利用血漿中游離DNA進行次世代定序檢測結核分枝桿菌感染

Detection of Mycobacterium Tuberculosis Infection by Next-generation Sequencing Using Plasma Cell-Free DNA

目的：約90％的結核分枝桿菌（M. tb）感染者無症狀，終生發展為活動性結核病的機率僅10％。現今的挑戰在於開發一種足夠靈敏且高效的活動性結核病檢測方法。耐酸性桿菌培養和後續的DNA檢測是確認致病性分枝桿菌存在常用的方法，被認為是實驗室診斷的黃金標準。然而由於M. tb生長緩慢，可能會延遲診斷和治療，因此需要新的檢測方法。過去十年，得益於次世代定序（NGS）技術的新應用，游離DNA（cfDNA）的研究取得了突破性進展。方法：本研究從17位活動性結核病患者的血漿樣本中分離出游離cfDNA。我們設計了利用NGS技術針對M. tb基因（embB、eis、gyrA、inhA、katG、pncA、rpoB和rpsL）的標靶定序方法，並評估NGS與其他結核病診斷方法在結核病診斷準確性方面的差異。結果：NGS可以檢測到游離DNA中的M. tb DNA片段，特別是rpoB基因中的AMPL1660333。與GeneXpert TB檢測類似，NGS檢測cfDNA的準確性高於一些傳統檢測方法。結論：結果表明NGS技術能夠檢測血漿中的游離M. tb DNA。此發現可為開發利用血液樣本檢測M. tb感染的cfDNA檢測方法提供支持。

Objectives. About 90% of people infected with Mycobacterium tuberculosis (M. tb) are asymptomatic, with only a 10% lifetime chance of developing active disease. The current challenge is to develop a sufficiently sensitive and efficient method for detecting active tuberculosis. Acid-fast bacilli cultures and subsequent tests with DNA currently are used to confirm the presence of pathogenic mycobacteria and are regarded as the gold standard for laboratory diagnosis. However, the slow growth of the bacteria may delay diagnosis and medical intervention, and new detection methods thus are needed. In the past decade, ground-breaking studies on circulating tumor DNA have been reported, facilitated by advances in cancer genome projects and new applications of next-generation sequencing (NGS) technology.
Methods. Cell-free DNA (cfDNA) was isolated from the plasma samples of 17 patients with active tuberculosis in this study. We designed target-sequencing methods for M. tb genes (embB, eis, gyrA, inhA, katG, pncA, rpoB, and rpsL) by NGS and evaluated the difference in diagnostic accuracy of tuberculosis between NGS and other diagnostic methods.
Results. We found that M. tb DNA fragments could be detected by NGS of cfDNA, especially AMPL1660333 in rpoB gene. Similar to the GeneXpert TB test, NGS of cfDNA had higher accuracy than some traditional tests.
Conclusion. These results suggest that NGS technology should offer the ability to detect cell-free M. tb DNA in plasma. The findings could support development of a cfDNA assay for detecting M. tb infection using blood samples.