細菌誘發炎性血栓造成導管相關中心靜脈阻塞之機轉

Abstract

在台灣，每年因醫療需求進行中心靜脈導管置放的次數超過數百萬次，不論是用於監測重症病患的血液動力學或藥物給予，甚至提供腎病患者血液透析途徑，以及癌症患者化學治療藥物的投遞，這些都仰賴通暢的靜脈導管。長期留置的中心靜脈管路除了會增加患者血液感染的風險，也會增加患者中心靜脈血栓發生的機會。留置導管導致血栓的種類可由最常見造成管路阻塞功能不良的導管周圍纖維鞘膜增生，也有從管壁延展至靜脈壁的整塊血栓，最嚴重的併發症會導致中央靜脈全阻塞（Central vein occlusion）。然而，並非所有置放長期留置中心靜脈導管的患者都會進展到最嚴重的靜脈全阻塞。多數患者存在導管周圍部分血栓以及局部靜脈狹窄。產生導管周圍血栓的患者在初期並沒有症狀，通常進展到靜脈血栓嚴重阻塞血液回流，才會出現臉部、肢體浮腫等上腔靜脈阻塞症候群。血栓延展的過程是緩慢漸進的，也因此，有許多患者是在接受其他相關疾病檢查治療時，才意外發現中心靜脈因之前留置導管導致阻塞及纖維化萎縮。靜脈內留置導管表面最常見的纖維鞘膜（Fibrin sheath），是靜脈血栓形成的危險因子。在血導管表面發生血栓延展擴大的進展中，纖維鞘膜扮演重要的基石。目前對於留置導管周圍產生纖維鞘膜的研究有限，早期組織學觀察推測其可能源自於受傷血管壁暴的露壁層平滑肌細胞，受發炎反應及細胞激素刺激後活化，進一步移行至導管表面增生及分泌細胞外基質形成包覆導管的結締組織。 纖維鞘膜產生的過程涉及發炎反應，而嗜中性細胞是體內面對外來物或致病原的第一道防線。嗜中性細胞可藉由吞噬作用或產生嗜中性球胞外網(Neutrophil extracellular trap, NETs)以對抗細菌。細菌感染對血管內植入物是巨大的威脅，臨床上有症狀的感染雖然嚴重，但反而容易及時發現給予適當處置及治療。然而，來自患者或照護者身上的伺機性細菌，常引起反覆無症狀感染，容易被乎略而導致慢性的傷害。我們過去的研究發現，口腔內鏈球菌引起的短暫菌血症，可透過嗜中性細胞球外網形成，造成感染性心內膜炎的贅生物擴大。過去也有許多研究指出，導管相關的細菌感染，會導致導管周圍血栓形成而造成中心靜脈阻塞。然而，臨床上許多患者即便在治療過程中並沒有出現相關細菌感染證據，仍可能發生管路相關中心靜脈阻塞，我們認為無症狀的暫時性菌血症可能是其根本原因。 血液透析患者是最容易暴露於暫時性菌血症的高危險族群，在本研究中，我們收集自五十位血液透析患者身上移除的長期靜脈透析留置管路，分析其表面包覆纖維鞘膜檢體。所有患者在過去治療期間雖然沒有臨床細菌感染的病史，然而，利用16S rRNA標記細菌，檢測纖維鞘膜內容物，我們發現有78%臨床檢體可偵測到微量細菌感染。在革蘭氏染色下，亦觀察到纖維鞘膜的層狀結構中有散在分佈的細菌菌落包覆其中。進一步利用基因定序分析，我們發現在纖維鞘膜內最常見無症狀細菌感染的菌種為金黃色葡萄球菌，約佔69%。在共軛焦顯微鏡觀察下，我們可以在纖維鞘膜內觀察到嗜中性細胞膜外網纏繞細菌，以及部分嗜中性細胞球吞噬細菌的現象。利用大鼠中心靜脈留置導管模型，我們給予微量（104 CFU/ml）金黃色葡萄球菌靜脈注射，引起短暫無症狀菌血症，可在留置靜脈管路表面偵測到微量細菌附著的發生。經由金黃色葡萄球菌刺激，進一步誘發免疫反應，引起嗜中性細胞球外網釋放，導致纖維鞘膜增厚以及造成靜脈狹窄。反之，利用DNase I靜脈注射分解嗜中性細胞球外網，則可以有效地減少導管上嗜中性細胞球外網沈積與抑制纖維鞘膜增厚導致的靜脈狹窄。 臨床上，為了避免靜脈留置管路造成血栓發生，除了常規給予抗凝血劑灌注，也建議避免長期留置血管內導管。在導管材質選擇上，則採用生物相容性較高或具表面塗特殊塗層的材料。然而，實證的結果顯示，目前的治療並沒有顯著降低血栓發生的機會。在此研究中，我們發現體表常見的金黃色葡萄球菌，其具備特殊的免疫逃脫及附著能力使它成為血管內導管常見的感染菌種。面對無法有效吞噬的病原體，嗜中性細胞球外網可箝制病原體避免感染擴散。然而，嗜中性細胞球外網本身亦具有刺激血栓生成的能力。透過活化凝血途徑、影響內皮細胞功能、調節循環纖維源細胞，可進一步造成導管表面纖維鞘膜的增長。血液透析患者是菌血症的高危險族群，照護者及病人對於潛在細菌感染的發生，都應提高警覺，確實遵守無菌清潔技術的規範，亦是降低留置靜脈導管造成靜脈內血栓的重要預防策略。

In Taiwan, there are over million times of central venous catheter placement annually in clinical practice. Adequate catheter function and hygiene are important in providing treatment for patient requiring hemodialysis, delivering chemotherapy drugs and monitoring hemodynamic status in intensive care units. Besides catheter related bloodstream infection, the catheter-related thrombosis (CRT) is another serious complication. The catheter related fibrin sheath is the most common type of CRT, it may cause catheter occlusion and dysfunction. In more severe situation, the thrombus may propagate from the surface of catheter to adjacent venous wall and cause symptomatic central vein occlusion syndrome. However, not all patients with central venous catheterization will develop venous occlusion disease. Most patients present with peri-catheter thrombus and partial luminal stenosis. Patients are often asymptomatic until the thrombus grow up and become big enough to occlude the vessel. The clinical presentation of central venous occlusion includes facial swelling, limb edema and sometimes headache. However, the development of venous occlusion is a slow process and asymptomatic. Most patients with catheter related venous occlusion or fibrotic vein following venous thrombosis are diagnosed during other intervention. The peri-catheter fibrin sheath is known as a risk factor for venous thrombosis. The fibrin sheath could become the basement for thrombus adherent. The pathophysiology of peri-catheter fibrin sheath formation is not well understood. In histological study, the fibrin sheath is composed of collagen and the possible mechanism of fibrin sheath formation might be migration and proliferation of adjacent vascular smooth muscle cells after local vessel wall injury. Neutrophil is the most abundant innate immune cell and the first line of defense against microbes. Neutrophil may utilize phagocytosis or produce neutrophil extracellular traps (NETs) to prevent disseminated infection. Systemic bacteremia will induce implant infection, especially for intravascular devices. Symptomatic bacteremia is easy identified and could be treated quickly and optimally. However, asymptomatic transient bacteremia, especially those caused by commensal pathogens, could usually be neglected. The frequently bloodstream infection will result in long-term sequels. Our previous study found oral commensal Streptococcus mutant could evoke transient bacteremia and induce NETs formation on the injured valve. This mechanism enlarged the size of valve vegetation after infectious endocarditis. Previous studies had identified catheter related infection as a risk factor for venous thrombosis. However, in our clinical investigation, some patients without infection also developed catheter related venous thrombosis. Transient bacteremia is common situation during dialysis therapy. In this study, we collected fifty fibrin sheath samples retrieval from dialysis patients without history of infection. Using 16S rRNA probe to detect bacteria, we found 78% (39 of 50) of specimens had bacteria colonization. The genome typing showed Staphylococcus aureus was the most predominant pathogens (69%). Under Gram staining, scattered distribution of bacteria inside the fibrin sheath was identified. Under confocal microscopic laser scanning, the NETs were found in human fibrin sheath samples. By fluorescence in situ hybridization, we found bacteria inside the sheath was simultaneously trapped by NETs and phagocyted by neutrophil. Therefore, we thought NETs structure was an important component of fibrin sheath and bacteria colonization may be the stimuli of NETosis in the mechanism peri-catheter sheath formation. In rat central venous catheterization model, we induced transient asymptomatic bacteremia with 104 CFU/ml S. aureus HG001 strain and catheter surface bacteria colonization was noted. The colonized S. aureus induced NETs formation and promoted the fibrin sheath thickening. On the other hand, administrating with DNase I to digest NETs could diminish the NETs on the surface of catheter, decrease the thickness of fibrin sheath and prevent catheter related venous stenosis. In this research, we identified the important role of NETs in catheter related venous thrombosis. In our study, catheter surface bacteria colonization in asymptomatic dialysis patients is a common situation. Under confocal microscopy, we identified bacteria imbedded in NETs structure inside the pericatheter sheath. Using a rat catheterization model, we induced transient asymptomatic S. aureus bacteremia and linked the interaction of bacteria-stimuli NETs formation and thickening fibrin sheath. Single DNase I treatment could significantly reduce NETs production and fibrin sheath formation surrounding the catheter. Therefore, transient bacteremia could be a silent trigger, which induces NETs associated immunothrombosis and results in catheter-related central venous stenosis. During peri-dialysis health care, skin commensal pathogen decolonization is an important strategy to prevent catheter associated bloodstream infection. According to our finding, it may also have benefit to reduce the development of catheter related venous thrombosis in dialysis patients.