Cribra orbitalia are a porotic or sieve-like lesions in the bony orbital roof. This characteristic has frequently been detected in palaeopathological skulls from many parts of the world and has been the object of extensive research. Our objective was to determine if high-resolution peripheral quantitative computed tomography (HR-pQCT) could produce reliable information in the study of cribra orbitalia. Seven skulls displaying cribra orbitalia were investigated by HR-pQCT. The two-dimensional slices were compared with histological sections. The HR-pQCT images and histological sections showed similar results, i.e. two groups of lesions with different characteristics. HR-pQCT can be of great value in palaeopathological research. It is a nondestructive, fast and precise technique that allows an easy evaluation of the bone architecture without destruction of the sample.

The major goal of the present study was to investigate the potential use of a novel single nucleotide polymorphism (SNP) genotyping technology, called iPLEX Gold (Sequenom), for the simultaneous analysis of 16 SNPs that have been previously validated as useful for identification of Mycobacterium tuberculosis complex (MTBC) species and classification of MTBC isolates into distinct genetic lineages, known as principal genetic groups (PGGs) and SNP cluster groups (SCGs). In this context, we developed a 16-plex iPLEX assay based on an allele-specific-primer single-base-extension reaction using the iPLEX Gold kit (Sequenom), followed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis on the commercially available Sequenom MassARRAY platform. This assay was tested on a panel of 55 well-characterized MTBC strains that were also genotyped for the same loci using the previously reported SNaPshot assay, as well as 10 non-MTBC mycobacteria and 4 bacteria not belonging to the genus Mycobacterium. All MTBC samples were successfully analyzed with the iPLEX assay, which yielded clear allelic data for 99.9% of the SNPs (879 out of 880). No false-positive results were obtained with the negative controls. Compared to the SNaPshot assay, the newly developed 16-plex iPLEX assay produced fully concordant results that allowed reliable differentiation of MTBC species and recognition of lineages, thus demonstrating its potential value in diagnostic, epidemiological, and evolutionary applications. Compared to the SNaPshot approach, the implementation of the iPLEX technology could offer a higher throughput and could be a more flexible and cost-effective option for microbiology laboratories.

Tuberculosis, one of the most ancient human diseases, was present in ancient Egypt and has been observed since predynastic times. Excavations in the predynastic to early dynastic necropolis of Adaima, Upper Egypt (3500-2700 BC) led to the discovery of a number of remarkably well-preserved skeletons of children. The skeletal remains of a 4.5-5-year-old child dated from Nagada III A2 (3200-3100 BC) displayed various lesions on the post-cranial skeleton: spondylitis on the thoracic (T12) and lumbar (L1) vertebrae, partial lytic destruction of the right radio-ulnar joint, lytic lesions on the scapula and a clavicle, dactylitis on the short bones of hands and feet, enlargement (spina ventosa) and periosteal new bone formation on the long bones. Radiographs show well-defined radiolucent (cyst-like) lesions in the metaphysis and the diaphysis of long tubular bones (ulna, radius, femur, tibia, fibula). The lesions recorded during macroscopic and radiological analysis strongly suggest a case of multiple bone tuberculosis. The occurrence of this case of tuberculosis in a child provides a picture of a period where tuberculosis must have been endemic throughout the population living during the origins of urban settlement in Upper Egypt during the predynastic period. Copyright (C) 2009 John Wiley & Sons, Ltd.

The aim of the present study was to investigate the use of the SNaPshot minisequencing method for the identification of Mycobacterium tuberculosis complex (MTBC) isolates to the species level and for further genotyping of M. tuberculosis isolates. We developed an innovative strategy based on two multiplex allele-specific minisequencing assays that allowed detection of eight species-specific and eight lineage-specific single nucleotide polymorphisms (SNPs). Each assay consisted of an eightplex PCR amplification, followed by an eightplex minisequencing reaction with the SNaPshot multiplex kit (Applied Biosystems) and, finally, analysis of the extension products by capillary electrophoresis. The whole strategy was developed with a panel of 56 MTBC strains and 15 negative controls. All MTBC strains tested except one M. africanum clinical isolate were accurately identified to the species level, and all M. tuberculosis isolates were successfully further genotyped. This two-step strategy based on SNaPshot minisequencing allows the simultaneous differentiation of closely related members of the MTBC, the distinction between principal genetic groups, and the characterization of M. tuberculosis isolates into one of the seven prominent SNP cluster groups (SCGs) and could be a useful tool for diagnostic and epidemiological purposes.

Connaître les localisations osseuses de la tuberculose. Connaître les différents aspects des lésions de la tuberculose osseuse chez l’enfant. Savoir reconnaître des lésions rares ou peu typiques et évoquer un diagnostic différentiel. La tuberculose, une maladie du passé toujours d’actualité. La tuberculose osseuse a de multiples localisations en particulier chez l’enfant. Les vertèbres ne sont pas les seules localisations de la tuberculose osseuse. Spina ventosa est un mot à ne pas oublier. La radiologie est un outil précieux pour la paléopathologie. La tuberculose, maladie ré-émergente, est aussi une maladie du passé qui a sévi dans les populations prédynastiques de la vallée du Nil. Les conditions locales d’inhumation ont permis la conservation du squelette et des lésions osseuses dont ont souffert adultes et enfants des nécropoles. Chez l’enfant, les localisations osseuses de tuberculose sont le plus souvent multiples et dominées par un aspect lytique. La radiologie complète l’étude de l’aspect macroscopique des lésions et permet de les comparer avec celles de la maladie évoluée de la période pré-antibiotique, de révéler des lésions kystiques métaphysaires ou corticales et de confirmer des réactions périostées.

In the last years, the use of single nucleotide polymorphisms (SNPs) has increased in numerous areas (i.e. medicinal, forensic and human population genetics). These genetic markers interest today an emerging field termed molecular palaeomicrobiology. In this context, we have developed a three-step assay based on the typing of SNPs using the SNaPshot minisequencing methodology for the detection of mycobacterial DNA, differentiation of species within the Mycobacterium tuberculosis complex (MTBC), and distinction of M. tuberculosis lineages. Screening of ancient human samples showing lesions suggestive of tuberculosis (TB) using this strategy should not only confirm the morphological diagnosis of TB but also provide a direct insight into the evolution of the MTBC.

In a previous study, we extracted, amplified and sequenced a DNA fragment from bone lesions similar to those of bone tuberculosis in a predynastic skeleton (Egypt, around 3400 BC). This 65 kDa gene fragment encodes the surface of the mycobacterium responsible for the lesions. In the present study, we reconstructed the phylogenetic tree of the Mycobacterium family using this fragment and 51 sequences of pathogens and environmental mycobacteria. This reconstruction enabled us to polarise the phylogenic tree and to confirm the originality of the sequence and its ancestral character. According to a recent evolutionary scenario for the Mycobacterium tuberculosis complex, these data suggest the pathogenicity of an archaic mycobacterium at the emergence of urban life. This result could lead to a better understanding of present day evolutionary processes, especially the emergence or re-emergence of non-tuberculosis mycobacteria infection and disease.