Design, development and validation of a series of multiplex real-time PCR diagnostics assays for the rapid and accurate detection and differentiation of the Mycobacterium tuberculosis complex
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Tuberculosis (TB) remains a major health concern both in developed and developing countries due to the high rates of morbidity and mortality associated with disease. In humans, TB is caused by members of the Mycobacterium tuberculosis complex (MTC) namely M. tuberculosis, M. canettii, M. africanum, M. bovis, M. caprae, M. microti, M. pinnipedii and the attenuated M. bovis BCG vaccine strain. This group of microorganisms are ~99% similar on a nucleotide sequence level and have a wide range of natural hosts. While M. tuberculosis is responsible for the majority of cases of human TB, accurate identification of other members of the MTC causing infection is not routinely performed. As a result, the global frequency and distribution of each member of the MTC remains largely unknown with some studies suggesting that TB caused by members of the MTC other than M. tuberculosis are in fact underreported. Therefore, the capability to accurately identify each member of the MTC causing human TB infection would be desirable. This would enable unambiguous TB epidemiological studies and monitoring of human to human and/or zoonotic TB transmission. Equally, differentiation of the MTC is clinically important for treatment management decisions due to the inherent natural resistance of some members of the complex to the first line anti-TB drug pyrazinamide (PZA). Currently there is only one commercially available diagnostics assay for differentiation of the MTC, the Genotype MTBC kit. This diagnostics kit is limited by its inability to accurately identify M. tuberculosis, M. canettii, M. africanum and M. pinnipedii. There are a number of molecular based assays for MTC differentiation described in the literature which are also limited by an inability to differentiate all members of the MTC and most require post amplification processing increasing method complexity, analysis time and potential contamination. The overall aim of this study was to design, develop, optimise and validate a robust, internally controlled, multiplex real-time PCR based method for the rapid and accurate identification of all members of the MTC. This was achieved using a sequential experimental design consisting of three main studies. In the first study, a multiplex real-time PCR diagnostics assay using novel molecular targets was designed to identify the MTC while simultaneously differentiating between M. tuberculosis and M. canettii. In the second study, a multiplex real-time PCR assay was developed for the identification and simultaneous differentiation of M. bovis, M. bovis BCG and M. caprae in one internally controlled reaction. In the third study two additional biomarkers were incorporated into the previous two multiplex assays specific for the two clades of M. africanum. By incorporation of these two additional targets, it was possible to devise a diagnostic algorithm to differentiate all 8 members of the MTC. The specificity of this method was optimised and validated against a panel of 119 MTC isolates which had been previously characterised using methods such as spoligotyping, mycobacterial interspersed repetitive units - variable number tandem repeats (MIRU-VNTR), IS6110-based typing methods, RD analysis, biochemical testing in addition to morphological examination. Specificity was also demonstrated against 44 Non Tuberculosis Mycobacteria (NTM) and 17 other bacterial species. Analytical sensitivities of less than 100 genome equivalents were determined for each diagnostics assay developed in multiplex format. Further evaluation of SeekTB was performed by blindly testing 125 Mycobacteria Growth Indicator Tube (MGIT) positive cultures. The results of SeekTB were compared to those obtained using the commercially available GenoType MTBC and TBc ID tests. SeekTB and GenoType MTBC test results were 100% concordant identifying 84 isolates as M. tuberculosis and 41 as non MTC. Nine discordant results were observed between the molecular methods and the TBc ID culture confirmation test, however, nucleotide sequencing of the discordant isolates confirmed the results obtained with SeekTB and GenoType MTBC tests In summary, SeekTB, the diagnostic method developed in this study, is the first description of an internally controlled multiplex real-time PCR based diagnostics method for the accurate identification of all eight members of the MTC. This method, designed for use on cultured patient samples, is specific, sensitive and rapid with a turnaround time to results of approximately 1.5 to 3.5 h, depending on which, if any, member of the MTC is present.
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