Drinking water sources, quality, and associated health outcomes in Appalachian Virginia: A risk characterization study in two counties.

OBJECTIVES: In the US, violations of drinking water regulations are highest in lower-income rural areas overall, and particularly in Central Appalachia. However, data on drinking water use, quality, and associated health outcomes in rural Appalachia are limited. We sought to assess public and private drinking water sources and associated risk factors for waterborne pathogen exposures for individuals living in rural regions of Appalachian Virginia. METHODS: We administered surveys and collected tap water, bottled water, and saliva samples in lower-income households in two adjacent rural counties in southwest Virginia (bordering Kentucky and Tennessee). Water samples were tested for pH, temperature, conductivity, total coliforms, E. coli, free chlorine, nitrate, fluoride, heavy metals, and specific pathogen targets. Saliva samples were analyzed for antibody responses to potentially waterborne infections. We also shared water analysis results with households. RESULTS: We enrolled 33 households (83 individuals), 82% (n = 27) with utility-supplied water and 18% with private wells (n = 3) or springs (n = 3). 58% (n = 19) reported household incomes of <$20,000/year. Total coliforms were detected in water samples from 33% (n = 11) of homes, E. coli in 12%, all with wells or springs (n = 4), and Aeromonas, Campylobacter, and Enterobacter in 9%, all spring water (n = 3). Diarrhea was reported for 10% of individuals (n = 8), but was not associated with E. coli detection. 34% (n = 15) of saliva samples had detectable antibody responses for Cryptosporidium spp., C. jejuni, and Hepatitis E. After controlling for covariates and clustering, individuals in households with septic systems and straight pipes had significantly higher likelihoods of antibody detection (risk ratios = 3.28, 95%CI = 1.01-10.65). CONCLUSIONS: To our knowledge, this is the first study to collect and analyze drinking water samples, saliva samples, and reported health outcome data from low-income households in Central Appalachia. Our findings indicate that utility-supplied water in this region was generally safe, and individuals in low-income households without utility-supplied water or sewerage have higher exposures to waterborne pathogens.

Time outdoors and residential greenness are associated with reduced systemic inflammation and allostatic load.

Contacts with nature are linked with reduced morbidity and mortality. Hypothesized pathways include relaxation, physical activity, and improved immune function. This cross-sectional study of 320 adults in central North Carolina assessed health benefits of residential greenness using allostatic load (AL) and systemic inflammation (INFL) indices, composite biomarker-based measures of physiological dysregulation and inflammation, respectively. Distance-to-residence weighted tree cover and vegetated land cover measures were estimated within 500 m of each residence; 37 biomarkers of immune, neuroendocrine, cardiovascular, and metabolic functions were dichotomized at distribution or health-based cut-offs. AL was calculated as a sum of potentially unhealthy values of all biomarkers; INFL was based on a subset of 18 immune biomarkers. Regression analysis used generalized additive models for Poisson-distributed outcome. An interquartile range (IQR) increase in tree cover was associated with 0.89 (95 % Confidence Limits 0.82; 0.97) and 0.90 (0.79; 1.03)-fold change in AL and INFL, respectively. Greater daily outdoor time was associated with reduced AL and INFL, while leisure screen time, problems with sleeping, and common chronic infections were linked with increased AL and INFL. Among 138 individuals spending more than 1 h outdoors daily, an IQR increase in tree cover was associated with 0.76 (0.67; 0.86) and 0.81 (0.65; 1.02)-fold changes in AL and INFL, respectively. Among individuals with residential tree cover above the 50th percentile, spending more than 3 h outdoors daily was associated with 0.54 (0.37; 0.78) and 0.28 (0.15; 0.54)-fold changes in AL and INFL, respectively, compared to spending less than 30 min outdoors; there were no significant effects in the low tree cover stratum. Consistent but weaker effects were observed for vegetated land cover. Interaction effects of tree and vegetative cover and time spent outdoors on AL and INFL were statistically significant. This biomarker-based approach can help to assess public health benefits of green spaces.

CRISPR/Cas9 mutagenesis of the Arabidopsis GROWTH-REGULATING FACTOR (GRF) gene family.

Genome editing in plants typically relies on T-DNA plasmids that are mobilized by Agrobacterium-mediated transformation to deliver the CRISPR/Cas machinery. Here, we introduce a series of CRISPR/Cas9 T-DNA vectors for minimal settings, such as teaching labs. Gene-specific targeting sequences can be inserted as annealed short oligonucleotides in a single straightforward cloning step. Fluorescent markers expressed in mature seeds enable reliable selection of transgenic or transgene-free individuals using a combination of inexpensive LED lamps and colored-glass alternative filters. Testing these tools on the Arabidopsis GROWTH-REGULATING FACTOR (GRF) genes, we were able to create a collection of predicted null mutations in all nine family members with little effort. We then explored the effects of simultaneously targeting two, four and eight GRF genes on the rate of induced mutations at each target locus. In our hands, multiplexing was associated with pronounced disparities: while mutation rates at some loci remained consistently high, mutation rates at other loci dropped dramatically with increasing number of single guide RNA species, thereby preventing a systematic mutagenesis of the family.

Recreational water exposure and waterborne infections in a prospective salivary antibody study at a Lake Michigan beach.

In a prospective observational study, seroconversion to a specific pathogen can serve as a marker of an incident infection, whether or not that infection is symptomatic or clinically diagnosed. While self-reported symptoms can be affected by reporting bias, seroconversion is likely to be free of this bias as it is based on objective measurements of antibody response. Non-invasive salivary antibody tests can be used instead of serum tests to detect seroconversions in prospective studies. In the present study, individuals and families were recruited at a Lake Michigan beach in Wisconsin in August 2011. Data on recreational water exposure and baseline saliva samples (S1) were collected at recruitment. Follow-up data on gastrointestinal symptoms were collected via a telephone interview approximately 10 days post-recruitment. Follow-up saliva samples were self-collected approximately 2 weeks (S2) and 30-40 days post-recruitment (S3) and mailed to the study laboratory. Samples were analyzed for immunoglobulin (Ig) G responses to recombinant antigens of three noroviruses and Cryptosporidium, as well as protein purification tags as internal controls, using an in-house multiplex suspension immunoassay on the Luminex platform. Responses were defined as ratios of antibody reactivities with a target protein and its purification tag. Seroconversions were defined as at least four-fold and three-fold increases in responses in S2 and S3 samples compared to S1, respectively. In addition, an S2 response had to be above the upper 90% one-sided prediction limit of a corresponding spline function of age. Among 872 study participants, there were seven (0.8%) individuals with seroconversions, including six individuals with seroconversions to noroviruses and two to Cryptosporidium (one individual seroconverted to both pathogens). Among 176 (20%) individuals who reported swallowing lake water, there were six (3.4%) seroconversions compared to one (0.14%) seroconversion among the remaining 696 individuals: the crude and age-standardized risk differences per 1000 beachgoers were 32.7 (95% confidence limits 5.7; 59.6) and 94.8 (4.6; 276), respectively. The age-adjusted odds ratio of seroconversion in those who swallowed water vs. all others was 49.5 (4.5; 549), p = 0.001. Individuals with a norovirus seroconversion were more likely to experience vomiting symptoms within 4 days of the index beach visit than non-converters with an odds ratio of 34 (3.4, 350), p = 0.003. This study contributed further evidence that recreational water exposure is associated with symptomatic and asymptomatic waterborne infections, and that salivary antibody assays can be used in epidemiological surveys of norovirus and Cryptosporidium infections.

A Multiplex Noninvasive Salivary Antibody Assay for SARS-CoV-2 Infection and Its Application in a Population-Based Survey by Mail.

Noninvasive salivary antibody immunoassays can enable low-cost epidemiological surveillance of infections. This study involved developing and validating a multiplex suspension immunoassay on the Luminex platform to measure immunoglobulin G (IgG) responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid and spike (S) proteins, and the spike protein's S1 and S2 subunits and receptor binding domain. Multiple versions of these recombinant proteins acquired from commercial and noncommercial sources were evaluated. Assay development and validation utilized saliva and serum samples from coronavirus disease 2019 (COVID-19) cases procured from commercial sources and negative controls from a prepandemic survey. Saliva was also collected in a demonstration survey by mail involving adult individuals in the United States who were diagnosed with SARS-CoV-2 infection 15 to 80 days prior to sample collection. The survey had an 83% valid sample return rate (192 samples from 38 states). Most COVID-19 cases (93%) reported mildly symptomatic or asymptomatic infections. The final salivary assay based on the best-performing spike and nucleocapsid proteins had a sensitivity of 87.1% (95% bootstrap confidence interval, 82.1 to 91.7%) and specificity of 98.5% (95.0 to 100%) using 227 and 285 saliva samples, respectively. The same assay had 95.9% (92.8 to 98.9%) sensitivity and 100% (98.4 to 100%) specificity in serum (174 and 285 serum samples, respectively). Salivary and serum antibody responses to spike and nucleocapsid proteins were strongly correlated in 22 paired samples (r = 0.88 and r = 0.80, respectively). Antibody responses peaked at approximately 50 days postonset; greater illness severity was associated with stronger responses. This study demonstrated that a salivary antibody assay can be used in large-scale population surveys by mail to better characterize public health impacts of COVID-19. IMPORTANCE Given the enormous impacts of the COVID-19 pandemic, developing tools for population surveillance of infection is of paramount importance. This article describes the development of a multiplex immunoassay on a Luminex platform to measure salivary immunoglobulin G responses to the spike protein, its two subunits and receptor binding domain, and the nucleocapsid protein of SARS-CoV-2. The assay validation utilized serum and saliva samples from prepandemic controls and recent COVID-19 cases. A survey by mail targeting recent COVID-19 cases across the United States also demonstrated the utility of safe, at-home self-collection of saliva. By incorporating multiple SARS-CoV-2 proteins, this assay may differentiate responses to natural SARS-CoV-2 infections from responses to most vaccines. Application of this noninvasive immunoassay in COVID-19 surveillance can help provide estimates of cumulative incidence rates of symptomatic and asymptomatic infections in various communities and subpopulations, temporal patterns of antibody responses, and risk factors for infection.