Multiplex real-time polymerase chain reaction for rapid detection of β-lactamase–negative, ampicillin-resistant Haemophilus influenzae

We evaluated a multiplex real-time quantitative polymerase chain reaction (PCR) method for quantification of Haemophilus influenzae and rapid detection of β-lactam–resistant strains. We designed 5 PCR primer sets to simultaneously detect the β-lactam–resistant genes and quantify the pathogen. To demonstrate the validity of this assay, we used 191 clinical isolates, including 141 H. influenzae strains, and 100 purulent sputum samples, including 30 samples from which H. influenzae had been isolated. This assay showed 92.9% sensitivity and 91.8% specificity for detecting β-lactam–resistant genes, relative to the conventional phenotypic method, and this assay correlated well with conventional quantitative culture counts. By using this assay, we could quantify H. influenzae and identify β-lactam susceptibility in only 3 h and with only one tube. This method will be helpful for the rapid detection of H. influenzae infections and the selection of appropriate antibiotics.