The global resurgence of treponematoses, particularly syphilis, poses a growing public health challenge. Despite recent advances in sequencing technologies, obtaining complete Treponema pallidum genome sequences for epidemiological studies remains time-consuming and challenging due to the difficulty related to procuring clinical samples with sufficient treponemal burden to fulfil the sequencing requirements. There is an urgent need for rapid, cost-effective and accessible typing methods suitable for laboratories with Sanger sequencing resources. Based on the analysis of 121 T. pallidum genomes from geographically diverse regions, we selected seven highly variable genes to form the basis of this new typing system. These seven genes show high discrimination capacity, identifying many allelic profiles among T. pallidum isolates. Importantly, the scheme employs a single-step PCR protocol for the amplification and sequencing of all seven targets enabling straightforward implementation in standard laboratory settings. The MLST was validated using a diverse set of T. pallidum clinical samples from across the globe. A significant proportion of the tested samples showed macrolide resistance, emphasizing the need for epidemiological surveillance. Utilizing this new tool, we have analyzed the genetic variation within and between populations of T. pallidum, considering the geographical origin of the samples. Population structure analysis revealed distinct genetic clusters, underlining complex transmission dynamics of T. pallidum, shaped by local epidemiological factors. The MLST scheme is publicly accessible through the PubMLST database, encouraging widespread adoption in standard laboratories due to this database being user-friendly, intuitive, and fast to implement. The novel MLST scheme offers a promising tool to advance the study of the molecular epidemiology of T. pallidum, facilitate tracking transmission, and establish a global surveillance network with the overall goal of strengthening public health interventions for syphilis control.

• Keywords
• MLST, T. pallidum, epidemiology, typing,
• Publication type
• Journal Article MeSH
• Preprint MeSH

Globally, 94% of Treponema pallidum subsp. pallidum (TPA) clinical strains belong to the SS14-like group and 6% to the Nichols-like group, with a prevalence of macrolide resistance of 90%. Our goal was to determine whether local TPA strain distribution and macrolide resistance frequency have changed significantly since our last report, which revealed that Buenos Aires had a high frequency of Nichols-like strains (27%) and low levels of macrolide resistance (14%). Swab samples from patients with suspected syphilis were collected during 2015-2019 and loci TP0136, TP0548, TP0705 were sequenced in order to perform multilocus sequence typing. Strains were classified as Nichols-like or SS14-like. The presence of macrolide resistance-associated mutations was determined by examination of the 23S rDNA gene sequence. Of 46 typeable samples, 37% were classified as Nichols-like and 63% as SS14-like. Macrolide resistance prevalence was 45.7%. Seven allelic profiles were found, five were SS14-like and two were Nichols-like. The frequency of Nichols-like strains increased between studies (26.8% vs. 37%, p = 0.36). A dramatic increase was found in the frequency of macrolide resistant strains between studies (14.3% vs. 45.7%, p = 0.005). Our results are in agreement with international trends and underscore the need to pursue further TPA molecular typing studies in South America.

• MeSH
• Anti-Bacterial Agents pharmacology   MeSH
• Drug Resistance, Bacterial genetics   MeSH
• Treponemal Infections * MeSH
• Humans MeSH
• Macrolides pharmacology   MeSH
• Multilocus Sequence Typing MeSH
• DNA, Ribosomal MeSH
• Treponema pallidum * genetics   MeSH
• Treponema MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Argentina epidemiology   MeSH
• Names of Substances
• Anti-Bacterial Agents MeSH
• Macrolides MeSH
• DNA, Ribosomal 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
• Humans MeSH
• Polymerase Chain Reaction * MeSH
• Retrospective Studies MeSH
• Syphilis Serodiagnosis methods   MeSH
• Syphilis blood   diagnosis   MeSH
• Treponema genetics   immunology   isolation & purification   physiology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't 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
• Alleles MeSH
• Anti-Bacterial Agents pharmacology   MeSH
• Drug Resistance, Bacterial genetics   MeSH
• DNA, Bacterial genetics   MeSH
• Adult MeSH
• Genotype MeSH
• Humans MeSH
• Young Adult MeSH
• Multilocus Sequence Typing * MeSH
• RNA, Ribosomal, 23S genetics   MeSH
• Syphilis genetics   microbiology   pathology   MeSH
• Treponema pallidum genetics   pathogenicity   MeSH
• Check Tag
• Adult MeSH
• Humans MeSH
• Young Adult MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Czech Republic epidemiology   MeSH
• Names of Substances
• Anti-Bacterial Agents MeSH
• DNA, Bacterial MeSH
• RNA, Ribosomal, 23S MeSH

BACKGROUND: Treponema pallidum subsp. pertenue (TPE) is the causative agent of yaws, a multistage disease endemic in tropical regions in Africa, Asia, Oceania, and South America. To date, seven TPE strains have been completely sequenced and analyzed including five TPE strains of human origin (CDC-2, CDC 2575, Gauthier, Ghana-051, and Samoa D) and two TPE strains isolated from the baboons (Fribourg-Blanc and LMNP-1). This study revealed the complete genome sequences of two TPE strains, Kampung Dalan K363 and Sei Geringging K403, isolated in 1990 from villages in the Pariaman region of Sumatra, Indonesia and compared these genome sequences with other known TPE genomes. METHODOLOGY/PRINCIPAL FINDINGS: The genomes were determined using the pooled segment genome sequencing method combined with the Illumina sequencing platform resulting in an average coverage depth of 1,021x and 644x for the TPE Kampung Dalan K363 and TPE Sei Geringging K403 genomes, respectively. Both Indonesian TPE strains were genetically related to each other and were more distantly related to other, previously characterized TPE strains. The modular character of several genes, including TP0136 and TP0858 gene orthologs, was identified by analysis of the corresponding sequences. To systematically detect genes potentially having a modular genetic structure, we performed a whole genome analysis-of-occurrence of direct or inverted repeats of 17 or more nucleotides in length. Besides in tpr genes, a frequent presence of repeats was found in the genetic regions spanning TP0126-TP0136, TP0856-TP0858, and TP0896 genes. CONCLUSIONS/SIGNIFICANCE: Comparisons of genome sequences of TPE Kampung Dalan K363 and Sei Geringging K403 with other TPE strains revealed a modular structure of several genomic loci including the TP0136, TP0856, and TP0858 genes. Diversification of TPE genomes appears to be facilitated by intra-strain genome recombination events.

• MeSH
• Genome, Bacterial * MeSH
• Humans MeSH
• Gene Order MeSH
• Recombination, Genetic MeSH
• Repetitive Sequences, Nucleic Acid MeSH
• Sequence Analysis, DNA * MeSH
• Treponema pallidum genetics   MeSH
• Computational Biology MeSH
• High-Throughput Nucleotide Sequencing MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Indonesia MeSH

Syphilis is an important public health problem and an increasing incidence has been noted in recent years. Characterization of strain diversity through molecular data plays a critical role in the epidemiological understanding of this re-emergence. We here propose a new high-resolution multilocus sequence typing (MLST) scheme for Treponema pallidum subsp. pallidum (TPA). We analyzed 30 complete and draft TPA genomes obtained directly from clinical samples or from rabbit propagated strains to identify suitable typing loci and tested the new scheme on 120 clinical samples collected in Switzerland and France. Our analyses yielded three loci with high discriminatory power: TP0136, TP0548, and TP0705. Together with analysis of the 23S rRNA gene mutations for macrolide resistance, we propose these loci as MLST for TPA. Among clinical samples, 23 allelic profiles as well as a high percentage (80% samples) of macrolide resistance were revealed. The new MLST has higher discriminatory power compared to previous typing schemes, enabling distinction of TPA from other treponemal bacteria, distinction between the two main TPA clades (Nichols and SS14), and differentiation of strains within these clades.

• MeSH
• Alleles MeSH
• Anti-Bacterial Agents pharmacology   MeSH
• DNA, Bacterial genetics   MeSH
• Phylogeny MeSH
• Genome, Bacterial MeSH
• Genotype MeSH
• Globus Pallidus MeSH
• Polymorphism, Single Nucleotide MeSH
• Macrolides pharmacology   MeSH
• Multilocus Sequence Typing methods   MeSH
• RNA, Ribosomal, 23S genetics   MeSH
• Sequence Analysis, DNA methods   MeSH
• Syphilis epidemiology   MeSH
• Treponema pallidum genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• France epidemiology   MeSH
• Switzerland epidemiology   MeSH
• Names of Substances
• Anti-Bacterial Agents MeSH
• DNA, Bacterial MeSH
• Macrolides MeSH
• RNA, Ribosomal, 23S MeSH

Treponema pallidum subsp. pallidum, the causative agent of sexually transmitted syphilis, detected in clinical samples from France, was subjected to molecular typing using the recently developed Multilocus Sequence Typing system. The samples (n = 133) used in this study were collected from 2010-2016 from patients with diagnosed primary or secondary syphilis attending outpatient centers or hospitals in several locations in France. Altogether, 18 different allelic profiles were found among the fully typed samples (n = 112). There were five allelic variants identified for TP0136, 12 for TP0548, and eight for TP0705. Out of the identified alleles, one, seven, and three novel alleles were identified in TP0136, TP0548, and TP0705, respectively. Partial allelic profiles were obtained from 6 samples. The majority of samples (n = 110) belonged to the SS14-like cluster of TPA isolates while 7 clustered with Nichols-like isolates. Patients infected with Nichols-like samples were more often older (p = 0.041) and more often diagnosed with secondary syphilis (p = 0.033) compared to patients infected with SS14-like samples. In addition, macrolide resistance caused by the A2058G mutation was found to be associated with allelic profile 1.3.1 or with strains belonging to the 1.3.1 lineage (p<0.001). The genetic diversity among TPA strains infecting the European population was surprisingly high, which suggests that additional studies are needed to reveal the full genetic diversity of TPA pathogens infecting humans.

• MeSH
• Alleles MeSH
• Biodiversity MeSH
• Child MeSH
• Adult MeSH
• Infant MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Multilocus Sequence Typing MeSH
• Infant, Newborn MeSH
• Child, Preschool MeSH
• Aged MeSH
• Syphilis epidemiology   microbiology   MeSH
• Bacterial Typing Techniques MeSH
• Treponema pallidum genetics   isolation & purification   MeSH
• Check Tag
• Child MeSH
• Adult MeSH
• Infant MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Male MeSH
• Infant, Newborn MeSH
• Child, Preschool MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• France epidemiology   MeSH

BACKGROUND: Treponema pallidum subsp. pertenue (TPE) is the causative agent of yaws, a multi-stage disease, endemic in tropical regions of Africa, Asia, Oceania, and South America. To date, four TPE strains have been completely sequenced including three TPE strains of human origin (Samoa D, CDC-2, and Gauthier) and one TPE strain (Fribourg-Blanc) isolated from a baboon. All TPE strains are highly similar to T. pallidum subsp. pallidum (TPA) strains. The mutation rate in syphilis and related treponemes has not been experimentally determined yet. METHODOLOGY/PRINCIPAL FINDINGS: Complete genomes of two TPE strains, CDC 2575 and Ghana-051, that infected patients in Ghana and were isolated in 1980 and 1988, respectively, were sequenced and analyzed. Both strains had identical consensus genome nucleotide sequences raising the question whether TPE CDC 2575 and Ghana-051 represent two different strains. Several lines of evidence support the fact that both strains represent independent samples including regions showing intrastrain heterogeneity (13 and 5 intrastrain heterogeneous sites in TPE Ghana-051 and TPE CDC 2575, respectively). Four of these heterogeneous sites were found in both genomes but the frequency of alternative alleles differed. The identical consensus genome sequences were used to estimate the upper limit of the yaws treponeme evolution rate, which was 4.1 x 10-10 nucleotide changes per site per generation. CONCLUSIONS/SIGNIFICANCE: The estimated upper limit for the mutation rate of TPE was slightly lower than the mutation rate of E. coli, which was determined during a long-term experiment. Given the known diversity between TPA and TPE genomes and the assumption that both TPA and TPE have a similar mutation rate, the most recent common ancestor of syphilis and yaws treponemes appears to be more than ten thousand years old and likely even older.

• MeSH
• Time Factors MeSH
• Escherichia coli genetics   MeSH
• Yaws epidemiology   microbiology   MeSH
• Genome, Bacterial * MeSH
• Humans MeSH
• Chromosome Mapping MeSH
• Mutation MeSH
• Papio microbiology   MeSH
• Sequence Analysis, DNA MeSH
• Treponema pallidum classification   genetics   isolation & purification   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Asia epidemiology   MeSH
• Ghana epidemiology   MeSH
• South America epidemiology   MeSH

BACKGROUND: Treponema pallidum subsp. endemicum (TEN) is the causative agent of endemic syphilis (bejel). An unusual human TEN 11q/j isolate was obtained from a syphilis-like primary genital lesion from a patient that returned to France from Pakistan. METHODOLOGY/PRINCIPAL FINDINGS: The TEN 11q/j isolate was characterized using nested PCR followed by Sanger sequencing and/or direct Illumina sequencing. Altogether, 44 chromosomal regions were analyzed. Overall, the 11q/j isolate clustered with TEN strains Bosnia A and Iraq B as expected from previous TEN classification of the 11q/j isolate. However, the 11q/j sequence in a 505 bp-long region at the TP0488 locus was similar to Treponema pallidum subsp. pallidum (TPA) strains, but not to TEN Bosnia A and Iraq B sequences, suggesting a recombination event at this locus. Similarly, the 11q/j sequence in a 613 bp-long region at the TP0548 locus was similar to Treponema pallidum subsp. pertenue (TPE) strains, but not to TEN sequences. CONCLUSIONS/SIGNIFICANCE: A detailed analysis of two recombinant loci found in the 11q/j clinical isolate revealed that the recombination event occurred just once, in the TP0488, with the donor sequence originating from a TPA strain. Since TEN Bosnia A and Iraq B were found to contain TPA-like sequences at the TP0548 locus, the recombination at TP0548 took place in a treponeme that was an ancestor to both TEN Bosnia A and Iraq B. The sequence of 11q/j isolate in TP0548 represents an ancestral TEN sequence that is similar to yaws-causing treponemes. In addition to the importance of the 11q/j isolate for reconstruction of the TEN phylogeny, this case emphasizes the possible role of TEN strains in development of syphilis-like lesions.

• MeSH
• Travel MeSH
• Adult MeSH
• Phylogeny MeSH
• Genetic Variation * MeSH
• Genetic Loci * MeSH
• Genotype MeSH
• Humans MeSH
• Evolution, Molecular MeSH
• Polymerase Chain Reaction MeSH
• Recombination, Genetic * MeSH
• Sequence Analysis, DNA * MeSH
• Sequence Homology MeSH
• Cluster Analysis MeSH
• Syphilis microbiology   MeSH
• Treponema pallidum genetics   isolation & purification   MeSH
• Check Tag
• Adult MeSH
• Humans MeSH
• Male MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• France MeSH
• Pakistan MeSH

A total of 54 clinical samples, including genital lesion swabs, whole blood and cerebrospinal fluid from patients diagnosed with syphilis were collected in 2006 and in 2013 in Buenos Aires, Argentina. Treponemal DNA was detected in 43 of the analyzed samples (79.6%) and further analyzed using Sequencing-based molecular typing (SBMT) and Enhanced CDC-typing (ECDCT). By SBMT, 10 different Treponema pallidum subsp. pallidum (TPA) genotypes were found, of which six were related to the TPA SS14 strain, and four to the TPA Nichols strain. The 23S rRNA gene was amplified in samples isolated from 42 patients, and in six of them (14.3%), either the A2058G (four patients, 9.5%) or the A2059G (two patients, 4.8%) mutations were found. In addition to Taiwan, Madagascar and Peru, Argentina is another country where the prevalence of Nichols-like isolates (26.8%) is greater than 10%.

• MeSH
• Anti-Bacterial Agents pharmacology   MeSH
• Drug Resistance, Bacterial genetics   MeSH
• DNA, Bacterial genetics   MeSH
• Adult MeSH
• Phylogeny MeSH
• Middle Aged MeSH
• Humans MeSH
• Macrolides pharmacology   MeSH
• Adolescent MeSH
• Young Adult MeSH
• Multilocus Sequence Typing MeSH
• DNA Mutational Analysis MeSH
• Base Sequence MeSH
• Aged MeSH
• Syphilis epidemiology   microbiology   MeSH
• Treponema pallidum genetics   isolation & purification   MeSH
• Cities MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Argentina epidemiology   MeSH
• Cities MeSH
• Names of Substances
• Anti-Bacterial Agents MeSH
• DNA, Bacterial MeSH
• Macrolides MeSH