Klotoe Bernice J, Molina-Moya Barbara, Gomes Harrison Magdinier, Gomgnimbou Michel K, Oliveira Suzarte Lorenna, Féres Saad Maria H, Ali Sajid, Dominguez José, Pimkina Edita, Zholdybayeva Elena, Sola Christophe, Refrégier Guislaine
Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud, Université Paris-Saclay, 91198, Gif-sur-Yvette cedex, France.
Servei de Microbiologia, Hospital Universitari Germans Trias i Pujol, Institut d'Investigació Germans Trias i Pujol, Universitat Autonoma de Barcelona, Badalona, Spain.
J Microbiol Methods. 2018 Sep;152:10-17. doi: 10.1016/j.mimet.2018.06.003. Epub 2018 Jun 15.
Several diagnostic tests are being developed to detect drug resistance in tuberculosis. In line with previous developments detecting rifampicin and isoniazid resistance using microbead-based systems (spoligoriftyping and TB-SPRINT), we present here an assay called TB-EFI detecting mutations involved in resistance to ethambutol, fluoroquinolones and the three classical injectable drugs (kanamycin, amikacin and capreomycin) in Mycobacterium tuberculosis. The proposed test includes both wild-type and mutant probes for each targeted locus. Basic analysis can be performed manually. An upgraded interpretation is made available in Excel 2016®. Using a reference set of 61 DNA extracts, we show that TB-EFI provides perfect concordance with pyrosequencing. Concordance between genotypic resistance and phenotypic DST was relatively good (72 to 98% concordance), with lower efficiency for fluoroquinolones and ethambutol due to some untargeted mutations. When compared to phenotypical resistance, performances were similar to those obtained with Hain MTBDRsl assay, possibly thanks to the use of automatized processing of data although some mutations involved in fluoroquinolone resistance could not be included. When applied on three uncharacterized sets, phenotype could be predicted for 51% to 98% depending on the setting and the drug investigated, detecting one extensively drug-resistant isolate in each of a Pakistan and a Brazilian set of 91 samples, and 9 XDR among 43 multi-resistant Kazakhstan samples. By allowing high-throughput detection of second-line drugs resistance and of resistance to ethambutol that is often combined to second-line treatments, TB-EFI is a cost-effective assay for large-scale worldwide surveillance of resistant tuberculosis and XDR-TB control.
目前正在开发几种诊断测试来检测结核病中的耐药性。与之前使用基于微珠的系统（spoligoriftyping和TB-SPRINT）检测利福平及异烟肼耐药性的进展一致，我们在此展示一种名为TB-EFI的检测方法，该方法可检测结核分枝杆菌中与乙胺丁醇、氟喹诺酮类药物以及三种传统注射用药物（卡那霉素、阿米卡星和卷曲霉素）耐药性相关的突变。所提议的检测方法针对每个目标位点均包含野生型和突变型探针。基本分析可手动进行。在Excel 2016®软件中有升级后的解读功能。使用一组61份DNA提取物作为参考，我们发现TB-EFI与焦磷酸测序结果完全一致。基因型耐药性与表型药物敏感性试验（DST）之间的一致性相对较好（一致性为72%至98%），由于存在一些非靶向突变，氟喹诺酮类药物和乙胺丁醇的检测效率较低。与表型耐药性相比，其性能与Hain MTBDRsl检测方法相似，这可能得益于数据的自动化处理，尽管氟喹诺酮类药物耐药性相关的一些突变未被纳入检测范围。当应用于三个未鉴定的样本集时，根据样本集情况和所检测的药物不同，可预测出51%至98%的表型，在巴基斯坦和巴西的各91份样本集中分别检测到1例广泛耐药分离株，在哈萨克斯坦的43份多重耐药样本中检测到9例广泛耐药结核（XDR-TB）。通过实现对二线药物耐药性以及常与二线治疗联合使用的乙胺丁醇耐药性的高通量检测，TB-EFI是一种具有成本效益的检测方法，可用于全球范围内大规模监测耐药结核病和控制广泛耐药结核。
