Association of pneumococcal carriage in infants with the risk of carriage among their contacts in Nha Trang, Vietnam: A nested cross-sectional survey.

BACKGROUND: Infants are at highest risk of pneumococcal disease. Their added protection through herd effects is a key part in the considerations on optimal pneumococcal vaccination strategies. Yet, little is currently known about the main transmission pathways to this vulnerable age group. Hence, this study investigates pneumococcal transmission routes to infants in the coastal city of Nha Trang, Vietnam. METHODS AND
FINDINGS: In October 2018, we conducted a nested cross-sectional contact and pneumococcal carriage survey in randomly selected 4- to 11-month-old infants across all 27 communes of Nha Trang. Bayesian logistic regression models were used to estimate age specific carriage prevalence in the population, a proxy for the probability that a contact of a given age could lead to pneumococcal exposure for the infant. We used another Bayesian logistic regression model to estimate the correlation between infant carriage and the probability that at least one of their reported contacts carried pneumococci, controlling for age and locality. In total, 1,583 infants between 4 and 13 months old participated, with 7,428 contacts reported. Few infants (5%, or 86 infants) attended day care, and carriage prevalence was 22% (353 infants). Most infants (61%, or 966 infants) had less than a 25% probability to have had close contact with a pneumococcal carrier on the surveyed day. Pneumococcal infection risk and contact behaviour were highly correlated: If adjusted for age and locality, the odds of an infant's carriage increased by 22% (95% confidence interval (CI): 15 to 29) per 10 percentage points increase in the probability to have had close contact with at least 1 pneumococcal carrier. Moreover, 2- to 6-year-old children contributed 51% (95% CI: 39 to 63) to the total direct pneumococcal exposure risks to infants in this setting. The main limitation of this study is that exposure risk was assessed indirectly by the age-dependent propensity for carriage of a contact and not by assessing carriage of such contacts directly.
CONCLUSIONS: In this study, we observed that cross-sectional contact and infection studies could help identify pneumococcal transmission routes and that preschool-age children may be the largest reservoir for pneumococcal transmission to infants in Nha Trang, Vietnam.

Introduction

Mathematical modelling is a key part of the evidence synthesis process that informs public health policy for infectious diseases and their mitigation, not least evident in its role in the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic [1,2]. An essential part of such infectious disease models is their adequate reflection of transmission in the community studied. For respiratory pathogens, social contacts and their age structure have been used predominantly as the key source of heterogeneity in infectious disease spread, and, hence, the accurate measurement of social contacts as a proxy for potential transmission events has been fundamental for the validity of such models [3,4].

While social contacts are a seemingly obvious proxy for transmission risk for pathogens transmitted via droplets or aerosols from the respiratory tract, there is limited direct evidence that the common definition of a transmission relevant contact, i.e., a 2-way conversation or skin-to-skin contact, is indeed a major risk factor for infection. Nested contact and infection studies in Fiji and Uganda have shown that the frequency of physical (skin-to-skin) household contacts of longer duration (>1 hour) was associated with higher rates of pneumococcal carriage [5,6], and a prospective cohort study in the United Kingdom showed that adults hospitalised with community acquired pneumonia had increased odds for close child contact within the 4 weeks prior to their illness [7]. Furthermore, during the SARS-CoV-2 pandemic, changes in contact behaviour as measured by social contact surveys have enabled real-time estimation of the impact of non-pharmaceutical interventions on pandemic transmission intensity [8,9].

Infants are typically at high risk for severe respiratory disease and are targeted for either direct or indirect vaccine protection. Their exposure to disease through their social contacts can also be an important guide for selecting different dosing schedules for these vaccines, including pneumococcal conjugate vaccines (PCVs) [10]. However, there is little evidence on what constitutes a transmission relevant contact for infants or the frequency and distribution of such contacts. Most contact studies include few infants [3,9] or are conducted in specific environments that capture only a portion of their contacts [11]. The most detailed study to date, which studied the contact patterns of 115 infants in the UK, found that almost two-thirds of infant contacts were not with household members, highlighting the potential importance of non-household transmission routes for infant infection [12].

We report the results of a large infant contact study nested into a cross-sectional pneumococcal carriage survey in Nha Trang, Vietnam. We investigate the correlation of infant exposure risk, approximated by reported social contacts and the prevalence of infection in such, with their risk of pneumococcal infection and the spatial and demographic structure of infant contacts and exposure in this setting.

Methods

Study population

The study was conducted in Nha Trang, a coastal city in south-central Vietnam with a total population of just over 426,958 and under-five population of 28,495 in the 2018 census (personal communication with Dr. Minh Xuan Bui, Khanh Hoa Health Service, October 2, 2021—see S3 File). Nha Trang city consists of 27 communes of similar population size. Each commune has a commune health centre, providing a range of basic health services. Similarly, educational services including nurseries and schools are largely provided on a commune basis.

In October 2016, a cluster randomised controlled trial was initiated to evaluate alternative PCV dosing schedules [13]. In the 24 communes that were allocated to receive PCV, routine infant vaccination was initiated according to the allocated schedule: 2 priming doses and 1 booster dose (2p+1), 3p+0, 1p+1, or 0p+1, and a catch-up campaign including children under 3 years old was conducted. The primary endpoint of the trial is dependent on pneumococcal carriage, and, as such, this is monitored in each commune through annual cross-sectional nasopharyngeal carriage surveys in 60 infants (4 months to 11 months), 60 toddlers (14 months to 23 months), randomly selected from an administrative population list, and their respective main carers. Completion rate of routine PCV vaccination in the community was 81.2%, 88.5%, 97.4%, and 87.0%, in the 2p+1, 1p+1, 0p+1, and 3p+0 arms, respectively, using the 18 months population as denominator for the 2p+1, 1p+1, and 0p+1 arms and the 12 months population for the 3p+0 arm, in 2018 when the infant contact survey was conducted. Due to serotype replacement, the trial did not affect overall carriage rates in the Nha Trang population.

Study design

In October 2018, 2 years after the start of the trial, an infant contact survey was nested into the cross-sectional carriage study. All infants enrolled for the carriage study were also eligible for enrolment into the contact survey.

For each commune health centre, a staff member was identified and trained to conduct the contact survey in their local commune. During an initial home visit, written informed consent was obtained for both the infant contact and carriage studies from parents or guardians before any collection of data or specimen.

Subsequently, a background questionnaire was filled in together with the parent or guardian on behalf of the infant, collecting information including sex, age, household composition, and the infant’s mobility (see S1 File for the full questionnaire). The parent or guardian was asked to monitor their infant’s contacts the following day, and another home visit was scheduled for the day after. For that visit, the interviewer would aid the carer’s memory by discussing their day and noting initials of the infant’s contacts. Subsequently, information on those contacts’ age, sex, duration, and location was filled in (see S2 File for the full questionnaire). A contact was defined as skin-to-skin contact with the infant, because the common conversational contact definition would be inappropriate to apply to infants and because physical contacts, as a proxy for close contacts, have been shown to better correlate with pneumococcal carriage risk [5,6].

Consent to participate in the contact study and the nasopharyngeal carriage study was sought from the carers by home visit. The date of keeping a contact diary was assigned, and field workers conducted a second visit 1 or 2 days after to record the contact information based on the diary. Participants were also given an appointment with the commune health centre during which a nasopharyngeal swab from the infant was obtained by a study nurse. Sample collection, handling and storage were performed according to the World Health Organization guidelines [14]. At the Pasteur Institute of Nha Trang, DNA extractions were performed on a QIAcube HT instrument using the QIAmp 96 DNA QIAcube HT Kit (QIAGEN, Hilden, Germany) with a lysis buffer (20 mg/ml lysozyme, 20 mM Tris/HCl, 2 mM EDTA, 1% v/v Triton) and RNase A treatment, as previously described [15]. Real-time quantitative PCR (qPCR) to detect the autolysin-encoding gene (lytA) of Streptococcus pneumoniae was performed. Final reaction volumes of 25 μl were run on an Applied Biosystems 7500 Fast real-time PCR instrument with 5 μl of template DNA, TaqMan GeneExpression Mastermix (Applied Biosystems, Massachusetts, USA), and 200 nM of primers and probe (forward primer: 5′-ACGCAATCTAGCAGATGAAGCA-3′, reverse primer: 5′-TCGTGCGTTTTAATTCCAGCT-3′, and lytA probe: 5′ FAM -TGCCGAAAACGCTTGATACAGGGAG- 3′ BHQ1) [16]. lytA positive (cycle threshold (Ct) value < 35) or equivocal (Ct value 35 to 40) samples were cultured on sheep blood agar plates with 5 μg/ml of gentamicin. Samples with alpha-hemolytic growth were harvested and DNA was extracted from the bacterial pellet on a QIAcube HT instrument as described previously [17]. Pneumococcal serotyping was performed by DNA microarray at the Murdoch Children’s Research Institute using Senti-SPv1.5 microarrays (BUGS Bioscience, London, UK) [18].

Pneumococcal carriage was defined as samples that were both lytA positive/equivocal and culture positive and subsequently confirmed by microarray. A small number of samples that were lytA qPCR positive but were nonculturable (n = 5) or had technical issues (n = 4) were also deemed pneumococcal positive.

This study is reported as per the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guideline (S1 STROBE Checklist).

Statistical analyses

The infant contact study is a cross-sectional study, and no prospective analysis plan exists. The plan for the current study combining contact and carriage data was conceived during a collaborative meeting that happened after the infant contact and pneumococcal carriage surveys were conducted, but without being informed by the data. We used negative binomial regression to estimate crude rate ratios of the mean number of contacts of participants for each characteristic of the participants, as defined in Table 1. We also calculated rate ratios adjusted for age (categorical: 4 to 7 months and 8 to 11 months of age) and for clustering of participants into communes.

Table 1

Mean number of contacts reported for infants and effect of each infant’s characteristic on incidence of contacts, estimated using a negative binomial regression model.

Characteristic			Number	Mean (SD)	Incidence rate ratio	p-Value	Adjusted incidence rate ratio*	p-Value
	Total		1,583	4.7 (1.8)
Demographics
	Sex
		Male	871	4.7 (1.8)	reference	0.376	reference
		Female	712	4.6 (1.8)	0.98 (0.94 to 1.03)		0.98 (0.94 to 1.01)	0.233
	Age at the time of enrolment (months)
		4 to 7 months	616	4.7 (1.8)	reference	0.56
		8 to 11 months	967	4.7 (1.8)	1.01 (0.97 to 1.06)
Family
	Siblings in the household
		No sibling	666	4.6 (1.9)	reference	0.256	reference
		One or more siblings	917	4.7 (1.7)	1.03 (0.98 to 1.08)		1.03 (0.99 to 1.06)	0.103
	Number of people in the household
		2 to 4	611	4.1 (1.7)	reference	<0.001	reference
		>4	972	5.1 (1.7)	1.25 (1.19 to 1.31)		1.25 (1.19 to 1.30)	<0.001
	Caretaker currently in a paid employment
		No	1,056	4.7 (1.9)	reference	0.673	reference
		Yes	527	4.7 (1.6)	1.01 (0.96 to 1.06)		1.01 (0.96 to 1.06)	0.725
	Highest level of education in the household
		Secondary school or lower	268	4.8 (1.8)	reference	0.568	reference
		Degree	1,315	4.7 (1.8)	0.98 (0.93 to 1.04)		0.98 (0.92 to 1.05)	0.582
Infant’s activity
	Sit
		Yes	1,334	4.7 (1.8)	1.04 (0.98 to 1.11)	0.177	1.05 (0.99 to 1.11)	0.094
		No	249	4.5 (1.7)	reference		reference
	Crawl
		Yes	873	4.7 (1.8)	1.01 (0.96 to 1.05)	0.752	1.00 (0.95 to 1.05)	0.977
		No	710	4.7 (1.8)	reference		reference
	Walk
		Yes	186	4.7 (2.0)	0.99 (0.93 to 1.07)	0.863	1.10 (0.98 to 1.24)	0.647
		No	1,397	4.7 (1.8)	reference		reference
Mobility
	Transport available for immediate travel
	Bicycle
		Yes	31	4.5 (1.4)	0.95 (0.80 to 1.12)	0.532	0.95 (0.86 to 1.04)	0.251
		No	1,552	4.7 (1.8)	reference		reference
	Motorbike
		Yes	1,549	4.7 (1.8)	1.15 (0.97 to 1.36)	0.1	1.15 (1.06 to 1.25)	0.001
		No	34	4.1 (1.2)	reference		reference
	Car
		Yes	99	4.9 (2.1)	1.04 (0.95 to 1.14)	0.376	1.04 (0.97 to 1.13)	0.286
		No	1,484	4.7 (1.8)	reference		reference
	Walk
		Yes	108	5.1 (2.0)	1.1 (1.01 to 1.20)	0.027	1.10 (0.98 to 1.24)	0.105
		No	1,475	4.7 (1.8)	reference		reference
	Public transportation
		Yes	40	4.8 (1.8)	1.03 (0.89 to 1.19)	0.695	1.03 (0.90 to 1.18)	0.65
		No	1,543	4.7 (1.8)	reference		reference
	Number of times caretaker left the commune in the last 7 days
		0 to 2	872	4.5 (1.7)	reference	0.002	reference	0.001
		3 or more	711	4.9 (1.9)	1.07 (1.03 to 1.12)		1.07 (1.03 to 1.12)
	Number of times infant left the commune in the last 7 days
		0	788	4.6 (1.7)	reference	0.012	reference	0.004
		1 or more	795	4.8 (1.8)	1.06 (1.01 to 1.11)		1.06 (1.02 to 1.10)
	Day care attendance
		Yes	86	5.0 (1.5)	1.07 (0.97 to 1.18)	0.16	1.07 (1.00 to 1.14)	0.053
		No	1,497	4.7 (1.8)	reference		reference
Pneumococcus carriage
		Yes	353	4.8 (1.8)	1.03 (0.97 to 1.09)	0.299	1.03 (0.98 to 1.08)	0.284
		No	1,229	4.7 (1.8)

*Incidence rate ratios adjusted for age group, considering clustering in each commune.

After completion of the nested contact survey, we also examined the association of infant infection and their contact behaviour but, in an extension to previous studies, also accounted for the risk of infection in each contact. We used logistic regression to estimate the crude ratio of odds for a contact being made outside the commune of residence, for each characteristic of the participants. In an adjusted analysis, we included the age group (4 to 7 and 8 to 11 months of age: the first 4 months and the second 4 months of the target age range) and day care attendance as covariates and accounted for clustering of participants into communes.

To explore whether an infant’s contact patterns are correlated with their probability to carry pneumococcus, we created the potential exposure index (PEI). For each infant participating in the study, PEI estimates the probability that at least one of the reported contacts was carrying pneumococcus and hence that the infant was exposed to the pneumococcus on that day at least once. We define PEI as where p represents the probability that the ith contact of the respective study participant is a pneumococcal carrier. We assume that p is solely dependent on the contact’s age. To estimate age-dependent probabilities for pneumococcal carriage in the study setting, we performed Bayesian penalised B-spline regression on logit-transformed carriage probability, obtained from previously reported low-granularity age-stratified pneumococcal carriage across age groups from Nha Trang [19]. These data were collected in October 2006 from 519 individuals in a cross-sectional survey. The age group stratifications were yearly from ages until 5 years old and then 6 to 10 years old, 11 to 17 years old, 18 to 30 years old, 31 to 49 years old, and over 50 years old. We visually validated our estimates against carriage prevalence observed in young children in the trial. The PEI estimate is based on the contact patterns recorded for a single day but were interpreted as a proxy for the general contact behaviour of the respective infant. The Mann-Whitney U test was used to assess whether there was a significant difference in average PEI value in carriers and noncarriers.

We used a hierarchical Bayesian logistic regression model (see Table B in S4 File) to estimate the correlation of PEI (covariate) and pneumococcal infection (outcome). Based on selection via a Bayesian belief network (see Fig A in S4 File), we included age in months as a covariate and the resident commune as a random effect. Hence, our logistic regression model was where

p is the probability that the ith infant in the study is a pneumococcal carrier;

b0 is the model intercept under a single-level regression model (fixed effects only);

C is the random effect on the intercept due to the influence of the commune to which the ith individual belongs;

b1 is the coefficient corresponding to the effect of PEI;

E is the value of PEI, for the ith individual;

b2 is the coefficient corresponding to the effect of the infant’s age; and

A is the age of the ith individual in months.

The model was implemented in JAGS [20]. The uncertainty in the individual level estimates for PEI in this analysis was retained from the posterior estimates in the previous regression (namely, the cubic spline on age) by assuming a normal distribution for each year of age and drawing samples from this distribution when calculating the posterior samples of PEI. We chose to assume a normal distribution for simplicity and since, by visual inspection, the distribution of carriage rates across all ages appeared to follow a Gaussian (see Fig C in S4 File). Subsequently, this individual-level uncertainty in PEI is further propagated by assuming a beta distribution for these PEI estimates, since PEI is bounded between 0 and 1, and each individual’s PEI distribution is right skewed (see Fig B in S4 File); this distribution then serves as an input to the logistic regression predicting carriage status. Potential correlations among the predictor variables were checked before model fitting using the variance inflation factor (VIF) [21].

The total pneumococcal exposure to an infant on the day that contacts were reported was approximated by the sum of each reported contact’s age specific probability of pneumococcal carriage. The contribution of a specific age group to that total was calculated as the pneumococcal exposure from contacts of that age group divided by the total exposure. This can be generalised to calculate the contribution of specific age groups to the total pneumococcal exposure across all infants by including all contacts in the study.

The descriptive statistics were done in STATA version 14.0 [22]. The analyses on the association of contact patterns and pneumococcal carriage were conducted in R 4.0.2 [23] and RJags [24]. The code is available on https://github.com/GeorgeYQian/Nha-Trang-Contact-Study-Data-and-Code.

Ethical approval

Ethical approval for the study was granted by the respective ethics boards of the National Institute of Hygiene and Epidemiology in Hanoi, Vietnam (4405/QD-BYT) and the London School of Hygiene & Tropical Medicine (15881).

Results

Characteristics of study participants and their contacts

The contact study was conducted between October 19 and November 7, 2018. Overall, 1,583 infants aged between 4 and 13 months (median: 9 and interquartile range: 7 to 11 months) were enrolled in both the contact survey and the carriage survey (Table 1). All children were aged 11 months or younger at the time of enrolment, although 122 children were 12 or 13 months by the time of data collection. An additional 11 infants were enrolled in the carriage survey but did not consent to the contact survey because it was not convenient for the carer; these infants were not included in any of the following analyses (response rate was 99%). There were no missing data in the contact survey except that S. pneumoniae carriage status could not be determined in one infant due to technical reasons. This infant was excluded from the analysis of pneumococcal carriage. Over half (917, 58%) of infants had siblings living in the same household and the average household size was 4.9 (SD 1.0). The majority of infants were able to sit on their own (84%), many could crawl (55%), and some could walk (12%). Most families (98%) had a motorbike; other means of transport were rarely used. Moreover, 50% of infants did not leave their commune of residence during the preceding week. Few infants (5%) attended day care. Pneumococcal carriage prevalence was 22% and increased from 17% at 4 to 7 months to 26% at 8 to 11 months.

A total of 7,428 contacts aged between 0 and 100 years (median: 33 years) were reported (Fig 1). Contacts mostly occurred at home (89%) and with family members (76%). They were mostly with other children, parents, and grandparents and lasted for more than 1 hour (55%). Few contacts (5%) were reported to occur outside the commune of residence or with other infants (0.4%) (Table 2).

Fig 1

The number of infant contacts.

Reported skin-to-skin contacts are shown disaggregated into annual age groups and stratified by contact duration.

Table 2

Characteristics of infants’ contacts.

Characteristics		Total, n = 7,428 (100%)
Sex
	Male	2,988 (40.2)
	Female	4,353 (58.6)
	Group contact	87 (1.2)
Family member
	Yes	5,672 (76.4)
	No	1,756 (23.6)
Place of contact (first contact)
	At home	6,600 (88.9)
	Day care	134 (1.8)
	Transport	1 (0.0)
	Office	17 (0.2)
	Leisure	115 (1.6)
	Other	561 (7.6)
Contact occurred in the infant’s residence commune
	Yes	7,074 (95.2)
	No	354 (4.8)
Contact duration
	<5 minutes	331 (4.5)
	6 minutes to 1 hour	3,009 (40.5)
	>1 hour	4,088 (55.0)
Contact frequency
	Daily or almost daily	6,466 (87.1)
	Once or twice a week	715 (9.6)
	Once or twice a month	162 (2.2)
	Less than once a month	52 (0.7)
	Never met before	33 (0.4)

Factors affecting the number of contacts

The number of skin-to-skin contacts an infant made on the day before the survey ranged from 1 to 15, and the mean was 4.7 (SD 1.8). Infants who lived in households with more than 4 people were reported to have 25% (95% confidence interval (CI): 19% to 30%) more contacts than infants in smaller households (adjusted incidence rate ratio 1.25, 95% CI: 1.19 to 1.30, p-value <0.001). The infant’s ability to crawl or sit was not found to be associated with their number of contacts; however, infants who had travelled beyond commune borders in the week preceding the survey were reported to have 6% (95% CI: 2% to 10%) more contacts (adjusted incidence rate ratio 1.06, 95% CI: 1.02 to 1.10, p-value 0.004). Infants who were found to carry pneumococci were reported to have slightly more contacts; however, this was not statistically significant (adjusted incidence rate ratio 1.03, 95% CI: 0.98 to 1.08, p-value 0.284) (Table 1).

Factors affecting the location of contacts

About 10% of infants had at least one contact outside of their commune of residence (Table A in S4 File). Infants at least 8 months old and those with caretakers in current employment were 67% (95% CI: 16 to 140) and 67% (95% CI: 19 to 134) more likely (according to the crude odds ratios 1.67, 95% CI: 1.16 to 2.40, p-value 0.006 and 1.67, 95% CI: 1.19 to 2.34, p-value 0.003) to have a contact outside their commune, respectively. Also, pneumococcal carriers were more likely to have had contact outside the commune; however, this was no longer the case if adjusted for day care centre attendance (adjusted odds ratio 1.25, 95% CI: 0.92 to 1.71, p-value 0.159), which was strongly associated with contacts outside the commune (adjusted odds ratio 6.56, 95% CI: 3.92 to 10.98, p-value <0.001). Infant-to-infant contact was rare; only 2% of infants were reported to have contacted another infant in another commune. Of such infant-to-infant contacts, the majority (19/31, 61%) happened in day care.

Validating contacts as a risk factor for carriage

Modelled carriage prevalence in Nha Trang based on carriage observations from a previous study [19] increased rapidly in infancy and peaked at about 4 years of age. Secondary school–age children and particularly adults were rarely carrying pneumococcus; about 10% and less than 5% respectively were infected. Carriage rates observed in 2008 and in 2018 were similar in the age groups observed in both surveys (Fig 2).

Fig 2

Age-stratified carriage prevalence in Nha Trang.

Grey dots indicate the positions of knots for the spline and the open circles and their corresponding 95% binomial CIs the carriage prevalence data that the model was fitted to. The black line and the region highlighted in blue represents the model’s median and 95% quantile estimates. The “x” symbols are added for visual comparison and indicate the carriage prevalence observed in the infant survey. To aid visualisation, we used quadratic scaling of the x-axis. Inset: the distribution of estimated PEI values in infants without (left) and with (right) pneumococcal carriage. CI, confidence interval; PEI, potential exposure index.

We estimate that most (61%) infants had less than a 25% probability of having had close contact with a pneumococcal carrier on the surveyed day; however, for 22% of infants, the probability of exposure was much higher at 50% to 70%. The estimated PEI for each infant was associated with substantial uncertainty (Fig B in S4 File). The median PEI value for pneumococcal carriers was 27% (95% CI: 5.1% to 70%), compared with 15% (95% CI: 4.9% to 61%) for noncarriers, and, from the Mann–Whitney U test, there was a statistically significant difference between PEI values from the 2 groups (p < 0.001).

When adjusting for age and locality, we find that the probability of exposure to pneumococci on the surveyed day and the risk of pneumococcal carriage were highly correlated: for every 10 percentage points increase in PEI, the odds of pneumococcal carriage increased by 22% (95% CI: 15 to 29), i.e., the odds of pneumococcal carriage were 7.1 (95% CI: 4.1 to 12.3) times higher in infants who had at least one effective contact with a pneumococcal carrier (PEI = 1), compared to those who had not (Table C in S4 File).

Age-specific infant exposure to pneumococci

Children between 1 and 4 years of age contributed 38% (95% CI: 22% to 56%) of the total pneumococcal exposure to infants; children 5 to 9 and adults 21 to 40 year olds contributed 22% (95% CI: 16 to 29) and 20% (95% CI: 9.2 to 32), respectively. We estimate that 2- to 6-year-old children are key pneumococcal transmitters to infants, contributing 51% (95% CI: 39 to 63) of the total exposure and that infection risk from other infants or older children and adults is likely low in this setting (Fig 3). The majority of potential pneumococcal exposures (80%) originated from household contacts.

Fig 3

The contribution of different age groups towards the total exposure of infants to pneumococcus.

Bars show the relative contribution of an age group to the total pneumococcal exposure (number of close contacts and their propensity to carry pneumococci) of infants in Nha Trang. Error bars indicate 95% credible intervals. Inset: the contribution of up to 15 year olds to total pneumococcal exposure of infants in Nha Trang.

Discussion

We described in detail the social contacts of infants in Nha Trang, Vietnam. Unlike other age groups, infant contacts are not age assortative and happen largely within the household. We show that close social contacts, as measured in contact surveys on a specific day, and in combination with age specific infection probabilities of such contacts, are highly correlated with the risk of pneumococcal infection of infants in Nha Trang. Thus, we estimate that 1 to 4 year olds and 5 to 9 year olds contribute most (39% (95% CI: 32% to 47%) and 22% (95% CI: 16 to 29), respectively) to the infection pressure to infants in this setting and that 80% of infections are acquired from household contacts.

We have previously shown that pneumococcal infection risk is correlated with the frequency of physical contacts [5,6]. Here, we expand that notion by including the probability that contacts are infected with pneumococci, similar to how age-stratified dynamic mathematical models are constructed [3,25,26], and thus provide direct evidence for the value of social contact structure as a proxy for disease transmission routes in mathematical models. Furthermore, we suggest that the combination of social contacts and age-dependent infection probabilities provides a simple and useful method to identify likely transmission routes, without the need for more complex mathematical modelling or the need for longitudinal data collection [27,28].

Similar to their key role in pneumococcal transmission generally [25,29-31], we find that 2- to 6-year-old children are the likely main direct transmitters of pneumococcal infection to infants. This has important implications for the optimal design of pneumococcal vaccination strategies, particularly those that aim to sustain herd protection while reducing the number of doses given in infancy [10]. While the exact duration of protection from PCVs remains relatively uncertain [32], booster dose schedules may induce longer lasting protection and hence may be preferred in settings with high carriage prevalence in older children and hence a potentially high contribution to the pneumococcal infection pressure from that age group [33].

We find that 76% of contacts and 80% of potential pneumococcal exposures to the infants in this study were with members of their households. In contrast, in the UK, the household contacts had a less dominant role and 11-week- to 12-month-old infants were reported to have less than 50% of their contacts at home [12]. This illustrates the importance of context in the evaluation of transmission routes for infants. For example, while in the UK many children will attend nursery from 6 to 8 months of age, in Vietnam, publicly funded nurseries will only accept children older than 12 months. Thus, infant infection routes may be substantially different in settings where childcare responsibilities are shared with older generations or siblings.

Our study suggests that pneumococcal exposure risk to infants stems mostly from household contacts and particularly siblings. In combination with the commune organised schooling system in Vietnam this suggests that the commune is a major spatial entity in determining pneumococcal infection in infants in Nha Trang. The main mode of transport for families in this community was travel by motorcycle, with the vast majority of families owning and using one for travel. The infants were mostly able to sit but fewer were able to crawl or walk (55% and 12%, respectively), and so for individuals who had not achieved these milestones, contact would likely be initiated by parents or siblings.

A limitation of this study is that there is large uncertainty around the carriage rates in teenagers. However, direct contact with teenagers was rare for our study population and hence the associated uncertainty in infection probability has little impact on the overall results. In settings where teenagers share childcare responsibilities and where high pneumococcal infection rates extend well into teenage years [34-36], such contacts may contribute a larger part to the infection pressure on infants. A further limitation is that contacts were only surveyed on a single day and that carriage status was only assessed about a week after that. The strong correlation between contact behaviour and pneumococcal carriage in our study suggests that, indeed, the contact behaviour on a given day is somewhat representative for the general contact behaviour of that infant. This is an important validation for the adequacy of social contact surveys for informing mathematical transmission models advising public health decision making. Our survey period did not include any major holidays such as the Tết Nguyên Đán, or Lunar New Year. Such events may well change contact patterns and accelerate the spread of pneumococci in infants. It is also worth noting that our estimates for the relative proportions of exposure to infants represent only the direct contributions of each age group. Our study does not allow us to ascertain additional indirect effects of exposure that would arise when considering second generation (or beyond) exposure patterns.

In summary, we provide direct evidence that for pneumococci, and thus likely other respiratory pathogens, social contact surveys indeed add a crucial part in our understanding of transmission pathways. We find that 2- to 6-year-old children are the most likely source of pneumococcal infection in infants in Nha Trang. Thus, it adds support to catch-up vaccination of preschoolers to accelerate impact upon introduction as well as supports schedules aimed at extending direct protection into this age group to establish pronounced herd protection. Similar studies in other settings can help evaluate local pneumococcal transmission routes and hence provide crucial evidence for the discussion on optimal dosing schedules for PCVs.

STROBE checklist of the infant pneumococcal carriage and contact studies.

STROBE, Strengthening the Reporting of Observational Studies in Epidemiology.

(PDF)

Click here for additional data file.

Background questionnaire.

(PDF)

Click here for additional data file.

Contact questionnaire.

(PDF)

Click here for additional data file.

Personal communications permission form.

(PDF)

Click here for additional data file.

Supporting information to the paper.

(DOCX)

Click here for additional data file.

6 Jul 2021

Dear Dr Qian,

Thank you for submitting your manuscript entitled "Pneumococcal exposure routes for infants, a nested cross-sectional survey in Nha Trang, Vietnam" for consideration by PLOS Medicine.

Your manuscript has now been evaluated by the PLOS Medicine editorial staff and I am writing to let you know that we would like to send your submission out for external peer review.

However, before we can send your manuscript to reviewers, we need you to complete your submission by providing the metadata that is required for full assessment. To this end, please login to Editorial Manager where you will find the paper in the 'Submissions Needing Revisions' folder on your homepage. Please click 'Revise Submission' from the Action Links and complete all additional questions in the submission questionnaire.

Please re-submit your manuscript within two working days, i.e. by Jul 08 2021 11:59PM.

Login to Editorial Manager here: https://www.editorialmanager.com/pmedicine

Once your full submission is complete, your paper will undergo a series of checks in preparation for peer review. Once your manuscript has passed all checks it will be sent out for review.

Feel free to email us at plosmedicine@plos.org if you have any queries relating to your submission.

Kind regards,

Caitlin Moyer, Ph.D.

Associate Editor

PLOS Medicine

15 Feb 2022

Dear Dr. Qian,

Thank you very much for submitting your manuscript "Pneumococcal exposure routes for infants, a nested cross-sectional survey in Nha Trang, Vietnam" (PMEDICINE-D-21-02876R1) for consideration at PLOS Medicine.

Your paper was evaluated by a senior editor and discussed among all the editors here. It was also discussed with an academic editor with relevant expertise, and sent to three independent reviewers, including a statistical reviewer. The reviews are appended at the bottom of this email and any accompanying reviewer attachments can be seen via the link below:

[LINK]

In light of these reviews, I am afraid that we will not be able to accept the manuscript for publication in the journal in its current form, but we would like to consider a revised version that addresses the reviewers' and editors' comments. Obviously we cannot make any decision about publication until we have seen the revised manuscript and your response, and we plan to seek re-review by one or more of the reviewers.

In revising the manuscript for further consideration, your revisions should address the specific points made by each reviewer and the editors. Please also check the guidelines for revised papers at http://journals.plos.org/plosmedicine/s/revising-your-manuscript for any that apply to your paper. In your rebuttal letter you should indicate your response to the reviewers' and editors' comments, the changes you have made in the manuscript, and include either an excerpt of the revised text or the location (eg: page and line number) where each change can be found. Please submit a clean version of the paper as the main article file; a version with changes marked should be uploaded as a marked up manuscript.

In addition, we request that you upload any figures associated with your paper as individual TIF or EPS files with 300dpi resolution at resubmission; please read our figure guidelines for more information on our requirements: http://journals.plos.org/plosmedicine/s/figures. While revising your submission, please upload your figure files to the PACE digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at PLOSMedicine@plos.org.

We expect to receive your revised manuscript by Mar 08 2022 11:59PM. Please email us (plosmedicine@plos.org) if you have any questions or concerns.

***Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.***

We ask every co-author listed on the manuscript to fill in a contributing author statement, making sure to declare all competing interests. If any of the co-authors have not filled in the statement, we will remind them to do so when the paper is revised. If all statements are not completed in a timely fashion this could hold up the re-review process. If new competing interests are declared later in the revision process, this may also hold up the submission. Should there be a problem getting one of your co-authors to fill in a statement we will be in contact. YOU MUST NOT ADD OR REMOVE AUTHORS UNLESS YOU HAVE ALERTED THE EDITOR HANDLING THE MANUSCRIPT TO THE CHANGE AND THEY SPECIFICALLY HAVE AGREED TO IT. You can see our competing interests policy here: http://journals.plos.org/plosmedicine/s/competing-interests.

Please use the following link to submit the revised manuscript:

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Your article can be found in the "Submissions Needing Revision" folder.

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Please ensure that the paper adheres to the PLOS Data Availability Policy (see http://journals.plos.org/plosmedicine/s/data-availability), which requires that all data underlying the study's findings be provided in a repository or as Supporting Information. For data residing with a third party, authors are required to provide instructions with contact information for obtaining the data. PLOS journals do not allow statements supported by "data not shown" or "unpublished results." For such statements, authors must provide supporting data or cite public sources that include it.

We look forward to receiving your revised manuscript.

Sincerely,

Caitlin Moyer, Ph.D.

Associate Editor

PLOS Medicine

plosmedicine.org

-----------------------------------------------------------

Requests from the editors:

1. Title: Please revise your title according to PLOS Medicine's style. Your title must be nondeclarative and not a question. It should begin with main concept if possible. "Effect of" should be used only if causality can be inferred, i.e., for an RCT. Please place the study design ("A randomized controlled trial," "A retrospective study," "A modelling study," etc.) in the subtitle (ie, after a colon).

2. Throughout: Please include line numbers with the revised version.

3. Abstract: Methods and Findings: Please explicitly mention the years during which the study took place and main outcome measures. Please specify the ages of the infants.

4. Abstract: Methods and Findings: In the last sentence of the Abstract Methods and Findings section, please describe the main limitation(s) of the study's methodology.

5. Abstract: Conclusions: Please address the study implications without overreaching what can be concluded from the data; beginning with the phrase "In this study, we observed ..." may be useful.

6. Author Summary: At this stage, we ask that you include a short, non-technical Author Summary of your research to make findings accessible to a wide audience that includes both scientists and non-scientists. The Author Summary should immediately follow the Abstract in your revised manuscript. This text is subject to editorial change and should be distinct from the scientific abstract. Please see our author guidelines for more information: https://journals.plos.org/plosmedicine/s/revising-your-manuscript#loc-author-summary

7. Methods: “The study was conducted in Nha Trang, a coastal city in south-central Vietnam with a total population of just over 426,958 and under-five population of 28,495 in the 2018 census (personal communication with Khanh Hoa Health Service).”

The information may be cited in the text as a personal communication with the author if the author provides written permission to be named. Please provide the name of the individual, the affiliation, and date of communication. The individual named must provide written permission to be named. Alternatively, please provide a different appropriate reference.

8. Methods: “In October 2016 a cluster randomised controlled trial was initiated to evaluate alternative pneumococcal conjugate vaccine (PCV) dosing schedules [13].” It would be helpful to describe relevant details of the trial, including recruitment and enrollment of participants. The Methods indicate that 24 of 27 communes were allocated to receive PCV vaccination. Please comment on how this would be expected to impact infant carriage and the PEI estimates, for example.

9. Methods: “In October 2018, two years after the start of the trial, an infant contact survey was nested into the cross-sectional carriage study.” Please provide some information on the PCV vaccine schedule and coverage at the time when the survey was carried out. Please define how the infant population was sampled for the carriage study/contact survey. Please report the response rates, clarify the dates when the contact survey was conducted.

10. Methods: “In an adjusted analysis we included the age group (4-7 and 8-11 months of age)” Please explain the rationale for the categorization of ages into these groups.

11. Methods: “Microbiological culture was subsequently attempted for lytA positive (cycle threshold (Ct) value < 35) or equivocal (Ct value 35-40) samples. Pneumococcal carriage was defined as samples that were both lytA positive/equivocal and culture positive, and subsequently confirmed by microarray [15] .” Please briefly describe the methods for qPCR detection, including primers, as well as for microbiological culture and microarray confirmation.

12. Methods: “We used negative binomial regression to estimate crude rate ratios of the mean number of contacts of participants in different population subgroups.” Please define the population subgroups.

13. Methods: “To estimate age dependent probabilities for pneumococcal carriage in the study setting, we performed Bayesian Penalised B-Spline Regression on logit-transformed carriage probability, obtained from previously reported low-granularity age-stratified pneumococcal carriage across age groups from Nha Trang [16]. We compared our estimates against carriage prevalence observed in young children in the trial. The PEI estimate is based on the contact patterns recorded for a single day but were interpreted as a proxy for the general contact behaviour of the respective infant.”

Please provide additional description of the low-granularity age-stratified carriage data from reference 16 (including years when those data were obtained), as well as more description of how age dependent probabilities obtained were compared to the carriage prevalence observed in young children in the trial (including a reference for those data).

14. Methods: Prospective analysis plan: Did your study have a prospective protocol or analysis plan? Please state this (either way) early in the Methods section.

a) If a prospective analysis plan (from your funding proposal, IRB or other ethics committee submission, study protocol, or other planning document written before analyzing the data) was used in designing the study, please include the relevant prospectively written document with your revised manuscript as a Supporting Information file to be published alongside your study, and cite it in the Methods section. A legend for this file should be included at the end of your manuscript.

b) If no such document exists, please make sure that the Methods section transparently describes when analyses were planned, and when/why any data-driven changes to analyses took place.

c) In either case, changes in the analysis-- including those made in response to peer review comments-- should be identified as such in the Methods section of the paper, with rationale.

15. Methods: STROBE: Please report your study according to the relevant guideline, which can be found here: http://www.equator-network.org/. Please ensure that the study is reported according to the STROBE guideline, if that is most appropriate, and include the completed STROBE checklist as Supporting Information. Please add the following statement, or similar, to the Methods: "This study is reported as per the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guideline (S1 Checklist)."

The STROBE guideline can be found here: http://www.equator-network.org/reporting-guidelines/strobe/

When completing the checklist, please use section and paragraph numbers, rather than page numbers.

16. Results: Please present numerators and denominators for percentages, at least in the Tables (not necessarily each time they're mentioned).

17. Results: For the results described, please report in the text the results of all statistical analyses, with both CIs and p values.

18. Results: “Modelled carriage prevalence in Nha Trang based on carriage observations from a previous study…” please provide a reference for the study.

19. Discussion: Please present and organize the Discussion as follows: a short, clear summary of the article's findings; what the study adds to existing research and where and why the results may differ from previous research; strengths and limitations of the study; implications and next steps for research, clinical practice, and/or public policy; one-paragraph conclusion.

20. Tables and Supporting Information Tables: Please report p values in addition to confidence intervals presented for all analyses. Please also present the unadjusted results for all analyses. In the legends, please note statistical tests used, and factors adjusted for in the analyses.

21. Table S4: Please define “DIC” and “PEI” in the legend.

22. Table S5: Please define “PEI” and explain “Commune ID” in the legend.

23. Table S6: Please define “ PEI” and “VIF” in the legend.

24. Figure S4: Please define “PEI” in the legend.

25. Figure S5: Please define “MCMC” and “PEI” in the legend.

Comments from the reviewers:

Reviewer #1: In the proposed paper, the authors report the results of a large pneumococcus carriage and contact tracing study from Vietnam. They develop an individual-level 'potential exposure index' based on the age-structure of contacts. The descriptions of the number, location and type of contacts is an important addition to the literature, as is the age-specific probabilities of carriage. I only have a few comments.

The authors claim that on average pneumococcal carriers had a higher PEI than non-carriers - however, the uncertainty with these number is (very) high. I do not believe the authors can make this statement - instead they should do a statistical test to see if the PEI levels were different between the two groups.

It would be useful to understand the distributions of PEIs - overall and within each age group. At a population level each age group is assumed to have a normally distributed PEI values - but it is unclear how appropriate this is.

The authors state that individuals that almost certainly had an exposure (PE=1) had a high odds of being positive - however, we do not know how common that is and what approximately 1 means (>0.95?). I assume that this implies that carriage within an age group is randomly distributed and that e.g., in a classroom of children each will have the same probability of being positive and their risks will not be correlated between members of the class - this should be stated and discussed.

I would move the logistic regression equation to the main text - it will make it clear what approach the authors have taken.

The authors assume a beta distribution for the PEI distributions - it wasn't clear how appropriate this assumption was - a histogram of the posterior with a beta distribution would help.

It would be good to include a discussion of ways to improve PEI estimation - in particular in reducing uncertainty. For example, would looking at serotype/GPSC distribution help?

Reviewer #2: In this study, the authors combine a high-quality dataset of pneumococcal carriage with detailed survey data to infer the contribution of different age groups to transmission of pneumococcus. The survey data are used to develop a novel index that quantifies an individual child's risk of exposure given their contacts and the prevalence of pneumococcus in those contacts (by age). This is a clean, intuitive approach that aids in understanding the importance of different age groups for transmission. The authors also provide a well-documented Github repository, which should allow others to replicate the analyses in other settings.

My only major comment is about the interpretation and directionality of the exposure index. For instance the authors "estimate that 2 to 6 year old children are key pneumococcal transmitters to infants, contributing 51% (95% CI: 39-63) of the total exposure, and that infection risk from other infants or older children and adults is likely low in this setting". Thinking about population-level effects, 51% of exposure is directly attributed to kids in that age group. But if the 2-6 year olds are also responsible for a large fraction of transmission to older children and adults, the effect would be even greater because the 2-6 year olds transmit to adults, who then transmit to infants. For example, in an extreme situation where all transmission in the population originated with 2-6 year olds, Is there a way to account for this type of dependency? If not, it should be clear that these estimates, which capture the direct contribution from the age group, represent a lower bound for the total contribution of that age group.

The authors could also add some discussion about the implications of their findings for vaccination strategies. What is the potential policy response to knowing that 2-6 year olds are important for transmission? Catch-up campaigns? different dose timing?

Reviewer #3: This is an interesting paper combining pneumococcal carriage and social contact data to assess the risk of pneumococcal transmission as a function of social contacts among infants, building on previous studies in Uganda and Fiji.

The authors have used an elegant statistical approach to estimate the potential exposure to a pneumococcal carrier (and hence infection risk) based on knowledge about the social contacts of infants and the probability of carriage of their contacts. The method allows to further explore the type of social contacts that matter for pneumococcal transmission. The methods are clear and the paper is well written

I have few very comments on the paper:

1. In their calculation of the Potential Exposure Index, authors are using age-specific carriage probabilities. Have they looked at sex too, particularly among adult populations (caregivers, mothers). While the carriage prevalence in that setting is very low across all age groups, and the carriage difference between adult age groups is likely small too, it would be useful to explore

2. It is a little unclear from the description whether there were variations among the study populations in vaccination schedule and/or coverage, given that this was nested within a trial, and whether carriage estimates differed between the different datasets used (pre-trial, trial). Please clarify

Any attachments provided with reviews can be seen via the following link:

[LINK]

29 Mar 2022

Submitted filename: Letter to Editors and Reviewers_Submission.pdf

Click here for additional data file.

26 Apr 2022

Dear Dr. Qian,

Thank you very much for re-submitting your manuscript "Pneumococcal exposure routes for infants, a nested cross-sectional survey in Nha Trang, Vietnam" (PMEDICINE-D-21-02876R2) for review by PLOS Medicine.

I have discussed the paper with my colleagues and the academic editor and it was also seen again by three reviewers. I am pleased to say that provided the remaining editorial and production issues are dealt with we are planning to accept the paper for publication in the journal.

The remaining issues that need to be addressed are listed at the end of this email. Any accompanying reviewer attachments can be seen via the link below. Please take these into account before resubmitting your manuscript:

[LINK]

***Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.***

In revising the manuscript for further consideration here, please ensure you address the specific points made by each reviewer and the editors. In your rebuttal letter you should indicate your response to the reviewers' and editors' comments and the changes you have made in the manuscript. Please submit a clean version of the paper as the main article file. A version with changes marked must also be uploaded as a marked up manuscript file.

Please also check the guidelines for revised papers at http://journals.plos.org/plosmedicine/s/revising-your-manuscript for any that apply to your paper. If you haven't already, we ask that you provide a short, non-technical Author Summary of your research to make findings accessible to a wide audience that includes both scientists and non-scientists. The Author Summary should immediately follow the Abstract in your revised manuscript. This text is subject to editorial change and should be distinct from the scientific abstract.

We expect to receive your revised manuscript within 1 week. Please email us (plosmedicine@plos.org) if you have any questions or concerns.

We ask every co-author listed on the manuscript to fill in a contributing author statement. If any of the co-authors have not filled in the statement, we will remind them to do so when the paper is revised. If all statements are not completed in a timely fashion this could hold up the re-review process. Should there be a problem getting one of your co-authors to fill in a statement we will be in contact. YOU MUST NOT ADD OR REMOVE AUTHORS UNLESS YOU HAVE ALERTED THE EDITOR HANDLING THE MANUSCRIPT TO THE CHANGE AND THEY SPECIFICALLY HAVE AGREED TO IT.

Please ensure that the paper adheres to the PLOS Data Availability Policy (see http://journals.plos.org/plosmedicine/s/data-availability), which requires that all data underlying the study's findings be provided in a repository or as Supporting Information. For data residing with a third party, authors are required to provide instructions with contact information for obtaining the data. PLOS journals do not allow statements supported by "data not shown" or "unpublished results." For such statements, authors must provide supporting data or cite public sources that include it.

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript.

Please note, when your manuscript is accepted, an uncorrected proof of your manuscript will be published online ahead of the final version, unless you've already opted out via the online submission form. If, for any reason, you do not want an earlier version of your manuscript published online or are unsure if you have already indicated as such, please let the journal staff know immediately at plosmedicine@plos.org.

If you have any questions in the meantime, please contact me or the journal staff on plosmedicine@plos.org.

We look forward to receiving the revised manuscript by Apr 28 2022 11:59PM.

Sincerely,

Caitlin Moyer, Ph.D.

Associate Editor

PLOS Medicine

plosmedicine.org

------------------------------------------------------------

Requests from Editors:

1. Title: Please place the study design in the subtitle (ie, after a colon) and please update this in the manuscript submission system in addition to the main text. We suggest revising to: “Association of pneumococcal carriage in infants with the risk of carriage among their contacts in Nha Trang, Vietnam: A nested cross-sectional survey”

2. Abstract: Background: Please add a final sentence that clearly states the study question.

3. Abstract: Methods and Findings, Line 10: Please add the sentence punctuation: “In October 2018, we conducted a nested cross-sectional contact and pneumococcal carriage survey in randomly selected 4 to 11 months old infants across all 27 communes of Nha Trang, Vietnam.”

4. Abstract: Methods and Findings: Line 16: Please report the actual numbers, in addition to percentages.

5. Author summary: Please reformat the author summary, providing 2-3 single sentence bullet points under each of the following headings. Why Was This Study Done?; What Did the Researchers Do and Find?; What Do These Findings Mean? Please see https://journals.plos.org/plosmedicine/s/revising-your-manuscript#loc-author-summary for guidelines and examples.

We suggest reformatting as follows, or similar:

Why was this study done?

-There is little information in the current literature on pneumococcal transmission to infants. -Since individuals in this age group are at major risk of pneumococcal disease and at potentially reduced protection in reduced dose pneumococcal vaccination schedules, we carried out a study to identify their close contacts and likely routes of transmission.

What did the researchers do and find?

-We identified all 7428 physical contacts of 1583 infants in Nha Trang, a coastal city in Vietnam. -We found that there was a high correlation between infant infection and their probability of contact with at least one pneumococcal carrier on the surveyed day.

-Preschool-age children contributed around half of the total direct pneumococcal exposure risk to infants in Nha Trang. Around 80% of the exposure risk came from contact with household members.

What do these findings mean?

-Social contact surveys can help to identify possible transmission pathways.

- Infants in Nha Trang may be likely to receive substantial indirect protection against pneumococcal infection from vaccinated older siblings.

6. Methods: Line 93-94: Please also include a reference to the personal communication letter in the Supporting Information.

7. Methods: Line 123, 128, and throughout: The terms gender and sex are not interchangeable (as discussed in http://www.who.int/gender/whatisgender/en/ ); please use the appropriate term.

8. Methods: Line 134-135: Please specify if this is “written informed consent” for both contact and carriage studies.

9. Methods: Line 250: Please change this to “National Institute of Hygiene and Epidemiology in Hanoi, Vietnam”

10. Methods: Line 163: Prospective analysis plan: Please explicitly state that the study had no prospective analysis plan. Please explicitly state when these analyses were planned, and when and why any data-driven changes to analyses took place, including those made in response to peer review comments.

11. Results: Line 315-316: Please report the p value in the text for: “...from the Mann-Whitney U test, there was a statistically significant difference between PEI values from the two groups.”

12. Lines 418-430: Please remove the “Author contributions” and “Funding” sections from the manuscript text, and please make sure all information is completely and accurately entered into the relevant sections of the manuscript submission system.

13. Figures and Tables: Please be sure that each text, table, and figure has a descriptive title, and a legend describing the figure/table. We suggest numbering the figures/tables within the supporting information separately from the main text (begin with S1 Table, for example).

14. Table S3: Please report p values in addition to 95% CIs.

15. References: Please check each reference and please use the "Vancouver" style for reference formatting, and see our website for other reference guidelines https://journals.plos.org/plosmedicine/s/submission-guidelines#loc-references

Please check journal title abbreviations. For example, reference 3 should be formatted as PLoS Med.

Comments from Reviewers:

Reviewer #1: I have no additional comments. I congratulate the authors on an excellent paper.

Reviewer #2: The authors have addressed my comments.

Reviewer #3: Comments have been thoroughly addressed

Any attachments provided with reviews can be seen via the following link:

[LINK]

6 May 2022

Submitted filename: Letter_To_Editors_May.pdf

Click here for additional data file.

9 May 2022

Dear Dr Qian,

On behalf of my colleagues and the Academic Editor, Mirjam Kretzschmar, I am pleased to inform you that we have agreed to publish your manuscript "Association of pneumococcal carriage in infants with the risk of carriage among their contacts in Nha Trang, Vietnam: A nested cross-sectional survey" (PMEDICINE-D-21-02876R3) in PLOS Medicine.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. Please be aware that it may take several days for you to receive this email; during this time no action is required by you. Once you have received these formatting requests, please note that your manuscript will not be scheduled for publication until you have made the required changes.

In the meantime, please log into Editorial Manager at http://www.editorialmanager.com/pmedicine/, click the "Update My Information" link at the top of the page, and update your user information to ensure an efficient production process.

Please also address the following editorial requests:

-Line 58-59: Please revise this sentence to: “...and that preschool age children may be the largest reservoir for pneumococcal transmission to infants in Nha Trang, Vietnam.”

-Line 68: Please change “vaccinate” to “vaccination”.

-Line 202: Please revise to: "The infant contact study is a cross sectional study and no prospective analysis plan exists. The plan for the current study combining contact and carriage data was conceived during a collaborative meeting that happened after the infant contact and pneumococcal carriage surveys were conducted, but without being informed by the data.” if this is accurate.

-References: Please update reference 4 with the journal publication information (https://doi.org/10.1371/journal.pcbi.100909). Please update the journal title abbreviation for reference 28 to PLoS One.

-Supporting information files list: Please submit files as part of the supporting information in the manuscript submission system. Please do not include links to the external versions of the files (e.g. https://drive.google.com/file…). Please include only one version of the Supplemental Material.

-Figure 2: In the legend, you describe red and green dots to indicate carriage prevalence data, however these points seem to be represented by open circle and “x” symbols, respectively. Please update the legend.

PRESS

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To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Thank you again for submitting to PLOS Medicine. We look forward to publishing your paper.

Sincerely,

Caitlin Moyer, Ph.D.

Associate Editor

PLOS Medicine