First direct dating of the Late Neanderthal remains from Subalyuk Cave in Northern Hungary.

The Subalyuk hominin remains were uncovered in 1932 in a cave of the same name in the Bükk Mountains, near the village of Cserépfalu in Borsod-Abaúj-Zemplén County, Northern Hungary. The remains represent two individuals, an adult and a young child who have been described in a few publications since their discovery, providing substantial anthropological data and general assessments of their Neanderthal affiliation. They were associated with Late Mousterian industry. Thus, the Bükk Mountains gain importance in the discussion concerning the contribution of East Central European sites to the debate on the peopling history of Europe during the Late Middle to Early Upper Palaeolithic transition. In this paper, we summarize the archaeological and chronological context of the two individuals, and publish the first direct dating results that place them among the Last Neanderthals of Central Europe.

More than one millennium (2nd-16th century CE) of the White Plague in the Carpathian Basin - New cases, expanding knowledge.

The causative agent of tuberculosis is still a widespread pathogen, which caused the death of ca. 1.6 million people globally in 2021. The paleopathological study of human remains revealed the antiquity of the disease and its continuous presence throughout the history of humankind. The Carpathian Basin has always been a biocultural melting pot, since it has seen several migrations over the centuries, and served as a location of admixture and interaction for numerous populations of different cultures. Thus, this geographical territory is ideal for the examination of the coevolutionary processes of hosts and their pathogens. We aimed to reveal the spatial and temporal distribution of tuberculosis cases excavated inside the borders of Hungary between the 2nd and 16th centuries CE. We established a comprehensive database by collecting 114 already published cases and introducing 39 new cases. The involved cases include those that have been confirmed by different molecular methods, as well as possible infections that were identified based on the presence of macromorphological and radiological alterations. The progress of future molecular and paleopathological studies can be facilitated by our dataset, as it presents spatial and temporal information concerning the spread of the disease in the Carpathian Basin, as well as the biological profile and detailed paleopathological description of lesions illustrated by photo- and radiographs.

Re-examination of the Subalyuk Neanderthal remains uncovers signs of probable TB infection (Subalyuk Cave, Hungary).

In 1932, skeletal remains of two Neanderthal individuals, a young adult female and a 3-4-year-old child, were discovered in Subalyuk Cave in Northern Hungary [1,2]. Results of the anthropological examination were published some years after this important discovery. Methodological progress encouraged re-examination of the material during the last few years. Radiocarbon dating revealed a chronological age of 39,732-39,076 cal. BP for the adult female and 36,117-35,387 cal. BP for the child [3]. Morphological paleopathological studies of these Neanderthal remains uncovered distinct evidence of skeletal infections. Alterations of the adult individual's sacrum suggest probable early-stage sacroiliitis, while several vertebral bodies indicate superficial osseous remodelling of infectious origin. Traces of pathological lesions were observed on the endocranial surface of the child's skull, reflecting a reaction of meningeal tissues, a consequence of a probable TB-related meningeal infectious process. Results of recent paleomicrobiological examinations - lipid biomarker and aDNA studies - support the morphological diagnosis of probable TB infections [4].

Sensitive lipid biomarker detection for tuberculosis in late Neanderthal skeletons from Subalyuk Cave, Hungary.

Skeletal remains of two Neanderthal individuals, a 25-35 year-old woman and a 3-4 year-old child, were discovered in a Subalyuk Cave in North-Eastern Hungary. Radiocarbon dating of the female and child remains revealed an age of 39,732-39,076 and 36,117-35,387 cal BP, respectively. Paleopathological studies of these Neanderthal remains revealed probable evidence of skeletal mycobacterial infection, including in the sacrum of the adult specimen and the endocranial surface of the child's skull. Application of PCR amplification to the juvenile cranium and a vertebra gave a positive result (IS6110) for tuberculosis, backed up by spoligotyping. Lipid biomarker analyses of the same two specimens revealed definitive signals for C32 mycoserosates, a very characteristic component of the Mycobacterium tuberculosis complex (MTBC). A vertebra from the adult provided weak evidence for mycocerosate biomarkers. The correlation of probable skeletal lesions with characteristic amplified DNA fragments and a proven lipid biomarker points to the presence of tuberculosis in these Neanderthals. In particular, the closely similar biomarker profiles, for two distinct juvenile cranial and vertebral bones, strengthen this diagnosis.

Tuberculosis in mummies - New findings, perspectives and limitations.

The molecular analysis of ancient pathogen DNA represents a unique opportunity for the study of infectious diseases in ancient human remains. Among other diseases, paleogenetic studies have been successful in detecting tuberculous DNA in ancient human remains. In the beginning of ancient DNA (aDNA) studies, the presence of tuberculosis (TB) DNA was assessed using a PCR-based assay targeting specific regions of the Mycobacterium tuberculosis (MTB) complex, such as the repetitive element IS6110. The advent of high-throughput sequencing has enabled the reconstruction of full ancient TB genomes in the field of paleomicrobiology. However, despite the numerous paleopathological and PCR-based studies on the presence of tuberculosis in historic human remains, full genome wide reconstructions are still limited to well-preserved specimens with low environmental contamination and connected with extensive screening efforts. This has led to some controversies regarding the evolutionary history of its causative agent Mycobacterium tuberculosis. In this context, mummies have been shown to be a good source for the detection of MTB complex DNA due to a low exposure to environmental influences and the overall good state of preservation of hard and soft tissues in the human remains. Here, we present the major findings on the presence of TB infections in the 18th century naturally mummified human remains from Vác, Hungary and the current status of the detection of MTB complex DNA in mummified human remains. The future perspectives of detecting tuberculosis in mummies will be discussed in the light of methodological aspects, as well as ethical and curational challenges.

Upper Paleolithic tuberculosis: A probable case illustrated by paleoimaging methods (Azilian site of Les Iboussières, France).

The aim of this paper is to present the results of µCT-scan and 3D imaging analyses of two skeletal lesions observed on human remains of one of the last European hunter-gatherers from the late Paleolithic (Azilian period): a sacroiliac osteoarthritis and a femoral lesion suggestive of a soft tissue abscess imprint. These two skeletal elements (fused left sacrum and coxal bone, and right femur) displayed osteometric criteria indicating that they belonged to the same individual. These two associated lesions are consistent with a low-grade osteoarticular infection, and suggest a diagnosis of pelvic tuberculosis with a cold abscess of the thigh. If molecular confirmation is obtained, this case would be the first evidence of tuberculosis among Upper Palaeolithic populations.

Hit or miss - A metagenomic evaluation of intra-bone variability of host pathogen load in tuberculosis-infected human remains.

Many sampling protocols have been established to successfully retrieve human DNA from archaeological remains, however the systematic detection of ancient pathogens remains challenging. Here, we present a first assessment of the intra-bone variability of metagenomic composition in human skeletal remains and its effect on the sampling success for Mycobacterium tuberculosis (MTB) and human endogenous DNA. For this purpose, four bone samples from published peer-reviewed studies with PCR-based evidence for ancient MTB DNA were selected. Two bone samples of a Neolithic individual from Halberstadt, Germany and two ribs of two 18th-century Hungarian church mummies were sampled at multiple locations for equal amounts, followed by DNA extraction and library construction. Shotgun sequencing data was generated for taxonomic profiling as well as quantitative and qualitative evaluation of MTB and human endogenous DNA. Despite low variance in microbial diversity within and across samples, intra-bone variability of up to 36.45- and 62.88-fold for authentic ancient MTB and human reads, respectively, was detected. This study demonstrates the variable sampling success for MTB and human endogenous DNA within single skeletal samples despite relatively consistent microbial composition and highlights how a multisampling approach can facilitate the detection of hotspots with highly concentrated pathogen and human endogenous DNA.

Leprosy: The age-old companion of humans - Re-evaluation and comparative analysis of Avar-period cases with Hansen's disease from the Danube-Tisza Interfluve, Hungary.

In recent years, our knowledge of leprosy in the past has substantially been enriched. Nonetheless, much still remains to be discovered, especially in regions and periods from where no written sources are available. To fill in some research gaps, we provide the comparative analysis of eight Avar-period leprosy cases from the Danube-Tisza Interfluve (Hungary). In every case, to reconstruct the biological consequences of leprosy, the detected bony changes were linked with palaeopathological and modern medical information. To reconstruct the social consequences of being affected by leprosy, conceptualisation of the examined individuals' treatment in death was conducted. In every case, the disease resulted in deformation and disfigurement of the involved anatomical areas (rhinomaxillary region, feet, and/or hands) with difficulties in conducting certain physical activities. These would have been disadvantageous for the examined individuals and limited or changed their possibilities to participate in social situations. The most severe cases would have required continuous support from others to survive. Our findings indicate that, despite their very visible disease and associated debility, the examined communities did not segregate leprosy sufferers but provided and cared for them, and maintained a strong enough social network that made their survival possible even after becoming incapable of self-sufficiency.

On some mummy findings from the Semmelweis Museum of Medical History, Hungarian National Museum, Budapest.

The permanent exhibition of the Hungarian National Museum, Semmelweis Museum of Medical History of Budapest, Hungary, displays some human remains believed to originate from ancient Egypt. Within the framework of the Nephthys Project, three objects consisting of two heads and one foot were studied via paleoimaging and archaeometric techniques in order to provide new anthropological and paleopathological data. It is argued here that even partial or fragmentary items are worthy of investigation as they can reveal new information about their owners, as well as their supposed authenticity.

Lumbosacral tuberculosis, a rare manifestation of Pott's disease - How identified human skeletons from the pre-antibiotic era can be used as reference cases to establish a palaeopathological diagnosis of tuberculosis.

The macromorphological examination of identified human osteological collections from the pre-antibiotic era (e.g., Terry Collection) can provide invaluable information about the skeletal manifestations of tuberculosis (TB) in individuals who did not receive pharmaceutical therapy. With analysis of such collections, new diagnostic criteria for TB can be recognised which can be used in palaeopathological interpretation. The aim of our paper is to provide a reference and aid for the identification of TB in past populations by demonstrating and discussing in detail the vertebral alterations indicative of one of its rare skeletal manifestations, lumbosacral TB. These changes were detected in two individuals from the Terry Collection (Terry No. 760 and Terry No. 1093). These two case studies furnish palaeopathologists with a stronger basis for diagnosing lumbosacral TB in skeletons which exhibit similar vertebral lesions from osteoarchaeological series. To illustrate this, an archaeological case from Hungary (KK146) is also presented, displaying vertebral alterations resembling that of the two cases from the Terry Collection. Through the demonstrated case studies, we can derive a better insight into the disease experience of people who lived in the past and suffered from TB.

Development of bilateral asymmetry of the upper limb in children from a medieval population in Central Europe, Hungary.

This study examined the emergence and characteristics of bilateral asymmetry of the upper limb during development in a medieval agricultural population from Hungary, and investigated the agricultural activity-types in a bioarchaeological and biomechanical context. The skeletal remains of 169 nonadult individuals were selected from the cemetery of Bátmonostor-Pusztafalu, providing 134 cases for humeral diameter and length, 70 cases for radial length and 62 cases for ulnar length measurements. Biological age was estimated by using tooth eruption. Age groups were defined on the basis of the development of motor skills in children. Statistical analyses included correlation, Chi-square test, Fisher analysis and ANOVA. Our investigation revealed that the frequency of asymmetry increased significantly during growth with a shift to the right side in all measurements and reached adult-like distribution in early childhood. The bilateral asymmetry increased with age in humeral length, while other measurements revealed no change. The magnitude of asymmetry decreased with age in humeral diameter, but remained constant in other measurements. Our observations strengthen the hypothesis that right-sided asymmetry develops gradually during growth. Our findings also illustrate the effects of medieval agricultural labour on upper limb asymmetry: mechanical loading has various effects during development both on the magnitude and on direction of asymmetry.

A rare case of calvarial tuberculosis from the Avar Age (8th century CE) cemetery of Kaba-Bitózug (Hajdú-Bihar county, Hungary) - Pathogenesis and differential diagnostic aspects.

The aim of our paper is to present and discuss in detail the bony changes indicative of tuberculosis (TB) that were identified in a skeleton (KB67), unearthed from grave 67 of the 8th-century-CE cemetery of Kaba-Bitózug (Hungary). Furthermore, to provide the differential diagnoses of the observed alterations, with special attention to the cranial osteolytic lesions. During the macro- and micromorphological examinations of KB67, the skull revealed three small, well-circumscribed, punched-out osteolytic lesions accompanied by endocranial granular impressions, abnormal blood vessel impressions, periosteal appositions, and cortical erosion. The postcranial skeleton exhibited osteolytic lesions, cortical remodelling and erosion, and signs of hypervascularisation in the spine. Based on the differential diagnosis of the cranial osteolytic lesions and their co-occurrence with endocranial and vertebral bony changes indicative of TB, they most likely resulted from tuberculous involvement of the frontal and left parietal bones. The morphologically established diagnosis was confirmed by a PCR analysis that provided evidence for the presence of Mycobacterium tuberculosis DNA in KB67. KB67, the first reported archaeological case with calvarial TB from the present-day territory of Hungary, gives us a unique insight into the occurrence of a rare manifestation of TB in the Avar Age of the Great Plain.

The two extremes of Hansen's disease-Different manifestations of leprosy and their biological consequences in an Avar Age (late 7th century CE) osteoarchaeological series of the Duna-Tisza Interfluve (Kiskundorozsma-Daruhalom-dulo II, Hungary).

To give an insight into the different manifestations of leprosy and their biological consequences in the Avar Age of the Hungarian Duna-Tisza Interfluve, two cases from the 7th-century-CE osteoarchaeological series of Kiskundorozsma-Daruhalom-dulo II (Hungary; n = 94) were investigated. Based on the macromorphology of the bony changes indicative of Hansen's disease, KD271 (a middle-aged male) and KD520 (a middle-aged female) represent the two extremes of leprosy. KD271 appears to have an advanced-stage, long-standing near-lepromatous or lepromatous form of the disease, affecting not only the rhinomaxillary region but also both upper and lower limbs. This has led to severe deformation and disfigurement of the involved anatomical areas of the skeleton, resulting in his inability to perform the basic activities of daily living, such as eating, drinking, grasping, standing or walking. The skeleton of KD520 shows no rhinomaxillary lesions and indicates the other extreme of leprosy, a near-tuberculoid or tuberculoid form of the disease. As in KD271, Hansen's disease has resulted in disfigurement and disability of both of the lower limbs of KD520; and thus, the middle-aged female would have experienced difficulties in standing, walking, and conducting occupational physical activities. KD271 and KD520 are amongst the very few published cases with leprosy from the Avar Age of the Hungarian Duna-Tisza Interfluve, and the only examples with detailed macromorphological description and differential diagnoses of the observed leprous bony changes. The cases of these two severely disabled individuals, especially of KD271 -who would have required regular and substantial care from others to survive-imply that in the Avar Age community of Kiskundorozsma-Daruhalom-dulo II there was a willingness to care for people in need.

The genetic origin of Huns, Avars, and conquering Hungarians.

Huns, Avars, and conquering Hungarians were migration-period nomadic tribal confederations that arrived in three successive waves in the Carpathian Basin between the 5th and 9th centuries. Based on the historical data, each of these groups are thought to have arrived from Asia, although their exact origin and relation to other ancient and modern populations have been debated. Recently, hundreds of ancient genomes were analyzed from Central Asia, Mongolia, and China, from which we aimed to identify putative source populations for the above-mentioned groups. In this study, we have sequenced 9 Hun, 143 Avar, and 113 Hungarian conquest period samples and identified three core populations, representing immigrants from each period with no recent European ancestry. Our results reveal that this "immigrant core" of both Huns and Avars likely originated in present day Mongolia, and their origin can be traced back to Xiongnus (Asian Huns), as suggested by several historians. On the other hand, the "immigrant core" of the conquering Hungarians derived from an earlier admixture of Mansis, early Sarmatians, and descendants of late Xiongnus. We have also shown that a common "proto-Ugric" gene pool appeared in the Bronze Age from the admixture of Mezhovskaya and Nganasan people, supporting genetic and linguistic data. In addition, we detected shared Hun-related ancestry in numerous Avar and Hungarian conquest period genetic outliers, indicating a genetic link between these successive nomadic groups. Aside from the immigrant core groups, we identified that the majority of the individuals from each period were local residents harboring "native European" ancestry.

Correction: Life and death of a leprosy sufferer from the 8th-century-CE cemetery of Kiskundorozsma-Kettoshatár I (Duna-Tisza Interfluve, Hungary)-Biological and social consequences of having Hansen's disease in a late Avar Age population from Hungary.

[This corrects the article DOI: 10.1371/journal.pone.0264286.].

Metagenomic analysis reveals mixed Mycobacterium tuberculosis infection in a 18th century Hungarian midwife.

The Vác Mummy Collection comprises 265 well documented mummified individuals from the late 16th to the early 18th century that were discovered in 1994 inside a crypt in Vác, Hungary. This collection offers a unique opportunity to study the relationship between humans and pathogens in the pre-antibiotic era, as previous studies have shown a high proportion of tuberculosis (TB) infections among the individuals. In this study, we recovered ancient DNA with shotgun sequencing from a rib bone sample of a 18th century midwife. This individual is part of the collection and shows clear skeletal changes that are associated with tuberculosis and syphilis. To provide molecular proof, we applied a metagenomic approach to screen for ancient pathogen DNA. While we were unsuccessful to recover any ancient Treponema pallidum DNA, we retrieved high coverage ancient TB DNA and identified a mixed infection with two distinct TB strains by detailed single-nucleotide polymorphism and phylogenetic analysis. Thereby, we have obtained comprehensive results demonstrating the long-time prevalence of mixed infections with the sublineages L4.1.2.1/Haarlem and L4.10/PGG3 within the local community in preindustrial Hungary and put them in context of sociohistorical factors.

Life and death of a leprosy sufferer from the 8th-century-CE cemetery of Kiskundorozsma-Kettoshatár I (Duna-Tisza Interfluve, Hungary)-Biological and social consequences of having Hansen's disease in a late Avar Age population from Hungary.

The aim of our paper is to demonstrate a middle-aged male (KK61) from the 8th-century-CE cemetery of Kiskundorozsma-Kettoshatár I (Duna-Tisza Interfluve, Hungary), who appears to represent the lepromatous form of Hansen's disease. Leprosy has affected not only the rhinomaxillary region of his face but also his lower limbs, with severe deformation and disfigurement of the involved anatomical areas (saddle-nose and flat-foot deformity, respectively). Consequently, he would have experienced disability in performing the basic activities of daily living, such as eating, drinking, standing or walking; and thus, he would have required regular and substantial care from others to survive. Despite his very visible disease and associated debility, it seems that KK61 was accepted as a member of the community in death, since he has been buried within the cemetery boundaries, among others from his community. In addition, his grave has conformed to the mortuary practices characteristic of the Kiskundorozsma-Kettoshatár I cemetery (e.g., burial orientation, position of the body in the grave, and type and quantity of accompanying grave goods). Although distinction or segregation in life do not preclude normative treatment in death, the long-lasting survival of KK61 with Hansen's disease implies that he would not have been abandoned but cared for by others. KK61 is one of the few published historic cases with leprosy from the Avar Age of the Hungarian Duna-Tisza Interfluve. His case gives us a unique insight into the biological consequences of living with Hansen's disease and illustrates the social attitude toward leprosy sufferers in early mediaeval Hungary.

DNA methylation profiling in mummified human remains from the eighteenth-century.

Reconstruction of ancient epigenomes by DNA methylation (DNAm) can shed light into the composition of cell types, disease states, and age at death. However, such analysis is hampered by impaired DNA quality and little is known how decomposition affects DNAm. In this study, we determined if EPIC Illumina BeadChip technology is applicable for specimens from mummies of the eighteenth century CE. Overall, the signal intensity on the microarray was extremely low, but for one of two samples we were able to detect characteristic DNAm signals in a subset of CG dinucleotides (CpGs), which were selected with a stringent processing pipeline. Using only these CpGs we could train epigenetic signatures with reference DNAm profiles of multiple tissues and our predictions matched the fact that the specimen was lung tissue from a 28-year-old woman. Thus, we provide proof of principle that Illumina BeadChips are applicable for DNAm profiling in ancient samples.