Detection of Cell-Dissociated Non-Typeable Haemophilus influenzae in the Airways of Patients with Chronic Obstructive Pulmonary Disease

Background Non-typeable Haemophilus influenzae (NTHi) is the most commonly found pathogen in the lower respiratory airways of patients with COPD. NTHi is predominantly regarded as an intracellular pathogen; however, like most pathogens, it can exist and co-exist in two broad forms: cell-associated (intracellularly or adhered to cells) or cell-dissociated (biofilm dispersed or planktonic). We sought to investigate if cell-dissociated NTHi can be detected from the sputum of COPD patients and assess this relationship to disease severity and airway inflammation. Methods DNA was extracted from the sputum plug and cell-free supernatant to quantify absolute (cell-associated and cell-dissociated NTHi) and cell-dissociated NTHi, respectively, from 87 COPD subjects attending an observational longitudinal COPD exacerbation study. NTHi was quantified using TaqMan hydrolysis probes, targeting the OMP P6 gene using qPCR. Results At stable state cell-dissociated NTHi was detected 56% of subjects with a median (IQR) of 9.95x102 gene copies (1.26x102 to 1.90x104). Cell-dissociated NTHi correlated with absolute NTHi levels (r=0.34, p<0.01) but not airway inflammation or spirometry at stable state. At exacerbation, cell-dissociated NTHi correlated with lung function (FEV1 r=0.629, p=0.005; FEV1%predicted r=0.564, p=0.015; FVC r=0.476 p=0.046) and sputum neutrophilic inflammation (% neutrophils r=0.688, p=0.002; total neutrophils r=0.518, p=0.028). Conclusion In patients with COPD, NTHi can exist in both cell-associated and cell-dissociated forms. Cell-dissociated NTHi is associated with neutrophilic airway inflammation during exacerbations of COPD and may be a driving factor in worsening lung function during these episodes.