Santo André Regional Center, Adolfo Lutz Institute, Santo André, São Paulo, Brazil.
PLoS One. 2022 Jun 14;17(6):e0269895. doi: 10.1371/journal.pone.0269895. eCollection 2022.
Meningitis caused by Streptococcus pneumoniae is still a disease of great impact on Public health, which requires immediate diagnosis and treatment. However, the culture of clinical specimens is often negative and antibiotic susceptibility testing (AST) must be performed with isolated strains. Multiplex real-time polymerase chain reaction (qPCR) has high sensitivity and specificity, produces faster results to identify the pathogen, and it can also be an important tool to identify resistance antibiotic genes earlier than AST, especially in the absence of an isolated strain. This study developed a multiplex qPCR assay, using SYBR Green as a nonspecific dye, to detect antibiotic resistance genes to predict pneumococcal susceptibility/resistance in cerebrospinal fluid (CSF) samples from meningitis patients. From 2017 to 2020, CSF samples were cultured and analyzed by qPCR to detect the main three bacteria causing meningitis. Isolated and reference strains were applied in SYBR Green qPCR multiplex to detect pbp2b, ermB, and mef genes, and the results were compared with the AST. Pneumococcal-positive CSF samples (lytA-positive gene) without isolated strains were also tested to evaluate the antimicrobial susceptibility profile in the region from 2014 to 2020. From the received 873 CSF samples; 263 were cultivated, 149 were lytA-positive in the qPCR, and 25 produced viable isolated pneumococci strains, which were evaluated by AST. Melting temperature for each gene and the acceptance criteria were determined (pbp2b: 78.24-79.86; ermB: 80.88-82.56; mef: 74.85-76.34 ºC). A total of 48/51 strains presented a genetic profile in agreement with the AST results. Resistant strains to erythromycin and clindamycin were ermB-positive, and two were also mef-positive, indicating both resistance mechanisms were present. In the retrospective study of the genetic profile of resistance, 82 lytA-positive CSF samples plus 4 strains were applied in the SYBR Green qPCR multiplex: 51% of samples presented the wild genotype (pbp2b positive and ermB/mef negative); 15% were negative for all the three evaluated, indicating pneumococci resistant to penicillin; and 17% represented the multidrug-resistant pneumococci (pbp2b negative and ermB positive or pbp2b negative and ermB and mef positive). Therefore, SYBR Green qPCR multiplex proved to be a reliable tool to identify resistance genes in S. pneumoniae and would be less expensive than multiplex qPCR using specific probes. This could be easily introduced into the routine of diagnostic laboratories and provide a strong presumption of pneumococcal resistance, especially in the absence of isolated strains.
由肺炎链球菌引起的脑膜炎仍然是对公共卫生有重大影响的疾病，需要立即进行诊断和治疗。然而，临床标本的培养往往呈阴性，必须对分离株进行抗生素敏感性试验 (AST)。多重实时聚合酶链反应 (qPCR) 具有高灵敏度和特异性，能够更快地识别病原体，并且还可以比 AST 更早地识别出耐药抗生素基因，尤其是在没有分离株的情况下。本研究开发了一种多重 qPCR 检测方法，使用 SYBR Green 作为非特异性染料，用于检测抗生素耐药基因，以预测脑膜炎患者脑脊液 (CSF) 样本中的肺炎链球菌敏感性/耐药性。2017 年至 2020 年，对 CSF 样本进行培养和 qPCR 分析，以检测引起脑膜炎的主要三种细菌。将分离株和参考株应用于 SYBR Green qPCR 多重检测 pbp2b、ermB 和 mef 基因，并将结果与 AST 进行比较。还对 2014 年至 2020 年无分离株的肺炎链球菌阳性 CSF 样本 (lytA 阳性基因) 进行了检测，以评估该地区的抗菌药物敏感性谱。从收到的 873 份 CSF 样本中; 263 个被培养，149 个在 qPCR 中呈 lytA 阳性，25 个产生了有活力的肺炎链球菌分离株，这些分离株通过 AST 进行了评估。确定了每个基因的熔解温度和接受标准(pbp2b: 78.24-79.86; ermB: 80.88-82.56; mef: 74.85-76.34°C)。共有 48/51 株的遗传特征与 AST 结果一致。对红霉素和克林霉素耐药的菌株 ermB 阳性，其中两株还 mef 阳性，表明存在两种耐药机制。在耐药性遗传特征的回顾性研究中，对 82 个 lytA 阳性 CSF 样本加上 4 个菌株进行了 SYBR Green qPCR 多重检测：51%的样本呈现野生基因型(pbp2b 阳性且 ermB/mef 阴性)；15%的三个评估结果均为阴性，表明对青霉素耐药的肺炎链球菌；17%的样本代表了多药耐药的肺炎链球菌(pbp2b 阴性且 ermB 阳性或 pbp2b 阴性且 ermB 和 mef 阳性)。因此，SYBR Green qPCR 多重检测被证明是一种可靠的工具，可以识别肺炎链球菌中的耐药基因，并且比使用特异性探针的多重 qPCR 更便宜。这可以很容易地引入诊断实验室的常规工作中，并提供对肺炎链球菌耐药性的有力推测，尤其是在没有分离株的情况下。
