Alanin Mikkel Christian
Dan Med J. 2017 May;64(5).
The respiratory tract is lined with motile cilia that transport respiratory mucus. Primary ciliary dyskinesia (PCD) is a chronic genetic disease caused by mutations in genes responsible for ciliary structure and function. Non-functional airway cilia impair the mucociliary clearance (MCC), causing mucostasis, lung infections and destruction, chronic rhinosinusitis (CRS) and hearing impairment. It is of paramount importance to postpone chronic lung infection mainly with Gram-negative bacteria (GNB) in patients with an impaired MCC. When successful, lung function can be stabilized and quality of life (QoL) improved. In this thesis, we evaluated whether PCD patients can benefit from the experience we have gained from our operative approach towards the paranasal sinuses in cystic fibrosis (CF) patients. In CF, it has been established that bacterial sinusitis can be a focus for initial lung colonization and chronic lung infection. Combined endoscopic sinus surgery (ESS) and adjuvant therapy can eradicate sinus bacteria, reduce pulmonary infections and improve quality of life (QoL). Currently, approximately 120 patients are diagnosed with PCD in Denmark and all are affiliated with the Danish PCD Centre. Patients included in this thesis were recruited from this cohort. In papers (I, IV), we found that the most frequent sinus pathogen was P. aeruginosa, which was isolated in 12 out of 31 (39%) patients who underwent ESS. In searching for a non-pulmonary infectious focus, we observed simultaneous sinus and lung infec-tions with identical pathogens in two out of three patients. This supports our hypothesis of a bacterial reservoir in the sinuses. Next (II), we examined the bacterial flora associated with acute and chronic pulmonary infections in PCD. A high prevalence of chronic infections encouraged a search for new treatment regimens, including ESS with adjuvant therapy, to impact the course of infection. We revealed that P. aeruginosa frequently colonizes the airways in PCD and during the 11-year study period a total of 42 out of 107 (39%) patients fulfilled the definition of chronic lung infection at some point. Importantly, 10 out of 12 patients (83%) with chronic lung infection had the same clone type of P. aeruginosa for years, as determined by pulsed field gel-electrophoresis (PFGE), thus substantiating factual chronic airway infection. Further, we found an increase in the prevalence of P. aeruginosa with age and observed a negative association between early PCD diagnosis and prevalence of P. aeruginosa. This indicates a positive effect of early diagnosis and initiation of therapy. In paper (III), we performed whole genome sequencing (WGS) of P. aeruginosa isolated from the same 12 patients who were included in the PFGE analysis in paper II. By sequencing and phenotypically characterizing multiple isolates from the same patients we were able to study the with-in host bacterial evolution for the first time in PCD. The analyses provided detailed insight into how P. aeruginosa evolves in PCD when they are stressed by the host immune system and antibiotics. We verified the persistence of clonal lineages and confirmed that different clone types of P. aeruginosa can establish persistent infections in PCD patients. Further, we showed that P. aeruginosa acclimatizes grad-ually to the PCD airway by accumulating pathoadaptive mutations and phenotypic characteristics similar to those of CF. Such information may provide valuable clinical information, and as an example we identified mutations in genes responsible for the development of antibiotic resistance. Based on our research we conclude that P. aeruginosa is a major pathogen in PCD and that future research should focus on pre-venting or eradicating these bacteria. Implementing ESS with adjuvant therapy to PCD patients (I, IV) significantly ameliorated CRS symptoms. Further, postoperatively patients tended to have fewer positive lower airway cultures and better lung function; approximately one out of four operated patients, in search of an infectious focus, remained free of lung colonization with P. aeruginosa during follow-up for at least six months. Based on these results, it is tempting to speculate that ESS with adjuvant therapy can eradicate sinus bacteria and thereby reduce lung re-colonization from the sinuses. However, further evidence is needed to support this hypothesis, preferably from a multicentre randomized controlled trial.
呼吸道内衬有可运动的纤毛，这些纤毛负责运输呼吸道黏液。原发性纤毛运动障碍（PCD）是一种慢性遗传疾病，由负责纤毛结构和功能的基因突变引起。无功能的气道纤毛会损害黏液纤毛清除功能（MCC），导致黏液停滞、肺部感染与破坏、慢性鼻窦炎（CRS）以及听力障碍。对于MCC受损的患者，推迟主要由革兰氏阴性菌（GNB）引起的慢性肺部感染至关重要。若成功，肺功能可得以稳定，生活质量（QoL）也会提高。在本论文中，我们评估了PCD患者是否能从我们在囊性纤维化（CF）患者鼻窦手术方法中所获得的经验中受益。在CF中，已证实细菌性鼻窦炎可能是初始肺部定植和慢性肺部感染的病灶。联合内镜鼻窦手术（ESS）和辅助治疗可根除鼻窦细菌、减少肺部感染并改善生活质量（QoL）。
目前，丹麦约有120名患者被诊断为PCD，他们均隶属于丹麦PCD中心。本论文纳入的患者即从该队列中招募。在论文（I、IV）中，我们发现最常见的鼻窦病原体是铜绿假单胞菌，在接受ESS的31名患者中有12名（39%）分离出该菌。在寻找非肺部感染病灶时，我们观察到三分之二的患者鼻窦和肺部同时感染了相同病原体。这支持了我们关于鼻窦中存在细菌储存库的假设。接下来（II），我们研究了与PCD急性和慢性肺部感染相关的细菌菌群。慢性感染的高患病率促使我们寻找新的治疗方案，包括ESS联合辅助治疗，以影响感染进程。我们发现铜绿假单胞菌经常在PCD患者的气道中定植，在11年的研究期间，107名患者中有42名（39%）在某个时间点符合慢性肺部感染的定义。重要的是，通过脉冲场凝胶电泳（PFGE）测定，12名慢性肺部感染患者中有10名（83%）多年来感染的是同一克隆型的铜绿假单胞菌，从而证实了实际存在的慢性气道感染。此外，我们发现铜绿假单胞菌的患病率随年龄增加，并且观察到PCD早期诊断与铜绿假单胞菌患病率之间呈负相关。这表明早期诊断和开始治疗具有积极作用。在论文（III）中，我们对从论文II中纳入PFGE分析的同一12名患者分离出的铜绿假单胞菌进行了全基因组测序（WGS）。通过对同一患者的多个分离株进行测序和表型特征分析，我们首次能够在PCD中研究宿主内细菌的进化。这些分析详细深入地揭示了铜绿假单胞菌在PCD中受到宿主免疫系统和抗生素压力时是如何进化的。我们验证了克隆谱系的持久性，并证实不同克隆型的铜绿假单胞菌可在PCD患者中建立持续感染。此外，我们表明铜绿假单胞菌通过积累与CF相似的致病适应性突变和表型特征，逐渐适应PCD气道。此类信息可能提供有价值的临床信息，例如我们鉴定出了负责抗生素耐药性发展的基因突变。基于我们的研究，我们得出结论，铜绿假单胞菌是PCD中的主要病原体，未来的研究应聚焦于预防或根除这些细菌。对PCD患者实施ESS联合辅助治疗（I、IV）可显著改善CRS症状。此外，术后患者下呼吸道培养阳性结果往往较少，肺功能更好；在寻找感染病灶的手术患者中，约四分之一在至少六个月的随访期间未出现铜绿假单胞菌肺部定植。基于这些结果，很容易推测ESS联合辅助治疗可根除鼻窦细菌，从而减少鼻窦对肺部的再定植。然而，需要进一步的证据来支持这一假设，最好来自多中心随机对照试验。
