Neonate Human Remains: A Window of Opportunity to the Molecular Study of Ancient Syphilis Rafael Montiel1,4, Eduvigis Solo´ rzano2¤, Nancy Dı´az2¤, Brenda A. A´ lvarez-Sandoval1, Mercedes Gonza´ lez- Ruiz2, Mari Pau Can˜ adas3, Nelson Simo˜ es4, Albert Isidro2,5, Assumpcio´ Malgosa2* 1 Laboratorio Nacional de Geno´ mica para la Biodiversidad, Centro de Investigacio´ n y de Estudios Avanzados del Instituto Polite´cnico Nacional (CINVESTAV-IPN), Irapuato, Guanajuato, Mexico, 2 Unitat d’Antropologia, Departament de Biologia Animal, Biologia Vegetal i Ecologia, Universitat Auto` noma de Barcelona, Bellaterra, Spain, 3 Departament de Biologia Molecular, General Lab, Barcelona, Spain, 4 Research Center for Natural Resources (CIRN), Department of Biology, University of the Azores, Ponta Delgada, Azores, Portugal, 5 Servei de Cirurgia Ortope`dica i Traumatologia (COT), Hospital Universitari del Sagrat Cor, Barcelona, Spain Abstract Ancient DNA (aDNA) analysis can be a useful tool in bacterial disease diagnosis in human remains. However, while the recovery of Mycobacterium spp. has been widely successful, several authors report unsuccessful results regarding ancient treponemal DNA, casting doubts on the usefulness of this technique for the diagnosis of ancient syphilis. Here, we present results from an analysis of four newborn specimens recovered from the crypt of ‘‘La Ermita de la Soledad’’ (XVI–XVII centuries), located in the province of Huelva in the southwest of Spain. We extracted and analyzed aDNA in three independent laboratories, following specific procedures generally practiced in the aDNA field, including cloning of the amplified DNA fragments and sequencing of several clones. This is the most ancient case, reported to date, from which detection of DNA from T. pallidum subspecies pallidum has been successful in more than one individual, and we put forward a hypothesis to explain this result, taking into account the course of the disease in neonate individuals. Citation: Montiel R, Solo´ rzano E, D´ıaz N, A´lvarez-Sandoval BA, Gonza´lez-Ruiz M, et al. (2012) Neonate Human Remains: A Window of Opportunity to the Molecular Study of Ancient Syphilis. PLoS ONE 7(5): e36371. doi:10.1371/journal.pone.0036371 Editor: David Caramelli, University of Florence, Italy Received December 22, 2011; Accepted March 30, 2012; Published May 2, 2012 Copyright: 2012 Montiel et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors acknowledge the following grants: Ministerio de Educaci o´n y Ciencia, Spain (MEC; www.educacion.gob.es) grant No. CGL2008-00800 to Dr. Malgosa; Generalitat de Catalunya (www.gencat.cat), grant No. PR2009-0128 to the Grup de Recerca en Antropologia Biolo´ gica (GREAB); Consejo de Ciencia y Tecnolog´ıa del Estado de Guanajuato, Mexico (CONCYTEG; www.concyteg.gob.mx) grant No. 08-03-K662-076 A04 to Dr. Montiel; Consejo Nacional de Ciencia y Tecnolog´ıa (CONACYT; www.conacyt.gob.mx) grant No. CB-2008-01-105481) to Dr. Montiel. Dr. Montiel received a postdoc grant (SFRH/BPD/32473/2006) from the Fundac¸a˜o para a Cieˆncia e a Tecnologia, Portugal (FCT; www.fct.pt). Dr. A´ lvarez-Sandoval is supported by CONACYT. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Assumpcio.Malgosa@uab.cat ¤ Current address: Departamento de Biopatolog´ıa, Universidad de los Andes, Me´rida, Venezuela Introduction Syphilis was the most important chronic infectious disease that spread in Europe in the late 15th and early 16th centuries. Historical chroniclers relate that in March 1493 Columbus returned in triumph to the royal court in Barcelona, and while a second voyage was being prepared, a strange affliction broke out in the city. Everything indicated that, with the discovery of America in 1492, Columbus’s sailors were infected there, and brought the disease back into Europe [1]. However, according to different scholars, syphilis appeared for the first time in Europe in 1493, in March in Rome, or in February in Naples [1]. Then, the period of time between the discovery of America and the outbreak of the epidemic, seems to be non tenable for developing the epidemic. In spite of the many publications on its origin, this issue is far from being resolved. While some authors claim a lack of evidence for syphilis in the Old World before the voyages of Columbus [2], a minority contend that syphilis occurred in Europe before the Christian Era [3], and some others find ancient bone remains that prove its ancient presence in Europe [4–8]. However, morphology-based diagnosis of treponemal disease in skeletal remains is not always reliable, and differentiating between different treponematoses, or even other entities such as leprosy, is very difficult. Ancient DNA (aDNA) analysis can be a useful tool in bacterial disease diagnosis in human remains [9] and, in the case of syphilis it can confirm the presence in the bones of DNA from Treponema pallidum subspecies pallidum, the causative agent of venereal syphilis [10]; helping to establish a reliable diagnosis. Nevertheless, while the recovery of DNA from Mycobacterium spp. has been widely successful [11–14], several authors report unsuccessful results regarding ancient treponemal DNA [15–17], casting doubts on the usefulness of this technique for the diagnosis of ancient syphilis. A hypothesis for this failure is based on the observation that in advanced stages of the disease, when bone marks are produced, Treponemal organisms ‘‘will most likely not be located in bone and the possibility of isolating them by PCR will be negligible’’ [17]. In general, syphilis is challenging to study in part because it cannot be cultured or genetically manipulated [18], and because the T. pallidum subspecies are morphologically indistinguishable by immunofluorescence or electron microscopy inducing similar histopathological changes and cross-reactive antibodies [19]. This makes diagnostic extremely difficult and therefore complicate further epidemiologic or phylogenetic analyses. However, molec- ular typing has proven to be useful in the specific detection of some of these subspecies [19–21]. In this sense, paleopathology could PLoS ONE | www.plosone.org 1 May 2012 | Volume 7 | Issue 5 | e36371