Paleomicrobiology of Leprosy 13 MARK SPIGELMAN1,2,3 and MAURO RUBINI4,5

Leprosy (or Hansen’s disease) is a chronic infectious disease caused by a slowly multiplying obligate pathogen, Mycobacterium leprae, an acid-fast, rod- shaped bacillus belonging to a single species with limited genetic variability (1). M. leprae has four types and 16 subtypes based on single-nucleotide polymorphisms (SNPs) and variations (InDels) in insertions and deletions (2). Although not highly infectious, it is transmitted via droplets from the nose and mouth during close and frequent contacts with untreated cases. The incubation period can be very long (in some cases up to 20 years) before clinical signs and symptoms become apparent (3, 4). Leprosy can affect all age groups and both sexes. To this day, we cannot grow the bacillus in the laboratory. The bacillus is almost specific to humans but does affect some armadillos (Dasypus novemcinctus) from the southeastern United States (Texas and Louisiana) (5) and a specific type of monkey. In the laboratory, rats, mice, and hamsters can be infected. The diagnosis of the disease in archaeological specimens can be problematic because the characteristic bony changes can occur in a number of other diseases. Thus, paleomicrobiology

1Centre for Clinical Microbiology, Division of Infection & Immunity, University College London, London, UK; 2Department of Anatomy and Anthropology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; 3The Kuvin Center for the Study of Infectious and Tropical Diseases and Ancient DNA, Hadassah Medical School, The Hebrew University, Jerusalem, Israel; 4Department of Archaeology Foggia University, Foggia, Italy; 5Anthropological Service of Soprintendenza per i Beni Archeologici del Lazio (Ministry of Culture), Rome, Italy. Paleomicrobiology of Humans Edited by Michel Drancourt and Didier Raoult © 2016 American Society for Microbiology, Washington, DC doi:10.1128/microbiolspec.PoH-0009-2015 Copyright @ 2016. ASM Press. All rights reserved. May not be reproduced in any form without permission from the publisher, except fair uses permitted under U.S. or applicable copyright law. 131

EBSCO : eBook Collection (EBSCOhost) - printed on 10/10/2018 5:02 PM via UNIVERSITY OF MANITOBA AN: 1402610 ; Drancourt, Michel, Raoult, Didier.; Paleomicrobiology of Humans Account: s5519424.main.ehost 132 SPIGELMAN AND RUBINI

can help to confirm a clinical incidence and, In an advanced stage, leprosy can affect along with genotyping, can trace the spread the skeleton, and a number of specific and of the disease and even human migration nonspecific bony and osteoporotic changes patterns responsible for its spread (6). occur during pathogenesis (8; M. Rubini and Today, leprosy is curable, and multidrug P. Zaio, submitted for publication). Specific therapy (MDT) that provides a simple yet bone changes are the result of invasion of highly effective cure for all types of leprosy the tissues by M. leprae, which is why facial has been made available by the WHO free changes are seen especially in the nose, of charge to all patients worldwide since where nerves are in the mucosa attached to 1995. The incidence of new cases, however, the bone. Secondary bone changes are a appears not to be decreasing according to consequence of peripheral nerve involvement official reports received from 115 countries; (hands, feet, and lower leg bones) and ulti- the global registered prevalence of leprosy mately lead to sensory nerve anesthesia with at the end of 2012 was 189,018 cases. The trauma to the hands and feet because the number of new cases reported globally in victim cannot feel pain, resulting in ulcera- 2014 was 249,657, compared with 232,857 in tion of the feet and hands and then secondary 2012. Pockets where the disease is highly infection of the bones of the hands and feet. endemic still remain in some areas of many Motor and autonomic nerve damage, causing countries, and statistics show that 220,810 lesions such as claw hands and feet, and bone new cases of leprosy (95%) were reported absorption and remodeling are all features of from 16 countries and only 5% of new cases advanced disease. Osteoporosis secondary to from the rest of the world (7). disuse results in a reduction of the bony mass Today, the clinical approach to leprosy and is caused by three factors: a reduction is multidisciplinary because the disease is of osteoblastic activity, a rise of osteoclastic of interest to specialists from various fields, activity, or both (simultaneous competition). including dermatology, infectious diseases, In leprosy, nerve damage and the conse- ophthalmology, neurology, microbiology, quent cessation of normal functional activ- and molecular biology. Clinical signs of dis- ity of the limb produce an imbalance in the ease can include relatively painless ulcers, osteoclastic–osteoblastic equilibrium in the skin lesions consisting of hypopigmented bones, with a rise of osteoclastic activity macules (flat, pale areas of skin), and eye (resorption of bone) that leads to osteoporo- damage (dryness and reduction in the blink- sis by disuse (3). Osteoporosis can produce ing reflex). However, leprosy mainly affects remodeling of the bone because of the col- the peripheral nervous system, especially in lapse of the cortical layer of the bone under the extremities (neural leprosy), with sec- the pressure of the muscles. This type of dam- ondary involvement of the skin and other age causes remodeling and is responsible for tissues. The Schwann cells, the principal the characteristic pencil shape of the bones support cells in the peripheral nervous of the fingers and toes (3, 9). However, the system, appear to be the major target of major classic foci of leprosy damage are in M. leprae in peripheral nerves. As a conse- the rhinomaxillary region of the face and the quence of long-term infection and the host tubular bones of the hands and feet, although immunological response, Schwann cells are other bones may be affected (10, 11). ultimately destroyed or functionally impaired The body response to the disease is highly in damaged nerves. Atrophy of myelinated variable. The immune status of each infected fibers and secondary demyelization have individual determines the type and severity been shown to occur as result of infection, of pathological changes (12). The different sometimes even in the absence of evidence clinical manifestations are the result of the of inflammation or acid-fast bacilli. immune status of patients, not the bacterial Copyright @ 2016. ASM Press. All rights reserved. May not be reproduced in any form without permission from the publisher, except fair uses permitted under U.S. or applicable copyright law.

variety (12). At one end of the immune course. In this case, age at death could have spectrum, there is low resistance to infection played an important role in the possibility or multibacillary (MeSH lepromatous) lep- of finding of skeletal changes in hominid rosy; at the other, there is high resistance to remains. Another possibility is that in the im- infection, resulting in paucibacillary (MeSH mune system of the Neanderthals, a balance tuberculoid) leprosy (13). Between these of immunity developed between M. leprae extremes are the borderline types. How- and host, in which the effects caused by the ever, such responses may help a clinician to mycobacterium were attenuated and better place the patient in the more detailed Ridley- tolerated. Leprosy was preferentially a rural Jopling immune spectrum classification (14). disease in the past, as it is today (16, 19). It is In addition to these groups manifesting clin- probably for this reason that in European ical leprosy, individuals may be infected with prehistoric remains it is very rare to find the bacteria but not develop clinical signs of leprosy cases. The most ancient European the disease; this is termed subclinical leprosy skeletal evidence may be the relatively recent (3). According to some authors (13, 15), the discovery in Scotland of an individual with incidence of subclinical leprosy is as high leprosy dating from 1600 to 2000 BC (20; today as it was in the past (16). In a general J. Roberts, personal communication). Fur- view of the disease, it is important also to thermore, aside from a case in northeastern consider the infectivity of the other forms of Italy (21), we do not know of many other leprosy (15) because in ancient times these cases in the first millennium BCE in Europe. probably played an important role in the During the first millennium AD, the docu- spread of the disease (16, 17). mented osteoarchaeological remains become significant. There are skeletal traces of leprosy during the Middle Ages in western BIOLOGICAL HISTORY OF LEPROSY Europe—for example, in Denmark (22, 23, 24, 25, 26), southern Germany (27), England Leprosy was described in osteoarchaeological (28, 29), Ireland (30), France (28), and Italy remains for the first time in India and dated (31, 32, 33, 34), whereas in central Europe, to 2000 BCE (18). Molecular studies on SNP there are cases in the Czech Republic (35) distribution have shown that the disease and Hungary (36). The presence of a case started following the migration paths of of lepromatous leprosy in central Asia the first human groups from East Africa in (Uzbekistan) dating to the 1st to 3rd centuries the direction of Asia and established itself AD (37) suggests that during the first millen- in eastern and central Europe and in the nium, the spread of leprosy was primarily Mediterranean Basin about 40,000 years ago terrestrial (1, 9). The disease was known in (1, 2). This result suggests that even the last the Roman Empire, as will be detailed later in Neanderthals and the first Homo sapiens this chapter. came into contact with M. leprae. The question is, why we do not find hominids with the bone changes of leprosy? The DIAGNOSIS OF LEPROSY answer is complex, with the main problem IN PALEOPATHOLOGY being the scarcity of hominid fossil remains showing changes in the rhinomaxillary re- Leprosy afflicts bones at the late stages gion and tubular bones of the hand and foot. of disease, and only a small percentage of The second could be linked to the long in- them. The typical changes can be best cubation period and slow development of the appreciated by looking at the figures (Fig. 1, disease. Leprosy involves the skeleton (only 2, 3, 4). The bone changes are highly varia- and not always) in the last stage of its clinical ble in intensity and location. Even though Copyright @ 2016. ASM Press. All rights reserved. May not be reproduced in any form without permission from the publisher, except fair uses permitted under U.S. or applicable copyright law.

bones producing the characteristic shape of a pencil tip (Fig. 5). Although the pathogenesis of the skeletal changes in leprosy is generally difficult to assess in osteoarchaeological remains, Ortner (10) observed that the presence of destructive and proliferative lesions could indicate the infectious phase (chronic or acute) of the disease, without specifying the pathological process that caused the bone changes. Evidence of a roughened, finely pitted cortical surface is uncommon in osteoarchaeological samples. Ortner suggests that the destructive phase is characterized by brief osteoclastic activity quickly followed by slight repair that smoothes the surface. Because in a number of pathological conditions there may be a considerable destruc- tion of bone in the rhinomaxillary region, FIGURE 1 Stages of bone inﬁltration. the differential diagnosis must take account of these conditions; fungal infections such as aspergillosis and mucormycosis (phyco- mycosysis), actinomycosis (a bacterial rather the first infiltration of the mycobacterium than a true fungal disease), lupus vulgaris involves the peripheral nerves of the limbs, (tuberculosis of the facial skin and soft tissue), the first region of the skeleton that is in- granulomatous diseases such as sarcoidosis, filtrated is the rhinomaxillary region be- and treponemal diseases such as syphilis can cause here the nerves are in close contact with bone and covered by a thin layer of nasal mucosa. The so-called facies leprosa, or rhinomaxillary syndrome (38), appears when the skeletal structure of the nose is remodeled and capped with new bone and the nasal spine disappears. In this stage, remodeling of the margins of the nasal aper- ture in conjunction with alterations to the intranasal structures results in the appearance of a wide, empty cavity. Regression of the inferior portion of the superior face is clearly visible. Alterations of the tubular bones of the hands and feet (especially the fifth metatarsal bone, where the same nerves that innervate the fibula are present) are secondary to pathological changes in the peripheral nerves, with consequent loss of sensory and motor functions (39). This leads to osteoporosis due to disuse, with consequences in the metacarpal and metatarsal FIGURE 2 Facial appearance of a victim. Copyright @ 2016. ASM Press. All rights reserved. May not be reproduced in any form without permission from the publisher, except fair uses permitted under U.S. or applicable copyright law.

an important development because as a result of the degradation of DNA over time, it is necessary to use such short target sequences of DNA from ancient bones in order to obtain positive results. The search for M. leprae DNA began with a metatarsal bone from a grave at the site of a massacre of Christians by the Persians in 614 AD (45). It was excavated from the Monastery of Saint John the Baptist on the Jordan River, at the spot where it is tradi- tionally believed that John the Baptist bap- tized Jesus. This is also the traditional site for the washing of the “leper” in Christian sources. The bone proved positive for M. leprae ancient DNA (aDNA) (46, 47). The set of primers and the technique developed

FIGURE 3 Rhinomaxillary syndrome.

cause similar changes in the rhinomaxillary region (10, 40). Psoriatic arthritis, septic arthritis, and other joint diseases may also cause identical changes in the hands and feet.

PALEOMICROBIOLOGY

This new branch of science was developed following the discovery of PCR by Mullis and Faloona (41), which enables the amplification of minute quantities of DNA and so makes it possible to sequence the results. The first paper on this topic was by Spigelman and Lemma (42) and was followed soon after by the paper of Salo et al. (43), who used a primer specific for Mycobacterium tuberculosis based on the insertion sequence found by Eisenach et al. (44). This amplified a specific sequence of human M. tuberculosis DNA that is only 123 bp in length. This was FIGURE 4 Nasal changes in leprosy. Copyright @ 2016. ASM Press. All rights reserved. May not be reproduced in any form without permission from the publisher, except fair uses permitted under U.S. or applicable copyright law.

in the examination of archaeological samples, in which the DNA is likely to be damaged and fragmented. Two new sets of M. leprae- specific nested primers were designed. These were based on the 18-kDa antigen, which gave an outer product of 136 bp and inner product of 110 bp, as well as primers based on the RLEP repetitive sequence, which yielded a 129-bp outer product and a 99-bp nested product. The 18-kDa protein outer primers were 100-fold more sensitive than those for the 36-kDa antigen, and the RLEP outer primers were 1000-fold more sensitive (51). Subsequently, by using the above primers, a problem of the differential diagnosis of Madura foot and leprosy was resolved, in part by showing that the lesions proved positive for the DNA of M. leprae; however, this did not totally exclude Madura foot because of the possibility of the presence of fungal co-infection, thus demonstrating the limits of the conclusion one can draw even when positive results are obtained (52, 53, 54, 55). The problems associated with the detec- FIGURE 5 (A, B) Characteristic pencil shape of the tion of aDNA by PCR are that amplification tubular (metatarsal) bones of the foot. requires strict protocols to prevent contamination. This led to the development of a strict set of standards by workers studying by Hartskeerl et al. (48) was used to amplify human and mammalian aDNA, summarized a nucleotide sequence of 530 bp of the gene in a paper by Cooper and Poinar (56). One encoding the 36-kDa antigen of M. leprae, recommendation was having independent and a positive result was obtained. The 530- duplication of data, but this was at times bp target sequence is a very large size for difficult. Bacterial DNA is not ubiquitous and aDNA, but it was successful from this and can be highly localized. Thus, specimens sent subsequently from other samples after strin- to an independent laboratory may prove neg- gent precautions had been taken to avoid ative, and at the same time, multiple sampling contamination. Therefore, this was deemed of bones may damage important paleopatho- an early sign of the good preservation that logical specimens. In part, this problem was can be found in M. leprae. It is believed overcome by the identification of species- that the lipid-rich cell wall of the organism specific mycolic acids for M. leprae in the cell was a significant contributing factor in this wall of the bacillus that allowed independent preservation. confirmation of the presence of the microbes. A comprehensive review of the develop- Because of the exquisite sensitivity of this ment of M. leprae paleomicrobiology was technique, amplification with all its attendant given in 1999 at a meeting in Bradford, UK problems is not necessary. Further, mycolic (“The Past and Present of Leprosy”) and acids are chemically more robust then DNA subsequently published (49, 50). The size of and have been detected in specimens in the primers was considered a disadvantage which the DNA was not found (57, 58). Copyright @ 2016. ASM Press. All rights reserved. May not be reproduced in any form without permission from the publisher, except fair uses permitted under U.S. or applicable copyright law.

Following the sequencing of the entire tests found the aDNA of M. leprae in the M. leprae genome and the recognition of in- bones of this individual (64). Another ques- formative SNPs, scientists were able to tion regarded the appearance and subsequent determine the molecular typing of the orga- disappearance of the almost plaguelike epi- nism (1). It was shown that the genome is demic of leprosy in medieval Europe. There remarkably stable over time, having only a is no question that the disease was present human host (3). There is a clonal relationship during the Roman Empire and even before between M. leprae and its human host, so (21, 65, 66). However, the skeletal evidence discovering the genetic profiles of modern indicates that these sporadic early reports and extinct or ancient strains of M. leprae were not in the same epidemic proportions can tell us about the migration and spread as in the subsequent medieval epidemic that of pathogen and host over time (1, 59, 60, 61). swept through Europe but then almost dis- As technology progressed, the development appeared over time. It has been suggested of high-throughput sequencing enabled the that the decline of leprosy in the Western sequencing of entire genomes from archaeo- World in the past was not monocausal but logical samples. The problem of low yields due to a complex web of social, legal, po- of aDNA was solved by next-generation se- litical, and biomedical causes (67). The latter quencing, which uses targeted enrichment is explained by the rise in tuberculosis. Stud- techniques with hybridization capture, fol- ies that began in the 20th century proposed a lowed by the use of bioinformatics software model of cross-immunity between these two to construct the whole genome. Monot et al. mycobacterial infections as a possible cause (2) were able to conduct comparative ge- of the decline of leprosy. Lowe and McNulty nomic and phylogeographical analyses of (68), following the Rogers hypothesis (1924) M. leprae. A more recent paper reported a and the overall work of Chaussinand (69), genome-wide comparison between medieval observed that the Bacille Calmette-Guérin and modern M. leprae specimens (61). Fur- (BCG) vaccine, prepared specifically for thermore, insights into ancient leprosy and inoculation against tuberculosis, was of ben- tuberculosis gained by using metagenomics, efit in preventing leprosy in some people. as reported in papers Taylor et al (62) and The origin of this cross-immunity is contro- Donoghue (63), demonstrated the progress of versial, but one fact is certain: in clinical the science. contexts, leprosy may be prevented following The use of paleomicrobiology in answer- BCG vaccination in 20% to 91% of individu- ing questions related to questions raised by als, based on different studies (70). There is archaeologists in leprosy can best be shown a close antigenic relationship between the by a few examples. An early-published case mycobacteria causing leprosy (M. leprae) and based on aDNA resulted from the discovery those causing tuberculosis (M. tuberculosis). of the Firsst-Century Tomb of the Shroud in The amino acid sequences of 65-kDa antigens Alkadema, Jerusalem. A body was found that of M. leprae, M. tuberculosis, and Mycobacte- had been buried in a wall niche, whereas all rium bovis BCG display greater than 95% the other burials in this tomb were reburials homology (71, 72). The higher reproductive after decomposition in an ossuary, as was the rate of tubercle bacilli, compared with that custom at the time. This raised the question, of leprosy bacilli, and their degree of cross- why? There was no convincing evidence of immunity do not normally allow both infec- leprosy in the skeletal remains, but it was tions to occur simultaneously, but there have considered that perhaps there was something been sporadic reports of the co-existence of about the person that had made the family tuberculosis and leprosy in the same patients wary of touching the remains. The possibility based on physical symptoms (73, 74). Fur- of leprosy was suggested, and subsequent thermore, a study based on the examination Copyright @ 2016. ASM Press. All rights reserved. May not be reproduced in any form without permission from the publisher, except fair uses permitted under U.S. or applicable copyright law.

of mycobacterial aDNA by PCR has also in- own leprosarium. Indeed, Manchester and vestigated the relationship between tubercu- Roberts (79) noted that in Britain the number losis and leprosy among individuals buried in of lazar houses for the care of leprosy patients the past, finding evidence for the co-existence increased dramatically during the 12th and of tuberculosis and leprosy (64, 74). This early 13th centuries. One possible sugges- supports an earlier report that tuberculosis tion, supported by aDNA and cell wall lipid can occur in people throughout the immune analysis, is that that the migration of tribes spectrum of leprosy (75). from central Asia during the 1st millennium Chaussinand (69) was the first to observe led to a separate introduction of leprosy into that in several different clinical settings, the eastern and central Europe (80). prevalence of leprosy was inversely related to that of tuberculosis. Therefore, it may be considered unusual to encounter their co- CONCLUSION existence. For example, an important epidemiological study from South Africa reported It can be seen that the use of paleomicro- an increased incidence of pulmonary tuber- biological techniques in the study of leprosy culosis among patients with leprosy, but has the potential to assist clinicians and pa- not vice versa (76). This theory of cross- leopathologists in many important aspects immunity between tuberculosis and leprosy of their work. How can it help clinicians? In led to it being proposed as the hypothesis for leprosy, because of the unique nature of the the disappearance of leprosy from western organism, paleomicrobiological techniques Europe in the 14th century AD, before the can help solve problems of differential diag- advent of chemotherapy (16, 77). However, nosis. In the case of co-infection with Myco- research on skeletal samples, albeit limited, bacterium tuberculosis, they can also suggest has more recently refuted the hypothesis that a cause of death and possibly even trace the this cross-immunity exists and has suggested migratory patterns of people in antiquity. an alternative explanation for the decline of leprosy in western Europe (74). Furthermore, Manchester (16) and Donoghue et al. (74) CITATION suggested that immunological changes seen Spigelman M, Rubini M. 2016. Paleomicro- in multibacillary leprosy (lepromatous/low biology of leprosy, Microbiol Spectrum 4(4): resistance), together with the socioeconomic PoH-0009-2015. impact of the disease, may have led to increased mortality due to tuberculosis that resulted in the decline of leprosy. Despite this REFERENCES long history of epidemiological, clinical, and 1. Monot M, Honoré N, Garnier T, Araoz R, microbiological studies, the exact relation- Coppée J-Y, Lacroix C, Sow S, Spencer JS, ship between tuberculosis and leprosy still Truman RW, Williams DL, Gelber R, Virmond remains unclear. The only certain fact is that M, Flageul B, Cho SN, Ji B, Paniz-Mondolfi A, there is homology between the two mycobac- Convit J, Young S, Fine PE, Rasolofo V, Brennan PJ, Cole ST. 2005. On the origin of teria, and that in Europe during the Middle leprosy. Science 308:1040–1042. Ages an increase of tuberculosis as a result 2. Monot M, Honoré N, Garnier T, Zidane N, of increasing urbanization co-incided with Sherafi D, Paniz-Mondolfi A, Matsuoka M, a decline in leprosy (78). Paleopathologists Taylor GM, Donoghue HD, Bouwman A, Mays have noted the presence of leprosy in Roman S, Watson C, Lockwood D, Khamispour A, Dowlati Y, Jianping S, Rea TH, Vera-Cabrera times and before, but not in the almost L, Stefani MM, Banu S, Macdonald M, Sapkota plague-level proportions found in medieval BR, Spencer JS, Thomas J, Harshman K, Singh Europe, when almost every town has its P, Busso P, Gattiker A, Rougemont J, Brennan Copyright @ 2016. ASM Press. All rights reserved. May not be reproduced in any form without permission from the publisher, except fair uses permitted under U.S. or applicable copyright law.

PJ, Cole ST. 2009. Comparative genomic and Brazil: an ecological study. Int J Ecol 33:262– phylogeographic analysis of Mycobacterium 269. leprae. Nat Genet 41:1282–1288. 20. Anonymous. 2002. Bones raise leprosy doubts. 3. Robbins SL, Cotran RS. 2002. Patho- BBC News, November 5. http://news.bbc.co.uk/ logic Basis of Disease. W.B. Saunders Co., 2/hi/uk_news/scotland/2406001.stm. Philadelphia, PA. 21. Mariotti V, Dutour O, Belcastro MG, Facchini 4. Jopling WH. 2004. Handbook of Leprosy, F, Brasili P. 2005. Probable early presence of 5th ed. Sheridan Medical Books, New York, NY. leprosy in Europe in a Celtic skeleton of 5. Maiden MCJ. 2009. Putting leprosy on the the 4th-3rd century BC (Casalecchio di Reno, map. Nat Genet 41(12):1264. Bologna, Italy). Int J Osteoarchaeol 15:311–325. 6. Rubini M, Zaio P, Roberts AC. 2014. Tuber- 22. Andersen JG. 1969. Studies on the Medieval culosis and leprosy in Italy. New skeletal Diagnosis of Leprosy in Denmark. An Oste- evidence. HOMO - J Comp Hum Biol 65:13–32. archaeological, Historical and Clinical Study. 7. WHO. 2016. Leprosy fact sheet. http://www. Costers Bogtrykkeri, Copenhagen, Denmark. who.int/mediacentre/factsheets/fs101/en. 23. Arcini C. 1999. Health and disease in early 8. Thappa DM, Sharma VK, Kaur S, Suri S. Lund: osteopathological studies of 3,305 indi- 1992. Radiological changes in hands and viduals buried in the first cemetery area of feet in disabled leprosy patients: a clinical- Lund 990-1536. Archaeologica Lundensia VIII. radiological correlation. Lepr India 64:58–66. Department of Community Health Sciences, 9. Kulkarni VN, Mehta JM. 1983. Tarsal disinte- Lund. gration (T.D.) in leprosy. Lepr India 55:338–370. 24. Boldsen JL. 2001. Epidemiological approach 10. Ortner DJ. 2003. Identification of Pathological to the paleopathological diagnosis of leprosy. Conditions in Human Skeletal Remains. Am J Phys Anthropol 115:380–387. Smithsonian Institution Press, Washington, DC. 25. Boldsen JL. 2005. Leprosy and mortality in 11. Roberts CA, Manchester K. 2005. The the medieval Danish village of Tirup. Am J archaeology of disease, 3rd ed. Sutton Publish- Phys Anthropol 126:159–168. ing, Stroud, Gloucestershire, UK. 26. Bennike P, Lewis ME, Schultkowski H, 12. Geluk A, Ottenhoff THM. 2006. HLA and Valentin F. 2005. Comparison of child mor- leprosy in the pre- and postgenomic eras. Hum bidity in two contrasting Medieval cemeteries Immunol 67:439–445. from Denmark. Am J Phys Anthropol 128:734– 13. Kumarasinghe SPW, Kumarasinghe MP, 746. Amarasinghe UTP. 2005. “Tap sign” in tuber- 27. Haas CJ, Zink A, Szeimies U, Nerlich G. coloid and borderline tubercoloid leprosy. 2002. Molecular evidence of Mycobacterium Int J Lepr 72:291–295. leprae in historical bone samples from South 14. Ridley DS, Jopling WH. 1966. Classification Germany, p 287–292. In Roberts CA, Lewis of leprosy according to immunity, a five-group ME, Manchester K (ed), The Past and Present system. Int J Lepr 34:255–273. of Leprosy. Archaeological, Historical, Palaeo- 15. Kampirapap K. 2008. Assessment of subclin- pathological and Clinical Approaches. British ical leprosy infection through the measure- Archaeological Reports International Series ment of PGL-1 antibody levels in residents of a (Book 1054). British Archaeological Reports, former leprosy colony in Thailand. Lepr Rev Oxford, UK. 79:315–319. 28. Roberts CA. 2000. Infectious disease in bio- 16. Manchester K. 1984. Tuberculosis and leprosy cultural perspective: past, present and future in antiquity: an interpretation. Med Hist 28: work in Britain, p 145–162. In Cox M, Mays S 162–173. (ed), Human Osteology in Archaeology and 17. Rubini M, Zaio P. 2009. Lepromatous leprosy Forensic Science. Greenwich Medical Media, in an early mediaeval cemetery in Central Italy London, UK. (Morrione, Campochiaro, Molise, 6th–7th cen- 29. Schultz M, Roberts C. 2002. Diagnosis of tury AD). J Archeol Sci 36:2771–2779. leprosy in skeletons from an English late 18. Robbins G, Mushrif Tipathy V, Misra VN, Medieval hospital using histological analy- Mohanty RK, Shinde VS, Gray KM, Schug sis, p 89–105. In Roberts CA, Lewis ME, MD. 2009. Ancient skeletal evidence for Manchester K (ed), The Past and Present of leprosy in India (2000 B.C.). Plos One 4:1–8. Leprosy. Archaeological, Historical, Palaeo- doi:10.1371/journal.pone.0005669. pathological and Clinical Approaches. British 19. Kerr-Pontes LR, Dorta Montenegro AC, Archaeological Reports International Series Lima Barreto M, Werneck GL, Feldmeier (Book 1054). British Archaeological Reports, H. 2004. Inequality and leprosy in Northeast Oxford, UK. Copyright @ 2016. ASM Press. All rights reserved. May not be reproduced in any form without permission from the publisher, except fair uses permitted under U.S. or applicable copyright law.

30. Murphy E, Manchester K. 2002. Evidence for 42. Spigelman M, Lemma E. 1992. The use of the leprosy in Medieval Ireland, p 193–199. In polymerase chain reaction to detect Mycobac- Roberts CA, Lewis ME, Manchester K (ed), terium tuberculosis in ancient skeletons. Int J The Past and Present of Leprosy. Archaeolog- Osteoarchaeol 3:137–143. ical, Historical, Palaeopathological and Clinical 43. Salo W, Aufterheide A, Buikstra J, Holcomb Approaches. British Archaeological Reports T. 1994. Identification of Mycobacterium tu- International Series (Book 1054). British Ar- berculosis DNA in a pre-Columbian Peruvian chaeological Reports, Oxford, UK. mummy. Proc Natl Acad Sci U S A 91:2091– 31. Belcastro MG, Mariotti V, Facchini F, Dutour 2094. O. 2005. Leprosy in a skeleton from the 7th 44. Eisenach KD, Cave MD, Bates JH, Crawford century Necropolis of Vicenne-Campochiaro JT. 1990. Polymerase chain reaction amplifi- (Molise, Italy). Int J Osteoarchaeol 15:431–448. cation of a repetitive DNA sequence specific 32. Rubini M. 2008. Disfiguring diseases and for Mycobacterium tuberculosis. J Infect Dis society in the fourth - fifth centuries A.D.: the 161:977–981. case of Palombara Sabina (Rome, Central Italy). 45. Gill M. 1992. A History of Palestine 634-1099. In Proceedings of the Annual Meeting Archae- Cambridge University Press, Cambridge, UK. ological Institute of America, Chicago, IL. 46. Rafi A, Spigelman M, Stanford J, Lemma E, 33. Rubini M, Dell’Anno V, Giuliani R, Favia Donoghue H, Zias J. 1994. Mycobacterium P, Zaio P. 2012. The first probable case of leprae from ancient bones detected by PCR. leprosy in Southeast Italy (13th -14th centu- Lancet 343:1360–1361. ries AD, Montecorvino, Puglia). J Anthropol. 47. Rafi A, Spigelman M, Stanford J, Lemma E, doi:10.1155/2012/262790. Donoghue H, Zias J. 1994. DNA of Mycobac- 34. Rubini M, Cerroni V, Zaio P. 2015. The Origin terium leprae detected by PCR in ancient bone. and Spread of Mycobacterium leprae in Int J Osteoarchaeol 4:287–290. Human Evolution. Konrad Lorenz Institute, 48. Hartskeerl RA, De Wit M, Yand Klaster PR. Vienna, Austria. 1989. Polymerase chain reaction for the detec- 35. Strouhal E, Ladislava H, Likovsky J, Vargova tion of Mycobacterium leprae. J Gen Microbiol L, Danes J. 2002. Traces of leprosy from the 135:2355–2364. Czech Kingdom, p 223–232. In Roberts CA, 49. Donoghue HD, Gladykowska-Rzeczycka J, Lewis ME, Manchester K (ed), The Past and Marcsik A, Holton J, Spigelman M. 2002. Present of Leprosy. Archaeological, Historical, Mycobacterium leprae in archaeological sam- Palaeopathological and Clinical Approaches. ples, p 271–285. In Roberts CA, Lewis ME, British Archaeological Reports International Manchester K (ed), The Past and Present of Series (Book 1054). British Archaeological Leprosy: Archaeological, Historical, Palaeopath- Reports, Oxford, UK. ological and Clinical Approaches. British Archae- 36. Palfi G. 1991. The first osteoarchaeological ological Reports Series. Archaeopress, Oxford, evidence of leprosy in Hungary. Int J Osteo- UK. archaeol 1:99–102. 50. Spigelman M, Donoghue HD. 2002. The study 37. Blau S, Yagodin V. 2005. Osteoarchaeological of ancient DNA answers a palaeopathological evidence for leprosy from Western Central question, p 287–296. In Roberts CA, Lewis ME, Asia. Am J Phys Anthropol 126:150–158. Manchester K (ed), The Past and Present of 38. Andersen JG, Manchester K. 1992. The Leprosy: Archaeological, Historical, Palaeopatho- rhinomaxillary syndrome in leprosy: a clinical, logical and Clinical Approaches. British Archae- radiological and paleopathological study. Int J ological Reports Series. Archaeopress, Oxford, Osteoarchaeol 2:121–129. UK. 39. Bullock WE. 1990. Mycobacterium leprae (lep- 51. Donoghue HD, Holton J, Spigelman M. 2001. rosy), p 1906–1914. In Mandell GL, Douglas RG, PCR primers that can detect low levels of Bennett JE (ed), Principles and Practice of Mycobacterium leprae DNA. J Med Microbiol Infectious Diseases. Churchill Livingston, New 50:177–182. York, NY. 52. Hershkovitz I, Spiers M, Katznelson A, 40. Job CK, Sushil M, Chandi SM. 2001. Differ- Arensburg B. 1992. Unusual pathological ential Diagnosis of Leprosy: a Guide Book for condition in the lower extremities of a skele- Histopathologists. Sasakawa Memorial Health ton from ancient Israel. Am J Phys Anthropol Foundation, Tokyo, Japan. 88:23–26. 41. Mullis K, Faloona F. 1987. Specific synthesis 53. Hershkovitz I, Spiers M, Arensburg B. 1993. of DNA in vitro via a polymerase catalysed Leprosy or Madura foot? The ambiguous chain reaction. Methods Enzymol 155:335–350. nature of infectious disease in paleopathology: Copyright @ 2016. ASM Press. All rights reserved. May not be reproduced in any form without permission from the publisher, except fair uses permitted under U.S. or applicable copyright law.

reply to Dr Manchester. Am J Phys Anthropol 63. Donoghue HD. 2013. Insights into ancient 91:251–253. leprosy and tuberculosis using metagenomics. 54. Manchester K. 1993. Unusual pathological Trends Microbiol 21:448–450. condition in the lower extremities of a skele- 64. Matheson CD, Vernon KK, Lahti A, Fratpietro ton from ancient Israel. Am J Phys Anthropol R, Spigelman M, Gibson S, Greenblatt CL, 91:249–250. Donoghue HD. 2010. Correction: Molecular 55. Spigelman M, Donoghue HD. 2001. Brief exploration of the First-Century Tomb of the communication: unusual pathological condi- Shroud in Akeldama, Jerusalem. PLoS One 5(4). tion in the lower extremities of a skeleton doi:10.1371/annotation/32ada7b9-3772-4c08- from ancient Israel. Am J Phys Anthropol 114: 9135-b5c0933f0b5e. Zissu, Boaz [added]. 92–93. 65. Molto JE. 2002. p 179–192. In Roberts CA, 56. Cooper A, Poinar HN. 2000. Ancient DNA: do Lewes ME, Manchester K (ed), The Past and it right or not at all. Science 289:1139. Present of Leprosy. Archaeological, Historical, 57. Lee OY-C, Bull ID, Molnár E, Marcsik A, Palaeopathological and Clinical Approaches. Pálfi G, Donoghue HD, Besra GS, Minnikin British Archaeological Research International DE. 2012. Integrated strategies for the use of Series (Book 1054). Archaeopress, Oxford, UK. lipid biomarkers in the diagnosis of ancient 66. Rubini M, Erdal YS, Spigelman M, Zaio P, mycobacterial disease, p 63–69. In Mitchell Donoghue HD. 2014. Paleopathological and PD, Buckberry J (ed), Proceedings of the molecular study on two cases of ancient Twelfth Annual Conference of the British childhood leprosy from the Roman and By- Association for Biological Anthropology and zantine empires. Int J Osteoarchaeol 24:570– Osteoarchaeology, Department of Archaeology 582. and Anthropology, University of Cambridge 67. Rawcliffe C. 2006. Leprosy in Medieval 2010. British Arachaeological Reports Inter- England. Boydell Press, Woodbridge, UK. national Series (Book 2380). Archaeopress, 68. Lowe J, McNulty F. 1953. Tuberculosis and Oxford, UK. leprosy: immunological studies in healthy 58. Minnikin DE, Besra GS, Lee O-YC, Spigelman persons. Br Med J ii:579–584. M, Donoghue HD. 2011. The interplay of NA 69. Chaussinand R. 1948. Tubercolose et lèpre, and lipid biomarkers in the detection of tuber- maladies antagoniques. Eviction de la lèpre culosis and leprosy in mummies and other parl la tubercolose. Int J Lepr 16:431–438. skeletal remains, p 109–114. In Gill-Frerking H, 70. Merle CSC, Cunha SS, Rodrigues LC. 2010. Rosendahl W, Zink A, Piombini-Mascali D. BCG vaccination and leprosy protection: re- Yearbook of Mummy Studies, vol. 1. Verlag Dr. view of current evidence and status of BCG in Friedrich Pfeil, Münich, Germany. leprosy control. Expert Rev Vaccines 9:209– 59. Watson CL, Popescu E, Boldsen J, Slaus M, 222. Lockwood DNJ. 2010. Correction: single nu- 71. Shinnick TM, Sweetser D, Thole J. 1987. The cleotide polymorphism analysis of European etiologic agents of leprosy and tuberculosis archaeological M. leprae DNA. PLoS One 5(1). share an immunoreactive protein antigen with doi:10.1371/annotation/1b400b6e-8883-436c- the vaccine strain Mycobacterium bovis BCG. b3c4-00e1ec2db101. Infect Immun 55:1932–1935. 60. Economou C, Kjellström A, Lidén K, 72. Prasad R, Verma SK, Singh R. 2010. Panagopoulos I. 2013. Ancient DNA reveals an Concomittant pulmonary tuberculosis and Asian type of Mycobacterium leprae in medieval borderline leprosy with type-II lepra reaction Scandinavia. J Archaeol Sci 40:465–470. in single patient. Lung India 27:19–23. 61. Shuenemann VJ, Singh P, Mendum TA, 73. Ravindra K, Sugareddy M, Ramachander T. Krause-Kyora B, Jäger G, Bos KI, Herbig A, 2010. Coexistence of borderline tuberculoid Economou C, Benjak A, Busso P. 2013. Hansen’s disease with tuberculosis verrucosa Genome-wide comparison of medieval and cutis in a child – a rare case. Lepr India 82:91– modern Mycobacterium leprae. Science 341: 93. 179–183. 74. Donoghue HD, Marcsik A, Matheson C, 62. Taylor GM, Tucker K, Butler R, Pike AWG, Vernon K, Nuorala E, Molto JE, Greenblatt Lewis J, Roffey PM, Lee OY-C, Wu HHT, CL, Spigelman M. 2005. Co-infection of Minnikin DE, Besra GS, Singh P, Cole ST, Mycobacterium tuberculosis and Mycobacteri- Stewart GR. 2013. Detection and strain typing um leprae in human archaeological samples: a of ancient Mycobacterium leprae from a medi- possible explanation for the historical decline eval leprosy hospital. PLoS One 8:e62406. of leprosy. Proc R Socy of Lond B Biol Sci doi:10.1371/journal.pone.0062406. 272:389–394. Copyright @ 2016. ASM Press. All rights reserved. May not be reproduced in any form without permission from the publisher, except fair uses permitted under U.S. or applicable copyright law.

75. Kumar B, Kaur S, Kataria S, Roy SN. tuberculosis co-infection. Math Biosci 241: 1982. Concomitant occurrence of leprosy 225–237. and tuberculosis – a clinical, bacteriological 79. Manchester K, Roberts C. 1989. The palaeo- and radiological evaluation. Lepr India 54:71– pathology of leprosy in Britain: a review. 76. World Archaeol 21:265–272. 76. Gartner EMS, Glatthaar E, Imkamp FMJH, 80. Donoghue G, Taylor M, Marcsik A, Molnár E, Kok SH. 1980. Association of tuberculosis and Pálfi G, Pap I, Teschler-Nicola M, Pinhasi R, leprosy in South Africa. Lepr Rev 51:5–10. Erdal YS, Velemínsky P, Likovsky J, Belcastro 77. Lietman T, Porco T, Blower S. 1997. Leprosy MG, Mariotti V, Riga A, Rubini M, Zaio P, and tuberculosis: the epidemiological con- Besra GS, Lee OYC, Wu HHT, Minnikin DE, sequences of cross-immunity. Am J Public Bull ID, O’Grady J, Spigelman M. 2015. A Health 87:1923–1927. migration-driven model for the historical spread 78. Hohmann H, Voss-Böhme A. 2013. The of leprosy in medieval eastern and Central epidemiological consequences of leprosy- Europe. Infect Genet Evol 31:250–256. Copyright @ 2016. ASM Press. All rights reserved. May not be reproduced in any form without permission from the publisher, except fair uses permitted under U.S. or applicable copyright law.