Spoligotyping of Mycobacterium tuberculosis isolates using Luminex®-based method in Lebanon

Abstract

Introduction: Data about the genotypes of circulating Mycobacterium tuberculosis isolates (MTB) in Lebanon are scarce. This study was undertaken to reveal the spoligotypes of MTB isolates recovered from patients in Lebanon.
 Methodology: MTB isolates from 49 patients living in Lebanon were recovered and identified. The samples were heat killed and subjected to DNA extraction. Spoligotyping was performed using microbeads from TB-SPOL Kit and the fluorescence intensity was measured using Luminex 200®. Generated patterns were assigned to families using the SITVIT2 international database of the Pasteur Institute of Guadeloupe and compared.
 Results: The spoligotyping of the 49 MTB isolates revealed that 31 isolates belonged to Lineage 4 (Euro-American, 63.3%), 12 to Lineage 3 (East- African Indian, 24.5%), 3 to Lineage 2 (East Asian, 6%) and 2 were unknown. Over half of the genotypes (16 of 30) harbored SIT127 supposed to belong to the L4.5 sublineage. One isolate belonging to the rare Manu-Ancestor SIT523 was recovered for the first time in Lebanon, being associated with highly virulent extensively drug-resistant (XDR) MTB phenotype.
 Conclusion: The application of the Spoligotyping Multiplex Luminex® method is an efficient, discriminatory and rapid method to use for first-lane genotyping of MTB isolates. Though humble numbers were tested, this study is one of the first to describe the genomic diversity and epidemiology of MTB isolates of Lebanon, and suggests an increasing prevalence of SIT127 in the country.

Molecular Epidemiology and Polymorphism Analysis in Drug-Resistant Genes in M. tuberculosis Clinical Isolates from Western and Northern India

“… 74 While some reports discuss Manu ancestral lineage as an artefact due to co-infection by strains of Lineage 2 and 4, we are not in a position to comment, as this study did not examine any co-infection or polyclonal infections. 75 , 76 …”

Recent progress in the genotyping of bovine tuberculosis and its rapid diagnosis via nanoparticle-based electrochemical biosensors

“…61 This method has the benet of being able to use DNA isolated from infected tissue samples without the need for bacterial isolation. 62 Further automatization and development of this method has been directed to employ recognition systems based on microbeads, like Luminex platforms, 63 multiplexed primer extension depending on spoligotyping assay using matrixassisted laser desorption ionization time of ight mass spectrometry (MALDI-TOF MS), 64 microarray, 65,66 melting curve analysis and ligation based amplication. 58 One of the most important advantages of spoligotyping is its ability to be used directly in clinical samples without the requirement for preparatory culture due to its true sensitivity estimated at 10 fg of chromosomal DNA, or the DNA of 2-3 bacterial cells.…”