title: Specific detection of common pathogens of acute bacterial meningitis using an internally controlled tetraplex-PCR assay.
abstract: 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.
authors.affiliation: Department of Microbiology and Immunology, Arak University of Medical Sciences, Arak, Iran.
authors.forename: Hamidreza
authors.initials: H
authors.lastname: Farahani
authors.affiliation: Department of Microbiology and Immunology, Arak University of Medical Sciences, Arak, Iran; Molecular and Medicine Research Center, Arak University of Medical Sciences, Arak, Iran.
authors.forename: Ehsanollah
authors.initials: E
authors.lastname: Ghaznavi-Rad
authors.affiliation: Molecular and Medicine Research Center, Arak University of Medical Sciences, Arak, Iran; Department of Biotechnology and Molecular Medicine, Arak University of Medical Sciences, Arak, Iran.
authors.forename: Mahdieh
authors.initials: M
authors.lastname: Mondanizadeh
authors.affiliation: Food and Drug Laboratory Research Center, Ministry of Health and Medical Education, Tehran, Iran.
authors.forename: Siamak
authors.initials: S
authors.lastname: MirabSamiee
authors.affiliation: Department of Microbiology and Immunology, Arak University of Medical Sciences, Arak, Iran; Molecular and Medicine Research Center, Arak University of Medical Sciences, Arak, Iran. Electronic address: Behzad.Khansarinejad@arakmu.ac.ir.
authors.forename: Behzad
authors.initials: B
authors.lastname: Khansarinejad
mesh_terms: D000208:Acute Disease; D003362:Cost-Benefit Analysis; D004587:Electrophoresis, Agar Gel; D006193:Haemophilus influenzae; D006801:Humans; D057230:Limit of Detection; D016920:Meningitis, Bacterial / Q000175:diagnosis / Q000191:economics / Q000382:microbiology*; D009345:Neisseria meningitidis / Q000235:genetics* / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D012680:Sensitivity and Specificity; D013296:Streptococcus pneumoniae
keywords: Haemophilus influenzae; Meningitis; Multiplex; Neisseria meningitidis; PCR; Streptococcus pneumoniae