[基于下一代测序技术检测痰液标本中耐药性的探索性研究]
[Exploratory study on detection of drug resistance of in sputum specimens by next-generation sequencing].
作者信息
Liu Y Y, Shi J, Chu P, Wu T Y, Li L, Pang Y, Lu J, Guo Y L
机构信息
Beijing Key Laboratory for Pediatric Diseases of Otolaryngology, Head and Neck Surgery, Beijing Pediatric Research Institute, Beijing Children's Hospital, Capital Medical University, National Center for Children's Health, Beijing 100045, China.
Department of Tuberculosis, Beijing Chest Hospital, Capital Medical University, Beijing 101149, China.
出版信息
Zhonghua Jie He He Hu Xi Za Zhi. 2022 Jun 12;45(6):552-559. doi: 10.3760/cma.j.cn112147-20211104-00775.
To compare the diagnostic performance of next-generation sequencing (NGS) detection methods in sputum samples and strains, in order to explore the feasibility of the NGS method to detect drug resistance in sputum specimens. In this retrospective study, the sputum specimens and corresponding clinical isolates of 50 pulmonary tuberculosis patients admitted to Beijing Chest Hospital from January 2017 to December 2017 were collected. The gene mutations of , , , , , , , , and in sputum specimens and corresponding clinical isolates were detected by NGS method. The phenotypic drug susceptibility test (DST) of the strains was carried out by the proportion method. Using DST results as a reference, the sensitivity, specificity, positive predictive value and negative predictive value of the NGS method for clinical strains and sputum specimens, as well as the consistency statistic () with phenotype DST were calculated respectively. The Chi-square test was used to compare the accuracy of the NGS testing in sputum samples and strain samples. The results showed that (63.83%, 30/47) and (57.45%, 27/47) were the most common mutated genes, followed by (46.81%, 22/47)(29.79%, 14/47), (27.66%, 13/47), (21.28%, 10/47), (12.77%, 6/47), (8.51%, 4/47), and promoter(19.15%, 9/47), promoter region (12.77%, 6/47) mutation. when the NGS method was compared with the resistance phenotype of isoniazid, rifampicin, ethambutol, second-line injectable drugs (streptomycin, capreomycin, kanamycin, amikacin), levofloxacin, the sensitivity were 85.71%, 91.67%, 77.78%, 81.82%, 100.00%, 87.50%, 100.00%, 69.23%, and the specificity were 100.00%, 94.12, 87.50%, 89.47%, 97.06%, 96.97%, 94.29%, 89.29% in sputum samples, while in strain samples, the sensitivity were 92.86%, 100.00%, 81.82%, 86.96%, 88.89%, 80.00%, 100.00%, 85.71%. The specificity were 100.00%, 92.86%, 87.10%, 94.74%, 100.00%, 100.00%, 97.14%, 92.86%. Compared with the phenotypic drug susceptibility results, the NGS method has better detection performance for isoniazid, rifampicin, capreomycin, kanamycin, and amikacin in sputum specimens (≥0.75); while among the strains, the NGS method had a good detection performance for isoniazid, rifampicin, streptomycin, capreomycin, kanamycin, amikacin and levofloxacin (≥0.75). With the accuracy of the NGS method for detecting strains as a reference, there was no statistically significant difference in the accuracy of all drug resistance detected between strains and sputum specimens. This study showed that the NGS technology was effective in predicting the resistance of isoniazid, rifampicin, and second-line injectable drugs (capreomycin, kanamycin and amikacin) by detecting sputum samples and strain genotypes, suggesting the feasibility and potential of direct detection of sputum samples by the NGS method as an early detection method for drug resistance.
比较下一代测序（NGS）检测方法在痰液样本和菌株中的诊断性能，以探讨NGS方法检测痰液标本中耐药性的可行性。在这项回顾性研究中，收集了2017年1月至2017年12月在北京胸科医院住院的50例肺结核患者的痰液标本及相应临床分离株。采用NGS方法检测痰液标本及相应临床分离株中、、、、、、、、和的基因突变。采用比例法对菌株进行表型药物敏感性试验（DST）。以DST结果为参照，分别计算NGS方法对临床菌株和痰液标本的敏感性、特异性、阳性预测值和阴性预测值，以及与表型DST的一致性统计量（）。采用卡方检验比较NGS检测在痰液样本和菌株样本中的准确性。结果显示，（63.83%，30/47）和（57.45%，27/47）是最常见的突变基因，其次是（46.81%，22/47）（29.79%，14/47），（27.66%，13/47），（21.28%，10/47），（12.77%，6/47），（8.51%，4/47），以及启动子（19.15%，9/47），启动子区域（12.77%，6/47）突变。当NGS方法与异烟肼、利福平、乙胺丁醇、二线注射用药物（链霉素、卷曲霉素、卡那霉素、阿米卡星）、左氧氟沙星的耐药表型进行比较时，痰液样本中的敏感性分别为85.71%、91.67%、77.78%、81.82%、100.00%、87.50%、100.00%、69.23%，特异性分别为100.00%、94.12、87.50%、89.47%、97.06%、96.97%、94.29%、89.29%；而在菌株样本中，敏感性分别为92.86%、100.00%、81.82%、86.96%、88.89%、80.00%、100.00%、85.71%。特异性分别为100.00%、92.86%、87.10%、94.74%、100.00%、100.00%、97.14%、92.86%。与表型药物敏感性结果相比，NGS方法对痰液标本中的异烟肼、利福平、卷曲霉素、卡那霉素和阿米卡星具有较好的检测性能（≥0.75）；而在菌株中，NGS方法对异烟肼、利福平、链霉素、卷曲霉素、卡那霉素、阿米卡星和左氧氟沙星具有良好的检测性能（≥0.75）。以NGS方法检测菌株的准确性为参照，菌株和痰液标本中所有耐药性检测的准确性差异无统计学意义。本研究表明NGS技术通过检测痰液样本和菌株基因型可有效预测异烟肼、利福平及二线注射用药物（卷曲霉素、卡那霉素和阿米卡星）的耐药性，提示NGS方法直接检测痰液样本作为耐药性早期检测方法的可行性和潜力。
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