Molecular evidence for different stages of tuberculosis in ancient bone samples from Hungary

This paleomicrobiologic study was conducted on osseous tissue specimens fro m ancient Hungarian skeletal samples from the 7-8th and the 17th centuries AD with typical macromorphologic evidence of osseous tuberculosis (n = 3), morphologic alterations probably due to tuberculosis (n = 6), or with nonty pical osseous changes of vertebral bodies suggestive of inflammatory reacti on (n = 5). From these bone samples, DNA was extracted and amplified by pol ymerase chain reaction (PCR) by using various primer pairs recognizing DNA segments of different mycobacterial species. To confirm specificity of the analysis, the amplification products of several samples were subjected to r estriction enzyme digestion and/or direct sequencing. Of the analyzed 14 ca ses, 8 were unambiguously positive for mycobacterial DNA of the Mycobacteri um tuberculosis complex, as shown by the amplification of the IS6110 sequen ce. In 13 cases we found a PCR product with primers specific for the 65-kDa antigen gene, including 2 cases without genomic DNA. We conclude that the application of other mycobacterial DNA primers may reveal contamination of bones with atypical saprophytic mycobacteria. A positive result for typical mycobacteria was seen in 2 of 3 cases with typical morphologic signs of tu berculosis and amplifiable DNA, in 3 of 6 probable cases, but also in 3 of 6 cases with nontypical bone changes. This indicates that minor osseous rea ctions of the surface of vertebral bodies may be due-at least in several ca ses-to infections with bacteria of the M. tuberculosis complex. Ln these ca ses the disease may have proceeded rapidly, and the morphologic osseous cha nges may represent "early" stages of tuberculous infection of the vertebrae . (C) 2000 Wiley-Liss, Inc.