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.

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.

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.

Mycobacterium tuberculosis complex drug-resistance, phylogenetics, and evolution in Nigeria: Comparison with Ghana and Cameroon.

In this article, we provide an in-depth analysis on the drug-resistance phenotypic characteristics of a cohort of 325 tuberculosis and characterize by Whole Genome Sequencing 24 isolates from Nigeria belonging to L4, L5 and L6. Our results suggest an alarming rate of drug-resistance of the L4.6.2.2 Mycobacterium tuberculosis complex (MTBC) lineage and a high diversity of L5. We compiled these new Sequence Read Archives (SRAs) to previously published ones from available Bioprojects run in Nigeria. We performed RAxML phylogenetic reconstructions of larger samples that include public NCBI SRAs from some neighboring countries (Cameroon, Ghana). To confront phylogenetic reconstruction to metadata, we used a new proprietary database named TB-Annotator. We show that L5 genomes in Northern Nigeria belong to new clades as the ones described until now and allow an update of the taxonomy of L5. In addition, we describe the L4.6.2.2 lineage in Nigeria, Cameroon and Ghana. We provide computations on the likely divergence time of L4.6.2.2 and suggest a new hypothesis concerning its origin. Finally we provide a short overview on M. bovis diversity in Nigeria. This study constitutes a baseline knowledge on the global genomic diversity, phylogeography and phylodynamics of MTBC in Nigeria, as well as on the natural history of this largely ignored but densely populated country of Africa. These results highlight the need of sequencing additional MTBC genomes in Nigeria and more generally in West-Africa, both for public health and for academic reasons. The likelihood of replacement of L5-L6 by L4.6.2.2 isolates, leave potentially little time to gather historical knowledge informative on the ancient history of tuberculosis in West-Africa.

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.

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.

Local adaptation in populations of Mycobacterium tuberculosis endemic to the Indian Ocean Rim.

Background: Lineage 1 (L1) and 3 (L3) are two lineages of the Mycobacterium tuberculosis complex (MTBC) causing tuberculosis (TB) in humans. L1 and L3 are prevalent around the rim of the Indian Ocean, the region that accounts for most of the world's new TB cases. Despite their relevance for this region, L1 and L3 remain understudied. Methods: We analyzed 2,938 L1 and 2,030 L3 whole genome sequences originating from 69 countries. We reconstructed the evolutionary history of these two lineages and identified genes under positive selection. Results: We found a strongly asymmetric pattern of migration from South Asia toward neighboring regions, highlighting the historical role of South Asia in the dispersion of L1 and L3. Moreover, we found that several genes were under positive selection, including genes involved in virulence and resistance to antibiotics. For L1 we identified signatures of local adaptation at the esxH locus, a gene coding for a secreted effector that targets the human endosomal sorting complex, and is included in several vaccine candidates. Conclusions: Our study highlights the importance of genetic diversity in the MTBC, and sheds new light on two of the most important MTBC lineages affecting humans.

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.

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.

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

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.

Prediction of the hidden genotype of mixed infection strains in Iranian tuberculosis patients.

BACKGROUND: Patients with mixed-strain Mycobacterium tuberculosis infections may be at a high risk of poor treatment outcomes. However, the mechanisms through which mixed infections affect the clinical manifestations are not well recognized. Evidence suggests that failure to detect the pathogen diversity within the host can influence the clinical results. We aimed to investigate the effects of different genotypes in mixed infections and determine their relationship with heteroresistance in the treatment of Iranian tuberculosis patients. METHODS: One of the genotypes was identified in the culture and another genotype pattern in the mixed infection was predicted by comparing the pattern of MIRU-VNTR between the clinical specimens and their respective cultures in each patient. For all patients, the drug susceptibility testing was carried out on three single colonies from each clinical sample. The follow-up of patients was carried out during six months of treatment. RESULTS: Based on MIRU-VNTR profiles of clinical samples, we showed that 55.6% (25/45) of the Iranian patients included in the study had mixed infections. Patients with mixed infections had a higher rate of treatment failure, compared to others (P=0.03). By comparing clinical sample profiles to profiles obtained after culture, we were able to distinguish between major and hidden genotypes. Among hidden genotypes, Haarlem (L4.1.2) and Beijing (L2) were associated to treatment failure (6/8 patients). CONCLUSIONS: To conclude, we propose a procedure using the MIRU-VNTR method to identify the different genotypes in mixed infections. The present findings suggest that genotypes with potentially higher pathogenicity may not be detected when performing experimental culture in patients with mixed infections.

NTF-RINT, a new method for the epidemiological surveillance of MDR Mycobacterium tuberculosis L2/Beijing strains.

The most widely discussed antibiotic-resistant tuberculosis strains ("W" and "B0/W148", "CAO") belong to L2/Beijing Lineage and are characterized by IS6110 insertion sequences at the NTF locus. We present a high-throughput, microbead-based method, called NTF-RINT for detection of IS in NTF and Rifampicin and Isoniazid Typing. This method provides tuberculosis diagnostic confirmation, screens for the so-called modern L2/Beijing sublineage and detects mutations involved in resistance to Rifampicin (RIF) and Isoniazid (INH).

Multicenter evaluation of TB-SPRINT 59-Plex Beamedex®: accuracy and cost analysis.

BACKGROUND: Molecular tests can allow the rapid detection of tuberculosis (TB) and multidrug-resistant TB (MDR-TB). TB-SPRINT 59-Plex Beamedex® is a microbead-based assay developed for the simultaneous spoligotyping and detection of MDR-TB. The accuracy and cost evaluation of new assays and technologies are of great importance for their routine use in clinics and in research laboratories. The aim of this study was to evaluate the performance of TB-SPRINT at three laboratory research centers in Brazil and calculate its mean cost (MC) and activity-based costing (ABC). METHODS: TB-SPRINT data were compared with the phenotypic and genotypic profiles obtained using Bactec™ MGIT™ 960 system and Genotype® MTBDRplus, respectively. RESULTS: Compared with MGIT, the accuracies of TB-SPRINT for the detection of rifampicin and isoniazid resistance ranged from 81 to 92% and 91.3 to 93.9%, respectively. Compared with MTBDRplus, the accuracies of TB-SPRINT for rifampicin and isoniazid were 99 and 94.2%, respectively. Moreover, the MC and ABC of TB-SPRINT were USD 127.78 and USD 109.94, respectively. CONCLUSION: TB-SPRINT showed good results for isoniazid and rifampicin resistance detection, but still needs improvement to achieve In Vitro Diagnostics standards.

Mycobacterium tuberculosis spoligotypes and drug-resistant characterization from Beira compared to genotypes circulating in Mozambique.

INTRODUCTION: Mozambique is one of three countries with high prevalence of tuberculosis (TB), TB/human immunodeficiency virus coinfection, and multidrug-resistant TB. We aimed to describe Mycobacterium tuberculosis spoligotypes circulating among drug resistant (DR) strains from Beira, Mozambique comparing them with genotypes in the country. METHODS: We performed spoligotyping of 79 M. tuberculosis suspected of DR-TB compared all spoligotype patterns published on the international database and PubMed. RESULTS: Both in Beira and Mozambique (n=578), the main clades were Latin-American-Mediterranean, East-African-Indian, Beijing and T, with no extensively DR TB cases. CONCLUSIONS: Beira and Mozambique share the same population genetic structure of M. tuberculosis.

Diversity of Mycobacterium tuberculosis in the Middle Fly District of Western Province, Papua New Guinea: microbead-based spoligotyping using DNA from Ziehl-Neelsen-stained microscopy preparations.

Tuberculosis remains the world's leading cause of death from an infectious agent, and is a serious health problem in Papua New Guinea (PNG) with an estimated 36,000 new cases each year. This study describes the genetic diversity of Mycobacterium tuberculosis among tuberculosis patients in the Balimo/Bamu region in the Middle Fly District of Western Province in PNG, and investigates rifampicin resistance-associated mutations. Archived Ziehl-Neelsen-stained sputum smears were used to conduct microbead-based spoligotyping and assess genotypic resistance. Among the 162 samples included, 80 (49.4%) generated spoligotyping patterns (n = 23), belonging predominantly to the L2 Lineage (44%) and the L4 Lineage (30%). This is consistent with what has been found in other PNG regions geographically distant from Middle Fly District of Western Province, but is different from neighbouring South-East Asian countries. Rifampicin resistance was identified in 7.8% of the successfully sequenced samples, with all resistant samples belonging to the L2/Beijing Lineage. A high prevalence of mixed L2/L4 profiles was suggestive of polyclonal infection in the region, although this would need to be confirmed. The method described here could be a game-changer in resource-limited countries where large numbers of archived smear slides could be used for retrospective (and prospective) studies of M. tuberculosis genetic epidemiology.

Mycobacterium tuberculosis lineage 1 genetic diversity in Pará, Brazil, suggests common ancestry with east-African isolates potentially linked to historical slave trade.

Lineage 1 (L1) is one of seven Mycobacterium tuberculosis complex (MTBC) lineages. The objective of this study was to improve the complex taxonomy of L1 using phylogenetic SNPs, and to look for the origin of the main L1 sublineage prevalent in Para, Brazil. We developed a high-throughput SNPs-typing assay based on 12-L1-specific SNPs. This assay allowed us to experimentally retrieve SNP patterns on nine of these twelve SNPs in 277 isolates previously tentatively assigned to L1 spoligotyping-based sublineages. Three collections were used: Pará-Brazil (71); RIVM, the Netherlands (102), Madagascar (104). One-hundred more results were generated in Silico using the PolyTB database. Based on the final SNPs combination, the samples were classified into 11 clusters (C1-C11). Most isolates within a SNP-based cluster shared a mutual spoligotyping-defined lineage. However, L1/EAI1-SOM (SIT48) and L1/EAI6-BGD1 (SIT591) showed a poor correlation with SNP data and are not monophyletic. L1/EAI8-MDG and L1/EAI3-IND belonged to C5; this result suggests that they share a common ancestor. L1.1.3/SIT129, a spoligotype pattern found in SNPs-cluster C6, was found to be shared between Pará/Brazil and Malawi. SIT129 was independently found to be highly prevalent in Mozambique, which suggests a migration history from East-Africa to Brazil during the 16th-18th slave trade period to Northern Brazil.

Exportation of MDR TB to Europe from Setting with Actively Transmitted Persistent Strains in Peru.

We performed a cross-border molecular epidemiology analysis of multidrug-resistant tuberculosis in Peru, Spain, and Italy. This analysis revealed frequent transmission in Peru and exportation of a strain that recreated similar levels of transmission in Europe during 2007-2017. Transnational efforts are needed to control transmission of multidrug-resistant tuberculosis globally.

E-DNA detection of rpoB gene resistance in Mycobacterium tuberculosis in real samples using Fe3O4/polypyrrole nanocomposite.

In this work, we achieved the selective detection of wild and mutated rpoB gene in M. tuberculosis using an electrochemical DNA (E-DNA) sensor based on polypyrrole/Fe3O4 nanocomposite bearing redox naphthoquinone tag on PAMAM (spaNQ/PAMAM/PPy/Fe3O4). The hybridization between a given probe and the complementary DNA target induced a large decrease in the naphthoquinone redox signal as measured by SWV and no cross-hybridization with single nucleotide mismatch DNA target occurred. Thanks to the catalytic properties of iron oxide nanoparticles combined with conducting properties of polypyrrole platform, we demonstrated that the transducing system allowed the detection of 1 fM of DNA target in a 50-µL drop corresponding to 3 × 104 copies of DNA. The sensor was able to detect the rpoB gene in PCR-amplified samples of genomic DNA and could also discriminate between the wild type rpoB gene and a single nucleotide mutated rpoB gene that provides resistance to rifampicin. Furthermore, the sensor could selectively detect the wild and mutant DNA in genomic samples without PCR amplification.