Two distinct phylogenetic lineages of TPA strains, namely Nichols-like and SS14-like, have been identified [14]. Upon reviewing previously analyzed data, it was observed that the SS14-like lineage predominated among the samples obtained in Japan (refer to PubMLST BIGSdb). A previous study, utilizing Sequence-Based Molecular Typing (SBMT) other than the MLST method, reported greater genetic diversity of T. pallidum in the MSM population compared to the heterosexual population. Kojima et al. found that in MSM, 63% were SS14-like, 37% were Nichols-like, whereas in heterosexuals, 100% were SS14-like [15]. Similarly, Kanai et al. reported that in MSM, 75% were SS14-like, 25% were Nichols-like, and 25% were macrolide-resistant, while 100% of the heterosexuals were SS14-like [16]. Given that the predominant demographic in our study comprised MSM, our MLST analysis generally aligns with findings from earlier studies that employed different typing methods. Furthermore, WGS analysis revealed a close relationship between SS14-lineage strains in Japan and China [17]. In our study, ST3 (1.1.8) was predominant among 20 blood samples, followed by ST6 (3.2.3). This aligns with the prevalence of ST3 in China [18]. We also identified a new allele belonging to the SS14 lineage, resulting in a new ST that differed from ST1 (1.3.1) by one substitution on TP0136. ST1(1.3.1) was frequently detected in Czech Republic, the Netherlands, France, and Cuba [9, 19,20,21]. Vrbová et al. conducted a large survey in the Czech Republic from 2004 to 2022, finding that SS14-like strains were predominant [22]. The first group of isolates included profiles ST 1 (1.3.1) and ST 25 (1.26.1), while the second group comprised ST 3 (1.1.8), ST 2 (1.1.1), and ST 11 (1.1.3). The two groups accounted for 57.5% and 25.3% of the total isolates, respectively. In our study, ST 3 (1.1.8), which represented 45% of the isolates, belongs to the second group found in the Czech Republic, suggesting a somewhat different predominant strain. The most common Nichols-like strain in the Czech Republic, ST 6 (9.7.3), accounted for only about 6% of the total in the Czech Republic [22], whereas in our study, the major Nichols-like strain, which is also ST 6 (9.7.3), represented 40% of the total, highlighting a significant difference in strain distribution. In summary, our results indicate an MLST profile similar to that of China, considering the geographical proximity to Japan. However, it’s important to note that our typing utilized only three loci, and there might be additional diversity within the SS14-lineage. Limited data for the Asian region in the database highlights the need for a more convenient method to collect data for detailed analysis.
We identified three cases strongly suspected to be caused by TEN, the pathogen associated with bejel. A recent study by Kawabata et al. conducted a sequence-based molecular epidemiological analysis of TPA, which is prevalent in Japan. They encountered instances of TPA that are not easily typed from specimens derived from MSM. Utilizing phylogenetic tree analysis with sequences from TP0548 [23] and TP0856 genes [24], which exhibit relatively low homology between TPA/TPE/TEN, they concluded that seven out of 70 cases were infected with TEN [25]. This report covers specimens collected from 2014 to 2019. When combined with the results of our study from 2019 to 2022, it strongly suggests that TEN, traditionally considered a non-sexually transmitted treponema, has been prevalent among Japanese MSM for at least a decade, following a transmission pattern similar to that of TPA in sexually transmitted syphilis.
We were able to detect TP0136, TP0548, and TP0705 in approximately one-third of the RPR-positive cases. According to a previous study, the PCR-positive rate when using blood samples was lower than that of swab samples [8]. However, in this study the TP0136 detection rate, encompassing the partial profile, was 32.9% among TPA. Importantly, this doesn’t imply that whole blood is unsuitable for molecular epidemiological analysis of syphilis. It’s essential to recognize that the gene detection rate serves as a clinical diagnosis within the population of RPR positives. Whole blood samples can easily be collected from all people with syphilis, and the higher sensitivity of PCR positivity in participants with elevated CRP levels suggests an efficient molecular epidemiological analysis by selectively choosing patients for whole blood samples. In this study, we used TP0136 to screen for PCR positivity as part of an efficient MLST method. However, some studies have utilized multiple alleles or shorter amplicons, such as polA, for the genetic diagnosis of syphilis [26, 27]. Vrbova et al. performed PCR on multiple alleles simultaneously and reported positive results in 34.8% of cases [28], which is comparable to the sequencing success rate for three alleles in this study. Wang et al. reported PCR positivity rates of 7.4% in latent syphilis and 62.9% in secondary syphilis [27]. In contrast, our study found no significant difference in detection rates between latent and secondary syphilis; however, differences in HIV status, MSM status, and other background factors between the study populations may have influenced the results.
Moreover, diagnosing and treating infectious diseases based on antibody titers, monitored through before-and-after comparisons, relies on relative evaluations. However, antibody titers take time to decline, posing challenges in determining whether the decline is a treatment effect or a natural process. Given that many syphilis cases are asymptomatic, and individuals who test RPR-positive may have already healed spontaneously, relying solely on antibody titers might not accurately reflect the disease status. Therefore, T. pallidum DNA detection rate may offer insights into some etiologies of the disease beyond the sensitivity of the test. A previous study explored the correlation between RPR titer and T. pallidum DNA detection rate, considering the syphilis stage [27]. In addition to RPR, we examine the association between PCR-positives and CRP values. CRP, as an indicator of inflammation [29], a typically remains below 0.3 mg/dL in most healthy individuals, with normal or minor elevations falling within the range of 0.3-1.0 mg/dL [30]. This observation may be linked to the bacterial load in the blood during early syphilis, serving as a potential pathogenetic factor alongside technical detection sensitivity. PCR-negatives may not necessarily indicate the absence of the pathogen, and therefore, the need for treatment cannot be ruled out. However, it’s important to note that antibody titers do not necessarily imply the presence of the pathogen. While pathogen testing is fundamental for treating infectious diseases, it has proven insufficient in syphilis. At this stage, it is difficult to use the results of genetic testing of blood samples to make clinical decisions, and further research is needed. However, in the field of syphilis care, there is a need for diagnosis and treatment based on antigen testing rather than antibody titers, as is commonly done for other infectious diseases.
The small sample size is one of the limitations of this study. The uniqueness of this study lies in its use of blood specimens from all stages of the disease, not limited to primary syphilis. In MSM, syphilis often presents only as a rosacea or remains asymptomatic, with only a small percentage exhibiting the skin symptoms characteristic of primary syphilis. However, this study demonstrates the feasibility of conducting molecular epidemiological analysis with blood specimens from any stage of the disease. With a larger sample size, statistical significance could have been established in the relationship between the RPR and TP0136 detection rate. Nonetheless, numerous cases exhibited low RPR levels with positive PCR results, enabling MLST. The observed discrepancy between the antibody response and the onset of clinical symptoms is a common phenomenon in infectious diseases. Therefore, it remains unclear whether the RPR and TP0136 detection rate hold clinical significance. Another limitation is the lack of comparison between WGS and MLST. However, given the challenges of applying WGS to TPA, a simpler method is required. This study establishes that, at the very least, MLST possesses the discriminatory power to identify novel epidemic strains and outbreaks of bejel among MSM.
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