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Objectives The incidence of syphilis, caused by Treponema pallidum (TP), has increased significantly in recent years in Western countries, particularly among men who have sex with men (MSM). Recent data suggest that ongoing transmission may be facilitated by undetected mucosal excretion of TP. This study focuses on patients who had incident syphilis during the DOXYVAC study in order to evaluate oral and anal excretion of TP by molecular biology before, during and after infection.Methods During the DOXYVAC study, incident syphilis was defined as new TP haemagglutinations assay positivity or increased Venereal Disease Research Laboratory (VDRL) titres. Quantitative PCR tests were performed on stored oral and anal samples from the visit preceding the diagnosis, the diagnostic visit of syphilitic infection and the follow-up visit. For each TP-positive PCR sample, detection of 23S ribosomal RNA (rRNA) (azithromycin resistance) and 16S rRNA (doxycycline resistance) mutations was performed.Results Among the 556 participants, 44 cases of incident syphilis occurred in 43 patients. 11 patients (25%) had at least one PCR-positive site, including nine patients in the non-postexposure prophylaxis (PEP) group and two in the doxycycline PEP group. Eight patients had positive TP PCRs at diagnosis, two before diagnosis and one after. A total of eight anal samples and six oral samples tested positive. PCR-positive patients were more likely to be VDRL-positive with higher titres, suggesting more active infection. No mutations associated with doxycycline resistance were detected, while 75% patients had azithromycin-resistant TP strains.Conclusions This study shows that a significant proportion of patients have oral or anal TP excretion, sometimes several months before serological diagnosis, suggesting potential early asymptomatic transmission. The integration of the TP PCR assay into routine screening of high-risk MSM could enable earlier detection and treatment of these patients. The absence of doxycycline resistance is reassuring, but continued monitoring remains essential.