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Objectives: Extrapulmonary tuberculosis (EPTB) presents with nonspecific symptoms which can pose a significant diagnostic challenge. Various factors, including age, sex, and HIV status, have been associated with an increased risk of developing EPTB. However, the influence of the lineage of the infecting Mycobacterium tuberculosis complex (Mtbc) strain remains controversial., Methods: Between 2008 and 2023, comprehensive clinical data from 1035 cases, along with whole genome sequencing (WGS) data of the respective Mtbc strains have been collected. To examine the association between Mtbc lineage and EPTB, we calculated crude and adjusted odds ratios (OR) using logistic regression and performed propensity score matching with a subset of the cohort., Results: Of the 1035 patients, 272 had exclusively extrapulmonary disease and 138 had both pulmonary and extrapulmonary disease. Patients infected with a lineage 1 strain had the highest odds of developing EPTB in the univariate analysis (OR: 3.30, 95% CI: 1.97-5.49). However, Mtbc lineage was not a significant predictor in the multivariable model, while the odds of developing extrapulmonary disease were higher among patients born in the South-East Asian region (adjusted OR: 6.00, 95% CI: 3.41-10.69) and the Eastern Mediterranean Region (adjusted OR: 5.95, 95% CI: 3.61-9.96) compared to those born in the European region. Further, female sex and age were significant positive predictors for EPTB., Conclusions: Our results demonstrate that host factors, such as geographic origin, age and sex are stronger predictors for EPTB than infection with a Mtbc strain of a particular lineage. Further investigation of this host-pathogen interaction is needed., Competing Interests: Conflicts of interests Natalia Rachwal, Viola Dreyer, Elvira Richter, Stefan Niemann, Nils Wetzstein, and Udo Götsch have nothing to disclose. Raja Idris received speaker fees from Tillotts Pharma GmbH outside the submitted work. Thomas A. Wichelhaus reports research grants from BMBF, JPIAMR, Deutsche Krebshilfe, MSD as well as speaker fees and consulting honoraria from Insmed, Osartis, all outside the submitted work., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Competing Interests: Declaration of competing interest RAN is a paid consultant for ModernaTX and BioNTech. SJP is a consultant for Next Gen Diagnostics. GGR is scientific advisor of Resitell (Muttenz, Switzerland), a start-up implicated in nanomotion-based measure of antimicrobial resistance. DH is managing director and shareholder of the company Ridom GmbH that develops the SeqSphere + tool. AE is scientific advisor of Sefunda (Muttenz, Switzerland).

Objectives: We evaluated the ability of FluoroType MTBDR version 2 (FTv2; Hain Lifescience), a second-step real-time PCR assay, to simultaneously detect Mycobacterium tuberculosis complex (MTBC) DNA and mutations conferring resistance to rifampicin (RIF) and isoniazid (INH), in pulmonary and extrapulmonary samples from patients and compared them with corresponding cultures., Methods: FTv2 MTBC was evaluated on 1815 and 432 samples from Denmark (DK) and Germany (DE), respectively. RIF and INH resistance mutations were assessed in the German samples and 110 samples from Sierra Leone and subsequently compared to phenotypic antimicrobial susceptibility testing and a composite reference DNA (CRD) based on the GenoType MTBDR line-probe assay and Sanger sequencing or whole-genome sequencing., Results: Of the 584 (557 smear-negative) Danish and 277 (85 smear-negative) German sputum samples, 42 (16) and 246 (54) were culture positive, and 44 (18) and 222 (35) were FTv2 positive, providing an FTv2 sensitivity and specificity of 0.86 (0.63) and 0.98 (DK), 0.90 (0.65) and 1.00 (DE), respectively. The count, sensitivities, and specificities for all pulmonary samples were 1434, 0.79, and 0.99 (DK) and 347, 0.86, and 1.00 (DE), respectively; for extrapulmonary samples, 381, 0.33, 0.99 (DK) and 83, 0.50, and 1.00 (DE). The valid count, sensitivity, and specificity compared with CRD for detecting resistance mutations were RIF 355, 0.99, 0.96, and INH 340, 1.00, and 0.98, respectively., Discussion: FTv2 reliably detects MTBC DNA in pulmonary and extrapulmonary samples and detects resistance mutations for INH and RIF resistance in inhA promoter, katG, and rpoB genes., (Copyright © 2024 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Objectives: Since 2013, heater-cooler unit (HCU) associated Mycobacterium chimaera infections linked to a global outbreak have been described. These infections were characterised by high morbidity and mortality due to delayed diagnosis, as well as challenges in antimycobacterial and surgical therapy. This study aimed to investigate the clinical characteristics and outcome of published cases of HCU-associated M. chimaera infections., Methods: We searched PubMed and the Web of Science until 15 June 2022 for case reports, case series, and cohort studies, without language restriction, on patients with M. chimaera infection and a prior history of cardiac surgery. In this systematic review of case reports, no risk of bias assessment could be performed. Clinical, microbiological, and radiological features were recorded. Logistic regression and time-to-event analyses were performed to identify the potential factors associated with better survival., Results: One hundred eighty patients from 54 publications were included. Most patients underwent surgical aortic valve (67.0%; 118/176 of patients with available data) or combined aortic valve and root replacement (15.3%; 27/176). The median period between the time point of surgery and the first symptoms was 17 months (interquartile range 13-26 months). The overall case fatality rate was 45.5% (80/176), with a median survival of 24 months after the initiation of antimycobacterial therapy or diagnosis. A reoperation (including the removal or exchange of foreign material) was associated with better survival in multivariate logistic regression (OR 0.32 for lethal events; 95% CI 0.12-0.79; p 0.015) and in time-to-event analysis (p 0.0094)., Discussion: This systematic review and meta-analysis confirm the high overall mortality of HCU -associated disseminated M. chimaera infections after cardiac surgery. A reoperation seems to be associated with better survival. Physicians have to stay aware of this infection, as patients might still be present today due to the long latency period., (Copyright © 2023 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Background: Data from a prospective molecular-epidemiologic study (1997-2021) in Hamburg, Germany, were evaluated to assess the transmission risk of Mycobacterium tuberculosis complex (Mtbc) by children <15 years in a low-incidence setting., Methods: Isolates of Mtbc were genotyped whole genome sequencing, applying a core genome multilocus sequence typing scheme. Close contacts of culture-confirmed children were examined for latent Mtbc infections (LTBI) with particular focus on IGRA testing., Results: Out of 3154 culture-confirmed tuberculosis (TB) cases, 79 (2.5%) were children <15 years. Of those, 52 (58%) had pulmonary TB. Genotyping revealed that 35 of the 52 children (67%) were epidemiologically confirmed secondary cluster members; all of their source cases were adults. Six immigrant children presented without a presumed source case; their TB diagnoses came on average 48 weeks (interquartile range [IQR] 71) after their arrival in Germany. Three German-born children were determined to have been infected by adult relatives while visiting their parents' home country. Of the 317 children's close contacts tested with QuantiFERON-TB Gold-In Tube for LTBI, only 21 (6.6%) were positive. Absent a history of prior exposure or immigration from a high-incidence country, none of the contacts of younger (<10 years) TB-afflicted children was latently infected, whereas 2 older children infected 12 of their contacts, children and adults. During a mean observational period of 551 weeks (IQR 735) on average, no secondary TB cases appeared., Conclusions: Children with pulmonary TB disease, especially those aged below 10 years, rarely transmit Mtbc to their close contacts in a low-incidence setting., Competing Interests: Declaration of competing interest None., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: With the increasing dimensions of the international cardiac surgery-associated Mycobacterium chimaera outbreak the hypothesis of a point source arose., Objectives: To review the published evidence of clonality among cardiac surgery-associated M. chimaera isolates evaluated by whole-genome sequencing (WGS) and to perform an integrative genomic analysis of available genome data., Data Sources: We searched PubMed and EMBASE for studies applying WGS on cardiac surgery-associated M. chimaera isolates., Study Eligibility Criteria: We included studies that applied WGS on more than a single M. chimaera isolate., Methods: Two authors independently extracted data from included studies. Available genome data from published studies were subjected to a joint analysis., Results: Of 121 identified articles, nine studies were included. M. chimaera isolates from LivaNova heater-cooler devices (HCDs) had a high level of genetic similarity, but were genetically distant from isolates from HCDs produced by other manufacturers. With the exception of a single (11.1%) study, the remaining eight (89.9%) studies reported a high level of genetic proximity between the majority of M. chimaera isolates derived from cardiac surgery-associated patients and LivaNova HCDs. In-depth analysis revealed involvement of three distinct M. chimaera subgroups in the outbreak (1.1, 1.8, 2.1), with 1.1 suggested as causative of the outbreak. Samples taken at the LivaNova production site supported contamination with strains of subgroups 1.1 and 1.8. In the combined analysis of 526 publicly available WGS data sets, nearly all isolates from cardiac surgery-associated patients contained strain 1.1 (50/52, 96.2%), and at least one of the outbreak strains was found in almost all LivaNova HCDs (241/257, 93.8%), with strain 1.1 in particular present in 198/257 (77.0%)., Conclusions: HCD contamination during production seems plausible as the predominant point source for the global M. chimaera outbreak. Although HCDs can be contaminated with mixed populations, M. chimaera strains of the subgroup 1.1 caused most infections., (Copyright © 2021 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Objective: Phenotypic (Sensititre Myco, pDST) and genotypic drug susceptibility testing (GenoType NTM DR, gDST) in M. avium complex (MAC) have become available as standardized assays, but comparable data is needed. This study aimed to investigate the phenotypic and genotypic drug susceptibility patterns in MAC clinical isolates., Methods: Overall, 98 isolates from 85 patients were included. pDST and gDST were performed on all isolates and results compared regarding specificity and sensitivity using pDST as a reference method. The impact of drug instability on pDST results was studied using a biological assay over 14 days. In addition, the evolution of antimicrobial resistance was investigated in sequential isolates of 13 patients., Results: Macrolide resistance was rare, 1.2% (95% CI 0.7-7.3) of isolates in the base cohort. No aminoglycoside resistances were found, but 14.1% of the studied isolates (95% CI 7.8-23.8) showed intermediate susceptibility. The GenoType NTM DR identified two out of four macrolide-resistant isolates. Antibiotic stability was demonstrated to be poor in rifampicin, rifabutin, and doxycycylin., Conclusions: pDST results in NTM for unstable antibiotics must be interpreted with care. A combination of pDST and gDST will be useful for the guidance of antimicrobial therapy in MAC-disease., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Tuberculosis (TB) remains a serious public health threat worldwide. Theoretically ultimate resolution of whole genome sequencing (WGS) for Mycobacterium tuberculosis complex (MTBC) strain classification makes this technology very attractive for epidemiological investigations., Objectives: To summarize the evidence available in peer-reviewed publications on the role and place of WGS in detection of TB transmission., Sources: A total of 69 peer-reviewed publications identified in Pubmed database., Content: Evidence from >30 publications suggests that a cut-off value of fewer than six single nucleotide polymorphisms between strains efficiently excludes cases that are not the result of recent transmission and could be used for the identification of drug-sensitive isolates involved in direct human-to-human TB transmission. Sensitivity of WGS to identify epidemiologically linked isolates is high, reaching 100% in eight studies with specificity (17%-95%) highly dependent on the settings. Drug resistance and specific phylogenetic lineages may be associated with accelerated mutation rates affecting genetic distances. WGS can be potentially used to distinguish between true relapses and re-infections but in high-incidence low-diversity settings this would require consideration of epidemiological links and minority alleles. Data from four studies looking into within-host diversity highlight a need for developing criteria for acceptance or rejection of WGS relatedness results depending on the proportion of minority alleles., Implications: WGS will potentially allow for more targeted public health actions preventing unnecessary investigations of false clusters. Consensus on standardization of raw data quality control processing criteria, analytical pipelines and reporting language is yet to be reached., (Crown Copyright © 2019. Published by Elsevier Ltd. All rights reserved.)

Objectives: Neither the liquid medium-based Bactec MGIT, nor commercial molecular assays such as the Xpert MTB/RIF and the MTBDRplus V2.0 assays are capable of detecting up to 30% of rifampicin-resistant Mycobacterium tuberculosis strains in Swaziland because of the large proportion of the rpoB Ile491Phe mutations. In other countries, the frequency of this mutation is thought to be low., Methods: We designed a real-time multiplex allele-specific PCR assay to identify the rpoB Ile491Phe mutation responsible for these undetected resistant M. tuberculosis strains., Results: The technique showed 100% similarity with rpoB sequencing on a panel of 78 strains from Swaziland., Conclusions: We propose that the detection of the rpoB Ile491Phe rpoB mutation should complement commercial assays for the diagnosis of rifampicin-resistant M. tuberculosis in routine conditions, particularly in countries where this specific mutation is frequent. The technique proposed in this paper is adapted for most reference laboratories., (Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Global tuberculosis incidence has declined marginally over the past decade, and tuberculosis remains out of control in several parts of the world including Africa and Asia. Although tuberculosis control has been effective in some regions of the world, these gains are threatened by the increasing burden of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis. XDR tuberculosis has evolved in several tuberculosis-endemic countries to drug-incurable or programmatically incurable tuberculosis (totally drug-resistant tuberculosis). This poses several challenges similar to those encountered in the pre-chemotherapy era, including the inability to cure tuberculosis, high mortality, and the need for alternative methods to prevent disease transmission. This phenomenon mirrors the worldwide increase in antimicrobial resistance and the emergence of other MDR pathogens, such as malaria, HIV, and Gram-negative bacteria. MDR and XDR tuberculosis are associated with high morbidity and substantial mortality, are a threat to health-care workers, prohibitively expensive to treat, and are therefore a serious public health problem. In this Commission, we examine several aspects of drug-resistant tuberculosis. The traditional view that acquired resistance to antituberculous drugs is driven by poor compliance and programmatic failure is now being questioned, and several lines of evidence suggest that alternative mechanisms-including pharmacokinetic variability, induction of efflux pumps that transport the drug out of cells, and suboptimal drug penetration into tuberculosis lesions-are likely crucial to the pathogenesis of drug-resistant tuberculosis. These factors have implications for the design of new interventions, drug delivery and dosing mechanisms, and public health policy. We discuss epidemiology and transmission dynamics, including new insights into the fundamental biology of transmission, and we review the utility of newer diagnostic tools, including molecular tests and next-generation whole-genome sequencing, and their potential for clinical effectiveness. Relevant research priorities are highlighted, including optimal medical and surgical management, the role of newer and repurposed drugs (including bedaquiline, delamanid, and linezolid), pharmacokinetic and pharmacodynamic considerations, preventive strategies (such as prophylaxis in MDR and XDR contacts), palliative and patient-orientated care aspects, and medicolegal and ethical issues., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Whole genome sequencing (WGS) can help to relate Mycobacterium tuberculosis genomes to one another to assess genetic relatedness and infer the likelihood of transmission between cases. The same sequence data are now increasingly being used to predict drug resistance and susceptibility. Controlling the spread of tuberculosis and providing patients with the correct treatment are central to the World Health Organization's target to 'End TB' by 2035, for which the global prevalence of drug-resistant tuberculosis remains one of the main obstacles to success. So far, WGS has been applied largely to drug-susceptible strains for the purposes of understanding transmission, leaving a number of analytical considerations before transferring what has been learnt from drug-susceptible disease to drug-resistant tuberculosis. We discuss these potential problems here, alongside some of the challenges to characterizing the Mycobacterium tuberculosis 'resistome'-the optimal knowledge-base required for WGS-based assays to successfully direct individualized treatment regimens through the prediction of drug resistance and susceptibility in the future., (Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

The rpoB gene codes for the RNA polymerase β subunit, which is the target of rifampicin, an essential drug in the treatment of tuberculosis and other mycobacterial infections. This gene is present in all bacteria, but its length and nucleotide sequence vary between bacterial species, including mycobacteria. Mutations in the rpoB gene alter the structure of this protein and cause drug resistance. To describe the resistance-associated mutations, the scientific and medical communities have been using, since 1993, a numbering system based on the Escherichia coli sequence annotation. Using E. coli reference for describing mutations in mycobacteria leads to misunderstandings, particularly with the increasing use of whole genome sequencing, which brought an alternative numbering system based on the Mycobacterium tuberculosis rpoB sequence. We propose using a consensus numbering system for the reporting of resistance mutations based on the reference genomes from the species interrogated (such as strain H37Rv for M. tuberculosis). This manuscript provides the necessary figures and tables allowing researchers, microbiologists and clinicians to easily convert other annotation systems into one common language., (Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Staphylococcus aureus causes very serious infections of vascular grafts. Knowledge of the molecular mechanisms of this disease is largely lacking because of the absence of representable models. Therefore, the aim of this study was to set up a mouse model of vascular graft infections that closely mimics the human situation. A catheter was inserted into the right carotid artery of mice, which acted as a vascular graft. Mice were infected i.v. using 8 different S. aureus strains, and development of the infection was followed up. Although all strains had varying abilities to form biofilm in vitro and different levels of virulence in mice, they all caused biofilm formation on the grafts. This graft infection was monitored using magnetic resonance imaging (MRI) and 18 F-fluordeoxyglucose positron emission tomography (FDG-PET). MRI allowed the quantification of blood flow through the arteries, which was decreased in the catheter after infection. FDG-PET revealed high inflammation levels at the site of the catheter after infection. This model closely resembles the situation in patients, which is characterized by a tight interplay between pathogen and host, and can therefore be used for the testing of novel treatment, diagnosis, and prevention strategies. In addition, combining MRI and PET with microscopic techniques provides an appropriate way to characterize the course of these infections and to precisely analyze biofilm development., (Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Host cell invasion is a major feature of Staphylococcus aureus and contributes to infection development. The intracellular metabolically active bacteria can induce host cell activation and death but they can also persist for long time periods. In this study a comparative analysis was performed of different well-characterized S. aureus strains in their interaction with a variety of host cell types. Staphylococcus aureus (strains 6850, USA300, LS1, SH1000, Cowan1) invasion was compared in different human cell types (epithelial and endothelial cells, keratinocytes, fibroblasts, osteoblasts). The number of intracellular bacteria was determined, cell inflammation was investigated, as well as cell death and phagosomal escape of bacteria. To explain strain-dependent differences in the secretome, a proteomic approach was used. Barrier cells took up high amounts of bacteria and were killed by aggressive strains. These strains expressed high levels of toxins, and possessed the ability to escape from phagolysosomes. Osteoblasts and keratinocytes ingested less bacteria, and were not killed, even though the primary osteoblasts were strongly activated by S. aureus. In all cell types S. aureus was able to persist. Strong differences in uptake, cytotoxicity, and inflammatory response were observed between primary cells and their corresponding cell lines, demonstrating that cell lines reflect only partially the functions and physiology of primary cells. This study provides a contribution for a better understanding of the pathomechanisms of S. aureus infections. The proteomic data provide important basic knowledge on strains commonly used in the analysis of S. aureus-host cell interaction., (Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Background: Slow and cumbersome laboratory diagnostics for Mycobacterium tuberculosis complex (MTBC) risk delayed treatment and poor patient outcomes. Whole-genome sequencing (WGS) could potentially provide a rapid and comprehensive diagnostic solution. In this prospective study, we compare real-time WGS with routine MTBC diagnostic workflows., Methods: We compared sequencing mycobacteria from all newly positive liquid cultures with routine laboratory diagnostic workflows across eight laboratories in Europe and North America for diagnostic accuracy, processing times, and cost between Sept 6, 2013, and April 14, 2014. We sequenced specimens once using local Illumina MiSeq platforms and processed data centrally using a semi-automated bioinformatics pipeline. We identified species or complex using gene presence or absence, predicted drug susceptibilities from resistance-conferring mutations identified from reference-mapped MTBC genomes, and calculated genetic distance to previously sequenced UK MTBC isolates to detect outbreaks. WGS data processing and analysis was done by staff masked to routine reference laboratory and clinical results. We also did a microcosting analysis to assess the financial viability of WGS-based diagnostics., Findings: Compared with routine results, WGS predicted species with 93% (95% CI 90-96; 322 of 345 specimens; 356 mycobacteria specimens submitted) accuracy and drug susceptibility also with 93% (91-95; 628 of 672 specimens; 168 MTBC specimens identified) accuracy, with one sequencing attempt. WGS linked 15 (16% [95% CI 10-26]) of 91 UK patients to an outbreak. WGS diagnosed a case of multidrug-resistant tuberculosis before routine diagnosis was completed and discovered a new multidrug-resistant tuberculosis cluster. Full WGS diagnostics could be generated in a median of 9 days (IQR 6-10), a median of 21 days (IQR 14-32) faster than final reference laboratory reports were produced (median of 31 days [IQR 21-44]), at a cost of £481 per culture-positive specimen, whereas routine diagnosis costs £518, equating to a WGS-based diagnosis cost that is 7% cheaper annually than are present diagnostic workflows., Interpretation: We have shown that WGS has a scalable, rapid turnaround, and is a financially feasible method for full MTBC diagnostics. Continued improvements to mycobacterial processing, bioinformatics, and analysis will improve the accuracy, speed, and scope of WGS-based diagnosis., Funding: National Institute for Health Research, Department of Health, Wellcome Trust, British Colombia Centre for Disease Control Foundation for Population and Public Health, Department of Clinical Microbiology, Trinity College Dublin., (Copyright © 2016 Pankhurst et al. Open Access article distributed under the terms of CC BY. Published by Elsevier Ltd.. All rights reserved.)

Skeletons of (C3H X 101)F1 mice have been examined in earlier studies of the induction of dominant skeletal mutations. The present experiment was done to determine whether the same criteria used to identify mutations in (C3H X 101)F1 mice could be applied to the offspring collected in specific-locus experiments. Offspring were obtained from an experiment of Hitotsumachi et al. in which (101 X C3H)F1 male mice were exposed to 0, 300 mg/kg or 400 mg/kg of ethylnitrosourea (ENU) injected i.p. in exposures of 100 mg/kg administered 7 days apart. The 3 skeletal non-breeding-test (NBT) methods were applied in evaluating skeletons. The frequencies of presumed dominant skeletal mutations found following exposure of stem-cell spermatogonia to 0, 3 X 100 mg/kg, and 4 X 100 mg/kg of ENU were 2/374, 10/243, and 10/180, respectively. At each exposure level there is a highly statistically significant increase over the control. At the higher exposure, the induced presumed mutation frequency is 5.0% and the induced frequency of presumed mutations likely to be of clinical importance is 4.5%. The indices of mutation were 0% in the control, 11.5% in the 300 mg/kg group, and 12.4% in the 400 mg/kg group. These results show that the skeletal NBT methods can easily be combined with specific-locus experiments to increase the yield of data useful in estimating genetic risk. It appears that the induction of dominant skeletal mutations by ENU is reasonably similar when measured in specific-locus or (C3H X 101)F1 offspring.