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.

Molecular identification of human tuberculosis in recent and historic bone tissue samples: The role of molecular techniques for the study of historic tuberculosis.

We describe the molecular identification of the M. tuberculosis complex DNA in bone tissue samples from recent and historic populations. In a first set, archival paraffin material from vertebral bodies of 12 recent cases with clinically/microbiologically proven tuberculosis was compared to 12 further cases without tuberculosis. While eight TB cases revealed a specific mycobacterial amplification product, none of the controls was positive. Interestingly, one case with tuberculous sepsis (Landouzy sepsis), five cases with tuberculous spread beyond the primarily affected organ (i.e., lymph node or miliar involvement), and also two of six cases with restricted pulmonary tuberculosis reacted positively in the vertebral specimens. This indicates that a molecular analysis can detect mycobacteria even in unremarkable bone tissue, proving that organ tuberculosis is present. In addition, the extent of spread is of high significance for the frequency of positive reactions. In addition, we investigated a series of vertebral samples coming from an Egyptian population of the necropolis of Thebes-West dating to approximately 1450-500 BC. In this group of 36 cases, three of five cases with typical macromorphological signs for tuberculous spondylitis, 2 of 12 cases with nonspecific alterations, and 2 of 19 cases without macroscopic pathology revealed a specific amplicon of the M. tuberculosis complex. This suggests a significant frequency of infected people in that ancient population. Finally, a fourth group of 51 long bone samples with pathological alterations coming form a southern German ossuary (between AD 1400-1800) was investigated, and 10 cases were positive for the M. tuberculosis complex. These studies of historic material clearly support the notion that tuberculous infections can be unequivocally identified by molecular techniques. The relatively high frequency of ancient bacterial DNA amplifications in unremarkable bone is well-explained by our analysis of the recent material. Our data form an important basis for the investigation of tuberculosis frequency and spread in historic periods.