Fluoroquinolones 類藥物對結核菌抗藥性之研究

Abstract

結核病（tuberculosis , TB）仍是現今影響全球人類健康的重要疾病，由於缺乏新的藥，以及有多重藥物呈抗藥性的菌株（MDRTB-是指同時對 isoniazid和rifampin第一線藥物呈抗藥性的M.tuberculosis）存在，使得每年有800-900萬人新感染TB，以及每年有200-300萬人死於TB。目前估計全世界約有三分之一人口感染TB。Fluoroquinolones（FQs）是抗TB的新藥。由於其副作用的毒性較低，藥效持久，且是在生物體內和體外對Mycobacterium tuberculosis（MTB）都有良好活性的廣效性口服藥物，因此已經用於預防和治療MDRTB的病人或對第一線用藥有不適應症的病人。自1986年以來FQs已用於治療社區感染的一般傳染性疾病，然而近年來卻已發現有MTB可能在短短7天的單一FQs處方用藥而產生抗藥性的情形 (NEJM 349;20, 2003) 。因為TB是一傳染性疾病，抗藥性的增加對於防疫工作政策是非常急迫，因此必須密切注意FQs對MTB藥物感受性的發展。過去所知道的資訊都是針對抗TB的第一線用藥，而抗TB的新藥FQs的有關數據在台灣仍然很有限，因此，本篇論文的主題是探討FQs在台大醫院分離菌株中的抗藥情形，與TB病人中同時對第一線用藥呈抗藥性；或是MDRTB病人；或過去用過抗TB的藥物；或使用過FQs治療病人的相關數據。 從2004-2005年間抽樣420株臨床分離的MTB菌，來自420位病人。先確定第一線用藥的藥物感受性和FQs的最小抑制濃度MIC分析與MDRTB的相關性；進一步分析FQs抗藥性決定基因gyrase 在QRDRs（quinolones resistance-determining regions）是否發生突變；與FQs抗藥性以及MDRTB的關係；再進行genotyping（spoligotyping）的分析，是否有菌株的散布或群聚。結果顯示：420株中對第一線用藥 isoniazid，rifampin，ethambutol，或對任何一種呈抗藥性，以及是MDR的比例分別是12.4, 6.2, 6.2, 17.1和5.0（％）；對FQs藥物ofloxacin，ciprofloxacin，levofloxacin，moxifloxacin的感受性分別是98.3, 98.6, 98.6, 97.6（％）。抗藥的原因發生在QRDRs GyrA 基因是A90V和 D94G的突變(35.7％)，在GyrB基因是N538D的突變(7.1％)。整體而言FQs的抗藥性在台大醫院的檢體中為3.3％，發生在QRDR的比率是42.8％。而spoligotyping顯示北京株佔42.8％，在 MDR 和FQR的菌株中都比在具感受性的菌株中佔較高的比率。對FQ呈抗藥性者與過去是否接觸抗TB藥物或對第一線藥物呈抗藥性有明顯相關，但是其間的抗藥機制並不相同，為何會有相關？在本論文中推測可能與病人的免疫力情況有關。對第一線用藥呈抗藥性或MDRTB病人，或過去用過抗TB藥物的病人，細菌在其體內比較佔優勢，MTB菌有時間活化efflux pump並將藥物排出菌體外，以及製造特殊蛋白抑制藥物的作用。或也可能是藥物的不適當使用有關，文獻上指出，治療TB不能用單一處方，因此，建議在臨床使用FQ藥物前應先檢測其藥敏試驗，並謹慎選用，以免提早產生對FQs呈抗藥性的菌株，徒增防疫工作的負擔。未來除了繼續評估抗藥性的發展，應積極開發快速的檢測抗藥性的方法，俾能儘早提供臨床醫師正確的診斷，以免延誤治療，製造讓細菌產生抗藥性的機會

Tuberculosis (TB), due to lack of new medicine to cure and the existence of MDRTB (multi-drugs resistance tuberculosis, resistance to both isoniazid and rifampin in M. tuberculosis) , remains as a vital disease threatening human health globally, causing around 8 to 9 million people being newly affected and dead at the number of 2 to 3 million annually. It is currently estimated that one third of people are suffering from this disease. Fluoroquinolones (FQs) are the new agents to anti M. tuberculosis (MTB), with its low side-effects, toxicities and good activities in vitro and vivo, FQs have been used to prevent and treat MDRTB, and are applied to test the agent tolerance of the first line patients. Backing in 1986, FQs have been used to treat the community-acquired infections, but until recent years, the situation of new anti-drug (NEJM 349;20,2003) on FQs prescription of MTB within a short period of seven days was noticed. In view that TB is an infectious disease, it is crucial to implement an urgent epidemic prevention, and take a close watch on the development of FQs vs. MTB. The previous information we have is limited on the anti-TB in first line usage, with little data on related FQs against TB. It is therefore, the purpose of this study is to investigate the anti-TB on clinical isolates of FQs used in NTUH as well as the drug resistance by the first line TB patients or MDRTB patients and also to study the data relevance of patients of past history. A total of of 420 clinical isolates of M. tuberculosis were randomly selected during 2004 to 2005 to determine the minimal inhibitory concentrantion (MICs) of FQs. We amplified the subunit of DNA gyrase quinolones resistance-determining regions (QRDR) by PCR and compared the nucleotide-sequences of the FQs resistance with that of the susceptible isolates followed by the analysis of genotyping (spoligotyping) to see its community scattering against clinical isolates. The resistance to isoniazid, rifampin, ethambutol, any one drug, and multi-drugs were 12.4, 6.2, 6.2, 17.1 and 5.0 (%) respectively; susceptibility to ofloxacin, ciprofloxacin, levofloxacin, moxifloxacin in FQs at 98.8, 98.6, 98.6, 97.6 (%) respectively. The reason of resistance lies in QRDRs GyrA being the acquired genetic mutation of A90V and D94G (35.7%), and GyrB being the acquired genetic mutation of N538D (7.1%). Generally speaking, the FQs resistance held in NTUH is 3.3% as compared to 19% of that in MDRTB. The Beijing strains percentage as shown in spoligotyping was at 43%, in both MDRTB and FQR isolates was relatively higher than in the susceptible ones. The FQ resistance has its definite association with the usage of anti-TB or anti-drug in first line medical contact, subject to the mechanism of drug resistance. To investigate the reason behind, it is reckoned in this study to be rather related with the immunity of patients, or very likely with the bacteriostasis of MTB making special protein , or the misusage of medicine. There is not single prescription against TB, therefore, it is suggested to make necessary medical experiment with caution in order not to cause early FQs resistance which will increase the overload of epidemic prevention. It is also recommended to explore with earnest the experiment of medical resistance by assessing the anti-drug development so as to provide the doctors with appropriate clinical diagnosis. Conclusively, FQs should be very conscious in usage of the clinical treatment, in order not to cause resistance strains.