Development, confirmation, and application of a seeded Escherichia coli process control organism to validate Salmonella enterica serovar Typhi environmental surveillance methods.

Salmonella enterica serovar Typhi (S. Typhi) is the causative agent of Typhoid fever. Blood culture is the gold standard for clinical diagnosis, but this is often difficult to employ in resource limited settings. Environmental surveillance of waste-impacted waters is a promising supplement to clinical surveillance, however validating methods is challenging in regions where S. Typhi concentrations are low. To evaluate existing S. Typhi environmental surveillance methods, a novel process control organism (PCO) was created as a biosafe surrogate. Using a previous described qPCR assay, a modified PCR amplicon for the staG gene was cloned into E. coli. We developed a target region that was recognized by the Typhoid primers in addition to a non-coding internal probe sequence. A multiplex qPCR reaction was developed that differentiates between the typhoid and control targets, with no cross-reactivity or inhibition of the two probes. The PCO was shown to mimic S. Typhi in lab-based experiments with concentration methods using primary wastewater: filter cartridge, recirculating Moore swabs, membrane filtration, and differential centrifugation. Across all methods, the PCO seeded at 10 CFU/mL and 100 CFU/mL was detected in 100% of replicates. The PCO is detected at similar quantification cycle (Cq) values across all methods at 10 CFU/mL (Average = 32.4, STDEV = 1.62). The PCO was also seeded into wastewater at collection sites in Vellore (India) and Blantyre (Malawi) where S. Typhi is endemic. All methods tested in both countries were positive for the seeded PCO. The PCO is an effective way to validate performance of environmental surveillance methods targeting S. Typhi in surface water.

Development of a computational model to inform environmental surveillance sampling plans for Salmonella enterica serovar Typhi in wastewater.

Typhoid fever-an acute febrile disease caused by infection with the bacterium Salmonella enterica serotype Typhi (S. Typhi)-continues to be a leading cause of global morbidity and mortality, particularly in developing countries with limited access to safe drinking water and adequate sanitation. Environmental surveillance, the process of detecting and enumerating disease-causing agents in wastewater, is a useful tool to monitor the circulation of typhoid fever in endemic regions. The design of environmental surveillance sampling plans and the interpretation of sampling results is complicated by a high degree of uncertainty and variability in factors that affect the final measured pathogens in wastewater samples, such as pathogen travel time through a wastewater network, pathogen dilution, decay and degradation, and laboratory processing methods. Computational models can, to an extent, assist in the design of sampling plans and aid in the evaluation of how different contributing factors affect sampling results. This study presents a computational model combining dynamic and probabilistic modeling techniques to estimate-on a spatial and temporal scale-the approximate probability of detecting S. Typhi within a wastewater system. This model may be utilized to inform environmental surveillance sampling plans and may provide useful insight into selecting appropriate sampling locations and times and interpreting results. A simulated applied modeling scenario is presented to demonstrate the model's functionality for aiding an environmental surveillance study in a typhoid-endemic community.

Air pollution and age-dependent changes in emotional behavior across early adolescence in the U.S.

Recent studies have linked air pollution to increased risk for behavioral problems during development, albeit with inconsistent findings. Additional longitudinal studies are needed that consider how emotional behaviors may be affected when exposure coincides with the transition to adolescence - a vulnerable time for developing mental health difficulties. This study investigates if annual average PM2.5 and NO2 exposure at ages 9-10 years moderates age-related changes in internalizing and externalizing behaviors over a 2-year follow-up period in a large, nationwide U.S. sample of participants from the Adolescent Brain Cognitive Development (ABCD) Study®. Air pollution exposure was estimated based on the residential address of each participant using an ensemble-based modeling approach. Caregivers answered questions from the Child Behavior Checklist (CBCL) at the baseline, 1-year follow-up, and 2-year follow-up visits, for a total of 3 waves of data; from the CBCL we obtained scores on internalizing and externalizing problems plus 5 syndrome scales (anxious/depressed, withdrawn/depressed, rule-breaking behavior, aggressive behavior, and attention problems). Zero-inflated negative binomial models were used to examine both the main effect of age as well as the interaction of age with each pollutant on behavior while adjusting for various socioeconomic and demographic characteristics. Against our hypothesis, there was no evidence that greater air pollution exposure was related to more behavioral problems with age over time.

Correlation between wastewater and COVID-19 case incidence rates in major California sewersheds across three variant periods.

Monitoring for COVID-19 through wastewater has been used for adjunctive public health surveillance, with SARS-CoV-2 viral concentrations in wastewater correlating with incident cases in the same sewershed. However, the generalizability of these findings across sewersheds, laboratory methods, and time periods with changing variants and underlying population immunity has not been well described. The California Department of Public Health partnered with six wastewater treatment plants starting in January 2021 to monitor wastewater for SARS-CoV-2, with analyses performed at four laboratories. Using reported PCR-confirmed COVID-19 cases within each sewershed, the relationship between case incidence rates and wastewater concentrations collected over 14 months was evaluated using Spearman's correlation and linear regression. Strong correlations were observed when wastewater concentrations and incidence rates were averaged (10- and 7-day moving window for wastewater and cases, respectively, ρ = 0.73-0.98 for N1 gene target). Correlations remained strong across three time periods with distinct circulating variants and vaccination rates (winter 2020-2021/Alpha, summer 2021/Delta, and winter 2021-2022/Omicron). Linear regression revealed that slopes of associations varied by the dominant variant of concern, sewershed, and laboratory (ß = 0.45-1.94). These findings support wastewater surveillance as an adjunctive public health tool to monitor SARS-CoV-2 community trends.

Air pollution and emotional behavior in adolescents across the U.S.

Recent studies have linked air pollution to increased risk for behavioral problems during development, albeit with inconsistent findings. Additional longitudinal studies are needed that consider how emotional behaviors may be affected when exposure coincides with the transition to adolescence - a vulnerable time for developing mental health difficulties. This study examines how annual average PM2.5 and NO2 exposure at ages 9-10 years relates to internalizing and externalizing behaviors over a 2-year follow-up period in a large, nationwide U.S. sample of participants from the Adolescent Brain Cognitive Development (ABCD) Study®. Air pollution exposure was estimated based on the residential address of each participant using an ensemble-based modeling approach. Caregivers answered questions from the Child Behavior Checklist (CBCL) at baseline and annually for two follow-up sessions for a total of 3 waves of data; from the CBCL we obtained scores on internalizing and externalizing problems plus 5 syndrome scales (anxious/depressed, withdrawn/depressed, rule-breaking behavior, aggressive behavior, and attention problems). Zero-inflated negative binomial models were used to examine both the main effect of age as well as the interaction of age with each pollutant on behavior while adjusting for various socioeconomic and demographic characteristics. Overall, the pollution effects moderated the main effects of age with higher levels of PM2.5 and NO2 leading to an even greater likelihood of having no behavioral problems (i.e., score of zero) with age over time, as well as fewer problems when problems are present as the child ages. Albeit this was on the order equal to or less than a 1-point change. Thus, one year of annual exposure at 9-10 years is linked with very small change in emotional behaviors in early adolescence, which may be of little clinical relevance.

Ambient fine particulate exposure and subcortical gray matter microarchitecture in 9- and 10-year-old children across the United States.

Neuroimaging studies showing the adverse effects of air pollution on neurodevelopment have largely focused on smaller samples from limited geographical locations and have implemented univariant approaches to assess exposure and brain macrostructure. Herein, we implement restriction spectrum imaging and a multivariate approach to examine how one year of annual exposure to daily fine particulate matter (PM2.5), daily nitrogen dioxide (NO2), and 8-h maximum ozone (O3) at ages 9-10 years relates to subcortical gray matter microarchitecture in a geographically diverse subsample of children from the Adolescent Brain Cognitive Development (ABCD) Study℠. Adjusting for confounders, we identified a latent variable representing 66% of the variance between one year of air pollution and subcortical gray matter microarchitecture. PM2.5 was related to greater isotropic intracellular diffusion in the thalamus, brainstem, and accumbens, which related to cognition and internalizing symptoms. These findings may be indicative of previously identified air pollution-related risk for neuroinflammation and early neurodegenerative pathologies.

Evaluation of Sampling and Concentration Methods for Salmonella enterica Serovar Typhi Detection from Wastewater.

Salmonella enterica serovar (Salmonella Typhi) is the causative bacterial agent of typhoid fever. Environmental surveillance of wastewater and wastewater-impacted surface waters has proven effective in monitoring various pathogens and has recently been applied to Salmonella Typhi. This study evaluated eight sample collection and concentration methods with 12 variations currently being developed and used for Salmonella Typhi surveillance globally to better understand the performance of each method based on its ability to detect Salmonella Typhi and its feasibility. Salmonella Typhi strains Ty21a and Ty2 were seeded to influent wastewater at known concentrations to evaluate the following methods: grab sampling using electropositive filters, centrifugation, direct enrichment, or membrane filtration and trap sampling using Moore swabs. Concentrated samples underwent nucleic acid extraction and were detected and/or quantified via quantitative polymerase chain reaction (qPCR). Results suggest that all methods tested can be successful at concentrating Salmonella Typhi for subsequent detection by qPCR, although each method has its own strengths and weaknesses, including the Salmonella Typhi concentration it is best suited for, with a range of positive detections observed as low as 0.1-0.001 colony-forming units (CFU) Ty21a/mL and 0.01 CFU Ty2/mL. These factors should be considered when identifying a method for environmental surveillance and will greatly depend on the use case planned.

Neurotoxicants, the Developing Brain, and Mental Health.

While life in urban environments may confer a number of benefits, it may also result in a variety of exposures, with toxic consequences for neurodevelopment and neuropsychological health. Neurotoxicants are any of a large number of chemicals or substances that interfere with normal function and/or compromise adaptation in the central and/or peripheral nervous system. Evidence suggests that neurotoxicant effects have a greater effect when occurring in utero and during early childhood. Recent findings exploring neural-level mechanisms provide a crucial opportunity to explore the ways in which environmental conditions may get "under the skin" to impact a number of psychological behaviors and cognitive processes, ultimately allowing for greater synergy between macro- and microlevel efforts to improve mental health in the presence of neurotoxicant exposures. In this review, we provide an overview of 3 types of neurotoxicants related to the built environment and relevant to brain development during childhood and adolescence: lead exposure, outdoor particulate matter pollution, and endocrine-disrupting chemicals. We also discuss mechanisms through which these neurotoxicants affect central nervous system function, including recent evidence from neuroimaging literature. Furthermore, we discuss neurotoxicants and mental health during development in the context of social determinants and how differences in the spatial distribution of neurotoxicant exposures result in health disparities that disproportionately affect low-income and minority populations. Multifaceted approaches incorporating social systems and their effect on neurotoxicant exposures and downstream mental health will be key to reduce societal costs and improve quality of life for children, adolescents, and adults.

Association of Outdoor Ambient Fine Particulate Matter With Intracellular White Matter Microstructural Properties Among Children.

Importance: Outdoor particulate matter 2.5 µm or less in diameter (PM2.5) is a ubiquitous environmental neurotoxicant that may affect the developing brain. Little is known about associations between PM2.5 and white matter connectivity. Objectives: To assess associations between annual residential PM2.5 exposure and white matter microstructure health in a US sample of children 9 to 10 years of age and to examine whether associations are specific to certain white matter pathways or vary across neuroimaging diffusion markers reflective of intracellular and extracellular microstructural processes. Design, Setting, and Participants: This cross-sectional study, the Adolescent Brain and Cognitive Development (ABCD) Study, was composed of 21 study sites across the US and used baseline data collected from children 9 to 10 years of age from September 1, 2016, to October 15, 2018. Data analysis was performed from September 15, 2020, to June 30, 2021. Exposures: Annual mean PM2.5 exposure estimated by ensemble-based models and assigned to the primary residential addresses at baseline. Main Outcomes and Measures: Diffusion-weighted imaging (DWI) and tractography were used to delineate white matter tracts. The biophysical modeling technique of restriction spectrum imaging (RSI) was implemented to examine total hindered diffusion and restricted isotropic and anisotropic intracellular diffusion in each tract. Hierarchical mixed-effects models with natural splines were used to analyze the associations between PM2.5 exposure and DWI. Results: In a study population of 7602 children (mean [SD] age, 119.1 [7.42] months; 3955 [52.0%] female; 160 [ 21.%] Asian, 1025 [13.5%] Black, 1616 [21.3%] Hispanic, 4025 [52.9%] White, and 774 [10.2%] other [identified by parents as American Indian/Native American or Alaska Native; Native Hawaiian, Guamanian, Samoan, other Pacific Islander; Asian Indian, Chinese, Filipino, Japanese, Korean, Vietnamese, or other Asian; or other race]), associations were seen between annual ambient PM2.5 and hemispheric differences in white matter microstructure. Hemisphere-stratified models revealed significant associations between PM2.5 exposure and restricted isotropic intracellular diffusion in the left cingulum, in the left superior longitudinal fasciculus, and bilaterally in the fornix and uncinate fasciculus. In tracts with strong positive associations, a PM2.5 increase from 8 to 12 µg/m3 was associated with increases of 2.16% (95% CI, 0.49%-3.84%) in the left cingulum, 1.95% (95% CI, 0.43%-3.47%) in the left uncinate, and 1.68% (95% CI, 0.01%-3.34%) in the right uncinate. Widespread negative associations were observed between PM2.5 and mean diffusivity. Conclusions and Relevance: The findings of this cross-sectional study suggest that annual mean PM2.5 exposure during childhood is associated with increased restricted isotropic diffusion and decreased mean diffusivity of specific white matter tracts, potentially reflecting differences in the composition of white matter microarchitecture.

Emergency Medical Service Utilization and Response Following COVID-19 Emergency and Stay-at-Home Policies: An Interrupted Time-Series Analysis.

Objective Examine changing emergency medical services (EMS) utilization and response patterns associated with coronavirus disease 2019 (COVID-19) emergency declaration and stay-at-home orders during the first year of the COVID-19 pandemic. Methods We conducted an uncontrolled interrupted time series analysis of EMS calls (January 1, 2019 - March 1, 2021) in Marin County, California analyzing call volume (All calls, n=46,055); patient refusal of EMS care or transport and patient care resolved on scene (Calls with opportunity for transport; n=37,401); and call severity (Transported calls; n=27,887). Results Pre-COVID-19 (1/1/2019-3/2/2020), EMS transported patients were predominately female (50.6%), 80+ years old (31.6%), and Marin County residents (68.0%). During COVID-19 (3/3/2020-3/1/2021), EMS transported patients were predominately male (52.7%), 35-64 years old (29.8%), and Marin County residents (70.4%). After the first stay-at-home order on 3/17/2020, call volume immediately decreased by 48% (adjusted incidence rate ratio [aIRR]=0.52; 95% CI=0.35,0.79) for children (0-15 years) and 34% for adults 80+ years (aIRR=0.66;95% CI=0.46,0.95). The odds of a transported call being prioritized as severe doubled (adjusted odds ratio [aOR]=2.26; 95% CI=1.11,4.59). Though transport refusals increased by 69% for children after the first order (aOR, 1.69 [95% CI, 1.13-2.52]), immediately following the second order on 12/8/2020, transport refusals decreased by 30% for children but increased 38-40% for adults 35-79 years (aOR=1.40 [95% CI=1.04-1.89] for 35-64 years; 1.38 [95% CI=1.02-1.87] for 65-79 years). Calls resolved on scene by EMS increased after the first order among all ages and after the second order for adults 16-79 years.  Conclusions Call volume reduced for children and older adults after the first COVID-19 stay-at-home order. Changes in call severity, patient care refusals, and on-scene care provided by EMS indicated a changing role for EMS during the outbreak.

A comparison of SARS-CoV-2 wastewater concentration methods for environmental surveillance.

Wastewater1 surveillance of SARS-CoV-2 may be a useful supplement to clinical surveillance as it is shed in feces, there are many asymptomatic cases, and diagnostic testing can have capacity limitations and extended time to results. Although numerous studies have utilized wastewater surveillance for SARS-CoV-2, the methods used were developed and/or standardized for other pathogens. This study evaluates multiple methods for concentration and recovery of SARS-CoV-2 and seeded human coronavirus OC43 from municipal primary wastewater and/or sludge from the Greater Seattle Area (March-July 2020). Methods evaluated include the bag-mediated filtration system (BMFS), with and without Vertrel™ extraction, skimmed milk flocculation, with and without Vertrel™ extraction, polyethylene glycol (PEG) precipitation, ultrafiltration, and sludge extraction. Total RNA was extracted from wastewater concentrates and analyzed for SARS-CoV-2 and OC43 with RT-qPCR. Skimmed milk flocculation without Vertrel™ extraction performed consistently over time and between treatment plants in Seattle-area wastewater with the lowest average OC43 Cq value and smallest variability (24.3; 95% CI: 23.8-24.9), most frequent SARS-CoV-2 detection (48.8% of sampling events), and highest average OC43 percent recovery (9.1%; 95% CI: 6.2-11.9%). Skimmed milk flocculation is also beneficial because it is feasible in low-resource settings. While the BMFS had the highest average volume assayed of 11.9 mL (95% CI: 10.7-13.1 mL), the average OC43 percent recovery was low (0.7%; 95% CI: 0.4-1.0%). Ultrafiltration and PEG precipitation had low average OC43 percent recoveries of 1.0% (95% CI: 0.5-1.6%) and 3.2% (95% CI: 1.3-5.1%), respectively. The slopes and efficiency for the SARS-CoV-2 standard curves were not consistent over time, confirming the need to include a standard curve each run rather than using a single curve for multiple plates. Results suggest that the concentration and detection methods used must be validated for the specific water matrix using a recovery control to assess performance over time.

ADAM23 is a negative regulator of Kv1.1/Kv1.4 potassium currents.

Background ADAM22 and ADAM23 are transmembrane proteins that bind the secreted synaptic protein LGI1 and associate with Kv1.1/Kv1.4 potassium channels. However, the roles of these proteins in regulated voltage-gated potassium currents are poorly understood. Methods Cultured cells were transfected to express ADAM22, ADAM23, and Kv1.1/Kv1.4. Voltage-gated potassium currents were measured by whole-cell patch-clamp. Immunostaining Kv1.1 with fluorescent antibodies and fluorescently tagged Kv1.1 subunits was used to measure the effects of ADAM proteins on cell-surface and total expression of Kv1.1 channels. LGI1-conditioned media was added to assess the effect on LGI1 on Kv1.1 currents. Results Cells transfected with Kv1.1/Kv1.4 showed voltage-gated potassium currents (Kv1.1 currents). ADAM23 was a powerful negative regulator of Kv1.1 currents and caused decreased surface expression of Kv1.1 subunits. This decrease in current was not mediated by clathrin-dependent endocytosis. LGI1-conditioned media did not affect the negative regulation of Kv1.1 currents by ADAM23. ADAM22 had no significant effect on Kv1.1 currents by itself, but in the presence of LGI1-conditioned media markedly potentiated Kv1.1 currents without changing channel activation kinetics. Conclusions ADAM22 and ADAM23 have opposite effects on Kv1.1 currents. The relative expression of these proteins, and the availability of LGI1 may shape the expression of Kv1.1 currents in different neuronal membrane domains.

Neurofascin antibodies in autoimmune, genetic, and idiopathic neuropathies.

OBJECTIVE: To measure the frequency, persistence, isoform specificity, and clinical correlates of neurofascin antibodies in patients with peripheral neuropathies. METHODS: We studied cohorts of patients with Guillain-Barre syndrome (GBS) or chronic inflammatory demyelinating polyneuropathy (CIDP) (n = 59), genetic neuropathy (n = 111), and idiopathic neuropathy (n = 43) for immunoglobulin (Ig) G and IgM responses to 3 neurofascin (NF) isoforms (NF140, NF155, and NF186) using cell-based assays. RESULTS: Neurofascin antibodies were more common in patients with GBS/CIDP (14%, 8 of 59) compared to genetic neuropathy controls (3%, 3 of 111, p = 0.01). Seven percent (3 of 43) of patients with idiopathic neuropathy also had neurofascin antibodies. NF155 IgG4 antibodies were associated with CIDP refractory to IV immunoglobulin but responsive to rituximab, and some of these patients had an acute onset resembling GBS. NF186 IgG and IgM to either isoform were less specific. A severe form of CIDP, approaching a locked-in state, was seen in a patient with antibodies recognizing all 3 neurofascin isoforms. CONCLUSIONS: Neurofascin antibodies were 4 times more frequent in autoimmune neuropathy samples compared to genetic neuropathy controls. Persistent IgG4 responses to NF155 correlated with severe CIDP resistant to usual treatments but responsive to rituximab. IgG4 antibodies against the common domains shared by glial and axonal isoforms may portend a particularly severe but treatable neuropathy. The prognostic implications of neurofascin antibodies in a subset of idiopathic neuropathy patients and transient IgM responses in GBS require further investigation.