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標題: 台灣中部地區分枝桿菌檢測,分子流行病學與藥物敏感性之研究
The species identification, molecular epidemiology and the drug susceptibility studies of mycobacteria in central Taiwan
作者: 沈光漢
Shen, Gwan-Han
關鍵字: mycobacteria
molecular epidemiology
drugs susceptibility test
出版社: 分子生物學研究所
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摘要: 研究目的: 非結核分枝桿菌的分離率日益增加,若沒有詳細加以鑑定,往往會被誤認為肺結核治療, 造成結核病疫情的誤差,另外由於呼吸照護病房的成立,產生許多疑似分枝桿菌群聚感染事件,也必須盡速將疫情釐清,以免造成社會大眾的恐慌。本計劃的目的在於提供非結核分枝桿菌的傳統生化鑑定方法與快速的分子鑑別方法,當台灣各地,包括北部,中部,南部及東部有疑似分枝桿菌群聚感染事件發生時,可以提供快速分枝桿菌的分離及診斷,並逐年建立各分枝桿菌尤其是非結核分枝桿菌的監測系統。並進一步與各醫院及代檢合約實驗室合作,收集各醫院分離出來的非結核分枝桿菌,並將資料匯整,以逐步了解台灣地區非結核分枝桿菌的分離率或盛行率。並進一步進行分子流行病學之研究,並建立藥物敏感試驗方法,了解是否有流行菌株,致病菌株,及抗藥菌株。非結核分枝桿菌的散佈與水的關係相當密切,台灣近來水患頻傳,除了腸道性傳染病之外,非結核分枝桿菌的病患分離率是否也隨著增加,而呼吸照護病房的非結核分枝桿菌的感染也相當嚴重,本研究不斷的持續於2004-2006三年間不斷的監測。 研究方法: 本計劃一開始收集中部各大醫院(包括醫學中心,區域醫院,與呼吸照護病房)的呼吸道檢體或菌株,一方面進行培養,培養菌株之後,由五個方面來判斷菌株的種類,包括菌落的表面特徵、菌株的抗酸性染色、菌的生長天數、菌落經光照後顏色的變化與菌株生化試驗,一方面進行rpoB duplex PCR 核酸探針檢查,希望能在一天內就能將結核分枝桿菌與非結核分枝桿菌分開,一旦分離為非結核分枝桿菌接下來進行hsp65聚合脢連鎖反應,反應產物再進行BstE II (10U/uL)、Hae III(10U/uL) 切割脢切割,將作用後的切割脢圖譜加以比對,用來鑑定屬於哪一種非結核分枝桿菌。另外並採集呼吸照護病房環境中的水檢體,進行分枝桿菌的培養與鑑定。另外進行胺基配醣體類抗生素與clofazimine等藥物的敏感試驗,試著找出病患可能可以接受的治療藥物。 主要發現 我們首先確定了非結核分枝桿菌的鑑定方法,我們融合了分子鑑定的快速與生化鑑定的準確,對快速生長菌而言,rpoB duplex-PCR RFLP的敏感度跟特異性分別為 98.3% 和100%。而針對慢生長分枝桿菌中的光照產色菌 (Photochromogen),暗產色菌(scotochromogen),非產色菌(nonchromogen)其敏感度分別為97.2%,94.4%,92.7%,而特異性為100%, 100%,98.9%。在本研究中,大部分為常見的分枝桿菌菌種為RGM、M. tuberculosis complex、M. kansasii、M. intracellulae、M. gordonae。這些五種菌種就佔了所有分枝桿菌菌種的97.3%。在台灣我們甚至可由簡要的流程圖中就可大致區別為何種分枝桿菌,再搭配兩種生化檢驗試驗來做進一步的確認,我們的方法可簡要且快速的鑑別42種常見的分枝桿菌。另外我們還合併rpoB duplex-PCR RFLP的分型與hsp65 RFLP組合成新的鑑定流程圖,運用這樣的鑑定流程我們可以快速且重複確定菌種的種類。 鑑定方法確立之後,我們首先進行非結核分枝桿菌較嚴重的醫院感染,我們選擇中部四家區域級以上醫院,而中部地區區域級以上,以台中榮0為例,非結核分枝桿菌的分離率為42.3% (402/951=42.3%),結核分枝桿菌僅佔57.7%,非結核分枝桿菌的分離率又較以往為高(2000-2002年本院,非結核分枝桿菌的分離率為35%,結核分枝桿菌佔65%),其中以M. intracellulare (29.9 %) 為最高其次為M. abscessus (19.7 %); M. fortuitum (15.8 %);M. gordonae (8.7 %);而 M. kansassi 佔5.5 %;其它的菌種如 M. malmoense (0.8 %); M. scrofulaceum (0.8 %); M. terrae (1.6 %); M. genavense (0.8 %);M. triviale (0.8 %)皆只佔少數。本研究再次證實台灣最重要的前五大分枝桿菌為結核分枝桿菌,快速生長分枝稈菌,鳥型分枝桿菌,戈登氏分枝稈菌與坎薩斯分枝桿菌。而每個醫院所收集到的非結核分枝桿菌的菌種分離總類又有不同,如中山醫學大學附設醫院非結核分枝桿菌分離鑑定結果。M. abscessus佔76.1 %; M. fortuitum (5.4 %); M. gordonae (6.5 %); M. intracellulare (7.6 %); M. kansassi (4.4 %)。可能因中山醫學大學附設醫院收了許多呼吸照護病房送來的檢體。 我們並鎖定中部11家的呼吸照護病房,同時進行病患痰液或呼吸道的檢體做檢查,由我們總共做了415位病患的檢體中,共培養出86株非結核分枝桿菌,其分離率為20.72%(分離率為20.72%=20,720/100,000),其中86株最終皆鑑定為M. abscessus,然而我們仍發現有三位病患無意中分離出結核分枝桿菌,其分離率為0.723%(分離率為0.723%=723/100,000)。非結核分枝桿菌在中部呼吸照護病房的分離率為結核分枝桿菌的30倍,值得注意的是有三位病患在這次的篩檢中發現結核分枝桿菌,且發生率為每十萬人口為723人對比全台灣92年新病患通報的發生率每十萬人口66.67人而言為10.8倍。 我們進一步的收集了六家醫院的自來水及醫療照護機構供水系統或儲存槽,並試著由水中分離非結核分枝桿菌。而由我們的脈衝式電泳分析顯示大部分的醫院的非結核分枝桿菌(以膿瘍分枝桿菌為主),大部分皆為同一種菌種的流行,而其中有兩家醫院水中的檢體,包括醫院用水,尤其是浴室用水及飲水機用水中,含有與病患相同的基因型,代表水中的膿瘍分枝桿菌,藉由洗澡或飲用水,傳播開來,故感染源是醫院的用水供應系統,藉由醫護人員或看護的手或手套來散播,而我們也可在醫療照護機構供水系統中,分離到兩種膿瘍分枝桿菌亞型(M. abscessus type I 及M. abscessus type II),但也有三家醫院有許多種分子流病型,而水中並無法分離出膿瘍分枝桿菌,此時醫療照護機構供水系統的污染並無法解釋呼吸照護病房膿瘍分枝桿菌的流行,此時我們也成功由病患的潮濕瓶中分離到膿瘍分枝桿菌,而這些潮濕瓶的消毒不確實,再拿到其他病患使用時,或直接由潮濕瓶中經由飛沫或空氣傳播,此時就容易造成其他病患的感染,此時的感染源就是病患,傳染途徑就是受污染的潮濕瓶,而呼吸照護病房病患的特色,就是可能病人會隨著病情的改變而轉院,如此就可能造成病患在各醫院中流動,而萬一這些病患身上帶著菌株,就可能將這些菌株帶往各個醫院,如此我們才能在某一家醫院中看到許多不同的基因型,這些基因型可能皆來自不同的醫院。 而較新的胺基配醣體類抗生素isepamicin對快速生長分枝桿菌也有極好的抑制作用。Isepamicin針對M. abscessus (94.9 %)、M. fortuitum (97.3 %) 興M. chelone (100 %) 幾乎皆落入MIC ≦16 μg/mL。Kanamycin同樣也對快速生長分枝桿菌有最好的抑制作用。Kanamycin針對50 %快速生長分枝桿菌 (M. abscessus, M. fortuitum, M. chelonae) 有效抑制之MIC結果(MIC50S)比amikacin好;Kanamycin針對90 % M. fortuitum與 M. chelonae有效抑制之MIC結果(MIC90S)比amikacin好 (表三十一)。Kanamycin與amikacin針對M. chelonae有效抑制之MIC結果相同。而對於clofazimine而言以往用於治療痲瘋病(leprosae),我們發現所有的(100%)的M. abscessus、M. fortuitum 其最小的抑菌濃度接小於≦1 μg/mL,且與amikacin有加乘效果(FIC<1),未來可使用clofazimine來治療快速生長分枝桿菌。 結論及建議事項 非結核分枝桿菌的分離率,尤其是快速生長分枝桿菌,逐年不斷的增加,不論是在醫學中心,區域醫院或地區醫院之呼吸照護病房,而這些非結核分枝桿菌的傳播與醫療機構的供水系統有關,需要相關單位更進一步的重視。與其事後亡羊補牢,不如於設計醫療照護機構供水系統網路時,應該考慮愈短愈好的供水管路長度;當供水系統有再循環使用時,應採用隔離的方式並且避免有管路的死角產生,以有效降低非結核分枝桿菌屬所喜好的水垢、沉澱物的累積與生物膜的形成。水龍頭與供水系統與管線須定期保養與更新。而醫護人員與看護工的無菌技術需進一步落實,病患所使用的器具,尤其是呼吸道管路與潮濕瓶的清消要確實。另外空氣的傳播也是另一個傳染途徑,當病患一但分離出快速生長分枝桿菌時,不管寄生或者已發病,都希望比照結核分枝桿菌一樣,隔離在一間單獨的房間或同一區域。若要投藥時胺基配醣體類抗生素,包括amikacin、isepamycin與kanamycin都是不錯的選擇。而clofazimine本身對於快速生長分枝桿菌就有很強的殺菌效果,搭配amikacin的效果更佳。
Aim: The isolation rate of nontuberculous mycobacteria (NTM) is increasing. If not identified well, they could be easily missed treated as M.tuberculosis. Many NTM pseudo-outbreak happened in Taiwan. We need quickly to differentiate MTB from NTM and realize whether tuberculosis outbreak or just NTM pseudo-outbreak. We hoped to develop to be a reference NTM laboratory. While suspicious tuberculosis or NTM outbreak happened, we could provide the service to rapid differentiation. We'll co-operate with other hospitals and help them to identify the NTM species try to find out the common endemic species or drug resistant strains. We also tried to define the problem of NTM outbreak or pseudo-outbreak in Central Taiwan including medical centers or respiratory care ward (RCW). Material and methods: We collected the clinical respiratory specimens and performed mycobacteria culture and further identify the speices with conventional biochemical methods such as the growth rate, micro-colonies, photo-reactivity, etc. In the other hand, we use the rpoB duplex PCR and try to differentiate the M.TB from NTM in one day. Two biochemistry tests combined with rpoB DPCR were used to differentiate the NTM into species levels. We further combined the patterns of rpoB DPCR-RFLP and hsp65 RFLP to develop a novel algorithm for differentiating the mycobacteria. We also collected the environmental specimens try to isolate NTM from the hospital water supply system, humidifier or patients' nasal cavity. Because aminoglycoside is the most important medication for treatmet of RGM, the drug susceptibility of aminoglycoside was performed. Clofazimine had been used to treat Leprosy for several decades and we also to evaluate the activity against rapid growing mycobacteria. Results: The presence of a 235-bp amplicon was identified as a Mycobacterium tuberculosis complex (MTBC) and a 136-bp amplicon as a nontuberculous Mycobacterium (NTM). NTM species were then divided into eight patterns by the MspI and HaeIII restriction enzyme analysis of a 136-bp rpoB gene segment, which could be easily differentiated by the naked eye without the need of computerized algorithm. The growth character, the presence or absence of pigmentation and the two key biochemical (nitrate reduction and Tween 80 or arylsulfatase) tests were further analyzed to identify Mycobacterium to the species level. A total of 440 clinical isolates of mycobacteria representing 17 Mycobacterium species were analyzed. The sensitivity and specificity were 96.2% and 98.8% for the MTBC, respectively. All 334 NTM strains had a 136-bp amplicon. Overall, 97.3% (428/440) were concordant with those obtained by conventional culture and biochemical tests and 96.8% (426/440) concordant with those obtained by conventional culture, biochemical testing, and BD probeTec ET tests. The most common five NTM isolates were MTB, RGM, M. intracellulae ,M. gordonae and M. kansasii. To realize the problem of NTM outbreak or pseudo-outbreak in the hospitals and RCWs in Central Taiwan, four hospitals were enrolled in our studies. The isolation rate of NTM also increased in the general hospital. It had increase to 42.4% and the TB isolation rate only 57.6% in TCVGH. In these NTMs, M. intracellulae (29.9%), M. abscessus(19.7%), M. fortuitum(15.8%), M. gordonae(8.7%) and M. kansasii(5.5%). There were the top five frequent isolated species,including MTB, M. intracellulae, RGM, M. gordonae and M. kansasii in Taiwan. There were another eleven RCWs surveyed. Total 415 patients were screened and 86 isolates of NTM were found. The isolation rate of NTM in RCW was 20.72%. Totally 86 isolates were identified as M. abscessus . But there were still three patients cultured with MTB. The isolation rate of MTB was 0.723%. The isolation rate of NTM is 30 folds higher than MTB. But the TB isolation rate of RCW is 10.8 folds higher than the newly reported TB incidence in 2003. We also collected the specimen from 6 hospital and two environmental M. abscessus isolates from the faucets of toilet room and drinking machines in the two hospitals. The two environmental M. abscessus strains were identical in PFGE patterns with some of the clinical isolates from patients. We also isolated M. abscessus strains from the humidifier of one RCW. The in vitro activity of isepamicin was compared to that of amikacin, gentamicin, kanamycin, streptomycin, capreomycin and colistin against rapidly growing mycobacteria (RGM) isolated from patients in Taiwan between November 2005 and July 2006. The minimal inhibitory concentrations (MICs) for 213 non-duplicate, consecutive RGM isolates, including 118 Mycobacteria abscessus, 75 Mycobacterium fortuitum, and 20 Mycobacterium chelonae, were determined by micro-dilution test for each antibiotic. Overall, isepamicin at a concentration of 16 μg/ml inhibited 94.9% of M. abscessus isolates, 97.3% of M. fortuitum isolates, and 100% of M. chelonae isolates. Isepamicin was found as active as amikacin and more active than gentamycin, streptomycin, capreomycin and colistin against these pathogenic RGM isolates. Although developed earlier, kanamycin also had the best activity against M. abscessus, M. fortuitum and M. chelonae. MIC distributions of isepamicin, amikacin and kanamycin against M. chelonae isolates are very similar. Colistin and capreomycin had very poor activities against RGM; the MICs at which 50% or 90% of the isolates tested are inhibited were both ≧128 μg/ml. Discussion &conclusion: The novel rpoB DPCR algorithm combined with the genotype and phenotype analyses allow us to identify M. tuberculosis complex, M. avium complex, the rapid growth mycobacteria, M. kansasii and M. gordonae in a straightforward way. The growth character, the presence or absence of pigmentation, and the two biochemical tests provide the option for double check, preventing some interpretation errors and can be used to supplement species identification. M. abscessus outbreaks are of particular concern in Taiwanese long-term respiratory wards. Strains from the environmental water faucets were the source of nosocomial infection. The chronic ventilator-dependent patients could be infected and spread via the Integrated Delivery System (IDS) transportation system that was instituted by the Bureau of National Health Insurance (NHI). The environmental control and implementation of an infection control program in RCWs is urgently needed to eliminate the spread of nosocomial M. abscessus infection. The aseptic techinique including the nurse and nurse assistant should be re-emphasized and monitored. As the susceptibility breakpoint of amikacin for RGM has been addressed by the Clinical and Laboratory Standards Institute (CLSI) as16 μg/ml, isepamicin, kanamycin and amikacin appear to be suitable agents for empiric therapeutic use and controlled trials in RGM infections in Taiwan. Clofazimine also had good activity against RGM and had synergic effect with amikacin. Clofazimine might be a potential drug to be used in the management of RGM infectin in the future.
其他識別: U0005-1408200715504100
Appears in Collections:分子生物學研究所



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