In recent years, many clinical laboratories switched to mass spectrometry (MALDI-TOF) to identify the bacterial species in clinical samples. This powerful technique rapidly compares the unique protein ‘fingerprint’ of a bacteria with a large database, allowing quick and reliable species identification. The implementation in mycobacteriology has always been difficult, mainly due to problems with their robust cell wall. Therefore, the National Reference Centre (NRC) for Mycobacteria teamed up with MALDI-TOF experts and optimised sample preparation. Moreover, we are testing the possibility to use the same technique to determine the susceptibility of Mycobacteria for selected antibiotics. The derived protocol is freely available for clinical laboratories, to implement in their workflow. Currently, the performance of this technique is being investigated in an European consortium of clinical mycobacterial laboratories.
The genus Mycobacterium comprises at least 175 recognized species with a wide spectrum of pathogenicity. M. tuberculosis (MTB), the leading cause of tuberculosis (TB), is among the greatest public health threats in low-income countries. According to the latest WHO report, TB ranks now alongside HIV as a primary cause of death worldwide, with an estimated 9.6 million new cases in 2014 and 1.5 million deaths. Whereas MTB is an obligate pathogen, non-tuberculous mycobacteria (NTMs) are environmental organisms which occasionally act as opportunistic pathogens, leading to a wide array of clinical syndromes. The number of reported cases of NTM infections has risen dramatically in the past decade, which has been linked to increasing numbers of immunocompromised patients, the implementation of advanced diagnostic tools, and the increased awareness of the role of NTMs in diseases. However, only 29 of the >160 recognized NTM species are associated with human disease; others (such as M. gordonae) are frequent contaminants from sample collection or processing. Therefore, diagnostic laboratories should perform species-level identification to establish the clinical significance of a mycobacterial isolate in clinical specimens. Traditional identification of mycobacterial species mainly relied on phenotypic and biochemical traits. More recent methods involve the chromatographic analysis of mycolic acid profiles, or the use of DNA probes which target the most common Mycobacterium spp. To date, the gold standard for species identification remains DNA sequencing of 16S rRNA, rpoB and/or hsp65 genes. However, these methods are costly, can be labour-intensive, require specific equipment and expertise and are often restricted to reference laboratories.
A similar complexity can be found in mycobacterial drug susceptibility testing (DST), which is culture-based and time-consuming. The current gold standard for MTB is the MGIT960/TBeXiST platform, which measures growth based on oxygen consumption. Recently developed molecular techniques can rapidly detect genetic resistance markers in MTB but do not eliminate the need for culture-based susceptibility testing for the full spectrum of available TB drugs. For NTM strains, broth microdilution has been identified as recommended method. However, ample differences between in vivo treatment outcome and in vitro susceptibilities have been reported, and the few breakpoints which have been established have little clinical evidence base. As a consequence, most diagnostic labs perform either the ancient agar proportion method, or use commercial platforms for broth microdilution to establish the minimal inhibitory concentrations (MIC) values for various antimicrobials. However, the interpretation of the latter is challenging for slow-growing mycobacteria, and prone to inter-laboratory variations. Moreover, the mere report of a MIC value without an accompanying clinical breakpoint is often useless for the physician.
In the last few years, important progress has been made in the application of matrix-assisted laser desorption ionization−time of flight mass spectrometry (MALDI-TOF MS) for species identification in mycobacteriology. Unlike most bacteria, mycobacteria require prior inactivation and disruption of the mycolic acid-rich cell wall, both for biosafety reasons and to liberate the cellular protein content.
In this project, we investigate the possibilities to use MALDI-TOF in species identification and DST testing of mycobacteria.