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.

Quantifying the Cost of Shigella Diarrhea in the Enterics for Global Health (EFGH) Shigella Surveillance Study.

Background: Comparative costs of public health interventions provide valuable data for decision making. However, the availability of comprehensive and context-specific costs is often limited. The Enterics for Global Health (EFGH) Shigella surveillance study-a facility-based diarrhea surveillance study across 7 countries-aims to generate evidence on health system and household costs associated with medically attended Shigella diarrhea in children. Methods: EFGH working groups comprising representatives from each country (Bangladesh, Kenya, Malawi, Mali, Pakistan, Peru, and The Gambia) developed the study methods. Over a 24-month surveillance period, facility-based surveys will collect data on resource use for the medical treatment of an estimated 9800 children aged 6-35 months with diarrhea. Through these surveys, we will describe and quantify medical resources used in the treatment of diarrhea (eg, medication, supplies, and provider salaries), nonmedical resources (eg, travel costs to the facility), and the amount of caregiver time lost from work to care for their sick child. To assign costs to each identified resource, we will use a combination of caregiver interviews, national medical price lists, and databases from the World Health Organization and the International Labor Organization. Our primary outcome will be the estimated cost per inpatient and outpatient episode of medically attended Shigella diarrhea treatment across countries, levels of care, and illness severity. We will conduct sensitivity and scenario analysis to determine how unit costs vary across scenarios. Conclusions: Results from this study will contribute to the existing body of literature on diarrhea costing and inform future policy decisions related to investments in preventive strategies for Shigella.

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.

Shigella Detection and Molecular Serotyping With a Customized TaqMan Array Card in the Enterics for Global Health (EFGH): Shigella Surveillance Study.

Background: Quantitative polymerase chain reaction (qPCR) targeting ipaH has been proven to be highly efficient in detecting Shigella in clinical samples compared to culture-based methods, which underestimate Shigella burden by 2- to 3-fold. qPCR assays have also been developed for Shigella speciation and serotyping, which is critical for both vaccine development and evaluation. Methods: The Enterics for Global Health (EFGH) Shigella surveillance study will utilize a customized real-time PCR-based TaqMan Array Card (TAC) interrogating 82 targets, for the detection and differentiation of Shigella spp, Shigella sonnei, Shigella flexneri serotypes, other diarrhea-associated enteropathogens, and antimicrobial resistance (AMR) genes. Total nucleic acid will be extracted from rectal swabs or stool samples, and assayed on TAC. Quantitative analysis will be performed to determine the likely attribution of Shigella and other particular etiologies of diarrhea using the quantification cycle cutoffs derived from previous studies. The qPCR results will be compared to conventional culture, serotyping, and phenotypic susceptibility approaches in EFGH. Conclusions: TAC enables simultaneous detection of diarrheal etiologies, the principal pathogen subtypes, and AMR genes. The high sensitivity of the assay enables more accurate estimation of Shigella-attributed disease burden, which is critical to informing policy and in the design of future clinical trials.

The Planetary Child Health & Enterics Observatory (Plan-EO): A protocol for an interdisciplinary research initiative and web-based dashboard for mapping enteric infectious diseases and their risk factors and interventions in LMICs.

BACKGROUND: Diarrhea remains a leading cause of childhood illness throughout the world that is increasing due to climate change and is caused by various species of ecologically sensitive pathogens. The emerging Planetary Health movement emphasizes the interdependence of human health with natural systems, and much of its focus has been on infectious diseases and their interactions with environmental and human processes. Meanwhile, the era of big data has engendered a public appetite for interactive web-based dashboards for infectious diseases. However, enteric infectious diseases have been largely overlooked by these developments. METHODS: The Planetary Child Health & Enterics Observatory (Plan-EO) is a new initiative that builds on existing partnerships between epidemiologists, climatologists, bioinformaticians, and hydrologists as well as investigators in numerous low- and middle-income countries. Its objective is to provide the research and stakeholder community with an evidence base for the geographical targeting of enteropathogen-specific child health interventions such as novel vaccines. The initiative will produce, curate, and disseminate spatial data products relating to the distribution of enteric pathogens and their environmental and sociodemographic determinants. DISCUSSION: As climate change accelerates there is an urgent need for etiology-specific estimates of diarrheal disease burden at high spatiotemporal resolution. Plan-EO aims to address key challenges and knowledge gaps by making and disseminating rigorously obtained, generalizable disease burden estimates. Pre-processed environmental and EO-derived spatial data products will be housed, continually updated, and made publicly available for download to the research and stakeholder communities. These can then be used as inputs to identify and target priority populations living in transmission hotspots and for decision-making, scenario-planning, and disease burden projection. STUDY REGISTRATION: PROSPERO protocol #CRD42023384709.

Viral gastroenteritis.

Since the discovery of norovirus in 1972 as a cause of what was contemporarily known as acute infectious non-bacterial gastroenteritis, scientific understanding of the viral gastroenteritides has continued to evolve. It is now recognised that a small number of viruses are the predominant cause of acute gastroenteritis worldwide, in both high-income and low-income settings. Although treatment is still largely restricted to the replacement of fluid and electrolytes, improved diagnostics have allowed attribution of illness, enabling both targeted treatment of individual patients and prioritisation of interventions for populations worldwide. Questions remain regarding specific genetic and immunological factors underlying host susceptibility, and the optimal clinical management of patients who are susceptible to severe or prolonged manifestations of disease. Meanwhile, the worldwide implementation of rotavirus vaccines has led to substantial reductions in morbidity and mortality, and spurred interest in vaccine development to diminish the impact of the most prevalent viruses that are implicated in this syndrome.

Short-term dynamics of linear growth among Peruvian infants in the first year of life in a population with linear growth faltering.

OBJECTIVES: Infant growth is recognized to vary over the short term, with periods of greater and lesser linear growth velocity. Our objectives were to (1) examine the potential differences in overall growth profiles between children who experienced cumulative growth faltering in the first year of life consistent with that seen by many children living in poverty in low- and middle-income countries, versus children without growth faltering and (2) test whether biological factors were associated with the timing of magnitude of growth saltations. METHODS: Thrice-weekly measurements of length were recorded for n = 61 Peruvian infants (28 boys and 33 girls) enrolled from birth to 1 year. A total of 6040 measurements were analyzed. We tested for the evidence of saltatory growth and used hurdle models to test whether the timing and magnitude of saltations varied between children with greater or lesser growth faltering. RESULTS: There were no differences in the duration of stasis periods or magnitude of growth saltations between children who were stunted at 1 year old (N = 18) versus those who were not stunted (N = 43). Children who experienced greater declines in LAZ in the first year of life trended toward longer periods between saltations than those with less of a decline (14.5 days vs. 13.4 days, p = .0512). A 1-unit increase in mid upper arm circumference for age Z-score in the 21 days prior was associated with 35% greater odds of a saltation occurring (p < .001), and a 0.128 cm greater saltation (p < .001). CONCLUSIONS: After characterizing infant growth into periods of saltation and stasis, our results suggest that increases in weight preceded increases in length.

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.

Unified real-time environmental-epidemiological data for multiscale modeling of the COVID-19 pandemic.

An impressive number of COVID-19 data catalogs exist. However, none are fully optimized for data science applications. Inconsistent naming and data conventions, uneven quality control, and lack of alignment between disease data and potential predictors pose barriers to robust modeling and analysis. To address this gap, we generated a unified dataset that integrates and implements quality checks of the data from numerous leading sources of COVID-19 epidemiological and environmental data. We use a globally consistent hierarchy of administrative units to facilitate analysis within and across countries. The dataset applies this unified hierarchy to align COVID-19 epidemiological data with a number of other data types relevant to understanding and predicting COVID-19 risk, including hydrometeorological data, air quality, information on COVID-19 control policies, vaccine data, and key demographic characteristics.

Giardia hinders growth by disrupting nutrient metabolism independent of inflammatory enteropathy.

Giardia lamblia (Giardia) is among the most common intestinal pathogens in children in low- and middle-income countries (LMICs). Although Giardia associates with early-life linear growth restriction, mechanistic explanations for Giardia-associated growth impairments remain elusive. Unlike other intestinal pathogens associated with constrained linear growth that cause intestinal or systemic inflammation or both, Giardia seldom associates with chronic inflammation in these children. Here we leverage the MAL-ED longitudinal birth cohort and a model of Giardia mono-association in gnotobiotic and immunodeficient mice to propose an alternative pathogenesis of this parasite. In children, Giardia results in linear growth deficits and gut permeability that are dose-dependent and independent of intestinal markers of inflammation. The estimates of these findings vary between children in different MAL-ED sites. In a representative site, where Giardia associates with growth restriction, infected children demonstrate broad amino acid deficiencies, and overproduction of specific phenolic acids, byproducts of intestinal bacterial amino acid metabolism. Gnotobiotic mice require specific nutritional and environmental conditions to recapitulate these findings, and immunodeficient mice confirm a pathway independent of chronic T/B cell inflammation. Taken together, we propose a new paradigm that Giardia-mediated growth faltering is contingent upon a convergence of this intestinal protozoa with nutritional and intestinal bacterial factors.

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.

Evolving Drivers of Brazilian SARS-CoV-2 Transmission: A Spatiotemporally Disaggregated Time Series Analysis of Meteorology, Policy, and Human Mobility.

Brazil has been severely affected by the COVID-19 pandemic. Temperature and humidity have been purported as drivers of SARS-CoV-2 transmission, but no consensus has been reached in the literature regarding the relative roles of meteorology, governmental policy, and mobility on transmission in Brazil. We compiled data on meteorology, governmental policy, and mobility in Brazil's 26 states and one federal district from June 2020 to August 2021. Associations between these variables and the time-varying reproductive number (R t ) of SARS-CoV-2 were examined using generalized additive models fit to data from the entire 15-month period and several shorter, 3-month periods. Accumulated local effects and variable importance metrics were calculated to analyze the relationship between input variables and R t . We found that transmission is strongly influenced by unmeasured sources of between-state heterogeneity and the near-recent trajectory of the pandemic. Increased temperature generally was associated with decreased transmission and increased specific humidity with increased transmission. However, the impacts of meteorology, policy, and mobility on R t varied in direction, magnitude, and significance across our study period. This time variance could explain inconsistencies in the published literature to date. While meteorology weakly modulates SARS-CoV-2 transmission, daily or seasonal weather variations alone will not stave off future surges in COVID-19 cases in Brazil. Investigating how the roles of environmental factors and disease control interventions may vary with time should be a deliberate consideration of future research on the drivers of SARS-CoV-2 transmission.