Genetic engineering employing MPB70 and its promoter enables efficient secretion and expression of foreign antigen in bacillus Calmette Guérin (BCG) Tokyo.

Vaccination is an important factor in public health. The recombinant bacillus Calmette Guérin (rBCG) vaccine, which expresses foreign antigens, is expected to be a superior vaccine against infectious diseases. Here, we report a new recombination platform in which the BCG Tokyo strain is transformed with nucleotide sequences encoding foreign protein fused with the MPB70 immunogenic protein precursor. By RNA-sequencing, mpb70 was found to be the most transcribed among all known genes of BCG Tokyo. Small oligopeptide, namely, polyhistidine tag, was able to be expressed in and secreted from rBCG through a process in which polyhistidine tag fused with intact MPB70 were transcribed by an mpb70 promoter. This methodology was applied to develop an rBCG expressing the receptor binding domain (RBD) of severe acute respiratory syndrome coronavirus 2. Immunoblotting images and mass spectrometry data showed that RBD was also secreted from rBCG. Sera from mice vaccinated with the rBCG showed a tendency of weak neutralizing capacity. The secretion was retained even after a freeze-drying process. The freeze-dried rBCG was administered to and recovered from mice. Recovered rBCG kept secreting RBD. Collectively, our recombination platform offers stable secretion of foreign antigens and can be applied to the development of practical rBCGs.

Case Report: Borderline Lepromatous Leprosy Therapy Complicated by Type 1 Leprosy Reaction and Adverse Reactions with Dapsone and Clofazimine.

Leprosy is a global health issue, causing long-term functional morbidity and stigma. Rapid diagnosis and appropriate treatment are important; however, early diagnosis is often challenging, especially in nonendemic areas. Here, we report a case of borderline lepromatous leprosy accompanied by dapsone-induced (neutropenia, anemia, and methemoglobinemia) and clofazimine-induced (skin discoloration and ichthyosis) side effects and type 1 leprosy reactions during administration of the multidrug therapy. The patient completely recovered without developing any deformities or visual impairment. To ensure early diagnosis and a favorable outcome, clinicians should be aware of the diminished sensation of skin lesions as a key physical finding and manage the drug toxicities and leprosy reactions appropriately in patients on multidrug therapy.

Evaluating anti-GPL-core IgA as a diagnostic tool for non-tuberculous mycobacterial infections in Thai patients with high antibody background.

Diagnosis of non-tuberculous mycobacterial (NTM) infection is difficult due to low sensitivity and time-consuming laboratory tests. Current serological assays fail in tropical countries due to high antibody background. This study aimed to investigate an appropriate method for detecting anti-glycopeptidolipid (GPL)-core antibodies to diagnose NTM infection in Thailand. Heparinized plasma samples were collected from 20 patients with NTM-pulmonary disease (NTM-PD) and 22 patients with disseminated NTM (dNTM) for antibody detection by ELISA. The results were compared with those from patients with tuberculosis, other bacterial pulmonary infections and healthy controls. Among the different antibody isotypes, anti-GPL-core IgA exhibited the highest suitability. Therefore, anti-GPL-core IgA and its subclass IgA2 were further investigated. A significant increase in antibody levels was observed during the active infection stage, whereas NTM-PD with culture conversion at the 6-month follow-up showed reduced IgA levels. The diagnostic cut-off for IgA and IgA2 was newly defined as 1.4 and 1.0 U/ml, respectively. Using our IgA cut-off, the sensitivity and specificity for diagnosing NTM-PD were 77.3% and 81.4%, respectively. The new IgA cut-off demonstrated significantly improved specificity compared to the manufacturer's cut-off. Thus, serological detection of anti-GPL-core IgA, with a cut-off of 1.4 U/ml, can be a valuable tool for supporting NTM diagnosis in Thailand.

Catheter-associated Mycobacterium intracellulare biofilm infection in C3HeB/FeJ mice.

Non-tuberculosis mycobacterial (NTM) diseases are steadily increasing in prevalence and mortality worldwide. Mycobacterium avium and M. intracellulare, the two major pathogens of NTM diseases, are resistant to antibiotics, and chlorine, necessitating their capacity to survive in natural environments (e.g. soil and rivers) and disinfected municipal water. They can also form biofilms on artificial surfaces to provide a protective barrier and habitat for bacilli, which can cause refractory systemic disseminated NTM disease. Therefore, preventing biofilm formation by these pathogens is crucial; however, not many in vivo experimental systems and studies on NTM biofilm infection are available. This study develops a mouse model of catheter-associated systemic disseminated disease caused by M. intracellulare that reproduces the pathophysiology of catheter-associated infections observed in patients undergoing peritoneal dialysis. In addition, the bioluminescence system enabled noninvasive visualization of the amount and distribution of bacilli in vivo and conveniently examine the efficacy of antimicrobials. Furthermore, the cellulose-based biofilms, which were extensively formed in the tissue surrounding the catheter insertion site, reduced drug therapy effectiveness. Overall, this study provides insights into the cause of the drug resistance of NTM and may guide the development of new therapies for NTM diseases.

Antibodies against native proteins of Mycobacterium tuberculosis can detect pulmonary tuberculosis patients.

Accurate point-of-care testing (POCT) is critical for managing tuberculosis (TB). However, current antibody-based diagnosis shows low specificity and sensitivity. To find proper antigen candidates for TB diagnosis by antibodies, we assessed IgGs responsiveness to Mycobacterium tuberculosis proteins in pulmonary TB (PTB) patients. We employed major secreted proteins, such as Rv1860, Ag85C, PstS1, Rv2878c, Ag85B, and Rv1926c that were directly purified from M. tuberculosis. In the first screening, we found that IgG levels were significantly elevated in PTB patients only against Rv1860, PstS1, and Ag85B among tested antigens. However, recombinant PstS1 and Ag85B from Escherichia coli (E. coli) couldn't distinguish PTB patients and healthy controls (HC). Recombinant Rv1860 was not checked due to its little expression. Then, the 59 confirmed PTB patients from Soetomo General Academic Hospital, Surabaya, Indonesia, and 102 HC were tested to Rv1860 and Ag85B only due to the low yield of the PstS1 from M. tuberculosis. The ROC analysis using native Ag85B and Rv1860 showed an acceptable area under curve for diagnosis, which is 0.812 (95% CI 0.734-0.890, p < 0.0001) and 0.821 (95% CI 0.752-0.890, p < 0.0001). This study indicates that taking consideration of native protein structure is key in developing TB's POCT by antibody-based diagnosis.

Clinical Characteristics of Corynebacterium ulcerans Infection, Japan.

Corynebacterium ulcerans is a closely related bacterium to the diphtheria bacterium C. diphtheriae, and some C. ulcerans strains produce toxins that are similar to diphtheria toxin. C. ulcerans is widely distributed in the environment and is considered one of the most harmful pathogens to livestock and wildlife. Infection with C. ulcerans can cause respiratory or nonrespiratory symptoms in patients. Recently, the microorganism has been increasingly recognized as an emerging zoonotic agent of diphtheria-like illness in Japan. To clarify the overall clinical characteristics, treatment-related factors, and outcomes of C. ulcerans infection, we analyzed 34 cases of C. ulcerans that occurred in Japan during 2001-2020. During 2010-2020, the incidence rate of C. ulcerans infection increased markedly, and the overall mortality rate was 5.9%. It is recommended that adults be vaccinated with diphtheria toxoid vaccine to prevent the spread of this infection.

PGL-III, a Rare Intermediate of Mycobacterium leprae Phenolic Glycolipid Biosynthesis, Is a Potent Mincle Ligand.

Although leprosy (Hansen's disease) is one of the oldest known diseases, the pathogenicity of Mycobacterium leprae (M. leprae) remains enigmatic. Indeed, the cell wall components responsible for the immune response against M. leprae are as yet largely unidentified. We reveal here phenolic glycolipid-III (PGL-III) as an M. leprae-specific ligand for the immune receptor Mincle. PGL-III is a scarcely present trisaccharide intermediate in the biosynthetic pathway to PGL-I, an abundant and characteristic M. leprae glycolipid. Using activity-based purification, we identified PGL-III as a Mincle ligand that is more potent than the well-known M. tuberculosis trehalose dimycolate. The cocrystal structure of Mincle and a synthetic PGL-III analogue revealed a unique recognition mode, implying that it can engage multiple Mincle molecules. In Mincle-deficient mice infected with M. leprae, increased bacterial burden with gross pathologies were observed. These results show that PGL-III is a noncanonical ligand recognized by Mincle, triggering protective immunity.

Massive and Lengthy Clonal Nosocomial Expansion of Mycobacterium abscessus subsp. massiliense among Patients Who Are Ventilator Dependent without Cystic Fibrosis.

Nontuberculous mycobacterial infections are generally believed to be independently acquired from the environment. Although person-to-person transmission of nontuberculous mycobacteria, especially Mycobacterium abscessus subsp. massiliense, is a serious concern among individuals with cystic fibrosis (CF), evidence of its spread among patients without CF has never been established. We unexpectedly found a number of M. abscessus subsp. massiliense cases among patients without CF in a hospital. This study aimed to define the mechanism of M. abscessus subsp. massiliense infection among patients who were ventilator dependent and without CF who had progressive neurodegenerative diseases in our long-term care wards from 2014 to 2018 during suspected nosocomial outbreaks. We conducted whole-genome sequencing of M. abscessus subsp. massiliense isolates from 52 patients and environmental samples. Potential opportunities for in-hospital transmission were analyzed using epidemiological data. M. abscessus subsp. massiliense was isolated from one air sample obtained near a patient without CF who was colonized with M. abscessus subsp. massiliense but not from other potential sources. Phylogenetic analysis of the strains from these patients and the environmental isolate revealed clonal expansion of near-identical M. abscessus subsp. massiliense isolates, with the isolates generally differing by fewer than 22 single nucleotide polymorphisms (SNPs). Approximately half of the isolates differed by fewer than nine SNPs, indicating interpatient transmission. Whole-genome sequencing revealed a potential nosocomial outbreak among patients who were ventilator dependent and without CF. IMPORTANCE The isolation of M. abscessus subsp. massiliense from the air, but not from environmental fluid samples, may suggest airborne transmission. This was the first report to demonstrate person-to-person transmission of M. abscessus subsp. massiliense, even among patients without CF. M. abscessus subsp. massiliense may spread among patients who are ventilator dependent without CF through direct or indirect in-hospital transmission. The current infection control measures should address potential transmission among patients without CF, particularly in facilities that treat patients who are ventilator dependent and patients with preexisting chronic pulmonary diseases, such as CF.

Unique genomic sequences in a novel Mycobacterium avium subsp. hominissuis lineage enable fine scale transmission route tracing during pig movement.

Mycobacterium avium subsp. hominissuis (MAH) is one of the most prevalent mycobacteria causing non-tuberculous mycobacterial disease in humans and animals. Of note, MAH is a major cause of mycobacterial granulomatous mesenteric lymphadenitis outbreaks in pig populations. To determine the precise source of infection of MAH in a pig farm and to clarify the epidemiological relationship among pig, human and environmental MAH lineages, we collected 50 MAH isolates from pigs reared in Japan and determined draft genome sequences of 30 isolates. A variable number of tandem repeat analysis revealed that most pig MAH isolates in Japan were closely related to North American, European and Russian human isolates but not to those from East Asian human and their residential environments. Historical recombination analysis revealed that most pig isolates could be classified into SC2/4 and SC3, which contain MAH isolated from pig, European human and environmental isolates. Half of the isolates in SC2/4 had many recombination events with MAH lineages isolated from humans in East Asia. To our surprise, four isolates belonged to a new lineage (SC5) in the global MAH population. Members of SC5 had few footprints of inter-lineage recombination in the genome, and carried 80 unique genes, most of which were located on lineage specific-genomic islands. Using unique genetic features, we were able to trace the putative transmission route via their host pigs. Together, we clarify the possibility of species-specificity of MAH in addition to local adaptation. Our results highlight two transmission routes of MAH, one exposure on pig farms from the environment and the other via pig movement. Moreover, our study also warns that the evolution of MAH in pigs is influenced by MAH from patients and their residential environments, even if the MAH are genetically distinct.

Mycobacterium kiyosense sp. nov., a scotochromogenic slow-glowing species isolated from respiratory specimens.

Scotochromogenic slow-growing mycobacteria were isolated from the sputum or bronchoalveolar lavage fluid of 12 patients in Japan. From a comparison of the whole-genome sequences, the representative strain IWGMT90018-18076T and the unknown strains obtained from the patients were found to represent a novel species related to the Mycobacterium gordonae complex. The average nucleotide identity values of IWGMT90018-18076T with Mycobacterium vicinigordonae, Mycobacterium paragordonae and M. gordonae were 86.7, 82.5 and 82.2 %, respectively. The genome size of the representative strain IWGMT90018-18076T was approximately 6.3 Mbp, and the genomic DNA G+C content was 67.1 %. The major fatty acid methyl esters were C16 : 0 (37.71 %), C18 : 1ω9c (29.5 %) and C16 : 1ω7c (10.32 %). In this study, we performed phylogenetic analyses, physiological and biochemical characteristic tests, drug susceptibility tests and fatty acid profiling of the clinical isolates. On the basis of the results obtained, we propose that the unknown clinical isolates represent a novel species, 'Mycobacterium kiyosense sp. nov,' with the type strain being IWGMT90018-18076T (=JCM 34837T =KCTC 49725T).

Epidemiological characteristics of nontuberculous mycobacteriosis and bronchiectasis: comparative study using national mortality statistics from 1970 to 2015 in Japan.

Background: This study assessed longitudinal national data on mortality due to nontuberculous mycobacteriosis (NTMosis) and bronchiectasis and the association between the two diseases. Methods: We analysed the national death statistics of Japan from 1970 to 2015. The International Classification of Disease (ICD) codes were used to extract the relevant data. Crude mortality, age-adjusted mortality and standardised mortality rates were calculated using vital statistics and the population in 2000. We also identified domestic publications related to NTMosis and bronchiectasis with an internet-based search system. Results: The total number of bronchiectasis-related deaths remained at the same level, which was approximately 1000, for 45 years, although the number of deaths has consistently decreased in males but increased in females since the mid-1990s. A substantial increasing trend in females was also observed for NTMosis in the same period. The age-adjusted mortality data showed an increase in mortality in women due to NTMosis and confirmed the trend in bronchiectasis in women. The patterns in the number of domestic reports showed a recent slight increase in bronchiectasis and a marked increase in NTMosis. Conclusions: The trends in bronchiectasis-related mortality differed by sex. The epidemiological trends in the two diseases were associated, especially in elderly females since the mid-1990s. It is suggested that pulmonary NTMosis without pre-existing bronchiectasis might be a leading cause of postinfectious bronchiectasis in Japan.

Proposed Algorithm for Integrated Management of TB-SARS-CoV-2 Co-Infection in a TB-Endemic Country.

Tuberculosis (TB) and COVID-19 have become significant health problems globally, especially in countries with high prevalence. Therefore, this research aims to examine all possibilities and predict the impact of TB-SARS-CoV-2 co-infection to anticipate the cascade effect of both diseases in all sectors. The conceptual strategy of the algorithm in TB-COVID-19 is needed to create an integrated management system. It includes the stages of early detection with accurate and effective methods, as well as the synchronization of TB-COVID-19 health services, starting from primary health facilities to secondary and tertiary referral centers. The algorithm in TB-COVID-19 is crucial to prepare future strategies for PTB co-infection viral respiratory infections other than SARS-CoV-2, ILI, ARI, and SARI. Since the implementation involves all health services, there is a need to integrate the governance of TB-COVID-19 and other comorbidities in good health services based on research and multicentre design.

Hansen's disease (leprosy) in Japan, 1947-2020: an epidemiologic study during the declining phase to elimination.

OBJECTIVES: Leprosy, or Hansen's disease was a major public health problem in Japan in the early 20th century. Today, the number of new cases has decreased significantly. We aimed to investigate the trends of leprosy in Japan over the past 73 years and the challenges faced in recent years. METHODS: We assessed the data on newly registered cases of leprosy from 1947 to 2020. RESULTS: A total of 10,796 newly registered cases of leprosy were reported during the study period, of which 7573 were registered in mainland Japan, 2962 in Okinawa, and 250 were of foreign origin. Most autochthonous cases were born before 1950 in mainland Japan and before 1975 in Okinawa. The number of nonautochthonous cases surpassed that of autochthonous cases in 1992. Nonautochthonous cases originated from 26 countries, particularly Brazil and the Philippines. Three cases of antimicrobial resistance have been detected among nonautochthonous cases since 2004. CONCLUSION: Our data suggest that ongoing transmission of leprosy likely ceased in the 1940s in mainland Japan and in the 1970s in Okinawa. With the recent rise of nonautochthonous cases with globalization, continuous surveillance and efforts to maintain leprosy services within the country are necessary even after reaching the state of elimination.

Potential Cross-Transmission of Mycobacterium abscessus among Non-Cystic Fibrosis Patients at a Tertiary Hospital in Japan.

Mycobacterium abscessus (M. abscessus) is a highly antimicrobial-resistant pathogen that causes refractory pulmonary disease. Recently, the possibility of M. abscessus cross-transmission among cystic fibrosis (CF) patients has been reported. CF is rare in Asia, but M. abscessus pulmonary disease is common. Therefore, we investigated the possibility of M. abscessus cross-transmission in a Japanese hospital setting. Of 104 M. abscessus isolates, 25 isolates from 24 patients were classified into four clusters based on their variable number of tandem repeat profiles and were subjected to whole-genome sequencing (WGS). The epidemiological linkages among our patients were investigated by integrating the WGS data of previously reported nosocomial outbreak-related M. abscessus clinical isolates in the United Kingdom and the United States. Eight transmissible clusters (TCs) were identified. The United Kingdom and United States isolates were assigned to four clusters (TC1, TC2, TC5, and TC8) and one cluster (TC3), respectively. A total of 12 isolates from our hospital belonged to 4 clusters (TC4, TC5, TC6, and TC7). Epidemiological linkage analysis inferred direct or indirect transmission between patients in our hospital in TC4 and TC5 but not in TC6 and TC7. In TC5, the single nucleotide polymorphism distance between isolates from Japanese and United Kingdom patients was less than 21; however, there was no contact. This study revealed that genetically closely related isolates exist, even in non-CF patients. However, the transmission route remains unclear, and further research is warranted to clarify whether cross-transmission is involved. IMPORTANCE Although the possibility of Mycobacterium abscessus (M. abscessus) cross-transmission in cystic fibrosis (CF) patients has often been reported, it is not clear whether similar events have occurred in Asian non-CF patients. Whole-genome sequencing analysis of M. abscessus isolates from Fukujuji Hospital in Japan indicated that genetically closely related M. abscessus isolates exist. In addition, according to epidemiological linkage analysis, some clusters were suspected of direct or indirect transmission between patients within our hospital. However, the transmission route of M. abscessus remains unclear, because interestingly, one cluster showed a single nucleotide polymorphism distance of less than 21 from the United Kingdom isolates, but no epidemiological linkage was identified.

Complete Chromosomal Genome Sequence of Mycobacterium sp. Strain IWGMT90018-18076.

We have isolated a strain that we believe is identical to strain IWGMT90018-an unidentified nontuberculous mycobacterium (NTM) species published in 1981-and named it IWGMT90018-18076. Here, we report its complete chromosomal genome sequence. This study will help us understand the diversity and pathogenicity of NTM.

Molecular Epidemiological Characteristics of Mycobacterium abscessus Complex Derived from Non-Cystic Fibrosis Patients in Japan and Taiwan.

Mycobacterium abscessus complex (MABC) is a group of emerging, highly antimicrobial-resistant non-tuberculous mycobacteria. Specific MABC clones are spreading globally in patients with cystic fibrosis (CF); however, associated genomic epidemiology is lacking in East Asia, with very few patients with CF. Here, we investigated MABC populations derived from non-CF patients in Japan and Taiwan. Analysis of whole-genome sequencing data of 220 MABC isolates revealed that 112, 105, and 3 were M. abscessus subsp. abscessus (ABS), M. abscessus subsp. massiliense (MAS), and M. abscessus subsp. bolletii (BOL), respectively. Moreover, >50% of ABS and >70% of MAS were related to four predominant clones in the region. Known mutations conferring macrolide resistance were rare (1.4%) and were not enriched in the predominant clones. Conversely, the macrolide-susceptible erm(41) T28C mutation was significantly enriched in one predominant ABS clone. The most predominant ABS clone was genetically related to the previously described dominant circulating clone (DCC)1 in patients with CF, whereas no isolates were related to DCC2; isolates related to DCC3 were not necessarily predominant in our sample set. We found that the erm(41) T28C mutants spread globally, and some of them reacquired the functional erm(41) gene through both point mutation and recombination. This study revealed predominant MABC clones in Japan and Taiwan and their relationship with the globally superadding clones in the patient community with CF. Our study provides insights into the genetic characteristics of globally dominant and area-specific strains isolated from patients with or without CF and differences between globally spread and regionally specific strains. IMPORTANCE Members of Mycobacterium abscessus complex (MABC) are frequently isolated from patients. Studies have reported that predominant clones of MABC (known as dominant circulating clones; DCCs) are distributed worldwide and transmitted from humans to humans in patients with cystic fibrosis (CF). However, associated genomic epidemiology has not yet been conducted in East Asia, including Japan and Taiwan, where there are only a few patients with CF. Using whole-genome sequencing data derived from non-CF patients in Japan and Taiwan, we revealed prevalent clones and the incidence of macrolide resistance-associated mutations in the MABC population in this region. We also clarified the associations between these predominant clones and DCCs in the global CF patient community. Our results would assist further studies in elucidating the genetic characteristics of strains isolated from patients with or without CF, the differences between globally spread and regionally specific strains, and the adaptive evolution of MABC within the host.

Attempt of thyX gene silencing and construction of a thyX deleted clone in a Mycobacterium bovis BCG.

Mycobacterium tuberculosis, the causative agent of tuberculosis, possess flavin-dependent thymidylate synthase, ThyX. Since thyX is absent in humans and was shown to be essential for M. tuberculosis normal growth, ThyX is thought to be an attractive novel TB drug target. This study assessed thyX essentiality in Mycobacterium bovis BCG strains using CRISPR interference based gene silencing and found that thyX is not essential in an M. bovis BCG Tokyo derivative strain. A thyX deletion mutant strain was successfully constructed from that strain, which reinforces the non-essentiality of thyX under a certain genetic background.

Simultaneous Determination of Mycobacterium leprae Drug Resistance and Single-Nucleotide Polymorphism Genotype by Use of Nested Multiplex PCR with Amplicon Sequencing.

Mycobacterium leprae is the predominant cause of leprosy worldwide, and its genotypes can be classified into four single-nucleotide polymorphism (SNP) types and 16 subtypes. Determining M. leprae drug resistance and genotype is typically done by PCR and Sanger DNA sequencing, which require substantial effort. Here, we describe a rapid method involving multiplex PCR in combination with nested amplification and next-generation sequence analysis that allows simultaneous determination of M. leprae drug resistance and SNP genotype directly from clinical specimens. We used this method to analyze clinical samples from two paucibacillary, nine multibacillary, and six type-undetermined leprosy patients. Regions in folP1, rpoB, gyrA, and gyrB that determine drug resistance and those for 84 SNP-InDels in the M. leprae genome were amplified from clinical samples and their sequences determined. The results showed that seven samples were subtype 1A, three were 1D, and seven were 3K. Three samples of the subtype 3K had folp1 mutation. The method may allow more rapid genetic analyses of M. leprae in clinical samples.

Production of antibiotic resistance gene-free urease-deficient recombinant BCG that secretes antigenic protein applicable for practical use in tuberculosis vaccination.

Mycobacterium bovis BCG has been the only practical vaccine for tuberculosis. However, BCG cannot fully prevent adult pulmonary tuberculosis. Therefore, the improvement of BCG vaccine is necessary. We previously produced recombinant (r) BCG (BCG-PEST) for the better control of tuberculosis. BCG-PEST was developed by introducing PEST-Heat Shock Protein (HSP)70-Major Membrane Protein (MMP)-II-PEST fusion gene into urease-deficient rBCG using antibiotic-resistant gene for the selection of rBCG. HSP70-MMPII fusion protein is highly immunogenic and PEST sequence was added to enhance processing of the fusion protein. Although BCG-PEST effectively inhibited intrapulmonary growth of Mycobacterium tuberculosis (MTB), BCG with antibiotic-resistant gene is not appropriate for human use. Therefore, we produced antibiotic-resistant gene-free rBCG. We generated leucine-biosynthetic gene (leuD)-deficient BCG and introduced the fusion gene with leuD as the selection marker and named this rBCG as BCG-LeuPH. BCG-LeuPH activated human naïve T cells of both CD4 and CD8 subsets and efficiently inhibited aerosol-challenged MTB in mice. These results indicate that leuD can replace antibiotic-resistant gene for the selection of vaccine candidates of rBCG for human use.

Genome-wide association study in patients with pulmonary Mycobacterium avium complex disease.

RATIONALE: Nontuberculous mycobacteria (NTM) are environmental mycobacteria that can cause a chronic progressive lung disease. Although epidemiological data indicate potential genetic predisposition, its nature remains unclear. OBJECTIVES: We aimed to identify host susceptibility loci for Mycobacterium avium complex (MAC), the most common NTM pathogen. METHODS: This genome-wide association study (GWAS) was conducted in Japanese patients with pulmonary MAC and healthy controls, followed by genotyping of candidate single-nucleotide polymorphisms (SNPs) in another Japanese cohort. For verification by Korean and European ancestry, we performed SNP genotyping. RESULTS: The GWAS discovery set included 475 pulmonary MAC cases and 417 controls. Both GWAS and replication analysis of 591 pulmonary MAC cases and 718 controls revealed the strongest association with chromosome 16p21, particularly with rs109592 (p=1.64×10-13, OR 0.54), which is in an intronic region of the calcineurin-like EF-hand protein 2 (CHP2). Expression quantitative trait loci analysis demonstrated an association with lung CHP2 expression. CHP2 was expressed in the lung tissue in pulmonary MAC disease. This SNP was associated with the nodular bronchiectasis subtype. Additionally, this SNP was significantly associated with the disease in patients of Korean (p=2.18×10-12, OR 0.54) and European (p=5.12×10-03, OR 0.63) ancestry. CONCLUSIONS: We identified rs109592 in the CHP2 locus as a susceptibility marker for pulmonary MAC disease.