全基因组测序技术在耐药结核病诊断中的应用价值

Application value of whole genome sequencing in the diagnosis of drug-resistant tuberculosis

目的:评估全基因组测序(WGS)技术在耐药结核病诊断中的应用价值。方法:收集2021年1月至2022年9月温州市中心医院收治的65例结核病患者的菌株,采用MGIT960液体法药敏试验及WGS同时对菌株进行链霉素、利福平、异烟肼、乙胺丁醇4种一线抗结核药物的耐药性检测。以MGIT960试验结果为参考标准评价WGS在耐药结核病中的诊断效能。结果:WGS检测后发现链霉素耐药基因突变类型以rpsL_K43R(62.07%,18/29)、rpsL_K43R+rrs_A1401G(13.79%,4/29)为主;利福平耐药基因突变类型以rpoB_S450L(48.15%,26/54)、rpoB_D435V(9.26%,5/54)为主;异烟肼耐药基因突变类型以katG_S315T(78.72%,37/47)、fabG1_C-15T(8.51%,4/47)为主;乙胺丁醇耐药基因突变类型以embB_M306V(52.38%,11/21)、emb B_M306I(14.29%,3/21)为主。以MGIT960试验结果为参考标准,WGS检测链霉素、利福平、异烟肼、乙胺丁醇耐药的灵敏度分别为93.10%、98.11%、95.92%、81.25%,特异度分别为94.44%、83.33%、100.00%、83.67%,kappa值分别为0.875、0.842、0.920、0.578。WGS诊断耐多药结核病(MDR-TB)的灵敏度为93.10%,特异度为100.00%,kappa值为0.887。结论:WGS诊断链霉素、利福平、异烟肼的耐药性具有较高的灵敏度和特异度,一致性较好,诊断MDR-TB效能较好,可为临床早期诊断耐药结核病提供帮助。

Objective:To evaluate the whole genome sequencing(WGS) in the diagnosis of drug resistant tuberculosis.Methods:The strains of 65 tuberculosis patients treated in Wenzhou Central Hospital from January 2021 to September 2022 were collected.MGIT960 liquid drug sensitivity test and WGS were used to detect the drug resistance of streptomycin,rifampicin,isoniazid and ethambutol.The result of MGIT960 liquid drug sensitivity test was used as reference criteria to evaluate the diagnostic efficacy of WGS in drug-resistant tuberculosis.Results:After WGS detection,it was found that the main mutation types of streptomycin resistance gene were rpsL_K43R(62.07%,18/29) and rpsL_K43R+rrs_A1401G(13.79%,4/29),while those of rifampicin resistance gene were rpoB_S450L(48.15%,26/54) and rpoB_D435V(9.26%,5/54).The main mutation types of isoniazid resistance gene were katG_S315T(78.72%,37/47) and fabG1_C-15T(8.51%,4/47).The main mutation types of ethambutol resistance gene were emb B_M306V(52.38%,11/21) and emb B_M306I(14.29%,3/21).Taking the MGIT960 test results as the reference standard,the sensitivity of WGS to streptomycin,rifampicin,isoniazid and ethambutol resistance was 93.10%,98.11%,95.92% and 81.25%,respectively and the specificity was 94.44%,83.33%,100.00% and 83.67%,respectively;and the kappa values were 0.875,0.842,0.920 and 0.578,respectively.The sensitivity of WGS in the diagnosis of multidrug resistant tuberculosis(MDR-TB) was 93.10%,the specificity was 100.00%,and the Kappa value was 0.887.Conclusion:WGS has high sensitivity,specificity and consistency in the diagnosis of streptomycin,rifampicin and isoniazid resistance,and effective in the diagnosis of MDR-TB,which can serve as reference for the early diagnosis of drug resistant tuberculosis.