ИСТИНА

Laser scanning confocal microscopy (LSCM) is acknowledged as a selective and highly sensitive method for the visualization in histological preparations. However, such a technique combined with immunofluorescent staining (IFS) has not been routinely applied for patients with tuberculosis (TB). The aim of the study was to reveal advantages and limitations the M. tuberculosis (Mtb) visualization in lung tissue preparations using IFS, thick histological slices and LSCM. Methods: Tissue samples were obtained from 23 patients with suspected lung tuberculomas. For polyclonal antibody production, rabbits were immunized with whole cells of irradiated Mtb H37Rv in incomplete Freund adjuvant. Commercial secondary antibodies labeled by Alexa were used. 20-70 μm frozen sections were examined using LSCM. Results: Spectral detection allowed to discriminate Mtb from bacteria-like structures in all obtained samples. Bacterial aggregates and solitary bacteria were detected, while 3D reconstruction gave additional information about Mtb morphology in various regions of TB inflammation. The number of revealed bacteria was 3-fold high in 20 μm and 7-folds high in 40 μm optical sections than in 5 μm sections. The best result was obtained on 20 μm optical sections, since bacterial-like structures with a non-specific spectrum are detected much less frequently than on thicker sections. Conclusions: The tested technique significantly increases the specificity of Mtb detection in the lung tissue in comparison with classical histochemical methods. Non-specific luminescence of bacteria-like structures is the limiting factor for above-mentioned technique.