Pathogenic microbial ancient DNA: a problem or an opportunity?

Copyright © Royal Society 2006Eske Willerslev, Alan Coope

Confirmation of the presence of Mycobacterium-tuberculosis complex-specific DNA in three archaeological specimens

This journal published the first reported identification of Mycobacterium tuberculosis complex (MTE) DNA in ancient human remains but CONCERNS were raised about the article two years after publication. These were based on methodology which, in the field of ancient DNA, was still developing. Here we present a re-examination of the 1993 research conducted on three specimens which exhibited palaeopathologies indicative of tuberculosis. The specimens were: an ulna from pre-European-contact Borneo, a spine from Byzantine Turkey, and a lumbar-sacral spine from 17th century Scotland. There was insufficient material to permit re-examination of all of the original samples. The earlier results were confirmed in two independent laboratories using different methodologies. MTB DNA complex-specific Dna amplicons were obtained, and sequenced in both laboratories, in a re-analysis of samples which supported the earlier findings

Widespread occurrence of Mycobacterium tuberculosis DNA from 18th-19th century Hungarians

A large number (265) of burials from 1731-1838 were discovered in sealed crypts of the Dominican Church, Vac, Hungary in 1994. Many bodies were naturally mummified, so that both soft tissues and bones were available. Contemporary archives enabled the determination of age at death, and the identification of family groups. In some cases, symptoms before death were described and, occasionally, occupation. Initial radiological examination of a small number of individuals had indicated calcified lung lesions and demonstrable acid-fast bacteria suggestive of tuberculosis infection. Tuberculosis was endemic in 18th-19th century Europe, so human remains should contain detectable Mycobacterium tuberculosis complex (MTB) DNA, enabling comparisons with modern isolates. Therefore, a comprehensive examination of 168 individuals for the presence of MTB DNA was undertaken. Specific DNA amplification methods for MTB showed that 55% of individuals were positive and that the incidence varied according to age at death and sampling site in the body. Radiographs were obtained from 27 individuals and revealed an association between gross pathology and the presence of MTB DNA. There was an inverse relationship between PCR positivity and MTB target sequence size. In some cases, the preservation of MTB DNA was excellent, and several target gene sequences could be detected from the same sample. This information, combined with MTB DNA sequencing data and molecular typing techniques, will enable us to study the past epidemiology of TB infection, and extends the timeframe for studying changes in molecular fingerprints. Am J Phys Anthropol 120:144-152, 2003. (C) 2003 Wiley-Liss, Inc

Molecular analysis of Mycobacterium tuberculosis DNA from a family of 18th century Hungarians

The naturally mummified remains of a mother and two daughters found in an 18th century Hungarian crypt were analysed, using multiple molecular genetic techniques to examine the epidemiology and evolution of tuberculosis. DNA was amplified from a number of targets on the Mycobacterium tuberculosis genome, including DNA from IS6110, gyrA, katG codon 463, oxyR, dnaA–dnaN, mtp40, plcD and the direct repeat (DR) region. The strains present in the mummified remains were identified as M. tuberculosis and not Mycobacterium bovis, from katG and gyrA genotyping, PCR from the oxyR and mtp40 loci, and spoligotyping. Spoligotyping divided the samples into two strain types, and screening for a deletion in the MT1801–plcD region initially divided the strains into three types. Further investigation showed, however, that an apparent deletion was due to poor DNA preservation. By comparing the effect of PCR target size on the yield of amplicon, a clear difference was shown between 18th century and modern M. tuberculosis DNA. A two-centre system was used to confirm the findings of this study, which clearly demonstrate the value of using molecular genetic techniques to study historical cases of tuberculosis and the care required in drawing conclusions. The genotyping and spoligotyping results are consistent with the most recent theory of the evolution and spread of the modern tuberculosis epidemic

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

Detection and characterization of Mycobacterium tuberculosis DNA in 18th Century Hungarians with pulmonary and extra-pulmonary tuberculosis

Skeletal and naturally mummified tissues from a previously archived group of 18th century Hungarian remains were examined for the presence of Mycobacterium tuberculosis complex (MTB) DNA, using specific nested PCR for the IS6110 locus. Paleopathological changes in bones and from radiographs were noted in a minority of cases. Overall, specimens from 157/232 (67.7 %) of individuals proved positive, ranging from 20/43 (46.5 %) in children, 26/29 in middle-age (89.7 %) and 32/46 individuals aged 65-95 years (69.6 %). Single samples gave a positive result in 67/120 of cases (55.8 %). Most were ribs where the surface adjacent to lungs and pleura was sampled. When multiple sites were examined, 73/93 (78.5 %) individuals were positive; most of these had MTB only in the pulmonary region but 26 had disseminated disease (35.6 %) and 12 (16.4 %) had extra-pulmonary disease only. To distinguish M. tuberculosis from Mycobacterium bovis, well-preserved positive samples were examined for several additional genetic loci including the TbD1 deletion – characteristic of modern European strains of M. tuberculosis, and spoligotyped. No evidence other than of human M. tuberculosis was found, but different strains were detected. Tuberculosis was widespread in this community and whilst some individuals succumbed early in life, the majority co-existed with the infection. Therefore, this study may lead to the identification of host alleles and MTB strains associated with active and latent disease