title: Comparison of Established Diagnostic Methodologies and a Novel Bacterial smpB Real-Time PCR Assay for Specific Detection of Haemophilus influenzae Isolates Associated with Respiratory Tract Infections.
abstract: Haemophilus influenzae is a significant causative agent of respiratory tract infections (RTI) worldwide. The development of a rapid H. influenzae diagnostic assay that would allow for the implementation of infection control measures and also improve antimicrobial stewardship for patients is required. A number of nucleic acid diagnostics approaches that detect H. influenzae in RTIs have been described in the literature; however, there are reported specificity and sensitivity limitations for these assays. In this study, a novel real-time PCR diagnostic assay targeting the smpB gene was designed to detect all serogroups of H. influenzae. The assay was validated using a panel of well-characterized Haemophilus spp. Subsequently, 44 Haemophilus clinical isolates were collected, and 36 isolates were identified as H. influenzae using a gold standard methodology that combined the results of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and a fucK diagnostic assay. Using the novel smpB diagnostic assay, 100% concordance was observed with the gold standard, demonstrating a sensitivity of 100% (95% confidence interval [CI], 90.26% to 100.00%) and a specificity of 100% (95% CI, 63.06% to 100.00%) when used on clinical isolates. To demonstrate the clinical utility of the diagnostic assay presented, a panel of lower RTI samples (n = 98) were blindly tested with the gold standard and smpB diagnostic assays. The results generated were concordant for 94/98 samples tested, demonstrating a sensitivity of 90.91% (95% CI, 78.33% to 97.47%) and a specificity of 100% (95% CI, 93.40% to 100.00%) for the novel smpB assay when used directly on respiratory specimens.
authors.affiliation: Nucleic Acid Diagnostics Research Laboratory (NADRL), Microbiology, School of Natural Sciences, National University of Ireland, Galway, Ireland.
authors.forename: Kate
authors.initials: K
authors.lastname: Reddington
authors.affiliation: Centre for Clinical Microbiology, Division of Infection and Immunity, University College London, London, United Kingdom.
authors.forename: Stefan
authors.initials: S
authors.lastname: Schwenk
authors.affiliation: Nucleic Acid Diagnostics Research Laboratory (NADRL), Microbiology, School of Natural Sciences, National University of Ireland, Galway, Ireland.
authors.forename: Nina
authors.initials: N
authors.lastname: Tuite
authors.affiliation: Centre for Clinical Microbiology, Division of Infection and Immunity, University College London, London, United Kingdom.
authors.forename: Gareth
authors.initials: G
authors.lastname: Platt
authors.affiliation: Centre for Clinical Microbiology, Division of Infection and Immunity, University College London, London, United Kingdom.
authors.forename: Danesh
authors.initials: D
authors.lastname: Davar
authors.affiliation: Nucleic Acid Diagnostics Research Laboratory (NADRL), Microbiology, School of Natural Sciences, National University of Ireland, Galway, Ireland.
authors.forename: Helena
authors.initials: H
authors.lastname: Coughlan
authors.affiliation: Centre for Clinical Microbiology, Division of Infection and Immunity, University College London, London, United Kingdom.
authors.forename: Yoann
authors.initials: Y
authors.lastname: Personne
authors.affiliation: Department of Medical Microbiology, University College London Hospitals NHS Foundation Trust, London, United Kingdom.
authors.forename: Vanya
authors.initials: V
authors.lastname: Gant
authors.affiliation: Centre for Clinical Microbiology, Division of Infection and Immunity, University College London, London, United Kingdom.
authors.forename: Virve I
authors.initials: VI
authors.lastname: Enne
authors.affiliation: Centre for Clinical Microbiology, Division of Infection and Immunity, University College London, London, United Kingdom.
authors.forename: Alimuddin
authors.initials: A
authors.lastname: Zumla
authors.affiliation: Nucleic Acid Diagnostics Research Laboratory (NADRL), Microbiology, School of Natural Sciences, National University of Ireland, Galway, Ireland thomas.barry@nuigalway.ie.
authors.forename: Thomas
authors.initials: T
authors.lastname: Barry
mesh_terms: D001431:Bacteriological Techniques / Q000379:methods*; D006192:Haemophilus Infections / Q000175:diagnosis*; D006193:Haemophilus influenzae / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012141:Respiratory Tract Infections / Q000175:diagnosis*; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*