Consistency of Mycobacterium tuberculosis Complex Spoligotyping between the Membrane-Based Method and In Silico Approach

Abstract

Whole-genome sequencing (WGS) has profoundly transformed the perspectives of tuberculosis (TB) diagnosis, providing a better discriminatory power to determine relatedness between Mycobacterium tuberculosis complex (MTBC) isolates. Previous genotyping approaches, such as spoligotyping consisting of an initial PCR step followed by reverse dot hybridization, are currently being replaced by WGS.

Development, Evaluation, and Implementation of a House-Made Targeted Next-Generation Sequencing Spoligotyping in a French Laboratory

“…Between January 2017 and February 2018, 144 MTBC isolates from specimens sampled from patients during routine care in the Hospices Civils de Lyon, France were retrospectively selected to be representative of the local epidemiology. MTBC strains were separated in two homogenous sets (in terms of lineage diversity, both representing the local epidemiology (Genestet et al 2022 [ 10 ]; Barbier et al 2018 [ 13 ])): A training set (80 strains) and a validation set (64 strains; Figure 1 ).…”

“…The MTBC CRISPR locus, which is the preferential insertion site for the IS6110 that possibly disrupts DR or adjacent spacer sequences [ 15 ] of both DR variations and IS insertion, may hamper primer affinity resulting in incomplete or abortive DNA amplification. These genomic alterations (the insertion of IS6110 within the DR sequence upstream or downstream of the spacers, the presence of mutated DR, or the presence of truncated spacers) modify the expected spoligotype patterns, despite the presence of spacers within the CRISPR locus [ 10 , 16 , 17 , 18 ]. As observed herein, the most discordant spacer due to the insertion of IS6110 between the in silico spoligotyping using WGS and the PCR-based spoligotyping (membrane-based method and tNGS spoligotyping) was spacer 31 [ 10 , 15 , 16 , 17 ].…”

“…These genomic alterations (the insertion of IS6110 within the DR sequence upstream or downstream of the spacers, the presence of mutated DR, or the presence of truncated spacers) modify the expected spoligotype patterns, despite the presence of spacers within the CRISPR locus [ 10 , 16 , 17 , 18 ]. As observed herein, the most discordant spacer due to the insertion of IS6110 between the in silico spoligotyping using WGS and the PCR-based spoligotyping (membrane-based method and tNGS spoligotyping) was spacer 31 [ 10 , 15 , 16 , 17 ]. This led to a significant change in the local epidemiology of the laboratory during the transition to in silico spoligotyping with WGS, e.g., the disappearance of MTBC strains belonging to the SIT 50.…”

“…This led to a significant change in the local epidemiology of the laboratory during the transition to in silico spoligotyping with WGS, e.g., the disappearance of MTBC strains belonging to the SIT 50. Indeed, it has been previously shown that, using membrane-based spoligotyping, the SIT number was 50 for all MTBC strains (i.e., no detection of spacer 31), whereas using in silico spoligotyping, the SIT number was actually 53 (i.e., with detection of spacer 31) due to the insertion of IS6110 within the DR sequence downstream of spacer 31 [ 10 ]. Due to the phylogenetic relevance of this insertion [ 5 , 19 , 20 ], the classification of the corresponding strains as SIT 50 rather than SIT 53 should be preferred.…”

“…Moreover, several pipelines have been developed to extract a spoligotype from WGS data, also called in silico spoligotyping [ 9 ], enabling a continuity of MTBC molecular surveys. Previously, it has been shown that a smooth transition from the membrane-based to the in silico WGS-based genotyping of MTBC isolates was possible for TB diagnosis and epidemiological survey despite discrepancies with the membrane-based method, notably related to the insertion of sequence (IS) 6110 in the DR region flanking the spacer 31 [ 10 ].…”