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The transition from MIRU-VNTR-based epidemiology studies in tuberculosis (TB) to genomic epidemiology has transformed how we track transmission. However, short-read sequencing is poor at analyzing repetitive regions such as the MIRU-VNTR loci. This causes a gap between the new genomic data and the large amount of information stored in historical databases. Long-read sequencing could bridge this knowledge gap by allowing analysis of repetitive regions. However, the feasibility of extracting MIRU-VNTRs from long reads and linking them to historical data has not been evaluated. In our study, an in silico arm, consisting of inference of MIRU patterns from long-read sequences (using MIRUReader program), was compared with an experimental arm, involving standard amplification and fragment sizing. We analyzed overall performance on 39 isolates from South Africa and confirmed reproducibility in a sample enriched with 62 clustered cases from Spain. Finally, we ran 25 consecutive incident cases, demonstrating the feasibility of correctly assigning new clustered/orphan cases by linking data inferred from genomic analysis to MIRU-VNTR databases. Of the 3,024 loci analyzed, only 11 discrepancies (0.36%) were found between the two arms: three attributed to experimental error and eight to misassigned alleles from long-read sequencing. A second round of analysis of these discrepancies resulted in agreement between the experimental and in silico arms in all but one locus. Adjusting the MIRUReader program code allowed us to flag potential in silico misassignments due to suboptimal coverage or unfixed double alleles. Our study indicates that long-read sequencing could help address potential chronological and geographical gaps arising from the transition from molecular to genomic epidemiology of tuberculosis., Importance: The transition from molecular epidemiology in tuberculosis (TB), based on the analysis of repetitive regions (VNTR-based genotyping), to genomic epidemiology transforms in the precision with which we track transmission. However, short-read sequencing, the most common method for performing genomic analysis, is poor at analyzing repetitive regions. This means that we face a gap between the new genomic data and the large amount of information stored in historical databases, which is also an obstacle to cross-national surveillance involving settings where only molecular data are available. Long-read sequencing could help bridge this knowledge gap by allowing analysis of repetitive regions. Our study demonstrates that MIRU-VNTR patterns can be successfully inferred from long-read sequences, allowing the correct assignment of new cases as clustered/orphan by linking new data extracted from genomic analysis to historical MIRU-VNTR databases. Our data may provide a starting point for bridging the knowledge gap between the molecular and genomic eras in tuberculosis epidemiology., Competing Interests: The authors declare no conflict of interest.

Introduction Mycobacterium caprae is a member of the Mycobacterium tuberculosis complex (MTBC) not routinely identified to species level. It lacks specific clinical features of presentation and may therefore not be identified as the causative agent of tuberculosis. Use of whole genome sequencing (WGS) in the investigation of a family microepidemic of tuberculosis in Almería, Spain, unexpectedly identified the involvement of M. caprae .AimWe aimed to evaluate the presence of additional unidentified M. caprae cases and to determine the magnitude of this occurrence.MethodsFirst-line characterisation of the MTBC isolates was done by MIRU-VNTR, followed by WGS. Human and animal M. caprae isolates were integrated in the analysis.ResultsA comprehensive One Health strategy allowed us to (i) detect other 11 M. caprae infections in humans in a period of 18 years, (ii) systematically analyse M. caprae infections on an epidemiologically related goat farm and (iii) geographically expand the study by including 16 M. caprae isolates from other provinces. Integrative genomic analysis of 41 human and animal M. caprae isolates showed a high diversity of strains. The animal isolates' diversity was compatible with long-term infection, and close genomic relationships existed between isolates from goats on the farm and recent cases of M. caprae infection in humans.DiscussionZoonotic circulation of M. caprae strains had gone unnoticed for 18 years. Systematic characterisation of MTBC at species level and/or extended investigation of the possible sources of exposure in all tuberculosis cases would minimise the risk of overlooking similar zoonotic events.

Costa Rica has a low incidence of tuberculosis. Thus, identifying transmission hotspots is key to implement interventions. A tuberculosis outbreak was suspected in a prison in Costa Rica. Given the suboptimal quality of the samples received in our laboratory in Madrid, we applied alternative schemes for their analysis. In the first scheme, we bypassed the standard approach of applying systematic mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) and used a strain-specific polymerase chain reaction (PCR) that allowed identifying a cluster involving six cases (C1). The second scheme followed the canonical MIRU-VNTR path coupled with a whole-genomic amplification step, by which a second unsuspected overlapping cluster (C2), was detected in the same prison. These findings justified the implementation of a surveillance programme adapted to local resources based on a tailored multiplex allele-specific oligonucleotide (ASO)-PCR targeting C1 and C2. Presence of the C2 strain at a different prison was determined. ASO-PCR was applied extensively and alerted to the active circulation of one of the strains within and beyond prisons. Our study shows that alternative methodological strategies may provide useful data in settings with lack of resources for performing systematic standard molecular epidemiology programmes and/or with suboptimal material for analysis., (© 2021 Wiley-VCH GmbH.)

Recurrent tuberculosis occurs due to exogenous reinfection or reactivation/persistence. We analysed 90 sequential MDR Mtb isolates obtained in Argentina from 27 patients with previously diagnosed MDR-TB that recurred in 2018 (1-10 years, 2-10 isolates per patient). Three long-term predominant strains were responsible for 63% of all MDR-TB recurrences. Most of the remaining patients were infected by strains different from each other. Reactivation/persistence of the same strain caused all but one recurrence, which was due to a reinfection with a predominant strain. One of the prevalent strains showed marked stability in the recurrences, while in another strain higher SNP-based diversity was observed. Comparisons of intra- versus inter-patient SNP distances identified two possible reinfections with closely related variants circulating in the community. Our results show a complex scenario of MDR-TB infections in settings with predominant MDR Mtb strains., (© 2021 Wiley-VCH GmbH.)

Genotyping tools help identify the complexity in Mycobacterium tuberculosis transmission clusters. We carried out a thorough analysis of the epidemiological and bacteriological complexity of a cluster in Almería, Spain. The cluster, initially associated with Moroccan migrants and with no secondary cases identified in 4 years, then reappeared in Spanish-born individuals. In one case, two Mycobacterium tuberculosis clonal variants were identified. We reanalyzed the cluster, supported by the characterization of multiple cultured isolates and respiratory specimens, whole-genome sequencing, and epidemiological case interviews. Our findings showed that the cluster, which was initially thought to have restarted activity with just a single case harboring a small degree of within-host diversity, was in fact currently growing due to coincidental reactivation of past exposures, with clonal diversity transmitted throughout the cluster. In one case, within-host diversity was amplified, probably due to prolonged diagnostic delay. IMPORTANCE The precise study of the dynamics of tuberculosis transmission in socio-epidemiologically complex scenarios may require more thorough analysis than the standard molecular epidemiology strategies. Our study illustrates the epidemiological and bacteriological complexity present in a transmission cluster in a challenging epidemiological setting with a high proportion of migrant cases. The combination of whole-genome sequencing, refined and refocused epidemiological interviews, and in-depth analysis of the bacterial composition of sputa and cultured isolates was crucial in order to correctly reinterpret the true nature of this cluster. Our global approach allowed us to reinterpret correctly the unnoticed epidemiological and bacteriological complexity involved in the Mycobacterium tuberculosis transmission event under study, which had been overlooked by the usual molecular epidemiology approaches.

Objectives: To characterize the clonal complexity in Mycobacterium tuberculosis (MTB) infections considering factors that help maximize the detection of coexisting strains/variants., Methods: Genotypic analysis by Mycobacterial Interspersed Repetitive-Unit-Variable-Number Tandem-Repeats (MIRU-VNTR) was performed directly on 70 biopsy specimens from two or more different tissues involving 28 tuberculosis cases diagnosed post-mortem in Mozambique, a country with a high tuberculosis burden., Results: Genotypic data from isolates collected from two or more tissues were obtained for 23 of the 28 cases (82.1%), allowing the analysis of within-patient diversity. MIRU-VNTR analysis revealed clonal diversity in ten cases (35.7%). Five cases showed allelic differences in three or more loci, suggesting mixed infection with two different strains. In half of the cases showing within-host diversity, one of the specimens associated with clonal heterogeneity was brain tissue., Conclusions: Direct MTB genotyping from post-mortem tissue samples revealed a frequent within-host Mycobacterium tuberculosis diversity, including mixed and polyclonal infections. Most of this diversity would have been overlooked if only standard analysis of respiratory specimens had been performed., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Beijing genotype Mycobacterium tuberculosis strains associate with increased virulence, resistance and/or higher transmission rates. This study describes a specific Beijing strain predominantly identified in the Panamanian province of Colon with one of the highest incidences of tuberculosis in the country. Retrospective mycobacterial interspersed repetitive unit/variable number of tandem repeats analysis of 42 isolates collected between January and August 2018 allowed to identify a cluster (Beijing A) with 17 (40.5%) Beijing isolates. Subsequent prospective strain-specific PCR-based surveillance from September 2019 to March 2020 confirmed the predominance of the Beijing A strain (44.1%) in this province. Whole-genome sequencing revealed higher-than-expected diversity within the cluster, suggesting long-term prevalence of this strain and low number of cases caused by recent transmission. The Beijing A strain belongs to the Asian African 3 (Bmyc13, L2.2.5) branch of the modern Beijing sublineage, with their closest isolates corresponding to cases from Vietnam, probably introduced in Panama between 2000 and 2012., (© 2020 Wiley-VCH GmbH.)

Background: Growing international migration has increased the complexity of tuberculosis transmission patterns. Italy's decision to close its borders in 2018 made of Spain the new European porte entrée for migration from the Horn of Africa (HA). In one of the first rescues of migrants from this region at the end of 2018, tuberculosis was diagnosed in eight subjects, mainly unaccompanied minors., Methods: Mycobacterium tuberculosis isolates from these recently arrived migrants were analysed by Mycobacterial Interspersed Repetitive-Unit/Variable-Number of Tandem Repeat (MIRU-VNTR) and subsequent whole genome sequencing (WGS) analysis. Data were compared with those from collections from other European countries receiving migrants from the HA and a strain-specific PCR was applied for a fast searching of common strains. Infections in a cellular model were performed to assess strain virulence., Results: MIRU-VNTR analysis allowed identifying an epidemiological cluster involving three of the eight cases from Somalia (0 single-nucleotide polymorphisms between isolates, HA cluster). Following detailed interviews revealed that two of these cases had shared the same migratory route in most of the trip and had spent a long time at a detention camp in Libya. To confirm potential en route transmission for the three cases, we searched the same strain in collections from other European countries receiving migrants from the HA. MIRU-VNTR, WGS and a strain-specific PCR for the HA strain were applied. The same strain was identified in 12 cases from Eritrea diagnosed soon after their arrival in 2018 to the Netherlands, Belgium and Italy. Intracellular replication rate of the strain did not reveal abnormal virulence., Conclusions: Our study suggests a potential en route transmission of a pan-susceptible strain, which caused at least 15 tuberculosis cases in Somalian and Eritrean migrants diagnosed in four different European countries., (© International Society of Travel Medicine 2021. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Systematic molecular/genomic epidemiology studies for tuberculosis surveillance cannot be implemented in many countries. We selected Panama as a model for an alternative strategy. Mycobacterial interspersed repetitive unit-variable-number tandem-repeat (MIRU-VNTR) analysis revealed a high proportion (50%) of Mycobacterium tuberculosis isolates included in 6 clusters (A-F) in 2 provinces (Panama and Colon). Cluster A corresponded to the Beijing sublineage. Whole-genome sequencing (WGS) differentiated clusters due to active recent transmission, with low single-nucleotide polymorphism-based diversity (cluster C), from clusters involving long-term prevalent strains with higher diversity (clusters A, B). Prospective application in Panama of 3 tailored strain-specific PCRs targeting marker single-nucleotide polymorphisms identified from WGS data revealed that 31.4% of incident cases involved strains A-C and that the Beijing strain was highly represented and restricted mainly to Colon. Rational integration of MIRU-VNTR, WGS, and tailored strain-specific PCRs could be a new model for tuberculosis surveillance in countries without molecular/genomic epidemiology programs.

BackgroundThe analysis of transmission of tuberculosis (TB) is challenging in areas with a large migrant population. Standard genotyping may fail to differentiate transmission within the host country from new importations, which is key from an epidemiological perspective.AimTo propose a new strategy to simplify and optimise cross-border surveillance of tuberculosis and to distinguish between recent transmission in the host country and new importationsMethodsWe selected 10 clusters, defined by 24-locus mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR), from a population in Spain rich in migrants from eastern Europe, north Africa and west Africa and reanalysed 66 isolates by whole-genome sequencing (WGS). A multiplex-allele-specific PCR was designed to target strain-specific marker single nucleotide polymorphisms (SNPs), identified from WGS data, to optimise the surveillance of the most complex cluster.ResultsIn five of 10 clusters not all isolates showed the short genetic distances expected for recent transmission and revealed a higher number of SNPs, thus suggesting independent importations of prevalent strains in the country of origin. In the most complex cluster, rich in Moroccan cases, a multiplex allele-specific oligonucleotide-PCR (ASO-PCR) targeting the marker SNPs for the transmission subcluster enabled us to prospectively identify new secondary cases. The ASO-PCR-based strategy was transferred and applied in Morocco, demonstrating that the strain was prevalent in the country.ConclusionWe provide a new model for optimising the analysis of cross-border surveillance of TB transmission in the scenario of global migration.

The introduction of genotypic tools to analyze Mycobacterium tuberculosis isolates has transformed our knowledge of the transmission dynamics of this pathogen. We discuss the development of the laboratory methods that have been applied in recent years to study the epidemiology of M. tuberculosis . This review integrates two approaches: on the one hand, it considers how genotyping techniques have evolved over the years; and on the other, it looks at how the way we think these techniques should be applied has changed. We begin by examining the application of fingerprinting tools to suspected outbreaks only, before moving on to universal genotyping schemes, and finally we describe the latest real-time strategies used in molecular epidemiology. We also analyze refined approaches to obtaining epidemiological data from patients and to increasing the discriminatory power of genotyping by techniques based on genomic characterization. Finally, we review the development of integrative solutions to reconcile the speed of PCR-based methods with the high discriminatory power of whole-genome sequencing in easily implemented formats adapted to low-resource settings. Our analysis of future considerations highlights the need to bring together the three key elements of high-quality surveillance of transmission in tuberculosis, namely, speed, precision, and ease of implementation.

Introduction: Mycobacterium tuberculosis Beijing lineage isolates are considered to be especially virulent, transmissible and prone to acquire resistances. Beijing strains have been reported worldwide, but studies in Latin America are still scarce. The only multinational study performed in the region indicated a heterogeneous distribution for this lineage, which was absent in Chile, Colombia and Ecuador, although further studies found the lineage in Chile and Colombia., Objective: To search for the presence of the Beijing lineage in Ecuador, the only country in the region where it remains unreported., Materials and Methods: We obtained a convenience sample (2006-2012) from two hospitals covering different populations. The isolates were genotyped using 24-MIRU-VNTR. Lineages were assigned by comparing their patterns to those in the MIRU-VNTRplus platform. Isolates belonging to the Beijing lineage were confirmed by allele-specific PCR., Results: We identified the first Beijing isolate in Ecuador in an unexpected epidemiological scenario: A patient was infected in the Andean region, in a population with low mobility and far from the borders of the neighboring countries where Beijing strains had been previously reported., Conclusion: This is the first report of the presence of the Beijing lineage in Ecuador in an unusual epidemiological context that deserves special attention.

Molecular epidemiology analysis of tuberculosis transmission is based mostly on the application of MIRU-VNTR. In certain isolates a complete 24-loci genotype is not obtained and these incompletely genotyped isolates can not be used in the definition of clusters. In a population-based molecular epidemiology study performed in Almería, Southeast Spain, a context with a high proportion of immigrants, we found that an 88-bp deletion in isolates of Mycobacterium africanum Lineage 5 hampers MIRU-VNTR analysis. A more extensive analysis revealed that this deletion was shared by all the Lineage 5 isolates in certain countries of origin of immigrants, such as Equatorial Guinea, and is likely present in several other African countries and also in the USA. A procedure is proposed to enable epidemiological analysis of these isolates., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Clonal variants of Mycobacterium tuberculosis (MTB) coexist in specific patients, although the dynamics of their emergence is unknown. We used MIRU-VNTR to detect microevolution leading to variants of MTB in 3 out of 19 patients (15%) soon after diagnosis (61-85days). Most harbored SNPs and for some of them a potential functional role was suggested. Microevolution in tuberculosis seems to occur sooner and more often than expected and could affect tracking of transmission., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

A persistent 8-year infection by a Beijing Mycobacterium tuberculosis strain from a previous outbreak after importation from West Africa obliged us to investigate secondary cases. We developed a multiplex PCR method based on whole-genome sequencing to target strain-specific single nucleotide polymorphisms (SNPs). In 1 week, we analyzed 868 isolates stored over 6 years. Only 2 cases (immigrants from Guinea Conakry) harbored the strain, which ruled out transmission-despite opportunities-and challenged some of the advantages associated with Beijing strains., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Background: It has been accepted that the infection by Mycobacterium tuberculosis (M. tuberculosis) can be more heterogeneous than considered. The emergence of clonal variants caused by microevolution events leading to population heterogeneity is a phenomenon largely unexplored. Until now, we could only superficially analyze this phenomenon by standard fingerprinting (RFLP and VNTR)., Methods: In this study we applied whole genome sequencing for a more in-depth analysis of the scale of microevolution both at the intrapatient and interpatient scenarios., Results: We found that the amount of variation accumulated within a patient can be as high as that observed between patients along a chain of transmission. Intrapatient diversity was found both at the extrapulmonary and respiratory sites, meaning that this variability can be transmitted and impact on the inference of transmission events. One of the events studied allowed us to track for a single strain the complete process of (i) interpatient microevolution, (ii) intrapatient respiratory variation, and (iii) isolation of different variants at different infected sites of this patient., Conclusions: Our study adds new data to the understanding of variability in M. tuberculosis in a wide clinical scenario and alerts about the difficulties of establishing thresholds to differentiate relatedness in M. tuberculosis with epidemiological purposes.

Clonal variants of Mycobacterium tuberculosis can emerge as a result of microevolution phenomena. The functional significance of these subtle genetic rearrangements is normally disregarded. We show that clonal variants from two patients had different infective behaviours in some in vitro cellular infection models but not in others. Microevolution may have a subtle impact on infectivity, but specific experimental conditions are needed to unmask it., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

We performed an in-depth analysis of a large Mycobacterium tuberculosis cluster involving nine isolates with identical or highly similar RFLP types. The strain involved in this transmission cluster microevolved and accumulated up to five differential IS6110 transposition events, several of them not revealed by standard genotyping approaches. Some of these events probably generated extended deletions. In two cases of tuberculosis within the cluster, we observed the simultaneous presence of both the reference and microevolved variants. A longitudinal genotypical survey of M. tuberculosis in a real clinical evolutionary scenario can reveal dynamic and complex microevolution events., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Clonally complex infections by Mycobacterium tuberculosis are progressively more accepted. Studies of their dimension in epidemiological scenarios where the infective pressure is not high are scarce. Our study systematically searched for clonally complex infections (mixed infections by more than one strain and simultaneous presence of clonal variants) by applying mycobacterial interspersed repetitive-unit (MIRU)-variable-number tandem-repeat (VNTR) analysis to M. tuberculosis isolates from two population-based samples of respiratory (703 cases) and respiratory-extrapulmonary (R+E) tuberculosis (TB) cases (71 cases) in a context of moderate TB incidence. Clonally complex infections were found in 11 (1.6%) of the respiratory TB cases and in 10 (14.1%) of those with R+E TB. Among the 21 cases with clonally complex TB, 9 were infected by 2 independent strains and the remaining 12 showed the simultaneous presence of 2 to 3 clonal variants. For the 10 R+E TB cases with clonally complex infections, compartmentalization (different compositions of strains/clonal variants in independent infected sites) was found in 9 of them. All the strains/clonal variants were also genotyped by IS6110-based restriction fragment length polymorphism analysis, which split two MIRU-defined clonal variants, although in general, it showed a lower discriminatory power to identify the clonal heterogeneity revealed by MIRU-VNTR analysis. The comparative analysis of IS6110 insertion sites between coinfecting clonal variants showed differences in the genes coding for a cutinase, a PPE family protein, and two conserved hypothetical proteins. Diagnostic delay, existence of previous TB, risk for overexposure, and clustered/orphan status of the involved strains were analyzed to propose possible explanations for the cases with clonally complex infections. Our study characterizes in detail all the clonally complex infections by M. tuberculosis found in a systematic survey and contributes to the characterization that these phenomena can be found to an extent higher than expected, even in an unselected population-based sample lacking high infective pressure.

Under certain circumstances, it is possible to identify clonal variants of Mycobacterium tuberculosis infecting a single patient, probably as a result of subtle genetic rearrangements in part of the bacillary population. We systematically searched for these microevolution events in a different context, namely, recent transmission chains. We studied the clustered cases identified using a population-based universal molecular epidemiology strategy over a 5-year period. Clonal variants of the reference strain defining the cluster were found in 9 (12%) of the 74 clusters identified after the genotyping of 612 M. tuberculosis isolates by IS6110 restriction fragment length polymorphism analysis and mycobacterial interspersed repetitive units-variable-number tandem repeat typing. Clusters with microevolution events were epidemiologically supported and involved 4 to 9 cases diagnosed over a 1- to 5-year period. The IS6110 insertion sites from 16 representative isolates of reference and microevolved variants were mapped by ligation-mediated PCR in order to characterize the genetic background involved in microevolution. Both intragenic and intergenic IS6110 locations resulted from these microevolution events. Among those cases of IS6110 locations in intergenic regions which could have an effect on the regulation of adjacent genes, we identified the overexpression of cytochrome P450 in one microevolved variant using quantitative real-time reverse transcription-PCR. Our results help to define the frequency with which microevolution can be expected in M. tuberculosis transmission chains. They provide a snapshot of the genetic background of these subtle rearrangements and identify an event in which IS6110-mediated microevolution in an isogenic background has functional consequences.

Costa Rica has a low incidence of tuberculosis. Thus, identifying transmission hotspots is key to implement interventions. A tuberculosis outbreak was suspected in a prison in Costa Rica. Given the suboptimal quality of the samples received in our laboratory in Madrid, we applied alternative schemes for their analysis. In the first scheme, we bypassed the standard approach of applying systematic mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) and used a strain-specific polymerase chain reaction (PCR) that allowed identifying a cluster involving six cases (C1). The second scheme followed the canonical MIRU-VNTR path coupled with a whole-genomic amplification step, by which a second unsuspected overlapping cluster (C2), was detected in the same prison. These findings justified the implementation of a surveillance programme adapted to local resources based on a tailored multiplex allele-specific oligonucleotide (ASO)-PCR targeting C1 and C2. Presence of the C2 strain at a different prison was determined. ASO-PCR was applied extensively and alerted to the active circulation of one of the strains within and beyond prisons. Our study shows that alternative methodological strategies may provide useful data in settings with lack of resources for performing systematic standard molecular epidemiology programmes and/or with suboptimal material for analysis. [ABSTRACT FROM AUTHOR]

Background: Mycobacteriumtuberculosis (MTB) has limited ability to acquire variability. Analysis of its microevolution might help us to evaluate the pathways followed to acquire greater infective success. Whole-genome sequencing (WGS) in the analysis of the transmission of MTB has elucidated the magnitude of variability in MTB. Analysis of transmission currently depends on the identification of clusters, according to the threshold of variability (<5 SNPs) between isolates. Objective: We evaluatedwhether the acquisition of variability inMTB, wasmore frequent in situations which could favor it, namely intrapatient, prolonged infections or reactivations and interpatient transmissions involving multiple sequential hosts. Methods: We used WGS to analyze the accumulation of variability in sequential isolates from prolonged infections or translations from latency to reactivation. We then measured microevolution in transmission clusters with prolonged transmission time, high number of involved cases, simultaneous involvement of latency and active transmission. Results: Intrapatient and interpatient acquisition of variability was limited, within the ranges expected according to the thresholds of variability proposed, even though bursts of variability were observed. Conclusions: The thresholds of variability proposed for MTB seem to be valid in most circumstances, including those theoretically favoring acquisition of variability. Our data point to multifactorial modulation of microevolution, although further studies are necessary to elucidate the factors underlying this modulation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Mycobacterium bovis is both the causative agent of bovine tuberculosis (TB) and a zoonotic pathogen. In humans, considerably fewer cases of TB are caused by M. bovis than M. tuberculosis; nevertheless, diagnostic limitations mean that currently available data on prevalence grossly underestimate the true dimension of the problem. The routes of transmission from animals to humans are well known and include direct exposure to infected animals or consumption of contaminated animal products. Application of fingerprinting tools facilitates analysis of the molecular epidemiology of M. bovis in animal-to-human and humanto- human transmission. Apart from cattle and M. bovis, other animal species and members within the M. tuberculosis complex can contribute to the zoonosis. Improvements in diagnostic techniques, application of more advanced discriminatory genotyping tools, and collaboration between veterinary and human health care researchers are key to our understanding of this zoonosis. [ABSTRACT FROM AUTHOR]

Abstract: Clonal variants of Mycobacterium tuberculosis can emerge as a result of microevolution in a single host or after sequential infection of different hosts. The significance of subtle genotypic variations is still unknown. In three of the four loci analyzed from clonal variants differing in only one MIRU-VNTR locus, we found that the expression of the adjacent genes was modulated differently. These data highlight the potential advantages that acquisition of subtle variability may have in M. tuberculosis. [Copyright &y& Elsevier]

Highlights: [•] We analyzed genotypic characteristics of a Mycobacterium tuberculosis strain with a high tendency to microevolve. [•] We analyzed 56 3R genes and the IS6110 insertion sites from this strain. [•] An SNP in alkA and an IS6110 copy located upstream of a transposase were found. [•] Both features were shared by other strains that were also involved in microevolution events. [•] These two genetic features could have a putative role as microevolution markers. [Copyright &y& Elsevier]