**5. Conclusion**

After a slow beginning, almost two decades ago, characterization of *M. tuberculosis* aDNA has been systematically developed in a limited number of laboratories. The analysis of aDNA can provide a wealth of information about the particular strain of *M. tuberculosis*  diagnosed, but the exact information is governed by the degree of preservation. The fact that aDNA analyses are now firmly established is due partly to technological advances but mainly due to established skills in key laboratories, built up over an extended period. Conclusive results can be obtained by aDNA analyses alone, but confirmatory biomarkers are valuable in completing the overall diagnosis. The tubercle bacillus and related mycobacteria are rich in unusual lipids, which are not produced in mammalian tissues. Building on sensitive methods, previously developed to detect lipid biomarkers for modern tuberculosis, and inspired by the initial aDNA results, mycolic acid biomarkers for tuberculosis were detected for the first time in archaeological bone just over a decade ago. After a flurry of initial studies, time was taken to explore more robust mycolic acid methods and expand the range of lipids to include characteristic mycocerosic and mycolipenic acids. Currently, aDNA and lipid analyses are established as a powerful combination to diagnose both tuberculosis and leprosy and, indeed, co-infections; these analyses can be performed on

Molecular Biomarkers for Ancient Tuberculosis 27

accelerated evolutionary changes to *M. tuberculosis* and the emergence of highly drugresistant and readily transmissible strains. Modern genomic approaches are also highlighting the great diversity existing within the inhomogeneous species labelled as *M. tuberculosis*. The developing science of paleogenomics is enabling approximate timelines for the evolution of *M. tuberculosis* to be laid down. The analysis of biomarkers is the prime way to verify the various proposed evolutionary pathways and to provide a direct timescale, rather than one inferred from bioinformatic analysis. In this review the contributions of ancient DNA (aDNA) and lipid biomarker analyses have been elaborated and critically

G.S.B. has a James Bardrick Personal Research Chair and a Royal Society Wolfson Research Merit Award. D.E.M. was a recipient of an Emeritus Fellowship from The Leverhulme Trust, who are also thanked for Project Grant F/00 094/BL (G.S.B., D.E.M., O.Y-C.L.).

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**6. Acknowledgement** 

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**7. References** 

the same sample. This is not a static situation, however, as new powerful methods are becoming available for analyses of aDNA and lipids; additionally, the range of lipids available for detection is by no means exhausted.

The availability of established aDNA and biomarker protocols and expertise provides avenues into a range of interlocking research areas. As noted above, evolutionary pathways can be verified for tuberculosis, also providing evidence about virulence as the key lipid biomarkers are implicated in this process. The very existence of apparently intact key lipids in ancient samples is also of basic chemical interest; for example, the mycolic acids from the 9,000 year old Atlit-Yam skeletons probably represent the oldest known cyclopropane rings! It is important also to study leprosy, as the evolutionary processes of these two mycobacterial diseases are fatally intertwined with many clear co-infections. The relative prevalence of tuberculosis and leprosy can give clear indications of the prevailing social environment. In tuberculosis/leprosy co-infections it is becoming possible to estimate the relative bacterial load in particular bones and thereby obtain indications regarding which disease was the immediate cause of death. For each disease and co-infections it will be instructive to examine bones throughout particular individual skeletons in order to gain information about dissemination. Evidence of tuberculosis and leprosy can be quite clear in the osteological record, with characteristic bone lesions and deformations and bone loss in the case of leprosy. Biomarker analysis can help illuminate and reinforce the diagnoses of skilled osteologists and compare bacterial loads in bones with and without disease indication. An appealing aspect of lipid biomarker analyses is the possibility of extracting the lipids with neutral solvents, avoiding any chemical or physical damage to particularly valuable bone samples. It must be borne in mind, however, that biomarker analyses for the diagnosis of ancient mycobacterial disease are currently sophisticated procedures, which cannot be easily attempted without time being spent in accumulating the necessary skills and experience.

The evolution of life on earth is a complex web of competitive and/or symbiotic interactions. Humans, related primates and all mammals are dependent on a symbiosis with many microorganisms, whose cells outnumber those of the host. From the perspective of the microorganism, the mammalian host provides an ecological niche in which it can multiply and evolve to improve its prospects for survival. In some cases, the interaction of microorganisms with mammals is a rapid, pathogenic process resulting in the demise of the host and the infecting agent passes on to a new subject. Early hominids and humans with a hunter/gatherer lifestyle had a low population density, so an alternative relationship with slow-growing organisms such as the pathogenic mycobacteria, emerged. Such pathogens have a long-term relationship with their host, thus enabling persistence of the organism until transmission is possible. This is likely to occur at the extremes of life, when the host immune response is immature or less effective, and at times of physical or mental stress, often associated with war, famine, poverty or social unrest. This latter scenario is typical of the ancient scourges of tuberculosis and leprosy, caused by *M. tuberculosis* and *M. leprae*, respectively.

Several decades ago, it appeared that BCG vaccination and combinations of effective drugs were conquering tuberculosis. However, declining vaccination efficacy, misuse of drug regimens and the rapid spread of HIV/AIDS-related immunodeficiency, together with increased urbanisation and population density, have provided the opportunity for accelerated evolutionary changes to *M. tuberculosis* and the emergence of highly drugresistant and readily transmissible strains. Modern genomic approaches are also highlighting the great diversity existing within the inhomogeneous species labelled as *M. tuberculosis*. The developing science of paleogenomics is enabling approximate timelines for the evolution of *M. tuberculosis* to be laid down. The analysis of biomarkers is the prime way to verify the various proposed evolutionary pathways and to provide a direct timescale, rather than one inferred from bioinformatic analysis. In this review the contributions of ancient DNA (aDNA) and lipid biomarker analyses have been elaborated and critically assessed.
