Biotyping of clinical mycobacterium tuberculosis isolates using MALDI-TOF MS.

Abstract

Tuberculosis continues to be a major threat in public health; 8.8 million incidence of TB has been reported and 2 million deaths every year. Diagnosis of TB is impeded by slow growth of an organism with a generation time of 21 days. The emergence of multidrug-resistant TB isolates which are resistant to rifampicin and isoniazid worsened the treatment programme. Furthermore, surfacing of extensively drug-resistant TB isolates especially in HIV positive patients has led to a treatment failure. Currently available diagnostic methods are time consuming and laborious. Polymerase chain reaction-based assay proved to have a better resolution for TB strain discrimination, nevertheless are costly and cannot be routinely employed in resource poor settings. As a result there is an urgent need of cheap, cost effective and rapid diagnostic methods that will reduce a turnaround time. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry potentially offers an alternative rapid and cheaper method for discrimination of TB isolates. Proper discrimination of TB isolates depends on the sample preparation method that is capable of yielding high protein content. Conventional formic/ethanol sample preparation was investigated for mycobacteria MALDI-TOF mass spectrometric analysis. Poor quality of spectra was obtained due to a complex cell wall structure of mycobacteria which released less amounts of proteins. Further attempts were made to optimize the sample preparation method by introducing glass beads for maximum cell wall disruption. Non-consistent spectra were obtained in some mycobacterial strain; therefore it was not used as a method of choice. Introduction of delipidation step using chloroform/methanol (1:1, v/v) before formic/ethanol sample preparation step, led to a generation of reproducible and consistent spectra. This newly developed method was selected to extract protein content from large number of clinical TB isolates. With MALDI-TOF MS and chloroform/methanol-based method, all mycobacterial isolates used in the proof-of-concept were correctly identified and clustered. Fifty six of sixty clinical TB isolates were correctly identified using Biotyper software. Four were incorrectly identified; it might be possible that they carry mutations in unknown regions in their genome which led to a translation of proteins that affected the overall spectra profile. MALDI-TOF MS showed the potential to be used in the clinical laboratories for discrimination of TB isolates at lower costs.