Application of Laser Scanning Confocal Microscopy for the Visualization of M. tuberculosis in Lung Tissue Samples with Weak Ziehl–Neelsen Staining

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.

Lipoarabinomannan as a Point-of-Care Assay for Diagnosis of Tuberculosis: How Far Are We to Use It?

“…This was observed through the use of specific fluorescent antibody labelling combined with confocal microscopy scanning (Sande et al, 2016). According to our hypothesis, it will open up new possibilities for immunoassay detection strategies for Mtb (Erokhina et al, 2019). Not only would detection of histological specimens be possible, but also the detection of LAM from peripheral blood.…”

Laser Scanning Confocal Microscopy Was Used to Validate the Presence of Burkholderia pseudomallei or B. mallei in Formalin-Fixed Paraffin Embedded Tissues

“…This technology may be diagnostic for melioidosis (or glanders) or complement the diagnosis of the disease with the isolation of the pathogen. In addition, it would be useful to have B. pseudomallei and B. mallei capsule-specific monoclonal antibodies, if there is a question between diagnosis of melioidosis or glanders [28][29][30]. One advantage of LSCM is that it can validate the diagnosis of melioidosis or glanders, but some disadvantages of LSCM are the cost of the system, the expertise to operate the system, and the preparation of the sample would limit its usefulness in an on-site clinical setting where diagnosis is needed.…”

Improved Conventional and New Approaches in the Diagnosis of Tuberculosis

“…This method improves the detection efficiency and saves valuable time for the laboratory staff. The specific fluorescent antibody labeling with laser confocal microscopy has been developed to improve the efficiency of M. tuberculosis detection in lung tissue samples, especially when these bacteria have weak ZN staining ( Erokhina et al, 2019 ). The Pat-Scan program constructed using digital pathology has been developed to detect and quantify the bacteria in the paraffin-embedded ZN-stained tissue, thus being helpful in reducing the diagnostic time ( Sua et al, 2021 ).…”