Prevalence of human respiratory pathogens and associated mucosal cytokine levels in young children and adults: a cross-sectional observational study in the Netherlands during the winter of 2012/2013

Abstract Respiratory pathogens can cause severe disease and even death, especially in the very young and very old. Studies investigating their prevalence often focus on individuals presenting to healthcare providers with symptoms. However, the design of prevention strategies, e.g. which target groups to vaccinate, will benefit from knowledge on the prevalence of, risk factors for and host response to these pathogens in the general population. In this study, upper respiratory samples (n = 1311) were collected cross-sectionally during winter from 11- and 24-month old children, their parents, and adults ≥60 years of age that were recruited irrespective of seeking medical care. Almost all children, approximately two-thirds of parents and a quarter of older adults tested positive for at least one pathogen, often in the absence of symptoms. Viral interference was evident for the combination of rhinovirus and respiratory syncytial virus. Attending childcare facilities and having siblings associated with increased pathogen counts in children. On average, children showed increased levels of mucosal cytokines compared to parents and especially proinflammatory molecules associated with the presence of symptoms. These findings may guide further research into transmission patterns of respiratory pathogens and assist in determining the most appropriate strategies for the prediction and prevention of disease.


Introduction
Respiratory infections are a major cause of morbidity and mortality, most pr ominentl y in the v ery young and v ery old.Wher eas SARS-CoV-2 has illustrated the havoc a r espir atory pathogen can wreak during a pandemic, endemic r espir atory pathogens suc h as influenza virus, Streptococcus pneumoniae , and r espir atory syncytial virus (RSV) have continuously posed a considerable threat to public health in the past, and will likely do so in the future (PERCH Study Group 2019 , Shi et al. 2019 ).A large number of human pathogens that target the respiratory tract are known, some of whic h can r eadil y cause se v er e disease while others mostly do not (Sarna et al. 2018, PERCH Study Gr oup 2019 ).Licensed v accines ar e pr esentl y onl y av ailable for a small number of these pathogens, including influenza virus, RSV, S. pneumoniae , and Haemophilus influenzae .
For knowledge-based decision making on the implementation of pr e v entativ e measur es suc h as risk gr oup-specific v accination against endemic respiratory pathogens, a thorough understanding of their pr e v alence in the general population is needed.Howe v er, the majority of studies relating to the occurrence of respiratory pathogens focus on medically attended infections.While these studies are important for estimating the burden of disease and determining which groups are at risk for se v er e illness, they mostly do not provide information on the general presence of these pathogens in the population.Collecting such data requires active sampling of mostly healthy participants from various subpopulations and the use of broad diagnostic panels, which is not often undertaken or, when they are, often focus primarily on infants (Jansen et al. 2011, Byington et al. 2015, Moe et al. 2016, Birger et al. 2018, Sarna et al. 2018, Galanti et al. 2019, Zoch-Lesniak et al. 2020, Po w ell et al. 2022 ).
In addition to population-based pr e v ention of r espir atory infections, morbidity and mortality might be decreased by the early identification of infected individuals that ar e likel y to de v elop sev er e disease.Cytokines and chemokines are important mediators of the imm une r esponse and are therefore potential biomarkers or e v en determinants for se v er e illness (Rot and von Andrian 2004, Garcia et al. 2012, Bohmwald et al. 2019, Glaser et al. 2019 ).Proper inter pr etation of the role of these molecules in the de v elopment of se v er e disease will likely benefit fr om incr eased knowledge r egarding their baseline presence during mild or asymptomatic infections (van Woudenbergh et al. 2023 ).
In this cross-sectional study performed in the Netherlands during the winter months, we have assessed the prevalence in the up-per r espir atory m ucosa of a wide v ariety of r espir atory pathogens, both viral and bacterial, in different age groups and in the absence of serious illness.In addition, we have assessed childcare and having siblings as risk factors associated with higher pathogen counts in young c hildr en.Finall y, we determined the concentration in nasopharyngeal swab samples of a range of (proinflammatory) cytokines/chemokines and assessed their association with symptoms of r espir atory infection.

Study design
Upper r espir atory s wab samples , both nasopharyngeal and oropharyngeal (adults only), were collected as part of two crosssectional, observational cohort studies, which were performed throughout the winter of 2012/2013 in the Netherlands.For the exact timing of sampling, see Supplementary Fig. S1A .The studies were designed and run in parallel to allow for combined analysis.Study A was primarily designed to assess carriage of S. pneumoniae in c hildr en and adults in r elation to c hanges in the national imm unization pr ogr am (Bosc h et al. 2016 ).Study B was primarily designed to identify the causativ e a gents of influenza-like illness in community-dwelling older adults (van Beek et al. 2017 ).Study participants belonged to one of four groups: 11-month old children ( n = 327, study A), 24-month old children ( n = 325, study A), parents of the 24-month old c hildr en ( n = 319, study A), and older adults ≥60 years of age ( n = 340, study B).Exclusion criteria for study A were known or suspected imm unodeficiency, cr aniofacial or c hr omosomal abnormalities, and coa gulation disorders or use of anticoagulant medication.There were no exclusion criteria for study B. Ho w e v er, the pr esent anal ysis includes onl y the baseline controls of study B and therefore does not include participants presenting with fever at the time of sampling, as these would have been designated as cases according to the study protocol.Throughout the study period, trained research nurses performed scheduled home visits that consisted of a structured interview and sample collection.Each participant was sampled once during the entire study period.

Interview
By means of a structured interview during the scheduled home visit data was obtained r egarding a ge, sex, body-mass index (BMI; adults onl y), br eastfeeding history (c hildr en onl y), c hildcar e attendance (c hildr en onl y), and having siblings (c hildr en onl y).In addition, participants were asked the following question during the intervie w (tr anslated fr om Dutc h): ar e you at this moment experiencing a common cold and/or do you have complaints of a respir atory infection, suc h as otitis , laryngitis , sinusitis , br onc hitis, or pneumonia?P ar ents wer e asked to answer the questions for their child.

Sampling
Nasopharyngeal (all groups) and oropharyngeal (adults only) samples were obtained according to World Health Organization standard pr ocedur es with a sterile swab with a floc ked n ylon tip and stored in 1 ml modified liquid Amies transport medium (Eswab; Copan, Br escia, Ital y).Swab samples wer e tr ansported at room temperature to the laboratory, where the samples were aliquoted and subsequently used within 12 hours after sampling for bacterial culturing or stored at −80 • C within 8 hours after sampling for subsequent vir al dia gnostics and cytokine analysis.Sample collection and processing were performed using the same pro-tocols and by the same teams of r esearc h nurses and labor atory personnel for both studies.

Sta tistical anal ysis and visualiza tion
Comparisons between categorical data were assessed using the Fisher's exact test.Cytokine concentrations for group comparisons are presented in boxplots with median and quartiles while showing individual data points.Differences in cytokine concentrations or pathogen counts between groups were assessed using a nonparametric Kruskal-Wallis test, follo w ed b y Dunn's multiple comparisons test.Corr elations wer e assessed using the Spearman rank order method.Interaction of risk factors for numerical data was assessed using a Kruskal-Wallis test and for categorical data using a gener alized Coc hr an-Mantel-Haenszel test, wher e for both tests the P -value was evaluated by permutation test.
Statistical analysis and graph design were performed with Gr a phP ad Prism 9.3.1 software or R Statistical Software v4.3.0 and Rstudio v2023.03.1.The cytokine heatmap was generated with log10-transformed data using the pheatmap package v1.0.12 in which the Euclidean method was used to calculate distance and complete linkage clustering was performed on both rows and columns .T he cytokine correlation matrix was generated based on Spearman rank order correlation using the rcorr function in the Hmisc pac ka ge v5.1-1.

Description of study population, sampling time, and diagnostics
To assess the pr e v alence of r espir atory pathogens in the general population, a study cohort consisting of 11-and 24-month old c hildr en, their par ents, and older adults ≥60 years of a ge was formed.Participant demographics can be found in Table 1 .Eac h gr oup consisted of 319-340 participants, with a ppr oximatel y equal female to male ratios except for parents where 82% of participants was female .T he mean a ge for par ents and older adults w as 35 y ears and 74 y ears, r espectiv el y.Br eastfeeding history, c hildcar e attendance, and having siblings was comparable between 11-and 24-month old c hildr en.Of the older adults, 77% had r eceiv ed an influenza vaccination in 2012, while this was ∼7% for parents .In the Netherlands , older adults of ≥60 years and individuals with specific medical conditions are eligible for influenza vaccination within the national immunization program.Dutch children do not routinely receive influenza vaccination as part of the national immunization program.
One sampling moment was r andoml y sc heduled for eac h study participant during the winter months (October-March) of 2012/2013, for an ov ervie w see Supplementary Fig. S1(A) .P athogen dia gnostics wer e performed on upper r espir atory swab samples to determine the presence of influenza viruses, RSV, hMPV, rhino/entero virus , AdV, PIV, hBoV, KIPyV, WUPyV, seasonal hCoV, S. pneumoniae , H. influenzae , M. catarrhalis , and S. aureus .Diagnostics data concerning viruses in older adults and bacteria in c hildr en/par ents hav e been published befor e (Bosc h et al. 2016, van Beek et al. 2017 ), but are reproduced here in more detail for compar ativ e pur poses.Virus dia gnostics wer e performed on nasopharyngeal swab samples for all groups.Bacterial diagnostic culturing was performed on nasopharyngeal swab samples for c hildr en and parents, and on oropharyngeal swab samples for parents and older adults.

Children and adults show differing patterns of viral and bacterial prevalence in the upper respir a tory mucosa
We first assessed the pr e v alence of vir al pathogens per a ge gr oup, irr espectiv e of viral species (Fig. 1 A).Notably, in the 11-month old a ge gr oup onl y 33 out of 327 c hildr en (10%) wer e negativ e for all of the viruses tested, while ∼50% was positive for at least two (and up to five) different viruses.In the 24-month old age group, slightly mor e c hildr en tested negativ e for vir al infections (52/325 or 16%), but still a ppr oximatel y half were positive for at least two (and up to six) different viruses.In contrast, in older adults the vast majority (87%) of individuals tested negative for viral infections and the occurrence of viral codetections was rare (1%).The parent group sho w ed an intermediate profile with 61% testing negative for viral infection and 9% of individuals displaying viral codetections.
Next, we assessed whether certain virus species are relatively mor e common compar ed to other species in specific a ge gr oups.For this, we calculated the r elativ e contribution of each virus to the total number of virus detections per group (Fig. 1 B).Of note, each individual potentially contributes more than one count to the total number of virus detections depending on the presence of codetections.From these data, it is clear that especially hBoV, but also adeno virus , ar e r elativ el y mor e common in c hildr en and par ents compar ed to older adults.In contr ast, endemic hCoVs a ppear to be r elativ el y mor e common in older adults, compar ed to c hildr en and par ents.Inter estingl y, WU pol y omavirus display ed the highest r elativ e contributions in older adults and 24-month old c hildr en.
For some viruses, more detailed classification of the exact viral species or strains was performed (Fig. 1 C).Data combined for all study participants sho w ed that of the four endemic coronaviruses circulating at that time, OC43 was r elativ el y most abundant (63%), follo w ed at a distance by NL63 (18%).Furthermore, PIV 4 (77%), RSV A (73%), and influenza virus A/H1N1 (62%), follo w ed b y B/YAM (24%), were the most abundant strains/species in their respective virus groups.
The pr e v alence of bacterial pathogens per a ge gr oup, irr espective of species (Fig. 1 D), sho w ed a similar pattern as the viral pathogen pr e v alence .T he v ast majority of c hildr en tested positiv e for bacterial pathogens ( > 92% in both groups) and bacterial codetections wer e fr equent ( > 72% for both gr oups) in nasopharyngeal s wabs .T he majority of older adults (86%) tested negative for bacterial pathogens and bacterial codetections wer e r ar el y seen ( < 1%) in oropharyngeal s wabs .Again, parents sho w ed an intermediate profile with 62% testing negative and 6% showing bacterial codetection in oropharyngeal s wabs , with a slightly higher bacterial pr e v alence in nasopharyngeal swabs ( Supplementary Fig. S1B ).When looking at bacterial species specifically, it is clear that S. aureus was r elativ el y mor e abundant in adults than in c hildr en, while S. pneumoniae and M. catarrhalis wer e r elativ el y mor e abundant in c hildr en than in adults (Fig. 1 E).The a ppar ent differ ence in H. influenzae pr e v alence between c hildr en and adults a ppears to reflect the different sampling sites, as parental oropharyngeal sw abs sho w a consider abl y higher r elativ e contribution of this species than nasopharyngeal swabs ( Supplementary Fig. S1C ).
Next, w e w er e inter ested in the distribution of vir al and bacterial pathogens between individuals (Fig. 1 F).As could be expected from the individual viral and bacterial pr e v alence data, the simultaneous presence of both viral and bacterial pathogens was most fr equentl y and indeed quite often observed among c hildr en (85% and 79% for 11-month old c hildr en and 24-month old c hildr en, r espectiv el y) and c hildr en that tested negative for all pathogens wer e v ery r ar e (1.5% for both gr oups).For older adults onl y a small proportion of individuals was simultaneously positive for both viral and bacterial pathogens ( < 2%) and this proportion was higher for parents (15%).Furthermore, 74% of older adults and 38% of parents tested negative for all pathogens.Nasopharyngeal and oropharyngeal swab bacterial data yielded largely similar results for the pathogen distribution pattern in parents ( Supplementary Fig. S1D ).

Se ver al pa thogen combina tions occur a t a different frequency than would be expected based on individual pathogen prevalence
Considering pr e vious r eports on pathogen interfer ence (Pir et andBoivin 2022 , Takashima et al. 2022 ), we used the diagnostics data fr om c hildr en to anal yze the fr equenc y of codetections betw een different pathogen species (Table 2 ).In line with other reports (Achten et al. 2017, Wu et al. 2020, Takashima et al. 2022 ), we observed that rhinoviruses appear to be less frequently detected in combination with RSV , hMPV , influenza virus, AdV , hCoV , and WUPyV than expected based on their frequency in the total study population.Ho w e v er, our sample onl y supports e vidence for the combination of rhinovirus with RSV (corrected P = .019,Fisher's exact test, FDR < 20%) and WUPyV (corrected P = .17,FDR < 20%).
In addition, our sample supports evidence for less frequent codetection than expected for the combination hBoV with hCoV (corrected P = .15,FDR < 20%).In contrast, PIV , hBoV , and WUPyV ar e mor e often detected in combination with each other than expected, as is the combination of PIV with KIPyV.Regarding bacterial codetections, S. aureus is detected together with M .catarrhalis , H. influenzae , and S. pneumoniae less fr equentl y than expected.In contrast, M .catarrhalis , H. influenzae , and S. pneumoniae ar e mor e often detected in combination with each other than expected.Finally, the combination of AdV with M .catarrhalis or H. influenzae occurs more frequently than expected.Corrected P -values and odds ratios for all codetection pairs with an FDR < 20% can be found in Table 3 .

Childcare attendance and having siblings strongl y associa te with higher pa thogen counts in children
Having established that the presence of multiple pathogens sim ultaneousl y in the nasopharyngeal mucosa is quite common in c hildr en, w e w er e inter ested in the factors that potentially contribute to the occurrence of such compounded detections.Based on the information obtained from the interview that was performed during the home visits, we assessed the association of infection status with c hildcar e attendance and having siblings (Table 4 and Supplementary Fig. S2A -D ).We found that both 11-and 24-month old c hildr en that attended c hildcar e facilities ( ≥4 hours per week) had a significantly higher mean number of pathogen species in their nasopharyngeal mucosa than children who did not attend c hildcar e facilities ( P < .001,Kruskall-Wallis with Dunn's multiple comparisons test).Of note, none of the children that tested negative for all pathogens ( n = 10) attended childcar e facilities.Similarl y, c hildr en that attended c hildcar e wer e significantl y mor e likel y to test positiv e for both bacterial and vir al pathogens sim ultaneousl y than c hildr en that did not attend c hild-care facilities ( P < .001,Fisher's exact test).Furthermore, we found that 11-month old c hildr en, but not 24-month old c hildr en, with at least one sibling had a significantly higher mean number of pathogens in their nasopharyngeal mucosa than c hildr en without siblings ( P = .004,Kruskal-Wallis).In addition, 11-month old c hildr en with siblings were significantly more likely to test positive for both bacterial and viral pathogens simultaneously than c hildr en without siblings ( P = .003,Fisher's exact test).Finally, the combination of c hildcar e and having siblings in 11-month old childr en a ppears to be an e v en mor e pr ominent risk factor than either of the two factors by itself ( Supplementary Fig. S2E and F ), for both pathogen counts ( P < .001,Kruskall-Wallis) and simultaneous detection of bacteria and viruses ( P < .001,generalized Coc hr an-Mantel-Haenszel Test).
As alr eady a ppar ent fr om Fig. 1 (A) and (D), consider able differences exist in the pr e v alence of r espir atory pathogens between parents and older adults.Parents had on average both more viruses and more bacteria in their upper r espir atory tr act than older adults ( P < .001,Kruskal-Wallis; Supplementary Fig. S2G ).In addition, parents were significantly more likely to test positive both for viruses and for bacteria than older adults ( P < .001,Fisher's exact test; Supplementary Fig. S2H ).Finally, we assessed whether sex or a BMI ≥25 associated with infection status in parents, but no differences were apparent ( Supplementary Fig. S2I  and J ).

T he pre v alence of symptoms of respir a tory infection differs between age groups
Next, w e w er e inter ested to see to what extent the observ ed pr esence of r espir atory pathogens is accompanied by the occurr ence of symptoms related to r espir atory infection.Fr om the data collected during the interview it was a ppar ent that, without taking infection status into account, c hildr en wer e significantl y mor e likely to present with symptoms of respiratory infection than parents and older adults ( P < .001,Fisher's exact test, Fig. 2 A).In addi- Guide for inter pr etation: cells show the number of codetections of column and row pathogen, with the detection frequency of the column pathogen as a percentage of the total number of row pathogen detections between br ac kets .For example , in 94 c hildr en both rhino virus and adeno virus were detected.Since ther e wer e a total of 231 c hildr en positiv e for adenovirus ( n in top ro w belo w AdV), this means that 40.7% (94/231 × 100%) of adenovirus-infected c hildr en also had a rhinovirus infection.As can be observed in the top row of the rhinovirus column, of all c hildr en ( n = 652) ther e wer e 298 (45.7%) positiv e for rhino virus infection.T his latter percentage is thus the frequency of rhinovirus infection in the total population under study, which is regarded as the "expected" frequency of codetections with rhinovirus in the absence of pathogen inter actions.Notabl y, a c hild that is infected with rhino virus , adeno virus , and boca virus will be counted twice in the rhinovirus column, which is the reason that the sum of counts per column is higher than the total number of c hildr en infected with the column pathogen.tion, par ents wer e significantl y mor e likel y to pr esent with symptoms than older adults ( P = .02,Fisher's exact test).These findings are in line with the differences observed in pathogen prevalence between c hildr en, par ents, and older adults.
Since our data r e v ealed that pathogen codetections in childr en ar e v ery common, w e w er e subsequentl y inter ested to see whether the number of pathogens per individual associated with the pr e v alence of symptoms of r espir atory infection.Indeed, we observed an increasing trend in the prevalence of symptoms with increasing pathogen numbers (Fig. 2 B), which was statistically significant when comparing the negative or 1 pathogen group to the ≥4 pathogens group ( P = .04and P = .03,r espectiv el y, Fisher's exact test).Notably, it is clear that e v en in the group with the highest percentage of symptomatic individuals , i.e .children ha ving ≥4 pathogens detected, the vast majority still presented as asymptomatic (69%).Whereas all of the children that were reported to show symptoms of r espir atory infection ( n = 181/181) tested positive for ≥1 pathogen, 15% of parents that reported to be symptomatic ( n = 6/40) tested negative for all assessed pathogens in both nasopharyngeal and oropharyngeal s wabs .
To obtain an indication of the association between the presence of certain pathogen species and the occurrence of symptoms of  r espir atory infection, we counted for each pathogen species the number of symptomatic and asymptomatic individuals.Because the manifestation of symptoms for specific pathogens might differ with age, we performed these analyses separately for childr en and adults.Notabl y, because of the low numbers of single infections in c hildr en, we did not corr ect for the presence of other pathogens in the same individuals.For example: of all c hildr en that tested positive for rhinovirus (and potentially other pathogens), 202 (68%) were asymptomatic and 96 (32%) were symptomatic at the time of sampling.For c hildr en, we found that infection with hMPV , RSV , and influenza virus was most fr equentl y associated with symptoms of r espir atory infection (Fig. 2 C).In the case of parents and older adults, it appears that influenza virus, rhino virus , and adeno virus infections wer e most fr equentl y associated with symptoms (Fig. 2 D).For both gr oups, the pr esence of S. aureus , hBoV, and KI poly omavirus w as least often associated with symptoms of r espir atory infection (Fig. 2 C and D).Children appear mor e likel y to be symptomatic than adults for all pathogens except influenza.Of note, for se v er al pathogens the detection numbers are very low and these should therefore be interpreted with caution.

Mucosal cytokine levels are generally higher in children compared to parents and associate with the presence of symptoms of respir a tory infection
Since cytokines and chemokines are important mediators of the imm une r esponse and potential biomarkers or e v en determinants of disease se v erity, w e w onder ed whether differ ences in their concentr ation r elating to the pr esence of symptoms of r espir atory infection could be observed in this cross-sectional cohort despite the absence of se v er e disease.To this end, we determined the concentr ation of pr oinflammatory c hemokines , interleukins , interferons, and se v er al cytotoxic effector molecules in a r andom selec- tion of nasopharyngeal swab samples ( n = 100 per group).As both viral and bacterial nasopharyngeal data were available for children and parents-but not for older adults-we focused on these groups for the cytokine analysis.Participant demographics for the selected subset can be found in Table 5 .
To obtain a general impression of the cytokine data we first generated a heatmap based on unsupervised hierarchical clustering of both cytokines and participants (Fig. 3 A).Participant clustering basicall y r esulted in a separ ation between c hildr en and parents, with some exceptions.Ov er all, c hildr en show higher mucosal cytokine levels than parents, in line with the observed differences in pathogen prevalence between these groups.IL-33 sho w ed an opposite pattern with gener all y higher le v els in par ents compar ed to c hildr en.Clustering of cytokines r esulted in grouping of functionally related molecules such as IL-1 β/6/8 and CXCL9/10/11, suggesting that the measured cytokine levels wer e r epr esentativ e of biological e v ents.For a mor e detailed ov ervie w of the various relations between each of the measured c ytokines, w e then pr epar ed a corr elation matrix based on the data of the c hildr en onl y (Fig. 3 B), as the ov er all differences between c hildr en and par ents might obscur e other biologicall y r ele v ant corr elations .T he corr elation matrix r e v ealed mostl y positiv e corr elations, except for IL-33 and IL-13 whic h sho w ed a positive correlation with each other but a negative correlation with several other molecules including for example CCL3 and IL-1 β.Similar to the heatmap clustering, several groups of functionally related molecules could be discerned within the correlation matrix, with some additional-mostly weaker-correlations between these groups.Fig. 3 (C) shows an example of the str ong positiv e corr elation between IL-1 β and CCL3 (Spearman rho = 0.83, P < .0001),within a group of several proinflammatory molecules.
Finally, we asked whether any of the measured cytokines associated with the presence of symptoms of r espir atory infection in c hildr en and/or par ents.Especiall y IL-6 sho w ed a clear association with the presence of symptoms, which was statistically significant in 11-and 24-month old c hildr en ( P < .05 and .01,respectiv el y, Kruskall-Wallis, follo w ed b y Dunn's multiple comparisons test) but not parents (Fig. 4 A).Both IL-1 β and CCL3 also sho w ed an association with symptoms, but this was only statistically significant in 24-month old c hildr en ( P < .05,Kruskall-Wallis; Fig. 4 B and  C).In line with its the negativ e corr elation with IL-1 β and CCL3 (Fig. 3 B), IL-33 sho w ed a trend to w ar d lo w er le v els in symptomatic compared to asymptomatic individuals, but this was not statistically significant (Fig. 4 D).The raw cytokine data are available as supplementary material for additional in-depth analysis.

Discussion
In this study, we show that r espir atory pathogens are widely present in the generally healthy population.At least one respiratory pathogen was detected in > 98% of 11-and 24-month-old c hildr en and in 26% of older adults, while parents sho w ed an intermediate percentage of positivity (62%-69% depending on pharyngeal sampling site).Although only assessed at the time of sam-  pling, it appears that a large part of these detections are not associated with clinical symptoms.Our analyses further confirm that c hildcar e attendance and having siblings associate with significantl y incr eased pathogen counts in young c hildr en.Finall y, we show that the concentration of proinflammatory molecules such as IL-6, IL-1 β, and CCL3 is increased in symptomatic compared to asymptomatic individuals.
The major strength of this study is that our sampling strategy did not depend on medically attended infections.For this reason, our data provide a snapshot of the prevalence of respiratory pathogens in the general population during the peak r espir atory infection season, which is important information that helps to increase our understanding of microbial reservoirs and pathogen exposure.In ad dition, stud y participants included young c hildr en as well as parents and older adults .T he inclusion of various subpopulations allo w ed for comparison of pathogen pr e v alence and species distribution across age groups.Finally, our analyses included an extensive diagnostic panel, consisting of both viruses and bacteria, and a broad range of cytokines and chemokines.
A limitation of our study is that it cov er ed onl y one winter (2012/2013), and ther efor e does not account for variability in the circulation of pathogens within and between years.Of note, based on available information on national respiratory infection surveillance, this r espir atory season was gener all y similar to other years (Teirlinck et al. 2015 ).Nevertheless, due to the nonpharmaceutical interventions implemented to contain the COVID-19 pandemic, circulation of a number of r espir atory viruses, including influenza, RSV , hMPV , and PIV , has dr amaticall y decr eased in 2020/2021 (Kuitunen et al. 2020, Redlberger-Fritz et al. 2021, Tang et al. 2021, Yeoh et al. 2021 ).As the resumed circulation of these pathogens has shown atypical patterns in some cases, it remains to be seen how their circulation will take shape again in the future (Baker et al. 2020, Foley et al. 2021 ).Furthermor e, the cr osssectional nature of the study does not allow for analysis of for example the duration of infection or the occurrence of repeated infections in the same individuals.Because of the lack of follow-up on the de v elopment of symptoms, we cannot draw firm conclusions on the percentage of asymptomatic infections.Probably for this reason, the percentage of individuals without symptoms in this study appears to be slightly higher than what has been found in earlier studies (Byington et al. 2015, Birger et al. 2018 ).
An important question in the context of infection pr e v ention is whic h gr oup of individuals form the main source of pathogen spread.Although we did not address this question directly in our study, our data supports the notion that, in general, young childr en ar e an important r eservoir of r espir atory pathogens.For example, c hildcar e attendance, having siblings, and being a parentall involving frequent exposure to young children-associate with an increased likelihood of being infected, which is in line with previous reports (Chu et al. 2013, Po w ell et al. 2022 ).In addition, the infection patterns observed in parents are in many ways intermediate between those in young c hildr en and older adults .T he latter have less frequent contact with young children than parents, although additional (e.g.lifestyle or immunological) differences might also play a r ole.Furthermor e, c hildr en a ppear to harbor more pathogen species per individual and to be symptomatic more often than adults, both of which potentially contribute to pathogen spread.For several pathogens, including bocavirus, adeno virus , and S. pneumoniae , their r elativ e contribution a ppears to be higher in young c hildr en than in adults.In contrast, the relative contribution of endemic coronaviruses and S. aureus appears to be lo w er in young c hildr en compar ed to adults .T hese observations suggest that while young c hildr en may be important reservoirs for the spread of respiratory infections in general, their actual role lik ely de pends on the specific pathogen in question.Additional data and modelling studies are needed to address these speciesspecific transmission dynamics.
The ubiquitous presence of both viral and bacterial respiratory pathogens, especiall y in v ery young c hildr en and often without clinical symptoms, begs the question whether and how their presence influences the de v elopment of local and systemic immunity.Man y r eports exist on the detrimental role of rhinovirus and RSV infections in the de v elopment and/or exacerbation of asthma, which is a chronic inflammatory disease of the lung (Busse et al. 2010, Lambr ec ht and Hammad 2015, Jartti and Gern 2017 ).Ho w e v er, (mild) infections might also play a beneficial role in the pr oper de v elopment of imm unity, as has been suggested for the gut microbiome (Virgin 2014 , Zheng et al. 2020 ).While these aspects of pathogen-host interactions are often difficult to investigate, the decrease in circulation of v arious r espir atory pathogens due to nonpharmaceutical interventions during the COVID-19 pandemic might provide a unique window of opportunity for this.It would be interesting to examine whether the local and systemic imm une r esponse of c hildr en gr owing up under these conditions differs from that observed during regular circulation of these respiratory pathogens.
In addition to r espir atory pathogen pr e v alence, we assessed the concentrations of numerous (proinflammatory) cytokines and chemokines in the nasopharyngeal mucosa of children and parents.Our data show that e v en in the absence of se v er e illness, marked differences in the levels of proinflammatory molecules can be observed that correlate with the presence of symptoms of r espir atory infection.While the majority of cytokines that were assessed sho w ed higher le v els in c hildr en compar ed to adults, suggestive of a positive association with the presence of increased numbers of r espir atory pathogens, the opposite was true for IL-33.This cytokine is a so-called "alarmin," which is constitutiv el y expressed by for example epithelial cells in the lung and released upon cellular necrosis (Johansson and McSorley 2019 ).IL-33 is mainly associated with type 2 immune responses and has been implicated in the de v elopment of asthma (Johansson and McSorley 2019 ).Because of the cr oss-sectional natur e of our data, it remains unclear whether the observed differences in IL-33 levels precede pathogen acquisition or are in fact a result of infection.Inter estingl y, Robinson et al. ( 2018 ) have shown in a mouse model that influenza infection results in decreased S. aureus -induced IL-33 pr oduction, potentiall y leading to incr eased susceptibility to this pathogen.The observed lo w er levels of IL-33 in children would be in line with a scenario in which an initial (viral) infection increases the susceptibility to subsequent (bacterial) pathogen acquisition by means of IL-33 inhibition, thereby providing a possible explanation for the large numbers of pathogens detected in these younger individuals, but further r esearc h is needed.
With this study, we further establish the widespread nature of r espir atory pathogens, both viral and bacterial, in the gener all y healthy population and their association with local cytokine and c hemokine le v els .T hese findings ma y guide further r esearc h that is needed to understand r espir atory pathogen transmission dy- Pathogen codetections in 11-and 24-month old children combined.

Figure 1 .
Figure 1.Detection of pathogens in the r espir atory m ucosa in differ ent a ge gr oups .(A) T he pr e v alence of r espir atory vir al infections in all participants ( n indicates group size) and (B) the relative contribution of viral species (d indicates the total number of individual pathogen detections, a single participant can be included multiple times with the occurrence of codetections) in differ ent a ge gr oups.(C) Further subdivision in species/subtypes for endemic human CoVs, PIV, RSV, and influenza virus for all age groups combined.(D) The prevalence of respiratory bacteria and (E) the relative contribution of bacterial species in different age groups.(F) Overview of the distribution of viral infections and bacteria between individuals in differ ent a ge gr oups.Gr oups consisted of 11-month old c hildr en ( n = 327), 24-month old c hildr en ( n = 325), par ents ( n = 319), and older adults ≥60 years of age ( n = 340).Bacterial presence was determined in (a) nasopharyngeal or (b) oropharyngeal s wabs .Abbreviations: AdV, adenovirus; hBoV, human bocavirus; hCoV, human coronavirus; hMPV, human metapneumovirus; KIPyV, Karolinska Institute polyomavirus; Rhino, rhinovirus; RSV, r espir atory syncytial virus; PIV, parainfluenza virus; and WUPyV, Washington University polyoma virus .Data concerning virus detections in older adults and bacteria in c hildr en/par ents hav e been pr e viousl y described in less detail and ar e shown her e for compar ativ e pur poses (Bosc h et al. 2016 , van Beek et al. 2017 ).

Figure 2 .
Figure 2. Occurrence of symptoms of respiratory infection in different age groups.Participants were asked whether, at the time of sampling, they experienced any symptoms of respiratory infection, e.g.cough, headache, or sneezing.(A) Ov ervie w of the pr e v alence of self-r eported symptoms per a ge gr oup irr espectiv e of infection status.(B) Ov ervie w of the pr e v alence of self-r eported symptoms by pathogen count for c hildr en ( n = 652).(C) and (D) Ov ervie w of the pr e v alence of self-r eported symptoms per pathogen for c hildr en ( n = 652, C) and (older) adults ( n = 659, D).Abbr e viations: AdV, adenovirus; H. inf , Haemophilus influenzae ; hBoV, human bocavirus; hCoV, human coronavirus; hMPV, human metapneumovirus; KIPyV, Karolinska Institute polyomavirus; M. cat , Moraxella catarrhalis ; PIV, parainfluenza virus; Rhino, rhinovirus; RSV, r espir atory syncytial virus; S. aureus , Staphylococcus aureus ; S. pneu , Streptococcus pneumoniae ; and WUPyV, Washington Univ ersity pol yoma virus .

Figure 3 .
Figure 3. Heatmap and correlation matrix of mucosal cytokines determined in nasopharyngeal s wabs .A multiplex immunoassay was used to determine the concentrations of several cytokines , chemokines , and effector molecules in nasopharyngeal swab samples of 11-and 24-month old c hildr en and parents ( n = 100 per group).(A) A heatmap was produced using scaled data from all children and parents by performing unsupervised hier arc hical clustering of both cytokines and participants based on Euclidean distance.Pathogen count (path_count), pathogen combination, presence of r espir atory symptoms, sex, and a ge gr oup of eac h participant ar e indicated on the left (a legend is pr esent on the right side of the heatma p).(B) A correlation matrix was produced based on Spearman rank order correlation analysis of data from 11-and 24-month old children.Only correlations with a P -value below .02are shown as dots, of which the size and color indicate the strength and direction of the correlation (also see legend on the right side of the matrix).(C) Example of a correlation plot for IL-1 β and CCL3 using data from 11-and 24-month old children ( r = 0.83, P < .0001,Spearman).

Figure 4 .
Figure 4. Cytokine concentrations in the nasopharyngeal mucosa of children and parents in the absence or presence of symptoms of respiratory infection.A multiplex immunoassay was used to determine the concentrations of among others IL-6 (A), IL-1 β (B), CCL3 (C), and IL-33 (D) in nasopharyngeal swab samples of asymptomatic and symptomatic 11-and 24-month old c hildr en and parents ( n = 100 per group).Samples below the limit of detection were set to 0.5 times the lowest detected concentration for analytical purposes.Boxplots depict median and quartiles.Statistical significance of the difference between symptomatic and asymptomatic individuals within age groups was assessed using a nonparametric Kruskal-Wallis test, follo w ed b y Dunn's multiple comparisons test.* P < .05;* * P < .01.

Table 1 .
P articipant c har acteristics, total study population.
a P ar ents of the 24-month old c hildr en.b Data are missing for one child in the 24 months group.c For those c hildr en that attend c hildcar e facilities.d For those c hildr en that have siblings.e Data are missing for two older adult participants.

Table 3 .
List of codetected pathogen pairs in c hildr en for whic h the observed frequency differs from the expected frequency at a FDR < 20%.

Table 4 .
Risk factors for infection in c hildr en and adults, univariate analysis.
2Fisher's exact test with Baptista-Pike for odds ratio CI.Abbr e viations: CI, confidence interval; na, not applicable; and OR, odds ratio.

Table 5 .
P articipant c har acteristics, selection for cytokine analysis.P ar ents of the 24-month old c hildr en.b Data are missing for one child in the 24 months group.c For those c hildr en that attend c hildcar e facilities.
a d For those c hildr en that have siblings.