Leprosy in a Lombard-Avar cemetery in central Italy (Campochiaro, Molise, 6th-8th century AD): ancient DNA evidence and demography.

BACKGROUND: The study of past infectious diseases increases knowledge of the presence, impact and spread of pathogens within ancient populations. AIM: Polymerase chain reaction (PCR) was used to examine bones for the presence of Mycobacterium leprae ancient DNA (aDNA) as, even when leprosy is present, bony changes are not always pathognomonic of the disease. This study also examined the demographic profile of this population and compared it with two other populations to investigate any changes in mortality trends between different infectious diseases and between the pre-antibiotic and antibiotic eras. SUBJECTS AND METHODS: The individuals were from a site in Central Italy (6th-8th CE) and were examined for the presence of Mycobacterium leprae aDNA. In addition, an abridged life mortality table was constructed. RESULTS: Two individuals had typical leprosy palaeopathology, and one was positive for Mycobacterium leprae aDNA. However, the demographic profile shows a mortality curve similar to that of the standard, in contrast to a population that had been subjected to bubonic plague. CONCLUSIONS: This study shows that, in the historical population with leprosy, the risk factors for health seem to be constant and distributed across all age classes, similar to what is found today in the antibiotic era. There were no peaks of mortality equivalent to those found in fatal diseases such as the plague, probably due to the long clinical course of leprosy.

Paleomicrobiology of Leprosy.

The use of paleomicrobiological techniques in leprosy has the potential to assist paleopathologists in many important aspects of their studies on the bones of victims, solving at times diagnostic problems. With Mycobacterium leprae, because of the unique nature of the organism, these techniques can help solve problems of differential diagnosis. In cases of co-infection with Mycobacterium tuberculosis, they can also suggest a cause of death and possibly even trace the migratory patterns of people in antiquity, as well as explain changes in the rates and level of infection within populations in antiquity.

Eighteenth-century genomes show that mixed infections were common at time of peak tuberculosis in Europe.

Tuberculosis (TB) was once a major killer in Europe, but it is unclear how the strains and patterns of infection at 'peak TB' relate to what we see today. Here we describe 14 genome sequences of M. tuberculosis, representing 12 distinct genotypes, obtained from human remains from eighteenth-century Hungary using metagenomics. All our historic genotypes belong to M. tuberculosis Lineage 4. Bayesian phylogenetic dating, based on samples with well-documented dates, places the most recent common ancestor of this lineage in the late Roman period. We find that most bodies yielded more than one M. tuberculosis genotype and we document an intimate epidemiological link between infections in two long-dead individuals. Our results suggest that metagenomic approaches usefully inform detection and characterization of historical and contemporary infections.

Two positive tuberculosis cases in the late Nigrovits family, 18th century, Vác, Hungary.

Two mummies of the Hungarian mummy collection from Vác were the subjects of anthropological, paleopathological, radiological, paleomicrobiological, paleohistological and paleoproteomic studies. Both individuals belonged to the same family. The father, József Nigrovits (No 29), died at the age of 55 on the 11th of November 1793; his son, Antal Nigrovits (No 54), died on the 16th of July 1803, at the age of 22. They lived in the 18th century in Vác, a small town in northern Hungary. The macroscopic examination of the son showed a severely deformed neck and back region; the father has no visible mark of any illnesses. As earlier researches showed that tuberculosis was widespread in the community, the etiology of these deformities was examined. The paleomicrobiological results found that both individuals were infected with tuberculosis. Although they suffered from TB, the CT scan data of the bodies and their 3D reconstructions showed no skeletal evidence of tuberculosis. The deformity of the son turned to be a developmental abnormality of unknown origin, but no Pott's gibbus was present.

Lipid biomarkers provide evolutionary signposts for the oldest known cases of tuberculosis.

Studies on the evolution of tuberculosis, and the influence of this disease on human and animal development and interaction, require the accumulation of indisputable biomarker evidence. Ideally, the determination of full genomes would provide all the necessary information, but for very old specimens DNA preservation may be compromised and only limited DNA amplification may be a possibility. Mycobacterium tuberculosis is characterised by the presence of unusual cell envelope lipids, with specific biomarker potential. Lipid biomarker recognition has been decisive in pinpointing the oldest known cases of human and animal tuberculosis; the former are a woman and child from a pre-pottery settlement at Atlit-Yam, Israel (∼9,000 ka) and the latter is an extinct Bison antiquus from Natural Trap Cave, Wyoming (∼17,000 ka). Including some new data, it is demonstrated how analysis of a combination of mycolic, mycocerosic and mycolipenic acid and phthiocerol biomarkers provide incontrovertible evidence for tuberculosis in these landmark specimens.

Tuberculosis origin: The Neolithic scenario.

This paper follows the dramatic changes in scientific research during the last 20 years regarding the relationship between the Mycobacterium tuberculosis complex and its hosts - bovids and/or humans. Once the M. tuberculosis and Mycobacterium bovis genomes were sequenced, it became obvious that the old story of M. bovis evolving into the human pathogen should be reversed, as M. tuberculosis is more ancestral than M. bovis. Nevertheless, the timescale and geographical origin remained an enigma. In the current study human and cattle bone samples were examined for evidence of tuberculosis from the site of Atlit-Yam in the Eastern Mediterranean, dating from 9250 to 8160 (calibrated) years ago. Strict precautions were used to prevent contamination in the DNA analysis, and independent centers used to confirm authenticity of findings. DNA from five M. tuberculosis genetic loci was detected and had characteristics consistent with extant genetic lineages. High performance liquid chromatography was used as an independent method of verification and it directly detected mycolic acid lipid biomarkers, specific for the M. tuberculosis complex. These, together with pathological changes detected in some of the bones, confirm the presence of the disease in the Levantine populations during the Pre-pottery Neolithic C period, more than 8000 years ago.

Ancient mycobacterial lipids: Key reference biomarkers in charting the evolution of tuberculosis.

Mycobacterium tuberculosis has a cell envelope incorporating a peptidoglycan-linked arabinogalactan esterified by long-chain mycolic acids. A range of "free" lipids are associated with the "bound" mycolic acids, producing an effective envelope outer membrane. The distribution of these lipids is discontinuous among mycobacteria and such lipids have proven potential for biomarker use in tracing the evolution of tuberculosis. A plausible evolutionary scenario involves progression from an environmental organism, such as Mycobacterium kansasii, through intermediate "smooth" tubercle bacilli, labelled "Mycobacterium canettii"; cell envelope lipid composition possibly correlates with such a progression. M. kansasii and "M. canettii" have characteristic lipooligosaccharides, associated with motility and biofilms, and glycosyl phenolphthiocerol dimycocerosates ("phenolic glycolipids"). Both these lipid classes are absent in modern M. tuberculosis sensu stricto, though simplified phenolic glycolipids remain in certain current biotypes. Dimycocerosates of the phthiocerol family are restricted to smaller phthiodiolone diesters in M. kansasii. Diacyl and pentaacyl trehaloses are present in "M. canettii" and M. tuberculosis, accompanied in the latter by related sulfated acyl trehaloses. In comparison with environmental mycobacteria, subtle modifications in mycolic acid structures in "M. canettii" and M. tuberculosis are notable. The probability of essential tuberculosis evolution taking place in Pleistocene megafauna, rather than Homo sapiens, is reemphasised.

Evolutionary changes in the genome of Mycobacterium tuberculosis and the human genome from 9000 years BP until modern times.

The demonstration of Mycobacterium tuberculosis DNA in ancient skeletons gives researchers an insight into its evolution. Findings of the last two decades sketched the biological relationships between the various species of tubercle bacilli, the time scale involved, their possible origin and dispersal. This paper includes the available evidence and on-going research. In the submerged Eastern Mediterranean Neolithic village of Atlit Yam (9000 BP), a human lineage of M. tuberculosis, defined by the TbD1 deletion in its genome, was demonstrated. An infected infant at the site provides an example of active tuberculosis in a human with a naïve immune system. Over 4000 years later tuberculosis was found in Jericho. Urbanization increases population density encouraging M. tuberculosis/human co-evolution. As susceptible humans die of tuberculosis, survivors develop genetic resistance to disease. Thus in 18th century Hungarian mummies from Vác, 65% were positive for tuberculosis yet a 95-year-old woman had clearly survived a childhood Ghon lesion. Whole genome studies are in progress, to detect changes over the millennia both in bacterial virulence and also host susceptibility/resistance genes that determine the NRAMP protein and Killer Cell Immunoglobulin-like Receptors (KIRs). This paper surveys present evidence and includes initial findings.

Ancient DNA analysis - An established technique in charting the evolution of tuberculosis and leprosy.

Many tuberculosis and leprosy infections are latent or paucibacillary, suggesting a long time-scale for host and pathogen co-existence. Palaeopathology enables recognition of archaeological cases and PCR detects pathogen ancient DNA (aDNA). Mycobacterium tuberculosis and Mycobacterium leprae cell wall lipids are more stable than aDNA and restrict permeability, thereby possibly aiding long-term persistence of pathogen aDNA. Amplification of aDNA, using specific PCR primers designed for short fragments and linked to fluorescent probes, gives good results, especially when designed to target multi-copy loci. Such studies have confirmed tuberculosis and leprosy, including co-infections. Many tuberculosis cases have non-specific or no visible skeletal pathology, consistent with the natural history of this disease. M. tuberculosis and M. leprae are obligate parasites, closely associated with their human host following recent clonal distribution. Therefore genotyping based on single nucleotide polymorphisms (SNPs) can indicate their origins, spread and phylogeny. Knowledge of extant genetic lineages at particular times in past human populations can be obtained from well-preserved specimens where molecular typing is possible, using deletion analysis, microsatellite analysis and whole genome sequencing. Such studies have identified non-bovine tuberculosis from a Pleistocene bison from 17,500 years BP, human tuberculosis from 9000 years ago and leprosy from over 2000 years ago.

A migration-driven model for the historical spread of leprosy in medieval Eastern and Central Europe.

Leprosy was rare in Europe during the Roman period, yet its prevalence increased dramatically in medieval times. We examined human remains, with paleopathological lesions indicative of leprosy, dated to the 6th-11th century AD, from Central and Eastern Europe and Byzantine Anatolia. Analysis of ancient DNA and bacterial cell wall lipid biomarkers revealed Mycobacterium leprae in skeletal remains from 6th-8th century Northern Italy, 7th-11th century Hungary, 8th-9th century Austria, the Slavic Greater Moravian Empire of the 9th-10th century and 8th-10th century Byzantine samples from Northern Anatolia. These data were analyzed alongside findings published by others. M. leprae is an obligate human pathogen that has undergone an evolutionary bottleneck followed by clonal expansion. Therefore M. leprae genotypes and sub-genotypes give information about the human populations they have infected and their migration. Although data are limited, genotyping demonstrates that historical M. leprae from Byzantine Anatolia, Eastern and Central Europe resembles modern strains in Asia Minor rather than the recently characterized historical strains from North West Europe. The westward migration of peoples from Central Asia in the first millennium may have introduced different M. leprae strains into medieval Europe and certainly would have facilitated the spread of any existing leprosy. The subsequent decline of M. leprae in Europe may be due to increased host resistance. However, molecular evidence of historical leprosy and tuberculosis co-infections suggests that death from tuberculosis in leprosy patients was also a factor.

A possible case of cherubism in a 17th-century Korean mummy.

Cherubism is a benign fibro-osseous disease of childhood limited specifically to the maxilla and mandible. The progressive replacement of the jaw bones with expansile multilocular cystic lesions causes eventual prominence of the lower face, and hence the classic "cherubic" phenotype reflecting variable extents of jaw hypertrophy. Histologically, this condition has been characterized as replacement of the normal bone matrix with multicystic pockets of fibrous stroma and osteoclastic giant cells. Because of radiographic features common to both, primarily the presence of multiloculated lucencies with heterogeneous "ground-glass" sclerosis on CT imaging, cherubism was long mistaken for a craniofacial subtype of fibrous dysplasia. In 1999, however, the distinct genetic basis for cherubism was mapped to chromosome 4p16.3 and the SH-3 binding protein SH3BP2. But while there are already three suspected cases of fibrous dysplasia amongst archaeological populations, no definitive cases of cherubism have yet been reported in historical populations. In the current study we describe micro- and macro-structural changes in the face of a 17th century Joseon Dynasty Korean mummy which may coincide with the clinic-pathologic and radiologic features of cherubism.

Mycobacterium tuberculosis complex lipid virulence factors preserved in the 17,000-year-old skeleton of an extinct bison, Bison antiquus.

Tracing the evolution of ancient diseases depends on the availability and accessibility of suitable biomarkers in archaeological specimens. DNA is potentially information-rich but it depends on a favourable environment for preservation. In the case of the major mycobacterial pathogens, Mycobacterium tuberculosis and Mycobacterium leprae, robust lipid biomarkers are established as alternatives or complements to DNA analyses. A DNA report, a decade ago, suggested that a 17,000-year-old skeleton of extinct Bison antiquus, from Natural Trap Cave, Wyoming, was the oldest known case of tuberculosis. In the current study, key mycobacterial lipid virulence factor biomarkers were detected in the same two samples from this bison. Fluorescence high-performance liquid chromatography (HPLC) indicated the presence of mycolic acids of the mycobacterial type, but they were degraded and could not be precisely correlated with tuberculosis. However, pristine profiles of C(29), C(30) and C(32) mycocerosates and C(27) mycolipenates, typical of the Mycobacterium tuberculosis complex, were recorded by negative ion chemical ionization gas chromatography mass spectrometry of pentafluorobenzyl ester derivatives. These findings were supported by the detection of C(34) and C(36) phthiocerols, which are usually esterified to the mycocerosates. The existence of Pleistocene tuberculosis in the Americas is confirmed and there are many even older animal bones with well-characterised tuberculous lesions similar to those on the analysed sample. In the absence of any evidence of tuberculosis in human skeletons older than 9,000 years BP, the hypothesis that this disease evolved as a zoonosis, before transfer to humans, is given detailed consideration and discussion.

Genetic characterization of Mycobacterium tuberculosis in the West Bank, Palestinian Territories.

BACKGROUND: The World Health Organization (WHO) declared human tuberculosis (TB) a global health emergency and launched the "Global Plan to Stop Tuberculosis" which aims to save a million lives by 2015. Global control of TB is increasingly dependent on rapid and accurate genetic typing of species of the Mycobacterium tuberculosis (MTB) complex including M. tuberculosis. The aim of this study was to identify and genetically characterize the MTB isolates circulating in the West Bank, Palestinian Territories. Genotyping of the MTB isolates from patients with pulmonary TB was carried out using two molecular genetic techniques, spoligotyping and mycobacterial interspersed repetitive units-variable number of tandem repeat (MIRU-VNTR) supported by analysis of the MTB specific deletion 1 (TbD1). FINDINGS: A total of 17 MTB patterns were obtained from the 31 clinical isolates analyzed by spoligotyping; corresponding to 2 orphans and 15 shared-types (SITs). Fourteen SITs matched a preexisting shared-type in the SITVIT2 database, whereas a single shared-type SIT3348 was newly created. The most common spoligotyping profile was SIT53 (T1 variant), identified in 35.5 % of the TB cases studied. Genetic characterization of 22 clinical isolates via the 15 loci MIRU-VNTR typing distinguished 19 patterns. The 15-loci MIT144 and MIT145 were newly created within this study. Both methods determined the present of M. bovis strains among the isolates. CONCLUSIONS: Significant diversity among the MTB isolates circulating in the West Bank was identified with SIT53-T1 genotype being the most frequent strain. Our results are used as reference database of the strains circulating in our region and may facilitate the implementation of an efficient TB control program.

Tracing hepatitis B virus to the 16th century in a Korean mummy.

UNLABELLED: A rare find of a mummified child from the 16th century AD, in Korea, with relatively preserved organs, enabled a search for ancient hepatitis B virus (aHBV) DNA sequences from laparoscopic-derived liver biopsies. Analysis of the complete aHBV genome (3,215 base pairs) revealed a unique HBV genotype C2 (HBV/C2) sequence commonly spread in Southeast Asia, which probably represents an HBV that infected the Joseon Dynasty population in Korea. Comparison of the aHBV sequences with contemporary HBV/C2 DNA sequences revealed distinctive differences along four open reading frames. Genetic diversity between contemporary and recovered aHBV/C2 DNA may be the result of immunologic, environmental, and/or pharmacologic pressures. The calculated time of most recent common ancestor suggests that the Korean HBV sequence origin dates back at least 3,000 years and possibly as long as 100,000 years. This isolate most likely represents the earliest human HBV sequence that colonized Southeast Asia by human migration. CONCLUSION: This study describes the complete sequence of the oldest HBV isolate and the most ancient full viral genome known so far.

Pulmonary tuberculosis in the West Bank, Palestinian Authority: molecular diagnostic approach.

OBJECTIVE: To compare the effectiveness and feasibility of an insertion sequence (IS6110)-based polymerase chain reaction (PCR) assay with conventional methods of detecting Mycobacterium tuberculosis and to analyse mutations present in the hot spot region of the RNA polymerase B subunit (rpoB) gene associated with rifampin resistance by DNA sequencing. METHODS: Ninety-five sputum samples from 84 clinically suspected cases of tuberculosis were tested for mycobacterial infections by Ziehl Neelsen smear examination, Lowenstein-Jensen culture and IS6110-based PCR assay. RESULTS: Sensitivity and specificity of the PCR were 94%; the sensitivity of culture was 65%, and of smear tests, 59%. Both smear microscopy and culture had 100% specificity. DNA sequencing data of the 305-bp fragment of the rpoB gene for nine clinical isolates revealed one point mutation at position I572F and double mutations at position S531F in two isolates obtained from two patients who did not respond to the anti-tuberculosis therapy. CONCLUSION: IS6110-based PCR can be used routinely in clinical laboratories for rapid detection of Mycobacterium tuberculosis and thus allow early diagnosis and treatment of any contacts by the cheapest method currently available in the Palestinian Authority region. Rapid detection of rifampin resistance isolates will enable efficient treatment of patients and assist in eradication of the disease in the Palestinian territories.

Rapid differentiation of Mycobacterium tuberculosis and M. bovis by high-resolution melt curve analysis.

Identification and characterization of the Mycobacterium tuberculosis strains are important for clinical and therapeutic management of tuberculosis. Real-time PCR with a high-resolution melt assay was found to improve the diagnostic process. The assay includes differentiation between M. tuberculosis and Mycobacterium bovis based on one single-nucleotide polymorphism (SNP) in the narGHJI and oxyR genes and determination of M. bovis based on the region of differences 1 (RD1). This assay correctly identified the 7 tested Mycobacterium reference strains and 52 clinical samples with a sensitivity of 2 pg DNA. This assay will help in prescribing adequate treatment and monitoring disease dynamics.

Tuberculosis in Dr Granville's mummy: a molecular re-examination of the earliest known Egyptian mummy to be scientifically examined and given a medical diagnosis.

'Dr Granville's mummy' was described to the Royal Society of London in 1825 and was the first ancient Egyptian mummy to be subjected to a scientific autopsy. The remains are those of a woman, Irtyersenu, aged about 50, from the necropolis of Thebes and dated to about 600 BC. Augustus Bozzi Granville (1783-1872), an eminent physician and obstetrician, described many organs still in situ and attributed the cause of death to a tumour of the ovary. However, subsequent histological investigations indicate that the tumour is a benign cystadenoma. Histology of the lungs demonstrated a potentially fatal pulmonary exudate and earlier studies attempted to associate this with particular disease conditions. Palaeopathology and ancient DNA analyses show that tuberculosis was widespread in ancient Egypt, so a systematic search for tuberculosis was made, using specific DNA and lipid biomarker analyses. Clear evidence for Mycobacterium tuberculosis complex DNA was obtained in lung tissue and gall bladder samples, based on nested PCR of the IS6110 locus. Lung and femurs were positive for specific M. tuberculosis complex cell-wall mycolic acids, demonstrated by high-performance liquid chromatography of pyrenebutyric acid-pentafluorobenzyl mycolates. Therefore, tuberculosis is likely to have been the major cause of death of Irtyersenu.

Molecular exploration of the first-century Tomb of the Shroud in Akeldama, Jerusalem.

The Tomb of the Shroud is a first-century C.E. tomb discovered in Akeldama, Jerusalem, Israel that had been illegally entered and looted. The investigation of this tomb by an interdisciplinary team of researchers began in 2000. More than twenty stone ossuaries for collecting human bones were found, along with textiles from a burial shroud, hair and skeletal remains. The research presented here focuses on genetic analysis of the bioarchaeological remains from the tomb using mitochondrial DNA to examine familial relationships of the individuals within the tomb and molecular screening for the presence of disease. There are three mitochondrial haplotypes shared between a number of the remains analyzed suggesting a possible family tomb. There were two pathogens genetically detected within the collection of osteological samples, these were Mycobacterium tuberculosis and Mycobacterium leprae. The Tomb of the Shroud is one of very few examples of a preserved shrouded human burial and the only example of a plaster sealed loculus with remains genetically confirmed to have belonged to a shrouded male individual that suffered from tuberculosis and leprosy dating to the first-century C.E. This is the earliest case of leprosy with a confirmed date in which M. leprae DNA was detected.