Determination of capsulation status in Haemophilus influenzae by multiplex polymerase chain reaction☆

Since the introduction of Haemophilus influenzae type b conjugate vaccines, there have been concerns regarding the emergence of invasive non–type b strains. Serotyping of H. influenzae with commercially available reagents is subjective. Definitive characterization of the capsulation status can be performed by polymerase chain reaction (PCR) amplification of capsular genes. However, PCR amplification of the conserved export locus in the 2 known phylogenic lines of type b strains and detection of serotype conferring genes in each of the 6 serotypes require multiple assays. To rapidly screen multiple isolates, we devised a multiplex method using 15 primers, which produced a serotype-specific, distinct pattern of amplicons with reference-encapsulated H. influenzae. We applied this technique to a panel of 35 clinical isolates that had been serotyped as type a, c, d, e, or f by slide agglutination; 15 strains lacked capsular genes. Conversely, of 69 invasive isolates that were not serotypeable, all but 11 contained capsule genes. We conclude that this technique will be useful in screening recently isolated H. influenzae for capsulation status.