INFECTIOUS DISEASES IN PALEOPATHOLOGY

MEDICINA NEI SECOLI 33/2 (2021) 229-260 Journal of History of Medicine and Medical Humanities Articoli/Articles INFECTIOUS DISEASES IN PALEOPATHOLOGY: A METHODOLOGICAL APPROACH TO EPIDEMIOLOGICAL SITUATIONS MAURO RUBINI1,2, NUNZIA LIBIANCHI1, ALESSANDRO GOZZI1, PAOLA ZAIO1, ELENA DELLÙ1,3 1 Servizio di Antropologia S.A.B.A.P.-RM-MET; S.A.B.A.P.-LAZ, 2 Università di Foggia - Dipartimento di Archeologia, 3Soprintendenza Archeologia Belle Arti e Paesaggio per la Città Metropolitana di Bari, I Corresponding author: Mauro Rubini, email: [email protected] SUMMARY INFECTIOUS DISEASES The study of infectious diseases in the past is a very interesting and important topic. In the last years a series of new tools were used in Paleopathology in order to increase the reliability of the results. The aim of our research was to present a suggestion for the approach to the study of infectious diseases in the past. This methodology is based on interdisciplinary bases and is divided into four steps: excavation, macroscopic analysis of bone or mummified remains, molecular analysis to confirm the presence of the pathogen or for its discovery from scratch and observation of mortality curves for evaluate the demographic impact of the disease. Through the complementary use of these four steps, results can be obtained that can increase the level of reliability of the clinical evaluation and of the spread of an infectious disease. 1. The evolution of ancient knowledge Human-environment and human-infection interactions have been strongly correlated for millennia, even more so as humans have become, albeit unknowingly, a useful host for pathogens. Human atKey words: Infectious diseases - Paleopathology - Molecular biology - Demography 229 Mauro Rubini, Nunzia Libianchi, Alessandro Gozzi, Paola Zaio, Elena Dellù tempts at interaction with, and knowledge of, nature have always been aimed at understanding, dominating, and directing it in our favor, such as using it to eradicate disease. It is therefore not surprising that there are many references in history, archaeology, and anthropology (e.g. Hippocrates and the De morbo Sacro1, but also in some modern popular contexts) that attest to widespread beliefs in the sacred component of diseases and epidemics. Disease is seen as brought about by divine entities and curable only by healers in direct contact with that divine source2. Historical sources (e.g., medical and non-medical texts, chronicles, biographies, or parish registers) are thus a useful tool for monitoring the presence of some diseases in the past and their perception by the community. However, their use must always account for the particular historical period in which they were written and the nature of medical knowledge at the time3, as well as the fact that not all epidemics would have been recorded4. Where records do survive to the present, there are intrinsic difficulties of description and interpretation5. Modern paleopathology emerged at the end of the 19th century (thanks to Shufeldt) as an important field for the study of ancient diseases6. The definition has varied over time and it has been often relegated to the study of extinct and fossilized remains7 or, as stated by Møller-Christensen, “[…] the science of very ancient diseases”8. Ruffer defined it more formally as the “[…] science of diseases which can be demonstrated on the basis of human and animal remains”9. The methodologies applied to the study of infectious diseases have, from this early period, provided glimpses of a potential non-exclusively medical-anthropological approach. Ruffer’s pioneering study of Schistosoma haematobium eggs in the remains of an Egyptian mummy10, for example, laid the foundations for what would become modern paleoparasitology. At the end of the 1960’s, with the work of Mirko Grmek, an interdisciplinary and innovative approach to the study of infectious diseases began to emerge in which the demographic sample is also analysed in its natural and cultural context. It 230 Infectious diseases in paleopathology is possible to define this new approach, so-called “pathocenosis”, in Grmek’s own words: “By pathocenosis, I mean the qualitatively and quantitatively defined group of pathological states present in a given population at a given time. The frequency and the distribution of each disease depend not only on endogenous-infectivity, virulence, route of infection, vector-and ecological factors-climate, urbanization, promiscuity-but also on frequency and distribution of all the other diseases within the same population”11. Therefore, with this new methodology, both the environmental and spatio-temporal contexts merit investigation. Starting from the 1970’s, the evolution of the discipline has been aimed not only at the analysis of single case studies, but has also been increasingly focused at the population level. The concept of epidemiological transition, developed by Omran12, integrates epidemiological data with demographic changes traceable throughout paleodemographic studies. This approach has been used to demonstrate that the so-called “First Epidemiological Transition” occurred in the Epi-Paleolithic period when the spread of infectious diseases coincided with the introduction of agriculture (Neolithic revolution) and crowded permanent settlements13. In fact, the attestation of a recent Third Epidemiological Transition, born as a result of industrialization, globalization, and fast and continuous human movement, has led to substantial epidemiological instability caused by antibiotic resistance among many microorganisms and pesticide resistance among carriers of disease (mosquitos, fleas, snails)14. While prompt antibiotic treatment is able to reduce mortality from 60% to 15% in many cases, the risk of infection and death in some developing countries is similar to those of the Medieval period15. In fact, some diseases which were considered eradicated, such as bubonic plague, have reappeared in areas where health systems are limited in scope or hygiene is insufficient (such as Africa and areas of Southern Asia and South America)16. It is possible to study the presence, frequency, and distribution of infectious diseases and their endogenous and environmental basis using 231 Mauro Rubini, Nunzia Libianchi, Alessandro Gozzi, Paola Zaio, Elena Dellù several research tools in a multidisciplinary approach (the ‘biocultural approach’17). We propose a methodological approach which combines traditional anthropological and paleopathological practices with the study of skeletal remains (from archaeological excavation) and historical sources. Recent studies conducted with this method have been successful. For example, one study on the spread of Yersinia pestis (plague) in Madagascar, following its reintroduction from China at the end of the 19th century, shows a cyclical re-emergence of the disease, especially in the last forty years18. In this case, the greatest risk of contagion is proximity and contact with the ground where hygiene is poor, such as the placement of beds on the floor. Jobs related to agriculture, trades or local manufacturing also lead people into frequent contact with rats and therefore plague19. For these reasons, paleopathology can produce useful results for understanding patterns of infection that can then be compared with recent outbreaks. Furthermore, it provides useful data on different risk factors and the evolutionary trends of individual pathologies. Therefore, the aim of this work is to suggest a methodological approach for studying infectious diseases and related evolutionary trends in past populations. 2. Methodological approaches to the study of pathogens in the past The study of infectious diseases in the past involves the elucidation of the etiology and the spread of pathogens within a population or groups of populations from different geographical and chronological contexts. The greatest difficulty is the identification of skeletal samples from victims of fast-moving infectious diseases (e.g., plague) as the pathogens quickly attack vital organs and do not lead to the formation of bone lesions detectable from a macroscopic analysis of the remains. Traditionally, the method used in such cases is to combine historical written sources with other literary description of a disease such as medical texts, histories, and legal documents20 which 232 Infectious diseases in paleopathology can suggest areas for further study, provide helpful comparisons, or act as a final validation of scientific investigations. For example, in the case of the 6th century CE plague in the Mediterranean (known as the Justinian plague), historical sources provided an accurate description of the epidemic that allowed only one obvious conclusion21, while molecular testing on mass graves from the era confirmed the suspected etiology22. The methodological approach to the study of infectious diseases in paleopathology which we propose involves the following four steps (Table 1): 1) excavation and archaeological identification; 2) macroscopic and X-ray investigation of the remains (mummies, skeletons); 3) immuno-histological detection and molecular analysis; 4) mortality curves (paleodemography). When combined, these complementary approaches lead to a diagnosis with higher reliability than any isolated approach. It should be remembered that the study of the past always involves the projection of patterns built in the present. Obviously, this represents the main limit. Table 1. Steps for approaching an epidemiological investigation Methodological approach Procedure 1) Excavation and archaeological identification The reaction of the population to the mortality crisis is detected through field excavations and the study of funerary complexes. In particular, crisis episodes are documented by multiple burials which can be deduced based on joint articulation and the existence of contact points between skeletons. The aim is to highlight the possibility of simultaneous burials. This is a good indicator of a “catastrophic” event. In diagnosing disease in palaeopathology, when the human skeletal remains preserve infectious lesions, careful description of the appearance and distribution of lesions is essential. The dominant study approach is providing a differential diagnosis and identifying bone alterations with a different aetiology in order to exclude other infections. Comparisons with skeletal reference group with recorded diseases known and the x-ray, CT and MCT methods are helpful. 2) Macroscopic and radio-diagnostic investigation 233 Mauro Rubini, Nunzia Libianchi, Alessandro Gozzi, Paola Zaio, Elena Dellù Methodological approach Procedure 3) Immunohistological detection and molecular analysis Immunohistological analyses, e.g., immunohistochemistry and immunofluorescence, based on detection of antigenic determinants in mummified tissues, faecal remains or bone samples, provide specific recognition of microorganisms. However, some limitations remain relating especially to preservation and sample access. The analysis of aDNA provides significant advances in the diagnosis of pathogens, particularly in confirming diagnoses. Recommended protocols and accurate procedure steps (pre-treatment of the sample, aDNA extraction, PCR amplification of products, sequencing and analysis) must be followed. Epidemic crises usually cause a selection in terms of age and sex. Estimating these parameters is fundamental as demographic anomalies may suggest an epidemic when compared with a natural population. All the collected data is used to reconstruct mortality trends, graphically represented, which can highlight crisis mortality or demographic shock. Study of the co-evolutionary relationship with infectious diseases allows the definition of mathematical patterns of infection dynamics in different geographical and historical contexts and the creation of theories about mobility and migration patterns of the human groups in the past. 4) Mortality curves (paleodemography) 2.1. Archaeological identification Since the 1980s, archaeological excavations have brought to light an increasing number of human skeletal remains (and, in some cases, mummies). Not all remains receive anthropological and paleopathological study, while in some cases material is selected on the basis of archaeological interests related to the grave contexts (e.g., types of tombs, grave goods, social importance of individuals). However, it is now possible to apply an innovative and interdisciplinary approach - “Archaeoanthropology or Archaeothanatology”23 - from the moment of excavation to reduce the loss of anthropological data. The archaeological excavation of a burial is destructive, and therefore is a unique and unrepeatable moment. For this reason, it is necessary to fully document the stratigraphic deposit as well as the taphonomic 234 Infectious diseases in paleopathology and diagenetic changes which would have impacted the body from the moment of death until its discovery and excavation. Modern technologies allow for the documentation of every detail of the burial for subsequent studies. Through the use of photogrammetry or laser scanning we can recreate the depositional context of the body using 3D models, and therefore observe the deceased and their grave from multiple angles and measure individual bone fragments in situ24. This data is not only useful for archaeologists but is also essential for anthropologists and paleopathologists who are able to access data that is otherwise lost forever after the removal of the skeleton. In particular, this method of investigation and documentation is useful in the study of multiple burials, where some mass death event can be suspected (Fig. 1) due to the articulated nature of the skeletons and the existence of contact points between skeletons which suggests a simultaneous deposition of bodies25. It is not always possible to find articulated or connected skeletons in certain situations, such as shallow mass graves which can be easily disturbed by later activities (ploughing, agricultural ditches, etc.) which alter the skeletal positioning. Even in such cases, the presence of multiple individuals in a single grave is still significant. However, it is necessary to identify “true” multiple graves, which correspond to the deposition of several corpses in the same place over a short period of time. These must be distinguished from so-called collective burials, which are not due to unusual mortality rates but rather are the result of successive burials on a longer time scale (e.g., family graves). In the former case, we can observe the maintenance of skeletal articulation even when several bodies are superimposed while, in the latter case, the various bones are likely to be disturbed26. Application of archaeothanatological methods leads to more reliable results, since the distinction between a collective or multiple burial is determined based on the degree of resistance of the joints to post-depositional disruption27. 235 Mauro Rubini, Nunzia Libianchi, Alessandro Gozzi, Paola Zaio, Elena Dellù Fig. 1. Plague. Simultaneous multiple burials with overlapping bodies - A over B - without alteration of anatomical topography. This generally changes over the course of a few weeks, but it may fluctuate considerably according to climatic conditions and funerary treatments; it is not always easy to differentiate between truly simultaneous deposits and those separated by a few days. Furthermore, poor preservation of the bones and/or physical separations between the bodies (caused by wood, shroud, cloth etc.) may hamper the recognition of the real degree of disarticulation28. Where the state of preservation of the remains is optimal, it is possible to identify si- 236 Infectious diseases in paleopathology multaneous mortality, attributable in some cases to social or natural events (such as wars, massacres, catastrophes, epidemics)29. We must not forget that many individuals who died naturally or as a result of epidemics may have been cremated, or otherwise had their bodies destroyed, which limits our view into certain populations and periods throughout history30. Therefore, for those cases with extant skeletal remains, detailed and innovative excavations are vital for providing sufficient data for such studies, from macroscopic study of the bones to microscopic and biomolecular studies, in order to understood both individual cases as well as wider demographic trends. 2.2. Macroscopic and radiological observations A careful macroscopic taphonomic and diagenetic screening of the lesions present on the osteological remains - aimed at discerning the pathological from the pseudopathological - is the basis from which to begin the analysis31. However, it should be noted that infectious diseases do not always produce observable traces on the bones and for this reason further laboratory investigations are necessary. While such laboratory methods identify the pathogens of a particular disease, it does not necessarily mean that the subject has had recognizable or significant clinical manifestations, as the disease may have remained latent. Similarly, some bacteria with low pathogenicity and a long incubation (such as leprosy which can have a 20-year incubation32) may lead to late clinical manifestations33. Therefore, it is likely that the number of people who are diagnosed on the basis of biomolecular tests is greater than the number showing obvious lesions or who may have been affected to such an extent that they die34. In order to provide an exact diagnosis, it is important to first exclude bone lesions relating to violent death and, consequently, to acts of war or massacres. The presence of wounds allows us to reject an epidemic as the cause of death35. However, occasionally some nonspecific pathological alterations may have caused similar reactions in 237 Mauro Rubini, Nunzia Libianchi, Alessandro Gozzi, Paola Zaio, Elena Dellù Fig. 2. Leprosy. Rhinomaxillary changes. Loss of the nasal spine (white arrow) and remodeling of the inferior margin of the piriform aperture with new bone formation (black arrow). the skeleton, such as periostitis or osteomyelitis lesions, complicating the diagnosis. The latter are documented in numerous burial sites and their aetiology can be traced back to either bacterial infections or traumatic events (based on traces of haematomas or blunt force trauma etc.). Once trauma is excluded, it is then necessary to distinguish between infection and other illnesses36 on the basis of clinical criteria37. In a differential diagnosis, the skeletal lesions potentially attributable to infectious disease can also be the result of metabolic diseases, tumours, haematopoietic diseases, and other pathological conditions. For instance, there are some alternative diagnoses that could lead to the destruction of bone in the rhinomaxillary area in the cases of leprosy (Fig. 2). These disorders include granulomatous disease, 238 Infectious diseases in paleopathology sarcoidosis, treponematoses, some infections such as aspergillosis, phycomycosis, and actinomycosis, and tuberculosis of the facial skin (Lupus vulgaris)38. Distinguishing between these diagnostic options will not always be possible on the basis of skeletal evidence. However, a careful identification of the type and distribution of lesions often allows identification of, at a minimum, a general category of diseases (Table 2). The two most common pathological processes that occur in bone are abnormal bone formation and abnormal bone destruction. Both these aspects can occur from the same infection and in the same lesion, as can be observed in treponematoses. In these cases, a central lytic focus that is the initial lesion is present, followed by the development of a crater-like depression surrounded by newly-formed bone39. Table 2. Main bone changes in some infectious diseases (from Ortner, 2008 modified) Manifestation level* Tuberculosis Leprosy Treponematosis Periostitis Osteomyelitis Brucellosis Smallpox Mycosis Abnormal bone formation 2 3 3 3 3 1 3 2 Abnormal bone destruction 3 3 2 1 3 3 1 2 Sclerosis on lytic margins 2 2 2 1 3 3 1 2 Central lytic/peripheral forming 0 1 3 1 2 1 0 2 Bilateral 1 2 3 1 1 2 3 0 Symmetrical ? 2 2 1 2 1 2 0 Rhinomaxillary remodelling 1 3 1 0 0 0 0 0 Axial involvement 3 2 2 1 3 3 0 3 Appendicular involvement 1 3 3 3 3 2 3 3 Clavicular involvement 0 0 1 0 0 0 0 1 Elbow predilection 0 0 0 0 0 0 3 0 * 3: common; 2: occasionally; 1: occurs but is uncommon; 0: does not occur or is rare; ?: insufficient. 239 Mauro Rubini, Nunzia Libianchi, Alessandro Gozzi, Paola Zaio, Elena Dellù Although the effect of infectious pathogens on the skeletal remains is an important source of information for identifying infectious disease, the skeleton is remarkably resistant to the effects of infectious pathogens40. Certain diseases, such as skeletal syphilis, tuberculosis, and leprosy produce specific bone lesions41 that are more easily recognisable as they consist of epidemic diseases that are not lethal in the short term42. However, leprosy, although it is a non-fatal infection that is associated with long-term close contact between individuals, often has non-specific indicators on the skeleton indicated only by new bone formation43. Furthermore, diagnosis on the basis of macroscopic observation is almost impossible when the rapid action of the infectious agents does not allow time for the development of osseous lesions, such as in acute infections44 of malaria45, trench fever, typhus46 and bubonic plague. The latter, a zoonosis caused by the bacterium Yersinia pestis, does not leave any skeletal lesions47. Bubonic plague, linked to two historical pandemics from the 6th to 18th centuries, was widespread in Eurasia from the Neolithic Age, as evidenced by the recent discovery and reconstruction of the Yersinia pestis genome in Neolithic farmers in Sweden, pre-dated and basal to all known strains of this pathogen48. The absence of bone lesions requires the use of historical data and/or biomolecular analysis49, that may provide to determine a possible cause for the mortality crisis. Importantly, the description and diagnosis of many infectious alterations is possible through X-ray, CT and mCT scans. Early applications of radiology in archaeology began in 1896 and were applied on Egyptian mummified remains in order to discern what lay within the wrappings50. However, in the 1970s when paleopathology became a recognised discipline, radiography became routine in skeletal analyses, allowing the morphological identification of more lesions by revealing those parts hidden by overlying bone. The bone alterations examined can then be compared with known lesion morphology in living patients and/or with the ample radiographic re- 240 Infectious diseases in paleopathology Fig. 3. Tuberculosis. CT scan and 3D application in Pott’s disease (A) and pulmonary tuberculosis (B). cord of diseases documented in the medical literature from the first half of the 20th century51. At present, the most efficient and useful source for the study of the lesions of infectious diseases is computer assisted tomography (CT). This technique, developed in 1974, consists of taking a large series of X-rays around a single axis of rotation in order to record a body in detail. CT scans continue to have all the advantages of conventional radiographs without the problems of superimposition, in addition to the ability to make separate slices through the body that can be combined to create a three-dimensional picture52 (Fig. 3). 241 Mauro Rubini, Nunzia Libianchi, Alessandro Gozzi, Paola Zaio, Elena Dellù 2.3. Supplementary analysis: histologic, immuno-detection and molecular biology In recent years, new histological analyses have emerged as a result of improvements in microscopic instrumentation, dramatically improving our ability to identify traces of infectious pathogens (viruses, bacteria, parasites, or fungi) in ancient organic tissues. Initial observations concerned specific histological samples, cytopathic effect, reliable patterns of inflammation, and evidences of microorganisms in hematoxylin and eosin (H&E) stained sections. However, the microscopic analyses soon revealed some limitations, specifically as a result of the variable size of the microorganisms themselves. While some microorganisms are too small to be clearly observed by light microscopy, larger specimens are difficult to identify in sections as they are often obscured by surrounding tissues53. Therefore six special stains were introduced which embed paraffin in the sample: Giemsa, Gram, Periodic Acid-Schiff (PAS), Grocott-Gomori methenamine silver (GMS), Warthin-Starry, and Ziehl-Neelsen stains54. In the 1960s, the development of the Transmission Electron Microscope (TEM) and the Scanning Electron Microscope (SEM), proved beneficial for the identification of detailed structures within skeletal remains. In paleopathology the most common application of the TEM and SEM has been microscopic observation of the connective fibers of ossein, collagen and parasites (Fig. 4). Electron microscopy has been used to diagnose the Variola virus in old formalin-fixed tissues, in an Italian mummy from the 16th century, and to identify treponemes in an Italian Renaissance mummy with syphilis55. In the 1980s, immunohistochemistry developed which allowed for the recognition of specific microorganism in tissue sections, such as in mummified tissue or fecal remains56. Most of the proteins are protected from degradation by bone so extracellular matrix proteins may be isolated from archaeological material and analyzed57. The lipids are less relevant but can be helpful for identifying microor- 242 Infectious diseases in paleopathology Fig. 4. SEM analysis – Exoskeleton of a flea in an ancient Roman dress (2nd-3rd century CE) ganisms such as Mycobacterium tuberculosis with its characteristic long-chain fatty acids and other cell-wall components which may more easily detect the molecules of mycolic acid able to protect the pathogens58. These techniques are based on the detection of antigenic determinants in tissue sections, and particularly on the use of monoclonal or polyclonal antibodies directed against specific microbial antigens. Immunofluorescence procedures use frozen samples while immunoperoxidase methods are applied on formalin-fixed and paraffin-embedded tissues. Both methods allow the analysis of fastidious or non-cultivatable microorganisms and well-fixed specimens in order to reveal differences between morphologically similar pathogens, cytopathic effects, and to recognize those infectious agents highly causative of outbreaks of infection. Two potential limitations of immunohistological techniques are the difficulty of detecting microorganism antigens because the tissue samples are often preserved 243 Mauro Rubini, Nunzia Libianchi, Alessandro Gozzi, Paola Zaio, Elena Dellù in fixatives such as formaldehyde and that the antigenic determinants in such histological sections are often damaged59. Nevertheless, such techniques are considered valid and have been used in a wide range of studies such as the detection of Salmonella antigens in a Peruvian mummy60, the Schistosoma antigen and the malaria protozoan parasite Plasmodium falciparum in the ancient Egyptian mummies61, as well as more recent studies that have detected Yersinia pestis in bone, demonstrating that this technique can be successfully applied to both skeletal samples and mummified remains62. Many tissue specimens can now be analysed using ancient DNA (aDNA). These new studies allow the identification of microorganisms present within ancient animal and human remains63. aDNA investigations include the sequencing of ancient genomes that are then compared with already-analysed modern genomes. There are many uses for this new technique, including assisting with differential diagnoses, confirming diagnoses, providing disease data for individuals without bone changes, providing genetic data to aid in the examination of species or strains of an organism, revealing carriers of disease, documenting soft tissue pathologies in skeletal remains, looking at susceptibility and resistance genes, and reconstructing the frequency rates of diseases in populations64. However, as seen in human remains and historical documentation65, aDNA techniques also have some limitations. This includes contamination with ‘foreign’ DNA and the destructive nature of the DNA fragments, as well as the high costs, time, and specialized structures needed for the analysis. The extraction process for the aDNA and the interpretation of the results also differ between different laboratories. The lack of preservation of the aDNA can be a substantial barrier to analysis66. The choice of samples is therefore often impacted by environmental factors. There are few geographical areas with climatic conditions suitable for the preservation of aDNA67. Good preservation occurs with a continuous low temperature, a dry environment, 244 Infectious diseases in paleopathology and an absence of sunlight68. Low temperatures and a lack of oxygen inhibit microbial degradation, and aDNA is also preserved in material submerged in water69. Unfortunately, most of the ancient infections documented in historical records did not spread in geographical areas characterized by these environmental conditions70. However, certain precautions can be used to obtain high quality samples, such as the Altai Neanderthal and Denisovan samples which show which regions of archaic hominin DNA have been preserved in the modern human genome (AMH). An interesting case study, only possible with new molecular biology techniques, is a recent analysis of a sub-adult individual with a nonspecific disease dating from 8th-9th-century AD from Byzantine Turkey71. The results obtained from this sample were positive for M. leprae DNA (Fig. 5) and subsequently leprosy within this human group was confirmed by specific morphology in three other adult individuals from the same burial area72. However, it should be noted that even when skeletal lesions are present (indicating the existence of an infectious disease) molecular tests of the same individual can be negative. This may be due to a lack of pathogens, to poor conservation of pathogenic DNA, or (in very ancient cases) to decay of the pathogenic DNA73. 2.4. Palaeodemography Paleodemography, the demographic comparison of intra- and intergroup populations is another area of interest. Indeed, mortality crises originating from epidemics usually do not affect different age groups within a population to the same degree; rather, a selection in terms of age and sex is inevitable, depending on the nature of the epidemic (Fig. 6). Thus, understanding such parameters74 is fundamental as demographic anomalies in the mortality curves may, when compared with a natural population75, suggest a probable death cause76. However, research has demonstrated that diseases do not 245 Mauro Rubini, Nunzia Libianchi, Alessandro Gozzi, Paola Zaio, Elena Dellù Fig. 5. Leprosy aDNA – PCR of a positive sample of leprosy in the absence of bone changes. always discriminate by sex77. The Black Death (bubonic plague) is one such case in which bioarchaeological research had confirmed a lack of sex-discrepancy in mortality. DeWitte has slightly complicated this picture; his analysis reveals that mortality was higher for males with osteological stress markers than for females78. This suggests two interpretations: 1) a higher number of stress markers increased the risk of death for men, or 2) the Black Death was killing more otherwise healthy women than healthy men, based on the 246 Infectious diseases in paleopathology Fig. 6. Mortality curves in a “normal” population (A) and in populations with plague (B). lower excess mortality of women with stress markers79. Additional findings and analysis would be necessary to confirm one of these interpretations. This is made more difficult by the limited nature of the samples (e.g., sex ratio data, lack of accuracy in sex identification80) and of the documentary source material81. It also unclear whether “[...] sex differentials in mortality during plagues were the result of inherent vulnerabilities to the disease itself, or instead caused by inequalities in exposure [...]”82. For example, the bioarchaeological 247 Mauro Rubini, Nunzia Libianchi, Alessandro Gozzi, Paola Zaio, Elena Dellù data from a burial site in Milan between 1452 and 1523, showed a higher mortality rate for women. This was attributed to poor hygiene and overcrowded living conditions for female immigrants83. The first stage of the analysis of the exhumed population should therefore be a detailed collection of individual biological data, sex, and age at death84. Sex estimation is conducted through the macroscopic observation of morphologic features of the os coxae85 and skull86, using standards suggested by Buikstra and Ubelaker87. While molecular determination is more reliable, the costs are still high for this type of analysis. Age at death is evaluated using standard anthropological and forensic methods for the analysis of adults88 and subadults89. Diagnostic reliability levels are higher for sub-adults. For adults, it is useful to attribute large age classes which can then be redistributed when constructing the mortality profile. While this reduces the error interval of the estimate of age to death, it does result in lower accuracy. The second stage of the analysis is the creation of a mortality profile and the calculation of the sex ratio (the theoretical rate is 50%)90. This makes it possible to test whether the sex distribution derived from the archaeological data reflect natural demographic distributions or if there are anomalies that require explanation91. The collected data is used to construct a life-table for the group. The mortality trends, graphically represented, may highlight a crisis relating to warfare, catastrophe, or an epidemic that can either confirm, or be confirmed by, results obtained by other methodical approaches. The abridged life-table consists of a rectangular matrix showing a set of life table measures (columns) alongside different ages (rows). In order to construct a graphical representation of mortality and life expectancy trends for the individuals examined, the key variables are mx (the percentage of people who died aged x) and ex (the life expectancy to x age)92. These two variables are chosen in order to represent the demographic impact of an infectious disease within a restricted human population93. 248 Infectious diseases in paleopathology In a paper about the recent Malagasy epidemic, the authors highlight how the risks of death (graphically represented by cyclical peaks) are not uniformly distributed across age in either modern or historic human groups exposed to the plague94. The study showed that, during the 2014-2015 outbreak, the highest risks of death were mainly recorded in two age groups (5-9 and 20-29 years of age) although this disease can usually affect all age classes. This trend is in accordance to those highlighted through the previous 50 years of Malagasy plague outbreaks95. It was also observed that risk factors in a plague-free population are uniformly distributed throughout life with only natural concentrations usually detected around birth (i.e., delivery, premature birth, stillbirth) and among the elderly (≥ 70 years). A graphic representation of this homogenous distribution of risk factors in plague-free populations reveals flat, linear mortality curves with two peaks in perinatal and older ages96. Conversely, demographic data relating to the effects of leprosy from a LombardAvar cemetery in central Italy (Campochiaro, Molise, 6th–8th century AD) showed a similar mortality trend to other comparison populations, with no mortality peaks equivalent to those observed in short-term diseases (i.e., plague), perhaps due to the long clinical course of leprosy97. As we are dealing specifically with infectious and parasitic diseases - with an exogenous or endogenous origin in the first case, or an endoparasitic or ectoparasitic origin in the second - it is necessary to look beyond the individual case to the larger population context98. The identification of pathogens, together with vehicles (such as water, air, soil, and food) and vectors (such as insects) of infection, can lead to considerations that exceed purely biological data and take on a historical character. In this case, in addition to defining the health status of past populations, we can create models for the diffusion of various pathologies which are strongly connected to migratory movements and commercial exchanges that took place throughout history. From this, 249 Mauro Rubini, Nunzia Libianchi, Alessandro Gozzi, Paola Zaio, Elena Dellù it is possible to project possible trends into the present and suggest risk factors stemming from contact with various pathogens. 3. Conclusions The method of study presented here is intended to be a suggestion for the study of infectious diseases in the past. In isolation, each of the four approaches cannot provide us with comprehensive and exhaustive information on the presence of a particular pathology. Moreover, the importance of studying infectious diseases in the past cannot be represented by a single case study as, by their nature, infections spread in a population. Thus, the ultimate goal of their study will require first the identification of the pathogen followed by contagion estimates and assessment of the probable impact on the population in terms of mortality/survival. The application of different approaches in this exploratory sequence allows us to correlate interdisciplinary information in order to arrive at the highest level of reliability with a global result. 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