What Immunological Defects Predispose to Non-tuberculosis Mycobacterial Infections?

Nontuberculous mycobacteria (NTM) are categorized as one of the large and diverse groups of environmental organisms which are abundant in water and soil.  NTM cause a variety of diseases in humans that mainly affect the lung. A predisposition to pulmonary NTM is evident in patients with parenchymal structural diseases including bronchiectasis, emphysema, tuberculosis (TB), cystic fibrosis (CF), rheumatologic lung diseases and other chronic diseases with pulmonary manifestations. Lung infections are not the only consequences of being infected by NTM as they can also infect skin and soft tissue and may also cause lymphadenitis (predominantly in young children) and disseminated disease in human immunodeficiency virus (HIV)-infected patients or those with severely compromised immune system. NTM are also found in many subjects without any known risk factors.  Although the recent advances in imaging and microbiologic techniques including gene sequencing have provided a better view of the problems caused by NTM and has enhanced our understanding of the disease, many uncertainties regarding the immunologic response to NTM still exist. There is also limited data on the immunogenetics of NTM infection. Here, the authors reviewed the main immunogenetic defects as well as other immunological conditions which are associated with an increased the risk of NTM infections

Nontuberculous Mycobacterial Pulmonary Diseases in Immunocompetent Patients

Nontuberculous mycobacterial (NTM) infections are an increasingly recognized cause of chronic lung disease in immunocompetent adults, and the M. avium complex, M. kansasii, and a rapidly growing mycobacteria such as M. abscessus, M. fortuitum, and M. chelonae account for most of the pathogens involved. Because the clinical features of NTM disease are not distinguishable from those of tuberculosis, and NTM are ubiquitous in the environment, diagnosis requires that the bacilli are isolated and identified. NTM diseases have been difficult to treat, though since the introduction of new macrolides, the outcome for patients with some NTM diseases has improved significantly. For correct diagnosis and the successful treatment of NTM pulmonary disease, a knowledge of the full spectrum of clinical and radiological findings is important

Clinical relevance of pulmonary non-tuberculous mycobacterial isolates in three reference centres in Belgium : a multicentre retrospective analysis

Background/objectives: Assessing the clinical relevance of non-tuberculous mycobacteria (NTM) isolated from respiratory samples can be challenging. The epidemiology and pathogenicity of NTM species vary geographically. We aimed to outline the clinical relevance and associated radiological patterns of NTM species isolated in Belgium. Methods: We performed a retrospective multicentre analysis of all patients identified from the laboratory database with >= 1 respiratory sample growing NTM from January 2010 through December 2017. We collected clinical, radiological and microbiological data through medical record review and assessed clinical relevance according to ATS/IDSA criteria for NTM pulmonary disease (NTM-PD). Results: Of the 384 unique patients, 60% were male, 56% had a smoking history and 61% had pre-existing lung disease. Mycobacterium avium complex (MAC), M. gordonae and M. xenopi were the most frequently isolated species: 53, 15 and 8% respectively. 43% of patients met ATS/IDSA criteria, of whom 28% presented with fibrocavitary disease. Weight loss, fever, nodular bronchiectatic and fibrocavitary lesions on chest CT, and a positive acid-fast bacilli (AFB) stain were significantly associated with NTM-PD. The species with the highest pathogenic potential were M. abscessus (11/12), M. malmoense (6/7) and M. intracellulare (41/64). Conclusion: In our study, MAC was the most commonly isolated NTM species, but M. abscessus and M. malmoense showed the highest probability of being clinically relevant. Clinical relevance varied not only by species but also by radiological findings on chest CT and AFB staining. Clinicians should consider these elements in their treatment decision making. Prospective data including clinical outcome are needed to provide more robust evidence

Non-tuberculous Mycobacteria in The Gambia: Prevalent Species, Carriage and Disease

Pulmonary tuberculosis-like disease caused by non-tuberculous mycobacteria (NTM) has gained attention, in part, because of its increased recognition especially in the elderly and immunocompetent population. This is an emerging problem of public health significance. With the increasing incidence of case reports and series from diverse countries and regions of the world, the distribution of NTM species isolated from clinical samples appear to vary significantly by region. However, very little is known about the contribution(s) of NTM to tuberculosis-like disease, and significant knowledge gaps exist regarding their geographical distribution, clinical and molecular epidemiology in low and middle income countries (LMIC) where there is a high burden of disease caused by Mycobacterium tuberculosis complex (MTBC). This dissertation provides a state of the art review of the current epidemiology of NTM carriage and disease in sub-Saharan Africa and presents results from a study of the epidemiology of NTM in The Gambia on pulmonary carriage and disease. I systematically searched electronic databases (PubMed, Embase, Popline, Ovid and Africa Wide Information) for English language articles on pulmonary NTM in sub-Saharan Africa published from Jan 1,1940 to Oct 1, 2016. The American Thoracic Society and Infectious Disease Society of America (ATS/IDSA) diagnostic criteria was applied to differentiate between colonisation of NTM and clinically relevant pulmonary NTM disease. Mycobacterium avium complex (MAC) species were the most frequently isolated NTM (15.0% - 57.8%) in colonisation while M. kansasii, was the most common (184 [69.2%] of 266) cause of confirmed pulmonary NTM disease. A significant proportion (2,623 [29.2%] of 8,980) of NTM isolates in the studies reviewed were not identified to species level. This and the absence of detailed clinical and radiological data hampered the assessment of the clinical relevance of all NTM isolates. A nationwide Tuberculosis prevalence survey provided a platform for me to investigate the population prevalence and molecular epidemiology of NTM in pulmonary samples in the general population of the Gambia. Here, I confirmed suspected NTM cultures from decontaminated sputa stored from the parent survey by 16S rRNA gene sequencing analysis and applied the ATS/IDSA diagnostic criteria to determine clinical relevance of identified NTM. The prevalence of NTM in pulmonary samples was 39.8% [95% Confidence Interval, CI: 35.8% – 44.0%). M. avium complex was by far the most commonly isolated NTM (71.0%), followed by M. fortuitum (9.5%) and M. nonchromogenicum (2.9%). All age groups were three times as likely to have NTM in their sputa as the 15-24 age group (p= 0.017), while urban compared to rural residents were 40 times less likely to be NTM positive (p =0.012). Collectively, these findings highlight the contribution of NTM to colonisation and the risk for over diagnosis of smear positive TB, given the prevalence in presumptive TB cases in The Gambia. I have highlighted the knowledge gap resulting from incomplete identification of NTM species in the sub-continent and the risk of misdiagnosis of pulmonary NTM as PTB when smear microscopy is the sole diagnostic modality. Additional research and surveillance is therefore required to investigate the full contribution of NTM to pulmonary disease, particularly in high risk groups and it is important to review the existing tuberculosis identification methods for presumptive tuberculosis suspects in The Gambia, and sub-continent

Overview of Non Tuberculosis Mycobacterial Lung Diseases

Nontuberculosis mycobacteria (NTM) are ubiquitous in nature and opportunistically infect different animals, including humans. Currently, NTM is emerging as an important cause of pulmonary infection among both immunocompromised and immunocompetent persons worldwide. The clinical relevance of pulmonary NTM varies among species while showing geographical heterogeneity in distribution as well as pathogenicity. The outcome of the respiratory NTM disease is a consequence of a complex interplay between microbial factors and host susceptibility. Furthermore, HIV infection, cystic fibrosis, cancer, underlying chronic lung disease and history of tuberculosis (TB) may be associated as risk factors for active nontuberculosis pulmonary diseases (NTMPD). The diagnosis of NTMPD requires the presence of symptoms, radiographic evidences, microscopic observations and definitive laboratory diagnostics. Lung infections resulted from a clinically significant NTM species should be treated with appropriate antimicrobial regimen

"Why me, why now?" Using clinical immunology and epidemiology to explain who gets nontuberculous mycobacterial infection

BACKGROUND: The prevalence of nontuberculous mycobacterial (NTM) disease is rising. An understanding of known risk factors for disease sheds light on the immunological and physical barriers to infection, and how and why they may be overcome. This review focuses on human NTM infection, supported by experimental and in vitro data of relevance to the practising clinician who seeks to understand why their patient has NTM infection and how to further investigate. DISCUSSION: First, the underlying immune response to NTM disease is examined. Important insights regarding NTM disease susceptibility come from nature's own knockouts, the primary immune deficiency disorders. We summarise the current knowledge surrounding interferon-gamma (IFNγ)-interleukin-12 (IL-12) axis abnormalities, followed by a review of phagocytic defects, T cell lymphopenia and rarer genetic conditions known to predispose to NTM disease. We discuss how these define key immune pathways involved in the host response to NTM. Iatrogenic immunosuppression is also important, and we evaluate the impact of novel biological therapies, as well as bone marrow transplant and chemotherapy for solid organ malignancy, on the epidemiology and presentation of NTM disease, and discuss the host defence dynamics thus revealed. NTM infection and disease in the context of other chronic illnesses including HIV and malnutrition is reviewed. The role of physical barriers to infection is explored. We describe how their compromise through different mechanisms including cystic fibrosis, bronchiectasis and smoking-related lung disease can result in pulmonary NTM colonisation or infection. We also summarise further associations with host factors including body habitus and age. We use the presented data to develop an over-arching model that describes human host defences against NTM infection, where they may fail, and how this framework can be applied to investigation in routine clinical practice

Diagnosis and Treatment of Nontuberculous Mycobacterial Pulmonary Diseases: A Korean Perspective

The incidence of pulmonary disease caused by nontuberculous mycobacteria (NTM) appears to be increasing worldwide. In Korea, M. avium complex and M. abscessus account for most of the pathogens encountered, whilst M. kansasii is a relatively uncommon cause of NTM pulmonary diseases. NTM pulmonary disease is highly complex in terms of its clinical presentation and management. Because its clinical features are indistinguishable from those of pulmonary tuberculosis and NTMs are ubiquitous in the environment, the isolation and identification of causative organisms are mandatory for diagnosis, and some specific diagnostic criteria have been proposed. The treatment of NTM pulmonary disease depends on the infecting species, but decisions concerning the institution of treatment are never easy. Treatment requires the use of multiple drugs for 18 to 24 months. Thus, treatment is expensive, often has significant side effects, and is frequently not curative. Therefore, clinicians should be confident that there is sufficient pathology to warrant prolonged, multidrug treatment regimens. In all of the situations, outcomes can be best optimized only when clinicians, radiologists, and laboratories work cooperatively

Comparative genomics of Mycobacterium avium complex reveals signatures of environment-specific adaptation and community acquisition

Nontuberculous mycobacteria, including those in the Mycobacterium avium complex (MAC), constitute an increasingly urgent threat to global public health. Ubiquitous in soil and water worldwide, MAC members cause a diverse array of infections in humans and animals that are often multidrug resistant, intractable, and deadly. MAC lung disease is of particular concern and is now more prevalent than tuberculosis in many countries, including the United States. Although the clinical importance of these microorganisms continues to expand, our understanding of their genomic diversity is limited, hampering basic and translational studies alike. Here, we leveraged a unique collection of genomes to characterize MAC population structure, gene content, and within-host strain dynamics in unprecedented detail. We found that different MAC species encode distinct suites of biomedically relevant genes, including antibiotic resistance genes and virulence factors, which may influence their distinct clinical manifestations. We observed that M. avium isolates from different sources-human pulmonary infections, human disseminated infections, animals, and natural environments-are readily distinguished by their core and accessory genomes, by their patterns of horizontal gene transfer, and by numerous specific genes, including virulence factors. We identified highly similar MAC strains from distinct patients within and across two geographically distinct clinical cohorts, providing important insights into the reservoirs which seed community acquisition. We also discovered a novel MAC genomospecies in one of these cohorts. Collectively, our results provide key genomic context for these emerging pathogens and will facilitate future exploration of MAC ecology, evolution, and pathogenesis

개에서의 Mycobacterium avium complex 감염에 대한 숙주 면역반응 규명

학위논문(박사) -- 서울대학교대학원 : 수의과대학 수의학과, 2022.2. 유한상.비결핵 항산균 (nontuberculous mycobacteria, NTM)은 토양 및 자연수 등 자연 환경에 널리 분포하고 있는 기회 감염균이다. M. avium 및 M. intracellulare는 NTM 폐질환을 일으키는 주요 원인균으로 Mycobacterium avium complex (MAC)의 대표적인 균주이다. MAC는 사람을 포함하여 다양한 동물에서 감염이 보고되었으며 개에서는 산발적인 발생이 보고되고 있다. MAC에 감염된 개의 경우 대부분 예후가 좋지 않아 안락사되거나 사망하였다. 또한, MAC 감염에 대한 개에서의 인수공통감염병의 가능성은 현재까지 밝혀진 바가 없지만 MTBC (Mycobacterium tuberculosis complex)중 M. tuberculosis, M. bovis 및 M. microti 등은 개에서 사람으로의 전파가 보고된 바 있다. 현재 가축과 야생동물에 의한 MAC의 감염 보고 또한 증가하고 있기 때문에 MAC 감염에 대한 개에서의 면역 반응을 조사하는 것은 진단 및 치료와 더불어 사람과 개 사이의 잠재적인 인수공통 감염을 제어하는 데에도 필수적인 요소라고 판단된다. 개에서의 MAC 감염은 주로 M. avium subsp. hominissuis (MAH)가 원인균으로 지목되어 왔다. 이에 본 연구에서는 개의 MAH 감염에 대한 숙주 면역반응을 개 말초 혈액 단핵세포 (peripheral blood mononuclear cells; PBMCs)의 전사체 분석을 통해 확인하였다. 전사체 분석은 MAH 감염시 Th1 및 Th17 반응과 관련한 T 세포 면역 반응이 유도됨을 보여주었다. Th1 관련 유전자의 발현은 감염 초기에 확인된 반면, Th17 관련 유전자의 발현은 감염 12시간 이후에 확인되었다. 또한 24시간 감염 이후 큰포식세포 (Macrophages) 내에서 세포 사멸 관련 유전자의 발현이 감소되었으며 MAH가 증식함을 확인하였다. 본 연구를 통해 MAH 가 개 말초 혈액 단핵세포에 침입시 Th1 및 Th17 면역 반응을 유도하고 세포 사멸 기작을 피하며 개의 큰포식세포 내에서 생존할 수 있음을 확인하였다. 개에서의 MAC 감염에 대한 원인균은 MAH 가 주로 지목되어 왔지만 대부분의 감염 사례에서는 종 또는 아종이 정확히 확인되지 않았다. 이는 샘플 수집의 어려움, 불특정 임상 징후 및 긴 잠복 기간으로 인하여 원인균 동정이 어렵기 때문인 것으로 생각된다. 그러나 MAC는 종마다 병원성이 다르고 항생제 내성이 다양하기 때문에 감염 시 원인균을 확인하는 것이 치료에 매우 중요하다. 따라서 본 연구에서는 MAH 와 더불어 반려견에서 감염 가능성이 높을 것으로 예상되는 M. intracellulare 감염에 대한 개에서의 숙주 면역 반응을 조사하였다. 개의 자가 단핵구 유래 큰포식세포 (monocyte-derived macrophages; MDMs) 및 림프구의 공동 배양을 통해 M. intracellulare 감염에 대한 숙주 면역 반응을 확인하였다. 전사체 분석 결과는 M. intracellulare 침입시 개 MDMs이 M1 유사 큰포식세포로 분화하고 Th1 및 Th17 세포의 분화를 유도하는 사이토카인을 분비하는 것을 보여주었다. 또한 감염된 MDMs 과 개 림프구의 공동 배양을 통해 Th17 세포가 M. intracellulare 감염시 우세하게 반응함을 확인하였다. 따라서 본 연구를 통해 M. intracellulare 감염 시 개의 큰포식세포 활성화에 의해 Th17 반응이 유도됨을 확인할 수 있다. 현재까지도 MAC의 새로운 종과 아종은 여전히 밝혀지고 있으며 이는 질병 통제에 새로운 위협이 되고 있다. 이러한 신종은 항생제 내성, 숙주 특이성 및 병원성과 관련한 유전적 다양성을 나타낸다. 따라서 MAC 감염에 의한 질병의 통제를 위해서는 신종 균주의 유전적 특성을 이해하는 것이 필수적이다. 현재 전장 유전체 분석의 발달로 MAC의 유전학적인 특성이 밝혀지고 있지만 아직까지도 M. intracellulare는 다른 MAC 종에 비해 관련 정보가 매우 부족한 실정이다. 국내 동물 보호소 및 반려동물 출입 가능 공원에서 새롭게 분리된 M. intracellulare 가 각 분리주 별로 다양한 항생제 감수성을 가지고 있음이 확인되었다. 이에 본 연구에서는 SMRT (single-molecule real-time) 시퀀싱을 통해 M. intracellulare 국내 분리주들의 유전적 특성을 조사하였다. M. intracellulare 분리주의 전장유전체 비교 분석은 Mammalian cell entry 및 Type VII secretion system 와 관련한 병원성 인자들의 유전적 다양성을 나타내었다. 또한 이러한 분리주들의 유전적인 차이는 마우스 폐 큰포식세포에서의 사이토카인 유도 및 세포내 생존에서도 차이를 나타내었다. 따라서 환경 분리주의 유전적 변이는 토양과 같이 환경에 쉽게 노출되는 반려견에게 새로운 위협이 될 수 있을 것으로 판단된다. 개에서의 MAC 감염이 꾸준히 보고됨에 따라 MAC에 대한 개에서의 숙주 면역반응 분석은 질병의 예방 및 치료법 개발에 큰 도움이 될 것이다. 또한, 전장 유전체 비교 분석은 MAC 환경 분리주의 특이적인 병원성 인자 및 유전적 특성을 이해하는 데 필수적인 요소이다. 따라서 본 연구는 개에서의 MAC의 병원성 및 감수성을 이해하고 잠재적인 인수공통감염병 전파를 억제하는 데에 기여할 수 있을 것으로 생각된다.Nontuberculous mycobacteria (NTM) are ubiquitous bacteria that are widely distributed in natural environments such as soil, water, and dust. Mycobacterium avium complex (MAC), to which M. avium and M. intracellulare belong, is a major opportunistic pathogen causing chronic lung disease in humans. MAC can infect a wide range of hosts, including dogs and there have been sporadic reports of MAC-induced mycobacteriosis in dogs. However, in most cases, infected dogs were euthanized or died due to clinical deterioration, a lack of response to therapy, and poor prognosis. Nevertheless, the underlying mechanisms of MAC infection in dogs have not yet been studied. In addition, dogs represent a reservoir of zoonotic diseases caused by Mycobacterium tuberculosis-complex (MTBC) such as M. tuberculosis, M. bovis and M. microti. The zoonotic potential of MAC has not been elucidated in dogs; however, the zoonotic aspects of MAC transmitted by domestic animals and wildlife have a major impact on human health. Therefore, investigating the canine immune response to MAC infection is important for diagnosis and treatment and can help to control potential zoonotic transmission between humans and dogs. M. avium subsp. hominissuis (MAH) has been identified as a major causative agent of canine mycobacteriosis by members of the MAC family. In this study, the host immune response against MAH infection was investigated by transcriptome analysis of canine peripheral blood mononuclear cells (PBMCs). Transcriptome profiling revealed that MAH infection induced a T cell immune response related to Th1 and Th17 cells. The expression of Th1-associated genes was identified in early infection, while that of Th17-associated genes increased 12 hours after infection. The expression of apoptosis-related genes decreased and the abundance of intracellular MAH increased within macrophages after 24 h. The results showed that MAH induces Th1 and Th17 immune responses and can survive within canine macrophages by avoiding apoptosis signaling. Although MAH appears to be the predominant pathogenic subspecies in canine cases, most case reports have not described the species and subspecies. The identification of species in canine mycobacteriosis is difficult due to sample acquisition, unspecific clinical signs and long incubation periods. However, different treatment regimens are required for each species of MAC because they exhibit differential pathogenicity and antibiotic susceptibilities. In particular, M. intracellulare is a major causative agent of MAC lung disease along with M. avium, and it mainly occurs from environmental sources rather than by infected individuals. In Korea, it was reported that M. intracellulare accounted for the majority of NTM distributed in animal shelters and parks. Therefore, companion animals might easily come in contact with M. intracellulare. In this study, the host immune response to M. intracellulare infection was investigated by coculture systems of canine T helper cells and autologous monocyte-derived macrophages (MDMs). Transcriptome analysis revealed that canine MDMs differentiated into M1-like macrophages and secreted molecules that induced Th1/Th17 cell polarization. The coculture systems showed that Th17 cells predominantly responded to M. intracellulare infection through macrophage activation in dogs. New species and subspecies of MAC are still being identified and pose new threats to disease control. These new species and subspecies of MAC exhibit genetic diversity and evolution with respect to antimicrobial susceptibility, host specificity, and pathogenicity. It is important to understand the genomic characteristics of newly identified species for the control of diseases caused by MAC. The genetic characteristics of MAC have been elucidated with the development of whole genome sequencing. However, M. intracellulare is poorly understood compared to the other MAC species. In Korea, M. intracellulare strains newly isolated from animal shelters and parks indicated various antimicrobial resistance patterns for each species. In this study, the genomic characteristics of these M. intracellulare isolates were investigated by single-molecule real-time (SMRT) sequencing. The comparative analysis of M. intracellulare isolates showed genetic diversity and evolution in relation to virulence factors such as mammalian cell entry and the type VII secretion system. These genetic differences were also associated with cytokine induction and survival in alveolar macrophages. Genetic variation of these environmental isolates will pose a new threat to dogs that are easily exposed to environmental sources. Investigating the host immune response is important for disease prevention and treatment development as MAC infection has been reported in dogs. The findings from this study will contribute to a better understanding of the pathogenesis and susceptibility of MAC in dogs and the control of potential zoonotic transmission.Contents Abstract I Contents V List of Figures VII List of Tables X List of Abbreviation XII General Introduction 1 Literature Review 7 Mycobacterium avium complex 7 I. Differentiation of Mycobacterium avium complex species 8 II. Host immune response to Mycobacterium avium complex 10 III. Mycobacterium avium complex infection in dogs 13 Chapter Ⅰ. Mycobacterium avium subsp. hominissuis modulates the protective immune response in canine peripheral blood mononuclear cells Abstract 20 1.1. Introduction 21 1.2. Materials and Methods 25 1.3. Results 31 1.4. Discussion 38 Chapter Ⅱ. Mycobacterium intracellulare induces a Th17 immune response via M1-like macrophage polarization in dogs Abstract 60 2.1. Introduction 61 2.2. Materials and Methods 64 2.3. Results 72 2.4. Discussion 78 Chapter Ⅲ. Whole genome analysis of Mycobacterium intracellulare isolates from environment reveals genetic diversity and the evolution of virulence Abstract 103 3.1. Introduction 105 3.2. Materials and Methods 107 3.3. Results 113 3.4. Discussion 121 General Discussion 146 General Conclusion 155 References 157 Abstract in Korean 187박