Scarpellini P, Braglia S, Brambilla A M, Dalessandro M, Cichero P, Gori A, Lazzarin A
Infectious Diseases Division, San Raffaele Scientific Institute, Milan, Italy.
J Clin Microbiol. 1997 Nov;35(11):2802-6. doi: 10.1128/jcm.35.11.2802-2806.1997.
Mutations in a 69-bp region of the rpoB gene of Mycobacterium tuberculosis are associated with rifampin resistance (Rif[r]). These have been detected with mycobacterial DNA extracted from bacterial suspensions or respiratory specimens that were acid-fast smear positive. We experimented with a strategy for the rapid detection of Rif(r) in cerebrospinal fluid (CSF) samples. The strategy involves the amplification of the 69-bp region of rpoB by means of PCR and the identification of nucleotide mutations by single-strand conformation polymorphism (SSCP) analysis of the amplification products. Sixty-five CSF specimens collected from 29 patients (19 patients were coinfected with human immunodeficiency virus) with culture or autopsy-confirmed (22 patients) or highly probable (7 patients) tuberculosis of the central nervous system (CNS-TB) were processed. Amplified products suitable for evaluation by SSCP analysis were obtained from 37 CSF specimens from 25 subjects (86.2%). PCR-SSCP of CSF correctly identified the rifampin susceptibility phenotype of isolates from all 17 patients for whom the results of susceptibility tests carried out with strains cultured from CSF or respiratory samples were available. Moreover, this assay revealed the rifampin susceptibility genotype of isolates from the eight patients (three patients with culture-confirmed CNS-TB and five patients in whom CNS-TB was highly probable) for whom no susceptibility test results were available; the PCR-SSCP data obtained for these patients were concordant with the outcome after a standard antituberculosis treatment. The evolution of a mutation in the rpoB gene was documented in a patient during the course of treatment. PCR-SSCP analysis of CSF seems to be an efficacious method of predicting Rif(r) and would reduce the time required for susceptibility testing from approximately 4 to 8 weeks to a few days.
结核分枝杆菌rpoB基因69bp区域的突变与利福平耐药性（Rif[r]）相关。这些突变已在从抗酸涂片阳性的细菌悬液或呼吸道标本中提取的分枝杆菌DNA中检测到。我们试验了一种快速检测脑脊液（CSF）样本中Rif[r]的策略。该策略包括通过PCR扩增rpoB基因的69bp区域，并通过对扩增产物进行单链构象多态性（SSCP）分析来鉴定核苷酸突变。对从29例患者（19例患者合并人类免疫缺陷病毒感染）收集的65份CSF标本进行了处理，这些患者患有经培养或尸检证实（22例患者）或高度疑似（7例患者）的中枢神经系统结核病（CNS-TB）。从25名受试者的37份CSF标本中获得了适合通过SSCP分析进行评估的扩增产物（86.2%）。CSF的PCR-SSCP正确鉴定了所有17例患者分离株的利福平敏感性表型，这些患者的CSF或呼吸道样本培养菌株的药敏试验结果可用。此外，该检测方法还揭示了8例患者（3例培养确诊的CNS-TB患者和5例高度疑似CNS-TB的患者）分离株的利福平敏感性基因型，这些患者没有药敏试验结果；这些患者的PCR-SSCP数据与标准抗结核治疗后的结果一致。在一名患者的治疗过程中记录了rpoB基因的突变演变。CSF的PCR-SSCP分析似乎是预测Rif[r]的有效方法，并且将药敏试验所需时间从大约4至8周缩短至几天。
