| 摘要: | 世界衛生組織(World Health Organization;WHO) 於2005年針對Mycobacterium tuberculosis 宣布了『STOP TB Program』做為一項重要的防疫政策，在台灣，M. tuberculosis也是一項高傳染性的致病菌，因此，台灣疾病管制局(Center for Disease Control ; CDC)也針對M. tuberculosis的感染制定了十年減半計畫，內容包含建立結核菌代檢系統及結核菌藥物感受性試驗的品質管制系統。我們自2002年6月起至2007年6月止共收集台灣西部共 163,758個檢體進行抗酸菌培養，培養結果為M. tuberculosis時再進行藥物感受性試驗及基因分型，分型結果並再評估是否有群聚現象的發生。
培養方法是以BACTEC™ MGIT™ 960儀器進行自動化培養偵測，結果平均陽性率為11.6% (19,012/163,758)，陽性檢體再觀察抗酸性染色並次培養至L-J medium 觀察生長狀態、生化試驗(nitrate reduction 、niacin test、arylsufatase test、tween 80 hydrolysis、catalase)等方法進行菌種鑑定，結果共分離出9,804株M. tuberculosis，結核菌:非結核分枝桿菌 (Non tuberculosis mycobacterium ；NTM)比率為52：48。這些進行菌種鑑定的原始檢體類別以痰液為主(7,714株，佔92％)，性別則以男性居大多數(5,383人次，佔69％)。
M. tuberculosis的藥物感受性試驗(Drug Susceptibility Test ；DST)是依照CLSI (Clinical and Laboratory Standards Institute)的建議以瓊脂比例法進行。第一線所用抗結核藥物包含四種抗生素isoniazid 、rifampin 、ethambutol 、streptomycin；另一項第一線藥物pyrazinamide則是在PH5.0的環境下則以BACTEC™ MGIT™ 960 儀器進行藥物感受性試驗，結果發現在台灣西部多重抗藥性結核菌MDR-TB(至少rifampin及isoniazid二種藥物同時產生抗藥性)的比率為3.67%，實驗中也同時進行結核菌的第二線抗生素的抗藥性試驗，包含五種抗生素：ofloxacin、levofloxacin、moxacillin、amikacin、capreomycin，結果發現廣泛性抗藥性結核菌XDR-TB(MDR-TB的患者針對第二線抗生素fluoroquinolone及aminoglycoside各至少一項抗生素產生抗藥性)的比率為7.1%。
本研究中利用MIRU-VNTR(Mycobacterial Interspersed Repetitive Unit–Variable-Number Tandem Repeat)的方法針對MDR-TB利用M. tuberculosis中15個基因位點所設計出15種引子進行基因分型，PCR結果以傳統瓊脂的方法及以自動化的方式進行相互比對，再以網路比對資料庫(http://mlva.u-psud.fr/)進行菌種分析，結果發現檢體中M. tuberculosis含有亞太地區最常見之北京株；將結果再以Bionumerics軟體進行統計分析，發現共有五個群聚，將各個群聚之檢體來源檢討其患者資料後發現並無相互關聯性，藉由此研究我們也建立了以毛細管電泳快速進行分子生物學分型的方法。
WHO has announced the “Stop TB” program since 2005 and the eradication of Mycobacterium tuberculosis is their important mission. The M. tuberculosis is the top one reported infectious disease in Taiwan. The CDC (Center for Disease Control) of Taiwan also makes a ten-year program for decreacing TB infection rate into half. The program includes to establish a laboratory referred system and improve the quality of drug sensitivity test of M. tuberculosis. From 2006/6 to 2007/6, we collected 163,758 specimens cultured for acid fast bacilli in western Taiwan area. If M .tuberculosis is identified, the tests for drug susceptibility and genotyping will be continued to see if there is clustering phenomenon existing or not.
The BACTEC™ MGIT™ 960 methods were used to detect acid fast bacilli. The average positive rate is 11.6% (19,012/163,758). Then acid fast bacilli were subcultured to L-J medium and observed growth condition. Biochemical tests, including Nitrate reduction, Niacin test, Arylsufatase test, Tween 80 hydrolysis, and Catalase test, were used for the Mycobacterium species. Totally, 9,804 M. tuberculosis strains were isolated. The M. tuberculosis / nontuberculosis mycobacteria ratio was 52/48 (9,804/8,072).The major specimens for bacteria strain identification were from sputum.The sex majority was male.
The drug susceptibility test (DST) of M. tuberculosis was performed agar proportion method according to CLSI (Clinical and Laboratory Standards Institute) guide line. First-line anti-tuberculosis agents contains four antibiotics (INH: isoniazid, R: rifampin, E: ethambutol, S: streptomycin). Another first-line agent is PZA (pyrazinamide) which DST is recommended adjusting the pH to 5.0 in the BACTEC™ 960 system. We found the MDR-TB (M. tuberculosis resists both of rifampin and isoniazid) ratio was 3.67% in western Taiwan. We further detected the DST of second-line anti-TB drugs which contained five antibiotics, including Ofloxacin, Levofloxacin, Moxacillin, Amikacin, and Capreomycin. The incidence of XDR-TB (MDR-TB isolates that is also resistant to one of the fluoroquinolone and one of the injectable agents in the second line anti-TB drugs) was 7.1%。
We used a new process, MIRU-VNTR (Mycobacterial Interspersed Repetitive Unit–Variable Number Tandem Repeat), for genotyping of MDR-TB isolates. These methods contained 15 sets of primers. We compared with PCR results from tradition agarose method and e-Gene automation method and used web on-line identification (http://mlva.u-psud.fr/) to analyze data. Beijing strains of M. tuberculosis were very common in Taiwan. Carrying on the statistical analysis with Bionumeric software, there were five clusters identified and the data of these two methods were consistent. In this study, we developed a high speed method in capillary electrophoresis for high throughout put molecular epidemiology. |
