Abstract
One of the key requirements for the diagnosis of pulmonary tuberculosis is the identification of M. tuberculosis in tissue. In this paper, we present the advantages of specific fluorescent antibody labelling, combined with laser scanning confocal microscopy (LSCM), for the detection of M. tuberculosis in histological specimens of lung tissues. We demonstrate that the application of LSCM allows: (i) The automatic acquisition of images of the whole slice and, hence, the determination of regions for subsequent analysis; (ii) the acquisition of images of thick (20–40 μm) slices at high resolution; (iii) single bacteria identification; and (iv) 3D reconstruction, in order to obtain additional information about the distribution, size, and morphology of solitary M. tuberculosis; as well as their aggregates and colonies, in various regions of tuberculosis inflammation. LSCM allows for the discrimination of the non-specific fluorescence of bacteria-like particles and their aggregates presented in histological lung samples, from the specific fluorescence of labelled M. tuberculosis, using spectrum emission analysis. The applied method was effective in the identification of M. tuberculosis in lung histological samples with weak Ziehl–Neelsen staining. Altogether, combining immunofluorescent labelling with the application of LSCM visualization significantly increases the effectiveness of M. tuberculosis detection.
Highlights
Tuberculosis (TB) has remained a major health problem: TB is still being reported as one of the top ten causes of death worldwide [1]
An overview of regions of caseous necrosis demonstrates a difference in the Ziehl–Neelsen staining of M. tuberculosis, within lung tissue with high (Figure 2A), and low (Figure 2B) bacterial content
Ziehl–Neelsen staining did not allow an unambiguous identification of M. tuberculosis and their colonies in samples of lung tissue with a low bacterial content (Figure 2B)
Summary
Tuberculosis (TB) has remained a major health problem: TB is still being reported as one of the top ten causes of death worldwide [1]. The pathomorphological landscape of TB-triggered lung inflammation may be difficult to identify because of the emergence of complications from drug-resistant TB and co-morbidities such as HIV/AIDS, fungal and parasitic infections, and alveolitis. One of the key requirements for TB diagnosis is the identification of Mycobacterium tuberculosis (M. tuberculosis) in sputum and tissue, and it remains one of the most important tasks in phthisiology. The method is fast and inexpensive, but its main limitations are low sensitivity (40–60%), especially in individuals co-infected with HIV [5,6]. Another drawback of sputum analysis is that the detectable number of mycobacterium tuberculosis does not indicate the activity of the inflammatory process in the lungs [7]. In 2002, the WHO declared that it is clear that and sensitive diagnostic tests for TB diagnosis are needed [8]
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