The NTM Host Research ConsortiumInternational workshop 2024
Disseminated NTM disease“Toward future international collaborative research for NTM”
February 24 2024
Time schedule : Japan Time
14:00 Video message: Hiroto Yasuura, Japan Science and Technology Agency
Welcome address: Yoko Yamaguchi, Japan Science and Technology Agency
14:05 opening remarks
Naoki Hasegawa, Keio University, Japan
Part1 Chair : Ho Namkoong, Keio University, Japan
14:10 「Genome-wide meta-analysis identifies novel loci for nontuberculous mycobacterial
pulmonary disease and a potential causal relationship with body mass index」
Kyungtaek Park, Institute of Health and Environment, Seoul National University, South Korea
14:30 「The Philippines NTRL and the TB Laboratory Network」
Ramon P. Basilio, Research Institute for Tropical Medicine, Department of Health, Philippines
14:50 「Respiratory Bacterial Microbiome in Nontuberculous Mycobacteria Pulmonary
Disease」
Byung Woo Jhun, Samsung Medical Center, South Korea
15:10 「Precision practice in NTM infection associated with anti-interferon-gamma
autoantibodies」
Doris Un-In Wu, National Taiwan University College of Medicine, Taiwan
15:30-35 short breakPart2 Chair : Ho Namkoong, Keio University, Japan
15:35 「Decoding the Mycobacterium tuberculosis (MTB) Genome:Optimizing Whole Genome Sequencing for Improved Drug-resistance profiling」

Ma. Angelica A. Tujan, Research Institute for Tropical Medicine, Department of Health,Philippines
15:55 「Genomic epidemiology of Mycobacterium tuberculosis in the Philippines, a highburden country and overview of NTM genomic diversity study in the Philippines」
Dodge R. Lim, Research Institute for Tropical Medicine, Department of Health, Philippines
16:15 「An Epidemiological Survey on NTM-PD in Southeast Asia」
Masaki Inoue, Keio University, Japan
16:35 「Single-cell transcriptomics of blood identified IFIT1+ Neutrophil subcluster
expansion in NTM-PD patients」
Wei Sha, Shanghai Pulmonary Hospital, School of Medicine, Tongji University, China
16:55 Closing Remarks
Ho Namkoong, Keio University, JapanAbstracts
Part1
14:05 ～
Genome-wide meta-analysis identifies novel loci for nontuberculous mycobacterial
pulmonary disease and a potential causal relationship with body mass index

Kyungtaek Park
Institute of Health and Environment, Seoul National University, Republic of Korea

This study investigates nontuberculous mycobacteria pulmonary disease (NTM-PD), acondition with a rising global incidence. Genome-wide association studies on Korean andJapanese cohorts reveal three genetic loci associated with NTM-PD. These loci impact thesplicing patterns of IL1R1 and expression levels of PDE8B and CHP2 genes in lung tissue.
Additionally, the study establishes a significant negative genetic correlation between
body mass index (BMI) and NTM-PD. Mendelian randomization analysis suggests a
potential causal relationship, with low BMI increasing the risk of NTM-PD. Overall, thisresearch identifies genetic factors and low BMI as potential risk factors for NTM-PD.

14:25 ～The Philippines NTRL and the TB Laboratory NetworkRamon P. Basilio
Medical Specialist IV
Head, National Tuberculosis Reference Laboratory
Research Institute for Tropical Medicine, Department of Health, Philippines
The National Tuberculosis Reference Laboratory (NTRL) at the Research Institute for Tropical
Medicine is pivotal in the Philippines' TB Laboratory Network and the Department ofHealth's TB eradication strategy. NTRL provides reference services, training, quality
assessment, and support for the National TB Control Program. Central to the TB Laboratory
Network Strategic Plan, NTRL aims to increase access to quality TB diagnostics, ensureservice continuity, strengthen laboratory quality management, and enhance the utilizationof TB laboratory information and research. It plays a crucial role in advancing the cascade ofcare for TB testing and diagnostics in the country's comprehensive strategy to combat
tuberculosis.

14:45 ～Respiratory Bacterial Microbiome in Nontuberculous Mycobacteria Pulmonary Disease
Byung Woo Jhun
Division of Pulmonary and Critical Care Medicine, Department of Medicine,
Samsung Medical Center, Sungkyunkwan University School of Medicine, Republic of Korea.
Nontuberculous mycobacteria (NTM) are ubiquitous organisms found in the environmentand can cause pulmonary disease (PD), with the global burden increasing. Among theapproximately 200 NTM species, Mycobacterium avium complex is the most common
causative agent, while Mycobacterium abscessus is known for its high treatment resistance,
making it a significant focus in clinical practice. NTM-PD can occur in immunocompromisedindividuals, but a significant proportion of NTM-PD patients show no clear evidence of
immunodeficiency and exhibit a typical phenotype known as 'Lady Windermere syndrome.'
Therefore, NTM-PD is currently recognized as a multifactorial disease, with its pathogenesis
assumed to involve interactions between NTM virulence, the host's immune system, and themicrobial environment.
Recently, the association between the respiratory tract's microbial environment anddiseases has been reported. The microbiome, composed of microbial communities, hasbeen employed to understand the progression and pathogenesis of various respiratory
diseases. Especially, culture-independent techniques, such as targeted sequencing of the16S rRNA gene, aid in identifying bacterial microbes and characterizing the taxa present in arespiratory bacterial microbiome. However, to date, there has been limited data on themicrobiome's role in influencing the course of NTM-PD.
In recent times, our research team at my institution has been conducting bacterial
microbiome analysis using lung tissue and sputum samples from NTM-PD patients. Some ofthis data has been published in research papers. Therefore, in this lecture, I will review
recent literature on the microbiome in NTM-PD and briefly share and explain the researchfindings conducted by my research team.

15:05 ～Precision practice in NTM infection associated with anti-interferon-gamma autoantibodies
Doris Un-In Wu
Associate Professor, Department of Internal Medicine,National Taiwan University College of Medicine
In addressing the challenge of frequent misdiagnosis in Adult-Onset Immunodeficiency
Syndrome associated with neutralizing anti-interferon-gamma autoantibodies (AIGA), Dr.
Wu and her research team have undertaken a comprehensive diagnostic approach. They
initially validated the QuantiFERON assay for efficient AIGA screening. This, coupled with theidentification of distinctive clinicopathological features, aim not only to facilitate thedifferentiation between AIGA-associated lymphadenopathy and lymphoma, but also to gain
insights into the disorder's pathogenesis. Ongoing efforts are directed towards exploring
serological and radiographic markers to predict clinical outcomes. This multifaceted
approach aims to guide precision management for AIGA patients.

Part2 Chair : Ho NAMKOONG, Keio University, Japan
15:35 ～
Decoding the Mycobacterium tuberculosis (MTB) Genome:
Optimizing Whole Genome Sequencing for Improved Drug-resistance profiling
Ma. Angelica A. Tujan
Senior Science Research Specialist
Head, Research and Development Section
Molecular Biology Laboratory
Research Institute for Tropical Medicine, Department of Health, Philippines
In response to the rising burden of drug-resistant tuberculosis (TB), we are optimizing awhole genome sequencing (WGS) protocol for TB research and clinical applications. By
analyzing 31 MTB samples using Oxford Nanopore Technology (ONT) and Illumina
sequencing platforms, we achieved >98% genome coverage and identified L1 and L4lineages. Importantly, this protocol detected mutations conferring resistance to key drugs in
80.6% (25/31) of samples. Notably, the most prevalent resistance mutations were isoniazid(67.7%; 21/31), ethionamide (64.5%; 20/31), streptomycin (48.4%; 15/31), and rifampicin(25.8%; 8/31). These results highlight the potential of WGS for drug resistant profiling andtreatment tailoring and with further optimization, this streamlined WGS protocol holds
promise for tackling the evolving challenges of TB control.

15:55 ～Genomic epidemiology of Mycobacterium tuberculosis in the Philippines, a high-burdencountry and overview of NTM genomic diversity study in the Philippines
Dodge R. LimScience Research Specialist II
National Tuberculosis Reference Laboratory
Research Institute for Tropical Medicine, Department of Health, Philippines
The Philippines faces a high burden of tuberculosis (TB), exacerbated by the rise in multidrug-resistant (MDR-TB) and extensively drug-resistant (XDR-TB) Mycobacterium
tuberculosis strains, posing challenges to disease control. Analyzing genome sequencing
data from 732 isolates collected during drug resistance surveys (2011-2019), we identified
prevalent lineages 1 (72.5%) and 4 (23.8%), notably the Manila strain (lineage 1.2.1.2.1). A
substantial proportion exhibited at least MDR-TB (66.0%), with emerging XDR-TB genotypicresistance (0.4%). Transmission clusters revealed prison-based transmission and mutations
linked to bedaquiline and previously unreported resistance mutations. Meanwhile, thescientific enigma of non-tuberculous mycobacteria in the Philippines remains unexplored. A
proposed collaboration between the Philippines and Japan seeks to unveil this scientificmystery, potentially accelerating TB and NTM research and control strategies.

16:15 ～An Epidemiological Survey on NTM-PD in Southeast Asia
Masataka INOUE
Keio University, JapanFor many years, NTM pulmonary disease (NTM-PD) has not been considered as seriously astuberculosis. However, it is a condition that can worsen if not treated properly. In order toestablish a system that allows for more accurate diagnosis and appropriate treatment ofNTM-PD in Southeast Asia, we conduct a survey using Google Form. Not only does it enable
us to estimate the incidence of NTM-PD, it would also reveal how the situations are different
among the hospitals. Even if NTM infection is not tested in a hospital, this survey is still
important; identifying the reasons for not testing can contribute to the improvement ofmedical services in the region.

16:35～Single-cell transcriptomics of blood identified IFIT1+ Neutrophil subcluster expansion in
NTM-PD patients
Wei ShaShanghai Pulmonary Hospital, School of Medicine, Tongji University, China
Objective
Non-tuberculous mycobacterial pulmonary disease (NTM-PD) is caused by animbalance between pathogens and impaired host immune responses. Mycobacterium avium
complex (MAC) and Mycobacterium abscessus (MAB) are the two major pathogens that cause
NTM-PD. In this study, we sought to dissect the transcriptomes of peripheral blood immune
cells at the single-cell resolution in NTM-PD patients and explore potential clinical markers
for NTM-PD diagnosis and treatment.
Methods
Peripheral blood samples were collected from six NTM-PD patients, including threeMAB-PD patients, three MAC-PD patients, and two healthy controls. We employed single-cell
RNA sequencing (scRNA-seq) to define the transcriptomic landscape at a single-cellresolution. A comprehensive scRNA-seq analysis was performed, and flow cytometry wasconducted to validate the results of scRNA-seq.
Results
A total of 27,898 cells were analyzed. Nine T-cells, six mononuclear phagocytes (MPs),and four neutrophil subclusters were defined. During NTM infection, naïve T-cells werereduced, and effector T-cells increased. High cytotoxic activities were shown in T-cells ofNTM-PD
patients. The proportion of inflammatory and activated MPs subclusters was enriched inNTM-PD patients. Among neutrophil subclusters, an IFIT1 + neutrophil subcluster wasexpanded in NTM-PD compared to healthy controls. This suggests that IFIT1+ neutrophil
subcluster might play an important role in host defense against NTM. Functional enrichment
analysis of this subcluster suggested that it is related to interferon response. Cell-cellinteraction analysis revealed enhanced CXCL8-CXCR1/2 interactions between the IFIT1+
neutrophil subcluster and NK cells, NKT cells, classical mononuclear phagocytes subcluster 1(classical Mo1), classical mononuclear
phagocytes subcluster 2 (classical Mo2) in NTM-PD patients compared to healthy controls.