Using cerebrospinal fluid nanopore sequencing assay to diagnose tuberculous meningitis: a retrospective cohort study in China

This study aimed to evaluate the efficiency of nanopore sequencing for the early diagnosis of tuberculous meningitis (TBM) using cerebrospinal fluid and compared it with acid-fast bacilli (AFB) smear, mycobacterial growth indicator tube (MGIT) culture, and Xpert MTB/Rifampicin (RIF). We enrolled 64 adult patients with presumptive TBM admitted to our hospital from August 2021 to August 2023. We calculated the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of AFB smear, culture, Xpert MTB/RIF, and nanopore sequencing to evaluate their diagnostic efficacy compared with a composite reference standard for TBM. Among these 64 patients, all tested negative for TBM by AFB smear. The sensitivity, specificity, PPV, and NPV were 11.11%, 100%, 100%, and 32.2% for culture, 13.33%, 100%, 100%, and 2.76% for Xpert MTB/RIF, and 77.78%, 100%, 100% and 65.52% for nanopore sequencing, respectively. The diagnostic accuracy of the nanopore sequencing test was significantly higher than that of conventional testing methods used to detect TBM.

Methods
We retrospectively screened patients with clinically presumptive TBM admitted to the Tuberculosis Diagnosis and Treatment Center of Zhejiang Chinese and Western Medicine Integrated Hospital from August 2021 to August 2023. The inclusion criteria were as follows: (1) patients aged 18–80 years; (2) those presenting with the symptoms of presumptive TBM (such as headache, high fever, nausea and vomiting, or altered consciousness) or signs of neurological disease; (3) those who completed lumbar puncture; and (4) those whose CSF samples were used for all four tests (AFB smear, culture, Xpert MTB/RIF, and nanopore sequencing). Patients whose CSF samples were not obtained for relevant testing and those lost to follow-up were excluded.

本研究旨在评估纳米孔测序（nanopore sequencing）应用脑脊液（cerebrospinal fluid）样本早期诊断结核性脑膜炎（tuberculous meningitis, TBM）的效能，并将其与抗酸杆菌（acid-fast bacilli, AFB）涂片、分枝杆菌生长指示管（mycobacterial growth indicator tube, MGIT）培养及Xpert MTB/利福平（Xpert MTB/Rifampicin, RIF）检测进行对比。本研究纳入2021年8月至2023年8月于本院就诊的64例疑似TBM成年患者。以TBM综合参考标准为参照，计算AFB涂片、培养、Xpert MTB/RIF及纳米孔测序的灵敏度、特异度、阳性预测值（positive predictive value, PPV）与阴性预测值（negative predictive value, NPV），以评估其诊断效能。64例患者的AFB涂片检测结果均为TBM阴性。培养检测的灵敏度、特异度、PPV及NPV分别为11.11%、100%、100%及32.2%；Xpert MTB/RIF检测分别为13.33%、100%、100%及2.76%；纳米孔测序检测分别为77.78%、100%、100%及65.52%。纳米孔测序检测的诊断准确度显著高于现有TBM常规检测方法。

方法
本研究回顾性筛选2021年8月至2023年8月于浙江中西医结合医院结核病诊疗中心收治的临床疑似TBM患者。纳入标准如下：（1）年龄18~80岁的成年患者；（2）存在疑似TBM相关症状（如头痛、高热、恶心呕吐或意识改变）或神经系统疾病体征者；（3）完成腰椎穿刺术者；（4）脑脊液样本可完成全部四项检测（AFB涂片、培养、Xpert MTB/RIF及纳米孔测序）者。排除未获取脑脊液样本进行相关检测及失访患者。