The nose is the best niche for detection of pneumococcal colonisation following experimental challenge in adults of all ages

The nose is the best niche for detection of pneumococcal colonisation following experimental challenge in adults of all ages | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The nose is the best niche for detection of pneumococcal colonisation following experimental challenge in adults of all ages Elissavet Nikolaou, Esther Lauryn German, Annie Blizard, Ashleigh Howard, and 16 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-513376/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Background Previous studies have suggested that the pneumococcal niche changes from the nose to the oropharynx with age. We use an Experimental Human Pneumococcal Challenge model to investigate pneumococcal colonisation in each anatomical niche with age. Methods Healthy adults (n = 112) were intranasally inoculated with Streptococcus pneumoniae serotype 6B (Spn6B) and were categorised as young 18-55yrs (n = 57) or older > 55yrs (n = 55). Colonisation status (frequency and density) was determined by multiplex qPCR targeting the lytA and cpsA -6A/B genes in both raw and culture-enriched nasal wash and oropharyngeal swab samples collected at 2-, 7- and 14-days post-exposure. For older adults, raw and culture-enriched saliva samples were also assessed. Results 64% of NW samples and 54% of OPS samples were positive for Spn6B in young adults, compared to 35% of NW samples, 24% of OPS samples and 6% of saliva samples in older adults. Many colonisation events were only detected in culture-enriched samples. Experimental colonisation was detected in 72% of young adults by NW and 63% by OPS. In older adults, this was 51% by NW, 36% by OPS and 9% by saliva. Conclusions The nose is the best niche for detection of experimental pneumococcal colonisation in both young and older adults. Applied & Industrial Microbiology Bacteriology Infectious Diseases Pathology pneumococcal niche colonisation Experimental Human Pneumococcal Challenge model age Figures Figure 1 Figure 2 Introduction The natural flora of the human upper respiratory tract (URT) is in constant interaction with the external environment, resulting in a diverse ecology of microorganisms colonising epithelial surfaces in the oral and nasal cavities and oropharynx 1 . Streptococcus pneumoniae (Spn, pneumococcus) can proliferate and establish colonisation as a non-pathogenic symbiont, which is asymptomatic for the host, particularly in adults 2 , 3 . Pneumococcus can however migrate to other organs, resulting in pneumonia, meningitis, and bacteraemia, causing significant morbidity and mortality worldwide, especially in the very young and very old. Colonisation of the URT has been shown to be the source of, and pre-requisite for, pneumococcal disease 4 and its transmission throughout the community 5 . Accurate detection of pneumococcal colonisation is crucial to assessing disease potential, as well as direct and indirect impact of vaccines. The World Health Organisation (WHO) recommends the use of nasopharyngeal swabs for pneumococcal colonisation detection in children and both nasopharyngeal and oropharyngeal swabs (OPS) in adults 6 , 7 . WHO recommendations were based on culture-based methods and more recent studies using sensitive molecular methods have encouraged other sampling methods such as saliva or nasal wash 8 , 9 , due to improved comfort and pneumococcal detection. Many studies have investigated pneumococcal colonisation prevalence in the first few years of life, showing that the nasopharynx of 40–95% of young children 10 , 11 is naturally colonised with pneumococcus. With the onset of adulthood, falling pneumococcal disease rates are accompanied by decreased nasopharyngeal colonisation rates at 10–25% 12–15 . In older adults, a population at increased risk of pneumococcal disease, colonisation prevalence and density are reported to be lower (19), but study results are heterogeneous with some reporting very low colonisation rates of 1.9–4.2% 16–18 . Our recent systematic review analysed 29 studies, 18 with participant-level data (representing 6290 participants), and reported prevalence of detected pneumococcal colonisation of 0–39% by conventional culture methods and 3–23% by molecular methods 19 . Various theories have been put forward to explain the paradox of increased disease risk with decreased colonisation prevalence, including a more transient colonisation dynamic in older adults 17 , the poor sensitivity of conventional culture methods to detect low density colonisation in polymicrobial samples 20 , 21 and a transition of the pneumococcal niche from the nasopharynx to the oral cavity with ageing 20 . The Experimental Human Pneumococcal Colonisation (EHPC) model is a safe, reproducible, and controlled method of studying colonisation dynamics in adult human participants, as the challenge dose and time of exposure is known 22 . We have recently expanded this model to older adults and reported that experimental colonisation was established in 39% of participants (25/64) with no adverse events. Colonisation occurred in 47% (9/19) of participants aged 50–59 and in 42% (13/31) of those aged 60–69 compared to 21% (3/14) in those aged ≥ 70 years. Colonisation density was similar between old and younger adults 23 . We used 780 nasal, oropharyngeal and saliva samples to define precisely whether the niche of experimental pneumococcal colonisation changes in adults with increasing age. Association with bacterial density was also analysed. Materials And Methods Clinical trial design, participant cohorts and sample analysis The methodology and inclusion/exclusion criteria for EHPC studies have been previously described 24 . Briefly, participants were healthy adults aged ≥ 18 years with no major risk factors for pneumococcal disease, colonisation, or transmission (such as: cigarette smoking; close contact with children aged < 5 years; healthcare work or caring responsibilities; steroid therapy and respiratory or immunosuppressive comorbidities). Studies were conducted in accordance with the Declaration of Helsinki and Good Clinical Practice procedures. All participants provided written informed consent and underwent a safety screening. Studies were submitted to and approved by a local NHS Research Ethics Committee. In this study, samples have been sourced from two separate EHPC clinical studies. The young vaccine study was a double-blind, randomised controlled trial conducted between September 2013 - April 2014 investigating the impact of PCV-13 vaccination on experimental pneumococcal colonisation in adults aged 18–50 (NHS REC number 12/NW/0873, ISRCTN number 45340436 registered 18/11/2013) 25 . In the present analysis, we include participants from the control arm only (vaccinated with Hepatitis A vaccine rather than PCV-13). The older adults study was an observational study of the effect of age on experimental pneumococcal colonisation in adults aged 50–80 conducted between June 2016 - February 2018 (NHS REC number 16/NW/0031, ISRCTN number 10948363 registered 08/11/2016) 23 . In total, 112 participants were analysed. The sample sets can be combined because the inoculation dose and strain, as well as the methods for inoculation, nasal wash collection and processing remained identical. Furthermore, pneumococcal colonisation rates and serotype distribution in healthy adults in Liverpool appears to be relatively stable, based on data collected between 2010–2017 26 . The low prevalence of naturally occurring Spn6 provides reassurance that detected Spn6B is the inoculated strain. Using the full sample set, participants were split into young 18–55 years (n = 57) and older adults > 55 years (n = 55), in keeping with the cut-off used by Marrie et al. 27 . In both studies, participants were inoculated with 80,000 colony-forming units (CFU) per nostril of live Spn6B pneumococcus (BHN418, GenBank accession number ASHP00000000.1) 25 . Nasal wash (NW) samples were collected before inoculation to screen for pneumococcal colonisation acquired from community. Depending on the study, NW, oropharyngeal swabs (OPS) and saliva samples were then collected post pneumococcal exposure. In the young vaccine study, NW and OPS samples were collected on days 2, 7, 14 (only culture-positives) and 21 post exposure. In the older adults study, NW, OPS and saliva samples were collected on days 2, 7, 9, 14, 22 (only culture-positives) and 29 post exposure. Pneumococcal colonisation status in both studies was determined by NW culture (not discussed here) and by multiplex real-time polymerase chain reaction (qPCR) targeting the lytA and cpsA -6A/B genes in raw and culture-enriched NW, OPS, and saliva samples. The raw and culture-enriched NW, OPS, and saliva qPCR data from days 2, 7 and 14 (covered in both studies) were included in this study. NW collection was performed as previously described 24 . Briefly, 20mL of 0.9% sodium chloride solution in total (10mL saline per nostril) was introduced using a syringe and held for a few seconds in the participant’s nose before being expelled into a sterile container. NW was centrifuged at 4000rpm for 10 minutes. Supernatant was collected and pellet was resuspended in STGG (Skimmed milk-Tryptone-Glucose-Glycerine) 20% glycerol before storage at -80°C. OPS samples were collected in 1 mL STGG and stored at -80°C, until further use. Saliva samples were collected using the salivette device (Sarstedt, UK). Following collection, each salivette was centrifuged at 4000rpm for 3minutes at 4 o C and after measuring the volume of saliva liquid retrieved, both pellet and supernatant were re-suspended in equal volume of STGG 50% glycerol and stored at -80°C, until further use. Preparation of raw and culture-enriched NW, OPS, and saliva samples Before DNA extraction, samples were thawed for 30 minutes at room temperature and vigorously vortexed for 20 seconds. 300µL raw NW pellet, 200µL raw OPS and 300µL raw saliva aliquots were prepared. For culture-enrichment, 50µL of NW pellet, OPS or saliva samples were plated on Columbia blood agar supplemented with 5% horse blood and 80µL gentamicin 1mg/mL. Plates were incubated overnight at 37°C in 5%CO 2 . Following incubation, 2mL of STGG 20% glycerol was added onto each plate and microbial growth scraped off. 300µL culture-enriched NW, 200µL culture-enriched OPS and 300µL culture-enriched saliva aliquots were prepared. Both raw and culture-enriched NW, OPS and saliva aliquots were stored at -20 o C until DNA extraction took place. DNA extraction on raw and culture-enriched NW, OPS, and saliva samples Bacterial genomic DNA was extracted from raw and culture-enriched NW, OPS, and saliva samples. On the day of the extraction, prepared aliquots were thawed at room temperature and vigorously vortexed for 20 seconds. Samples were pelleted at 20,238xg for 10 minutes. Following centrifugation, 300µL of lysis buffer with protease (Agowa Mag mini-DNA extraction kit; LGC Genomics, Germany), 100µL of zirconium beads (diameter of 0.1 mm), and 300µL of phenol pH 8.0 (toxic, performed in a cabinet with charcoal filter) were added to the pellets. Samples were mechanically disrupted at 50 Hz for 3 minutes in a tissue homogenizer followed by 3 minutes on ice, twice. The samples were then centrifuged for 10 minutes at 9,391xg, and the upper aqueous phase was transferred to a sterile 1.5mL Eppendorf tube pre-filled with 600µL binding buffer and 10µL magnetic beads. The samples were incubated in a mixing machine (~ 265 rpm) for 1 hour at room temperature, then washed twice with 200µL of wash buffers 1 and 2. Magnetic beads were dried at 55°C for 10 minutes, eluted in 63µL of elution buffer and stored at -20°C until further use. Quantification of pneumococcal DNA by multiplex qPCR in NW, OPS, and saliva pellet samples We used a multiplex qPCR targeting the lytA 28 and cpsA -6A/B 29 genes as previously described 30 . The reaction mixture of 25µL contained 0.6µM of each lytA primer, 0.3µM of lytA probe, 0.4µM of each cpsA -6A/B primer, 0.2µM of cpsA -6A/B probe, 12.5µM of Taqman Gene Expression Master Mix (Applied Biosystems, USA) and 2.5µL of extracted DNA. The qPCR reaction was run on a Mx3005P machine (Agilent Technologies, USA) on the following programme: 10 minutes at 95°C followed by 40 cycles of 15 seconds at 95°C and 1 minute at 60°C. For standard curve, Spn6B DNA was extracted using the QIAamp DNA mini kit (Qiagen, Germany) and serially diluted 1:10 from 4.14x10 6 copies in 2.5 µL. A sample was considered positive if duplicates had a CT value less than 40. Quantification and Statistical analysis Statistical analysis was performed by GraphPad Prism version 5.0. Contingency tables were used to assess the differences in the pneumococcal frequency in raw and culture-enriched extracted samples in both niches in each age group and in the colonisation frequency between the two niches in each age group or between the two age groups on all study days. The association was tested using Fisher’s exact test and considered significant if P < 0.05 (two-sided). Unpaired t-tests were used to compare colonisation densities in experimentally colonised participants, calculated from cpsA-6A/B gene copies in raw samples, between niches and age groups. Samples that were negative in the raw sample but positive in the corresponding culture-enriched sample were imputed a density of 1 copy/ml. A Generalized Linear Model (GLM) with binomial distribution was also used to explore the relationship between age and pneumococcal frequency in both niches. Results Culture-enrichment increased pneumococcal detection in all sample types in both age groups. To detect pneumococcal presence, DNA was extracted from both raw and culture-enriched samples. Samples positive for both lytA and cpsA-6A/B genes were defined as Spn6B+, whereas those positive only for the lytA gene as lytA+ . For young adults (n = 57, Supplementary Table S1), 147 NW samples (57 D2, 57 D7 and 33 D14) and 146 OPS samples (57 D2, 57 D7, 32 D14) were assessed. 94/147 (64%) NW samples were Spn6B+:13 only when analysed raw, 20 only when culture-enriched, and 61 by both methods (Supplementary Table S2A: P = 0.25 Fisher’s test). 79/146 (54%) OPS samples were Spn6B+: 45 only when culture-enriched and 34 by both methods (Supplementary Table S2B: P < 0.0001 Fisher’s test). Culture-enrichment increased the detection of SPN6B + samples in both niches with significant difference in OPS samples. For older adults (n = 55, Supplementary Table S1), 163 NW samples (55 D2, 55 D7 and 53 D14), 163 OPS samples (55 D2, 55 D7 and 53 D14) and 161 saliva samples (55 D2, 54 D7, 52 D14) were assessed. 57/163 (35%) NW samples were Spn6B+: 3 only when analysed raw, 20 only when culture-enriched, and 34 by both methods (Supplementary Table S3A; P = 0.0001 Fisher’s test). 39/163 (24%) OPS samples were Spn6B+: 1 only when analysed raw, 25 only when culture-enriched, and 13 by both methods (Supplementary Table S3B: P < 0.0001 Fisher’s test). 9/161 (6%) saliva samples were Spn6B+: 5 only when culture-enriched and 4 by both methods (Supplementary Table S3C: P = 0.0294 Fisher’s test). Culture-enrichment significantly increased the detection of Spn6B + samples in all three niches. There was greater additional benefit in culture-enriching oropharyngeal samples (OPS and saliva) than NW in both age groups. In young adults, 21% (20/94) and 57% (45/79) of Spn6B + samples were detected by culture-enrichment only in NW and OPS respectively, indicating that culture-enrichment increased pneumococcal detection 2.7 times more in OPS samples than NW. In older adults, 35% (20/57), 64% (25/39) and 56% (5/9) of Spn6B + samples were detected by culture-enrichment only in NW, OPS and saliva respectively, indicating that culture-enrichment increased pneumococcal detection 1.1–1.8 times more in oropharyngeal (OPS and saliva) samples than in NW. Moreover, in case of the oropharyngeal niche, pneumococcal detection rates in OPS were 1.6 times higher than in saliva. Pneumococcal colonisation frequency and density with ageing in both nose and oropharynx. To investigate the kinetics of experimental colonisation in both niches, we assessed the colonisation frequency and density of pneumococcal DNA in both age groups on days 2, 7 and 14 post pneumococcal exposure as shown in Fig. 1 . For D14, only data from culture-positive participants in the older adults study was analysed to ensure comparability with the young vaccine study data. Pneumococcal colonisation frequency was significantly higher in young than older adults at all study days post exposure (Fig. 1 A) in both NW (Young vs Older: Day 2 36/57 (63%) vs 22/55 (40%), P = 0.014, Day 7 34/57 (60%) vs 19/55 (34.5%), P = 0.008 and Day 14 23/29 (79.3%) vs 11/19 (57.9%), P = 0.1931) and OPS (Young vs Older: Day 2 28/57 (49.1%) vs 10/55 (18.2%), P = 0.0007, Day 7 29/57 (50.9%) vs 14/55 (25.5%), P = 0.007 and Day 14 22/28 (78.6%) vs 11/19 (57.9%), P = 0.1946). Comparing the two niches in both age groups separately, pneumococcal frequency was higher in the nose than the oropharynx at days 2 and 7 and similar at day 14 post pneumococcal exposure in both age groups (Fig. 1 A, older adults NW 22/55 (40%) vs OPS 10/55 (18.2%) D2, P = 0.020). Pneumococcal colonisation density was higher in young adults than in older adults for OPS ( P = 0.008) but not for NW ( P = 0.30) (Fig. 1 B). Pneumococcal colonisation density was higher in NW than in OPS for older adults ( P = 0.016) but not for young adults ( P = 0.09) (Fig. 1 B). Pneumococcal presence is higher in the nose (NW) than oropharynx (OPS) within and between young and older adults. In order to compare overall pneumococcal detection rates between the nasal and oropharyngeal niches, an overall nasal (combined raw and culture-enriched NW) and oropharyngeal (combined raw and culture-enriched OPS) profile were created for each participant and plotted on a heat map as shown in Fig. 2 . When extraction from raw and CE samples yielded different results, the positive result was retained regardless of the method. Participants with qPCR-negative samples on all study days were defined as negative (shown in white). Those with a Spn6B + sample on any study day were classified as experimentally colonised (shown in black). Participants with a lytA + but cpsA-6A/B - sample on any study day were classified as colonised with a lytA -carrying streptococcus (shown in grey). Participants with Spn6B + samples and lytA + ( cpsA-6A/B -) samples on different study days were classified as co-colonised (shown with hatched shading). The nose of 41/57 (72%) young adults was colonised with Spn6B (Fig. 2 A). Using NW, 12/57 (21%) participants were negative, 37/57 (65%) experimentally colonised, 4/57 (7%) colonised with a lytA -carrying streptococcus and 4/57 (7%) co-colonised. The oropharynx of 36/57 (63%) young adults was colonised with Spn6B (Fig. 2 A). Using OPS, 16/57 (28%) participants were negative, 29/57 (51%) experimentally colonised, 5/57 (9%) colonised with a lytA -carrying streptococcus and 7/57 (12%) co-colonised. The carriage rate in older adults was lower than in younger adults. The nose of 28/55 (51%) older adults was colonised with Spn6B (Fig. 2 B). Using NW, 22/55 (40%) participants were negative, 24/55 (44%) experimentally colonised, 5/55, (9%) colonised with a lytA -carrying streptococcus and 4/55 (7%) co-colonised. The oropharynx of 20/55 (36%) older adults, as assessed by OPS, was colonised with Spn6B (Fig. 2 B). Using OPS, 22/55 (40%) participants were negative, 19/55 (35%) experimentally colonised, 12/55 (24%) colonised with a lytA -carrying streptococcus and 1/55 (2%) co-colonised. Because there were more participants colonised with lytA -carrying streptococci in their oropharynx in the older cohort, we analysed these samples by microarray. In all 12 cases, non-pneumococcal streptococci were identified (data not shown). Using combined raw and culture-enrichment methods, higher pneumococcal presence was detected in the nose than the oropharynx in both age groups with statistical significance in older adults (Supplementary Table S4, P = 0.016). Pneumococcal presence was significantly different between young and older adults in both NW (Supplementary Table S4, P = 0.026) and OPS (Supplementary Table S4, P = 0.004). OPS is more sensitive than saliva for pneumococcal detection in older adults. Both OPS (described above, combined raw and culture-enriched OPS) and saliva (combined raw and culture-enriched saliva) samples were used to assess pneumococcal colonisation in the oropharynx of older adults. In saliva, 36/55 (65%) participants were negative, 5/55 (9%) experimentally colonised, 14/55 (25%) colonised with a lytA -carrying streptococcus and no co-colonised were detected. Therefore, overall, the oropharynx of 20/55 (36%) older adults, as assessed by both OPS and saliva, were colonised with Spn6B (Fig. 2 B). Only 5 of these (25%) were detected in saliva compared to 19 (95%) in OPS, (Fig. 2 B), indicating that in the older age group, saliva is a less sensitive method of assessing pneumococcal colonisation than OPS. Discussion This study investigated whether the pneumococcal colonisation niche alters with increasing age following experimental human challenge. Our findings show that regardless of age, the nasal niche had the highest percentage of experimentally colonised participants. Experimental colonisation involves the direct inoculation of pneumococcus into the nose and therefore may not precisely imitate natural colonisation dynamics. Nevertheless, we have described in a series of independent studies that participants who become colonised following inoculation develop a consistent colonisation episode of 1–3 weeks of similar density to natural colonisation 24 , 31 . Our findings agree with studies of natural colonisation where a higher incidence of pneumococcal growth is found in individuals’ nasopharyngeal samples compared with their oropharyngeal samples 15 , 32 . Young adults showed a higher percentage of colonised participants than their older counterparts at each study day following inoculation, in both the nose and the oropharynx. The relationship between age and prevalence of colonisation has been well documented 33 – 35 , in agreement with our findings that prevalence of colonisation decreases with age. This decrease did not reach statistical significance when samples were analysed using classical microbiology methods ( P = 0.19), in keeping with the increased sensitivity of molecular methods. Culture-enrichment of samples increased pneumococcal detection in both niches in both age groups. This extra step has been shown previously to increase pneumococcal detection in saliva 20 , 36 and our group now uses it routinely when analysing clinical trial samples. Although labour-intensive, we believe that its added value justifies recommending it as standard practice in combination with analysing raw samples 37 . The strength of our study is the collection of paired longitudinal nasal and oropharyngeal samples before and after pneumococcal inoculation of a known strain. Coupled with culture enrichment and molecular methods for capsular polysaccharide-specific detection, this allows the precise determination of the frequency and density of bacteria for each study day and niche, according to age. A weakness of our work is the lack of saliva in the young cohort and that collection of samples in the older and young cohorts was conducted during different studies. However, the strain used for inoculation as well as the methods for inoculation, nasal wash collection and processing remained identical, allowing for direct comparison of cohorts as done previously 23 . A further limitation could extend to the methods used for nasal sampling and saliva collection. Nasal wash is more comfortable and more sensitive than nasopharyngeal swab for pneumococcal detection in adults 9 , however it is not always feasible outside of a clinic setting. It may be that we would not have seen such a difference between the nose and oropharynx if we had used nasopharyngeal swabs. Our saliva detection levels were also much lower than those reported elsewhere when a spitting method was used for sample collection 20 , 37 . In older adults, with drier mouths, low sample volumes could be obscured by the use of the Salivette device. Our results indicate age-related host factors could affect colonisation prevalence in these age groups. The percentage of participants showing experimental carriage fell between days 2 and day 14 in the nose of older age group, as assessed by NW. This could be evidence of pneumococcal colonisation clearance, which is influenced by host immune responses such as local phagocytic function and acute mucosal inflammatory responses 37 as well as mechanisms known to be affected by immunosenescence (toll like receptors and reduction in the function of host signalling pathways) 38 . Unlike pneumococcal colonisation prevalence, pneumococcal density in NW was unaffected by sampling age. Several studies have found an opposite relationship between nasopharyngeal colonisation density and age indicating higher pneumococcal densities in younger subjects 39 – 41 . It has been established that the complexity and diversity of the microbiome increases from the nasopharynx to the oropharynx to saliva 42 . Investigation of NW, OPS and saliva samples in this study showed a higher percentage of participants colonised with a lytA -carrying streptococcus in OPS (and saliva in older adults) in contrast to NW in both age groups. Due to higher levels of species diversity within the oral cavity, and the capacity of pneumococci to exchange genes with other streptococci, the use of lytA as a PCR target needs to be treated with caution. Positive lytA PCR results may indicate the presence of non-pneumococcal species in addition to pneumococcal species, leading to false positives, which was indeed the case here. In addition, in our experience the addition of saliva sampling in older adults was not beneficial as it is less sensitive when compared with OPS. In summary, this study has shown that the optimal sampling niche to detect experimental pneumococcal colonisation is the nose regardless of age, as assessed by NW. However, individuals show different colonised niches, so reducing sampling to only the nose would exclude detection of pneumococcal colonisation in some patients (both young and older adults). Future studies could investigate the site of pneumococcal colonisation over a longer time-period following experimental inoculation. The current study examined a short period of time, and studies in the literature of natural carriage are snapshots in time. Declarations Funding This work was supported by the Medical Research Council/FAPESP (grant number MR/M011569/1) and the Bill & Melinda Gates Foundation (grant number OPP1117728). Acknowledgements We would like to thank all participants for their participation. Author contributions JR, AMC, HA, SBG and DMF designed and supervised the two clinical trials; EN, ELG, SP, EM and JFG processed clinical trial samples; EN, ELG, CSG and EM designed and optimised the multiplex qPCR; EN, ELG, AB, AH, LH, SS, FD and JFG performed DNA extractions and qPCRs; JH and KAG performed microarray analysis; EN, ELG and JC performed data analysis; TC performed statistical analysis; EN and ELG prepared the manuscript text, tables and figures; All authors approved the manuscript. Data availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Additional Information The authors declare no competing interests. References Costello, E. K., Stagaman, K., Dethlefsen, L., Bohannan, B. J. M. & Relman, D. A. The application of ecological theory toward an understanding of the human microbiome. Science. 336 , 1255–1262 https://doi.org/10.1126/science.1224203 (2012). Hales, C. et al. 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The Journal of infection. 81 , 540–548 https://doi.org/10.1016/j.jinf.2020.06.028 (2020). Krone, C. L. et al. Carriage of Streptococcus pneumoniae in Aged Adults with Influenza-Like-Illness. PLOS ONE. 10 , e0119875 https://doi.org/10.1371/journal.pone.0119875 (2015). van Deursen, A. M., van den Bergh, M. R. & Sanders, E. A. Carriage of Streptococcus pneumoniae in asymptomatic, community-dwelling elderly in the Netherlands. Vaccine. 34 , 4–6 https://doi.org/10.1016/j.vaccine.2015.11.014 (2016). Gritzfeld, J. F. et al. Experimental human pneumococcal carriage. Journal of visualized experiments: JoVE. https://doi.org/10.3791/50115 (2013). Adler, H. et al. Experimental Human Pneumococcal Colonisation in Older Adults is Feasible and Safe, Not Immunogenic. Am J Respir Crit Care Med. https://doi.org/10.1164/rccm.202004-1483OC (2020). Ferreira, D. M. et al. Controlled human infection and rechallenge with Streptococcus pneumoniae reveals the protective efficacy of carriage in healthy adults. 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Azzari, C. et al. Realtime PCR is more sensitive than multiplex PCR for diagnosis and serotyping in children with culture negative pneumococcal invasive disease. PloS one. 5 , e9282–e9282 https://doi.org/10.1371/journal.pone.0009282 (2010). German, E. L. et al. Protective effect of PCV vaccine against experimental pneumococcal challenge in adults is primarily mediated by controlling colonisation density. Vaccine. 37 , 3953–3956 https://doi.org/10.1016/j.vaccine.2019.05.080 (2019). Gritzfeld, J. F. et al. Density and duration of experimental human pneumococcal carriage. Clinical microbiology and infection: the official publication of the European Society of Clinical Microbiology and Infectious Diseases. 20 , O1145–1151 https://doi.org/10.1111/1469-0691.12752 (2014). Lieberman, D. et al. Nasopharyngeal versus oropharyngeal sampling for isolation of potential respiratory pathogens in adults. Journal of clinical microbiology. 44 , 525–528 https://doi.org/10.1128/jcm.44.2.525-528.2006 (2006). Grant, L. R. et al. Impact of the 13-Valent Pneumococcal Conjugate Vaccine on Pneumococcal Carriage Among American Indians. Pediatr Infect Dis J. 35 , 907–914 https://doi.org/10.1097/INF.0000000000001207 (2016). Mackenzie, G. A., Leach, A. J., Carapetis, J. R., Fisher, J. & Morris, P. S. Epidemiology of nasopharyngeal carriage of respiratory bacterial pathogens in children and adults: cross-sectional surveys in a population with high rates of pneumococcal disease. BMC Infectious Diseases. 10 , 304 https://doi.org/10.1186/1471-2334-10-304 (2010). Scott, J. R. et al. Impact of more than a decade of pneumococcal conjugate vaccine use on carriage and invasive potential in Native American communities. The Journal of infectious diseases. 205 , 280–288 https://doi.org/10.1093/infdis/jir730 (2012). Wyllie, A. L. et al. Molecular surveillance on Streptococcus pneumoniae carriage in non-elderly adults; little evidence for pneumococcal circulation independent from the reservoir in children. Sci Rep. 6 , 34888 https://doi.org/10.1038/srep34888 (2016). Nikolaou, E. et al. Experimental Human Challenge Defines Distinct Pneumococcal Kinetic Profiles and Mucosal Responses between Colonized and Non-Colonized Adults. mBio 12 , doi: 10.1128/mBio.02020-20 (2021). Krone, C. L., van de Groep, K., Trzciński, K., Sanders, E. A. & Bogaert, D. Immunosenescence and pneumococcal disease: an imbalance in host-pathogen interactions. The Lancet. Respiratory medicine. 2 , 141–153 https://doi.org/10.1016/s2213-2600(13)70165-6 (2014). Roca, A. et al. Effect of age and vaccination with a pneumococcal conjugate vaccine on the density of pneumococcal nasopharyngeal carriage. Clin Infect Dis. 55 , 816–824 https://doi.org/10.1093/cid/cis554 (2012). Trzciński, K. et al. Superiority of trans-oral over trans-nasal sampling in detecting Streptococcus pneumoniae colonization in adults. PloS one. 8 , e60520–e60520 https://doi.org/10.1371/journal.pone.0060520 (2013). Sutcliffe, C. G. et al. Association of Laboratory Methods, Colonization Density, and Age With Detection of Streptococcus pneumoniae in the Nasopharynx. American journal of epidemiology. 188 , 2110–2119 https://doi.org/10.1093/aje/kwz191 (2019). Tavares, D. A. et al. Identification of Streptococcus pneumoniae by a real-time PCR assay targeting SP2020. Sci Rep. 9 , 3285 https://doi.org/10.1038/s41598-019-39791-1 (2019). Additional Declarations No competing interests reported. Supplementary Files SupplementaryTables.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revision 05 Jul, 2021 Reviews received at journal 23 Jun, 2021 Reviews received at journal 04 Jun, 2021 Reviewers agreed at journal 25 May, 2021 Reviewers agreed at journal 20 May, 2021 Reviewers invited by journal 12 May, 2021 Editor assigned by journal 12 May, 2021 Editor invited by journal 12 May, 2021 Submission checks completed at journal 12 May, 2021 First submitted to journal 10 May, 2021 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-513376","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":27731634,"identity":"1e05b703-e6c1-4fcf-9d10-a1119af58ae7","order_by":0,"name":"Elissavet Nikolaou","email":"data:image/png;base64,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","orcid":"","institution":"Liverpool School of Tropical Medicine","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Elissavet","middleName":"","lastName":"Nikolaou","suffix":""},{"id":27731635,"identity":"42e99f91-5d33-4803-af21-1ac2ff412f4e","order_by":1,"name":"Esther Lauryn 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London","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jason","middleName":"","lastName":"Hinds","suffix":""},{"id":27731652,"identity":"fab3a9eb-62cd-4253-a16f-64af6af251c0","order_by":15,"name":"Katherine A Gould","email":"","orcid":"","institution":"St George's, University of London","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Katherine","middleName":"A","lastName":"Gould","suffix":""},{"id":27731654,"identity":"86dff07e-63ea-4716-b656-ddfbaeba2236","order_by":16,"name":"Jamie Rylance","email":"","orcid":"","institution":"Liverpool School of Tropical Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jamie","middleName":"","lastName":"Rylance","suffix":""},{"id":27731656,"identity":"ef175789-2437-4b73-bc2a-13e48673a4d6","order_by":17,"name":"Andrea M Collins","email":"","orcid":"","institution":"Liverpool School of Tropical Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Andrea","middleName":"M","lastName":"Collins","suffix":""},{"id":27731657,"identity":"40546ff3-3fd6-4a3a-8abb-77065cdd93cb","order_by":18,"name":"Stephen B Gordon","email":"","orcid":"","institution":"Queen Elizabeth Central Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Stephen","middleName":"B","lastName":"Gordon","suffix":""},{"id":27731658,"identity":"e697ff39-e0e5-4d89-8e2d-08f00d59aba4","order_by":19,"name":"Daniela M Ferreira","email":"","orcid":"","institution":"Liverpool School of Tropical Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Daniela","middleName":"M","lastName":"Ferreira","suffix":""}],"badges":[],"createdAt":"2021-05-10 14:59:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-513376/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-513376/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":9356698,"identity":"4b59aa9b-615b-492b-8b61-1b1782f9fab0","added_by":"auto","created_at":"2021-05-19 19:42:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":22570,"visible":true,"origin":"","legend":"Experimental pneumococcal colonization in young (18-55yrs, n=57) and older adults (\u003e55yrs, n=55). A. Frequency of Spn6B pneumococcus in nose (NW) and oropharynx (OPS) in both age groups. Detection of pneumococcal DNA was determined by multiplex qPCR. Participants with qPCR results positive for Spn6B (CT \u003c40) in the nose and oropharynx per day post-exposure per age group were: Young NW: D2 n=36/57, D7 n=34/57, D14 n=23/29, Young OPS: D2 n=28/57, D7 n=29/57, D14 n=22/28, Older NW: D2 n=22/55, D7 n=19/55, D14 n=11/19, Older OPS: D2 n=10/55, D7 n=14/55, D14 n=11/19. The number of volunteers with Spn6B+ sample (CT \u003c40) in each day post-exposure is expressed as a percentage (%) of the total number of volunteers. Statistical significance was assessed by Fisher’s contingency test: Young vs Older adults: NW D2 P=0.014, D7 P=0.008, D14 P=0.1931 and OPS D2 P=0.0007, D7 P=0.007, D14 P=0.1946, Older adults NW vs OPS: D2 *P=0.020. B. Density of Spn6B pneumococcus in nose (NW) and oropharynx (OPS) in both age groups. Each time point represents the average density of SPN6B+ per study day per niche. Data was log transformed. ","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-513376/v1/1d7a9f4a1f4fea77ceb8a340.png"},{"id":9356699,"identity":"6b46d4e1-e3c9-471d-86e5-4edf39143223","added_by":"auto","created_at":"2021-05-19 19:42:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":53819,"visible":true,"origin":"","legend":"Heatmap showing individual nasal and oropharyngeal profiles in A. young (18-55yrs, n=57) and B. older (\u003e55yrs, n=55) adults. Participants are colour coded; white squares qPCR-negative, black squares Spn6B+, grey squares lytA+ and hatched squares co-colonisers. Data is broken down by age group and gender. More Spn6B+ participants were detected in nose (NW) than oropharynx (OPS) in both age groups (Young: NW n=41 vs OPS n=36, Older: NW n=28 vs OPS n=20). Pneumococcal presence in both NW (Young n=41 vs Older n=28, *P=0.026) and OPS (Young n=36 vs Older n=20, **P=0.008) was statistically significantly different between young and older adults (GLM model). In older adults, fewer Spn6B+ participants were detected in saliva (n=5).","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-513376/v1/431a2cc1623369a38d513db1.png"},{"id":13693771,"identity":"da3a9b64-6110-4b45-91f3-a4d1dc9cacd5","added_by":"auto","created_at":"2021-09-17 12:49:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":562552,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-513376/v1/5581e0e9-5902-4a25-82aa-79daf516a4e6.pdf"},{"id":9356873,"identity":"e406aef0-0a09-4367-8ec3-8db5c3824786","added_by":"auto","created_at":"2021-05-19 19:45:59","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":24760,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTables.docx","url":"https://assets-eu.researchsquare.com/files/rs-513376/v1/611a9378b8a11d1d89465691.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The nose is the best niche for detection of pneumococcal colonisation following experimental challenge in adults of all ages","fulltext":[{"header":"Introduction","content":" \u003cp\u003eThe natural flora of the human upper respiratory tract (URT) is in constant interaction with the external environment, resulting in a diverse ecology of microorganisms colonising epithelial surfaces in the oral and nasal cavities and oropharynx \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e (Spn, pneumococcus) can proliferate and establish colonisation as a non-pathogenic symbiont, which is asymptomatic for the host, particularly in adults \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Pneumococcus can however migrate to other organs, resulting in pneumonia, meningitis, and bacteraemia, causing significant morbidity and mortality worldwide, especially in the very young and very old.\u003c/p\u003e \u003cp\u003eColonisation of the URT has been shown to be the source of, and pre-requisite for, pneumococcal disease \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e and its transmission throughout the community \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Accurate detection of pneumococcal colonisation is crucial to assessing disease potential, as well as direct and indirect impact of vaccines. The World Health Organisation (WHO) recommends the use of nasopharyngeal swabs for pneumococcal colonisation detection in children and both nasopharyngeal and oropharyngeal swabs (OPS) in adults \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. WHO recommendations were based on culture-based methods and more recent studies using sensitive molecular methods have encouraged other sampling methods such as saliva or nasal wash \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, due to improved comfort and pneumococcal detection.\u003c/p\u003e \u003cp\u003eMany studies have investigated pneumococcal colonisation prevalence in the first few years of life, showing that the nasopharynx of 40\u0026ndash;95% of young children \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e is naturally colonised with pneumococcus. With the onset of adulthood, falling pneumococcal disease rates are accompanied by decreased nasopharyngeal colonisation rates at 10\u0026ndash;25% \u003csup\u003e12\u0026ndash;15\u003c/sup\u003e. In older adults, a population at increased risk of pneumococcal disease, colonisation prevalence and density are reported to be lower (19), but study results are heterogeneous with some reporting very low colonisation rates of 1.9\u0026ndash;4.2% \u003csup\u003e16\u0026ndash;18\u003c/sup\u003e. Our recent systematic review analysed 29 studies, 18 with participant-level data (representing 6290 participants), and reported prevalence of detected pneumococcal colonisation of 0\u0026ndash;39% by conventional culture methods and 3\u0026ndash;23% by molecular methods \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eVarious theories have been put forward to explain the paradox of increased disease risk with decreased colonisation prevalence, including a more transient colonisation dynamic in older adults \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, the poor sensitivity of conventional culture methods to detect low density colonisation in polymicrobial samples \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e and a transition of the pneumococcal niche from the nasopharynx to the oral cavity with ageing \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe Experimental Human Pneumococcal Colonisation (EHPC) model is a safe, reproducible, and controlled method of studying colonisation dynamics in adult human participants, as the challenge dose and time of exposure is known \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. We have recently expanded this model to older adults and reported that\u003c/p\u003e \u003cp\u003eexperimental colonisation was established in 39% of participants (25/64) with no adverse events. Colonisation occurred in 47% (9/19) of participants aged 50\u0026ndash;59 and in 42% (13/31) of those aged 60\u0026ndash;69 compared to 21% (3/14) in those aged\u0026thinsp;\u0026ge;\u0026thinsp;70 years. Colonisation density was similar between old and younger adults \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe used 780 nasal, oropharyngeal and saliva samples to define precisely whether the niche of experimental pneumococcal colonisation changes in adults with increasing age. Association with bacterial density was also analysed.\u003c/p\u003e "},{"header":"Materials And Methods","content":" \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eClinical trial design, participant cohorts and sample analysis\u003c/h2\u003e \u003cp\u003eThe methodology and inclusion/exclusion criteria for EHPC studies have been previously described \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Briefly, participants were healthy adults aged\u0026thinsp;\u0026ge;\u0026thinsp;18 years with no major risk factors for pneumococcal disease, colonisation, or transmission (such as: cigarette smoking; close contact with children aged\u0026thinsp;\u0026lt;\u0026thinsp;5 years; healthcare work or caring responsibilities; steroid therapy and respiratory or immunosuppressive comorbidities). Studies were conducted in accordance with the Declaration of Helsinki and Good Clinical Practice procedures. All participants provided written informed consent and underwent a safety screening. Studies were submitted to and approved by a local NHS Research Ethics Committee. In this study, samples have been sourced from two separate EHPC clinical studies. The young vaccine study was a double-blind, randomised controlled trial conducted between September 2013 - April 2014 investigating the impact of PCV-13 vaccination on experimental pneumococcal colonisation in adults aged 18\u0026ndash;50 (NHS REC number 12/NW/0873, ISRCTN number 45340436 registered 18/11/2013) \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. In the present analysis, we include participants from the control arm only (vaccinated with Hepatitis A vaccine rather than PCV-13). The older adults study was an observational study of the effect of age on experimental pneumococcal colonisation in adults aged 50\u0026ndash;80 conducted between June 2016 - February 2018 (NHS REC number 16/NW/0031, ISRCTN number 10948363 registered 08/11/2016) \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. In total, 112 participants were analysed. The sample sets can be combined because the inoculation dose and strain, as well as the methods for inoculation, nasal wash collection and processing remained identical. Furthermore, pneumococcal colonisation rates and serotype distribution in healthy adults in Liverpool appears to be relatively stable, based on data collected between 2010\u0026ndash;2017 \u003csup\u003e26\u003c/sup\u003e. The low prevalence of naturally occurring Spn6 provides reassurance that detected Spn6B is the inoculated strain. Using the full sample set, participants were split into young 18\u0026ndash;55 years (n\u0026thinsp;=\u0026thinsp;57) and older adults\u0026thinsp;\u0026gt;\u0026thinsp;55 years (n\u0026thinsp;=\u0026thinsp;55), in keeping with the cut-off used by Marrie et al. \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn both studies, participants were inoculated with 80,000 colony-forming units (CFU) per nostril of live Spn6B pneumococcus (BHN418, GenBank accession number ASHP00000000.1) \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Nasal wash (NW) samples were collected before inoculation to screen for pneumococcal colonisation acquired from community. Depending on the study, NW, oropharyngeal swabs (OPS) and saliva samples were then collected post pneumococcal exposure. In the young vaccine study, NW and OPS samples were collected on days 2, 7, 14 (only culture-positives) and 21 post exposure. In the older adults study, NW, OPS and saliva samples were collected on days 2, 7, 9, 14, 22 (only culture-positives) and 29 post exposure. Pneumococcal colonisation status in both studies was determined by NW culture (not discussed here) and by multiplex real-time polymerase chain reaction (qPCR) targeting the \u003cem\u003elytA\u003c/em\u003e and \u003cem\u003ecpsA\u003c/em\u003e-6A/B genes in raw and culture-enriched NW, OPS, and saliva samples. The raw and culture-enriched NW, OPS, and saliva qPCR data from days 2, 7 and 14 (covered in both studies) were included in this study.\u003c/p\u003e \u003cp\u003eNW collection was performed as previously described \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Briefly, 20mL of 0.9% sodium chloride solution in total (10mL saline per nostril) was introduced using a syringe and held for a few seconds in the participant\u0026rsquo;s nose before being expelled into a sterile container. NW was centrifuged at 4000rpm for 10 minutes. Supernatant was collected and pellet was resuspended in STGG (Skimmed milk-Tryptone-Glucose-Glycerine) 20% glycerol before storage at -80\u0026deg;C. OPS samples were collected in 1 mL STGG and stored at -80\u0026deg;C, until further use. Saliva samples were collected using the salivette device (Sarstedt, UK). Following collection, each salivette was centrifuged at 4000rpm for 3minutes at 4\u003csup\u003eo\u003c/sup\u003eC and after measuring the volume of saliva liquid retrieved, both pellet and supernatant were re-suspended in equal volume of STGG 50% glycerol and stored at -80\u0026deg;C, until further use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of raw and culture-enriched NW, OPS, and saliva samples\u003c/h2\u003e \u003cp\u003eBefore DNA extraction, samples were thawed for 30 minutes at room temperature and vigorously vortexed for 20 seconds. 300\u0026micro;L raw NW pellet, 200\u0026micro;L raw OPS and 300\u0026micro;L raw saliva aliquots were prepared. For culture-enrichment, 50\u0026micro;L of NW pellet, OPS or saliva samples were plated on Columbia blood agar supplemented with 5% horse blood and 80\u0026micro;L gentamicin 1mg/mL. Plates were incubated overnight at 37\u0026deg;C in 5%CO\u003csub\u003e2\u003c/sub\u003e. Following incubation, 2mL of STGG 20% glycerol was added onto each plate and microbial growth scraped off. 300\u0026micro;L culture-enriched NW, 200\u0026micro;L culture-enriched OPS and 300\u0026micro;L culture-enriched saliva aliquots were prepared. Both raw and culture-enriched NW, OPS and saliva aliquots were stored at -20\u003csup\u003eo\u003c/sup\u003eC until DNA extraction took place.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eDNA extraction on raw and culture-enriched NW, OPS, and saliva samples\u003c/h2\u003e \u003cp\u003eBacterial genomic DNA was extracted from raw and culture-enriched NW, OPS, and saliva samples. On the day of the extraction, prepared aliquots were thawed at room temperature and vigorously vortexed for 20 seconds. Samples were pelleted at 20,238xg for 10 minutes. Following centrifugation, 300\u0026micro;L of lysis buffer with protease (Agowa Mag mini-DNA extraction kit; LGC Genomics, Germany), 100\u0026micro;L of zirconium beads (diameter of 0.1 mm), and 300\u0026micro;L of phenol pH 8.0 (toxic, performed in a cabinet with charcoal filter) were added to the pellets. Samples were mechanically disrupted at 50 Hz for 3 minutes in a tissue homogenizer followed by 3 minutes on ice, twice. The samples were then centrifuged for 10 minutes at 9,391xg, and the upper aqueous phase was transferred to a sterile 1.5mL Eppendorf tube pre-filled with 600\u0026micro;L binding buffer and 10\u0026micro;L magnetic beads. The samples were incubated in a mixing machine (~\u0026thinsp;265 rpm) for 1 hour at room temperature, then washed twice with 200\u0026micro;L of wash buffers 1 and 2. Magnetic beads were dried at 55\u0026deg;C for 10 minutes, eluted in 63\u0026micro;L of elution buffer and stored at -20\u0026deg;C until further use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eQuantification of pneumococcal DNA by multiplex qPCR in NW, OPS, and saliva pellet samples\u003c/h2\u003e \u003cp\u003eWe used a multiplex qPCR targeting the \u003cem\u003elytA\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e and \u003cem\u003ecpsA\u003c/em\u003e-6A/B \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e genes as previously described \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. The reaction mixture of 25\u0026micro;L contained 0.6\u0026micro;M of each \u003cem\u003elytA\u003c/em\u003e primer, 0.3\u0026micro;M of \u003cem\u003elytA\u003c/em\u003e probe, 0.4\u0026micro;M of each \u003cem\u003ecpsA\u003c/em\u003e-6A/B primer, 0.2\u0026micro;M of \u003cem\u003ecpsA\u003c/em\u003e-6A/B probe, 12.5\u0026micro;M of Taqman Gene Expression Master Mix (Applied Biosystems, USA) and 2.5\u0026micro;L of extracted DNA. The qPCR reaction was run on a Mx3005P machine (Agilent Technologies, USA) on the following programme: 10 minutes at 95\u0026deg;C followed by 40 cycles of 15 seconds at 95\u0026deg;C and 1 minute at 60\u0026deg;C. For standard curve, Spn6B DNA was extracted using the QIAamp DNA mini kit (Qiagen, Germany) and serially diluted 1:10 from 4.14x10\u003csup\u003e6\u003c/sup\u003e copies in 2.5 \u0026micro;L. A sample was considered positive if duplicates had a CT value less than 40.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eQuantification and Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed by GraphPad Prism version 5.0. Contingency tables were used to assess the differences in the pneumococcal frequency in raw and culture-enriched extracted samples in both niches in each age group and in the colonisation frequency between the two niches in each age group or between the two age groups on all study days. The association was tested using Fisher\u0026rsquo;s exact test and considered significant if \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (two-sided). Unpaired t-tests were used to compare colonisation densities in experimentally colonised participants, calculated from \u003cem\u003ecpsA-6A/B\u003c/em\u003e gene copies in raw samples, between niches and age groups. Samples that were negative in the raw sample but positive in the corresponding culture-enriched sample were imputed a density of 1 copy/ml. A Generalized Linear Model (GLM) with binomial distribution was also used to explore the relationship between age and pneumococcal frequency in both niches.\u003c/p\u003e \u003c/div\u003e "},{"header":"Results","content":"\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003eCulture-enrichment increased pneumococcal detection in all sample types in both age groups.\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eTo detect pneumococcal presence, DNA was extracted from both raw and culture-enriched samples. Samples positive for both \u003cem\u003elytA\u003c/em\u003e and \u003cem\u003ecpsA-6A/B\u003c/em\u003e genes were defined as Spn6B+, whereas those positive only for the \u003cem\u003elytA\u003c/em\u003e gene as \u003cem\u003elytA+\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eFor young adults (n\u0026thinsp;=\u0026thinsp;57, Supplementary Table S1), 147 NW samples (57 D2, 57 D7 and 33 D14) and 146 OPS samples (57 D2, 57 D7, 32 D14) were assessed. 94/147 (64%) NW samples were Spn6B+:13 only when analysed raw, 20 only when culture-enriched, and 61 by both methods (Supplementary Table S2A: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.25 Fisher\u0026rsquo;s test). 79/146 (54%) OPS samples were Spn6B+: 45 only when culture-enriched and 34 by both methods (Supplementary Table S2B: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 Fisher\u0026rsquo;s test). Culture-enrichment increased the detection of SPN6B\u0026thinsp;+\u0026thinsp;samples in both niches with significant difference in OPS samples.\u003c/p\u003e\n\u003cp\u003eFor older adults (n\u0026thinsp;=\u0026thinsp;55, Supplementary Table S1), 163 NW samples (55 D2, 55 D7 and 53 D14), 163 OPS samples (55 D2, 55 D7 and 53 D14) and 161 saliva samples (55 D2, 54 D7, 52 D14) were assessed. 57/163 (35%) NW samples were Spn6B+: 3 only when analysed raw, 20 only when culture-enriched, and 34 by both methods (Supplementary Table S3A; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0001 Fisher\u0026rsquo;s test). 39/163 (24%) OPS samples were Spn6B+: 1 only when analysed raw, 25 only when culture-enriched, and 13 by both methods (Supplementary Table S3B: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 Fisher\u0026rsquo;s test). 9/161 (6%) saliva samples were Spn6B+: 5 only when culture-enriched and 4 by both methods (Supplementary Table S3C: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0294 Fisher\u0026rsquo;s test). Culture-enrichment significantly increased the detection of Spn6B\u0026thinsp;+\u0026thinsp;samples in all three niches.\u003c/p\u003e\n\u003cp\u003eThere was greater additional benefit in culture-enriching oropharyngeal samples (OPS and saliva) than NW in both age groups. In young adults, 21% (20/94) and 57% (45/79) of Spn6B\u0026thinsp;+\u0026thinsp;samples were detected by culture-enrichment only in NW and OPS respectively, indicating that culture-enrichment increased pneumococcal detection 2.7 times more in OPS samples than NW. In older adults, 35% (20/57), 64% (25/39) and 56% (5/9) of Spn6B\u0026thinsp;+\u0026thinsp;samples were detected by culture-enrichment only in NW, OPS and saliva respectively, indicating that culture-enrichment increased pneumococcal detection 1.1\u0026ndash;1.8 times more in oropharyngeal (OPS and saliva) samples than in NW. Moreover, in case of the oropharyngeal niche, pneumococcal detection rates in OPS were 1.6 times higher than in saliva.\u003c/p\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003ePneumococcal colonisation frequency and density with ageing in both nose and oropharynx.\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the kinetics of experimental colonisation in both niches, we assessed the colonisation frequency and density of pneumococcal DNA in both age groups on days 2, 7 and 14 post pneumococcal exposure as shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. For D14, only data from culture-positive participants in the older adults study was analysed to ensure comparability with the young vaccine study data. Pneumococcal colonisation frequency was significantly higher in young than older adults at all study days post exposure (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA) in both NW (Young vs Older: Day 2 36/57 (63%) vs 22/55 (40%), \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.014, Day 7 34/57 (60%) vs 19/55 (34.5%), \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.008 and Day 14 23/29 (79.3%) vs 11/19 (57.9%), \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.1931) and OPS (Young vs Older: Day 2 28/57 (49.1%) vs 10/55 (18.2%), \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0007, Day 7 29/57 (50.9%) vs 14/55 (25.5%), \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.007 and Day 14 22/28 (78.6%) vs 11/19 (57.9%), \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.1946). Comparing the two niches in both age groups separately, pneumococcal frequency was higher in the nose than the oropharynx at days 2 and 7 and similar at day 14 post pneumococcal exposure in both age groups (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA, older adults NW 22/55 (40%) vs OPS 10/55 (18.2%) D2, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.020).\u003c/p\u003e\n\u003cp\u003ePneumococcal colonisation density was higher in young adults than in older adults for OPS (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.008) but not for NW (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.30) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). Pneumococcal colonisation density was higher in NW than in OPS for older adults (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.016) but not for young adults (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.09) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003ePneumococcal presence is higher in the nose (NW) than oropharynx (OPS) within and between young and older adults.\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eIn order to compare overall pneumococcal detection rates between the nasal and oropharyngeal niches, an overall nasal (combined raw and culture-enriched NW) and oropharyngeal (combined raw and culture-enriched OPS) profile were created for each participant and plotted on a heat map as shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. When extraction from raw and CE samples yielded different results, the positive result was retained regardless of the method. Participants with qPCR-negative samples on all study days were defined as negative (shown in white). Those with a Spn6B\u0026thinsp;+\u0026thinsp;sample on any study day were classified as experimentally colonised (shown in black). Participants with a \u003cem\u003elytA\u003c/em\u003e\u0026thinsp;+\u0026thinsp;but \u003cem\u003ecpsA-6A/B\u003c/em\u003e - sample on any study day were classified as colonised with a \u003cem\u003elytA\u003c/em\u003e-carrying streptococcus (shown in grey). Participants with Spn6B\u0026thinsp;+\u0026thinsp;samples and \u003cem\u003elytA\u003c/em\u003e+ (\u003cem\u003ecpsA-6A/B -)\u003c/em\u003e samples on different study days were classified as co-colonised (shown with hatched shading).\u003c/p\u003e\n\u003cp\u003eThe nose of 41/57 (72%) young adults was colonised with Spn6B (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). Using NW, 12/57 (21%) participants were negative, 37/57 (65%) experimentally colonised, 4/57 (7%) colonised with a \u003cem\u003elytA\u003c/em\u003e-carrying streptococcus and 4/57 (7%) co-colonised. The oropharynx of 36/57 (63%) young adults was colonised with Spn6B (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). Using OPS, 16/57 (28%) participants were negative, 29/57 (51%) experimentally colonised, 5/57 (9%) colonised with a \u003cem\u003elytA\u003c/em\u003e-carrying streptococcus and 7/57 (12%) co-colonised.\u003c/p\u003e\n\u003cp\u003eThe carriage rate in older adults was lower than in younger adults. The nose of 28/55 (51%) older adults was colonised with Spn6B (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). Using NW, 22/55 (40%) participants were negative, 24/55 (44%) experimentally colonised, 5/55, (9%) colonised with a \u003cem\u003elytA\u003c/em\u003e-carrying streptococcus and 4/55 (7%) co-colonised. The oropharynx of 20/55 (36%) older adults, as assessed by OPS, was colonised with Spn6B (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). Using OPS, 22/55 (40%) participants were negative, 19/55 (35%) experimentally colonised, 12/55 (24%) colonised with a \u003cem\u003elytA\u003c/em\u003e-carrying streptococcus and 1/55 (2%) co-colonised. Because there were more participants colonised with \u003cem\u003elytA\u003c/em\u003e-carrying streptococci in their oropharynx in the older cohort, we analysed these samples by microarray. In all 12 cases, non-pneumococcal streptococci were identified (data not shown).\u003c/p\u003e\n\u003cp\u003eUsing combined raw and culture-enrichment methods, higher pneumococcal presence was detected in the nose than the oropharynx in both age groups with statistical significance in older adults (Supplementary Table S4, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.016). Pneumococcal presence was significantly different between young and older adults in both NW (Supplementary Table S4, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.026) and OPS (Supplementary Table S4, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.004).\u003c/p\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003eOPS is more sensitive than saliva for pneumococcal detection in older adults.\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eBoth OPS (described above, combined raw and culture-enriched OPS) and saliva (combined raw and culture-enriched saliva) samples were used to assess pneumococcal colonisation in the oropharynx of older adults. In saliva, 36/55 (65%) participants were negative, 5/55 (9%) experimentally colonised, 14/55 (25%) colonised with a \u003cem\u003elytA\u003c/em\u003e-carrying streptococcus and no co-colonised were detected. Therefore, overall, the oropharynx of 20/55 (36%) older adults, as assessed by both OPS and saliva, were colonised with Spn6B (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). Only 5 of these (25%) were detected in saliva compared to 19 (95%) in OPS, (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB), indicating that in the older age group, saliva is a less sensitive method of assessing pneumococcal colonisation than OPS.\u003c/p\u003e"},{"header":"Discussion","content":" \u003cp\u003eThis study investigated whether the pneumococcal colonisation niche alters with increasing age following experimental human challenge. Our findings show that regardless of age, the nasal niche had the highest percentage of experimentally colonised participants.\u003c/p\u003e \u003cp\u003eExperimental colonisation involves the direct inoculation of pneumococcus into the nose and therefore may not precisely imitate natural colonisation dynamics. Nevertheless, we have described in a series of independent studies that participants who become colonised following inoculation develop a consistent colonisation episode of 1\u0026ndash;3 weeks of similar density to natural colonisation \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Our findings agree with studies of natural colonisation where a higher incidence of pneumococcal growth is found in individuals\u0026rsquo; nasopharyngeal samples compared with their oropharyngeal samples \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eYoung adults showed a higher percentage of colonised participants than their older counterparts at each study day following inoculation, in both the nose and the oropharynx. The relationship between age and prevalence of colonisation has been well documented \u003csup\u003e\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, in agreement with our findings that prevalence of colonisation decreases with age. This decrease did not reach statistical significance when samples were analysed using classical microbiology methods (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.19), in keeping with the increased sensitivity of molecular methods.\u003c/p\u003e \u003cp\u003eCulture-enrichment of samples increased pneumococcal detection in both niches in both age groups. This extra step has been shown previously to increase pneumococcal detection in saliva \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e and our group now uses it routinely when analysing clinical trial samples. Although labour-intensive, we believe that its added value justifies recommending it as standard practice in combination with analysing raw samples \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe strength of our study is the collection of paired longitudinal nasal and oropharyngeal samples before and after pneumococcal inoculation of a known strain. Coupled with culture enrichment and molecular methods for capsular polysaccharide-specific detection, this allows the precise determination of the frequency and density of bacteria for each study day and niche, according to age. A weakness of our work is the lack of saliva in the young cohort and that collection of samples in the older and young cohorts was conducted during different studies. However, the strain used for inoculation as well as the methods for inoculation, nasal wash collection and processing remained identical, allowing for direct comparison of cohorts as done previously \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. A further limitation could extend to the methods used for nasal sampling and saliva collection. Nasal wash is more comfortable and more sensitive than nasopharyngeal swab for pneumococcal detection in adults \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, however it is not always feasible outside of a clinic setting. It may be that we would not have seen such a difference between the nose and oropharynx if we had used nasopharyngeal swabs. Our saliva detection levels were also much lower than those reported elsewhere when a spitting method was used for sample collection \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. In older adults, with drier mouths, low sample volumes could be obscured by the use of the Salivette device.\u003c/p\u003e \u003cp\u003eOur results indicate age-related host factors could affect colonisation prevalence in these age groups. The percentage of participants showing experimental carriage fell between days 2 and day 14 in the nose of older age group, as assessed by NW. This could be evidence of pneumococcal colonisation clearance, which is influenced by host immune responses such as local phagocytic function and acute mucosal inflammatory responses \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e as well as mechanisms known to be affected by immunosenescence (toll like receptors and reduction in the function of host signalling pathways) \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Unlike pneumococcal colonisation prevalence, pneumococcal density in NW was unaffected by sampling age. Several studies have found an opposite relationship between nasopharyngeal colonisation density and age indicating higher pneumococcal densities in younger subjects \u003csup\u003e\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIt has been established that the complexity and diversity of the microbiome increases from the nasopharynx to the oropharynx to saliva \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Investigation of NW, OPS and saliva samples in this study showed a higher percentage of participants colonised with a \u003cem\u003elytA\u003c/em\u003e-carrying streptococcus in OPS (and saliva in older adults) in contrast to NW in both age groups. Due to higher levels of species diversity within the oral cavity, and the capacity of pneumococci to exchange genes with other streptococci, the use of \u003cem\u003elytA\u003c/em\u003e as a PCR target needs to be treated with caution. Positive \u003cem\u003elytA\u003c/em\u003e PCR results may indicate the presence of non-pneumococcal species in addition to pneumococcal species, leading to false positives, which was indeed the case here. In addition, in our experience the addition of saliva sampling in older adults was not beneficial as it is less sensitive when compared with OPS.\u003c/p\u003e \u003cp\u003eIn summary, this study has shown that the optimal sampling niche to detect experimental pneumococcal colonisation is the nose regardless of age, as assessed by NW. However, individuals show different colonised niches, so reducing sampling to only the nose would exclude detection of pneumococcal colonisation in some patients (both young and older adults). Future studies could investigate the site of pneumococcal colonisation over a longer time-period following experimental inoculation. The current study examined a short period of time, and studies in the literature of natural carriage are snapshots in time.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Medical Research Council/FAPESP (grant number MR/M011569/1) and the Bill \u0026amp; Melinda Gates Foundation (grant number OPP1117728).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank all participants for their participation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJR, AMC, HA, SBG and DMF designed and supervised the two clinical trials; EN, ELG, SP, EM and JFG processed clinical trial samples; EN, ELG, CSG and EM designed and optimised the multiplex qPCR; EN, ELG, AB, AH, LH, SS, FD and JFG performed DNA extractions and qPCRs; JH and KAG performed microarray analysis; EN, ELG and JC performed data analysis; TC performed statistical analysis; EN and ELG prepared the manuscript text, tables and figures; All authors approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCostello, E. 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Epidemiology of nasopharyngeal carriage of respiratory bacterial pathogens in children and adults: cross-sectional surveys in a population with high rates of pneumococcal disease. \u003cem\u003eBMC Infectious Diseases.\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e, 304 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/1471-2334-10-304\u003c/span\u003e\u003c/span\u003e (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eScott, J. 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A. \u003cem\u003eet al.\u003c/em\u003e Identification of Streptococcus pneumoniae by a real-time PCR assay targeting SP2020. \u003cem\u003eSci Rep.\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e, 3285 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-019-39791-1\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"pneumococcal niche, colonisation, Experimental Human Pneumococcal Challenge model, age","lastPublishedDoi":"10.21203/rs.3.rs-513376/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-513376/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003ePrevious studies have suggested that the pneumococcal niche changes from the nose to the oropharynx with age. We use an Experimental Human Pneumococcal Challenge model to investigate pneumococcal colonisation in each anatomical niche with age.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eHealthy adults (n\u0026thinsp;=\u0026thinsp;112) were intranasally inoculated with \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e serotype 6B (Spn6B) and were categorised as young 18-55yrs (n\u0026thinsp;=\u0026thinsp;57) or older\u0026thinsp;\u0026gt;\u0026thinsp;55yrs (n\u0026thinsp;=\u0026thinsp;55). Colonisation status (frequency and density) was determined by multiplex qPCR targeting the \u003cem\u003elytA\u003c/em\u003e and \u003cem\u003ecpsA\u003c/em\u003e-6A/B genes in both raw and culture-enriched nasal wash and oropharyngeal swab samples collected at 2-, 7- and 14-days post-exposure. For older adults, raw and culture-enriched saliva samples were also assessed.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003e64% of NW samples and 54% of OPS samples were positive for Spn6B in young adults, compared to 35% of NW samples, 24% of OPS samples and 6% of saliva samples in older adults. Many colonisation events were only detected in culture-enriched samples. Experimental colonisation was detected in 72% of young adults by NW and 63% by OPS. In older adults, this was 51% by NW, 36% by OPS and 9% by saliva.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe nose is the best niche for detection of experimental pneumococcal colonisation in both young and older adults.\u003c/p\u003e","manuscriptTitle":"The nose is the best niche for detection of pneumococcal colonisation following experimental challenge in adults of all ages","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-05-19 19:42:57","doi":"10.21203/rs.3.rs-513376/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2021-07-05T16:17:08+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-06-23T20:56:35+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-06-04T17:05:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"a6c8b2bd-bb31-471c-b791-096a7f808f6f","date":"2021-05-25T11:45:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"9fdcb5f3-8052-405c-8cca-652ae919ba9d","date":"2021-05-20T15:30:52+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-05-12T12:44:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2021-05-12T12:35:42+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2021-05-12T12:22:21+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2021-05-12T11:55:52+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2021-05-10T14:53:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"20bcbda9-5016-4d01-8857-f3b04c0518f0","owner":[],"postedDate":"May 19th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":4437141,"name":"Applied \u0026 Industrial Microbiology"},{"id":4437142,"name":"Bacteriology"},{"id":4437143,"name":"Infectious Diseases"},{"id":4437144,"name":"Pathology"}],"tags":[],"updatedAt":"2021-08-10T07:29:04+00:00","versionOfRecord":[],"versionCreatedAt":"2021-05-19 19:42:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-513376","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-513376","identity":"rs-513376","version":["v1"]},"buildId":"ApUGefWb6u5IBVtyqm6d5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}