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Background The effect of chronic sputum (CS) symptoms on health-related quality of life (HRQOL) in patients with nontuberculous mycobacterial pulmonary disease (NTM-PD) has not been studied. The aim of this study was to clarify the differences in the clinical characteristics of NTM-PD patients with and without CS and to investigate the effect of CS on HRQOL. Methods This cross-sectional study included patients with NTM-PD who were prescribed pulmonary rehabilitation at the Fukujuji Hospital from March 2016 to June 2019. HRQOL was evaluated using the MOS 36-Item Short-Form Health Survey (SF-36). Results Of the 99 subjects studied, 71 had CS (CS+) (71.7%), and 28 (28.3%) did not have CS (CS-). Patients in the CS + group had a lower body mass index, forced vital capacity percent predicted, and forced expiratory volume in 1 s percent predicted. Regarding the radiological evaluation, the proportion of patients with the fibrocavitary form and the radiological score were significantly higher in the CS + group. The mental component summary (MCS) score of the SF-36 were significantly lower in the CS + group. Multiple regression analysis showed that the presence of CS was independently associated with a lower MCS score of the SF-36. Conclusions NTM-PD patients with CS had more severe disease, with reduced pulmonary function and severe radiological findings. CS was shown to independently affect HRQOL, especially mental status.

Background: In the CONVERT study, treatment with amikacin liposome inhalation suspension (ALIS) added to guideline-based therapy (GBT) met the primary end point of increased culture conversion by month 6 in patients with treatment-refractory Mycobacterium avium complex lung disease (ALIS plus GBT, 29% [65/224] vs GBT alone, 8.9% [10/112]; P < .0001). Research Question: In patients who achieved culture conversion by month 6 in the CONVERT study, was conversion sustained (negative sputum culture results for 12 months with treatment) and durable (negative sputum culture results for 3 months after treatment) and were there any additional safety signals associated with a full treatment course of 12 months after conversion? Study Design and Methods: Adults were randomized 2:1 to receive ALIS plus GBT or GBT alone. Patients achieving culture conversion by month 6 continued therapy for 12 months followed by off-treatment observation. Results: More patients randomized to ALIS plus GBT (intention-to-treat population) achieved conversion that was both sustained and durable 3 months after treatment vs patients randomized to GBT alone (ALIS plus GBT, 16.1% [36/224] vs GBT alone, 0% [0/112]; P < .0001). Of the patients who achieved culture conversion by month 6, 55.4% of converters (36/65) in the ALIS plus GBT treated arm vs no converters (0/10) in the GBT alone arm achieved sustained and durable conversion (P = .0017). Relapse rates through 3 months after treatment were 9.2% (6/65) in the ALIS plus GBT arm and 30.0% (3/10) in the GBT alone arm. Common adverse events among ALIS plus GBT-treated patients (dysphonia, cough, dyspnea, hemoptysis) occurred mainly within the first 8 months of treatment. Interpretation: In a refractory population, conversion was sustained and durable in more patients treated with ALIS plus GBT for 12 months after conversion than in those treated with GBT alone. No new safety signals were associated with 12 months of treatment after conversion. Trial Registry: ClinicalTrials.gov; No.: NCT02344004; URL: www.clinicaltrials.gov

The nontuberculous mycobacterial pulmonary disease (NTM-PD) caused by Mycobacterium species has increased in prevalence all over the world. The distributions of NTM-PD are possibly determined by the westerly wind traveling at high altitudes over East Asia. However, the long-range transport of Mycobacterium species has not been demonstrated by analyzing the bacterial communities in aerosols such as desert mineral particles and anthropogenic pollutants transported by the westerly wind. Here, airborne bacterial compositions were investigated including Mycobacterium species in high-elevation aerosols, which were captured in the snow cover at 2,450 m altitude on Mt. Tateyama. This was further compared to the ground-level or high-altitude aerosols collected at six sampling sites distributed from Asian-dust source region (Tsogt-Ovoo) to downwind areas in East Asia (Asian continental cities; Erenhot, Beijing, Yongin, Japanese cities; Yonago, Suzu, Noto Peninsula). The cell concentrations and taxonomic diversities of airborne bacteria decreased from the Asian continent to the Japan area. Terrestrial bacterial populations belonging to Firmicutes and Actinobacteria showed higher relative abundance at high-elevation and Japanese cities. Additionally, Mycobacterium species captured in the snow cover on Mt. Tateyama increased in relative abundance in correspondence to the increase of black carbon concentrations. The relative abundance of Mycobacterium sequences was higher in the aerosol samples of Asian continental cities and Japanese cities than in the desert area. Presumably, anthropogenic pollution over East Asia carries potential Mycobacterium species, which induce NTM-PD, thereby impacting upon the public health.

Mycobacterium europaeum (M. europaeum) was recently identified as a nontuberculous mycobacterium belonging to the Mycobacterium simiae complex. There have been only a few reported cases of M. europaeum lung disease, all of which occurred in patients with immunodeficiency or prior lung disease. We herein report a case of M. europaeum lung disease in an otherwise healthy Japanese individual. A 70-year-old woman who had no apparent immunodeficiency or medical history was diagnosed with M. europaeum lung disease by multiple positive sputum cultures. The patient was successfully treated with clarithromycin, rifampin, ethambutol, and amikacin. This report is the first case of M. europaeum lung disease occurring in an individual without predisposing risk factors.

Introduction The details of the practice of treating nontuberculous mycobacterial pulmonary disease (NTMPD) have not been studied in Japan. Methods We studied a random sample of 2% (184) of the 9,200 patients with incident NTM-PD in 2010 who received standard three-drug therapy for at least some of their treatment between 2010 and 2014. Results The median duration of the standard treatment period was 248 days (IQR 56–540 days). Although 59% of the patients were treated with standard therapy for more than 6 months, only 41% were treated for 12 months. Fifty-three patients (29%) initiated treatment with substandard regimen, and 18 (34%) of those patients received treatment regimens that can lead to the development of macrolide resistance (MR)(CLR monotherapy or CLR + RIF). Furthermore, initially, 184 receiving the standard treatment, 49 patients (27%) eventually deviated from it, and 31 patients (63%) received regimens increasing the risk of developing MR. The sporadic administration of macrolide monotherapy was observed before and after the administration of the standard treatment for 50 patients (27.7%) and 41 patients (27.2%), respectively. Conclusions Approximately 60% of the treated patients did not continue the standard regimen for more than 12 months and 42% were at risk for developing MR before and after receiving the standard treatment. It is important to educate physicians and patients about the correct and safe management of NTMPD.

Background The optimal duration of antimicrobial therapy for Mycobacterium avium complex lung disease (MAC-LD) is unknown, and recurrence rates are high after treatment discontinuation. Intermittent therapy is recommended for the initial treatment of non-cavitary nodular/bronchiectatic MAC-LD. We hypothesized that intermittent maintenance therapy (IMT) could effectively prevent recurrence after successful treatment of MAC-LD. Methods Adult patients diagnosed with MAC-LD who received IMT after successful daily therapy (DT) between January 1, 2006 and December 31, 2016 were identified from clinical databases at three institutions in Japan. Treatment outcomes were evaluated for all patients. Results Of 38 patients (median age, 66 years; 29 women; nodular/bronchiectatic form, 29 patients) who received IMT after successful treatment, one was excluded due to death from an unknown cause, 1 month after IMT initiation. Finally, treatment outcomes were evaluated for 37 patients. Twenty-eight (76%) patients had sustained negative culture results over a median follow-up duration of 2.7 (interquartile range [IQR], 1.9–6.0) years, while six (16%) required switching to DT because of clinical deterioration over a median follow-up duration of 2.7 (IQR, 1.6–4.1) years. Favorable clinical outcomes were achieved for all patients who exhibited clinical deterioration. All patients tolerated the antimicrobials without discontinuation, and follow-up drug susceptibility testing showed negative results for clarithromycin-resistant MAC in the patients who experienced clinical deterioration. Conclusions IMT after successful treatment may be a feasible option for patients with MAC-LD. Further studies should determine the population that would benefit from this strategy.

Rationale No comprehensive analysis has previously been performed to evaluate the clinical aspects of and microbiological evidence associated with Mycobacteroides abscessus complex (MABC) infection in a region, such as Japan, with a low MABC incidence. Objectives This study aimed to clarify the clinicopathological characteristics of MABC, which included clinical relatedness to erm(41) sequevar, phenotype (as colony morphology and minimum inhibitory concentration), and genotype. Methods A total of 121 MABC patients (68 with M. abscessus subsp. abscessus and 53 with M. abscessus subsp. massiliense) were recruited into this retrospective clinical-biological study from tertiary hospitals in Japan between 2004 and 2014. Results Approximately 30% of MABC patients had a history of previous nontuberculous mycobacterium (NTM) disease. Furthermore, 24.8% of the patients had another concomitant NTM infection after they were diagnosed with MABC. Fewer than 10% of the patients in the M. abscessus group had T28C in erm(41). While we observed a higher conversion rate for M. massiliense than for M. abscessus (72.4% and 34.8%, respectively, p = 0.002), recurrence remained relatively common for M. massiliense (31.0%). In the M. abscessus patients, the MIC of clarithromycin (CLR) was significantly lower on day 3 in patients with a better treatment response than in refractory patients (The median MIC; 0.75 μg/ml v.s 2.0 μg/ml, p = 0.03). There was no significant relation between clinical manifestations and variable number of tandem repeat genotypes. Conclusions Because the history and simultaneous isolation of other NTM in MABC infection are relatively common, these information should be carefully translated into clinical actions. The evaluation of early CLR resistance in M. abscessus and the erm(41) functions should be important to improve the treatment strategy.

Background Mycobacterium lentiflavum is a slow-growing non-tuberculous Mycobacterium that is often associated with an immunocompromised state and cervical lymphadenitis in young children. However, little is known about the clinical importance of pulmonary infection with M. lentiflavum in adults. Methods The medical records of all adults who met the diagnostic criteria of pulmonary M. lentiflavum disease at Keio University Hospital and Fukujuji Hospital from 2001 to 2015 were reviewed. In addition, the PubMed database was searched to identify further reported cases in non-HIV adults. Results Five cases of pulmonary M. lentiflavum disease were identified in the medical records search and 11 additional cases were identified in the literature review. Eleven of the total 16 cases were female, and 15 of 16 cases showed a nodular/bronchiectatic pattern on chest computed tomography imaging. No cases showed an aggressive clinical course of pulmonary M. lentiflavum disease, although one patient died of an exacerbation of underlying vasculitis and bacterial pneumonia. Conclusions The clinical characteristics of pulmonary M. lentiflavum disease in adult patients were identified. This disease mainly affects females, displays a nodular/bronchiectatic pattern on chest computed tomography imaging, and does not demonstrate an aggressive clinical course. Further larger studies are needed to reveal detailed clinical features.

Early detection and treatment are paramount for the timely control of Mycobacterium avium infections. Herein, we designed a LAMP assay targeting a widely used species-specific marker IS1245 for the rapid detection of M. avium and evaluated its applicability using human (n = 137) and pig (n = 91) M. avium isolates from Japan. The developed assay could detect as low as 1 genome copy of M. avium DNA within 30 minutes. All 91 (100%) M. avium isolates from pigs were detected positive while all other tested bacterial species were negative. Interestingly, among the 137 clinical M. avium isolates, 41 (30%) were undetectable with this LAMP assay as they lacked IS1245, the absence of which was revealed by PCR and whole-genome sequencing. These findings highlighted genotypic differences in M. avium strains from humans and pigs in Japan and how this diversity can influence the applicability of a detection tool across different geographic areas and hosts.

Xpert MTB/RIF is an automated nucleic acid amplification test (NAT) that can detect the presence of Mycobacterium tuberculosis complex (MTC) in clinical specimens as well as rifampicin (RIF) resistance resulting from rpoB mutation. Despite its high sensitivity and specificity for diagnosing tuberculosis (TB) with or without RIF resistance, the clinical performance of the test is variable. In this study, we evaluated the performance of Xpert MTB/RIF in a setting of moderate TB burden and high medical resources. A total of 427 sputum specimens were obtained from 237 suspected TB cases. Of these, 159 were identified as active TB, while the other 78 were non-TB diseases. The overall sensitivity and specificity of MTC detection by Xpert MTB/RIF using culture results as a reference were 86.8% [95% confidence interval (CI): 81.8%-90.6%] and 96.8% (95% CI: 93.1%-98.5%), respectively. Among MTC-positive culture specimens, Xpert MTB/RIF positivity was 95.2% (95% CI: 91.2%-97.5%) in smear-positive and 44.7% (95% CI 30.1-60.3) in smear-negative specimens. Xpert MTB/RIF was similar to other NATs (TaqMan MTB and TRCRapid M.TB) in terms of performance. Xpert MTB/RIF detected 25 RIF-resistant isolates as compared to 22 with the mycobacterial growth indicator tube antimicrobial susceptibility testing system, yielding a sensitivity of 100% (95% CI: 85.1%-100%) and specificity of 98.3% (95% CI: 95.1%-99.4%). These results indicate that although sensitivity in smear-negative/culture-positive specimens was relatively low, Xpert MTB/RIF is a useful diagnostic tool for detecting TB and RIF resistance even in settings of moderate TB burden.

Background Pulmonary nontuberculous mycobacteria (NTM) disease is a chronic progressive lung disease caused by environmental mycobacteria. Although epidemiological data indicate a potential genetic predisposition to NTM disease, the nature of this remains unclear. The present study aimed to identify host genes associated with susceptibility to Mycobacterium avium complex (MAC), the most common NTM pathogen. Methods We conducted a genome-wide association study (GWAS) in Japanese patients with pulmonary MAC and healthy controls, followed by genotyping of candidate SNPs in another group of patients and controls. For verification in European populations, we performed imputation to estimate genotypes of a candidate SNP from exome sequencing data. Findings The GWAS discovery set included 475 pulmonary MAC patients and 417 healthy subjects. After quality control filtering of genotyped variants, 622,723 autosomal variants were analysed. Both GWAS and replication analysis of 591 pulmonary MAC patients and 718 controls identified strongest association with chromosome 16p21, and particularly with rs109592 (p = 1.60E-13, odds ratio = 0.54). This SNP is located in an intronic region of the calcineurin like EF-hand protein 2 (CHP2) gene, and expression quantitative trait locus analysis showed association with lung CHP2 expression. Further, patient radiological findings showed that this SNP was associated with the nodular bronchiectasis disease subtype. This SNP is also significantly associated in European populations (p = 5.08E-06, odds ratio = 0.23). Interpretation These findings identify CHP2 as a candidate gene for further investigation of the pathogenesis of pulmonary MAC disease. Funding: JSPS KAKENHI, AMED, Keio Gijuku Academic Development Funds, NIH. Declaration of Interest: The authors declare no conflict of interest. Ethical Approval: This study was reviewed and approved by the research ethics committees of the Keio University School of Medicine (2012-0336), the Graduate School of Medicine, the University of Tokyo (G3579), and other collaborating institutions (UMIN000021692). Regarding European cohort, the subjects were enrolled in studies under National Institute of Allergy and Infectious Diseases Institutional Review Board-approved protocol (93-I-0119). All adult subjects provided written informed consent and all the experiments were performed in accordance with the relevant guidelines and regulations.

Summary Mycobacterium tuberculosis is the major causative agent of tuberculosis in humans. It is well known that Mycobacterium bovis and other species in the M. tuberculosis complex (MTC) can cause respiratory diseases as zoonosis. We analyzed the MTC isolates collected from tuberculosis patients from Japan in 2002 using a multiplex PCR system that detected cfp32, RD9 and RD12. A total of 970 MTC isolates that were representative of the tuberculosis cases throughout Japan, were examined using this method. As a result, 966 (99.6%) M. tuberculosis, two Mycobacterium africanum and two Mycobacterium canettii were identified using a multiplex PCR system, while no M. bovis was detected. Two isolates that lacked RD9 were initially considered to be M. canettii, but further analysis of the hsp65 sequence revealed them to be M. tuberculosis. Also two M. africanum were identified as M. tuberculosis using the −215 narG nucleotide polymorphism. Though PCR-linked methods have been used for a rapid differentiation of MTC and NTM, from our cases we suggest careful interpretation of RD based identification.

CnfU, a key iron-sulfur (Fe-S) cluster biosynthetic scaffold that is required for biogenesis of ferredoxin and photosystem I in chloroplasts, consists of two tandemly repeated domains in which only the N-terminal domain contains a conserved CXXC motif. We have determined the crystal structure of the metal-free dimer of AtCnfU-V from Arabidopsis thaliana at 1.35 A resolution. The N-terminal domains of the two monomers are linked together through two intermolecular disulfide bonds between the CXXC motifs. At the dimer interface, a total of four cysteine sulfur atoms provide a Fe-S cluster assembly site surrounded by uncharged but hydrophilic structurally mobile segments. The C-terminal domain of one monomer interacts with the N-terminal domain of the opposing monomer and thereby stabilizes dimer formation. Furthermore, Fe K-edge X-ray absorption spectroscopic analysis of the holo-CnfU dimer in solution suggests the presence of a typical [2Fe-2S]-type cluster coordinated by four thiolate ligands. Based on these data, a plausible model of the holo-AtCnfU-V dimer containing a surface-exposed [2Fe-2S] cluster assembled in the dimer interface was deduced. We propose that such a structural framework is important for CnfU to function as a Fe-S cluster biosynthetic scaffold.

It has been shown that the so-called scaffold proteins are vital in Fe-S cluster biosynthesis by providing an intermediate site for the assembly of Fe-S clusters. However, since no structural information on such scaffold proteins with bound Fe-S cluster intermediates is available, the structural basis of the core of Fe-S cluster biosynthesis remains poorly understood. Here we report the first Fe-S cluster-bound crystal structure of a scaffold protein, IscA, from Thermosynechococcus elongatus, which carries three strictly conserved cysteine residues. Surprisingly, one partially exposed [2Fe-2S] cluster is coordinated by two conformationally distinct IscA protomers, termed alpha and beta, with asymmetric cysteinyl ligation by Cys37, Cys101, Cys103 from alpha and Cys103 from beta. In the crystal, two alphabeta dimers form an unusual domain-swapped tetramer via central domains of beta protomers. Together with additional biochemical data supporting its physiologically relevant configuration, we propose that the unique asymmetric Fe-S cluster coordination and the resulting distinct conformational stabilities of the two IscA protomers are central to the function of IscA-type Fe-S cluster biosynthetic scaffold.