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International Journal of Medical Microbiology 301 (2011) 303–309

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International Journal of Medical Microbiology
journal homepage: www.elsevier.de/ijmm

Detection of bacterial pathogens in Mongolia meningitis surveillance with a new real-time PCR assay to detect Haemophilus influenzae
Xin Wang a,∗ , Raydel Mair a , Cynthia Hatcher a , M. JordanTheodore a , Karen Edmond b , Henry M. Wu a , Brian H. Harcourt a , Maria da Gloria S. Carvalho c , Fabiana Pimenta c , Pagbajab Nymadawa d , Dorjpurev Altantsetseg d , Mariah Kirsch e , Sarah W. Satola f , Amanda Cohn a , Nancy E. Messonnier a , Leonard W. Mayer a
a Meningitis and Vaccine Preventable Diseases Branch, Division of Bacterial Diseases, National Center of Immunization and RespiratoryDiseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA b Infectious Disease Epidemiology Unit, London School of Hygiene and Tropical Medicine, UK c Respiratory Diseases Branch, Division of Bacterial Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA d National Center of Communicable Diseases,Ministry of Health, Ulaanbaatar, Mongolia e Centers for Disease Control and Prevention, Atlanta, GA 30333, USA f Dept. of Medicine, Emory University School of Medicine, Atlanta, GA 30324, USA

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Since the implementation of Haemophilus influenzae (Hi) serotype b vaccine, other serotypes and nontypeable strains have taken on greater importance as a cause ofHi 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 fluidspecimens 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 rtPCR assays, and the new hpd assays. The rt-PCR assays were more sensitive for detection of meningitis pathogens thanother 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. Published by Elsevier GmbH.

Article history: Received 29September 2010 Received in revised form 12 November 2010 Accepted 14 November 2010 Keywords: Real-time PCR Haemophilus influenzae Bacterial meningitis Surveillance Diagnosis

Introduction Haemophilus influenzae (Hi) is a common commensal organism in the human upper respiratory tract and an important pathogen that causes a spectrum of infectious diseases (St Geme, 1993; Foxwell et al., 1998; Cody etal., 2003). Encapsulated or typeable Hi strains produce one of 6 structurally and antigenically distinct capsules (a–f). The typeable Hi, particularly serotype

∗ Corresponding author at: Meningitis Laboratory, Centers for Disease Control and Prevention, 1600 Clifton Rd NE, MS D-11, Atlanta, GA 30033, USA. Tel.: +1 404 639 5474; fax: +1 404 639 4421. E-mail address: xwang2@cdc.gov (X. Wang)....