Paleopathological and metagenomic study of a XIIth cetury Perucian mummy: an ancient case of Chagas disease

Among the results obtained from this study there is the only known complete paleopathological study of Chagas’ disease (American Trypanosomiasis), comprising macroscopic, microscopic and ultrastructural data, as well as information on atherosclerosis, anthracosis, emphysema and pneumonia. We characterized the gut microbiome of two pre-Columbian Andean mummies dating to the 10–15th centuries using 16S rRNA gene high-throughput sequencing and metagenomics, and compared them to a previously characterized gut microbiome of an 11th century AD pre-Columbian Andean mummy. Our previous study showed that the Clostridiales represented the majority of the bacterial communities in the mummified gut remains, but that other microbial communities were also preserved during the process of natural mummification, as shown with the metagenomics analyses. Metagenome analyses showed the presence of other microbial groups that were positively or negatively correlated with specific metabolic profiles. The presence of sequences similar to both Trypanosoma cruzi and Leishmania donovani could suggest that these pathogens were prevalent in pre-Columbian individuals

The mummy of Ferdinando Orsini, 5th Duke of Gravina (†1549): a paleopathological study

In the monumental Sacristy of the Abbey of Saint Domenico Maggiore, Naples, 37 wooden sarcophagi contain the well preserved bodies of ten Aragonese kings, princes and other Neapolitan nobles who died between the 15th and 16th centuries. One of the arks revealed the natural mummy of Ferdinando Orsini, 5th Duke of Gravina, identified by an epigraph with his name and date of death (1549), in good condition, with the exception of the face, completely skeletonized. The skull suffers from an extensive destructive lesion that afflicted the medial wall of the orbit right, the root of the nose and, partly, the ethmoid without osteitic reaction. The histological examination performed on the bone showed wide lacunae with, inside, epithelial-like cells, partially necrotic, positive for the immunohistochemical stain for PanCK. The border between the bone and the surrounding neoplasia were clear; the brownish fleshy appearance mass had darker margins (like a palisade) and was separated from the bone by clefting artifacts. In our opinion, the pathology that affected Orsini 500 years ago was the basal cell carcinoma in an advanced stage, in fact it is the most frequent form of skin cancer and occurs predominantly on the sun-exposed skin of adults. Microscopically the tumour tends to infiltrate the subcutaneous tissue with a peripherical palisade surrounded by loose of stroma and cleft-like retraction spaces of artifactual nature. It grows in a slow and indolent fashion, but can ulcerate and may invade skull, nares, orbit or temporal bone with wide osteolithic lesion, enough to deserve the Latin name of ‘ulcus rodens’, i.e. erosive ulcer. Immunohistochemically, the cells are positive for keratin and distant metastases are very rare. This case is very important because it represents one of the only four cases of malignant soft tissue tumor diagnosed in paleopathology

Paleogenomic and ancient DNA

Introduction The DNA is a nucleic acid that contains the genetic information necessary for RNA and protein biosynthesis. DNA extracted from past samples (teeth, bones, faeces, etc.) is defined ancient DNA (aDNA) and needs, to preserve, low level of oxygen, fast decrease in water content and, above all, according to the Arrhenius equation, low temperature. From 1985 (first aDNA extracted from a mummy) with the introduction of the Polymerase Chain Reaction (PCR) several studies of paleogenomic were born, but it is essential to identify some possible errors such as fragmentations, contaminations and post-mortal mutations [1]. The applications of the paleogenomic are: evolutionary biology, population studies, studies of the pathogens and microorganism. Materials and methods We report 3 cases studied by the Division of Paleopathology of Pisa: a) Ferrante I, king of Naples (1431-1494). The natural mummy showed round white formations of the pelvis infiltrating the abdominal wall ( 1a-b). b) Maria of Aragon (1503-1568). The well-preserved artificial mummy had a small peduncolate arborescence neoformation in the right inguinal region. c) Andean female mummy (so-called ‘Fi9’) dated 10th–11th century A.D by radiocarbonium analyses. The natural young mummy presented a marked megavisceral syndrome characterized by megacolon, megaoesophagus and cardiomegaly. It was possible to perform complete autopsies and collect tissue samples utilized for histological analyses and DNA extraction. Results a) Histology performed on the round formations confirmed the diagnosis of colorectal adenocarcinoma. Amplification of aDNA highlighted a point mutation of the codon 12 in K-Ras oncogene responsible for the cancer [2] (fig. 1c). b) Macroscopic and histological aspects seemed peculiar of condyloma acuminatum, a papillomavirus-induced squamous lesion also called “venereal wart”. Molecular study revealed the presence of HPV 18, a virus with high oncogenic potential. Automated sequencing of several clones revealed 100% similarity sequences of both HPV 18 and JC9813 DNA, a putative novel HPV with low oncogenic potential [3] (fig. 2c) c) Analysis of the gut microbiome (paleofeces, descending, transverse and ascending colon) underlined the massive presence of Clostridiceae. Sequences homologous to HPVs in the mummified gut (descending colon) was particularly surprising. It was detected also the Tripanosoma cruzi; by comparing a partial sequence homologous to the large ribosomal subunit alpha of the presumptive ancient T. cruzi with modern strains, we suggest that this pathogen may have a more remote origin than previously expected. We also found sequences associated with putative beta-lactamases, penicillin-binding proteins, resistance to fosfomycin, chloramphenicol, aminoglycosides, macrolides, sulfa, quinolones, tetracycline and vancomycin, and multi-drug transporters [4] (fig. 3c). Conclusion a) The alimentary “environment” of the Neapolitan court of the XV century, with its abundance of natural alimentary alkylating agents (red smoked meat), well explains the acquired mutation of K-Ras. b) This represented the first molecular diagnosis of HPV in mummies. HPV is a very old virus that evolved together with man. c) Streptococcus, Staphylococcus, Bacillus and Pseudomonas sequences were identified in the mummified gut, opening the opportunity to investigate possible mechanisms by which these bacteria are preserved. The detection of sequences homologous to those of pathogens such as T. cruzi and HPV indicate their presence in the Americas prior to European colonization. The presence of antibiotic-resistance genes in an 11th century pre-Columbian Andean mummy is intriguing as antibiotics were introduced recently. The presence of beta-lactam antibiotic resistance is certainly not unexpected in any culture, as would be in the case of resistance to any natural rather than a semi- or completely synthetic antibiotic as a result of exposure to natural antibiotic-producing microbiota originating from the environment (e.g. soil); however, vancomycin, particularly, was discovered more than 50 years ago, and vancomycin-resistance genes have been mainly implicated with the increased use of this antibiotic. The presence of antibiotic-resistance genes in the ancient human gut microbiome clearly indicates that these genes pre-date therapeutical use of these compounds and that they are not necessarily associated to a selective pressure of antibiotics use. Identification of pathogens and antibiotic-resistance genes in ancient human specimens will aid in the understanding of the evolution of pathogens as a way to treat and prevent diseases caused by bacteria, microbial eukaryotes and viruses. References Willerslev E, Cooper A. Review paper. Ancient Dna. Proceedings of the Royal Society of London B: Biological Sciences. 2005; 272(1558):3– 16. Marchetti A, Pellegrini S, Bevilacqua G, Fornaciari G. K-RAS mutation in the tumour of Ferrante I of Aragon, King of Naples. The Lancet; 1996, May 4;347(9010):1272. Fornaciari G, Zavaglia K, Ciranni R. Human papillomavirus in a 16th century mummy. The Lancet; 2003, Oct 3, vol 362. Santiago-Rodriguez TM, Fornaciari G, Luciani S, Dowd SE, Toranzos GA, Marota I, Cano RJ. Gut Microbiome of an 11th Century A.D. PreColumbian Andean Mummy. PLoS One, 2015 Sep 30;10(9)

Paleopathological study of Mammuthus meridionalis of Madonna della Strada (L’Aquila)

A skeleton of a male, 50-55 years old Mammuthus meridionalis, dated to the Pleistocene and conserved at the Spanish Fort in L’Aquila (Italy), showed a broken left tusk, in association with the presence of a deep (15x20 cm) bone erosion, involving the dental alveolus and the premaxillary bone, in close proximity to the maxillary sinus and the nasal cavity. During gross examination, small samples from three representative areas of the eroded bone were obtained. Thin sections were made and the specimens were examined under plane and polarized light, using a high resolution microscope with an incorporated digital camera. Microscopical study revealed the intra vitam origin of the lesions, characterized by the presence of woven bone fibers, typical of the early phases of bone remodeling, and lamellar bone with dilated and remodeled Haversian systems. The gross and histological findings were consistent with an osteomyelitis with bone sequestration, caused by a localized blunt trauma or, more likely, resulting from an ascending, post-traumatic chronic pulpitis, due to the tusk fracture occurred during an accident or interspecies fights. The histological exam excluded the involvement of granulomatous inflammation (e.g. tuberculosis) or neoplasia. A disease process of at least several months in duration may be hypothesized, as suggested by the histologically visible bone remodeling. A long survival of the animal after tusk loss may also be supposed, since alteration of masticatory function with altered molar teeth consumption and postural changes (i.e. atlantoaxial fusion), resulting from asymmetric weight distribution, were observed. In this study, the application of (paleo)histological techniques proved to be fundamental in order to establish the nature of bone lesions detected on archeological samples, also providing a good case for studying skull trauma and shedding light on the life history of these large mammals

A Case of Brachymetatarsia From Medieval Sardinia (Italy)

Archaeological excavations carried out in the Medieval village of Geridu (Sardinia) uncovered several burials dating to the late 13th or the first half of 14th century. Among these individuals, the skeleton of an adult female showing a bilateral abnormal shortness of the fourth metatarsal bone was identified. Bilaterality and absence of other skeletal anomalies allow to rule out an acquired aetiology of the disease and to support a diagnosis of congenital brachymetatarsia. Such a rare deformity has a clinical incidence of 0.02% to 0.05%, with strong predominance of the female gender. To our knowledge, no other cases of brachymetatarsia have been reported in paleopathology so far

Sclerosing bone dysplasia from 16th century Sardinia (Italy): a possible case of Camurati-Engelmann disease

The skeletal remains of a male aged 45–55 years displaying several bone anomalies were unearthed from the Alghero (Sardinia) plague cemetery ‘lo Quarter’, a burial site dating back to the 1582–1583 AD outbreak. The skeleton, whose stature is about 165 cm, presents a bilateral hyperostosis with increased diameter of the diaphyses of all the long bones of the upper and lower limbs; the metaphyses appear to be involved, while the epiphyses are spared. Marked thickening of the cranial vault is also evident. Radiological study showed irregular cortical thickening and massive endoperiosteal bone apposition; sclerotic changes are observed in the diaphysis of some metacarpals. Computed tomography (CT) cross sections of the long bones displayed a thickening of the cortical portion and endoperiosteal bone apposition. The individual was affected by a sclerosing bone dysplasia, a genetic disease characterized by increased bone density. In differential diagnosis, several sclerosing bone dysplasia, such as hyperostosis corticalis generalisata, craniodiaphyseal dysplasia, craniometadiaphyseal dysplasia, pachydermoperiostosis and Camurati–Engelmann disease, as well as other disorders characterized by sclerosing manifestations, such as Erdheim–Chester disease, mehloreostosis and skeletal fluorosis, need to be considered. The anomalies observed in skeleton 2179 fit with the features of Camurati–Engelmann disease, which is the most likely candidate for final diagnosis. It is highly challenging to evaluate how such a condition may have influenced the individual’s lifestyle in terms of development, mobility and quality of life. This individual was probably symptomatic and must have experienced common clinical symptoms, such as pain in the limbs and fatigability. However, the strong development of the muscular insertions and the degenerative changes in the upper limbs suggest that the mobility problems should not have prevented him from reaching a mature age and from performing essential daily activities. The presented case is the unique paleopathological evidence of Camurati–Engelmann disease so far diagnosed

Segnalazione di un nuovo ittiosauro dal Cretaceo dell’Appennino modenese (Pavullo nel Frignano)

Alcuni frammenti vertebrali di ittiosauro sono stati recentemente rinvenuti in un calanco presso Pa- vullo nel Frignano (MO). I fossili sono stati trovati entro la Formazione delle Argille Varicolori di Cassio, datate al Cenomaniano superiore-Campaniano (Cretaceo). Tra i reperti rinvenuti è impor- tante segnalare un tratto di colonna vertebrale ancora parzialmente articolata. Si tratta del primo ritrovamento di questo tipo in Italia per quanto riguarda gli ittiosauri del Cretaceo. Alcuni dei corpi vertebrali in oggetto sono di dimensioni relativamente grandi, paragonabili a quelle già note per alcune specie di Platypterygiinae quali Platypterygius australis e P. americanus.Some vertebral fragments of ichthyosaur have recently been found in a clay badland near Pavullo nel Frignano (Modena Province). The finds were discovered in the “Argille Varicolori di Cassio” Formation, which dates from the upper Cenomanian-Campanian (Cretaceous). Among the fossils recovered, a portion of vertebral column, still partially articulated, is of outstanding importance. This is the first discovery of an articulated portion of a skeleton of a Cretaceous ichthyosaur in Italy. Some of the centra are relatively large, comparable to those already known for some species of Platypterygiinae such as Platypterygius australis and P. americanus

Enlarged vascular foramina and lytic lesions in vertebral bodies: a diagnostic dilemma

Among the skeletal material from the sites of Alghero, Mesumundu and Sant’Antioco di Bisarcio (Sassari, Sardinia) and dated back to the period comprises between the 13th and the late 16th century 5 subadult individuals aged between 5 and 15 years and a mature male showed peculiar osteolytic phenomena of the vertebral bodies. These lesions have the appearance of enlarged vascular foramina, affecting several vertebrae mainly of the thoracic and lumbar spine, sometimes with involvement of the sacrum; on the same vertebral body several lesions are generally visible. In the literature similar features have been attributed to brucellosis or tuberculosis. As for the Sardinian skeletal material, an imaging study on the vertebrae of the adult individual was carried out in order to evaluate the appearance of the lesions within the body. Computed Tomography evidenced internal irregular elongated cavitations, sometimes joined together; erosive rounded lesions, whose presence is not detectable externally, were also showed. The molecular analysis has so far been performed on the subadult from Sant’Antioco di Bisarcio, but at initial analysis the DNA resulted degraded. Therefore, the nature of these lesions remains unclear, as it is not sure if they should be referred to tuberculosis, brucellosis or other pathological conditions [hemolytic anemias (eg. Thalassemia), lymphomas, multiple myeloma and infection by Echinococcus]. Further molecular analyses will be carried out on the remains belonging to the other five individuals in an attempt to clarify the etiology of the above mentioned lesions

Enlarged vascular foramina and lytic lesions in vertebral bodies: a diagnostic dilemma

Among the skeletal material from the sites of Alghero, Mesumundu and Sant’Antioco di Bisarcio (Sassari, Sardinia) and dated back to the period comprises between the 13th and the late 16th century 5 subadult individuals aged between 5 and 15 years and a mature male showed peculiar osteolytic phenomena of the vertebral bodies. These lesions have the appearance of enlarged vascular foramina, affecting several vertebrae mainly of the thoracic and lumbar spine, sometimes with involvement of the sacrum; on the same vertebral body several lesions are generally visible. In the literature similar features have been attributed to brucellosis or tuberculosis. As for the Sardinian skeletal material, an imaging study on the vertebrae of the adult individual was carried out in order to evaluate the appearance of the lesions within the body. Computed Tomography evidenced internal irregular elongated cavitations, sometimes joined together; erosive rounded lesions, whose presence is not detectable externally, were also showed. The molecular analysis has so far been performed on the subadult from Sant’Antioco di Bisarcio, but at initial analysis the DNA resulted degraded. Therefore, the nature of these lesions remains unclear, as it is not sure if they should be referred to tuberculosis, brucellosis or other pathological conditions [hemolytic anemias (eg. Thalassemia), lymphomas, multiple myeloma and infection by Echinococcus]. Further molecular analyses will be carried out on the remains belonging to the other five individuals in an attempt to clarify the etiology of the above mentioned lesions