Two new rapid SNP-typing methods for classifying Mycobacterium tuberculosis complex into the main phylogenetic lineages

There is increasing evidence that strain variation in Mycobacterium tuberculosis complex (MTBC) might influence the outcome of tuberculosis infection and disease. To assess genotype-phenotype associations, phylogenetically robust molecular markers and appropriate genotyping tools are required. Most current genotyping methods for MTBC are based on mobile or repetitive DNA elements. Because these elements are prone to convergent evolution, the corresponding genotyping techniques are suboptimal for phylogenetic studies and strain classification. By contrast, single nucleotide polymorphisms (SNP) are ideal markers for classifying MTBC into phylogenetic lineages, as they exhibit very low degrees of homoplasy. In this study, we developed two complementary SNP-based genotyping methods to classify strains into the six main human-associated lineages of MTBC, the 'Beijing' sublineage, and the clade comprising Mycobacterium bovis and Mycobacterium caprae. Phylogenetically informative SNPs were obtained from 22 MTBC whole-genome sequences. The first assay, referred to as MOL-PCR, is a ligation-dependent PCR with signal detection by fluorescent microspheres and a Luminex flow cytometer, which simultaneously interrogates eight SNPs. The second assay is based on six individual TaqMan real-time PCR assays for singleplex SNP-typing. We compared MOL-PCR and TaqMan results in two panels of clinical MTBC isolates. Both methods agreed fully when assigning 36 well-characterized strains into the main phylogenetic lineages. The sensitivity in allele-calling was 98.6% and 98.8% for MOL-PCR and TaqMan, respectively. Typing of an additional panel of 78 unknown clinical isolates revealed 99.2% and 100% sensitivity in allele-calling, respectively, and 100% agreement in lineage assignment between both methods. While MOL-PCR and TaqMan are both highly sensitive and specific, MOL-PCR is ideal for classification of isolates with no previous information, whereas TaqMan is faster for confirmation. Furthermore, both methods are rapid, flexible and comparably inexpensive

Diversity of Mycobacterium tuberculosis genotypes circulating in Ndola, Zambia

<p>Abstract</p> <p>Background</p> <p>Tuberculosis (TB) is one of the major public health problems in Zambia. However, information about lineages of <it>M. tuberculosis </it>complex (MTBC) isolates useful for epidemiology investigations is unknown. In this study, we investigated the diversity of MTBC isolates from Ndola, a typical Zambian urbanized city with a documented high HIV prevalence.</p> <p>Methods</p> <p>This was part of a prospective cohort study in subjects with sputum smear-positive pulmonary TB. Spoligotyping was used to genotype the MTBC isolates and establish the circulating lineages. The 15-locus Mycobacterial Interspersed Repetitive Units - Variable Number Tandem Repeats (MIRU-VNTR) typing was used to study recent transmission.</p> <p>Results</p> <p>A total of 98 different spoligotypes were identified among 273 MTBC isolates. The majority (64.8%) of the isolates belonged to 9 known families, while 96 (35.2%) of the isolates were orphans. While LAM (41.8%) was the largest spoligotype family observed, most of the isolates (87.7%) belonging to the SAF1 family, with a significant portion coming from the T (13.6%), and X (5.9%) families. A few isolates (3.6%) belonged to the CAS, EAI, H, S, X1-LAM9 or U families. MIRU-VNTR typing was highly discriminatory (h = 0.988) among the 156 isolates tested in our sample, and increased the discrimination among 82 SAF1 isolates from 6 to 46 distinct patterns. In addition, 3.2% (5/156) of cases with available MIRU-VNTR results harbored more than one MTBC strain.</p> <p>Conclusions</p> <p>Our findings show a limited diversity of MTBC in Ndola with a high clustering rate (37.7%), which indicates that recent transmission plays an appreciable role in the dynamics of TB disease in this setting. This conclusion emphasizes the importance of early diagnosis and timely treatment. The results also confirm that MIRU-VNTR typing is suitable for studying the molecular epidemiology of TB in Ndola.</p

Influence du mode d'élevage, du type de test et des caractéristiques humaines sur la réaction des veaux à l'homme

*INRA, Centre de Theix Diffusion du document : INRA, Centre de Theix Diplôme : Maîtris

Bone density and functional results after femoral revision with a cementless press-fit stem

International audienceINTRODUCTION:The influence of radiographic bone density changes in the area surrounding a total hip arthroplasty (THA) revision with a cementless press-fit stem is unknown, notably in terms of functional results. We have therefore conducted a study aiming to (1) propose a radiographic method to assess bone density, (2) measure the functional effects of reduced bone density, and (3) determine the factors contributing to these modifications.HYPOTHESIS:A reduction in radiographic bone density has a negative influence on the functional result after revision using a cementless press-fit stem.MATERIAL AND METHODS:We retrospectively assessed 150 THA revisions at a mean follow-up of 6.3 ± 3.2 years (range, 2-15 years). The clinical assessment was based on the Harris Hip Score. Bone density modifications were measured radiographically and the method was evaluated. The change in bone density was classified into two groups: (1) bone density not reduced or < 2 Gruen zones (118 cases [79%]); (2) bone density reduced ≥ 2 zones (32 cases [21%]). The variables showing a potential influence were the Cortical Index (CI), the type of primary stability with the press-fit system, and the femoral implant length.RESULTS:Inter- and intraobserver reliability of radiographic bone density measurement was evaluated as moderate or good (Kappa, 0.58; 0.60 and 0.67, respectively). For the Harris Hip Score at follow-up, there was a borderline statistical relation between stages 1 and 2: for the 118 stage 1 patients, this score was 83.62 ± 11.54 (range, 27-99) versus 78.34 ± 15.98 (range, 62-91) for stage 2 patients (P = 0.09). A CI ≤ 0.44 showed mediocre bone quality contributing to decreased bone density (P < 0.02). On the other hand, there was no statistically significant relation with the type of primary fixation (P = 0.34) or the length of the implant (P = 0.23).CONCLUSIONS:A cementless revision femoral stem can induce a reduction in bone density with possible functional effects. The negative role played by bone scarcity on the functional score is confirmed, and even though the difference is not statistically significant, we suggest using a short stem when this is possible