ObjectivesThe prevalence of syphilis, caused by the spirochete Treponema pallidum subsp. pallidum (TPA), remains high despite the availability of effective antibiotics. In the Netherlands, most syphilis cases are found among men who have sex with men (MSM). We studied the distribution of TPA strain types by molecular characterisation and related this to available characteristics. In addition, resistance to macrolides was assessed.MethodsTPA DNA was extracted from 136 genital ulcer swab or skin lesions samples deriving from 135 patients diagnosed with syphilis in 2016 and 2017 at the Public Health Service in Amsterdam, the Netherlands. Molecular typing was done according to the enhanced CDC method (E-CDC), in which three genetic regions of the arp, tpr and tp0548 genes are analysed by gel electrophoresis of the arp and tpr regions and by sequence analysis for the tp0548 region. Part of the 23S rDNA locus was sequenced to determine the presence of macrolide resistance-associated mutations.ResultsFull E-CDC strain types could be determined for 99/136 (73%) DNA samples, which tested positive in a diagnostic PCR targeting the polA gene. Types differed within one patient of whom two samples were available. No association was found between the demographic and clinical characteristics and the TPA types. The most prevalent type was 14d/g, found in 23 of the 99 (23%) fully typed samples. Part of the 23S rDNA locus was successfully sequenced for 93/136 (68%) samples and 83 (88%) contained the A2058G mutation. No A2059G mutation was found.ConclusionsA broad strain distribution was found. Few subtypes were clonally expanded, and most other subtypes were rare. Detection of the most prevalent strain type, 14d/g, is in concordance with other TPA typing studies. The high prevalence of genetic macrolide resistance indicates that azithromycin is not an alternative treatment option.

Typing of Chlamydia trachomatis (CT) is traditionally performed by characterising the ompA gene, resulting in more than a dozen different genovars, A to L. Type L is associated with Lymphogranuloma venereum (LGV) and commonly screened for using PCR, targeting the chromosomal pmpH gene. We aimed to develop and validate a new CT/LGV plasmidbased typing assay targeting the pgp3 gene, to increase sensitivity and thus reduce the number of non-typeable results. Methods The new pgp3 PCR assay using LNA probes to detect point mutations was analytically and prospectively validated in a routine diagnostic laboratory setting. For the analytical tests, quantified nucleotide constructs (gBlocks) were used to perform limit of detection analyses. Quality control panel samples from 2018 and 2019 for CT were also tested. For the clinical study patient samples which were collected in two months in 2018 were tested simultaneously using the pmpH PCR and the pgp3 PCR. Results Analytically, the assay proved to be 100% specific relative to the previously used LGV typing assay targeting the single copy pmpH gene but it was much more sensitive to detect non-LGV CT. In the quality control panel 2 nonLGV samples and 7 LGV samples were solely positive with the pgp3 PCR and not with the pmpH PCR. None of the samples from analytical specificity panels were positive, indicating 100% specificity. In a prospective panel of 152 clinical samples, 142 (93%) were successfully typed with the pgp3 PCR compared to 78% with the pmpH PCR. The pgp3 PCR was fully concordant with the pmpH PCR to