Denmark, a tuberculosis low burden country, still experiences significant active Mycobacterium tuberculosis (Mtb) transmission, especially with one specific genotype named Cluster 2/1112–15 (C2), the most prevalent lineage in Scandinavia. In addition to environmental factors, antibiotic resistance, and human genetics, there is increasing evidence that Mtb strain variation plays a role for the outcome of infection and disease. In this study, we explore the reasons for the success of the C2 genotype by analysing strain specific polymorphisms identified through whole genome sequencing of all C2 isolates identified in Denmark between 1992 and 2014 (n = 952), and the demographic distribution of C2. Of 234 non-synonymous (NS) monomorphic SNPs found in C2 in comparison with Mtb reference strain H37Rv, 23 were in genes previously reported to be involved in Mtb virulence. Of these 23 SNPs, three were specific for C2 including a NS mutation in a gene associated with hyper-virulence. We show that the genotype is readily transmitted to different ethnicities and is also found outside Denmark. Our data suggest that strain specific virulence factor variations are important for the success of the C2 genotype. These factors, likely in combination with poor TB control, seem to be the main drivers of C2 success.

Four novel nontuberculous mycobacteria were discovered from a historical strain collection at the International Reference Laboratory of Mycobacteriology at Statens Serum Institut in Copenhagen, Denmark. Phylogenetic analysis combining the 16S rrs, internal transcribed spacer and 23S rrl elements, as well as a single-copy core-gene (hsp65, rpoB+C, secA and tuf) analysis of these freeze-dried mycobacteria, clinically isolated from gastric lavage samples between 1948 and 1955, showed to be associated with type strains grouping within the Terra and Fortuitum-Vaccae clade. Phenotypic characteristics, biochemical properties and fatty acid and mycolic acid profiles supported the classification as novel strains. A genomic comparison to the closest related type strain was done by calculating average nucleotide identity and in silico DNA:DNA hybridization values, which showed 87.9% and 33.0% for Mu0050, 85.2% and 27.4% for Mu0053, 85.3% and 27.6% for Mu0083 and 93.3% and 50.1% for Mu0102, respectively. The names proposed for the new species are Mycobacterium wendilense sp. nov. (Mu0050^T=ITM 501390^T=CCUG 77525^T), Mycobacterium burgundiense sp. nov. (Mu0053^T=ITM 501391^T=CCUG 77526^T), Mycobacterium kokjensenii sp. nov. (Mu0083^T=ITM 501392^T=CCUG 77527^T) and Mycobacterium holstebronense sp. nov. (Mu0102^T=ITM 501393^T=CCUG 77528^T).

Infection with Mycobacterium tuberculosis remains one of the biggest causes of death from a single microorganism worldwide, and the continuous emergence of drug resistance aggravates our ability to cure the disease. New improved resistance detection methods are needed to provide adequate treatment, such as whole genome sequencing (WGS), which has been used increasingly to identify resistance-conferring mutations over the last decade. The steadily increasing knowledge of resistance-conferring mutations increases our ability to predict resistance based on genomic data alone. This study evaluates the performance of WGS to predict M. tuberculosis complex resistance. It compares WGS predictions with the phenotypic (culture-based) drug susceptibility results based on 20 years of nationwide Danish data. Analyzing 6,230 WGS-sequenced samples, the sensitivities for isoniazid, rifampicin, ethambutol, and pyrazinamide were 82.5% [78.0%–86.5%, 95% confidence interval (CI)], 97.3% (90.6%–99.7%, 95% CI), 58.0% (43.2%–71.8%, 95% CI), and 60.5% (49.0%–71.2%, 95% CI), respectively, and specificities were 99.8% (99.7%–99.9%, 95% CI), 99.8% (99.7%–99.9%, 95% CI), 99.4% (99.2%–99.6%, 95% CI), and 99.9% (99.7%–99.9%, 95% CI), respectively. A broader range of both sensitivities and specificities was observed for second-line drugs. The results conform with previously reported values and indicate that WGS is reliable for routine resistance detection in resource-rich tuberculosis low-incidence and low-resistance settings such as Denmark.

Denmark is a low-incidence country for tuberculosis (TB) and multidrug-resistant (MDR) TB at 5 and 0.05 cases per 100,000 population, respectively. Until 2018, the transmission of MDR-TB was nonexistent except for a few pairwise related family cases. In this study, we describe the first MDR-TB outbreak in Denmark. On the basis of genotyping of all Mycobacterium tuberculosis (Mtb) culture-positive cases in Denmark spanning 3 decades, 6 molecular- and epidemiologically linked Danish-born cases were identified as the first cluster of an MDR-TB in Denmark. The primary case was diagnosed posthumously in 2010 followed by 5 epidemiologically linked cases from 2018 to 2019. Through a combination of routine Mtb genotyping and clinical epidemiological surveillance data, we identified the first Danish MDR-TB outbreak spanning 10 years and were able to disclose the specific transmission pathways in detail, which helped guide the outbreak investigations. The occurrence of an MDR-TB outbreak in a resource-rich low TB incidence setting such as Denmark highlights the importance of a collaborative control system combining classic contact tracing; timely identification of drug-resistant TB through rapid diagnostics; and a close collaboration between clinicians and classical- and molecular epidemiologists for the benefit of TB control.

Patients with pulmonary tuberculosis (PTB) may face challenges in providing sputum specimens for diagnosis. Mycobacterium tuberculosis (Mtb) is potentially present in the stool due to swallowed sputum. Therefore easyto-obtain stool could be used as an alternative to sputum sampling. However, the evidence for using stool specimens for PTB diagnosis is sparse, especially in resource-rich settings. In this study, spanning two years, the results of 562 stool specimens were evaluated alongside those of respiratory specimens sent to the International Reference Laboratory of Mycobacteriology in Denmark. Despite the potential advantages of stool culture, only five out of 19 patients with PTB had Mtb culturepositive stool, all of whom also had positive respiratory specimens. Conversely, relying solely on stool specimens could lead to missed diagnoses of PTB. While stool analysis may offer additional value in specific settings or populations, such as those unable to produce sputum, this study discourages its general use for PTB testing in resource-rich, TB low-incidence settings like Denmark. Instead, we advocate for prospective trials in specific subpopulations to elucidate the role of stool as a complementary diagnostic tool for PTB. The study underscores the importance of tailoring diagnostic approaches based on the setting and patient characteristics.