Matrix assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF MS) proteomic profiling of mycobacteria.
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Abstract
Mycobacterium tuberculosis still remains a major cause of tuberculosis(TB) and death. In
addition, nontuberculous mycobacteria (NTM) play a significant role in the aetiology of
tuberculosis-like syndromes. A diagnosis of M. tuberculosis as the causative agent of
pulmonary TB using only clinical symptoms may be inadequate and inaccurate in some cases
since M.bovis, M.avium, M.kansasii, and M.intracellulare may all produce a pulmonary
disease in humans that may be indistinguishable from that caused by M.tuberculosis.
For diagnostic purposes, current mycobacterial identification strategies to the species level
primarily rely on phenotypic analysis, complex biochemical tests, cultivation on specific
growth media and genotype analysis. However, the main disadvantages that are associated
with these techniques include extensive processing times (6-12 weeks to confirm a positive
identification) and misidentification. Both factors severely hinder the control and
management of diseases associated with mycobacteria, particularly tuberculosis. Apart from
an increased patient mortality, interpersonal transmission of mycobacterial infections may
increase. This scenario will also result in the prescription of inappropriate anti-tubercular
treatment regimes.
The present study assesses the feasibility of using MALDI-TOF MS proteomic profiling as a
rapid and precise technology to identify mycobacteria in Kwazulu-Natal, South Africa which
is currently a global epicentre of mycobacterial-associated diseases. In this study a modified
EFA protein extraction protocol that incorporates an initial cell envelope delipidation step was
formulated and shown to produce mass spectra that were unique and highly reproducible. The
spectra were used to create an independent main spectral profile reference library (CMEFAMSP)
representing clinically relevant American Type Culture Collection (ATCC)
mycobacterial strains. Interestingly, this proof of concept study clearly demonstrates that
MALDI-TOF MS-based biotyping of mycobacteria using the CMEFA-MSP reference library
correctly identified 11 blind-coded ATCC strains sourced from an autonomous facility to the
genus and species level with 100% accuracy.In addition the CMEFA-MSP reference library
was employed to differentiate 39 blind-coded clinical mycobacterial isolates. Importantly all
CMEFA-derived samples of the 39 clinical mycobacterial isolates displayed log score values
of ≥ 2.3 and were correctly identified to the species level. This strongly suggests that MALDITOF
MS when used in conjunction with the CMEFA sample preparation protocol has
potential as a simple and cost-effective alternative for the unambiguous identification of
clinically important mycobacteria. Moreover, individual members of the MTBC and NTM
groups used in this study were distinguished from each other.
Description
M. Med. Sc. University of KwaZulu-Natal, Durban 2015.
Keywords
Mycobacterium tuberculosis., Mycobacteria--Identification., Mycobacterial diseases., Proteomics., Theses--Pharmacy and pharmocology.