Bridging the gap between molecular and genomic epidemiology in tuberculosis: inferring MIRU-VNTR patterns from genomic data.

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.

Microevolution, reinfection and highly complex genomic diversity in patients with sequential isolates of Mycobacterium abscessus.

Mycobacterium abscessus is an opportunistic, extensively drug-resistant non-tuberculous mycobacterium. Few genomic studies consider its diversity in persistent infections. Our aim was to characterize microevolution/reinfection events in persistent infections. Fifty-three sequential isolates from 14 patients were sequenced to determine SNV-based distances, assign resistance mutations and characterize plasmids. Genomic analysis revealed 12 persistent cases (0-13 differential SNVs), one reinfection (15,956 SNVs) and one very complex case (23 sequential isolates over 192 months), in which a first period of persistence (58 months) involving the same genotype 1 was followed by identification of a genotype 2 (76 SNVs) in 6 additional alternating isolates; additionally, ten transient genotypes (88-243 SNVs) were found. A macrolide resistance mutation was identified from the second isolate. Despite high diversity, the genotypes shared a common phylogenetic ancestor and some coexisted in the same specimens. Genomic analysis is required to access the true intra-patient complexity behind persistent infections involving M. abscessus.

A One Health approach revealed the long-term role of Mycobacterium caprae as the hidden cause of human tuberculosis in a region of Spain, 2003 to 2022.

IntroductionMycobacterium 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.

Insights into the Complexity of a Dormant Mycobacterium tuberculosis Cluster Once Transmission Is Resumed.

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.

Assessment of closely related Mycobacterium tuberculosis variants with different transmission success and in vitro infection dynamics.

Whole genome sequencing (WGS) is able to differentiate closely related Mycobacterium tuberculosis variants within the same transmission cluster. Our aim was to evaluate if this higher discriminatory power may help identify and characterize more actively transmitted variants and understand the factors behind their success. We selected a robust MIRU-VNTR-defined cluster from Almería, Spain (22 cases throughout 2003-2019). WGS allowed discriminating, within the same epidemiological setting, between a successfully transmitted variant and seven closely related variants that did not lead to secondary cases, or were involved in self-limiting transmission (one single secondary case). Intramacrophagic growth of representative variants was evaluated in an in vitro infection model using U937 cells. Intramacrophage multiplication ratios (CFUs at Day 4/CFUs at Day 0) were higher for the actively transmitted variant (range 5.3-10.7) than for the unsuccessfully transmitted closely related variants (1.5-3.95). Two SNPs, mapping at the DNA binding domain of DnaA and at kdpD, were found to be specific of the successful variant.

Integrative transnational analysis to dissect tuberculosis transmission events along the migratory route from Africa to Europe.

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.

Whole genome sequencing-based analysis of tuberculosis (TB) in migrants: rapid tools for cross-border surveillance and to distinguish between recent transmission in the host country and new importations.

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.

A deletion hampering appropriate typing of Mycobacterium africanum.

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.

Mycobacterium tuberculosis Acquires Limited Genetic Diversity in Prolonged Infections, Reactivations and Transmissions Involving Multiple Hosts.

Background:Mycobacterium tuberculosis (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 evaluated whether the acquisition of variability in MTB, was more 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.

Urgent Implementation in a Hospital Setting of a Strategy To Rule Out Secondary Cases Caused by Imported Extensively Drug-Resistant Mycobacterium tuberculosis Strains at Diagnosis.

Current migratory movements require new strategies for rapidly tracking the transmission of high-risk imported Mycobacterium tuberculosis strains. Whole-genome sequencing (WGS) enables us to identify single-nucleotide polymorphisms (SNPs) and therefore design PCRs to track specific relevant strains. However, fast implementation of these strategies in the hospital setting is difficult because professionals working in diagnostics, molecular epidemiology, and genomics are generally at separate institutions. In this study, we describe the urgent implementation of a system that integrates genomics and molecular tools in a genuine high-risk epidemiological alert involving 2 independent importations of extensively drug resistant (XDR) and pre-XDR Beijing M. tuberculosis strains from Russia into Spain. Both cases involved commercial sex workers with long-standing tuberculosis (TB). The system was based on strain-specific PCRs tailored from WGS data that were transferred to the local node that was managing the epidemiological alert. The optimized tests were available for prospective implementation in the local node 33 working days after receiving the primary cultures of the XDR strains and were applied to all 42 new incident cases. An interpretable result was obtained in each case (directly from sputum for 27 stain-positive cases) and corresponded to the amplification profiles for strains other than the targeted pre-XDR and XDR strains, which made it possible to prospectively rule out transmission of these high-risk strains at diagnosis.

Preliminary evaluation of a new kit for differentiation of Mycobacterium tuberculosis complex species using Speed-Oligo MTBC.

We present the first evaluation of a novel molecular assay, the Speed-Oligo Mycobacterium tuberculosis complex (SO-MTBC), which is based on PCR combined with a dipstick for the differentiation of M. tuberculosis complex (MTBC) members. The results of this assay were compared with findings obtained using the Genotype MTBC assay. In this study, 189 strains of MTBC isolates from 2011 to 2014 were evaluated to determine the MTBC species. Most (174, 92 %) of the strains were identified as M. tuberculosissensu stricto, 7 (3.7 %) as Mycobacteriumbovis, 5 (2.6 %) as M. bovis bacillus Calmette-Guérin, 2 (1.1 %) as Mycobacteriumafricanum and 1 (0.5 %) as Mycobacteriumcaprae; no strains belonged to Mycobacteriummicroti and Mycobacteriumcanettii subsp. The concordance κ coefficient obtained was 0.96 with the results of the Genotype MTBC assay. SO-MTBC may represent a fast and easy-to-use alternative for differentiating among MTBC subspecies in laboratories with standard equipment.

Usefulness of Genotype MTBDRplus assay in acid-fast bacilli positive smear specimens in Almeria, Spain.

INTRODUCTION: The urgent need for operational research evaluating test performance in a real-world setting has been highlighted. The purpose of this study was therefore to evaluate the performance of MTBDRplus assay. MATERIALS: According to the reference method, of the 155 clinical specimens with valid results, 147 were susceptible to rifampicin (RIF) and isoniazid (INH), with 4 being multi-drug resistant (MDR) and 4 with isolated resistance to isoniazid (INH). RESULTS: The results of the MTBDRplus assay were 100% concordant for the MDR and mono-resistant to INH specimens. However, the MTBDRplus assay showed a resistance pattern to RIF in one specimen which was classified as susceptible by the reference method. The majority of the specimens (118/75.6%) were also tested using the MTBDRplus method after culture on Lowenstein-Jensen media, showing 100% agreement with the results of the test directly from the specimens. An MTBDRplus test result was available within an average of 8 days. CONCLUSIONS: Overall, MTBDR results showed excellent results when compared with the reference method and achieved a significant time-reduction.

Different penetrance of disseminated infections caused by nontuberculous Mycobacteria in Mendelian susceptibility to mycobacterial disease associated with a novel mutation.

Deficiency in the interleukin12/INFgamma pathway is a genetic condition that predisposes to some infections, including nontuberculous mycobacteria infection and extraintestinal salmonellosis. We report 2 cases in sisters who were diagnosed with a genetic defect caused by a new mutation in Interleukin-12 receptor ß1 chain (IL12Rß1) leading to different clinical presentations and responses to therapy.

Evaluation of the potential role of a new mutation in mabA in modifying the response of Mycobacterium tuberculosis to isoniazid.

A new mutation in mabA, Thr4Ile, was identified in a Mycobacterium tuberculosis isolate from a patient whose culture remained positive after treatment. The same mutation was found in another 5 patients infected by different strains. A putative role for this mutation in the process of diminishing susceptibility to isoniazid is evaluated.

Selecting criteria for improving results of an immunochromatographic assay for Mycobacterium tuberculosis complex identification from BacT/ALERT cultures.

We describe a combined macro and microscopic criteria for rapid presumptive differentiation between Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) evaluated by two independent observers. This strategy achieved rapid MTC identification in most cases (95.8% expert observer and 91.6% novice observer) with significant savings compared to more expensive and unnecessary tests.

Evaluation of the speed-oligo direct Mycobacterium tuberculosis assay for molecular detection of mycobacteria in clinical respiratory specimens.

We present the first evaluation of a novel molecular assay, the Speed-oligo Direct Mycobacterium tuberculosis (SO-DMT) assay, which is based on PCR combined with a dipstick for the detection of mycobacteria and the specific identification of M. tuberculosis complex (MTC) in respiratory specimens. A blind evaluation was carried out in two stages: first, under experimental conditions on convenience samples comprising 20 negative specimens, 44 smear- and culture-positive respiratory specimens, and 11 sputa inoculated with various mycobacterium-related organisms; and second, in the routine workflow of 566 fresh respiratory specimens (4.9% acid-fast bacillus [AFB] smear positives, 7.6% MTC positives, and 1.8% nontuberculous mycobacteria [NTM] culture positives) from two Mycobacterium laboratories. SO-DMT assay showed no reactivity in any of the mycobacterium-free specimens or in those with mycobacterium-related organisms. Compared to culture, the sensitivity in the selected smear-positive specimens was 0.91 (0.92 for MTC and 0.90 for NTM), and there was no molecular detection of NTM in a tuberculosis case or vice versa. With respect to culture and clinical data, the sensitivity, specificity, and positive and negative predictive values for the SO-DMT system in routine specimens were 0.76 (0.93 in smear positives [1.0 for MTC and 0.5 for NTM] and 0.56 in smear negatives [0.68 for MTC and 0.16 for NTM]), 0.99, 0.85 (1.00 in smear positives and 0.68 in smear negatives), and 0.97, respectively. Molecular misidentification of NTM cases occurred when testing 2 gastric aspirates from two children with clinically but not microbiologically confirmed lung tuberculosis. The SO-DMT assay appears to be a fast and easy alternative for detecting mycobacteria and differentiating MTC from NTM in smear-positive respiratory specimens.

Real-time molecular epidemiology of tuberculosis by direct genotyping of smear-positive clinical specimens.

We applied MIRU-VNTR (mycobacterial interspersed repetitive-unit-variable-number tandem-repeat typing) to directly analyze the bacilli present in 61 stain-positive specimens from tuberculosis patients. A complete MIRU type (24 loci) was obtained for all but one (no amplification in one locus) of the specimens (98.4%), and the allelic values fully correlated with those obtained from the corresponding cultures. Our study is the first to demonstrate that real-time genotyping of Mycobacterium tuberculosis can be achieved, fully transforming the way in which molecular epidemiology techniques can be integrated into control programs.