Nontypeable Haemophilus influenzae (NTHi) and Haemophilus haemolyticus are residents of the human respiratory tract. These species are indistinguishable by traditional culture methods and molecular differentiation has proven difficult. NTHi is a common cause of acute and chronic ear and lung disease, whilst H. haemolyticus is considered a commensal and is rarely associated with disease. The impact of misidentification can be far-reaching leading to incorrect antibiotic prescriptions, and a failure to correctly determine the efficacy of preventative treatments (such as vaccines) on NTHi carriage and disease.
We have optimised quantitative (q) duplex PCR assays for NTHi (hpd#3 and fucP) and H. haemolyticus (Hh hpd and hypD), which cover known variants of each species. Testing on 103 isolates of H. influenzae, H. haemolyticus and NTHi-like Haemophili and 16 other respiratory species has demonstrated that the NTHi and H. haemolyticus qPCRs are highly specific (100% and 95%) and sensitive (100% and 100%). Using these qPCR assays on nasopharyngeal swabs from 40 children with recurrent acute otitis media and 40 healthy controls, we demonstrated that these assays can measure the density of NTHi and H. haemolyticus in the same nasopharyngeal swab. We also observed a positive correlation between NTHi and H. haemolyticus densities (r=0.51) in nasopharyngeal swabs, suggesting a synergism between NTHi and H. haemolyticus within the nasopharynx (or possibly just the ideal environment for Haemophilus growth). We will investigate this further as part of our in vivo evaluation of H. haemolyticus as a potential therapy to block NTHi colonisation and prevent NTHi disease.
In summary, we have optimised specific qPCR assays to detect and quantify NTHi and H. haemolyticus in nasopharyngeal swabs. These assays are suitable for use in clinical and research laboratories to improve the accuracy of NTHi and H. haemolyticus identification and quantification for enhanced disease and vaccine surveillance.