Combined Species Identification, Genotyping, and Drug Resistance Detection of Mycobacterium tuberculosis Cultures by MLPA on a Bead-Based Array

Abstract

The population structure of Mycobacterium tuberculosis is typically clonal therefore genotypic lineages can be unequivocally identified by characteristic markers such as mutations or genomic deletions. In addition, drug resistance is mainly mediated by mutations. These issues make multiplexed detection of selected mutations potentially a very powerful tool to characterise Mycobacterium tuberculosis. We used Multiplex Ligation-dependent Probe Amplification (MLPA) to screen for dispersed mutations, which can be successfully applied to Mycobacterium tuberculosis as was previously shown. Here we selected 47 discriminative and informative markers and designed MLPA probes accordingly to allow analysis with a liquid bead array and robust reader (Luminex MAGPIX technology). To validate the bead-based MLPA, we screened a panel of 88 selected strains, previously characterised by other methods with the developed multiplex assay using automated positive and negative calling. In total 3059 characteristics were screened and 3034 (99.2%) were consistent with previous molecular characterizations, of which 2056 (67.2%) were directly supported by other molecular methods, and 978 (32.0%) were consistent with but not directly supported by previous molecular characterizations. Results directly conflicting or inconsistent with previous methods, were obtained for 25 (0.8%) of the characteristics tested. Here we report the validation of the bead-based MLPA and demonstrate its potential to simultaneously identify a range of drug resistance markers, discriminate the species within the Mycobacterium tuberculosis complex, determine the genetic lineage and detect and identify the clinically most relevant non-tuberculous mycobacterial species. The detection of multiple genetic markers in clinically derived Mycobacterium tuberculosis strains with a multiplex assay could reduce the number of TB-dedicated screening methods needed for full characterization. Additionally, as a proportion of the markers screened are specific to certain Mycobacterium tuberculosis lineages each profile can be checked for internal consistency. Strain characterization can allow selection of appropriate treatment and thereby improve treatment outcome and patient management.

Tuberculosis-Spoligo-Rifampin-Isoniazid Typing: an All-in-One Assay Technique for Surveillance and Control of Multidrug-Resistant Tuberculosis on Luminex Devices

“…Results are in progress (Gomgnimbou et al, unpublished data). Because methods developed with the Luminex 200 or MagPix system are highly flexible, local improvements are possible (33). This flexibility will also allow us to adapt a panel of new drug resistance targets, including different markers for different regions.…”

A cross-sectional study of sputum handling by and supervision of patients with pulmonary tuberculosis treated at home in China

“…Therefore, this study suggests that providing PTBTHs with relevant health education and encouraging a willingness to be supervised can help improve their sputum disposal. This might be explained by the fact that patients who have received health education are better able to manage TBrelated waste on their own (25). More relevant health education must be developed so that PTBTHs are better able to manage TB-related waste on their own.…”

Simplified Microarray System for Simultaneously Detecting Rifampin, Isoniazid, Ethambutol, and Streptomycin Resistance Markers in Mycobacterium tuberculosis

“…However, the number of known genes and mutations conferring resistance to first-and second-line drugs greatly exceeds the multiplexing capacity of these platforms, which may limit their clinical efficacy in the treatment and control of multidrug-resistant or extensively drug-resistant TB. Planar and suspension microarrays are well suited to address the multiple-gene, multiple-mutation challenge of diagnosing drug-resistant TB (4)(5)(6)(7)(8)(9)(10)(11)(12), but clinical adoption of microarray technology is hampered by poor reproducibility (13)(14)(15), complex workflows, and/or extensive user subjectivity and involvement in image and data analysis (16). In order to translate microarrays into efficacious TB diagnostics at the point of need, it is therefore necessary to simplify user interaction with the technology while retaining the ability to detect multiple genes and multiple mutations in a timely manner.…”