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Evidence of probable tuberculosis in Lithuanian mummies
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Evidence of probable tuberculosis in Lithuanian mummies a ¯ D. Piombino-Mascali a,∗, R. Jankauskas a, A. Tamoˇsiunas , a b c,d R. Valanˇcius , H. Gill-Frerking , M. Spigelman , S. Panzer e,f a
Faculty of Medicine, Vilnius University, Vilnius, Lithuania NTK Services, Concord, NH, USA Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel d Medical School, University College London, London, UK e Trauma Center Murnau, Murnau, Germany f Paracelsus Medical University, Salzburg, Austria b c
a r t i c l e
i n f o
Article history: Received 26 April 2014 Accepted 6 January 2015 Available online xxx
a b s t r a c t Tuberculosis has affected Europe for millennia and continues to be a burden upon modern society. It is estimated that one-third of the world’s population is infected with Mycobacterium tuberculosis, the causative agent of this condition. Despite the introduction of control strategies, the disease continues to be one of the most common causes of death globally. Within the framework of the Lithuanian Mummy Project, seven spontaneously mummified human bodies from a church crypt in Vilnius, dating from the 18th and 19th century, were CT-scanned to assess the presence of tuberculosis or other lung diseases. We encountered pulmonary lesions suggestive of cases of pulmonary tuberculosis. In addition, one case might have been affected by extra-pulmonary tuberculosis. This report replicates the image findings from previous studies on ancient mummies that provided evidence of tuberculosis in soft tissues, thus helping reconstruct the history of this disease over time. © 2015 Elsevier GmbH. All rights reserved.
∗ Corresponding author at: Department of Anatomy, Histology and Anthropology, Faculty of Medicine, Vilnius University, ˇ Street 21, LT-03101 Vilnius, Lithuania. Tel.: +370 52398709; fax: +370 52398705. M.K. Ciurlionis E-mail address: [email protected] (D. Piombino-Mascali). http://dx.doi.org/10.1016/j.jchb.2015.01.004 0018-442X/© 2015 Elsevier GmbH. All rights reserved.
Please cite this article in press as: Piombino-Mascali, D., et al., Evidence of probable tuberculosis in Lithuanian mummies. HOMO - J. Comp. Hum. Biol. (2015), http://dx.doi.org/10.1016/j.jchb.2015.01.004
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Introduction Tuberculosis is one of the re-emerging infectious diseases that has been a major cause of mortality in previous centuries and continues to be a burden upon the population of Europe today. The improvement of living standards, better healthcare and specific therapies caused a decline of the disease toward the late 19th/early 20th centuries (Ferlinz, 1999); however, a resurgence in the last decades has caused it to be declared a global emergency (WHO, 2012). For many years, research into the natural history of this condition was based upon skeletal pathologies of past populations and historical documents; only during the last 20 years has the analysis of ancient DNA been successfully employed (Spigelman and Lemma, 1993; Faerman et al., 1997; Donoghue et al., 2009; Chan et al., 2013; Müller et al., 2014). In this regard, human mummies are a very significant source of scientific information, in that they contain remains of soft tissues and thus can provide significant data on past diseases, living conditions and ancient pathogens (Aufderheide, 2003). Computerized tomography (CT) has been regularly employed on mummies since 1979, and allows assessment of human remains through its cross-sectional approach, the superior contrast and the possibility to remove superimposed materials. Furthermore, post-processing software allows for cutting along different planes and creating three-dimensional models (Chhem and Brothwell, 2008; Lynnerup, 2010). The present paper, applying the aforementioned imaging techniques, assessed the presence of pulmonary disease in a set of seven natural mummies from Vilnius, Lithuania, and has identified lesions compatible with human tuberculosis in six of the cases. Imaging lung tuberculosis Tuberculosis is a bacterial infection predominantly located in the lungs, which can disseminate to other areas, including kidneys, bones, meninges and lymph nodes (Aufderheide and Rodríguez Martín, 1998; Kumar et al., 2013). It is traditionally described as a biphasic disease that begins with a primary lesion in a previously unexposed host; the focus of bacterial infection is usually in the lungs, though occasionally other organs may be involved. A caseating or non-caseating granuloma is formed usually in the periphery of the upper or middle lobe of the lungs (Ghon focus); in addition it can initiate the innate immune response and is then phagocytosed and transported to the hilar lymph nodes (Ghon complex) (Bezuidenhout and Schneider, 2009; Hunter, 2011; Piccini et al., 2014). The primary lesion can progress to what is now referred to as ‘progressive primary disease’, which follows directly on the primary lesion. A Ghon focus with latent tuberculosis is present in one-third of the world’s population but less than 10% of infected individuals develop active disease: the remainder develop an effective immune response that contains bacteria within the granulomas and these persist in a dormant state for long periods (Bezuidenhout and Schneider, 2009). A secondary form may be caused by a reinfection or reactivation of the dormant disease in a Ghon focus and may occur years later, following some form of immuno-suppression. However, not all primary lesions are activated, as was observed in a 94-year-old 18th century Hungarian mummy (Spigelman and Donoghue, 2003). In clinical radiology, primary tuberculosis is demonstrated by a dense, homogeneous, parenchymal consolidation that can be located in any lobe (Burrill et al., 2007). The parenchymal focus can leave a radiological calcified scar, creating a Ghon lesion, but other foci, called tuberculomas, can also cavitate and calcify. Lymph-nodal involvement is common, and generally unilateral and right-sided, affecting the hilum and the paratracheal region (Andreu et al., 2004; Burrill et al., 2007). The combination of a calcified node and a Ghon lesion is known as ‘Ranke complex’ (Andreu et al., 2004). The presence of the latter often represents the only radiological evidence of the primary condition, since the nodes alone are not pathognomonic (Van Dyck et al., 2003). Secondary tuberculosis by contrast occurs most often in the lungs but may be widely disseminated, showing a predilection for the upper lobes, the absence of lymphadenopathy, and the presence of cavitations (Andreu et al., 2004). Radiologically, its main feature is parenchymal disease in the form of patchy and poorly defined consolidation, especially in the apical and posterior segments of the upper lobes (Curvo-Semedo et al., 2005; Lee et al., 2008). Furthermore, cavitation with thick and irregular walls occurs in areas of consolidation, while pleural extensions resulting in thickening and calcification can also be seen (Burrill et al., 2007). In Nigeria, a study of tuberculosis in 423 sufferers found that the most common radiological feature in adults was Please cite this article in press as: Piombino-Mascali, D., et al., Evidence of probable tuberculosis in Lithuanian mummies. HOMO - J. Comp. Hum. Biol. (2015), http://dx.doi.org/10.1016/j.jchb.2015.01.004
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cavitation occurring primarily in lung apices and in the posterior segments of the upper lobes and the superior segments of the lower lobes (Piccini et al., 2014). The radiological diagnosis of tuberculosis in mummies requires a consideration of both the clinical manifestations of the condition seen in modern, living patients described above and acknowledgment of post-mortem changes to the soft tissue of a body during mummification. Post-mortem changes in soft tissue may lead to misidentification or misinterpretation of soft tissue structures during radiological examination (Chhem and Brothwell, 2008). In the past, definitive diagnosis of tuberculosis in mummies was dependent upon histological analysis of preserved lung tissues (Allison et al., 1973; Zimmerman, 1979). This method is destructive, generally requiring a complete autopsy of the mummy, although samples may be obtained by endoscopy in some cases. More recently, the presence of tuberculosis was conclusively identified through analysis of pathogen DNA detected in tissue samples extracted via autopsy (Arriaza et al., 1995) or other physical sampling methods (Konomi et al., 2002; Fletcher et al., 2003). Studies that rely solely on radiological analysis for the diagnosis of tuberculosis in mummies are rare, and those that do exist consider both soft tissue and osseous evidence for the presence of the disease (Friedrich et al., 2010). Radiological evidence of tuberculosis in mummies may include the presence of pulmonary, intrapulmonary or mediastinal calcifications, similar to those seen in clinical images of living patients. Osseous evidence may include vertebral collapse and radiolucent lesions in vertebral bodies. These same characteristics also occur as a result of a Staphylococcus infection, so differential diagnosis must be considered and radiological analysis is insufficient, even in a clinical setting (Chhem and Brothwell, 2008). Radiolucent lesions may also occur in long bones, and there may be evidence of erosion of bony margins and narrowing of the joint space, suggestive of tuberculous arthritis (Chhem and Brothwell, 2008). In general, however, while radiological analysis may indicate the presence of tuberculosis, further analysis through microbiological studies may be necessary to provide a definitive diagnosis. Recently, museums and other institutions often prohibit destructive physical sampling of mummies, even on a limited basis, so radiological analysis is the only method of analysis available for paleopathological diagnoses. Materials and methods The Dominican Church of the Holy Spirit of Vilnius overlies a number of subterranean chambers which hold spontaneously mummified and skeletonized human remains of clergymen and laypeople of middle-upper socioeconomic status, dating to the 18th and 19th centuries (Fig. 1a) (PiombinoMascali and Jankauskas, 2014). Within the framework of the Lithuanian Mummy Project, seven mummies out of a total sample of 23 individuals were selected for this study, owing to their remarkable external preservation status (Fig. 1b) (Piombino-Mascali et al., 2014a,b). Sadly, age at death could not be determined with precision in most of the cases (Cox and Mays, 2000). Additionally, no parish record or labels could be located in order to identify the investigated individuals. These remains were examined at the Central Branch of Vilnius University Hospital using a Philips Mx8000 Dual CT scanner (Best, The Netherlands) with a slice thickness of 1.3 mm, an increment of 1.3 mm, a tube voltage of 120 kV, and a tube current of 90 mA. DICOM data were later processed using a workstation (ADW 4.3, General Electrics, Milwaukee, USA) in the Department of Radiology, Trauma Center Murnau, Germany. Lung lesions encountered in this study were described according to the Radiology Review Manual (Dähnert, 1999). Individual soft tissue findings Six out of the seven mummies investigated showed lesions consistent with pulmonary tuberculosis. The results are itemized in Table 1. Mummy VD #3, an adult male, showed desiccated lungs providing a collapsed appearance on both sides, as well as an adherence of the right upper lobe. The right lower lobe appeared thickened and with a small calcification, while another small calcification was seen on the right side of the mediastinum and at the right hilum. The left upper and lower lobes appeared thickened and coherent, and dried fluid in the lower parts of the trachea and central bronchi was also noted. Remnants of the spleen demonstrating a capsule with increased density were Please cite this article in press as: Piombino-Mascali, D., et al., Evidence of probable tuberculosis in Lithuanian mummies. HOMO - J. Comp. Hum. Biol. (2015), http://dx.doi.org/10.1016/j.jchb.2015.01.004
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Fig. 1. (a) Exterior of the Dominican Church and Convent of the Holy Spirit, Vilnius, where the subterranean chambers hold mummified and skeletonized remains tentatively dated to the 18th and 19th centuries. The investigated mummies are spontaneously mummified. (b) Image of one of the mummies featured in this study. In all likelihood, the deceased belonged to the middle-upper social classes, as corroborated by contemporaneous historical sources.
Table 1 Results of the CT investigation. Individual
Features
Pathological stigmata suggesting tuberculous infection
Illustrations
VD #3 VD #9 VD #10 VD #12 VD #14 VD #17
Adult male Adult female Adult female Adult male Adult male Female child
Present Present Present Present Present Present
Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7
visible (Fig. 2a–d). VD #9, an adult female, showed calcification of the trachea and central parts of the bronchi, as well as mediastinal and hilar calcifications on the right side. The right upper and middle lobes appeared to be fibrotic with adherence to the mediastinum, while an additional calcification was also observed in the desiccated right upper lobe. On the left side, the upper and lower lobes were desiccated and appeared slightly fibrotic. In addition, a density that suggested dried fluid with formation of levels, detectable sedimentation effect and fragmentation was seen inside the distal trachea (Fig. 3a–c). A young adult female, VD #10, demonstrated a desiccated lung on the right side, while the one on the left side appeared desiccated but thickened with some calcifications. Additionally, calcifications of the mediastinum of the right side and a calcification of the left hilum were observed (Fig. 4a–c). Mummy VD #12, an adult male, revealed a high seat of the left diaphragm, as well as Please cite this article in press as: Piombino-Mascali, D., et al., Evidence of probable tuberculosis in Lithuanian mummies. HOMO - J. Comp. Hum. Biol. (2015), http://dx.doi.org/10.1016/j.jchb.2015.01.004
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Fig. 2. Case VD #3. (a) Axial CT image shows adherence of the right upper lobe to the chest wall (long arrow) and a calcification in the region of the right hilum (small arrow). On the left side thickening and coherence of the upper and lower lobe is visible (dotted arrow). (b) Axial CT image reveals a calcification on the right side of the mediastinum (arrow). (c) Axial CT image illustrates a thickened right lower lobe with a small calcification (arrow). All other visible calcifications represent coronary calcifications and distinct calcification of the mitral anulus. (d) Axial CT image reveals increased density of the capsule of the spleen (arrows).
calcifications in the mediastinum on the left side and in the left hilum. The left upper lobe was widely preserved, with a detectable pleura and with a broad adherence to the thorax wall. Calcifications were seen both centrally and in the periphery. In addition, preserved parts of the left lower lobe suggesting fibrotic/scarred remnants were seen, together with a shift of the mediastinal remnants to the left side (Fig. 5a and b). Mummy VD #14, an adult male, showed bilateral desiccated remnants of the lungs, and the right upper lobe revealed two small calcifications. Several calcifications in the mediastinum on the right side were also observed (Fig. 6a and b). Finally, mummy VD #17, a female child, showed an increased density in the desiccated left upper lobe, with adhesions to the ventral and lateral thorax and calcifications centrally and in the periphery. The desiccated right upper lobe and middle lobe appeared fibrotic with central calcifications, while the mediastinum revealed three calcifications (Fig. 7a–c). Discussion Tuberculosis, caused by the pathogen Mycobacterium tuberculosis, remains a major factor of mortality in present-day society: it is reported that one third of the world population is affected, with two million people dying each year (Roberts and Buikstra, 2003). In this context, Lithuania still has a very high incidence of the disease, being among the 27 high multi-drug-resistant tuberculosis burden countries in the world (Dewan et al., 2005; WHO, 2012). Like many other parasitic diseases, tuberculosis is thought to have emerged in the period of transition from the Mesolithic to the Neolithic, with a settled population and the formation of foodproducing communities based on animal domestication, agriculture, and a sedentary way of life Please cite this article in press as: Piombino-Mascali, D., et al., Evidence of probable tuberculosis in Lithuanian mummies. HOMO - J. Comp. Hum. Biol. (2015), http://dx.doi.org/10.1016/j.jchb.2015.01.004
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Fig. 3. Case VD #9. (a) Axial CT image shows a small calcification in the desiccated right upper lobe (long arrow) and a dense right middle lobe (short arrow). On the left side a desiccated upper lobe with increased density is visible (dotted arrow). (b) Axial CT image reveals mediastinal calcifications on the right side (arrow). (c) Axial CT image illustrates what appears to be dried fluid with formation of levels, sedimentation effects and partial fragmentation in the right and left ventricle, the trachea, on the left lung remnants and on both sides dorsally (arrows).
Please cite this article in press as: Piombino-Mascali, D., et al., Evidence of probable tuberculosis in Lithuanian mummies. HOMO - J. Comp. Hum. Biol. (2015), http://dx.doi.org/10.1016/j.jchb.2015.01.004
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Fig. 4. Case VD #10. (a) Axial CT image demonstrates two mediastinal calcifications on the right side (short arrows), a desiccated right lung (long arrow), and a desiccated but thickened left lung (dotted arrow). (b) Axial CT image shows a small calcification in the left lung (arrow). (c) Axial CT image illustrates a calcification in the region of the left hilum (arrow).
(Inhorn and Brown, 1990; Roberts and Buikstra, 2003). The earliest known case of tuberculosis scientifically proven with DNA and mycolic acids came from the 9000-year-old community of Atlit Yam off the coast of Israel (Hershkovits et al., 2008; Donoghue et al., 2009). Osteological evidence for the condition was also reported from about the same period at ‘Ain Gazal (El-Najjar et al., 1997). Lithuania provides important historical evidence consisting of bio-archeological materials that document the history of tuberculosis in the area dating to the first millennium. As is often the case with Please cite this article in press as: Piombino-Mascali, D., et al., Evidence of probable tuberculosis in Lithuanian mummies. HOMO - J. Comp. Hum. Biol. (2015), http://dx.doi.org/10.1016/j.jchb.2015.01.004
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Fig. 5. Case VD #12. (a) Axial CT image demonstrates a calcification in the region of the left mediastinum (long arrow) and a large calcification centrally in the left lung (short arrow). The left upper lobe is widely preserved (asterisks) and shows broad adherence to the thorax wall. (b) Coronal reconstruction CT image illustrates asymmetry of the thorax, high seat of the left diaphragm (long arrow) in contrast to the right diaphragm (dotted arrow), and shift of the mediastinum and heart (asterisks) to the left side. The left upper lobe reveals a large calcification in the periphery (small arrow).
Fig. 6. Case VD #14. (a) Axial CT image reveals a small calcification in the right desiccated upper lobe (arrow). (b) Axial CT image demonstrates two mediastinal calcifications on the right side (arrows).
ancient remains, this disease was diagnosed in Lithuanian skeletal materials through the presence of vertebral lesions that can cause angular kyphosis (Pott’s disease) or fusion of the vertebrae, indicating an advanced stage of the condition (Holloway et al., 2011). In addition, other accompanying lesions were used in combination to obtain a diagnosis, such as those on the long and flat bones and those at the joints. However, some of these lesions can present themselves in other diseases, which lead to uncertainties in diagnosis (Roberts and Buikstra, 2003). Based on the skeletal observations, it was concluded that during the first centuries of the first millennium, the area saw an increase in population density and an intensification of agriculture, which likely caused the disease to spread. Our skeletal material suggests that younger individuals did not experience a high mortality rate, but developed chronic lesions (Jankauskas, 1999). Numerous additional cases were recovered from sites dated to the second millennium and more specifically in the Medieval and Early Modern periods, when agriculture was further intensified and trade networks had increased, leading to population expansion as well as enhanced social stratification and the beginning of urbanization. By 1803, records report that the disease was the major cause of mortality in the south of the country, representing 10% of all registered Please cite this article in press as: Piombino-Mascali, D., et al., Evidence of probable tuberculosis in Lithuanian mummies. HOMO - J. Comp. Hum. Biol. (2015), http://dx.doi.org/10.1016/j.jchb.2015.01.004
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Fig. 7. Case VD #17. (a) Axial CT image shows an increased density in the left upper lobe with adherence to the ventral and lateral chest wall and a central (long arrow) and peripheral calcification (short arrow). (b) Coronal reconstruction CT image illustrates intrapulmonary calcifications on the right (long arrow) and left side (short arrow) with adjacent increased density of the lung parenchyma. (c) Axial CT image reveals a small mediastinal calcification on the right side (long arrow) and central calcifications of the left lung (short arrow).
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deaths (Jankauskas, 1999). Comparable numbers can also be seen in 18th/19th century Finland, where pulmonary tuberculosis mortality reached 2–4 per thousand inhabitants, being roughly twice as high in towns (Vuorinen, 1999). Health figures from Russia dating from the end of the 19th century also provide a clear geographic trend, indicating that mortality from tuberculosis was higher in northern ¯ e, ˙ 1999). cities (Mieˇzutaviˇciut For this present study, possible evidence of the disease was found in soft tissues, rather than in bones (Lombardi and Cáceres, 2000; Friedrich et al., 2010). In fact, it should be noted that in the overwhelming majority of cases this is a disease of the lungs, with bone involvement only being around 3% of the total and 30% of extra-pulmonary infection (Ortner, 2003). It is possible that these individuals did not survive long enough to develop skeletal manifestations of the disease, or that the form of tuberculosis contracted by these individuals did not lead to osteological change. It is a significant advantage to be able to study preserved soft tissue and not rely solely on skeletal material for analysis and interpretation. In this research, most of the investigated individuals showed lesions consistent with a tuberculosis diagnosis, comprising calcific parenchymal lesions corresponding to caseous granulomas in a healing phase, as well as calcific lymph nodes located in the hilum and mediastinum, typical of the primary stage of the disease (Van Dyck et al., 2003). These lesions are generally asymptomatic, and only in a minority of cases is the host defense impaired to allow progression of the primary infection (Aufderheide and Rodríguez Martín, 1998). Some of the samples, in addition, displayed signs of postprimary tuberculosis, such as fibrotic-like changes, and possible involvement of the tracheobronchial tree (Andreu et al., 2004). As in all likelihood these individuals were already primed, their organism must have responded promptly to the infection, creating tissue necrosis and accompanying inflammatory reaction (Aufderheide and Rodríguez Martín, 1998). Finally, one of the investigated individuals, VD #3, also showed calcification of the splenic capsule that might possibly be the result of extrapulmonary tuberculosis (Sharma et al., 2007). In summary, the historical context of these remains and the localization and appearance of the lesions described in this paper seem to suggest tuberculosis as the most probable cause. Pulmonary tuberculosis was also evident in a radiological study of 18th/19th century spontaneous crypt mummies from Vác, Hungary, which showed lesions similar to those observed in our study, later confirmed by molecular investigation (Donoghue et al., 2011). In fact, of the 168 mummified bodies found in the Dominican Church in Vác, over 50% had DNA evidence of tuberculosis infection (Fletcher et al., 2003). However, as tissue samples were not obtained from the Lithuanian mummies in the current study, we were unable to provide a definitive answer on the nature of the observed lesions. Any differential diagnosis should bear in mind that the tubercular parenchymal and lymph nodal lesions are also common in other bacterial or fungal infectious diseases. Pathologies with comparable lesions include histoplasmosis, coccidiomycosis, pneumoconiosis, sarcoidosis, and some other miscellaneous conditions such as hypercalcemia, mitral stenosis and alveolar microlithiasis, which make a radiological diagnosis alone challenging (Dähnert, 1999; Andreu et al., 2004). Finally, it should also be acknowledged that post-depositional changes may also alter the tissues in a way to mimic actual lesions (Chhem and Brothwell, 2008). The presence of textiles and the position of the body can affect the preservation of soft tissue during some processes of mummification (Aturaliya and Lukasewycz, 1999). Furthermore, the specific mechanism of mummification, such as the natural desiccation of the mummies in this study, also greatly impacts the preservation of the body, and affects different types of tissue differently. Any change in environment from the original mummification context, or transportation, storage or museum conservation efforts can also cause substantial changes to the tissues of the mummy, possibly leading to potential misinterpretation. Any radiological diagnosis of tuberculosis in mummies must also consider the potential effects of taphonomy on the individuals in the study. Concluding remarks Despite limitations, which include taphonomic changes and the fact that to date we have not analyzed soft tissue samples, the radiological investigation of human mummies from Vilnius has provided possible evidence for the presence of pulmonary tuberculosis in individuals dating from the 18th and Please cite this article in press as: Piombino-Mascali, D., et al., Evidence of probable tuberculosis in Lithuanian mummies. HOMO - J. Comp. Hum. Biol. (2015), http://dx.doi.org/10.1016/j.jchb.2015.01.004
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19th centuries. This finding would add to our current knowledge of the presence of the disease in the country, previously only assessed through observation of skeletal material. As indicated by historical sources, this condition reached epidemic proportions in Europe during this period (Daniel, 2006). Notwithstanding the fact that these individuals belonged to the middle and upper social classes, and thus would have enjoyed better living conditions, the Holy Spirit community may have suffered from tuberculosis in different stages of clinical evolution, providing clues to its widespread diffusion. Future research should be based on confirming the presence of tuberculosis DNA and mycolic acids (Minnikin et al., 2011), in order to shed light on the host resistance to the disease and compare it with present-day data. 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Evidence of probable tuberculosis in Lithuanian mummies a ¯ D. Piombino-Mascali a,∗, R. Jankauskas a, A. Tamoˇsiunas , a b c,d R. Valanˇcius , H. Gill-Frerking , M. Spigelman , S. Panzer e,f a
Faculty of Medicine, Vilnius University, Vilnius, Lithuania NTK Services, Concord, NH, USA Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel d Medical School, University College London, London, UK e Trauma Center Murnau, Murnau, Germany f Paracelsus Medical University, Salzburg, Austria b c
a r t i c l e
i n f o
Article history: Received 26 April 2014 Accepted 6 January 2015 Available online xxx
a b s t r a c t Tuberculosis has affected Europe for millennia and continues to be a burden upon modern society. It is estimated that one-third of the world’s population is infected with Mycobacterium tuberculosis, the causative agent of this condition. Despite the introduction of control strategies, the disease continues to be one of the most common causes of death globally. Within the framework of the Lithuanian Mummy Project, seven spontaneously mummified human bodies from a church crypt in Vilnius, dating from the 18th and 19th century, were CT-scanned to assess the presence of tuberculosis or other lung diseases. We encountered pulmonary lesions suggestive of cases of pulmonary tuberculosis. In addition, one case might have been affected by extra-pulmonary tuberculosis. This report replicates the image findings from previous studies on ancient mummies that provided evidence of tuberculosis in soft tissues, thus helping reconstruct the history of this disease over time. © 2015 Elsevier GmbH. All rights reserved.
∗ Corresponding author at: Department of Anatomy, Histology and Anthropology, Faculty of Medicine, Vilnius University, ˇ Street 21, LT-03101 Vilnius, Lithuania. Tel.: +370 52398709; fax: +370 52398705. M.K. Ciurlionis E-mail address: [email protected] (D. Piombino-Mascali). http://dx.doi.org/10.1016/j.jchb.2015.01.004 0018-442X/© 2015 Elsevier GmbH. All rights reserved.
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Introduction Tuberculosis is one of the re-emerging infectious diseases that has been a major cause of mortality in previous centuries and continues to be a burden upon the population of Europe today. The improvement of living standards, better healthcare and specific therapies caused a decline of the disease toward the late 19th/early 20th centuries (Ferlinz, 1999); however, a resurgence in the last decades has caused it to be declared a global emergency (WHO, 2012). For many years, research into the natural history of this condition was based upon skeletal pathologies of past populations and historical documents; only during the last 20 years has the analysis of ancient DNA been successfully employed (Spigelman and Lemma, 1993; Faerman et al., 1997; Donoghue et al., 2009; Chan et al., 2013; Müller et al., 2014). In this regard, human mummies are a very significant source of scientific information, in that they contain remains of soft tissues and thus can provide significant data on past diseases, living conditions and ancient pathogens (Aufderheide, 2003). Computerized tomography (CT) has been regularly employed on mummies since 1979, and allows assessment of human remains through its cross-sectional approach, the superior contrast and the possibility to remove superimposed materials. Furthermore, post-processing software allows for cutting along different planes and creating three-dimensional models (Chhem and Brothwell, 2008; Lynnerup, 2010). The present paper, applying the aforementioned imaging techniques, assessed the presence of pulmonary disease in a set of seven natural mummies from Vilnius, Lithuania, and has identified lesions compatible with human tuberculosis in six of the cases. Imaging lung tuberculosis Tuberculosis is a bacterial infection predominantly located in the lungs, which can disseminate to other areas, including kidneys, bones, meninges and lymph nodes (Aufderheide and Rodríguez Martín, 1998; Kumar et al., 2013). It is traditionally described as a biphasic disease that begins with a primary lesion in a previously unexposed host; the focus of bacterial infection is usually in the lungs, though occasionally other organs may be involved. A caseating or non-caseating granuloma is formed usually in the periphery of the upper or middle lobe of the lungs (Ghon focus); in addition it can initiate the innate immune response and is then phagocytosed and transported to the hilar lymph nodes (Ghon complex) (Bezuidenhout and Schneider, 2009; Hunter, 2011; Piccini et al., 2014). The primary lesion can progress to what is now referred to as ‘progressive primary disease’, which follows directly on the primary lesion. A Ghon focus with latent tuberculosis is present in one-third of the world’s population but less than 10% of infected individuals develop active disease: the remainder develop an effective immune response that contains bacteria within the granulomas and these persist in a dormant state for long periods (Bezuidenhout and Schneider, 2009). A secondary form may be caused by a reinfection or reactivation of the dormant disease in a Ghon focus and may occur years later, following some form of immuno-suppression. However, not all primary lesions are activated, as was observed in a 94-year-old 18th century Hungarian mummy (Spigelman and Donoghue, 2003). In clinical radiology, primary tuberculosis is demonstrated by a dense, homogeneous, parenchymal consolidation that can be located in any lobe (Burrill et al., 2007). The parenchymal focus can leave a radiological calcified scar, creating a Ghon lesion, but other foci, called tuberculomas, can also cavitate and calcify. Lymph-nodal involvement is common, and generally unilateral and right-sided, affecting the hilum and the paratracheal region (Andreu et al., 2004; Burrill et al., 2007). The combination of a calcified node and a Ghon lesion is known as ‘Ranke complex’ (Andreu et al., 2004). The presence of the latter often represents the only radiological evidence of the primary condition, since the nodes alone are not pathognomonic (Van Dyck et al., 2003). Secondary tuberculosis by contrast occurs most often in the lungs but may be widely disseminated, showing a predilection for the upper lobes, the absence of lymphadenopathy, and the presence of cavitations (Andreu et al., 2004). Radiologically, its main feature is parenchymal disease in the form of patchy and poorly defined consolidation, especially in the apical and posterior segments of the upper lobes (Curvo-Semedo et al., 2005; Lee et al., 2008). Furthermore, cavitation with thick and irregular walls occurs in areas of consolidation, while pleural extensions resulting in thickening and calcification can also be seen (Burrill et al., 2007). In Nigeria, a study of tuberculosis in 423 sufferers found that the most common radiological feature in adults was Please cite this article in press as: Piombino-Mascali, D., et al., Evidence of probable tuberculosis in Lithuanian mummies. HOMO - J. Comp. Hum. Biol. (2015), http://dx.doi.org/10.1016/j.jchb.2015.01.004
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cavitation occurring primarily in lung apices and in the posterior segments of the upper lobes and the superior segments of the lower lobes (Piccini et al., 2014). The radiological diagnosis of tuberculosis in mummies requires a consideration of both the clinical manifestations of the condition seen in modern, living patients described above and acknowledgment of post-mortem changes to the soft tissue of a body during mummification. Post-mortem changes in soft tissue may lead to misidentification or misinterpretation of soft tissue structures during radiological examination (Chhem and Brothwell, 2008). In the past, definitive diagnosis of tuberculosis in mummies was dependent upon histological analysis of preserved lung tissues (Allison et al., 1973; Zimmerman, 1979). This method is destructive, generally requiring a complete autopsy of the mummy, although samples may be obtained by endoscopy in some cases. More recently, the presence of tuberculosis was conclusively identified through analysis of pathogen DNA detected in tissue samples extracted via autopsy (Arriaza et al., 1995) or other physical sampling methods (Konomi et al., 2002; Fletcher et al., 2003). Studies that rely solely on radiological analysis for the diagnosis of tuberculosis in mummies are rare, and those that do exist consider both soft tissue and osseous evidence for the presence of the disease (Friedrich et al., 2010). Radiological evidence of tuberculosis in mummies may include the presence of pulmonary, intrapulmonary or mediastinal calcifications, similar to those seen in clinical images of living patients. Osseous evidence may include vertebral collapse and radiolucent lesions in vertebral bodies. These same characteristics also occur as a result of a Staphylococcus infection, so differential diagnosis must be considered and radiological analysis is insufficient, even in a clinical setting (Chhem and Brothwell, 2008). Radiolucent lesions may also occur in long bones, and there may be evidence of erosion of bony margins and narrowing of the joint space, suggestive of tuberculous arthritis (Chhem and Brothwell, 2008). In general, however, while radiological analysis may indicate the presence of tuberculosis, further analysis through microbiological studies may be necessary to provide a definitive diagnosis. Recently, museums and other institutions often prohibit destructive physical sampling of mummies, even on a limited basis, so radiological analysis is the only method of analysis available for paleopathological diagnoses. Materials and methods The Dominican Church of the Holy Spirit of Vilnius overlies a number of subterranean chambers which hold spontaneously mummified and skeletonized human remains of clergymen and laypeople of middle-upper socioeconomic status, dating to the 18th and 19th centuries (Fig. 1a) (PiombinoMascali and Jankauskas, 2014). Within the framework of the Lithuanian Mummy Project, seven mummies out of a total sample of 23 individuals were selected for this study, owing to their remarkable external preservation status (Fig. 1b) (Piombino-Mascali et al., 2014a,b). Sadly, age at death could not be determined with precision in most of the cases (Cox and Mays, 2000). Additionally, no parish record or labels could be located in order to identify the investigated individuals. These remains were examined at the Central Branch of Vilnius University Hospital using a Philips Mx8000 Dual CT scanner (Best, The Netherlands) with a slice thickness of 1.3 mm, an increment of 1.3 mm, a tube voltage of 120 kV, and a tube current of 90 mA. DICOM data were later processed using a workstation (ADW 4.3, General Electrics, Milwaukee, USA) in the Department of Radiology, Trauma Center Murnau, Germany. Lung lesions encountered in this study were described according to the Radiology Review Manual (Dähnert, 1999). Individual soft tissue findings Six out of the seven mummies investigated showed lesions consistent with pulmonary tuberculosis. The results are itemized in Table 1. Mummy VD #3, an adult male, showed desiccated lungs providing a collapsed appearance on both sides, as well as an adherence of the right upper lobe. The right lower lobe appeared thickened and with a small calcification, while another small calcification was seen on the right side of the mediastinum and at the right hilum. The left upper and lower lobes appeared thickened and coherent, and dried fluid in the lower parts of the trachea and central bronchi was also noted. Remnants of the spleen demonstrating a capsule with increased density were Please cite this article in press as: Piombino-Mascali, D., et al., Evidence of probable tuberculosis in Lithuanian mummies. HOMO - J. Comp. Hum. Biol. (2015), http://dx.doi.org/10.1016/j.jchb.2015.01.004
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Fig. 1. (a) Exterior of the Dominican Church and Convent of the Holy Spirit, Vilnius, where the subterranean chambers hold mummified and skeletonized remains tentatively dated to the 18th and 19th centuries. The investigated mummies are spontaneously mummified. (b) Image of one of the mummies featured in this study. In all likelihood, the deceased belonged to the middle-upper social classes, as corroborated by contemporaneous historical sources.
Table 1 Results of the CT investigation. Individual
Features
Pathological stigmata suggesting tuberculous infection
Illustrations
VD #3 VD #9 VD #10 VD #12 VD #14 VD #17
Adult male Adult female Adult female Adult male Adult male Female child
Present Present Present Present Present Present
Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7
visible (Fig. 2a–d). VD #9, an adult female, showed calcification of the trachea and central parts of the bronchi, as well as mediastinal and hilar calcifications on the right side. The right upper and middle lobes appeared to be fibrotic with adherence to the mediastinum, while an additional calcification was also observed in the desiccated right upper lobe. On the left side, the upper and lower lobes were desiccated and appeared slightly fibrotic. In addition, a density that suggested dried fluid with formation of levels, detectable sedimentation effect and fragmentation was seen inside the distal trachea (Fig. 3a–c). A young adult female, VD #10, demonstrated a desiccated lung on the right side, while the one on the left side appeared desiccated but thickened with some calcifications. Additionally, calcifications of the mediastinum of the right side and a calcification of the left hilum were observed (Fig. 4a–c). Mummy VD #12, an adult male, revealed a high seat of the left diaphragm, as well as Please cite this article in press as: Piombino-Mascali, D., et al., Evidence of probable tuberculosis in Lithuanian mummies. HOMO - J. Comp. Hum. Biol. (2015), http://dx.doi.org/10.1016/j.jchb.2015.01.004
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Fig. 2. Case VD #3. (a) Axial CT image shows adherence of the right upper lobe to the chest wall (long arrow) and a calcification in the region of the right hilum (small arrow). On the left side thickening and coherence of the upper and lower lobe is visible (dotted arrow). (b) Axial CT image reveals a calcification on the right side of the mediastinum (arrow). (c) Axial CT image illustrates a thickened right lower lobe with a small calcification (arrow). All other visible calcifications represent coronary calcifications and distinct calcification of the mitral anulus. (d) Axial CT image reveals increased density of the capsule of the spleen (arrows).
calcifications in the mediastinum on the left side and in the left hilum. The left upper lobe was widely preserved, with a detectable pleura and with a broad adherence to the thorax wall. Calcifications were seen both centrally and in the periphery. In addition, preserved parts of the left lower lobe suggesting fibrotic/scarred remnants were seen, together with a shift of the mediastinal remnants to the left side (Fig. 5a and b). Mummy VD #14, an adult male, showed bilateral desiccated remnants of the lungs, and the right upper lobe revealed two small calcifications. Several calcifications in the mediastinum on the right side were also observed (Fig. 6a and b). Finally, mummy VD #17, a female child, showed an increased density in the desiccated left upper lobe, with adhesions to the ventral and lateral thorax and calcifications centrally and in the periphery. The desiccated right upper lobe and middle lobe appeared fibrotic with central calcifications, while the mediastinum revealed three calcifications (Fig. 7a–c). Discussion Tuberculosis, caused by the pathogen Mycobacterium tuberculosis, remains a major factor of mortality in present-day society: it is reported that one third of the world population is affected, with two million people dying each year (Roberts and Buikstra, 2003). In this context, Lithuania still has a very high incidence of the disease, being among the 27 high multi-drug-resistant tuberculosis burden countries in the world (Dewan et al., 2005; WHO, 2012). Like many other parasitic diseases, tuberculosis is thought to have emerged in the period of transition from the Mesolithic to the Neolithic, with a settled population and the formation of foodproducing communities based on animal domestication, agriculture, and a sedentary way of life Please cite this article in press as: Piombino-Mascali, D., et al., Evidence of probable tuberculosis in Lithuanian mummies. HOMO - J. Comp. Hum. Biol. (2015), http://dx.doi.org/10.1016/j.jchb.2015.01.004
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Fig. 3. Case VD #9. (a) Axial CT image shows a small calcification in the desiccated right upper lobe (long arrow) and a dense right middle lobe (short arrow). On the left side a desiccated upper lobe with increased density is visible (dotted arrow). (b) Axial CT image reveals mediastinal calcifications on the right side (arrow). (c) Axial CT image illustrates what appears to be dried fluid with formation of levels, sedimentation effects and partial fragmentation in the right and left ventricle, the trachea, on the left lung remnants and on both sides dorsally (arrows).
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Fig. 4. Case VD #10. (a) Axial CT image demonstrates two mediastinal calcifications on the right side (short arrows), a desiccated right lung (long arrow), and a desiccated but thickened left lung (dotted arrow). (b) Axial CT image shows a small calcification in the left lung (arrow). (c) Axial CT image illustrates a calcification in the region of the left hilum (arrow).
(Inhorn and Brown, 1990; Roberts and Buikstra, 2003). The earliest known case of tuberculosis scientifically proven with DNA and mycolic acids came from the 9000-year-old community of Atlit Yam off the coast of Israel (Hershkovits et al., 2008; Donoghue et al., 2009). Osteological evidence for the condition was also reported from about the same period at ‘Ain Gazal (El-Najjar et al., 1997). Lithuania provides important historical evidence consisting of bio-archeological materials that document the history of tuberculosis in the area dating to the first millennium. As is often the case with Please cite this article in press as: Piombino-Mascali, D., et al., Evidence of probable tuberculosis in Lithuanian mummies. HOMO - J. Comp. Hum. Biol. (2015), http://dx.doi.org/10.1016/j.jchb.2015.01.004
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Fig. 5. Case VD #12. (a) Axial CT image demonstrates a calcification in the region of the left mediastinum (long arrow) and a large calcification centrally in the left lung (short arrow). The left upper lobe is widely preserved (asterisks) and shows broad adherence to the thorax wall. (b) Coronal reconstruction CT image illustrates asymmetry of the thorax, high seat of the left diaphragm (long arrow) in contrast to the right diaphragm (dotted arrow), and shift of the mediastinum and heart (asterisks) to the left side. The left upper lobe reveals a large calcification in the periphery (small arrow).
Fig. 6. Case VD #14. (a) Axial CT image reveals a small calcification in the right desiccated upper lobe (arrow). (b) Axial CT image demonstrates two mediastinal calcifications on the right side (arrows).
ancient remains, this disease was diagnosed in Lithuanian skeletal materials through the presence of vertebral lesions that can cause angular kyphosis (Pott’s disease) or fusion of the vertebrae, indicating an advanced stage of the condition (Holloway et al., 2011). In addition, other accompanying lesions were used in combination to obtain a diagnosis, such as those on the long and flat bones and those at the joints. However, some of these lesions can present themselves in other diseases, which lead to uncertainties in diagnosis (Roberts and Buikstra, 2003). Based on the skeletal observations, it was concluded that during the first centuries of the first millennium, the area saw an increase in population density and an intensification of agriculture, which likely caused the disease to spread. Our skeletal material suggests that younger individuals did not experience a high mortality rate, but developed chronic lesions (Jankauskas, 1999). Numerous additional cases were recovered from sites dated to the second millennium and more specifically in the Medieval and Early Modern periods, when agriculture was further intensified and trade networks had increased, leading to population expansion as well as enhanced social stratification and the beginning of urbanization. By 1803, records report that the disease was the major cause of mortality in the south of the country, representing 10% of all registered Please cite this article in press as: Piombino-Mascali, D., et al., Evidence of probable tuberculosis in Lithuanian mummies. HOMO - J. Comp. Hum. Biol. (2015), http://dx.doi.org/10.1016/j.jchb.2015.01.004
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Fig. 7. Case VD #17. (a) Axial CT image shows an increased density in the left upper lobe with adherence to the ventral and lateral chest wall and a central (long arrow) and peripheral calcification (short arrow). (b) Coronal reconstruction CT image illustrates intrapulmonary calcifications on the right (long arrow) and left side (short arrow) with adjacent increased density of the lung parenchyma. (c) Axial CT image reveals a small mediastinal calcification on the right side (long arrow) and central calcifications of the left lung (short arrow).
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deaths (Jankauskas, 1999). Comparable numbers can also be seen in 18th/19th century Finland, where pulmonary tuberculosis mortality reached 2–4 per thousand inhabitants, being roughly twice as high in towns (Vuorinen, 1999). Health figures from Russia dating from the end of the 19th century also provide a clear geographic trend, indicating that mortality from tuberculosis was higher in northern ¯ e, ˙ 1999). cities (Mieˇzutaviˇciut For this present study, possible evidence of the disease was found in soft tissues, rather than in bones (Lombardi and Cáceres, 2000; Friedrich et al., 2010). In fact, it should be noted that in the overwhelming majority of cases this is a disease of the lungs, with bone involvement only being around 3% of the total and 30% of extra-pulmonary infection (Ortner, 2003). It is possible that these individuals did not survive long enough to develop skeletal manifestations of the disease, or that the form of tuberculosis contracted by these individuals did not lead to osteological change. It is a significant advantage to be able to study preserved soft tissue and not rely solely on skeletal material for analysis and interpretation. In this research, most of the investigated individuals showed lesions consistent with a tuberculosis diagnosis, comprising calcific parenchymal lesions corresponding to caseous granulomas in a healing phase, as well as calcific lymph nodes located in the hilum and mediastinum, typical of the primary stage of the disease (Van Dyck et al., 2003). These lesions are generally asymptomatic, and only in a minority of cases is the host defense impaired to allow progression of the primary infection (Aufderheide and Rodríguez Martín, 1998). Some of the samples, in addition, displayed signs of postprimary tuberculosis, such as fibrotic-like changes, and possible involvement of the tracheobronchial tree (Andreu et al., 2004). As in all likelihood these individuals were already primed, their organism must have responded promptly to the infection, creating tissue necrosis and accompanying inflammatory reaction (Aufderheide and Rodríguez Martín, 1998). Finally, one of the investigated individuals, VD #3, also showed calcification of the splenic capsule that might possibly be the result of extrapulmonary tuberculosis (Sharma et al., 2007). In summary, the historical context of these remains and the localization and appearance of the lesions described in this paper seem to suggest tuberculosis as the most probable cause. Pulmonary tuberculosis was also evident in a radiological study of 18th/19th century spontaneous crypt mummies from Vác, Hungary, which showed lesions similar to those observed in our study, later confirmed by molecular investigation (Donoghue et al., 2011). In fact, of the 168 mummified bodies found in the Dominican Church in Vác, over 50% had DNA evidence of tuberculosis infection (Fletcher et al., 2003). However, as tissue samples were not obtained from the Lithuanian mummies in the current study, we were unable to provide a definitive answer on the nature of the observed lesions. Any differential diagnosis should bear in mind that the tubercular parenchymal and lymph nodal lesions are also common in other bacterial or fungal infectious diseases. Pathologies with comparable lesions include histoplasmosis, coccidiomycosis, pneumoconiosis, sarcoidosis, and some other miscellaneous conditions such as hypercalcemia, mitral stenosis and alveolar microlithiasis, which make a radiological diagnosis alone challenging (Dähnert, 1999; Andreu et al., 2004). Finally, it should also be acknowledged that post-depositional changes may also alter the tissues in a way to mimic actual lesions (Chhem and Brothwell, 2008). The presence of textiles and the position of the body can affect the preservation of soft tissue during some processes of mummification (Aturaliya and Lukasewycz, 1999). Furthermore, the specific mechanism of mummification, such as the natural desiccation of the mummies in this study, also greatly impacts the preservation of the body, and affects different types of tissue differently. Any change in environment from the original mummification context, or transportation, storage or museum conservation efforts can also cause substantial changes to the tissues of the mummy, possibly leading to potential misinterpretation. Any radiological diagnosis of tuberculosis in mummies must also consider the potential effects of taphonomy on the individuals in the study. Concluding remarks Despite limitations, which include taphonomic changes and the fact that to date we have not analyzed soft tissue samples, the radiological investigation of human mummies from Vilnius has provided possible evidence for the presence of pulmonary tuberculosis in individuals dating from the 18th and Please cite this article in press as: Piombino-Mascali, D., et al., Evidence of probable tuberculosis in Lithuanian mummies. HOMO - J. Comp. Hum. Biol. (2015), http://dx.doi.org/10.1016/j.jchb.2015.01.004
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19th centuries. This finding would add to our current knowledge of the presence of the disease in the country, previously only assessed through observation of skeletal material. As indicated by historical sources, this condition reached epidemic proportions in Europe during this period (Daniel, 2006). Notwithstanding the fact that these individuals belonged to the middle and upper social classes, and thus would have enjoyed better living conditions, the Holy Spirit community may have suffered from tuberculosis in different stages of clinical evolution, providing clues to its widespread diffusion. Future research should be based on confirming the presence of tuberculosis DNA and mycolic acids (Minnikin et al., 2011), in order to shed light on the host resistance to the disease and compare it with present-day data. 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Please cite this article in press as: Piombino-Mascali, D., et al., Evidence of probable tuberculosis in Lithuanian mummies. HOMO - J. Comp. Hum. Biol. (2015), http://dx.doi.org/10.1016/j.jchb.2015.01.004