Benjamin F. Arnold

Treatment effects in randomized trials may vary geographically, and estimating this variation is a broadly generalizable problem for large-scale, cluster-randomized trials. We conducted a secondary analysis of AVENIR, a cluster-randomized, placebo-controlled trial of biannual azithromycin distribution among children aged 1–59 months in Dosso, Niger (2020–2023). We assessed whether the survival benefit of mass azithromycin distribution varied geographically using Poisson models to estimate spatially continuous child mortality and average treatment effects (ATE). Among 1390 villages (276,452 person-years; 3635 deaths), the overall mortality rate was 13.1 and the ATE was −2.6 deaths per 1000 person-years. Mortality was highest in the south, whereas treatment benefit was concentrated in the southeast, following a northwest–southeast gradient. Aggregation of data to 160 primary health catchment areas preserved broad spatial patterns in mortality but attenuated local heterogeneity, suggesting that health catchment monitoring is sufficient for broad mortality surveillance. Examining unit-level, spatially predicted treatment effects as a function of mortality rate demonstrated that underlying mortality captured much, but not all, of the heterogeneity in treatment effects. The analysis demonstrates how to estimate localized treatment effects in large cluster-randomized trials and provides new insights into spatial heterogeneity in mortality and benefits of azithromycin distribution in Niger.

Trachoma is the leading infectious cause of blindness worldwide and is targeted for global elimination as a public health problem by 2030. As we approach this target, defining when population-level trachoma control interventions can be stopped, and determining how post-intervention surveillance should be performed, are becoming increasingly important. Molecular diagnosis of ocular Chlamydia trachomatis (Ct) using polymerase chain reaction (PCR) provides a highly sensitive and specific indicator of infection, yet there are currently no programmatic guidelines based on Ct infection. We assembled 86 cross-sectional surveys of ocular Ct infection from 13 countries, representing data from 46,914 children aged 1-5 years. Our analyses suggest that infection prevalence of 0.81% in this age group corresponds to a threshold below which population-level interventions can be stopped with at least 90% confidence. Correspondingly, with confidence exceeding 90%, we propose that interventions should continue when infection prevalence is greater than 1.99%. Interpretation of infection surveillance data is a generalisable challenge in low-prevalence settings for many infectious diseases. This study proposes a robust framework for how Ct infection surveillance could support accurate population-level assessments of trachoma endemicity and could allow a step-change in decision-making precision for programmes as we approach the endgame for trachoma elimination.

/Summary: Background: We evaluated whether water, sanitation, and handwashing (WASH) and nutrition interventions reduced enteric pathogen seroprevalence during the first two years of life among children in rural Kenya. Methods: We analysed the 2×2 factorial subset of the WASH Benefits Kenya cluster-randomised trial (control, WASH, nutrition, and WASH plus nutrition). Dried blood spots were collected from all children in the birth cohort after two years of intervention and at 6 and 12 months within a longitudinal substudy. Immunoglobulin G (IgG) responses were measured using a multiplex bead assay that included 12 pathogens. The primary outcome was IgG seroprevalence at 2 years. We estimated intention-to-treat differences, comparing WASH with no WASH and nutrition with no nutrition across the factorial design. Trial registration: NCT01704105 . Findings: The primary analysis included 3030 children in 384 clusters (median age 23 months). Seroprevalence increased rapidly with age and exceeded 90% for norovirus, Campylobacter , and LT-ETEC by the final visit, with several pathogens at moderate prevalences (24% were seropositive to typhoidal S. enterica HlyE). Differences between intervention groups were small: lower T. solium (−3.3 percentage points [95% CI −6.3 to −0.2]) and higher E. histolytica (3.2 [0.1 to 6.3]) with WASH versus control, and lower V. cholerae (−1.1 [−1.8 to −0.3]) and Shigella /EIEC (−2.7 [−5.5 to 0.0]) with nutrition versus no nutrition. No comparison met the 5% Benjamini– Hochberg false discovery rate threshold. Secondary analyses and age-seroprevalence curves suggested modest reductions for some pathogens but increased norovirus seroprevalence around 12 months, when intervention adherence was highest. Interpretation: At achieved adherence levels, household-level WASH and nutrition interventions were insufficient to reduce widespread early-life enteric pathogen exposure in this high-transmission setting, supporting the development of complementary vaccine and transmission-control strategies. Funding: National Institutes of Health (R01AI166671, R01AI106878, R01AI134814). The original WASH Benefits trial was funded by the Gates Foundation (OPPGD759).

/Summary: Background: Improvements to water quality, sanitation, handwashing (WASH) and nutrition may prevent enteric pathogen infection during the first two years of life. Few randomised controlled trials have measured pathogen-specific effects of WASH and nutritional interventions. Methods: Within a 2×2 factorial cluster-randomized trial of WASH and nutritional interventions in Bangladesh, we tested dried blood spot specimens with multiplex assay for IgG responses to 12 enteric pathogens: Norovirus , Campylobacter , LT-producing enterotoxigenic E. coli (LT-ETEC), Shigella , non-typhoidal and typhoidal Salmonella enterica , Vibrio cholerae , Cryptosporidium , Giardia , Entamoeba histolytica , Taenia solium , and Strongyloides stercoralis . Primary analysis included children with IgG measurements at ∼24 months, and secondary analysis assessed early effects in a sub-study at ∼14 months. We estimated age-specific seroprevalence and marginal seroprevalence differences for WASH and nutrition effects. Trial is registered with ClinicalTrials.gov ( NCT01590095 ). Findings: Across the four arms (450 clusters), 4817 samples were tested for IgG responses, 2453 samples at the primary endpoint. Seroprevalence increased with age, consistent with intense early-life transmission. For highest transmission pathogens, >94% of children had seroconverted to norovirus , LT-ETEC and Campylobacter by 12-18 months. WASH effects were modest and pathogen-specific but generally protective. At 24 months, children that received WASH interventions had less antibody against Shigella (–4.5%, 95% CI –7.6% to –1.5%), non-typhoidal S. enterica (–4.0%, 95% CI –6.5% to –1.5%), and Giardia (–3.4%, 95% CI –6.7% to –0.2%), although not after multiple-testing correction. Nutrition had limited effects on most enteric pathogens, but reduced non-typhoidal S. enterica at 14 months (–6.4%, 95% CI: –9.8% to –3.0%). Interpretation: Multiplex serology provided evidence that WASH may modestly reduce cumulative exposure to selected enteric pathogens, but household-level interventions were insufficient to substantially disrupt transmission in this high-exposure setting. Funding: National Institutes of Health (R01AI166671, R01AI106878, R01AI134814). The original WASH Benefits trial was funded by Gates Foundation (OPPGD759).

Enteric pathogens account for a substantial global disease burden, yet population-based surveillance remains limited by the transient nature of pathogen shedding, which constrains the sensitivity of stool-based molecular testing to a narrow detection window. Detection of IgG responses in serological surveys could enable new insights into enteric pathogen transmission, but benchmarking serological measures relative to stool-based measures of infection remains a critical evidence gap. We compared measures of disease transmission in longitudinal birth cohort samples using multiplex IgG (1,575 dried blood spots, 369 children) and PCR assays (2,229 stool samples, 369 children) that overlapped for nine pathogens (norovirus GI/GII, Shigella /enteroinvasive Escherichia coli (EIEC), Campylobacter spp., enterotoxigenic Escherichia coli (LT-ETEC), Salmonella enterica , Giardia spp., Cryptosporidium spp., Entamoeba histolytica ). Campylobacter and LT-ETEC ranked highest while E. histolytica and S. enterica ranked among the lowest by both IgG and PCR measures of infection. Measures of infection were less aligned for norovirus GI, norovirus GII, Cryptosporidium , Shigella /EIEC, and Giardia . Despite this, for most pathogens, and across both measures, force of infection was lowest in the urban city of Esmeraldas and substantially higher in more rural populations, with relative risks in assay measures (seroconversion rate and PCR detected prevalence) of approximately 2.0 for pathogens with the largest differences ( Shigella /EIEC and norovirus GI). Together, our results suggest that although absolute levels of infection differed between assays, IgG and PCR captured consistent relative risk of infection across geographic strata, suggesting either method can identify high-versus low-transmission settings.

High-density peptide arrays are useful for mapping linear antibody epitopes and resolution of antibody specificity in a single-assay platform. Here, we used peptide library screening to evaluate magnitude and breadth of humoral responses to enteric pathogens in the context of public health interventions. We characterized the epitope landscape to known immunogenic proteins to several viruses, bacteria, and parasites and used that to infer immunological profiles at age 3, 14, and 28 months. The peptide libraries detected immune signatures better for viruses compared with bacteria and parasites and captured distinct dynamics of protein-level variation in immune signatures over time. We found limited sensitivity for bacterial and protozoan pathogens whose humoral responses may depend more on conformational or natively modified epitopes than linear-peptide binding. Unbiased peptide libraries have potential utility for broad analysis of antibody responses in integrated serosurveillance, but our results reveal pathogen-specific limitations where peptide-based serology would be less predictive of true infection status and where it would provide greater surveillance value. Beyond assay performance, peptide arrays are useful for studying epitope architecture, and a practical implication would require augmentation with alternative antigen formats (whole-protein or folded antigens) in a complementary hybrid approach.

Programmatic decisions regarding surveillance and intervention for trachoma are made at the district level, reflecting an implicit assumption that transmission within districts is sufficiently homogeneous. However, as transmission declines, residual pockets of transmission may become spatially heterogeneous at sub-district scales. Using cluster-level data from 12 districts in Amhara, Ethiopia (2019–2023), we assess the spatial structure of Pgp3 antibody responses, a sensitive measure of infection transmission. Spatial clustering was evident in higher prevalence districts but diminished as populations approached elimination. We show that as districts approach elimination sub-district heterogeneity likely disappears, and district-level summaries may be sufficient to guide decision-making.

Background: Giardia is the most common enteric parasite among children in low-resource settings, causing diarrhea and leading to prolonged infection or asymptomatic carriage. We assessed whether the effect of water, sanitation, and handwashing (WSH) interventions on Giardia infection among rural Bangladeshi children varies with seasonal conditions. Methods: We conducted a secondary analysis of the WASH Benefits Bangladesh cluster-randomized trial, with 450 clusters assigned to 4 arms in a 2 × 2 factorial design (WSH: WSH, WSH + Nutrition; no WSH: Control, Nutrition). Giardia infection was measured by multiplex real-time polymerase chain reaction in stool samples after 2 years of intervention. Effects were estimated by marginal treatment and assessed for heterogeneity by season when Giardia was measured. We also assessed heterogeneity by cumulative exposure to dry and monsoon seasons from birth to measurement age to estimate cumulative seasonal exposure history. Results: Giardia prevalence, measured among 2773 children (median age: 30 months, range: 22–38 months), was higher in the dry seasons (32%) than in the monsoon (21%). Improved WSH was associated with a consistent 20% relative reduction and a modest absolute reduction that was slightly greater during the dry season (−6.1%; 95% confidence interval: −10.1 to −2.1), although interaction test did not show strong evidence of effect modification by season. Prevalence remained lower in the WSH group across increasing dry season exposure, with the largest differences among children with more than 17 months of dry-season exposure. Conclusions: We demonstrate how WSH provides resilience to seasonal variation in infection risk and mitigates climate-driven, seasonally varying Giardia transmission.

Enteric pathogen infections are a major global health challenge, influenced by a variety of host and environmental factors, and their clinical presentation and treatment can be complicated by the presence of co-infections. The prevalence of enteric infections and co-infections tend to vary between rural and urban contexts, likely driven by underlying environmental, geographic, and demographic characteristics. To improve understanding of urbanicity and age on enteric pathogen prevalence and on co-infection risk, we measured 22 enteric pathogens in fecal samples collected from children aged 6, 12, and 18 months across a rural-urban gradient within the ECoMiD birth cohort study ( n =473). Enteric pathogen burden was high and increased with age, with at least one pathogen detected in 91% of children at 6 months, 97% at 12 months, and 98% at 18 months. However, prevalence of some pathogens— notably Salmonella enterica, enterovirus, and rotavirus— decreased with age. Co-infections were also common (88%), and children were infected with as many as 11 pathogens simultaneously. The most frequently observed co-infection profiles included enteroaggregative E. coli and atypical enteropathogenic E. coli , followed by combinations with diffusely adherent E. coli , enterovirus, enterotoxigenic E. coli , and/or adenovirus. Enteric pathogen detection generally was higher in more rural settings, though patterns varied by pathogen. These results provide useful information for future examination of pathogen dynamics of co-occurrence. Given the ubiquity of enteric infections in high transmission settings, strategies that aim to reduce overall microbial exposure may be needed to supplement interventions targeting control of individual pathogens. AUTHOR SUMMARY: Children living in low-resource settings are frequently exposed to pathogens that cause enteric infections, which can contribute to diarrhea, poor growth, and long-term health problems. Many children are infected with more than one pathogen at the same time, but we still know relatively little about how these infections occur together or how they vary across different environments. We examined stool samples collected from children during their first 18 months of life in communities ranging from urban to rural areas of northern Ecuador. We found that nearly every child carried at least one pathogen, and most carried several simultaneously. The number of pathogens increased as children grew older, and children living in more rural communities generally had a higher burden of infection. We also found that some pathogens occurred together more often than expected by chance, suggesting they may share common transmission routes or interact in ways that deserve further study. Our findings show that children in high-transmission settings experience a substantial burden of multiple simultaneous infections from an early age. These results support the need for interventions that reduce overall exposure to enteric pathogens, alongside strategies that target individual pathogens such as vaccines.