SARS-CoV-2 seropositivity and COVID-19 among 5 years-old Amazonian children and their association with poverty and food insecurity.

BACKGROUND: The epidemiology of childhood SARS-CoV-2 infection and COVID-19-related illness remains little studied in high-transmission tropical settings, partly due to the less severe clinical manifestations typically developed by children and the limited availability of diagnostic tests. To address this knowledge gap, we investigate the prevalence and predictors of SARS-CoV-2 infection (either symptomatic or not) and disease in 5 years-old Amazonian children. METHODOLOGY/PRINCIPAL
FINDINGS: We retrospectively estimated SARS-CoV-2 attack rates and the proportion of infections leading to COVID-19-related illness among 660 participants in a population-based birth cohort study in the Juruá Valley, Amazonian Brazil. Children were physically examined, tested for SARS-CoV-2 IgG and IgM antibodies, and had a comprehensive health questionnaire administered during a follow-up visit at the age of 5 years carried out in January or June-July 2021. We found serological evidence of past SARS-CoV-2 infection in 297 (45.0%; 95% confidence interval [CI], 41.2-48.9%) of 660 cohort participants, but only 15 (5.1%; 95% CI, 2.9-8.2%) seropositive children had a prior medical diagnosis of COVID-19 reported by their mothers or guardians. The period prevalence of clinically apparent COVID-19, defined as the presence of specific antibodies plus one or more clinical symptoms suggestive of COVID-19 (cough, shortness of breath, and loss of taste or smell) reported by their mothers or guardians since the pandemic onset, was estimated at 7.3% (95% CI, 5.4-9.5%). Importantly, children from the poorest households and those with less educated mothers were significantly more likely to be seropositive, after controlling for potential confounders by mixed-effects multiple Poisson regression analysis. Likewise, the period prevalence of COVID-19 was 1.8-fold (95%, CI 1.2-2.6-fold) higher among cohort participants exposed to food insecurity and 3.0-fold (95% CI, 2.8-3.5-fold) higher among those born to non-White mothers. Finally, children exposed to household and family contacts who had COVID-19 were at an increased risk of being SARS-CoV-2 seropositive and-even more markedly-of having had clinically apparent COVID-19 by the age of 5 years.
CONCLUSIONS/SIGNIFICANCE: Childhood SARS-CoV-2 infection and COVID-19-associated illness are substantially underdiagnosed and underreported in the Amazon. Children in the most socioeconomically vulnerable households are disproportionately affected by SARS-CoV-2 infection and disease.

Introduction

Children account for a relatively small fraction of total COVID-19 cases, hospitalizations, and deaths due to SARS-CoV-2 infection worldwide [1], but this fraction has recently increased as more adults receive their COVID-19 vaccines and become partially protected from infection and disease [2]. Although severe illness may occasionally develop, children with SARS-CoV-2 infection typically remain asymptomatic or have mild symptoms [3-5]. Nevertheless, both symptomatic and asymptomatic children can carry relatively high concentrations of viral mRNA [6] and replicating SARS-CoV-2 [7], constituting a potential infectious reservoir of public health importance. Young children, who are not routinely vaccinated, appear to be more likely to transmit SARS-CoV-2 to household contacts once infected, compared with older children and adolescents [8].

The COVID-19 crisis in Brazil has been most dramatic across the Amazon, a region that covers 60% of the country′s territory, where public health facilities were already operating near full capacity before the pandemic and very few intensive care unit beds were available. The emergence of the Gamma (formerly known as P.1) variant of concern in Manaus [9], the largest city in the Amazon, was followed by a dramatic upsurge in mortality across the region during the second COVID-19 epidemic wave in early 2021 [10,11]. Official COVID-19 case notification statistics in the Amazon are imprecise due to the shortage of diagnostic molecular and antigen-detection tests, but SARS-CoV-2 antibody data can be used to estimate the proportion of mild or asymptomatic infections that remained undetected and the proportion of infections that led to overt disease, health facility visits, and hospitalization.

The epidemiology of SARS-CoV-2 infection and COVID-19 in young children remains relatively understudied [1], especially in hard-hit communities in tropical, low to middle-income countries. To address this knowledge gap, this exploratory study examines the prevalence of SARS-CoV-2 antibodies and the period prevalence of COVID-19 at the age of 5 years among participants in an ongoing population-based birth cohort study in Amazonian Brazil [12]. We show that nearly half of the children had serological evidence of prior SARS-CoV-2 infection, but very few had COVID-19 episodes previously diagnosed, suggesting that SARS-CoV-2 infections in young children are substantially underreported in this high-transmission setting.

Materials and methods

Ethics statement

All mothers or children’s parents or guardians (if mothers were <18 years old) provided written informed consent. The study protocol was approved by the institutional review board of the School of Public Health, University of São Paulo (# 872.613, 2014; #2.358.129, 2017).

Study design and population

The Maternal and Child Health and Nutrition in Acre, Brazil (MINA-Brazil) study is a prospective, population-based birth cohort set-up in 2015 to examine the impact of a wide range of early exposures on child growth and development in the Amazonian municipality of Cruzeiro do Sul, next to the Brazil-Peru border [12]. The study site and population are described in detail in S1 File. Fig 1 shows the study flow diagram. Briefly, mother-baby pairs were enrolled at pregnancy in public antenatal clinics, or at birth in the Women and Children’s Hospital of Juruá Valley, the only maternity hospital of Cruzeiro do Sul, where 96% of all local deliveries take place [13]. At delivery, mothers were interviewed to obtain sociodemographic information [12] such as the duration of mother′s schooling self-reported mother′s skin color, mother′s occupation, and whether the family is currently supported by the Bolsa Família conditional cash transfer program [14]. An assets-based wealth index [15] was used as a proxy of socioeconomic status.

Fig 1

Study flowchart.

Between July 2015 and June 2016, pregnant women attending antenatal clinics or admitted for delivery to the maternity ward of the Women and Children’s Hospital of Juruá Valley in Cruzeiro do Sul, Brazil, were invited to participate. Reasons for exclusion and the final number of subjects analyzed for SARS-CoV-2 antibodies at the age of 5 years are indicated.

Here we focus on sociodemographic and morbidity data obtained during the 5-year follow-up visit. We searched for evidence of household food insecurity, defined when household members have “no access to sufficient safe and nutritious food to meet their dietary needs and food preferences for an active and healthy life” [16], by using a validated, 5-question version of the Brazilian Food Insecurity Scale [17]. History of prior COVID-19 episodes was collected for children and their household and family contacts (parents, siblings, grandparents, or neighbors). Venous blood was collected for SARS-CoV-2 antibody testing. Information was obtained from 695 (56.0%) out of 1240 eligible children. Children participating in the 5-year assessment and those lost to follow-up (n = 514) had similar perinatal characteristics regarding sex, gestational age, preterm birth, and birth weight, but differed significantly in the proportion of children from poorest families (36.2% vs. 44.1%) and of mothers with ≤9 years of schooling (26.4% vs. 47.5%), respectively, with P <0.01 (χ2 test) for both.

Follow-up assessment of 5 years-old children was carried out in two rounds. The first took place between 15 and 31 of January 2021 and targeted children born between July and December 2015; the second, between 18 of June and 4 of July 2021, targeted children born between January and June 2016. At the time of the follow-up assessment, children′s age ranged between 58.5 and 71.9 months (average, 63.5 months), with a significant difference between the first and the second round (63.2 vs. 63.8 months respectively, P = 0.003, Student′s t test). No study participant had been vaccinated against COVID-19 at the time of the study rounds; vaccination of children aged 5–11 years started in Brazil on 14 January, 2022. Child weight and height were measured in duplicate by trained health professionals [18] as described in detail in S1 File. Anthropometric data were processed with the Anthro software (https://www.who.int/childgrowth/en/), which uses the World Health Organization standard curves [19]. Stunting (stature-to-age: <-2 z-scores) and overweight (body mass index [BMI]-to-age: >2 z-scores) were included as covariates in our initial analyses.

SARS-CoV-2 antibody measurement

Plasma samples from 660 study children (95.0% of those interviewed) were tested for SARS-CoV-2 antibodies; 330 of them were from the first study round and 330 from the second round. Importantly, an upsurge in COVID-19 cases was recorded in the study site between the study rounds, lasting between February and March 2021, when the Gamma variant became widespread across Juruá Valley [20].

Total antibodies (including IgG and IgM) that recognize a recombinant protein representing the nucleocapsid antigen of SARS-CoV-2 were detected in 20-μL plasma aliquots using the Elecsys anti-SARS-CoV-2 double-antigen sandwich electrochemiluminescence assay (Roche Diagnostics, Penzberg, Germany) on a Cobas 411 analyzer (Roche Diagnostics). The Elecsys assay provides a qualitative result (reactive vs. nonreactive) but with a quantitative signal cut-off index (COI) value; a COI ≥1.0 is interpreted as reactive and a COI <1.0 is interpreted as nonreactive. Owing to its antigen sandwich format, the Elecsys assay shows good signal stability, with little COI decay over time and rare seroreversions following a natural infection, being ideally suited for serosurveillance [21].

Outcome definitions

Two primary outcomes were considered: (1) presence of SARS-CoV-2 antibodies in the 5-year survey, regardless of any clinical signs and symptoms, as a proxy of SARS-CoV-2 infection anytime since the COVID-19 epidemic onset; and (2) presence of SARS-CoV-2 antibodies in the 5-year survey combined with at least one new or increased clinical sign or symptom experienced since April 2020, as a proxy of prior clinically apparent COVID-19. We specifically asked mothers or guardians whether the child had presented since April 2020 (when the first COVID-19 cases were diagnosed in Juruá Valley) at least one of the following symptoms that were among the most suggestive of COVID-19 before the emergence of the Omicron variant: cough, shortness of breath, and loss of taste or smell. We define the proportion of children fulfilling the above criteria as the “period prevalence” of clinically apparent COVID-19, since children may have experienced one or more COVID-19 episodes anytime between the pandemic onset and the follow-up visit at 5 years of age. Our analysis does not consider other common signs and symptoms such as fever, diarrhea, and vomiting, which are often reported in childhood COVID-19 [22], because they are little specific and may be found in several other locally prevalent febrile illnesses.

Data analysis

Data were collected using tablets programmed with CSPro software (https://www.census.gov/programs-surveys/international-programs.html) and transferred to STATA 15.1 (StataCorp, College Station, TX, USA) for statistical analysis.

We report raw seroprevalence rates as point estimates and 95% confidence intervals (CIs), as well as sensitivity- and specificity-adjusted prevalence estimates along with 95% highest density intervals (HDIs). To this end, we used a Bayesian framework that propagates uncertainty in the sensitivity and specificity estimates of the test [23]–reported as 97.2% (95% CI, 95.4–98.4%) and 99.8% (95% CI, 99.3–100%), respectively [24].

Separate multiple Poisson regression models were built to identify predictors associated with each of two binary outcomes: (1) SARS-CoV-2 infection (using seropositivity as a proxy) regardless of any clinical signs and symptoms, and (2) clinically apparent COVID-19 upon serologically documented SARS-CoV-2 infection. Because children are nested into two study rounds (grouping variable “round”), which introduces dependency among observations, for each outcome we built mixed-effects Poisson regression models with random effects at the “round” level and robust variance. Variables associated with the outcome at a significance level <20% in unadjusted analysis were entered in multiple Poisson regression models. We next used a hierarchical approach based on conceptual frameworks [25] to select covariates that were retained in the final adjusted models (S1 File). Participants with missing values in categorical covariates were maintained in the model by creating a new missing-value category. Statistical significance was defined at the 5% level. Prevalence ratio (PR) estimates are provided along with 95% CIs to quantify the influence of each predictor on the outcome while controlling for all other covariates.

Results

High SARS-CoV-2 seroprevalence but low COVID-19-related morbidity in Amazonian children

Overall, 297 (45.0%; 95% CI 41.2–48.9%) of the 660 study participants had serological evidence of prior SARS-CoV-2 infection (Fig 2B). Not surprisingly, given the dramatic increase in COVID-19 incidence in the study site in February-March 2021 (Fig 2A), antibody positivity rates were significantly higher during the second study round (June-July 2021: 52.1%, 95% CI, 46.6–57.6%), which was carried out after the case upsurge (June-July 2021), when compared with the first round (January 2021: 37.9%, 95% CI, 32.6–43.4%), with P < 0.01 by the χ2 test. The sensitivity- and specificity-adjusted seroprevalence estimates were 53.6% (95% HDI, 48.0–59.2%) for June-July 2021 and 38.9% (95% HDI, 33.5–44.4%) for January 2021. Only 15 (5.1%; 95% CI, 2.9–8.2%) seropositive children had a prior medical diagnosis of COVID-19 reported by their mothers or guardians at the time of the interview. Previous laboratory confirmation of SARS-CoV-2 infection was reported for only 11 (3.7%; 95% CI, 1.9–6.5%) seropositive children, 8 by antigen-based rapid diagnostic test, and 3 by reverse-transcriptase (RT)-PCR.

Fig 2

COVID-19 cases in Cruzeiro do Sul, Brazil, between April 2020 and July 2021 and period prevalence of SARS-CoV-2 infection and clinically apparent COVID-19 in 5 years-old children as measured in January 2021 (first study round) and June-July 2021 (second study round).

(A) Monthly cases of COVID-19 notified in the municipality of Cruzeiro do Sul, between April 2020 and July 2021. Light purple and light blue shaded areas represent the dates of follow-up assessment in January 2021 and June-July 2021, respectively. Data source: State Secretary of Health, Acre. Data available daily at: http://saude.acre.gov.br/. (B) Circles show the proportions (%) of children positive for anti-SARS-CoV-2 antibodies in the first study round (light purple), in the second round (light blue), and in both rounds combined (grey). Squares show the period prevalence (%) of clinically apparent COVID-19 (see the main text for definition) among these same children, as estimated in the first study round (light purple), in the second round (light blue), and in both rounds combined (grey). A total of 330 children were assessed during each study round. Error bars indicate 95% confidence intervals.

COVID-19-related morbidity appeared to be infrequent in this population. Only 48 (16.2%; 95% CI, 12.2–20.8%) of the 297 seropositive children had cough, shortness of breath, and/or loss of taste or smell, according to their mothers or guardians. The relative frequencies of reported symptoms were: cough (11.4%; 95% CI, 8.1–15.6%), loss of taste or smell (3.7%; 95% CI, 1.8–6.5%), and shortness of breath (2.7%; 95% CI, 1.2–5.2%). S2 Fig shows the extent of overlap of symptoms experienced by SARS-CoV-2 seropositive children. Among the 363 SARS-CoV-2 seronegative children, only 19 (5.2%; 95% CI, 3.2–8.1%) had a report of cough, shortness of breath, and/or loss of taste or smell during the study period.

The period prevalence of clinically apparent COVID-19, defined as the presence of specific antibodies and one or more of the symptoms listed above experienced since the pandemic onset, was estimated at 7.3% (95% CI, 5.4–9.5%) by the age of 5 years. COVID-19 period prevalence estimates were similar between study rounds: 7.9% (95% CI, 5.2–11.3%) in January 2021, and 6.7% (95% CI, 4.2–9.9%) in June-July 2021 (Fig 2B).

Higher risk of SARS-CoV-2 infection and COVID-19 among socioeconomically vulnerable children

Children living under conditions of socioeconomic vulnerability–those from the poorest households, exposed to food insecurity, and with non-White and less educated mothers–were significantly more likely to be seropositive and/or to have experienced COVID-19-related morbidity. Results of the unadjusted analysis are shown in Table 1. Children from the poorest households and those with less-educated mothers remained significantly more likely to be seropositive at the age of 5 years after controlling for potential confounders (Table 2). Similarly, children from households experiencing food insecurity and those born to non-White mothers were more likely to have had COVID-19-related morbidity by the age of 5 years, after adjustment for confounders (Table 2). Importantly, the presence of these same clinical symptoms (cough, shortness of breath, and/or loss of taste or smell) was not significantly associated with household food insecurity or mother′s skin color among SARS-CoV-2 seronegative children (n = 363; S1 Table). We found evidence that the socioeconomic status (children in the intermediate, but not the highest tertile, compared with the first tertile of wealth index) and >12 years of mother′s schooling were predictors of reduced risk of COVID-19-unrelated respiratory symptoms, with no clear dose-response relationship (S1 Table).

Table 1

Prevalence (%) of SARS-CoV-2 antibodies and prevalence ratio (95% confidence interval) of clinically apparent COVID-19 in 5 years-old Amazonian children according to sociodemographic, neonatal, nutritional, and morbidity history variables.

		SARS-CoV-2 antibodies		Clinically apparent COVID-19
Variable	Total	Prevalence (%)	Prevalence ratio (95% CI)*	Prevalence (%)	Prevalence ratio (95% CI)*
Household wealth index1 (poorest)23 (wealthiest)	222219219	52.347.535.2	10.90 (0.65–1.23)0.67 (0.54–0.84)P < 0.001	8.67.36.0	10.85 (0.63–1.16)0.69 (0.31–1.56)P = 0.369
Support from the Bolsa Família programNo Yes	360300	40.850.0	11.23 (1.10–1.38)P < 0.001	5.39.7	11.83 (0.66–5.08)P = 0.245
Household food insecurityNoYes	306354	43.846.1	11.06 (0.94–1.19)P = 0.334	4.99.3	11.90 (1.18–3.06)P = 0.008
Mother′s schooling≤ 9 years10 to 12 years>12 years	172327146	48.349.532.2	11.05 (0.95–1.15)0.68 (0.67–0.69)P < 0.001	9.96.75.5	10.68 (0.31–1.47)0.55 (0.30–1.02)P = 0.141
Mother′s skin colorWhiteNon-white	79567	30.447.3	11.59 (0.99–2.53)P = 0.053	2.57.9	13.13 (2.67–3.68)P < 0.001
Mother′s occupationNo paid jobPaid job	409237	48.739.2	10.82 (0.79–0.86)P < 0.001	8.15.9	10.73 (0.62–0.86)P < 0.001
Child′s age (months)	660	--	0.97 (0.96–0.99)**P < 0.001	--	0.91 (0.91–0.92)**P < 0.001
Child′s sexFemaleMale	326334	44.245.8	11.03 (0.95–1.12)P = 0.411	6.87.8	11.15 (0.59–2.27)P = 0.680
Birth weight (kg)2.5 -- 4.0< 2.5> 4.0	5694842	45.250.038.1	11.14 (0.73–1.78)0.84 (0.73–0.96)P = 0.069	7.410.42.4	11.41 (1.12–1.77)0.32 (0.40–2.58)P = 0.266
PrematurityNoYes	60159	45.639.0	10.85 (0.78–0.93)P = 0.001	7.28.5	11.18 (0.88–1.59)P = 0.255
Exclusive breastfeeding≥ 90 daysNoYes	389214	4.046.7	11.02 (0.89–1.18)P = 0.738	8.26.5	10.80 (0.48–1.32)P = 0.373
Total breastfeeding ≥12 monthsNoYes	219429	42.045.2	11.06 (0.83–1.35)P = 0.626	9.65.6	10.58 (0.33–1.03)P = 0.063
Total breastfeeding ≥12 monthsNoYes	219429	42.045.2	11.06 (0.83–1.35)P = 0.626	9.65.6	10.58 (0.33–1.03)P = 0.063
Stunting at 5 years of ageNoYes	64515	44.853.3	11.20 (0.87–1.65)P = 0.259	7.36.7	10.91 (0.09–9.15)P = 0.940
Overweight at 5 years of ageNoYes	57189	44.846.1	11.01 (0.57–1.82)P = 0.963	7.27.9	11.10 (0.22–5.43)P = 0.911
Malaria infection within the past 12 monthsNoYes	64317	44.852.9	11.14 (0.90–1.46)P = 0.283	7.211.8	11.64 (1.08–2.51)P = 0.021
Pneumonia within the past 12 monthsNoYes	6555	45.040.0	10.87 (0.44–1.73)P = 0.687	7.30	-
Anemia at 5 years of ageNoYes	62434	44.455.9	11.25 (1.12–1.39)P < 0.001	7.45.9	10.80 (0.11–5.87)P = 0.825
Immunization status#CompleteIncomplete	513147	44.646.3	11.03 (0.95–1.12)P = 0.417	7.46.8	10.92 (0.65–1.29)P = 0.624

Clinically apparent COVID-19 was defined in children with SARS-CoV-2 antibodies who reported at least one of the following symptoms/signs: cough, shortness of breath, and loss of taste or smell. Totals differ due to missing values.

*95% CI = 95% confidence interval.

**PR variation per month.

# Immunization status was considered complete when the participant received all doses of the recommended vaccines up to 5 years of age.

Table 2

Adjusted prevalence ratios (PR) and 95% confidence intervals (95% CI) for predictors of SARS-CoV-2 infection and clinically apparent COVID-19 in 5-year-old Amazonian children, as estimated by mixed-effects multiple Poisson regression models (n = 660).

Variables	SARS-CoV-2 infection			COVID-19
	PR	95% CI	P	PR	95% CI	P
Child’s age (months)	0.97	0.96–0.98	<0.001	0.90	0.90–0.92	<0.001
Mother′s skin color*WhiteNon-white	11.54	0.93–2.54	0.091	13.05	2.67–3.49	<0.001
Household wealth index1 (poorest)23 (wealthiest)P for trend	10.920.74	-0.72–1.180.57–0.97	-0.4960.0290.045	-	-	-
Mother′s schooling* ≤9 years10 to 12 years>12 yearsP for trend	11.090.81	-0.98–1.200.66–0.99	-0.1260.0380.012	10.680.66	-0.32–1.460.42–1.06	-0.3240.0860.205
Household food insecurityNoYes	-			11.76	-1.17–2.65	-0.007

Clinically apparent COVID-19 was defined in children with SARS-CoV-2 antibodies who reported at least one of the following symptoms/signs: cough, shortness of breath, and loss of taste or smell.

*Missing values: mother’s skin color, n = 14; mother’s schooling, n = 15.

Familial and household aggregation of SARS-CoV-2 infection and COVID-19

Consistent with substantial household-level SARS-CoV-2 transmission [8,26], children whose household and family contacts (parents, other close relatives, or neighbors) reported one or more prior COVID-19 episodes were significantly more likely to be seropositive and have experienced COVID-19 morbidity by the age of 5 years (Fig 3). Separate models were built for each child’s contact (mother, father, siblings, grandparents, etc.). Positive associations were stronger for child contacts who usually live inside the child’s household, such as parents and siblings. Nevertheless, significant associations were also found for grandparents and more distant relatives, as well as neighbors, who usually do not share the household with the study children (Fig 3).

Fig 3

Association between self-reported prior COVID-19 in household and family contacts (close relatives or neighbors) and the risk of SARS-CoV-2 infection and clinically apparent COVID-19 in 5 years-old Amazonian children.

Adjusted prevalence ratios (PR) indicate the magnitude of association between exposure to COVID-19-reporting household or family contacts and two outcomes, SARS-CoV-2 seropositivity and COVID-19, among MINA-Brazil study participants (n = 660), after controlling for the following potential confounders: child′s age, mother′s self-reported skin color and schooling, and household wealth index tercile for SARS-CoV-2 seropositivity; and child′s age, mother′s self-reported skin color and schooling, and presence of household food insecurity for clinically manifest COVID-19. Note that the denominators (numbers of children at risk) are the same in analyses with different outcomes (SARS-CoV-2 infection in the upper panel and clinically apparent COVID-19 in the lower panel). PR estimates and their respective 95% confidence intervals (95% CIs) and P values were derived from separate multiple mixed-effects Poisson regression models which each child contact type tested at once as an explanatory variable.

Discussion

The present cross-sectional serosurveys in the Amazon Basin of Brazil provide evidence for a substantial underreporting of SARS-CoV-2 infection among young children from this hard-hit region, with clear public health implications. For example, the misperception that young children are less susceptible to infection has been identified as a major cause of parental vaccine hesitancy [27,28], defined as parents’ delay in acceptance or refusal of vaccines despite their availability [29]. In our study, 45% of the participants had SARS-CoV-2 antibodies at the age of 5 years but only 5% of them had a prior episode of COVID-19 reported by their mothers or guardians, suggesting that 8 in 9 infections remained undiagnosed–and, therefore, were not notified. As expected [3-5], most childhood infections were subclinical and only 16% of the seropositive children had reportedly experienced cough, shortness of breath, and/or loss of taste or smell since the pandemic started.

Importantly, children with household or family contacts who reported prior COVID-19 were substantially more likely to be infected and to present COVID-19-related illness (Fig 3). The positive association between COVID-19 risk in children and past infection in their mothers was particularly strong. We acknowledge that our cross-sectional analysis does not allow us to infer the direction of SARS-CoV-2 transmission between children and their close contacts. However, literature data suggest that adults are more likely than children to acquire COVID-19 from a household or family index case [26]. We hypothesize that asymptomatic young children [8], especially those with relatively high viral loads [6,7], may be a significant source of household transmission to adults in settings characterized by poor housing conditions, little compliance with social distancing, and limited availability of diagnostic testing to identify subclinical SARS-CoV-2 carriage. Whether this applies to our study participants remains to be investigated by considering (currently unavailable) information on time of infection of each household member. Nursery school attendance does not account for the high SARS-CoV-2 prevalence in our population, since <1% of the children have reportedly attended childcare facilities since the school closures in early April 2020.

The COVID-19 pandemic has exacerbated pre-existing socioeconomic and health disparities worldwide [30] which, in turn, increase the risk of SARS-CoV-2 infection and COVID-19 morbidity in children. This is due to poverty-related comorbidities (e.g., malnutrition), reduced access to healthcare, low-quality and overcrowded housing, and parents′ exposure to high-risk occupations [31]. We note, however, that some SARS-CoV-2 seropositive children may have been misclassified as having experienced clinically apparent COVID-19 in our study if they had other poverty-related respiratory infections leading to cough, shortness of breath, or loss of taste or smell. Importantly, socially vulnerable people are less likely to be tested for SARS-CoV-2 infection, but more likely to be positive once tested [32]. Here we present further evidence that, as in the United States and United Kingdom [33], the socioeconomic and ethnic status are associated with SARS-CoV-2 infection and COVID-19 risk in Brazil [34,35]. The most vulnerable Amazonian children have higher SARS-CoV-2 seropositivity rates and are more likely to have experienced COVID-19-related morbidity by the age of 5 years (Table 2). Notably, study children from the poorest families are at increased risk of many other poverty-related childhood infections in addition to COVID-19 –including malaria, dengue, and intestinal and respiratory infections by other viruses, bacteria, and parasites–that may also severely affect their growth and development.

Children exposed to household food insecurity, which affects 54% of our study participants (Table 1), had their COVID-19 risk increased by 76% (Table 2). We note that food insecurity rates have increased worldwide since the onset of the COVID-19 pandemic [36,37], most markedly in low- and middle-income countries [38,39]. Compared with 2019, approximately 14 million more people in Latin America and the Caribbean were affected by hunger in 2020 (https://data.unicef.org/resources/sofi-2021/). Over one-third of households in Brazil already experienced some degree of food insecurity before the COVID-19 pandemic [40] and this proportion is estimated to have increased to 55% in 2020 (http://olheparaafome.com.br/VIGISAN_Inseguranca_alimentar.pdf), due to the reduced economic activity and increased food prices during the pandemic [38-41]. Importantly, the economic crisis aggravated by the pandemic has led families to rely on cheaper foods, most of which ultra-processed products that are rich in sugar, sodium, and fat but lack essential nutrients [40]. Strategies to mitigate poverty and food insecurity are urgently needed in the context of the current COVID-19 pandemic [36,40].

The present study has some limitations. First, only 53% of the original MINA cohort participants enrolled at birth (n = 1246) and 56% of those alive at the age of 5 years (n = 1240) had SARS-CoV-2 antibodies measured (Fig 1). Children lost to follow-up differ from those participating in the present serosurvey in key correlates of socioeconomic vulnerability, such as socioeconomic status and mother′s education, suggesting that SARS-CoV-2 infection and disease could be even more common in the original study population. Second, antibody measurements were carried out during two separate study rounds, with substantial SARS-CoV-2 transmission occurring between them (Fig 2A). Moreover, the time elapsed between potential exposure and detection of seroconversion varies widely among study participants and seroprevalence rates are not directly comparable between surveys. We used two approaches to cope with this limitation. We used a serological test with stable signal over time and rare seroreversions following a natural infection [21] to minimize the risk of differential misclassification (participants infected during the first pandemic wave might be more likely to yield a false-negative serology due to waning antibody responses). In addition, we used mixed-effects regression models to account for the clustering of participants within study rounds, as participants in each round are expected to differ in their previous exposure to (and time elapsed since) SARS-CoV-2 infection. Third, the symptoms investigated retrospectively are not necessarily specific for childhood COVID-19 and may be affected by recall bias. Some symptoms may have been caused by other locally prevalent infections experienced by their children since the COVID-19 pandemic started, such as malaria. Only the unadjusted analysis showed that the 17 children with self-reported malaria within the past 12 months were more likely to report one or more clinical signs and symptoms associated with COVID-19 (Table 1), suggesting that our definition of clinically apparent COVID-19 may have been, at least in part, confounded by malaria-associated symptoms. We acknowledge respiratory symptoms caused by diseases other than COVID-19, but also associated with poverty (S1 Table), as a potential source of residual confounding in the association between clinically apparent COVID-19 and socioeconomic status. However, only 5.2% of the SARS-CoV-2 seronegative had COVID-19-like symptoms (cough, shortness of breath, and/or loss of taste or smell) during the study period and their prevalence was not significantly associated with mother′s skin color of household food insecurity (S1 Table). Fourth, to minimize the risk of false positives, we excluded from our symptom/sign list other clinical manifestations that, although not specific, are commonly reported in childhood COVID-19. These include fever, vomiting, diarrhea, and headache [22]. For example, seropositive children with reported fever but without cough, shortness of breath, and loss of taste or smell have not been classified as having experienced clinically apparent COVID-19. Moreover, symptom reports are prone to recall bias and mild clinical manifestations may have been overlooked by mothers and guardians. Consequently, we surely have missed some clinically apparent COVID-19 cases in our study population.

Conclusion

The present study provides new insights into the epidemiology of SARS-CoV-2 infection and COVID-19 and its association with social inequalities in young Amazonian children. Our results indicate that SARS-CoV-2 infections are frequent but substantially underreported among 5 years-old children in the Brazilian Amazon and possibly in other similar high-prevalence tropical settings. Children in the most socioeconomically vulnerable households are disproportionately affected by SARS-CoV-2 infection. Importantly, children living in households experiencing food insecurity and born to non-White mothers are more likely to have COVID-19-related morbidity once infected, further contributing to socially determined health disparities in the Amazon.

STROBE checklist.

(DOCX)

Click here for additional data file.

Study site.

The map shows Brazil and Peru in South America (panel A) and the location of the municipality of Cruzeiro do Sul (dark green) in the Upper Juruá Valley region, Acre State (light green), northwestern Brazil (panel B). The urban area of Cruzeiro do Sul is shown in greater detail in panel C. Other cities and towns in the region (Mâncio Lima, Guajará, and Rodrigues Alves) are also indicated by triangles. Roads and streets are represented in light gray. Rivers are represented in blue. Figure created with QGIS software version 3.14, an open-source Geographic Information System (GIS) licensed under the GNU General Public License (https://bit.ly/2BSPB2F). Publicly available shapefiles were obtained from the Brazilian Institute of Geography and Statistics (IBGE) website (https://bit.ly/34gMq0S). Roads, streets, and rivers were obtained from the Open Street Map Foundation website (https://bit.ly/3pzh4xp). All utilized geographical data are under the Creative Commons Attribution License (CC BY 4.0). Reproduced from: Pincelli A, Cardoso MA, Malta MB, Johansen IC, Corder RM, Nicolete VC, et al. Low-level Plasmodium vivax exposure, maternal antibodies, and anemia in early childhood: Population-based birth cohort study in Amazonian Brazil. PLoS Negl Trop Dis. 2021;15:e0009568. doi: 10.1371/journal.pntd.0009568.

(PDF)

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Venn diagram of the four considered symptoms experienced by SARS-CoV-2 seropositive children since the pandemic onset, as reported by mothers and guardians.

(PDF)

Click here for additional data file.

Supplementary methods.

(DOCX)

Click here for additional data file.

Crude prevalence ratios (PR) and 95% confidence intervals (95% CI) for predictors of respiratory symptoms suggestive of respiratory infection (cough, shortness of breath, and/or loss of taste or smell in SARS-CoV-2-seronegative 5-year-old Amazonian children, as estimated by mixed-effects multiple Poisson regression models (n = 363).

(DOCX)

Click here for additional data file.

29 Mar 2022

Dear Dr. Cardoso,

Thank you very much for submitting your manuscript "SARS-CoV-2 infection and COVID-19 among 5 years-old Amazonian children and their association with poverty and food insecurity" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

Both Reviewers brought up important comments about the experimental design, the analysis and interpretation of data, and the significance of results. These points must be addressed. One reviewer had excellent suggestions for re-organizing the manuscript, particularly to ensure the manuscript is focused on very well-defined research objectives, eliminating or moving less-important information, and making the statistical and analytical approaches very clear.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. 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.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Georgios Pappas

Associate Editor

PLOS Neglected Tropical Diseases

Jeremy V. Camp, PhD

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer comments about design and results' significance need to be addressed.

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: Overall, it appears authors have an ongoing birth cohort from which they are using to assess the SARS-CoV-2 antibodies in children in Amazonian Brazil. This type of study is needed to fill the current gaps in the COVID literature, but I do recommend some revisions before full manuscript acceptance.

-Currently as the manuscript stands there is a lot of information about the birth cohort and variable acquisition that may potentially benefit from paraphrasing and including these methods in a supplementary file for individuals who are more interested in the cohort conception. Authors should primarily focus on the methods specific to this analysis of prevalence of SARS-CoV-2 antibodies among children from Brazil, this way it is more clear and concise for readers.

-Data Analysis: Authors decided to use poisson regression to identify factors associated with: 1 SARS-CoV-2 infection and 2 COVID-19 with clinical symptoms and serological positive test. Authors state that they created a separate category for missingness within covariates. This type of approach is not ideal, I suggest authors revise their analysis to include multiple imputation of the missing data for the regression models.

- Data Analysis: Authors use an alpha level of <0.20 to select variables to adjust for in the model. This approach is typically not ideal and leads to residual confounding. Ideally authors should use another methods such as change in estimate or directed acyclic graphs, unless authors are trying to truly identify "predictors". This is not clear in the methods section of the true goal of the multivariable model. Throughout the results it appears authors were trying to build more of a causal model. This lack of clarity has made my enthusiasm for the paper decrease.

-Laboratory methods for serological evidence: Currently authors have only stated that the nucleocapsid antigen is what was detected, was any method to detect spike protein or RBD?

Reviewer #2: -Are the objectives of the study clearly articulated with a clear testable hypothesis stated? - NO

-Is the study design appropriate to address the stated objectives? - NO

-Is the population clearly described and appropriate for the hypothesis being tested? - No

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested? - NO

-Were correct statistical analysis used to support conclusions? - yes

-Are there concerns about ethical or regulatory requirements being met? - yes

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

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: The results of the multivariable model do not match the goal of the analysis plan, recommend authors address whether they are trying to identify a causal model or identify predictors.

Figure/Table placeholders are currently placed in the methods section, recommend authors consider putting these placeholders near the results they pertain to.

Reviewer #2: Does the analysis presented match the analysis plan? - yes

-Are the results clearly and completely presented? -yes

-Are the figures (Tables, Images) of sufficient quality for clarity? - too many

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

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: Overall, nice discussion and public health relevance is addressed. However, I do a couple of concerns that authors should address in the limitations section:

-Authors do not address the time between potential exposure and seroconversion. We know that individuals can wane in detection overtime and this is a bias/flaw of the study design. This needs to be addressed of how this would impact the effect estimates; along if its differential/non-differential misclassification; and the degree to which the effect estimates may be impacted

Reviewer #2: Are the conclusions supported by the data presented? - NO

-Are the limitations of analysis clearly described? -yes

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study? -Yes

-Is public health relevance addressed? - yes

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

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

Reviewer #2: None

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

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: (No Response)

Reviewer #2: Author’s efforts to conduct research work and publish this study entitled “SARS-CoV-2 infection and COVID-19 among 5 years-old Amazonian children and their association with poverty and food insecurity” are appreciable. However, there are many issues related to this study. Some of the comments are as follows.

1. selected study population from ongoing enrolled cohort cannot be equated with general population as the opportunities to interact with care provider, counselling and heath care seeking behaviour are likely to be different.

2. Inclusion of a control group from general population was desirable.

3. This data pertains to the pandemic survivors only. There is no information about those children who lost their life due to covid or related complications.

4. Based on the reports from other countries, most vulnerable children for covid complications and adverse outcome are in the preschool age group (especially infants) or adolescence. Rationale for screening at 5 years of age is not clear.

5. Basic population characteristics with regard to immunisation status, anemia, chronic infections and other comorbidities are not addressed

6. Title of the study is not representative of work done and need modifications with regard to Covid sero-positivity

7. Conclusions are not supported by the study and represent more of general statement.

8. Author have listed many limitations of this study, substantiating a fact that it may not be possible to draw a meaningful conclusion from this study and possibly does not add much to existing knowledge.

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

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Reviewer #1: No

Reviewer #2: No

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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

7 Apr 2022

Submitted filename: Reviewer comments.docx

Click here for additional data file.

29 Apr 2022

Dear Dr. Cardoso,

Thank you very much for submitting your manuscript "SARS-CoV-2 seropositivity and COVID-19 among 5 years-old Amazonian children and their association with poverty and food insecurity" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Due to the mixed reviews on the first evaluation, an additional reviewer was invited. The previous reviewer (1) noted that all issues were addressed, and it is in the editors' opinions that the issues raised by Reviewer 2 were adequately addressed. However, there are still some issues to be addressed, as specified by a new reviewer. As you have extensively revised the first submission, and the revision is now much more clear about the study design, analysis, and limitations, we believe the remaining issues may be easily resolved.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

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

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Georgios Pappas

Associate Editor

PLOS Neglected Tropical Diseases

Jeremy V. Camp, PhD

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Please note editorial comments above when responding to the additional comments from reviewers.

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: (No Response)

Reviewer #3: 1. The retrospective study design is a limitation.

2. It is not clear how the study was planned and carried out. Is the design overall explorative? Specifically, was there any pre-plan of outcomes, or based on what were they selected? If the outcome selection was based on preliminary results, the quality (i.e. replicability) of the results will be low because of high risk of Type 1 error. In the same line of thought, were there any pre-planned statistical analysis plan?

3. It is essential to address whether the findings are SARS-CoV-2/COVID19-specific, or in fact pertinent to airway infections in general: The analyses must include an (exploratory) analyses with another composite outcome of any cough, shortness of breath, loss of taste or smell (and no regard to +/- SARS-CoV-2 results). Such approach will test if (the same? or other?) socioeconomic factors determine symptoms often experienced by individuals having airway infections in general. Using symptoms of gastrointestinal infection as the explorative composite outcome, the same approach would test if (the same? or other?) socioeconomic factors determine infections in general. Such input is of outmost importance to the overall message: If socioeconomic factors determine any infection (which is likely), specific vaccination programs will not suffice when it comes to securing better health globally.

4. Based on what was predictors selected? Also after reading the authors' answer to prior reviewer comments, the processes of predictor selection and statistical modelling are not clearly described and argued for. For each round of analyses (crude, adjusted, final adjusted), please provide information on assumptions, criteria and evidence base, inclusive considerations about each factor (for example is household food insecurity considered a socioeconomic factor, or a somatic factor (hunger and malnutrition leading to less efficient organ function and immune responses)?.

5. Figure 2: Information on deaths in the overall population is irrelevant and may be misleading. Please delete information on mortality from the figure.

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

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: (No Response)

Reviewer #3: 1. Figure 3: How can the percentages be the excact same in the two analyses?

2. Any considerations about multiplicity?

3. Line 265 - 267: Why "interestingly"? It is well known - also for SARS-CoV-2 - that crowding, especially indoor crowding, increases the risk of transmission. Please note, as mentioned above, that transmission patterns cannot be inferred from the present study where no information on index case is available (please see Discussion below).

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

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: (No Response)

Reviewer #3: Lines 290 - 293 the authors conclude that their data point towards small children being a source of SARS-CoV-2-household transmission. That cannot be concluded based on the present study, since data on the timing of infection (i.e. index cases) were not available. It may be most likely that the SARS-CoV-2 is introduced to the family by the adolescents or adults who have more contacts outside the family.

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

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

Reviewer #3: (No Response)

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

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: Thank you for addressing all of my comments/feedback, the manuscript reads much clearer.

I just have two very minor revisions:

1. Throughout the text, some sentences appear in a lighter text and different font. Recommend revising.

2. From my prior concern: "Authors do not address the time between potential exposure and seroconversion. We know

that individuals can wane in detection overtime and this is a bias/flaw of the study design. This

needs to be addressed of how this would impact the effect estimates; along if its

differential/non-differential misclassification; and the degree to which the effect estimates may

be impacted."

It is recommended authors add a summarized version of their response/rationale to this comment in the limitations section of the manuscript. Ensure you state regardless of the method used the type of misclassification bias and the degree this may impact your effect estimates.

Reviewer #3: Thank you for letting med review this interesting and relevant study.

Major revisions are needed to Methods, Results and Discussion.

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

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Reviewer #1: No

Reviewer #3: Yes: Lone Graff Stensballe

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (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 figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

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

References

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. If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice.

10 May 2022

Submitted filename: Reviewer comments 2.docx

Click here for additional data file.

30 May 2022

Dear Dr. Cardoso,

Thank you very much for submitting your manuscript "SARS-CoV-2 seropositivity and COVID-19 among 5 years-old Amazonian children and their association with poverty and food insecurity" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic.

The manuscript will be evaluated upon the next revision by the editorial staff and will not go to reviewers again. It can be considered accepted, provided that the minor issues raised by Reviewer 3 will be addressed.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

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

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Georgios Pappas

Associate Editor

PLOS Neglected Tropical Diseases

Jeremy Camp

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

The manuscript will be evaluated upon the next revision by the editorial staff and will not go to reviewers again. It can be considered accepted, provided that the minor issues raised by Reviewer 3 will be addressed.

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: (No Response)

Reviewer #3: OK

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

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: (No Response)

Reviewer #3: Please note that the new Table S1 presenting predictors of symptoms suggestive of respiratory infection in the SARS-CoV-2 negative children show significant associations between the socioeconomic factors "household wealth index being beyond 1 (the poorest)", and "mothers schooling being beyond 9 years" and a significantly decreased risk of symptoms suggestive of respiratory infection. Thus, the concept socioeconomic status certainly predicts respiratory symptoms in the children under study, and this prediction is not SARS-CoV-2 specific. Although in the analysis of the subgroup of SARS-CoV-2 positive children, the associations were significant for other socioeconomical factors, namely "non-white mothers" and "food insecurity".

And residual confounding is - of course - present in the present observational study and must be acknowledged.

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

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: (No Response)

Reviewer #3: Significant associations were found between socioeconomic factors and respiratory symptoms, no matter if the children were SARS-CoV-2 positive or negative. This means that for example covid19-vacicination is not the cure. Improved socio-economy is a much better cure! I find that message of utmost importance. The point of socio-economy predicting transmission and severity of a broad range of infections, in the present study defined as respiratory symptoms, is indeed important and should be elaborated upon in the discussion.

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

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

Reviewer #3: (No Response)

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

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: Thank you for addressing the comment on misclassification bias. I have no further recommendations/revisions.

Reviewer #3: Thank you for letting me read this interesting and relevant manuscript again.

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

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References

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7 Jun 2022

Submitted filename: Rebuttal 3.docx

Click here for additional data file.

13 Jun 2022

Dear Dr. Cardoso,

We are pleased to inform you that your manuscript 'SARS-CoV-2 seropositivity and COVID-19 among 5 years-old Amazonian children and their association with poverty and food insecurity' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Georgios Pappas

Associate Editor

PLOS Neglected Tropical Diseases

Jeremy Camp

Deputy Editor

PLOS Neglected Tropical Diseases

***********************************************************

1 Jul 2022

Dear Dr. Cardoso,

We are delighted to inform you that your manuscript, "SARS-CoV-2 seropositivity and COVID-19 among 5 years-old Amazonian children and their association with poverty and food insecurity," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

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Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases