Factors associated with pneumococcal carriage and density in children and adults in Fiji, using four cross-sectional surveys.

This study describes predictors of pneumococcal nasopharyngeal carriage and density in Fiji. We used data from four annual (2012-2015) cross-sectional surveys, pre- and post-introduction of ten-valent pneumococcal conjugate vaccine (PCV10) in October 2012. Infants (5-8 weeks), toddlers (12-23 months), children (2-6 years), and their caregivers participated. Pneumococci were detected and quantified using lytA qPCR, with molecular serotyping by microarray. Logistic and quantile regression were used to determine predictors of pneumococcal carriage and density, respectively. There were 8,109 participants. Pneumococcal carriage was negatively associated with years post-PCV10 introduction (global P<0.001), and positively associated with indigenous iTaukei ethnicity (aOR 2.74 [95% CI 2.17-3.45] P<0.001); young age (infant, toddler, and child compared with caregiver participant groups) (global P<0.001); urban residence (aOR 1.45 [95% CI 1.30-2.57] P<0.001); living with ≥2 children <5 years of age (aOR 1.42 [95% CI 1.27-1.59] P<0.001); low family income (aOR 1.44 [95% CI 1.28-1.62] P<0.001); and upper respiratory tract infection (URTI) symptoms (aOR 1.77 [95% CI 1.57-2.01] P<0.001). Predictors were similar for PCV10 and non-PCV10 carriage, except PCV10 carriage was negatively associated with PCV10 vaccination (0.58 [95% CI 0.41-0.82] P = 0.002) and positively associated with exposure to household cigarette smoke (aOR 1.21 [95% CI 1.02-1.43] P = 0.031), while there was no association between years post-PCV10 introduction and non-PCV10 carriage. Pneumococcal density was positively associated with URTI symptoms (adjusted median difference 0.28 [95% CI 0.16, 0.40] P<0.001) and toddler and child, compared with caregiver, participant groups (global P = 0.008). Predictors were similar for PCV10 and non-PCV10 density, except infant, toddler, and child participant groups were not associated with PCV10 density. PCV10 introduction was associated with reduced the odds of overall and PCV10 pneumococcal carriage in Fiji. However, after adjustment iTaukei ethnicity was positively associated with pneumococcal carriage compared with Fijians of Indian Descent, despite similar PCV10 coverage rates.

Introduction

Pneumococcal disease is a leading cause of childhood morbidity and mortality worldwide [1]. Pneumococcal disease is preceded by pneumococcal nasopharyngeal carriage, and the severity of pneumococcal pneumonia is associated with bacterial load (density) of pneumococci in the nasopharynx [2, 3]. Public health interventions to prevent pneumococcal disease can be improved by identifying the factors associated with pneumococcal carriage. Determining factors associated with higher pneumococcal carriage density could aid estimation of pneumococcal pneumonia prevalence in childhood pneumonia studies [2].

In low- and middle-income countries, risk factors for pneumococcal nasopharyngeal carriage vary [4-8] and few studies have investigated factors associated with the density of pneumococcal carriage in healthy populations [2, 9–11]. Common factors positively associated with pneumococcal carriage in low- and middle-income countries include indigenous ethnicity, passive smoking; co-colonisation with Haemophilus influenzae, childcare attendance, poverty, acute respiratory infection, living with young children, and being under five years old [4, 8, 12]. In studies from low- and middle-income countries, higher pneumococcal density has been positively associated with the symptoms of upper respiratory tract infection, presence of a febrile acute respiratory infection in children [13], rainy season, severe pneumonia, viral co-infection, radiologically confirmed pneumococcal pneumonia, and encapsulated serotypes (compared with non-encapsulated serotypes) [2, 9–11, 13–16].

Pneumococcal conjugate vaccines (PCV) reduce vaccine-type carriage and disease [17, 18]. However, serotype replacement with non-vaccine-type carriage occurs following the introduction of PCV which can result in non-vaccine-type disease [19, 20]. In the post-PCV era, it is largely unknown what the impact of PCV is on the risk factors for pneumococcal carriage and density in low- and middle-income countries in the Asia-Pacific region.

In 2012, Fiji introduced the ten-valent PCV (PCV10). Six years before PCV10 was introduced, factors associated with pneumococcal carriage in healthy 3–13 month old Fijians included indigenous iTaukei (iTaukei) ethnicity and having symptoms of an upper respiratory tract infection (URTI)[4]. iTaukei ethnicity was also associated with higher median pneumococcal density among 17 month old Fijians [21]. Fiji provides an opportunity to investigate factors associated with pneumococcal carriage and density in the post-PCV10, in a tropical, upper middle-income setting. As part of a PCV10 impact evaluation on pneumococcal carriage, we previously reported that the prevalence of vaccine-type carriage declined in both iTaukei and Fijians of Indian Descent (FID) three years after the introduction of PCV10, but carriage of non-vaccine-type carriage increased in iTaukei infants and toddlers [22]. The aim of this study is to determine the factors associated with carriage and density of pneumococci (overall, vaccine-type, and non-vaccine-type) in Fiji up to three years following PCV10 introduction.

Materials and methods

Setting

The majority of the Fijian population (81%) lives on Viti Levu, the bigger of Fiji’s two main islands. Greater than half (56.8%) of the population are iTaukei and 37.8% are FID. This study was conducted in Suva, and the surrounding areas, where over one-third of the population lives [23]. PCV10 was introduced nationally in October 2012 to be administered at 6, 10, and 14 weeks of age, with no catch-up campaign. The national coverage of the third dose of PCV10 one, two, and three years post-introduction was 84.9%, 84.9%, and 89.0%, respectively [24-26].

Cross-sectional carriage studies

The design and methods for these cross-sectional studies have been described elsewhere [22]. Briefly, four annual cross-sectional carriage surveys were undertaken: one pre-PCV10 (2012), and then annually thereafter (2013–2015). Purposive quota sampling achieved a sample proportionate to the national iTaukei: FID (3:2) and rural: urban (1:1) ratios [4, 23, 27]. Each year, approximately 500 participants were recruited into each of the following four groups: infants 5–8 weeks (infants), toddlers 12–23 months (toddlers), children 2–6 years (children), and their parents/guardians (caregivers). Age groups were selected in order to best answer the primary research question regarding impact of PCV10 on pneumococcal carriage prevalence in Fiji, and as described previously, were based on those most likely to benefit from direct and indirect effects of PCV10; those likely to have the highest pneumococcal carriage prevalence; those age-eligible for PCV10 vaccination; and those most likely to transmit or be in contact with transmitters of pneumococci [22]. This analysis used the same study population.

Participants were recruited from two health centres in the Greater Suva area, and from surrounding communities. Eligibility criteria included age or being a caregiver, and for non-infant participants, that they had lived in the area for at least three months. Those with an axillary temperature ≥37.0°C were excluded. For the pre-PCV10 survey, any receipt of PCV10 was an exclusion criterion for all participant groups. For subsequent surveys, only infants who had ever received PCV10 were excluded.

Study staff interviewed caregivers and recorded individual level participant characteristics on data collection forms. Variables collected included: self-reported ethnicity, sex, residential location, antibiotic use in the fortnight preceding the survey, exposure to household cigarette smoke, coryza, symptoms of allergic rhinitis, cough, ear discharge, number of children less than five years in the household, and weekly family income. Caregivers reported their own, and their participating child’s or children’s ethnicity, according to options defined by the investigator (iTaukei, FID, or other) and recognized by Fijian population[23]. PCV10 vaccination status for infants and children was obtained from written records. As PCV10 was unavailable privately, caregivers were assumed to be PCV10 unvaccinated. A binary variable for symptoms of URTI was derived from the presence of one or more of the following: coryza, allergic rhinitis, cough, or ear discharge. A binary variable for low family income was derived, defined as family income on / above or below the basic needs poverty line (28].

Trained study nurses collected nasopharyngeal samples using flocked nylon swabs (COPAN FLOQSwabsTM), which were transported and stored according to standard methods, as described previously [22, 29]. Microbiological analyses were undertaken at the Murdoch Children’s Research Institute in Melbourne, Australia as described previously [22]. In brief, pneumococci were detected using real-time quantitative-polymerase chain reaction targeting the lytA gene, with molecular serotyping by microarray [29-31]. Laboratory staff were blinded to participant characteristic data. Detection of any pneumococci in swab samples, including non-encapsulated lineages, was defined as overall pneumococcal carriage. Detection of serotypes included in PCV10 (serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F) was defined as PCV10 carriage, and detection of serotypes not included in PCV10, including non-encapsulated lineages, was defined as non-PCV10 carriage. Detection by microarray of a PCV10 serotype and a non-PCV10 serotype from the same swab sample was recorded as positive for both PCV10 and non-PCV10 serotype carriage [22]. Any detection of a serotype by microarray was considered positive, regardless of relative abundance [22]. Non-encapsulated lineages were classified based upon previously described genetic variants [32]. We determined pneumococcal density only for pneumococcal positive samples, and reported it in genome equivalents per ml (GE / ml). Participant characteristic data were double entered, and validated, in an EpiData 3.1 database [33]. Microbiological outcome data were entered into Microsoft Excel (Excel 2013) and merged with characteristic data in Stata 15.1 [34].

Statistical analyses

Participant characteristics were summarised using counts and percentages. We built logistic and quantile regression models to investigate the factors associated with pneumococcal carriage and density, respectively. Empirical univariable results (p<0.2) and a priori selection informed variable choice for multivariable models. Factors assessed empirically included residential location, participant sex, two or more children under five years living in the household, low family income, exposure to household cigarette smoke, and recent antibiotic use. Variables selected a priori included PCV10 vaccination, survey year, ethnicity, participant group, and URTI symptoms [4, 14]. Interaction terms for ethnicity with PCV10 vaccination status, and with survey year, were assessed to account for the potential differential effect of PCV10 vaccination, or number of years post-PCV10 introduction, on pneumococcal carriage and density by ethnicity. We also assessed potential interaction between ethnicity and other socio-demographic factors in the models. Interaction terms were included as indicated, with significance level set at P<0.05. Estimates of the association of participant characteristics with carriage and density were reported as odds ratios and differences in medians, respectively, with 95% confidence intervals (95% CI) and P-values. Pneumococcal density data were log10 transformed prior to analyses, and analyses of pneumococcal density were restricted to pneumococcal carriers. Only 14/38 participants who identified as “other” ethnicity had pneumococcal positive samples, so were excluded from inferential analyses. Merged data were cleaned and analysed in Stata 15.1 [34].

Ethics statement

This study was carried out in accordance with the protocols approved by the Fijian National Health Research and Ethics Review Committee (201228), and The University of Melbourne Health Sciences Human Ethics Sub-Committee (1238212). Study staff discussed the study with caregivers, and written informed consent was completed prior to any study procedures. Participants were not offered any incentive to participate.

Results

Participant characteristics

There were 8,109 participants, with characteristics shown in Table 1. The overall vaccination rate of 13.6% reflects the pooled participant group, most (85.8%) of which were not age-eligible to receive PCV10. Similar numbers of people participated per survey year and by participant group [22]. Few had used antibiotics in the preceding two weeks. Forty-eight participant swab samples were excluded from microbiological analysis due to insufficient volume, sample loss, or labelling errors. A further 61 pneumococcal positive samples were excluded from serotyping due to biological reasons or technical issues. Of the 8,061 participants for whom swab sample results were available, 30.5% tested positive for pneumococci. Among the 8,000 serotyped samples, PCV10 carriage was uncommon (8.9%), and 23.9% of participants carried non-PCV10 pneumococci. Carriage of non-encapsulated pneumococci was rare (390 / 8,000, 4.9%). Density data was unavailable for one pneumococcal carrier. Overall carriage median density was 5.0 log10 GE/ml (4.2–5.7), while those for PCV10, non-PCV10 carriage, and non-encapsulated lineages were 4.9 log10 GE/ml (4.1–5.6), 4.9 log10 GE/ml (4.1–5.7), and 4.3 log10 GE/ml (3.7–4.9), respectively [22].

Table 1

Characteristics of participants in four annual cross-sectional community nasopharyngeal carriage surveys, 2012–2015, Fiji (n = 8,109 ).

Characteristics		Summary statistic
PCV10 vaccinatedb, n (%)		1105 (13.6)
Survey year, n (%)
	Pre-PCV10 (2012)	2025 (25.0)
	1 year post-PCV10 (2013)	2042 (25.2)
	2 years post-PCV10 (2014)	2022 (24.9)
	3 years post PCV10 (2015)	2020 (24.9)
Ethnicity, n (%)
	Fijian of Indian Descent	3236 (39.9)
	iTaukei	4835 (59.6)
	Other	38 (0.5)
Participant group, n (%)
	Infants (5–8 weeks)	2006 (24.7)
	Toddlers (12–23 months)	2004 (24.7)
	Children (2–6 years)	2052 (25.3)
	Caregivers	2047 (25.3)
Residential location, n (%)
	Rural	3944 (48.6)
	Urban	4165 (51.4)
Female sex, n (%)		4683 (57.8)
Two or more children under five years in the household, n (%)		n = 8106
		4004 (49.4)
Low family incomec, n (%)		n = 7831
		4599 (58.7)
Symptoms of URTI, n (%)		2092 (25.8)
Exposure to household cigarette smoke, n (%)		4353 (53.7)
Antibiotic use in past fortnight, n (%)		n = 8105
		357 (4.4)
Pneumococcal carriage, n / N (%)
	Overalld	n = 8061
		2456 (30.5)
	PCV10 serotypese	n = 8000
		713 (8.9)
	Non-PCV10 serotypesf	n = 8000
		1915 (23.9)
	Non-encapsulated pneumococcig	n = 8000
		390 (4.9)
Pneumococcal densityh, n, median log10 GE/ml (IQR)
	Overall	2455, 5.0 (4.2–5.7)
	PCV10 serotypes	713, 4.9 (4.1–5.6)
	Non-PCV10 serotypes	1915, 4.9 (4.1–5.7)
	Non-encapsulated pneumococci	390, 4.3 (3.7–4.9)

Abbreviations: PCV10, ten-valent pneumococcal conjugate vaccine; URTI, upper respiratory tract infection; IQR, interquartile range.

a Unless otherwise specified

b Two doses of PCV10 given before the age of 12 months, or one or more doses of PCV10 given at or after 12 months of age[35]

c Weekly family income below the basic needs poverty line (

d Any pneumococci, including non-encapsulated lineages

e Pneumococcal serotypes included in PCV10 (serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F)

f Pneumococcal serotypes not included in PCV10, including non-encapsulated lineages

g Includes carriage of any of the following non-encapsulated lineages: NT, NT1, NT2, NT2/NT3b, NT3a, NT3b, NT4a, NT4b

h Only includes participants who were carriers of indicated pneumococcal types

c Weekly family income below the basic needs poverty line (28]

Factors associated with overall carriage

ITaukei ethnicity, young age (infant, toddler, and child participant groups vs caregivers), urban residence, living with two or more children under five years, low family income, and URTI symptoms were positively associated with overall carriage (Table 2). Survey year was negatively associated with overall carriage. There was evidence of protection from PCV10 vaccination against overall carriage, however the confidence interval crossed the null value. There was evidence of an interaction between survey year and ethnicity (global P<0.001), but no evidence of an interaction between PCV10 vaccination status and ethnicity (global P = 0.880).

Table 2

Unadjusted and adjusted odds ratios of overall pneumococcal carriage in association with participant characteristics in four cross-sectional carriage surveys pre-PCV10 (2012) and annually thereafter (2013–2015) in Fiji (n = 8,023).

Exposure		Overall carriagea n/N (%)	Unadjusted odds ratio (95% CI)	P-value	Adjusted odds ratio (95% CI)	P-value
PCV10 vaccination status				<0.001		0.065
	Not vaccinated	2027 / 6931 (29.3)	ref		ref
	Vaccinatedb	415 / 1092 (38.0)	1.48 (1.30–1.69)		0.82 (0.66–1.01)
Survey year				<0.001		<0.001
	Pre-PCV10 (2012)	708 / 2001 (35.4)	ref		ref
	1 year post-PCV10 (2013)	655 / 2033 (32.2)	0.87 (0.76–0.99)		0.67 (0.51–0.88)
	2 years post-PCV10 (2014)	433 / 1997 (21.7)	0.51 (0.44–0.58)		0.49 (0.36–0.66)
	3 years post PCV10 (2015)	646 / 1992 (32.4)	0.88 (0.77–1.00)		0.62 (0.46–0.83)
Ethnicity				<0.001		<0.001
	Fijian of Indian Descent	496 / 3218 (15.4)	ref		ref
	iTaukei	1946 / 4805 (40.5)	3.74 (3.34–4.18)		2.74 (2.17–3.45)
Participant group				<0.001		<0.001
	Caregivers	193 / 2035 (9.5)	ref		Ref
	Infants (5–8 weeks)	516 / 1974 (26.1)	3.38 (2.82–4.04)		4.15 (3.40–5.06)
	Toddlers (12–23 months)	845 / 1986 (42.6)	7.07 (5.95–8.40)		8.88 (7.13–11.07)
	Children (2–6 years)	888 / 2028 (43.8)	7.43 (6.26–8.83)		8.48 (6.99–10.29)
Residential location				<0.001		<0.001
	Rural	1070 / 3911 (27.4)	ref		ref
	Urban	1372 / 4112 (33.4)	1.33 (1.21–1.46)		1.45 (1.30–2.57)
Participant sex				<0.001		0.300
	Male	1167 / 3385 (34.5)	ref		ref
	Female	1275 / 4638 (27.5)	0.72 (0.65–0.79)		1.06 (0.95–1.19)
Number of children < 5 years living in the householdc				<0.001		<0.001
	Less than two	955/4067 (23.5)	ref		ref
	Two or more	1487 / 3953 (37.6)	1.96 (1.78–2.16)		1.42 (1.27–1.59)
Family income leveld				<0.001		<0.001
	Not low	801 / 3193 (25.1)	ref		ref
	Low	1523 / 4558 (33.4)	1.50 (1.35–1.66)		1.44 (1.28–1.62)
Symptoms of URTI				<0.001		<0.001
	Not present	1529 / 5955 (25.7)	ref		ref
	Present	913 / 2068 (44.2)	2.29 (2.06–2.54)		1.77 (1.57–2.01)
Household cigarette smoke				0.105		0.555
	No exposure	1099 / 3729 (29.5)	ref		ref
	Exposure	1343 / 4303 (31.2)	1.08 (0.98–1.19)		0.97 (0.87–1.08)
Antibiotic use in previous fortnighte				0.350
	Not used	2325 / 7667 (30.3)	ref
	Used	115 / 352 (32.7)	1.11 (0.89–1.40)

Abbreviations: CI, confidence interval; PCV10, ten-valent pneumococcal conjugate vaccine; URTI, upper respiratory tract infection.

a Any pneumococci, including non-encapsulated lineages

b Two doses of PCV10 given before the age of 12 months, or one or more doses of PCV10 given at or after 12 months of age[35]

c Data on number of children under five years living in the household were missing for three participants, of whom none were pneumococcal carriers

d Weekly family income below the basic needs poverty line (

e Data on antibiotics use were missing for four participants, of whom two were pneumococcal carriers.

d Weekly family income below the basic needs poverty line (28]; data on family income were missing for 272 participants, of whom 118 were pneumococcal carriers

Factors associated with PCV10 carriage

iTaukei ethnicity, young age (infant, toddler, and child participant groups), urban residence, living with two or more children under five years, low family income, symptoms of URTI, and exposure to household cigarette smoke were positively associated with PCV10 carriage (Table 3). PCV10 vaccination and survey year were negatively associated with PCV10 carriage. There was no evidence of an interaction between PCV10 vaccination status (P = 0.902) or survey year (P = 0.171) and ethnicity with regard to PCV10 carriage.

Table 3

Unadjusted and adjusted odds ratios of PCV10 pneumococcal carriage in association with participant characteristics in four cross-sectional carriage surveys pre-PCV10 (2012) and annually thereafter (2013–2015) in Fiji (n = 7,962).

Exposure		PCV10 carriagea n / N (%)	Unadjusted odds ratio (95% CI)	P-value	Adjusted odds ratio (95% CI)	P-value
PCV10 vaccination status				0.043		0.002
	Not vaccinated	629 / 6875 (9.2)	ref		ref
	Vaccinatedb	79 / 1087 (7.3)	0.78 (0.61–0.99)		0.58 (0.41–0.82)
Survey year				<0.001		<0.001
	Pre-PCV10 (2012)	275 / 1975 (13.9)	ref		ref
	1 year post-PCV10 (2013)	216 / 2022 (10.7)	0.74 (0.61–0.89)		0.74 (0.60–0.91)
	2 years post-PCV10 (2014)	102 / 1987 (5.13)	0.33 (0.26–0.42)		0.40 (0.30–0.53)
	3 years post PCV10 (2015)	115 / 1978 (5.8)	0.38 (0.30–0.48)		0.46 (0.35–0.61)
Ethnicity				<0.001		<0.001
	Fijian of Indian Descent	147 / 3206 (4.6)	ref		ref
	iTaukei	561 / 4756 (11.8)	2.78 (2.31–3.36)		2.70 (2.21–3.30)
Participant group				<0.001		<0.001
	Caregivers	41 / 2029 (2.0)	ref		ref
	Infants (5–8 weeks)	121 / 1946 (6.2)	3.21 (2.24–4.61)		3.60 (2.45–5.30)
	Toddlers (12–23 months)	277 / 1972 (14.1)	7.92 (5.67–11.07)		9.76 (6.67–14.37)
	Children (2–6 years)	269 / 2015 (13.4)	7.47 (5.34–10.44)		7.65 (5.32–11.00)
Residential location				0.001		0.001
	Rural	304 / 3880 (7.8)	ref		ref
	Urban	404 / 4082 (9.9)	1.29 (1.11–1.51)		1.34 (1.13–1.58)
Participant sex				<0.001		0.772
	Male	352 / 3358 (10.5)	ref		ref
	Female	356 / 4604 (7.7)	0.72 (0.61–0.84)		0.98 (0.82–1.16)
Number of children < 5 years living in the householdc				<0.001		0.040
	Less than two	283 / 4035 (7.0)	ref		ref
	Two or more	425 / 3924 (10.8)	1.61 (1.38–1.88)		1.20 (1.01–1.43)
Family income leveld				<0.001		0.004
	Not low	207 / 3175 (6.5)	ref		ref
	Low	463 / 4518 (10.3)	1.64 (1.38–1.94)		1.31 (1.09–1.57)
Symptoms of URTI				<0.001		<0.001
	Not present	435 / 5903 (7.4)	ref		ref
	Present	273 / 2059 (13.3)	1.92 (1.64–2.26)		1.42 (1.19–1.70)
Household cigarette smoke				<0.001		0.031
	No exposure	283 / 3689 (7.7)	ref		ref
	Exposure	425 / 4273 (10.0)	1.33 (1.14–1.56)		1.21 (1.02–1.43)
Antibiotic use in previous fortnighte				0.012		0.367
	Not used	663 / 7610 (8.7)	ref		ref
	Used	44 / 348 (12.6)	1.52 (1.09–2.10)		0.84 (0.58–1.22)

Abbreviations: CI, confidence interval; PCV10, ten-valent pneumococcal conjugate vaccine; URTI, upper respiratory tract infection.

a Pneumococcal serotypes included in PCV10 (serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F)

b Two doses of PCV10 given before the age of 12 months, or one or more doses of PCV10 given at or after 12 months of age [35]

c Data on number of children under five years living in the household were missing for three participants, of whom none were PCV10 pneumococcal carriers

d Weekly family income below the basic needs poverty line (

e Data on antibiotics use were missing for four participants, of whom one was a PCV10 pneumococcal carrier.

d Weekly family income below the basic needs poverty line (28]; data on family income were missing for 269 participants, of whom 38 were PCV10 pneumococcal carriers

Factors associated with non-PCV10 carriage

iTaukei ethnicity, young age (infant, toddler, and child participant groups), urban residence, living with two or more children younger than five years, low family income, and URTI symptoms were positively associated with non-PCV10 carriage (S1 Table). As with overall carriage, there was evidence of an interaction between survey year and ethnicity, as the two ethnic groups had differential odds of non-PCV10 carriage (P<0.001), but no evidence of interaction between PCV10 vaccination status and ethnicity (P = 0.856).

Factors associated with overall pneumococcal density

Toddler and child participant groups, and symptoms of URTI were positively associated with density of overall pneumococcal carriage (Table 4). There was evidence of an association between iTaukei ethnicity and overall pneumococcal density, however the confidence interval included the null value. Although the adjusted median difference in overall pneumococcal carriage density increased in the first two years after the introduction of PCV10, this was not sustained into the third year (Table 4). There was no indication of an interaction between PCV10 vaccination status (P = 0.864) or survey year (P = 0.347) with ethnicity.

Table 4

Unadjusted and adjusted differences in medians of overall pneumococcal carriage density in association with participant characteristics in four cross-sectional carriage surveys pre-PCV10 (2012) and annually thereafter (2013–2015) in Fiji (n = 2,441).

Exposure		Density of overall pneumococcal carriagea (log10 GE/ml) n, median / IQR	Unadjusted mean difference (95% CI)	P-value	Adjusted mean difference (95% CI)	P-value
PCV10 vaccination status				0.408		0.210
	Not vaccinated	2026, 4.9 (4.2–5.8)	ref		Ref
	Vaccinatedb	415, 4.9 (4.0–5.7)	-0.06 (-0.21, 0.08)		-0.13 (-0.35, 0.08)
Survey year				0.004		0.001
	Pre-PCV10 (2012) b	707, 4.9 (4.2–5.7)	ref		ref
	1 year post-PCV10 (2013)	655, 5.2 (4.3–5.9)	0.28 (0.13, 0.43)		0.33 (0.18, 0.47)
	2 years post-PCV10 (2014)	433, 5.0 (4.1–5.8)	0.11 (-0.06, 0.27)		0.18 (0.00, 0.36)
	3 years post-PCV10 (2015)	646, 4.9 (4.0–5.7)	0.00 (-0.15, 0.15)		0.01 (-0.17, 0.18)
Ethnicity				0.012		0.053
	Fijian of Indian Descent	495, 4.8 (4.1–5.7)	ref		ref
	iTaukei	1946 5.0 (4.2–5.8)	0.17 (0.04, 0.31)		0.14 (0.00, 0.28)
Participant group				0.003		0.008
	Caregivers	193, 4.7 (4.0–5.4)	ref		ref
	Infants (5–8 weeks)	515, 4.9 (4.1–5.7)	0.17 (-0.06, 0.39)		0.17 (-0.06, 0.40)
	Toddlers (12–23 months)	845, 5.0 (4.1–5.8)	0.33 (0.11, 0.55)		0.32 (0.08, 0.55)
	Children (2–6 years)	888, 5.0 (4.3–5.8)	0.34 (0.12, 0.55)		0.34 (0.12, 0.55)
Residential location				0.612
	Rural	1070, 5.0 (4.1–5.7)	ref
	Urban	1371, 5.0 (4.2–5.7)	0.03 (-0.08, 0.14)
Participant sex				0.597
	Male	1166, 5.0 (4.2–5.7)	ref
	Female	1275, 5.0 (4.2–5.8)	0.03 (-0.08, 0.14)
Number of children < 5 years living in the household				0.708
	Less than two	954, 5.0 (4.2–5.8)	ref
	Two or more	1487, 5.0 (4.2–5.7)	-0.02 (-0.13, 0.09)
Family income levelc				0.943
	Not low	801, 5.0 (4.1–5.7)	ref
	Low	1522, 5.0 (4.2–5.8)	0.00 (-0.11, 0.12)
Symptoms of URTI				<0.001		<0.001
	Not present	1528, 4.9 (4.1–5.6)	ref		Ref
	Present	913, 5.2 (4.3–5.9)	0.31 (0.19, 0.42)		0.28 (0.16, 0.40)
Household cigarette smoke				0.540
	No exposure	1098, 5.0 (4.1–5.7)	ref
	Exposure	1343, 5.0 (4.2–5.8)	0.03 (-0.08, 0.15)
Antibiotic use in previous fortnightd				0..084		0.752
	Not used	2324, 5.0 (4.2–5.7)	ref		ref
	Used	115, 5.2 (4.5–5.7)	0.22 (-0.03, 0.48)		0.04 (-0.23, 0.30)

Abbreviations: CI, confidence interval; PCV10, ten-valent pneumococcal conjugate vaccine; URTI, upper respiratory tract infection.

a Density of overall, including non-encapsulated, pneumococci

b Two doses of PCV10 given before the age of 12 months, or one or more doses of PCV10 given after 12 months of age [35]

c Weekly family income below the basic needs poverty line (

Data on antibiotic use were missing for two pneumococcal carriers.

c Weekly family income below the basic needs poverty line (28]; data on family income were missing for 118 pneumococcal carriers

Factors associated with PCV10 pneumococcal density

Symptoms of URTI were positively associated with median density of PCV10 carriage (S2 Table). Although there was an initial increase in the adjusted median difference in PCV10 carriage density in the first year after PCV10 introduction, it was not sustained through the second and third year post-PCV10 introduction (S2 Table). There was no indication of an interaction between PCV10 vaccination status (P = 0.170) or survey year (P = 0.686) with ethnicity.

Factors associated with non-PCV10 pneumococcal density

Symptoms of an URTI and young age (infant, toddler, and child participant groups) were positively associated with median density of non-PCV10 carriage (S3 Table). As with density of overall and PCV10 pneumococcal carriage, the increase in density of non-PCV10 carriage in association with survey year was not sustained to the third year post-PCV10 introduction, and there was no evidence of interaction between PCV10 vaccination status (P = 0.156) survey year (P = 0.138) with ethnicity.

Discussion

This study provides new findings on the factors associated with overall, PCV10, and non-PCV10 pneumococcal carriage and density, in the years surrounding PCV10 introduction in a tropical, upper middle-income country in the Asia-Pacific region.

In this study, iTaukei ethnicity was an independent predictor of carriage (overall, PCV10, and non-PCV10), after adjustment for PCV10 vaccination status and survey year post-PCV10 introduction. We also checked the potential interaction between ethnicity and other socio-demographic variables, and found no evidence of interaction. Previously, we found that prior to PCV10 introduction, iTaukei ethnicity was associated with increased odds of overall pneumococcal carriage in children aged 3–13 months (aOR 2.81 [95% CI 1.76–4.49] P<0.001) [4]. The reason for this association is unknown. Further, we have reported that differences in social contact patterns by ethnicity partially account for higher prevalence of pneumococci among iTaukei, compared with FID, but that differences in carriage prevalence are also likely related to ethnic differences in host or environmental factors [36]. Few studies have described associations between pneumococcal carriage and indigenous and non-indigenous populations living in the same area, with similar access to healthcare, and with similar and high PCV coverage rates. Our findings are consistent with other studies comparing indigenous and non-indigenous populations in the same setting. In a pre- and very early post-PCV7 longitudinal study of 280 indigenous and non-indigenous children in remote Australia, who were followed from birth to 2 years, indigenous status was positively associated with pneumococcal carriage (OR 3.3 [95% CI 2.6–4.2] P<0.001)[37]. In Israel, a longitudinal study of 369 Bedouin and 400 Jewish children enrolled in a trial of PCV7, found Jewish children to have significantly lower odds of pneumococcal carriage, compared with Bedouin children (aOR 0.14 [0.10–0.21]) [38]. In contrast, a post-PCV13 cross-sectional study of 352 children aged less than six years in Greenland, found indigenous ethnicity was not associated with pneumococcal carriage (aOR 0.7 [95% CI 0.3–1.5] P = 0.32) [39]. Likewise, a cross-sectional study in Alaska post-PCV7 involving 1,275 children aged 3–59 months, found no association between indigenous ethnicity and overall or PCV7 carriage (OR 1.0 [95% CI 0.8–1.3] and (OR 1.1 [95% CI 0.75–1.6], respectively) [40]. Unlike our study, however, both the Greenland and Alaska studies occurred in high income settings, which may comparatively reduce the impact of ethnicity based socio-environmental differences that might be related to pneumococcal carriage. Further, the Greenland study included few non-Inuit participants, and may have been underpowered for analysis by ethnicity [39].

Other factors associated with pneumococcal carriage in this study, were largely consistent with studies from the pre-PCV10 era. For example, we observed young age, residential location, living with young children, low family income, and symptoms of URTI to be risk factors for all types of carriage [4, 8, 12]. Similarly, the majority of studies assessing factors associated with pneumococcal carriage after the introduction of PCV into national immunization schedules have reported age, poverty or proxies of poverty, number of young children living in the household, and symptoms of URTI as positively associated with pneumococcal carriage [41-45]. Previous studies have heterogenous findings regarding exposure to cigarette smoke and recent antibiotic use and their associations with pneumococcal carriage [5, 11, 46–53]. We found exposure to household cigarette smoke was a risk factor, but only for PCV10 carriage. However, levels of exposure to cigarette smoke require detailed monitoring, which was not incorporated in this study. We also found no association between antibiotic use and pneumococcal carriage, which may reflect very low prevalence of recent antibiotic use in our sample.

In this study, PCV10 vaccination status and survey year were protective against overall and PCV10 carriage, but were not associated with non-PCV10 carriage. We found only three studies undertaken after PCV was introduced into national immunization programs that assessed factors associated with pneumococcal carriage, and included PCV vaccination status as a variable [16, 54, 55]. In a cross-sectional study of 361 children under five years of age in Jimma, Ethiopia, the odds of overall pneumococcal carriage increased in association with having siblings under five years old (aOR 1.80 [95% CI 1.17–2.77] P = 0.008), and malnutrition (aOR 2.07 [95% CI 1.24–3.44] P = 0.005), but PCV vaccination was not associated with a decrease in carriage (three doses aOR 1.08 [95% CI 0.60–1.89] P = 0.82, one or two doses aOR 1.06 [95% CI 0.40–2.83] P = 0.90) [54]. This may have been due to serotype replacement and capsular switching of pneumococci by recombination, such that the immune pressure from PCV selected for non-vaccine serotypes [54]. However, in a cross-sectional study of 1,668 healthy children aged 12–23 months in Brazil, the odds of vaccine-type carriage declined in association with three (aOR 0.073 [95% CI 0.026–0.204] P<0.001) and four (aOR 0.027 [95% CI 0.007–0.113] P<0.001) doses of PCV10, and increased in association with day care attendance (aOR 2.358 [95% CI 1.455–3.821] P<0.001) and colonization with H. influenzae (aOR 2.454 [95% CI 1.529–3.939] P = 0.0006) [55]. Similarly, in pre and post-PCV13 pneumococcal carriage surveys involving 999 infants aged 5–8 weeks and 1,010 toddlers aged 12–23 months in Lao PDR, two or three doses of PCV13, compared with zero or one, was protective against PCV13 carriage among toddlers (aOR 0.60 [95% CI 0.44–0.83] P = 0.002) [16].

Although our findings are consistent with the Brazilian and Lao PDR studies regarding PCV being protective against PCV carriage, broader comparisons between these and other studies is hampered by the heterogeneity of settings, populations sampled, and the factors and definitions used. For example, in our study participants were community based, healthy, from four different age groups, did not attend childcare, and we did not include co-colonisers or malnutrition as exposures. Comparatively, the studies from Ethiopia, Brazil, and Lao PDR included child and infant participants only, including those attending childcare / school, and those suffering malnutrition, pneumonia, sinusitis, and otitis media.

Our observations regarding no association between PCV10 and non-PCV10 carriage may be due a lack of selection pressure towards an overall increase in non-PCV10 carriage early post-PCV10 introduction, due to serotype replacement occurring only in iTaukei infants and toddlers, rather than across all participant groups, as described previously [22]. In contrast, increases in non-vaccine type carriage have been reported following the introduction of PCV7 in England, PCV10 in Kenya, and PCV13 in Malawi and The Gambia [56-59]. However, it should be noted that the studies from Kenya, England, The Gambia, and Malawi were in vastly different contexts from our study, one notably in a high-income setting, rendering comparisons difficult.

Pneumococcal density has previously been found to be positively associated with microbiologically confirmed pneumococcal pneumonia, and could be used to improve estimates of pneumococcal pneumonia prevalence in childhood pneumonia studies [2].Our study contributes to the understanding of factors associated with pneumococcal carriage density. We found that symptoms of an URTI were associated with increased median density of carriage (overall, PCV10, and non-PCV10), consistent with cross-sectional carriage surveys from Peru, Lao PDR, and Indonesia [9, 11, 13, 16]. We found PCV10 vaccination was not associated with pneumococcal density, and that although differences in median density of all types of pneumococci increased one to two years following PCV10 introduction, this was not sustained into the third year. There are relatively few risk factor studies describing the association between pneumococcal density and PCV vaccination status. Those that do, have heterogeneous findings. A double-blind, randomized controlled trial of PCV13 and Hepatitis A vaccine (control arm) in adults, using the Experimental Human Pneumococcal Challenge model, found that pneumococcal density in the PCV13 arm was significantly lower compared with the control arm (P<0.0001) [60]. In a cluster-randomized trial of PCV7 and Meningococcal C vaccine (control arm) in rural Gambia, density of PCV7 pneumococcal carriage was lower in PCV7 villages compared with control arm villages [61]. However, this was not attributed to PCV7, as density of non-PCV7 carriage also declined in both vaccine and control villages [61]. Similarly, a decline observed in both PCV13 and non-PCV13 pneumococcal density in Laotian infants and toddlers was attributed to secular trends rather than PCV13 directly [16].

Limitations to our study should be noted. Firstly, because participants were recruited from Greater Suva and the surrounding areas, generalizability to the wider Fijian population may be limited. The non-random sampling method may have introduced sample selection bias, such results may not be generalizable to the Fijian population. However, the purposive quota sampling method rendered the sample representative of the Fijian population with regard to ethnicity and residential location, which are associated with pneumococcal carriage [4, 27]. The cross-sectional nature of this observational study precludes causal, associations from being drawn between participant characteristics and pneumococcal carriage or densities. Finally, we did not collect data on co-colonizing bacterial or viral species, and are therefore unable to investigate the association of such factors with pneumococcal carriage or density, which have been identified as risk factors in other studies [9-11].

These limitations notwithstanding, this study documents the factors associated with pneumococcal carriage and density post-PCV10 introduction in an upper middle-income country. Introduction of PCV10 was negatively associated with the odds of overall and PCV10 pneumococcal carriage in Fiji. However, iTaukei ethnicity remains positively associated with pneumococcal carriage in Fiji, despite high and similar PCV10 coverage rates across iTaukei and FID populations. Further research is warranted regarding the factors underlying the observed ethnicity based differences in pneumococcal carriage, and whether the impact of PCV10 on pneumococcal disease incidence differs by ethnicity in Fiji.

Unadjusted and adjusted odds ratios of non-PCV10 pneumococcal carriage in association with participant characteristics in four cross-sectional carriage surveys pre-PCV10 (2012) and annually thereafter (2013–2015) in Fiji (n = 7,962).

(DOCX)

Click here for additional data file.

Unadjusted and adjusted differences in medians of PCV10 pneumococcal carriage density in association with participant characteristics in four cross-sectional carriage surveys pre-PCV10 (2012) and annually thereafter (2013–2015) in Fiji (n = 708).

(DOCX)

Click here for additional data file.

Unadjusted and adjusted differences in medians of non-PCV10 pneumococcal carriage density in association with participant characteristics in four cross-sectional carriage surveys pre-PCV10 (2012) and annually thereafter (2013–2015) in Fiji (n = 1,905).

(DOCX)

Click here for additional data file.

27 Jan 2020

PONE-D-19-35241

Factors associated with pneumococcal carriage and density in children and adults in Fiji, using four cross-sectional surveys

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Reviewer #1: Manuscript Number: PONE-D-19-35241

Title: Factors associated with pneumococcal carriage and density in children and adults in Fiji, using four cross-sectional surveys.

Excellent work describing the factors associated with pneumococcal nasopharyngeal carriage and density in children and caregivers in Fiji, pre and post-introduction of PCV10. This article is a continuation of the previously published article of the same authors, and reference 22 of the present one: Dunne EM, Satzke C, Ratu FT, Neal EF, Boelsen LK, Matanitobua S, et al. Impact of 10-valent pneumococcal conjugate vaccine introduction on pneumococcal carriage in Fiji: results from four annual cross sectional carriage surveys. Lancet Glob Health. 2018;6(12):PE1375-E85.

The article is well written, methodology is, in general, sound (see comment 16 on pneumococcal density) and support the results obtained.

I only have some minor comments:

General comments

1. Vaccinated subjects in each age group are different: from non-vaccinated to different percentages of vaccinated depending on the year of the survey. However data have not been stratified by age-groups and analysed globally, so that the direct effect of vaccination on carriage might be somehow “diluted”.

2. In many places of the article it is stated that “young age was positively associated with pneumococcal carriage”. However overall, PCV10 serotypes, and non-PCV10 serotypes carriage was lower in infants than in toddles and children. The authors should better specify to what age groups they are refereeing when talking of young age” as a general word.

3. In the tables there is a P-value that in some items (survey year and participant group) is given in total for the other 3 categories. Please, explain if this P-value was calculated comparing the reference value (caregivers) with the addition of the results of all the other groups or if the comparison of each group with the reference one was the P-value expressed.

Detailed comments

Abstract

4. Line 23. I suggest adding “in October 2012” after “…post-introduction of ten-valent pneumococcal conjugate vaccine (PCV10).”

5. May be the authors should differentiate in the abstract between factors positively or negatively associated with carriage, in order to be clearer for the reader. For instance, in line 26 it is stated that “Factors associated with pneumococcal carriage were years post-PCV10 introduction (global P<0.001) …” and in line 31: “…PCV10 carriage was also associated with PCV10 vaccination (0.58 [95% CI 0.41 – 0.82] P=0.002)”.

As PCV10 vaccination and survey year were negatively associated with PCV10 carriage (line 184) and survey year was negatively associated with overall carriage (line 165), but other factors were positively associated to carriage I recommend to write this clearly in the abstract to avoid possible misunderstandings.

6. Abstract and end of discussion. Although the study is mostly descriptive, it would be desirable some conclusions of the study, not just the report of the data obtained.

Material and methods

7. Children between 8 weeks and 12 months were not sampled, probably because they were in the vaccination period. However I would suggest an explanation of this decision in material and methods.

8. Line 141. Percentage of vaccinated subjects was different in each age-group (no vaccinated in infants and caregivers, 23% vaccinated children in 2015 and nearly 100% toddlers vaccinated in 2014-2015). In fact, 1105 (13.6%) participants had been vaccinated. Could these differences in the vaccination rates have biased the percentage of carriage in the different age-groups? The direct effect of vaccination in carriage could only be measured in 13.6% of participants, most of them in the 12-23 months age- group.

9. Line 147. In the previous publication authors detailed that “19.8% of pneumococcal-positive samples contained more than one serotype.” I suppose that some of these samples contained PCV10 and non-PCV10 serotypes. In this case, to which group (PCV10 carrier or not) were assigned the subjects?

10. Table 1. I suggest to put in this table and in the following, the year in the “Survey year” line, next to “pre-PCV10 (2012)”, “1 year post-PCV10 (2013)” … Also, I suggest adding the age of the subjects in each of the groups in the tables (“Infants (5-8 weeks)”, “Toddlers (12-23 months)”… and remove it from the footnote.

11. Table 1. Footnote. Please, explain in Material and Methods how were NT-lineages established.

Results

12. Line 165. “Survey year was negatively associated with overall carriage.”. However, percentage of carriage in the 3rd year was higher than in the 2nd year (p<0.001).

Discussion

13. Lines 234-242. In this paragraph the authors resume the main findings of their work. As they have been already described in the abstract and in the results section I recommend considering the possibility of deleting this paragraph. In the same way, I recommend to delete all the figures of “OR and P” through the discussion, to make easier its reading.

14. Line 262. “Unlike our study, however, both the Greenland and Alaska studies occurred in high income settings, which may comparatively reduce the impact of ethnicity based socio-environmental differences that might be related to pneumococcal carriage.”

If in the studies of Greenland and Alaska the carriage ORs were adjusted to other factors (as number of children <5 years living on the house, smoking, and other related socio-environmental factors) the statement would be speculative.

In the same way as the authors have studied the interaction of survey year and vaccination with ethnicity, maybe they should have also analysed if the two different ethnic groups of their study had different socio-environmental conditions (Young children living in the same house, low incomes, exposure to smoke, …) to see if ethnicity by itself was associate to carriage or were these conditions the responsible for the increased carriage.

15. Line 301. “…lack of selection pressure towards an overall increase in non-PCV10 carriage early post-PCV10 introduction, due to serotype replacement occurring only in iTaukei infants and toddlers, rather than across all participant groups [22].

However iTaukei infants had not received PCV10, so a direct effect of vaccination on serotype replacement (increase in non PCV-10 serotypes) was not expected. And if serotype replacement in iTaukei infants had been a consequence of the indirect effect of PCV10 on, iTaukei caregivers would had also suffered this serotype replacement…

16. Line 325. Limitations. From my point of view another limitation of the study are the data on the carriage density because of diferent factors. First, it can greatly depend on the sampling. Nasopharyngeal sampling is relatively uncomfortable and sampling in older children and adults can be more complicated than in younger children because the accession to the nasopharynx is more difficult. Besides, PCR can detect non-viable bacteria. Other studies have shown that there are serotype-dependent variations when detecting pneumococcal load (Messaoudi M, et al. PLoS One. 2016;11:e0151428). Other studies have shown a specificity of lytA PCR for detection of pneumococcal carriage of 75.9% (Gritzfeld JF, et al. Clin Microbiol Infect. 2014;20:O1145-51). I suggest that, although real time PCR is a good technique for measuring pneumococcal carriage especially in studies comprising thousands of samples, it has some limitations that have to be considered by the researches.

Reviewer #2: The manuscript by Neal et al addresses the factors associated with pneumococcal carriage and density in children and adults in Fiji, using data from cross-sectional studies that encompass the introduction of PCV10.

Pneumococcal carriage and carriage density were determined by qPCR targeting the lytA gene and serotyping was done by microarray. Factors associated with pneumococcal carriage and density were determined using logistic and quantile regressions, respectively. Variables included in the model were selected from an empirical univariate analysis and from an informed selection of variables. The latter were properly justified.

This paper is well structured and very well written. The methods are sound and the conclusions are properly supported by the results described. Some recognizable limitations to the study are acknowledged by the authors in the discussion section. All references are appropriate and were cited.

Please find below one minor comment:

Tables 2, 3 and 4: Each variable should include information regarding the number of samples that were excluded (presumably due to lack of information on that specific variable). The totals do not add up.

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9 Mar 2020

24 February 2020

Dear Dr Melo-Cristino,

We thank the Editor and the reviewers for their thorough assessment of our manuscript, and the detailed and insightful comments provided. A point-by-point response to editorial requirements, comments, and reviewer comments is below.

To enhance the reproducibility of your results, we recommend that if applicable 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. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Authors’ response

We thank the Editor for this opportunity, however note that our laboratory methods are described in detail in our previous publications (Dunne et al Lancet Glob Health 2018; Satzke et al Vaccine 2019; von Mollendorf et al Vaccine 2019) and are in accordance with the overarching guidelines of the WHO (Satzke et al Vaccine 2013).

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Authors’ response

We thank the Editor and have amended our manuscript in line with the PLOS ONE style requirements

2. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For more information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions.

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Authors’ response

While our data has been fully de-identified, there remains an ethical restriction on making our data available publicly. The protocol approved by the Fijian Ministry of Health and Medical Services National Health Research and Ethics Review Committee, and by the University of Melbourne Health Sciences Human Ethics Sub-Committee, specified the purpose of the study and what the data would be used for. This information was conveyed during the informed consent process. Therefore, reasonable requests for data will need to include details on how data would be used, and will be subject to approval by the Fijian Ministry of Health and Medical Services National Health Research and Ethics Review Committee, which is the overseeing ethics committee for this study. This process considered the sensitivities regarding data from indigenous populations.

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Authors’ response

Thank you for the opportunity to amend this. We have responded via return email with the updated Competing Interests Statement, which now reads: “I have read the journal's policy and the authors of this manuscript have the following competing interests: CDN, CS, EKM, and EMD are investigators on a research projected funded by Pfizer for research in Mongolia. This does not alter our adherence to PLOS ONE policies on sharing data and materials. All other authors have no declarations of competing interests to declare.”

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Reviewer #1: Manuscript Number: PONE-D-19-35241

Title: Factors associated with pneumococcal carriage and density in children and adults in Fiji, using four cross-sectional surveys.

Excellent work describing the factors associated with pneumococcal nasopharyngeal carriage and density in children and caregivers in Fiji, pre and post-introduction of PCV10. This article is a continuation of the previously published article of the same authors, and reference 22 of the present one: Dunne EM, Satzke C, Ratu FT, Neal EF, Boelsen LK, Matanitobua S, et al. Impact of 10-valent pneumococcal conjugate vaccine introduction on pneumococcal carriage in Fiji: results from four annual cross sectional carriage surveys. Lancet Glob Health. 2018;6(12):PE1375-E85.

The article is well written, methodology is, in general, sound (see comment 16 on pneumococcal density) and support the results obtained.

I only have some minor comments:

General comments

1. Vaccinated subjects in each age group are different: from non-vaccinated to different percentages of vaccinated depending on the year of the survey. However data have not been stratified by age-groups and analysed globally, so that the direct effect of vaccination on carriage might be somehow “diluted”.

Authors’ response

We thank Reviewer 1 for their time and consideration of our manuscript. We agree that the number and percentage of vaccinated subjects differs by age group, depending on the year of survey, and that the direct effect of vaccination on carriage might be diluted. However, the aim of the current study was to determine the factors associated with pneumococcal carriage in Fiji, taking into account the introduction of PCV10, so we explored the adjusted odds of carriage by potential risk factor. In our previously published paper, our aim was to measure the impact of PCV10 on pneumococcal carriage prevalence in Fiji, so we investigated adjusted prevalence rates by year post-PCV10 introduction and age group. We have not presented stratified analyses here, but these can be found in our previously published paper (Dunne et al Lancet Glob Health 2018).

2. In many places of the article it is stated that “young age was positively associated with pneumococcal carriage”. However overall, PCV10 serotypes, and non-PCV10 serotypes carriage was lower in infants than in toddles and children. The authors should better specify to what age groups they are refereeing when talking of young age” as a general word.

Authors’ response

We have amended our manuscript for clarity

3. In the tables there is a P-value that in some items (survey year and participant group) is given in total for the other 3 categories. Please, explain if this P-value was calculated comparing the reference value (caregivers) with the addition of the results of all the other groups or if the comparison of each group with the reference one was the P-value expressed.

Authors’ response

In this study, we presented odds ratios and 95% confidence intervals for comparison between reference groups and other levels of categorical variables with multiple categories, such as survey year and participant group. The reported P-values for such variables were calculated to determine inclusion of the variable in the model, i.e., global P-values.

Detailed comments

Abstract

4. Line 23. I suggest adding “in October 2012” after “…post-introduction of ten-valent pneumococcal conjugate vaccine (PCV10).”

Authors’ response

We have amended our abstract accordingly.

5. May be the authors should differentiate in the abstract between factors positively or negatively associated with carriage, in order to be clearer for the reader. For instance, in line 26 it is stated that “Factors associated with pneumococcal carriage were years post-PCV10 introduction (global P<0.001) …” and in line 31: “…PCV10 carriage was also associated with PCV10 vaccination (0.58 [95% CI 0.41 – 0.82] P=0.002)”.

As PCV10 vaccination and survey year were negatively associated with PCV10 carriage (line 184) and survey year was negatively associated with overall carriage (line 165), but other factors were positively associated to carriage I recommend to write this clearly in the abstract to avoid possible misunderstandings.

Authors’ response

We have amended our abstract for clarity.

6. Abstract and end of discussion. Although the study is mostly descriptive, it would be desirable some conclusions of the study, not just the report of the data obtained.

Authors’ response

We have amended our abstract and conclusion to highlight the findings regarding ethnicity,

Material and methods

7. Children between 8 weeks and 12 months were not sampled, probably because they were in the vaccination period. However I would suggest an explanation of this decision in material and methods.

Authors’ response

The age groups were the same as reported for our study to measure the impact of PCV10 in Fiji on pneumococcal carriage prevalence (Dunne et al Lancet Glob Health, 2018). For the vaccine impact study, age groups were selected in order to best answer the primary research question based on those most likely to benefit from direct and indirect effects of PCV10; those likely to have the highest pneumococcal carriage prevalence; those age-eligible for PCV10 vaccination; and those most likely to transmit / be in contact with transmitters of pneumococci. The 5 – 8 week old infants were selected as they represent a vulnerable age group for pneumococcal disease, yet are too young to be vaccinated. The 12-23 month age group aligns with peak pneumococcal carriage and the majority of this age group would be fully vaccinated in the latter survey years. As such, the 5-8 week and 12-23 month old age groups were selected based on their potential to evaluate the indirect and direct effects of PCV10 introduction in Fiji, rather than a deliberate non-inclusion of children aged between 8 weeks to 12 months.

This study used the same samples for convenience. For clarity, we have amended our manuscript to include a statement to this effect.

8. Line 141. Percentage of vaccinated subjects was different in each age-group (no vaccinated in infants and caregivers, 23% vaccinated children in 2015 and nearly 100% toddlers vaccinated in 2014-2015). In fact, 1105 (13.6%) participants had been vaccinated. Could these differences in the vaccination rates have biased the percentage of carriage in the different age-groups? The direct effect of vaccination in carriage could only be measured in 13.6% of participants, most of them in the 12-23 months age- group.

Authors’ response

In Dunne et al, Lancet Glob Health, 2018, our aim was to measure the impact of PCV10 vaccination and introduction on pneumococcal carriage prevalence in Fiji. In that study, we calculated adjusted prevalence rates by year post-PCV10 introduction, taking into account that there was no unbiased opportunity to measure direct effects alone, as vaccinated participants would be subject to both direct and indirect effects of PCV10. We found that prevalence of PCV10 carriage in both vaccinated and unvaccinated participants declined after the introduction of PCV10, with evidence of indirect effects in infants too young to be vaccinated and among older unvaccinated age groups. In this study, we aimed to investigate the host and demographic factors associated with pneumococcal carriage (such as age), not the direct effect of vaccination on carriage prevalence, so we explored the adjusted odds of carriage by potential risk factor.

9. Line 147. In the previous publication authors detailed that “19.8% of pneumococcal-positive samples contained more than one serotype.” I suppose that some of these samples contained PCV10 and non-PCV10 serotypes. In this case, to which group (PCV10 carrier or not) were assigned the subjects?

Authors’ response

In our revised manuscript, we have amended the Methods section to clarify that “detection by microarray serotyping of a PCV10 serotype and a non-PCV10 serotype from the same swab sample was recorded as positive for both PCV10 serotype and non-PCV10 serotype carriage (Dunne ED et al, Lancet Glob Health 2018). Any detection of a serotype by microarray was considered positive, regardless of relative abundance (Dunne ED et al, Lancet Glob Health 2018).

10. Table 1. I suggest to put in this table and in the following, the year in the “Survey year” line, next to “pre-PCV10 (2012)”, “1 year post-PCV10 (2013)” … Also, I suggest adding the age of the subjects in each of the groups in the tables (“Infants (5-8 weeks)”, “Toddlers (12-23 months)”… and remove it from the footnote.

Authors’ response

We have amended our manuscript accordingly.

11. Table 1. Footnote. Please, explain in Material and Methods how were NT-lineages established.

Authors’ response

In our revised manuscript, we have explained that non-encapsulated lineages were classified based upon previously described genetic variants (Salter et al Microbiol 2012).

Results

12. Line 165. “Survey year was negatively associated with overall carriage.”. However, percentage of carriage in the 3rd year was higher than in the 2nd year (p<0.001).

Authors’ response

In our manuscript, we note that compared with the first year of survey (pre-PCV10) the adjusted odds of overall carriage were lower after the introduction of PCV10. We agree that the prevalence of pneumococcal carriage was notably low in the second year post-PCV10 introduction (2014) compared with other years. In addition, our 2015 sample collection period coincided with a local influenza outbreak (WHO FluNet http://apps.who.int/flumart/Default?ReportNo=7), which may have affected pneumococcal carriage prevalence that year. However, these details and discussion of them have been published by us previously in a paper regarding the impact of PCV10 on pneumococcal carriage prevalence in Fiji (Dunne ED et al Lancet Glob Health 2018); and the aim of this analysis was to determine factors associated with pneumococcal carriage, rather than analysis of prevalence rates.

Discussion

13. Lines 234-242. In this paragraph the authors resume the main findings of their work. As they have been already described in the abstract and in the results section I recommend considering the possibility of deleting this paragraph. In the same way, I recommend to delete all the figures of “OR and P” through the discussion, to make easier its reading.

Authors’ response

We have amended our manuscript accordingly.

14. Line 262. “Unlike our study, however, both the Greenland and Alaska studies occurred in high income settings, which may comparatively reduce the impact of ethnicity based socio-environmental differences that might be related to pneumococcal carriage.”

If in the studies of Greenland and Alaska the carriage ORs were adjusted to other factors (as number of children <5 years living on the house, smoking, and other related socio-environmental factors) the statement would be speculative.

In the same way as the authors have studied the interaction of survey year and vaccination with ethnicity, maybe they should have also analysed if the two different ethnic groups of their study had different socio-environmental conditions (Young children living in the same house, low incomes, exposure to smoke, …) to see if ethnicity by itself was associate to carriage or were these conditions the responsible for the increased carriage.

Authors’ response

Previous analysis of pneumococcal carriage reported an ethnicity based disparity in pneumococcal carriage prevalence and invasive pneumococcal disease (Russell et al Ann Trop Paediatr 2006; Russell et al Pediatr Infect Dis J 2010; Dunne et al Lancet Glob Health 2018). Investigation of factors associated with pneumococcal carriage prior to the introduction of PCV10 found ethnicity to be an independent predictor of pneumococcal carriage (Russell et al, Paediatr Infect Dis J 2010; Neal et al Pneumonia 2014). Socio-demographic factors potentially associated with the ethnicity based disparity in pneumococcal carriage epidemiology in Fiji were explored in Neal et al Vaccine, 2019. In that study, we found indigenous iTaukei had larger household sizes (more people), more social contacts, and more frequent contacts of longer duration compared with Fijians of Indian Descent, but that observed differences in pneumococcal carriage prevalence by ethnicity were not explained by differences in socio-demographic patterns ethnicity. In the current study, our statistical models permitted evaluation of the association of ethnicity with pneumococcal carriage, adjusting for demographic factors such as number of young children living in the same house, low incomes, exposure to smoke etc., and found that ethnicity remained an independent predictor of pneumococcal carriage.

We appreciate the reviewer’s suggestion, and as a result, explored possible interactions between ethnicity and sociodemographic factors. In our revised Discussion, we have indicated that we confirmed this, by checking the potential interaction between ethnicity and other variables included in models, with P<0.05 considered significant, and that found no evidence of interaction (as indicated by P values in Table 1 in our Response to Reviewers letter).

15. Line 301. “…lack of selection pressure towards an overall increase in non-PCV10 carriage early post-PCV10 introduction, due to serotype replacement occurring only in iTaukei infants and toddlers, rather than across all participant groups [22].

However iTaukei infants had not received PCV10, so a direct effect of vaccination on serotype replacement (increase in non PCV-10 serotypes) was not expected. And if serotype replacement in iTaukei infants had been a consequence of the indirect effect of PCV10 on, iTaukei caregivers would had also suffered this serotype replacement…

Authors’ response

We agree with Reviewer 1 that a direct effect of vaccination on serotype replacement among an unvaccinated cohort is not to be expected. Previously we have reported that in this early post-PCV10 setting, serotype replacement in carriage occurred solely in iTaukei infants and children, potentially due to the higher baseline carriage of non-PCV10 serotypes among infants and children, compared with adults (Dunne et al Lancet Glob Health 2018).. In our revised manuscript, we have clarified that serotype replacement in infants and toddlers rather than all age groups was the observed data, rather than speculation.

16. Line 325. Limitations. From my point of view another limitation of the study are the data on the carriage density because of diferent factors. First, it can greatly depend on the sampling. Nasopharyngeal sampling is relatively uncomfortable and sampling in older children and adults can be more complicated than in younger children because the accession to the nasopharynx is more difficult. Besides, PCR can detect non-viable bacteria. Other studies have shown that there are serotype-dependent variations when detecting pneumococcal load (Messaoudi M, et al. PLoS One. 2016;11:e0151428). Other studies have shown a specificity of lytA PCR for detection of pneumococcal carriage of 75.9% (Gritzfeld JF, et al. Clin Microbiol Infect. 2014;20:O1145-51). I suggest that, although real time PCR is a good technique for measuring pneumococcal carriage especially in studies comprising thousands of samples, it has some limitations that have to be considered by the researches.

Authors’ response

The methods used in detecting and quantifying pneumococcal carriage in the current study are globally accepted as the gold standard (Satzke et al Vaccine 2013). In addition, previous studies (including Gritzfeld CMI 2014) have shown a high degree of concordance with lytA qPCR and culture, and conducting viable counts on thousands of samples would be impractical.

Importantly, the lytA real-time PCR used in our study is highly specific for nasopharyngeal samples. However, we agree that specificity can be affected if oropharyngeal flora contaminate the specimen (e.g. during sampling) given the high abundance of closely-related streptococci in this niche. Indeed, we hypothesise that the lower specificity reported in Gritzfeld CMI 2014 in comparison with other studies, may be due to ‘contaminating’ oral flora in the nasal wash, consistent with the higher levels of alpha-haemolytic streptococci obtained from nasal washes (compared with nasopharyngeal swabs) in uninfected healthy adults (Gritzfeld et al., BMC Res Notes 2011).

Detection of non-viable pneumococci may arguably be advantageous, but in any case was relatively uncommon in our study, where there were 2,456 swabs that were lytA positive, and of these, 2,395 (97.5%) were culture-positive. The paper by Messaoudi et al. is informative for the field, but its implications for our study are less obvious. The authors developed serotype-specific real-time multiplex PCRs and found that the nasopharyngeal density in hospitalised/pneumonia patients was around 3-fold higher for serotypes that were also isolates from the blood of the patients tested. They also reported that some serotype-specific qPCRs had different sensitivity, but the reason was unclear. The authors presented some reasons, including primer-probe interactions in the multiplex PCR, as well as differences in extraction efficacy between serotypes. Of note, our focus is on risk factors with overall, vaccine-type, and non-vaccine type carriage and density, rather than serotype specific carriage and density. We agree that there are likely some differences in the cell lysis between serotypes of pneumococci, but we are not aware of evidence that this characteristic differs between vaccine vs non-vaccine serotypes more generally.

Reviewer #2: The manuscript by Neal et al addresses the factors associated with pneumococcal carriage and density in children and adults in Fiji, using data from cross-sectional studies that encompass the introduction of PCV10.

Pneumococcal carriage and carriage density were determined by qPCR targeting the lytA gene and serotyping was done by microarray. Factors associated with pneumococcal carriage and density were determined using logistic and quantile regressions, respectively. Variables included in the model were selected from an empirical univariate analysis and from an informed selection of variables. The latter were properly justified.

This paper is well structured and very well written. The methods are sound and the conclusions are properly supported by the results described. Some recognizable limitations to the study are acknowledged by the authors in the discussion section. All references are appropriate and were cited.

Please find below one minor comment:

Tables 2, 3 and 4: Each variable should include information regarding the number of samples that were excluded (presumably due to lack of information on that specific variable). The totals do not add up.

Authors’ response

We thank Reviewer 2 for their reading and consideration of our manuscript. Our study included 8,109 participants. As noted in our Methods section, 38 participants who identified as “other” ethnicity were excluded from inferential analyses due to small numbers. A further 48 samples were excluded from microbiological analysis due to technical reasons such as insufficient volume, sample loss, and/or labelling errors. A further 61 pneumococcal positive samples were not able to be serotyped, because they were culture-negative (n = 51) or had low DNA yield from culture (n =6), or due to technical reasons (n = 4). In our revised manuscript, we have added footnotes to Tables 2, 3, and 4 for those variables where the pneumococcal carriage denominators do not total 8,023 (table 2) or 7,962 (Tables 3 and 4).

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We hope the revised version is now suitable for publication, and look forward to hearing from you in due course.

Yours sincerely,

Eleanor Neal, on behalf of all co-authors.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

16 Mar 2020

Factors associated with pneumococcal carriage and density in children and adults in Fiji, using four cross-sectional surveys

PONE-D-19-35241R1

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18 Mar 2020

PONE-D-19-35241R1

Factors associated with pneumococcal carriage and density in children and adults in Fiji, using four cross-sectional surveys

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