Molecular characterisation of Haemophilus influenzae type a and untypeable strains isolated simultaneously from cerebrospinal fluid and blood: novel use of quantitative real-time PCR based on the cap copy number to determine virulence.

This study investigated the genetic structure of the cap region of an isolate of Haemophilus influenzae serotype a (Hia) from the cerebrospinal fluid (CSF) of a child with meningitis. In addition, the genetic structure of the cap region of a non-serotypeable H. influenzae isolate, obtained simultaneously from the blood of the same patient, was determined. According to restriction fragment length polymorphism analysis, the CSF and blood isolates were identical, with the exception of a single band shift of c. 35 kb. PCR analyses suggested that the CSF isolate possessed the IS1016-bexA gene and cap region II, whereas the blood isolate only had the IS1016 element. Furthermore, Southern analysis of DNA from both isolates showed that the CSF isolate carried the cap gene(s), while the blood isolate did not. Using a novel quantitative real-time PCR approach for determining the cap copy number, it was demonstrated that the CSF isolate had two intact tandem repeats of the cap gene containing three copies of IS1016, whereas the blood isolate had only one copy of IS1016. This study provided evidence that H. influenzae serotypes other than serotype b can cause serious disease, and that the virulence of these non-serotype b strains relates primarily to the cap gene copy number and the structure of the cap locus. Therefore, the quantitative real-time PCR assay described in this study should be useful for the rapid and definitive identification of strains of H. influenzae type a that represent a risk for serious disease.