PCR for capsular typing of Haemophilus influenzae

Abstract

A PCR method for the unequivocal assignment of Haemophilus influenzae capsular type (types a to f) was developed. PCR primers were designed from capsule type-specific DNA sequences cloned from the capsular gene cluster of each of the six capsular types. PCR product was amplified only from the capsular type for which the primers were designed. Product was confirmed by using either an internal oligonucleotide or restriction endonuclease digestion. A total of 172 H. influenzae strains of known capsular type (determined genetically) comprising all capsular types and noncapsulate strains were tested by PCR capsular typing. In all cases the PCR capsular type corresponded to the capsular genotype determined by restriction fragment length polymorphism analysis of the cap region. When used in conjunction with PCR primers derived from the capsular gene bexA, capsulate, noncapsulate, and capsule-deficient type b mutant strains could be differentiated. PCR capsular typing overcomes the problems of cross-reaction and autoagglutination associated with the serotyping of H. influenzae strains. The rapid and unequivocal capsular typing method that is described will be particularly important for typing invasive H. influenzae strains isolated from recipients of H. influenzae type b vaccine. Capsulate Haemophilus influenzae isolates express one of six chemically distinct capsular polysaccharides (types a to f) (17). Until the implementation of H. influenzae type b vaccination, more than 95% of H. influenzae disease was caused by H.

Differential susceptibility of invasive Haemophilus influenzae serotype a and serotype b to ampicillin and other commonly prescribed antibiotics

“…Isolates of H. influenzae were serotyped by bacterial slide agglutination using commercial antisera (Difco, Oakville, Ontario, Canada) and confirmed by PCR detection of serotype-specific genes and the capsule transport gene, bexA (Falla et al 1994). Biotypes were determined according to Kilian (1976), and MLST was carried out as previously described (Meats et al 2003) using the MLST online database, http://haemophilus.mlst.net, for allele number and sequence type (ST) assignment.…”

Characterization of nontypeableHaemophilus influenzaecollected from respiratory infections and invasive disease cases in Manitoba, Canada

“…The nontypeable nature of all 125 isolates was confirmed by slide agglutination test using antisera against all six serotypes purchased from commercial sources (Difco, Oakville, ON, Canada; Denka Seiken, Tokyo, Japan). The absence of both the serotype-specific and the capsule transport, bexA, genes was confirmed by PCR using primers described by Falla et al (1994).…”

Development and Evaluation of a Novel Multiplex PCR Technology for Molecular Differential Detection of Bacterial Respiratory Disease Pathogens

“…It is not intended for clinical diagnostic use. The assay amplifies and detects gene-specific DNA sequences of six respiratory bacterial pathogens: S. pneumoniae (lytA), Neisseria meningitidis (ctrA), encapsulated or nonencapsulated Haemophilus influenzae (bexA, ompP2), L. pneumophila (mip), Mycoplasma pneumoniae (ATPase), and C. pneumoniae (ompA) (4,6,9,11,13,17,18). The objective of this study was to design the ResPlex I assay using modifications of CDC real-time PCR primers and probes to C. pneumoniae, L. pneumophila, M. pneumoniae, N. meningitidis, and S. pneumoniae and examine the application of this assay for the detection of bacteria associated with respiratory infections.…”