Farahani Hamidreza, Ghaznavi-Rad Ehsanollah, Mondanizadeh Mahdieh, MirabSamiee Siamak, Khansarinejad Behzad
Department of Microbiology and Immunology, Arak University of Medical Sciences, Arak, Iran.
Department of Microbiology and Immunology, Arak University of Medical Sciences, Arak, Iran; Molecular and Medicine Research Center, Arak University of Medical Sciences, Arak, Iran.
Mol Cell Probes. 2016 Aug;30(4):261-265. doi: 10.1016/j.mcp.2016.07.002. Epub 2016 Jul 9.
Accurate and timely diagnosis of acute bacterial meningitis is critical for antimicrobial treatment of patients. Although PCR-based methods have been widely used for the diagnosis of acute meningitis caused by bacterial pathogens, the main disadvantage of these methods is their high cost. This disadvantage has hampered the widespread use of molecular assays in many developing countries. The application of multiplex assays and "in-house" protocols are two main approaches that can reduce the overall cost of a molecular test. In the present study, an internally controlled tetraplex-PCR was developed and validated for the specific detection of Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae in cerebrospinal fluid (CSF) samples. The analysis of a panel of other human pathogens showed no cross-reactivity in the assay. The analytical sensitivity of the in-house assay was 792.3 copies/ml, when all three bacteria were presentin the specimens. This value was calculated as 444.5, 283.7, 127.8 copies/ml when only S. pneumoniae, N. meningitidis and H. influenzae, respectively, were present. To demonstrate the diagnostic performance of the assay, a total of 150 archival CSF samples were tested and compared with a commercial multiplex real-time PCR kit. A diagnostic sensitivity of 92.8% and a specificity of 95.1% were determined for the present tetraplex-PCR assay. The results indicate that the established method is sensitive, specific and cost-effective, and can be used particularly in situations where the high cost of commercial kits prevents the use of molecular methods for the diagnosis of bacterial meningitis.
准确及时地诊断急性细菌性脑膜炎对于患者的抗菌治疗至关重要。尽管基于聚合酶链反应（PCR）的方法已广泛用于诊断由细菌病原体引起的急性脑膜炎，但其主要缺点是成本高昂。这一缺点阻碍了分子检测方法在许多发展中国家的广泛应用。多重检测和“内部”方案的应用是两种可降低分子检测总体成本的主要方法。在本研究中，开发并验证了一种内控四重PCR方法，用于特异性检测脑脊液（CSF）样本中的肺炎链球菌、脑膜炎奈瑟菌和流感嗜血杆菌。对一组其他人类病原体的分析表明该检测方法无交叉反应。当标本中同时存在所有三种细菌时，内部检测方法的分析灵敏度为792.3拷贝/毫升。当标本中仅分别存在肺炎链球菌、脑膜炎奈瑟菌和流感嗜血杆菌时，该值分别计算为444.5、283.7、127.8拷贝/毫升。为证明该检测方法的诊断性能，共检测了150份存档脑脊液样本，并与一种商业多重实时PCR试剂盒进行比较。本四重PCR检测方法的诊断灵敏度为92.8%，特异性为95.1%。结果表明，所建立的方法灵敏、特异且具有成本效益，尤其可用于商业试剂盒成本高昂而阻碍使用分子方法诊断细菌性脑膜炎的情况。
