title: Development of Real-Time PCR Methods for the Detection of Bacterial Meningitis Pathogens without DNA Extraction.
abstract: Neisseria meningitidis (Nm), Haemophilus influenzae (Hi), and Streptococcus pneumoniae (Sp) are the lead causes of bacterial meningitis. Detection of these pathogens from clinical specimens using traditional real-time PCR (rt-PCR) requires DNA extraction to remove the PCR inhibitors prior to testing, which is time consuming and labor intensive. In this study, five species-specific (Nm-sodC and -ctrA, Hi-hpd#1 and -hpd#3 and Sp-lytA) and six serogroup-specific rt-PCR tests (A, B, C, W, X, Y) targeting Nm capsular genes were evaluated in the two direct rt-PCR methods using PerfeCTa and 5x Omni that do not require DNA extraction. The sensitivity and specify of the two direct rt-PCR methods were compared to TaqMan traditional rt-PCR, the current standard rt-PCR method for the detection of meningitis pathogens. The LLD for all 11 rt-PCR tests ranged from 6,227 to 272,229 CFU/ml for TaqMan, 1,824-135,982 for 5x Omni, and 168-6,836 CFU/ml for PerfeCTa. The diagnostic sensitivity using TaqMan ranged from 89.2%-99.6%, except for NmB-csb, which was 69.7%. For 5x Omni, the sensitivity varied from 67.1% to 99.8%, with three tests below 90%. The sensitivity of these tests using PerfeCTa varied from 89.4% to 99.8%. The specificity ranges of the 11 tests were 98.0-99.9%, 97.5-99.9%, and 92.9-99.9% for TaqMan, 5x Omni, and PerfeCTa, respectively. PerfeCTa direct rt-PCR demonstrated similar or better sensitivity compared to 5x Omni direct rt-PCR or TaqMan traditional rt-PCR. Since the direct rt-PCR method does not require DNA extraction, it reduces the time and cost for processing CSF specimens, increases testing throughput, decreases the risk of cross-contamination, and conserves precious CSF. The direct rt-PCR method will be beneficial to laboratories with high testing volume.
authors.affiliation: Meningitis and Vaccine Preventable Diseases Branch, Centers for Disease Control and Prevention, Atlanta, United States of America.
authors.forename: Jeni
authors.initials: J
authors.lastname: Vuong
authors.affiliation: Scientific Institute of Public Health, Brussels, Belgium.|Centre de Recherche Médicale et Sanitaire, Niamey, Niger.
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Collard
authors.affiliation: Meningitis and Vaccine Preventable Diseases Branch, Centers for Disease Control and Prevention, Atlanta, United States of America.
authors.forename: Melissa J
authors.initials: MJ
authors.lastname: Whaley
authors.affiliation: Centre de Recherche Médicale et Sanitaire, Niamey, Niger.
authors.forename: Issaka
authors.initials: I
authors.lastname: Bassira
authors.affiliation: Centre de Recherche Médicale et Sanitaire, Niamey, Niger.
authors.forename: Issaka
authors.initials: I
authors.lastname: Seidou
authors.affiliation: Institut National de Recherche en Santé Publique, Bamako, Mali.
authors.forename: Seydou
authors.initials: S
authors.lastname: Diarra
authors.affiliation: Centre Hospitalier Universitaire Pédiatrique Charles De Gaulle, Ouagadougou, Burkina Faso.
authors.forename: Rasmata T
authors.initials: RT
authors.lastname: Ouédraogo
authors.affiliation: Centre Hospitalier Universitaire Pédiatrique Charles De Gaulle, Ouagadougou, Burkina Faso.
authors.forename: Dinanibè
authors.initials: D
authors.lastname: Kambiré
authors.affiliation: Meningitis and Vaccine Preventable Diseases Branch, Centers for Disease Control and Prevention, Atlanta, United States of America.
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Taylor
authors.affiliation: Instituto Adolfo Lutz, São Paulo, Brazil.
authors.forename: Claudio
authors.initials: C
authors.lastname: Sacchi
authors.affiliation: Meningitis and Vaccine Preventable Diseases Branch, Centers for Disease Control and Prevention, Atlanta, United States of America.
authors.forename: Leonard W
authors.initials: LW
authors.lastname: Mayer
authors.affiliation: Meningitis and Vaccine Preventable Diseases Branch, Centers for Disease Control and Prevention, Atlanta, United States of America.
authors.forename: Xin
authors.initials: X
authors.lastname: Wang
mesh_terms: D004269:DNA, Bacterial / Q000302:isolation & purification*; D006801:Humans; D057230:Limit of Detection; D016920:Meningitis, Bacterial / Q000134:cerebrospinal fluid / Q000175:diagnosis*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D004269:DNA, Bacterial
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