Autofluorescence of Mycobacteria as a Tool for Detection of Mycobacterium tuberculosis

Abstract

The diagnosis of tuberculosis in developing countries still relies on direct sputum examination by light microscopy, a method that is easy to perform and that is widely applied. However, because of its poor sensitivity and requirement for significant labor and training, light microscopy examination detects the bacilli in only 45 to 60% of all people whose specimens are culture positive for Mycobacterium tuberculosis. Therefore, new diagnostic methods that would enable the detection of the undiagnosed infected population and allow the early commencement of antituberculosis treatment are needed. In this work, the potential use of mycobacterial cyan autofluorescence for the detection of Mycobacterium tuberculosis was explored. The tubercle bacilli were easily visualized as brilliant fluorescent bacilli by microscopy and were easily tracked ex vivo during macrophage infection. Assays with seeded sputum and a 96-well microplate reader fluorimeter indicated that <10(6) bacilli ml(-1) of sputum could be detected. Moreover, the use of microplates allowed the examination of only 200 microl of sputum per sample without a loss of sensitivity. Treatment with heat or decontaminating chemical agents did not interfere with the autofluorescence assay; on the contrary, they improved the level of bacterial detection. Autofluorescence for the detection of bacilli is rapid and easy to perform compared to other methodologies and can be performed with minimal training, making this method suitable for implementation in developing countries.

The Mycobacterium tuberculosis SecA2 System Subverts Phagosome Maturation To Promote Growth in Macrophages

“…To detect bacilli in macrophages, we took advantage of the recently described autofluorescence of mycobacteria (45). Using scanning spectrophotometry, we first experimentally deter- mined the optimal peak excitation and emission wavelengths (415 nm and 470 nm, respectively) for autofluorescence of paraformaldehyde-fixed M. tuberculosis.…”

Autofluorescence as a Tool for Structural Analysis of Biofilms Formed by Nonpigmented Rapidly Growing Mycobacteria

“…It has been hypothesized that coenzyme F 420 is involved in autofluorescence of mycobacteria (16). According to our results, this molecule must be secreted by the bacterial components of the biofilm and can be detected in the extracellular matrix using autofluorescence.…”

“…Autofluorescence has been found in different mycobacterial species (16,19). It has been hypothesized that coenzyme F 420 is involved in autofluorescence of mycobacteria (16).…”

“…Intrinsic autofluorescence, including the presence of autofluorescence in the cyan range in Mycobacterium species (16), is a characteristic that has been found previously in several microorganisms (15). In this study, we aimed to analyze the structure of mycobacterial biofilms, with a special focus on detection of autofluorescence.…”

Quantitative Comparison of the Autofluorescence of Bacteria and Polystyrene Microspheres under Violet Wavelength Excitation for Verification of Fluorescence-based Bioaerosol Detector Results

“…In fermentation engineering, the microbial metabolic state can be monitored by measuring the autofluorescence intensity, particularly the fluorescence originating from NAD P H Scheper et al, 1987;Scheper and Shügel, 1986 . In diagnostic research, autofluorescence has been used in d e t e c t i o n o f t h e m a c r o p h a g e s i n f e c t e d b y Mycobacterium Patiño et al, 2008 . Recently, it has also been used in bioaerosol detection, leading to the development of fluorescence-based bioaerosol detectors Hill et al, 1999;Pinnick et al, 1995 . Bioaerosols have been studied in the fields of environmental and industrial health, and in the quality control of pharmaceuticals and food products Albrecht et al, 2007;Bernasconi et al, 2010;Pacheco and Pinto, 2010;Schlosser et al, 2009 .…”