more often positive when collected from syphilis patients in the primary and secondary stages, compared to the latent or unknown stage. There was no significant difference in PCR positivity between the primary and secondary stages (p = 0.099). In IgM-positive patients, a statistically significant association with PCR-positivity was found in samples from seropositive (p = 0.033) and serodiscrepant (RPR negative) patients (p = 0.0006). When assessing our laboratory-defined cases of syphilis, the RPR, IgM, and PCR tests were similarly effective (within the range of 80.1-86.1 %). However, parallel testing with these methods was even more effective, i.e., RPR + PCR was 96.1 % effective and RPR + IgM + PCR was 97.8 % effective. A combination of RPR + PCR, or a combination of all three tests (RPR, IgM, and PCR) can therefore be used to reliably detect active syphilis cases, including reinfections. Our findings show that the reverse algorithm for detecting syphilis could be substantially improved by adding IgM and PCR testing.
- MeSH
- DNA bakterií genetika MeSH
- dospělí MeSH
- imunoglobulin M * krev MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- polymerázová řetězová reakce * metody MeSH
- protilátky bakteriální krev MeSH
- senzitivita a specificita MeSH
- sérologická diagnostika syfilis metody MeSH
- syfilis * diagnóza mikrobiologie MeSH
- Treponema pallidum * genetika izolace a purifikace imunologie MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
Syphilis is a multistage sexually transmitted disease caused by Treponema pallidum ssp. pallidum. In the Czech Republic, there are around 700-800 new syphilis cases annually, continuously increasing since 2012. This study analyzed a total of 1228 samples from 2004 to 2022. Of the PCR-positive typeable samples (n = 415), 68.7% were fully-typed (FT), and 31.3% were partially-typed. Most of the identified isolates belonged to the SS14-clade and only 6.3% were the Nichols-like cluster. While in the beginning of sample collection isolates have been macrolide-susceptible, recent isolates are completely resistant to macrolides. Among the FT samples, 34 different allelic profiles (APs) were found. Most of the profiles (n = 27) appeared just once in the Czech population, while seven profiles were detected more than twice. The most frequent APs belonged to two separate groups of SS14-like isolates, including group of 1.3.1 (ST 1) and 1.26.1 (ST 25) profiles, and the second group containing 1.1.8 (ST 3), 1.1.1 (ST 2), and 1.1.3 (ST 11) (representing 57.5%, and 25.3% of all detected APs, respectively). Both groups consistently differed in 6 nucleotide positions in five genes (TP0150, TP0324, TP0515, TP0548, and TP0691) coding amino-acid replacements suggesting that one or more of these differences could be involved in the higher success of the first group.
- MeSH
- alely * MeSH
- dospělí MeSH
- genotyp MeSH
- lidé středního věku MeSH
- lidé MeSH
- makrolidy farmakologie MeSH
- multilokusová sekvenční typizace * MeSH
- syfilis * mikrobiologie epidemiologie genetika MeSH
- Treponema pallidum * genetika izolace a purifikace MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
Syphilis, caused by Treponema pallidum ssp. pallidum (TPA), is a persisting global health problem. Although syphilis diagnostics relies mainly on serology, serological tests have some limitations, and it is recommended that the final diagnosis be supported by additional tests. The purpose of this study was to analyze the relationship between serology and PCR in syphilis diagnostics. From the year 2004 to May 2019, a total of 941 samples were taken from 833 patients suspected of having syphilis, in Czech Republic. In all these samples, both nested PCR detection of TPA and serology testing were performed. Of the 941 samples, 126 were seronegative, 651 were seropositive, and 164 were serodiscrepant. Among seronegative samples (n = 126), 11 were PCR-positive (8.7%). Among seropositive samples (n = 651; i.e., samples positive for both non-treponemal and treponemal serology tests), 368 samples were PCR-positive (56.5%). The remaining 164 serodiscrepant samples included RPR negative and treponemal serological test-positive samples (n = 154) and a set of 10 RPR-positive samples negative in treponemal serological tests. While the first group revealed 73 PCR-positive samples (47.4%), the second revealed 5 PCR positive samples (50.0%). PCR detection rates were highest in primary syphilis, with lower rates in the secondary and undetermined syphilis stages. As shown here, the nested PCR can improve diagnostics of syphilis, especially in seronegative patients and in patients with discrepant serology.
- MeSH
- lidé MeSH
- polymerázová řetězová reakce * MeSH
- retrospektivní studie MeSH
- sérologická diagnostika syfilis metody MeSH
- syfilis krev diagnóza MeSH
- Treponema genetika imunologie izolace a purifikace fyziologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Syphilis, caused by Treponema pallidum subsp. pallidum (TPA), remains an important public health problem with an increasing worldwide prevalence. Despite recent advances in in vitro cultivation, genetic variability of this pathogen during infection is poorly understood. Here, we present contemporary and geographically diverse complete treponemal genome sequences isolated directly from patients using a methyl-directed enrichment prior to sequencing. This approach reveals that approximately 50% of the genetic diversity found in TPA is driven by inter- and/or intra-strain recombination events, particularly in strains belonging to one of the defined genetic groups of syphilis treponemes: Nichols-like strains. Recombinant loci were found to encode putative outer-membrane proteins and the recombination variability was almost exclusively found in regions predicted to be at the host-pathogen interface. Genetic recombination has been considered to be a rare event in treponemes, yet our study unexpectedly showed that it occurs at a significant level and may have important impacts in the biology of this pathogen, especially as these events occur primarily in the outer membrane proteins. This study reveals the existence of strains with different repertoires of surface-exposed antigens circulating in the current human population, which should be taken into account during syphilis vaccine development.
- Publikační typ
- časopisecké články MeSH
A recently introduced Multilocus Sequence Typing scheme for Treponema pallidum subsp. pallidum was applied to clinical samples collected from 2004 to 2017 from the two largest cities (Prague and Brno) in the Czech Republic. Altogether, a total of 675 samples were tested in this study and 281 of them were found PCR-positive for treponemal DNA and typeable. Most of the typed samples (n = 281) were swabs from primary or secondary syphilis lesions (n = 231), and only a minority were whole blood or tissue samples (n = 50). Swab samples from patients with rapid plasma regain (RPR) values of 1-1024 were more frequently PCR-positive (84.6%) compared to samples from patients with non-reactive RPR test (46.5%; p-value = 0.0001). Out of 281 typeable samples, 136 were fully-typed at all TP0136, TP0548, and TP0705 loci. Among the fully and partially typed samples, 25 different allelic profiles were identified. Altogether, eight novel allelic variants were found among fully (n = 5) and partially (n = 3) typed samples. The distribution of TPA allelic profiles identified in the Czech Republic from 2004 to 2017 revealed a dynamic character with allelic profiles disappearing and emerging over time. While the number of samples with the A2058G mutation was seen to increase (86.7% in 2016/2017), the number of samples harboring the A2059G mutation was found to have decreased over time (3.3% in 2016/2017). In addition, we found several allelic profile associations with macrolide resistance or susceptibility, the gender of patients, as well as patient residence.
- MeSH
- alely MeSH
- antibakteriální látky farmakologie MeSH
- bakteriální léková rezistence genetika MeSH
- DNA bakterií genetika MeSH
- dospělí MeSH
- genotyp MeSH
- lidé MeSH
- mladý dospělý MeSH
- multilokusová sekvenční typizace * MeSH
- RNA ribozomální 23S genetika MeSH
- syfilis genetika mikrobiologie patologie MeSH
- Treponema pallidum genetika patogenita MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
Treponema pallidum subsp. pallidum (TPA) is the infectious agent of syphilis, a disease that infects more than 5 million people annually. Since TPA is an uncultivable bacterium, most of the information on TPA genetics comes from genome sequencing and molecular typing studies. This study presents the first complete TPA genome (without sequencing gaps) of clinical isolate (UZ1974), which was obtained directly from clinical material, without multiplication in rabbits. Whole genome sequencing was performed using a newly developed Anti-Treponemal Antibody Enrichment technique combined with previously reported Pooled Segment Genome Sequencing. We identified the UW074B genome, isolated from a sample previously propagated in rabbits, to be the closest relative of the UZ1974 genome and calculated the TPA mutation rate as 2.8 x 10(-10) per site per generation.
- MeSH
- fylogeneze MeSH
- genetická variace MeSH
- genom bakteriální genetika MeSH
- králíci MeSH
- lidé MeSH
- mutační rychlost MeSH
- sekvenční analýza DNA MeSH
- sekvenování celého genomu * MeSH
- syfilis genetika mikrobiologie MeSH
- Treponema pallidum genetika patogenita MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
