Comparison of Sample Preparation Methods, Instrumentation Platforms, and Contemporary Commercial Databases for Identification of Clinically Relevant Mycobacteria by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

Abstract

When mycobacteria are recovered in clinical specimens, timely species-level identification is required to establish the clinical significance of the isolate and facilitate optimization of antimicrobial therapy. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently been reported to be a reliable and expedited method for identification of mycobacteria, although various specimen preparation techniques and databases for analysis are reported across studies. Here we compared two MALDI-TOF MS instrumentation platforms and three databases: Bruker Biotyper Real Time Classification 3.1 (Biotyper), Vitek MS Plus Saramis Premium (Saramis), and Vitek MS v3.0. We evaluated two sample preparation techniques and demonstrate that extraction methods are not interchangeable across different platforms or databases. Once testing parameters were established, a panel of 157 mycobacterial isolates (including 16 Mycobacterium tuberculosis isolates) was evaluated, demonstrating that with the appropriate specimen preparation, all three methods provide reliable identification for most species. Using a score cutoff value of >1.8, the Biotyper correctly identified 133 (84.7%) isolates with no misidentifications. Using a confidence value of >90%, Saramis correctly identified 134 (85.4%) isolates with one misidentification and Vitek MS v3.0 correctly identified 140 (89.2%) isolates with one misidentification. The levels of accuracy were not significantly different across the three platforms (P ‫؍‬ 0.14). In addition, we show that Vitek MS v3.0 requires modestly fewer repeat analyses than the Biotyper and Saramis methods (P ‫؍‬ 0.04), which may have implications for laboratory workflow.T he genus Mycobacterium has undergone tremendous taxonomic revision in recent decades. There are currently 170 recognized species (http://www.bacterio.net/mycobacterium.html; accessed 20 February 2014) with a wide range of pathogenic potential from benign environmental contaminants to the pathogenic Mycobacterium tuberculosis complex, which was estimated to be responsible for 9 million cases of disease and 1.5 million deaths worldwide in 2013 (1). While M. tuberculosis remains the most clinically significant species and public health threat within this genus, many non-tuberculosis mycobacteria (NTM) are wellestablished pathogens and may be increasing in prevalence in part due to increased numbers of immunocompromised individuals as well as to the increasing prevalence of medical hardware and indwelling devices (2).When mycobacteria are recovered from clinical specimens, establishment of a species-or complex-level identification is critical to distinguish pathogenic species from common environmental contaminants (such as Mycobacterium gordonae) and to guide antimicrobial therapy, when indicated. Species-level identification has classically relied on a variety of characteristics and methodologies, including growth rate, pigmentation, enzymatic properties (3, 4), and high-performance liquid chromatography (HPLC), ...

Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Combined Species Identification and Drug Sensitivity Testing in Mycobacteria

“…As it allows pathogen-specific culture and protein extraction protocols (34,35), we first optimized our in-house mycobacterial cell disruption and protein extraction protocol for MALDI-TOF MS analysis. Upon comparison of various described protocols using M. tuberculosis H37Rv cells (22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34), we found that in our hands, bead beating in the presence of 1.2 ml of 70% ethanol gave the best output in terms of peak intensity and identification scores (Table S1).…”

“…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. Numerous suitable procedures have been proposed, combining heat-killing, ethanol, sonication, and/or silica-based bead beating preceding the regular formic acid/acetonitrile extraction (21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33). The reported success rates vary between 55% and 98% correct species-level identifications, but the large variety in strain collections, culture conditions, extraction protocols, hardware, and databases hinder data comparison between publications.…”

“…The reported success rates vary between 55% and 98% correct species-level identifications, but the large variety in strain collections, culture conditions, extraction protocols, hardware, and databases hinder data comparison between publications. However, most studies agree that a very reliable distinction between NTM and M. tuberculosis complex can be made using MALDI-TOF MS (23)(24)(25)(26)(27)(28), which is arguably the most important task of a clinical mycobacterial lab (29).…”

Evaluation of the Vitek MS Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry System for Identification of Clinically Relevant Filamentous Fungi

“…Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been utilized in the clinical microbiology laboratory for rapid and accurate identification of bacteria, mycobacteria, and yeasts (2)(3)(4)(5)(6)(7)(8)(9). For filamentous fungi, databases have been limited, and unlike bacteria, filamentous fungi require additional processing steps to disrupt the cell wall, extract proteins, and inactivate the isolate (10).…”

Multicenter Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Study for Identification of Clinically Relevant Nocardia spp

“…Several laboratories have previously published descriptions of the use of this standard for the identification of Mycobacterium spp. (11,12). Further supplementation of existing libraries will be necessary to improve the identification of select species.…”