Astrovirus Infection and Diarrhea in 8 Countries

Home , Astrovirus, Norovirus, Rotavirus

Maribel Paredes Olortegui, BS,a Saba Rouhani, MSc,b Pablo Peñataro Yori, MPH,a, b Mery Siguas Salas, BS,a Dixner Rengifo Trigoso,Astrovirus BS,a Dinesh Mondal, PhD,c LadapornInfection Bodhidatta, MD,d James and Platts-Mills, MD,e Amidou Samie, PhD,f Furqan Kabir, MSc, MBA,g Aldo Lima, MD, PhD,h Sudhir Babji, MD,i Sanjaya Kumar Shrestha, MD,d,j Carl J. Mason, MD, MPH,d Adil Kalam, MSc,g Pascal Bessong, PhD,f Tahmeed Ahmed, MBBS, PhD,c Estomih Mduma, MD,k Zulfiqar A. Bhutta, MBBS, PhD,g IlaDiarrhea Lima, PhD,h Rakhi Ramdass, in MSc, i 8Lawrence Countries H. Moulton, PhD,b Dennis Lang, PhD,l,m Ajila George, MSc,i Anita K.M. Zaidi, MD,g Gagandeep Kang, MD, PhD,i Eric R. Houpt, MD,e Margaret N. Kosek, MD,a, b on behalf of the MAL-ED Network

BACKGROUND AND OBJECTIVES: abstract

Astroviruses are important drivers of viral gastroenteritis but remain understudied in community settings and low- and middle-income countries. We present data from 8 countries with high prevalence of diarrhea and undernutrition to describe astrovirus epidemiology and assess evidence for protective immunity among METHODS: children 0 to 2 years of age. We used 25898 surveillance stools and 7077 diarrheal stools contributed by 2082 children for enteropathogen testing, and longitudinal statistical analysis to describe RESULTS: incidence, risk factors, and protective immunity. Thirty-five percent of children experienced astrovirus infections. Prevalence in diarrheal stools was 5.6%, and severity exceeded all enteropathogens except rotavirus. Incidence of infection and diarrhea were 2.12 and 0.88 episodes per 100 child-months, – P respectively. Children with astrovirus infection had 2.30 times the odds of experiencing diarrhea after adjustment for covariates (95% confidence intervalz [CI], 2.01 2.62; < .001). Undernutrition was a risk factor: odds of infection and diarrhea were reduced by – P – P 10% and 13%, respectively, per increase in length-for-age score (infection: odds ratio, 0.90 [95% CI, 0.85 0.96]; < .001; diarrhea: odds ratio, 0.87 [95% CI, 0.79 0.96]; = .006). – P Some evidence of protective immunity to infection was detected (hazard ratio, 0.84 [95% CI, 0.71 1.00], = .052), although this was heterogeneous between sites and significant in CONCLUSIONS: India and Peru. Astrovirus is an overlooked cause of diarrhea among vulnerable children worldwide. With the evidence presented here, we highlight the need for future research as well as the potential for astrovirus to be a target for vaccine development.

What’s Known on This Subject: Astroviruses commonly aAsociación Benéfica PRISMA, Iquitos, Peru;b Department of International Health, Johns Hopkins Bloomberg cause viral gastroenteritis among young children. Researchers School of Public Health, Baltimore, Maryland; cicddr,b, Dhaka, Bangladesh; dArmed Forces Research Institute traditionally focus on clinical outbreaks in hospital-based or of Medical Sciences, Bangkok, Thailand; eDivision of Infectious Diseases and International Health, University high-income settings, and a more complete description of of Virginia, Charlottesville, Virginia; fUniversity of Venda, Thohoyandou, South Africa; gAga Khan University, asymptomatic infection, disease burden, and immunogenicity in Naushahro Feroze, Pakistan; hUniversidade Federal do Ceará, Fortaleza, Brazil; iChristian Medical College, vulnerable communities is needed. Vellore, India; jCentre for International Health, University of Bergen, Bergen, Norway; kHaydom Lutheran Hospital, Haydom, Tanzania; lFogarty International Center, National Institutes of Health, Bethesda, Maryland; and What This Study Adds: We present the most comprehensive mFoundation for the National Institutes of Health, Bethesda, Maryland description of astrovirus epidemiology in young children to date. With our longitudinal, community-derived estimates Ms Olortegui, Ms Yori, Ms Salas, Ms Trigoso, Dr Mondal, Dr Bodhidatta, Dr Samie, Mr Kabir, Dr in 8 low- to middle-income settings, we demonstrate high Lima, Dr Babji, Dr Shrestha, Dr Mason, Mr Kalam, Dr Bessong, Dr Ahmed, Dr Mduma, Dr Bhutta, Dr prevalence, clinical episodes of comparable severity to other Lima, Ms Ramdass, Dr Lang, Ms George, Dr Zaidi, Dr Kang, Dr Houpt and Dr Kosek conceptualized enteropathogens, key risk factors, and evidence of protective and designed the study; Ms Rouhani led the initial analysis, and she and Ms Olortegui contributed immunity. equally to the drafting and revision of the manuscript; Dr Platts-Mills and Dr Moulton provided To cite: Olortegui MP, Rouhani S, Yori PP, et al. Astrovirus Infection and Diarrhea in 8 Countries. Pediatrics. 2018; 141(1):e20171326

Downloaded from www.aappublications.org/news by guest on September 29, 2021 PEDIATRICS Volume 141, number 1, January 2018:e20171326 Article Astroviruses are a common cause infection and symptoms. Lack of were collected monthly in the first of viral gastroenteritis– across evidence from LMIC highlights year and quarterly thereafter for geographic1 3 and developmental an important gap in knowledge surveillance of enteric infections. settings. ‍‍ In temperate settings, considering the undue burden Active surveillance of breastfeeding, infection is associated with seasonal of childhood diarrhea in these diarrhea, and symptoms of other outbreaks in hospitals, child care communities and the increasing illnesses (including fever, vomiting, centers, cruise ships, and hotels. In appreciation of complex interactions and respiratory problems) was tropical settings with poor water between undernutrition, enteric conducted twice weekly in the and sanitation infrastructure, infections with or without symptoms, home. Additional stool samples were ≥ astroviruses contribute to the and child growth and cognitive obtained during diarrheal episodes, burden of childhood diarrhea, which development. Multisite cohort defined as 3 loose stools in a accounts for considerable loss of studies in LMIC can help characterize 24-hour period and separated by at life and human potential in low- and the patterns of asymptomatic and least 2 diarrhea-free days. Children middle-income countries (LMIC). symptomatic infection and protective experiencing moderate to severe immunity against astrovirus in diarrhea were referred to local health Human astroviruses are small, community-based settings, where services. nonenveloped, single-stranded Astroviridae the majority of the consequences RNA viruses belonging to the For incidence calculations, distinct 2 of childhood diarrhea occur. In this family. They are infections were defined as astrovirus- longitudinal study, we describe the ≥ estimated to account for 2% to 9% positive surveillance stool samples epidemiology of astrovirus infection of acute, nonbacterial childhood separated by 1 negative sample 2 among infants 0 to 24 months of diarrhea, with novel strains from the previous infection. Distinct age in 8 geographically distinct sites linked to central nervous system diarrheal episodes were defined as – with a high incidence of childhood consequences (including meningitis astrovirus-positive samples preceded 2 7 diarrhea and undernutrition. and encephalitis) in children. ‍‍ by either an astrovirus-negative Methods Astrovirus gastroenteritis commonly diarrheal stool sample or a 14-day manifests as an acute, self-limiting Study Population lag from the previous astrovirus- watery diarrhea, sometimes positive diarrheal sample. Duration accompanied by vomiting, anorexia, of exclusive breastfeeding (EBF) and fever. Type 1 astroviruses are the The Etiology, Risk Factors, and was defined as time from birth until most prevalent of 8 serotypes8 across Interactions of Enteric Infection and introduction of clear liquids, solids, epidemiologic contexts. Disease Malnutrition and the Consequences or milk formula. Diarrheal severity occurs mostly in young children and for Child Health and Development was calculated by using a community immunocompromised adults, and (MAL-ED) Study enrolled children in diarrheal assessment scale that with although symptoms in adults are 8 countries: Bangladesh, India, Nepal, which scores out of 15 points were rare, seroprevalence8,9 to common Pakistan, Brazil, Peru, Tanzania, and assigned according to presence and strains is high. ‍ This implies that South Africa. Healthy infants were duration of fever, vomiting, anorexia, infection confers some protection enrolled within 17 days of birth liquid stools, and maximum stool14 among humans,9 although evidence and were managed through 2 years outputMicrobiology within a 24-hour period. is conflicting. Severity of symptoms of age; details of our study12 design has been negatively correlated are described elsewhere. All sites with the presence of antibodies 10,11 received ethical approval and written A diagnostic and quality assurance to astrovirus. ‍ However, our informedData Collection consent. protocol was harmonized across understanding of the mechanisms all 8 sites for the detection of of protective immunity remains enteropathogens. The ProSpecT incomplete, with the majority of Sex, birthdate, and anthropometric (Remel, obtained from Fisher evidence generated from high- data were recorded at Scientific, Pittsburgh, PA) enzyme- income settings and a paucity of data enrollment, and data collection of linked immunosorbent assay on seroconversion and immunity in z anthropometrics was conducted was used to detect astrovirus, LMIC. monthly. Length-for-age scores rotavirus, and adenovirus within In studies of astrovirus epidemiology, (LAZs) to assess growth faltering 1 week of stool collection and ’ researchers have generally focused were calculated according to the suspension in diluent. This on children exhibiting diarrhea World Health Organization s assay employs a combination of or acute gastroenteritis, with few Multicentre Growth13 Reference Study genus-specific monoclonal and reporting longitudinal history of Group guidelines. Stool samples polyclonal antibodies and detects all Downloaded from www.aappublications.org/news by guest on September 29, 2021 2 Olortegui et al TABLE 1 Sample Population by Age and Site No. Children Diarrheal Samples Surveillance Stools Contributed Contributed Across sites, 550 (26.4%) children experienced at least 1 astrovirus Total No. children 2082 7077 25 868 No. children recruited at infection in the first year of life; by each site the end of follow-up, 732 (35.2%) Asia had documented infections. Bangladesh 260 1525 3331 India 243 615 3212 Astrovirus detection in diarrheal Nepal 238 860 3322 stools exceeded that in surveillance Pakistan 274 1793 3232 stools across the full cohort: 5.6% Africa (394) of diarrheal stools and 2.2% South Africa 288 172 3758 Tanzania 259 171 3448 (573) of surveillance stools were South America positive for astrovirus infection, Brazil 221 98 2076 respectively. In contrast with Peru 299 1843 3489 asymptomatic astrovirus detection, No. children contributing prevalence during diarrhea samples within each age group, mo increased with age in the first year 0–5 2071 1810 9154 of life, peaking at 12 to 14 months 6–11 1938 2273 9516 and declined thereafter, with the 12–17 1814 1700 3001 exception of a second peak from 18 24 1759 1294 4197 – 21 to 24 months (Fig 2A). We observed heterogeneity in astrovirus infection and symptoms by country. 5 Children in Peru exhibited the described genotypes of astrovirus. (PAF), with which differences highest prevalence of astrovirus- Microbiologic methods of detection in prevalence and strength of positive diarrhea. Prevalence ratios for additional enteropathogens15 are association with diarrheal symptoms of astrovirus infection in diarrhea Sdescribedtatistical inAnalyses detail elsewhere. are incorporated and LAZ, EBF, and relative to surveillance stools also age are adjusted for to estimate differed by site (Fig 2B). the astrovirus-specific burden of Mean severity of astrovirus-positive diarrhea. – Cumulative incidence and incidence Results diarrhea was 3.15 points (95% rates were calculated by using confidence interval [CI], 2.90 3.40) out of a 15-point community Kaplan-Meier curves and survival Astrovirus Epidemiology Across 8 – analysis, allowing for multiple Sites diarrheal severity scale, compared with 2.89 (95% CI, 2.84 2.95) failures per child. Associations – points for all-cause diarrhea, 3.92 between astrovirus, diarrhea, and – undernutrition were modeled (95% CI 3.66 4.18) for rotavirus, Across 8 sites, 1036 boys and by using linear regression with a and 2.95 (95% CI, 2.83 3.06) for 1046 girls were managed between generalized estimating equations norovirus. Overall, 77% (744) of November 2009 and February 2012. approach, adjusting for within-child all astrovirus-positive diarrheal These 2082 children contributed clustering. Adjustment for EBF was samples were coinfected with at 7077 diarrheal samples and 25868 included in all analyses because least 1 other pathogen. Prevalence surveillance stools through 24 of the well-described relationship of common coinfections is shown months of age (Table 1). between breastfeeding, immunity, in the full cohort in Table 3, and and growth in early life. A Cox Children experienced 2.1 distinct by siteCampylobacter in Supplemental Table 6. proportional hazards model was astrovirus infections per 100 child- The most common coinfection was used to calculate hazard rates of months across sites, with a peak of with spp., which ï infection and diarrheal episodes 3.1 detections at 6 to 11 months of was present in 28.7% and 40.1% in exposed versus na ve children. age. There were marked differences of all surveillance and diarrheal Countries with at least 5 children between countries, with overall stools, respectively. Other prevalent who were experiencing at least 10 incidence of infection highest in Peru, coinfections with diarrhealEscherichia coli events (subsequent infections or India, and Bangladesh and lowest in symptomsGiardia included norovirus, diarrheal episodes) were selected for Brazil and South Africa. Incidence enteroaggregative , site-specific estimation of protective rates and cumulative incidence of and . These findings were immunity. We calculated the adjusted infection and diarrhea are shown consistent across surveillance and population attributable fraction in Table 2 and Fig 1, respectively. diarrheal samples. Downloaded from www.aappublications.org/news by guest on September 29, 2021 PEDIATRICS Volume 141, number 1, January 2018 3 TABLE 2 Incidence Rates of Astrovirus Infection Among 2082 Children Across 8 MAL-ED Sites by Age Group and Country Child-Months Total Episodes With Incidence of Infection Diarrheal Episodes Incidence of Diarrhea at Risk Astrovirus Detected (95% CI) (95% CI) Age group, mo 0–5 11 025.2 265 2.40 (2.13–2.71) 75 0.68 (0.54–0.85) 6–11 11 235.0 344 3.06 (2.76–3.40) 127 1.13 (0.95–1.35) 12–17 8391.6 177 2.11 (1.82–2.44) 121 1.44 (1.21–1.72) 18–24 13 443.2 150 1.11 (0.95–1.31) 65 0.48 (0.38–0.62) Country Asia Bangladesh 5476.8 185 3.38 (2.92–3.90) 80 1.46 (1.17–1.82) India 5535.4 92 3.67 (1.36–2.04) 32 0.58 (0.41–0.82) Nepal 5497.0 117 2.13 (1.78–2.55) 40 0.73 (0.53–0.99) Pakistan 6181.6 156 2.52 (2.16–2.95) 74 1.20 (0.95–1.50) Africa South Africa 6001.35 50 0.83 (0.63–1.10) 5 0.08 (0.03–0.20) Tanzania 5441.7 112 2.06 (1.71–2.48) 12 0.22 (0.13–0.39) South America Brazil 4309.2 9 0.21 (0.11–0.40) 4 0.09 (0.03–0.25) Peru 5651.8 215 3.80 (3.33–4.35) 141 2.49 (2.11–2.94) Total 44 094.9 936 2.12 (1.99–2.26) 388 0.88 (0.80–0.97) Incidence expressed as number of detections per 100 child-months. All detections, whether asymptomatic or diarrheal, were considered episodes for calculation of incidence rates for infection. Only episodes of diarrhea with astrovirus detected were included as events for calculation of incidence rates for diarrhea. New episodes were defined as separated from previous episodes by either astrovirus-negative stool samples or a period of ≥14 d from previous astrovirus-positive stools.

Evidence of Protective Immunity

have a significantly reduced hazard Evidence for protective immunity – P of future infections (HR, 0.97 [95% was found in India and Peru. In Protective immunity was assessed by CI, 0.77 1.21]; = .766) nor future India, previous infection was – P comparing the hazard of subsequent associated with a 50% decrease in diarrheal episodes (HR, 1.01 [95% – infections and diarrhea between CI, 0.74 1.37]; = .975) relative to Pthe subsequent hazard of infection children with and without previous children without. HRs for each model (HR, 0.50 [95% CI, 0.26 0.96]; exposure to infection. Models were are shown in Table 4. We could not = .037), suggesting a reduced risk adjusted for EBF, LAZ, and site. The explore dose responses because of among children with past exposure. results revealed a reduced incidence insufficient events across the full No evidence of protection against of infection among children with cohort. subsequent diarrheal episodes or of previous infection, although this was protection from previous astrovirus – marginally nonsignificant (hazard diarrhea was detected (Table 5). P Immunity was also explored at each ratio [HR], 0.84 [95% CI, 0.71 1.00]; Children living in Peru had a 46% site separately. Brazil and South = .052). When the number of and 43% reduced risk of future Africa were excluded from this previous infections was categorized, infections and diarrheal episodes, portion of the analysis because the the same trend was seen among respectively, on the basis of previous – number of reinfections at these sites – P ≤ children with 1 previous infection infection (infection: HR, 0.54 [95% P ≥ was insufficient (none in Brazil, 3 – P (HR, 0.84 [95% CI, 0.70 1.01]; CI, 0.38 0.75]; .001; diarrhea: = .065) as well as those with 2 in South Africa). When modeling HR, 0.57 [95% CI, 0.38 0.87]; = – P previous infections (HR, 0.84 [95% protection incurred by previous .009). A protective trend against CI, 0.59 1.20]; = .334), although astrovirus diarrhea, only 3 sites future infections and symptoms neither was statistically significant. met the inclusion criteria of 10 was seen among children who No relationship between previous events: Bangladesh, Pakistan, and experienced at least 1 clinical episode infection and risk of subsequent Peru (15, 11, and 25 subsequent of astrovirus diarrhea, although this – diarrheal episodes at each site, – diarrheaP was detected in the full was notP significant at the 95% level cohort (HR, 0.94 [95% CI, 0.74 respectively). Site-specific HRs are (infection: HR, 0.74 [95% CI, 0.53 – P 1.20]; = .613). When explored by shown in Table 5. We found no 1.04]; = .082; diarrhea: HR, 0.64 modeling the hazard of infections evidence that differences in overall [95% CI, 0.41 1.00]; = .051). and diarrheal episodes among coinfection or coinfection by specific children with previous astrovirus pathogens explained the observed On the basis of these findings, diarrhea, no protective immunity was heterogeneities in protective evidence of a dose response was detected. Children with exposure to immunity by site (M.P.O., S.R., P.P.Y., assessed in Peru. Of the 163 children astrovirus-positive diarrhea did not et al, unpublished data). infected with astrovirus during Downloaded from www.aappublications.org/news by guest on September 29, 2021 4 Olortegui et al ≥

The 7 children with 2 previous detections experiencedP an additional 20% reduction in theP subsequent risk of future infections ( = .013) and diarrheal episodes ( = .032) relative to children with 1 previous detection. Associations Between Astrovirus Infection, Diarrhea, and Undernutrition

Across sites, there was a strong relationship between astrovirus and diarrheal symptoms. After adjustment for age, EBF, and LAZ, odds of astrovirus detection were 2.30 times higher in diarrheal – P samples than in surveillance stools (95% CI, 2.01 2.62; < .001). This association was statistically significant at all sites except South Africa, where only 5 episodes of astrovirus-positive diarrhea were reported among 285 children – P throughout follow-up (odds ratio [OR], 2.51 [95% CI, 0.96 6.54]; = .061). Using estimates from the global cohort, we found that the adjusted PAF was 3.44%.

An additional analysis was run to investigate whether the association with symptoms was because of coinfection.Campylobacter After adjustment for the E4 colimost prevalent coinfectionsGiardia ( spp., enteroaggregative , norovirus, FIGURE 1 and ), the strength of Cumulative incidence of astrovirus in (A) surveillance stools and (B) diarrheal samples in the association between infection and MAL-ED cohort. Time to first infection and clinical episode of diarrhea is shown globally (black dotted diarrheal symptoms was attenuated line) and separately by site. BGD: Mirpur, Bangladesh; BRF: Fortaleza, Brazil; INV: Vellore, India; NEB: but remained significant and was Bhaktapur, Nepal; PEL: Iquitos, Peru; PKN: Naushero Feroze, Pakistan; SAV: Limpopo, South Africa; TZH: stronger for astrovirus than other Heydom, Tanzania. – P pathogens considered (OR, 1.88 [95% CI, 1.57 2.34]; < .001). When a binary indicator of any other – P coinfection was considered, the follow-up, 39 experienced (HR, 0.37 [95% CI, 0.17 0.81]; odds of astrovirus detection were ≥ 1 reinfection, and 7 experienced = .013). Theren were insufficient 1.48 times higher in diarrheal stools 2 reinfections. Children with instances of tertiary or quaternary relative to surveillance stools among – P 1 previous infection had a 42% infection ( = 7) to meet the inclusion children infected with only astrovirus – P reduced incidence of infection (HR, threshold of 10 events. Nonetheless, (95% CI, 1.09 2.00; = .012) and ≥ – 0.58 [95% CI, 0.40 0.84]; = .004), this model produced the expected P2.61 times higher among children whereas those with 2 previous direction of effect, demonstrating with coinfections (95% CI, 2.22 2.99; exposures experienced a 63% a gradient of protection as the < .001) relative to pathogen- reduction in subsequent infections number of infections increased. negative stools. Downloaded from www.aappublications.org/news by guest on September 29, 2021 PEDIATRICS Volume 141, number 1, January 2018 5 P growth insultsP at 3, 6, or 9 monthsP postinfection (3 months: = .389; 6 months: = .339; 9 months: = .335), even after adjustment for EBF or site. Discussion

Gastroenteritis is an important cause of morbidity and hospitalizations across developmental contexts and of childhood morbidity in

LMIC. Etiologies include bacterial,16,17 protozoal, and viral agents ‍ ; among these, the astrovirus burden is poorly recognized. With the findings reported here and elsewhere, an impetus for greater attention to astrovirus as an important pathogen and potential vaccine target is provided. Astrovirus was strongly associated with diarrhea of equal or greater severity thanShigella well-recognizedE coli enteropathogens, including

norovirus,18,19 spp, and spp. ‍ It has been noted that astroviruses are more frequently associated with symptoms than

many other pathogens1 receiving research investments. The PAF of astrovirusCampylobacter is exceeded only by norovirus genogroup II, rotavirus,

and 18 spp. in the MAL-ED cohort. Here we report a PAF of 3.4%, which if applied to the estimated 1.731 billion annual FIGURE 2 20 Astrovirus prevalence by (A) age and (B) site across 2082 children in the MAL-ED cohort. Surveillance diarrheal episodes suggests that stools are shown in red, and diarrheal samples are shown in blue. BGD: Mirpur, Bangladesh; BRF: astrovirus may account for up to Fortaleza, Brazil; INV: Vellore, India; NEB: Bhaktapur, Nepal; PEL: Iquitos, Peru; PKN: Naushero Feroze, 5.96 million cases each year. Pakistan; SAV: Limpopo, South Africa; TZH: Heydom, Tanzania. Astroviruses are estimated to account for 2.3% to 8.9% of children presenting with diarrhea– in ambulatory, clinical, or hospital- Undernutrition was a risk factor for was observed. At the site level, a 1,21 23 based settings worldwide ‍ ‍ ‍ and astrovirus. Odds of infection were significant reduction in astrovirus 4% to 5% of diarrheal disease in diminished by a mean of 10% per diarrhea with increasing LAZ was – P – P community24,25 settings in Egypt and increase in LAZ (OR, 0.90 [95% CI, detected only in Nepal (OR, 0.68 Mexico. ‍ In our community-based 0.85 0.96]; < .001), and odds of [95% CI, 0.51 0.92]; = .013). assessment, astrovirus was detected experiencing astrovirus-positive Increased odds of experiencing in 5.6% of study-defined diarrhea diarrhea decreased by 13% per astrovirus diarrhea were observed – P – and up to 7.9% in Peru, where increase in LAZ (OR, 0.87 [95% CI, Pwith increasing LAZ in Tanzania prevalence was highest. We also 0.79 0.96]; = .006) after accounting (OR, 1.73 [95% CI, 1.05 2.88]; observed a high overall incidence for EBF. However, when adjusted = .032). There was no association of astrovirus diarrhea relative to for site, no significant relationship between astrovirus and subsequent reports from longitudinal community Downloaded from www.aappublications.org/news by guest on September 29, 2021 6 Olortegui et al TABLE 3 Astrovirus Coinfections in Surveillance and Diarrheal Stools Astrovirus-Positive Diarrhea Astrovirus-Positive Surveillance Samples (N = 394), % (n) Stools (N = 573), % (n) vast epidemiologic heterogeneity by site. We found previous astrovirus Viruses exposure to be associated with a Rotavirus 5.6 (22) 3.7 (21) Norovirus 24.9 (98) 26.3 (26)a 40% to 50% reduction in the hazard Adenovirus 4.8 (19) 3.8 (22) of subsequent infection in Peru Bacteria and India. In contrast, South Africa, Campylobacter 40.1 (158) 34.4 (197) Tanzania, and Brazil had 1 or fewer Shigella 1.3 (5) 0.7 (4) occurrences of reinfection and no EAEC 28.2 (111) 31.4 (180) EPEC 4.6 (18) 3.8 (22) diarrheal episodes reported after ETEC 11.9 (47) 7.7 (44) initial exposure. Whether this is Aeromonas 3.3 (13) 1.9 (11) a function25 of rapid acquisition of Protozoa immunity or of low incidence of Giardia 17.5 (69) 16.2 (93) astrovirus cannot be inferred from Cryptosporidium 5.6 (22) 3.8 (22) this study. Nonetheless, we believe EAEC, enteroaggregative E coli; EPEC, enteropathogenic E coli; ETEC, enterotoxigenic E coli. a Norovirus testing was conducted on a subsample of children in the global cohort and thus only available for 99 of the 573 this work provides suggestive surveillance stools positive for astrovirus. evidence for protective immunity to astrovirus among children living in TABLE 4 Protection Associated With Astrovirus Across MAL-ED Sites poverty. Exposure No. Previous Astrovirus HRs (95% CI) We note a lack of serotype-level data Infections Subsequent Subsequent Diarrheal Infections Episodes as a limitation, especially in light of observed disparities in evidence for Previous detection None (reference) protective immunity. That serotypes Any 0.84 (0.71–1.00) 0.94 (0.74–1.20) P = .054 P = .613 exhibit different27 epidemiology is well No. previous detections None (reference) described, and their distribution 1 0.84 (0.70–1.01) 0.95 (0.73–1.23) is likely to differ between sites. .065 .682 Furthermore, it precludes this 2+ 0.84 (0.59–1.19) 0.91 (0.58–1.42) P = .334 P = .663 work from contributing to our Previous astrovirus None (reference) understanding of homotypic and diarrhea Any 0.97 (0.77–1.21) 1.01 (0.74–1.36) heterotypic immunity. Serotyping P = .766 P = .975 of astrovirus is an important recommendation for future work. Another limitation of this study is

24,25 the diagnostic technique. Enzyme- cohorts. ‍ The heterogeneity in evidence of protection, although linked immunosorbent assays are infection and diarrhea between they did report possible homotypic a popular method for detection of sites demonstrates that estimates immunity. Across 8 sites and with viruses in large samples and has vary considerably on the basis of 2082 participants, we report comparable sensitivity to electron geographic setting and across urban suggestive evidence of immunity to microscopy, but a greater proportion– and rural contexts. infection (but not diarrheal disease) of positive samples can be28 identified31 among children in the first 2 years by molecular diagnostics. ‍ In observational studies, researchers of life. Lack of protection against However, assuming the decreased have shown that infection is symptomatic disease may be partially sensitivity is nondifferential between associated with antibody responses explained by coinfection; because asymptomatic and diarrheal stools, that coincide with reduction11,26 astrovirus incidence was lower, this would translate into the in disease severity. ‍ To our there is a greater risk that clinical reporting of conservative estimates knowledge, in only 1 other study symptoms were driven by other here. Finally, high prevalence of have researchers modeled protective pathogens in diarrheal samples. coinfection makes it impossible to immunity to astrovirus in a 25 The inclusion of asymptomatic attribute episodesCampylobacter exclusively to longitudinal cohort. Naficy et al infection as an indicator of exposure astrovirus. However, we note that conducted active surveillance for may have allowed us to capture aside from spp., the diarrhea in rural Egypt and modeled upstream evidence of immunity most prevalent coinfections were the hazard of clinical disease with that a study in which researchers with pathogens less frequently and without previous exposure observed only clinical disease would associated 32with diarrhea than to diarrhea. They did not find any not detect. Furthermore, we report astrovirus, and a strong statistical Downloaded from www.aappublications.org/news by guest on September 29, 2021 PEDIATRICS Volume 141, number 1, January 2018 7 TABLE 5 Protection Associated With Astrovirus in MAL-ED by Site Site Previous Astrovirus HRs (95% CI) disease of a pathogen that, despite its Exposure Subsequent Infection Subsequent Diarrheal Episodes contribution to the diarrheal burden, Asia has received little attention to date. Bangladesh Any infection 0.94 (0.65–1.36) 1.22 (0.76–1.95) We also report evidence of protective n = 131 P = .739 P = .416 immunity and a strong relationship Diarrheal episode 1.13 (0.67–1.92) 1.63 (0.85–3.12) n = 65 P = .654 P = .139 between infection and diarrheal India Any infection 0.50 (0.26–0.96) 0.52 (0.15–1.80) symptoms of considerable severity. n = 81 P = .037 P = .302 We believe that these findings provide Diarrheal episode — — impetus for continued studies into the n = 31 development of protective immunity Nepal Any infection 0.99 (0.59–1.66) 1.12 (0.56–2.23) n = 93 P = .964 P = .743 to astrovirus in different settings to Diarrheal episode — — elucidate the potential public health n = 37 merit of combination vaccines that Pakistan Any infection 0.84 (0.56 1.25) 1.11 (0.63, 1.96) – would include astrovirus as well as n = 120 P = .386 P = .727 36 Diarrheal episode 0.79 (0.48–1.31) 1.17 (0.64–2.13) rotavirus and to broaden protection n = 63 P = .356 P = .608 against viral gastroenteritis in Africa children. Tanzania Any infection 1.11 (0.70–1.78) 2.74 (0.86–8.71) Acknowledgments n = 88 P = .655 P = .088 Diarrheal episode — — n = 12 South America We thank the staff and participants Peru Any infection 0.54 (0.38–0.75) 0.57 (0.38–0.87) of the MAL-ED Network for their n = 163 P < .001 P = .009 important contributions, patience, Diarrheal episode 0.74 (0.53–1.04) 0.64 (0.41–1.00) n = 115 P = .082 P = .051 and tireless work. South Africa and Brazil were excluded because of an insufficient number of subsequent infections (<10). Sites where there were sufficient infections but <10 subsequent diarrheal episodes are included, with —( ) denoting the excluded Abbreviations components of the analyses.

CI: confidence interval association between astrovirus to 90% of adults were reported HR: hazard ratio z infection and diarrheal diseases has to be seropositive in studies EBF: exclusive breastfeeding been demonstrated after adjustment 1 in the United8,33, 34 States and Great LAZ: length-for-age score for coinfections, here and elsewhere. Britain. ‍ ‍ A vaccine could LMIC: low- and middle-income therefore have applications in a countries With 72.6% of detected infections range of populations. MAL-ED: The Etiology, Risk occurring after 6 months of age, Factors, and we suggest that astrovirus may Conclusions Interactions of Enteric be an appropriate vaccine target. Infection and This would not only be relevant Malnutrition and the to children living in poverty. The This study is a significant contribution Consequences for Child persistence of astrovirus in high- to our understanding of an important Health and income settings suggests that, like cause of pediatric gastroenteritis 3,30, 35 Development norovirus, prevention of morbidity worldwide. ‍ ‍ Through the use of OR: odds ratio is unlikely to be achieved through a large, multicountry birth cohort, PAF: population attributable water, sanitation, and hygiene we provide a description of the fraction improvements alone. Indeed, 65% epidemiology of infection and clinical critical review and contributions to the analysis and revision of the manuscript; and all authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work. DOI: https://doi.org/10.1542/peds.2017-1326 Accepted for publication Oct 12, 2017 Address correspondence to Margaret N. Kosek, MD, Department of International Health, Johns Hopkins Bloomberg School of Public Health, 615 N Wolfe St, Baltimore, MD 21205. E-mail [email protected]

Downloaded from www.aappublications.org/news by guest on September 29, 2021 8 Olortegui et al PEDIATRICS (ISSN Numbers: Print, 0031-4005; Online, 1098-4275). Copyright © 2018 by the American Academy of Pediatrics FINANCIAL DISCLOSURE: The authors have indicated they have no financial relationships relevant to this article to disclose. FUNDING: The Etiology, Risk Factors and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development Project (MAL-ED) is carried out as a collaborative project supported by the Bill & Melinda Gates Foundation, the Foundation for the National Institutes of Health, and the National Institutes of Health Fogarty International Center. Dr Margaret Kosek was supported in part by the the Sherrilyn and Ken Fisher Center for Environmental Infectious Diseases at the Johns Hopkins School of Medicine. POTENTIAL CONFLICT OF INTEREST: The authors have indicated they have no potential conflicts of interest to disclose.

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Downloaded from www.aappublications.org/news by guest on September 29, 2021 10 Olortegui et al Astrovirus Infection and Diarrhea in 8 Countries Maribel Paredes Olortegui, Saba Rouhani, Pablo Peñataro Yori, Mery Siguas Salas, Dixner Rengifo Trigoso, Dinesh Mondal, Ladaporn Bodhidatta, James Platts-Mills, Amidou Samie, Furqan Kabir, Aldo Lima, Sudhir Babji, Sanjaya Kumar Shrestha, Carl J. Mason, Adil Kalam, Pascal Bessong, Tahmeed Ahmed, Estomih Mduma, Zulfiqar A. Bhutta, Ila Lima, Rakhi Ramdass, Lawrence H. Moulton, Dennis Lang, Ajila George, Anita K.M. Zaidi, Gagandeep Kang, Eric R. Houpt, Margaret N. Kosek and on behalf of the MAL-ED Network Pediatrics 2018;141; DOI: 10.1542/peds.2017-1326 originally published online December 19, 2017;

Updated Information & including high resolution figures, can be found at: Services http://pediatrics.aappublications.org/content/141/1/e20171326 References This article cites 36 articles, 4 of which you can access for free at: http://pediatrics.aappublications.org/content/141/1/e20171326#BIBL Subspecialty Collections This article, along with others on similar topics, appears in the following collection(s): Infectious Disease http://www.aappublications.org/cgi/collection/infectious_diseases_su b Epidemiology http://www.aappublications.org/cgi/collection/epidemiology_sub Permissions & Licensing Information about reproducing this article in parts (figures, tables) or in its entirety can be found online at: http://www.aappublications.org/site/misc/Permissions.xhtml Reprints Information about ordering reprints can be found online: http://www.aappublications.org/site/misc/reprints.xhtml

Downloaded from www.aappublications.org/news by guest on September 29, 2021 Astrovirus Infection and Diarrhea in 8 Countries Maribel Paredes Olortegui, Saba Rouhani, Pablo Peñataro Yori, Mery Siguas Salas, Dixner Rengifo Trigoso, Dinesh Mondal, Ladaporn Bodhidatta, James Platts-Mills, Amidou Samie, Furqan Kabir, Aldo Lima, Sudhir Babji, Sanjaya Kumar Shrestha, Carl J. Mason, Adil Kalam, Pascal Bessong, Tahmeed Ahmed, Estomih Mduma, Zulfiqar A. Bhutta, Ila Lima, Rakhi Ramdass, Lawrence H. Moulton, Dennis Lang, Ajila George, Anita K.M. Zaidi, Gagandeep Kang, Eric R. Houpt, Margaret N. Kosek and on behalf of the MAL-ED Network Pediatrics 2018;141; DOI: 10.1542/peds.2017-1326 originally published online December 19, 2017;

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