Association between breastfeeding, host genetic factors, and calicivirus gastroenteritis in a Nicaraguan birth cohort

Background Norovirus and sapovirus are important causes of childhood acute gastroenteritis (AGE). Breastfeeding prevents AGE generally; however, it is unknown if breastfeeding prevents AGE caused specifically by norovirus and sapovirus. Methods We investigated the association between breastfeeding and norovirus or sapovirus AGE episodes in a birth cohort. Weekly data on breastfeeding and AGE episodes were captured during the first year of life. Stools were collected from children with AGE and tested by RT-qPCR for norovirus and sapovirus. Time-dependent Cox models estimated associations between weekly breastfeeding and time to first norovirus or sapovirus AGE. Findings From June 2017 to July 2018, 444 newborns were enrolled in the study. In the first year of life, 69 and 34 children experienced a norovirus and a sapovirus episode, respectively. Exclusive breastfeeding lasted a median of 2 weeks, and any breastfeeding lasted a median of 42 weeks. Breastfeeding in the last week did not prevent norovirus (HR: 1.09, 95% CI: 0.62, 1.92) or sapovirus (HR: 1.00, 95% CI: 0.82, 1.21) AGE in a given week, adjusting for household sanitation, consumption of high-risk foods, and mothers and childs histo-blood group phenotypes. Maternal secretor-positive phenotype was protective against norovirus AGE, whereas childs secretor-positive phenotype was a risk factor for norovirus AGE. Interpretation Exclusive breastfeeding in this population was short-lived, and no conclusions could be drawn about its potential to prevent norovirus or sapovirus AGE. Non-exclusive breastfeeding did not prevent norovirus or sapovirus AGE in the first year of life. However, maternal secretor-positive phenotype was associated with a reduced hazard of norovirus AGE.


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WHAT IS KNOWN

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-It is unknown if breastfeeding prevents AGE caused by norovirus or sapovirus, which are leading 50 causes of childhood AGE worldwide.

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-Host genetic factors can influence AGE risk, and the mediating effects of breastfeeding are 52 unclear.

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-Exclusive breastfeeding was rare, and breastfeeding did not prevent norovirus (hazard ratio 1.09,

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-Secretor-positive phenotype of the child and the mother may be more important than 57 breastfeeding in determining norovirus and sapovirus AGE risk in this setting.
. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted April 17, 2022. ;https://doi.org/10.1101/2022 The main outcome was the first episode of AGE in which norovirus or sapovirus was detected in 110 the stool. AGE surveillance occurred weekly, and an AGE episode was characterized by diarrhea, defined 111 as ≥3 stools that were loose or looser than normal in a 24-hour period, including notable changes to the 112 stool, such as presence of blood or excessive liquid; and/or vomiting. This analysis was restricted to the 113 first detected episode of norovirus or sapovirus. Breastfeeding practice was ascertained using a weekly 114 questionnaire administered to the mothers. Children for whom the answer to the question "Did you 115 breastfeed your child yesterday?" was "Yes" were considered breastfed. The child was considered to be 116 exclusively breastfed until the first week the mother answered "Yes" to question "In the past week, has 117 your child received any food, formula, or liquids, including water or tea or liquid in a bottle, in addition to 118 breast milk?"; the child was considered non-exclusively breastfed thereafter. Children were considered to susceptibility to infections, including in a prior study conducted in the same birth cohort, 17 we adjusted . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted April 17, 2022. ;https://doi.org/10.1101https://doi.org/10. /2022 134 for the child's secretor and Lewis phenotypes. Due to the additional potential for mother's HBGAs 135 phenotypes to be associated with AGE risk, 16 we also adjusted for mother's secretor and Lewis 136 phenotypes.

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Sample collection and laboratory methods

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Stool samples were collected from children with AGE within 10 days of the onset of symptoms.

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Specimens were collected from a plastic container or a soiled diaper retrieved from the household within  Figure 1).

Severity of norovirus and sapovirus infections
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The copyright holder for this preprint this version posted April 17, 2022.  (Table 3).

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Our study sample had a higher proportion of children who were secretor-positive compared to 202 mothers, which was unexpected given that this salivary phenotype is inherited as a dominant Mendelian 203 trait. When the analysis was restricted to mothers of secretor-positive children, the protective association . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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between mother secretor-positive phenotype showed slightly greater protection against norovirus AGE

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In a post-hoc analysis, we estimated the association between breastfeeding and severity of AGE is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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that travel time to a health center greater than one hour was associated with exclusive breastfeeding in the 229 first six months of life. 23 In León, most women have access to a health post in their neighborhood,

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suggesting that exclusive breastfeeding is more common among women living in remote or rural areas.

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Children experienced their first norovirus and sapovirus episodes at approximately 8 months of 232 age, long after most mothers had stopped breastfeeding exclusively and began introducing supplementary 233 foods, suggesting that some maternally-acquired immunity prevented infections in early infancy.

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However, Nicaraguan women are guaranteed eight weeks of paid maternity leave after delivery, which

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We were also surprised that consumption of high-risk foods and eating food outside of the home 245 were associated with protection against sapovirus AGE. We found that consumption of seafood and fresh 246 fruits and vegetables were positively associated with having piped water in the home, which is a marker 247 of higher socioeconomic status. If nutritional practices were associated with other factors that were 248 protective against sapovirus AGE, the relationship between high-risk foods and sapovirus AGE could be 249 confounded.

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Our study, like others, shows that a higher risk of norovirus AGE in the first year of life is 251 associated with child's secretor-positive phenotype but not with Lewis-positive phenotype. 16,17,25 252 However, an important finding in our study is the protective association of mother's secretor-positive . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted April 17, 2022. possible that breastfeeding is associated with protection against other enteric pathogens that we did not 276 assess in the current analysis.
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The copyright holder for this preprint this version posted April 17, 2022. ; https://doi.org/10.1101/2022.04.14.22273890 doi: medRxiv preprint Despite these limitations, our study is one of the few studies to focus on breastfeeding as a

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primary exposure to understand its association with norovirus and sapovirus AGE. In contrast to previous 279 studies that explored breastfeeding as a fixed variable, our study provides more granular insight into 280 breastfeeding treated as a weekly varying exposure, an approach that closely mirrors actual practices. Our 281 findings reinforce the importance of continuing to study the protective benefits of breastfeeding and the 282 mechanisms by which breastfeeding can prevent early childhood infections. Our findings also suggest that  298 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted April 17, 2022. ;https://doi.org/10.1101https://doi.org/10. /2022  . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted April 17, 2022. ;https://doi.org/10.1101https://doi.org/10. /2022  . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted April 17, 2022. ; https://doi.org/10.1101/2022.04.14.22273890 doi: medRxiv preprint  9.9) . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted April 17, 2022. ; https://doi.org/10.1101/2022.04.14.22273890 doi: medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted April 17, 2022. ; https://doi.org/10.1101/2022.04.14.22273890 doi: medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.