Newly Identified Mycobacterium africanum Lineage 10, Central Africa.

Analysis of genome sequencing data from >100,000 genomes of Mycobacterium tuberculosis complex using TB-Annotator software revealed a previously unknown lineage, proposed name L10, in central Africa. Phylogenetic reconstruction suggests L10 could represent a missing link in the evolutionary and geographic migration histories of M. africanum.

The future of CRISPR in Mycobacterium tuberculosis infection.

Clustered Regularly Interspaced Short Palindromic repeats (CRISPR)-Cas systems rapidly raised from a bacterial genetic curiosity to the most popular tool for genetic modifications which revolutionized the study of microbial physiology. Due to the highly conserved nature of the CRISPR locus in Mycobacterium tuberculosis, the etiological agent of one of the deadliest infectious diseases globally, initially, little attention was paid to its CRISPR locus, other than as a phylogenetic marker. Recent research shows that M. tuberculosis has a partially functional Type III CRISPR, which provides a defense mechanism against foreign genetic elements mediated by the ancillary RNAse Csm6. With the advent of CRISPR-Cas based gene edition technologies, our possibilities to explore the biology of M. tuberculosis and its interaction with the host immune system are boosted. CRISPR-based diagnostic methods can lower the detection threshold to femtomolar levels, which could contribute to the diagnosis of the still elusive paucibacillary and extrapulmonary tuberculosis cases. In addition, one-pot and point-of-care tests are under development, and future challenges are discussed. We present in this literature review the potential and actual impact of CRISPR-Cas research on human tuberculosis understanding and management. Altogether, the CRISPR-revolution will revitalize the fight against tuberculosis with more research and technological developments.

CRISPRbuilder-TB: "CRISPR-builder for tuberculosis". Exhaustive reconstruction of the CRISPR locus in mycobacterium tuberculosis complex using SRA.

Mycobacterium tuberculosis complex (MTC) CRISPR locus diversity has long been studied solely investigating the presence/absence of a known set of spacers. Unveiling the genetic mechanisms of its evolution requires a more exhaustive reconstruction in a large amount of representative strains. In this article, we point out and resolve, with a new pipeline, the problem of CRISPR reconstruction based directly on short read sequences in M. tuberculosis. We first show that the process we set up, that we coin as "CRISPRbuilder-TB" (https://github.com/cguyeux/CRISPRbuilder-TB), allows an efficient reconstruction of simulated or real CRISPRs, even when including complex evolutionary steps like the insertions of mobile elements. Compared to more generalist tools, the whole process is much more precise and robust, and requires only minimal manual investigation. Second, we show that more than 1/3 of the currently complete genomes available for this complex in the public databases contain largely erroneous CRISPR loci. Third, we highlight how both the classical experimental in vitro approach and the basic in silico spoligotyping provided by existing analytic tools miss a whole diversity of this locus in MTC, by not capturing duplications, spacer and direct repeats variants, and IS6110 insertion locations. This description is extended in a second article that describes MTC-CRISPR diversity and suggests general rules for its evolution. This work opens perspectives for an in-depth exploration of M. tuberculosis CRISPR loci diversity and of mechanisms involved in its evolution and its functionality, as well as its adaptation to other CRISPR locus-harboring bacterial species.

Molecular epidemiology of Mycobacterium tuberculosis in Brazil before the whole genome sequencing era: a literature review.

Molecular-typing can help in unraveling epidemiological scenarios and improvement for disease control strategies. A literature review of Mycobacterium tuberculosis transmission in Brazil through genotyping on 56 studies published from 1996-2019 was performed. The clustering rate for mycobacterial interspersed repetitive units - variable tandem repeats (MIRU-VNTR) of 1,613 isolates were: 73%, 33% and 28% based on 12, 15 and 24-loci, respectively; while for RFLP-IS6110 were: 84% among prison population in Rio de Janeiro, 69% among multidrug-resistant isolates in Rio Grande do Sul, and 56.2% in general population in São Paulo. These findings could improve tuberculosis (TB) surveillance and set up a solid basis to build a database of Mycobacterium genomes.

Unexpected diversity of CRISPR unveils some evolutionary patterns of repeated sequences in Mycobacterium tuberculosis.

BACKGROUND: Diversity of the CRISPR locus of Mycobacterium tuberculosis complex has been studied since 1997 for molecular epidemiology purposes. By targeting solely the 43 spacers present in the two first sequenced genomes (H37Rv and BCG), it gave a biased idea of CRISPR diversity and ignored diversity in the neighbouring cas-genes. RESULTS: We set up tailored pipelines to explore the diversity of CRISPR-cas locus in Short Reads. We analyzed data from a representative set of 198 clinical isolates as evidenced by well-characterized SNPs. We found a relatively low diversity in terms of spacers: we recovered only the 68 spacers that had been described in 2000. We found no partial or global inversions in the sequences, letting always the Direct Variant Repeats (DVR) in the same order. In contrast, we found an unexpected diversity in the form of: SNPs in spacers and in Direct Repeats, duplications of various length, and insertions at various locations of the IS6110 insertion sequence, as well as blocks of DVR deletions. The diversity was in part specific to lineages. When reconstructing evolutionary steps of the locus, we found no evidence for SNP reversal. DVR deletions were linked to recombination between IS6110 insertions or between Direct Repeats. CONCLUSION: This work definitively shows that CRISPR locus of M. tuberculosis did not evolve by classical CRISPR adaptation (incorporation of new spacers) since the last most recent common ancestor of virulent lineages. The evolutionary mechanisms that we discovered could be involved in bacterial adaptation but in a way that remains to be identified.

Assessment of tuberculosis spatial hotspot areas in Antananarivo, Madagascar, by combining spatial analysis and genotyping.

BACKGROUND: Tuberculosis (TB) remains a public health problem in Madagascar. A crucial element of TB control is the development of an easy and rapid method for the orientation of TB control strategies in the country. Our main objective was to develop a TB spatial hotspot identification method by combining spatial analysis and TB genotyping method in Antananarivo. METHODS: Sputa of new pulmonary TB cases from 20 TB diagnosis and treatment centers (DTCs) in Antananarivo were collected from August 2013 to May 2014 for culture. Mycobacterium tuberculosis complex (MTBC) clinical isolates were typed by spoligotyping on a Luminex® 200 platform. All TB patients were respectively localized according to their neighborhood residence and the spatial distribution of all pulmonary TB patients and patients with genotypic clustered isolates were scanned respectively by the Kulldorff spatial scanning method for identification of significant spatial clustering. Areas exhibiting spatial clustering of patients with genotypic clustered isolates were considered as hotspot TB areas for transmission. RESULTS: Overall, 467 new cases were included in the study, and 394 spoligotypes were obtained (84.4%). New TB cases were distributed in 133 of the 192 Fokontany (administrative neighborhoods) of Antananarivo (1 to 15 clinical patients per Fokontany) and patients with genotypic clustered isolates were distributed in 127 of the 192 Fokontany (1 to 13 per Fokontany). A single spatial focal point of epidemics was detected when ignoring genotypic data (p = 0.039). One Fokontany of this focal point and three additional ones were detected to be spatially clustered when taking genotypes into account (p < 0.05). These four areas were declared potential TB transmission hotspots in Antananarivo and will be considered as priority targets for surveillance in the future. CONCLUSION: This method, combining spatial analysis and TB genotyping will now be used for further focused clinical and epidemiological studies in Madagascar and will allow better TB control strategies by public health authorities.

Correlation between the BACTEC MGIT 960 culture system with Genotype MTBDRplus and TB-SPRINT in multidrug resistant Mycobacterium tuberculosis clinical isolates from Brazil.

BACKGROUND: The accurate detection of multidrug-resistant tuberculosis (MDR-TB) is critical for the application of appropriate patient treatment and prevention of transmission of drug-resistant Mycobacterium tuberculosis isolates. The goal of this study was to evaluate the correlation between phenotypic and molecular techniques for drug-resistant tuberculosis diagnostics. Molecular techniques used were the line probe assay genotype MTBDRplus and the recently described tuberculosis-spoligo-rifampin-isoniazid typing (TB-SPRINT) bead-based assay. Conventional drug susceptibility testing (DST) was done on a BACTECTM MGIT 960 TB. METHOD: We studied 80 M. tuberculosis complex (MTC) clinical isolates from Minas Gerais state, of which conventional DST had classified 60 isolates as MDR and 20 as drug susceptible. FINDINGS: Among the 60 MDR-TB isolates with MGIT as a reference, sensitivity, specificity, accuracy, and kappa for rifampicin (RIF) resistance using TB-SPRINT and MTBDRplus, were 96.7% versus 93.3%, 100.0% versus 100.0%, 97.5% versus 95.0% and 0.94 versus 0.88, respectively. Similarly, the sensitivity, specificity, accuracy, and kappa for isoniazid (INH) resistance were 85.0% and 83.3%, 100.0% and 100.0%, 88.8% and 87.5% and 0.74 and 0.71 for both tests, respectively. Finally, the sensitivity, specificity, accuracy, and kappa for MDR-TB were 85.0% and 83.3%, 100.0% and 100.0%, 88.8% and 87.5% and 0.74 and 0.71 for both tests, respectively. MAIN CONCLUSIONS: Both methods exhibited a good correlation with the conventional DST. We suggest estimating the cost-effectiveness of MTBDRplus and TB-SPRINT in Brazil.

Genetic diversity and molecular epidemiology of multidrug-resistant Mycobacterium tuberculosis in Minas Gerais State, Brazil.

BACKGROUND: We aimed to characterize the genetic diversity of drug-resistant Mycobacterium tuberculosis (MTb) clinical isolates and investigate the molecular epidemiology of multidrug-resistant (MDR) tuberculosis from Minas Gerais State, Brazil. METHODS: One hundred and four MTb clinical isolates were assessed by IS6110-RFLP, 24-locus mycobacterial interspersed repetitive units variable-number tandem repeats (MIRU-VNTR), TB-SPRINT (simultaneous spoligotyping and rifampicin-isoniazid drug-resistance mutation analysis) and 3R-SNP-typing (analysis of single-nucleotide polymorphisms in the genes involved in replication, recombination and repair functions). RESULTS: Fifty-seven different IS6110-RFLP patterns were found, among which 50 had unique patterns and 17 were grouped into seven clusters. The discriminatory index (Hunter and Gaston, HGDI) for RFLP was 0.9937. Ninety-nine different MIRU-VNTR patterns were found, 95 of which had unique patterns and nine isolates were grouped into four clusters. The major allelic diversity index in the MIRU-VNTR loci ranged from 0.6568 to 0.7789. The global HGDI for MIRU-VNTR was 0.9991. Thirty-two different spoligotyping profiles were found: 16 unique patterns (n = 16) and 16 clustered profiles (n = 88). The HGDI for spoligotyping was 0.9009. The spoligotyped clinical isolates were phylogenetically classified into Latin-American Mediterranean (66.34 %), T (14.42 %), Haarlem (5.76 %), X (1.92 %), S (1.92 %) and U (unknown profile; 8.65 %). Among the U isolates, 77.8 % were classified further by 3R-SNP-typing as 44.5 % Haarlem and 33.3 % LAM, while the 22.2 % remaining were not classified. Among the 104 clinical isolates, 86 were identified by TB-SPRINT as MDR, 12 were resistant to rifampicin only, one was resistant to isoniazid only, three were susceptible to both drugs, and two were not successfully amplified by PCR. A total of 42, 28 and eight isolates had mutations in rpoB positions 531, 526 and 516, respectively. Correlating the cluster analysis with the patient data did not suggest recent transmission of MDR-TB. CONCLUSIONS: Although our results do not suggest strong transmission of MDR-TB in Minas Gerais (using a classical 100 % MDR-TB identical isolates cluster definition), use of a smoother cluster definition (>85 % similarity) does not allow us to fully eliminate this possibility; hence, around 20-30 % of the isolates we analyzed might be MDR-TB transmission cases.

Validation of a microbead-based format for spoligotyping of Legionella pneumophila.

A 42-plex clustered regularly interspaced short palindromic repeat (CRISPR)-based typing technique (spoligotyping) was recently developed at the French National Reference Center for Legionella. It allows the subtyping of the Legionella pneumophila sequence type 1/Paris pulsotype. In this report, we present the transfer of the membrane-based spoligotyping technique to a microbead-based multiplexed format. This microbead-based high-throughput assay uses devices such as Luminex 200 or the recently launched Magpix system (Luminex Corp., Austin, TX). We designated this new technique LP-SPOL (for L. pneumophila spoligotyping). We used two sets of samples previously subtyped by the membrane-based spoligotyping method to set up and validate the transfer on the two microbead-based systems. The first set of isolates (n = 56) represented the whole diversity of the CRISPR patterns known to date. These isolates were used for transfer setup (determination of spacer cutoffs for both devices). The second set of isolates (n = 245) was used to validate the transfer to the two microbead-based systems. The results obtained by the Luminex 200 system were 100% concordant with those obtained by the Magpix system for the 2 sets of isolates. In total, 10 discrepant results were observed when comparing the membrane-based method to the microbead-based method. These discrepancies were further resolved by repeating either the membrane-based or the microbead-based assay. This new assay is expected to play an emerging role for surveillance of L. pneumophila, starting with one of the most frequent genotypes, the sequence type 1/Paris pulsotype. However, the generalization of this typing method to all L. pneumophila strains is not feasible, since not all L. pneumophila strains contain CRISPRs.

Molecular epidemiology of tuberculosis in Sicily, Italy: what has changed after a decade?

BACKGROUND: We aimed to investigate the molecular epidemiology of Mycobacterium tuberculosis complex (MTBC) isolates in the province of Palermo, Sicily, Italy, by characterizing 183 isolates identified in the years 2004-2012. A comparison with 104 MTBC strains identified in the same geographic area in the years 1994-2000 was also carried out. METHODS: One hundred eighty-three MTBC isolates identified in Palermo, Italy, in the years 2004-2012 were analyzed by spoligotyping and the 24 mycobacterial interspersed repetitive unit (MIRU)-variable-number tandem-repeat (VNTR) method typing. Susceptibility testing to streptomycin, isoniazid, rifampin and ethambutol was also performed. Furthermore, the spoligotyping dataset obtained from 104 MTBC isolates identified from 1994 to 2000 was reanalyzed. Distribution into lineages and clustering of isolates in the two periods was compared. RESULTS: One hundred seventy-seven out of the 183 isolates of MTBC submitted to molecular typing were fully characterized. Of these, 108 were from Italian-born and 69 from foreign-born individuals. Eleven different lineages and 35 families-subfamilies were identified with the most represented lineages being Haarlem (26.5%), T (19.2%), LAM (13.6%) and S (8.5%). Except for the Haarlem lineage, where isolates from foreign-born patients were overrepresented, the distribution of isolates in the families belonging to the Euro-American clone reflected the proportions of the two subpopulations. A total of 27 (15.2%) strains were clustered and three clusters were mixed. Approximately 25% of the 183 MTBC isolates under study proved to be resistant to at least one antiTB drug, with only three isolates categorized as multidrug resistant (MDR). When MTBC isolates identified in the years 1994-2000 were reanalyzed, lineages T (30.8%), LAM (29.8%), Haarlem (16.3%) and S (13.5%) proved to be predominant. No MTBC isolates belonging to CAM, U, CAS, Turkish and Ural lineages were identified. CONCLUSIONS: A wide heterogeneity was detected among the MTBC strains isolated in the years 2004-2012. Six lineages were not present among the isolates of the period 1994-2000. Comparison between distribution of lineages in the two consecutive periods depicts rapid and deep changes in the TB epidemiology in Palermo, Italy. An universal and continued laboratory-based surveillance of TB in Sicily is required.

Tuberculosis-spoligo-rifampin-isoniazid typing: an all-in-one assay technique for surveillance and control of multidrug-resistant tuberculosis on Luminex devices.

As a follow-up of the "spoligoriftyping" development, we present here an extension of this technique which includes the detection of isoniazid resistance-associated mutations in a new 59-plex assay, i.e., tuberculosis-spoligo-rifampin-isoniazid typing (TB-SPRINT), running on microbead-based multiplexed systems. This assay improves the synergy between clinical microbiology and epidemiology by providing (i) mutation-based prediction of drug resistance profiles for patient treatment and (ii) genotyping data for tuberculosis (TB) surveillance. This third-generation microbead-based high-throughput assay for TB runs on the Luminex 200 system and on the recently launched MagPix system (Luminex, Austin, TX). Spoligotyping patterns obtained by the TB-SPRINT method were 100% (n = 85 isolates; 3,655/3,655 spoligotype data points) concordant with those obtained by microbead-based and membrane-based spoligotyping. Genetic drug susceptibility typing provided by the TB-SPRINT method was 100% concordant with resistance locus sequencing (n = 162 for rpoB gene sequencing and n = 76 for katG and inhA sequencing). Considering phenotypic drug susceptibility testing (DST) as the reference method, the sensitivity and specificity of TB-SPRINT regarding Mycobacterium tuberculosis complex (n = 162 isolates) rifampin resistance were both 100%, and those for isoniazid resistance were 90.4% (95% confidence interval, 85 to 95%) and 100%, respectively. Used routinely in national TB reference and specialized laboratories, the TB-SPRINT assay should simultaneously improve personalized medicine and epidemiological surveillance of multidrug-resistant (MDR) TB. This assay is expected to play an emerging role in public health in countries with heavy burdens of MDR TB and/or HIV/TB coinfection. Application of this assay directly to biological samples, as well as development for extensively drug-resistant (XDR) TB detection by inclusion of second-line antituberculosis drug-associated mutations, is under development. With bioinformatical methods and data mining to reduce the number of targets to the most informative ones, locally adapted formats of this technique can easily be developed everywhere.

Towards a better understanding of the long-lasting evolutionary history of Mycobacterium tuberculosis.

The daily increasing sequencing of Mycobacterium tuberculosis has made it possible to establish an advanced phylogeny of this bacterium. It currently includes 9 lineages mainly affecting humans, completed by animal lineages, which form the Mycobacterium tuberculosis complex. Inherited from various historical approaches, this phylogeny is now based on Single Nucleotide Polymorphisms (SNPs), of which updates are frequently proposed. We present here evidence that the task needs refinements: some lineages have currently suboptimal defining SNPs, and many sublineages still need to be named and characterized. These findings are based on a new tool specifically designed to index the entire existing sequencing data. In this article, we focus on lineages 4.5, 4.7, 6 and 7. We take the opportunity to present some evidence that TB-annotator shows strong relevance, identifying well supported sublineages, as well as good global agreement with previous findings.

Comparison of in silico predicted Mycobacterium tuberculosis spoligotypes and lineages from whole genome sequencing data.

Bacterial strain-types in the Mycobacterium tuberculosis complex underlie tuberculosis disease, and have been associated with drug resistance, transmissibility, virulence, and host-pathogen interactions. Spoligotyping was developed as a molecular genotyping technique used to determine strain-types, though recent advances in whole genome sequencing (WGS) technology have led to their characterization using SNP-based sub-lineage nomenclature. Notwithstanding, spoligotyping remains an important tool and there is a need to study the congruence between spoligotyping-based and SNP-based sub-lineage assignation. To achieve this, an in silico spoligotype prediction method ("Spolpred2") was developed and integrated into TB-Profiler. Lineage and spoligotype predictions were generated for > 28 k isolates and the overlap between strain-types was characterized. Major spoligotype families detected were Beijing (25.6%), T (18.6%), LAM (13.1%), CAS (9.4%), and EAI (8.3%), and these broadly followed known geographic distributions. Most spoligotypes were perfectly correlated with the main MTBC lineages (L1-L7, plus animal). Conversely, at lower levels of the sub-lineage system, the relationship breaks down, with only 65% of spoligotypes being perfectly associated with a sub-lineage at the second or subsequent levels of the hierarchy. Our work supports the use of spoligotyping (membrane or WGS-based) for low-resolution surveillance, and WGS or SNP-based systems for higher-resolution studies.

Towards the reconstruction of a global TB history using a new pipeline "TB-Annotator".

Mycobacterium tuberculosis complex (MTBC) has a population structure consisting of 9 human and animal lineages. The genomic diversity within these lineages is a pathogenesis factor that affects virulence, transmissibility, host response, and antibiotic resistance. Hence it is important to develop improved information systems for tracking and understanding the spreading and evolution of genomes. We present results obtained thanks to a new informatics platform for computational biology of MTBC, that uses a convenience sample from public/private SRAs, designated as TB-Annotator. Version 1 was a first interactive graphic-based web tool based on 15,901 representative genomes. Version 2, still interactive, is a more sophisticated database, developed using the Snakemake Workflow Management System (WMS) that allows an unsupervised global and scalable analysis of the content of the USA National Center for Biotechnology Information Short Read Archives database. This platform analyzes nucleotide variants, the presence/absence of genes, known regions of difference and detect new deletions, the insertion sites of mobile genetic elements, and allows phylogenetic trees to be built, imported in a graphical interface and interactively analyzed between the data and the tree. The objective of TB-Annotator is triple: detect recent epidemiological links, reconstruct distant phylogeographical histories as well as perform more complex phenotypic/genotypic Genome-Wide Association Studies (GWAS). In this paper, we compare the various taxonomic SNPs-based labels and hierarchies previously described in recent reference papers for L1, and present a comparative analysis that allows identification of alias and thus provides the basis of a future unifying naming scheme for L1 sublineages. We present a global phylogenetic tree built with RAxML-NG, and one on L2; at the time of writing, we characterized about 200 sublineages, with many new ones; a detail tree for Modern L2 and a hierarchical scheme allowing to facilitate L2 lineage assignment are also presented.

Spoligotyping of Mycobacterium africanum, Burkina Faso.

Using Ziehl-Neelsen-positive slides collected from tuberculosis diagnostic centers in Burkina Faso, we showed that 20% of 80 spoligotyping-positive DNA samples had a characteristic Mycobacterium africanum-specific genomic signature. This result suggests that M. africanum is still present in Burkina Faso at almost the same prevalence as 15-20 years ago.

"Spoligoriftyping," a dual-priming-oligonucleotide-based direct-hybridization assay for tuberculosis control with a multianalyte microbead-based hybridization system.

We developed "spoligoriftyping," a 53-plex assay based on two preexisting methods, the spoligotyping and "rifoligotyping" assays, by combining them into a single assay. Spoligoriftyping allows simultaneous spoligotyping (i.e., clustered regularly interspaced short palindromic repeat [CRISPR]-based genotyping) and characterization of the main rifampin drug resistance mutations on the rpoB hot spot region in a few hours. This test partly uses the dual-priming-oligonucleotide (DPO) principle, which allows simultaneous efficient amplifications of rpoB and the CRISPR locus in the same sample. We tested this method on a set of 114 previously phenotypically and genotypically characterized multidrug-resistant (MDR) Mycobacterium tuberculosis or drug-susceptible M. tuberculosis DNA extracted from clinical isolates obtained from patients from Bulgaria, Nigeria, and Germany. We showed that our method is 100% concordant with rpoB sequencing results and 99.95% (3,911/3,913 spoligotype data points) correlated with classical spoligotyping results. The sensitivity and specificity of our assay were 99 and 100%, respectively, compared to those of phenotypic drug susceptibility testing. Such assays pave the way to the implementation of locally and specifically adapted methods of performing in a single tube both drug resistance mutation detection and genotyping in a few hours.

Mycobacterium tuberculosis Diversity Exploration: A Way to Serve the Three Main Weapons against Epidemics, Hygiene, Vaccine Development and Chemotherapy.

As highlighted by the SARS pandemic which is still ongoing, the battle against pathogens relies on three main "weapons": hygiene, vaccine development and chemotherapy strategies [...].

Investigating the Diversity of Tuberculosis Spoligotypes with Dimensionality Reduction and Graph Theory.

The spoligotype is a graphical description of the CRISPR locus present in Mycobacterium tuberculosis, which has the particularity of having only 68 possible spacers. This spoligotype, which can be easily obtained either in vitro or in silico, allows to have a summary information of lineage or even antibiotic resistance (when known to be associated to a particular cluster) at a lower cost. The objective of this article is to show that this representation is richer than it seems, and that it is under-exploited until now. We first recall an original way to represent these spoligotypes as points in the plane, allowing to highlight possible sub-lineages, particularities in the animal strains, etc. This graphical representation shows clusters and a skeleton in the form of a graph, which led us to see these spoligotypes as vertices of an unconnected directed graph. In this paper, we therefore propose to exploit in detail the description of the variety of spoligotypes using a graph, and we show to what extent such a description can be informative.

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

To tackle the spread of tuberculosis (TB), epidemiological studies are undertaken worldwide to investigate TB transmission chains. Clustered regulatory interspaced short palindromic repeats (CRISPR) locus diversity, also called spoligotyping, is a widely used genotyping assay for the characterization of Mycobacterium tuberculosis complex (MTBC). We compared herein the spoligotyping of MTBC clinical isolates using a membrane-based method (following an initial PCR step) and whole-genome sequencing (WGS)-based method (i.e., in silico spoligotyping). All MTBC strains isolated at the Lyon University Hospital, France, between November 2016 and December 2020 were included (n = 597). Spoligotyping profiles were also used for species identification among the MTBC. Outputs of both methods were analyzed, and discrepant results were investigated thanks to CRISPRbuilder-TB. The overall agreement was 85.7%. Spacer discrepancies observed between the methods were due to the insertion of IS6110 within the direct repeat (DR) sequence upstream or downstream of spacers, mutated DR sequences, or truncated spacers. Discrepancies did not impact species identification. Although spoligotyping-based species identification was inconclusive for 29 isolates, SNP-based phylogeny conducted after WGS allowed the identification of 23 M. tuberculosis (Mtb), 2 M. canettii, and 4 mixed MTBC infections. WGS yielded very few discrepancies compared to membrane-based spoligotyping. Overall agreement was significantly improved (92.4%) by the CRISPR locus reconstruction using CRISPRbuilder-TB for the MTBC isolates with the shared international type 53 in silico spoligotyping. A smooth transition from the membrane-based to the in silico-based genotyping of M. tuberculosis isolates is, therefore, possible for TB diagnosis and epidemiologic survey. IMPORTANCE 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. Several pipelines have been developed to extract a spoligotype from WGS data (in silico spoligotyping) allowing for the continuity of MTBC molecular surveys before and after WGS implementation. The present study found very good overall agreement between hybridization to membrane-based spoligotyping and in silico spoligotyping, indicating the possibility of a smooth transition from the traditional to the in silico-based genotyping of MTBC isolates for TB diagnosis and epidemiological survey.