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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 . Mycobacterium abscessus is considered an emerging pathogen, given its prevalence in patients with pulmonary diseases, such as cystic fibrosis. Here, authors perform a genomic analysis on sequential isolates obtained from patients with persistent infections of M. abscessus .

Background. An increase in the incidence of tuberculosis (TB) in immigrants has changed the socioepidemiologic scenario in Spain. It is generally assumed that TB in immigrants is the result of importation of infection, but the role of recent transmission is rarely considered. Standard contact tracing is not suitable for the survey of transmission in this complex scenario. Methods. During the study period (2003–2006), we genotyped 356 (90.4%) of 394 isolates from patients with microbiologically confirmed TB in Almería, the province with the highest percentage of TB cases among immigrants in Spain. The epidemiologic survey of TB transmission was performed by active data collection using standardized interviews of the patients with TB and subsequent interviews of the clustered patients (who were clustered on the basis of the restriction fragment–length polymorphism types of their isolates) to identify transmission locations (supported by nominal and/or photographic recognition by the clustered patients). Results. Of all 356 genotyped isolates, 131 (36.8%) were clustered, suggesting recent transmission. The difference between the clustering rate for immigrants (32.8%) and that for native patients (41.6%) was not statistically significant (P=.087); of the 45 clusters, 15 (33.3%) involved only immigrants, 17 (37.8%) involved only autochthonous patients, and 13 (28.9%) involved both immigrants and autochthonous patients. The advanced system to investigate the clustered patients succeeded in detecting links in 10 of the 12 clusters that involved >4 patients, whereas the conventional approach, based on contact tracing, could detect links in only 2 clusters. Conclusions. Recent transmission among immigrants and transmission permeability between the immigrant and autochthonous populations were found. Epidemiologic strategies that combine universal genotyping and refined surveys of the clustered patients are needed to investigate transmission patterns in complex scenarios.

Abstract 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 (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.

Whole-genome sequencing (WGS) enhances precision in predicting antimicrobial resistance and tracking (MTB) transmission. Due to MTB's slow-growing nature, genomic results are delayed; however, few efforts have sought to accelerate them by performing WGS directly on respiratory specimens. Most culture-free efforts have focused on accelerating resistance prediction. The present study provides further evidence to the only preceding study aiming to accelerate precise delineation of transmission, coupling culture-free WGS to a surveillance programme. Our study is distinguished from its predecessor by being the first to apply flexible nanopore sequencing to further accelerate the process. A total of 71 sputa were selected, in which we applied only a procedure to deplete human DNA, thus avoiding costly and cumbersome capture-bait alternatives. Optimal results (>90% genome covered, mean coverage >45× and >70% genome covered >20×) were obtained from 33.8% of cases, allowing the assignment to transmission clusters close to diagnosis of every new case. A further 12.6% of samples yielded suboptimal results (15.5%-90.92% at >10×), which were exploited through a rescue pipeline. This approach was based on identifying informative SNPs acting as markers for relevant transmission clusters in our population. The pipeline enabled pre-allocation of new cases to pre-existing clusters and, in some cases, precise genomic relationships with the preceding cases in the cluster. In summary, this study demonstrates that epidemiologically valuable information can be obtained directly from sputum in approximately half the samples analysed. It represents a new advancement in the pursuit of faster comparative genomics, with epidemiological purposes, at diagnosis.