Novel rpsK / rpsD primer-probe assay improves detection of Campylobacter jejuni and Campylobacter coli in human stool.

Campylobacter causes bacterial enteritis, dysentery, and growth faltering in children in low- and middle-income countries (LMICs). Campylobacter spp. are fastidious organisms, and their detection often relies on culture independent diagnostic technologies, especially in LMICs. Campylobacter jejuni and Campylobacter coli are most often the infectious agents and in high income settings together account for 95% of Campylobacter infections. Several other Campylobacter species have been detected in LMIC children at an increased prevalence relative to high income settings. After doing extensive whole genome sequencing of isolates of C. jejuni and C. coli in Peru, we observed heterogeneity in the binding sites for the main species-specific PCR assay (cadF) and designed an alternative rpsKD-based qPCR assay to detect both C. jejuni and C. coli. The rpsKD-based qPCR assay identified 23% more C.jejuni/ C.coli samples than the cadF assay among 47 Campylobacter genus positive cadF negative samples verified to have C. jejuni and or C. coli with shotgun metagenomics. This assay can be expected to be useful in diagnostic studies of enteric infectious diseases and be useful in revising the attribution estimates of Campylobacter in LMICs.

Microbiological Methods Used in the Enterics for Global Health Shigella Surveillance Study.

Background: Shigella is a major cause of diarrhea in young children worldwide. Multiple vaccines targeting Shigella are in development, and phase 3 clinical trials are imminent to determine efficacy against shigellosis. Methods: The Enterics for Global Health (EFGH) Shigella surveillance study is designed to determine the incidence of medically attended shigellosis in 6- to 35-month-old children in 7 resource-limited settings. Here, we describe the microbiological methods used to isolate and identify Shigella. We developed a standardized laboratory protocol for isolation and identification of Shigella by culture. This protocol was implemented across all 7 sites, ensuring consistency and comparability of results. Secondary objectives of the study are to determine the antibiotic resistance profiles of Shigella, compare isolation of Shigella from rectal swabs versus whole stool, and compare isolation of Shigella following transport of rectal swabs in Cary-Blair versus a modified buffered glycerol saline transport medium. Conclusions: Data generated from EFGH using culture methods described herein can potentially be used for microbiological endpoints in future phase 3 clinical trials to evaluate vaccines against shigellosis and for other clinical and public health studies focused on these organisms.

Evaluation of Fecal Inflammatory Biomarkers to Identify Bacterial Diarrhea Episodes: Systematic Review and Protocol for the Enterics for Global Health Shigella Surveillance Study.

Background: The measurement of fecal inflammatory biomarkers among individuals presenting to care with diarrhea could improve the identification of bacterial diarrheal episodes that would benefit from antibiotic therapy. We reviewed prior literature in this area and describe our proposed methods to evaluate 4 biomarkers in the Enterics for Global Health (EFGH) Shigella surveillance study. Methods: We systematically reviewed studies since 1970 from PubMed and Embase that assessed the diagnostic characteristics of inflammatory biomarkers to identify bacterial diarrhea episodes. We extracted sensitivity and specificity and summarized the evidence by biomarker and diarrhea etiology. In EFGH, we propose using commercial enzyme-linked immunosorbent assays to test for myeloperoxidase, calprotectin, lipocalin-2, and hemoglobin in stored whole stool samples collected within 24 hours of enrollment from participants in the Bangladesh, Kenya, Malawi, Pakistan, Peru, and The Gambia sites. We will develop clinical prediction scores that incorporate the inflammatory biomarkers and evaluate their ability to identify Shigella and other bacterial etiologies of diarrhea as determined by quantitative polymerase chain reaction (qPCR). Results: Forty-nine studies that assessed fecal leukocytes (n = 39), red blood cells (n = 26), lactoferrin (n = 13), calprotectin (n = 8), and myeloperoxidase (n = 1) were included in the systematic review. Sensitivities were high for identifying Shigella, moderate for identifying any bacteria, and comparable across biomarkers. Specificities varied depending on the outcomes assessed. Prior studies were generally small, identified red and white blood cells by microscopy, and used insensitive gold standard diagnostics, such as conventional bacteriological culture for pathogen detection. Conclusions: Our evaluation of inflammatory biomarkers to distinguish diarrhea etiologies as determined by qPCR will provide an important addition to the prior literature, which was likely biased by the limited sensitivity of the gold standard diagnostics used. We will determine whether point-of-care biomarker tests could be a viable strategy to inform treatment decision making and increase appropriate targeting of antibiotic treatment to bacterial diarrhea episodes.

Population Enumeration and Household Utilization Survey Methods in the Enterics for Global Health (EFGH): Shigella Surveillance Study.

Background: Accurate estimation of diarrhea incidence from facility-based surveillance requires estimating the population at risk and accounting for case patients who do not seek care. The Enterics for Global Health (EFGH) Shigella surveillance study will characterize population denominators and healthcare-seeking behavior proportions to calculate incidence rates of Shigella diarrhea in children aged 6-35 months across 7 sites in Africa, Asia, and Latin America. Methods: The Enterics for Global Health (EFGH) Shigella surveillance study will use a hybrid surveillance design, supplementing facility-based surveillance with population-based surveys to estimate population size and the proportion of children with diarrhea brought for care at EFGH health facilities. Continuous data collection over a 24 month period captures seasonality and ensures representative sampling of the population at risk during the period of facility-based enrollments. Study catchment areas are broken into randomized clusters, each sized to be feasibly enumerated by individual field teams. Conclusions: The methods presented herein aim to minimize the challenges associated with hybrid surveillance, such as poor parity between survey area coverage and facility coverage, population fluctuations, seasonal variability, and adjustments to care-seeking behavior.

Data Management in Multicountry Consortium Studies: The Enterics For Global Health (EFGH) Shigella Surveillance Study Example.

Background: Rigorous data management systems and planning are essential to successful research projects, especially for large, multicountry consortium studies involving partnerships across multiple institutions. Here we describe the development and implementation of data management systems and procedures for the Enterics For Global Health (EFGH) Shigella surveillance study-a 7-country diarrhea surveillance study that will conduct facility-based surveillance concurrent with population-based enumeration and a health care utilization survey to estimate the incidence of Shigella--associated diarrhea in children 6 to 35 months old. Methods: The goals of EFGH data management are to utilize the knowledge and experience of consortium members to collect high-quality data and ensure equity in access and decision-making. During the planning phase before study initiation, a working group of representatives from each EFGH country site, the coordination team, and other partners met regularly to develop the data management systems for the study. Results: This resulted in the Data Management Plan, which included selecting REDCap and SurveyCTO as the primary database systems. Consequently, we laid out procedures for data processing and storage, study monitoring and reporting, data quality control and assurance activities, and data access. The data management system and associated real-time visualizations allow for rapid data cleaning activities and progress monitoring and will enable quicker time to analysis. Conclusions: Experiences from this study will contribute toward enriching the sparse landscape of data management methods publications and serve as a case study for future studies seeking to collect and manage data consistently and rigorously while maintaining equitable access to and control of data.

The Enterics for Global Health (EFGH) Shigella Surveillance Study in Peru.

Background: The Enterics for Global Health (EFGH) Peru site will enroll subjects in a periurban area of the low Amazon rainforest. The political department of Loreto lags behind most of Peru in access to improved sources of water and sanitation, per capita income, children born <2.5 kg, and infant and child mortality. Chronic undernutrition as manifested by linear growth shortfalls is common, but wasting and acute malnutrition are not. Methods: The recruitment of children seeking care for acute diarrheal disease takes place at a geographic cluster of government-based primary care centers in an area where most residents are beneficiaries of free primary healthcare. Results: Rates of diarrheal disease, dysentery, and Shigella are known to be high in the region, with some of the highest rates of disease documented in the literature and little evidence in improvement over the last 2 decades. This study will update estimates of shigellosis by measuring the prevalence of Shigella by polymerase chain reaction and culture in children seeking care and deriving population-based estimates by measuring healthcare seeking at the community level. Conclusions: Immunization has been offered universally against rotavirus in the region since 2009, and in a context where adequate water and sanitation are unlikely to obtain high standards in the near future, control of principal enteropathogens through immunization may be the most feasible way to decrease the high burden of disease in the area in the near future.

Diarrhea Case Surveillance in the Enterics for Global Health Shigella Surveillance Study: Epidemiologic Methods.

Background: Shigella is a leading cause of acute watery diarrhea, dysentery, and diarrhea-attributed linear growth faltering, a precursor to stunting and lifelong morbidity. Several promising Shigella vaccines are in development and field efficacy trials will require a consortium of potential vaccine trial sites with up-to-date Shigella diarrhea incidence data. Methods: The Enterics for Global Health (EFGH) Shigella surveillance study will employ facility-based enrollment of diarrhea cases aged 6-35 months with 3 months of follow-up to establish incidence rates and document clinical, anthropometric, and financial consequences of Shigella diarrhea at 7 country sites (Mali, Kenya, The Gambia, Malawi, Bangladesh, Pakistan, and Peru). Over a 24-month period between 2022 and 2024, the EFGH study aims to enroll 9800 children (1400 per country site) between 6 and 35 months of age who present to local health facilities with diarrhea. Shigella species (spp.) will be identified and serotyped from rectal swabs by conventional microbiologic methods and quantitative polymerase chain reaction. Shigella spp. isolates will undergo serotyping and antimicrobial susceptibility testing. Incorporating population and healthcare utilization estimates from contemporaneous household sampling in the catchment areas of enrollment facilities, we will estimate Shigella diarrhea incidence rates. Conclusions: This multicountry surveillance network will provide key incidence data needed to design Shigella vaccine trials and strengthen readiness for potential trial implementation. Data collected in EFGH will inform policy makers about the relative importance of this vaccine-preventable disease, accelerating the time to vaccine availability and uptake among children in high-burden settings.

Exploring Natural Immune Responses to Shigella Exposure Using Multiplex Bead Assays on Dried Blood Spots in High-Burden Countries: Protocol From a Multisite Diarrhea Surveillance Study.

Background: Molecular diagnostics on human fecal samples have identified a larger burden of shigellosis than previously appreciated by culture. Evidence of fold changes in immunoglobulin G (IgG) to conserved and type-specific Shigella antigens could be used to validate the molecular assignment of type-specific Shigella as the etiology of acute diarrhea and support polymerase chain reaction (PCR)-based microbiologic end points for vaccine trials. Methods: We will test dried blood spots collected at enrollment and 4 weeks later using bead-based immunoassays for IgG to invasion plasmid antigen B and type-specific lipopolysaccharide O-antigen for Shigella flexneri 1b, 2a, 3a, and 6 and Shigella sonnei in Shigella-positive cases and age-, site-, and season-matched test-negative controls from all sites in the Enterics for Global Health (EFGH) Shigella surveillance study. Fold antibody responses will be compared between culture-positive, culture-negative but PCR-attributable, and PCR-positive but not attributable cases and test-negative controls. Age- and site-specific seroprevalence distributions will be identified, and the association between baseline antibodies and Shigella attribution will be estimated. Conclusions: The integration of these assays into the EFGH study will help support PCR-based attribution of acute diarrhea to type-specific Shigella, describe the baseline seroprevalence of conserved and type-specific Shigella antibodies, and support correlates of protection for immunity to Shigella diarrhea. These insights can help support the development and evaluation of Shigella vaccine candidates.

Genomic resistant determinants of multidrug-resistant Campylobacter spp. isolates in Peru.

OBJECTIVES: Antimicrobial resistant (AMR) Campylobacter is a global health threat; however, there is limited information on genomic determinants of resistance in low- and middle-income countries. We evaluated genomic determinants of AMR using a collection of whole genome sequenced Campylobacter jejuni and C. coli isolates from Iquitos, Peru. METHODS: Campylobacter isolates from two paediatric cohort studies enriched with isolates that demonstrated resistance to ciprofloxacin and azithromycin were sequenced and mined for AMR determinants. RESULTS: The gyrA mutation leading to the Thr86Ile amino acid change was the only gyrA mutation associated with fluoroquinolone resistance identified. The A2075G mutation in 23S rRNA was present, but three other 23S rRNA mutations previously associated with macrolide resistance were not identified. A resistant-enhancing variant of the cmeABC efflux pump genotype (RE-cmeABC) was identified in 36.1% (35/97) of C. jejuni genomes and 17.9% (12/67) of C. coli genomes. Mutations identified in the CmeR-binding site, an inverted repeat sequence in the cmeABC promoter region that increases expression of the operon, were identified in 24/97 C. jejuni and 14/67 C. coli genomes. The presence of these variants, in addition to RE-cmeABC, was noted in 18 of the 24 C. jejuni and 9 of the 14 C. coli genomes. CONCLUSIONS: Both RE-cmeABC and mutations in the CmeR-binding site were strongly associated with the MDR phenotype in C. jejuni and C. coli. This is the first report of RE-cmeABC in Peru and suggests it is a major driver of resistance to the principal therapies used to treat human campylobacteriosis in this setting.

Nutrient intakes from complementary foods are associated with cardiometabolic biomarkers among undernourished Peruvian children.

Relatively little is known about how the diet of chronically undernourished children may impact cardiometabolic biomarkers. The objective of this exploratory study was to characterise relationships between dietary patterns and the cardiometabolic profile of 153 3-5-year-old Peruvian children with a high prevalence of chronic undernutrition. We collected monthly dietary recalls from children when they were 9-24 months old. At 3-5 years, additional dietary recalls were collected, and blood pressure, height, weight, subscapular skinfolds and fasting plasma glucose, insulin and lipid profiles were assessed. Nutrient intakes were expressed as average density per 100 kcals (i) from 9 to 24 months and (ii) at follow-up. The treelet transform and sparse reduced rank regress'ion (RRR) were used to summarize nutrient intake data. Linear regression models were then used to compare these factors to cardiometabolic outcomes and anthropometry. Linear regression models adjusting for subscapular skinfold-for-age Z-scores (SSFZ) were then used to test whether observed relationships were mediated by body composition. 26 % of children were stunted at 3-5 years old. Both treelet transform and sparse RRR-derived child dietary factors are related to protein intake and associated with total cholesterol and SSFZ. Associations between dietary factors and insulin were attenuated after adjusting for SSFZ, suggesting that body composition mediated these relationships. Dietary factors in early childhood, influenced by protein intake, are associated with cholesterol profiles, fasting glucose and body fat in a chronically undernourished population.

Etiology of Acute Febrile Illness in the Peruvian Amazon as determined by modular formatted quantitative PCR: A Protocol for RIVERA, a Health Facility-Based Case-Control Study.

Background: The study of the etiology of acute febrile illness (AFI) has historically been designed as a prevalence of pathogens detected from a case series. This strategy has an inherent unrealistic assumption that all pathogen detection allows for causal attribution, despite known asymptomatic carriage of the principal causes of acute febrile illness in most low- and middle-income countries (LMICs). We designed a semi-quantitative PCR in a modular format to detect bloodborne agents of acute febrile illness that encompassed common etiologies of AFI in the region, etiologies of recent epidemics, etiologies that require an immediate public health response and additional pathogens of unknown endemicity. We then designed a study that would delineate background levels of transmission in the community in the absence of symptoms to provide corrected estimates of attribution for the principal determinants of AFI. Methods: A case-control study of acute febrile illness in patients ten years or older seeking health care in Iquitos, Loreto, Peru, was planned. Upon enrollment, we will obtain blood, saliva, and mid-turbinate nasal swabs at enrollment with a follow-up visit on day 21-28 following enrollment to attain vital status and convalescent saliva and blood samples, as well as a questionnaire including clinical, socio-demographic, occupational, travel, and animal contact information for each participant. Whole blood samples are to be simultaneously tested for 32 pathogens using TaqMan array cards. Mid-turbinate samples will be tested for SARS-CoV-2, Influenza A and Influenza B. Conditional logistic regression models will be fitted treating case/control status as the outcome and with pathogen-specific sample positivity as predictors to attain estimates of attributable pathogen fractions for AFI. Discussion: The modular PCR platforms will allow for reporting of all primary results of respiratory samples within 72 hours and blood samples within one week, allowing for results to influence local medical practice and enable timely public health responses. The inclusion of controls will allow for a more accurate estimate of the importance of specific, prevalent pathogens as a cause of acute illness. Study Registration: Project 1791, Registro de Proyectos de Investigación en Salud Pública (PRISA), Instituto Nacional de Salud, Perú.

Etiology of acute febrile illness in the peruvian amazon as determined by modular formatted quantitative PCR: a protocol for RIVERA, a health facility-based case-control study.

BACKGROUND: The study of the etiology of acute febrile illness (AFI) has historically been designed as a prevalence of pathogens detected from a case series. This strategy has an inherent unrealistic assumption that all pathogen detection allows for causal attribution, despite known asymptomatic carriage of the principal causes of acute febrile illness in most low- and middle-income countries (LMICs). We designed a semi-quantitative PCR in a modular format to detect bloodborne agents of acute febrile illness that encompassed common etiologies of AFI in the region, etiologies of recent epidemics, etiologies that require an immediate public health response and additional pathogens of unknown endemicity. We then designed a study that would delineate background levels of transmission in the community in the absence of symptoms to provide corrected estimates of attribution for the principal determinants of AFI. METHODS: A case-control study of acute febrile illness in patients ten years or older seeking health care in Iquitos, Loreto, Peru, was planned. Upon enrollment, we will obtain blood, saliva, and mid-turbinate nasal swabs at enrollment with a follow-up visit on day 21-28 following enrollment to attain vital status and convalescent saliva and blood samples, as well as a questionnaire including clinical, socio-demographic, occupational, travel, and animal contact information for each participant. Whole blood samples are to be simultaneously tested for 32 pathogens using TaqMan array cards. Mid-turbinate samples will be tested for SARS-CoV-2, Influenza A and Influenza B. Conditional logistic regression models will be fitted treating case/control status as the outcome and with pathogen-specific sample positivity as predictors to attain estimates of attributable pathogen fractions for AFI. DISCUSSION: The modular PCR platforms will allow for reporting of all primary results of respiratory samples within 72 h and blood samples within one week, allowing for results to influence local medical practice and enable timely public health responses. The inclusion of controls will allow for a more accurate estimate of the importance of specific prevalent pathogens as a cause of acute illness. STUDY REGISTRATION: Project 1791, Registro de Proyectos de Investigación en Salud Pública (PRISA), Instituto Nacional de Salud, Perú.

Spatiotemporal variation in risk of Shigella infection in childhood: a global risk mapping and prediction model using individual participant data.

BACKGROUND: Diarrhoeal disease is a leading cause of childhood illness and death globally, and Shigella is a major aetiological contributor for which a vaccine might soon be available. The primary objective of this study was to model the spatiotemporal variation in paediatric Shigella infection and map its predicted prevalence across low-income and middle-income countries (LMICs). METHODS: Individual participant data for Shigella positivity in stool samples were sourced from multiple LMIC-based studies of children aged 59 months or younger. Covariates included household-level and participant-level factors ascertained by study investigators and environmental and hydrometeorological variables extracted from various data products at georeferenced child locations. Multivariate models were fitted and prevalence predictions obtained by syndrome and age stratum. FINDINGS: 20 studies from 23 countries (including locations in Central America and South America, sub-Saharan Africa, and south and southeast Asia) contributed 66 563 sample results. Age, symptom status, and study design contributed most to model performance followed by temperature, wind speed, relative humidity, and soil moisture. Probability of Shigella infection exceeded 20% when both precipitation and soil moisture were above average and had a 43% peak in uncomplicated diarrhoea cases at 33°C temperatures, above which it decreased. Compared with unimproved sanitation, improved sanitation decreased the odds of Shigella infection by 19% (odds ratio [OR]=0·81 [95% CI 0·76-0·86]) and open defecation decreased them by 18% (OR=0·82 [0·76-0·88]). INTERPRETATION: The distribution of Shigella is more sensitive to climatological factors, such as temperature, than previously recognised. Conditions in much of sub-Saharan Africa are particularly propitious for Shigella transmission, although hotspots also occur in South America and Central America, the Ganges-Brahmaputra Delta, and the island of New Guinea. These findings can inform prioritisation of populations for future vaccine trials and campaigns. FUNDING: NASA, National Institutes of Health-The National Institute of Allergy and Infectious Diseases, and Bill & Melinda Gates Foundation.

Antibiotic Use and Stewardship Practices in a Pediatric Community-based Cohort Study in Peru: Shorter Would be Sweeter.

BACKGROUND: There is a need to evaluate antibiotic use, duration of therapy, and stewardship in low- and middle-income countries to guide the development of appropriate stewardship programs that are global in scope and effectively decrease unnecessary antibiotic use. METHODS: We prospectively collected information on illness occurrence and antibiotic use from a cohort of 303 children. We evaluated the incidence, duration of therapy, and appropriateness of antibiotic prescriptions by 5 main antibiotic prescribers (physicians and nurses, pharmacists, nursing assistants, self-prescriptions, and neighbors or family members). RESULTS: Ninety percent of children received an antibiotic during follow-up, and on average, by the end of follow-up a child had spent 4.3% of their first 5 years of life on antibiotics. The most frequent prescribers were physicians/nurses (79.4%), followed by pharmacists (8.1%), self-prescriptions (6.8%), nursing assistants (3.7%), and family or neighbors (1.9%). Of the 3702 courses of antibiotics prescribed, 30.9% were done so for the occurrence of fever, 25.3% for diarrhea, 2.8% for acute lower respiratory disease, 2.7% for dysentery, and 38.2% for an undetermined illness. Courses exceeding the recommended duration were common for the principal diseases for which treatment was initiated, with 27.3% of courses exceeding the recommended length duration, representing a potential reduction in 13.2% of days on which this cohort spent on antibiotics. CONCLUSIONS: Stewardship programs should target medical personnel for a primary care stewardship program even in a context in which antibiotics are available to the public with little or no restrictions and appropriate duration should be emphasized in this training.

Shotgun metagenomics of fecal samples from children in Peru reveals frequent complex co-infections with multiple Campylobacter species.

Campylobacter spp. are a major cause of bacterial diarrhea worldwide and are associated with high rates of mortality and linear growth faltering in children living in low- to middle-income countries (LMICs). Campylobacter jejuni and Campylobacter coli are most often the causative agents of enteric disease among children in LMICs. However, previous work on a collection of stool samples from children under 2 years of age, living in a low resource community in Peru with either acute diarrheal disease or asymptomatic, were found to be qPCR positive for Campylobacter species but qPCR negative for C. jejuni and C. coli. The goal of this study was to determine if whole-genome shotgun metagenomic sequencing (WSMS) could identify the Campylobacter species within these samples. The Campylobacter species identified in these stool samples included C. jejuni, C. coli, C. upsaliensis, C. concisus, and the potential new species of Campylobacter, "Candidatus Campylobacter infans". Moreover, WSMS results demonstrate that over 65% of the samples represented co-infections with multiple Campylobacter species present in a single stool sample, a novel finding in human populations.

"Candidatus Campylobacter infans" detection is not associated with diarrhea in children under the age of 2 in Peru.

A working hypothesis is that less common species of Campylobacter (other than C. jejuni and C. coli) play a role in enteric disease among children in low resource settings and explain the gap between the detection of Campylobacter using culture and culture independent methods. "Candidatus Campylobacter infans" (C. infans), was recently detected in stool samples from children and hypothesized to play a role in Campylobacter epidemiology in low- and middle-income countries (LMIC). This study determined the prevalence of C. infans in symptomatic and asymptomatic stool samples from children living in Iquitos, Peru. Stool samples from 215 children with diarrhea and 50 stool samples from children without diarrhea under the age of two were evaluated using a multiplex qPCR assay to detect Campylobacter spp. (16S rRNA), Campylobacter jejuni / Campylobacter coli (cadF gene), C. infans (lpxA), and Shigella spp. (ipaH). C. infans was detected in 7.9% (17/215) symptomatic samples and 4.0% (2/50) asymptomatic samples. The association between diarrhea and the presence of these targets was evaluated using univariate logistic regressions. C. infans was not associated with diarrhea. Fifty-one percent (75/146) of Campylobacter positive fecal samples were negative for C. jejuni, C. coli, and C. infans via qPCR. Shotgun metagenomics confirmed the presence of C. infans among 13 out of 14 positive C. infans positive stool samples. C infans explained only 20.7% of the diagnostic gap in stools from children with diarrhea and 16.7% of the gap in children without diarrhea. We posit that poor cadF primer performance better explains the observed gap than the prevalence of atypical non-C. jejuni/coli species.

The Epidemiology of Sapovirus in the Etiology, Risk Factors, and Interactions of Enteric Infection and Malnutrition and the Consequences for Child Health and Development Study: Evidence of Protection Following Natural Infection.

BACKGROUND: Sapovirus is one of the principal agents of acute viral enteritis in children. Because it has not been routinely included in diagnostic evaluations, the epidemiology and natural history remain poorly described. METHODS: A birth cohort of 1715 children from 8 countries contributed surveillance samples (n = 35 620) and diarrheal specimens (n = 6868) from 0 to 24 months of age. Sapovirus was detected by quantitative polymerase chain reaction concurrently to other enteropathogens using multiarray cards. Logistic regression was used to identify risk factors, and longitudinal models were employed to estimate incidence rates and evaluate evidence of protective immunity. RESULTS: Sapovirus was detected in 24.7% (n = 1665) of diarrheal stools and 12.8% (n = 4429) of monthly surveillance samples. More than 90% of children were infected and 60% experienced sapovirus diarrhea in the first 2 years of life. Breastfeeding and higher socioeconomic status were associated with reduced incidence of infection and illness. Specimens with sapovirus detected had an increased odds of coinfection with rotavirus (odds ratio [OR], 1.6 [95% confidence interval {CI}, 1.3-2.0]), astrovirus (OR, 1.5 [95% CI, 1.3-1.7]), adenovirus (OR, 1.3 [95% CI, 1.1-1.5]), and Shigella (OR, 1.4 [95% CI, 1.3-1.6]). Prior infection with sapovirus conferred a risk reduction of 22% for subsequent infection (hazard ratio [HR], 0.78 [95% CI, .74-.85]) and 24% for subsequent diarrhea (95% CI, 11.0%-35.0%; HR, 0.76). CONCLUSIONS: Sapovirus is a common cause of early childhood diarrhea. Further research on coinfections is warranted. Evidence of acquired immunity was observed even in the absence of genotype-specific analysis for this pathogen of known genetic diversity.

Enteropathy Markers in Early Life Were Associated with Adipokine, Apolipoprotein, and Cytokine Profiles Consistent with an Adverse Cardiometabolic Disease Risk Profile Later in Childhood in a Peruvian Birth Cohort.

Metabolic syndrome is a cluster of risk factors for cardiovascular disease afflicting more than 1 billion people worldwide and is increasingly being identified in younger age groups and in socioeconomically disadvantaged settings in the global south. Enteropathogen exposure and environmental enteropathy in infancy may contribute to metabolic syndrome by disrupting the metabolic profile in a way that is detectable in cardiometabolic markers later in childhood. A total of 217 subjects previously enrolled in a birth cohort in Amazonian Peru were monitored annually from ages 2 to 5 years. A total of 197 blood samples collected in later childhood were analyzed for 37 cardiometabolic biomarkers, including adipokines, apolipoproteins, cytokines, which were matched to extant early-life markers of enteropathy ascertained between birth and 2 years. Multivariate and multivariable regression models were fitted to test for associations, adjusting for confounders. Fecal and urinary markers of intestinal permeability and inflammation (myeloperoxidase, lactulose, and mannitol) measured in infancy were associated with later serum concentrations of soluble CD40-ligand, a proinflammatory cytokine correlated with adverse metabolic outcomes. Fecal myeloperoxidase was also associated with later levels of omentin-1. Enteric protozoa exposure showed stronger associations with later cardiometabolic markers than viruses, bacteria, and overall diarrheal episodes. Early-life enteropathy markers were associated with altered adipokine, apolipoprotein, and cytokine profiles later in childhood consistent with an adverse cardiometabolic disease risk profile in this cohort. Markers of intestinal permeability and inflammation measured in urine (lactulose, mannitol) and stool (myeloperoxidase, protozoal infections) during infancy may predict metabolic syndrome in adulthood.

Genomic Characterization of Campylobacter jejuni Adapted to the Guinea Pig (Cavia porcellus) Host.

Campylobacter jejuni is the leading bacterial cause of gastroenteritis worldwide with excessive incidence in low-and middle-income countries (LMIC). During a survey for C. jejuni from putative animal hosts in a town in the Peruvian Amazon, we were able to isolate and whole genome sequence two C. jejuni strains from domesticated guinea pigs (Cavia porcellus). The C. jejuni isolated from guinea pigs had a novel multilocus sequence type that shared some alleles with other C. jejuni collected from guinea pigs. Average nucleotide identity and phylogenetic analysis with a collection of C. jejuni subsp. jejuni and C. jejuni subsp. doylei suggest that the guinea pig isolates are distinct. Genomic comparisons demonstrated gene gain and loss that could be associated with guinea pig host specialization related to guinea pig diet, anatomy, and physiology including the deletion of genes involved with selenium metabolism, including genes encoding the selenocysteine insertion machinery and selenocysteine-containing proteins.

Homotypic and Heterotypic Protection and Risk of Reinfection Following Natural Norovirus Infection in a Highly Endemic Setting.

BACKGROUND: Norovirus is a leading cause of acute gastroenteritis worldwide, yet there is limited information on homotypic or heterotypic protection following natural infection to guide vaccine development. METHODS: A total of 6020 stools collected from 299 Peruvian children between 2010 and 2014 were tested by norovirus real-time reverse-transcription polymerase chain reaction followed by sequence-based genotyping. Cox proportional hazards models were used to derive adjusted hazard ratios (HRs) of infection among children with vs without prior exposure. RESULTS: Norovirus was detected in 1288 (21.3%) samples. GII.4 (26%), GII.6 (19%), and GI.3 (9%) viruses accounted for 54% of infections. Homotypic protection for GI.3 (HR, 0.35; P = .015), GI.7 (HR, 0.19; P = .022), GII.4 (HR, 0.39; P < .001), and GII.6 (HR, 0.52; P = .006) infections was observed. Hazard analysis showed that children with prior GII.4 infection exhibited heterotypic protection with a 48% reduction of subsequent GI.3 infection (HR, 0.52; P = .005). Prior exposure to GI.3, GII.2, and GII.17 infections enhanced susceptibility to subsequent infections with several other norovirus genotypes. CONCLUSIONS: Children up to 2 years of age infected with GII.4 noroviruses demonstrated both homotypic and heterotypic protection to reinfection with other genotypes. These data support the need for ongoing vaccine development efforts with GII.4 as the main component and caution the inclusion of genotypes that may enhance susceptibility to infections.