Rapid, whole genome sequencing-based diagnosis of drug-resistant Mycobacterium abscessus complex and new options for treatment

非结核分枝杆菌(NTM)是除结核分枝杆菌复合体和麻风分枝杆菌外的分枝杆菌。它们在环境中无处不在，通常与水、土壤和医院病房隔绝。随着越来越多的免疫受损患者(包括艾滋病毒感染和血液系统疾病患者)，以及囊性纤维化和慢性肺部疾病患者的数量增加，NTM作为人类疾病，特别是肺部疾病的病因的作用已经变得明显，最近的报告表明，全球范围内的发病率正在上升。特别是，脓肿分枝杆菌复合体由一组快速生长的、多药耐药的非结核分枝杆菌组成，这些分枝杆菌导致广泛的皮肤和软组织疾病、中枢神经系统感染、菌血症和眼部及其他感染。目前的治疗指南主要基于有限的数据，主要来自专家意见、病例系列和少数随机临床试验。由脓肿分支杆菌复合体引起的感染是出了名的难以治疗。虽然没有标准的治疗方法，但指南建议将大环内酯类药物与其他静脉注射的抗菌剂联合使用；然而，该方案已被证明对患者有很大的副作用。此外，药敏试验是有问题的，目前仍未就标准化方法达成共识，在实验室测量的药物敏感性与在患者身上观察到的药物活性之间存在重大差异。新的抗分枝杆菌药物(即贝达奎林、地拉曼德)和用于耐药结核病的其他化合物(即氯氮咪胺)在参考实验室中没有进行常规的抗NTM测试，尽管最近的研究表明有一些良好的活性。全基因组测序(WGS)已应用于广泛的临床方案，英国是世界上第一个在全国范围内率先将其用于结核分枝杆菌诊断、耐药性检测和分型的国家。这大大缩短了最终诊断的时间，一线易感数据仅需8天(从几周甚至几个月)。然而，NTM长期以来一直处于研究之中，仍需做大量工作才能通过药敏试验进行快速诊断。亚种分化的进展使得对脓肿分支杆菌复合体引起的肺部疾病有了更有效的治疗。例如，与脓肿分支杆菌亚种不同。脓肿，脓肿支原体亚种。马氏杆菌对克拉霉素不具有诱导性抗性。Erm基因的发现导致了大环内酯类药物的耐药性，这是一个例子，说明快速诊断将如何使医生能够自信地使用克拉霉素并优化早期治疗。而脓肿分支杆菌(M.abscessus subsp.脓肿仍然是一个诊断和治疗的挑战，因为80%的分离株对大环内酯类药物具有耐药性。目前还没有能够快速诊断的检测方法，而且在最佳抗菌剂和联合治疗方面缺乏共识，再加上可用药物的缺乏。因此，需要对脓肿的快速诊断和治疗方案进行进一步的研究。本研究项目的主要目的是调查WGS在脓肿分支杆菌复合体中的应用，特别是快速检测耐药性突变(从而使早期优化治疗成为可能)。第二个目的是研究各种化合物对脓肿分支杆菌复合体的抗菌活性，包括抗菌肽和噬菌体(从而为治疗耐药性提供额外的选择)。

Nontuberculous mycobacteria (NTM) are mycobacterial species other than the Mycobacterium tuberculosis complex and Mycobacterium leprae. They are ubiquitous in the environment and often isolated from water, soil, and hospital wards. With increasing numbers of immunocompromised patients (including those with HIV infection and haematological disorders), as well as patients with cystic fibrosis and chronic lung disorders, the role of NTM as a cause of human, and in particular pulmonary, disease has become apparent, with recent reports indicating a worldwide increase. In particular, Mycobacterium abscessus complex comprises a group of rapidly growing, multidrug-resistant, nontuberculous mycobacteria that are responsible for a wide spectrum of skin and soft tissue diseases, central nervous system infections, bacteraemia, and ocular and other infections.Current treatment guidelines are based on limited data derived mostly from expert opinion, case series, and few randomized clinical trials. Infections caused by M. abscessus complex are notoriously difficult to treat. Although there is no standard treatment, the guidelines suggest the administration of macrolide-based therapy in combination with other antimicrobial agents administered intravenously; however, this regimen has been shown to have substantial side effects for patients. Additionally, susceptibility testing is problematic and there is still no consensus on a standardized method and there are important discrepancies between drug susceptibility measured in the laboratory and the activity of the drug observed in patients. New antimycobacterial drugs (i.e. bedaquiline, delamanid) and other compounds used in case of drug resistant tuberculosis (i.e. cloafazimine) are not routinely tested against NTM in the reference laboratory, despite recent research studies suggesting some excellent activity. Whole genome sequencing (WGS) has been applied to a wide range of clinical scenarios and England is the first country in the world to pioneer its use on a national scale for the diagnosis of M. tuberculosis, detection of drug resistance, and typing. This has drastically reduced the time to final diagnosis with first line susceptibilities data available in only 8 days (from weeks and sometime months). However, NTM have long been under investigated and much work still needs to be done to allow a rapid diagnosis with susceptibility testing. The advancement of subspecies differentiation has allowed for more effective management of pulmonary disease caused by M. abscessus complex. For example, unlike M. abscessus subsp. abscessus, M. abscessus subsp. massiliense does not have inducible resistance to clarithromycin. The discovery of the erm gene, which is responsible for macrolide resistance, is an example of how rapid diagnosis would enable the physician to confidently administer clarithromycin and optimize treatment early. However, M. abscessus subsp. abscessus still remains both a diagnostic and treatment challenge as 80% of isolates are macrolide resistant. There is no current test to allow a rapid diagnosis and there is a lack of consensus on the optimal antimicrobial agents and combination therapy, combined with the paucity of available drugs. Hence, further research in both rapid diagnosis and treatment options of M. abscessus is needed. The main aim of this research project is to investigate the utility of WGS applied to M. abscessus complex, with a particular focus on rapid detection of drug resistance mutations (thus, allowing early optimization of treatment). The secondary aim is to investigate the antimicrobial activity of various compounds against M. abscessus complex, including anti-microbial peptides and bacteriophages (thus, providing additional options to treat drug resistance).