Generation of a database of mass spectra patterns of selected Mycobacterium species using MALDI-ToF mass spectrometry

[摘要] The genus Mycobacterium is a group of acid–fast, aerobic, slow- growing organisms which includemore than 90 different species. A member of this genus, Mycobacterium tuberculosis, belonging tothe Mycobacterium tuberculosis complex (MTB), is the causative agent of tuberculosis (TB). Thisdisease is currently considered a global emergency, with more than 2 million deaths and over 8million new cases annually. TB is the world's second most common cause of death afterHIV/AIDS. About one-third of the world's population is estimated to be infected with TB. Thiscatastrophic situation is further compounded by the emergence of Multi Drug Resistant tuberculosis(MDR-TB) and in more recent times, Extensive Drug Resistant tuberculosis (XDR-TB). Earlydiagnosis is critical to the successful management of patients as it allows informed use ofchemotherapy. Also, early diagnosis is also of great importance if the menace of MDR-TB andXDR-TB is to be curbed and controlled.As MTB is highly infectious for humans, it is of paramount importance that TB be diagnosed asearly as possible to stop the spread of the disease. Traditional conventional laboratory proceduresinvolving microscopy, culture and sensitivity tests may require turnaround times of 3-4 weeks orlonger. Tremendous technological advancement over the years such as the advent of automatedliquid culture systems like the BACTEC® 960 and the MGITTM Tube system, and the developmentof a myriad of molecular techniques most of which involves nucleic acid amplification (NAA) forthe rapid identification of mycobacterial isolates from cultures or even directly from clinicalspecimens have contributed immensely to the early diagnosis of tuberculosis. Most of these NAAtests are nevertheless fraught with various limitations, thus the search for a rapid, sensitive andspecific way of diagnosing tuberculosis is still an active area of research. The search has expanded to areas that would otherwise not have been considered 'conventional' in diagnosticmycobacteriology. One of such areas is mass spectrometry.This study joins the relatively few studies of its kind encountered in available literature to establishthe ground work for the application of mass spectrometry, specifically Matrix Assisted LaserDesorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-ToF MS) in the field ofdiagnostic mycobacteriology. This is an area which is in need of the speed, sensitivity andspecificity that MALDI-ToF technique promises to offer. Since this technology is still in itsinfancy, the use of utmost care in the preparation of reagents, and the handling and storage of theorganisms used to generate reference mass spectra for the database cannot be overemphasized.Similarly, the optimization of certain crucial experimental factors such as inactivating method andchoice of matrix is of paramount importance.The main aim of this thesis was to generate a database of reference mass spectra fingerprints ofselected (repository) Mycobacterium species. This necessitated the standardization of anexperimental protocol which ensured that experimental factors and the various instrumentparameters were optimized for maximum spectra generation and reproducibility. A standardoperating procedure (SOP) for generating the database of reference mass spectra finger print ofselected Mycobacterium species was developed and used to investigate the ability of the database todifferentiate between species belonging to the same clinical disease complex as well as the nontuberculosiscomplex.The findings of this study imply that if the defined protocol is followed, the database generated hasthe potential to routinely identify and differentiate (under experimental conditions) more species of Mycobacterium than is currently practical using PCR and its related techniques. It is therefore arealistic expectation that when the database is clinically validated and tested in the next phase of thestudy, it will contribute immensely to the diagnosis of tuberculosis and other mycobacterioses. Itwill also aid in the identification of emerging pathogens particularly amongst the non-tuberculousmycobacteria.

[发布机构] Stellenbosch University