MALDI-TOF mass spectrometry from nucleic acid: development and evaluation of a novel platform for identification of mycobacteria and detection of genetic markers of resistance

Abstract

ABSTRACT Complete identification methods are critical for evaluating nontuberculous mycobacteria (NTM). Here, we describe a novel diagnostic method for identification of eight NTM, Mycobacterium tuberculosis complex, and three drug resistance markers using PCR/matrix-assisted, laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) from cultured organisms. With this technology, a multiplex end-point PCR is performed for targets of interest. Detection probes that are extended in the presence of a target are added. The extended probes have greater molecular weight and can be detected by MALDI-TOF MS. An AFB Primary Panel was designed to differentiate Mycobacterium avium; Mycobacterium intracellulare subsp. chimaera; Mycobacterium avium complex (other); Mycobacterium abscessus subsp. abscessus, bolletii, and massiliense; Mycobacterium kansasii, and M. tuberculosis complex. This design should cover 90% (3,483/3,691) of mycobacteria seen onsite. A development set of unblinded isolates (n = 217) was used to develop PCR primers, detection probes, and probe barcodes. It demonstrated 99.1% (215/217) agreement with reference methods. An evaluation set using blinded isolates (n = 320) showed an overall sensitivity of 94.3% (range by target: 90.0–100%). Overall specificity from negative media, non-target mycobacteria, and bacteria was 99.1% (108/109; range by target: 94.4–100%). Three drug resistance markers erm (41), rrl, and rrs demonstrated 100%, 91%, and 100% sensitivity, respectively, and >99% specificity. Limit of detection per target ranged from 2.2 × 103 to 9.9 × 106 CFU/mL. The AFB Primary Panel allows for mycobacterial speciation, subspeciation, and resistance mutation detection, which is essential for diagnosis, appropriate therapy, identifying outbreaks, and managing treatment-refractory disease. It can perform with high-throughput and high specificity and sensitivity from isolates.IMPORTANCEEven closely related mycobacteria can have unique treatment patterns, but differentiating these organisms is a challenge. Here, we tested an innovative platform that combines two commonly used technologies and creates something new: matrix-assisted, laser-desorption ionization time-of flight mass spectrometry was performed on PCR amplicons instead of on proteins. This created a robust system with the advantages of PCR (high discriminatory power, high throughput, detection of resistance) with the advantages of mass spectrometry (more targets, lower operational cost) in order to identify closely related mycobacterial organisms.

Keywords

• PCR
• MassArray
• MALDI-TOF MS
• mycobacteria
• rrs
• rrl