Rapid Identification of Mycobacteria by Raman Spectroscopy

Abstract

A number of rapid identification methods have been developed to improve the accuracy for diagnosis of tuberculosis and to speed up the presumptive identification of Mycobacterium species. Most of these methods have been validated for a limited group of microorganisms only. Here, Raman spectroscopy was compared to 16S rRNA sequencing for the identification of Mycobacterium tuberculosis complex strains and the most frequently found strains of nontuberculous mycobacteria (NTM). A total of 63 strains, belonging to eight distinct species, were analyzed. The sensitivity of Raman spectroscopy for the identification of Mycobacterium species was 95.2%. All M. tuberculosis strains were correctly identified (7 of 7; 100%), as were 54 of 57 NTM strains (94%). The differentiation between M. tuberculosis and NTM was invariably correct for all strains. Moreover, the reproducibility of Raman spectroscopy was evaluated for killed mycobacteria (by heat and formalin) versus viable mycobacteria. The spectra of the heat-inactivated bacteria showed minimal differences compared to the spectra of viable mycobacteria. Therefore, the identification of mycobacteria appears possible without biosafety level 3 precautions. Raman spectroscopy provides a novel answer to the need for rapid species identification of cultured mycobacteria in a clinical diagnostic setting.Mycobacteria cause a variety of infections in humans. Classically defined lung tuberculosis (TB) is predominantly caused by M. tuberculosis complex. The number of new cases is estimated at nine million per year worldwide, and the disease causes more than two millions deaths annually (17). In addition, the incidence of pulmonary disease caused by nontuberculous mycobacteria (NTM) appears to be increasing worldwide (1, 6). The clinical features of NTM-derived pulmonary disease are in some cases indistinguishable from those of tuberculosis. Because the treatment and the epidemiology of NTM-derived infections differ significantly from TB caused by M. tuberculosis complex bacteria, the timely and correct identification of causative organisms is mandatory for diagnosis, therapy, and control.Conventional approaches to the diagnosis of mycobacterial infection rely on tests that are far from optimal. For example, sputum smear microscopy is insensitive, laborious, and timeconsuming. Culture is technically complex and time-consuming, has a sensitivity of only 80 to 85%, and is scarcely available in high-prevalence settings. Chest radiography is nonspecific and not widely implemented either. Tuberculin skin testing is imprecise, and the results are often nonspecific (3). In the last decade, a number of rapid diagnostic tests have been developed in an effort to improve the diagnostic accuracy for TB and to speed up presumptive identification. PCR and other molecular amplification techniques are the most prominent among these new tools. Although promising, none are more than adjunctive to the diagnosis of TB, since the sensitivities of these tests vary widely. The most reliable results are fou...

Tuberculosis - Current Issues in Diagnosis and Management

“…This vibrational spectroscopy-based detection method can detect and differentiate various molecular compositions of microorganism [15][16][17][18] and therefore is suitable to identify the species and strains of microorganism. Buijtels technique is also are much faster; results can be obtained within 3 hours since positive automated cultured system is obtained [18]. In view of the importance of early diagnosis to prevent further spread of tuberculosis in the community, this time efficiency is the most significant contribution of Raman spectroscopy.…”

Automatic identification of novel bacteria using Raman spectroscopy and Gaussian processes

“…A large set of techniques [1,2] was developed to access as much information as possible from samples. Among these, vibrational techniques [3,4,5] such as Infrared [3] and Raman spectroscopy [6] showed to be especially suitable for many applications due to their non-destructive and fast methodology [7,8]. Moreover, it has been shown that very reliable recognition results can be obtained without timeconsuming cultivation steps, since it is possible to extract informative spectra from single cells [9,10,11,12].…”

Biological and Medical Applications of Multivariate Curve Resolution Assisted Raman Spectroscopy

“…Indeed, we have demonstrated that it is possible to study ergosterol metabolism in detail at the single cell level using fission yeast model. 8 Figure 3 shows the space-resolved lipid Raman spectra of S. pombe in mixed 12 C and 13 C isotope media. The band at 1602 cm -1 , corresponding to C=C-C=C symmetric stretch of the 5,7-diene structure of ergosterol in a pure 12 C medium redshifts to 1542 cm -1 in the 13 C medium.…”

“…Raman spectroscopy, when combined with a microscope, achieves sub-μm spatial resolution, which can provide detailed spaceresolved molecular information and visualize their intracellular distribution. Owing to its ability to detect biochemical changes at the molecular level, Raman spectroscopy has gained much attention as a bio-analytical tool in recent years, and has been successfully applied in myriad different problems in biological and medical sciences, which include, but not limited to, structural identification and label-free biomolecular imaging of living cells, [1][2][3][4][5][6][7] cellular metabolic pathways, 8,9 cellular discrimination, [10][11][12][13] tissue characterization, 14,15 disease diagnosis, prognosis and therapy. component analysis (PCA) and clustering analysis (CA) are two popular methods.…”