title: Detection of bacterial pathogens in Mongolia meningitis surveillance with a new real-time PCR assay to detect Haemophilus influenzae.
abstract: Since the implementation of Haemophilus influenzae (Hi) serotype b vaccine, other serotypes and non-typeable strains have taken on greater importance as a cause of Hi diseases. A rapid and accurate method is needed to detect all Hi regardless of the encapsulation status. We developed 2 real-time PCR (rt-PCR) assays to detect specific regions of the protein D gene (hpd). Both hpd assays are very specific and sensitive for detection of Hi. Of the 63 non-Hi isolates representing 21 bacterial species, none was detected by the hpd #1 assay, and only one of 2 H. aphrophilus isolates was detected by the hpd #3 assay. The hpd #1 and #3 assays detected 97% (229/237) and 99% (234/237) of Hi isolates, respectively, and were superior for detection of both typeable and non-typeable Hi isolates, as compared to previously developed rt-PCR targeting ompP2 or bexA. The diagnostic sensitivity and specificity of these rt-PCR assays were assessed on cerebrospinal fluid specimens collected as part of meningitis surveillance in Ulaanbaatar, Mongolia. The etiology (Neisseria meningitidis, Hi, and Streptococcus pneumoniae) of 111 suspected meningitis cases was determined by conventional methods (culture and latex agglutination), previously developed rt-PCR assays, and the new hpd assays. The rt-PCR assays were more sensitive for detection of meningitis pathogens than other classical methods and improved detection from 50% (56/111) to 75% (83/111). The hpd #3 assay identified a non-b Hi that was missed by the bexA assay and other methods. A sensitive rt-PCR assay to detect both typeable and non-typeable Hi is a useful tool for improving Hi disease surveillance especially after Hib vaccine introduction.
authors.affiliation: Meningitis and Vaccine Preventable Diseases Branch, Division of Bacterial Diseases, National Center of Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA. xwang2@cdc.gov
authors.forename: Xin
authors.initials: X
authors.lastname: Wang
authors.forename: Raydel
authors.initials: R
authors.lastname: Mair
authors.forename: Cynthia
authors.initials: C
authors.lastname: Hatcher
authors.forename: M Jordan
authors.initials: MJ
authors.lastname: Theodore
authors.forename: Karen
authors.initials: K
authors.lastname: Edmond
authors.forename: Henry M
authors.initials: HM
authors.lastname: Wu
authors.forename: Brian H
authors.initials: BH
authors.lastname: Harcourt
authors.forename: Maria da Gloria S
authors.initials: Mda G
authors.lastname: Carvalho
authors.forename: Fabiana
authors.initials: F
authors.lastname: Pimenta
authors.forename: Pagbajab
authors.initials: P
authors.lastname: Nymadawa
authors.forename: Dorjpurev
authors.initials: D
authors.lastname: Altantsetseg
authors.forename: Mariah
authors.initials: M
authors.lastname: Kirsch
authors.forename: Sarah W
authors.initials: SW
authors.lastname: Satola
authors.forename: Amanda
authors.initials: A
authors.lastname: Cohn
authors.forename: Nancy E
authors.initials: NE
authors.lastname: Messonnier
authors.forename: Leonard W
authors.initials: LW
authors.lastname: Mayer
mesh_terms: D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification*; D001431:Bacteriological Techniques / Q000379:methods*; D002555:Cerebrospinal Fluid / Q000382:microbiology; D002675:Child, Preschool; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D006193:Haemophilus influenzae / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D007223:Infant; D008583:Meningitis, Haemophilus / Q000453:epidemiology* / Q000382:microbiology; D008969:Molecular Sequence Data; D008986:Mongolia / Q000453:epidemiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial