The clinical significance of Haemophilus haemolyticus: identification, discrimination and host interaction

[Truncated] Nontypeable Haemophilus influenzae (NTHi) is a major cause of respiratory infections including otitis media (OM), which is the most common reason for Western children to visit a General Practitioner, be prescribed antibiotics and undergo surgery. Vaccines have had limited impact on the rates of OM, and the rise of antibiotic resistance in NTHi reduces the effectiveness of antibiotic therapies. Consequently, new therapeutics for the treatment and prevention of NTHi infections are required, and would be of significant global benefit. Accurate disease surveillance is necessary for assessing new intervention strategies and determining populations most at risk to disease. Surveillance of NTHi is complicated by H. haemolyticus, a respiratory tract commensal that can be misidentified as NTHi by culture. This thesis further investigates H. haemolyticus misidentification, methods for differentiation from NTHi, and H. haemolyticus interaction with the host.

Aboriginal Australian children have some of the highest rates of OM in the world. In this thesis, the misidentification and diversity of H. haemolyticus and NTHi was quantified in the Kalgoorlie otitis media research project, the largest longitudinal carriage study of Aboriginal and non-Aboriginal children in Australia. Aboriginal children were found to be colonised by different and more unique NTHi ribotypes than non-Aboriginal children. Molecular methods for NTHi surveillance (16SrDNA PCR and ribotyping) were unable to distinguish some strains of NTHi and H. haemolyticus.

This finding prompted the development of a new molecular method for NTHi/H. haemolyticus differentiation. A sensitive and specific target (hpd which encodes Protein D in H. influenzae) was chosen for the developing the assay. H. haemolyticus also possesses the hpd gene, and comparative sequencing determined that the species differences in hpd DNA sequences could be exploited for species differentiation. The resulting PCR high-resolution-melt assay that was developed was robust and reduced the time, cost and expertise required to distinguish NTHi from H. haemolyticus. Validation of the assay on a larger set of isolates lead to the discovery of H. haemolyticus isolates that lacked hpd, highlighting the possibility of immune evasion following vaccination with PHiD-CV- (pneumococcal conjugate vaccine coupled to protein D carrier) as well as the limits of this discriminative assay. To further investigate the dynamics of H. haemolyticus carriage, a H. haemolyticus specific qPCR assay was developed to quantify the density of H. haemolyticus in nasopharyngeal swabs from otitis prone and healthy children. Low density of H. haemolyticus was found in healthy and otitis prone children, whilst NTHi density correlated with OM status.