Japan Anti Tuberculosis Association

We aimed to investigate a nosocomial outbreak of Mycobacterium abscessus subspecies massiliense (MAM) and to track its transmission route via genomic analyses and environmental surveys at a hospital in Osaka, Japan. The outbreak was initially detected in two patients (M1 and M2) with severe disability in 2020 and then expanded to five other patients (M3‒M7). The 34-month observation period was divided into three phases separated by two interventions. Mycobacterial culture screening was performed for 294 clinical and environmental samples. We confirmed that five patients (M1‒M5) had infections in phase 1 (March 2020 to July 2021) and implemented an initial intervention. In phase 2 (November 2021 to May 2022), new patients (M6 and M7) were identified, wherein an environmental survey identified MAM strains, prompting a second intervention. No patients were identified in phase 3 (September to December 2022). However, MAM was isolated from the environment during follow-up surveys. A total of 52 MAM isolates were analysed, including 11 clinical isolates (one from M1 and M2 in each phase and one each from M3-M7) and 41 environmental isolates obtained from care gloves, medical devices, and room equipment surrounding patients. We sequenced the isolate genomes and identified 15 subclone clusters with a threshold of 24.5 single-nucleotide variants (SNVs). The overall SNV distribution of the clinical and environmental strains was within 61 SNVs, showing near-identical genomes. Clinical strains from patient M2 with persistent positivity were classified as the same subclone, with 0-6 SNVs. The isolate from a wagon brought into patient rooms showed the lowest number of SNVs (3-8) compared with isolates from M2. This subclone cluster, involving M2 and wagon isolates, formed the hub of the intercluster connection of 11 clusters comprising other clinical and environmental strains. M. abscessus could persist in dry environments and might be indirectly transmitted via fomites.

In genetic testing of non-tuberculous mycobacteria, false positives due to cross-reactions and differences in detection performance among strains are problematic. A rapid genetic testing method with high specificity to distinguish only Mycobacterium avium and Mycobacterium intracellulare and high detection performance against genetic diversity among strains is desirable. We identified completely novel genes (MAA44156_02837 for M. avium and KN251_15665 for M. intracellulare) showing strong identity with M. avium and M. intracellulare respectively. We then developed a new M. avium/M. intracellulare simultaneous detection assay targeting these genes and evaluated their specificity and sensitivity. To evaluate cross-reactivity, the newly developed assay correctly identified all Mycobacterium spp. type strains, except for one examination of a duplicated evaluation with a Mycobacterium kyorinense type strain as a false positive at McFarland 1.0. All M. avium-positive and M. intracellulare-positive clinical isolates used in this study were accurately detected using the newly developed assay. The limits of detection for the newly developed assay were estimated to be 100CFU/mL for M. avium and 30CFU/mL for M. intracellulare. In addition, the detection performance was maintained even in samples containing both M. avium and M. intracellulare. The M. avium/M. intracellulare simultaneous detection assay developed in this study demonstrated high specificity, detection performance, and sensitivity for detecting M. avium and M. intracellulare.

Objective: Mongolia is among the 30 high-burden countries for TB and DR-TB. Whole-genome sequencing (WGS) offers precise insights into TB transmission, strain diversity, and antimicrobial resistance (AMR), which are vital for TB control strategies. This study aimed to investigate the molecular epidemiology, lineage distribution, transmission clustering, and drug resistance patterns of M.tuberculosis collected from rural areas of Mongolia using NGS.Methods: A total of 50,194 individuals were screened in a national TB prevalence survey conducted according to WHO guidelines. From rural participants, 102 TB isolates were obtained; 100 with sufficient DNA quality were subjected to WGS. Bioinformatics analyses included lineage identification, AMR mutation profiling, and phylogenetic and clustering analyses. Results: Among the 100 isolates, lineage 2 (Beijing genotype) accounted for 72.0% and lineage 4 (Euro-American) for 28.0%. The Beijing type was dominant across all regions, especially the central region (84.6%) and showed high clustering (56.4%). A total of 13 clusters (≤12 SNVs) were identified; 86.6% were Beijing strains. MDR-TB comprised 10% of isolates, with 100% of MDR-TB strains belonging to the Beijing genotype. Resistance to isoniazid (23%) was common. Mutations associated with resistance were mainly found in katG, inhA, rpoB, pncA, rpsL, rrs, and embB genes. No resistance was observed to new drugs such as bedaquiline, linezolid, or clofazimine. Conclusions: In rural of Mongolia, M. tuberculosis Lineage 2 (modern Beijing genotype) is predominant, accounting for 72% of cases, with consistent distribution across geographic locations. Beijing strains demonstrated higher drug resistance compared to Euro-American types. Importantly, no resistance was detected to newly introduced TB drugs.

Mycobacterium intracellulare is a major etiological agent of the recently expanding Mycobacterium avium-intracellulare complex pulmonary disease (MAC-PD). Therapeutic regimens that include a combination of macrolides and antituberculous drugs have been implemented with limited success. To identify novel targets for drug development that accommodate the genomic diversity of M. avium-intracellulare, we subjected eight clinical MAC-PD isolates and the type strain ATCC13950 to genome-wide profiling to comprehensively identify universally essential functions by transposon sequencing (TnSeq). Among these strains, we identified 131 shared essential or growth-defect-associated genes by TnSeq. Unlike the type strain, the clinical strains showed increased requirements for genes involved in gluconeogenesis and the type VII secretion system under standard growth conditions, the same genes required for hypoxic pellicle-type biofilm formation in ATCC13950. Consistent with the central role of hypoxia in the evolution of M. intracellulare, the clinical MAC-PD strains showed more rapid adaptation to hypoxic growth than the type strain. Importantly, the increased requirements of hypoxic fitness genes were confirmed in a mouse lung infection model. These findings confirm the concordant genetic requirements under hypoxic conditions in vitro and hypoxia-related conditions in vivo and highlight the importance of using clinical strains and host-relevant growth conditions to identify high-value targets for drug development.

Mycobacterium intracellulare is a major etiological agent of the recently expanding Mycobacterium avium–intracellulare complex pulmonary disease (MAC-PD). Therapeutic regimens that include a combination of macrolides and antituberculous drugs have been implemented with limited success. To identify novel targets for drug development that accommodate the genomic diversity of M. avium–intracellulare, we subjected eight clinical MAC-PD isolates and the type strain ATCC13950 to genome-wide profiling to comprehensively identify universally essential functions by transposon sequencing (TnSeq). Among these strains, we identified 131 shared essential or growth-defect-associated genes by TnSeq. Unlike the type strain, the clinical strains showed increased requirements for genes involved in gluconeogenesis and the type VII secretion system under standard growth conditions, the same genes required for hypoxic pellicle-type biofilm formation in ATCC13950. Consistent with the central role of hypoxia in the evolution of M. intracellulare, the clinical MAC-PD strains showed more rapid adaptation to hypoxic growth than the type strain. Importantly, the increased requirements of hypoxic fitness genes were confirmed in a mouse lung infection model. These findings confirm the concordant genetic requirements under hypoxic conditions in vitro and hypoxia-related conditions in vivo, and highlight the importance of using clinical strains and host-relevant growth conditions to identify high-value targets for drug development.

Delirium is common among patients with advanced illness. The prevention of delirium and management of its symptoms are important to the palliative care of patients with interstitial lung diseases (ILDs). We investigated the incidence of delirium among patients with terminally ill ILDs and risk factors for its development. This retrospective observational study included patients with ILDs who were admitted to the National Hospital Organization (NHO) Kinki Chuo Chest Medical Center between April 2020 and September 2022 and died during hospitalization. The primary and secondary outcomes were the percentage of patients with delirium and risk factors associated with delirium. A total of 85 patients were included in this study. Delirium was present in 38 of these 85 patients (44.7%). Patients with dementia had a significantly higher risk of delirium compared to those without dementia [risk ratio, 2.38; 95% confidence interval (CI): 1.84-3.08], and patients who were physically restrained also had a higher risk of delirium compared to those without restraint (risk ratio, 2.32; 95% CI: 1.53-3.52). Delirium is prevalent in terminally ill patients with ILDs. Dementia and physical restraint may be risk factors for delirium. Physicians should recognize the risk factors for delirium and prepare for its development in terminally ill patients with ILDs.

PurposeTo investigate area under diaphragm (AUD) obtained by dynamic digital radiography (DDR) for the differentiation between normal subjects and chronic obstructive pulmonary disease (COPD) patients.MethodsThis retrospective study included healthy volunteers and COPD patients recruited from 2009 to 2014 at Fukujuji Hospital, who received DDR and pulmonary functional test. AUD was defined as an area under a hemidiaphragm and above the line connecting the ipsilateral costophrenic angle to the top of the hemidiaphragm on DDR image. AUD in full inspiration minus AUD in full expiration (ΔAUD) was also calculated. The diaphragmatic surface was demarcated manually on DDR image to calculate AUD. Three-group comparison of AUD and ΔAUD among normal, mild COPD, and severe COPD subjects was tested with one-way analysis of variance, followed by multiple comparison with Tukey-Kramer method. The diagnostic accuracy of COPD by ΔAUD was assessed using receiver-operating-characteristics (ROC) curve.ResultsSixty-eight participants (36 men, 29 COPD patients) were enrolled. AUD in full inspiration was larger in healthy volunteers than in COPD patients (right, p < 0.001; left, p = 0.02). ΔAUD were different in the three-group comparison (right, normal, 208.7 ± 184.6 mm2, mild COPD, −18.1 ± 117.5 mm2, severe COPD −97.5 ± 150.0 mm2, p < 0.001; left, normal, 254.9 ± 131.5 mm2, mild COPD, −12.5 ± 136.5 mm2, severe COPD, −100.7 ± 134.1 mm2, p < 0.001). ROC curve showed high diagnostic performance of COPD by unilateral ΔAUD (right, area-under curve 0.942; left, area-under-curve 0.965).ConclusionThe value of ΔAUD was smaller according to the severity of COPD. ΔAUD can be helpful in distinguishing healthy subjects from COPD patients.