Large bladder stone and severe arthritis in a natural mummy of the early 19th century from borgo cerreto (spoleto, central italy)

Valentina Giuffra*, Lorenzo Costantini**, Davide Caramella***, Loredana Costantini Biasini** Gino Fornaciari*

*Division of Paleopathology, Department of Oncology, Transplant and New Technologies in Medicine, University of Pisa

**Bioarchaeological Research Centre, National Museum of Oriental Art and IsIAO, Rome
***Division of Diagnostic Radiology and Interventistic, Department of Oncology, Transplant and New Technologies in Medicine, University of Pisa

Fig.1 the mummy

Abstract
The natural mummy of an adult woman was found in the crypt of the church of “Saints Jesus and Mary” of Borgo Cerreto, a village near Spoleto (Umbria, central Italy). The mummy, in a poor state of preservation, dates back to the beginning of the 19th century. A very large bladder calculus was observed in the pelvis. The stone is round-shaped and has a diameter of 7.5 cm. Chemical analysis shows a high level of P and Mg (Magnesium ammonium phosphate) and suggests an infectious aetiology of this calculosis. Furthermore, radiological examination evidenced scoliosis and degenerative changes of the spine, with osteophytosis of the lumbar tract and flattening of L4. Diffused osteoporosis and a bilateral calcification of the femoral artery are also evident.

Key words: natural mummy, Modern Age, bladder stone, arthritis, aterosclerosis.
Palabras clave: momia natural, etad Moderna, calculo vejigal, poliartritis, aterosclerosis

Introduction
In 2001 the exploration of the crypt of the “Saints Jesus and Mary” church of Borgo Cerreto, a village near Spoleto (Umbria, central Italy), revealed six natural mummies dated back to the 18th and beginning of 19th century.
The body of an adult woman was placed in a rough wooden coffin of a classical anthropoid shape.
The woman was wearing a long skirt decorated with flowers, woollen socks and a petticoat of raw fabric, probably linen. A striped silk scarf was used to support the chin and a stripe to tie the wrists together. A rosary was found underneath her folded hands. The style of the garment made it possible to date the mummy back to the early 19th century.
The body was lying on its back with its arms along the sides and hands joined over the pubic region. The mummy, in poor state of preservation, was partly skeletonized, in particular at the level of the skull and of the thorax, which exposes the column (Fig.1).
Dental wear, osteoarthritis changes and osteoporosis allowed us to identify a mature woman, with a large calculus in the pelvis.

Materials and Methods
The mummy was submitted to X-ray examination, performed with a portable device: Minibloc 100/70 designed for animal studies (voltage 220 V, frequency 50 MHz). The radiographs were acquired at 90 cm focus-film distance. The films – all manually processed in situ – were Du Pont, CRONEX 4 Blue Base, 30x 40 cm, and 3M R2, 30×24 cm.
The bladder stone consists in a solid nuclear mass of a rather spherical shape, slightly compressed at the poles, with maximum diameter of about 7.5 cm and total weight of 110 g. The stone was characterized by three fracture lines that crossed it throughout, making it possible to divide it into three parts. The surface was rough, grey in colour with yellowish shadings (Fig.2).
The calculus was studied by scanning electron microscopy (SEM), microchemical analysis (EDS), and X-ray diffraction (XRD), to define its mineral composition and to obtain information on the internal structure. The combined approach of stone analysis by SEM, EDS and XRD are able to provide more information than the conventional techniques, since the latter tend to underestimate the complexity of stones that can contain more than one type of mineral.
Scanning electron microscopy and microanalysis
The stone was examined under scanning electron microscopy with detectors recording secondary and back-scattered electrons, as well as a microanalytical system by energy-dispersive X-rays. Samples from the surfaces were examined using a Leo435VP SEM microscope, equipped with SE and BSE detectors and equipped with an energy-dispersive spectroscopy (EDS) unit of a microanalytic system (Link Oxford ISIS 300). This first step of investigation excluded all possible post-mortem contaminations. Analysis was performed on five areas of the outer layers.
A more detailed analysis was carried out on the inner structure of one of the three parts (ca half of the stone) into which the calculus was naturally divided. The analysis was performed on four spots and three areas in the central portion of the calculus. Chemical composition of each EDS microanalysis is reported in Table 1.

Tab.1 Elements detected on the structure of the calculus (all results in weight %, normalized).

X-ray diffraction
X-ray diffractional powder analysis of human calculi is a very simple method to determine the proportional rate of the crystalline components. Four samples from different locations of the inner and outer structure were studied using a Philips x´Pert diffractometer with CuKalfa radiation. XRD analysis yielded the mineral composition for each sample allowing the identification of crystalline components, as summarized in Table 2.

Results
Macroscopic examination
The head is detached from the spine. The skull, thorax, abdomen and hands are skeletonized. The skin is partly present on the arms, forearms, legs and feet and is well-preserved on the external genitalia. Dehydration occurred slowly, as demonstrated by the large decomposition of the cutaneous surface in the sloping regions; the internal organs are present only in traces.
The woman is completely edentulous (Fig.3a). The cervical vertebrae, the ribs, the left elbow joint are disarticulated; a disconnection is evident between the eighth and the eleventh thoracic vertebrae.
The elbow joints show severe degenerative changes, in particular at the level of the olecranum (Fig.3b). The spine is affected by arthritis, with osteophytosis and porosity of the vertebral plates (Fig.3c). 
A rounded 7.5 cm-diameter formation, broken into 4 parts, inserted in the pelvis and identifiable with a bladder stone, is visible in the abdomen (Fig.3d). Before its removal, the vagina, uterus, intestinal ansae, perivesical tissue and the fibrotic bladder which enveloped the calculus were sampled.
Imaging studies
X-ray examination showed degenerative changes of the spine, with osteophytosis and flattening of the body of the fourth lumbar vertebra. The column is also affected by slight scoliosis (Fig.4a). The woman suffered from diffuse osteoporosis, caused by immobilisation in which she was forced because of her bladder calculosis.
A subluxation between the scafoid and the semilunar bone of the right hand is evidenced. The bladder stone is well visible in the pelvis (Fig.4b). Bilateral calcification of the femoral artery is also evidenced.
Chemical analysis of the calculus
Chemical analysis of a urinary stone is usually aimed at the identification of primary mineral content in the diagnosis and management of urolithiasis. This method is often inadequate for identification of the urinary calculi, as it provides only limited information on the general composition of the stone: i.e. calcium oxalate monohydrate (COM), uric acid, cystine, etc. The chemical composition is obvious but the stone structure can be important as well, because the compositional variability of uroliths has different aetiologies. The composition of the nucleus and of the individual layers is very important to establish the pathogenesis of a urinary calculus.
EDS study of the five areas of the outer layers reveals contents of a mean of oxygen (O) = 46.65%, carbon (C) = 22.33%, nitrogen (N) = 18.51%, phosphorus (P) = 4.85%, calcium (Ca) = 2.89% and magnesium = 2.37%. Other elements such as sodium (Na), sulphur (S), potassium (K), chlorine (Cl) and silicon (Si) are also present in smaller proportions.
The analysis performed on four spots and three areas on the inner structure of the calculus revealed a concentration of oxygen (O) = 33.54 %, carbon (C) = 25.36%, nitrogen (N) = 37.28%, with smaller proportion of calcium (Ca), magnesium (Mg), phosphorus (P), and potassium (K), while other elements, such as silicon (Si), sodium (Na), sulphur (S) and chlorine (Cl), found in the outer structure were absent.
XRD analysis shows that the outer layers present a marked and more evident crystallization than the internal structure, as a result of diagenetic phenomena connected to continuous and repeated exchange of humidity with the external environment. Most of the inner layers, in which less sensitive was the contact with the external atmosphere, appear in amorphous state.
The diffractogram (Fig.5) clearly suggests that the external layers were mainly composed by magnesium ammonium phosphate hexahydrate, in the crystalline phase known as struvite, while the central laminate structure and the core contain ammonium acid urate (Table 2).

Tab. 2 Main crystalline components of the urinary calculus.

Discussion
Morphological details and statistics on the composition of ancient bladder stones were examined by Steinbock (1989).
Ammonium acid urate is one of the most common components of bladder stones, whereas  magnesium ammonium phosphate is less frequent (Gershoff & Prien, 1963).
Epidemiological studies indicate the diet as a major factor in the incidence of bladder stone disease. It has been ascertained that low animal protein intake combined with high grain carbohydrate consumption are responsible for an increase in bladder calculosis formation. A diet of this type produces more acidic urine and decreased urinary phosphate excretion, which decreases the solubility of calcium oxalate and uric acid. The bladder stone under examination is therefore to be referred to environmental factors, at least in its initial stage.
In the Umbria region the presence of calculosis seems to be endemic, judging from the tradition of lithotomy, which was the main specialization of the chirurgical school of Preci, famous from the Middle Ages onwards (Fabbi, 1974; Cecchini, 1997).
The magnesium ammonium phosphate external composition is due to repeated urinary infections. This component represents secondary infection, with the development of alkaline urine and subsequent crystallization of struvite. The struvite calculi may originate ex novo or complicate a lithiasis when pre-existing stones are colonized with urea-splitting bacteria. According to modern clinical data, they represent about 2-3% of calculoses (Rieu, 2005).

References
Cecchini, L. (ed.), 1997. La chirurgia Preciana. Ponte S. Giovanni: Provincia di Perugia.
Fabbi, A., 1974. La scuola chirurgica di Preci. Preci (Perugia): Arti Grafiche Panetto & Petrelli.
Gershoff, S. N. and E. L. Prien, 1963. Urinary Stones in Thailand, Journal of Urology, 90, 285-288.
Rieu, P., 2005. Lithiases d’infection, Annales d’Urologie, 39 (1), 16-29.
Steinbock, R. T., 1989. Studies in Ancient Calcified Soft Tissues and Organic Concretions. II: Urolithiasis (Renal and Urinary Bladder Stone Disease), Journal of Paleopathology, 3(1), 39-59.