Erik Michael Rasmussen

Tuberculosis (TB) remains a major global health challenge, requiring rapid, reliable molecular tests to detect the Mycobacterium tuberculosis complex (MTBC) and resistance to the drugs that cause multidrug resistance, i.e., rifampicin and isoniazid. Although many moderate-complexity automated nucleic acid amplification tests use multiple targets to improve sensitivity, previous versions of FluoroType MTBDR (FTv2old; Bruker-Hain) relied on a single MTBC DNA target, often necessitating the use of an initial multilocus MTBC assay in the workflow to achieve comparable sensitivity. This prospective head-to-head study compared FTv2old with an updated version (FTv2new) that includes the multi-copy IS 6110 insertion sequence to improve MTBC detection. Using 1,483 clinical specimens from pulmonary and extrapulmonary sources, with culture and whole-genome sequencing (WGS) as reference standards, FTv2new showed a significant 17-percentage-point increase in overall sensitivity, reaching 87% compared with 70% for FTv2old ( P = 0.008). The improvement was particularly notable in microscopy-negative, culture-positive samples, where the sensitivity increased from 50% to 78% ( P = 0.008). Specificity remained high and comparable between versions at 99%. Because the assay’s resistance-conferring targets remained unchanged, the capability for resistance profiling did not improve significantly. These findings indicate that inclusion of the IS 6110 target closes the performance gap of the FluoroType platform, eliminating the need for reflex testing and streamlining laboratory workflows for high-throughput tuberculosis screening. IMPORTANCE: Tuberculosis remains a major global health threat, and rapid, accurate diagnosis is essential for disease control. This study evaluates an updated molecular assay for detecting the Mycobacterium tuberculosis complex (MTBC). Earlier versions of this assay relied on a single genetic target and were less sensitive than competing methods. The inclusion of a multi-copy target significantly improves MTBC detection, particularly in specimens with low bacterial loads that are frequently microscopy-negative. This improvement is clinically relevant, enabling faster, more reliable diagnosis while eliminating the need for additional, time-consuming reflex testing, thereby streamlining laboratory workflows and supporting timely initiation of appropriate treatment, thereby supporting global efforts to eliminate TB.

The global burden of human disease caused by nontuberculous mycobacteria (NTM) is steadily increasing. Using our unique historical collection of freeze-dried mycobacterial isolates at the Statens Serum Institut, Copenhagen, we investigated long-term trends in NTM clinical relevance and species distribution in Denmark over the past seven decades. A total of 666 historical NTM isolates from human samples collected between 1948 and 1978 were compared with 1,820 contemporary isolates identified during routine diagnostics from 2013 to 2022. Species identification was performed for all isolates using the GenoType CM and AS line-probe assays. Among historical isolates, 16 different species were identified, with the majority classified as Mycobacterium sp. (32.1%, n = 214). In contrast, recent isolates were dominated by Mycobacterium avium (39.6%, n = 721), while Mycobacterium sp. accounted for only 7.8% ( n = 142). Furthermore, notable changes in prevalence over time at the single species level were observed for M. avium and Mycobacterium abscessus . Further genomic analyses of historical Mycobacterium sp. isolates revealed several uncommon or previously unrecognized species rarely detected in current diagnostic settings. In conclusion, our findings suggest a significant shift in the spectrum of species responsible for human NTM infections in Denmark over the past seven decades, with possible implications for diagnostic strategies, clinical management, and public health surveillance. IMPORTANCE: Infections caused by nontuberculous mycobacteria (NTM) are increasingly recognized worldwide, yet the long-term epidemiology of these human pathogens remains poorly understood. By leveraging a unique historical collection of mycobacterial isolates, this study suggests a shift in the species spectrum responsible for NTM infections in Denmark over the past seven decades, with Mycobacterium avium now predominating. Sequencing of historical isolates further uncovered several rare or previously unrecognized Mycobacterium species, illustrating the dynamic nature of NTM epidemiology over time. These findings underscore the need for sustained surveillance, precise species-level identification, and heightened clinical awareness to guide effective management of NTM infections amid a continually evolving disease landscape.

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).

Despite the fact that the majority of people in tuberculosis (TB)–endemic areas are vaccinated with the Bacillus Calmette–Guérin (BCG) vaccine, TB remains the leading infectious cause of death. Data from both animal models and humans show that BCG and subunit vaccines induce T cells of different phenotypes, and little is known about how BCG priming influences subsequent booster vaccines. To test this, we designed a novel Mycobacterium tuberculosis–specific (or “non-BCG”) subunit vaccine with protective efficacy in both mice and guinea pigs and compared it to a known BCG boosting vaccine. In naive mice, this M. tuberculosis–specific vaccine induced similar protection compared with the BCG boosting vaccine. However, in BCG-primed animals, only the M. tuberculosis–specific vaccine added significantly to the BCG-induced protection. This correlated with the priming of T cells with a lower degree of differentiation and improved lung-homing capacity. These results have implications for TB vaccine design. Key Points Pre-existing BCG immunity influences T cell responses of subunit booster vaccines. M. tuberculosis–specific subunit vaccines bypass this mechanism and improve protection.

Background: Nontuberculous mycobacteria belonging to the Mycobacterium avium complex are recognized as opportunistic pathogens to humans. Mycobacterium arosiense is one of the novel members of the Mycobacterium avium complex. The organism has only rarely been reported in human clinical cases and may be routinely misidentified. Case presentation: An adult male with a history of a discus prolapse and sarcoidosis presented with high fever and a strong back pain with projection to the extremities. A Magnetic Resonance Imaging scan of columna revealed a tumor suspect process at thoracic vertebrae 11/12 with changes at the second lumbar vertebra, which was partly removed by laminectomy. Biopsy smears revealed acid-fast bacilli and turned out to be Mycobacterium tuberculosis complex PCR negative. The routine line probe assay INNO-LiPa v2 (INNOGENETICS NV, Gent), which differentiates 16 mycobacterial species indicated the presence of a not readily identifiable NTM species. Whereas, the GenoType Mycobacterium CM v2.0 (HAIN Lifescience GmbH) that routinely differentiates 14 clinically relevant mycobacteria revealed a Mycobacterium intracellulare species. However, additional diagnostic sequencing of the 16S rRNA gene confirmed the presence of a Mycobacterium arosiense species. Conclusions: This is the second unusual case of osteomyelitis with clinical significance ever to be reported, caused by Mycobacterium arosiense and complicated by an underlying sarcoidosis. Mycobacterium arosiense has rarely been reported clinically and the first description of the species was identified as the cause of osteomyelitis in a child with a hereditary partial interferon gamma deficiency. Symptoms attributed to sarcoidosis waned on Mycobacterium arosiense treatment and it is inconclusive whether the patient ever suffered from sarcoidosis. Mycobacterium arosiense was misidentified by the GenoType as Mycobacterium intracellulare and implicates that the diagnosis requires supplemental sequencing of the 16S rRNA gene.