Molecular Serotype-specific Identification of Haemophilus influenzae Using the Loop-mediated Isothermal Amplification Method

Abstract

BackgroundOver the past several decades, the incidence of meningitis caused by Haemophilus influenzae in children has decreased due to wide-spread vaccination of H. influenzae type b (Hib). The incidence of invasive diseases due to H. influenzae that were not included in the vaccines, however, has increased. We developed a rapid, simple, and cost-effective method for detecting serotypes of H. influenzae.MethodsWe designed LAMP primer sets based on the sequences available for the capsular types a, c, d, e, and f. The assays were evaluated regarding reactivity, specificity and sensitivity of the assays. To be used for the patients suspected meningitis, we evaluated the detection limit of the non-Hib serotype specific LAMP assay using DNAs spiking CSF specimens.ResultsThe reactivity and specificity of the LAMP assays were confirmed using 6 serotypes and nontype of H. influenzae strains, plus 21 strains representing other Haemophilus species and non-Haemophilus genera. The sensitivity of the LAMP assays was confirmed from 10 to 103 copies per reaction that was 1 to 103 times of sensitivity of the non-Hib serotype specific PCR that demonstrated the sensitivity 103 or 104 genome copies per reaction. The sensitivity of the LAMP was also the same, 10 to 103 genome copies per reaction.ConclusionThis is the first report of the serotype-specific identification assay for H. influenzaeusing the LAMP method. Our results suggest potential usage for children or adults with suspected meningitis in resource limited laboratories or public health surveillance systems.Funding sourceJSPS Bilateral Open Partnership Joint Research ProjectsDisclosures K. Takahashi, Kaneka Corp.: Employee, Salary; S. Hayakawa, Kaneka Corp.: Collaborator, Research grant