Evaluation of the new advanced 15-loci MIRU-VNTR genotyping tool in Mycobacterium tuberculosis molecular epidemiology studies

Background. During the last few years, PCR-based methods have been developed to simplify and reduce the time required for genotyping Mycobacterium tuberculosis (MTB) by standard approaches based on IS6110-Restriction Fragment Length Polymorphism (RFLP). Of these, MIRU-12-VNTR (Mycobacterial interspersed repetitive units- variable number of tandem repeats) (MIRU-12) has been considered a good alternative. Nevertheless, some limitations and discrepancies with RFLP, which are minimized if the technique is complemented with spoligotyping, have been found. Recently, a new version of MIRU-VNTR targeting 15 loci (MIRU-15) has been proposed to improve the MIRU-12 format. Results. We evaluated the new MIRU-15 tool in two different samples. First, we analyzed the same convenience sample that had been used to evaluate MIRU-12 in a previous study, and the new 15-loci version offered higher discriminatory power (Hunter-Gaston discriminatory index [HGDI]: 0.995 vs 0.978; 34.4% of clustered cases vs 57.5%) and better correlation (full or high correlation with RFLP for 82% of the clusters vs 47%). Second, we evaluated MIRU-15 on a population-based sample and, once again, good correlation with the RFLP clustering data was observed (for 83% of the RFLP clusters). To understand the meaning of the discrepancies still found between MIRU-15 and RFLP, we analyzed the epidemiological data for the clustered patients. In most cases, splitting of RFLP-clustered patients by MIRU-15 occurred for those without epidemiological links, and RFLP-clustered patients with epidemiological links were also clustered by MIRU-15, suggesting a good epidemiological background for clustering defined by MIRU-15. Conclusion. The data obtained by MIRU-15 suggest that the new design is very efficient at assigning clusters confirmed by epidemiological data. If we add this to the speed with which it provides results, MIRU-15 could be considered a suitable tool for real-time genotyping.

Histological and histochemical evaluation of human oral mucosa constructs developed by tissue engineering

Reconstruction of large oral mucosa defects is often challenging, since the shortage of healthy oral mucosa to replace the excised tissues is very common. In this context, tissue engineering techniques may provide a source of autologous tissues available for transplant in these patients. In this work, we developed a new model of artificial oral mucosa generated by tissue engineering using a fibrin-agarose scaffold. For that purpose, we generated primary cultures of human oral mucosa fibroblasts and keratinocytes from small biopsies of normal oral mucosa using enzymatic treatments. Then we determined the viability of the cultured cells by electron probe quantitative X-ray microanalysis, and we demonstrated that most of the cells in the primary cultures were alive and had high K/Na ratios. Once cell viability was determined, we used the cultured fibroblasts and keratinocytes to develop an artificial oral mucosa construct by using a fibrin-agarose extracellular matrix and a sequential culture technique using porous culture inserts. Histological analysis of the artificial tissues showed high similarities with normal oral mucosa controls. The epithelium of the oral substitutes had several layers, with desmosomes and apical microvilli and microplicae. Both the controls and the oral mucosa substitutes showed high suprabasal expression of cytokeratin 13 and low expression of cytokeratin 10. All these results suggest that our model of oral mucosa using fibrin-agarose scaffolds show several similarities with native human oral mucosa.