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. 1A) 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. 1A, 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. 1B). 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. 1B).
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. 2A). 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. 2A). 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. 2B). 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. 2B). 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. 2B). Only 5 of these (25%) were detected in saliva compared to 19 (95%) in OPS, (Fig. 2B), indicating that in the older age group, saliva is a less sensitive method of assessing pneumococcal colonisation than OPS.