結核分枝桿菌快速核酸檢測試劑之開發及抗藥性研究

Abstract

造成結核病的病菌統稱為Mycobacterium tuberculosis complex（MTBC），包含有M. tuberculosis（MTB）、M. bovis、M. africanum，M. microti和M. canetti，其中在人體中造成結核病的最主要病菌為M. tuberculosis，而造成結核病的最主要部位為肺部。目前商業化的套組，以IS6110或是16S rRNA為偵測標的物，其所主要偵測的是結核分枝桿菌群而非結核分枝桿菌。經由比對基因體DNA可提供一些RD存在於MTB但是卻不在於MTBC中。其中RD9區域更是特異性地存在於MTB而不存於MTBC中。在這次的實驗當中，利用IS6110 和 Rv3618 (屬於 RD9)當做鑑定結核分枝桿菌群和結核分枝桿菌的標的物。我們發展了一種multiplex nested PCR-ICT (免疫色層分析法)來同時偵測1,500臨床痰液檢體中所存在的MTBC及MTB。試驗結果與傳統培養與生化試驗相比，這個試驗在偵測MTBC時有95.5%敏感性，97.9%特異性，2.1%偽陽性和4.5%偽陰性，在偵測MTB時有93.0%敏感性，99.8%特異性，0.2%偽陽性和7.0% 偽陰性。所以，結果顯示Multiplex PCR-ICT 試驗是一個方便、低成本、容易操作且對MTB的偵測上擁有高度的敏感性及特異性。 隨著多重抗藥性菌株的產生，一線抗結核藥物（INH、RIF）已不敷使用。這個部分的實驗是利用自台大醫院從2003~2004中所分離出的475株的結核分枝桿菌群進行moxifloxacin （MXF）、levofloxacin （LVX）、ciprofloxacin （CIP）、ofloxacin（OFX）、linezolid（LZD）及clarithromycin（CLR）等6種藥物最低抑菌濃度（MIC）測試。除了CLR外，其餘藥物的MIC90皆小於2μg/mL，結果顯示這些藥物對結核分枝桿菌皆有良好的治療效果。由實驗中，篩選了16株對linezolid非感受型菌株，為了研究這些菌株抗藥的機制，我進一步地分析23S rRNA domain V的位置，但並未發現有G2576U此一最常見突變形式的發生。

The M. tuberculosis complex comprises M. tuberculosis, M. bovis, M. bovis BCG, M. africanum, M. microti and M. canneti. The major pathogen for tuberculosis among M. tuberculosis complex is M. tuberculosis. The major syndrome is pulmonary tuberculosis and the most often clinical specimens are sputum. The currently commercially available molecular assays which use either IS6110 or 16S rRNA fragment as identification targets are mainly designed for identifying MTBC but not for MTB. Comparative genomic DNA analysis has provided valuable information on regions of difference (RD) present in MTB but not in other members of the MTBC. RD9 region is further suggested to be a potential target for differential identification of MTB from MTBC. In this study, using IS6110 and Rv3618 (belong to RD9) as the specific identification targets for MTBC and MTB, respectively, we developed and tested a multiplex nested PCR-ICT (immuno-chromatography test) assay for simultaneously and directly detecting not only MTBC but also MTB from 1500 clinical sputum specimens. The results were compared with traditional culture and biochemical identification results together with patients' clinical assessments. This assay showed a 95.5% sensitivity, 97.9% specificity, 2.1% false positive rate and 4.5% false negative rate towards detection of MTBC, and 93.0% sensitivity, 99.8% specificity, 0.2% false positive rate and 7.0% false negative rate for detection of MTB. Our results showed that the multiplex nested PCR-ICT assays is a convenient, low-cost and easy-to-use detection system for identification of MTB with high sensitivity and specificity. Following the production of the multiple drug resistant bacteria strains, the first-lane of antituberculosis drug cannot be satisfied. The tests of this part were used 475 MTB strains isolated from NTUH from 2003~2004. MICs of moxifloxacin （MXF）, levofloxacin （LVX）, ciprofloxacin（CIP）, ofloxacin（OFX）, linezolid （LZD） and clarithromycin（CLR）were tested for all of MTB strains. Beside of the CLR, the MIC90 of all other drugs were smaller than 2μg/mL. The result suggests that those drugs have better inhibitory effect on MTB. On the other hand, We had screend 16 LZD-non-sensitived strains in these tests. To further adressed the resistence mechnism of these MTB strains, We had sequenced the 23S rRNA of domain V of these strains but no mutation, G2576U, the most common mutation types was observed.