761 Molecular detection of Treponema pallidum subspecies pallidum in 150-year-old foetal remains, southeastern France Avril Mefray 1 , Marie Perrin 1 , Anne Richier 1,2 , Aurore Schmitt 1 , Yann Ardagna 1 and Philippe Biagini 1, * RESEARCH ARTICLE Mefray et al., Journal of Medical Microbiology 2019;68:761–769 DOI 10.1099/jmm.0.000978 Received 24 July 2018; Accepted 25 March 2019; Published 17 April 2019 Author afliations: 1 Aix-Marseille Univ, CNRS, EFS, ADES, Marseille, France; 2 INRAP Mediterranee, Marseille, France. *Correspondence: Philippe Biagini, philippe.biagini@efs.sante.fr Keywords: ancient DNA; congenital syphilis; palaeomicrobiology; palaeopathology; PCR; skeletal treponematosis. Abbreviations: aDNA, ancient DNA; ML, maximum-likelihood; NJ, neighbour-joining; PCR, polymerase chain reaction; TEN, Treponema pallidum subsp. endemicum); TPA, Treponema pallidum subsp. pallidum; TPE, Treponema pallidum subsp. pertenue. 000978 © 2019 The Authors INTRODUCTION Syphilis has been known since the late ffeenth century for being a chronic, ubiquitous infectious disease among ancient populations and, consequently, a severe health burden. Bacterial microrganisms involved in this infec- tion are spirochetes belonging to the genus Treponema, which includes diferent pathogenic subspecies: Treponema pallidum subsp. endemicum (TEN), the causative agent of bejel; T. pallidum subsp. pertenue (TPE) responsible for yaws; and T. pallidum subsp. pallidum (TPA) for syphilis [1]. Te TPA Nichols prototype genome (Washington DC, USA; 1912) is a single circular chromosome of approxi- mately 1 114 000 bp, frst sequenced in 1998 [2]. Pathogenic organisms of the Treponema group exhibit nearly identical genomes, difering by only a few base pairs concentrated in a relatively small number of repetitive genes or sequences [1]. Genetic transfers among diferent treponemal subspe- cies have thus been proved to occur, which can some- times complicate identifcation and characterization of treponemal strains [1, 3]. Venereal syphilis is described as the most destructive of all treponematosis, with various clinical manifestations including osseous alterations in the late stage of the disease. Possibility of congenital infection by subspecies pallidum via transpla- cental contamination has long been demonstrated. Diagnosis of skeletal congenital syphilis may involve dental modifca- tions, osteochondritis, osteomyelitis and osteoperiostitis [4]. In theory, the presence of treponemal spirochetes in bones allows their DNA detection in ancient skeletal material by molecular approaches. Treponema p. subsp. pallidum was indeed frst isolated on an adult archaeological specimen from Easter Island in 1999 using PCR methods [5]. Abstract Purpose. Syphilis, caused by Treponema pallidum subspecies pallidum, is considered as an old disease afecting humans; traces of such infections, including congenital syphilis, are potentially identifable in archaeological samples. The aim of this research was to perform macroscopic and molecular investigations of T. pallidum on six infant remains, buried between 1837 and 1867, from the cemetery of ‘Les Crottes’ in Marseille city (southeastern France). Methodology. Pathological analysis of bones from individuals, aged from the twenty-ninth week of amenorrhea to 4–9 months, was performed. Samples served also as a source of ancient DNA (aDNA) for PCR-based molecular investigations targeting T. pallidum DNA; all samples were also tested for Mycobacterium tuberculosis and Plasmodium falciparum DNA. Sequences char- acterized were cloned and sequenced, and compared to those available in databases. Results/Key findings. All samples tested displayed widespread osteoporotic lesions across the skeleton possibly related to some metabolic or infectious disorders. Subsequent molecular analysis revealed that one individual, SP332 (unborn, 29 amen- orrhea weeks, inhumation date 1864–1866), exhibited positive signals for the fve T. pallidum amplifcation systems tested; sequence analysis provided strong evidence for the efective detection of T. pallidum subspecies pallidum DNA. Conclusions. Individual SP332 is the frst PCR-confrmed palaeopathological case of syphilis identifed in France, and the youngest specimen ever to be diagnosed with certainty for congenital syphilis. Future research aimed at better characterizing this 150-year-old treponeme genome and exploring new archaelogical cases of syphilis in the very young should contribute to a better comprehension of the disease's history.