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The morbidity and mortality related to respiratory tract diseases is enormous, with hundreds of millions of individuals afflicted and four million people dying each year. Understanding the immunological processes in the mucosa that govern outcome following pathogenic encounter could lead to novel therapies. There is a need to study responses at mucosal surfaces in humans for two reasons: (i) Immunological findings in mice, or other animals, often fail to translate to humans. (ii) Compartmentalization of the immune system dictates a need to study sites where pathogens reside. In this manuscript, we describe two novel non-invasive nasal mucosal microsampling techniques and their use for measuring immunological parameters: 1) using nasal curettes to collect cells from the inferior turbinate and; 2) absorptive matrices to collect nasal lining fluid. Both techniques were well tolerated and yielded reproducible and robust data. We demonstrated differences in immune populations and activation state in nasal mucosa compared to blood as well as compared to nasopharyngeal lumen in healthy adults. We also found superior cytokine detection with absorptive matrices compared to nasal wash. These techniques are promising new tools that will facilitate studies of the immunological signatures underlying susceptibility and resistance to respiratory infections.

IntroductionPneumococcal colonisation is regarded as a pre-requisite for developing pneumococcal disease. In children previous studies have reported pneumococcal colonisation to be a symptomatic event and described a relationship between symptom severity/frequency and colonisation density. The evidence for this in adults is lacking in the literature. This study uses the experimental human pneumococcal challenge (EHPC) model to explore whether pneumococcal colonisation is a symptomatic event in healthy adults. MethodsHealthy participants aged 18-50 were recruited and inoculated intra-nasally with either Streptococcus pneumoniae (serotypes 6B, 23F) or saline as a control. Respiratory viral swabs were obtained prior to inoculation. Nasal and non-nasal symptoms were then assessed using a modified Likert score between 1 (no symptoms) to 7 (cannot function). The rate of symptoms reported between the two groups was compared and a correlation analysis performed. ResultsData from 54 participants were analysed. 46 were inoculated with S. pneumoniae (29 with serotype 6B, 17 with serotype 23F) and 8 received saline (control). In total, 14 became experimentally colonised (30.4%), all of which were inoculated with serotype 6B. There was no statistically significant difference in nasal (p = 0.45) or non-nasal symptoms (p = 0.28) between the inoculation group and the control group. In those who were colonised there was no direct correlation between colonisation density and symptom severity. In the 22% (12/52) who were co-colonised, with pneumococcus and respiratory viruses, there was no statistical difference in either nasal or non-nasal symptoms (virus positive p = 0.74 and virus negative p = 1.0). PLOS ONE PLOS ONE | https://doi.org/10.1371/journal.pone.0229558 March 10, 2020 1 / 12 OPEN ACCESS Citation: Trimble A, Connor V, Robinson RE, McLenaghan D, Hancock CA, Wang D, et al. (2020) Pneumococcal colonisation is an asymptomatic event in healthy adults using an experimental human colonisation model. PLoS ONE 15(3): e0229558. https://doi.org/10.

Current diagnostic tests are ineffective for identifying the etiological pathogen in hospitalized adults with lower respiratory tract infections (LRTIs). The association of pneumococcal colonization with disease has been suggested as a means to increase the diagnostic precision. We compared the pneumococcal colonization rates and the densities of nasal pneumococcal colonization by (i) classical culture and (ii) quantitative real-time PCR (qPCR) targeting lytA in patients with LRTIs admitted to a hospital in the United Kingdom and control patients. A total of 826 patients were screened for inclusion in this prospective case-control study. Of these, 38 patients were recruited, 19 with confirmed LRTIs and 19 controls with other diagnoses. Nasal wash (NW) samples were collected at the time of recruitment. Pneumococcal colonization was detected in 1 patient with LRTI and 3 controls (P = 0.6) by classical culture. By qPCR, pneumococcal colonization was detected in 10 LRTI patients and 8 controls (P = 0.5). Antibiotic usage prior to sampling was significantly higher in the LRTI group than in the control group (19 versus 3; P < 0.001). With a clinically relevant cutoff of >8,000 copies/ml on qPCR, pneumococcal colonization was found in 3 LRTI patients and 4 controls (P > 0.05). We conclude that neither the prevalence nor the density of nasal pneumococcal colonization (by culture and qPCR) can be used as a method of microbiological diagnosis in hospitalized adults with LRTI in the United Kingdom. A community-based study recruiting patients prior to antibiotic therapy may be a useful future step.

IntroductionPneumococcal colonisation is regarded as a pre-requisite for developing pneumococcal disease. In children previous studies have reported colonisation to be a symptomatic event and described a relationship between symptom severity/frequency and colonisation density. The evidence for this in adults is lacking in the literature. This study uses an experimental human pneumococcal challenge model to explore whether pneumococcal colonisation (or co-colonisation with a respiratory virus) is a symptomatic event in healthy adults.MethodsHealthy volunteers aged 18-50 were recruited and inoculated intra-nasally with either Streptococcus pneumoniae (serotypes 6B, 23F) or saline as a control. Respiratory viral swabs were obtained prior to inoculation. Nasal and non-nasal symptoms were then assessed using a modified Likert score between 1 (no symptoms) to 7 (cannot function). The rate of symptoms reported between groups was compared and a correlation analysis performed.ResultsData from 54 participants were analysed. 46 were inoculated with S. pneumoniae (29 with 6B, 17 with 23F) and 8 received saline. In total, 14 became experimentally colonised (30.4%), all of which were inoculated with 6B serotype. There was no statistically significant difference in nasal (p= 0.45) or non-nasal symptoms (p=0.28) between the pneumococcal inoculation group and the saline group. There was no direct correlation between colonisation density and symptom severity in those who were colonised. In the 22% (12/52) who were co-colonised with pneumococcus and respiratory viruses there was no statistical difference in either nasal or non-nasal symptoms (virus positive p=0.74 and virus negative p=1.0).ConclusionPneumococcal colonisation is asymptomatic in healthy adults, regardless of bacterial density or viral co-colonisation.